Annual Review of Immunology Volume 1 1983

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Annual Reviews

Annual Reviews


Ant~ Rev. ImmunoL 1983. 1.’1-32 Copyright©1983by .4nnualReviewsInc. All rights reserved

GETTING STARTED 50 YEARS AGOmEXPERIENCES, PERSPECTIVES, AND PROBLEMSOF THE FIRST 21 YEARS Elvin A. Kabat Departmentsof Microbiologyand HumanGenetics and Development,and the CancerCenter/Institute for CancerResearch, ColumbiaUniversity College of Physicians and Surgeons, NewYork, NewYork10032; and the National Institute of Allergyand Infectious Diseases,NationalInstitutes of Health, Bethesda, Maryland20205

This first volumeof the Annual Review of Immunologywill appear just 50 years after I began working in immunochemistryin Michael Heidelberger’s laboratory at ColumbiaUniversity’s College of Physicians and Surgeons. The differences between then and nowin the path for a graduate student, postdoctoral, and beginning independent investigator are striking, and I thought for this prefatory chapter I wouldrecount someof the experiences of myfirst 21 years in the field, considering not only the workbut also the outside economicand political influences on mycareer, as well as World War II and its aftermath of loyalty and security investigations and the Senator Joseph McCarthy period. On January 1, 1933, I began working in Michael Heidelberger’s laboratory as a laboratory helper. Thedefinitive paper on the quantitative precipitin method by Heidelberger and Forrest E. Kendall had appeared in the Journal of Experimental Medicine in 1929 and the laboratory was well on its way to providing analytical chemical methodsfor measurementof antigens and antibodies. This paper furnished the. key to modernstructural immunologyand immunochemistry, which, not without strong resistance from the then classical immunologists, changed our way of thinking. 1 0732-0582/83/0410-0001 $02.00

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I had received a BSdegree in September, 1932, from the City College of NewYork and had majored in chemistry. I was 18 years old. The country was in the depths of the depression and myfamily had suffered acutely. I had been walking the length and breadth of NewYork visiting universities and hospitals looking for a job, literally walking,since I rarely had a nickel to take the subwayand if I had only a nickel I used it for lunch. Mymother had started selling dresses in our apartment; by chance Mrs. Nina Heidelberger had becomeone of her customers and she suggested I go up to see Michael. At the end of October or early Novemberof 1932, he offered me a job at a salary of $90 a monthto begin on the first of January, and the possibility of a future for me and for myfamily began. Michaelhad hesitated about giving me the job since I had been to college and the previous incumbenthad only been a high school graduate. I assured him that I would do the routine, which involved makingsolutions, keeping the laboratory clean, and washing glassware when MaryO’Neill, our halftime glassware worker, was ill, etc, provided he would let me do as much technical work and research as I was capable of doing. Hans T. Clarke, the Chairman of the Biochemistry Department at P and $, had interviewed me in connection with myapplication to do graduate work and had accepted me as a PhDcandidate. I had explained that I would be unable to begin until I found a job, but this was the norm during the depression. Many graduate students had part- or full-time jobs; somewere teaching at City College or elsewhere and were doing their graduate studies part-time at Columbia. Of the $90 a month, I had to give myparents $50 toward paying the rent and I had to pay the Columbia tuition, then $10 a point per semester, for mycourses, plus registration and laboratory fees. Myhours were 8:30 A.~. to 5:00 P.~., and beginning in February, 1933, I took an evening course in experimental physical chemistry with Professor Charles O. Beckman.Mygraduate courses were taken in the evening or late afternoon, or during the summersession, except for those given at P and S. During the summerof 1933 I took the course in medical bacteriology given by Calvin B. Coulter. Webecame good friends and he, Florence Stone, and I collaborated in a study of the ultraviolet absorption spectra of a numberof proteins I had prepared in Michael’s laboratory. Throughhim I met Arthur Shapiro, then a medical student, whowas full of ideas. We becamevery close friends; he was one of the very first to see the potentialities of bacterial genetics and several years later Sol Spiegelmanworkedwith him. One of the required courses, which was given only in the daytime at the downtowncampus, was quantitative organic analysis and students had to spend lots of extra time in the lab. Myjob madethis ditficult, so Hans Clarke, whohad written the classical text in the field, gave me 18 compounds to identify and I did these in Michael’s lab.

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The Department of Biochemistry was at its peak in reputation and productivity with Michael, Rudolf Schoenheimer, Karl Meyer, Erwin Chargaff, Erwin Brand, SamGurin, David Rittenberg, and SamGraft. Graduate students workedin a large laboratory on the fifth floor and included Joe Fruton, Lew Engel, David Shemin, De Witt Stetten Jr, Sarah Ratner, Konrad Bloch, William H. Stein, Abe Mazur, and Ernest Borek, and somewhat later SeymourCohen and David Sprinson. I got to know W. E. van Heyningen,whocameto us as a postdoctoral fellow. Since I workeddirectly in Michael’s lab mycontacts with the other students were muchless close. Myinterests also led me to be close to the Bacteriology Department, of which Frederick P. Gay was Chairman and whose members included Beatrice Seegal, Theodor Rosebury, James T. Culbertson, Claus Jungeblut, MaximSteinbach, Calvin Coulter, Florence Stone, Sidney J. Klein, and Rose R. Feiner. I attended both the weekly biochemistry and microbiology seminars. Professor Gay, whohad been closely associated with Jules Bordet and was still waging the Paul Ehrlich-Jules Bordet war of antibodies as substances rather than as vague properties of immuneserum, was strongly anti-immunochemical and became very impatient with my questions at seminars. This view came across clearly to the medical students and Edward H. Reisner, P and S ’39, wrote the following little ditty, which I learned about from Oscar Ratnoff. PasteurinspiredMetchnikoff andMetchnikoff wasnuts, Hehada pupil namedBordetwhohated Ehrlich’sguts, Now Ehrlichhad the goodsyouknow,but this wedare not say, For wedescendfromMetchnikoff throughBordetout of Gay. The Heidelberger laboratory in 1933-37 consisted of Forrest E. Kendall, who was working on pneumococcal polysaccharides and on the mechanism of the precipitin reaction, Arthur E. O. Menzel, who was studying the proteins and polysaccharides of the tubercle bacillus, Check M. Soo Hoo, our bacteriologist, myself, and MaryO’Neill. Several visiting scientists, among whomwere Henry W. Scherp, Torsten Teorell, D. L. Shrivastava, Alfred J. Weil, and Maurice Stacey, camefor varying periods, the longest being 1 year. WhenForrest left in 1936 to go to Goldwater Memorial Hospital with David Seegal, Henry P. Treffers, whohad just completed his PhD with Louis P. Hammettat Columbia, replaced him. Forrest Kendall worked opposite me and taught me the micro-Kjeldahl method, which in those days was the basic tool in the laboratory since all quantitative precipitin assays on the washedprecipitates were finally analyzed for total nitrogen. Digestions were carried out in 100-mlflasks. The workingrange was between 0.1 and 1.0 mgof N. I generally was responsible for doing all of the digestions for Michaeland Forrest, and after I acquired

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sufficient skill I also carriedout the distillations andtitrations for Michael. It wasnot unusualto run 40 distillations a day. I also ran manyquantitative iodine determinations on thyroglobulin, using the methoddevelopedby Professor G. L. Foster in the BiochemistryDepartment,until Herbert E. Stokinger becamea graduate student with Michael and was given the thyroglobulin problem. Thelaboratorywasa very exciting place in whichto workandI generally kept Forrest busyaskinghimquestions, no doubtinterfering often with his train of thought. Hewasvery patient and helpful, but Michaeloncesuggested that I had better put someof myquestions to him rather than to Forrest. I wasalwaysable to get answersor wastold whereto find them. After a few months,as Michaelhas described (2), I suggestedthat one might use a well-washedsuspension of heat-killed bacteria of knownN content to removeantibody and measureantibodyN as agglutinin. Michael said that I could do this so I started with type I pneumococci. Themethod wasvery successful, provided one took care in growingthe organismsto avoid autolysis. Whathappenswhenthis procedureis not strictly adhered to has already beenpublished(3). Thecombinedapplication of the quantitative precipitin and quantitative agglutinin methodspermittedus to establish that the antibodyto the typespecific polysaccharideof pneumococci was the samewhethermeasuredas precipitin or as agglutinin. Naturally, the bacterial suspensionwasable to removeantiprotein, and this wasmeasuredindependentlywith a suspension of rough unencapsulatedpneumococci.In those days wewere only beginning to be awareof antibodyheterogeneity,that our type-specific antibody was a complexmixture of antibodies, someof which were non- or coprecipitating, and that all of these werebeing measuredas agglutinin or as precipitin. MichaelandI also studiedthe courseof the quantitativeagglutinin reaction and showedthat it followed the empirical equation he and Forrest had described for the quantitative precipitin reaction, and which they later derived from the Lawof MassAction. Thequantitative agglutinin workwassupposedto be myPhDdissertation; the first two paperson the methodand on the identity of agglutinin and precipitin had beenpublishedin the Journalof ExperimentalMedicine in 1934and 1936. These,as reprints, and the third part on the mechanism of agglutination were submittedearly in 1937. However,the University’s rules had been changedso that the student was required to be the first author on any publication and Michaelhadbeenthe first author on the two papers. I couldof coursehaveused the third part as the thesis, but I had also been studying the quantitative precipitin reaction betweenR-saltazobiphenylazo-serum albuminand rabbit antibody to serumalbumin.This systemhadcertain unsuspecteduniqueaspects since the introductionof the

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haptenic group had not altered the reactivity with anti-serum albumin. Michaelsuggestedthat I write this up and use it as myPhDdissertation, whichI did in about 3 or 4 weeks. It created a minorsensation at the Faculty of Pure Science office whenI broughtdownthe copies of the new dissertation and took backthe other. ArneTiselius had suggestedsometime earlier that it wouldbe nice to have someonefrom Michael’s laboratory to study the physicoehemical properties of purified antibodiesat Uppsalaand that the RockefellerFoundation might provide a Fellowship. He had just developed the moving boundarymethodof electrophoresis with the rectangular cell, whichfirst madepossibleprecise quantitativeestimatesof purity andpermittedresolution of mixtures of proteins. Michaelhad spent two summersin Uppsala workingwith KaiO. Pedersenand with Tiselius and had suggestedme. This wasan extraordinaryopportunityto learn the newermethodsof fractionating and characterizing macromolecules in the Svedberglaboratory. Frank Blair Hansoncameto interview mefromthe Foundationand I wasawarded the Fellowship. The Fellowshippaid $125per month.Onthe original application there wasa questionas to whetherone hadany special financial obligations and I had noted that I gavemyparents $50a month.WhenI received the award, it specified $125per month.I wentto the Foundation’soffices to discuss whetherI could get along on $75 per month.Theylooked at myoriginal application and assured methat $50 wouldbe sent to myparents and that I wouldreceive the full $125.I madesure that I did not spendmuchof my first month’sstipenduntil several weekslater whena letter frommymother arrived(this wasbeforetransatlantic airmail)sayingthat the first checkhad beenreceivedpromptlyon September 1, 1937.After that I wasable to live quite comfortably. TheRockefellerFoundationalso paid for cabin class transportation from NewYorkto Uppsala.Since I wasentitled to a monthvacation for mywork with Michael,I wantedto spend it in Europeand especially to visit the SovietUnion,to whichat that time I wasvery favorablyinclined, predominantly becauseof the great economicsuffering of myfamily during the depressionand also becauseof Russia’s UnitedFront policy in opposition to Hitler andtheir supportof the Spanishloyalists. TheRockefellerFoundation agreed to give mewhat it wouldcost themto send medirectly in cabinclass and I could go anywhere I chose. In those days if one purchased a tourist class ticket on the QueenMary,onecould go by rail secondclass anywhere in Europefor $10 extra. I choseLeningrad,spendingseveral days in Paris and Warsaw.For $50 one could spend 10 days in Leningradand Moscow with all hotels, meals, travel betweencities, and guidedtours to places of interest included.In ParAsI visited the Institut Pasteur, sawthe

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Pasteur Museum,and met Gaston Ramon, who invited me to lunch at his home where I met Andr6 Boivin and Lydia Mesrobeanu. In the Soviet UnionI was unable to see any scientists and so I was essentially a tourist visiting museums,factories, etc, on standard guided tours. I then spent a day or two in Helsinki, took the boat to Stockholmand the train to Uppsala, and arrived at the Institute early in September. Before leaving and in anticipation of mybeing able to go to Uppsala, Michael had suggested that we immunize several different species with suspensions of pneumococci, so Soo Hoo and I injected two pigs and a monkey.These immunizations were less traumatic than those we had done earlier, injecting two goats (4). Rabbit and horse antisera were available the lab and a cow was immunized at Sharpe and Dohme.Torsten Teorell with Forrest and Michael had found that a given quantity of pneumococcal polysaccharide precipitated less antibody in 15 %salt than in physiological saline, due to a shift in the combiningproportions at equilibrium whenthe reaction was carried out in high salt. Michael and Forrest then used this finding to purify antibodies to polysaccharidesby precipitating the antibody from a large volumeof antiserum with polysaccharide under physiological conditions of 0°C, washing repeatedly with cold saline, and extracting the washedprecipitate with 15%NaCI at 37°C to dissociate a portion of the antibody. With individual antisera as much as 15-30%purified antibody could be obtained and over 90%was precipitable by polysaccharide. This was the first time substantial amounts,tens of milligrams, of antibody could be prepared. Michael and I also extended the methodby using a suspension of bacteria to remove the antibody followed by washing and elution with 15 %salt. Thesepurified antibodies plus various antisera and antigens were shipped to Uppsala and were available when I arrived. The laboratory was at a peak in its productivity with Professor Svedberg popping in and out, with Arne Tiselius, then a docent, and with Kai O. Pedersen and Ole Lamm.Numerous visitors came for various periods, including Basil Record from Haworth’s laboratory, Frank L. Horsfall from the Rockefeller Institute, whoseinterests were similar to mine, J. B. Sumner with crystalline urease and concanavalin A, G. Bressler from Leningrad, G. S. Adair from Cambridge, and Gerhard Schramm from Germany. Professor Svedberg was very anxious to test his new separation cell, which had a membranedividing the cell into an upper and a lower compartment. Using some of myhorse antipneumococcal serum, he centrifuged it until the 18S peak had gone below the membrane.I tested both proteins and found all of the antibody in the lower compartment. I had also brought someof the crystalline horse serum albumin used in myPhDthesis. Tiselius was very surprised whenwe examinedit by electrophoresis at a concentration of 0.5 or 1%and saw only a single peak. He

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said it wasthe first time he had seen a protein that wasa single component electrophoretically. Althoughnewtechniques provide moreand often better criteria for establishing purity, they frequently only showthat not enoughcare wastakenin purifyingthe material. Theneedfor such care was most forcefully demonstrated years later whenKnight (8) examined paper chromatography Emil Fischer’s collection of 34 synthetic peptides madehalf a century earlier, whichhis son Hermann had broughtto Berkeley. All but three, whichcontaineda trace of oneof the constituent amino acids, gavea single spot. I got to workright awaywith the help of Kai Pedersenlearning to run the oil turbine ultracentrifuge and the diffusion apparatusto measurethe molecularweightsof the antibodies I had brought. Surprisingly, the horse antibodies werequite polydisperse. Wewereat a loss to understandthis. Fortunately, the State SerumInstitute had a horse that had been under immunizationfor a short time, and on purifying its antibody by the salt dissociation methodwefounda single 18Speak. All of the other antibodies gave multiple peaks, as did a second samplefrom the same horse after further immunization.Therapeuticuse of antipneumococcal horse sera in Swedenhad not been very successful, so I was able to get serum from humansrecovering from lobar pneumoniawhohad not received antibody, throughthe courtesyof Jan Waldcnstrtim.I tested the serumof quite a few convalescents, found one with about 1 mgof antibody protein per ml, purified the antibodyfrom about 40 ml of serum, and measuredits molecular weight. ArneTiselius andI studied the electrophoretieproperties of antibodies. Oneof the rabbit hyperimmune anti-ovalbumin sera I had brought had 36.4%precipitable antibody. Weexaminedits electrophoretic pattern before andafter removalof the antibodyand found, by the decrease, 37.2%, in area of the gamma globulin peak, that wehad removedthe sameproportion of the total serumprotein. This definitely establishedthat these antibodies were gamma globulins [nowimmunoglobulin (Ig) G, one of the five majorimmunoglobulin classes]. Theelectrophoretic patterns wepublished appear in most textbooks of immunology. I also spent a considerableamountof time studyingspecific precipitates of ovalbuminrabbit anti-ovalbumin and horse serumalbumin-rabbit antiserumalbumindissolvedin excessantigen both by ultracentrifugation and by electrophoresis, calculating the compositionof the soluble complexes. This was all done by the Lamm scale method,whichwas very tedious and especially hard on the eyes. Beforemyreturn to the USI left a manuscript with ArneTiselius, which, becauseof the war, never waspublished, althoughhe cited our workand an electrophoretic pattern showingthe soluble complexesas a schlieren band migrating behind the ovalbuminband

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appears in Tiselius’ HarveyLecture (10). It remainedfor JonathanSinger and DanCampbellto do the definitive study. Bythat time, the automated Schlieren methodhad replaced the laborious scale methodand work was mucheasier. I have already described myexperiences at the Nobel Ceremonyin December1937 (6). The terms of the Rockefeller FoundationFellowshiprequired that the sponsorpermit the Fellowto return to a position in the laboratory from whichhe cameand Michael had assured the Foundationhe would do so. However,he told mehe woulddo his best to find mea moreindependent position, and somemonthsafter I had arrived in UppsalaI receiveda letter from Jacob Furth offering me a position as Instructor in Pathology at Cornell University MedicalCollegeat $2400per year beginningSeptember 1, 1938, to work on viruses causing tumors and leukemias in chickens. Michaeladvised meto accept and I did. I believed it wasimportantfor meto visit certain laboratories before returning to the USand discussedthis with HarryMiller of the Rockefeller Foundation.Theytended to discouragesuch travel but finally agreed that I could leave Uppsalaabout July 15 to visit Linderstrom-Lang and the State SerumInstitute in Copenhagen;J. D. Bernal, I. Fankuchen,and John Marrack in London; F. G. Hopkins in Cambridge; W. N. Haworth and MauriceStacey in Birmingham; HansKrebs in Shettield, whereI spent 4 days learning to do someenzymereactions in the Warburgapparatus; Gorter in Leyden;and den Doorende Jong in Amsterdam, finally going to Zurichto attend the International PhysiologicalCongressbefore returning to the US.All of these visits established lasting friendships andcontacts with junior as well as senior investigators, andin myreport to the Rockefeller FoundationI emphasized the desirability of providingsimilar opportunities to their other fellows. In Zurichin August,1938,everyonewasvery disturbed about the situation in Spain wherethe Franco forces were about to or had already cut Catalonia from the rest of loyalist Spain. Manyof the membersof the Loyalist government were physiologists, including Juan Negrin, the Prime Minister, and Cabrera, the ForeignMinister, and a good-sizeddelegation attended the Congress.Agroupof us felt weshoulddo somethingto express our support and organized a dinner at the Congress,whichwasvery well attendedand raised a considerablesum.I wasanxiousto go to Spainto find out whatpeople in the UScould do to help, and it wasarrangedfor meand LewEngel to go to Spainfor a few days before sailing for the US.MyUS passport wasnot valid for travel to Spainso Dr. Cabrerawroteon a sheet of paper that all border patrols shouldpermit meto enter and leave Spain without makingany marks in mypassport. Wetook a train from Zurich

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to Perpignan,France,andto Cerb~re,the usual crossing point, thinkingit wouldbe easy to get across. I very soonhada Frenchsoldier with a bayonet proddingmealong until I got on the train backto Perpignan. Wondering whatto do wedecided to visit the Spanishconsul in Perpignan. Whenhe sawthe Cabreranote, he told us to go out andget pictures taken, andwhen wereturned he at~xed themto a SpanishCarte d’Identit6 on the back of whichhe had written the contents of the Cabreraletter (Figure 1), summonedhis car and chauffeur, andhad us driven fromPerpignanto the Hotel Majesticin Barcelona.TheHotel Majesticwasthe center for foreign correspondents and there I met Herbert L. Matthews, the NewYork Times correspondent.Foodwasvery scarce and I washungryall the time, most especiallyafter the rationedmeals.Indeed,whenI left a Frenchdiningcar, on mywayback, i emptiedthe bread tray into mypockets. Mostof mytime was spent visiting hospitals wherethe woundedAmericansof the Lincoln Brigadeand other International Brigadevolunteers werebeing treated. I vividly remember speakingto one wounded anti-fascist Germansoldier who felt his problemswerenot being understoodbecausehe spokeonly German; I wasable to translate his wishesinto Englishto oneof the doctors. Onreturning to NewYork, I immediatelybeganto workat Cornell with Jacob Furth. EugeneOpie, the Chairmanof the Pathology Department, had built up an important departmentwith Jacob, MurrayAngevine,Robert A. Moore,and Richard Linton, whohad been at Columbiaand with whoseworkin India on cholera antigens I had becomefamiliar. Vincent du Vigneaudwas Chairmanof Biochemistry and permitted me to attend their weeklyseminars. I becameclose friends with W.H. Summerson and soon collaborated in studies with DeanBurk, Otto K. Behrens, Herbert Sprince, and Fritz Lipmann,whohad left Germanyand arrived shortly thereafter from Denmark.Cornell MedicalSchoolwasnot anxious to have too manyJewson its staff and du Vigneaud,whohad madea definite offer to Lipmann,indicated that he wouldresign if the appointmentdid not go through. At Cornell the mainproblemJacob Furth wishedmeto workon was to purify the virus of oneof his chickenstrains that causedleukosisif injected intravenouslyandtumorslike the Roussarcomaif injected subcutaneously. Theapproachwasessentially to centrifuge crude extracts in the Pickels air-drivenultracentrifuge,followingthe biologicalactivity by assaysin baby chicks, as well as bynitrogento estimatethe extent of purification. I wanted to buy a micro-Kjeldahlapparatus, but a previouspostdoctoral fellow with Jacob had purchaseda Dumasapparatus. Jacob, considering mea chemist and wishing to save money,wantedto get the Dumasworking. I had to insist that this wasnot a suitable methodif one hadto run manydeterminations and he finally consentedto buy a micro-Kjeldahlapparatus; he was


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Annual Reviews GETTING STARTED 50 YEARS AGO

Figure 1 SpanishCarte d’Identit6, with the Foreign Minister’s letter.

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very happywhenhe saw 40 to 50 analyses a day comingout immediately and told methat they had tried to use the Dumasfor a wholeyear without a single successfulresult. Albert Claudehad essentially done a study with Rous sarcomavirus similar to whatwewere planningand had foundthat high-speedsedimentable materialswerepresentin normaltissues. This wasall in the daysbefore subcellular organdies, mitochondria,microsomes, etc, and it took mea long time to convinceJacobthat onecouldnot get a purevirus just by centrifugation of tumorextracts. Among our interesting findings at that time werethe demonstrationthat alkaline phosphatase and the Forssmanand Wassermannantigens wereassociated with these high-molecular-weight particles. Wealso preparedrabbit antisera, whichweused to try to characterizethese materials. Jacob also suggestedthat I try to workout somehistochemicalmethods for localizing enzymesin tissues. I wasjust getting started whenGeorge Gomoripublishedhis elegant methodfor localizing alkaline phosphatasein paratfin sections of alcohol-fixedtissues. Weadoptedit andcarried out a study of the distribution of alkaline phosphatasein normaland neoplastic tissues. Thestained sections werevery impressive;coloredlantern slides werejust cominginto use and Jacobthoughtweshouldhavea coloredplate for our paper in the American Journalof Pathology,whichwouldcost $400. This wasconsideredtoo expensive.It wasfinally arrangedthat the Departmentof Pathologywouldpay $100, the research fund wouldpay $200, and Jacob and I would each pay $50. It was a very worthwhile investment becauseit stimulated muchworkon the subject. In EuropeI had hopedto visit KSgl’s laboratory at Utrecht; he had startled the world by announcingthat malignanttumorshad large amounts of D-aminoacids instead of L-aminoacids. Muchof his data could be accounted for by racemization, but from one tumor several grams of t~-glutamic acid had been isolated. Naturally, everyonebeganto try to checkthis and Jacobgot mesometumors, thinking that after I established that I could isolate D-aminoacids I could then workon leukemiccells, whichwere available in muchsmaller amounts. However,I workedup a numberof tumors but only found L-glutamic acid. Twoconfirmations of KiSgl’s workwere soonreported and it seemedclear that I wasnot a very goodchemist. Theproblemwas solved by the genius of Fritz Lipmann,who suggested that after hydrolysis, one could add D-aminoacid oxidase and estimate D-aminoacid concentrations by using the Warburgapparatus. He, DeanBurk,Otto Behrens,and I soonshowedthat there wereno significant amountsof D-aminoacids. DavidRittenberg at Columbiaapplied the isotope dilution methodand also failed to find D-amino acids. This turned out to be oneof the instancesof falsification of data, someone havingapparently

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added o-glutamic acid to K/Jgl’s hydrolysates. It led to mycoining an aphorism:"Everyincorrect discoveryhas alwaysbeenindependentlyverified." In our efforts to purify the tumorviruses, wefrequentlyfoundthat saline extracts of tumors were extremelyviscous, and from one chicken with a tumor,wewereable to get a considerablequantityof this very viscousfluid. The viscosity reminded me of the pneumococcalpolysaccharides I had prepared. I addedsomesodiumacetate to the fluid and then two volumes of ethanolandgot an extraordinarilystringy precipitate, almostall of which adheredto the stirring rod and could be removed.Karl Meyerat P and S had reported the isolation of hyaluronicacid as behavingsimilarly and I said to Jacob, whowaswatchingmeprecipitate the polysaccharide,that it looked like hyaluronic acid. Hebecamesomewhatupset that I could make sucha statementfromso little evidence,but whenI addeda little hyaluronidase providedby Karl Meyer,the Viscosity disappeared.Welearned it was essential to treat our tumorextracts with hyaluronidasebefore ultracentrifugation. DavidShemin,whohad been at City College with me, had received his PhDin biochemistryat Columbia,and had taken a job there in the Departmentof Pathologywith JamesJobling, the Chairman,working on Roussarcoma,had similar findings. DeanBurkwasalso very interested in tumormetabolismand he, Jacob, Herbert Sprince, Janet Spangler,Albert Claude,andI carried out studies in the Warburgon chicken tumors. On the international scene mystay at Cornell was the period of the Munichagreement, the conquest of Czechoslovakiaby Hitler, the NaziSoviet pact, the partition of PolandbetweenGermany and Russia, and the Finnish-Sovietwar. Theseevents shookmeand I began to worryabout my political views. Aboutthe spring of 1940,TracyJ. Putnam,Director of the Neurological Institute at Columbia, wantedan immunochemistto work on multiple sclerosis and asked MichaelHeidelbergerto suggest someone.Michaelgave hima choice, assuring himthat if he picked mehe ffould have a strong personality on his hands. Heinvited mefor an interview and said that he understood that I had lots of myownideas and that I could work on whateverI wishedexcept he hopedI wouldnot discriminate’againstneurological problems. The salary was $3600 per year and Hans Clarke had agreed that I wouldhave an appointmentas ResearchAssociate in Biochemistry(assigned to Neurology).Fundswereavailable for a technician; two laboratories were available in the NeurologicalInstitute, whichhad formerlybeenintern’s bedrooms,plus somespace sharedwith the clinical laboratory. It took little thoughton mypart to decideto accept. RobertF. Loeb, for whomI had the greatest admiration and affection, was then

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Associate Director of the Neurological Institute and was a great attraction for me. I was especially thrilled when I met him at WoodsHole in the summerof 1940 and he came over to me saying, "Elvin, we need you, we can hardly wait until you get to Neuro." I arranged to leave Cornell at the end of May, 1941, taking June as my terminal vacation. I started at P and S and the Neurological Institute on June 1, 1941, and took myvacation later in the summer. Mylaboratories were temporarily occupied by NormanWeissman, a friend who had gotten his degree in biochemistry, and Murray Glusman, with whomI was to collaborate years later when he returned from a Japanese prison camp. Harold Landow,a resident in neurology, was interested in collaborating. Wedecided to look at the electrophoretic patterns of cerebrospinal fluid in the Tiselius electrophoresis apparatus. Dan H. Moore, who had come from Lederle laboratories where he had studied horse antisera electrophoretically, had set up a laboratory for electrophoresis and had an air-driven ultracentrifuge. For the Tiselius electrophoresis, ¯ the cerebrospinal fluids had to be concentrated by pressure dialysis to a small volumeand had to be run in a 2-ml micro cell. Large amountsof fluid were available from pneumoencephalograms and we arranged to obtain these. Wereadily obtained good patterns on concentrated cerebrospinal fluid and showedthat patients with multiple sclerosis often had substantial increases in the gamma globulin fraction, as did patients with neurosyphilis, and that positive colloidal gold tests correlated with increased gamma globulin. This led naturally to a study of serum proteins with Franklin M. Hanger, which showedhigh levels of gammaglobulin to be responsible for positive cephalin flocculation tests in various sera. Thesefindings provided someinsight into the mechanismof these two clinical diagnostic tests. At the same time, Harold Landowand I began a quantitative study of passive anaphylaxis in the guinea pig. Oddlyenough, although the antibody N content of rabbit antisera could be determined by quantitative precipitin analysis, nobodyhad measuredhowlittle antibody was required to sensitize a guinea pig so that fatal anaphylaxis wouldresult on subsequent administration of antibody. Our data showed, with anti-ovalbumin and antipneumococcalserum that 30/.tg of antibody N sensitized 250-g guinea pigs, so that fatal anaphylaxis wouldresult if 1 mgof ovalbuminor of pneumococcal polysaccharide were given 48 hr later. This was the beginning of a series of studies on quantitative aspects of allergic reactions. In the summer of 1940, while still at Cornell, MaryLoveless and I had workedat Woods Hole, trying to measure uptake of skin-sensitizing antibody (later recognized as IgE) in sera of ragweed-sensitive patients, using a suspension of formalinized pollen with negative results. The skin sensitizing powerof the sera was removed,but no increase in N in the washedpollen was detectable.

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As we nowknowfrom the work of the Ishizakas and Bennich, IgE is so muchmoreactive per unit weightthan rabbit IgGthat the methodwasnot suitable. For similar reasonsI also obtainednegativeresults trying to measure uptakeof bacteriophageby suspensionsof bacteria. Also while at Cornell Professor du Vigneaudhad given mepermission to use the Tiselius electrophoresisapparatushe hadjust purchased.I examined numerous leukemicsera for changesin protein pattern, but significant changeswere not seen. However,a BenceJones protein he also provided gave a beautiful homogeneous peak. Duringthe first weeksafter myreturn to Columbiain June, 1941, I stopped in to see Alexanderand Ethel Gutman,whoselaboratories were opposite Michael’sand from whomI had received advice while with Michael. They were studying myelomasera in the Tiselius apparatus and showedmetheir patterns with their sharp peaks movingbetween/32and gamma globulin, the mobilityof the peaksdiffering amongindividual sera. I asked themwhythey were not studying BenceJones proteins since the mobility of mypeakhad also beenbetweenthat of B2and gamma globulins. AI took out a pattern of a BenceJones protein he had sent to someone several years earlier. Therewasa beautiful sharp peaklike the one I had observed--it was labeled "boundary disturbance." Wedecided to add BeneeJones proteins from a myeloma patient to normalserumand compare the pattern with that of the patient’s serumby itself; wefoundwecould reproducethe patient’s serumpattern in a numberof instances by using BenceJones proteins of different mobility. Wethen used a rabbit antiserum to urinary BenceJones protein from a myelomapatient to measurethe levels in the serumof the samepatient. I also continued the histochemicallocalization of enzymes,studying normaland neoplastic tissues of the nervous system with Harold Landow and WilliamNewman, whocameto us as a technician, later studied medicine, and becameProfessor of Pathologyat GeorgeWashington University. AbnerWolf, whowasProfessor of Neuropathology,also wasinterested in this field. Wecollaborated closely on histochemicalstudies on acid and alkaline phosphatases and especially on producing and studying disseminatedencephalomyelitisin monkeys by injection of brain tissue emulsified in Freundadjuvants, until the grant for this workwassummarily terminated in 1953 during the hysteria generated by Senator Joseph McCarthy. Onthe eveningof June22, 1941,I wasat a party--everyonewasdiscussing the rumorsthat Germany wasgoing to attack Russia. I arguedvigorously that Hitler wouldnot, andwhenI hadjust about convincedeveryone, weturned on the radio. This markedmyretirement as a political prognosticator. It wasfar moretragic for mankindthat Stalin wastaken in than

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that I was. I joined with manyothers to set up a group for Russian War Relief at the MedicalCenter. The doubts generated by the Nazi-Soviet pact werestilled. On Sunday, December7, 1941, I was working in the laboratory and heard the news about Pearl Harbor. No one knew what was happening but we decided to move acids and hazardous chemicals to a storeroom in the basement. It was clear that medical scientists, immunologists, and immunochemists were going to make important contributions to the war effort. Michael Heidelberger, whoI saw very frequently since myreturn to Columbia, had been working for the PneumoniaCommissionof the Board for the Control of Epidemic Diseases, USArmy, on immunization against lobar pneumonia, using pneumococcalpolysacchaddes. I proposed that the type I (now group A) meningococcal polysaccharide, which had been purified by Geoffrey Rake and Henry W. Scherp, might be used for immunization in man. Meningitis had been a serious problem during recruiting in World WarI. The Meningitis Commissionunder the Chairmanship of J. J. Phair was centered at Johns Hopkins and gave me an initial subcontract for $1000, renewedfor a year at $2500, to purify type I meningococcalpolysaccharide and study its antigenicity in man. I needed to order someequipment, which I did before going on a trip. WhenI returned I found that the order had not been processed. I phoned Dean Willard C. Rappleye, who said they were having a problem about whether my subcontract would receive overhead. I asked how much the overhead was and when he said $40, I shrieked into the. telephone, "Andfor forty dollars you are holding up my work on immunization against meningitis with a war going on!" There was a momentof deathly silence and I expected to hear that I was no longer employedby Columbia.Instead, he said, "I’m sorry. I guess you are right. We’ll order it right away." Fromtoday’s perspective it is easy to see howthe Universities’ attitudes on getting the incredible amounts of moneythey demandin indirect costs developed, to understand what happensto the reputation of the investigator who cannot bring in what they consider his share, and to marvel at the unanimity administrators exhibit in lobbying against any proposed reductions whenthey can agree on almost nothing else. I hired Hilda Kaiser, whose husband, Samuel Kaiser, had been dismissed from Brooklyn College as a consequence of the NewYork State Legislature’s Rapp Coudert Committee and later suggested to Michael that he hire Samfor the pneumoniawork, which he did. All of the individuals fired from the City Colleges during that period were reinstated with apologies 40 years later, manyposthumously. Webegan to immunize medical student volunteers with type I meningococcal polysaccharide following Michael’s schedule for pneumococcal

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polysaccharides. In these and all mylater studies on blood group substances, dextrans, levans, etc, I generally injected myself first with any new materials unless they were expected to cross-react with antibodies to antigens I had already received. Weused Michael’s precipitin assay with about 4 ml of serum per test and with Hilda Kaiser and Helen Sikorski were able clearly to demonstrate that precipitins were formed; C. Philip Miller and Alice Foster at the University of Chicago assayed the sera for protective antibody and we demonstrated that the polysacchadde was antigenic. Only 4 of 38 individuals, however,produced a significant antibody response and this seemed poor by comparison with the pneumococcal polysaccharides, so immunization was not considered promising. Whenimmunization with meningococcal polysaccharides was taken up years later, radioimmunoassay was available and detection of an antibody response becamethousands of times more sensitive. Wetoo would have found detectable antibody by radioimmunoassayin a substantial proportion of the 38 subjects. Mylaboratory was involved in two other projects during the war: one with the Ottice of Scientific Research and Developmentand the Committee on MedicalResearchon false-positive serological tests for syphilis; and the other for the National Defense Research Committee on the plant toxin dcin, a possible chemical-biological warfare agent, with a view toward developing methodsof detecting it and protecting against its toxic effects. Our experiences in buying and immunizingtwo horses as part of this work have already been described (5). Bernard D. Davis was assigned to the laboratory by the USPublic Health Service to work on serological tests for syphilis. With Ad Harris and Dan Moore we studied the anticomplementary action of humangammaglobulin and succeeded in purifying Wassermannantibody by absorption on and elution from lipid floccules. For the studies on ricin, which were classified secret, Michael Heidelberger and I joined forces as co-responsible investigators. Ada E. Bezer cameto work in mylaboratory on this problem and we began an association that was to last over 20 years until she went to work with WHO in Ibadan, Nigeria. Wewere able to demonstrate by immunochemicalmethods that the toxic and hemagglutinating properties of dcin were due to different substances. This was later confirmed by Moses L. Kunitz and R. Keith Carman,whoindependently succeeded in crystallizing ricin. One very puzzling observation was that the protective powerof anti-dcin sera could be assayed readily in animals but did not correlate with the amountof precipitable antibody. Wenowknowof course that ricin has a combiningsite for carbohydrates and therefore was reacting with and precipitating non-antibody gammaglobulin and probably other serum glycoproteins, as well as antibody.

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During the war years Manfred Mayer had replaced me in Michael’s laboratory. He continued the study of the cross-reaction of types III and VIII antipneumococcal antibodies Michael, D. L. Shrivastava, and I had begun earlier. Webecamevery close friends. The war years had seen important changes in mypersonal life. Harold Landow,a brilliant neurologist and close friend, had committed suicide in 1940 because of his parent’s objections to his marrying outside of his religion. In 1940 at a literary meeting I had been introduced to a young Canadian named Sally Lennick. She had come to NewYork from Toronto to study painting. Wemet again a year later. Then in the summer of 1942 we met at a party, began seeing one another frequently, and were married on November28, 1942; our oldest son, Jonathan, was born on June 5, 1944. It had becomeclear at this time that a book outlining the thinking and methodology of quantitative immunochemistry was sorely needed. Most workers using the quantitative precipitin methodhad learned it directly from Michael or from someone who had learned it from Michael and this waslimiting growthof the field. I had been invited to write a review entitled "Immunochemistry of the Proteins" for the Journal of Immunology by Alfred J. Weil; it appeared in December,1943, and I was astonished at the hundreds of requests for reprints. Manfred Mayerand I decided to write a text called Experimental Immunochemistry, which would not only give the methodsin detail but wouldalso outline principles and concepts so that it would be useful to students and workers who might think immunochemistry of value in attacking their problems.It wouldalso include the preparation of materials needed for work in immunochemistry. Weprepared an outline and submitted it to John Wiley, who did a survey and assured us that it would never sell even a thousand copies. Fortunately, Charles C Thomascameto visit Michael and immediatelyagreed to take it and invited Michael to write a preface. Weset right to work by dividing up the chapters and beginning to write. Weusually met on Saturday or Sunday at one or another’s apartment and read aloud what had been written, correcting, revising, and reordering sections. Our wives were left to entertain each other or to be bored by listening to what we had written. The book was sent to the publisher at the end of 1945, but because of paper shortages after the war, and various other delays, it did not appear until 1948. Fortunately, we were aware of many studies during the war years that were just being prepared for publication and insisted on doing extensive revisions in proof so that ~the bookwas up to date. It had a substantial influence on the field and the first edition went through four printings, the last being in 1958. The second edition, which appeared in 1961, also went through four printings. After Pearl Harbor, a group of us at Columbiawho were membersof the

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AmericanAssociationof Scientific Workersdevoteda considerable amount of time to consideringwhatcould be doneto aid the wareffort, especially with respect to defense and improvingmethodsof immunization.Theuse of bacterial andviral warfareagentsby the Naziswasconsidereda possibility and TheodorRoseburyand I undertookto prepare from the literature a report of the agents that mightbe used; wewereassisted by MartinH. Boldt, then a medical student. Wespent several monthspreparing the review, and AlphonseDochez,whowasan adviser to the Secretary of War, gaveit to him.Naturally,it waswithheldfrompublicationby us voluntarily during the war. The Armywas also very concerned about the possible military use of infectious agents and set up CampDetrick near Frederick, Maryland,as a large-scale military research facility. TedRoseburystarted workingthere full time and I becamea consultant, spendingseveral days there each month.Ourreport was amongthe first items newpersonnel at Fort Detrick were given to read. At the end of the war, whenthe SmytheReport on the AtomicBomb was published, wefelt it was in the public interest that our report on bacterial warfareshould also be publishedand werequestedclearance from the WarDepartment.Several incidents arose. In our original request for clearance westated in a footnote that wehad withheld the report from publication during the war but that it wouldnowbe published with the approval of the WarDepartment.In their letter stating that wecould submitthe reviewfor publication, they requestedthat the wordswith the approval of the WarDepartmentbe changedto "in view of the removalof war time restrictions" (Figure 2). Wenaturally complied.TheJournal of Immunology indicated it wouldconsider publishing it. In accordancewith Universitypolicy wethen gavethe report for approvalto Dr. Dochez,who wasChairmanof the BacteriologyDepartment.Althoughhe had originally takenthe report to the Secretaryof War,he said it hadnonscientificimplications and that he wouldhave to take it up with DeanRappleye.Rosebury and I werecalled to the Dean’sot~ce andweretold the report mightoffend religious groupsandthat if weinsisted on publishingit weshouldwrite our resignationson the spot. Sincewewerein no position to do this, and since our appeals about Freedomof Speechandthe Press wereunavailing, weput the report in a drawer. Wealso wentto see Osmond K. Frankel, a leading civil liberties attorney, whotold us essentially that wehadunlimitedfreedomto publish but havingdone so wedid not have the right to workfor ColumbiaUniversity. About6 monthslater, DeanRappleyecameto Ted Rosebury’soffice and said that he had madea mistake, that the University did not intend to limit our freedomto publish, and wecould submitthe report for publication. Wedid, andit waspublishedin the May,1947,issue of Journal of Immunology (9), whichalso arranged to sell reprints. The report created a world-widesensation (Figure 3).


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9e~art~t oF Bacteriology 6:~ ~est l~thStreet Dea~ I have looked over the revi~

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~Little,

in The Nashville Tennessean

Figure3 Political cartoon(by TomLittle, in The Tennessean)resulting fromthe published bacterial warfarearticle. (Reprintedwith permission.)

Whenit appeared, Timemagazineimplied that wewere procommunist and interpreted the statementin the footnote to indicate that wehad not obtainedclearance but had publishedit on our own"in viewof the removal of wartimerestrictions." MichaelHeidelbergerwrotea letter supporting our loyalty, whichthey published. At the sametime HowardJ. Mueller, Professor of Bacteriologyand Immunology at Harvard, whohad also been a consultant at CampDetrick, wrote DeanRappleyesuggesting that Rosebury and Kabatshould be fired becausethey had timed the publication of their report to appear whenCongresswas considering the WarDepartment’s budgetso that they wouldget moremoneyfor biological warfare, although the war was over. Dean Rappleye knewthat if any one had

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GETTING STARTED50 YEARS AGO

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determined if and whenthe report would appear it was he and not Rosebury and Kabat. However, we learned that our clearances for CampDetrick were cancelled just after the report was published. The FBI was assigned to investigate us and the following episode with mylandlord was told to me. Sally and I were leaving for Europe on June 20, 1947, to attend the International Cytology Congress in Stockholm and the International Microbiology Congress in Copenhagen. I wrote mylandlord, giving him the dates we would be away and enclosing two rent checks dated July 1 and August 1. Weleft our two children, Jonathan, age 3, and Geoffrey, born May11, 1946, with myparents. Shortly after our departure, an FBI agent visited the landlord. The conversation as recounted to me went about as follows. Did you know the Kabats were leaving for Europe? Do you think they are ever comingback? He showedthem the letter and the checks. The questions continued. If you were plann’ing to leave the country wouldn’t this be a good way to hide your intentions? Mylandlord naturally was very surprised when we returned on August 20. In Stockholmat the CytologyCongress, there was substantial interest in the report on bacterial warfare and a group of Swedish microbiologists invited Sally and meto an elegant lunch at the Operkellaren; several of the guests were evidently assigned to entertain Sally while the others turned the topic of conversation to myreport. Since Roseburyand I had been involved in research subsequent to our report, all of whichwas still secret, it would have been almost impossible for meto discuss anything without creating the impressionthat it represented the report plus additional classified information. Fortunately, suspecting what the lunch was about, ! had put a reprint of the report in mypocket and in reply to each specific question, I merely read the pertinent sections written in 1941 and finally gave the reprint to one of the microbiologists sitting next to me. At the end of the war, I took up two major lines of investigation. One, on the immunochemistryof blood group A and B substances, was to continue to the present day. The backgroundand details of work in this area and the personal aspects have been described by merecently (7). The second was an attempt, together with AbnerWolf and Ada Bezer, to produce acute disseminated encephalomyelitis in monkeysbecause of its possible relation to multiple sclerosis. The use of the Pasteur treatment for rabies had been knownto result in what were termed paralytic accidents, which involved disseminated demyelinating lesions in the brain and spinal cord. This had been shown by TomRivers and Francis Schwenkter to be due to the nervous tissue and they succeeded in producing the disease in monkeysby injecting suspensions of spinal cord tissue. Manyinjections were neededand the disease did not appear for a long period. Tracy Putnamhad always been most interested in multiple sclerosis and was delighted when we got back

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KABAT

to this problem. Jules Freund had shownthat one could get a very enhanced and protracted antibody response, as well as delayed type hypersensitivity, by incorporating antigens with mineral oil and killed mycobactedumby using an emulsifying agent, and I was anxious to see if this procedurecould be used to produce disseminated encephalomyelitis in monkeysmore rapidly and reproducibly. Wefound that after only one to three injections of brain tissue emulsified in the Freund adjuvant, the monkeysdeveloped the disease and we sent a short note to Science early in 1946. While having dinner with Isabel Morganduring the Federation meetings I learned that she had made similar observations in monkeysshe had been injecting with spinal cord tissue containing poliomyelitis virus with Freund adjuvants and then had omitted the virus with similiar results. She was to present her work at the Society of American Bacteriologists meetings in May. Wedecided that we wouldwrite up our detailed papers completely independently, then send themto one another to criticize and request that they be published side by side. Since her paper arrived a few days before ours had been typed, I gave the unopened envelope to Robert Loeb for safekeeping until I had mailed our manuscript. The results were completely concordant and both papers appeared together in the Journal of Experimental Medicine in January 1947. The National Multiple Sclerosis Society had just been founded and sometime after our paper appeared I received a phone call from them saying they wantedme to apply for a grant. I said that I really was not interested in applying and didn’t need the money.Theyreplied that I "just have to" since they had received an anonymouscontribution on condition that they used it to support our work. WhenI inquired as to the amountof the contribution and was told it was $10,000, I said that this was not really sufficient to makethe more intensive effort they wished. I drew up an application for $64,350 for 3 years of support, which they showed the anonymousdonor whogenerously provided the entire sum. This was the first grant madeby the National Multiple Sclerosis Society. I was able to increase the size of my monkeycolony from about 10 or 12 to 40. In 1950, the Public Health Service gave mea grant to continue this work, but it was cancelled in 1953. By the end of the war mysalary had increased to $4500per year, but with the inflation following the removal of price controls, plus my growing family, I had to take a part-time job teaching elementary chemistry at City College. The multiple sclerosis grant provided an equivalent sum and made it possible for meto give up this outside work. Wespent the summer of 1948 in WoodsHole where we first met Fred and Sally Karush and their children. This was the beginning of a life-long friendship between the two families and Fred spent 6 months in mylaboratory in 1950. The laboratory was then so crowded that he had to work a second shift beginning in the late afternoon.

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Towardthe end of that summerI saw a note on the bulletin board at the MBLthat a house was for sale by the Professor of Microbiology at Washington University, Jacques Bronfenbrenner, whomI knewquite well. It took only a few minutes to arrange to buy the house. Our children becamevery attached to it and we spent part of each summerthere during the years when they were growingup, except whenI was on sabbatical. I wouldeither work in the library or rent a laboratory bench. In 1949, I rented a laboratory bench, which then cost $50 for the summer, so that I could prepare an enzymefrom snail hepatopancreas that split blood group substances. The business office at Columbiarefused to pay the bill, saying that grants stipulated that the institution wasto provide laboratory facilities. I assured them that the $50 entitled me to order and receive snails collected by the MBL boats and that if they wouldput snails on mydesk in 48 hr I woulddo the work in NewYork--they paid "the $50. WoodsHole was, of course, a marvelous place for makingscientific contacts. In 1949, ShlomoHestrin of the Hebrew University had the laboratory bench opposite me and we becamevery close friends. Around this time a problem developed in myrelationship to the Biochemistry Department at Columbia. For several years Tracy Putnam, the Professor of Neurology, had been asking Hans Clarke to promote me to Assistant Professor, but to no avail. This was in no way directed towards me personally, nor did it reflect any doubts about mywork. Hans Clarke was a very fine person, but he was completely unaware of the need to promote people. He did not consider persons in other departments with titles in biochemistry as real membersof his department, although he did not promote the regular memberseither. A substantial numberof Research Associates had been in these positions for years, requests of the chairmen of the departments in which they workedbeing unavailing. Indeed, Walter W. Palmer, the Professor of Medicine, told me he had asked Hans Clarke to promote Michael Heidelberger to full Professor for 17 years before he agreed. I broke the log jam of Research Associates by transferring to the Department of Bacteriology. The Chairman, Dr. Dochez, was very anxious to introduce immunochemistryinto the medical teaching. In discussing my situation he suggested that he would be willing to give me an Assistant Professorship. The appointmentwas delayed for a year by the objections of Hans Clarke, whoevidently didn’t want to promote me or lose me, but it finally took effect on July 1, 1946. Louis Levin, whofor about 10 years had been Research Associate in Biochemistry assigned to Anatomy,an appointment similar to mine, had left a year earlier for the University of Chicago because the AnatomyDepartment could not arrange his promotion. He was brought back as Assistant Professor of Anatomyand later went to the Office of Naval Research and the National Science Foundation.

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KABAT

After this, Hans Clarke agreed to several promotions of other Research Associates. As Assistant Professor of Bacteriology, I was entitled to have graduate students and my first was SamM. Beiser, who had been in the Navy and came to Columbia under the GI Bill. He was recommended by Arthur Shapiro. His PhDdissertation was on blood group substances from bovine gastric mucosa. The Academyof Allergy was just setting up a fellowship program(1). Even though Beiser was not working in allergy, at mysuggestion that the fellowship might stimulate his interest in allergy, he received it and later devoted sometime to work in this area. After two postdoctoral years with Bernard Davis, he returned to Columbia,rising to Professor and was Acting Chairmanat the time of his death from cancer in 1972. Among his most important contributions were studies on antibodies to nucleotides with Bernard F. Erlanger. His bovine blood group glycoproteins have remained important standards and Michael and I are still using the pneumococcal C-polysaccharide he prepared. Myposition in the Department of Bacteriology, the title of which was changed to Microbiology, and in the Neurology Department was eminently satisfactory over all these years. Tracy Putnamleft in 1946 and was followed by EdwinG. Zabriskie, and in 1948 by H. HoustonMerritt. Professor Dochez retired, and Beatrice Seegal became Acting Chairman of the Department of Bacteriology until Harry Rose was appointed in 1951. Drs. Zabriskie and Dochez had recommended my promotion to Associate Professor in 1947 and I was promoted in 1948. Harry Rose was also very supportive. Indeed, when he was offered the position as Chairmanof Microbiology in 1951 he laid downtwo conditions--that I be promotedto full Professor and that mysalary, which until then had come from grants, be paid out of departmental funds. This took effect on July 1, 1952. By 1947, the laboratory had three major lines of investigation immunochemistry of blood group substances (see 7), acute disseminated encephalomyelitis, and quantitative studies on allergic reactions. EdwardE. Fischel, who was in the Department of Medicine, and I measured the amounts of antibody needed to produce Arthus reactions passively in the rabbit, and GrangeCoffin, a medical student, and David M. Smith, a dental student, continued studies on passive anaphylaxis. Baruj Benacerraf came to see mein 1946, having completed medical school and a 1-year internship before serving in the Armyand wanting to spend a postdoctoral year in the laboratory. There were nb funds, but he told me he had independent means and came, as he later put it, on the "Benacerraf Fellowship." He was a very intense and dedicated worker and we studied quantitative aspects of the latent period in passive anaphylaxis and also the passive Arthus reactions in the guinea pig. John H. Vaughanspent two postdoctoral years in the

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laboratorystudyingthe skin-sensitizing properties of rabbit antibodies to ovalbuminand conalbuminin humanskin. Wehad foundincreases in the gamma globulin in the cerebrospinalfluid of patients withmultiplesclerosis by electrophoresis,but this wasimpractical for diagnostic purposes. MurrayGlusman,whohad returned to the NeurologicalInstitute after the war, VestaKnaub,and I decidedto measure the amountsof gamma globulin by using a microquantitative precipitin reaction. Wealso measuredalbuminimmunochemically at the same time. It becameclear that the methodwouldgive moreinformative results than the colloidal gold tests, and it becamepossible to followgamma globulin levels in cerebrospinalfluid in patients over prolongedperiods. DavidA. Freedman, Jean Murray, and Vesta Knaubmeasured the albumin and gamma globulinlevels in 100 cases of multiple sclerosis and found85 with elevatedgamma globulinlevels. In carryingout sucha studyit wasessential not to report our findings until the diagnosis hadbeenmadein the absence of these data, otherwiseclinicians whoavidly grasp for objectivedata would soongain a clinical impressionof the valueof the test andwouldnot make their diagnosisuntil the test wasin the patient’s chart. Subsequent studies were carried out with MelvinD. Yahrand Sidney S. Goldensohn,and the quantitative precipitin methodfor determininggamma globulin in the cerebrospinal fluid of patients at the NeurologicalInstitute becameone of my routine responsibilities for 30 years, until it wassupersededby moresensitive and automatedimmunochemical methods. I had manyclose friends and other colleaguesat the NeurologicalInstitute during this period, ineluding Saul R. Korey, Harry Grundfest, and David Nachmansohn. At the end of the war AbnerWolf had becomean attending consultant in Neuropathologyat the BronxVeterans Administration Hospital. The VeteransHospitals wereencouragingresearch and weresetting up substantial laboratory facilities. WilliamNewman had received his MDand had gone there, as had another MD,Irwin Feigen, and Abnersuggested that wecontinuestudies on histochemicallocalization of enzymesthere. I was appointedattending consultant and spent one afternoon a weekthere for severalyears. WhileI was at the VA, President Trumanissued an Executive Order initiating loyalty andsecurity investigationsof Federalemployees with the followingcriterion: "Reasonablegroundsmustexist for the belief that one is disloyal to the UnitedStates." JamesB. Sumner,with whomI had been very friendly at Uppsalain 1937-1938and whoI had later met briefly on only one occasionin the US,wentto the FBIto tell themthat I had been a communist while in Uppsala.This initiated a series of FBIinvestigations on the basis of whichI was presented with charges about the various organizationsto whichI hadbelongedor contributedduringthe late 1930s.

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KABAT

I had a hearing before the VAloyalty board, whichdismissed me. I appealedfinally to the Presidential LoyaltyReview Board,whichreversedthe decision and reinstated me. I returned to the VA,but it wasobviousthat there werepressures to reducefurther the rigidity of the criteria andthe quality of the evidenceuponwhichan individual could be dismissed, so I decidedto resign. This essentially led to mygiving up workon the histochemicallocalization of enzymesin tissues. ThePresidential LoyaltyReviewBoardwasabolishedby President Eisenhower,respondingto pressures that it had beentoo lenient. Indeed, it wasthe only board whosemembers were not Federal employeesand thus was not subject to pressures from Congressand fromwithin the Executivebranch. I wasvery pleased that I had carried out all myappeals without a lawyer. The Bronx VAHospital Loyalty Board that dismissed me had also written a letter to the passport office telling themthat I should not be allowedto travel, and so mypassport wascancelled; it was not returned whenI was reinstated. I had been invited to address the First International Congressof Allergists in Zurich in the summer of 1950but was unable to obtain a passport. I wentto the passport office to discuss the matter and was told that if I gave the namesof anyoneI knewor thought to be a communistI could get a passport. Needlessto say I was unable to travel until the decision of the USSupremeCourt in 1955that every Americancitizen had an unlimited right to travel, whenI attended the International Congressof Allcrgologyin Pctropolis, Brazil. Baruj Bcnacerraf broughtmysituation to the attention of the Zurich Congressin his address. I received the invitation to the AllergologyCongresswhile in Woods Hole. It provided$1000for travel expenses.I wrotea letter sayingthat I could not accept becauseI could not get a passport. I walkedinto townand droppedthe letter in the boxandwalkedacross the street to the drug store to buy the NewYork Times. The headline announced the US Supreme Court Decision. I rushed to the Post Office and was able to retrieve my letter. Sanford Elberg, with whomI becamefriendly at CampDerrick, invited meto teach two coursesat the Universityof California at Berkeleyduring the summerof 1950.Sally, Jonathan,Geoffrey,and I droveacross country, stoppingin Springfield,Illinois, to see the Lincolniana,in Denver,in Salt LakeCity, and at Boulder(nowHoover)Dam.I had a very enjoyable time teaching--amongthe students or auditors were A. A. Benedict, MelHerzberg, Fred Aladjem, and Keith Smart. I met and becamefriends with Ed Adelberg, RogerStanier, MikeDoudoroffat Berkeley, and K. F. Meyer, Professor of Bacteriologyat San Francisco.Whilein BerkeleyI wasinvited to speakat the NavalBiologicalLaboratory,the Navy’sequivalentof Camp Derrick. In this highly classified institution andat a time of considerable

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hysteria, Keith Smart, in trying to shorten an obituary-like, overly long introduction, brought downthe house by saying, "Dr. Kabatis a member of a large numberof organizations, whichI had better not mention." Of coursehe then felt obligatedto list them. In 1952Congressappropriated $I00,000for research in immunochemistry in the budgetof the Office of NavalResearch(ONR).To evaluate what problems were worthy of support, William V. Consolazio, whomI had never met, and Louis Levincalled a conferenceat P and S. About10 or 15 people were present, including myself, Michael Heidelberger, and Dan Campbell.Everyonearound the table indicated what problemshe thought were important. I stressed use of immunochemical criteria of purity of proteins and polysacch.arides. The meetinglasted until lunch time. When it wasover andeveryonewasleaving, Bill Consolaziosaid to me, "Youare staying here," and then asked, "To whomwould you give moneyon the basis of the suggestions?" F’outlined the programsI thought should be supported. Hethen said, "Youhave to take somemoney."I responded,"I don’t need any money,I’m loaded with money."He gave mea yellow pad, saying, "Youare not leaving here until you write out a proposal. Youcan hold on to the moneyand you can activate the contract any time within the next five years." I wrote out a title, "Immunochemical Criteria of Purity of Proteins and Polysaccharides," myname, the University’s nameand address, a short abstract, and a budgetthat providedfor a postdoctoral fellow and supplies. WhenI cameto overheadI asked, "Whatare you going to do about overhead?"At that time ONR was havinga battle with Columbia-they wouldonly pay 10%overhead, believing that the investigator wasinterested in doing the workanyway.TheDeanwasvery dissatisfied with 10%and had threatened to throw ONRout completely. Consolazio replied, "Oh, put down25%overhead--youdon’t want the moneyand I had to twist your arm." Several weekslater two Navycontract negotiators cameto see DeanRappleyewith the piece of paper containing myalmost illegible handwritingsaying, "We’vecometo give you this contract." The Deansaid, "Whatcontract?"Theyreplied, "Its all written on this piece of yellowpaper." I wasin the processof havingit typed. TheDeanthen said, "Whatabout overhead?"Theyreplied, "Twenty-fiv~percent." Mystock at Columbiarose enormously.I only activated the contract a year later when the Public Health Service cancelled mygrants at the height of the McCarthyhysteria. It was like having moneyin the bank. ONR supported mefor 17 years. Consolazioand Levin left ONR whenthe National Science Foundation wasset up andBill suggestedthat I applyfor a grant. I received oneof the first awards-S60,000 for 3 years, appropriatedout of the first year’s molecularbiologybudget;it representedabout 8%of the total. This replaced myblood group grant, whichthe Public Health Service cancelled in 1953. NSFhas been the mainsupport of mylaboratory since then.

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KABAT

In 19511 wasaskedto serve on the NationalResearchCouncil’sSubcommittee on Shock,whichwasconcernedabout the severe allergic reactions being found on administration of dextran, whichhad beendevelopedas a plasmaexpanderin Sweden.It wasgenerally believed that the reactions weredue to contamination with bacterial protein. I naturally suggestedthat the dextran itself, like the pneumococcal polysaccharides,mightbe antigenic in man.Althoughclinicians werepreparedto inject 30 g of dextran intravenously, no one on the committeewouldinject 1 mgto see if it was antigenic. I did an initial skin test, had a bloodsampletaken, and gave myselftwo injections of 0.5 mgof dextran a day apart. A secondskin test 3 weeks later showeda typical wheal and erythema, and quantitative precipitin assays on the pre- and post-immunizationsera showedthat my antidextran level had risen from1.5 to 25/.tN/ml. At the next meetingof the Subcommittee on Shock, I demonstratedmyprecipitates and also did a skin test on myselfand on DougLawrason,the Secretary of the Committee, as a control. DeborahBergand I studied the quantitative precipitin reaction of dextran and antidextran producedin medicalstudent volunteers. I suggested to the NationalResearchCouncilthat Paul Maurerbe asked to do a study to confirmour findings, whichhe did. To prove that the antibodies were indeedantidextranweused biosyntheticallylabeled [~4C]dextran to precipitate the antibody, and 14Canalyses by DavidRittenberg, LauraPontecorvo at P and S, and LeonHellmanand MaxwellEidinoff at Sloan-Kettering established that the dextran wasprecipitated by the antibody. Theantigenicity of dextranmadepossible studies probingthe size of the antibody combiningsite. Oneof the dextrans, B512, developedat the NorthernRegionalResearchLaboratoryof the Departmentof Agriculture at Peoria, Illinois, wasbuilt of 96%c~1~6-and 4%ctl~3-1inkedglucoses andso had very long stretches of ctl-~6-1inkedglucoses. AlleneJeanesat Peoria and Turveyand Whelanin England were isolating the series of al-~6-1inked isomaltose oligosaccharides. Thesystem, al-~6 dextran and humanantidextran and the c~1~6oligosaccharides, essentially provideda molecularruler, since they permitted one to comparethe potency on a molarbasis of the various oligosaccharidesin inhibiting precipitation of antidextranby dextran. This becamea fourth area of interest of the laboratory, whichcontinuesto the present day. In the summerof 1952,Sally and I again droveacross country with our three sons (David, born August7, 1951). I wishedto learn morecarbohydrate chemistry and decided to workwith HermanO. L. Fischer at Berkeley, havingbecomevery attracted to the BayArea frommyearlier visit. Hermanasked me what I wished to do and whenI said somethingabout learning methylation of sugars, he took out a sampleof galactinol, an

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a-galactoside of inositol, and said that methylation and hydrolysis would show where galactose was linked to the inositol. Clinton E. Ballou and Donald L. MacDonald collaborated on the problem and taught me the technics; fortunately, the methylated compound crystallized. The sabbatical was a wonderful experience and we were able to renew old friendships and make manynew ones that.have lasted. One unexpected consequence of the methylation study was that galactinol turned out to be one of the best inhibitors of the blood group B-anti-B reaction until the disacchadde DGal al-~3DGal and larger oligosaccharides were isolated. Mysabbatical ended in February, 1953, and we drove back to NewYork via the southern route, visiting various friends at universities and doing some sightseeing, arriving in NewYork early in March. It was hard to adjust to the rest of the NewYork winter after Berkeley. The laboratory was thriving; the allergic encephalitis, spinal fluid gammaglobulin, blood group substance, and dextran problems were all going well. I had been cleared by the top Presidential Loyalty Review Board and presumably could carry on with my activities normally. However, rumors of grants being cancelled were becomingmore frequent. Linus Pauling had his Public Health Service Grants cancelled and had to appoint others as Responsible Investigator so that the work could continue. My3-year grant for the monkeystudies was running out and I had naturally applied for a renewal. I received a letter from Frederick L. Stone, Chief, Extramural Programs, National Institute of Neurological Diseases and Blindness, dated December 14, 1953, which read, DearDr. Kabat: Your application for a research grant, identified as B-9(C4), and entitled "Immunochemical Studies on Acute Disseminated Encephalomyelitis and on Multiple Sclerosis" has nowhad a final review. I regret to report that this request falls in the group of applications for which grants cannot be made.

This was followed by a visit to Houston Merritt saying that this was because of the political climate, that they didn’t knowexactly why,etc. He then madethe suggestion that the work could go on if someoneelse’s name was substituted as Responsible Investigator. Althoughmanyothers in this situation complied, I refused, responded with the appropriate four-letter words, and began a boycott of the US Public Health Service, which had imposed the policy on NIH. Anyonewishing to work in mylaboratory had to agree not to accept any funds from any unit of the USPHS for the period he was working with me, nor could any employee of the USPHSset foot in my laboratory unless he was coming for some unrelated purpose. I received muchsupport from scientists at NIH, but the top administration did nothing.

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KABAT

The reactions of my colleagues were varied. Most expressed support. Columbia University through Dean Rappleye, Houston Merritt, and Harry Rosesupported meunequivocally. The scientific societies fought the policy vigorously. Michael and manyothers refused to review grant applications. Unfortunately, not all actions were of this type: One person, whomI had considered one of myclosest friends, avoided me and on one occasion even hid in the stacks of the WoodsHole library when he saw me comingtoward him. Abner Wolf, Ada Bezer, and I had to kill off the only~monkeycolony in the world then being devoted to the multiple sclerosis problem. Shortly thereafter I was informed that the tenth committedyear of myBlood Group Grant was cancelled. I shall have to leave the story at this point. The suspense will not be dreadful. Youall knowthat I survived and continued to work actively, that I spent a year at NIHas a Fogarty Scholar, and that I have been spending 2 days a weekat NIHfor the past 7 years. The rest of the story must wait for another occasion. ACKNOWLEDGMENT

I thank Daniel M. Singer and Harvey N. Bernstein of Fried, Frank, Harris, Shriver, and Kampelman,Washington,D. C., for their advice, assistance,and direction in obtaining various government files about me under the Freedom of Information/Privacy Act. Literature Cited 1. Cohen, S. D. 1979. The American Academyof Allergy--An historical review. J. Allergy Clin. Immunol. 64:332466 2. Heidelberger, M. 1979. A "pure" organic chemist’s downwardpath: Chapter 2--The years at P .and S. Ann. Rev. Biochem. 48:1-21 3. Kabat, E. A. 1978. Life in the laboratory. Trends tliochem. Sci. 3:N87 4. Kabat, E. A. 1978. Close encounters of the odoriferous kind. Trends Biochem. Sc£ 3:N256 5. Kabat, E. A. 1979. Horse sense? Trends Biochem. ScL 4:N21M 6. Kabat, E. A. 1980. Wrongdoor at the

7.

8. 9. 10.

Nobel banquet. Trends Biochem. Sci. 6:VI Kabat, E. A. 1982. Contributions of quantitative immunochemistry to knowledge of blood group A, B, H, Le, I, and i antigens. Am. J. Clin. Pathol. 78:281-92 Knight, C. A. 1951. Paper chromatography of some lower peptides. J. Biol. Chem. 190:753-62 Rosebury, T., Kabat, E. A. 1947. Bacterial warfare. £ lmmunol. 56:7-96 Tiselius, A. 1939-1940. Electrophoretic analysis and the constitution of native fluids. Harvey Lectures 35:37-70



Annual Reviews www.annualreviews.org/aronline

An~ Rev. Immunot1983. 1:33-62 Copyright©1983by AnnualReviewsIn~ .4ll rights reserved


CELLULAR MECHANISMS OF IMMUNOLOGIC TOLERANCE G. J. V. Nossal TheWalterandEliza Hall Institute of MedicalResearch,Post Office,Royal Melbourne Hospital,Victoria3050,Australia INTRODUCTION Immunologictolerance, the phenomenon wherebyantigen interacts with the lymphoid systemto impairits later capacity to respondto that antigen, remainsone of the most fascinating problemsin cellular immunology. The capacity of individuals to discriminate"self" from"not self" has assumed a newdimensionsince the realization that T lymphocytesonly see foreign antigens in the context of "self" moleculesof the majorhistocompatibility complex(MHC)(131), and so the newer field of MHC restriction becomeintertwined with immunologic tolerance and indeedwith all aspects of immunoregulation.Althoughthe central interest of students of immunologictolerance lies in the establishment and maintenanceof selfrecognition, experimentalrealities mandatethat mostworkon tolerance in fact is performedwith modelsystemswhereforeign antigens, rather than autologousconstituents, are presented to the lymphoidsystemto induce a state of non-reactivity. It then becomesa matterof considerabledit~culty to judge whetherthe effects observedmimicthe physiologicalsituation of lymphocytescomingto grips with the need to avoid horrorautotoxicus, or whether the modelillustrates somefacet of immunoregulation neededto limit the cascadeof immunoproliferation that follows the introduction of antigen. Facedwith this constraint, investigators of immunologic tolerance fall into three groups.Thefirst, numericallythe largest, describespartienlar modelsystems and refrains from speculation as to howmajor a role the cdlular mechanism revealed by the study plays in physiologicalself-recognition. Somemembersof this group form a "tolerance is dead" subgroup, whobelieve nothing is all that special about self antigens, and that the avoidance of autoimmunity depends on myriad immunoregulatoryand 33 0732-0582/83/0410-0033502.00

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34 NOSSAL feedbackmechanisms, none of whichis uniquely importantto self-recognition and all of whichmayapply in particular circumstancesto foreign antigens as well. Thesecondgroup considers suppressorT lymphocytesas central to immunologictolerance. Onthis view, the repertoire of immunocytes is perfectly competentto recognizeself antigens, but the capacity to threaten health by actually switching on these immunocytesis thwartedby a powerfulactivation of the appropriatesuppressors.Thethird groupsees the mostimportantbulwarkagainst auto-reactivity as lying in somepurging of the immunologicdictionary, effector precursors with receptors for self being either destroyedor inactivated in someway. Whatis not revealedin mostarticles on toleranceis that these three major approachesare not mutuallyexclusive, nor are the conceptualdemarcations betweenthemas rigid as mayfirst appear. Giventhe importanceof avoiding uncontrolledautoimmunity,mayit not makesense to buttress a repertoirepurgingmechanism with a suppressorsystemthat acts as a fail-safe device? Giventhe immunopathological consequencesthat can result from continued antibody productionin the face of persisting antigen, could one not envisagethe bodyutilizing a trick developedprimarilyfor self-recognition to limit the immuneattack against foreign antigens, such as intestinal commensal organisms?Is the thought that an anti-idiotypic suppressor T cell stops a particular responseso far fromthe thoughtthat the idiotype is silenced by clonal purgingof the cell bearing it? Thetemptationto place tolerance phenomena into tight compartmentsmustbe resisted for another reasonas well. Aselective immune system, gearedto react to the unknown, must be degenerate and redundant(47), otherwise the repertoire of unispecific cellular elementswouldhaveto be infinitely large. A necessary consequence of this fact is that the at~nity of an.tibodiesvaries greatly, and that cross-reactivities amongunrelated antigens and antibodies are common.This beingso, andthe numberanddiversity of self antigensbeing very large, clonal deletion of all cells with any reactivity .to anyself antigen cannotbe absolutelycorrect, becausethere wouldbe a real risk of deleting the total repertoire! Thusany thought of clonal purgingas the mechanism of tolerance must immediatelybe qualified by exemptionsfor sequestered antigens, antigenspresentin lowconcentrationsin extracellular fluids, and (for antigens present at intermediateconcentrations)perhapswith respect to antibody alfinity thresholds, belowwhichthe purging mechanism could not be expectedto operate. Bythe sametoken, a limited amountof autoantibodyproduction,e.g. to heart muscleantigensafter a myocardialinfarction, neither transgresses the rules of tolerance nor causesany harm. It appears, therefore, that immunologic tolerance cannotbe describedor explainedsimplistically or in isolation fromimmune responsesas a whole. Fromthe viewpointof this review, it is fortunate that twoother chapters

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IMMUNOLOGIC TOLERANCE 35 in this volumedeal with idiotypic regulation (100) andsuppressorpathways (53a), whichthereforereceiveless emphasishere. Theplan of this review as follows. A brief historical introduction describes the mainstreamsof thinking in the field. Themajor section of the reviewdeals with the B lymphocyte,becausemoredetailed knowledge about its receptors for antigenand moreadvanced technologyfor its studyat the single cell level have given us a clearer picture than is available for T cells of howit can be signaled followingencounterwith antigen. Present knowledge of howactivation takes place is presentedto contrast with the various mechanisms of negativesignaling. Akeyfindingthat emergesis that not all B cells react equally to a given signal. The more complexproblem of T lymphocyte tolerance is then addressed, with special emphasison tolerance to MHC antigens. HISTORICAL

PERSPECTIVES

Thewordtolerance wasfirst applied to this field by Owen(89, 90), in his classical studies on binovular twin cattle sharing a common placenta. He noted that "erythrocyte precursors fromeach twin fetus had becomeestablished in the other and had conferred on their newhost a tolerance towards.... foreign ceils that lasted throughoutlife" (89). Evenearlier, Traub(122) had noted that miceinfected in utero with lymphocyticchoriomeningitisvirus carried the virus in their bloodandtissues throughoutlife, without an apparentimmune response, whereasmice first infected in adult life madea brisk, typical antibody response. Onthe basis of these two findings, Burnet& Fenner(19) predicted that an antigen introducedinto the bodyduring embryoniclife, before the immuneapparatus had developed, wouldbe mistakenfor self, and that it wouldnot evoke antibody formationthenor if reencountered later in life. It is of interest to recall that the somewhat awkwardself-marker theory of antibody production proposedin that workin fact postulatedself-recognitionunits in antibodyproducingcells that gavethemthe capacityto recognizeself. Thepostulated characteristics of these receptors are somewhat similar to those popular today for self-MHC recognition by proponentsof the two-receptortheory of MHC restriction. Burnet’s ownattempts to provide experimental evidencefor the theory wereunsuccessful(20), but experimentalinduction tolerance wasachievedsoonafter by Billinghamet al (9), whousedliving cells as the sourceof MHC antigens andskin graft rejection as the readout system. Theseauthors coined the phrase "actively acquired immunological tolerance." This study usheredin an avalancheof research on the subject. It wasperhapsfortunate that mostearly workerswhosoughtto validate the conceptof tolerance by using non-livingantigens, and antibodyproduc-

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36 NOSSAL tion as a readout, usedxenogeneicserumproteins as the test toleragen(24, 32, 56, 109, 110), as had they followedTraub’slead and used microorganisms, the results wouldhavebeenless successful(20, 82). In the event, the conclusionthat antigens introducedin the perinatal period preferentially caused tolerance quickly becamewidespread.Toleranceinducedin adult animalsby lethal doses of X-irradiationand allo- or xenografting(69) was explicable on the postulate that a repopulating lymphoidsystemrecapitulates the (unknown)events of early ontogeny.Thefinding of Felton (44) that small but supraimmunogenicamountsof pneumococcalpolysaccharide could cause nonreactivity even in normal, adult mice was generally placedinto a separate conceptualbasket until muchlater. A major stride towardsa frameworkfor the understandingof tolerance wasthe developmentof Burner’s (18) clonal selection theory. If immune recognition were indeed encodedin a population of immunocytes with one specificity per cell, then deletion of self-reactive clones could elegantly accountfor self-tolerance. Thenotion wasfurther elaboratedby Lederberg (65), whopostulated that lymphocytespassed throughan obligatorily paralyzable state early in their ontogeny,during whichantigenic encounters wouldsilence or delete them;if, this milepostwerepassedwithoutantigenic encounter,the cell wouldbecomeinducible,i.e. susceptibleto activation by clonal selection processes.Thediscoverythat onecell alwaysmadeonly one antibodyspecificity gavean early boost to clonal selection (83), and the discoveryof great heterogeneityin antigen binding amgnglymphocyteslent strong support (80). It wasnot until the late 1960s:thatclonal selection cameto be almostuniversally accepted, at least for the B lymphocyte.In the meantime,the notion of somethinguniqueabout the newbornstate that facilitated tolerance induction had received somerude shocks. In 1962, Dresser found that if care was taken to free preparations of serumproteins of all aggregatedmaterial, intravenousinjections of minute amountsinto normaladult animals could induce tolerance (37). This was an importantdiscovery, becauseit raised the possibility that evenmature immunocytes could receive negative signals from antigen. Graduallythe perception dawnedthat unprocessedantigen, acting alone, wasinsut~cient to trigger an immune response, but that someaccessorystimulusor characteristic wasrequired. Theterm adjuvanticity wascoined. Thebelief grew that direct encounters betweenlymphocytesand soluble antigens lacking this characteristic werelikely to deliver a negativestimulus(46). Yetthe nature of this signal remainedentirely unclear. Until the middle1960s, writers on tolerance considered phenomena involvingpredominantly T cells (e.g. graft rejection) andthose depending reducedantibody responsesmoreor less together. Thencametwin revolutions in rapid succession, namelythe clear separation of lymphocytes into

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IMMUNOLOGIC TOLERANCE

37

two great families, T and B cells, and the realization that these families had to collaborate in antibody formation (reviewed in 73). The impact on research into tolerance was immediate and profound. Weigle’s group (23, 127-129) soon reached the view that humangammaglobulin injected into adult mice produced tolerance in the T cell compartmentmore rapidly and at a muchlower dose of antigen than for the B cells. The greater ease of tolerance induction in T cells was confirmedin several other studies (74, 76, 99, 116). Nevertheless, one probable cause at workin these experimentsstill remained to be uncovered by independent work. The discovery of suppressor T cells by McCullagh(70) and t3ershon & Kondo(49) gave new thrust to tolerance research. It is in someways remarkablethat suppressor T cells had been missed until then. In retrospect, the ultra-low dose tolerance caused by femtogramamountsof flagellar antigens in our laboratory (107) could hardly have had any other explanation. In any event, once discovered, suppressor T cells were found to be active in manysituations. As early as 1973, a review by Droege(38) cited no fewer than 81 references on suppressor T cells, and the pace of research has certainly not slackened since. The key role of regulatory T cells in both antibody formation and T cell-mediated immunereactions raised another issue of great importance to the tolerance field. Giventhat the precursors of effector cells, particularly B cells, might not be tolerant of certain self components,the possibility was raised that autoimmunization might follow if a self molecule came into association with a foreign molecule that could act as a carrier, thus being capable of inaugurating T cell help (3, 127). A similar mechanismcan invoked for breakdown of experimentally induced tolerance in some models. Thoughthe emphasis of research in the early 1970s swungheavily towards regulatory T cells, the concept that B cells might be negatively signaled through direct contact with antigen was kept alive. Animportant experimental contribution was that of Borel’s group (1 l, 50), whoshowed that haptenic antigens coupled to autologous immunoglobulin acted as powerful, direct toleragens for adult B cells. A seminal theoretical work was the articulation by Bretscher &Cohn (17) of their two-signal theory lymphocyteactivation. This states that lymphocytesrequire two inductive signals to becomeactivated. One is provided by antigen occupying the receptor for antigen; the other is provided by the action of a helper T cell. If the first signal acts alone, in the absence of the second, the result is a negative signal or tolerance. In manyways, this formulation still provides a useful framework. By the mid-1970s,the last of the crucial issues mentionedin the introduction, namelythat of MHC restriction, entered the arena, profoundly affecting all our concepts of the T cell and therefore also of tolerance. AsDoherty

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& Benninkhaveput it (33), the tolerance problemcould no longer be seen simplyas a needto distinguish betweenself and non-self, but rather as a requirementthat the T cell be capableof distinguishingself fromself plus other. Theintriguing possibility emergesthat within the thymuscells with a high affinity for self-MHC components are eliminatedby a clonal abortion mechanism(10, 33), whereasthose with low affinity for self-MHCare positively selected. Theextensiveliterature on MHC restriction is, however, outsidethe scopeof this review. SIGNALS INVOLVED TRIGGERING

IN

B LYMPHOCYTE

Before considering the possible mechanismsof induction of tolerance amongB lymphocytes,it is essential to summarize whatis knownabout the induction of immunityin this. population. Studies with purified B cells culturedat lowdensity(40, 58) or singly (97, 114,124, 130)havematerially enlargedour understanding.It is nowclear that a cross-linkingof the cell’s membrane immunoglobulin (mIg) receptors alone, whetherby anti-Ig antibodiesor by a multivalent,T-independent antigen, doesnot suffice to trigger the cell. Additionalsignals are required. For someB cells, a T-independent antigenandoneor moreB cell growthanddifferentiation factors(s) initiate proliferation and subsequentantibodyproduction. For others, an antigenspecific, MHC-restricted, T cell-derivedhelper signal is also needed(4, 81). Furthermore,the stimulatory co-factors able to movethe B cell from a resting Gostate to active blastogenesis maynot be identical to those requiredfor continuedcycles of multiplication,and these, in turn, maydiffer from factors that promotedifferentiation to antibody-producingstatus. Someevidencesuggests that macrophage-derived, interleukin l-like molecules are neededearly in the cascadeandat least twotypes of T cell-derived factors are neededsomewhat later. Thenature of these various co-stimulatory factors is under active investigation at the moment.Frommyviewpoint, the most important point is that this recent workvalidates the requirementfor somethingother than Signal 1 alone, openingthe door to the possibility that Signal1 withoutthis complexof factors that constitute Signal 2 indeedmayexert a negativeeffect on the cell. Somefurther interesting points warrant mentionwith respect to Signal 1. Althoughit appearsthat receptorcross-linkingis an essential elementin immuneinduction, not every cross-linker behavesequally. Dintzis’ group (30, 31), by using size-fractionated linear polymersof acrylamidesubstituted with hapten, reached the conclusion that a minimumnumberof receptors (about 12 to 16) mustbe connectedas a spatially compactcluster before an immunogenic signal is delivered. Polymerssubstituted with fewer

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IMMUNOLOGIC TOLERANCE 39 haptenmoleculesexert an inhibitory effect. Theimportanceof somecritical degreeof epitope spacingin immune signaling has also beennoted in other systems(42, 43). Weshouldtherefore be awarethat subtle differences may exist betweenthe Signal1 that combineswith Signal 2 in immune induction, and the Signal 1 only, whichmaybe operative in someformsof tolerance. Anotherimportantquestion in the creation of an appropriate micropatch on the B lymphocytesurface involves the cell Fc receptor. This must concernus, particularly as so manyof the toleragens used in current research feature Ig as the carrier molecule.Isolated Fc portions of humanor murine gammaglobulin are capable of polyclonally activating murine splenic B ceils to proliferation and antibodyproduction(8), mimicking the effects of lipopolysaccharides(LPS). Althoughthe effect wasclaimed involve only Fc-receptor-positiveB cells and not macrophages or T cells, the point is difficult to provein high-densitycultures. Multivalentantigens attached to the B cell surface could easily engagethe Fc receptors in a micropatch through capturing secreted antibody. Furthermore, antigen attached to dendritic follicle cells is complexedto natural or acquired antibody and so could lead to the formation of a mixedmlg-Fcreceptor patch on the lymphocytesurface. Weneed to knowmuchmore about the characteristics of the macromolecular interactions that constitute Signal1, which maybe more complexthan we currently imagine.

NEGATIVE SIGNALS TRANSDUCEDVIA THE B LYMPHOCYTEMIG RECEPTOR Manystudies showthat an engagement of the B cell mlgreceptors can also lead to negativesignaling. This section reviewsevidenceof this important processin immunologic tolerance. EveryB cell with mIgis susceptible to this negativesignal, but a dominantfeature that will engageour attention is the fact that cells at different maturationstages in B cell development displaymarkedly differing sensitivities to this influence.B lymphocytes are derivedfromproliferating pre-Bprecursor cells and exit fromthe mitotic cycles that generate themas small, non-dividinglymphocyteslacking mlg (88). Theythen developmlg, first IgMand later IgMplus IgD, during non-mitoticmaturationphase. I arguethat cells encounteringantigenas the first receptors emergeare exquisitelysensitive to negativesignaling. This phaseis whena potentially anti-self B cell wouldfirst meetantigen. Immature B cells already possessingmIgMcan be isolated from newbornspleen or adult bone marrow,and can be confrontedwith antigen in vitro. Note that this is subtly different fromallowingthe mlgreceptor coat to develop on the cell surface in the constant presenceof antigen. AnimmatureB cell is less susceptible to negative signals than one caught in the pre-Bto B

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transition, but it is muchmoresusceptible than is a mature mIgMplus mIgDopositive B cell. Finally, evenan activated B cell, engagedin largescale antibodysynthesis, can be switchedoff by antigen, but only at high concentration. AsI reviewthe data on whichthese assertions are made,the picture that emergesis not a dramatic,all-or-nonedifference conferringuniqueproperties on the immaturecell, but rather a carefully programmed quantitative difference geared to help self-recognition and also to limit appropriate immuneresponses. Negative Signaling During the Pre-B to B Transition The first evidence that receptor cross-linking could have profoundand long-lasting effects on B lymphocytedifferentiation was producedby an elegant series of studies by Cooper’sgroup(reviewedin 64). Whenantibodies to IgMare introducedinto an animalbefore B cells havedeveloped,B cell maturation can be prevented, and an agammaglobulinemic, B celldeprivedindividualresults. However, such animalsstill possesspre-Bcells. Whensuch lymphocytesare cultured in the absence of further anti-/.t antibody, mIg-positive B cells can emerge. Thesefindings could be explained by postulating that as soonas the first few mIgM receptors appear on the maturingB cell, contactwith anti-kt immediately initiates the patching-capping-endocytosis cycle and thus the cell never really developsinto a B cell. This viewgainedfurther credenceby two independentstudies (98, 108), whichshowedthat maturingB cells were muchmore susceptible to modulationby anti-Ig than were mature B cells. Furthermore,although mlgreceptor deprivationin matureB cells wasreadily reversible, a considerable proportionof immatureB cells failed to recovertheir receptor coat in tissue culture followinganti-Ig treatment.It seemslikely that suchcells woulddie in vivo without ever becomingmlg-positive, especially in the continuingpresenceof the modeluniversal toleragen, anti-Ig. Wewishedto take this line of investigationfurther. In somerecentstudies (93), Pike et al set out to determinewhetheror not concentrationsof anti/.t chain antibodiesinsufficient to modulatethe totality of the mlgM receptor coat couldneverthelessexert functionaleffects on B cells. In particular, what wouldhappenif very low concentrationsof anti-/.t were present as immaturesmall lymphocyteschangedfrom being mlgM-negativeto mIgMpositive? Theydevelopedan anti-bt chain monoclonalantibody, termedE4. Thefluorescence-activated cell sorter (FACS)wasused to select small, mIgM-negative cells from adult murinebone marrowor spleen. Thesewere cultured for 1-2 days, and in the absenceof E4, a populationconsisting of 30-40%mlg-positiveB cells resulted. Aspredicted, E4 wasable to inhibit this pre-Bto B transition. At a concentrationof 10/.tg/ml,virtually no cells with high mlgdensitydeveloped.At 1/.tg/ml, a slight inhibition of receptor

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IMMUNOLOGICTOLERANCE 41 emergencewas noted. At concentrations of 0.1 #g/ml or below, the E4 failed to affect mIgappearance. With these low concentrations, FACS analysispost-cultureshowed the mIgstatus of the cells to be exactlyequivalent to that of controls. Accordingly, Pike et al (93) allowedB cell maturation fromthe pre-Bpool to proceedin the presenceof 0.1 #g/mland still lowerconcentrationsof E4. Following1 or 2 days of such preculture, the resulting B cells weresubjected to functional tests. Theywerestimulated with the potent mixtureof polyclonal B cell activators, LP$plus dextran sulfate. Thecapacityof single B cells to giverise to proliferating clusters wasstudied, as wastheir capacity to produceIg, measuredby the protein Areverse plaque technique. It wasfound that 0.1 #g of E4 present during maturation completelyabrogated response capacity. With10-2/xg/ml, 97100%inhibition wasinduced, and even with 10-4 #g/ml, only half as many B cells respondedin the donal cultures as in controls with no E4 present duringmaturation.Yet, in all these groups,the number of B ceils appearing duringpreculture, andthe averagenumberof mIgreceptors per B cell, were perfectly normal. Thus, the B cells must have received and stored some negative signal, which,however,did not impair the insertion of mlginto the plasmamembrane of the cell. If this experimentaldesignis a modelfor whatmaybe occurringin vivo duringthe induction of self-tolerance, it oughtto be possible to produce similar effects on antigen-specificB cells. Wehaveexamined this possibility in vivoby introducingtolerageninto the tissues of fetal micevia the maternal placenta(94), at a time beforeanyB cells werepresentin the fetus. The hapten fluorescein (FLU)linked to the carder humangammaglobulin (HGG)waschosen. Isotope tracer studies indicated free passage into the fetus and helpedto establish tissue concentrations.Togaugethe effects on the emergingB cell pool in a strictly quantitative manner,Pike et al (94) onceagainmadeuse of B cell cloningtechniques,on this occasionusing the T-independent antigen FLU-polymerized flagellin to trigger the B cells. The results led to very similar conclusionsto those describedabove.It wasfound that with a fetal serumconcentrationof around80 pg/ml(8 X 10-5 ptg/ml) or 5 X 10-13 M, half the FLU-responsiveB cells wererendered incapable of responding,indicatingan extraordinarysusceptibility to negativesignaling. Oneinteresting feature of this studywasthat fully tolerant mice,given muchlarger doses of toleragen in utero, possessedin their spleens mlgnegative pre-B cells on the verge of receptor emergence.Whenthese were placed in vitro in a highly stimulatory environment,they acquired their receptors and, then confrontedwith Signal 1 plus Signal 2, reacted normally. This is at least onereasonwhyantigen mustpersist for tolerance to be maintained,and it soundsa warningfor studies in whichlarge numbers of cells are adoptivelytransferred to environments full of Signal 2. Thecapacityof suchvery lowconcentrationsof antigento affect the cells

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functionally suggests that some mechanismother than modulation of the mIgreceptor coat must be at work. It is therefore of considerable interest to ask whether the antigen, despite its low concentration, delivers some death blowto the maturingantigen-specific cell, or whetherthe functionally impaired cell can persist for sometime. Nossal &Pike studied this question in vivo by inducing tolerance via the placental route and subsequently searching for antigen-binding cells (85). Whensmall doses of toleragen were used, no significant reduction in FLU-binding cell numbers was noted. Furthermore, the antigen avidity profile of tolerant and control populations was identical.

The Concept of Clonal Anergy, and Competition between Signals Takentogether, these results suggested that clonal abortion was not really an accurate description of the events induced, at least not whencritically low concentrations of antigens were used. Rather, they are more consistent with the induction of a potentially reversible anergic state in the maturing B cell. I havetherefore coined the phrase clonal anergy to denote that Signal 1 only can render a cell non-responsivewithout actually killing it. I believe this finding settles an old controversy. Tolerant cells do seemto exist, and this allows one to pose the question of whether or not somereversal of this intracellular anergy might form part of the spectrum of autoimmunity. Important evidence exists that even at the earliest phase of B cell neogenesis tolerance induction is not the only possible outcomeof an encounter with antigen. The first hint of this was the work of Metcalf &Klinman(71, 72), who used unfractionated cell sources rich in immature B cells in T-dependent B cell cloning system, the splenic microfocus assay. They found that whenthese cells were confronted with specific multivalent antigen in the absence of T cell help, the cells were rendered tolerant. However, when they saw antigen for the first time simultaneously with a strong activation of helper T cells present in the immediatevicinity, they formed antibody-forming clones. Thus even here, Signal 1 plus Signal 2 resulted in immunity.Teale et al weresutficiently interested in this result to repeat it, using FACS-fractionated mIg-negative pre-B cells from either newborn spleen or (in small numbers) adult spleen (118). In the absence of T help, hapten-HGGpresent during their maturation rendered the immature B cells tolerant. In the presence of help, a clonal response was obtained. An analogous result was obtained in experiments where fractionated pre-B cells were placed in culture simultaneously with hapten-HGGand the polyclonal B cell activator LPS (96). This mitogen has been seen by some as representing a mixture of Signal 1 and Signal 2. In this situation, about two thirds of the calls of relevant specificity wereindeed rendered tolerant,

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but one third wenton to clonal proliferation and antibodyproduction. In that substantial minority, the tolerance signal hadbeenoverridden.Asomewhatsimilar intervention of a polyclonalB cell activator duringtolerance induction had been reported previously (68). Negative Signaling of Immature B Cells If the line of reasoningpostulatinga gradationof progressivelydecreasing sensitivity to negativesignalingas the B cell maturesis correct, interest attaches to the behaviorof B cells with mlgreceptors that maystill not be fully mature.Alikely sourcefor suchcells is the mlg-positivecell pool from newbornspleen or adult marrow,whereimmaturecells predominate. To study suchceils, onecan either resort againto the modeluniversal toleragen, anti-/~ antibody,or seek to identify, isolate, andstudy the immature B cells of a givenspecificity. Nossalet al (84, 85, 87, 92-96)havedoneboth and havedocumented that such B cells do represent an intermediatestage in sensitivity to toleranceinductionbetween the exquisitesensitivity of cells in the pre-Bto B transition state and matureB cells. Let us consider,first, the behaviorof B cells fromnewborn spleenfollowing a brief encounterwith anti-~ chain antibody(87). Thesewerefound be about 30-fold moresusceptible to negative signaling than wereadult B cells similarly treated. To achieve60%inhibition of clonal proliferation followingmitogenstimulation, 0.1 /~g of anti-/~ antibodyper ml wasrequired, acting for 24 h on the cells, whereas3 #g/ml only reducedthe proliferative potential of maturecells by 40%.A similar difference was noted for antigenic stimulation and antibody formation as the readout. Note, however,that the threshold concentration delivering an effective negativesignal is about1000-foldhigherthan for the pre-Bto B transition. As immatureB cells, thus defined, possess adequateamountsof mlg, it is possible to prepareantigen-specificimmature B cells by the sameantigenaffinity fractionationproceduresas for maturecells (55). Onecan then react such cells with multivalent antigen, e.g. for 24 h, and subsequentlycan challenge with antigen in immunogenic form. Their analysis (84) showed that with an oligovalent FLU-HGG that has 3.6 haptens per molof cartier, hapten-specific newbornB cells could be tolerized with 1000-foldlower antigenconcentrationsthan couldadult 13 cells, the thresholdbeing around 0.1 /zg/ml. In other words,the sensitivity is muchlowerthan that of cells that first see antigenbeforeachievingfull receptorstatus, but muchhigher than that of fully maturecells. Pike et al haveperformedan extensiveexplorationof negative signaling amongthis intermediatepopulationto determinethe importanceof molecular arrangementof haptens and of the cartier molecule(92). Spleencells from newbornor adult mice were incubated for 24 h with FLUcoupled to

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HGG,the F(abl)2 fragment of HGG,or bovine serum albumin (BSA). Conjugates were prepared that possessed a meanof 2, 5, 8, or 12 FLU residues per moleculeof protein. After this putative tolerance-inducing regime,the cells wereplacedinto a limit dilution cloningassaywith FLUPOL(polymerized ttagellin) as the antigenicstimulus.It wasfoundthat with all conjugates tested, the newborncells were moresensitive to negative signaling than were adult cells. However,the gap betweenimmatureand maturecell, in terms of sensitivity, varied widelyfromconjugateto conjugate. For all three carriers, the higher the haptendensity, the lowerthe molarity required for tolerance induction. TheFc piece appearedto play somerole in makingHGG the mosteffective of the three carriers. At a given hapten substitution rate, FLUconjugatedto the F (abe)2 fraction of HGG (FLU-FAB)was less toleragenic than was FLU-HGG. This difference could be overcome by crowding on more haptens. FLU-BSA was less toleragenic than either FLU-HGG or FLU-FAB. Dependingon the conjugate, the sensitivity difference factor betweenimmatureand maturecells varied from6 to 53. Withhighly multivalentconjugatesit is easy to see how susceptibility differencescouldhavebeenmissedby other investigators, as the differences are less pronounced than with oligovalent antigen. Thoughfew authors havesoughtto dissect the pre-Bto B transition from slightly later stages of ontogeny,the sensitivity of the immature B cell to negative signaling has beenwidelyconfirmed(21, 39, 71, 72, 105). Nevertheless, the adult B cell is not completelyrefractory, and wemust now examinemlgas a transducerof negativesignals in morematurecells of the Bseries. Negative Signaling of Mature B Cells and their Progeny I havealready madereference to the workof Borel (11, 50), to studies Nossalet al (see above), and to workby Weigle’sgroup (35, 36, 91, 128), whichamplydemonstratethat a sufficient (and by no meansunphysiologically high) concentrationof antigen can deliver a negative signal evento matureB cells. Conjugateswith Ig carriers are particularly effective, which brings us backto the questionof the role of the Fc receptor. This has been addressedin an interesting series of studies by Taylor’s group(117, 119121). Theypreparedstable covalent antigen-antibodycomplexes,for example linking haptendirectly to antibody, or hapten-proteinsto anti-hapten antibody. The hapten-antibody conjugates were profoundly suppressive whenT-independentanti-hapten responses were later measured.Evidence suggesteda direct effect on B cells rather than a suppressorT cell effect, and the Fc piece of the antibody in the conjugatewasrequired. This was closely analogousto earlier studies with non-covalentflagellin-antibody complexes(43). However,whenthe animalhad beenpre-primedagainst the

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rabbit antibody in the conjugate, the end result was an enhancedresponse, suggesting that the conjoint action of (normally toleragenic) conjugate and active T cell help was immunogenic. Whenribonuclease was linked to antibody in this fashion, this normally poor immunogeninduced priming or immunologicmemory,but this immunogenicitywas lost if recipient mice had been previously madetolerant to the rabbit IgG carrier. In that case the complexeswere frequently toleragenic. Again, the suggestion is strong that an engagementof the Fc receptor within the micropatch on the B cell surface somehowcauses the generation of a more effective Signal 1, which requires the Signal 2 of T cell help to becomeimmunogenicand in its absence acts to produce tolerance. It wouldbe of considerable interest to quantitate the effects of these stable antigen-antibody conjugates on immature B cells. The regulatory role of the mlg receptors as signal transducers does not end at the momentof lymphocyteactivation. In fact, activated B blasts and early antibody-forming cells including manyplasma cells still bear mlg. Presumablythis indicates that such cells are. still under the regulatory influence of antigen. Certainly premature removalof antigen halts expansion of immunocyteclones (95). There may be circumstances, however, such as the existence of immunecomplexes in the circulation, where it might be wise to halt the ongoing immuneproliferation, or even antibody formation at the single cell level. Someyears ago, Schrader & Nossal discovered that an interaction of a single antibody-forming cell with multivalent, specific antigen can result in profound inhibition of that cell’s secretory rate (104). This represents an unexpected late down-regulatory signal mediated via mlg, which may represent, though at a much less sensitive level, a mechanismanalogous to the tolerance induction just described. Independent confirmation ~ was provided by Klaus (62), whose group also soon showed that antibody production in monoclonal plasmacytomacell cultures could be similarly inhibited (1). Antigen-antibody complexeswere particularly effective in this regard (2). Mylaboratory has recently sought to characterize this phenomenon, termed effector cell blockade, more fully. As a first step (12), a mouse hybridoma cell line that secretes IgMspecific for FLUwas developed. Treatment of these cells with highly multivalent FLUconjugates (e.g. FLU20-gelatin) resulted in inhibition of hemolytic plaque formation. Biosynthetic labeling studies indicated this was a result of reducedIg synthesis. Total protein synthesis and cell proliferation were not affected, documenting the specificity of the inhibitory effect. Fluorescencestudies showedthat FLUconjugates capable of causing blockade aggregated on the cell surface, and clearance of cell-associated antigen correlated with recovery from blockade. Reversibility has also been documentedin other systems (78).

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In later studies, Boyd&Schraderhaveprovidedfurther valuableinsights (14, 15). Colchicinereverses effector cell blockade, providedexogenous antigen wasremoved.In the presenceof colchicine, cappingoccurs, presumablyaiding antigen removal. However,in the continued presence of exogenousantigen, colchicine wasnot able to reverse the negative signal, arguingagainst the notionthat colchicine-sensitivestructures, suchas microtubules, are essential for signal transmission. It wasalso found that antibody-forming cells (AFC)fromthe spleens of 10-day-oldmiceare more susceptible to blockade than are adult AFC.Furthermore, blockade in neonatal AFCcould not be completely reversed by enzymaticremovalof surface antigen. This increased susceptibility of neonatal AFCis reminiscent of the greater sensitivity of newborn splenic B cells to negativesignaling by antigen. It supportsthe notionof someintrinsic extra susceptibility in mlg-transducedsignaling in the neonatalstate. Anothermodelof a relatively mature B cell is a B lymphoma, such as the cell line WEHI 231, whichresemblesa B cell in bearing large amounts of IgMon its surface but not in secreting Ig. It wasfound(13) that anti/.t chain antibodies, including E4, profoundlysuppressedthe growthof WEHI 231 in both soft agar and liquid culture. Other antibodies binding to non-Igcell surface antigens lackedthis effect, and LPS,whichinduces antibody formation in normalWEHI 231 cells, could neither prevent nor reversethe growthinhibition. This negativesignal differs fromthat involved in effector cell blockadein affecting cellular life span. It provokesthe thought of whetheror not receipt of the anergic signal in normalB cells mayaid their early demise. Thepicture that emerges,then, is a graduallydiminishingsusceptibility to negative signaling as the B cell matures. Themonoclonalanti-/~ chain antibody, E4, has allowedsomequantitative comparisonsin this regard (93). It takes approximately 5.5 log~omoreE4to causeeffector cell blockade in matureantibody-forming cells than it raises to cause clonal anergyduring the pre-B to B transition. Operationally, this could well meanthat many exogenousantigens never reach the concentrations that might impair the B cell responseto them. CONSTRAINTS ON THE UNIVERSAL APPLICABILITY OF CLONAL ANERGY IN SELF-TOLERANCE Althougha strong case can be madefor ~he plausibility of the clonal anergy modelas beingrelevantto self-tolerance, particularlyfor antigens(e.g. cell surface molecules)present in multivalentform, it is importantto mention someof the constraints on the universality of the mechanism. Perhapsthe

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most obvious of these is that monovalent antigens do not induce clonal anergy in vitro. Most of the evidence suggests that the negative signal requires mlg receptor cross-linking. One could imagine in vivo matrixgenerating mechanisms,e.g. self antigens associating with macrophagesurfaces, or even one B cell presenting toleragenic antigen to another. Given the extreme sensitivity during the pre-B to B transition, even someselfassociation through protein-protein interactions at the B cell surface could not be entirely excluded. It is also of interest that despite the enormous amount of work on autoimmunediseases in both experimental animals and man, no one has yet reported an autoimmunedisease that involves antibody production to autologous albumin, transferrin, complementcomponents, or other soluble monomericserum proteins present in high concentration (rheumatoid factors are an interesting exception). If tolerance to albumin did not exist within the B cell compartment,it is hard to see howantibody production could be avoided on occasions of abnormal induction (e.g. endotoxins present in septicemia). There are no albumin-binding cells in humanperipheral blood (5), though B cells capable of binding autoantigens present in low concentrations in serumdo exist (5, 41, 102). This suggests that for antigens present in high concentration, someclonal purging mechanism may be at work in vivo, perhaps one we have not yet been able to modelin the artificial in vitro models. Nevertheless, the possibility must be faced that someself antigens cannot evoke the clonal anergy/clonal abortion pathway, but cause tolerance through other mechanisms.From that viewpoint, recent studies from Dienet’s groupare of interest (29, 125, 126). The sensitivity of the fetal immune system to tolerance induction in vivo was investigated by injecting various antigens into the maternal circulation. Transplacental passage was monitored, as was persistence of antigen in serum and lymphoidtissues. Despite adequate exposure, the fetuses failed to becometolerant in most instances. Of seven antigens, only two gammaglobulins (HGGand bovine) induced significant tolerance. Furthermore, whenhaptens were introduced multivalently onto the proteins, the various non-tolerageniccarriers failed to induce hapten-specific tolerance. This was despite fetal concentrations of up to 10-6 M. In other studies it was found that some carbohydrates, but not others, can act as a toleragenic carrier for hapten-specific B cell tolerance, and there appeared to be no clear correlation betweenthe capacity to induce tolerance and either the molecular weight of the carrier or its epitope density. Indeed, rather subtle chemical changes were found to abolish an antigen’s tolerag~nic potential. Thesesomewhatsurprising findings are interpreted as suggesting that the toleragenie potential of certain extdnisic antigens reflects someobscure carrier-related property. To account for the results, Diener’s group has

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postulated (28, 125) a dual recognition event in self-tolerance induction that bears somesimilarities to the dual recognition mechanismresponsible for immuneinduction in T cells. Consider an organ-specific cell membraneself antigen, S, and a ubiquitous, polymorphicself marker or interaction structure, Z. The latter is postulated to be present on all cells including lymphocytes. Whenan anti-S lymphocyteadzes in ontogeny its anti-S receptor meets S, and its self-marker Z meets the Z present on the S-bearing cell This interaction is postulated to cause clonal abortion. Extrinsic, non-self antigens are seen as incapable of inserting into cell membranesin such a way as to allow correct dual recognition, with the exception of somemolecules with special properties. This novel speculation, however, does not account for mechanismsby which animals become tolerant of self components not associated with cell surfaces, e.g. serumproteins. Preliminary studies in our laboratory (B. L. Pike, G. J. V. Nossal, unpublished data) have sought to use transplacental FLUoBSA in vivo as an agent to induce clonal anergy in FLU-specific B cells emergingin fetal and early postnatal life. Even large doses achieved only moderate reduction in the numbersof clonable anti-FLU B cells. This contrasts with the relatively poor but still definite potential of FLU-BSA to give a negative signal to immatureB cells in vitro (92). The result confirms that the carrier molecule is not a neutral entity in tolerance induction. Obviouslythis constraint will require muchfurther investigation.

B CELL TOLERANCE AND AUTHENTIC SELF ANTIGENS There would obviously be many advantages if students of immunologic tolerance, rather than introducing foreign modeltoleragens into animals or cultures, could investigate interactions betweenauthentic autologous antigens and the lymphoid system of the body. However,surprisingly little workhas been done in this way, probablybecause of the practical diti~culties posed by omnipresent antigen, e.g. through neutralizing any antibody that may be formed or through blockading immunocytereceptors. I have already mentioned some of the studies (5, 41, 102) seeking autoantigenbinding B cells, whichshowedpresence of B cells for self antigens occurring at low molarity but absence for those in serum at high concentration. This finding seems consistent with the rules of clonal anergy/clonal abortion. Twoearly studies that are frequently quoted are those of Triplett (123), who claimed that extirpating an organ early in ontogenyled to a loss of tolerance to the organ-specific antigens so that later transplantation led to rejection, and the hemoglobinstudies of Reichlein (101), whoconcluded that rabbits could only form antibodies to such determinants of hemoglobinchains as

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IMMUNOLOGICTOLERANCE 49 are not represented in rabbit hemoglobin.In this section, three recent studies are reviewed,two of whichargue against, while one argues for, clonal anergy/clonal abortion as an important mechanism. The first study comesfrom Borel’s group (57) and exploits a pair congenicstrains of mice, one of whichlacks the fifth component of complement, C5, a serumprotein normallypresent at a concentration of 50-85 /.tg/ml. Lymphoid cells fromC5-deficientmicewereadoptivelytransferred into X-irradiatedhosts of the normalstrain andvice versa. C5-deficientcells were not tolerant to C5. They formedantibody whentransferred to C5sufficient (normal)irradiated hosts. Cells fromnormalmiceweretolerant, failing to form antibody to C5on primary or secondaryimmunizationof the C5-deficient hosts with C5. T and B cell mixingexperimentsdemonstrated that the tolerance appearedto reside in the T cell compartment of the CS-sufficient(normal)lymphoid cells. T cells fromCS-deficient(nontolerant) donorscould collaborate either with B cells fromnontolerant (C5deficient) donorsor fromtolerant (normal)donors.This arguesthat anti-C5 B cells had not beeneliminatedin normalmice, nor werethey functionally silenced. This thought-provokingstudy exhibits two features that are somewhat worrying.First, the adoptivehosts receivedonly 760--780rads of irradiation in a split dose. In myexperience,this wouldallow considerableendogenous reconstitution. Second,70 million cells weretransferred, a very large number, whichraises the possibility that T cell sourcesmightbe contaminated by significant numbersof B cells and vice versa. Takenat face value, the results provide another exampleof a monovalentantigen not leading to B cell tolerance. Asa general point, caution mustbe taken in adoptivetransfer studies to excludethe possibility that effects attributed to B cells in fact are not ascribableto mlg-negative pre-Bcells or evenearlier precursors.It is clear that such occur in spleen and bone marrowin substantial numbers,and if they maturein the concomitantpresence of antigen and T cell help, they will immediately be triggered. Of course, pre-Bcells that lack mlghavenot yet beensubjectedto self-censorship. Thesecondstudies concernan alloantigen, the cytoplasmicliver protein F. This moleculeof 40,000 mol wt occurs in two forms in mice, F1 and F2. Serumconcentrationsare around10-8 to 10-9 M.It has long beenknown that in certain strains of micealloimmunization can lead to an autoantibody responsedirected against determinantscommon to F~ and F2, implyingthat self-tolerance is normallymaintainedby T cells (59). This lack of B cell tolerance is not too worrying,given the relatively lowserumconcentration. Detailedinvestigationof cellular interactions in this systemare nowpossible becauseof the recent development of an in vitro assay(113). Thesystem

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measuresthe proliferation of F-primedT cells exposedto F-pulsedsplenic adherentcells. SyngeneicF antigen fails to activate undercircumstances whereallo-F workswell. It has beensuggested(27) that this is dueto vivoeliminationof self-F reactiveT cells. If so, this wouldbe oneinteresting exampleof clonal deletion affecting T cells but not B cells, an interesting counterpointto the usual explanationsinvolvingsuppressorT cells. Thethird exampleinvolves tolerance to antigenic determinantson mouse cytochrome c (60). This study fromKlinman’sgroupis of particular interest, not only becauseit supports clonal anergy/clonalabortion modelsof tolerance,but also becauseit dependson the careful, quantitativeanalytical technologyso vital in tolerance research. To understandthe study, it is necessaryagain to recall that pre-B cells can form antibody-forming cell clonesif artificially transplantedinto a milieuwheretheymatureinto B cells in the concomitantpresenceof antigen andT cell help (Signal 1 plus Signal 2 overridingthe toleragenicpotential of Signal 1 alone). It is possible to prepare pre-B cell-rich cell populations, e.g. by using bone-marrow depletedof mlg-positivecells. Onecan then contrast this pre-Bcell repertoire of clonotypes with that of mature splenic B ceils. If clonal anergy or analogousmethodsof repertoire purging are importantin self-tolerance, oneshouldfind autoreactiveB cells presentin the pre-Bcell repertoire but absent fromthe matureB cell pool. Jemmerson et al (60) synthesizedtwo peptides from mousecytochromec and probed the T-dependent immune response against each in the clonal splenic microfocusassay. Withone peptide it wasfoundthat the pre-Bcell pool containedmanyclonal precursors against the self component,the numbersbeing analogousto those for a foreignpeptideof equivalentsize. MatureB cells showed far fewerprecursors, indicating an in vivo repertoire purgingduringthe pre-Bto B transition. Withthe other peptide, whichincluded an evolutionarily highly conservedregion at the N-terminalend of cytochrome c, neither cell population madean anti-self response.Thisraises the fascinatingpossibility that V genes whoseproducts mighthave reacted with this region mayhave been eliminated fromthe specificity repertoire by evolutionarymeans. In somerespects, wefind this examplemoreilluminating than the other two. Wehavealready stated that clonal abortion/clonal anergycannot be absolutelytrue for everyself antigen, for fear of purgingthe wholerepertoire throughcross-reactivity. Thedegreeof the contributionof this mechanism to self tolerance therefore becomesa quantitative question. The existenceof someundetermined numberof anti-self B cells, as there appear to be in the C5and F antigen examples,cannot invalidate clonal silencing as an importantquantitative contribution to the immune balance. Whatone needsto knowis whetheror not there are feweranti-self C5or anti-self F antigen B cells than there wouldbe for a foreign antigen of comparable size

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IMMUNOLOGIC TOLERANCE 51 andshapeand whetheror not such cells display a lowermedianavidity for the self antigen than might be expected. Until such information comes forward,the possibility of a partial Bcell contributionto the final tolerant state in these modelsremainsopen.


TOLERANCE PHENOMENAWITHIN THE T CELL COMPARTMENT The subject of tolerance within the T cell compartmentis muchmore difficult to addressat either the cellular or the molecular level. Westill know relatively little about the T cell receptor for antigen. Wehaveno method of enumeratingeffector T cells that can comparewith the Jerne plaque technique.Aboveall, wehaveto face the complexitythat whereasthe B cell can encounterand bind antigen free in solution, and evidently can receive signals from such an event, the T cell appears to be programmed to see antigenonly at cell surfaces. Furthermore,both the triggering of resting T cells into activation andthe deliveryby the effector progenyof someeffect (cytotoxickilling, help, suppression)requireboth a unionof T cell receptor with foreign antigen and reeogrtition by the T cell of certain self-MHC determinants, the nature of that "self-hood" having been somatically learned by the T cell pool. Theonly exceptionto these rules appearsto be the effector cell engagedin mediatingsuppression, whichcan adhere to antigen-coated surfaces that lack any MHC molecules. Cell interactions involvingproductsof the I-J genesare probablyactive in getting resting suppressor precursors moving,so evenfor this category of cells, signal inductioninvolveslinked recognition. Conceptually,this extra complexityposes no major dilemmafor clonal anergy/clonalabortion theories. Onewouldsimplyhave to postulate that for those self antigens not already on a cell surface, mechanisms exist for presentingthemin cell-associatedformto the maturingT cell, e.g. by means of accessorycells in the thymus.Onthe other hand,a large bodyof opinion prefers to attribute self-recognition within the T cell compartment to the induction and maintenance of an anti-self-suppressor T cell population.If this special kind of immune responseis placedat a post-thymicstage of T cell development, it is hard to see howthe peripheralpool of (not clonally purged)T cells couldoperationallydistinguishself fromnot self, activating exclusivelyor predominantly a suppressorresponseonly against the former. If, on the other hand, one were to postulate an intrathymic preferential genesis of suppressorsshouldthe maturingT cell see antigen, a formulation that wouldgive ever-presentself antigens the edgethey possesson a clonal abortion model, then weare left with the need to explain whyforeign antigens,in the face of a partial blood:thymus barrier, can so readily gener-

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ate suppressor cells under somecircumstances, even in adult animals. For these reasons, I prefer to regard the generation of suppressor cells as a regulatory mechanism,an ancillary device in self-tolerance, rather than as the primary cause. The main point, however,is that the existence of a family of immunocytes knownas suppressor T cells cannot explain self-recognition. It merelyshifts the problemto another level. Furthermore, the existence of an (undefined) numberof suppressor cells in a T cell population that behaves as tolerant does not necessarily implythat these cells are the sole cause of the tolerance. Clonal silencing of effector precursors maybe present concfirrently, but they escape detection unless sought. Onceagain, only strictly quantitative methodscan address the relative contributions of the various mechanisms. Suppressor T Cells in Tolerance Measured by Antibody Formation It is surprising that someantigens that are good at negatively signaling B cells in vitro or in vivo also seemto be excellent at inducingsuppressorcells. For example, HGGhas been used in manystudies of suppression (6, 7, 35), although it has also been claimed that suppressor cells may not be the essential element in keeping the animal as a whole unresponsive (36, 91, 126). Most authors study induction of suppressor cells in adult mice, but some(66, 126) have used fetuses and the transplacental route of antigen administration and find it effective in initiating suppressor cell generation. Aninteresting recent series of studies (66, 67) documentsthat HGG can not only produce suppressor cells, but also can set up a state of suppressor T cell memory.Thus, whenthe first effective waveof active suppression has passed, animals maybe left with memorysuppressor T cells ready to spring into a secondary wave of multiplication and active suppression should antigen be encountered again. HGGin vitro can generate memorysuppressors that even 6 monthslater can be reactivated by antigen. Such cells may have been missed in previous studies, possibly explaining some examples where tolerance appeared to persist though active suppressors could not be detected. The memorysuppressor cells were antigen specific and were found to have the same phenotype as those mediating primary suppression, namelyLy - 1-23 +, Thy- 1 +, I a+. Moreover,thoughit requires morethan 100 ktg of HGGto generate primary suppression, as little as 1 ktg induces suppressor T cell memory.Exposure to very small concentrations of self antigens throughoutlife might give a continuing series of boosts to the pool of memorycells without necessarily generating detectable active suppressor activity. Anyunexpectedpulsatile rele~ase of self antigen, e.g. from organ damage, could quickly lead to emergenceof active suppressors, preventing autoimmunity. Thus, a powerful case has been made for a mechanismwith

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physiological relevance. Wecan only conclude that this co-exists with B cell-silencing mechanismsas demonstrated in our studies.


Suppressor T Cells in MHCReactions A large literature exists on suppressor T cells in various modelsof T cell tolerance involving MHC antigens (e.g. 34, 45, 52, 53, 61, 75, 103). This is beyond the scope of this review. The view has been expressed (77) that in this case clone elimination is the prime process and suppressor cells operate as a fail-safe mechanism. Functional Clonal Deletion in T Cell Tolerance I wish nowbriefly to review some evidence supporting the view (16) that functional clonal deletion does occur amonganti-allo-MHC T lymphocytes. Given that T cells of a given specificity cannot be enumeratedby an antigenbinding technology, it is difficult to ask howmanyT cells active against a given self antigen exist in the body. However,the point can be approached by cloning technology. Wecannot visualize the specific T cells nor isolate themby antigen-affinity binding techniques as in the case of the B cell, but we can enumerate specific cells capable of yielding progeny in vitro whose effects can be measured. The best knownsuch technology is the enumeration of precursors of cytotoxic T lymphocytes(CTL-P), which develop from single cells by appropriate stimulation with irradiated allogeneic cells acting as an antigen source, and T cell growth factor as a co-stimulus, a methodology now in use in many laboratories. Nossal & Pike have applied this methodto the classical tolerance modelof newbornmice receiving semiallogeneic spleen cells (86). FUNCTIONAL CLONALDELETION OF ANTI-ALLO-MHC CYTOTOXIC T LYMPHOCYTE PRECURSORS CBA(H-2 k) mice were rendered tolerd ant to H-2 antigens by injection of (CBA× BALB/c)F 1 spleen cells on the day of birth (86). At intervals of 2 days to 12 weeks, the frequencies anti-H-2 d CTL-Pin the thymus and spleen were determined by limiting dilution cloning technology. A profound and long-lasting functional clonal deficit was noted. This wasfirst clear-cut by day 5 of life in the thymusand by day 8-10 in the spleen, as if to suggest that the clonal purging occurred in the thymus and movedto the spleen only as that organ came to be dominated by purged thymus migrants. The functional clonal deletion reduced the observed proportion of anti-H-2 d cells in adult spleens from a normallevel of about 1 in 500 spleen cells (1 in 150 T cells) to 3%of that figure. This showsthat whether or not suppression is also induced concomitantly, clonal silencing is a mostimportant feature of the tolerant state. Its rapid occurrence in the thymus makes a clonal abortion/clonal anergy mechanismhighly plausible.

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Althoughthis strict quantitative approach awaits confirmation, there is a considerable amountof indirect evidence for functional clonal deletion emergingfrom bulk culture studies. For example, Streilein’s group (54, 112) failed to detect lymphocytesreactive with tolerated H-2 alloantigens in a wide battery of tests, including mixedlymphocytereactions, graft versus host reactions, cell-mediated lympholysis, or concanavalin A-mediated, polyclonal activation of tolerant T cell populations. Suppressor cells were not found in these studies, and the inference was of an active process achieving specific clonal deletion of an early stage in the ontogenyof the alloreactive T cells. Proponents of the suppressor T cell might then postulate that suppressor T cells (anti-idiotype in nature?) might actually be responsible for this functional or actual clonal deletion. A hint in this direction was provided by Gorczynski&MacRae (52, 53). Streilein (11 l) investigated this possibility in an original way by using MHCrecombinant strains to induce tolerance. Newbornmice were rendered tolerant to F1 semiallogeneic spleen cells in such a mannerthat donor and host differed over the entire H-2 region, over a region including the K or D locus alone, or over a region embracing either K or D and the mid-I (probably IJ) region. Althoughthe first and last groups developedexcellent tolerance, groups differing at the Class I antigens alone showedlittle or no tolerance as judged by skin graft rejection. It is as if isolated K or D antigens acted as poor tolerogens, requiring some kind of activation (presumably of suppressor cells) dependent on IJ disparity to reach the final tolerant state. If the K or D antigen is regarded as a hapten, and the mid-I (presumably IJ) antigen is regarded as a carrier, then active suppression is induced only whenthe immunogen differs from the host in both hapten and carrier. Howthis activation of suppression finally achieves clonal deletion is not clear. These somewhatunexpected results prompted Nossal & Pike to investigate the effects of anti-IJ sera on tolerance induction in their modeldepending on injecting F1 spleen cells differing at the full MHC into newbornmice (86). Repeated high doses of anti-IJ injected into newborns during the period of toleragenesis partially inhibited clonal deletion, an effect, however, that proved to be quite transient. This has been a frustrating finding to follow further. Thoughthe phenomenonwas readily reproducible, it dependedon a particular batch of anti-IJ serum, which ’so far has not been reproduced with several other batches. The role of suppressor cells in functional clonal deletion therefore remains obscure. It must be remembered that the injection of semiallogeneic cells into newbornmice at birth represents an imperfect modelfor self-tolerance. The toleragen-beadng cells enter the body at a time whensmall but significant numbersof alloreactive T cells are already present in the thymicmedullaand the periphery. It could

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be that suppressor cells are moreinvolved in preventing these mature cells from getting out of hand than in clonal abortion/anergy within the thymus. FUNCTIONALCLONAL DELETION OF ANTI-HAPTEN CYTOTOXICT LYMPHOCYTE PRECURSORS Another model of T lymphocyte physiology extensively explored in recent years is the reaction of T lymphocytes to haptenic antigens presented in association with syngeneic cells (106) isolated cell membranes (25). Tolerance can be induced in such systems and is usually ascribed to suppressor T cells (e.g. 25, 115). Alerted to the fact that suppression mayco-exist with an underlying clonal deletion or anergy, Good& Nossal studied anti-hapten T cells in a model system involving adult tolerance (51). Reactive haptens were administered intravenously adult mice and doses were carefully chosen to provide convincing nonreactivity. Presumablythe operative toleragen was hapten coupled to autologous cells. The anti-hapten CTL-Pnumbers of control and tolerant animals were studied in a limiting dilution microculture system. After apprbpriate correction for anti-self activity within the population, the antihapten CTL-Pnumbers in tolerant mice were found to be reduced by 90%. In other words, functional clonal deletion was present. Kinetic studies showedthat a substantial degree of tolerance existed already at 24 h. These studies documentthat tolerance within the T lymphocyte system, even in models where suppressor T cells have been noted, maycontain an element of functional clonal deletion. Again, the relative importanceof each process in physiological self-tolerance must await the developmentof modelsthat moreclosely mimicthe real-life situation. Other Possibilities for Clonal Abortion of T Cells So far it has appearedas thoughelonal purging of the T cell repertoire must take place in the thymus. However,there are somehints that bone marrow precursors of thymic cells already bear receptors for antigen (26, 63) and are susceptible to toleragenesis by self antigens. Althoughthis woulddestroy the elegant notion of the thymusas the chief generator of diversity amongT cells, it wouldnot offend the central tenets of clonal abortion/clonal anergy. Anintriguing recent study (79) involves radiation chimeras in which bone marrowcells repopulated a thymus graft syngeneic to them, but within the body of a lethally irradiated semiallogeneic thymectomized host. The intrathymic lymphocytes, proven to be of bone marrowgenotype, were nevertheless tolerant not only to themselves, but also to the peripheral alloantigens. Noalloantigenic material was detected intrathymically, nor were suppressor cells found. Althoughthe results were interpreted as favoring pre-thymic functional inactivation of pre-T cells, the presence of sub-

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detectable but still toleragenic alloantigen in the thymus cannot be excluded. An interesting example of what appears to be clonal abortion/anergy of T cells, as judged by a limiting dilution assay, has been reported by ChiecoBianchi et al (22). Mice inoculated at birth with Moloneymurine leukemia virus were challenged as adults with Moloneymurine sarcoma virus. Although other adults (regressors) rejected the resulting sarcomas, these dually infected mice (progressors) died with rapidly growing sarcoma. Progressor mice were found to lack virus-specific CTL,and this was not due to suppressor cells. A limit dilution study showeda severe defect of antiviral CTL-Pin the Moloneymurine leukemia virus carrier mice, as well as in the dually infected progressors. This study has reached similar conclusions to Nossal & Pike involving MHC antigens (86). On the basis of these results, it maybe concludedthat functional clonal deletion is well established as one mechanismof T lymphocytenon-responsiveness. Whetherwe are dealing with clonal abortion or clonal anergy will not be clear until knowledgeof T cell receptors increases materially.

CONCLUSIONS This review has concentrated on" recent studies supporting the notion that functional silencing of particular elements in the immunologicrepertoire is an important mechanismin immunologic tolerance for both B and T lymphocyte classes. It has stressed, however,that it is not the only mechanism, and, in particular, a substantial role is seen for suppressor T cells, not perhaps as the primary mechanism, but as an important ancillary one. For the B cell, the view has been promotedthat mIg is the vital signal transducer, and that each stage in the B cell life history that exhibits mIg displays its ownparticular sensitivity to such signals. Effective signaling may involve the aggregation of receptors into an array with particular characteristics, e.g. a critical minimalnumberbefore signaling can occur. Involvement of the Fc receptor, although not essential, mayamplify the signal. Formation of an mIg receptor micropatch is a prelude either to activation or to a down-regulatory event. The former requires conjoint activity of T cells and perhaps macrophages, with B cell growth and differentiation-promoting factors of an antigenically nonspecific, MHCunrestricted nature prominently involved. The latter is the end result when non-immunogeniccarriers with particular characteristics multivalently present antigenic determinants to the B cell in the absence of co-stimulatory signals comingfrom T cells or artificial mitogens. Thehierarchy of sensitivities to such negative signaling is as follows, in decreasingorder of susceptibility: cells in transition frompre-B to B cell status (self antigens obviously

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first interact with devdoping B cells at this stage); immature B cells; mature B calls; activated, antibody-forming B ceils. The negative signal, particularly if delivered by small concentrations of antigen, insutt~cient to modulate the mIg receptor coat, does not kill the cell but rather renders it anergic. Functional clonal deletion is also a possibility within the T lymphocyte class. This has been formally demonstrated for anti-hapten, anti-viral, and anti-allo-MHC CTL-P. Whether an actual deletion or the induction of an anergic state is involved is not known. Functional deletion can co-exist with induction of suppressor cells. Although some evidence suggests that functional deletion occurs in the thymus, a pre-thymic repertoire purging remains a possibility. The complex process of self-tolerance is probably an amalgam of repertoire purging, suppression, sequestration of self antigens, and blockade of receptors by monovalent, non-immunogenie self molecules and other regulatory loops, possibly including anti-idiotype networks. Evolutionary elimination of V genes with self-reactive potential is also conceivable, but chimeric states amply testify that muchof tolerance is somatically acquired. Carefully quantitated experiments with cloning techniques for enumeration of competent B and T cells should continue to enlarge our understanding. More experimentation on authentic self antigens is badly needed. ACKNOWLEDGMENTS Original work reviewed in this chapter was supported by the National Health and Medical Research Council, Canberra, Australia; by U.S. Public Health Service Grant AI-03958; and by specific donations to The Walter and Eliza Hall Institute. Literature Cited 1. Abbas, A. K., Klaus, G. G. B. 1977. Inhibition of antibody production in plasmacytoma cells by antigen. Eur. J. lmmunol. 7:667-74 2. Abbas, A. K., Klaus, G. G. B. 1978. Antibody-antigen complexessuppress antibody production by mouseplasmacytoma cells in vitro. Eur. J. IrnmunoL8:217-20 3. Allison, A. C. 1971. Unresponsiveness to self antigens. Lancetii:1401-3 4. Andersson, J., Melchers,F. 1981.T cell dependentactivation of resting B cells: requirementfor both nonspecifie,unrestricted and antigen-specific, Iarestricted soluble factors. Proc.Natl. Acad. ScL USA78:2497-501 5. Bankhurst,A. D., Tordgiani,G., Allison, A. C. 1973. Lymphoeytes binding humanthyroglobulin mhealthy people

andits relevanceto tolerance for autoantigens. Lanceti:226-29 6. Basten,A., Miller, J. F. A. P., Sprent, J., Cheers,C. 1974.Cell-to-cellinteraction in the immune response.X. T-celldependentsuppressionin tolerant mice. Z Exp. Med. 140:199-217 7. Benjamin,D. C. 1975. Evidencefor specific suppressionin the maintenanceof immunologictolerance. Z Exp. Med. 141:635--46 8. Berman,M. A., Weigle, W.O. 1977. B lymphocyteactivation by the Fc region of IgG. J. Exp. Meal 146:241-56 9. Billingham,R. E., Brent, L., Medawar, P. B. 1953.Activelyacquiredtolerance of foreign ceils. Nature172:603-6 10. Blanden,R. V., Ada,G. L. 1978.A dual recognitionmodelfor eytotoxie T cells basedon thymicselection of precursors

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with lowd.affinity for 7:181-90 self H-2antigens. Scand. Immunol. 11. Borel, Y., Kilimm,L. 1974. Carrierdeterminedtolerancein various strains of mice:the role of isogonicIgGin the inductionof haptenspecific tolerance. Proc. Soc. Exp. Biol. Med.145:470-74 12. Boyd, A. W., Schrader, I. W. 1980. Mechanism of effector cell blockade.I. Antigen-induced suppressionof Ig syathesis in a hybridoma cell line, andcorrelation withcell-associatedantigen. £ Exp. Med. 151:1436-51 13. Boyd,A. W., Schrader,J. W.1981. The regulationof growthanddifferentiation of a murine B cell lymphoma. II. The inhibition of WEHI231 by antiimmunoglobulln antibodies. Z Immunol. 126:2466-69 14. Boyd, A. W., Sckrader, J. W. 1983. Mechanism of effector cell blockade,II. Colchicinefails to block effector cell blockadebut enhancesits reversal. Cell Immunol.In press 15. Boyd, A. W., Schrader, j. w. 1983. Mechanism of effector cell blockade. IIL Theincreasedsensitivity of neonatad PFCto ¢ffcetor cell blockade.Cell ImmunoLIn press 16. Brent, L., Brooks, C., Lubling, N., Thomas,A. V. 1972. Attemptsto demonstrate an in vivorole for serumblock. ing factors in tolerant mice.Transplantation 14:3~2-87 17. Brgtacher,P,, Cohn,M.1970. A theory of self-nouselfdiscrimination:paralysis and inductioninvolvethe recognitionof one and two determinants on an antigen, respectively. Science169:1042-49 18. B~rnet, F, M. 1957. A modification of erne s theory of antibody production using the conceptof clonal selcetion, Aust. £ Sc~ 20:67-69 19. But’net, F. M., Fetmer, F. 1949. The Productionof Antibodies, 2nded. London: Macmillan 20. Burnet,F. M., Stone, J. D., Edney,M. 1950. Thefailure of antibodyproduction in the chick embryo,dust. d. Exp. Biol. Med. Sc£ 28:291-97 21. Cambier,L C., Vitetta, E. S., Uhr, J. W., Kettman,J. R. 1977. B cell tolerance. II. Triultrophenyl humangamma globulin-inducedtolerance in adult and neonatal mnrineB cells responsive to thymus dependent and independent formsof the samehapten, d. Exp. Med. 145:778-83 22. Chieco-Bianchi, L., Collavo,D., Biasi, G., Zanovello,P., Ronchese,F. 1983. T lymphocyte tolerance in RNAtumor VLrUSoncogenesis: a model for the

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IMMUNOLOGIC 34. Dorsch,S., Roser,B. 1977. Recirculating, suppressorT cells in transplantation tolerance. J. Exp. Med. 145:1144-57 35. Doyle,M.V., Parks, D. E., Weigle,W. O. 1976.Specificsuppressionof the immuneresponse by HGG-tolerantspleen cells. I. Parameters affectingthe level of suppression. J. ImmunoL116:1640-45 36. Doyle,M. V, Parks, D. E., Weigle,W. O. 1976.Specific,transient suppression of the immune response by HGG-tolerant spleencells. II. Effector cells and target cells. J. ImmunoL 117:1152-58 37. Dresser,D. W.1962.Specificinhibition of antibodyproduction.I. Protein over loading paralysis. Immunology5: 161-68 38. Droege,W.1973.Five questions on the suppressive effect of thymns-derived cells. In Curt. Titles ImmunoL, Transplant. Allergy, 1:95-134 NewYork: Pergamon 39. Elson,C. J. 1977.Tolerancein differentiating B lymphocytes. Eur. J. ImmunoL7:6-10 40. Farrar, J. J., Benjamin, W.R., Hiifiker, M., Howard,M., Farrar, W.L., FullerFarrar, J. 1982. The biochemistry, biologyandrole of interleukin2 in the inductionof cytotoxic T cell and antibody-formingB cell responses. ImmunoLRev. 63:129-66 41. Feizi, T., Wernet,P., Kunkel,H. G., Douglas, S. D. 1973. Lymphocytes formingred cell rosettes in the cold in patients chronic cold agglutinin cfisease. with Blood 42:753-62 42. Feldmann,M., Howard,J. G., Desaymard,C. 1975. Role of antigen structure in the discriminationbetweentolerance and immunityby B cells. Trans. plant. Re~.23:78-97 43. Feldmann,M., Ntr~al, G. J. V. 1972. Tolerance, enhancementand the regulation of interactionsbetween T cells, B cells and macrophages.Transplant. Rev. 13:3-34 44. Felton,L. D.1949.Signilicanceof antigen in animal tissues. J.. Immunol. 61:10%17 45. Fitch, F. W.,Engers,H. D., Cerrottini, J.-C., Brunner,K. T. 1976. Generation of cytotoxic T lymphocytesin vitro. VII. Suppressiveeffect of irradiated MLCcells on CTLresponse. J. Immunog116:716-23 46. Frei, P. C., Bcnacerraf,B., Thorbecke, G.J. 1965.Phagocytosis of the antigen, a crucial step in the inductionof the primaryresponse.Proc.Natl. Acad.Sci. USA53:20-23

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47. Gaily, J. A., Edelman,G. M. 1972.The genetic control of immunoglobulin synthesis. An~Rev. Genet. 6:1-46 48. deleted in proof 49. Gershon,R. K., Kondo,K. 1971. Infectious immunologicaltolerance. ImmunoL21:903-14 50. Golan, D. T., Borel, Y. 1971. Nonantigenicity and immunologic tolerance: the role of the carderin the inductionof tolerance to the hapten. Z Exp. Med. 134:1046-61 51. Good, M. F., Nossal, G. J. V. 1983. Characteristics of tolerance induction amongstadult hapten-spccific T lymphocyte precursors revealed by clonal analysis. Z Immuno~ In press 52. Gorczynski, R. M., MacRae,S. 1979. Suppressionof cytotoxic response to histoincompatible cells. I. Evidencefor the two types of T lymphocyte-derived suppressorsactingat different stagesin the induction of a cytotoxicresponse.Z Immunol. 122:737-46 53. Gorczynski, R. M., MacRae,S. 1979. Suppressionof cytotoxic response to histoincompatible ceils. II. Analysisof the role of two independentT suppressor pools in maintenanceof neonatally inducedallograft tolerance in mice. Z ImmunoL122:747-52 53a. Green, D. R., Flood, P. M., Gershon, R. K. 1983. ImmunoregulatoryT Cell Pathways. Ann. Rev. Immuno~ 1:439-63 54. Gruchalla,R. S., Streilein, J. W.1982. Analysis of neonatally inducedtolerance of H-2alloantigem.II. Failure to detect alloantigenspecific T lymphocyte precursors and suppressors. Immunogenetics 15:111-27 55. Haas, W., Layton, J. E. 1975. Separation of antigen-specificlymphocytes. I. Enrichment of antigen-bindingcells. J. Exp. Med. 141:1004-14 56. Hanan,R., Oyama,J. 1954. Inhibition of antibodyformationin maturerabbits by contact with the antigen at an early age. ,~. Immunol.73:49-53 57. Harris, D. E., Cairns,L., Rosen,F. $., Borel, Y. 1983. A natural modelof immunologictolerance: tolerance to mufine C5is mediatedby T cells andantigen is required to maintain unresponsiveness. Z Exp. Med.In press 58. Howard,M., Farrar, J., Hilflker, M., Johnson,B., Takatsu,K., Hamaoka, T., Paul, W.E. 1982.Identification of a T cell-derived B cell growthfactor distinct fromInterleukin 2. J. Exp. Med. 155:914-23

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59. Iverson, G. M., Lindenmann, J. 1972. Therole of a carrier determinantandT cells in the induction of liver-speciticantoantibodies in the mouse.Eur. d. Immunol. 2:195-97 60. Jemmerson,R., Morrow,P., Klinman, N. 1982. Antibodyresponses to synthetic peptides correspondingto antigenic determinants on mouse, cytochromec. Fed. Proc. 41(3):420 61. Kilshaw,P. J., Brent, L., Pinto, M. 1975. SuppressorT ceils in mice made unresponsiveto skin allografts. Nature 255:489-91 62. Klans, O. O. B. 1976.B cell tolerance inducedby polymericantigens. IV. Antigen-mediatedinhibition of antibodyforming cells. Eur. J. ImmunoL6: 200-7 63. Kubara,T., Hosono,M., Fujiwara, M. 1981.Studiesonthe resistanceto tolerance induction against humanIgG in DDD mice. IV. Transienttolerant state of T-cell precursors in bone marrow. Cell IramunoL57:377-88 64. Lawton,A. R., Cooper, M. D. 1974. Modificationof B lymphocyte differentiation by anti-immunoglobulin.Contemp. Top. ImmunobioL3:193-225 65. Lederberg,J. 1959. Genesand antibodies: doantigensbearinstructionsfor antibodyspecificity or dothey select cell lines that arise by mutation. Science 129:1649-53 66. Loblay,R. H., Fazekas,B., PritchardBriscoe, H., Basten, A. 1983. Suppressor T cell memory. II. Therole of memory suppressorT cells in tolerance to humangammaglobulin..L Exp. Meal In press 67. Loblay,R. H., Pritchard-Briscoe, H., B~ten, A. 1983. Suppressor T cell memory.L Induction and recall of HGG-specific memorysuppressor T cells andtheir role in regulationof antibodyproduction.,L Exp. MecLIn press 68. Louis,J., Chiller, J. M., Weigle,W.O. 1973. Fate of antigen-bindingcells in unresponsiveand immune mice..L Exp. Med. 137:461-69 69. Loutit, J. F. 1959. Ionizing radiation and the whole animal. Sc£ /lt~ 201:117-34 70. McCullagh,P. J. 1970. The immunological capacity of lymphocytesfrom normaldonors after their transfer to rats tolerant of sheep erythrocytes. ~lust. d. Exp. BioLMed.ScL 48:369-79 71. Metcalf,E. S., Klinman,N. R. 1976.In vitro tolerance induction of neonatal murine spleen cells. ,L Exp. Med. 143:1327-40

72. Metealf,E. S., Klinman,N. R. 1977.In vitro tolerance of bonemarrow cells: a markerfor B cell maturation, d. ImmunoL118:2111-16 73. Miller, J. F. A. P. 1972. Lymphocyte interactionsin antibodyresponses.Int. Rev. CytoL33:77-130 74. Miller, J. F. A. P., Basten,A., Sprent, J., Cheers,C. 1971.Interactionbetween lymphocytes in immune responses. Cell Immunol. 2:469-95 75. Miller, S. D., Sy, M.-S.,Claman,H. N. 1977.Theinduction of hapten-specific T cell tolerance using hapten-moditied lymphoidmembranes. II. Relative roles of suppressorT cells and doneinhibition in the tolerant state. Eur. Z lmmunoL7:165-70 76. Mitchison, N. A. 1971. The relative ability of T and B lymphocytesto see protein antigens. In Cell Interactions and Receptor/lntibodies in lmmuneResponses,ed. O. M~ikel~,A. Cross,T. U. Kostmen,pp. 249--60. NewYork: Academic 77. Mitchison,N. A. 1978.Newideas about self-tolerance and autoimmunity. Clinics RheumaticDR4:539--48 78. Moreno,C., Hale, C., Hewett,R., Esdaile, J. 1981.Inductionandpersistence of B cell tolerance to the thymnsdependent componentof the a(1-,6) glucosyl determinantof dextran. Recovery inducedby treatment with dextranase in vitro. Immunology44: 517-27 79. Morrissey, P. J., Kruisbeek, A. M., Sharrow,S. O., Singer, A. 1982.Tolerance of thymiccytotoxic T lymphocytes to allogeneic H-2determinantsencountered prethymically: Evidencefor expression of anti-H2 receptors prior to entry into the thymus.Proc.NatLAcad. ScL USA79:2003-7 80. Naor,D., Sulitzeanu,D. 1967.Bindi~.g of radioiodinated bovine serumalbumm to mousespleen cells. Nature 214: 68%88 E., Singh,B., Diener,E. 81. Nisbet-Brown, 1981. AntigenRecognitionV: Requirementfor hlstocompatibilitybetweenantigen-presentingcell and B cell in the response to a thymus-dependentantigen, andlack of allogeneicrestriction between T and B cells. £ Exp. Meg 154:676-87 82. Nossal, G. J. V. 1957. Theimmunological responseof foetal miceto influenza virus. ~lust. d. Exp. BioL Med. 35:549-58 83. Nossal, G. J. V., Lederberg,J. 1958.

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Antibodyproduction by single cells. 96. Pike, B. L., Nossal, G. J. V. 1979. Mechanisms of clonal abortion toleroNature 181:1419-20 84. Nossal, G. J. V., Pike, B. L. 1978. genesis. III. Antigenabrogates funcMechanisms of clonal abortion tolerotional maturationof surfaceIg-negative genesis. I. Responseof immature,haptadult bone marrow lymphocytes. Eur. J. lmmunol. 9:708-14 en-specific B lymphocytes.Z Exp. Med. 148:1161-70 97. Pike, B. L., Vanx,D. L., Clark-Lewis, 85. Nossal, G. J. V., Pike, B. L. 1980. I., Schrader, J. W., Nossal, G. J. V. Clonalanergy: persistence in tolerant 1983. Proliferation and differentiation mice of antigen-bindingB lymphocytes of single, hapten-specifieB lymphocytes incapable of respondingto antigen or promotedby T cell factor(s) distinct mitogen. Proc. NatL Acad, Scl. USA fromT cell growth factor. Proc. NatL Acad.Scl. USA.In press 77:1602-6 86. Nossal,G. J. V., Pike, B. L. 1981.Func- 98. Raft, M.C., Owen,J. J. T., Cooper,M. tional clonal deletion in immunological D., Lawton,A. R., Megson,M., Gathtolerance to major histocompatibllity ings, W.E. 1975.Differencesin suscepcomplex antigens.Proc. Natl. Acad.Scl. tibility of matureand immaturemouse USA78:3844-47 B lymphocytesto anti-immunoglobulln 87. Nossal,G. J. V., Pike,B. L., Battye,F. suppression in vitro. ,L Exp. Med. L. 1979. Mechanisms of ¢lonal abortion 142:1052-64 tolerogenesis. II. Clonalbehaviourof 99. Rajewsky,K. 1971. The carrier effect immatureBcells followingexposureto and cellular cooperationin the indueanti-~ chain antibody. Immunology tion of antibodies. Proc.R. Soc. London 37:203-15 Ser. B. 176:385-92 88. Osmond, D. G., Nossal, G. J. V. 1974. 100. Rajewsky,K., Takemori,T. 1983. GeDifferentiation of lymphocytes in netics, Expression,andFunctionof Idiotypes. Ann. Rev. Imraunol.1:569-606 mousebone marrow. II. Kinetics of maturationandrenewalof antiglobulln- 101. Reiehlein, M.1972. Localising antibindingcells studiedbydoublelabeling. genie determinants in humanhacmoCell. Immunol.13:132-45 globin with mutants:molecularcorrela89. Owen,R. D. 1945. Immunogenetic contions of immunologic tolerance, d. Mol. sequencesof vascular anastomosesbeBiol. 64:485-96 tweenbovine twins. Science 102:400-1 102. Roberts, I. M., Whittingham, S., 90. Owen,R. D. 1956. Erythrocyte antiMackay,I. R. 1973. Tolerance to an gens and tolerance phenomena. Proc. R. autoantigen-thyroglobulin. AntigenSoc. LondonSer. B 146:8-18 binding lymphocytes in thymus and 91. Parks, D. E., Doyle,M.V., Weigle,W. blood in health and autoimmunedisO. 1978. Induction and modeof action ease. Lancetii:936-40 of suppressor cells generatedagainst 103. Rouse,B. T., Warner,N. L. 1974. The human gammaglobulin. I. An imrole of suppressorcells in avian allomunologic unresponsivestate devoidof geneic tolerance: implications for the demonstrablesuppressorcells, d. Exp. pathogenesis of Marek’sdisease. Z Iramunol. 113:904-9 Med. 148:625-38 92. Pike, B. L., Battye,F. L., Nossal,G.J. 104. Schrader,J. W.,Nossal,G. J. V. 1974. V. 1981. Effect of haptenvalency and Effector cell blockade. A newmechacarrier compositionon the tolerogenic nism of immune hyporeactivity induced potential of hapten-proteinconjugates. by multivalent antigens. Z Exp. Med. Z Immunol. 126:89-94 139:1582-98 93. Pike, B. L., Boyd,A. W., Nossal,G. J. 105. Scott, D. W., Venkataraman,M., JanV. 1982.Clonalanergy:the universally dinski, J. J. 1979.Multiplepathwaysof anerglcB lymphocyte. Proc. Natl. Acad. B lymphocytetolerance. Iramunol.Rez Scl. USA79:2013-17 43:241-80 94. Pike, B. L., Kay,T. W.,Nossal,G.J. V. 106. Shearer, G. M. 1974. Cell-mediated 1980.Relativesensitivity of fetal and cytotoxicityto trinitrophenyl-modified newbornmice to induction of haptensyngeneic lymphocytes. Eur. ,L Immunol. 4:527-33 specific Bcell tolerance, d. Exp. Med. 152:1407-12 107. Shellam,G. R., Nossal,G. J. V. 1968. 95. Pike, B. L., Nossal,G. J. V. 1976.ReMechanismsof induction of immunoquirementfor persistent extracellular logical tolerance.IV. Theeffects of ulantigen in cultures of antigen-binding B tra-low doses of flagellln. Immunology lymphocytes,d. Exp. Med.144:568-72 14:273-84

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108. Sidman, C. L., Unanue,E. R. 1975. Receptor-mediated inactivation of early B limphocytes. Nature257:149-51 109. Smith,R. T., Bridges, R. A. 1958.Immunologicalunresponsivenessin rabbits producedby neonatal injection of defined antigens. ,L Exp. MecL108: 227-50 110. Stevens, K. M., Pietryk, H. C., Ciminera, J. L. 1958. Acquiredimmunological tolerance to a protein antigen in chickens. Br. d. Extx PathoL39:1-7 111. Streilein, I. W.1979./Neonataltolerance: towardsan immunogenetic defmition of self. ImmunoL Rev. 46:125-46 112. Streilein, J. W.,Gruchalla,R. S. 1981. Analysisof neonatally induced tolerance of H-2alloantigens. I. Adoptive transfer indicatesthat toleranceof Class I and Cla~II antigensis maintainedby distinct mechanisms.Immunogenetics 12:161-73 113. Sunshine, G. H., Cyrus, M., Winchester, G. 1982.In vitro responsesto the liver antigen F. Immunology 4:357-63 114. Sw?aJn,S. L., Wetzel,G. D., Saubiran, P./, Dutton,R. W.1982.T cell replacing factors in the Bcell responseto antigen. ImmunoLRe~. 63:111-28 115. Tagart, V. B., Thomas,W.IL, Asherson, G. L. 1978. Suppressor T cells whichblock the inductionof cytotoxic T cells in vivo. Immunology 34:1109-16 116.Taylor, R. B. 1969. Cellular cooperation in the antibody responseof mice to two serumalbumins:specific function of thymuscells. Transplan~Re~. 1:114-49 117. Taylor, R. B., Tire, J. P. 1979. Immunoregulatory effects of a covalentantigen-antibody complex.Nature 281: 488-90 118. Teale, J. M., Layton,J. E., Nossal,G. J. V. 1979. In vitro modelfor natural toleranceto self antigens, d. Exp.Med. 150:205-17 119. Tire, J. P., Morrison,C. A., Taylor,R. B. 1981. Immunoregulatory effects of covalent antigen-antibodycom.plexes. III. Enhancementor suppres~on dependingon the time of administration of complexrelative to a T-independent antigen. Immunology42:355-62 120. Tite, J. P., Morrison,C. A., Taylor,R. B. 1982. Immunoregulatory effects of covalent antigen-antibodycomplexes. IV. Primingandtolerance in T-depend-

ent responses. Immunology46:809-17 121. Tite, J. P., Taylor, R. B. 1979. Immunoregulationby covalent antigenantibody complexes.II. Suppressionof a T cell-independent anti-hapten response. Immunology38:325-31 122.Traub,E. 1938.Factors influencingthe persistenceof choriomeningitis virus in the bloodof miceafter clinical recovery. d. Exp. Med~68:229-50 123. Triplett, E. L. 1962, Onthe mechanism of immunogenic self-recognition, d. ImmunoL89:505-10 124. Vanx,D. L., Pike, B. L., Nussal,G. J. V. 1981.Antibodyproductionby single, hapten-specificB lymphocytes: an antigen-drivencloningsystemfree of filler or accessorycells. Proc NatLAcad.ScL USA78:7702-6 125. Waters, C. A., Diener, E., Singh, B. 1981. Antigen-relatedsusceptibility to toleranceinductionin utero: an objection to the current dogma.In Cellular and MolecularMechanismsof Immunological Tolerance, ed. T. Hraba, M. I-Iasek, pp. 481-91. NewYork:Dekker 126. Waters, C. A., Pilarski, L. M., Wegmann,T. G., Diener, E. 1979. Tolerance induction during ontogeny. I. Presenceof active suppressionin mice rendered tolerant to human~-globulin in utero correlates with the breakdown of the tolerant state, d. Exp. Med. 149:1134-51 127. Welgle, W.O. 1971. Recent observations and concepts in immunological unresponsiveness and autoimmunity. Cli~ Exp. ImmunoL9:437-47 128. Weigle, W. O. 1973. Immunological unresponsiveness. Adv. ImmunoL16: 61-122 129. Weigle,W.O., Chiller, J. M., Habicht, G. S. 1972. Effect of immunological unresponsiveness on different cell populations. Tmn~plan~ Rez 8:3-25 130. Wetzel, G. D., Kettman, J. R. 1981. Activation of routine B lymphocytes. III. Stimulationof B lymphocyte clonal growth with lipopolysaccharide and dextran sulfate. .L ImmunoL126: 723-28 131. Zinkernagel, R. M., Doherty, P. C. 1974.Imunologicalsurveillance against altered self components by sensitised T lymphocytes in lymphocytic choriomeningitis. Nature 251:547-48





An~ Rev. lmmunol 1983. 1:63-86 Copyright © 1983 by Annual Reviews Inc. All right reserved

T-CELL AND B-CELL RESPONSES TO VIRAL ANTIGENS AT THE CLONAL LEVEL L. A. Sherman, A. Vitiello,

and N. R. Klinman

Departmentof Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

INTRODUCTION Over the years it has becomeapparent that the immunesystem can invoke a multiplicity of mechanismswith which to combatinfectious agents. These include the specific activation of both phagocytic and lymphoid cell subpopulations. Althoughthe action of antibodies in neutralizing infectious agents and facilitating opsonization is well known,in recent years the central role of cellular immunity in defense mechanismshas becomemore apparent. With respect to immunologicdefenses against viral infection per se, it has long seemedevident that even relatively low concentrations of antibodies in the serum or target organs could neutralize or immobilize virus and thus, if present, could protect against primary infection and subsequent viremic spread of the infectious agent. Under certain normal infectious circumstances, however,it appears that T-cell responses, by limiting or abolishing primary or secondary infectious foci, mayserve as the main weaponin the self-defense armamentarium(9). Since, in the course of a natural infection, it is likely that the primary presentation of viral antigens is in the context of the surface of infected cells, an understanding of immunerecognition of infected cells will be fundamentalto our understanding of both natural and prophylactically induced anti-viral responses. This review focuses on the recognition of viral determinants, particularly as they are expressed on infected cells, by both the B- and T-cell systems. Recent findings have indicated that B cells as well as T cells can recognize viral determinants that are uniquely expressed in the context of the infected cell. This finding provides a comparativeanalysis ofT-cell and B-cell recognition not heretofore available. Weattempt, wherever possible, to empha0732-0582/83/0410-0063502.00

63

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size studies carried out at the clonal level since it is probablethat an unambiguous study of recognition will ultimately dependon the relative simplicity of monoclonalresponses. Noattempt is madeto be comprehensive in termsof serologicalrecognitionof viruses per se, since the plethora of informationconcerningthis subject has already been reviewedextensively (20, 46). Rather, wetry to assess emergingconceptsof recognition to elucidaterelevant issues. ~iventhe enormousselective pressure that survival of virus infections must haveplayed in the evolution of the immune system, it is likely that both mammalianviruses and the mammalianimmunesystem, as we perceivethemtoday,in large part, are the productof these selective forces. For example,it has been shownthat encounters with the immunesystem are probablythe selective force for variations in influenzaantigens (34, 52). Similarly, since majorhistoeompatibility(MHC) antigens are so integrally involvedin immune recognition of foreign antigens expressedon cell surfaces, it is likely that the structure and function of MHC alloantigens has beeninfluenced over evolutionary time by their essential involvementin immune recognitionof infected cells. In this context, therefore, it maybe inappropriate to attempt to separate immune recognition of viral determinants per se from the recognition of such determinantsin the context of givenMHC alloantigens. Asthe studies presentedin this reviewreveal, both antigensystemsincreasinglyappearto be integrally related in terms of the structure of determinants perceived by the immunesystem. Nonetheless, elements of the immunesystem, primarily B cells, can recognize virion determinantsper se, and in the first section wereviewaspects .of that recognition that mayfurther our understandingof defense mechanisms in general andthe specific recognitionof viral determinantsin the context of MHC alloantigens, whichwill be discussed subsequently. THE RECOGNITION OF VIRION DETERMINANTS PER SE Thecapacity of serumantibodies to recognize various virion components is well established. Overthe past few years, these studies have beenadvancedmarkedlyby the advent of monoclonaltechnology(33, 48). Studies with monoclonal antibodies, derivedeither by the stimulation of individual B cells in culture (33) or by hybddoma technology(48), haveallowed detailed dissection of viral determinantsexpressed both on the isolated vidonand on infected cell surfaces. Mostimpressivein this regard, and a paradigmfor such studies, has beenthe analysis of influenzahemagglutinin (HA)by large panels of monoclonalanti-influenza antibodies (18, 31-33, 47). Thefirst studies that successfullygeneratedmonoclonal antibodyre-

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sponses to viral determinants and used such antibodies to delineate viral determinants were carried out with the fragment culture system in which isolated B cells were stimulated in vitro and the antibody product of their clonal progeny was collected over a period of several weeks (17, 18, 31, 33, 47). This technique can provide microgram quantities of monoclonal antibodies which have proven sut~cient for detailed specificity analysis. These studies also introduced the use of a large panel of influenza variants to study both the diversity of recognition of monoclonal antibodies and to delineate the determinants recognized by such antibodies. Such an analysis is shown in Table 1. This table shows a comparative analysis of monoclonal antibodies derived from primary versus secondary B cells. The antibodies were prescreened on viral strains that share only HAwith PR8 versus those that Table 1 Relative frequency of reactivity patterns in primary and secondary anti-PR8 aHAresponses Heterologousvirus W E I S S

C A M

+

+ + +

+ + -

+ +

+ +

+ -

+ -

+

-

b 7.0 4.5

1.6 5.3

.c

+

*

1.6 1.5

* *

* *

* *

* *

÷

3.1 ¯

* 3.0

* *

0.8 *

* *

0.8 *

* *

2.3 *

+

-

1.6 3.8

* 0.8

0.8 0.8

* 0.8

0.8 *

* 6.0

* *

1.6 0.8

-I-

÷

2.3 12.0

* *

2.3 5.3

* 3,0

* 0.8

* 3.0

¯ ¯

* 0.8

0.8 0.8

* *

* *

2.3 *

* 0.8 0.8 0.8

* * 2.3 1.5

0.8 0.8

0.8 0.8

0.8 0.8

* *

* *

2.3 0.8

4.7 0.8

1.6 1.5

1.6 ¯

* 0.8

* *

* 0.8

0.8 0.8

5.5 0.8

1.6 2.3

1.6 1.5

--

B E L

-

BH WSE MEL

5.5 2.3 1.6 7.8 3.1 6.2 5.5 14.0 1.5 * * * * * 12.8 23.3 a Relative frequencies are given as a percentage of total response and are based upon 129 primary and 134 secondary monoclonalanti-HA antibodies (from 18). bUppernumberrefers to primary response; lower numberrefers to secondaryresponse. c*, RPnot observed.

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share other virion determinants, including neuraminidase(NA),and those that share only chickenhost component. Thetable, therefore, includes only those antibodies that presumablyrecognize determinants on the HAof PR8.This andsimilar analysesestablished several highly importantgeneral principles.First, it is clear that a panelof viral variantsis a verypowerful tool in the dissectionof availableantibodyspecificities. Second,these studies give definitive evidenceof the enormous degreeof diversity availablein the B cell repertoire. Byexpandingsuch panels (26, 84), it has nowbeen foundthat morethan 103 different recognitionpatterns are available within the Balb/cmouse.Since B cells responsiveto the influenzaHAare approximately1 in l0 s of all B cells (18), such studies haveestablished that the slowerlimit to the size of the antibodyrepertoire is of the order of 10 specificities. Third, if eachindividualreactivity pattern canbe equatedwith the recognition of a given determinant,then it maybe concludedthat the B cell repertoire is not redundantin its recognitionof mostdeterminants. Thus, for manydeterminants a fully mature mousemayhave only one or no B cell clones capable of recognition. Fourth, since subsequentstudies haveshownthat only a few limited regions of the HAmoleculeare responsible for this complex pattern of binding(32, 81), it is clear that smallnuances in the structure of a regionof a protein can be recognizedanddiscriminated by monoclonalantibodies. Althoughin someinstances such nuances may be createdby the substituted aminoacid per se it is equallylikely that in someinstances aminoacid substitutions maybe recognizedas perturbations in other regions of the molecule. Subsequentstudies with monoclonalantibodies derived from hybridomas haveconfirmedall of the abovefindings(26, 32, 35, 84). In addition, it has beenpossible, by the use of hybridoma antibodies, to select for influenzaHA variants that represent a single mutationaldifference (34, 52). Suchnewly selected variants haveservedas invaluabletools in further discriminating the B cell repertoire and further delineating regions of the HAmolecule whereinsequencevariation affects immunologic recognition (81). _ Figure1 is a schematicrepresentationof the three-dimensional structure of influenza HAas determinedby X-ray crystallography by Wyley& Wilson (81). Includedin this figure are point mutationsselected by monoclonal antibodies.It is apparentthese mutationaldifferencescluster in immunologically relevant regions of the HAmolecule(81). Thecombinedanalysis monoclonalantibodies, selection of mutants, and crystal structure represents an extraordinarily powerfulnewset of approachesfor the understanding of determinantrecognition and the powerof selective forces on both immune expressionand viral expression.Recently,several other viral systemshave beensubjected to analysis by monoclonalantibodies (49, 56, 58). Someof these studies havebeenthe subject of extensiverecent reviews, including comprehensiveanalyses of the antigenic structures of murine

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67

leukemia viruses (58), the 70S glycoproteins of mudneretroviruses (56), and rabies viruses (49). In each of these instances, it has been possible the use of monoclonalantibodies to more clearly delineate structural relationships and variations amongviral strains.


VIRAL COMPONENTS RECOGNIZED ON INFECTED CELLS It is nowclear that both T cells and B cells can function, and mayprimarily function, by recognizing viral determinants as they are expressed on infected cells. Suchrecognition is particularly relevant to viruses that repli-

HA2 N

Figure 1 Schematicdrawingof a monomer of the AICHI-1968 hemagglatininshowing locations of aminoacid substitutions on HA,.¯ Site A; ¯ site B; ¯ site C; ¯ site D (subunitinterface); nT’ 2

(a). (b).

RestingT cells (T) require twosignals for their conversionto the differentiated state (T’); T’ cells canexpresscytotoxicactivity. T’ cells proliferate in the presenceof lymphokiiae(elonal expansion).Interleukin 1 (ILl) beenshownto providethe secondsignal for reaction (a) and interleukin (IL2 or T cell growthfactor) will induceclonal expansionof the activated T cell, reaction (b) (2a); see appendix. Mitogen-induced activation of T cells has also been shownto be a twosignal processin whichmitogen(ConA) providessignal oneand + spleen cells are requiredfor the generationof the secondsignal (2, 66), whichalso appears to be providedby ILl (66).

Role of Class I and Class H MHCAntigens Bach et al (7), arguing from an immunogeneticviewpoint, developed alternative two-signalmodalfor allogeneicT cell activation in whichrecognition of the Ia epitope on the allogeneic cell activated the helper T cell subset, whichthen providedthe secondsignal for cytotoxic T cell activation. Thereis nowevidence,however, that recognitionof the class II antigen is not requiredfor eytotoxieT cell activation, the clearest beingthe demonstration of cytotoxicT cell activation in the situation wherethe stimulator and responderstrains differ only in the K region of MHC (6-8, 82). Also, Batchelor’s group (10) has examinedthe response of lymphocytesto the class II MHC antigen and shownthat recognition of the Ia epitope alone is not a sufficient requirementfor activation of the helper lymphocyte subset. + T cells responsive to class I MHC antigens belong to the Lyt 1+2+3 cytotoxic subset whereasthose responsiveto class II MHC antigens are part of the Lyt 1+2-3- helper subset (24). Similar signalling requirementsare requiredfor the activation of cells of either subset (59). Thatis, reactions

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(a) and (b) describe the activation of both cytotoxicandhelper T cells. nowalso knowthat cells of both the cytotoxie and the helper subsets produceequivalent amountsof lymphokine whentriggered by antigen alone (3, 72). Theclearly establishedsynergybetweenclass I andclass II reactive T cells for the in vitro generation of cytotoxic T cell response can be attributed to the augmentationof IL2 production in the culture system. However,such synergistic effects havenot been clearly demonstratedin vivo and allografts are promptlyrejected whenclass I antigenic differences only are recognized(100, 105). This does not meanthat Ia + ceils are unimportantin the process of allogeneicT cell activation. Inactivation of these cells with anti-I-region antibody and complementremovesthe stimulating capacity of the cell population(93, 114). However, not all + cells st imulate (37, 67, 115), an someIa- cells, suchas P815,do stimulate(Table1). Thatis, althoughthere + is a strong correlation betweenIa positivity and the expressionof the S phenotype,exceptionsto the correlation showthat recognitionof Ia antigen itself cannotprovidethe sourceof the secondsignal required for cytotoxic Tcell activation. Nevertheless,the Ia+ call is probablythe cell responsible for the stimulationof both class I andclass II MHC-reaetive T cells in vivo. Themostlikely candidatefor the physiologicallyrelevant stimulatorcell is the Ia-rich dendriticcell (67). Sucha modelfor immunocyte induction has a profoundinfluence on the wayweviewallogeneicinteractions. In the case of allogeneicT-cell activation, the antigenis built into the surfaceof the allogeneiccell. Cells of the S+ phenotylaehave the capacity to produceCoSactivity; other cells are S-. Thus,only antigen-bearing S+ cells will stimulatethe allogeneicT cells, since it is only in this situation that the two signals required for T cell activation can be provided.Thus, alloantigen on the surface of metabolically active S+ cells will be highlyimmunogenic for T cells in vitro, whereas the sameantigen presentedon the surface of cells of the S- phenotypewill not be immunogenic (see Appendixfor detailed treatment). Since the + phenotypeis only expressedby cells of lymphoreticularorigin (42, 43, 63, 111), wecan see whyleukocytesare strong stimulatorsof allogeneicT cells in vitro. Passenger Leukocyte and Graft Rejection It follows fromthe abovediscussion that leukocytes carried within the grafted tissue play a major role in regulation of tissue immunogenicity. Stimulatorcells (dendriticcells?) will activate recipient T ceils to generate a graft-specific response(see Appendix).Antigencardedon graft parenchymal cells will not activate T cells directly becausethe parenchymal cells cannotprovidea sourceof CoSactivity. Free antigen, shed fromthe graft,

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could be carried to the immunesystem of the recipient and there presented on recipient stimulator cells to recipient T cells. Such indirect antigen presentation wouldnot lead to the generation of graft-specific T cell response; T cells activated by indirect antigen presentation wouldbe specific for graft antigen in association with the MHC of the recipient (see Appendix). Thus, it is stimulator cells in the graft that constitute the major immunogenicstimulus of the graft and therefore represent the major barrier to tissue grafting. Since stimulator cells of the graft, such as dendritic cells, are Ia +, it follows that Ia + cells carried within the grafted tissue will constitute a major immunogeniccomponentof the graft. This is not because Ia antigen itself is highly immunogenic (9, 10), but rather that the class antigen is a marker for the cell that expresses the S+ phenotype(93, 114), i.e. the cell that can provide a source of CoSactivity. Againstthis theoretical background it makes practical sense to attempt a reduction of tissue immunogenicityby removal of leukocytes from the graft prior to transplantation (see Appendix).

MODULATION OF TISSUE

IMMUNOGENICITY

OrganCulture Prior to Transplantation In the early 1970s, several groups investigated the effect of organ culture on the immunogenicityof endocrine organs and certain tumor tissue (65, 79). The idea that organ culture might reduce tissue immunogenicity was not new. There were several reports in the 1930s suggesting clinical benefit when parathyroid tissue was held in organ culture for a period prior to transplantation to patients with hypoparathyroidism (104). However,these studies were not genetically controlled, and without the support of an adequate theoretical base, enthusiasm for the experiments quickly waned. Our interest in organ culture was stimulated by a report from Summerlin et al subsequently not confirmed--that organ culture prior to transplantation could facilitate the grafting of skin to normal allogeneic recipients (108). Jacobs (49) suggested that antigen expression was modified during the period in organ culture. Such an explanation did not seem very likely, and against the above theoretical background, the effect of organ culture could be readily explainedif bloodcells in the tissue died or wereinactivated during the culture period. The organ culture technique has proved spectacularly successful in the case of endocrine organ transplantation. Mousethyroid can be transplanted under the kidney capsule of isogeneic thyroidectomized recipients, where its ability to concentrate radioactive iodine can be used as a measureof graft function (60). Organ culture of thyroid tissue in an atmosphere of 95%

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O2and 5%CO2for 14 days extends allograft survival (61), and after culture period of 3-4 weeks,allografts showno evidenceof rejection over an observation period of 100 days (60). Pretreatmentof the tissue donor with cyclophosphamide (300 mg/Kg)4 and 2 days prior to the harvest tissue reducedthe period of organculture required to facilitate allograft survival; after this treatment, tissue cultured for approximately2 weeks could be transplanted to normalallogeneie recipients whereit functioned indefinitely (65). Underthese conditionsboth thyroid andparathyroidshow indefinite survival in allogeneic recipient mice(65). Similar results were reported with other species (84) and the procedurehas beenshownto work for xenotransplantationof thyroid andislet tissue fromrat to mouse(58, 101). Whatis the explanation for this phenomenon? Doesantigen modulation occurin organculture as suggestedby Jacobs(49), or doesthe effect result from the postulated loss of passenger leukocytes? Duringorgan culture there is a rapid degenerationof the vascular bed and bloodelementswithin the cultured tissue (88). Thetissue retains antigen, recognizableby immunoferritinlabeling (88), and the tissue is rejected whenas few as 103 viable peritonealcells of donororigin are injected into the recipient animal at the time the cultured tissue is transplanted(110). Clearly, the cultured allograft carries functionallyrecognizableantigen. Establishedthyroidallografts are also rejected whena secondunculturedthyroid of donororigin is transplantedto the recipient (65). Thiseffect is antigen-specificsince unculturedthird party graft can be rejected, but its rejection doesnot affect the integrity of the establishedculturedallograft (65). Hirschberg & Thorsby (46) suggested that vascular endotheliummay provide a major stimulus of allograft immunity. Vascular endothelium degeneratesduringorganculture (88) and its destruction could accountfor the reduction in tissue immunogenicity achievedby organ culture. Endothelial cells express MHC antigens and would,therefore, providea primary target for the attack of activatedgraft-specificT cells or graft-specificantibody(see above). The question to be addressed is whether or not endotheliumprovidesa majorsourceof stimulatingactivity for recipient T cells. Cyclophosphamide treatment of animals for 2 days prior to the harvest of tissues causesa profounddropin the capacityof spleencells to stimulate allogeneie T cells in culture (60). This treatment of the donorwith eyelophosphamide has no obviouseffect on thyroid endothelium.However,after this treatment alone, approximately30%of thyroid allografts function normallyover a prolongedobservation period (65). If donor endothelium were a major source of tissue immunogenicitywe wouldnot expect to observesuch an effect. Batehelor’sstudies using kidneytransplantation(9, 11) led to similar conclusions(see below).

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Pancreatic lslet

Transplantation

Initial attemptsto applythe organculture pretreatmentto pancreaticislet transplantationwerefrustrated by technicaldifficulties associatedwith this procedure(16, 56). Theloss of tissue immunogenicity during organculture is an oxygen-dependent phenomenon, thought to result fromthe sensitivity of leukocytesto oxygentoxicity (109). Pancreatic islet tissue cannot treated in the sameway.Singleislets, isolated fromthe adult pancreas,are extremelysensitive to oxygentoxicity andrapidly degeneratewhencultured in 95%oxygen.This toxicity problemcan be overcomeby allowing groups of approximately 50 islets to aggregateand fuse together (16, 18, 58). Theislet clusters are moreresistant to oxygentoxicity andcan be successfully allotransplantedin miceafter 1 weekof culture underthese conditions (16, 18, 58). Followingorganculture in 95%oxygen,islet allografts of to 7 islet clusters havebeenshownto reverse streptozotocin-induceddiabetes in mice. Bloodsugar levels of transplantedanimalsrapidly return to normal, and the animals becomeaglycosuric and respond normally to a glucosechallenge(17, 18). Unculturedislets, on the other hand, temporarily reverse diabetes, but recipient animalsreturn to the diabetic state within4 weeksof transplantation(17). Lacy’sgroup(56) achievedsimilar results the rat. However, in their initial studies, islets werenot culturedin 95%O2 and allograft acceptancewas only achieved whenrecipient animals were treated with a single dose of ALSat the time of transplantation. Similar results havenowbeenreported in the case of rat islet xenotransplantation to mice(57, 58). Faustmanet al (32) demonstrateda dependenceof islet immunogenicity on the presenceof Ia + cells in the transplanted tissue, by achievingallograft survival followinganti-Ia serumandcomplement treatmentof the donor tissue prior to transplantation. However,Ia antigen recognitionis not required;Morrow et al (80) haveshown that islet allograft rejection occurs whenthere is I region compatibility betweendonor and recipient. Thefact that rejection is dependenton the presenceof an la+ cell in the tissues is consistentwiththe notionthat the S+ stimulatorcell carries Ia antigenon its surface (93, 114). Fetal Pancreas Transplantation Fetal pancreascan be used as a sourceof islet tissue for transplantation. Followingorgan culture or transplantation, the exocrinepancreasdegenerates whereasthe endocrinetissue continues to differentiate and develop endocrinefunction. In rodents, transplantationof onefetal pancreasto the kidneycapsulehas beenshownto reversediabetes (20, 47, 73, 95). However, early attemptsto conditionfetal pancreasfor allotransplantationby organ

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culture prior to transplantationmet with little or no success(73). When comparedthe immunogenicity of fetal pancreasand islets isolated fromthe adult pancreas,in the samestrain combinations,wefoundthe fetal pancreas muchmoredifficult to preparefor allotransplantation(94). This difference in behaviorof different tissues wasrelated to the large amountof lymphoid tissue associatedwith the fetal pancreas(94). It is possibleto reversediabetes in CBAmice with tissue from two BALB/c fetal pancreases cultured for 20 days in 95%02 (Figure 3). However,the success rate of allografts is low; approximately 40%of recipients reverse their diabetes. Tissuecultured in this wayis slowto reversediabetes evenin an isogeneicsituation (95). However, the fact that culturedtissue can reversediabetesin isogeneic animals, sometimesas late as 6 monthspost-transplantation, providesevidencethat the cultured fetal pancreashas a capacity for continuedgrowth anddifferentiation after transplantation(95). Immature precursorsof islet tissue, pro-islets, can be isolated fromthe pancreasby collagenasedigestionand culture in air for 4 days (96). Following transplantation, this tissue has the capacity to growanddifferentiate into functionalislet tissue that will reversestreptozotocin-induced diabetes (97). In the digestion procedurethe fetal pro-islets appearto be partially separated fromlymphoidelementsassociated with the fetal pancreas. Pro-

¯ ~ 30

o o 10 GRAF~REMOVEO

~o

~6o

1~o

260

TIMEPOST-TRANSPLANT (Days) Figure 3 Noafasting blood glucose respoa~of diabetic CBAmice following allotransplantation of two 20-day cultured BALB/cfetal pancreases (six 20-day cultured fetal pancreas segments) under the kidney capsule. The shaded region defines the nonfasting blood glucose range of normal CBAmice (mean _+95%confidence interval). Grafts were removed excision of the graft-bearing kidney. Approximately 40%of 20-day cultured fetal pancreas allog~s are successful in reversing streptozotocin-induccd diabetes.

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islets are less immunogenic than the wholefetal pancreasand mayprovide a moresuitable sourceof tissue for clinical transplantation(97). Reversal of Juvenile-Onset Diabetes Thereare suggestionsthat spontaneousjuvenile-onsetdiabetes mayhavean autoimmune etiology (23, 86) and, if this is the case, a transplant could succumb to the samefate as the animal’sownislet tissue. Naji et al (85), workingwith the spontaneousdiabetic BBrat, found that MHC-compatible transplants failed whenanimalsweregrafted soonafter the onset of overt diabetes. Transplants appearedto fare better whenanimals were grafted somemonthsafter the onset of disease. These findings suggest that the transplant maybe morevulnerableto attack whengrafted duringthe acute phaseof the disease. Inflammatorydamageof islet tissue maybe virally mediated(86). Thus, if a virus growsspecifically in the B cells of the pancreas, the immune responseto the virus-infected calls could irreversibly damagethe infected B cells. If such a process wasinvolved in the induction of spontaneous diabetes, one wouldexpect an allogeneic graft to be less susceptible to damagethan the MHC-compatible graft. MHC restriction of the T cell responseto virally infected host ceils wouldprotect the allograft froma cross-reactiveattack mediatedby activated T cells of the diabetic animals. In our laboratory wehavestudied a case of spontaneousjuvenile-onset diabetes that occurred in a youngCBA mouse(Figure 4). This animal was transplanted with a cultured BALB/callograft soon after the onset of diabetes. Withina weekof transplantation, the blood sugar levd returned to normal and remainednormal for a further 180-odddays (Figure 4). Duringthis period, the animalunderwenttwo normalpregnancieswith no evidenceof abnormalbloodsugar responses. This animalwaskilled at the completionof the study and both its ownpancreas and the transplanted tissue were examinedhistologically. Theanimal’s ownpancreas contained fewislets of Langerhans andthose islets that wereseen weregrossly abnormal with few granulated/3cells (Figure 5). Thetransplantedtissue, on the other hand, showedno evidenceof damage(Figure 5). Thesefindings are very promisingfromthe clinical viewpoint;althoughthere maybe technical problemsassociated with the transfer of this technologyto the treatment of juvenile-onset diabetes, there are no conceptualproblems. Organ Transplantation Initial attemptsto removepassengerleukocytesfromtissues prior to transplantation werenot successful. Theseattempts (56, 78, 107) involved the induction of leukopeniain the tissue donor by proceduressuch as whole

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32

16

0

~

-20

~

~

0

20

~

I

40

60

!

I

I

80 100 120

I

I~0 160 180

TIME (days) Figure 4

Nonfasting blood glucose concentrations (open squares) and body weight (closed

circles) of a spontaneously diabeticCBA/H femalemouse transplantedon day0 witheight clustersof culturedBALB/c islets. Pregnancy is indicatedbythe sharpbodyweightincrease. Theshadedarea showsthe rangeof normalbloodglucoseconcentrations(mean4-2 SD). body irradiation, cydophosphamidetreatment, or treatment with anti-lymphocyte serum. At best, only marginal effects were observed for tissues transplanted across a major histocompatibility barrier (38, 55, 107), and rat heart allografts transplanted across multiple minor differences (34). Stuart et al (107) attempted to removepassenger leukocytes by first allografting rat kidneys to passively enhanced intermediate hosts. At 60 to 300 days post-transplantation, the kidney grafts were retransplanted to naive recipient rats isologous to the intermediate host. Only a delay in the onset of rejection was achieved. These marginal or weak effects led to someconfu-


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sion over the extent to whichpassengerleukocytes contributed to tissue immunogenieity (12). Nevertheless,the clinical successreported with renal allografts taken from cadaver donors pretreated with cyclophosphamide and methylprednisolonehelped maintain someinterest in the passenger leukocyteconcept(37a). Studies fromBatchelor’slaboratoryhavenowclarified the role passenger leukocytesplay in renal allografting (9-11, 67). This group demonstrated that long-surviving, immunologicallyenhancedMHC-incompatible rat kidney grafts, whentransplanted froma primaryto a secondaryrecipient of the samegenotype, do not elicit T-cell alloimmunityin the secondary recipient. In contrast, normalprimarykidney allografts in the relevant donor/recipientcombinationare regularly rejected in 12 days. Thefailure of long-survivingkidneygrafts to activate a T-cell responsecould not be attributed to a lack of either class I or II MHC antigens (67), and Batchclot’s group suggestedthat the effect resulted from a lack of .passenger leukocytes. Theythen went on to show, quite convincingly, that donor strain dendritic cells in very lownumberswouldtrigger rejection of kidneys taken froman intermediateenhancedrecipient (67). Theearlier failure see such a dramaticeffect probablyresulted fromthe strain combinations usedin these studies (107). When discussingthis strain-dependentvariation in survival time, Lechler &Batchelor(67) emphasize,and wewouldagree, that acute graft destruction is not a precise measureof a recipient’s response. In those strain combinationswhereacute graft destruction occurs, no quantitative comparison has been madeto determine the relative strength of responsesinducedby normalallografts and those depleted of passenger leukocytes. Theygo on to predict that minimalimmunosuppression at levels ineffective for normalgrafts wouldmaintainpassengercelldepleted allografts in goodfunction. This proposal deserves careful investigation. Development of Tolerance Culturedallografts of both thyroid andpancreaticislet tissue carry recognizable antigen and are promptlyrejected if recipient animals are challenged with donor leukocytes at the time of transplantation. However, animals carrying allografts for a prolongedperiod (~>100days) become progressivdymoreresistant to challengewith donorcells (18, 28, 116). the case of the thyroid wehavedemonstrated that the grafted tissue retains antigen, andthat the adaptation of the graft to its host results fromthe development of tolerance in the adult recipient (28). Wearguedthat this phenomenon mightdevdopas the result of the slow leakage of free antigen into the immunesystem of the recipient, possibly leading to tolerance induction in a mannerakin to active enhancement.Somesupport for this

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TISSUE TRANSPLANTATION 161 notion has beenobtainedin the pancreatic islet system.Whentransplanted animalsare injected with UV-killeddonorspleen cells, a sourceof alloantigen on nonstimulatingcells, around 30 days post-transplantation, graft rejection is not stimulated;administrationof viable donorcells at this time promptlyactivates a rejection reaction. Thatis, the UV-irradiated cells are not immunogenic.Whenanimals that received UV-irradiated cells were subsequentlychallengedwith viable donorspleen cells, the allografts were not rejected. TheUV-irradiatedcells, whichprovide a source of antigen alone, have stimulated the developmentof tolerance in the allografted animals.Thespecificity of this tolerance wasdemonstrated by the fact that these animalswouldaccept an unculturedthyroid allograft of donororigin but wouldreject a third-party thyroid grafted to the samerecipient. We haveshown,in the case of both thyroid- and islet-transplantedanimals,that this phenomenon is not a deletion form of tolerance. Lymphocytes from "tolerant" animals respondnormallyto donor alloantigen in vitro. More recently, Faustmanet al (33) havesuggestedsuch unresponsivenessmay dueto the actionof suppressorcells; the role of suppressorcells in allograft tolerance has beenreviewedelsewhere(91). A similar phenomenon has been observedfollowing heart allografting under cover of immunesuppression with cyclosporin A (81). Whenthe cyclosporin Ais withdrawn,the heart continuesto function but can be rejected if the recipient is appropriatdy challenged withdonortissue; at this stage, the heart allograft is in a metastable condition. Withthe further passage of time, a stable form of donor tolerance gradually devdops(81). In summary, wecan nowsay it is possible to either eliminate or reduce tissue immunogenieity by removalof leukoeytes(dendritic cells) from the transplant prior to grafting. This phenomenon has beendemonstratedto be effective with both endocrinetissue andlarger organs, such as the kidney. Thereis somevariation in the effectivenessof the techniquewith different tissues, whichcan be related to the degree of leukocyte contamination. Long-standing allografts of pretreatedtissue inducea state of specific unresponsivenessin the adult recipient. Althoughthe mechanism of this "tolerance"is not fully understood,the existenceof the phenomenon is oneof the most encouragingrecent developmentsin transplantation biology. THEORETICAL

APPENDIX

Immunobiology requires a theoretical frameworkon whichto mangethe experimental findings of cellular immunology.Such a frameworkmust comfortablyexplain whyit is the MHC complexantigens play the dual role of immune regulation andcontrol of allograft rejection. In the past, this relationship has been explained by the "immunesurveillance" concept,

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whichpostulated that the immunesystem developedto recognize components closely related to "self," including foreign MHC antigens, so that tumors wouldbe readily detected and eliminated from the system. The experimental observation that tissues expressing foreign MHC antigens survive in non-immunosuppressed recipients and showno sign of lymphocyticinfiltration (65) points out the inadequacy of the surveillanceidea. Thefollowinganalysis attempts a solution to this problem. Theory Ourtheoretical development accepts the postulates of "clonal selection" (22), namely,that lymphocyte receptor diversity is generatedby a random process,and that self-reactivity is forbiddenby a mechanism learned during developmentand enforced throughout the life of the animal. Wedevelop a theoretical system based on a further two postulates that govern the processof clonal selection andexpansion.Wethen use a symbolicterminology, whichis abstract yet precise, to derive the characteristics of the immunesystem that follow from these postulates.

Terminology In the followingdevelopmentthe accessory cell required for immunocyte ¯ activationis called the stimulatorcell, S. S.~) is a stimulatorcell that carries on its surfacea control structure, c, anda receptorfor antigen, x; the term (x) is read as: receptor for x. This receptor maybe a product of the stimulator cell or could be a cytophylic productof someother cell. When S(Cx)interacts with x, it can behaveas antigen-presentingcell Sx~, whichpresents x to the potentially responsive immunocyte.Wecan write this reaction as S ~ e. + x------>S (x)

x

Thecontrol structure is a genetically determinedproductandcan be used to define the genotypeof the animalproducingthe cell; weuse c to define a genotypein the samewaythat blood group antigens are used for this purpose. T~x)is a resting T cell that carries a receptorfor x. T’~x)is an activated cell that carries a receptorfor x. Thegenotypeof the resting or activated ¢ ! ¢ T cell can be represented as ¯Tf and T ,. (x) respectively; these are resting x~ and activated cells of x spec~fictty, derived from animalsof c genotype. ActivatedT calls expresseffector functionssuchas the expressionof cellular cytotoxicity or lymphokine production. ActivatedT cells release a number of lymphokine activities (3, 53), and lymphokinerelease is triggered

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TISSUE TRANSPLANTATION 163 signal 1 alone(59). Thatis, 1 > T~ + l.


T’(x) +

Herel is usedas a generictermto covera number of lymphokine activities. Cell surface antigens other than those that formthe control structure, c, are representedby y. Thatis, (c + y) representsthe sumof all cell surface antigens. Theterms c, x, and y can be used in a generic sense. Particuhr examples of eachare representedas c, c1, c2, c3 ... etc.; x, Xl, x2, x3 ... etc.; y, Yl, Y2,Y3... etc.

Postulatesof the theory Thefirst postulate maintainsthat T cells do not respondto contact with antigen alone, but respondto antigen in conjunctionwith a secondsignal providedby an inductivemolecule,said to expressCoSactivity (Figure 2). FIRSTPOSTULATE Twosignals-antigen activity and CoSactivity-are requiredfor initiation of T cell activation. T

2

1 v. )T

T, the resting T cell, is convertedto the activatedstate Tv followingantigen bindingby the T-cell receptor (deliveryof signal 1) in the presenceof CoS activity, whichprovidessignal 2. Since CoSactivity is a cellular product, a corollaryof the first postulateis the idea that a stimulatorcell is required for T cell activation (Figure2). Thesecondpostulate concernsthe regulation of stimulator cell function. SECOND POSTULATE: A+ control structure on the surface of the S (stimulator) cell regulatesthe release of CoSactivity; CoSactivity is only releasedwhenthis structure is engagedby the potentially responsiveT cell.

Initiation of the lmmuneResponse In the resting immune system, spontaneousT cell activation does not occur becauseT cells do not expressa functional receptor for self-antigens, and c, the controlstructure, is a self-antigen.Thus,the resting situation in the immunesystem can be written c +T(C ) S(x )

> -Ve,

whereS(~) is a stimulator cell that carries a control structure c and

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receptor for x. T.( ~ is a T cell of the samegenotypethat has a receptor specificity definedaf this stage only as beingunresponsive to self-antigens such as c. Thatis, T c is unableto interact with the control structure of S(~. andso trigger ~o~release. ~onsider, now,the entry of foreign antigen, x, into the system. Antigen x), can interact with S<xc the antigen-presentingcell:


S(Cx) x

> S~,

if Sxc c. ~S Thatis, ff x interacts with c in sucha waythat c.x (the interaction between c and x on the antigen-presentingcell) is no longer equivalent to c, then the followingsituation will hold: c s;+r(.x)

1

2

~c

Thatis, T cells of c’x specificity will interact with c.x on the surface of the antigen-presenting call. This will providesignal 1 for the T ceil. Signal 2, CoSactivity, is released from the stimulator cell becausethe control structure is nowengaged in the interactionwith the T cell of c" x specificity. FIRSTINFERENCE The first inference we draw from our postulates is that the specificity of the T cell responseto exogenous antigen, x, is defined by both x and the control structure, c. That is, T cell responsesto x are restricted by c.

Alloreactivity Ananalysis of alloreactivity definesthe nature of c, the control structure. Considerin vitro allogeneicinteractions in whichT cells of one genotype interact with allogeneicstimulator cells (Figure6). Thereare basically two different classes of suchallogeneieinteractions: Thosethat involvedifferences in the control structure (differences in c), and those that involve differencesin other cell surfaceantigens (differencesin y) (Figure Allogeneicinteractions betweenceils of different c-type havethe form YsC + "T(~) 1 21

..,YT*Cl

: - (¢)’

andT cells specific for the control structure on the stimulatorcell, c, are generated. Onthe other hand, allogen¢icinteractions betweencells of the samec-type, but differing in y-type antigens, do not proceedbecausethe

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165

responsive T cell is unable to interact with the control structure on the stimulator cell (Figure 6). That is,


ysc + yl T c -~)

1

~ -Ve.

The potentially responsive T cell maycarry a functional receptor for y, the alloantigen on the stimulator ceil, but an interaction with the stimulator cell via y will not involve the control structure c. As a result, CoSactivity will not be released from the S+ cell, thus depriving the system of a source of signal 2 (see Figure 6). There is another important characteristic of c-type antigens. Allogeneic T cells respond to these antigens in an unrestricted manner: st+ T(~)I

1 T, tl 2 ) (c) Responses to other antigens, whether they be minor histocompatibility antigens (y-type) or other foreign antigens, x, will alwaysbe restricted the control structure of the antigen-presenting cell: c S~v) c+ y ---->S

If c, SCy ~ S

Figure 6 Alloreactivity.

T cells are activated when they interact with c-type antigens (closed blocks) on the surface of viable + cells b ecause t his i nteraction p rovides s ignals 1 and 2 required for lymphocyte activation, y-Type antigens (closed circles) on the surface of metabolically active S+ ceils are not immunogenic under these conditions because CoS activity is not released from the S+ cell. The symbofic representation of each interaction is set out below the diagrammatic sequence.

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LAFFERTY ET AL

1 ~ T’ c (c.x)" 2 ! ¢ Similarly, T cell responses to x antigens are of the type T (c.x)"


then Sc~ + ) T(c~

SECOND INFERENCEThe second inference we derive from the postulates is that there are twoclasses of alloantigen, c-type and y-type antigens. c-Type antigens are highly immunogenicfor allogeneic T cells in vitro, whenthey are carried on the surface of metabolically active stimulator cells. y-Type antigens can be seen by T cells in an unrestricted manner, whereas y-type antigens are not immunogenicunder these conditions, c-Type antigens are seen in association with the control structure of the antigen presenting cell. Thus, we can use alloreactivity to define experimentally the nature of c, the control structure, c-Type structures will be that group of alloantigens that are highly immunogenicfor T cells in vitro. Experimentally we know that the MHC antigens behave in this way. That is, MHC antigens constitute the control structures of the immunesystem. Complexity of the Control System The MHC of the species is complexand consists of at least two classes of cell surface antigens that showstrong homologieswithin the class and also display a characteristic tissue distribution. Klein (54) has called these antigens class I and class II MHC antigens. In this analysis we refer to class I antigens as K-type antigens and class II antigens as I-type antigens. Weknowfrom the analysis of alloreactivity amongcell populations that differ only in K-type or I-type antigens that K-type and I-type antigens control the activation of different T cell subsets (24): 1 T, ~ ~ 2 > (K)

Sx + T(~I

+ T cell of the cytotoxic subset (24). In the mouse, T’ ~ ~ is an Lyt 1+2+3 Similarly: 1 2 ~T,(~I, wherein the mouseT’(~)~ is an Lyt 1+2-3- T cell of the helper subset (24). Both T’(~1 and T’¢~1 release lymphokine (l) when they are triggered antigen ~il6ne (3, 53~72). That is: SI + Tc(1) ~

1

T’f~ l + K

’el d- l, >T -- K

and T’eI+ (1)

I

1

> T’el -I--I

1.

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Response to MHC-IncompatibleTissue Grafts The above analysis showsthat the control structures of the immunesystem behave as MHCantigens in the mixed leukocyte reaction. Let us now consider howthese antigens behave in the case of tissue transplantation. The in vitro response to a tissue graft is more complexthan the in vitro unidirectional mixed leukocyte interactions. The transplanted tissue will contain antigen-bearing parenchymalcells (Figure 7). The tissue will also carry lymphoreticular cells, either as fixed tissue elements or as passenger leukocytes. Thesecells provide a source of antigen-bearing S+ cells (Figure

7). The simplest way to analyze the allograft situation is to consider separately the types of interaction that mayoccur in the recipient’s immune systemand in the transplanted tissue itself.

Interactions in the Recipient’s ImmuneSystem Antigen-bearing S+ cells from the grafted tissue will be carried to lymph nodes draining the transplantation site by way of the lymphatic system. In the node, the control structure (i.e. MHC antigens) of the donor (c genotype) will be presented directly to the recipient’s T cells (ct genotype): 1 T, C l > (c) 2 If the tissue donor differs from the recipient in both K- and I-type control structures, the activated T cells will be specified as T’ .c_l and T’.C..l This ( direct stimulation of the recipient, s T cells will therefor~tead to the generation of T cells reactive to both the K- and I-type antigens of the donor. S c + T100 kb) multi-CH-genetranscript (4). Given the large sizes of the predicted /~ 71, # + ~, or /z + ’~2b nuclear RNAprecursors (100-200 kb), and the experimental inability to detect even the 25-kb # + 8 co-transcript, experimental confirmation of these large precursors will require ingenious approaches. It also remains to be established that such cells represent

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IMMUNOGLOBULINS 411 transitional stages in heavy chain class switching. Immunoglobulin gene introduction into lymphoidcells together with definedconditionsthat induceclass switching(mitogens,T cell factors, etc) shouldprovidefurther insights into the molecularmechanisms and dynamicsof class switching.


QUANTITATIVE CHANGES IN LEVEL OF IMMUNOGLOBULIN EXPRESSION DURING DEVELOPMENT Asnotedabove,there is differential expressionof H, L, andJ chainsduring development. In addition, the expressionof all three productsis amplified substantially during B cell maturation. In general, whenboth H and L chains are expressed(i.e. in immatureB cells and later stages of development), they are synthesizedat roughlyequimolarlevels evenas the. levels of expressionare increasedseveral orders of magnitude (86, 101, 105a,113, 128,134,153b,154).In contrast, the level of J chainexpression is not tightly coordinatedwith the level of HandL chainexpression,despite the fact that in plasmaceils, J chain is expressedat levels roughlycomparableto the levels of Hand L chain expression(85, 100, 134). Clonedmurinepre-B-cell lines and someimmatureB-cell lines maysynthesizeas little as 0.01-0.1% of their protein as # only or as # plus L chains (101, 153b,154), whereas murinemyeloma cells maysynthesize as muchas 20-30%of their protein as H and L chains (86, 113). Similar amplification of H and L chain expressionin late stages of B lymphocyte development is also seen in studies of normalcells (109, 155,). Thelevels of HandL chainexpressionin most immatureB-cell, B-cell, and memoryB-cell lines appear to be roughly intermediate betweenthe extremescited above, although there are few critical measurements for the intermediate stages of B-cell development (100, 134, 188). VariousB-cell lines and normalB cells havebeenshownto amplifyboth H and L chain impressionapproximatelytwo to ten times whenstimulated in vitro with mitogens(e.g. lipopolysaccharide),anti-immunoglobulins, appropriate combinationsof antigen and lymphocytesupernatants (24, 60, 101, 109, 122b, 154). Anumberof pre-B cell lines stimulated with LPS beginto expressL chainat levels comparable to Hchain, with little stimulation of the level of Hchain synthesis(101, 122b,142a). In contrast, after LPSstimulation, several Abelsonvirus-transformedpre-B-cell lines are stimulated to synthesize H chains at an approximately10-fold increased level but still do not expressL chains (4, 5). It has beennoted for these Abelsonvirus-transformedpre-B-cell lines that LPSstimulation of H-chain synthesis occurs only in sublines or clones that spontaneouslyhave decreased the level of H-chainsynthesis substantially (about 10-fold). The

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WALL& KUEHL

decreasein the unstimulatedlevel of H-chainsynthesis is correlated with a substantial deletion in the Jn-C~,intron 5’ of the/z switchregion. Based on these results, it has been suggested that LPSstimulation somehow compensatesfor the loss of a DNA sequencethat has a positive effect on H-chain production. Myeloma cells with smaller deletions in the JH-Ct, intron seemto be unaffected in their levd of H-chainexpression. Somaticcell hybrids betweenmousemyelomacells and mouseor human pre-B cells, immatureB cells, B cells, or memory B cells co-dominantly express H and L chains at approximatelythe samehigh level of expression observedin the myeloma fusion partner. In all reported studies of this type, the overall phenotypeof immunoglobulin expression(i.e. quantity of Hand L chains and secreted versus membrane form of H chain) is determinedby the moredifferentiated myeloma cell fusion partner (50, 91, 96, 134, 134b, 188). A numberof studies with murinecell lines have demonstratedthat the qualitative andquantitativelevels of H, L, andJ chainexpressionat different stages of B cell development are determinedlargely, if not entirely, by the amountof cytoplasmicH, L, and J chain mRNA’s, respectively (38, 56, 104, 128, 134, 147). Thefew studies on normalB lymphocytes are consistent with this conclusion(168). The rates of in vivo H and L chain RNAsynthesis and processing has been studied extensively in MOPC 21 and MPC11 murine myelomacell lines by the laboratories of Walland Perry, respectively (56, 104, 147). Thesestudies, together with other studies on the turnover of cytoplasmic mRNA in MOPC 21 myelomacells (38), provide insight into howa single functional H- or L-chain gene can account for as muchas 10%of the protein synthesized by a rapidly growingmyelomacell. The following conclusionsapplyto both myeloma cell lines: (a) Therate of transcription is greater than 20-30 transcripts per minute(whichis comparableto the apparent maximalrates of transcription observed for ribosomal RNA); (b) processing of the putative primarytranscripts into mRNA and transo port of mRNA from the nucleus to cytoplasm is rapid and essentially quantitative; (c) cytoplasmicmRNA is very stable, so that it is minimally metabolizedduring the 20-hr cell generation time; and (d) the fraction cellular mRNA encodingH and L chains is comparableto the fraction of cellular protein synthesis comprisedby Hand L chains. Thus, it appears that the level of H- and L-chaingeneexpressionin myeloma cells maywell approachthe maximum level possible for a higher eukaryoticcell, with an approximate20-hr generation time. Weknowmuchless about regulatory steps that result in a 10- to 1000fold lower expression of H and L chain mRNA (and protein) in B cells representing earlier stages of B lymphocytedevelopment.However,some preliminary informationis available from a study comparingsteady-state

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IMMUNOGLOBULINS 413 nuclear and cytoplasmic H chain mRNA contents of MPC11 murine myelomacells and the 70Z murine pre-B cell line (128). MPC11 cells contain about 30,000 copies of Hchain mRNA or about 300 times as much cytoplasmicH-chainmRNA as 70Zcells, consistent with the fact that H chain comprises about 10-12%and 0.02-0.10%of protein synthesis in MPC 11 and 70Zcells, respectively (86, 101). In contrast to the large difference in cytoplasmicH-chainmRNA content, only six times as much nuclear H-chain mRNA occurs in MPC11 myelomacells as in 70Zcells. The50-fold higher ratio of nuclear to cytoplasmicH-chainmRNA in 70Z cells comparedto myeloma cells is probablya consequenceof differences in one or moreof the followingpost-transcriptional events: (a) rate H-chain nuclear RNAprecursor processing; (b) extent of intranuclear H-chain mRNA degradation; (c) rate of transport of mature H-chain mRNA from the nucleus to the cytoplasm; and (d) rate of cytoplasmic chainmRNA turnover.It is of interest that the ratio of nuclearto cytoplasmic nonimmunoglobulinpoly(A)-containing RNAin either 70Z or MPC 11 myeloma cells is essentially the sameas the ratio of nuclearto cytoplasmic H-chainmRNA in the 70Zcells. In view of the apparent differential post-transcriptional events specifically affecting H-chainmRNA metabolism, it seemsunlikely that differencesin transcription rates are fully responsible for the vastly different levels of cytoplasmicH-chainmRNA in these two cell lines. Obviouslymorestudies of the transcription rates and metabolismof H- and L-chain mRNA’s are required to understand howthe levels of H- and L-chain mRNA (and thus H and L chains) are regulated during B lymphocytedevelopment. TRANSLATIONAL AND POST-TRANSLATIONAL REGULATION IMMUNOGLOBULIN EXPRESSION

OF

Initiation of translation of H- and L-chain mRNA’s appears to be much moreefficient than the averagecell mRNA in that Hand L chains represent a larger fraction of the newlysynthesizedprotein whenthere is a nonspecific decreasein initiation of translation(e.g. mitosis,starvation,viral infection) (119, 120, 160). However,there is no convincingevidencefor regulation the efficiency of mRNA translation in B cells of different developmental stages. Microsomal localization of H, L, and J chain mRNA’s to the rough endoplasmicreticulum and translocation of H, L, and J chains into the cisterna of the roughendoplasmicreticulumseemstq be determinedby the aminoterminal signal sequence,whichis proteolytically removedfromthe primarytranslation productprior to chainterminationand release fromthe ribosome(reviewedin 86). Thesignal sequencefunctions normallyeven

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an unusual situation where the signal sequence is contiguous with the CK sequence rather than a V~ sequence, as found for normal r L-chain mRNA’s (51, 141). There are no knownexamples of either cell variants or normal regulatory events that alter binding of mRNA to the rough endoplasmic reticulum or the vectorial transport of immunoglobulinchains into the cisterna. Glycosylation of all normal H chains as well as L chains that contain the Asn-X-Thr(or Ser) tripeptide N-glycosylation acceptor site in the amino terminal V region domainusually occurs by transfer of the core oligosaccharide (glucose3-N acetyl-glucosamineE-mannosetl)from a lipid carrier to a nascent polypeptide (15, 16). The transfer of the core oligosaccharide to a growing nascent H chain apparently slows polypeptide elongation sufficiently so that the net rate of H chain synthesis is decreased 10-25%, a phenomenonthat maycontribute to the slight imbalance of H and L chain synthesis observed in both normal and malignant B lymphocytes(9, 16, 86). Parameters that affect the efficiency of N-glycosylationor potential tripeptide acceptor sites of immunoglobulinchains are reviewed elsewhere (16). There is no evidence indicating that core glycosylation is regulated differentially in B cells of different developmentalstages or in different physiological states. Most processing of the core oligosaccharide, as well as addition of terminal sugars, is thought to occur during the brief period the glycosylated immunoglobulinchain traverses the Golgi apparatus (16, 164). The decision process for generating simple versus complex asparagine-linked oligosaccharides is poorly understood. The best exampleof possible regulation of glycosylation involves a block in converting a core oligosaccharide to a complexoligosaccharide on the secreted H chains in less differentiated B cells (see belowfor a morecompletediscussion of this interesting regulatory event). Intramolecular folding, including covalent disulfide bond formation, occurs to a significant extent on nascent L chains, and presumablyon nascent H chains as well (14). Intermolecular assembly of immunoglobulinchains also begins on nascent H chains, with some classes of immunoglobulin forming H-Hdisulfide bonds and other classes of immunoglobulinforming H-L disulfide bonds on nascent H chains (15, 146). However,most assembly occurs between completed chains, with the pathwayof assembly dependent on the class of Ig synthesized by the cell (19, 86, 146). Assemblyof chains is restricted to the chains of the sameclass, so .that cells expressing /.t and T H chains, for example, do not form covalent /.t- y heterodimers (105). The co-expression of secretory and membraneforms of H chain (e.g. ~mand /.ts) in various ratios in the same cell at different B lymphocyte developmentalstages raises the question as to whether or not the secretory

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IMMUNOGLOBULINS 415 and membraneforms of H chains form heterodimers. The best studies to address this issue involve B lymphomas and myeloma cells, whichsimultaneously express 7mand Ts. In two different murine B lymphomasthat express~2amand 72as in roughlyequivalentamounts,no "Y2am’T2as heterodimerswereseen (121, 188). Analysisof labded cell surface Ig of MPC myeloma cells revealed no detectable T2bschains expressed on the cell surface (88). Theseresults then suggest that membrane and secreted chainheterodimercovalentassemblyis rare or non-existentin B cells where the membrane and secreted chains are co-expressed. Lackof significant heterodimerassemblyof these chains could be due to one or moreof the following: (a) preferential assemblyof Hchains synthesizedby the ribosomeson the same mRNA molecule; (b) preferential assemblyof H chains selectively partitioned into the membrane (Hm)instead of into the cisterna (Hs) of the rough endoplasmicreticulum; and (c) preferential assembly homodimers due to intrinsic properties of the Hchains. In contrast to the results cited above,recent worksuggests that covalentheterodimerformation and membrane insertion of ~ls and )’lm H chains occurs in MOPC 21 myeloma cells, a cell line in which),1~ is synthesizedin a vast molarexcess compared to 7~rn(58). This result mustbe questionedin viewof the results in B lymphomaand other myelomacells cited above. The putative chains in labeled surface Ig of MOPC 21 cells could, for example,represent partially degraded~/~mchains. Differential intracellular degradation of mutant and normalH and L chains represents a well-documented exampleof post-translational regulation. For example, MOPC 173 and MOPC 21 myelomalines synthesize a molar excess of L chains over H chains, but degrade the unassembledL chains (9). AMOPC 21 variant that synthesizes L chains but not H chains does not secrete the L chains, but quantitatively degradesthemwithin the cell (184). Fusionof this MOPC 21 L chain-producing variant to a different myeloma cell permits assemblyof the MOPC 21 L chain to a heterologous H chain. This assembly prevents the MOPC 21 L chain from being degradedintracellulady (184). However,in most lymphoidcells, unassembled L chains are not degradedintracellularly (9). Variantor normalcells that express H chain in the absence of L chain expression mayor maynot rapidly degradethe Hchains within the cell (113, 154). Variant myeloma cells that express L chains and J chains, but not Hchains, degradethe J chains(113-115).Finally, there is very little informationregardingregulation of H, L, or J chain degradationin ceils representing different Blymphocytedevelopmentalstages. Thefinal step in expressionof Ig by B lymphocytes is cell surface insertion of membrane Ig or secretion of secreted Ig. In general, free L chains are secreted, whereasnormalHchains are not secreted unless linked to L

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chains (86, 113). Thus,pre-Bcells generallydo not secrete their Hchains (101, 122b,142, 154). Asingle report of human pre-Bcells activelysecreting H chain in the absenceof L chain (95) needs to be rigorously documented by further experiments. In contrast, there are a numberof examplesof lymphoidcells that secrete mutant H chains in the absence of L chain expression (113). As noted above, unassembledMOPC 21 L chains are not secreted, but are degradedintracellularly. However,translation of MOPC 21 L chain mRNA in Xenopusoocytes results in an L chain that is neither secreted nor degraded (172). However,the MOPC 21 L chain is secreted if MOPC 21 H chain mRNA is simultaneously translated in oocytes (172). If another L chain MRNA is co-injected into oocytes with MOPC 21 mRNA, only the non-MOPC 21 L chain is secreted (172). Thus, it appears that the lack of secretion of free MOPC 21 L chain from MOPC 21 myeloma cells maynot result from the rapid intracellular degradationof this L chain. Severalvariant myeloma cell lines synthesizemutantL chains or Hchains that are not secreted evenwhenassemblednormally(113, 115). Themutationsare not localized to a uniqueregion. This suggeststhat these mutations maycause conformational changes in immunoglobulinchains that are incompatiblewith secretion. Expressionof membrane Ig seemsto require simultaneousexpressionand co-assemblyof Hand L chains. Pre-B cells generally express cytoplasmic bt but no surfaceor secretedbt chains(95, 101, 122b,153c, 154). Thereare several recent reports of murineand humanpre-B lymphomas that express #mon the external call surface in the absenceof L chain expression(58a, 122a). Themurinecell line (70Z)usually expressescytoplasmic/z,but expresscell surface/z spontaneously or after dextransulfate stimulation. If this result can be validatedfor normalB cells, it has importantimplications for potential regulationof pre-Bcells by anti-idiotypic antibodyor antigen. Aplethora of studies has attemptedto determinewhetherglycosylation of normalor variant Hchainsis necessaryfor cell surface expressionand/or secretion of Ig. In general, glycosylationof Hchains is not necessaryfor cell surface expression(67, 153b). Theresults are morecomplicatedfor secretion of nonglycosylated Ig: (a) Secretionis blockedor markedlyinhibited for tunicamycin-treatedmyelomas that synthesize IgM,IgE, and IgA, but a tunicam.ycin-treatedhybridomasecreted a different IgAmoleculeat apparently normalrates; (b) secretion from tunicamycin-treatedmyelomas is little affected for IgG or IgD; (c) a B lymphoma (WEHI279) secretes lowlevels of IgMat only a marginallyslowerrate after tunicamycintreatment(20, 65, 66, 82, 113, 153b,155, 156, 157, 187). Someof the results obtainedabovedo not dependon the cells per se, since the samehybridcell is shownto secrete nonglycosylatedIgGbut not to secrete nonglycosylated IgM.In sum,whetheror not nonglycosylatedIg is secreted dependson the class of.Ig, the amountof Ig synthesized,and possiblythe VHgenesegment

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IMMUNOGLOBULINS 417 in the H chain. Asnoted above,there is no evidencefor regulation of core glycosylation in B lymphocytes. Themostintriguing exampleof differential post-translational regulation of Ig at different stages of B cell developmentinvolves expressionof H chains in secreted and surface Ig. As noted previously, B lymphocytes that synthesize H chain mRNA contain both membraneand secreted H-chain mRNA’s.The ratio of secreted/membranemRNA increases during B-cell development (140). Fromlimited data, it appearsthat the ratio of intracellular membrane andsecreted polypeptidechains reflects the ratio of the two H-chain mRNA’s.Approximately20-50%of the H chain is kq in pre-B or immatureB cell lines and small resting B lymphocytesfromanimals(2, 4, 5, 48, 101,136, 122b,153b).Yetthese cells secrete little (or none)of /zs polypeptidetranslation product (101, 122b,155). In contrast, the translation product is quantitatively secreted frommyeloma cells, B-cell lines and normalresting B cells stimulatedwith mitogensor T cell factors (86, 113, 122b). Thelack of secretion of Ps at early B cell developmental stages is not dueto a lack of core glyeosylation,since the unseeretedkts chains contain core oligosaccharide(but lack terminal sugars). Neither it due to a block in H and L chain assembly,since IgMwith Ps chains is formedin these cells. Similar results havebeenfoundfor a B cell line that expressesboth as and am(82, 159), althoughthe lack of secretion here less well documented than the examplescited above. Themechanism of this specific post-translational regulation of secretion is unclear. It mightbe due to any or a combinationof the following:(a) differential oligosaccharideprocessingand terminal glycosylation of the core oligosaccharide; (b) failure of transport into the Golgi apparatus, whichwouldexplainthe glycosylationdeficiencies; (c) lack of J chain(100). Theapparentlack of quantitative secretion of as froma B lymphoma argues against a role for J chainsince IgAcan be secretedas a monomer (82, 159). Similarly, the normalsecretion of nonglycosylatedIgMfroma B lymphoma cell line (153b)arguesthat glycosylationdoesnot explainthis situation. Studieson certain lines of 70 Z and18-81cells suggestthat the expression of surface IgMmayrequire morethan the expression of Pmand L chains (26, 122a, 142). The molecularmechanisms that control IgMsecretion early B cells represent interesting questionsyet to be resolved. FUTURE

DIRECTIONS

The studies reviewedhere dearly provide considerable insights into the events in immunoglobulin gene expression and regulation. Nonetheless, manyof the molecularmechanisms affecting the changingpatterns of immunoglobulin geneexpressionin B-cell development remainto be resolved.

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The powerful resolution of recombinant DNAcloning and sequencing will continue to provide insights into immunoglobulin gene structure. Several exciting recent developments provide molecular immunologists even more penetrating approaches for dissecting immunoglobulin gene control. These include (a) the establishment of cloned normal pre-B cell and B cell lines that undergo developmental changes in culture (183a), (b) the successful introduction and expression of cloned immunoglobulin genes transfected into lymphoid cells (42a), and (c) the increasing refinement of culture conditions and factors that stimulate B cell growth and affect changes in immunoglobulin gene expression (55a, 93a, 124a). ACKNOWLEDGMENTS Michael Kuehl has been supported by USPHSgrants AI 12525, AI 17748, and AI 00293 at the University of Virginia Medical School, Department of Microbiology. Randolph Wall has been supported by USPHS grants AI 13410 and CA 12800, and by NSF grant PCM79-24876. Literature Cited 1. ~tcad. Alt, F., ScL Baltimore, D. 1982.Proc.Natl. USA79:4118-22 2, Alt, F. W., Bothwell,A. L M., Knapp, M., Siden,E., Mather,E., et al. 1980. Cell 20:293-301 3. Alt, F., Enea, V., Bothwell,A. L. M., Baltimore,D. 1980. Cell 21:1-12 4. Alt, F. W., Rosenberg,N., Casanova, R. J., Thomas,E., Baltimore,D. 1982. Nature 296:325-31 5. Alt, F. W., Rosenberg,N., Enea, V., Siden, E., Baltimore, D. 1982. Mol. Cell. Biol. 2:386-400 6. Alt, F. W., Rosenberg,N., Lewis, S., Thomas,E., Baltimore, D. 1981. Cell 27:391-400 7. Altenburger, W., Steinmetz, M., Zachau, H. G. 1980. Nature 287:603-7 8. Amara,S., Jonas, V., Rosenfeld,M.G., Ong,E. S., Evans, R. M. 1982. Nature 298:240-44 9. Baumal, Scharff,M.D. 1973.J.. Immunol. R., 111:448-56 10. Banerji, J., Rusconi,S., Schaffner,W. 1981. Cell 27:299-308 11. Benoist, C., Chambon, P. 1981. Nature 290:304-10 12. Bentley,D.L., Farrell, P. J., Rabbitts, T. H. 1982. Nucl. Acids Rez 10: 1841-56 13. Bentley, D. L., Rabbitts, T. H. 1980. Nature 288:730-33 W.,254:8869-76 Kuehl, W. M. 1979. 14. Bergman, J. Biol,. L. Chem.

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141. Rose, S. M., Kuehl, W.M., Smith, G. P. 1977. Cell 12:453-62 142. Rosenberg, N. Personal communication 142a. Rosenberg,N., Siden, E., Baltimore, D. 1979. In B Lymphocytesin the lmmune Response, ed. M. Cooper, D. Mosier,A.Scher,E. S. Vitetta, pp. 37986. NewYork: Elsevier 143. Sakano,H., Huppi,K., Heinrieh, G., Tonegawa,S. 1979. Nature 280:288-94 144. Sakano,H., Maki,R., Furasaw,Y., Roeder, W., Tonegawa,S. 1980. Nature 286:676-83 145. Saragosti, S., Moyne,G., Yaniv, M. 1980. Cell 20:65-73 146. Sehartf, M. D. 1974. HarveyLecture 69:125--42 147. Schibler,U., Marcu,K. B., Perry, R. P. 1978. Cell 15:1495-509 148. Schwartz, R. C., Sonenshein, G. E., Bothwell, A., Gefter, M.L. 1981.J. Immunol. 126:2104-8 149. Seidman,J. G., Leder, P. 1978. Nature 276:790-95 150. Seidman,J. G., Leder, P. 1980. Nature 286:779-83 151. Self, I., Khoury,G., Dhar, R. 1979. Nucl. Acids~Rez 6:3387-98 152. Sekikawa,K., Levine,A. J. 1981.Proc. Natl. Acad. Sci, USA78:1100-4 153. Setzer, D., McGrogan, M., Nunberg,J. H., Schimke, R. T. 1980. Cell 22: 361-70 153a.Sibley, C. H., Ewald,S. J., Kehry,M. R., Douglas, R. H., Raschke, W.C., Hood, L. E. 1980. J.. lmmunoL125: 2097-105 153b.lmmunol. Sibley, C.126:1868-73 H., Wagner,R. A. 1981. J. 153c.Siden, E., Alt, F. W., Shinefeld, L., Sato, V., Baltimore,D. 1981.Proc.NatL Acad. ScL USA78:1823-27 154. Siden,E. J., Baltimore,D., Clark, D., Rosenberg,N. E. 1979. Cell 16:389-96 155, Sidman,C. 1981. Cell 23:379-89 156. Sidman,C. 1981. J. Biol. Chem.256: 9374-76 157. Sidman,C., Potash, M. J., KiShler,G. 1981. J. Biol. Chem.256:13180-87 158. Singer, P. A., Singer, H. H., Williamson, A. R. 1980. Nature 285:294-300 159. Sitia, R., Kikutani,H., Rubartelli, A., Bushkin, Y., Stavnezer, J., Hammerling, U. 1982. J. lmmunol.128:712-16 160. Sonenshein, G. E., Brawerman, G. 1976. Biochemistry.15:5497-506 161. Southern, E. 1975. J. Mol. Biol. 98: 503-17 162. Storb, U., Arp, B., Wilson,R. 1981.Nature 294:90-92

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163. Storb, U., Wilson,R., S¢lsing,E., WalImmuneResponse, ed. M. Cooper, D. fidd, A. 1981. Biochemistry20:990-96 Mosier,I. Sober,E. Vitetta, pp. 371-78. 164. Tartakoff,A., Vassalli, P. 1979.J. Cell Amsterdam:Elsevier/North Holland: BioL 83:284-99 371-378 165. Temin,H. M. 1981. Cell 27:1-3 181. Weintraub, H., Groudine, M. 1976. Science 193:848-56 166. Temin,H. M. 1982. Cell 28:3-5 182. Weischet,W.O., Glotoz, B., Schnell, 167. Tonegawa,S., Maxam,A. M., Tizard, R., Bernard,O., Gilbert, W.1978.Proc. H., Zachau,H. 1982. Nucl. Acids Res. Natl. Acad. Sc~ USA75:1485-89 10:362742 168. Tsuda, M., Honjo, T., Shimizu, A., 183. Whitlock,C., Witte, O. N. 1982. Proc. Mizuno, D., Natod, S. 1978. Z BioNatl. Acad. Sc~ USA79:3608-12 chen~ 84:1285-90 183a.Whitlock,C. A., Ziegler, S., Treiman, 169. Tucker,P. W., Lin, C., Mushinski,J. L., Stafford,J., Witte, O.Submitted for F., Blattner, F. R. 1980. Science publication 209:1353-60 184. Wilde,C. D., Milstein, C. 1980.Eur. J. 170. Tucker,P. W.,Slighton,J. L., Blattner, Immunol. 10:462-67 F. R. 1981. Proc. Natl. Acad.ScL USA 185. Wilks,A. F., Cozens,P. J., Mattaj, I. W.,Jost, J. 1982.Proc.Natl. Acad.Sci. 78:7684-88 171. Tyler, B. M., Cowman, A. F., GerondaUSA79:4252-55 kis, S. D., Adams,J. M., Bernard, O. 186. Williams,P. B., Kubo,R. T., Grey,H. 1982. Proc. Natl. Acad. Sci USA M. 1978. J. lmmunol.121:2435-39 79:2008-12 187. Williamson, A. R., Singer, H. H., 172.Valle, G., Besley,J., Colman, A. 1981. Singer, P. A., Mosmann,T. R. 1980. Biochem.Soc. Trans. 8:168-70 Nature 291:338-40 173. VanNess, B. G., Weigert, M., Cole- 188. Word,C. J., Kuehl, M. W.1981. Mol. dough,C., Mather,E., Kelley, D. E., Immunol. 18:311-22 Perry, R. P. 1981. Cell 27:593-602 189. Word,C., Mushinski,J. F., Tucker,P. 174. Varshavsky, A. J., Sundin,O. H., Bohn, W.1983. Cell 32: In press M.J. 1978. Nucl. Acids Rez 5:3469-77 190. Yagi, M., Koshland,M. E. 1981. Proc. A. J., Sundin,O. H., Bohn, Natl. Acad. ScL USA78:4907-11 175. Varshavsky, M. J. 1979. Cell 16:453-66 191. Yamawaki-Kataoka,Y., Kataoka, T., Takahashi, N., Obata, M., Honjo, T. 176. Vassalli, P., Tedghi,K., Lisowski-Bernstein, B., Tartakoff, A., Jaton, J. C. 1980. Nature 283:786-89 1979. Proc. NatL Aca~LSc£ USA76: 192. Yamawaki-Kataoka,Y., Miyata, T., 5515-19 Honjo, T. 1981. Nucl. Acids. Res. 9:1365-81 177. Vitetta, E. S., Uhr,J. W.1975.Science 193. Yamawaki-Kataoka,Y., Nakai, S., 189:964-68 Miyata,T., Honjo,T. 1982.Proc. Natl. 177a. Walfield, A., Sdsing, E., Alp, B., Storb, U. Unpublished results Acad. Sci. USA79:2623-27 178. Wall,R., Choi,E., Carter, C., Kuelil, 194. Yaoita, Y., Kumagai,Y., Okumura,K., Honjo, T. 1982. Nature297:697-99 M., Rogers, J. 1980. CoMSpring Harbor Syrup. Quan~BioL 45:879-85 195. Yang,V. W., Lerner, M.R., Steitz, J. 179. Wallach,M., Ishay-Michaeli,R., Givol, A., Flint, S. J. 1981.Proc.Natl. Acad. D., Laskov, R. 1982. J. lrnmunol. Sci. USA78:1371-75 128:684-90 196. Yaun,D., Uhr, J. W., Vitetta, E. S. 1980. J. lmmunol.125:40-46 180. Warner,N. L., Leary, J. F., MeLaughlin, S. 1979. In B Lymphocytesin the 197. Ziff, E. B. 1980. Nature287:491-99





An~Rev. lmrauno£ 1983. 1:423-38 Copyright ©1983by AnnualReviewsInc All rightsreserved

THE BIOCHEMISTRY OF ANTIGEN-SPECIFIC T-CELL FACTORS D. R. Webb Roche Institute

of Molecular Biology, Nutley, NewJersey 07110

Judith A. Kapp and Carl W. Pierce Department of Pathology and Laboratory of Medicine, The Jewish Hospital of St. Louis, and the Departments of Microbiology and Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

INTRODUCTION Soluble factors producedby immunoeompetent cells in response to antigenie stimulation represent one communications link by whichthe immune systemregulatesits activities. Thesefactors maybe spedtic for the inducing antigen or maybe non-antigen-speeitic.Otherchapters in this volumediscuss in detail the cellular aspects of immune regulation. This communication focuseson the biochemicalandserological nature of antigen-specific regulatory molecules.Thesemoleculesare exclusively the products of Tcells (immunoglobulins areobviously antigcn-spccilic andmayalsoserve regulatory function, buttheyarcdiscussed elsewhere). Theymaybedivided into twocategories: helper factors (Tr~F), which helpT cffector cells or cells torespond (e.g. become cytotoxic ormakeantibody); andsuppressor factors (TsF), which suppress T and/or B cellresponses. These factors are extremely potent interms oftheir specific activity, andarepresent in picogram quantities inlymphoid tissues. Forthese reasons, studies onthe biochemical nature of these substances haveprogressed veryslowly. A limited numbcr ofthcsc factors havc been purified toanysignificant degree andonlya fewlaboratories havereported purilication tohomogeneity (II, 21).This state ofaffairs haschanged recently with application ofsomatic 423 0732-0582/83/1M I0-0423502.00

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call hybidizationmethodology to T cells (3) and the use of microanalytical protein purification andanalysis to characterizethe hybddoma T-cell produet (16, 21, 49, 50). SomaticT-cell hybrids provide reasonably stable, rapidly growingcell lines, whichconstituitively produceT-cell factors. PrimaryT-cell clones havealso beenproduced,but so far havebeen less useful for the extensivebiochemicalcharacterizationof T-cell factors, due to culture limitations and stability (12). Mostof the studies discussedhere havenot beencarried out on fully puritied factors. Thus,conclusionsregarding the precise biochemicalproperties of most of these substances should be regarded as tentative. The reason for such caution becomes apparentwith the serological evidenceimplicatingIgVH region genesas the sourceof a portionof antigen-specificT-cell factors. Whatfollows,then, is in the nature of a progress report on whatis knownabout antigen-specific factors, both their structure and their genetic origin. Weclose with a consideration of what questions need to be addressed in the immediate future. ANTIGEN-SPECIFIC

HELPER FACTORS (THF)

In 1980, several very excellent reviews appeared, whichdiscussed the progress that had been madeon understandingthe biology, genetics, and biochemistryof antigen-specificfactors (1, 37, 45). In the ensuing2 years it is safe to say the progresshas beenuneven.This is particularly true of the antigen-specific helper factors. Despite the developmentof T-cell somatic hybrid technology, it is still di~cult to makeantigen-specific THF-producing T-cell hybrids. Becauseof this, and the fact that those hybridomasthat have been madestill produce relatively small amounts (micrograms/liter) of THF,biochemicalcharacterization has proceeded slowly. TheTnFmostextensively characterized are presented in Table 1. Wehavenot attemptedto list all of the THFthat havebeenreported;rather, wehavetried to include those factors for whichthere are moredata than wereavailablein 1980(1, 37, 45). Asmaybe seen, the list is short. However, somegeneralities about structure andfunction are beginningto emerge,and someconclusionsreached fromearlier studies seemto be holding up. First, those factors that havebeentested react with an antisera specific for Vn-framework determinants; manyTBFalso react with anti-idiotypespecific antisera (Id+). This impliesthat T cells use VHgenesor a portion of Vii genesto specify TnF.Recentresults fromseveral laboratories raise somedoubtabout the validity of this conclusion. Followingthe observations of Tonegawa and his colleagues (32), as well as others (33), mature B cells contain rearranged VLand Vri genes, several groups analyzed T-cell DNA (7, 19). Theresults havebeenquite consistent. T cells


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mayrearrange D-region and J-region sequences, but no T cell has yet been found that has a productively rearranged (e.g. functional) VRor VLgene. This leads to the conclusion that whatever T cells use to make antigenspecific proteins, they do not need rearranged VH or VL genes. Furthermore, molecular probes that represent the gene sequences specifying certain idiotypes have bcen utilized in studies of RNAand DNAfrom s¢rologically idiotype-positive (Id +) T cells. Using these probes, the two groups conducting these studies have failed to detect functional rearrangements of the VH genes in Id+ T cells; neither have they been able to showthe existence of mRNA coding for Id + proteins in the serologically Id + T-cell lines studied so far (27; E. Kraig, personal communication).This wouldsuggest that the Id + T cells maybear a determinant that cross-reacts with antiidiotype antisera but is not encoded by the gene specifying the original idiotype. A fascinating result is the observation by Lambet al (22) that the streptococcal antigen (SA)-specific THFfrom monkey and mouse react with xenoantisera against human//2 microglobulin. Since//2 microglobulin is homologouswith constant region domains, once again there is a suggestion of commongenetic origin for these T-cell factors and Ig (not to mention Thyl, H-2, etc). It is also possible that in the case of the SA-specific TriF, //2 microglob.ulin constitutes part of the molecule. In the case of mouse SA-specific THF,it reacts with chicken, rat, and guinea pig anti-//2 microglobulin but not monoclonal anti-human//2 microglobulin; monkeySAspecific Tt.IF react with chicken, rabbit, and rat anti-//2 microglobulin, as well as with monoclonalanti-human f12 microglobulin (22, 53). These data cannot discriminate between the possibility that the SA-specific THFare associated with//2 microglobulin or that they have sequence homologywith //2 microglobulin,although Lambet al favor the latter possibility. All of the factors tested react with antisera specific for H-2loci. This raises the issue as to whether or not the mixing of putative gene products of chromosome 12 with those of chromosome17 can occur. Since anti-Id antisera may not be detecting a chromosome12 gene product, there may be no problem at all. Also, if somefactors contain two chains, then one can easily concoct a two-gene two-polypeptide chain hypothesis to explain the results. This maybe the case for one class of two-chain polypeptide suppressor factors (see below). One-chain THF, containing apparently both VHand H-2 determinants, would present the biggest problem, but no THFof this type have yet been reported. The solution is simply to wait until more data become available concerning the nature of the genes that specify these molecules. It is notable that none of the studies summarizedin Table 1 has assigned a specific activity to the factor being examined. This is unfortunate. The measurementof biological activity by dilution analysis, coupled with careful estimates of protein concentration, give one. of the best measures of

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relative purity whenone calculates activity/unit weight (e.g. milligrams or micrograms).Purity, or its lack, represents the biggest obstacle to clear-cut interpretations of data. This is particularly important whendrawingconclusions about molecular weight or structural composition. For this reason, the data summarizedin Table 1 must be regarded as tentative, pending more complete purification.


ANTIGEN-SPECIFIC

SUPPRESSOR

FACTORS

In contrast to the antigen-specific helper factors, more progress has occurred in the biochemical characterization of antigen-specific suppressor factors. Reports are beginning to appear on purification and concommitant biochemicalanalyses of the isolated factors (11, 21). In addition, at least two groups have begun studies aimed at the cloning of the C-DNA derived from the mRNA that codes for TsF, which would for the first time allow us to look directly at the genes responsible for these factors (44, 51). A large number of TsF have been described, and new reports continue to appear (1, 37, 45). The progress has been made possible both by the continued improvementin our capacity to grow cloned T cells that maintain their function and by the use of hybridomatechnology (6). Most reports factor-producing hybridomas that have appeared since 1980 are concerned with suppressor factors rather than antigen-specific helper factors (6). Whether this is related to the almost exclusive reliance on BW5147 as a fusion partner or to other reasons is not clear. In addition to these techniques, the successful production of radiation leukemia virus-transformed T-cell lines with suppressor function has been presented recently (30, 31). The fact that such lines produce large amountsof antigen-specific TsF, are easy to grow, and have apparently a normal chromosomecomplement may makethem increasingly attractive to workers in this field. Since other authors in this volumecover the regulatory aspects of suppressor and helper factors, we confine ourselves, as with TnF, to a consideration of the biochemistry of TsF. It is known that antigen-specific suppressor factors could be elaborated to manykinds of antigens. For example, several different synthetic polypeptides, the immuneresponse to which is knownto be under Ir gene control, induce suppressor cells. In manycases these cells have been shownto release suppressor factors that block the appearanceof antibody-formingcells (18, 20, 34, 48). Suppressor factors that block the humoral immuneresponse to natural proteins such as KLHor HGGhave also been reported (39, 40). Cellular immuneresponses to alloantigens or tumor antigens maybe shownto be regulated by suppressor cells and TsF (15, 38). Also, haptens, either on proteins or autologous cells, mayinduce specific TsF (14, 54). It maybe concludedthat virtually every type of immuneresponse results in the generation of suppres-

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sor cells that mayrelease antigen-or determinant-specificTsF.It wasalso shownthat such factors could be idiotype specific rather than antigen specific and still be able to induceantigen-specificsuppression(36). Many of the factors reactedwith both anti-Vregion antisera (underwhichheading for convenienceweinclude anti-idiotype) and anti-I-region-specific antisera; andnonehad Ig constant-regionmarkers,as they are definedserologitally (1, 37, 45). Rather than present a completelisting of all of the suppressorfactors describedsince 1980,onceagain wefocus on those factors for whichwenow have substantial biochemicalinformation. Acompilationof these factors and their characteristics is listed in Table 2. There are four groups of investigators whohave been carrying out the most extensive biochemical analyses. Theyinclude(a) our group, whichis studyingfactors specific for the synthetic copolymersGAT,GA,and GT(16, 21, 49); (b) Cantor his associates, studyinga SRBC-specific factor (10-12); (c) Taniguehi Tadaand their co-workers,studying KLH-specificsuppressor factors (4143, 46, 47); and (d) Dorf, Greene,Benacerraf, and their associates, havedoneextensive serological studies and perhapshavethe mostcomplete set of suppressorfactors specific for NP(26, 28, 29). Manyother investigators have performedstudies on suppressor factors, but they have emphasized the morebiological aspects of these agents andfor that reasonthese other factors are not listed here (1, 37, 45). Fresnoet al have studied TsF obtainedfrom a cloned T-cell line using radiolabeled TsF. TheTsF in Table 2 have beenshownto suppress primary humoralimmuneresponses, secondaryhumoralimmuneresponses, or cellmediatedresponses. All of the factors react with antisera specific for one or another determinant on antibody Vr~ chains. All but one factor, the glycophorinTsF, react with H-2Iregion-specific antisera. Wherestudy has beenpossible, manyof the factors showMHC restriction or Igh restriction, or both. In terms of molecular weights, these molecules range between 19,000-80,000and maybe composed of one or two chains. Oneof the more interestingfeaturesof the table is the listing of specificactivities. Of the four research groups,only two havemadeestimates of specific activity. In our opinion, such estimates are crucial to any biochemicalstudy, since such estimates represent the only wayof assessing the degree of purity. Verylittle is knownconcerningstructure-function relationships of the majority of the TsF. This is no doubt largely becausebiochemicalstudies are quite recent. Fresnoet al (10) have reported that the glycophodnTsF maybe dissociated into an antigen-binding peptide of 25,000-MW and a 45,000-MW protein that retains the capacity to be suppressive; however,it nowlacks antigenic specificity. Thestudies of MHC and immunoglobulin


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allotype restriction suggest that both H-2 determinants and Igh determinants present on TsF are required for function. In this regard, the recent results of Sorensen et al are of interest (17). They have obtained two-chain GAT-TsFfrom a hybridoma (372D6.5) in which they have confirmed the observations of Taniguchi et al (41-43, 46, 47). That is, one chain carries the antigenic specificity and one chain bears an I-J determinant(I-Jb). When the two chains are dissociated by reduction and alkylation, they maystill be shownto have suppressive activity when added together. What is most surprising, the antigen-binding chain maybe added to spleen cell cultures given antigen; 24-48 hrs later, the I-J-boring chain may be added and suppression of the anti-GATresponse is still observed (17). This result suggests that in tissue culture, at least, antigen-binding chains mayfunction in the absence of I-J chain and the I-I chain may perform its function possibly without the antigen-binding chain. That such complex structurefunction relationships are the normrather than the exception is also suggested by recent data reported by Gershonand his associates (9, 52), who propose that suppressor-inducer factors may form molecular associations with "schlepper" or carder moieties which help to direct them to the proper cellular receptor. Anotheraspect of structure-function is specificity. In the synthetic polypeptide system (GAT,GA,or GT), it maybe seen that hybridomas derived from fusions with GATas the immunizing, antigen may be either GAT/{3A- or GAT/GT-specific. Of interest are the two hybridomas derived from fusions with H-2s splenic T cells. Whether the immunizing antigen was GT or GAT,the binding specificity to GAT-, GA-, or GT-Sepharose is the same: both 342.B1.11 and 395A4.4 TsF bind GATor GT. In fact, 395A4.4 may be analyzed using GAT-MBSA-pdmed cells with identical results to GT-MBSA-primed cells. The amino terminal sequences currently available through residue 30 show few differences between GAT-or GTspecific TsF (D. Webb,manuscript in preparation). Wherethe specificitydetermining regions are in these molecules remains to be determined. The SRBC-induced TsF-producing clone C1Ly23/4 was shown by Fresno et al (11) to have specificity for glycophodn.Interestingly, these TsF can discriminate (apparently) glycophorins from different species red blood cells (e.g. sheep versus burro). To date, no studies have been F. performed to determine the precise specificity of the KLH-Ts In addition to the results of studies shownin Table 2, two groups have also characterized to a similar extent the mRNA that codes for their respective suppressors. A summaryof these results is presented in Table 3. Wieder, in our laboratory (50, partially purified polyA+ RNAfrom hybddoma258CA.4by subjecting it to sucrose density gradient centrifugation. Fractions were collected and were translated in a rabbit reticulocyte lysate


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translation system, and the translated products were assayed for OATspecific suppression. RNAthat had a sedimentation coefficient of 16S was able to code for GAT-TsF.The molecular properties of this translated product indicate that it is similar if not identical to supernatant OATTsF from 258C4.4. Other investigators in our group (35; K. Krupen, unpublished data; E. Oerassi, unpublisheddata; C. Healy, submitted for publi+ cation; C. Sorensen, manuscript in preparation) have translated polyA RNAfrom T-cell hybridomasand have obtained similar results. Anintriguing feature in somehybridomasis the appearancein the cell-free translation products of a larger molecular weight form of TsF (80,000). Whatrelationship this has to the lower molecular weight form remains to be determined. In another study, Taniguchi and co-workers (44) isolated polyA+ RNA from a KLH-TsF-producinghybfidoma and subjected it to sucrose density centrifugation. They were able to isolate three species of RNA.Twoof these RNAs,13S and 18S, when translated in a frog oocyte system, yielded the antigen-binding chain, and the I-J-bearing chain was derived from an mRNA with a sedimentation coefficient of 11S. The properties of the translated products are similar if not identical to those of TsF obtained from culture supernatants of the hybddomas.Both our group and that of Taniguchi are involved in the molecular cloning of their respective mRNAs. This suggests that in the not-too-distant future we should be able to study directly the genes involved in the synthesis of regulatory T-cell products. Such molecular probes will undoubtedlybe useful in deciphering the nature of the antigen receptor on T cells. Having outlined the biochemical data currently available on antigenspecific TsF, it is necessaryto address the significance of these results. Some notable differences shownin Table 2 are the different molecularweights that have been determined for the different factors and the differing structures (e.g. one chain or two). A plausible reason for these differences may derived from studies in the OTsystem as well as the data obtained with the NPhapten, which suggest that several types of suppressor factors exist. Indeed, Germain&Benacerraf, in a recent review (13), have concluded that a suppressor cell "cascade" exists in which TsF serve as the links between different suppressor cell subclasses, as well as acting on nonsuppressor targets, T-helper cells, or B cells. Althoughit maybe that the details of the Germain& Benacerraf"consensus pathway" are incorrect, the general idea seemsto fit the available biochemicaldata. In the GTsystem, it is possible to show that single-chain, non-MHC-restricted, antigen-specific factors (TsF1) induce the appearance of MHC-restficted (presumably two chain) antigen-specific suppressor factors (TsF2). In the NPsystem, a cascade has been established involving an antigen-specific idiotype-positive suppressor inducer TsFl, which induces an anti-idiotype TsF2 which induces antigen-

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ANTIGEN-SPECIFIC T-CELL FACTORS 433 It is the TsF3 that directly interacts withTri or B cells. If one accepts this kind of scheme,it is possible to assign the KLH-TsF to the TsF2 class, based on homology to GAT-TsF currently being studied by C. 2 Sorensen, D. Webb,and C. Pierce (manuscriptin preparation). This TsF2 is composedof two chains, one antigen-specific and one I-J +, and is inducedby TsF~.Fresnoet al (12) havepreliminarydata that suggestthat the glyeophorinTsFcan directly affect B cells, whichwouldplace themin the TsF3 class. Notethat the TsF2 maybe either antigen-specificor idiotypespecific. Althoughthese explanationsremainhypotheticalto a large extent, they serveto illustrate the point that someof the different factors presented in Table2 almostcertainly represent different classes of TsF. Anissue rdated to molecularand structural heterogeneityis the question of serological or immunoehemieal analysis. Virtually all the serological evidencefor Igh, VH,idiotype determinants, or H-2determinantsmust be regardedas tentative. Theprincipal reasonfor makingso sweepinga statementis that for eachcase mentioned above,the antisera wereinitially raised with another antigen. Thus, reactions of TsF with these sera are crossreactive. In mostinstances it is not knownwhetherthe antisera recognize sequence-specificdeterminantsor conformationallyderived determinants. Asmentionedearlier in the discussionof TIFF,it is possible to demonstrate expressionof idiotype-positivematerialby T cells serologicallyin the absence of any detectable mRNA that can hybridize to the IgVri gene probes that encodeidiotype-positive Ig. In the case of the GAT-TsF, for example, E. Kraig (personal communication)has shownthat RNAextracted from several GAT-TsF-producing hybridomaswill not hybridize a molecularprobe that codes for idiotype-bearinganti-GATantibody (IgG). Nonetheless, the GAT-TsF will react with xenoantisera specific for this idiotype (GAT).Furthermore, preliminary sequence analysis of three GAT-TsF from three different hybridomas(258C4.4, 372B3.5, 395A4.4; Table 2) shows no homologyso far, with either anti-OATmonoelonal antibody or any knownV~. or VHregion sequences. Suchdata suggest one cannot be too cautious about drawingconclusionsfromserological data in the absenceof corroboratinginformation. In someof the cases shownin Table2, corroborationdoes exist. In the NPsystem,the existenceof I-J reactivity on the TsFand an I-J restriction at the cell target level supportsthe notion of a true I-J determinanton NPTsF. Likewise, the Igh determinants and Igh restriction imply a strong likelihoodthat an Igh-linkedgeneor genesplays a role in the structure and function of TsF. Thefact that both Tada(42) and Taniguchi(47) can by differential absorptionthe presenceof uniqueI-region geneproductsas well as Igh geneproductson T cells stronglysuggeststhat both sets of genes are importantin determiningthe structure and function of TIfF and TsF.


specific TsF3.

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Thepossible existence of two determinantson a single polypeptide(GATTsF), eachspecified by genesthat lie on separatechromosomes, is a dit~cult problem.Onepossible solution, particularly whereanti-idiotypic antisera are used, is that the apparent presenceof the idiotype on the factor is misleading.In the case of the GAT-TsF, the anti-idiotypic antisera, anticOAT and anti-GA-1,have not beenadsorbedwith idiotype first and then tested for binding to GAT-TsF; thus, the idiotype-positive GAT-TsF may turn out either to be cross-reactiveor to reflect the limited heterogeneityof aminoacids allowedin the combiningsite for any GAT-specificprotein. Thecase for the I-J determinantreflecting a real rather than a cross-reacting epitopeis strongerin those cases whereI-J restriction has beennoted(e.g. NPand KLH).It is also moreconvincingthat the epitope is at least "I-J like" in those instances wheremonoclonal antibodies are available. This is the case for two of the GAT-TsF,the KLH-TsF,and the NP-TsFmolecules. Theexperimentswith the cell-free translation product of (3AT-TsF mRNA are also of interest in this regard. Wiederet al (51) showedthat GAT-TsF generated from mRNA translated in a rabbit reticulocyte lysate systemwereboundby anti-Iq-Sepharose.Since little if any glycosylation takes place in the microsome-free, rabbit reticulocyte lysate system,these results suggest that the I-region determinantrecognizedis not carbohydrate. These results were confirmedby Sorensenet al (35) using GATTsF (H-2b) translated in a rabbit reticulocytc lysate system, whichwas boundby monoclonalanti-I-Jb-Sepharose (,provided by C. Waltenbaugh).

THE T-CELL ANTIGEN RECEPTOR AND ANTIGEN-SPECIFIC FACTORS Part of the reason for studying TsF or TnFis the possibility that such factors will be related to the elusive T-cell receptor for antigen. Indeed, there arc manyserological similarities betweenTsFand T-cell membrane moieties. For example,both T cells and factors maybear idiotype, I-J, or Igh determinants(1, 37, 45), and Binz and Wigzclland their colleagues (3-5) haveimplicatedidiotypc-positivemoleculesas beingpart of the T-cell antigen receptor. Several factors (e.g. all of the (3AT-TsF and the KLHTsF)can be isolated from membrane extracts ofT-cell hybridomas.Indeed, Matsuzawa &Tada(37) showedthat I-J + T cells could bind soluble antigen andthat anti-I-J antisera couldblockantigen-bindingto I-J-positive cells. Theauthors interpreted these results as implicatingI-J-bearing molecules in involvement at antigen-binding sites. Amajordifficulty that arises, however, is the structural heterogeneityof T-cell factors in comparison to the Ig on B cells. Setting aside for the moment the fact that Ig mayformdimers or pcntamcrs,the basic structure of Ig is the samefromisotype to isotype,

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e.g. two light chains andtwo heavychains linked to one anotherby disulfide bridges. By contrast, amongthe antigen-specific TsF, there are single polypeptide chain moleculesof 29,000, an apparentlysingle chain of 70,000, and two-chain TsF, which mayhave disulfide bonds or not dependingon their cellular location. Do these molecules reflect the antigen receptor on the respective T cells? The question must remain unanswered.It should be noted that C. Healyet al (submittedfor publication) have foundin a GATTsF-producinghybridomaa membrane form of GAT-TsFthat exists primarily as a dimer of 60,000-70,000 molecular weight (with minorspecies of higher molecular weight polymers). Since its amino acid content is identical (in moles percent) to the 29,000-molecular-weight form, the 29,000- and 60,000-MWGAT-TsFappear to be identical. C. Sorensen (manuscriptin preparation), in a related study of GAT-TsF froma responder strain (H-2b) finds that GAT-TsF in their pure state have a strong propensity to aggregate. Thus, mulfimersof the basic 29,000-MW protein begin to appearandultimately cause the protein to precipitate fromsolution even thoughthe solution contains a hydrophobicsolute (propanol). All of the characteristics outlined aboveshouldserve as a caution to the unwarythat considerable workremains to bc done to unravel the nature of the T-cell antigenreceptor. Thebest predictionat this time suggests that such a receptorwill reflect the natureof the cell fromwhichit is derived, as well as reflecting the natureof that cell’s products.It is unlikely that different T-cell subsets use the sameantigen receptor, if we use the antigenspecific T-cell factors as a guide. Additional questions concerningT-cell productand receptor diversity, andthe extent of T-cell repertoires, the influence of haplotypeon these, and their relationship to the T-cell antigen receptor mustawait the biochemical analysis that is just beginning.It is likely in the next 5 yearsthat manyof the remainingquestions will have foundat least partial answers. Literature Cited 1. Altman, A., Katz, D. H. 1980. Production andisolation of helperandsuppressor factors. J. Immunol.Metl~ 38:9-41 2. Apte, R. N., L~wy,I., DeBaetscher, P., Mozcs,E. 1981. Establishment and characterization of continuous helper T celllines spcciiic topoly (L tyr, L glu)poly(DL ala)~poly(L lys).Z Immunol. 127:25-30 3. Binz, H., Soots, A., Nemlander, A., Wight, E., Fenner, M., et al. 1982. Induction of speeitie transplantationtolerance via immunization with donorspeeitie idiotypes. Ann. NY Acad. Sci. 392:360-74

4. Binz, H., Wigzell, H. 1976. Spccitic transplantation tolerance induced by autoimmunizationagainst the individual’s ownnaturally occurring idiotypic antigen-bindingreceptors. J. Exp. Med. 144:1438-57 5. Binz, H., Wigz¢ll, H. 1981. T-cellreceptors with all0-major histocompatability complexspcciticity fromrat andmouse. J. Exp. Med. 154:1261-78 6. Boehmer,H. V., Haas, W., KiJhler, G., Melehers, F., Zeuthcn, T., eds. 1982. T-cell hybridomas. Curr. Top. crobio£ ImmunoL 100 7. Cory, S., Adams, J. M., Kemp, D. 1980. Somatic rearrangements forming

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active immunoglobulin/z genes in B and T lymphoid lines. Proc.Natl. Acad. Sci. USA77:4943-47 8. Eshhar, Z., Woks, T., Zinger, H., Mozes,E. 1982.T-cell hybridomasproducingantigen-specificfactors express heavy chain-variable-region determinants. Curt. Topics Microbiol. Immunol. 100:103-9 9. Flood, P., Yamanchi,K., Gershon,R. 1982. Analysisof the interactions betweentwo moleculesthat are required for the expression of Ly-2 suppressor cell activity. Threedifferenttypesof focusing events maybe neededto deliver the suppressive signal. J. Exp. Med. 156:361-71 L., Can10. Fresno, M., McVay-Boudreau, tor, H. 1982. Antigen-specificT-lymphocyteclonesIII. Papainsplits purified T suppressor moleculesinto two functional domains. J. Exp. Med. 155:981-93 11. Fresno, M., McVay-Boudreau, L., Nabel, G., Cantor, H. 1981. Antigenspecific T lymphocyte clones. II. Purification and biological characterization of an antigen-specificsuppressiveprotein synthesizedby clonedT-cells. J. ¯ Exp. Med. 153:1246-59 12. Fresno, M., Nabel, G., McVay-Boudreau, L., Furthmayer,H., Cantor, H. 1981. Antigen-specific T lymphocyte clones. I. Characterizationof a T lymphocyte clone expressing antigenspecific suppressiveactivity. J. Exp. Med. 153:1260-74 13. Germain,R. N., Benacerraf,B. 1981. A single major pathwayof T lymphocyte interactions in antigen-specificimmune suppression.Scand. J. ltnraunol. 13: 1-10 14. Greene,M.I., Bach,B. A., Benacerraf, B. 1979. Mechanismsof regulation of cell mediatedimmunity.III. Thecharacterization of Azobenzenearsonatespecific suppressorT-cell derivedsuppressor factors. J. Exp. Med. 149: 1069-83 15. Greene,M. I., Fujimoto,S., Sehon,A. H. 1977. Regulationof the immune responseto tumorantigens. III. Characterization of thymicsuppressor factor(s) produced by tumorboring hosts, J. lmmunol. 119:757~i3 16. Healy, C. T., Kapp,J. A., Stein, S., Brink, L., Webb,D. R. 1983. Comparative analysis of a monoelonalantigenspecific T suppressor factor obtained from supernatant, membrane or cytosol of a T-cellhybridoma. In lr Genes."Past, PresentandFuture,ed. C. W.Pierce, S.

E. Cullen, J. A. Kapp,B. D. Schwartz, and D. C. Schretiter. Clifton, NJ: Humana.In press 17. Kapp,J. A., Araneo,B. A., Sorensen, C. M., Pierce, C. W.1982. Identification of H-2restricted suppressorT-cell -factor s~cific for L-glutamicacid~°-L tyrosine~° ~° _(GT)and L-glutamicacid -L-alanine~-L-tyrosine~(GAT).In Ir Genes:Past, Presentand Future,ed. C. W.Pierce, S. E. Cullen, J. A. Kapp,B. D. Schwartz,and D. C. Shreflter. Clifton, NJ: Humana.In press 18. Kapp,J. A., Pierce, C. W., DeLaCroix, F., Benacerraf, B. 1976. Immunosuppressive factor(s) extracted fromlymphoid cells of nonresponder mice -Pnirimedwith L-glutamic t° (GAT). acid~°-L-ala ne~°-L-tyrosine I. Activity and antigenic specificity. ,L. Immunol. 16:306-15 19. Kemp,D. J., Wilson,A., Harris, A. W., Shortman,K. 1980. The immunoglobulin fl constantregiongeneis expressed in mouse thymocytes. Nature 286: 169-70 20. Kontiainen,S., Howie,S., Maurer,P. H., Feldmann,M.1979. Suppressorcell induction in vitro. VI. Production of suppressor factors to synthetic poly peptide GATand (T,G)-A-Lfrom cells of and non-respondermice. J. responder lmmunol.122:253-59 21. Krupen, K., A_raneo,B., Brink, L., Kapp,J. A., Stein,S., et al. 1982.Purification and characterization of a monoelonal T cell suppressorfactor specific for L-glutamicacid~°-L-alanine~°-L. tyrosinet° (GAT).Proc.Natl. dcad.Sc£ USA79:1254-59 22. Lamb,J. R., Zanders,E. D., Sanderson, A. R., Ward,P. J., Feldmann, M., et al. 1981. Antigen-specifichelper factors reacts with antibodies to humanf12 microglobulin. £ ImmunoL127:231-34 23. Lonai, P., Arman,E., Bitton-Grossfield, J., Grooten,J., Hammerling, G. 1982. H-2restricted hdper hybddomas: Onelocus or twocontrol dual specificity? Curt. Top. Microbiol. 100:97-102 24. Lonai, P., Puri, J., Bitton, S., BenMeriah, Y., Givol, D., Hammeding, G. 1981.H-2restricted helper factor secreted by cloned hybridomacells, d. Exp. Med. 154:942-49 25. Lonai, P., Puri, J., Hammerling,G. 1981.H-2restricted antigen-bindingby a hybridomaclone that producesantigen-specifichelper factor. Proc. Natl. Acad. Sci. USA78:549-53 26. Minami,M., Okuda,K., Furusawa,S., Benacerraf, B., Dorf, M.E. 1981. Ana-

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ANTIGEN-SPECIFIC lysis of T-cell hybridomas. I. Characterization of H-2andIgh-restficted monoclonal suppressorfactors. J. Exp.Med. 154:1390-401 27. Nakanishi, K., Sugimura,K., Yaoita, Y., Maeda, K., KashiWamura,S., Honjo,T., KishimotoT., 1982. A T15 idiotype .positive T suppressor hybddomadoes not use the T15 VHgene segment. Proc. Natl. Acad. Sci. USA 79:6984-8 28. Okuda,K., Minami,M., Furusanra, S., Doff, M. E. 1981. Analysis of T cell hybridomas. II. Comparisonsamong three distinct types of monoclonal suppressor factors. J. Exp. Med. 154: 1838-51 29. Okuda,K., Minami,M., Sherr, D. H., Doff, M.E. 198EHapten-specificT cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybddoma suppressor factors. J. Exp. Med. 154:468-79 30. Ricciardi-Castaguoli, P., Doria, G., Adorini, L. 1981. Productionof antigen-specificsuppressiveT cell factor by radiation leukemiavirus-transformed suppressorT ceil. Proc.Natl. Acad.Sc£ USA78:3804-8 31. Rieciardi-Castagnoli,P., Robliati, F., Barbanti, E., Doda,G., Adorini, L. 1982.Establishment of functional,antigen-speclfic T cell lines by RadLVinduced transformation of murine T lymphocytes. Curt. Top. Micro. Immunol. 100:89-96

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36. Sy, M-S.,Dietz, M.H., Germain,R. N., Benacerraf,B., Greene,M.I. 1980.Antigen and receptor driven regulatory + mechanisms. IV. Idiotype bearing I-J suppressorT-cell factors inducesecond ordersuppressorcells whichexpressanti-idiotype receptors. J. Exp.Med.151: 1183-95 37. Tada, T., Okumura,K. 1980. Therole of antigen-specificT cell factors in the immuneresponse. Adv. Immunol. 28: 1-87 38. Takei,F., Levy,J. G., Killburn, D. G. 1978.Characterizationof a solublefactor that specificallysuppresses the in vitro generationof cells cytotoxicfor syngeneictumorceils in mice.J. Immunol. 120:1218-24 39. Takemori,T., Tada,T. 1975.Properties of antigen-specific suppressiveT-cell factor in the regulationof antibodyresponsein the mouse.I. In vivoactivity and immunochemicalcharacterizations. J. Exp. Med.142:1241-53 40. Taniguchi,M., Miller, J. F. A. P. 1978. Specific suppressionof the immune responseby a factor obtainedfromspleen cells of micetolerant to human ,/-globuhn. J. lmmunol.120:21-26 41. Taniguchi, M., Saito, T., Takei, I., Tokuhisa,T. 1981. Presenceof interchain disulfide bondsbetweentwo gene products that composethe secreted form of an antigen-specific suppressor factor. J. Exp. Med.153:1672-77 42. Taniguchi,M., Saito, T., Tada,T. 1979. 32. Sakano, H., Maki, R., Kurosama,Y., Antigen-specificsuppressivefactor proRoeder, W., Tonegawa,S. 1980. Two duced by a transplantable I-J bearing types of somaticrecombination are neeT-cell hybridoma.Nature 278:555-58 essary for the generation of complete 43. Taniguchi,M., Takei,I., Tada,T. 1980. immunoglobulinheavy chain genes. Functional and molecularorganization Nature 286:676-83 of an antigen-specificsuppressorfactor 33. Seidman,J. G., Leder, A., Nan, M., from a T-cell hybridoma.Nature283: Norman,B., Leder, P. 1978. Antibody 227-28 diversity. Science 202:11-17 44. Taniguehi, M., Tokuhisa, T., Tanno, 34. Sorensen, C. M., Pierce, C. W.1982. M., Yaoita, Y., Shimizu,A., Honjo,T. Antigen-specificsuppressionin genetic -respondermice to L-glutamicacid~°-L 1982.Reconstitutionof antigen-specific t° suppressor activity with translation alanine~°-L-tyrosine (GAT).Characproducts of mRNA.Nature 298: terization of conventional and hybridomaderived factors producedby 172-74 45. Taussig, M.1980. Antigen-specific TsuppressorT cells frommiceinjected as cell factors. Immunology 41:759-85 neonates with syngeneic (}AT-macro46. Tokuhisa, T., Taniguchi, M. 1982a. phages. J. Exp. Med.156:1691-710 Twodistinct allotypic determinantson 35. Sorensen,C. M., Webb,D. R., Pierce, C. W.1982.Characterizationof an antithe antigen-specificsuppressorand engen-speeifiesuppressorfactor generated hancingT-cell factors that are encoded by cell-free translation. In Ir Genes." by genes linked to the immunoglobulin Past, Present and Future, ed. C. W. heavy chain locus. Z Exp. Med. 155: Pierce, S. E. Cullen, J. A. Kapp,B. D. 126-39 Schwartz,and D. C. Shretiter. Clifton, 47. Tokuhisa, T., Taniguchi, M. 1982b. NJ: Humana.In press Constant region determinants on the

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antigen-bindingchain of the suppressor tion of a biologicallyactive, antigenT-cell factor. Nature298:174-76 specific suppressorT-cell factor. Proc. 48. Waltenbough, C., Debre,P., Theze, L, Natl./lcad. Sci. USA79:3599-603 Benacerraf, B. 1977. Immunosuppres- 52. Yamauchi,K., Chao, N., Murphy,D. sive factor(s) specific for L-glutamic B., Gershon, R. K. 1982. Molecular ~° (GT).I. Production, acid~°-L-tyrosine composition of an antigen-specific,Ly-1 characterizationandlack of H-2restricT suppressorinducer factor: Onemoletion for activity in recipient strain. J. cule binds antigenandis I4-; anotheris Immunol. 118:2073-77 I-J + does not bind antigen, andimparts an Igh-vaxiableregion-linkedrestric49. Webb,D. R., Araneo,B. A., Healy,C., Kapp,J. A., Krupen,K., et al. 1982. tion. J. Exp. Med.155:655-65 Purificationand biochemical analysis of 53. Zanders, E. D., Lamb, J. R., Konantigen-specificsuppressorfactors isotiainen, S., Lehner,T. 1980. Partial lated from T-cell hybridomas.Curt. characterization of murineand monkey Top. Micro. and Immunol. 100:53-59 helper factor to a Streptococcalantigen. Immunology41:587-96 50. Webb,D. R., Araneo,B. A., Gerassi, E., Healy,C., Kapp,J. A., et al 1983. 54. Zimbala, M., Asherson, G. L 1974. T cell suppressionof contactsensitivity in Purification and analysis of antigenspecific suppressor proteins derived the mouse.II. Therole of soluble supfrom T-cell hybridomas.SecondInt. pressor factor and its interaction with Conf. on Immunopharmacol. In press macrophagcs. Euro. Z Imrnunol. 4: 794-804 51. Wieder,K. J., Araneo,B. A., Kapp,J. A., Webb,D. R. 1982.Cell-free transla-




Ann.Rev. Immuno£ 1983.1:439-63 Copyright ©1983by AnnualReviews lnc. All rights reserved


IMMUNOREGULATORY T-CELL PATHWAYS Douglas R. Green, Patrick M. Flood, and Richard K. Gershon Department of Pathology,TheHoward HughesMedicalInstitute for Cellular Immunology, YaleUniversitySchoolof Medicine,NewHaven,Connecticut 06510 INTRODUCTION Towardsthe end of the 18th and the beginning of the 19th century an alteration occurred in Westernthinking that allowedradical changesin manyfields of study,includingthe life sciences.1Prior to this time, the study of nature was predominantlyhermeneutic,concernedwith classification and interpretation of "signs" that wouldshowone the relationship of a plant or animalto its place in the universe(for example,its role in a medical cure). This perception of the order of reality persisted throughthe 18th century, until a newview emerged,in whichunderlying causes and processes wereexamined.Thus,nature and life movedinto the abstraction; the word biology was coined in 1800by Burdachand extendedin 1802to the study of living bodies by Treviranus and again by Lamark.It wasalso at this time (1803)that the germtheoryof disease first appeared(Med.J. 484). In 1826,Fidele Bretonneau concludedthat diseases are specific, developingthroughspecific reproducingagents. AgostinoBassi (1836)isolated a parasitic fungusas the causative agent in a silkwormdisease, and John Snow(1849) postulated similar agents operative in the communication cholera. Pasteur beganhis studies in fermentationin 1854, whichled to those studies that, together with those of Koch,resulted in the acceptance of the germtheory. Thus, disease cameto be understoodas an invasion of a healthy bodyby an infectious agent. Our understandingof immunology is firmly based upon this view. AlthoughPasteur generalizedthe vaccinationprocedureof Jenner to include ~Theviewspresentedin this introductionwerelargely influencedby Foueault(1). Other referencesto these laistodcal perspectivesare foundelsewhere(2,3).

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manydiseases, it was Kitasato and von Behringwhoproposedthe humoral theory of immunity,whichwasgiven a molecular basis by Paul Ehdich. Thus, the view of immunityas recognition and response to an invasion by a foreign substancehas dominatedthe study of the immunesystem. The mechanism by whichself and non-self arc discriminatedhas only recently becomea pressing one in spite of Ehrlich’s conceptionof "horror autotoxicus," the body’s(usual) failure to react againstitself. Mostimmunologiststoday would agree with this view of immunology. Yet, science in the 20th century is undergoinganotheralteration in view. Not only can nature be unified by virtue of underlying processes, but processesthemselvescan be understoodin abstract terms (4). Information theory and cybernetics present waysof looking at complexsystems in a novel way. The immunesystem can be viewedas being in a complexhomeostasis, in whichrecognition of self (rather than non-self) is a key feature. This conceptrepresentsa paradigm shift, with resulting shifts in emphasis.Thus, the immune response can be viewedas a heuristic process in whichforeign antigenin the contextof particular self antigens perturbsequilibrium,producinga regulatory shift leading ultimately to positive responses(immunity) or negative ones (tolerance). Immune regulation emerges,then, as central area in immunology. It is the complexset of regulatoryinteractions that allows the immunesystem to "think" about its primary decision to generate a response that produces maximum destruction of a potential pathogenwith minimum damageto self tissues. Immune regulation is a youngfield of study, and as usually happensin newareas, terminologyis not yet standardized. Different groups have adopteddifferent terms for the systemsunderstudy, and attempts to reconcile all of these terms and systems(5) maybe prematureuntil they are better understood. In this review we consider only one of several outlooks on immune regulation, that of our laboratoryand perhapsa few others. It is our hope that this will aid the commendable attempts to unify immunoregulatory theory. IMMUNOREGULATORY DISSECTION

CIRCUITS

AND THEIR

Theregulation of the immune responseoccursby the interactions of different subsets of lymphocytes,especially T cells. Regulationmaybe negative (suppressive)or positive, as is discussed. EachregulatoryT-cell function stemsfrominteractions of cells that formcircuits with a consistent structure. Activationof a circuit involvesan inductionevent, usually a signal froman inducer-cellsubset. This signal is acceptedby anothersubset, which

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IMMUNOREGULATORY T-CELL PATHWAYS 441 transducesthat signal (thus "transducer"subset) into an effect, via effector cells. Functionally,then, inducercells act indirectly andthus are defined by the requirementfor transducerand/oreffector ceils for their activity to become manifest. Transducercells, in turn, are definedas those cells that acceptthe inducercell signals. This admittedlycircular definition is nonetheless useful. Effectorcells are definedby their productionof active molecules that containthe final informationsignal of the circuit. In each case, functionscan be ascribedto T cells that maycorrelate with discrete subpopulationsof cells, or whichmayrepresent multiplecapabilities of a single T-cell set. Todistinguishbetween these possibilities, it is necessaryto developmeansto separate T-cell populations and matcheach to its function. In multicellularorganisms, cells differentiateto a point wheretheir activity extendsbeyondcellular survivalto that of the overall individual. Acell achievesthis by the activationof discrete families of genesdesignedfor its specific function ("luxury"genes, becausethey are not vital to the cell’s immediatelivelihood and therefore they tend to be shut downduringcellular crisis). In the courseof this activation, a profile of molecularentities encodedby these genes mayappear on the cell surface. Thus, the surface phenotypeof a cell (such as a particular T lymphocyte) correlates with its specializedfunctionin the wholeorganism.Withthe use of specific reagents directed againstsuchsurfacelabels, uniqueprofiles of cellular antigenscan be associated with distinct functional populations. Notethat there is no requirementin such an approachfor knowledgeof whateach surface label is actually doingthere, only that it is there on onetype of cell andnot on another (of course such knowledgemaybe extremely valuable in understanding the mechanisms of a cellular activity). Examplesof surface markersthat are importantto the study of regulatory circuits in the murineimmune systemare the Lyantigens (6), particularly Ly-1 and Ly-2; the Qa antigens (7), particularly Qa-1; antigen associatedwith the I regionof of the H-2,such as I-A (8) and I-J (9); specificleetin receptors(10). Throughoutthis reviewa dominantthemeis the correlation of a unique cell surfaceprofile witha uniquefunctionto definecell subsetsin regulatory circuits. ORGANIZATION OF IMMUNOREGULATORY

CIRCUITS

In this review,regulatoryT-cell circuits havebeenorganizedinto levels of activity. This has been done acording to a simple scheme.Whenantigen perturbs homeostasis,there are cellular interactions that are necessaryand

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sufl[icient for a responseto occur(e.g. helperT-cell activationof a Bcell to producespecific antibody). In the absenceof one of these necessarycell populationsbecauseof, say, a genetic deficiency, no responsewill occur. This, then, wouldbe a failure to respondat the groundstate (level 0). Many theoriesof Ir genefunctionpostulategeneticcontrolat this level (e.g. failure in ability to presentantigen)(11). Activationby antigen perturbationraises the systemto level 1. At this level, failure to respondmaybe due to concomitantactivation of suppressor T cells (via the level 1 suppressorcircuit). This, again, agrees with many modelsof Ir gene function (in whichunresponsiveness is mediated by suppressorT cells) (12). Responsiveness can proceedin the face of active suppressorcell function via the activation of contrasuppressor T cells, whichprotect the targets of suppressorcell signals (13). Suchcontrasuppressor cell activationraises the systemto level 2. At this level, the suppressorcells mentioned above(level 1 suppressors)are relatively ineffective (14, 15). Unresponsiveness at this level is postulatedto be mediatedprincipally by an amplificationof suppression that werefer to as the level 2 suppressorcircuit. Suchan activity has beenidentified andseemsto correlate with a previouslydescribedsuppressor cell circuit. Thisis discussedin somedetail in a sectiondevotedto level 2 regulation. Theseimmunologic eigen states (anteigen states?) form the basis of broad view of immuneregulation. This quantumview suggests that energy is required to go to higher levels; informationtheory dictates that energy is similarly requiredto increaseorder. Thehigherlevels ,are those required to tune the immune responseexquisitely to protect maximallywith minimal autoimmune dysfunction.Thegreater the threat, the moremetabolicenergy will be expended in the processof this tuning. In the final sectionwediscuss someevidencethat is compatiblewith these conclusions. LEVEL 1 SUPPRESSION:

CELLULAR ASPECTS

The Level 1 Suppressor Cell Circuit (Feedback Suppression) Both the afferent and efferent componentsof the "feedback suppressor circuit" havebeendescribedextensively(16, 17). Basically, a signal produeedby an antigen-stimulatedinducercell is transducedand amplifiedby a secondpopulationof non-immune regulatory cells, resulting in the generation of effector cells of level 1 suppression(Figure1). Thenewdesignation "level 1 suppression"for the feedbacksuppressioncircuit is basedupona moreunified conceptof immunoregulation, discussed in the previous section. In the in vitro response to sheep red blood cells (SRBC),addition feedbackinducercells results in eliminationof plaque-forming cell genera-

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T-J

443

[SUPPRESSION 1 ( )=Restrictions Qa-I TSIF INDUCER (VH~ ~ T-d


I-J

I.

TRANSDUCER

+, Ly-1T inducercell with Figure1 Level1 suppression.Theinteractionof an I-J +, Qa-1 +, Ly-l,2T transducer an I-J+, Qa-1 cell resultsin an Ly-2T effectorcell (l-J-). Thelatter, togetherwitha naiveI-J+, Ly-1T cell acts to inactivatetargethelperT cells. tion (16). The activity of this cell population requires a transducer population that transfers the suppressorsignal from the inducer cell to the effector population. In cultures that lack transducer cells, the decrease in the amplitude of the response by the inducer cells is very inefficient. The functional activity of suppressor induction at this level is correlated with an Ly-1 cell (Ly-l+,2 -) with an I-J +, Qa-1+ surface profile. The Ly-1 marker is also found on other inducer T cells, such as those that activate B cells to makeantibody (18), activate macrophagesin inflammation (17), and activate cytotoxic T cells (19). In addition to the Ly-l+,2- phenotype, the above suppressor inducer cells also bear products of the I-J subregion and the Qa-1 locus, a profile that distinguishes them from the helper cell in the in vitro antibody response to SRBC(20). The population that transduces the induction signal into one of direct suppression is an Ly-l,2 cell (Ly-l+,2 +) with the I-J +, Qa-1+ phenotype (20). Although the mechanismby which these cells convert the inducing signal into suppression is not well understood, at least one modeof action maybe the differentiation of these cells into maturesuppressoreffector cells in the presence of the inducing signal (21). The interaction between the Ly-1 inducer cell and the Ly-1,2 transducer cell is antigen specific and is restricted by genes linked to the V region of the immunoglobulinheavy chain locus. That is, these populations must be matched at this locus as a minimumrequirement for the induction of

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GREEN, FLOOD & GERSHON

suppression. Howthe interaction between these cells relates to idiotypeanti-idiotype regulation (22) is discussed in somedetail below and in the next section. The effector cells of the level 1 suppressorcircuit are functionally defined by their ability to suppress Ly- 1 T cells directly. In addition to exerting their effects uponhelper cells, these cells can also act uponthe feedbackinducer cells themselves and thus cause the inactivation of both new antibody synthesis (which requires T-cell help) and the recruitment of additional suppressorcell function (23). The latter fact illustrates the appropriateness of the term feedback as applied to this circuit. The level 1 suppressor effector cells bear the Ly-2 surface marker, but unlike the inducer and transducer subsets do not carry the same I-J gene product, at least in the case of the suppressor of the primary response to SRBCin vitro (14). The interaction between the suppressor effector cell and its target is restricted by genes mappingto the major histocompatibility locus, especially the I-J subregion. The molecular basis for this restriction is understood and is best explained in a subsequent section. Recentstudies have indicated that an I-J +, Ly-1 T cell is required for the action of the suppressor effector cell on its targets (24). This requirement is also best understood in molecular terms to be discussed in a subsequent section. The Nature of V~ Restriction Methylcholanthrene A (MCA)has been used to induce a large number sarcomas, all of which possess unique transplantation antigens (TSTA).Old et al (25) have managedto generate an antiserum to a unique antigen the surface of the MCAsarcoma and subsequently mapped the antigen to the short arm of chromosome12 in the mouse(26). This region also houses the immunoglobulin heavy chain complex, a fact that prompted Flood et al (27) to investigate the activity of anti-MCAsera in cellular interactions between T cells restricted to VH-linkedgenes. Antisera raised in homozygous syngeneic recipients all possessed the ability to block the interaction between the level 1 inducer and transducer cell. This ability was only dependent on the VHlOCUSof the interacting cells: if the cells camefrom mice that shared only Igh-linked genes and no other polymorphismswith Balb/c mice (the strain in which the antiserum was produced), the antiMCA serum blocked the reaction. In addition, the activity of the serum was independent of the antigen used to induce the suppressor cells. The MCAantigens were found to be present on all methylcholanthrene tumors tested. Taken together, these observations argue strongly that shared surface antigens on methylcholanthrene-induced sarcomas are identical to or highly cross-reactive with determinants found on molecules

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IMMUNOREGULATORY T-CELL PATHWAYS 445 involvedin Vri-restricted T-cell interactions. Sinceit is veryunlikelythat MCA-induced tumorscarry the entire array of"idiotypes" expressedin the mouse,one mustpostulate that these cross-reactive determinantsrepresent constant regions that are independentof the Vr~-encodedhypervariable regions used by antibodyand (probably)T-cell derived moleculesto recognize andbind antigen.This introducesthe possibility that Vii restriction is not alwaysdependenton individual recognition of a wide variety of idiotypes, but maybe accomplishedby recognition of a muchsmaller number of moleculardeterminants. Vr~restriction andthe activity of the anti-MCA antisera is considered further in somewhatmoremolecularterms in the following section. The question of whya cellular interaction moleculefor immune function should appearon a fibrosarcoma is openfor speculation(27), but certainly a closer examinationof this genetic region shouldyield fascinating information. LEVEL 1 SUPPRESSION:

MOLECULAR ASPECTS

Suppressor Factors In this section wediscuss the components andaction of twofactors believed to mediatethe functions of the inducer and effector cells of the level 1 suppressor circuit. Splenic T cells from animals hyperimmunized with xenogeneicred blood cells served as a source of these factors. Theimmunized T cells weretreated with antisera plus complement to produceactive, secreting populations. Theproduction of Ly-1 TsiF (suppressor inducer factor) involvedtreatmentof the T cells with anti-Ly-2.Alternatively,Ly-2 TsF(suppressorfactor) required treating the T cells with anti-Ly-1.In both cases, the enriched subsets were then cultured without antigen for 48 hr, after whichtime the supernatant (containing the molecularactivity) was harvested(28, 29). In addition to this procedure,a moleculewith the chemicalandbiological properties of the Ly-2 TsF was isolated from serumof hyperimmunized animals(30). This materialwasfoundto possessa uniqueidiotypic determinant (Shld) that allowedits purification. Since the preparationof the Ly-2 TsFcontains, in addition to the ShId+ material, other regulatoryfactors (to be discussed), the serummaterial has been designated Shld+ TsF. It is likely, however,that the Ly-2 TsF (level 1 suppressor factor) and the Shld+ TsFare identical. Ly-1 TsiF The Ly-1TsiF is dependentuponI-J + Ly-1T cells for its generation and (like the suppressorinducercell) requires I-J +, Ly-l,2 transducercells for suppressionto occur. This interaction is restricted by genesmappingto the

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Vregion, andthe effects are specific for the primingantigen(28). TheLy-1 TsiFmustbe addedto cultures prior to day 2 if suppressionis to be seen. Basedon such observations, it wasconcludedthat this material mediates the activity of the suppressorinducercell. The Ly-1 TsiF was found to be composedof two separate molecules (Figure 2). Onecan be removedby absorption with the appropriate antigen (hencedubbedantigen-bindingchain). Theother binds to and can be eluted from a columncoupled with a genetically appropriate anti I-J reagent (I-J + chain). TheI-J + chain has no antigen specificity and cannotbe absorbed with antigen. Both chains of the Ly-1 TsiF appear to be 68-72 kilodaltons on SDS-PAGE, have a low pI, and are hydrophobic. Recent evidencesuggeststhat each chainis producedby a different cell (or a cell in twostages of differentiation), one I-J + (producingthe I-J + chain), the other l-J- (producingthe antigen-bindingchain) (31). In experiments with I-J + chains and antigen-binding chains from Ly-1TsiF’s of different specificities andproducedby different strains of mice, it wasfoundthat althoughthe antigen-bindingchain determinedthe antigenspecificity of the factor, it wasthe 1-J+ chainthat determinedthe Vnrestriction of the induceractivity (32). This observationhelps to confirm thosedescribedin the previoussection in that the restriction has no antigen specificity (antigen specificity mightbe expectedfor an idiotype-anti-idiotype interaction). In support of these ideas, the anti-MCAantiserum described above was found to bind the I-J + chain but not the antigenbinding chain (unpublishedobservation). Thus,apparentlyone moleculeof Ly-1TsiF (I-J + chain) seemsto possess a determinant encodedby a locus on the 17th chromosome (I-J) and one on the 12th chromosome (Vn). These canffot be separated by SDS-PAGE or reduction and "alkylation (unpublishedobservations), whichraises the possibility that an excitingly novel genetic mechanism maybe at work. Either the structural genefor I-J is actually on the 12th chromosome (under control by the 17th), or else geneproductsfromtwo separate chromosomes

~

I-d

(VH Encoded Restriction) Meth A Morker

AntigenBinding (No Restriction Identified)

Figure 2 Biologically activemolecules mediating the induction of level1 suppression (Ly-1 TsiF).Seetextfordetails.

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can be linked by translocation, RNAprocessing, or a specific covalent protein interaction [such as can be observed in the complementsystem (33)]. The possibility that the MCAstructural gene is on chromosome has been ruled out by somatic cell genetics (26). These speculations are further complicated by observations of Ly-1 TsF made in F1 animals. (Balb/c X B6)FI animals (CB6F0 make an Ly-1 TsF that suppresses either Balb/c (I-J d, Igh-Va), or C57B1/6(I-Jb,Igh-Vb). Surprisingly, however, removal of the I-J b chain from the CB6Fronlyfactor removed activity for C57B1/6cells, whereas removal of the I-J d chain resulted in loss of activity on Balb/c cells (31). This indicates that no (or fewer) I-J b Chains were constructed with Igh-Va activity, neither were detectable I-J d chains produced with the Igh-Vb restriction (recall that the factors are Vri restricted, showingno H-2 restrictions). This paradox is illustrated in Figure 3.

Ly-2 TsF The Ly-2 TsF is produced by I-J-, Ly-2 T cells and is capable of directly suppressing helper T cells in the absence of Ly-1,2 T cells. The suppression is antigen specific. These observations support the idea that this factor mediates the function of levd one suppressor effector cells. The suppressor effector molecule appears to be a single chain of 68 kilodaltons. In SDS-PAGE, it has a low pI (4.9), is hydrophobic, and possesses a blocked N-terminus. It binds antigen and, in the case of SRBCspecific factor, bears a recognizable idiotype (Shld). This material had been identified in Ly-2 TsF, ShId+ material from hyperimmuneserum, and in ~ I-j

~

T-Jb i~

o Restriction WH

FOUND

b Restriction VH

~ ~ VH b Restriction

Figure 3 cis association of parental gene products from different chromosomesin Ly- 1 TsiF from an F~ animal.

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supernatants of an Ly-2 T suppressor cell clone (30;unpublished observation). In hyperimmune serum, ShId+ material can be found up to concentrations of approximately 0.1 mg/ml, and to be precipitable in high salt (28% NH3S04). This raises the possibility that some of the suppressive activity attributed to antibody in hyperimmune serum (34)may actually be due to products of suppressor T cells. As observed for level 1 suppressor effector cells, the Ly-2 TsF requires I-J+, Ly-1 T cells to effect suppression on I-J-, Ly-1 T cells (24). The Ly-2 TsF, in the presence of antigen, induces the production of a molecule from I-J+, Ly-1 cells, which represents the second functional chain of the suppressor factor. The induction of this second chain requires H-2 homology between the I-J+ Ly- 1 T cells and the Ly-2 TsF (manuscript in preparation). This induced molecule is antigen-nonspecific (and does not bind antigen) and bears an I-J subregion-encoded determinant. To function, it must genetically match the target helper T (I-J- cell) at VH. The functional interaction of the induced I-J+ chain and the Ly-2 TsF is restricted by the I-J subregion of the H-2. A minor number of anti-MCA antisera discussed above will block the suppressive activity of the Ly-2 TsF (27). A number of anti-I-J monoclonal antibodies have also been found to block Ly-2 TsF activity (35) (Figure 4).

H - 2 restricted specified antigen required

can also be provided by Ly-l TsiF preparation

TARGET L y - 1 T HELPER

Figure 4 Biologicallyactive effector moleculesmediating level 1 suppression (Ly-2 TsF). See text for details.

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An I-J+ chain that will function with the Ly-2 TsF can also be found in Ly-1 TsiF (24). This suggests either that the I-J+ molecule for both factors is interchangeable or that there is more than one type of I-J+ molecule in the Ly-1 TsiF. The latter case is supported by three lines of evidence: (a) Certain anti-MCA serum preparations are capable of blocking only Ly-1 TsiF activity and not Ly-2 TsF, and vice versa; (b) the target cell of the two factors is different, an Ly-1,2 cell for Ly-1 TsiF and an Ly-1 cell for the Ly-2 T s F and ( c ) an I-J+ induced from Ly-1 cells by the Ly-2 TsF was found to function with the Ly-2 TsF to effect suppression but not with the antigen-binding chain of the Ly-1 TsiF to induce suppression (unpublished observation). The latter observation may be due to the quantity of I-J+ chain required by each factor rather than the existence of two different I-J+ chains. Further work is required to characterize the differences in the two I-J+ molecules. Speculation on the subsequent action of the Ly-2 TsF, following binding of both chains to the helper cell target, points toward the possibility that the active molecule is internalized. Similar internalization has been observed for certain bacterial toxins (36), ribosome inhibitory proteins from plants (37), and several peptide hormones, including insulin (36), and suggests a similarity of action between these and the Ly-2 TsF. This similarity with toxin is enticing. Many sophisticated hormones that function in man have been identified as communication molecules in prokaryotes, an observation that suggests an evolutionary continuity in the development of hormones for different compartments of the physiology of higher organisms. Toxins may serve as defense mechanisms in some prokaryotes (38) and as a form of transplantation rejection against interspecies grafts in certain plants (37). With the appearance of the vertebrate immune system, the role of toxin may have inverted, becoming directed at elements of the immune system itself. If so, then the mechanism of action of Ly-2 TsF may well be the same as that of many bacterial toxins and plant ribosomeinactivating proteins (RIPS), i.e. inhibition of protein translation. TsF “toxicity” depends, like other toxins, upon targeting the active fragment (antigen-binding chain) with a binding chain (I-J+ chain) directed at the target (via VH recognition). It may be that specificity depends solely upon this targeting. This would explain why the cells that produce the active chain (Ly-2 TsF) seem to be resistant to its toxic effects. It does not appear, however, that target cells, in the case of Ly-2 TsF, are actually killed. Cantor et a1 (39) present evidence that suppressor factor inhibits the secretion of certain proteins from helper-cell clones, without affecting others. One possibility is that translation of certain luxury gene products depends upon specialized or specially compartmentalized ribosomes, and

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that it is only these ribosomesthat are suppressed.Specializedtranslational mechanisms for small numbersof inducedproteins have beendemonstrated in eukaryoteheat shock(40). Plant RIPsare knownto be increasinglytoxic as one movesawayfromthe species of origin (the ribosomesof whichare completelyresistant), to closely related species(partially susceptible), distant species (totally susceptible) (37). This modelpredicts, among other things, that whereasmurineTsF is knownto suppress murinecells without killing them(unpublished observations), humanTsF(suppressor effector factor), properlytargeted, mightbe foundto be toxic to the appropriate mousecells. HumanTsF has been shownto suppress murine responses(41). It should be a simple matter to test it upona mousehelper cell clone if the appropriate "schlepper" (I-J +, Vnrestricted) chain can interact withit.

Genetically ConstructedCompetitivelnhibitors of Ly-2 TsF Onetest of the modelof two-chaininteraction presentedaboveinvolvesthe construction of competitiveinhibitors for the Ly-2TsF. Accordingto the model, the l-J-, antigen-binding Ly-2 TsF induces the production of an I-J + moleculewith whichit interacts. This interaction is restricted by polymorphic I-J subregiongeneproducts. Thei-J + chain, in turn, interacts with its helper cell target via a VH-controlled interaction (Figure4). If one were to add another I-J + chain, bearing only one appropriate and one inappropriate restriction element, then this should act as a competitive inhibitor of Ly-2 TsF-mediatedsuppression(Figure 5). COMPETITOR

~

COMPETITOR

T-d°[~

VH b

Restr.

~,~..~~o

TARGET

~

I-dbwill not compete

Figure 5 Genetically constructed competitive inhibitorsof Ly-2TsF.

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IMMUNOREGULATORY T-CELL PATHWAYS 451 This is exactly the case (42). Witha C57B1/6Ly-2 TsF(SRBC)acting + uponC57B1/6Ly-1 T cells, suppression wasblocked by addition of I-J chain (antigen-absorbed Ly-1 TsiF) from CB20(matchedat Vn but mismatchedat H-2) or from BC9(matchedat H-2 but mismatchedat Vn). effect of a Balb/cfactor wasseen, as this is mismatched at both loci and therefore could not complete. Themodelpresented abovewas therefore employedto construct effects that otherwisewouldbe unexplainable(i.e., the inhibition of an Ly-2TsF by a genetically altered Ly-1 TsF). Althoughthis demonstrationdoes not provethe modelof Ly-2TsFaction, it does illustrate its predictivepower.

AdaptiveDifferentiation of GeneticRestrictions In the previous sections we described two, genetically restricted immunoregulatoryinteractions betweenT-cell subsets in the generation of level 1 suppression.Ly-1TsiF, undernormalcircumstances,cannot induce suppressiveactivity in the transducercells if these cells expressIgh-V-linked polymorphisms that are different from those of the Ly-1 T cell used to producethe factor. Ly-2TsFis also genetically restricted, requiring H-2 homology betweenthe Ly-2 cells that producedthe factor and the Ly-1 T calls uponwhichthe factor will act (to both induceand interact with the cooperatingI-J + chain). Thegenotypesof the factor-producing cells do not completelygovern these restrictions. Whenhomozygous("A") T cells mature in an (A B)F1 radiation chimeraor in an (A × B)F1thymusgrafted to a homozygous Anude mouse,these cells will producefactors restricted to both A and B (43, 44). This applies not only to the H-2restriction of the Ly-2TsF, but also to the Igh-Vrestriction of the Ly-1TsiF. Additionally, (A X B)F~ cells differentiating in an A-typethymusproducefactors restricted to A only andare unableto interact with B-typecells. Anextensiveliterature exists on the role of the thymusin the generation of H-2restrictions (45), but the adaptive differentiation of Igh-V-linked recognition in the thymusis novel. TheFrinto-parent chimeraexperiments makeit unlikely that the thymusis passively"armed"by circulating molecules (such as antibody) that mightact as the selection elements. Given these striking similarities in the development of recognition of Igh-Vand MHC geneproductsas self, one shouldconsider the possibility that Igh-Vlinked geneproducts, expressedby cells in the thymus,function to select for those Ly-1 TsiFproducercells that can recognizethese Igh-V-linkedcell interactionstructuresas self. Thismightthenbe addedto the list of similarities betweengenetic products of the MHC and Igh regions, whichinclude roles in graft rejection and cellular interaction, andgenetichomology (46).

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Althoughthe aboveobservations are certainly remarkable,they are not particularly ditficult to envision. Anevenmoresurprising observationwas madein examiningthe interaction of Ly-2TsFand its target cells in the types of chimeras mentionedabove. It was found that A cells that had maturedin an A XB thymushad also gained the ability to be suppressed by both Ly-1 TsiF and Ly-2 TsF madeby B animals. Thus, not only did the factor-producing cells learn a newself recognitionin the F1 thymus,but these cells "taught" their transducercells this newself specificity. This phenomenon has been called tandemadaptive differentiation. Theimplications have been discussed in detail (44). Oneparticularly extraordinary implication derives from the analysis that has shownthat the Ly-2 TsF recognizesprivate regions of I-J on the I-J + molecule.Since anti-I-J antibodiesdirected against the B-typeI-J determinantsdo not detect any alteration in I-J, the worksuggeststhat a variable (adaptable)portion of the I-J moleculeis recognizedby the Ly-2TsFbut not by antibodies(44). Attempts are nowbeing madeto produce antibodies to this adaptable region of I-J-encodedproducts.

CONTRASUPPRESSION: CELLULAR AND MOLECULARASPECTS Contrasuppressionand the regulatory T-cell interactions that produceit havebeenreviewedin depth (47, 48). Nevertheless,this activity serves a distinguishing feature betweenlevel 1 and level 2 suppression, and we believe it to be a crucial feature of immunoregulation,whichshould be discussedherein. Contrasuppression is definedas an activity that interferes with level 1 suppressionto allow dominantimmunity.Technically, then, the competitive inhibition experimentsdescribedin the last section couldbe considered as a formof contrasuppression (one that may,perhaps,give us insights into the mechanism of physiologiccontrasuppression).In this section, however, weare concernedwith an immunoregulatory T cell circuit that possesses this activity (withsyngeneicaction). Thecircuit is illustrated in Figure Thefirst cell identified as a member of this circuit is an immune I-J +, Ly-2 T cell, the contrasuppressor inducer(14). It is likely that other regulatory cells mightbe involvedin activating this cell, but this is currently only speculation. Thecontrasuppressorinducer cell producesa factor (TesiF) that bears an I-J + subregion-encoded determinantand binds antigen (49). Whetherthe TcsiF is composedof one or two chains is unknown.The antigen-bindingcapability is morecross-reactivethan that of the Ly-2TsF, such that a SRBC-induced factor can be absorbedon horse red blood cells (leaving the SRBC-specificTsFbehind).

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ICONTR~SUPPRESSIONI ( ) : Restrictions ~-J INDU~[R TcsIF/ I-J

~ AO ~" ~


#/ (VH)

F ~cs

EFFECTOR

LEVELZ

~

//?/

......’?"

Figure6 Contrasuppression. Theinteractionof an I-J +, +, Ly-2T inducercell andan I-J Ly-l,2T transducer cell resultsin an I-J+, Ly-1effectorcell. Thelatter acts to renderthe targetsof level1 suppression (e.g. helperTcells) resistantto suppressor cell signals. Both the contrasuppressor inducer cell and factor interact with an I-J +, Ly-l,2 T contrasuppressor transducer cell to activate contrasuppression (14, 49). Recentexperimentsindicate that this interaction is restricted by genes mappingto the Ig-VH region, and some anti-MCAantisera (discussed previously) are capable of blocking this induction (50). Theseantisera, unlike those discussed that block induction of suppression, are more commonlyproduced in FI animals, which are semi-syngeneic for the fibrosarcoma. MCAfibrosarcomas grown in FI animals have been found to express muchhigher levels of surface antigens, which absorb this contrasuppressor blocking activity, than those grownin the parental strain. Although this elevation might account for the appearance of the antibody predominantly in F1 animals, it leaves the puzzle of the elevation itself. This might be a phenomenonrelated to that previously described by Barret & Derringer (51) in which tumors appeared to acquire antigens whenpassaged F~ animals. The effector cell of contrasuppressionis an I-J +, Ly-1 T cell that adheres to the Vicia villosa lectin. This cell is present in the spleens of hyperimmune animals (15) and can be generated in cultures of spleen cells from neonatal animals (13), old animals (52), adult animals treated so as to removecertain inhibitory populations (discussed later), and animals manipulated become autoimmune (53).

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The contrasuppressor effector cell acts directly upon helper T cells to render them resistant to subsequent suppressor T-cell signals (13, 15). cell-free product of the effector cells with this activity has been identified and has been found to bear an I4 subregion-encoded determinant (52). Its mechanism of action is unknown. The I-J-encoded determinant found on all of the cells and factors of the contrasuppressor circuit can be distinguished from that on the cells and factors of the level 1 suppressor circuit by the use of monoclonalanti-I-J antibodies (54). The Physiologic Role of Contrasuppression Wehave suggested that contrasuppression exists as a sort of bridge between level 1 and level 2 immunoregulation,interferring with the former and being regulated, in turn, by the latter. At this point, therefore, we propose to examinethe role of contrasuppression in the regulation of immunity, which will also serve to underline the potential importanceof level 2 suppression (in balancing contrasuppression), for which relatively little information exists. Basically, contrasuppression is viewedas functioning in at least three areas of immunoregulation.These are (a) as a sequd to general suppression (leading to responsiveness), (b) in the localization of immuneresponses discrete regions, and (c) in the establishment of suppression-resistant states. A state of general unresponsivenessassociated with suppressor-cell activity (probably level 1 suppression) accompaniesthe earliest stages of neonatal life (55), parasitic infections (56), manytumors(57), and severe (58). In each case, recovery of immunefunction and establishment of dominant immunityoften requires activity of contrasuppressor cells (reviewed in 48). A similar contrasuppressive activity might serve to produce a local resistance to general suppression, thus focusing an immuneresponse into one discrete area. The Peyer’s patches of the gut-associated lymphoid tissue (GALT)contain cells of both the suppressor and the contrasuppressor circuits (59). Contrasuppression might permit GALTresponses to proceed against ingested antigen, whereas suppression prevents systemic immunity to any antigen that reaches the circulation. Such GALTimmunity in the face of systemic tolerance has been observed (60). The skin (61-63) bone marrow(64) are two other environments in which local activation contrasuppression mayhave an important role of maintaining immunityin the presence of suppression. Specialized antigen-presenting cells, such as Langerhans cells and dendritic cells, have been implicated in this local activation (63, 65). Other local mediators that might serve to activate contrasuppression are interferon (66) and histamine (67).

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Several immuneconditions lead to what can be observed as a suppressionresistant state. Although hyperimmune animals show no sign of suppression or unresponsiveness, it is well established that serum from such animals contains a potent suppressive substance (34) that is antigen specific and T-cell derived (30). A similar situation is seen in someforms of autoimmunity, i.e. autoimmunitymayproceed in the face of systemic nonspecific suppression. Examples are the MRLmurine model of systemic lupus erythematosis (68), old age (69), and autoimmunity associated with malaria (70). In each case of the suppression-resistant state contrasuppression has been implicated in some way (48,52), and it remains to be seen whether not a cause and effect relationship can be established.

LEVEL 2 SUPPRESSION Evidence for a Second "Level" of Suppression THE REGULATION OF CONTRASUPPRESSOR CELL GENERATION Manyinvestigators have observed the generation of potent suppressor activity uponculture of normal, adult spleen cells (71, 72). Whencells from very young animals are cultured, however, dominant contrasuppression, rather than suppression, is produced (13). A mixture of adult and neonatal cells was found to produce no eontrasuppressor cells, an effect requiring a Thy-1+, I-J +, Ly-l,2 cell in the adult population (73). The presence in the adult population of a cell that prevents the generation of contrasuppression raised the possibility that specific removalof this population wouldallow contrasuppressor cells to appear in cultures of adult spleen. A novel monoelonal anti-Thy-1 (F7D5) was employed, which at low concentrations reacted with a small population of adult spleen cells, including the cells responsible for the aboveinhibitory effects on contrasuppressorcell generation. Treated adult spleen cells were found to produce 1-J +, Ly-1+ contrasuppressor cells uponculture (73). Thus, the presence of one or more cell populations (including an I-J +, Ly- 1,2 population, but perhapsother ceils as well) prevents the spontaneous generation of contrasuppression by adult cells in culture (and allows dominantsuppressor activity). This activity appears ontogenetically with the loss of ability to producecontrasuppressor cells in untreated cell cultures (increasing from around day 8 to day 15 of life). PRODUCTION

OF DOMINANT

CONTRASUPPRESSOR

CELL

EFFECTS

WhenLy-2 TsF is produced and tested on cells of most strains of mice, suppression tends to be the dominant activity observed, even when the factor contains Ly-2 TcsiF and the cultures contain contrasuppressor trans-

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ducer cells. Exceptionsto this are the B10congenicmousestrains [notable becausethese were used to characterize the Ly-2TcsiF (49)] and the strain congenics. Anygenetically matchedLy-2factor will producedominant contrasuppression on B10cells (such that suppression mayonly be seen uponremovalof the TcsiF or the contrasuppressortransducer cell), whereasfactors producedin Astrain miceseemto be contrasuppressiveon otherwisesuppressiblerecipients (for example,an F1 recipient) (74). These effects were mimickedby treatment with FTD5. Cells from C57BL/6animals were treated with F7D5,then were used to produce an Ly-2factor. This factor suppressedcultures in the absenceof Ly-2+ cells in the recipients, or in the presenceof monoclonal anti-I-J reagents known to blockeontrasuppression,but otherwiseit producedno effects in cultures + cell fromthe Ly-2 of unfractionatedcells (73). Thus,removalof an FTD5 populationresulted in the productionof an Ly-2factor resemblingthe one madeby cells from untreated A strains. Similarly, treatment of C57BL/6 spleen ceils with a low concentration of F7D5causedthese cells to be resistant to suppressionunless the contrasuppressor circuit wasquenched.Thus, FTD5caused B6strain cells to behavelike B10strain cells (unpublishedobservations). + cells exist that function to override the contrasuppresTherefore,F7D5 sor circuit, in the absenceof whichthe induction of contrasuppressionby the Ly-2 TcsiF is dominant.

Contrasuppression Versus the Amplification of Suppression Aninteresting symmetryhas appeared betweensystems that under some conditionsare deficient in certain cellular members of the suppressorcell circuit describedby Tadaet al (75) and those with a dominantcontrasuppressive activity. Thesuppressorcell circuit involvesan Ly-2inducercell that is I-J +. This acts via an I-J +, Ly-l,2 transducerpopulation,resulting in active I-J +, Ly-2suppressoreffector cells. This interaction producesan amplificationof suppression(Figure 7). Miceof the A strain backgroundfail to producethis KLH-specificsuppressor-including factor (76). As previously discussed, Astrain mice producea suppressor factor for primaryanti-SRBCresponse, but one in whichcontrasuppressiondominates.Similarly, B10strains fail to accept this Ly-2 suppressorinducer factor and also showdominanteontrasuppression in the SRBCsystem. Experimentson absorption of the KLHsuppressor inducer factor demonstratedthat B10miceare indeeddeficient in this acceptor(transducer)cell (76). Theexaminationof the cell that accepts the inducer factor in Tada’s systemrevealed an I-J +, Ly-l,2 cell, whichbore a high density of Thy-1 antigen, as detected with a heteroantiserum(75). It is possible, therefore,

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LEVEL 1 LEVEL ~

/"

EFFECTOR

l-J

TRANSDUCER

INDUCER

ISUPPRESSION I Figure7 Level2 suppression.Thissuppressor-amplification circuit involvesan I-J +, Ly-2 +, inducercell, an I-J Ly-l,2transducer (acceptor)cell, andan I-J+, Ly-2effectorcell. that this cell (at leas0 will prove to be sensitive to a low concentration of F7D5. Observations similar to the above were madein another system. Peyer’s patch T cells from mice bearing the d allele at the H-2Dlocus will suppress the primary SRBCresponse of normal spleen cells in vitro. This suppression required the interaction of an Ly-2 cell in the Peyer’s patch population and an Ly-2+ cell (presumably Ly-l,2 cell) in the splenic population (77). Peyer’s patch T cells from non-H-2Dd animals induced a dominant contrasuppression (induced by an I-J +, Ly-2 cell), but also suppressed if the contrasuppressor circuit was severed (59). The resulting suppression was effected by an I-J-, Ly-2cell (level 1 suppressor). Thus, again, in cases where suppressor amplification did not seem to operate, contrasuppression was dominant. Whenthis contrasuppression was removed, level 1 suppression was found to produce dominant unresponsiveness. The resulting symmetry, i.e. contrasuppression dominates where the "Tada" suppressor circuit is absent, resemblesthe situation discussed in the last section. This leads to the possibility that the regulation of contrasuppression and the amplification of suppression are mediated by the same cellular circuit, i.e. the level 2 suppressorcircuit (Figure 7).

Regulatory ~lspects of Level 2 Suppression Wehave preliminary evidence that the level 2 suppressor circuit starts to appear at about day 8 post-birth in the mouse. Its appearancemayrepresent

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a control for the contrasuppressionthat appearsearlier (aroundday2) and maybe required for the maturation of normalresponsiveness(suppressed in neonatesby level 1 suppressorcells). Prematureintroduction of adult spleencells (whichcontainthe cells of the level 2 circuit) at or beforeday 8 blockednormalimmunedevelopment(52, 78). Treatmentof these adult cells with a low concentration of F7D5completelyremovedthis blocking activity (52). Thus,it seemslikely that the sequential appearanceof level 1 suppression,then eontrasuppression,then level 2 suppressionmustproteed on this schedule for normalimmunefunction to mature. PolyclonalB-cell activators generally inducesuppressionin vivo. When animalswere thymectomized on day 3 of life, however,they were found to produce normal T cell-dependent immuneresponses, but becameautoimmunein response to polyelonal B cell activators (53). Thethymectomized animalswerecapableof producinglevel 1 suppressorcells in vitro, but the autoimmune animals also possessed a potent contrasuppressive activity, sensitive to treatment with anti-I-J (79). Thus,removalof the thymusat time after the maturationof cells of the levd 1 suppressorcircuit andthe eontrasuppressorcircuit, but before the appearanceof level 2 regulatory cells, leads in maturity to a state prone to autoimmunity.Thepotential importanceof the level 2 suppressor circuit cannot be overemphasized. CONCLUSION:

LEVELS

UPON LEVELS

In the introduction weproposedthat the regulatory levels wediscussed becomeactivated at the expenseof increased metabolicenergy. Thus, the greater the threat, the greater the activation of a higher levd. This might explain the well-known(but poorly understood) dose response of the immunesystem (Figure 8, top). Generally, immuneresponses have an optimal antigen dose range. Suband supra-optimaldoses of antigen producesuppressionand unresponsiveness. Recentstudies of antigen-inducedregulatoryactivities in mouse(80) and man(81; T. Lehner, personal communication)have indicated that dominantcontrasuppressionis activated only in the optimal dose range. This contrasuppressiveactivity can overcomelow-dosesuppression,but not high dose (80). Wepostulate that low-dosesuppressionis associated with level 1, whereashigh-dosesuppressionis due to activation of the level 2 circuit (Figure 8, bottom).Experiments are in progressto test this notion. If higher antigen dose can be consideredan increased threat, then this is compatiblewith our notionof higher level activation. High-affaaityantibodyis morecross-reactive than low-atfinity antibody(82, 83), thus maximal, high-affinity responsesentail an increasedrisk of self destruction. Activationof the immune responsein the absenceof level 2 regulatorycells

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459

PATHWAYS

,,"

//

\\


l ANTIGEN DOSE

LEVELI SUPPRESSION

CONTRASUPPRESSION

Ly-I -\

csE

~

ANTIGEN DOSE

CST I

LEVEL 2 SUPPRESSION

\ ~ /

SA,

"

SAT I

i\ / \

~

Figure 8 (Top) Dose response and immunoregulation.(Bottom) Dose response and immunoregulation. I, inducer; T, transducer;E, effector; FB,feedback(level 1) suppression; CS,contrasuppression,SA,(level 2) suppressionamplification.

can lead to a potent autoimmune response(53, 79). Further studies on the relationship betweenlevel 2 regulation andautoimmunity are needed,as are studies Onthe role of contrasuppressionandlevel 2 suppressionin maturation of high-affinity responseswithout autoimmunity. The view that autoimmunitymaybe producedby a failure to control high-affinity responsesto foreign antigens properly is not a newone (84). Theschemeput forwardhere seeks to ascertain those regulatory sites at whichcontrol maygo awry.In addition, our viewplaces a greater emphasis

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on homeostasis,so that althoughantigensmayperturba defective system towardautoimmunity, disruptions other than those producedby antigen mayalso produceautoimmunity. Arethere morelevels? Perhaps so, butstudies on relatively simpleantigen challengesmayfail to reveal these additional complexities. As we increase our understanding of immune responsesto tumorsandparasitic infections,however, wemightwell find that suchlevels exist. Alternatively, it maybe that the immune system’sfailure to protectsuccessfullyagainst suchintrusions maybe linked to the absenceof higherlevels. Thus,the immune systemmaynot be capable of producingan effectively strong responsein someeases, preciselybecanseit lackssut~eientheuristicpower in its regulatorycircuitry.If so, thenby gaininginsight into the levels of regulationthat do exist, wemaybe able artificially to addour mental processesto those of the immune systemin producingtherapeuticmanipulations. Theprospectsof suchimmunoengineering are not far off. ACKNOWLEDGMENT

D. R. GreenandP. M.Floodare supported by NationalInstitutes of Health Training GrantnumberAI 07019. Literature Cited 1. Foucanlt, M. 1973. The Order of Things‘ NewYork: Vintage Books 2. OxfordEnglish Dictionary. 1971. Oxford: OxfordUniv. 3. Bynum,W.F., Browne,E. J., Porter, R., eds. 1981.Dictionaryof the History of Scienc~Princeton: Princeton Univ. 4. Weiner, N. 1948. Cybernetics, Cambridge: TechnologyPress 5. Germain¢,R. N., Benacerraf, B. 1981. A single major pathway of T-lym-

10. Kimura,A. K. 1982.Thefmespecificity of lectins applied to the study of lymphocyte membranestructure and immunologicalreactivity. Riv. Itnmunol. Immunofarm.3:117 11. Shevach,E. M., Rosenthal,A. S. 1973. Function of macrol?hagesin antigen recognition by guinea pig T-lymphocytes.II. Roleof the macrophage in the regulationof genetic control of the immuneresponse, d. Exp. Med. 138: 1213 PmunhOCyte interactionsin antigen-specific une13:1 suppression. Scand. £ Im12. Debre, P., Kapp,J. A., Dorf, M. E., munol. Benacerraf,B. 1975.Geneticcontrol of immunesuppression. II. H-2 linked 6. Cantor, H., Boyse, E. A. 1975. Functional subclassesof T lymphocytes beardominant genetic control of immune ing different Lyantigens,d.. F~xp.Med. suppression by the randomcopolymer GT. d.. Exp. Med.142:1447 141:1376 7. Stanton, T. H., Boyse, E. A. 1976. A 13. Green, D. R., Eardley, D. D. Kimura, newserologicallydefinedlocus, Qa-l, in A., Murphy, D. B., Yamauchi, K., Gershon, R. K..1981. Immunoregulathe Tin region of the mouse.Iraraunotory circuits whichmodulaterespongenetics 3:525 8. Shreflter, D. C., David,C. S. 1975.The siveness to suppressor cell signals: Characterizationof an effector cell in H-2 majorhistocompatibility complex the contrasuppressor and the I immune res~ponseregion: GeIraraunol. 11:973 circuit. Eur. d. netic variation, functionand organiza14. Gershon, R. K., Eardley, D. D., tion. Adz IraraunoL20:125 Durum,S., Green, D. R., Shen, F. W., 9. Murphy,D. B. 1978. The LJ Subregion Yamauchi,K., Cantor, H., Murphy,D. of the Murine H-2 Gene Complex. Springer Sera. Imraunopat& 1:111 B. 1981. Contrasuppression: A novel

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immunoregulatoryactivity. J. Exp. 26. Pravtcheva,D. D., DeLeo,A. B., Ruddie, F. H., Old, L. J. 1981.Chromosome Med. 153:1533 assignmentof the tumorspecific anti15. Green, D. R., Gershon, R. K. 1982. Hyperimmunity and the decision to bc gen of a 3-methylcholanthrene-induced mousesarcoma. ~. Exp. Me¢£154:964 intolerant. Ann. N. Y. Acad.Sc~ 392: 27. Flood,P. M., DeLeo,A. B., Old, L. J., 318 Gershon,R. K. 1983. The relation of 16. Eardley, D. D., Hugenberger, J., McVay-Boudreau,L., Shen, F. W., cell surface antigens on methylcholanthrene-indueedflbrosareomasto Gershort R. K., Cantor, H. 1978. Imcell Igh-Vlinked cell interaction molemunoreguiatorycirenits amongT-cell cules. Proc. Nat Acad. ScL USA. In sets. I. T-helpercells induceotherT-cell sets to exertfeedback inhibition.,/. Exp. press 28. Yamauchi,K., Murphy,D. B., Cantor, Med. 147:1106 17. Cantor, H., Gershon,R. K. 1979. ImH., Gershon,R. K. 1981. Analysisof an antigenspecificH-2restricted, cell free munological circuits: Cellular composition. Fed. Proc.38:2058 product(s) madeby "I-J" Ly-2 cells 18. Davies, A. J. S. 1969. Thethymusand (Ly-2TsF)that suppressesLy-2cell dethe cellular basis of immunity.Transpleted cell activity. Eur.~. lmmunol.spleen 11:913 plant. Rev. 1:43 19. Cantor, H., Boyse, E. A. 1977. Lym- 29. Yamauchi,K., Murphy,D. B., Cantor, phoeytes as modelsfor the study of H., Gershon,R. IC 1981. Analysis of antigenspecific, Ig restricted cell free mammalian cellular differentiation, lmmunoLRev. 33:105 material made by I-J + L~I cells (Ly-1 TsiF) that inducesLy-2 cells 20. Eardley, D. D., Murphy,D. B., Kemp, expresssuppressiveactivity. Eur.~. lmJ. D., Shen, F. W., Cantor, H., Gershon, R. K. 1980. Ly-1 inducer and munoL11:905 Ly-l,2 acceptor cells in the feedback 30. Iverson, G. M., Eardley, D. D., Janesuppressioncircuit bear an I-J subreway, C. A., Gershon,R. K. 1983. The gion controlled determinant, lmmunouse of anti-idiotype immunosorbents to genetics 11:549 isolate circulatingantigenspecificT-cell J. S., Shen, F. W., Cort, S. derived molecules from hyperimmune 21. MeDougal, P., Bard, J. 1980. FeedbacksuppresSera. Proc. Nat. Acad. Sc£ USA.In sion: Phenotypesof T cell subsets inpress volvedin the Ly-1 T-cell induced im31. Chao, N. 1981. Analysis of an antigen munoregulatorycircuit. J. lmmunol. specific, Ig restricted cell free material madeby I-~, FI Ly-1 cell~ thesis. Yale 125:1157 22. Jerne, N. 1974. Towardsa networktheUniv., NewHaven, CT ory of the immunesystem. Ant~ lra32. Yamauehi,K., Chao, N., Murphy,D. munol. 125:373 B., Gershon, R. K. 1982. Molecular 23. Green,D. R., Gershon,R. K., Eardley, composition of an antigen-specific,Ly-1 D. D. 1981. Functional deletion of T suppressorinducer factor: Onemoledifferent Ly-1T cell inducersubset acculebindsantigenandis I-J-; anotheris tivities by Ly-2 suppressor T lymI-J +, does not bindantigen, andimparts phocytes. Prec. NatL Acad. Sc~ USA an Igh-variable region linked restriction. J. Exp. Med.155:655 78:3819 24. Flood, P., Yamanehi,K., Gershon,R. 33. Hostetter, M. K., Thomas,M. L., RoK. 1982.Analysisof the interactionsbesen, F. S., Tack,B. F. 1982.Bindingof tween2 moleculesthat are requiredfor C3bproceedsby a transesterifieationrethe expressionof Ly-2suppressorcell action at the thiolester site. Nature activity; 3 different types of focusing 298:72 eventsmaybe neededto deliver the sup34. Hanghton,G., Nash, D. R. 1969. Spepressive signals. J. Exp. Med.156:361 cific immunosuppressionby minute 25. DeLeo,A. B., Shiku, H, Takakarki,T., doses of passive antibody. Transplant John, M., Old, L. J. 1977.Cell surface Proc. 1:616 antigens of chemically induced sar35. Golde, W. T., Green, D. R., Waltencomasof the mouse.I. Murineleukemia bangh, C. R., Gershon, R. K. 1982. virus-related antigens andalloantigens DifferentI-J speeifieitiesdefinedifferent on cultured fibroblasts and sarcoma T-cell regulatory pathways.Fed. Proc. cells: Descriptionof a uniqueantigenon 41:190 (ABSTR.) Balb/c MethA sarcoma.,/. Exp. Meal. 36. Middlebrook,J. L., Kohn,L. D., eds. 146:720 1981. Receptor-MediatedBinding and

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Internalization of Toxins and Hormodulateresponsivenessto suppressor mone~ NewYork: Academic cell signals: Thefailure of B10miceto 37. Stirpe, F. 1982.Onthe action of riborespond to suppressor factors can be someinactivating proteins: Are plant overcomeby quenchingthe contrasupribosomes species specific7 Biocher~ pressor circuit, d. Exp. Meal153:1547 202:279 50. Flood, P. M., DeLeo,A. B., Old, L. J., 38.Janzcn, D.1977. Whyfruits rot, seeds Gershon,R. K. 1983. Antisera against mold, andmeat spoils. Am.Naturalist tumor-associated surface antigens on 111:691 meth~,lcholanthrene-inducedsarcomas 39.Clayberger, C.,Cantor, H.1982. Geninhibit the inductionof contrasuppreseration, specificity andfunction of4sion. Mod. Trends Hun Leukemia 5: hydroxy-3-nitrophenyl (acetyl) specific In press clones. Fea~Proc. 41:1213 (ABSTR.) 51. Barter, M. K., Deringer, M. K. 1950. 40.Storti, R.V.,Scott, M.P.,Rich, A., Aninducedadaptation in a transplantaParduc, M.L.1980. Translational conble tumorof mice. d. NatZCancerInst trol ofprotein synthesis inresponse to 11:51 heatshock inD.mclanogastcr cells. 52. Green, D. R. 1981. Contrasuppresaion: Cell 22:825 an immunoreguiatory T cell activi~y. 41.Kontiaincn, S.,Feldmann, M.1979. PhDthesis. Yale Univ., NewHaven, Structural characteristics ofantigenspecific suppressor factors: Definition of 53. Smith,H. R., Green,D. R., Raveche,E. "constant" region and"variable" S., Snmthers,P. A., Oershon,R. K., giondeterminant. Thymus 1:59 Steinberg,A. D. 1983.Inductionof au42.Green, D.R.,Flood, P.M.,Gcrshon, toimmunityin normal mice by thymecR.K.Genetically constructed competitomyand administration of polyclonal tive inhibitors ofsuppressor activity. B cell activators: Associationwith conManuscript inpreparation trasuppressor function. Clin. Exp. lmK.,Flood, P.,Singer, A., 43.Yamauchi, munoLIn press Gcrshon, R.K.1983. Homologies be- 54. Yamauchi,K., Taniguehi, M., Green, tween cell-interaction molecules conD., Gershon,R. K. 1982. The use of a trolled byMHCandIgh-V linked gcnes monoclonal I-J specific antibodyto disthatT ceils useforcommunication: tinguish cells in the feedbacksuppresbothmolecules undergoadaptivc sion circuit fromthosein the contrasupdifferentiation in the thymus.Fur. pr.essor circuit. Immunogenetics 16:551 ImmunoZIn press 55. Mosier, D. E., Johnson, B. M. 1975. 44. Flood, P., Yamanchi,K., Singer, A., Ontogenyof mouselymphocytefuncGershon, R. K. 1982. Homologiesbetion. II. Development of the ability to roduce antibody is modulated by T tweencell interaction moleculescontrolled by MHC and Igh-Vlinked genes phocytes. J. Exp. Med.141:216 that T cells use for communication: 56. Jayawardena, A. N. 1981. ImmunereTandem"adaptive differentiation" of sponsesin malaria.Adv. Parasitic Di~ producer and acceptor cells. £ Exp. 1:85 MecL156:1390 57. Bluestone,J. A., et al. 1979.Suppres45. Moiler, G., ed. 1979. Acquisition of sion of the immuneresponse in tumor the T cell repertoire. ImmunoZRev. bearing mice. Z NatL Cancer Ins~ Vol. 42 63:1215 46. Steinmetz,M., Frelinger, J. G., Fisher, 58. McLoughlin,G. A., Wu,A. V., et al. 1979. Correlationbetweenanergyand a D., et al. 1981. ThreeeDNA clones encodingmousetransplantation antigens: circulating immunosuppressive factor Homologyto immunoglobulingenes. following major trauma. Ann. Surgery Cell 24:125 190:297 47. Green, D. R. 1982. Contrasuppression: 59. Green,D. R., Gold,J., St. Martin,S., Its role in immunoregulation.In The Gershon,R., Gershon,R. K. 1982. Mierocnvironmental Immunoregulation: Potential Role of T.Cells in Cancer Therapy, ed. A. Fefer, A. Goldstein. The possible role of contrasuppressor cells m maintaining immune responses NewYork: Raven in gut associated lymphoid tissues 48. Green, D. R., Gershon, R. K. Con(GALT)(immunosuppression/pcyers trasuppression. Submittedfor publicapatches). Proc. NatL Aca~LScL USA tion 79:889 49. Yamauchi,K., Green, D. R., Eardley, 60. Challacombe, $. J., Tomasi,T. B. 1980. D. D., Murphy,D. B., Gershon,R. K. Systemictolerance and secretory immu1981. Immunoregulatory circuits that

~ym

Annual Reviews


IMMUNOREGULATORY T-CELL pity after oral immunization.J. Exp. Med. 152:1459 61. Iverson, G. M., Ptak, W.,Green,D. R., Gershon,R. K. Therole of contrasuppressionin the adoptivetransfer of immuuity.Submitttedfor publication 62. Ptak, W., Green, D. R., Durum,S. K., Kimura,A., Murphy,D. B., Gershon, R. K. 1981.Iznmunoregulatory circuits that modulateresponsivenessto suppressor cell signals: Contrasuppressor cells can convertan in vitro toleragenic signal into11:980 an immunogemic one. Fur. J. Immunol. 63. Ptak, W., Rozycka,D., Askenase,P. W., Gershon,R. K. 1980.Roleof antigen-presentingcells in the development and persistence of contact hypersensitivity. J. Exp. Med.151:362 64. Michaelson,J.D. 1982. Thecharacterization and functional analysis of immunoregulato.rffcelis in murin¢bone marrow.Medicalschool thesis. Yale Univ. NewHaven, 65. Britz, J. S., Askenase,P. W.,Ptak, W., Steinman,R. M., Gershon,R. K. 1982. Specialized antigen-presentingcells: Splenicdendritic cells, andperitoneal exudatecells inducedby mycobacteria activateeffector T cells that are resistant to suppression. J. Exp. Med. 155:1344 66. Knop,J., Stremmer,R., Neumann, C., DeMaeyer, E., Macher,E. 1982. Interferoninhibits the suppressorT cell responseof delayedtype hypersensitivity. Nature 296:757 67. Seigel, J. N., Schwartz, A., Askenase, P. W., Gershon,R. K. 1983. Suppression and contrasuppressioninducedrespectively by histamine-2and histamine-1 receptoragnpists. Proc.NatL,4cad.Sc£ USA.In press 68. Gershon, R. K., Horowitz, M., Kemp, J. D., Murphy,D. B., Murphy,D. 1978. TheCellular site of immunoregulatory breakdown in the lpr mouse.In Genetic Controlof Autoimmune Disease,ed. N. R. Rose,R. E. Bigazzi., N. L. Warner, pp. 223-27. NewYork: Elsevier 69. Makinodan,T., Kay, M. B. 1980. Age influence on the immunesystem. Adz ImmunoL29:287 70. Poeis, L. G., vanNiekerk,C. C. 1977. Plasmodinmberghei: Immunosuppression and hyperimmunoglobulinemia. Exp. ParasitoL42:235 71. Janeway,C. A.et al. 1975.T cell population with different functions. Nature 253:544 72. Hodes,R. J., Hathcock,K. S. 1976. In vitro generationof suppressorcell activ-

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ity: Suppression of in vitro inductionof cell mediatedcytotoxicity. J. lmmunoL 116:167 73. Green,D. R., Chue,B., Gershon,R. K. 1983. Twotypes of T cell suppression discriminated by action on the contrasuppressioncircuit. J. MoLCeILImmunoLIn press 74. Chue,B., Gershon,R. K., Green,D. R. Apparentfailure of A strain mice to rOduceT suppressor effector factor y-2 TsF) is due to dominant contrasuppressor T cell activity. Manuscript mpreparation 75. Tada, T., Okumura,K. 1979. The role of antigen-speeificT cell factors in the immuneresponse. ,~dv. ImmunoL28:1 76. Taniguchi,M., Tada, T., Tokuhisa,T. 1976.Propertiesof the antigen-specific T-cell factor in the regulationof antibodyresponse of the mouse.III. Dual genecontrol to the T-cell mediatedsuppression of the antibodyresponse. J. Exp. Med. 144:20 77. Green,D.R., St. Martin,S., 1983.Su~ppression and contrasuppressionin ~e regulation of gut assoc/atedimmune response. An~NYAcad. ScL In press 78. Raybourne,R., Arnold, L. W., Hanghton, G. 1981.Perturbationof early development of the immunesystem by normal adult lymphocytes. Y.Immunol. 127:1142 79. Smith,H. R., Green,D. R., Raveche,E. S., Smathers,P. A., Gershon,R. K., Steinberg, A. D. 1982. Studies of the induction of 129:2332 anti-DN& in normal mice. J.. ImmunoL 80. Schitf, C. 1980. Regulationof the murine immune responseto sheepred blood cells: The effects of educating lymphocytesin vitro with glycoproteinsiaolated fromthe sheep erythrocytesmembrane. Thesis. Yale Univ., NewHaven, CT. 81. Lehner,T. 1982. Therelationship between humanhelper and suppressor factors to a streptococcalprot~nantigen. J. ImmunoL129:1936 82. Little, J. R., Eisen,H.H.1969.Specificity of the immune responseto the 2,4dinitrophenyl and 2,4,6-trinitrophenyl groups. Ligand binding and fluorescencepropertiesof cross-reactingantibodies. J. Exp. Med.129:247 83. Underdown, B. J., Eisen, H. N. 1974. Cross-reactionsbetween2,4-dinitrophenyl and106:1431 5-acetourscil groups. J. Immunol. 84. Asherson,.G. L. 1968. Therole of micro-orgamsms in autoimmune responses. Prog. Allergy 12:192

~L





An~ Rev. Immuno£1983. 1:465-498 Copyright©1983by AnnualReviewsInc. All rights reserved

THE COMPLEXITY OF STRUCTURES INVOLVEDIN T-CELL ACTIVATION Joel W. Goodman Department of Microbiology

and Immunology, University

of California

Medical

School, San Francisco, California 94143

Eli E. Sercarz Departmentof Microbiology,University of California, Los Angeles, California 90024

FOREWORD Whereasthe discussion of epitope-antigen structure relating to B cells wouldnot be very different from a similar consideration a decade ago, the nature of T-cell recognition and activation by "antigen" encompasses a completely altered area of concern. Within the past 10 years, concepts of restriction to major histocompatibility-complex MHCmolecules and involvementof T cells in idiotypie interactions have revolutionized the view of the "antigen" that activates the T calls. In this article we use a special definition for the term antigen structure, whichincludes all elements shownto be recognized by the diversity of T-cell subpopulations: epitopes (from the nominalantigen), idiotopes (on secreted Ig or on other cell surfaces), and at least two types of MHC determinants, whichwill be defined later. Wedo not undertake a review of the basis and evidence for MHC restriction, or T-cell idiotypic interactions: earlier excellent reviews on the former (57, 139, 148, 186) and latter (47, 82, 83, 175) exist, together with several in the present volume.Our focus is confined to current information describing the complexity of structures involved in T-cell activation. 465 0732-0582/83/0410-0465 $02.00

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GOODMAN AND SERCARZ


INTRODUCTION Theantigen specificity of T lymphocyteshas beenintensively investigated since their recognitionas a distinct class of cells, but a dear understanding of the subject has yet to be attained. Early in the game,it wasnatural enoughto assumethat T cells recognizedantigen using conventionalimmunoglobulins of perhapsa distinctive subclass, but that suppositionhas not stood the test of time. There are a numberof reasonsfor the obduracy of the T-cell antigen specificity problem.Until recently, homogeneous Tcell lines analogousto the myeloma cell, whichwasso crucial to the solution of antibody structure, were not available. Evennow,with the advent of antigen-specificnormaland neoplasticT-cell lines, it is still difficult to obtain antigen-specific soluble products(secreted proteins) in quantities sufficient to do the essential experiments.This has forced a dependency on assays basedon cellular responseto antigen rather than on simpleligandreceptor interaction. It is paramount,therefore, at the outset to acknowl. edgethe distinctions betweenthe specificity of ligand bindingand the consummaterequirementsfor a cellular response. Considerthe revolutionary contribution that hapten binding has madeto our understandingof antibodyspecificity. Considerfurther that haptens, althoughthey bind readily to the surface receptors of B cells of the appropriatespecificity, fail to activate the cells. Withsomenotable exceptions to be dealt with later, haptens also fail to activate T cells, so it has generally beennecessary to workwith antigens of epitypic complexity, complicating the is, sue further. Anothergraphic illustration of the non-identity betweenbinding and activation is the ostensibly equivalent binding of the mitogens coneanavalin A (ConA) and phytohem-agglutinin(PHA)by B and T cells (63), althoughactivation of eachcell type occursunderdistinctly different circumstances (7). Thebottomline is that ligand-receptorinteraction at the cell surface cannotbe equatedwith cell triggering, the latter involvinga different order of complexity. Anotherterm in the obduracyequationis the subdivisionof T cells into functionally distinct subsets with differing activation requirementsand, possibly, different antigen receptors as well. A majorunresolvedquestion is the mechanism by whichmostcells, particularly those with cytotoxic or helper functionactivity, manifesta dualspecificity for both nominalantigen and a product of the MHC such that activation takes place only whenthe T cell is presentedwith nominalantigen in the context of the appropriate MHC product (18, 90, 138, 153, 157, 188). Suppressor T cells can bind naked nominalantigen under somecircumstances and have been enriched by adherenceto antigen-coated polystyrene surfaces (109, 166), but the requirementsfor activation of this class of T cells havenot beenelucidated (but see below).

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T-CELL ACTIVATION 467 Themajorexperimentalevidencethat T-cell antigen receptors are related to immunoglobulinsrests on shared idiotypy. A body of indirect data supports the sharing of Vuregions betweenantibodymoleculesand surface receptors or soluble productsfromT cells of the sameantigen specificity. Sharedidiotypes havebeenfoundon receptors for alloantigens (19, 135), carbohydrates(20), proteins (32) and haptens(42, 131, 133, 166), as as on antigen-specifichelper andsuppressorfactors (10, 52, 59, 124, 163). Thereare twocritical limitations to the interpretation of these experiments. Thefirst is that serological cross-reactivitycannotbe extrapolatedto suggest V-regionidentity. Thesecondis that heterogeneous anti-idiotypic antisera havebeenemployed and it is possible that immunoglobulin and T-cell products are reacting with different componentsof the reagents. Amore convincingcase could be madeby demonstratingidiotypic sharing using monoclonal anti-idiotypic reagents, althougheventhis wouldnot constitute definitive proof of a common gene pool. The nature of the T-cell antigen receptor, howeverobscureandfascinating, is not the central themeof this review.Ourmissionis to summarize the bodyof evidenceidentifying structural features of moleculesimplicated in T-lymphocyte activation and to describe whatis knownabout the nature of the "activating moiety,"which, in at least mostinstances, is not simplynominalantigenitself.

MHC RESTRICTION AND T-CELL RECOGNITION OF ANTIGEN Thediscoverythat T cells respondto antigen in a genetically restricted fashion changedforever our concept of T-cell antigen recognition. The cytotoxie T cell attacks only targets that express allogeneic MHC markers coded by the K, D, or other class I genes of the MHC or targets that combinenominalantigen with self class I products(18, 153, 189). Avariety of helper (or inducer) T cells (Lyt-1+ 23- cells) respondto antigen only whenit is presentedin a modified,presumably"processed"formassociated with products of the I-region of the MHC (92, 138, 157). Presentation requires the participation of Ia-bearing antigen-presentingcells (APC), whichin most, but not all, cases possessthe properties of macrophages. The I-region wasquickly recognizedas the common denominatorbetweenantigen recognition by Lyt-1+ T cells and specific immune response(Ir) genes (16). Theobservationsthat Ir genesapparentlyfunction only in responses to thymus-dependent antigens (16) and manifestremarkablespecificity that readily distinguishesbetweenantigens with similar primarystructures (71, 136, 168, 169) promptedthe hypothesisthat the Ir gene product mightbe the T-cell receptoritself (16). Thereare compellingarguments opposingthis proposal, manyof whichhave been summarizedby Benacerraf & Germain (15). Themostpervasiveof these wouldappearto be the functional expres-

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sion of manyIr genes in macrophagesand B ceils (see 15) and evidence implicating Ia antigens, whichare found in greater abundanceon macrophageand B cells than on T cells, as the productsof Ir genes (108, 155). Especially illuminating in this regard havebeenstudies with the B6.C-H2bin12strain of mutantmice, whichpresumptivelydiffer fromthe parental strain only in the Ab chain of the I-A molecule(120, 104). The immune responsesof mutantmice to some,but not all, antigens are selectively impairedrelative to the parental H-2b strain (112, 113, 122, 100), furnishing vivid evidencefor a causal association betweenIr genes, Ia antigens, and T-cell responses. Recognition by Single or Dual Receptors A paramountquestion, then, is the mannerin which Ia molecules on macrophages and B cells, or K/Dmoleculeson target cells together with exogenous (nominal)antigen, producean activating signal for T cells. Various modelshave been proposedto address this question. Theysegregate accordingto whetherthey postulate independentrecognition of antigen and MHC by twodistinct receptors on the T cell (dual receptor hypothesis)(21, 38, 84, 96, 105, 177, 187) as opposedto recognition of an interaction product between antigen and MHC by a single receptor (modified self hypothesis)(127, 136, 189). It has beendifficult to designexperiments that can unambiguouslydistinguish betweenthe models. Perhaps the closest approachthus far has been the generation of T-cell hybddomas between cells that had different antigen/MHC specificities (92). Thus,hybridsspecific for KLH plus H-2f werefused with T-cell blasts specific for ovalbumin a. plus H-2 "Double"hybrids were obtained that recognizedboth parental combinations,but noneweredetected with either of the cross-specificities. In other words, none responded to KLHplus H-2a or to ovalbuminplus H-2f. Becausethe cells expressedreceptors for each of the two antigens and the two MHC products, the findings clearly argue against orthodoxdual recognition modelsof MHC restriction. However,the authors carefully point out that dual recognition of an interaction product betweenantigen and MHC cannot be excluded inasmuch as a given antigen might form different interaction productswith MHC moleculesof differing haplotype. Adefinitive decision will probablyrequire direct biochemicalcharacterization of T-cell receptors. STRUCTURES RECOGNIZED BY T-HELPER AND T-PROLIFERATIVE CELLS Macrophage-Derived Antigen-Ia Complexes If the T-cell receptorhas resisted all assaults on its character,the molecular entity recognizedby the receptor has been of comparableobstinacy. After

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469

the basic tenet of genetic restriction in macrophage:T-call interactions had been extended from the guinea pig to the mouse, it was reported that cell-free supernatants from histocompatible, and only histocompatible, macrophagespulsed with antigen were able to replace intact cells in the generation of helper T cells (50, 51). The macrophage-replacingfactor was shownto be removable by either anti-I-A, or antibodies to the antigen, suggestingthat it contains elementsof both. Its molecularweightis surprisingly uniform (approximately 50-60,000 daltons) considering that antigens of larger and smaller size have been used in its preparation. Lonai and his collaborators have described a similar Ia-antigen complex(IAC) released by antigen-fed macrophages(114, 132). IACwas boundby Lyt-1 +2- T cells in an H-2 restricted fashion, could suicide antigen-specific helper cells in vitro, and displayed much greater immunogenicity than free antigen in histocompatible recipients (132). Competitive antigen binding by enriched T and B cells with (T,G)-A-L in both free and Ia-coupled forms demonstrated muchhigher attinity by T cells, but not by B cells, for complexed antigen (114). Furthermore, C3H.SW B cells cross-reacted with a variety of other branched polypeptides, whereas T cells boundonly (T,G)-A-L and (Phe,G)-A-L, to both of which they are Ir gene-controlled high responders. The authors concluded that IACis recognized by T cells with high affinity and mayplay a major role in defining the specificity, H-2 restriction, and Ir-gene control of T cells. Moredetailed analysis of the provocative IAC complex or the genetically related factor (GRF) (50, 51) is awaited. Right now, the T-cell recognition problem, remains plagued by the complexity systems used and the paucity of analyzable material. Antigen Presentation by B Cells It has recently been found that Ia-positive B cells mayalso present antigen in a genetically restricted fashion to sensitized T cells. Chesnut&Grey(35) used normal mouse splenic B cells coated with rabbit anti-mouse Ig to stimulate rabbit IgG-primedT cells in a proliferation assay. A variety of controls minimized the likelihood that sources other than B cells might have been responsible for the observed stimulation. This represented a special case inasmuchas anti-mouse Ig will bind to the surface Ig of all B cells. Indeed, protein antigens that lack this property (e.g. ovalbumin)were not effectively presented by normalB cells. However,a series of H-2a B-cell tumor lines were capable of presenting ovalbumin, KLH,or HGGto Iregion-restricted, antigen-specific cloned T-cell hybridomas(177). For the most part, antigen presentation correlated with the expression of Ia antigens on the B call lines, although a few positive lines failed to present antigen. Functional activity could not be correlated with the stage of differentiation of the lines. All the available tumor lines were of H-2d haplotype, but the principle of B-cell antigen presentation was extended by Kappler et al (93)

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by fusing oneof the Ia+, H-2d tumorlines to T-cell-depletedspleen cells from mousestrains of other H-2 haplotypes. Someof the resultant hybfidomaspresentedvariousantigensto a groupof antigen-specific,I-regionrestricted T-cell hybridomaswith the phenotypeof the normalspleen cell partner. In a recent fascinatingstudy, antigen-presentingcell variants with lesions in particular Ia antigenic determinantshavebeeninducedby mutation and selected from hybfidomascreated by fusion of a B-lymphoma line which can present antigen, with normal B cells. Such hybridomaAPC mutantsnowfail to present certain antigens but remain able to present others (61). The availability of homogeneous rapidly growingAPCfines (includingmonocytelines) (177) shouldgreatly expedite the exploration I-region-restrictedantigen presentationto T cells. Theabovefindings, whichprovide a plausible scenario wherebyT cells mightdirectly collaboratewith B cells that havecapturedantigen, do raise questions about requirementsfor antigen processing pursuant to T-cell triggering. It mightnot be anticipated, a priori, that macrophages and B cells wouldhandleantigens identically in terms of degradationand subsequentassociation with surface Ia molecules.However, it has beensuggested that B cells that bind an exogenousantigen mightintefiorize, degrade,and process it in the mannerof macrophages (174). Final judgmentwill depend ondirect characterizationof the structuresresponsiblefor T-cell activation. Cross-Reactivity for Protein Antigens by T Cells and Antibodies Oneapproachto understandingthe antigen specificity of T lymphocyteshas beento compare the relative cross-reactivities for protein antigensin helper or delayedhypersensitivity responseswith those exhibitedby antibodies. A general consensushas not emergedfromthese studies, althoughmostof the findingsare potentially explicableon the basis of T cells recognizingsmall linear fragmentsof processednominalantigen presentedin association with MHC gene products on the surfaces of APC,as proposed by Benacerraf (14). Anextensiveanalysisof the specificity of helperT cells for a series cross-reactingserumalbuminsled to the conclusionthat the specifieities of virgin and primedhelpers weresimilar, if not identical, to eachother and to that of humoralantibody(134). Onthe other hand, several studies of the e_ross-reaetivitiesof erythroeyteantigenssuggesteda greater discriminatory powerfor antibodies than for hdperT cells, both in vivo (43, 130) and vitro (53, 74, 75), althoughthe particular antigens involvedin the assays werenot identified. However, there is a difficulty inherentin this type of comparison:since T cells respondto a morerestricted set of epitopes, discrimination on the part of any one T cell maybe as acute. Similar findings were gleaned from a comparisonof native and extensively denatured(urea plus reduction andalkylation) formsof the globular

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T-CELL ACTIVATION 471 proteins ovalbuminand bovineserumalbumin, using proliferation as the indicator of T-cell responsiveness(34). This study wenta step further formallyestablishing that the sameT cells respondedto both native and denaturedforms, an importantadjunct in that it eliminatedthe possibility that a subset of cells recognizedthe native protein exclusivelyand, hence, mightbe specific for conformationalfeatures. It is also apparent from studies with T-cell clones specific for lysozymethat certain clones can be propagated on either lysozymeor reduced, carboxymethylatedlysozyme (F. Manca,N. Shastri, A. Miller, and E. Sercarz, unpublisheddata). However, moreinteresting are the clones that will only growon one of these forms of the molecule. Protein Antigens are Recognized as Fragments by T Cells Thecomparative speeificities of T cells and antibodiesfor native anddenatured formsof the sameprotein antigen haveseeminglypointedto a broader specificity for T ceils, in agreement with the results obtainedwith red ceils mentionedabove. In retrospect, the evidencedoes not support the notion that T-cell receptorsare actually less precisethan their B-call counterparts. Thus, early experimentsdemonstratedthat guinea pig delayed hypersensitivity reactivity, in contrast to antibody,failed to distinguish between native and extensivdydenaturedprotein antigens (56). In another investigation, chemically modifiedBSAcross-reacted extensively with the unmodifiedprotein at the helperT-cell level, but scarcelyat all at the humoral antibodylevd (146). Thelist of eases of this kind is long. Othernoteworthy examplesincludedantigen E, whichin denaturedforminduceshelper T-cell responsesto the native antigen withoutserological cross-reactivity (81); antibodies to staphylococcalnuclease(142) and lysozyme(145) are highly conformation-dependentwhereasT-cell responses are not; and helper T cells primed with the unfolded VHand VLdomainsof MOPC-315 crossreacted with the native myeloma protein (87). All the findings indicate that the portion of the nominalantigenrecognizedby helper T cells is sequential and not conformationalin character. (It is, however,possible that two nonsequentialfragmentedpeptides derived fromthe native protein might remain boundto each other and be stabilized by disulfide or other local forces.) Theseobservations, in concert with the MHC-restrieted character of T-cell responses,inspired the notion that T cells recognizefragmentsof antigen followingprocessing and association of antigen to MHC products by the APCs(14). This proposalrests on the likelihood that proteins will lose their native conformationin the acidic environmentof the lysosome, resulting in generation of the samepeptide fragments from native and denaturedantigens. Indeed, eventhe very few studies that implya conformationaldependence of antigen-specific Th/Tpcell activation can be interpreted alongthese lines. Considerthe proliferative responsesof T-cell blasts from BDF1mice primed to beef apoeytoehromee and tested with the

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homologous apoproteins, the native protein, and cyanogenbromidecleavage productsfromeach protein (28). Cells maintainedin long term culture with apocytochrome c respondedmorevigorouslyin the proliferative assay to the apoantigcnand its cleavage products than to the native protein, suggestingat first glancethe possibility of conformationaldependence for T-cell recognition. However, a comparisonof responsesusing three different sets of APCs(the syngeneicand two parental types) led to the conclusion that the observeddifferenceswereassociatedwith different capacities of the APCto processand present the two formsof antigen. As the authors pointedout, absenceof T-cell cross-reactivity betweennative and denatured formsof a protein mightbe expectedfor proteins that are relatively stable in the acidic environmentof lysosomesand unlikely to unfold prior to proteolytic degradation. In such instances, the proteolytic fragmentsobtained will dependon the three-dimensionalstructure of the substrate. Thus,the differencesseen are likely to reside in the APCrather than in the antigen-recognizing properties of the T cell. Referencewasmadeearlier to the exquisite T-cell specificity manifested towardantigens underIr gene control. This has been particularly evident with small immunogenic peptides of defined structure and sets of closely related analoguesof the immunogen. Thus, guinea pig T lymphocytessensitized to the octapeptideangiotensinII could readily distinguish single aminoacid substitutions, whichhad no effect on antibody binding (169), whenthe assayusedwasthe ability to stimulateT-cell proliferation. Similar results were obtained in the samelaboratory with humanfibrinopeptide B anda series of structural analogues(168). Thesefindingsare consistentwith the idea that the epitopes recognizedby proliferating T cells are dictated primarily by aminoacid sequencerather than by conformation,of which theremightbe expectedto be little, in anycase, in peptidesof this size range. Theseinvestigators reasonedthat if such be the case, inverting the sequence of aminoacid residues in a small peptide antigenshouldmakelittle difference to the T cell, becausethe sameaminoacids are presentedin the same spatial order (166). Twoassumptionsare implicit in this reasoning. One that there is no polarity or directionality in the presentationof an immunogenic epitope to the T cell. Theother is that peptides of the dimensionof eight aminoacids are not degradedduringthe courseof processingby APC. Shouldadditional degradationoccur, then it can be readily foreseen that altering the sequenceof a peptide mightalter the course of degradation, leading to the formationof different neoantigens.Theprediction wastested with a fibdnopeptidefragmentof eight aminoacids and its inverted sequencepeptide in a guineapig T-cell proliferative assay (166). Strain guinea pig T cells sensitized to the native peptide werecompletelynon-

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cross-reactiveto the invertedpeptide; in fact, the invertedpeptidewasnot immunogenic in strain 2 animals. Theauthors interpreted this to meanthat the ordering of the aminoacids within the antigen is critical, perhaps because the antigen becomesassociated with MHC products in a nonrandomfashion. Thepossibility remainsthat degradation of even small peptide antigens is an intrinsic part of processingand will certainly be affected by aminoacid sequence. Studies with T-Cell Clones T-cell clones reactive with soluble protein andsynthetic polypeptideantigens haverecently beenpropagatedin long-termculture (73, 78, 97, 159). Someof these cloned lines havebeenshownto function as antigen-specific helpers and to obey the rules of MHC restriction. For example,clones reactive to the multichainsynthetic polypeptide(T,G)-A-Lprovidedspecific help to TNP-pdmed B cells whenchallengedin vitro with TNP-(T,G)A-Land respondedin proliferation assays in an I-region-restricted fashion (73). Anaspect of the workwith cloned, self MHC-restricted,antigenspecific T cells that has an importantbearingon the questionof the nature of T-cell specificity has been their cross-reactivity with allogeneic MHC determinants. Oneinterpretation of the high frequencyof alloreactive T cells, originally notedby Simonsen (156), has beenan overlapping of specificities of the alloreactive andnominalantigen-reactiveT-cell populations. This issue has beenaddressedin the past by searchingfor cross-stimulation of alloreactive cells by antigen and vice versa with heterogeneousT-cell populations(17, 30, 69, 107). Althoughcross-stimulationwasobserved these studies, interpretation wasdifficult becausethe possibility of nonspecific recruitmentcould not be excluded.This ambiguitycan be circumvented,or at least minimized, by usingextensivelyreclonedantigen-reactive T cells. Thus,a DNP-ovalbumin-specific clone froma B 10.Amouseproliferated in responseto addedantigenin conjunctionwith syngeneicirradiated antigen-presenting cells as well as in responseto allogeneicspleencells from B10.S mice in the absence of DNP-ovalbumin or B10.Aaccessory cells (158). Thesefindings do providecompellingevidencefor dually specific cells responsiveto both nominalantigen andalloantigens, but they do not yet permit distinction between, hypothesesconcerningthe receptors involvedin antigen recognition. Thus,the findings are compatiblewith completely independentsets of receptors for alloantigens and for nominal antigens randomlyexpressedon all T cells (181), with alloantigen recognition throughhigh affinity cross-reactivity with the anti-self MHC receptor (84) or the receptor for nominalantigen (38, 105) in dual receptor models, or byhighaffinity cross-reactivitywith a single receptorfor alteredself (17, 30). Additionalstudies with antigen-specific T-cell clones mayeventually

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distinguish betweenthe various modelsfor antigen recognitionby T cells. ClonedT cells will also be useful for settling other unresolvedissues concerning antigen recognition and MHC restriction. For example, the specificity of individualT cells for definedepitopesof antigenmolecules has beenobscuredby using heterogeneouspopulations of cells and relatively complexantigens. Theresponsivenessof T cells to the compound L-tyrosine-p azobenzenearsenate(ABA-T) furnishes a systemfor investigating the nature of T-cell antigen recognitionin whichthe antigen is reducedto its simplest possible form. Different functional types of T cells have been inducedthat are specific for ABA includinghelpers (5, 6, 62), suppressors (109, 161), killers (154), andmediatorsof delayedhypersensitivityreactions (11, 169). Veryrecently, long-termcultures of T cells specifically reactive to ABA-T were established from the lymphnode cells of A/J mice, which had been immunizedwith ABA-T,and the cells were cloned by limiting dilution (70). The MHC restriction of the antigen-inducedproliferation response of the clones wasassayed with accessorycells fromappropriate recombinantstrains of miceand monoclonalanti-Ia antibodies as blocking reagents. Fourteenof 15 clones tested provedto be I-A restricted in their response,whereasthe other clonewasI-E restricted. Thesefindingsindicate that the sameepitope (ABA-T) can be presented to T cells in the context of more than one MHC product. Although the bulk population of lymph nodecells respondedwhenaccessorycells compatibleat either the I-A or I-E loci werepresent, individualcloneswererestricted in their recognition to a single locus. Thefine antigenspecificity of the cloneswasanalyzedwith structural analoguesof ABA-T to induce proliferation in conjunctionwith syngeneieaccessorycells. Theclonescould be dividedinto three types with respect to responsivenessto ABA-histidine(ABA-H).Onetype responded about equally strongly to ABA-H and ABA-T.A second group responded to both, but significantly better to ABA-T, whereasthe third set did not respondat all to ABA-H. In all cases, the ABA-H-responsive clones discriminatedexquisitely betweenthe 2-azo and 4-azo isomersof histidine, respondingonly to the 4-azo derivative. Thus,at the level of individual clones, T cells display a remarkablespecificity for definedepitopes of the nominalantigen in situations wheredifferential processingof antigens is unlikely to be a factor, as it maybe whencomparingcomplexantigens differing in primarystructure.

The T Proliferative Responseis not Identical to the T Helper Cell Response It is often assumedthat cell populationsthat proliferate in responseto a particular epitope in the context of the appropriate MHC-APC are always helper cells. This is surely not the case. For example,in recent workin the

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T-CELL ACTIVATION 475 beta-galactosidase (GZ)system, T proliferation could be inducedin vitro by GZin most of the populations primedby individual cyanogen-bromide peptides derived from GZ(from 23-95 aminoacid residues in length). However, only two of a dozenpeptides tested could inducea T-helpereffect for the anti-hapten (FITC)response to GZ-FITC (101). The ability to inducedto proliferate by antigen maybe sharedby manyLyt-l+2-cells and conceivablyby Lyt-1-2+ cells [although in heterogeneouscell populations the suppressorT-cell proliferation mightbe missedin that the expressed activity of the Ts wouldprecludethe detectionof its target population(s).] Surely at the clonal level, manycellular membersof the enormousTs "regulatoryarmy,"as well as the Ts itself, mightproliferate in conjunction with antigen and/or IL-2. The secretion of IL-2 by a T inducer cell in responseto its activation stimulusmayrepresent a majorpathwayin Ts-cell and other T-effeetor-cell recruitment. It has beenshownthat manyof the cells recruited by the proliferating cell are B cells (172). It is possiblethat uponrecruitmentandproliferation, a subsequentdisplayof antigenby these B cells will back-recruita fewmore antigen-specific T calls. Surely, underthe usual Corradin&Chiller (40) conditions with whole, primedlymphnodes, a myriadof recruiting and proliferative events mustbe occurring. Determinant Selection on Multideterminant Antigens Theparticular choice of determinantson a potentially multideterminant antigen moleculecan be attributed to selectivity exercised by a component of the MHC.This concept of determinant selection was elaborated by Rosenthaland his colleaguesusing the modelof insulin recognition by the guinea pig and the mouse(136). Strain 2 and strain 13 guinea pig T cells eachresponded to a distinct regionof beefinsulin (137), andthis discrimination couldalso be observedin the mouse(140). In the case of insulin, and others subsequentlystudied, in whichmammals are challenged to respond to related mammalian proteins, it couldbe arguedthat, in fact, there is very little "selection": that is, whatevermechanisms purge the repertoire of responseagainst self antigens leave only a few regions that are different betweenthe two proteins, against whicha potential response might be directed. This is onereason whyresults with the bird lysozymesare noteworthy,in that as a groupthey differ by about 50 residues out of 129 from the mammalian congeners. In fact, almost all of the surface aminoacid residuesare different. Nevertheless, it is stalkingthat withall this potential "foreignness"only one to three determinantareas are selected by mice of differing haplotypesin the triggering of T-call hdper/proliferativesubpopulations (115, 116). In a sense, this reminds us of the concept of immunodominance of certain epitopes in the induction of antibody (88).

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At the T-cell level, it has beenpostulated(14) that whatmakesa particular determinantarea dominant or, rather, respondedto at all is the existence of a stretch of aminoacids on the nominalantigen that has an at~nity for a structure on the MHC restriction element. A useful nomenclaturefor consideringeachof the entities involvedin the recognitionhas recentlybeen proposed(144). Thus, the nominalantigenic entity that contacts the MHC has twodistinct moieties, at least conceptually:an epitope in contact with subsites on a T-cell receptor, and an agretope(antigen recognition), which acts as a site of attachmentto the MHC desetope(determinantselection). (Wemight suggest from the limited data available that roughly one agretope exists for every 50 aminoacid residues on a protein for Th/Tp cells.) Finally, the additional elementon ClassI or ClassII MHC molecules that is recognizedin additionto the epitope is designateda histotope. In workon pigeon cytochrome(64-66) by Schwartzand his colleagues, a potential agretope has been defined as giving the quality of immunogenicityto the C terminal peptide 81-104.Usingsynthetic peptides, it was shownthat residues 100 and 103 were important for antigenic strength, whichcould be related to interactions of the antigen and MHC structures on the APC.Grantingthe closeness of the agretope, residue 99 could be consideredpart of the T-cell contactingepitope related to T-cell memory,since a set of amidinatedC-terminalpeptides derivatized at position 99 were completelynon-cross-reactive with the native peptides but nevertheless showedthe same immunogenicity/non-immunogenicity discdmination amongstrains, whichis presumablydependenton the nature of the agretope. In the lysozymemodel,a completelyanalogousresult has been reported, in whichonly one dominant, circumscribedregion of the moleculenear aa 113-114is utilized in the B10.D2 haplotypefor presentation of the epitope at 113-114to T cells respondingto parallel sets of non-crossreactive bird lysozymes(96). A case in whichan agretope mayhave been synthetically manufactured appearedin the recent literature. As mentionedearlier, an octafibrinopeptide is immunogenic for strain 2 guineapig T cells (166). However,strain 13 animals are completelyunresponsiveto the peptide. Whena glycine residue wasaddedto the carboxylend of the octapeptide, the newnonapeptide inducedstrain 13 T-cell responses. Althoughthe mechanismof the immunogenic conversionis uncertain, a plausible interpretation is that the glycine residue formedpart of an agretopewhichcould productively associate with a strain 13 desetope. AnotherCandidatefor a critical componentof an agretopeis the side chain of tyrosine in ABA-T. Increasingthe hydrophobicityof the side chain decreasedimmunogenicity without palpably influencing specificity. Thus,

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T-CELL ACTIVATION 477 the tyrosyl end of the moleculecan be visualized as the agretopeand the ABA endthe epitope, althoughthe precise structures of these entities have not beendefined. If agretope scarcity were responsible for the very limited responseto certain antigens, the followingconsequencesmightbe expected: (a) when an agretopeis defined,a diversity ofTcells shouldbe availablefor combination with epitopes associated with that agretope; (b) the distribution agretopesrather than the repertoire diversity of ambientcells shouldbe the prime factor in determiningwhetheror not a particular antigen will be respondedto by the haplotype;and(c) the paucity of appropriateagretopes should lead to somenull peptides on a typical immunogen whichare not usedfor addressingany T-cell subpopulation.Someof these corollaries have beenobservedin the lysozymesystem(117). Thus, only a single lysozyme agretopeis presumablyavailable for H-2b strains near aa residue 91, but nevertheless,a heterogeneous array of T proliferative clonesof differing fine specificity for epitope can be detected, all of whichseeminglyutilize the unique agretope. TheC-terminalpeptide of lysozyme,aa106-129,makesno impactat all on H-2b mice, neither inducinghelper nor suppressorT cells; likewise, no other tryptic peptides than T11(aa74-96)are immunogenic reactive in H-2b strains. Agretope Limitations and Hierarchies Hints are available that the scarcity of agretopesmaysometimes be apparent rather than real. Accordingly,there maybe two or three immunogenic regions on a protein molecule,but a hierarchy of choice can obscurethe reactivity to all but a dominantagretope-desetopepair. Anexamplein the lysozymesystem (96) involves the subdominantC-terminal peptide in the B10.Astrain, reactivity to whichcannot be demonstratedafter immunization with native lysozymeand challengewith L3. However,initial in vivo immunizationwith L3 reveals strong responsivenessin this haplotype to either L3 or HELsubsequentlyused for in vitro challenge. Apparently,a competitivesituation exists in whichan N-terminalagretopenear residues 13-15 takes precedenceover two other agretopesthat are moreC-terminal, or alternatively,T cells possessgreater at~nities, in generalfor the dominant epitopes. SUPPRESSOR OF ANTIGEN

T CELLS

AND THE RECOGNITION

Does suppressor T-cell activation depend on the same concepts of desetope/agretope interaction we have just discussed for Lytl+ Th/Tp cells? DoS-agretopesexist that are distinct fromH-agretopes?Theconsid-

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erations relevant to this section hinge on the relationship of the MHC to suppressor cell induction and activity, an area that still has manyunknowns.


T Suppressor Cell-Inducing Determinants SuppressorT cells (Ts) play an important role in the expression of the Tp/Threpertoire, in somecases obscuringa latent capacity to producea responseto the native molecule.In Ir gene-controlledresponses,it has been reported in a variety of systemsthat nonrespondermicehavethe capacity to respondto the antigen in the absenceof Ts, or to presentthe antigento syngeneicor to FI(Rx NR)T-helperceil populations(9, 90, 98). Disclosure of this latent potential mightrequire the dissociation of the suppressorT cells fromthe system.In the ease of lysozyme,this wasachievedby amputation of the suppressor-inducingepitope at the N-terminusof the protein (185). In fact, removalof as little as the terminaltripeptide lys-val-phe aminopeptidaseconverts the non-immunogenic lysozymeinto a derivative that no longer inducessuppression,but rather a helper effect, and T-cell proliferation (A. Miller, L. Wicker,M. E. Katz, and E. Sercarz, unpublished results). Bythis simple transformation, actually two newdeterminants were revealed from what would have simply been regarded as a non-immunogenic molecule for H-2b mice: a suppressor determinant and the previouslysuppressed, latent responsivenessto the helper epitope on tryptic peptide 11. Of course, the samegeneral problemof paired suppressor andhelper epitopes mayexist on any "null peptide" such as the C-terminal peptide of lysozyme,and additional experimentscan be performedto examine this possibility. Latentresponsivenesshas also beendemonstrated in the eytochromesystem(41), and to one of the cyanogen-bromide peptides from beta-galaetosidase (U. Krzych,A. Fowler, E. Sercarz, unpublisheddata). Thefact that a single suppressor-inducingdeterminantcould affect the entire response to a molecule had been recognized with lysozyme(HEL) earlier (1,152). Morerecently, it has beenpossible to induce Ts with the CNBr-derived,reduced and earboxymethylatedN-terminal dodeeapeptide from HEL(N. Shastri, A. Oki, M. Kaplan, D. Kawahara,A. Miller, and E. Sercarz, unpublishedresults). Earlier evidenceof suppressordeterminants with beta-galactosidase (150, 173) and poly-glu-ala-tyr (147) carried the implication that the suppressor- and helper-inducingdeterminants mightbe different.

Suppressor and Helper Epitopes are Non-Overlapping In all protein systemsexamined,epitopes addressed to the Lyt-l-2+ Tsp population and those activating the Lyt-l+2-Th/Tpsubpopulation are different! This wastrue in the case of the small globular lysozymemolecule

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T-CELL ACTIVATION 479 (HEL=129aa)and likewise for the large tetrameric B-galactosidase (GZ=1021an/monomer). Whenthe second cyanogen bromide peptide from the N terminus (aa3-92) of GZwasassessed for induction of Th Ts, both subpopulations were activated. Further analysis showedthat a tryptic nonapeptide,aminoacids 44 to 52, inducedTh but not Ts, and the reverse wastrue for an 1 l-amino-acidpeptide 27-37. Thusthe principal of non-overlapremainedintact (102). Similarly, in the response to myelin basic protein in the guineapig (160)it appearsthat the suppressor-inducing peptide is different fromthe encephalitogenic peptide in this species. However, the possibility that the determinantsare very close couldbe concluded fromexperimentsin whichspecific residue alteration causeda reversal of functionaleffects inducedby the peptides(68, 94). In a similar vein, Thand Ts that recognize the sameor very similar epitopes can be induced by ABA-T(109). Althoughthe picture is not complete,manyresults can be accommodated by a general modelcited belowthat suggests that functionally and chemically uniqueantigen-presentingentities (desetopes) are employedin the activation of different T-cell subpopulations.Consequently,it maybe the desetope-agretope interaction that controls whichT cells will be triggered. Interactions

Within the Suppressive Cell Circuitry

Theintereractions amongthe varied members of the regulatory T-suppressor family have illustrated somenewprinciples in T-call activation and interaction. In consideringthe productionof antibody, it is evident that membersof a single AgThclone can collaborate with the appropriately specific B cell, leadingto B-cell proliferation, differentiation andantibody formation.This is the tip of a cellular iceberg, whichhides a multitudeof competingregulatory cells, each of whichmust knowwith whichpartner cell it mustinteract. Theserules of intercellular communication, or as Tada has termedit, "immunosemiotie interaction" (162), musteventuallylead cell collaboration that is purposefulrather than haphazardand chaotic. Evidenceindicates that the T-Tinteractions that accomplishthe regulation of suppressionare restricted by three types of elements--antigen,MHC and immunoglobulin. Moreexplicitly, these are the suppressorepitopes on the antigen, MHC-presentation elements, and regulatory idiotopes, but in addition there maybe MHC-interaction elementsand T-cell receptor constant-region determinants that must be recognized. Each memberof the systemis inducedby a specific cell or a factor derivedfromit: the factors have componentsthat recognize epitopes or idiotopes, in addition to a constant region, coded for by genes on the 12th chromosome in the mouse (170, 171); on two-chainfactors a separate peptide bears the MHC element involved,whichin the suppressorcircuit is a type of I-J.

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Assuggestedin the last paragraph,one of the critical questionsthat is still undergoingexploration is whether suppressor T cells are MHC restricted at either oneof twolevels: (a) at the point of presentationof antigen or (b) in the interactionbetween regulatoryandtarget cells in the collaborative milieu.


Effector-Cell

Induction Pathways

T cells whichwill eventuallydevelopinto effectors (Th, Ts, and To), seem to be activated by a particular inducer cell (augmenting cells, suppressor inducersandCTLhelpers, respectively). It can be predictedthat the rules of each partnership are immunosemiotieally defined to prevent anomalous induction. TheTs pathwayshavebeenmostextensively studied and will be discussedin a general, summary way,as other reviewshavedescribedtheir cellular constituentsin detail (31, 57). -, I-J+suppressorinducercells (Tsi) weredemonstrated Lyt-1+2 (45) to necessary in interacting with Ts precursor cells, usually Lyt-l+23+, to promotethemto functional effector status (9, 119). Morerecently, signiticant progresshas beenmadein assigningantigenicand idiotypic specificities to these cells or to factors derivedfromthem(46, 164, 184). In general, the restriction in interactions betweenTsi andTs cells appearsto be owing to Igh rather than to MHC structures, whereasTs effector cell interaction with Th targets is MHC-restricted. It has beensuggestedthat in all suppressorsystems, a cascadeof three sequential Ts calls (Ts 1, Ts2, andTs3) are induced,originally by the nominal antigen (58). In fact, in the systemswhichreveal eachof the three cell types, the next cell in the sequenceis usually inducedby a factor derived from the previous cell. However,antigen can also induce Ts3 directly, whichthen requires a Ts2-derivedsignal to achieveeffector status (123). appearsthat the systemwill employthe effector-cell specificity it needsto control the regulee: if it mustdown-regulate an idiotype-beadng cell, it will somehow raise idiotype-recognizingT-suppressorcells. Onsuch occasions, if a particular suppressorcell is in short supply,complex circuits involving amplifyingcells, whoserelationship to the effector cells is based upon idiotype, MHC, or antigenic recognition (31, 162) maybe resorted to. The Ease of Triggering Ts in Non-Responder Strains Certain well-characterizedcases of genetic nonresponsiveness,those concerning the terpolymer glu60-ala30-tyrl0 (GAT)and lysozyme,owetheir existenceto readily activated T-cell suppression(60, 151). In the lysozyme case, a Ts cell is inducedin H-2b miceby chickenlysozyme(HEL)but not

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T-CELL ACTIVATION 481 by anyvariant lysozymethat substitutes tyrosine for the phenylalanineat residue 3, suchas REL(4). Likewise,H-2~’ strains respondto RELbut not to HEL,at the antibody formation level. It can be asked whetheror not sometype of suppressiveelementexists in regulating the responseto REL in the H-2b mouse?If so, are these Ts simplyless readily activated by REL in the non-responder strain, or activated by a parallel but distinct mechanism? Althoughsuppressive circuitry mayoccur universally, the routes taken to achievethe suppressionmaybe quite different in the nonresponder andresponderstrains. Possibly,Ts inductionto certain epitopesis rapid and effective in the non-responderbecause of an MHC-restrictedactivation process that can proceedat a low level in the absenceof a T-suppressor inducer cell. In responderstrains, stimulation of the idiotype-bearingTs mightoccur at a later point, throughthe parallel mechanism involving a Tsi-delivered induction signal. Sucha circuit, for example,mightdepend on the appearanceof the predominantidiotype during B cell maturation, whichwouldarouse idiotype-reeognizing Tsi to stimulate suppression (103). Differences in Th and Ts Recognition of Antigen At the outset, antigen recognitionby Ts cells appearsto be quite different from that of Th or Tp cells. Evidencethat Ts can bind to native antigen has beenused to argue that nominalantigen recognition is independentof the MHC. Isolation of Ts on plates coated with antigen (109, 125, 165) indicates that Ts can recognizenative antigen. However, there are several publishedinstances (3, 39, 72, 118) in whichThhavelikewise beendemonstrated to bind to antigen or antigenic peptides. Endres and Grey (49) found that Ts induced by native or extensively denaturedovalbumindid not functionally cross-react with the alternative formof antigen, in contrastto helper T cells (34). Furthermore, the suppressor cells could be depletedon plastic dishes coatedwith the homologous but not with the heterologousantigen. Thesefindings suggesta closer kinship of Ts cells to B calls than to Th cells in terms of antigen recognition. However,in the lysozymesystem, the native enzymeor its reduced, carboxy-methylatedderivative or its fragments (including the N-terminal dodecapeptide)can each induceTs, althoughthe derivatives surely are not in the native conformation,nor are they cross-reactiveat the B-cell level. Certain authors (34) postulate that Ts cells recognize conformational determinantsandthat the differences in antigen recognitionbetweenTs and Th are related to the requirementsfor antigen processingandpresentation by accessorycells to Th; this argumentneglectsthe presentationof antigen to Ts. Theapparent lack of dependence on adherentcells in the induction of suppression(54, 80, 129)seemsto supportthe idea that antigen process-

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ing is not required for the activation of suppressorT cells. However, the existenceof special types of APCfor different T-cell subpopulations,with different adherenceproperties, has been documented (29) and wouldseem a relevant consideration. Theevidenceis still concordantwith the notion that Ts recognizeexternal epitopes on antigens, owingto the superficial placementof S-agretopes (also see below).This wouldpermitbindingto antigen alone, if the affinity of interaction were high enough,but possibly Ts activation wouldrequire an additional signal derivedvia associative recognition. MHCRestrictedness of Ts Cells Althoughthe evidence wehave sketched suggests that effector Ts maybe MHC restricted in the interactions with their inducers andtarget cells, we will nowexamineexperimentsthat convergeto suggest MHC restriction at the point of presentation of antigen and the induction of Ts precursors. In the lysozymesystem, the ability to induce Ts in the nonresponder H-2b mouseis limited to an epitope at the N-terminusof several gallinaceous lysozymes,those of hen, bobwhitequail, guinea hen and any other lysozymehavingphenylalanineat aminoacid residue 3. Interestingly, humanlysozyme(HUL)whichis almost completelynon-cross-reactive at the B cell level, apparently can induce the sameTs, bearing the predominant idiotype, IdXL,whichthen is equally active on the anti-HELand anti-HUL in vitro responses.This has a rationale in that not only pbe-3but also eight of the nine N-terminal residues are identical in HELand HUL.On the other hand, although the H-2q mousepossesses the same cross-reactive IdXL-bearingTs, it nevertheless can respond to HELwhile still being nonresponsiveto HUL.This failure to induce Ts by HELcan be attributed to an inability of HELto interaet with the appropriate MHC dementin antigen presentationto Ml-IC-restrictedTs (2). Othermoredirect evidence for MHC restriction in this systemwasobtainedin positive sdeetion of the IdXL+ Ts on HEL-pulsed, presenting-cell monolayers. Only syngeneic monolayerswere capable of antigen presentation to Ts, and this positive sdection could be inhibited by anti-IdXLserum(8). In recent workby Jan Klein andhis colleagues(12, 13), evidencehas been presentedfor restrietion of Ts induction by lactate dehydrogenase B in the eontext of an I-Ek restriction dement. Using nonresponderB10.A(2R) cells, antigen-presentation by B10.A(4R) lacking the I-Ek dement,gives rise to proliferation, but not suppression.Furthermore,in vivo treatment with monodonalanti-I-E antibody converts the B10.A(2R)into an LDH responder. Takentogether, the evidence points to MHC restriction at the time of initial antigen presentation, and suggeststhat further study of suchearly events mightreveal additional examples.

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DESETOPES AND HISTOTOPES IN CYTOTOXIC T-CELL RECOGNITION Theproblemsraised by recognition of class I and class II MHC proteins are really quite parallel. Severalclass I proteins (includingK, D, L, Mand R) havebeenimplicatedin associative recognition with nominalantigens. Just as in the case of Ia-restriction, it is apparentthat certain haplotypes utilize particular molecule(s)of the class I set for restricting their CTL responses. Oneinteresting exampleis providedby the recent workof Ciavarra andForman (36), studyingthe restriction elementsused for reactivity to vesicular stomatitis virus (VSV)in the H-2d haplotype. VSVwasonly d molecule[This was shownby coldrecognizedin the context of the H-2L dtarget inhibition experimentsas well as inhibition studies with H-2L directed monodonalantibodies. However,H-2L-restricted responses are not foundagainst trinitrophenol (111)or minorhistocompatibilityantigens (22). Wecan discuss these results by employingthe sameconventions describing the relationships betweenMHC class I moleculesand nominal antigenas wereusedearlier in conjunctionwith Ia restriction elements(see p. 476). Therequirementsfor association betweenan agretopeon the viral antigen and any particular class I desetope maybe unusuallydemanding. Therefore,eventhougha dozenor moredifferent class I "restriction sites" maybe available within a haplotype(if each is unique), a particular viral capsid antigen maynot be able to associate productivelywith mostclass I molecules.Likewise,hierarchical preferences also seemto be evident in class I T-cell activation. A related question of someimportanceis whethera distinct region (a histotope) on the class I or II moleculesis necessarilyrecognizedby the cell in addition to that desetopemakingcontact with the nominalepitope at an agretopic site. Oneexperimentalsystemthat seemsrelevant to this questionis the recognitionof haptenin conjunctionwith self-class I molecules by Te cells. Earlier workshowedthat hapten coupling to class I moleculesthemsdveswas unnecessaryin achieving a restricted response (143). Moleculesof TNP-BSA could insert into the cell membraneand TNP-selfrecognizingTc wouldstill respond. This lends somecredenceto the viewthat the histotope recognizedon a class I moleculemaybe some distance fromthe hapten. It has beenrecently pointed out that the hapten or nominalantigen could also be coupledto a self non-MHC protein (110). Studies of the sites of allogeneicrecognitionare also connectedto this question. Allogeneicrecognition by CTLmaybe limited to very few areas on the target class I molecules.In a recent report (77), cross-reactivity mutant mouseclass I proteins of knownsequencewasthe device utilized to approachthis problem.The70%cross-reactivity of b anti-a CTLwith bmltargets couldbe attributed to a tyrls~-tyrt~6 alteration of Kbm~.Since

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the identical alteration was uniquely found on the Lo molecule, it apparently accounts for the prominent cross-reactivity. Cold target inhibition studies verified the point. Whetherthe site functioning as an allogeneic target is related to a desetopeor a histotope is an importantarea of investigation. In this context, evidence provided by Weyandet al (178, 179) shows that 2 distinct polymorphicclusters, A and B, are recognized, both by CTL and monoclonalantibodies. It is interesting but perhaps circumstantial that both alloreactive H-2k-as well as H-2d-restricted self CTL’sstrongly prefer cluster B. It appears that a crystal ball is still neededto sort out howmanyseparate elements are recognized by the T cell. No experiments exist limiting the recognition decisively to a fixed numberof elements. Withinthe various cell subpopulafions examined, this could range from (a) epitope alone (b) tope plus somemixture of desetope/agretope to (c) histotope alone or (d) either (a) or (b) plus a distant and distinct histotope.

THE RECOGNITION OF IDIOTYPIC DETERMINANTS BY T CELLS During the past decade, voluminous evidence has implicated the T cell in intercellular idiotypic interactions (reviewed in 47, 82, 83, 175). Weshall consider the nature of the T-cell recognition and in what form the idiotopes are recognized (MHC-restricted, together with antigen, etc.). Although have discussed someof the Id-anti-Id interactions in the suppressive cell circuitry, we will focus here on the predominant idiotypic determinants displayed on the B-cell surface that seemto play a critical role in establishing the quality of the antibody response.

Predominant Idiotypes An idiotope on an immunoglobulin molecule can be recognized by an antigen-specific Th cell (AgTh)just as any other immunogenicepitope, the context of the MHC.Somestrains will be high responders to such idiotopes, and others will be low responders, as in any other immune response gene-control system (86). Jorgensen and Hannestadused different portions of MOPC-315 as T-cell priming agents,~ehallenging subsequently with 315 coupled to DNP. However, we would like to explore the nature of predominant idiotypes on B cells. Whyare there certain idiotopes that gain such prominenceas the T15 idiotope which appears on more than 90%of the anti-phosphocholine antibodies in the BALB/e(37) strain, and also in several others? many cases this maybe a consequence of the germ-line gene composition (55). The predominanceof certain idiotypes can be contrasted to the diver-

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T-CELL ACTIVATION 485 sity of private idiotypes that appearon individual hybridomas in manysuch systems.In several cases (3, 25, 182)it has beendemonstrated that T cells that recognizeidiotype and can be adsorbedon Id-coated petri dishes are implicated in causing the predominance.Onepossible scenario wouldinvolve a sequential activation of the B cells specific for the antigen by an AgThthat is MHC-restricted.Such AgThclones (73) have been shown activate B cells regardless of their idiotype (S. Cammisuli, personalcommunication), or isotype (149). In the absenceof an additional IdX-recognizing cell, this wouldlead to a nonselectivedistribution of idiotypes in the expressedrepertoire. Subsequently,dueto the displayof the special predominant idiotope(IdX)on the activatedB cell surface, a set of IdX-specific cells (IdXTh)wouldbe activated whichthen wouldpositively select those B cells for expansionthat display IdX. The kinetics of appearance of IdX amongthe B cells in a variety of systemssuggests that in cases wherethe antigen moleculeis ubiquitousor at least availablein the self-environment, a prior equih’brinm is established betweenIdX-recognizingTh and Id-bearing Ts whichdownregulate them. The Concept of a Regulatory Idiotope Evidenceseemsoverwhelmingly to favor the proposalby Jerne (85) that the lymphocytescomprisingthe immune systemare linked by an "’intricate web of V domains."A balance is established by the historical accidents of extrinsic exposureto immunogens and the receipt of passive maternal Ig transplacentally (180), both playingon the available genetic repertoire germ-lineidiotypes. The original suggestion by Jerne portrayed an ever-expandingnetwork of idiotype-1 (Ab-1)inducinganti-idiotype (Ab-2), whichthen influenced the expansionof both Ab-1and Ab-3anti-(anti-idiotype)-bearing cells. Ab-3resembledAb-1in idiotypybut not necessarilyantigen specificity. An alternative notion of regulatory idiotopes wasset forth by Paul and Bona (23, 128) whichdirectly implicatesa receptoron an idiotype-reeognizing (IdXTh)in interactions whichdeterminethe nature of the maturingB cells. Accordingto this concept, the networkessentially oscillates betweenidiotype-recognizingand idiotype-bearing lymphocytes. It is probablethat initial triggering of the B cell by antigen ~andAgTh is necessarybefore it will respondsuitably to ambientIdXTh:otherwise, virgin B cells wouldbe perpetually confrontedanddistracted by idiotyperecognizingT cells. Since the IdXidiotope mustbe clearly available for bindingby the IdXThor its specific effector factor, evenin the presenceof antigen, it has beenpostulated that such idiotopes mustreside outside of the antibodyactive site (128). Theseregulatory idiotopes mightrepresent a set of determinantswithin the framework that are quasi-isotypic, appearing on antibodies with a variety of paratopes.

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Starting with the seminalobservationsby Oudinand Cazenave(33, 126) that antibodiesof differentspecificitycan still shareidiotypy,this themehas reappearedand has been relined with the study of monoclonalantibodies (98, 106, 121). In these systemsit has beenshownthat monoclonal antibodies with differing peptidespecificity for the sameantigencan eachbear the predominant idiotype. This represents a slightly different aspect of Jerne’s conceptof a non-specificparallel set of antibodieswith sharedidiotypybut specificity for distinct antigens, evidencefor whichhas accumulatedrecentlyin severaldifferent anti-haptensystems(44, 46, 48, 75, 76, 183).What is morerdevant in our context is whetherthe perpetuation of antibodies bearing a predominantor partially predominantidiotope is a direct consequenceof activation of an IdXThwhichthen selects fromthe entire population, those B cells bearingparatopesof various distinct speciiicities for antigen. ~ It is probablethat the wavesof Ab-1,Ab-2,Ab-3,etc. havea role to play in regulating the response, but it remainsto be learned howT-cell control and serumantibody control contribute to the final outcome. Mostrecently, the question has beenaddressedof whetherany idiotope, even a private one, could subsumethe function of being "regulatory." Neonatalanimalswere treated with a particular silent Id, A48(141) and this idiotypesubsequentlybecame a significant portion of the anti-bacterial levan response. Thecapacity to promotea silent Id into a predominant one belongedto an idiotype-recoguizingLyt-1+ cell. The Idiotope as a Restriction

Element

Onthe basis of evidenceindicating a concurrentrequirementfor antigen in the activation of IdXTh(27), Bottomlyhas suggestedthat a receptor for antigenas well as for predominant idiotope exists on this cell (26). In such circumstances,the idiotope mayserve as a restriction dementsubstituting for the histotope on an MHC molecule. Does the idiotope resemble a histotope? Indeed, it is possible that a receptor for nominalantigen must exist on all T cells in the system, although Janewayremindsus (79) that the MHC-recoguizing Th cell can substitute an idiotope for the epitope. Self-idiotopes, as internal imagesof epitopes, can therebyplay a role in repertoire development. The existence of so manypredominantidiotypic systems suggests an evolutionaryreasonto focus on a limited numberof idiotopes, such as the need to allow coordinatedand simplified regulation of suppressorT cells (whichoften bear the predominantIdXof the B-cell pathway(67)). Thus, the involvement of B cells in the triggering of suppressorT cells mightserve to initiate the feedbackregulation of a well-establishedresponseby stimulating an idiotype-recoguizingT-suppressorinducercell (103).

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CONCLUDING

REMARKS

Perhapsthe mostintriguing unresolvedissues concerningthe antigen specificity of T cells center on: (a) the mechanism(s) underlying determinant selection; (b) the chemistryand genetics of T-cell receptors, whichshould providethe structural basis of geneticrestriction; and(c) a structural comparison of receptors fromdifferent T-ceil subsets with eachother and with immunoglobulins.For a complete understanding of the latter two, the receptors themselves(or the genescodingfor them)mustbe delineated. for determinant selection, the agretope-desetopeconcept, in whichthe agretope can be perceived as the "carder" moiety of T-cell immunogens, provides an attractive frameworkwithin whichexperimentaltests can be designed. Theenormouschangein our viewof T-cell activation in the past decade is almostentirely dueto the conceptsof MHC restriction and the idiotypic network.It is no longer possibleto considerthe problemof antigen activation of T cells in a context separate fromthe recognition of someother element, codedfor by the responder’sowngenome.Thedi~culty in achieving a completeunderstandingof T-cell activation is partly due to the tremendousdiversity of T regulatory ceils that has just recently been unearthed.In the considerationof antibodyspecificity, andB-cell activation, althoughit appearsthat there maybe increasingspecialization manifested by a large variety of B-cell subpopulations, there is nodoubtthat each member of the diverse array of B cells bears recognitionspecificity for a determinantregion on the nominalantigen. This statementcannotbe madeabout the T cell for twoseparate reasons: (a) the epitope whichis recognizedmaybe intricately associated with MHC moleculeand both moieties are simultaneouslyrecognized; (b) some interactions maybe restricted by idiotypic elementsin conjunctionwith MHC or antigenic moieties. In fact, wehave mentionedthat the feature whiehmakesT recognition so varied is the large numberof regulatory T cells each of whichmust circumscribeits interactions with surroundingcells. Todetermineits partner unambiguously, principles of simplicity can be invokedto assure coordinated control. Onesuch principle mightbe that recognitionby T cells and specific targeting by T cells can be accomplished by havingeach cell bind to a combinationof elements: an epitope plus idiotope, an epitope plus histotope, or an idiotope plus histotope. A second motif whichseemsto permeatethe relationships amongT ceils is that antigen-specific T cells throughoutthe system, whichthus bear idiotypic elementsin their receptors, will be activated by other T cells whichrecognizethese idiotypie elements.Aconsequence of this schemeis an alternation of antigen-specific, MHC-restricted cells with idiotype-specific T cells in sequential pathways.

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Therules of activation at the molecularlevel and details about "second messenger"function are boundto be the samefor manytypes of cells, and wehavenot consideredthemin this account. Wehaverestricted ourselves to attemptingto assemblethe informationaboutthe state in whichepitopes, idiotopes, and histotopes confront the T-cell machinery.If there is a rule about this confrontationwhichcan be discerned, it maybe that the choice of epitopewhichwill be available for a T-cell subpopulationwill dependon an "antigen-recognitionepitope," an agretope, whicha presenting cell (or entity such as a soluble factor) can recognize on the antigen. Thus, Hagretopes on the Ag will be recognized by Class IIIa molecules on APC presenting to Th, C-agretopeswill interact with an appropriate group or receptor on the Class I moleculefoundon the surface of cell types recognized by Tc, and S-agretopeswill associate with desetopes on MHC molecttles whichexist on APCof the type whichpresentsantigen to Ts. Thelist could be extended. Therefore, the basis for determinantsdection wouldreside in still unknownchemicalprinciples uniting desetopewith agretope: desetopeson the MHC molecules used for triggering each important T-cell subpopulation would seek out their complementaryagretopes based on a unique but non-clonal chemistry. An exampleof such a principle might be that the desetopesfor H-agretopesrecognizea particular groupingof hydrophobic aminoacids only, whereas the desetopes for S-agretopes combinewith hydrophilieaminoacids. This alignmentwouldhave utility in explaining two vexing problems. First, howcan the system maintain self-non-self discrimination in the face of the enormousexcess of self H-agretopes? Probablythese H-agretopesremain unexposeduntil antigenic fragmentation and "processing."Prior T-cell recognitionmaybe required to trigger processing: such maybe the role of augmentingor amplifier T calls which wouldthen haveto be able to recognizesuperficial residues on the antigen. Second,since S-agretopesare hydrophilicandexternal on the antigen, the nearbyepitope will be comparablysituated: this wouldexplain whyTs can often combinewith free antigen. Oneof the critical questionsthat can be approached experimentallyis the definition of agretopesfor particular haplotypes.Will any chemicalstructure associatedwith the agretopein the correct spatial relationship act as an epitope for T cells? This question is being experimentallyapproachedby synthetic chemistry, using as a starting point, for example,the putative agretope at the C-terminusof pigeon cytochromec, for the B10.Amouse strain. Other H-agretopeson L-tyr-ABAor lysozymecould also be used. It is important to realize that although wemaybe at the threshold of defining agretopesand synthesizing completeimmunogens with a particular agretopeas a universal carrier for a series of epitopes, difficulties may

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abound. Not all epitopes will be permissive for agretope function, as older observations seem to suggest for the small molecule, L-tyr-ABA (6). Finally, it would seem unlikely that a particular determinant area on any antigen could be designated as immunodominant for T cells in general. However, we may soon arrive at an understanding of the commonfeatures of attachment sites (agretopes) chosen by MHCelements for presentation to different T-cell subpopulations. Understanding the nature of specific epitope immunodominance for different T-cell subpopulations that might result from study of the features and constraints of desetope-agretope interaction should permit the logical manipulation of T-cell responsiveness. ACKNOWLEDGMENTS

This workwas supportedby NIHgrants CA-24442,AI-056Ot, and AI11183, and by grants IM-263and IM-323from the AmericanCancer Society. Literature Cited 1. Adorini, L, M. A. Harvey,A. Miller, gen recognition and the immunereandE. E. Sercarz. 1979.Thefine specisponse: humoraland cellular immune responses to small mono-andbifuncficity of regulatoryT cells. II. Suppressor and helper T cells are inducedby tional antigen molecules.J. Exp. Med. different regionsof henegg-whitelyso135:1228-1246 zyme(HEL)in a genetically nonrespon- 7. Andersson, J., G. M. Edelmun, G. der mousestrain. Z Exp. Med. 150: M6ller, and O. Sj~berg. 1972. Activa293-306 tion of B lymphocytesby locally con2. Adorini, L., M. Harvey, D. Rozyekacentrated concanavalinA. Eur. J. IraJackson,A. Miller, and E. E. Sercarz. munoL2:233-235 1980. Differential majorhistocompat8. Araneo,B. A., D. W.Metzger, R. L. bility complex related activationofidioYowelL and E. E. Sercarz. 1981. Positypic suppressor T cells. Z Exp. Med. tive selection of maj~histocompatibil152:521-531 ity complex restricted suppressorT cells 3. Adorini, L, M.Harvey,and E. E. Serbearingthe predominant idiotype in the carz. 1979.Thefine specificityof regulaimmuneresponse to lysozyme. tory T ceils. IV. Idiotypic complemenNatL Acad~Sci USA.78:499-503 tarity andantigen-bridginginteractions 9. Araneo,B. A., R. L. Yowell,E. E. Serin the anti-lysozyme carz. 1979.Ir genedefects mayreitect a Immunol. 9:906-909 response. Eur. Z regulatory imbalance.I. Helper T cell 4. Adorini, L., A. Miller, and E. E. Seractivity revealedin a strain whoselack carz. 1979.Thefine specificityof regulaof responseis controlledby suppression. tory ceils. I. Hen-~gg-white lysozymeZ Immunol. 123:961-7 inducedsuppressorT cells in a genet10. Bach,B. A., M. L Greene,B. Benacerically non-respondermousestrain do raf, and A. Nisonoff. 1979. Mechanisms not recognizea closely related immunoof regulation of cell mediatedimmugeneic lysozyme.J. ImmunoL 122:871nity. IV. Azobenzencarsunate(ABA) 877 specific suppressorfactor(s) bear cross5. Alkan,S. S., D. E. Nitecki, and J. W. reactive idiotype (CRI) determinants Goodman.1971. Antigen recognition the expressionof whichis linked to the and the immune response: the capacity heavy chain allotyp¢ linkage group of of L-tyrosine-azobenzenearsonateto genes. Z Exp. Med~149:1084-1098 serve as a carder for a macromolecular 11.Bach,B. A., L. Sherman,B. Benacerraf, hapten. J. Immunol.107:353-8 and M.I. Groene.1978. Mechanisms of 6. Alkan,S. S., E. B. Williams,D. E. Niregulation of cell-mediated immunity. tecki, and J. W.Goodman. 1972. AntiII. Induction and suppression of de-

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polymorphicMHC self determinants? ImmunologyToday. 3:204-205 111. Levy,R. B., G. M. Shearer, and T. H. Hausen.1978. Properties of H-2Llocus products in allogeneic and H-2 restricted, trinitrophenyl specific cytotoxic responses. J. ImmunoL 121:2263 112. Lin, C.-C. S., A. $. Rosenthal, H. C. Passmore,and T. H. Hausen.1981. Selective loss of antigen-specificIr gene b~n is function in I-d mutantB6.C-H-2 an antigen presentingcell defect. Proc. NatL ,4cad. ~cL U~A78:6406-6410 113. Lin, C. S., A. S. Rosenthal,and T. H. Hausen.1981. I-A mutationresulted in a selectedloss of an antigen-specificIr gene. J. SupramoLStmc~ 16:115 114. Lonai, F., L. Steinman,V. Friedman, G. Drizlikh, andJ. Purl. 1981.Specificity of antigen bindingby T cells; competition between soluble and Iaassociated antigen. Fur. J. Immunol. 11:382-387 115. Maizels, R. M., J. A. Clarke, M. A. Harvey,A. Miller, and E. E. Sercarz. 1980.Epitop¢specificity of the T cell proliferative responseto lysozyme:Proliferative T cells react predominantly to diferent determinantsfromthose recognized by B cells. Eur. J. Immunol.10: 509-515 116. Maizeis, R. M., J. A. Clarke, M. A. Harvey,A. Miller, and E. E. Sercarz. 1980.Ir-genecontrolofTcell proliferative responses:twodistinct expressions of the£genetically unresponsivestate. Eur. ImmunoL10:516-520 117. Manca,F., J. A. Clarke, E. E. Scrcarz, andA. Miller.1983.T cells withdiffering specificity exist fora single determinant on lysozyme.In Ir Genet"Past, Presentand Future,ed. C. W.Pierce, S. E. Cullen, J. A. Kapp,B. D. Schwartz, and D. C. Shrcflier. Clifton, NJ: Humana.In press 118. Maoz, A., M. Feldmann,and S. Kontialnen. 1976. Enrichmentof antigenspecific helper andsuppressor T cells. Nature. 260:324-326 J. S., F. W.Shen,S. P. Cort 119. McDougal, and I. Bard. 1980. Feedbacksuppression: phenotypesof T cell subsets involved intheLylT callinduced immunorcgulatory circuit. Z Immunol. 125:1157-1163 120. McKean,D. I., R. W.Mclvold,and C. S. David. 1981. Trypfic peptidc comparison of Ia antigen alpha and beta polypcptides from the I-A mutant B6.C-H-2~=12 and its parental strain B6. Immunogeneticz14:41-51

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T CELL 121. Metzger, D. W., A. Miller, E. Sercarz, 1980. Sharing of an idiotypic marker by monoclonalantibodies specific for distinct regions of hen lysozyme. Nature. 287:541-542 122. Michaelldes, M. M., M. S. Sandrin, G. M. Morg~an, I.F.C. McKenzie, R. Ashman, and R. W. Melvold. 1981. Ir gene function in an I-A mutant B6.C-H2bml2. .L Exp. Med. 153:464-469 123. Minami, M., N. Honji, and M. E. Doff. 1982. Mechanismresponsible for the induction of I-J restrictions on Ts3 suppressor cells, d. Exp. Med. 156:1502t515 124. Mozes, E., andJ. Haimovich. 1979. Antigen-speciilc T-cell helper factor cross reacts idiotypieally with antibodies of the same specificity. Nature 278:56-57 125. Okumura, K., T. Takemod, T. Tokuhisa, and T. Tada. 1977. Specific enrichment of the suppressor T cell bearing I-J determinants. Parallel functional and serological characterizations. Z Exp. Med. 146:1234-1245 126. Oudin, J., P. A. Cazenave. 1971. Similar idiotypic specificities in immunoglobulin fractions with ditferent antibody functions or even without detectabie antibody function. Proc. NatL ~4cad. Scg US~4 68:2616-2620 127. Paul, W. E., and B. Benacerraf. 1977. Ftmctional specificity of thymusdependent lymphocytes. Science. 195: 1293-1300 128. Paul, W. E., and C. A. Bona. 1982. Kegulatory idiotopes and immune networks: a hypothesis. ImmunoLToday. 3:230-234 129. Pierres, M, and R. N. Germain. 1978. Antigen-specific T cell-mediated suppression. IV. Role of macrophages in generation of L-gintamic acid -L-alanine -L-tyrosine (GAT)-specific suppressor T cells in responder mouse strains, d.. IramunoL121:1306-1314 130. Playfair, J. H. L. 1972. Response of mouse T and B lymphocytes to sheep erythrocytes. Nature, 235:115-18 131. Prange, C. A., J. Fiedler, D. E. Nitecki, and C. J. Bellone. 1977. Inhibition oft antigen-binding cells by idiotypic antisera. Z Exp. Med. 146:766-778 132. Puri, J. and P. Lonai. 1980. Mechanism of antigen binding by T cells. H-2 (I-A) -restricted binding of antigen plus Ia by helper cells. Eur. d. ImmunoL 10:273-281 133. Rajewsky, K., and K. Eichmatm. 1977. Antigen receptors of T helper cells. Contemp. Top. ImraunobioL 7:69-112

ACTIVATION

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134. Rajewsky, K., and R. Mohr. 1974. Specificity and heterogeneity of helper T cells in the response to serum albumins in mice. Eur. Z ImmunoL4:111119 135. Ramseier, H., M. Aguet, andJ. Lindenmann. 1977. Sitnilarity of idiotypi¢ determinants of T- and B-lymphocyte receptors for alloantigens. Immunol. Rev. 34:50-88 136. Rosenthal, A. S., 1978. Determinantselection and macrophagefunction in genetic control the immuneresponse. Immunol. Rev. of40:136-152 137. Kosenthal, A. S., M. A. Barcinsld, and J. T. Blake. 1977. Determinant selection: A macrophage dependent immune response gene function. Nature. 267: 156-158 138.Rosenthal, A. S., andE. M. Shevach. 1973. Function of macrophages in antigen recognition b~ guineapig T lymphocytes. Z Exp.Med.138:1194-1212 139.Rosenthul, A. S., J. W. Thomas,J. Schroer, andJ. T. Blake. 1980. Therole of macrophages in genetic control of the immuneresponse. In FourthInternational Congress of lmmunoLProgress in Immunology IV. ed. M. Fougerean and J. Dansset. New York: Academic p. 458-477 140. Rosenwasscr,L. J., M. A. Barcinski, R. H. Schwartz, and A. S. Rosenthal. 1979. Immuneresponse gene control of determinant selection. II. Genetic control of the murine T lymphocyteproliferative response to insulin, d. ImmunoL 123:471-476 141. Rubinstein, L. J., M. Yeh, and C. A. Bona. 1982. Idiotype-anti-idiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotypespecific helper T cells. J. Exp. Med. 156:506-521 142. Sachs, D. H., J. A. Berzofsky, C. G. Fatlmlan, D. S. Pisetsky, A. N. Schechter, and R. H. Schwartz. 1976. The immuneresponse to Staphylococcal unclease: A probe of cellular and humoral antigen-specific receptors. Cold Spring Harbor Syrup. Quan~BioL 41:295-306 143. Sckmitt-Verhulst, A.-M., C. B. Pettinelli, P. A. Henkart, J. K. Luuneyand G. M. Shearer. 1978. H-2 restricted toxic e.ffectors generated invitro by addition of trinitrophenyl-conjuated soluble proteins, d. Exp. Med. 47:352-368 144. Schwartz, R. H. 1983. Distinct structures on nominal and MHCantigens

~e ~

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involved in antigen presentation and T cell recognition. In Ir Genes: Past, Present andFuture, ed. C. W. Pierce, J. A. Kapp, B. D. Schwartz, and D. C. Shreflier, S. E. Cullen. Clifton NJ: HumanaIn press 145. scibienski, R. J., V. Klingmmm,C. Leung, K. Thompsonand E. Benjamini. 1977. In Recognition of lyso~me by lymphocyte subset& ed. M. Z. Atassi and A. B. Stavitsky. Adv. in Exp. MecL and Biol. 98:305-317 146. Schirrmacher, V., and H. Wigzell. 1974. Immuneresponses against native and chemically modified albumins in mice. II. Effect of alteration of electric charge and conformation on the humoral antibody response and on helper T ceil responses. £ ImmunoL 113:1635-1643 147. Schwartz, M., W. C. Waltenbangh, M. Dorf, R. Cesla, M. Sela, B. Bena~rraf. 1976. Determinants of antigen molecules responsible for genetically controlled regulation immune Proc. Natl. Acad.ofSc~ USAresponses. 73:28622866 148. Schwartz, R. H. 1982. Functional properties of I region geneproducts and theories of immune response (I 0 gene function. In Ia Antigens and their Analogues in Manand Other Animal£ ed. S. Ferrone and C. David, Boca Raton, Florida: CRCIn press 149. scman, M., and J. Theze. 1981. H-2 restricted carrier specific T cell clones do not select immunogiobalinisotypes. In Immunoglobulin Idiotypes, ed. C. A. Janeway Jr., E. Sercarz, and H. Wigzell,, p. 623-632. NewYork: Academic 150. Sercarz, E. E., D. T. Corenzwit, D. E. Eardley, and K. M. Morris. 1975. Suppressive versus helper effects in carrier priming. In Suppressor Cells in ImmunRy, ed. S. K. Singhal. Univ. Western Ontario p. 19-25 151. Sercarz, E. E., R. L. Yowell, and L. Adorini. 1977. Immuneresponse genes control the helper suppressor balance. In The Immune System: Genetics and Regulation. ed. E. E. Sercarz, L. Herzenberg, and C. F. Fox, 6:497-506. New York: Academic 152. Sercarz, E. E., R. L. Yowell, D. Turkin, A. Miller, B. Axaneo, and L. Adorini. 1978. Different functional specificity repertoires for suppressor and helper T ceils. Immunological Rev. 39:109-137 153. Shearer, G. M., T. G. Relm, and C. A. Garbarino. 197~. Cell-mediated lympholysis of trinitrophenylated-modified autologous lymphocytes. Effect cell

154.

specificity to modifiedcell surface components controlled by the H-2K and H-2Dserological regions of the major kistocompatibility complex. J. Exp. Med. 141:1348-1364 Sherman, L. A., S. J. Burakoff, and B. Benacerraf. 1978. The induction of cytolytic T-lymphocyteswith specificity for .p-azophenylarsonate coupled synenelc cells. J. ImmunoL 121:1432436 Shevach, E. M. 1976. The function of macrophagesin antigen recognition by guinea pig T lymphocyte,. III. Genetic analysis of the antigens mediating macrophage.T lymphocyte interaction, d. Immunol. 116:1482-1489 Simonsen, M. 1967. The clonal selection hypothesis evaluated by grafted cells reacting against their hosts. Cold Spring Hath. Symtx Quant. Biol. 32:517-523 Sprent, J. 1978. Restricted helper function of Ft hybrid T cells positively selected to heterologons erythrocytes in irradiated parental strain mice. II. Evidence for restrictions affecting helper cell induction and T-B collaboration, both mapping to the K-end of the H-2 complex. J. Exp. Med. 147:1159-1174 Sredni, B., and R. H. Schwartz. 1980. Alloreactivity of an antigen-specific T cell clone. Nature 287:855-857 Sredni, B., H. Y. Tse, C. Chen, and R. H. Schwartz. 1980. Antigen-specific clones of proliferating T lymphocytes.I. Methodology, specificity and MHCrestriction. J. Immunol. 126:341-347 Swanborg, R. H. 1972. Antigeninduced inhibition of experimental allergic encephalomyelitis. I. Inhibition in guinea pigs injected with non-encephalitogenic modified myelin basic protein. J. ImmunoL109:540-546 Sy, M.-S., M. H. Dietz, R. N. Germain, B. Benacerraf, and M. I. Greene. 1980. Antigen and receptor-driven regulatory + mechanisms. IV. Idiotyt?e-bearing I-J suppressor T ceil factor tnduce secondorder suppressor T cells which express anti-idiotype receptors. £ Exp. Med. 151:1183-1195 Tada, T. 1983. I region determinants expressedon different subsets of T-cells: Their role in immunecircuits. In Immunogenetic£ed. B. Benacerraf, Paris: Masson. In press Tada, T., K. Hayakawa, K. Okumura, and M. Taniguchi. 1980. Coexistence of variable region of immuno#obulin heavy chain and I region gene products on antigen-specific suppressor T cells

~ 155.

156.

157.

158. 159.

160.

161.

162.

163.

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andsuppressorT cell factor. A miaimal 174. Unanue,E. R. 1978. Theregulation of modelof functional antigen receptor of lymphocyte function by the macroT ceils. Mol. lmmunoL17:867-875 phage. Immunol.Rev. 40:227-255 164. Takaoki,M., M. S. Sy, B. Whitaker,J. 175. Urbain, J., C. Wuiimart, and P.-A. Nepom,R. Finberg, R. N. Germaln,A. Cazenave.1981. Idiotypic regulation in Nisonoif, B. Benacerraf, and M. I. immunenetworks. Contemp.Top. MoGreene. 1982. Biologicactivity of an lec. ImmunoL8:113-48 idiotype-beadngsuppressorT cell fac- 176. yon Boehmer,H., W.Haas, and N. K. tor producedby a long-termT cell hyJeme. 1978. Majorhistocompatibility bridoma. J. ImmunoL128:49-53 complex-linkedimmune-responsiveness 165.Taniguchi,M., andJ. F. A. P. Miller. is acquired b.y lymphoctyesof low1977.Enrichment of specific suppressor responder n~ce differentiating in T cells andcharacterizationof their surthymusof high responder mice. Proc. face markers. Z Exp. Med. 146:1450Nat£ Acad. ScL USA75:2439-2442 1454 177. Walker,E., N. C. Warner,R. Chesnut, 166. Thomas,D. W., M. D. Hoffman,and G. J. Kappler,and P. Marrack.1982. AntiD. Wilner.1982. T lymphocyterecognigen-specific,I region-restrictedinteraction of pcptideantigens:evidencefavortions in vitro betwceen tumorceil lines ing the formationof ncoantigenicdeterand T cell hybridomas. Z ImmunoL minants. J. Exp. Med.156:289-293 128:2164-2169 167. Thomas,D. W., K.-H. Hsieh, J. L. 178. Weyand,C., J. Goronzy, and G. J. Schanstcr, M.S, Mudd,and G, D. WilHammerling. 1981. Recognition of ncr. 1980. Nature ofT lymphocyterecpolymorphic H-2 domains by T lymognitionof macrophage-associated antiphocytes.I. Functionalrole of different gcns. V.Contributionof individualpepH-2domainsfor the generation of altide residues of humanfibrinopeptideB loreactive eytotoxie T lymphoeytesand to T lymphocyteresponses. J. Exp. determinationof precursorfrequencies. Med. 152:620--632 J. Exp. Med.54:1717-1731 168. Thomas, D. W., K.-H. Hsieh, J. L. 179. Weyand,C., G. J. Hammeding, and J. Schauster,andG. D. Wilncr.1981.Fine Goronzy.1981. Recognitionof H-2dospecificity of genetic regulation of mainsby cytotoxic T lymphocytes.Naguinea pig T lymphocytcresponses to ture 292:627-629 angiotensinII and related peptides. J. 180. Wilder, M., C. Demeur,G. Dewasme, Exp. Mea~153:583-594 and J. Urbain. 1980. Immunoregulatory 169. Thomas, W. R., F. I. Smith, I. D. role of maternalidiotypes: Ontogeny of Walkerand I. F. A. P. Miller. 1981. immunenetworks. J. Exp, Met~ 152: Contactsensitivity to azobenzenarson1024-1035 ate andits inhibitionafter interactionof sensitizedcells withantigen-conjugated 181. Woodland,R., and H. Cantor. 1978. Idiotypespecific T helper cells are recells. J.. Exp. Med.153:1124-1137 quired to induce idiotyp¢ positive B 170. Tokuhisa,T., and M.Taniguchi.1982. memory cells 8:600-606 to secrete antibody.Eur. Twodistinct allotypic determinantson J. Immuno£ the antigen specific suppressorandenreachancingT cell factors that are encoded 182. Wilson, D. B. 1974. Immunologic tivity to majorhistocompatibilityalby genes linked to the immunoglobulln loantigens: HARC, effector cells and heavy chain locus. J. Exp. Med. the problemof memory.In Progressin 155:126-139 Immunology, Vol. 2, ed. L Brentand J. 171. Tokuhisa,T., Y. Komatsu,Y. Uchida, Holborow,p. 145-156. Amsterdam:Eland M. Taniguchi. 1982. Monoclonal sevier alloantibodiesspecific for the constant 183. Wysoeki,L. J., and V. L. Sato. 1981. region of T cell antigen receptors. J. Thestrain A anti-p-azophenylarsonate Exp. Med. 156:888-897 majorcross-reactiveidiotype familyin172. Tse, H. Y., J. J. Mond,andW.E. Paul. eludes memberswith no reactivity to1981.T lymj[~_hocyte-dcpcndcnt B-lymwardp-azophenylarsonate. Eur. Z Imphocyteprottt~rative responseto antiCn.I. Geneticrestriction of the stimumunoL11:832-839 tion of B lymphocyte proliferation. J. 184. Yamanehi,K., N. Chao, D. B. Murphy, Exp. Med. 153:871-82 and R. K. Gershon. 1981. Molecular 173. Turkin, D., and E. E. Sercarz. 1977. compositioa of an antigenspecific, Ly-1 Keyantigenic determinantsin the regusuppressor inducer factor. Onemolelation of the immuneresponse. Proc. culebindsantigenandis I-J-, anotheris Nat. Sci. USA74:3984-3987 I-J +, doesnot bind antigen,andimparts

~a

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an IgI-I-variabl¢regionlinkedrestricdifferentiation of"H-2self-recognition" tion. d. ExtT. Med.155:655-665 by T cells: evidencefor dual recognition? Z Exp. MecL147:882-896 185. Yowell,R. L., B. A. Araneo,A. Miller, and E. E. Serearz. 1979.Amputation of 188. Zinkemagel,R. M., and P. C. Doherty. a suppressor determinant on lysozyme 1975. H-2 compatibility requirement reveals underlyingT cell reactivity to for T-cell mediatedlysis of target cells other determinants. Nature279:70-71 infected with lymphocytic chori186. Zinkemagel,R. M., and P. C. Doherty. omeningitis virus: different eytotoxicT1979.MHC-restdeted cytotoxic T cells: cell specificities are associated with structures coded from H-2Kor H-2D. Studies on the biological role of polyZ Exp. Med. 141:1427-1436 morphicmajortransplantation antigens determiningT-cell restriction-specifi189. Zinkernagel,R. M., and P. C. Doherty. city, ftmetion,andresponsiveness.Adv. 1977. Majortransplantation antigens, ImmunoL27:51-177 virus and specilicity of surveillance T cells. The"altered self" hypothesis. 187. Zinkernagel,R. M., G. N. Callahan,A. Althage,S. Cooper,P. A. Klein, and J. Comtemp.Top. Immunbiol. 7:179 Klein. 1978. On the thymus in the




Ann Rev. ImmunoL1983. 1:499-528 Copyright©1983by AnnualReviewsInc. All rights reserved

IMMUNOGLOBULIN GENES


Tasuku Honjo Department of Genetics,OsakaUniversityMedicalSchool,Kita-kuOsaka, 530 Japan

INTRODUCTION Theimmunoglobulin gene systemis comprisedof three separate gene loci of L~, Lx, andH(light andheavy)chaingenes,each containingvariable (V) and constant (C) genes. TheLK(157), Lx (36), and H (35) chain genes located on mousechromosomes6, 16, and 12, respectively [on human chromosomes 2 (95), 22 (104), and 14 (48, 84), respectively)]. Molecularcloning and structural characterization of the immunoglobulin gene haveshownthat DNA rearrangementplays essential roles in the somaticamplificationof the immunoglobulin diversity that manifestsin two aspects: the ability to bind an enormous numberof antigens, andthe ability to trigger a variety of immunologic reactions. Suchstudies madelongdebating immunologistsmoreor less unanimouson howthe immunoglobulin diversity is generated. Structural analysesof the immunoglobulin gene havealso given newinsights into the evolutionaryprocessthat contributes to the creation of the antibodydiversity. It is nowclear that organization and structure of the immunoglobulin gene per se incorporate the moleculardrive that increases the immunoglobulin diversity through the somatic and evolutionary processes. The basic principles that underlie the genetic mechanism to create the antibody diversity are summarizedas flexible, random,somatic DNArearrangementsaccompaniedby deletion and evolutionary mechanisms dependingon the frequent recombinationand rapid drift. Theantibodies thus created contain not only those that are specific to current antigens, but also those that appearuseless yet mightbe reactive to antigens to be encounteredin future. Thus, the immunoglobulin gene system has Prometheanforesight (116), preparing for unknownantigens by the flexible and randomDNA rearrangement. This system has strong advantages over the fixed rigid geneticsystemthat determinesall the specifities andphysiologicalfunctions a priori in the genome, becausethe latter makesit difficult for the organism to adapt to the drastic changeof the environment. 499 0732-0582/83/0410-0499502.00

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HONJO


In this review I focus on the organizational and structural features of the cloned immunoglobulingenes and on the implications derived from these studies. Transcription of the immunoglobulingene is not discussed in this review. The encyclopedic citation of references is not the object of this reviewin view of the limited space for such a broad subject. Unlessspecified, described information was derived from studies on mousegenes. Several aspects of this gene were reviewed previously (33, 40, 52, 64, 97).

VARIABLE REGION GENES Structure and Organization THE VARIABLE

REGION

IS

ENCODED BY TWO OR THREE

SEPARATE

DNASEGMENTS The V region was defined originally by the comparison of amino acid sequences of immunoglobulins. The length of the L-chain V region is rather constant (usually aminoterminal 107 residues) whereasthat of the H-chain V region is variable, ranging from 107 to 132 residues (74). Cloning and characterization of the Vx2region gene first demonstratedthat the Vx region is encoded by two separate DNAsegments, i.e. the Vx and Jx segments, as shownin Figure 1 (1 l, 160). The Vxsegment has two exons separated by a 93-bp intron; the 5’ exon codes for a major portion of the leader sequence (residues -19 to -5), which is eventually cleaved off, and the other exon codes for the remaining portion of the leader sequence and the major portion of the V region (residues -4 to 97). The rest of the region (residues 98-110) is encoded by the Jx segment, which is located

Vx2 II

dx2

C7t2

d~4

Cx4

~

Vta V~z ..... V~n

O~

---II-//-tl-//----#l-",;’ I IIII

C~

-"

Figure 1 Organization of immunoglobulin L-chain genes. Each exon is shown by a wide rectangle.

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close to the Cx gene. As described bdow, the expressed V gene is created by the fusion of the germline V and J segments. To distinguish the germline and the complete rearranged forms of the V gene, I refer to the germline form as the V segment throughout this review. The V~ region is also encoded by the V~ and JK segments with lengths similar to the Vx and Jx segments, respectively (102, 137). By contrast, the Vn region is encoded by the three separate DNAsegments, namely, the VH, D, and JH segments (38). Consequently, the complete active VHgene is generated by two somatic recombinations, VH-Dand D-Jn joinings. The D segment encodes primarily the CDR3and varies considerably in sequence as well as in length (88, 89). STRUCTUREAND ORGANIZATIONOF THE V SEGMENTThere are only two V~ segments (Vxl and Vx2) in the mousegenome, whereas a large number of V), segments are expected to be in the human genome. The V~l segment is genetically linked to the C~3and C~,l genes, and the V~2 segmentis linked to the C),2 and Cx4 genes in the mousegenome(14, 105). The VT, segments have not been physically linked to any of the C~genes. The germline V~ segment has the structure essentially similar to the Vx segment. Multiple V~ segments are cloned from mouseand man (9, 113, 142). One of them is a pseudogenecontaining substitutions, deletions, and insertions (9). About 31- to 43-kb DNAfragments carrying two V~ segments were cloned in a cosmid vector, but the distance between two adjacent VKsegments is not known precisely (21). Two human genomic fragments carrying two V~ segmentswith 12.5- to 15-kb spacers were cloned (126). Zeelon et al (78) measuredthe proportion of a distinct V~sequencein the total V~ sequences expressed in mousespleen and estimated the numberof the V~group as 280, assumingthat all the groups are equally expressed. The total numberof the V~ segment is simply calculated to be about 2000 on the assumption that each group has seven members. Cory et al (30) measured numbers of restriction fragments hybridized with 10 probes and concluded that the total numberof the V~segments is between 90 and 320. Bentley &Rabbitts (9) estimated that there are only 15-20 V~segments the humangenome on the basis of the number of hybridized bands with V~probes of three different subgroups. In view of the presence of pseudogenes and the absence of dear knowledge about the extent of cross-hybridization, these values can not be taken seriously. In addition, the ambiguityabout the definition of the V group has to be taken into account. Althoughthe subgroup was originally defined by the aminoacid sequence similarity of the V region framework,analysis at the DNAlevel is consistent with the view that each V subgroup is deter-

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HONJO

minedby a single germline V segment. The definition of.the group was somewhat arbitrary. For example,VKregions varying at three or moreof the amino-terminal 23 aminoacids wereclassified into different VKgroups. Thedefinition of the group at the DNA level is also arbitrary. Several investigators considerthat all the V segmentscross-hybridizingto a probe belong to the same group. However,cross-hybridization dependson the conditions of hybridization and washing,especially temperatureand salt concentration.Evolutionallyclosely related V segmentsshoudforma group of Vsegments.In somecases, however,there wouldbe no discrete boundary betweendifferent groups because extensive recombinatodalhomogenization of Vsegmentshas taken place, as is discussedlater. TheVnsegmentis also split into twoexonsby an intron at the identical position to the VLsegment.The lengths of two exons are also similar to those of the VLsegments.Several Vii segmentsare physically linked (31, 76, 80). The distance betweentwo adjacent Vnsegmentsis 12-15 kb. The averageVII-VI-Idistanceappearsto be longer(at least 25 kb) thanthe above value, becauseL. Hoodand his associates (personal communication) found 21 VH segmentsin cloned germline DNA fragments that total 492 kb. The total numberof the Vii segmentsis not knowneither. Probablyit wouldfall somewhere between100 and 1000. Kemp et al (81) estimatedthat each group contains 15 members by Southernblot hybridization using three probes of different groups. Assuming that the total numberof VrI groups is about10, they calculatedthat the total number of the Vii segmentsis 160. Three VHsegmentswere mappedin the order Vn76(inulin binding), (phosphorylcholinebinding), and Vm/A8 segmentsby deletion in myeloma cells expressingdifferent Vnsegments(81). Themembersof the sameVii grouptend to cluster (31, 76, 80, 131). However,Hoodand his associates (personal communication) were unable to link the four Vnmembersof the VT~5group even by cloning the germlinc DNA segmentsof 500 kb. It is possible that somemembers of the groupweretranslocated elsewherein the Vncluster. It is importantto stress that members of the sameVngroupdo not always encodethe sameantigen specificity. For example,only one of the four members of the Vx~5groupis used for anti-phosphorylcholinc antibody, the secondis usedfor anti-hemagglutininof influenza virus, the third is used for unknownspecificity, and the fourth is a pseudogene(31; L. Hood, personal communication). STRUCTURE AND ORGANIZATION OF THE J SEGMENT The JK segment is located 1.2-1.3 kb 5’ to each C~gene of mouse(Figure 1). From

the genetic linkage betweenthe Vxand Cxgenes, the Vxl segmentis able to associate with either JK3 or Jkl segment,whereasthe V~2segmentis to

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be joined with either the Jk2 or the Jh4 segment. However,the VX2 was shownto be associated with J)d and Cx3in a hybridoma,suggesting that the Vx2Jx2 Cx2Jx4 Cx4duster is 5’ to the Vx~Jx3 CX3 JM C~,l duster (43). TheJx4 segmentis probably inactive becauseof a 2-bp deletion in the splicing signal. Anotherpseudo-Jsegmentwasidentified betweenthe Jh3 segmentand the Cx3gene (14, 105). There are four JK segments and a pseudo-JK segment in the mouse genome.TheJr segmentsare clustered 2.6 kb 5’ to the CKgene(102, 137). Thefour J~ segmentsare presumedto be capableof joining with any of the V~segmentsfrom amino acid sequence analyses. The humanJ~ cluster contains active five J segmentsabout 2.5 kb 5’ to the C~gene (61). The absenceof the mouse Jr3 equivalentsequencewasexplainedby a duplication event in the humanJ~ duster by unequalcrossing over. In the Hchaingenefamilythere is only oneset of the Jn cluster for eight or nine Clt genes. Thereare four JH andone pseudo-JI.I segmentsin mouse and six Jn and three pseudo-Jnsegmentsin man(130, 139). Althoughthe length of the mouse JL segmentis constant (13 residues), the Jn segments of mouseand manhave varied lengths ranging from 16 to 21 residues. AND ORGANIZATION OF THE D SEGMENT The D segmentis characterizedbyits variability in sequenceas well as in length (88, 89). Theshortest germlineDsegmentidentified so far encodessix amino acid residues, and the longest one encodes17 residues in mouse.At least someDsegmentsare arranged in a relatively small region of the genome. Clusters of 11 and 4 Dsegmentswerefound in mouseand man,respectively (88, 150). Butnot all the aminoacid sequencesof the Hchainsare explained by knownD segments. Worsethan that, the four humanD segments reported are not found in knowncomplementarity-determiningregion (CDR)3sequences. It is possible that moreunknown germline D segments are present in the mouseand humangenomes.Kurosawa& Tonegawa(88) proposedthe D-Djoining as a mechanism to create unexplainedexpressed D region sequences.However,there is not a single case in whichthe D-D joining can explain the knownD region without the point mutation, insertion, or deletion. Thelocation of the D segmentrelative to the Cnand Vnis not known except for one Dsegmentlocated immediately5’ to the JIt segment(138). Evolutionaryconsideration led to a proposal that the D segmentdusters maybe dispersed amongVr~ dusters (65). STRUCTURE

V-(D)-J Recombination Structural comparisonof the germlineand expressedV genes clearly demonstrated that a completeV geneis created by recombinationaljoining of V, (D), and J segments.This recombinationalreaction has three important

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characteristics. First, the combination between different DNAsegments seems to be randombecause the same Vr~segmentsare able to associate with all the Jn segments in the anti-phosphorylcholine antibody H-chain of mouse(53). Secondly, the recombination reaction has flexibility at the joining nucleotide in spite of the strong requirement for the accurate inframe joining. Thirdly, the recombination is accompaniedby deletion. RECOMBINATIONAL MECHANISM Nucleotide sequence determination of the germline V and J segments and their flanking regions has revealed that conserved inverted repeat sequences are located immediately3’ and 5’ to the V and J segments, respectively (38, 102, 137, 139), The D segment is flanked by the similar sequences at both sides (89, 138). The conserved sequence is comprised of a nonomerand a heptamer separated by 12+1- or 23+l-bp essentially random nucleotides. Sequences of the nonomers and heptamers are not identical but are conserved enough to form stable complementary base pairs between the combining pair of segments, as shown in Figure 2. The nonomerand heptamer sequences are, therefore, considered to be at least a part of the recognition signal for putative V-(D)-J recombinase(s). The recognition sequence of the VKand JK segments have 23+l-bp and 12+l-bp spacers, respectively. By contrast, the nonomerand heptamer of the Vn and Jn segments are separated by 23+-l-bp spacers. The D segment has a 12+ 1-bp spacer on each side. Thus, the paired recognition signals have different lengths of the spacer sequences, which is called 12/23-bp spacer rule as originally proposed by Early et al (38). Since one turn of the DNA helix requires about 10.4 bp (168), the conserved spacing of 12 or 23 corresponds to either one or two turns of the helix. The two blocks of conserved nucleotides (nonomer and heptamer) are thus located on the 12

22

w,

A

B

C

D

C

D

12 A

B

22 A

B

C

D

A

B

C

O

Figure 2 Putative recombinational signal for V-(D)-J recombination. Solid rectangles show nonomer and heptamer recognition signals. The pair A and B form complementarybase pairs with C and D. Consensus sequences are A, CACAGTG; B, ACAAAAACC;C, GGTTTTTOT; D, CACTGTG.Numbers indicate average spacer nucleotides.

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sameside of the helix as if they formeda continuousstretch of 16 conserved nucleotides(38, 139). The intervening DNA segmentbetween the V and J segmentswas shown to be deleted in myelomas (27, 137, 145) in the samemanneras in the chain C gene rearrangement(66). Thedeletion is consistent with a stemloop structural modelbasedon the inverted repeat structure (102, 137). alternative modelto explain deletion is a sister chromatidexchangemodel (165) as originally proposedfor the H-chainC-genedeletion (115). modelwas proposedto account for the fact that DNA segments5’ to the JK segmentwere retained in somemyelomas,both of whoseJK segments were rearranged (156, 165). Most(76%)of the region upstreamof J~ retained in the populationof the C~-expressinglymphocytes,eventhough 68%of the J~ loci are rearranged. An inversion model was recently proposedto explain the unusual presenceof the upstreamregion of the J segment(see below). TheV-(D)-Jjoining, in general,is expectedto be preciseat the nucleotide level. Otherwise,the recombinationwouldoften result in the frame-shift mutation. However,the aminoacids at position 96 of somemyeloma kappa chains differ from that encodedin the correspondinggermline DNA segment(102, 137). The V-J joining seemsto have flexibility in the exact nucleotideposition of the recombination as long as it is in phasewith the codingsequence.Thefour possiblealternative sites of the in-framerecombination are shownin Table1. This flexibility providesfurther diversity at junctions of germline DNAsegments, which are located in the CDR. In case of VHgenesthere are manyinsertions or deletions at junctions with Dsegments.Theseinserted portions, termedN regions, wereproposed to be ascribed to the activity of terminal nucleotide transferase during YH’D-JH recombination(1). ABERRANT REARRANGEMENTS The flexibility at the V-J joining costs the generationof missensegenesto the organism.Theaberrant V-J joining wasreported in the kappaas well as lambdalocus (5, 10, 15, 26, 69, 90, 103,167).Eitherdeletionor insertion of a fewbasesresultedin out-of-~phase J sequences.In manyr-producingmyelomas two alleles of kappagenesare rearranged, one of whichis usually aberrantly rearranged. A cell line of MPC11 myelomaproduces an unusual fragment of the kappa chain in addition to a normal kappa chain. Aminoacid sequence determinationson an in vitro synthesized fragmentprecursor showedthat it consisted of a hydrophobicleader sequencejoined directly to the CK region and that the sequencecorrespondingto the Vregion, wascompletely lacking (19, 135). The gene encodingthis fragmentwasanalyzed, which

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at the recombination site

Germline genes

Codon generated ¯ - - AGT-TCT-CCT-CCCACAGTG ......

V~41


J

1

l I I[ CACTGTGG-TGG-ACG ........

......

Vt¢41

¯ ".AGT-TCT--CCT-CCCACAGTG ......

J2

....

I Ill CAGTGTGTG-TAC-ACG.........

TGG Trp CGG Arg CCG Pro CCC Pro TAC Tyr CAC His CCC Pro

Trc CTC Leu

J4

¯ .. o - c -cc -cccc oro...... I[il ...... CACTG.GA-TTC-ACG .........

V~41

¯ ¯ ¯ AGT-TCT--CCT-CCCACAGTG ......

CTC Leu

Vt¢41

Illl ......

CCC Pro

ccc ~ro

CACTGTGG-CTC-ACG.........

aTaken from Max et al (102). Codons are shown by hyphens. Recombination signal underlined; vertical lines indicate possible recombinationsites.

showed that it was generated by aberrant recombination of a V~ segment with the middle of the intervening sequence between the J~ segments and the Cr gene (140, 143). The resulting deletion of the Jr segment, including the splicing signals, allowed an abnormal RNAsplicing to create mRNA containing the leader and CKregion sequences. This abnormal rearrangement seems to be due to homologousrecombination between the putative recombinational signal 3’ to the V segment and the homologoussequence in the intervening sequence (IVS) between the Jr segment and the C~gene. Alt & Baltimore (1) found that the inverted Di-Jm and its 3’ flanking sequence is linked to the 5’ side of the JH3 segment in a spontaneously rearranged clone of Abelson routine leukemia virus-transformed cells. In addition, the inverted segment extends through the inverted Jn2 nonomer recognition sequence and ends abruptly at the terminus of the inverted Jn2 heptamer recognition sequence. The inverted Jm sequence is replaced by the noninverted 5’ flanking sequence of a D segmentat this point (Figure 3). Such an unusual structure has to be explained by intrachromosomal recombination because inverted joinings between sister chromatids would have resulted in one dicentric chromosomeand another lacking a centromere. The authors favor the looping-out model for deletion, and argue that the retention of the reciprocal product described above (the region upstream of J) can be explained by inversion. Lewis et al (92) provided further evidence

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D,

J~


I

02 5*flonldng "

J2

Ja

Ji DI J3 I

507

J4

J4

¯

Figure 3 Inverted joining of D and J segments. Top and bottom lines indicate germline and rearranged DNAs,respectively. ~ andS, 5’ and 3’ recognition nanomer, respectively. ( and ~, 5’ and 3’ recognition heptamer, respectively. Arrows indicate direction of DNAin germline configuration. Rearranged from Alt & Baltimore (1).

to support the inversion model using a spontaneously rearranging Abelson virus-transformed cell line, which seemsto be useful in analyzing differentiation steps. SomeT lymphomas,T hybridomas, and T-cell lines were shownto have rearrangement at the Jx segment (28, 50). Most of them seem to be D-Jn rearrangement ($9), indicating that D-Jx recombination initiates at about the same time as the differentiation of T and B lymphocytes.

Somatic Mutation Studies on the murine Lxl chains produced by myelomacells provided the first evidence for somatic point mutation of the V-region gene. Aminoacid sequences of 19 Vxl chains were determined and were divided into seven groups that differed from a common sequence (V~0) by one to three residues (24, 169). Subsequent DNAsequence determination of a rearranged Vxl gene from H2020 myelomaand the germline Vxl gene demonstrated that the two sequencesdiffer at two positions in CDRsand at one in the flanking region (11). More extensive studies on V~,, V~, and VI~ genes, however, indicate that the substitution is not restricted to either the CDRor the coding region and seems to spread into IVS and flanking regions (16, 31, 54, 69, 76, 83, 123, 139, 146). But no mutations were found in C genes linked to V genes. The C genes of both ~l and ),2a chains of the $43 hybridomas are germline DNAsequences, although both V genes contain mutations (16, 141). These results are unable to settle the long argument of whether there exists a special enzymicmechanismthat preferentially introduces mutations to the V gene or whetherthe selection at the cellular level is responsible. However, there seems to be some mechanism that accumulates mutations only around the V-coding sequence and not in the C gene. The Vxl gene of MOPC315 myeloma also shows somatic mutations, although it is an aberrantly rearranged gene (69). Similarly, both of the rearranged V~genes,

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whether expressed or unexpressed, showed similar levels of somatic mutation (123). There are several expressed V genes that have no somatic mutations (144). It is not clear whether inactive germline V segments in antibody-producing cell have substitutions or not, although everybody assumes that DNArearrangement is obligatory for the introduction of somatic mutation into the V segment. Many VL and Vn genes in IgM-producing lymphocytes are germline sequence, whereas most of VLand VI~ genes in IgG- or IgA-producing lymphocytes have substitutions (16, 53). These authors claim that class switching maytrigger somatic mutation. Since the B lymphocyte is stimulated and divides extensively during or after class switching, the chance of somatic mutation is expected to be higher for non-IgM-producing lymphocytes. It is not knownwhether or not class switching is coupled with somatic mutation. Genetic Basis for Variable-Region Diversity After many years of controversy, DNAcloning succeeded in convincing immunologists unanimously on how V-region diversity is generated in a broad view. Nature seems to be generous to everybody who has proposed any mechanismin such a way that everybodyis right in one way or the other (132). Four types of mechanismsgenerate the V-region diversity. First of all, the numberof the germ-line V segment is reasonably great, probably somewhere between 100 and 1000. This is due to the evolutionary process. Secondly, the recombinational joining of the V, (D), and J segments plays a very important role in amplifying the V-region diversity. The combination of each segment is assumed to be random. Thirdly, the flexibility at the recombination sites augmentsdiversity significantly. Finally, the somatic mutation also contributes to increase the V-regiondiversity. It is diiticult, however, to assess the extent of contribution of each mechanism. The minimal possible diversity due to the three mechanismsother than somatic mutation can be calculated as follows: Vr diversity of mouse= number of V~ (100) X numberof JK (4) × flexibility of joining (2) ---- 800; diversity of mouse = number of VH(200) X number of D (20) X number of Jn (4) X flexibility of joining (2 X 2) = 64,000. Provided any V~and n can associate to form a different antigen binding activity, the possible diversity would be 5.1 X l07 (= 800 X 64,000). The basic principle for all the genetic mechanismsto amplify the antibodydiversity is the randomexpression of all the possible genetic information by the enormous flexibility inherent in the immunoglobulin gene system. Obviously, the selection of the randomly expressed information at the cellular level is a key to constructing the useful defense systemfor the

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organism [clonal selection theory (18)]. The number and sequence of segmentsare out of control at the somatic level but certainly are controlled by the balance between evolutionary selection and changes (drift, gene conversion, unequal crossing over, etc).


Allelic Exclusion In general, only one species of the immunoglobulin is synthesized and secreted by a lymphocyte. The failure of expression of the immunoglobulin genes from more than one chromosomeis knownas allelic exclusion, the mechanismof which has been the subject of somedebate (2, 40, 124). Either a r or k light chain is produced, but not both, in a single lymphocyte (isotype exclusion). It is clear that the exclusion operates at the functional protein but not at transcription of the immunoglobulin genes, as MOPC104E produces both r and k chain mRNA’s,of which only ~ chain mRNA is translated into a stable protein (2, 75, 121). Since manylymphocytes, hybridomas, and myelomascontain at least one unrearranged Jr allele (72, 124), there must be some mechanismthat terminates further V~-J~ rearrangement on the other chromosome.In case the JK segments of both chromosomesare rearranged, one of themis usually defective (10, 15, 26). Theseobservations are consistent with a stochastic model: that the V-(D)-J recombination, which has a certain chance of failure, continues until the complete V genes are formed. The formation of the complete V gene, in turn, wouldshut off the activity of a putative recombinase. In the case of myelomaS107, however, both of the VKalleles are rearranged and transcribed, and two different kappa chains are produced (90). Only one of them is able to form a complex with the alpha chain and is secreted into the medium.The result is also explained by the stochastic model, assumingthe shut-off signal is not the appearance of the L chain in the cytoplasm but the appearance of a complete immunoglobulin on the membraneor in the cytoplasm. The H-chain genes have a higher proportion of nonproductive rearrangements in B lymphocytes as well as in hybddomasand myelomas(25, 26, 70, 114). It is not knownwhether the lower chance of successful joining of the VI~ locus is simply due to one additional recombinational event (V-D and D-J), or to the difference in the recombinatodal machineryas compared with the VL gene. Recently, Wahl& Steinberg (166) found an H-chain-binding protein that binds to free immunoglobulinH chains in pre-B cells, and proposed that the displacement of the H-chain’ binding protein by the L chain terminates the enzymesystem that catalyzes DNArearrangement at the L-chain loci. To explain exclusion of the H-chain locus they also proposed that the H

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chains are so toxic that those cells producingHchains fromboth alleles of the H-chainlocus are killed. Order of the V Gene Rearrangement During Differentiation Duringontogeny,the VHlocus is the first to be rearranged,resulting in the expression of cytoplasmic~ chains in the pre-B cell. Subsequently,the VLlOCUSundergoesthe rearrangement, and IgMmolecules are formedto serve as surface receptors. TheV~geneseemsto be rearrangedbefore the Vxgene, as the J~ geneswereeither ddetedor rearrangedin all 10 of human leukemiacell lines producing h chains (60). By contrast, the Jx genes remainedunrearrangedin eight humanB-cell leukemialines producing r chains. In mice, the hierarchyis less evident, as a nonproductive Vxrearrangementin a r-producing cell line was seen (26). The rearrangement order of the V~and Yx genes maybe regulated developmentally (60). Alternatively, the efficiency of recombination maysimplyfavor the V~gene locus. Evolution

of V-Region Genes

ORGANIZATION The $2

mieroglobulin L chain of the histoeompatibility antigen was shownto have homologousstructure with immunoglobulinL chains and was considered to be evolved from a common ancestor. The /32 microglobulingenewasrecently clonedand wasshownto be interrupted by IVSsat the aminoacid positions +3 and -t-95 (122). Thefirst intron the/32 microglobulingene corresponds to that betweenthe exons of the signal peptide and the V segment. Thelocation of the second IVSin the /32 microglobulingene is similar to the junction betweena VI. and a J~. segmentor betweena Vn and a D segment. The results not only suggest that the prototypeof the immunoglobulin geneis a split genelike V-J, but they also implythat the third exonof the/32 microglobulingenemaybe the evolutionary ancestor to both D and J segments.The V segmentmayhave evolved from the first and second exons. If so, one can assumethat a prototypeV-Jgenerepeatedduplicationeither as a set or as a separateexon. PrototypeJr~ segmentslocated within the Vr~cluster mighthave diverged into D segments, whereas prototype J segmentsclosest to the Ct, gene changedinto the present Jn segments. Fromthis evolutionary consideration, the homologousV segmentsare expected to cluster, as shownin several systems (31, 76, 81, 131). UnknownD segments maybe found dispersedin the Vri cluster. numbers of the V, D, and J segmentsand their variability contribute to the V-region diversity enormously.Thesefactors dependentirely on the evolutionary EVOLUTIONARY ORIGIN OF V-REGION DIVERSITY The

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process. Whatis the mechanismthat allows the accumulationof a large numberof variables and yet maintains the basic frameworkstructure amongthe hundredsof related genes9. This problemhas been discussed theoretically as concertedevolution(51, 154). Ohta(117) showedthat the rate of the aminoacid substitution in CDR is as high as that of the fibrinopeptide,the mostrapidly divergingprotein so far known.Thesubstitution rate in the other portion of the Vregion is one third of that in CDR,in agreementwith the DNA sequence analysis of closely related VHsegments(55). This result suggeststhat the variability in the Vsegmentis mostlydue to the accumulationof randomand neutral base changes. In other words, the V segmentis under weakest selection pressureat the proteinlevel, as the nucleotidedivergencerate at the synonymousposition of the V segmentis the sameas manyother genes(107). The homologyin the frameworkregion is maintained by domain(or segment) transfer betweendifferent loci by either geneconversionor doubleunequal crossing over, as indicated by computersimulationstudies ’ ’ (13, 153). Assuming that the V segmentdiverges by the balance of drift andrecombinational homogenization, Ohta(117) has evaluateddivergencerates of the V segmentusing a populationgenetics approach,and her calculated values fit well with observedvalues. Ohta(118) indicated that a high degree polymorphism and non-allelic diversity in the major histocompatibility complexcan be explained by a similar domaintransfer mechanism. The comparisonof the nucleotide sequencesof the closely related VH segmentsindicates that the rate of the replacementsubstitutions in CDR1 and CDR2are greater than in frameworkregions, indicating that the diversity of the hypervariableregionis generated,at least in part, through an evolutionaryprocess (55). TheVsegmentseemsto movequickly during evolution since restriction fragmentshybridizingto a VHor VKprobeare very different amonglaboratory mousestrains and wild mice (30, 77). Wu& Kabat (171) found that CDR2of a humanV segment contains nucleotides identical to a humanD segment.They proposedthat a minigene-like D segmentmaybe involved in the generation of CDRdiversity by a gene conversion mechanism.The minigene theory was originally proposedas a somatic mechanism to explain V-regiondiversity (73), and it predicted the presence of the Dsegment.Seidmanet al (142) proposed that recombinationand mismatchrepair mightbe a likely mechanismto explain extensive variability in CDR.Theyhaveshownthere is extensive homologyin the flanking regions of related V segments,whichwouldbe consistent with frequent recombinationamongV segments(23). In summary, two factors are involvedin the evolutionarydrive to amplify the V-segmentdiversity: (a) the frequent recombination(gene conversion and/or doubleunequalcrossing over) facilitated by the organizationof the

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V-segment family;and (b) very lowselection pressure at the protein level. Thesetwo factors workin opposite directions and reach the equilibrium. V-REGION GENESIN OTHER SPECIESThe rabbit Vn sequence of a cDNAclone was determined and was shownto contain cDNAsequence homologousto a humanD1 in CDR2 (12). The implication wouldbe that the similar Dsegmentis presentin the rabbit genome andis involvedin gene conversion. Rat J~ segmentswere cloned, and their structural analyses showedthat six JK segmentsseemedto be evolvedby recent two-sequential crossing-overevents as comparedwith the mouseJK cluster (20, 147). CONSTANT-REGION Structure

GENES

and Organization

ORGANIZATION OF CONSTANT-REGION GENESOnly one copy of the C~gene is represented in the mouseand humangenomes.TheC~ genes of mouseand manare located 2.1-2.3 kb 3v to the J segment(61, 102, 137). The distance between the V~segments and C~gene is unknown.The Cx locus is muchmorecomplexthan the C~ locus because there are four Cx genes in the mousegenome.Thepair of the Cx3and Cx~genes and that of the Cx2and Cx4genesare separately linked (14, 105). EachCxgenehas its ownJx segment1.2-1.3 kb upstream. TheCx4gene seemsto be a pseudogene becausethe Jh4 has 2-bp deletion in the heptamerof the recombinational signal. MultipleCa genesare also found in the humangenome(59). Six humanCxgenes were physically linked within a 30-kb region. Each Cxgeneis supposedto haveits ownJx segment.At least five other clones hybridizing with the Cxprobe were isolated, although none of themare linked to the majorCxcluster. Oneof themwasidentified as a processed gene not located on chromosome 22 (63). ThemouseCngene locus consists of eight genes (/x, 8, y3, yl, y2b, y2a, e, and a) that cluster in a 200-kbregion as shownin Figure 4 (149). Theorder of the mouseCagenes5’-Ct~-C~-C~,3-Cyl-C~,2b-C~,2a-C~-Cg-3’ is consistent with the order proposedby the deletion profile of the Cngene myelomas producingdifferent classes of the immunogloblin (66). Unlikethe CLgene, the CHgenes share one set of JH segments(148), whichprovides a genetic basis for H-chainclass switching, as discussedbelow.Themouse Cn gene locus does not contain any well-conservedpseudogene. ThehumanCa genefamily consists of at least nine genes, (/.t, 8, yl, 72, 73, 74, e, al, and or2). Thegeneral organizationof the humanCngene duster is similar to mousein that the C8geneis located several kilobases 3’ to the Ct, gene and that at least someC~, genes are linked (45, 87,

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0

50

I00

150 kiloba~es

513

~00

Figure 4 CH gene organization.

127, 159). However,the Ce-C~region is duplicated in the humangenome (100, 111). As it is not knownwhether or not all the Cy genes form one cluster, we should consider the possibility that the C~,-C,-C~region might be duplicated as a set. In contrast to the mouse CHgene duster, the human CHgene family contains at least three pseudogenes, one Cy (159) and two Ce (7, 49, 100, 112, 164). The S region of the r pseudogene i s r eplaced by c ompletely different reiterated sequences,althoughthe defect in the codingregion is not known.The C~2pseudogenehas deleted the first two exons and the Ce3 is a processed gene. SEQUENCES OF CONSTANT-REGION GENES All the mouse constant-region genes except for the Cx3 and Cx4 genes were sequenced. The C domainof the L chain is encoded by a single exon (4, 11, 15, 101, 170). As shownin Figure 5, the CH genes are composedof three (~, ~) or four (yl, "g2a, y2b, y3, ~, and bt) exons, each encoding a functional and structural unit of the H chain, namelya domainor a hinge region (22, 68, 71, 78, 120, 161, 162, 172, 173; F. Blattner, unpublisheddata). an exception, the hinge region of the C~chain is coded for by the Cmexon (162). Nucleotide sequences of humanCK(62) and Cx (59, 63) genes determined. The presence of the membrane-bound form of the immunoglobulin was suggested by analyses of the surface immunoglobulin of the B lymphocyte (79, 119, 151). These results indicate that the membrane-bound form of the immunoglobulinis larger than the secreted form. Exons encoding the hydrophobic transmembrane segment and the short intracytoplasmic segment were found about 2 kb 3’ to the exons encoding the last secreted domains of most mouse CHgenes: C~, (39, 134), Cy (133, 163, 174), C, (71), and C~ (22). All the membrane domains are encoded by two exons. The hydrophobic segment of the 26 amino acid residues is highly conserved amongall the H chains, suggesting that the membrane NUCLEOTIDE

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291

3~)6 39~ 321

291

306 A8107330 112 321 103

281

542

IZI 321 ~

LSItb I~1

L38kb 131

800

501

81

81

324 81 324 ?8 350 107 1~1~12~8181

Figure 5 Structures of CH genes. Oblique-lined, solid, dotted, and lined rectangles indicate domainexons, membraneexons, hinge exons, and 3’ untranslated regions, respectively. Numbers indicate nucleotides.

form of the immunoglobulinis anchored by a commonmembraneprotein (163, 174). Theintracytoplasmicsegmentsfall into two groups: one group 0,1, T2a, 72b, ~,3, and ¢) has a long segmentof 27 residues; the other (/~ and8) has a short two-residuesegment.If this portion has anything do with transmission of the triggering signal of the antigen-antibodyinteraction, the quality of the signal could be different betweentwo groups. Someof the humanCa genes werecompletelysequenced:~1 (46), ~2 (45), 74 (44), and¢ (49, 100, 164). S-S Recombination DELETION MODEL During differentiation of a single B lymphocyte, a given VHgeneis first expressedin combinationwith the C~geneof the same allelic chromosome, and later in the lineage of the B lymphocyte,the same VHgeneis expressedin combinationwith a different Ca gene. This phenomenonis called H-chainclass switch. Hybridization kinetic analyses in whichcDNA is used have shownthat specific Ca genes are deleted in mousemyelomas,dependingon the Cn genesexpressed(66). Theorder of the Cagenes, 5’-VHgenefamily-spacerCla-Cy3-Cyl-Cy2b-Cy2a-Ca-3’ wasconsistent with the assumptionthat the DNA segment between a Va and the Ca gene to be expressed is deleted, bringing these genesclose to each other. Deletionof the intervening DNA

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segment during H-chain class switch was confirmed by Southern blot hybridization analyses of myelomaand hybridoma DNAs,in which cloned immunoglobulingenes were used (25, 29, 70, 125, 175, 176). Such studies also support the proposed order of Co genes. Comparisonof germline and rearranged H-chain genes led to the proposal that a complete H-chain gene is formed by at least two types of recombinationalevents, as shownin Figure 6 (32, 75, 94, 158). The first type of recombinationtakes place between a given V~I segment, a D segment, and a Jn segment, completing a V-region sequence. After such recombination, referred to as V-D-Jrecombination, the V-region sequence is expressed as a # chain because the Cv is located closest to the JH segment. The second type of recombination is called S-S recombination, which takes place between two S regions located at the 5’ side of each CHgene. The initial S:S recombination always involves the Sv region as one of the pair, and the S region of another class is involved as the other partner, resulting in the switch of the expressed CHgene from C~, to another Ca gene without alteration of the V region sequence. The subsequent switch, such as one from Cy to C, or Co, will require another S-S recombination. Both types of rearrangements are accompanied by deletion of the intervening DNA segment from the chromosome. This model postulates several important structural features in the H-chain gene organization: (a) the order of genes 5’-C#-Cs-Cy3-CTI-CTzb-Cyza-Ce-Ca-3’; (b) the presence of only one set of the JH region segment at the 5’ side of the C~, gene; and (c) the presence of the S region before each Cr~ gene. Studies on the organization of the C~I gene cluster provided direct evidence for the predictions (a) and (b), as described above.

V.

D d.

~ ~ ~3 "~ T2b ~f2o

:-’-"-"-"-"-"---" -m -u -n -m S~.

Sr~ S~ S~zb S~rz,,

.u chcz,’n synthesis V D d

E ~X -m -N S, So~

~ I

~ S- S recombination

synmes/s

S.~ s~a~ ¯ Figure6 Twotypes of DNA rearrangements in H-chaingenelocus. TakenfromHonjoet ~ (6s).

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STRUCTURE OFS REGIONS The S region was originally defined as functionally responsiblefor the class switch(S-S) recombination (75). Nucleotide sequence determination of the region surrounding the S-S recombinationsite showedthat the structural basis of the S region is the tandemrepetition of related nucleotidesequences,Thenucleotidesequence of the St‘ region was shownto be represented by [(GAGCT)nGGGGT]m, wheren varies between1 and 7 (most frequently 3) and m is approximately 150 (109). Theother S regions havealso tandemrepetition of unit sequences that are different from each other but that havein common with the St‘ region the short sequences GAGCT and GGGGT (37, 77, 110). Tandem repetitive sequencesof the S regionare often deletedpartially in different strains of mice(65, 67, 96, 149)and duringcloning(109). TheS region found 1-4 kb upstreamof each codingregion except for the C8gene. The length of S region, whichvaries from about 10 kb (SrI) to about 1 (S,), rather than the sequencehomology with the St, region, is related the amountof the immunoglobulin class in mousesera (110). TheS regions are generally conserved between humanand mouse(112, 129, 158). The St‘, So and S~regions, especially, are highly homologous betweenthe two species. MECHANISM OF S-S RECOMBINATION There are two different interpretations of the S-regionstructure. Onegroup(33, 34) considersthat the S-regionsequences of different CHgenes,say St‘ andS,,, are so different that the homologousrecombination is an unlikely mechanismfor the class switch. Instead, they proposethat class-specific "switching"proteins recognize the St, and Sn regions and bind to one another, thus juxtaposingthe St‘ and S,, regions, and thereby allowinga recombinationalevent to join them.The other (77, 109) puts moreweighton the short common sequences shared amongall the S regions and considers that the recognition of the homologoussequences is the basic mechanismresponsible for the class switch, althoughthey do not excludethe presenceof specific recombinases for class switching. The nucleotide sequences surrounding the S-S recombinationsites of hybridomasand myelomashave revealed the presence of TGGG or TGAG adjacent to the recombinationsite (110). Anotherrelatively conservedsequence, YAGGTTG, was found in the vicinity of the recombination site and wasdispersed in S regions (98). The S-regionsequencecontains many Chi sequences,promotersof the generalizedrecombinationin h phage(82), but the functional significance of such a sequencein the immunoglobulin geneis yet to be seen. Recentstudies with the clonedSt‘ andSa regionshave shownthat preferential recombinationcan take place betweenthe St‘ and S,, regions in Escherichiacoli extracts (T. Kataokaet al., submittedfor publication). Somecultured cell lines switch classes in the presence or

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absence of mutagens(42, 128, 155). The mechanismseems to involve recombination between homologouschromosomes,at least in one case (136). Twoalternative mechanisms could be proposedto explain the deletion of the interveningDNA segmentassociated with the class switch. Thefirst modelpostulates that the DNA segmentis looped out from a chromosome (looping-outmodel).In this case, the S-Srecombination takes place within a single chromosome, whichcan occur at any stage of the cell cycle. The resultant daughtercells are identical, unless the looping-outoccursbetween the S andMphasesof the cell cycle. Theother modelexplains the deletion of the DNAsegment by an unequal crossing-over event between sister chromatids [sister chromatid exchange model (115)]. This mechanism brings about asymmetricsegregation, giving rise to onedaughtercell with the deletion of Ca genesandto anotherwith the duplication. Mostcompelling evidencefor the sister chromatidexchangemodelis the presence of a short S~ sequence between the S~ and Sy~ sequences upstream from the expressed y~ gene in MC101 myeloma(67, 115). Since the S region is located in the IVS, the S-S recombinationdoes not haveto be highly specific to the nucleotidesthat are joined together. Instead, the S region-mediated recombination is expectedto be efficient becausethe class switch takes place within a short period of time after encounteringan antigen. Therefore,not a single exampleof the aberrant S-S recombination results in cripplingthe functionof the gene. However, a numberof abortive S-S recombinations that take place on the unexpressed chromosome are found in myelomasand hybridomas(29, 125, 176). The CH-genedeletion on the unexpressedchromosome appears to be a secondaryreaction because an examplein whichonly an expressed chromosome underwentS-S recombination was clearly demonstrated in BKC~:15 myelomaderived from a BALB/c-C57BL F1 animal (175). The myelomaproduces the y2b chain of BALB/callotype. Using restriction site polymorphismbetween two parental chromosomes, we have shownthat the expressed BALB/c-derived chromosome has deleted the C~ and C~ genes, whereas the CHgenes of the unexpressed C57BL-derivedchromosome are retained. IgG2b-producingMPC11 myelomacarries two rearranged Cy2bgenes, one of which is the expressed gene. The unexpressed chromosomeof MPC11 myelomaunderwent an abortive S-S recombination between the Sy3 andSy2bregions, resulting in the deletionof the Cy3andCr~genes(91). IgA-producingS107 myelomaalso has two rearranged Ca genes, one of whichcorrespondsto the ct chain producedby the S107cell, whereasthe other is an aberrant recombinantin whicha fragmentof DNA,not obviously related to the immunoglobulin Ca gene, has been joined to the S~ ABERRANT RECOMBINATION IN OR AROUND S REGIONS

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region (85). A similar DNA fragment, presumablyunrelated to the immunoglobulinCa locus, was found to be linked to the S,~ regions of an unexpressedCogeneof an IgA-produeingmyeloma J558 (57). Harris et also found that the rearrangementof the DNA segmentoccurs in 15 out of 20 plasmacytomas tested, including 73, 71, y2b, y2a, and ~r producers. Mostof them,however,do not involveS regions. Harris et al (57) suggested that this non-immunoglobulin-associated rearranging DNA maybe related to the frequent translocation of chromosome 15 to chromosome12 in plasmacytomas (86). Reciprocal translocation betweenchromosomes 8 and 14 in the Burkitt lymphoma cell line (Daudi)wasshownto take place near the Vacluster (47). Role of RNASplicing in Class Switching It is knownthat manyB lymphocytescarry morethan one isotype on their surface. Themost frequent double-bearerscarry both/~ and 8 chains with the sameV region on their surface. TheC~and C8genesare not rearranged in /~+8+ lymphomacells, suggesting that the RNA-splicingmechanism rather than the S-S recombinationmaybe responsible for the simultaneous synthesis of the/~ and 8 mRNA’s (93, 108). The completedVr~ gene may be transcribed as a large mRNA precursor containing both C~, and C8 sequences. Subsequentprocessing of the RNAwouldgive rise to mRNA’s codingfor either/~ or ~ chain, both havingthe identical Vasequence.A 8-producinglymphoma has deleted the C~, gene (108), although there is typical S region sequencein the 5’ flanking region of the C8gene(110). Yaoita et al (177) analyzedthe Ca geneorganizationof the/~+e+B cells that weresorted fromspleens of Nippostrogylus-stimulated SJA/9mice and found that none of the Ca genes or their flanking regions had undergone DNA rearrangements.Theresults also suggest that .the simultaneousexpression of the C~and C, genes is mediated by an RNA-splicingmeehanlsm. Yaoita et al proposed that class switching wouldproceed by two differentiationsteps: the first step involvesthe activationof the differential splicing, and the secondinvolves the S-S recombination,as shownin Figure 7. Thesecondstep is a likely place for T cells to affect B-cell switching, probablyby selection. AY2bchain-producingvariant of an Abelsonvirustransformedcell line was also shownto retain the Ct, gene (3). These observationsare consistent with the abovemodel.Noneof the abovestudies, however,haveprovidedthe direct evidencefor the primarytranscript longer than 90 kb, whichis presumablyin scarce quantity.

Evolution of C~Genes IVS-MEDIATED DOMAINTRANSFER Extensive

nucleotide

sequence

data allowed the tracing of the evolutionary history of immunoglobulin Ca genes. Miyata et al (106) comparednucleotide sequences of the

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cell

Figure 7 Order of DNArearrangements during B-cell differentiation.

mouseC~,l and Cy2b genes and found an intriguing homologyregion containing the Cmdomainand the 5’ half of the IVSbetweenthe Cmdomain and hinge region exons. Since they comparedonly the divergence at the synonymous position, this conservationof the nucleotidesequencesis free fromselective constraint at the protein level. Theyproposedthat this homology region is not due to the selection pressure at the nucleotide sequence,but to the correction mechanism through recombinationsuch as doubleunequalcrossing over. Theyhave predicted fromthe comparisonof the aminoacid sequenc.esof the 72b and 72a chains that a similar mechanismmust haveoperated, betweenthese two genes. Subsequently,the comparison of the nucleotide sequencesof the Cy1, C3,2b, and C~,2agenes revealed, as expected, the different homologyregions dependingon the comparedpair (120, 141, 173). The homologyregions consist of domains and the adjacent IVS. Similarly, the M1exonand the adjacent intron seem to have undergoneIVS-mediateddomaintransfer between the C~,~ and C~,2bgenes (174). There are two possible mechanisms for the recombinational correction: unequalcrossing-overevents, andgeneconversion(6, 41, 152). Unfortunately,it is almostimpossibleto distinguish the two mechanisms experimentally in mammals. Another exampleof dynamicexon shuffling has been obtained from structural analysesof humanC~, genes(159). ThehumanC~,3genehas four hinge exons, one of whichis most homologousto that of the humanC~, pseudogene.Theremaining three hinge exons are most homologousto the C~,~gene. The results suggest that the humanC73gene was created by unequalcrossing over betweenthe C~,~gene and the C~, pseudogene,followedby the successive duplication of the C71-typehinge exon.

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PSEUDOGENE Unlike the mouse CL and CHgenes, the humanC gene families contain several pseudogenes.One C×pseudogene(63) and one C, pseudogene(7, 164) are processed genes that lack entire introns and movedto different chromosomes.The evolutionary origin of such genes is an intriguing problem.In viewof the fact that the introns are removedprecisely at the splicing site, the RNAintermediate seemsto be a likely process. A retro-viral, long-terminal, repeat-like structure wasfoundadjacent to the processedC~pseudogene,suggestingthat retrovirus might have been involved in the creation of the processed gene (164). DIVERGENCEOF EPSILON GENE Comparison of

nucleotide sequences of the humanand mouseC~genes has shownthat the epsilon gene is under the weakestselection pressure at the protein level amongthe immunoglobulin and related genes, although the divergence at the synonymous position is similar (71). Theresults maysuggest that only a small portion of the epsilon chain is important, or that the epsilon gene is dispensable in

accordancewith the fact that, while IgE has only obscure roles in selfdefense,it has an undesirablerole as a mediatorof hypersensitivity.Another explanation proposed is that the humanand murine epsilon genes were derived fromdifferent ancestors duplicated a long time ago (paralogous). A rabbit 9/ chain eDNA clone was isolated from a eDNA library of hyperimmunized rabbit spleen mRNA (58, 99). There are at least two C~, genes in the rabbit genome. A Xenopus C~ chain cDNAdone was isolated from a eDNA library of Xenopusspleen mRNA (17). Theportion of the H-chainsequence contained in the plasmid (22-amino-acidresidues) is 35%homologous mammalian/zand "y sequences. The mRNA hybridized with this plasmid is 2.5 kb, in agreementwith the size of mousebt mRNA. CONSTANT-REGIONGENES OF OTHER SPECIES

ACKNOWLEDGMENTS

I amgrateful to Y. Nishida,T. Kataoka,andA. Shimizufor critical reading of the manuscript. I thank F. Oguni, S. Takeda, and T. Nomafor their assistance in preparingthis manuscript.This investigation wassupported by a special grant-in-aid from the Ministry of Education, Science and Culture of Japan,

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globulin heavy chains. J. Exp. Med. 151:1545-50 36. D’eustachio, P., Bothwell, A. L. M., Takaro, T. K., Baltimore, D., Ruddle, F. H. 1981. Chromosomal location of structural genes encodingmurineimmunoglobulinh light chains. J. Exp. Meal 153:793-800 37. Dunnick,W., Rabbitts, T. H., Milstein, C. 1980. Animmunoglobulindeletion mutantwith implicationsfor the heavychain switch and RNA splicing. Nature 286:669-75 38. Early, P., Huang, H., Davis, M., Calame, K., Hood, L. 1980. An immunoglobulin heavy chain variable region gene is generatedfromthree segments of DNA:Cn, D and Jn. Cell 19:981-92 39. Early, P., Rogers, J., Davis, M., Calame,K., Bond,M., Wall, R., Hood, L. 1980. TwomRNAs can be produced from a single immunoglobulin/~ gene by alternative RNAprocessing pathways. Cell 20:313-19 40. Early, P., Hood,L. 1981.Allelic exclusion and nonproductive immunoglobulin gene rearrangements.Cell 24:1-3 41. Egel, R. 1981. Intergenic conversion and reiterated genes. Nature 290: 191-92 42. Eckhardt,L. A., Tilley, S. A., Lang,R. B., Marcu,K. B., Birshtein, B. K.1982. DNArearrangements in MPC-11immunoglobulinheavychain class-switch variants. Proc. Natl. Acad. ScL USA 79:3006-10 43. Elliott, B. W.Jr., Eisen,H.N., Steiner, L. A. 1982.Unusualassociation of V, J and C regions in a mouse immunoglobulin h chain. Nature 299:559-61 44. Ellison, J., Buxbanm, J., Hood,L. 1981. Nucleotide sequence of a humanimmunoglobulinCT4gene. DNA1:11-18 45. Ellison, J., Hood,L. 1982.Linkageand sequence homologyof two humanimmunoglobulinT heavy chain constant regiongenes. Proc.Natl. Acad.ScLUSA 79:1984-88 46. Ellison, J., Berson,B. J., Hood,L. E. 1982.Thenueleotide sequenceof a humanimmunoglobulinCr~gene. Nucleic Acids Res~ 10:4071-79 47. Erikson, J., Finan, J., Nowell,P. C., Croce,C. M.1982. Translocationofimmunoglobulin VHgenes in Burkitt lymphoma.Proc. Natl. Acad. Sci. USA79: 5611-15 48. Erikson,J., Martinis, J., Croce,C. M. 1981. Assignmentof the genes for human h immunoglobulin chains to chromosome22. Nature 294:173-75

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and Welfare, NIH Publication No. 80:2008 75. Kataoka,T., Kawakami, T., Takahashi, N., Honjo, T. 1980. Rearrangementof immunoglobulin Tl-chain gent and mechanism for heavy-chain class switch. Proc. Natl. Acad.ScL USA77: 919-23 76. Kataoka,T., Nikaido, T., Miyata,T., Moriwaki,K., Honjo,T. 1982. Thenucleotide sequencesof rearranged and germlineimmunoglobulin VHgenes of a mouse myelomaMCI01and evolution of VHgenes in mouse.J. Biol. Chem. 10:277-85 77. Kataoka, T., Miyata, T., Honjo, T. 1981. Repetitive sequences in classswitch recombination regions of immunoglobulinheavy chain genes. Cell 23:357-68 78. Kawakami,T., Takahashi, N., Honjo, T. 1980. Completenucleotide sequence of mouseimmunoglobulinbt gene and comparisonwith other immunoglobulin heavychain genes. Nucleic Acids Res. 8:3933-45 79. K¢lmy,M., Ewald,S., Douglas,R., Sibley, C., Raschke,W., Fambrough,D., Hood, L. 1980. The immunoglobulin/x chains of membrane-boundand secreted IgMmoleculesdiffer in their Cterminal segments.Cell 21:393--406 80. Kemp,D. J., Cory, S., Adams,J. M. 1979. Clonedpairs of variable region genes for immunoglobulin heavychains isolated froma clone library of the entire mousegenome.Proc. Natl. Acad. Sci. USA76:4627-31 81. Kemp,D. I., Tyler, B., Bernard, O., Gough,N., Gerondakis,$., et al. 1981. J. Mol.AppliedGenet. 1:245-61 82. Kenter, A. L., Birshtein, B. K. 1980. Chi, a promotorof generalizedrecombination in )~ phage, is present in immunoglobulingenes. Nature 293:402-4 83. Kim,S., Davis,M., Sinn,E., Patten, P., Hood, L. 1981. Antibody diversity: somatic hypermutation of rearranged Vngenes. Cell 27:573-81 84. Kirsch,I. R., Morton,C. C., Nakahara, K., Leder, P. 1982. Humanimmunoglobulin heavy chain genes mapto a regionof translocations in malignantB lymphocytes.Science 216:301-3 85. Kitsch, I. R., Ravetch, J. V., Kwan, S.-P., Max,E. E., Ney,R. L., Leder,P. 1981. Multiple immunoglobulin switch region homologiesoutside the heavy chain constant region locus. Nature 293:585-87 86. Klein, G. 1981. Therole ofgenedosage

and genetic transpositions in carcinogenesis. Nature294:313-38 87. Krawinkel,U., Rabbitts, T. H. 1982. Comparisonof the hinge-coding segments in human immunoglobulm gammaheavychain genes and the linkage of the gamma2 and gamma4 subclass genes. EMBOZ 1:403-7 88. Kurosawa,Y., Tonegawa,S. 1982. Organization, structure, and assemblyof immunoglobulin heavy chain diversity DNAsegments. J. Exp. Med. 155: 201-18 89. Kurosawa,Y., yon Boehmer,H., Haas, W., Sakano, H., Trauneker, A., Tonegawa, S. 1981. Identification of D segments of immunoglobulinheavychain genes and their rearrangementin T lymphocytes. Nature 290:565-70 90. Kwan,S-P., Max,E. E., Seidman,J. G., Leder, P., Scharff, M. D. 1981. Two kappa immunoglobulingenes are expressed in the myeloma S107.Cell 26: 57-66 91. Lang,R. B., Stanton, L. W., Marcu,K. B. 1981. On immunoglobulin heavy chain gene switching: Two~/2b genes are rearrangedvia switch sequencesin MPC11 cells but onlyoneis expressed. Nucleic.4cids Res. 10:611-30 92. Lewis,S., Rosenberg, N., A.lt, F., Baltimore, D. 1982. Continuing kappa-gene rearrangement in a cell line transformed by abelsoinmurineleukemiavirus. Cell 30:807-16 93. Maki, R., Roeder,W., Traunecker,A., Sidman,C., Wabl,M., et al. 1981. The role of DNA rearrangementand alternative RNAprocessing in the expression of immunoglobulindelta genes. Cell 24:353-65 94. Maki,R., Traunecker,A., Sakano,H., Roeder, W., Tonegawa,S. 1980. Exon shuffling generates an immunoglobulin heavychain gene. Proc.Natl. Acad.ScL USA 77:213842 95. Malcolm,S., Barton, P., Murphy,C., Ferguson-Smith, M.A., Bentley, D. L., Rabbitts, T. H. 1982. Localization of humanimmunoglobulinK light chain variableregiongenesto the short annof chromosome by inSci. situ USA hybridization. Proc. NatL2Acad. 79:4957-61 96. Marcu,K. B., Banerji, J., Penneavage, N. A., Lang,R., Arnheim,N. 1980. 5’ Flanking region of immunoglobulin heavychain constant region genes displays length heterogeneityin germlines of inbred mousestrains. Cell 22:187-96 97. Mareu, K. B. 1982. Immunoglobulin heavy-chainconstant-regiongenes. Cell 29:719-21

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98. Marcu,K. B., Lang,R. B., Stanton, W. 109. Nikaido,T., Nakai,S., Honjo,T. 1981. L., Harris, L. J. 1982.A modelfor the Theswitch (S) region of the immunomolecular requirements of immunoglobulin Cu geneis composed of simple globulin heavychain class switching. tandemrdpetitive sequences. Nature Nature 298:87-89 292:845-48 99. Martens,C. L., Moore,K. W., Stein- 110. Nikaido, T., Yamawaki-Kataoka, Y., metz, M., Hood, L., Knight, K. L. Honjo,T. 1982. Nueleotide sequences 1982.Heavychain genesof rabbit IgG: of switch regions of immunoglobulin C, genes257:7322-29 and their comparison. Isolation of a eDNA encodingV heavy J. and Biol.C~Chem. chain and identification of twogenomic C~genes. Proc. NatLAcad. Sci. USA 111. Nishida, Y., Hisajima, H., Ueda, S., 79:6018-22 Takahashi,N., Honjo,T. 1982. In Organization and Structure of Humanim100. Max,E. E., Battey,J., Ney,R., Kirsch, munoglobulinGenes: Proc. TakedaScL I. R., Leder, P. 1982. Duplicationand Found. Symp., ed. Y. Yamamura.In deletion in the humanimmunoglobulin press. NewYork: Academic ¢ genes. Cell 29:691-99 101. Max,E. E., Maizel,J. V. Jr., Leder,P. 112. Nishida, Y., Miki, T., Hisajima, H., Honjo,T. 1982. Cloningof humanim1981.Thenucleotidesequenceof a 5.5munoglobulin~ chain genes: Evidence kilobase DNAsegmentcontaining the for multipleC, genes.Proc.Natl. Acad. mouseK immunoglobulinJ and C reSc£ USA79:3833-37 gion genes. J. Biol. Chem.256:5116-20 113. Nishioka,Y., Leder,P. 1980.Organiza102. Max,E. E., Seidman,J. G., Leder, P. tion andcompletesequenceof identical 1979.Sequences of five potential recomembryonic and plasmacytoma ~ Vbination sites encodedclose to an imregion genes. Z Biol. Chem. 255: munoglobulin K constant region gene. Proc. NatL Acad. ScL USA76:3450-54 3691-94 103. Max,E. E., Seidman,J. G., Miller, H., 114. Nottenburg,C., Weissman,I. L. 1981. C~, gene rearrangement of mouseimLeder, P. 1980.Variationin the crossmunoglobulingenes in normalB cells over point of kappa immunoglobulin occurs on both the expressed and nongene V-J recombination:Evidencefrom expressed chromosomes.Proc. Natl. a cryptic gene. Cell 21:793-99 Acad. Sci. USA78:484-88 104. Mcbride,O. W.,Hieter, P. A,, Hollis, G. F., Swan,D., Otey,M.C., Leder, P. 115. Obata, M., Kataoka, T., Nakal, S., Yamagishi,H., Takahashi, N., et al. 1982. Chromosomal location of human 1981. Structure of a rearranged Tl kappa and lambda immunogiobulin chain geneand its implication to imlight chain constant region genes. J. munoglobulin class-switch Exp. Med. 155:1480-90 Proc. Natl. Acad. ScL USAmechanism. 78:2437-41 105. Miller, J., Selsing,E., Storb,U. 1982. 116. Ohno,S., Epplen,J. T., Matsunaga, T., Structural alterations in J regions of Hozumi,T. 1981. The curse of Promemouseimmunogiobulin h genes are astheus is laid uponthe immune system. sociated with differential geneexpresProg..411ergl,28:8-39 sion. Nature295:428-30 T. 1981.In Evolutionand Faria106. Miyata, T., Yasunaga,T., Yamawaki- 117. Ohta, tion of MultigeneFamilies,ed S. Lewin. Kataoka, Y., Obata, M., Honjo, T. Berlin: Springer 1980. Nucleotide sequencedivergence 118.Ohta, T. 1982. Allelie and nonallelic of mouseimmunoglobulin ~/1 and ~/2b homology of a supergenefamily. Proc. chain genes and the hypothesis of inNatl. Acad.Sci. USA79:3251-54 tervening sequence-mediateddomain 119. Oi, V. T., Bryan,V. M., Herzenberg,L. transfer. Proc. Natl. Acad. Sci. USA A. 1980. LymphocytemembraneIgG 77:2143-47 and secreted IgGare structurally and 107. Miyata,T., Yasunaga,T., Nishida, T. allotypieally distinct. J. Exp. Med. 1980. Nucleotide sequencedivergence 151:1260-74 and functional constraint in mRNA 120. Ollo, R., Autfray, C., Morchamps, C., evolution. Proc. Natl. Acad. Sc~ USA Rougeon, F. 1981. Comparison of 77:7328-32 mouse immunoglobulinT2a and ),2b 108. Moore,K. W., Rogers,J., Hunkapiller, chain genes suggests that exonscan be T., Early, P., Nottenburg,C., et al. exchangedbetweengenes in a multi1981. Expressionof IgD mayuse both genic family. Proc.Natl. Acad.Sc£ USA DNArearrangement and RNAsplicing 8:2442-46 mechanisms. Proc. Natl. dcad. Sc£ USA 121. Ono, M., Kawakami,M., Kataoka, T., 78:1800-4 Honjo, T. 1977. Existence of both

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kappaand lambdah.’ght chain messenger RNAsequences m mousemyeloma, MOPC-104E, knownas a lambda chain producer. Biochem.Biophyz gez Commun. 74:796-802 122. Parnes, J. R., Seidman,J. G. 1982. Structure of wild-type and mutant mousefl2-microglobulingenes. Cell 29: 661-69 123. Pech, M., Hoehtl, J., Schnell, H., Zathan, H. G. 1981. Differences between ~ermlineand rearranged immunoglobulin Vxcodingsequencessuggesta localized mutation mechanism. Nature 291:668-70 124. Perry, R., Kelley, D., Coleclough,C., Seidman, J., Leder,P., et al. 1980.Transcription of mouseKchain genes:implicationsSci. for allelic exclusion.Proc.Natl. Acad. USA77:1937-41 125. Rabbitts, J. H., Forster, A., Dunniek, W., Bentley, D. L. 1980. The role of gene delection in the immunoglobulin heavy chain switch. Nature283:351-56 126. Rabbitts, T. H., Bentley, D. L., Milstein, C. P. 1981. Human antibody genes: V gene variability and Cn gene switching strategies, lmmunol.Rev. 59:69-91 127. Rabbitts, T. H., Forster, A., Milstein, C. P. 1981. Humanimmunoglobulin heavychain genes: evolutionary comparisons of C~, C~ and C~genes and associated switch sequences. Nucleic Acids Res. 9:4509-24 128. Radbruck,A., Liesegang,B., Rajewsky, K. 1980.Isolation of variants of mouse m~,elomaX63that express changedimmunoglobulin class. Proc. Natl. Acad. Sci. USA77:2909-13 129. Ravetch,J. V., Kirsch,I. R., Leder,P. 1980. Evolutionary approach to the question of immunoglobulin heavy chain switching: Evidencefromcloned humanand mousegenes. Proc. Natl. Acad. ScL USA77:6734-38 130. Ravetch,J. V., Siebenlist, U., Korsmeyer, S., Waidmann,T., Leder, P. 1982. Structure of the humanimmunoglobulin /~ locus: Characterizationof embryonic and rearranged J and D genes. Cell 27:583-91 131. Reehavi,G., Bienz, B., Ram,D., BenNeriah,Y., Cohen,J. B., et al. 1982. Organizationand evolution of immunoglobulinVn genesubgroups.Proc.Natl. Acad. Sci. USA79:4405-9 132. Robertson, M. 1981. Genes of lymphocytesI: Diversemeansto antibody diversity. Nature290:625-27 133. Rogers,J., Choi,E., Souza,L., Carter, C., Word,C., et al. 1981. Geneseg-

merits encoding transmembranecarboxyl termini of immunoglobulinT chains. Cell 26:19-27 134. Rogers, J., Early, P., Carter, C., Calame,K., Bond,M., et al. 1980. Two mRNAs with different 3) ends encode membrane-bound and secreted forms of immunoglobulin ~u chain. Cell 20:303-12 135. Rose, S. M., Kuehl, W.M., Smith, 13. P. 1977. ClonedMPC11 myelomacells express two kappagenes: a gene for a completelight chain and a gene for a constant region polypeptide. Cell 12:453-62 136. Sablitzky, F., Radbruch,A., Rajewsky, K. 1982, Spontaneousimmunoglobulin class switching in myelomaand hybridomacell lines differs froml~hysiological class switching. Immunol.Rev. 67:59-72 137. Sakano,H., Huppi,K., Heinrich, G., Tonegawa,S. 1979. Sequencesat the somaticrecombinationsites ofimmunoglobulin light-chain genes. Nature 280:288-94 138. Sakano, H., Kurosawa,Y., Weigert, M., Tonegawa,S. 1981, Identification andnueleotide sequenceof a diversity DNAsegment (D) of immunoglobulin heavy-chaingenes. Nature 290:562-65 139. Sakano, H., Maki, R., Kurosawa,Y., Roeder, W., Tonegawa,S. 1980. Two types of somaticrecombinationare necessary for the generation of complete immunoglobulinheavy-chain genes. Nature 286:676-83 140. Schnell, H., Steinmetz,M., Zachan,H. G. 1980. Anunusual translocation of immunoglobulin gene segmentsin variants of the mouse myelomaMPCll. Nature 286:170-73 141. Schreier, P. H., Bothwell, A. L. M., Mueller-Hill, B., Baltimore, D. 1981. Multipledifferences betweenthe nucleic acid sequences of the IgG2# andIgG2ab alleles of the mouse.Proc. Natl. Acad. Sci. USA78:4495-99 142. Seidman,J. G., Leder, A., Nan, M., Norman.B., Leder, P. 1978. Antibody diversity. Science 202:11-17 143. Seidman,J. G., Leder, P. 1980. A mutant immunoglobulinlight chain is formedby aberrant DNA-andRNAsplicing events. Nature 286:779-83 144. Scidman, J.G.,Max,E.E.,Lcdcr, P. 1979.A K-immunoglobulin gencis formed bysite-specific recombination without furthcr somatic mutation. Nature280:370-75 145. Seidman,J. G., Nan, M. M., Norman, B., Kwan,S. P., Seharff, M.andLeder,

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IMMUNOGLOBULIN GENES P. 1980. Immunoglobulin V/J recombination is accompaniedby deletion of joining site and variable region segments. Proc. Natl. Acad. Sc~ USd77: 6022-26 146. Selsing,E., Storb, U. 1981.Somaticmutation of immunoglobulin light-chain variable-regiongenes. Cell 25:47-28 147. Sheppard,J. W., Gutman,G. A. 1982. Rat kappa-chainJ-segment genes: Two recent gene duplications separate rat and mouse. Ce1129:121-27 148. Shimizu, A,, Takahashi, N., Yamawaki-Kataoka, Y., Nishida, Y., Kataoka, T., Honjo,T. 1981. Orderingof mouse immunoglobulin heavy chain genes by molecularcloning. Nature289:49-53 149. Shimizu,A., Takahashi,N., Yaoita, Y., Honjo,T. 1982. Organizationof constant region genefamily of the mouse immunoglobulin heavy chain. Cell 28: 499-506 150. Siebenlist, U., Ravctch,J. V., Korsmcycr, S., Waldmann,T., Ledcr, P. 1981. HumanimmunoglobulinD segments encoded in tandem multigenic fatuities. Nature294:632-35 151. Singer, P. A., Singer, H. H., Williamson, A. R. 1980. Different species of messenger RNAencode receptor and secretory IgMp chains differing at their carboxytermini. Nature285:294300 152. Slighton,J. L., Blechl,A.E., Smithies, O. 1980. Humanfetal oy. and ~T" globin genes: completenucleotidc sequences suggest that DNAcan be exchanged between these duplicated genes. Cell 21:627-38 153. Smith, G. P. 1974. Unequalcrossover andthe evolutionof multigenefamilies. Cold SpringHarborSyrnp. QuantBiol. 38:507-13 154. Smith, G. P., Hood,L., Fitch, W.M. 1971. Antibodydiversity. ~nn. Re~. Biocherr~40:969-1012 155. Stavnezer,J., ~Marcu, K. B., Sirlin, S., Alhadeff, B., Hammering, V. 1982. Rearrangementsand deletions of immunoglobulinheavychain genesin the double-producing B cell lymphomaI29. MoLCell. Biol. 2:1002-13 156. Steinmctz, M., Altenburger, W., Zachau, H. O. 1980. A rearranged DNA sequencepossiblyrelated to the translocat~on ofimmunogiobulingene segments. Nucleic//cids Rex 8:1709-20 157. Swan,D., D’eustachio, P., Leinwand, L., Seidman,J., Keithley, D., Ruddle, F. H. 1979. Chromosomal assignment of the mouset~ light chain genes.Proc. NatL Acad. Scl USA76:2735-39

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Cold Spring HarborSymp.QuantBiol. 41:837-46 170. Wu,G. E., Govindji, N., Hozumi,N., Murialdo, H. 1982. Nucleotide sequence of a chromosomalrearranged k2 immunoglobulin gene of mouse.Nucleic Acids Rex 19:3831-43 171. Wu,T. T., Kabat,E. A. 1982.Fourteen nucleotides in the secondcomplementarity-deterrnining region of a human heavy-chainvariable region gene are id.en.tieal witha sequencein a human D numgene.Proc. Natl. Acad. ScL USA 79:5031-32 172. Yamawaki-Kataoka, Y., Kataoka, T., Takahashi, N., Obata, M., Honjo, T. 1980. Completenucleotide sequenceof immunoglobulin "~2bchain genecloned from mouse DNA.Nature 283:786-89 173. Yamawaki-Kataoka,Y., Miyata, T., Honjo, T. 1981. Thecompletenucleotide sequenceof mouseimmunoglobulin "~2a gene and evolution of heavy chain genes:further evidencefor interveninlg sequence-mediated domain transl~r. Nucleic AcidzRes. 9:1365-81

174. Yamawaki-Kataoka,Y., Nakai, S., Miyata,T., Honjo,T. 1982.Nucleotide sequences of gene segments encoding membranedomains of immunoglobulin ~/ chains. Proc. NatLAcad.Sci. USA 79:2623-27 175. Yaoita, Y., Honjo,T. 1980.Deletionof immunoglobulin heavy chain genes fromexpressedallelic chromosome. Nature 286:850-53 176. Yaoita, Y., Honjo,T. 1980.Deletionof immuno.globulin heavychain genes aecompames the class switch rearrangement. Biomed.Res. 1:164-75 177. Yaoita, Y., Kumagai,Y., Okumura, K., Honjo, T. 1982. Expression of lymphocyte surface IgE does not require switch recombination. Nature 297: 697-99 178. Zeelon,E. P., Bothwell,A. L. M., Kantot, F., Schechter,I. 1981. Anexperimental approach to enumerate the genes coding for immunoglobulin variable-regions. Nucleic Acids Res. 9: 3809-20





Ann.Rev.Immunol.1983.1:529-68 Copyright ©1983by AnnualReviews Inc. All rightsreserved

GENES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX OF THE MOUSE Leroy Hood, Michael Steinmetz,

and Bernard Malissen

Divisionof Biology,CaliforniaInstitute of Technology, Pasadena, California91125 INTRODUCTION In the past 2 years our understanding of the major histocompatibility complexhas advanceddramatically becauseof the rapid progress madeby moleculargenetics. Biologists can nowbeginto dissect the molecularnature of one of the mostfundamentalproblemsin eukaryoticbiology--theability of organismsto discriminatebetweenself and nonself. Eventhe mostprimitive of metazoa,the sponges,exhibit cell-surface recognitionsystemscapable of identifying and destroying nonself, presumablyto preserve the integrity of individuals growingin densely populatedenvironments(34). For example,whentwo genetically identical spongesare apposed,the individuals fuse to form a single organism. However,whentwo genetically dissimilarspongesare joined, there is a reactionleadingto tissue destruction at the boundarybetweenthe two individuals (35, 36). Presumably,cellsurface structures recognizenonself and trigger effector mechanisms that lead to the destruction of the foreign tissue. Perhapsthe most striking feature of the moleculesinvolvedin these cell-surface recognitionphenomena is their enormousdiversity or polymorphism. Morethan 900 genetically distinct spongeshavethe capacity to reject oneanother after apposition (35). Self/nonselfrecognitionsystemsin other invertebratesandvertebrates appearto displaysimilar characteristics. Thus,three features are fundamental to self/nonselfrecognitionsystems---cell-surface recognitionstructures, effector mechanisms that lead to the destruction of nonself, and a high degree of polymorphism in the recognition structures. Mammals have self/nonself recognition systems encodedby a chromosomal region termed the major histocompatibility complex(MHC)with pre529 0732-0582/83/0410-0529502.00

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cisely these features. These systems regulate various aspects of the mammalianimmuneresponse. The same recognition systems were first identified in micebecauseof the existenceof inbred (geneticallyidentical) and congenic(genetically identical but for a single chromosomal region) strains of mice. Bythe grafting of tumors or skin amongsuch mice and followingrejection or acceptanceof the graft, it waspossible to mapthe rejection of nonself to a region on chromosome 17, whichwasthen denoted the MHC (30, 31). Theinitial characterizationof the cell-surface structures responsiblefor graft rejection utilized alloantibodies producedby crossimmunization of micewith spleen cells of micediffering only at the MHC. Later, recombinant congenic mice, which had undergonerecombination within the MHC,were produced. With these recombinantcongenic mice, specific alloantisera to even smaller portions of the MHC could be produced. Thus, inbred, congenic, and recombinantcongenicmice with their attendant potential for r~combinationalanalyses and the generation of highly specific alloantisera permitted immunogeneticists (45, 10Ci), later protein chemists(81, 90, 101), and very recently molecularbiologists characterize the MHC of the mousein considerabledetail. In this review, wediscuss the results obtained primarily from molecularanalyses of the MHC of the mouse. From the data available, the MHC of the mouse appears to be a general modelfor the MHCs of other mammals and perhaps other vertebrates. THE MURINE

MHC

Three Classes of Molecules Alloantisera specifie for gene productsof the routine MHC havepermitted the identification of three classes or familiesof moleculesdenotedI, II, and III (Figure 1). There are two categories of class I genes. Class I genes located in the left-hand side of the MHC or the H-2region encodecellsurface moleculestermed transplantation antigens, denotedK, D, and L, whichmediatethe graft rejection assay initially used to define the MHC (Figure 1). Theclass I geneslocated in the right-handportion of the MHC, denotedthe Qa-2,3and Tla regions, encodecell-surface antigens that are structurally related to the transplantationantigens,but that differ in tissue distribution and presumablyfunction (24, 107) (Figure 1). Class II genes, designatedA~, A~, E~, and E~, encodecell-surface Ia antigens that later werefoundto be identical to the immune response,or Ir genesthat control the magnitudeof the murineimmuneresponsesto different antigens (49). Class III genesencodeseveral components of the activation stages of the complement cascade (1). The availability of recombinantcongenicstrains and specific alloantisera to various geneproductsof the MHC of the mouse

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MHCGENES 531 has permitted the construction of a detailed genetic map(Figure 1). The MHC of manon chromosome 6 is remarkablysimilar to that of its mouse counterpart, exceptfor a presumedtranslocation in mice, whichled to the separationof the Kfromother class I loci and the existenceof additional class II loci in manwhosehomologueshave not yet been found in mice (Figure 1). Class I and class II moleculesand genes isolated from both murineand humancells are closely related.


Polymorphism Themost striking feature of the MHC in mouseand manis the extensive genetic polymorphism exhibited by certain class I and class II genes. For example,the Kand D class I genes appear to have 50 or morealldes in both wild and inbred populations of mice (46). Theclass I genes of the Qa-2,3andTla regions are far less polymorphic (24). Certain of the class II genes, Aaand Ea, also appear quite polymorphic, whereasanother, E~, is muchless polymorphic(47). Thefact that somegenesin the class and II families exhibit extensive polymorphism, whereasothers do not, is an interesting feature that mayreflect functionalrequirementsfor diversity

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see text.

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HOODET AL


in the polymorphic loci. Individual inbred micewill havedistinct combinations or constellations of these alleles. Eachindividual combinationof alleles is termed a haplotype. For example, the BALB/cmousewhose MHC has been most thoroughlystudied at the molecularlevel is of the d haplotypeand its gene productsare generally denotedby a capital letter d, Ada, w~th a superscript for the haplotypedesignation~e.g. Kd, Da, A~ E , and E_. Theextensive polymorphisms as well as certain other features h~e led t~ the hypothesisthat interesting genecorrection mechanisms are operating within the class I and II genefamilies (see below)(101, 102). Evolution The MHC has been found in all mammals and vertebrates studied to date. In each case with adequateinformation,there appearto be closely linked class I andclass II genes,andin those fewcases wherethe analysis has been carded out, associated class III genes. A controversy has arisen as to whetherthe class III genes should be regardedas an integral part of the MHC complexor, alternatively, as an inadvertent addition comparableto the MHC-linked genes encodingseveral apparently unrelated enzymes(48). Since weknowlittle about the structure, organization,or numbersof class III genesin contemporary or primitive vertebrates, it is dit~cult to argue either sideof this issue. Thelinkageof the class I, class II, andpossiblyclass III genesover the 500 millionyears of vertebrate divergenceposesinteresting questions about the selective constraints that havemaintainedlinkage amongthese genefamilies. Size The MHC spans an enormouschromosomalregion, as indicated by the followingsimplistic calculation. Thehaploid mousegenome includes about 1600cMof DNA by genetic analysis and contains 3 X 109 base pairs by biochemicalanalysis. Therefore, 1 centimorganequals approximately2000kb pairs of DNA.The MHC of the mouseis approximately 1-2 cM in length and, accordingly, should contain about 2000-4000kb pairs of DNA. The cloning of the MHC has involved a variety of approaches. Various eDNAprobes were used to isolate class I and II genes from genomic libraries. Restriction enzymemappingand chromosomal walkinghave defined clusters of linked MHC genes. These genes have been mappedinto regions of the MHC by gene transfer experimentsand restriction enzyme site polymorphisms--as is described subsequently. In the past 2 years, 25-50%of the mouse MHC has been cloned and several new insights concerningl~he structure and organization of these gene families have emerged.

Annual Reviews MHC GENES 533


CLASS I MOLECULES AND GENES ClassI Polypeptides The most thoroughly characterized class I molecules are the mousetransplantation antigens. These molecules are comprisedof a class I polypeptide that is 45,000 daltons and is an integral membraneprotein noncovalently associated with fl2-microglobulin, a 12,000-dalton polypeptide encoded by a gene located on chromosome2 in the mouse (26, 75, 94) (Figure Aminoadd sequence analyses have demonstrated that the transplantation antigen is divided into five domainsor regions (14, 114). The three external domains, ~1, a2, and a3, are each about 90 residues in length. The transmembraneregion is about 40 residues and the cytoplasmic domainis about 30 residues in length. The ~2 and ~3 domains have a centrally placed disulfide bridge spanning about 60 residues and up to three N-linked glycosyl units bound to attachment points in the el and e2 (Kb, Da) and also in the a3 (Kd, Ld, and Db) domains (68). The transmembrane region contains a hydrophobic and uncharged core of about 25 residues, which presumablytransverses the membrane.Binding studies with peptide fragments from class I molecules have shownthat the fl2-mieroglobulin subunit associates with the a3 domain (128). Prdiminary X-ray analyses of crystals from a proteolytic fragment of a humantransplantation antigen containing the al, ~2, a3, and fl2-microglobulin domainsdemonstrate that this class I molecule has a twofold axis of symmetry(P. Bjorkman,J. Strominger, and D. Wiley, personal communication)and, accordingly, maybe folded so that its four domains are paired in a symmetric manner similar to antibody ANTIBODY

THY-I

YY

~’e~

£

~

YYY, YYYYYY, YYYYYYYY

YYYYY

Figure 2 A schematic representation of the membrane orientations of Thy 1, class I, class II, and antibody molecules. Thy 1 is a T-cell differentiation antigen. Shaded domains indicate sequence homologies that suggest a commonevolutionary ancestry (see text).

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domains(Figure 2). Aminoacid sequence analyses suggest that the domain(114) and ~2-microglobulin(88) showhomologyto the constant region domainsof immunoglobulins, thus posingthe provocativepossibility that the genes encodingantibodies and transplantation antigens share a commonancestry. As mentionedabove, the class I genes fall into two categories--those mappinginto the H-2 region and encodingtransplantation antigens and those mappinginto the Qa-2,3 and Tla regions and encodinghomologous antigens that appearto be expressedin a tissue-specific manner(Figure 1). Transplantationantigens are found on virtually all nucleatedcells of the mouse. Somemice, such as the inbred BALB/c,have three or even more distinct transplantation antigens (K, D, L, and perhaps Mor R), whereas other miceappear to express fewer transplantation antigens (21, 33). The cell-surface antigens encodedin the Qa-2,3andTla regions can further be distinguishedfromthe classical transplantation antigens becausethey are significantly less polymorphic (24). Someof the Tla genes encode hematopoieticdifferentiation antigens, whichare found on thymocytesat an early developmental stage as well as on leukemiccells, whereasothers appear to be expressedonly on leukemiccells. TheQaantigens are found on manydifferent types of lymphoidcells. Wedenotethose class I antigens encodedby the Qaand Tla regions as hematopoieticdifferentiation antigens, recognizingthe provisional nature of this designation. Class I cDN/t Clones Twoprocedures were used to isolate mouseand humanclass I eDNA clones. First, eDNA clones wereselected for their ability to bind class I mRNA, which could be identified by in vitro translation and immunoprecipitation of the synthesizedpolypeptides(54, 89). As an alternative approach,short oligonucleotidesweresynthesizedafter reverse translation of the aminoacid sequencesof class I polypeptidesinto DNA languageand wereused as hybridization probes for the identification of class I cDNA clones or as primers to synthesize eDNA’s from unpurified mRNA’s (91, 108). Furthermore, mouseeDNA clones and humangenomicclones also wereisolated by cross-species hybridization with eDNA clones (44, 109). Essentially the sameapproacheswereemployedfor the isolation of mouse and humaneDNA clones for g2-microglobulin(85, 115) and class II polypeptides(3, 32, 51, 60, 64, 113, 118, 124), whichis discussedsubsequently. The class I eDNA clones were characterized by DNA sequenceanalysis to verify their authenticity. Mostof the mouseeDNA clones havenot been correlated with knownclass I moleculesmainlybecauseof the paucity of aminoacid sequencedata currently available. However,the eDNA clones d (56, 126) molecules have been identified. for the Kb (91), b ( 93), and K

Annual Reviews MHC GENES 535 The class I cDNAprobes were then used to screen genomic libraries to obtain clones that could be used to analyze the structure and organization of class I genes. Structure

ORGANIZATION Three mouse class I genes have been completely sequenced, and their structural features are depicted in Figure 3A. d) encode Ld transplantation Two of these genes (27.5 and CH4A-H-2L antigens (23, 76), whereas a third (27.1) has been mappedinto the Qa-2,3 region (111). It is not knownwhether gene 27.1 is expressed. The exonintron organization of these class I genes is remarkablysimilar. Each gene is divided into eight exons, which correlate precisely with the domainsof the class I polypeptide (111). The first exon encodes the leader sequence, whereas the second, third, and fourth exons encode the t~l, a2, and a3 domains. The fifth exon encodes the transmembranedomainand the sixth, seventh, and eighth exons encode the cytoplasmic domain and 3’ untranslated region of these genes (Figure 3.4 .) The exon-intron organization of a human class I gene is similar to the mouse genes, with the only difference being that the humanclass I gene contains two rather than three cytoplasmic exons (67). Additional sequence data on humanclass I genes will be necessary to determine whetheror not this feature is characteristic of all humanclass I genes. The general features of the class I genes resemble those of other eukaryotic genes. The upstream and downstream intron boundaries typically have the classical GT/AGnucleotides, which appear to be important RNA splicing signals (99). The 5’ flanking sequenceof gene 27.1 appears to have sequence elements characteristic of other eukaryotic promoter regions-CAATand TATAboxes at positions 81 and 53 upstream of the AUG initiation codon(111 ). The consensus recognition sequence, whichprecedes the site of polyadenylation, AATAAA, appears twice, adjacent to one another approximately 390 nucleotides downstreamfrom the stop codon in exon 8 of the 27.1 gene (111). According to the nucleotide sequence, the two reported Ld genes are functional in that there are no stop codons at inappropriate positions, the RNAsplicing junctions are all characterized by the appropriate signal nucleotides, and there are no deletions or insertions that changethe reading frames of the exons (23, 76). In contrast, gene 27.1 appears to be a pseudogene by several criteria (111). First, there is a charged aspartic acid codon in the middle of the highly hydrophobic transmembrane exon. Second, there are termination codons near the end of the transmembraneand seventh exons. Third, the upstream RNAsplice signal for the sixth exon does SEQUENCE


of Class I Genes

Annual Reviews 536

HOOD ET AL ~s

~m TM 3’UT

C~I C~2 C~3 CH.4 3’UT

ANTIBODY GENE (C,,,)

L C~l

a2

c~5 TM CYT 3’UT

CLASSI GENE ~) (L


L

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2

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~YPE I 0

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Figure3 (A) A schematicdiagramof the exon-intronorganizationof antibody,class I, class II, and #2-microglobulin genes.L denotesthe exonsencodingthe signal or leader peptides; el, e2, and e3 represent exonsencodingthe external domains;TMdesignates the transmembrane exons; CYTsymbolizesthe exonsencodingthe cytoplasmicdomains;and 3’ UTdenotes the 3’ untranslated region. Darkboxesrepresent coding sequencesand hatchedboxes3’ UT regions. (B) Schematicdiagramof the exon-intronorganizationof two mouseclass I genes. Nonhomologous sequencesbetweenthe L~ and Qa(27.1) genes are indicated by stippling. Exonsare numbered;3’ untranslatedregions are hatched.Arrowsshowthe localization of type I andtype II Alu-like repeat sequences. not agree with the consensus sequence. Thus, it is clear that gene 27.1 cannot encode a full-length class I polypeptide. However, with the exception of the aspartic acid codon in the transmembrane exon, the aberrations in gene 27.1 are all in the 3’ portion downstream from the stop codon at the end of the transmembrane exon. Thus, gene 27.1 may encode a membrane-bound class I polypeptide that is missing the cytoplasmic domain, or alternatively, it may be expressed as a soluble transplantation antigen (39) (see below). PATTERNS OF VARIABILITY FOR CLASS I GENES AND POLYPEPTIDES A detailed comparison of 12 class I amino acid and translated nucleotide sequences has appeared recently (68). The overall homology

Annual Reviews


MHC GENES 537 between six polypeptides for which extensive sequence information exists [Kb, Db, L°, Kd, D°, Qa (27.1)] is 76-94%(Table 1). From these comparisons it is obvious that the alleles Kb and Kd or Db and Dd are not more related to each other than to other class I polypeptides. This has been denoted a general lack of "D-ness" and "K-ness" and is discussed below. However,close inspection of the sequences reveals the existence of short allele-speeitie sequences located mostly in exons 4 to 8 (68). Particularly interesting is the close relationship betweenthe Ld band D amino acid sequences (94%), whereas theDb and Dd polypeptides are only 84%homologous.This observation raises the possibility that the Ld and Db genes maybe alleles (68). Alternatively, the gene correction mechanisms that operate to generate polymorphisms (see below) may be capable correcting nonallelic genes extensively so that they resemble each other. The variability in the extracellular part of the analyzed class I molecules appears to be clustered in three regions in the first two external domains (residues 62-83, 95-121, and 135-177)(68). In contrast, the third external domainwhich interacts with fl2-microglobulin is more highly conserved. Exon6, encoding part of the cytoplasmic domain, is completely identical in seven functional class I sequences that can be compared(gene 27.1 has a different exon 6 sequence, but also contains a premature stop codon upstream of exon 6). The significance of this finding is unknown. THE Qa (27.1)

GENE SHOWS STRIKING

HOMOLOGY TO CLASSICAL

TRANSPLANTATION ANTIGENSThe overall homology between gene 27.1, which mapsinto the Qa-2,3 region, and the classical transplantation antigens (K, D, L) is 78-84%(Table 1). Therefore, the Qa-2,3 gene is closely related to the genes encoding transplantation antigens as they are to one another. These homologies suggest that the Qa and transplantation antigens descended from a commonancestor. It will be interesting to find out whether or not the structural homologyis reflected in similar functions as well. Peptide mapanalyses of Qa-2,3 antigens also have revealed strucaTable 1 Percent homology between mouse class I molecules Molecules bH-2K bH-2D dH-2L dH-2K dH-2D

bH-2K

bH-2D

dH-2L

dH-2K

dH-2D

Qa (27.1)

83

83 94

77 76 76

89 84 87 76

79 80 79 78 84

Qa (27.1) aAminoacid sequences compared ranged from 113 to 342 residues. Maloy & Coligan (68).

Table adapted from

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tural homology to transplantation antigens (107), whereasTLpolypeptides appearto be very different (127). Onthe other hand, the similar size and binding to/32-microgiobulin of the TLmolecules, as well as the strong cross-hybridization of these genes to a probe for the exonencodingthe /82-microglobulin-binding domainagain suggest homologyand a shared ancestry (I 12). DNA sequenceanalyses of the Tla genesare nowunderway.


ALU REPEATS AND CLASS I GENES

The human genome has an element

repeated300,000times that is termedthe Alu repeat becausethe restriction enzymeAlu I cleaves within the repeat and thus permits the visualization of this DNAon Southern blots. The mousegenomehas an homologous elementtermedan Alu-like repeat. The3’ untranslated regions of class I genescan be distinguished by the presence or absenceof Alu-like repeats (11, 109). Alu-likesequenceshavethe general characteristic of transposons with terminal direct repeats encompassinga central sequencecomposed of the Alu-typeand an A-rich sequence(43). Theycan be classified into two groupscalled type I (described above)and type II sequences.TypeII Alu sequencescontain an additional non-Alurepetitive sequence.Alu-like repeat sequencesof type II havebeenfoundat the 3’ endof the 3’ untranslated region of three class I eDNA clones [pH-2II(109), pH-2d-1(56),and 203(93)] and include the AATAAA polyadenylationsignal as a part of the A-rich sequence. One of these eDNA clones (pH-203)has been shown encodethe Db polypeptide(68, 93). ThepH-2IIclone encodesa polypeptide very similar (except for five positions) to the d polypeptide 7( 6, 109) a clone pH-2d-1appears to encodethe Dd polypeptide (10, 68). Atype Alu-like repeat also has been found at the 3’ end of the 3’ untranslated region of the L~ gene (76). Thus, class I genes encodedin the D region appearto contain Alu-likerepeat sequencesin their 3’ untranslatedregions. The eDNA clones encoding the Kb (92) and the d ( 126) polypeptides apparentlylack Alu-like repeat sequences.Their 3’ untranslatedregions are highly homologous(80-90%)to the D region eDNA clones for the first -,-300 bp, whereasthe remainingportion of ~120-160nucleotides encompassingthe Alu-like repeat in the Dgroupof genesis completelydifferent for the Kgenes (93, 126). Indeed, this region of the eDNA clone homologousto the Kd polypeptidehas beenusedas a specific probefor the identification of the Kd gene(126). Gene27.1 also does not contain the Alu-like repeat at its 3’ end(111) but a sequencethat is distantly related to the corresponding portion of the Kgene, possiblyreflecting a closer evolutionary relationship between Qa and K region genes than between K and D region genes. Cross-hybridizationof 5’ flanking sequencesbetweenKand Qa-2,3 genes but not betweenK and D genes of the b haplotype support this notion(27).



A comparison of the two genomicsequences of the genes 27.1 and Ld has shownthat the introns and exons have diverged about equally from one another apart from the insertion of a sequence of approximately 1000 nucleotides in the intron between exons 3 and 4 of gene 27.1 (76)(Figure 3B). This insertion is boundedat its 3’ end by a type II Alu-like repeat sequence and contains a type I Alu-like repeat sequence close to its 5’ end (Figure 3B). Thus, transposon-like Alu-like repeat sequences may have mediated this insertion of 1000 nueleotides. ARETHERETWOLa dGENES?The translated sequences of the two L genes correspond to the Ld protein sequence at 77 of 77 positions that can be compared(23, 76) and encode a class I polypeptide that can be detected with anti-L a monoclonalantibodies after gene transfer into mouseL cells (23, 29) (see below). However,these two genes appear to differ from another by 20 to 30 nucleotides. These differences might be explained in different ways. First, someof the differences might be due to DNA sequencing errors. However,the differences appear to be too extensive to be explained exclusively in this manner. Second, since these Ld genes were derived from two distinct populations of BALB/cmice, perhaps some genetic polymorphismhas already occurred since their separation. Finally, there maybe two distinct class I genes that both encode molecules recognized by anti-L d monoclonal antibodies. This latter hypothesis is being tested by chromosomalwalking techniques, which are discussed in a subsequent section. POSSIBLE SECRETORY CLASS I MOLECULES An unusual category of class I mRNA’s with two interesting features has recently been described (16). First, these mRNA’s contain several codons for charged aminoacids in the transmembraneregion, which is usually hydrophobic and uncharged. Second, a termination codon is found at the end of the membraneexon. Gene27.1 has precisely these same characteristics (111) and thus may capable of synthesizing a similar mRNA.These unusual class I mRNA’s are slightly smaller than the mRNA’s encoding full-length class I polypeptides and can be identified by a distinctive probe that again has been derived from the 3’ end of the 3’ untranslated sequence (17). It appears that these RNAmolecules are expressed only in liver cells and not in other mouse tissues that have been tested (17). Although there is to date no formal evidence that these mRNA’s are translated into proteins, it is attractive to speculate that they maydirect the synthesis of soluble class I gene products. ALTERNATIVE PATTERNS OF RNA SPLICING AT THE 3’ END OF THE CLASS I GENES Antibody heavy chain genes can be transcribed and then

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differentially processedby RNA splicing at their 3~ ends to generate the membraneand secreted forms of immunoglobulins (22) (Figure 3A). eral intriguing observations suggest that class I genes also mayundergo alternative patterns of RNA splicing at their 3’ ends(Figure 4). First, the C-terminal aminoacid sequences of class I polypeptides and translated eDNA clones appear to be heterogeneousin length and they exhibit blocks of aminoacids whichdiffer (111) (Figure 4A). Theseproperties would expectedif different codingregions (exons) could be employedfor the termini of these polypeptides. Second, two mouseclass I cDNA clones (pH-2Iand pH-2II) reveal a notable sequencedifference. The 3’ ends these two cDNA clones are homologousto one another, apart from an insertion of 139 bp in the pH-2IIdone (111) (Figure 4B). This 139 pair sequencecorrespondspreciselyto the seventhintron of the class I gene. Thus,it is attractive to suggest that the seventh intron wasnot removed fromthe mRNA represented by clone pH-2II, whereasit wasfor the pH-2I and Ld-like mRNA’s (Figure 4C). Third, comparison of the cDNAsequenceof pH-202encodingthe Kb moleculewith the sequenceof gene27.1 suggestsa third pattern of RNA splicing for the seventhintron (92). Togive rise to an H-2Kb-likemRNA, exon 7 wouldbe spliced to a sequence 27 nucleotidefurther upstreamof the splicing point used for the generation of pH-2I or La-like mRNA’s (Figure 4C). These alternative patterns RNAsplicing wouldlead to C-terminal sequences differing in size and sequence.This particular pattern of RNA splicing also has beendescribed for the gamma geneof fibrinogen(18). It remainsto be seen whetherthese alternative formsof mRNA’s result fromthe transcription of the sameclass I gene. Nevertheless,the heterogeneitygeneratedby different RNA splicing mechanismsin the cytoplasmic domainsof class I molecules might be importantfor different effector functionsthroughinteractions withdifferent componentsof the cytoskeleton.

Organizationof the Class I GeneFamily A MULTIGENE FAMILY The possible homologiesof the class I and antibodygeneshaveraised several questions about the organizationof class I genes. First, howmanyclass I genesare encodedin individual mice?Second, do class I genes undergosomaticrearrangementsas do their antibody genecounterparts duringthe differentiation of cells that expressclass I molecules?Southernblot analyses of the DNAs fromvarious strains of mice with class I cDNA probes display 12-15 bands (13, 69, 86, 100, 104, 109, 111). Therefore,it is presumedthat there are 12 or moreclass I genesin mice. Althoughmostcells including liver express class I molecules,sperm do not express transplantation antigens. There are no gross DNArearrangements of class I genesin cells actively synthesizingthese molecules,


b H2-K pH-2l]

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41 voriotions in272bp 85%homotogous

3 wriofions in 85bp 96%homologous

d L

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b K

Figure 4 Differential RNAsplicing patterns at the 3’ end of class I genes. (,4) Comparison b polypeptide and the polypeptides encoded by the of the C-terminal sequences of the H-2K class I, pH-2II, and pH-2I eDNAclones. The shaded area indicates the sequences encoded b sequence. (B) Comparison by exon 7. A straight line indicates identity to the H-2K of the DNAsequencesat the 3’ end of the class I eDNAclones pH-2II and pH-2I, indicating close homologyexcept for a 139-bp insertion in clone pH-2II. Termination codons are boxed. Vertical bars indicate a nacleotide substitution. Gapswere introduced to achieve maximum homology.(C) Threepossible differential RNA splicing patterns of class I genes consistent with the structures of the Ld polypeptide, the pH-2II-like mRNA’s,andthe K~ polypeptide. Numbered black boxes indicate exons. The 3’ untranslated regions are hatched, andtermination codons are shownas black boxes at the mRNA level. (Adapted from 111.)

since liver and spermDNAshave the samehybridization patterns in Southern blots (109). Furthermore, completeclass I genes, uninterruptedby long stretches of noncodingDNA,are present in spermDNA,as shownby gene cloning and sequencing. Therefore, it is unlikely that majorDNArearrangementsof widely separated exons occur in class I genes as they do in antibodygenes. Multiple class I genes also have been identified in human and pig DNA(10, 83, 105). CLUSTERS OFCLASSI GENES Several laboratories have used the cloning of large DNAfragments (30 to 50 kb in size) in cosmidvectors to study the complexity and linkage relationship of class I genes in mice and man (27, 66, 112). The most extensive study so far has been carried out with

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BALB/cDNA(112, 125). Some56 cosmidclones were identified in library of BALB/cspermDNAwith class I cDNAclones (112). These clones weremapped with10 infrequentcutting restriction enzymes to permit the identification of cosmidclones with overlappingDNA inserts. On the basis of these analyses, these clones couldbe orderedinto 13 gene clusterscontaining 36distinct class I genesin a total of 840kbpairsof DNA (Figure 5). Comparison of genomicSouthernblots with pooled cosmid DNA analyzedwith class I cDNA clones as hybridization probesshowed that most of the class I genes of the BALB/c mousehadbeencloned. Thelargestof thesegeneclusters, duster1, containssevenclass I genes spreadover 191 kb of DNA(Figure 6) andmapsinto the Qa-2,3 region Cluster

Location Mapping Expression Lenglh{kb)

Organization

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36 ¢105sI genes Figure 5 A schematic

representation

of the 13 cosmid clusters

and their

locations.

The

locations were determined by genetic mapping through restriction enzyme site polymorphisms and by expression in DNA-mediatedgene transfer experiments (see text). The class I genes are indicated as dark boxes. (Adapted from 111.)

Annual Reviews MHC GENES 543 ~ 5’ 3 C~oI KpnI Sma I Sac~I XhoI XmoTrr


Scale

27.1

~ ,

I 0

I 25

I 50

I 75

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Figure 6 Restriction map of the class I cosmid gene cluster 1 isolated from BALB/cDNA. Class I genes are indicated as black boxes. Homologousregions according to the restriction mapare indicated graphically by ovals, rectangles, and wedges(see text). Arrowsindicate the 5’ to 3’ directions of transcription. Sal I, Cla I, etc, are restriction enzymes.The location of gene 27.1 is indicated. (From111.)

(112) (see below). The spacing between these genes ranges between 7 28 kb. Thestructure of cluster 1 is interesting in that the three class I genes to the 3’ side of the cluster as well as their flanking sequencesare indistinguishable from one another by restriction mapanalysis. These genes probably arose by relatively recent duplication events, presumably by homologousunequal crossing-over. The two genes located at the 5’ end of cluster 1 also are related to one another, as revealed by cross-hybridization of their 3’ flanking sequences.Thus, the genesin cluster 1 suggest that class I genes can be ordered into closely related subgroupsof class I genes that are continually undergoing expansion and contraction by homologousunequal crossing-over. In this example,moreclosely related class I genes seem to be located adjacent to one another. Seventeen distinct class I genes located on seven cosmid clusters have been isolated and characterized from B10 mouse DNA(27). One of these gene clusters, cluster 3, containing five class I genes mapsinto the Qa-2,3 region and appears to be the homologto cluster 1 isolated from BALB/c DNA.Further characterization of the class I genes of B10mice is necessary to determine whether B10 mice contain only half as manyclass I genes as BALB/cmice. Such a difference is not apparent from Southern blot analyses (13, 69, 86, 109). If such a difference in class I genes amonginbred mice is real, extensive gene expansion and contraction must occur. Somemousestrains fail to express the L polypeptide at the serological level (e.g. B6, AKR,A.SW).To determine whether or not this failure expression results from deletion of the L gene, a DNAprobe has been isolated from the 5’ flanking sequence of the Ld gene (112). According Southern blot analyses, this sequence is missing in the DNAfrom mice of the b (B6) and the k (AKR)haplotypes, indicating that nonexpression might arise from ddetion of the L gene. Likewise, in mutant BALB/cmice denoted dml and din2, which also fail to express the Ld gene, the same 5’ flanking sequence is ddeted (H. Sun, personal communication). It will

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interesting to determinethe precise 5’ and3’ endpoints of these deletions by chromosomalwalking experiments, since a hybrid class I molecule comprisingthe N-terminalpart of the Dd moleculeand the C-terminalpart of the Ld moleculeappears to be present in BALB/c dm1 mice(123). These data also suppo~the hypothesisthat class I genesare undergoingcontinual gene expansionand contraction. GENETIC MAPPINGBY RESTRICTION ENZYMESITE POLYMORPHISMS Strategy Themosteffective approachfor mappingthe class I clusters to their respective locations in the MHC has beengenetic mappingby restriction enzymesite polymorphisms(27, 125). This approach employsthree steps. First, single- or low-copyprobes,whichwill hybridizeonly to oneor a few genomicrestriction fragments,are isolated from each of the cosmid clusters. Second,these probes are used to examinethe DNAs of congenic and recombinantcongenicmice for restriction enzymesite polymorphisms. Theoretically, three types of polymorphisms can be observeda changein restriction fragmentsize (mutation),a loss of the restriction fragment(deletion), or an increase in the numberof restriction fragments(duplication). Third, these restriction enzymesite polymorphisms are then correlated with the serological polymorphismsof the various congenic and recombinant congenicmousestrains analyzedto allow the mappingof a particular site to one of the four class I regions--K, D, Qa-2,3and Tla (Figure 7). Molecularmapof class Igenes Single- or low-copyprobes were isolated fromeach of the 13 class I geneclusters fromthe BALB/c mouseand were used to mapthe geneclusters with respect to the genetic mapof the MHC (125)(Figure 8). Fourstriking results emerged.First, all 36 class I genes could be mappedto the MHC.Noclass I genes have been found so far mappingto chromosomes other than 17. In contrast, processed pseudogenes fromother multigenefamilies such as the antibody (6, 37), globin (59), and tubulin (121) genefamilies mapto chromosomes other than those containingtheir functional counterparts.Second,31 of 36 class I genesmap to the Qa-2,3and Tla regions. Thus, these two regions contain morethan 80%of the class I genes. Threegeneclusters containingfive class I genes mapto the K and D regions, whichencodethe classical transplantation antigens. Thesedata are in agreementwith mappingstudies of class I gene clusters carried out for B10mouseDNA (27) and with previous conclusions derived from Southernblot analyses that most of the class I genes are located in the Qa-2,3and Tla regions (69, 86). However,these DNA blot


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analysesalso suggestedthat one or moreclass I geneswerelocated outside of the MHC (27, 86), whereasthe restriction enzymemappingstudies with cloned BALB/c genesfailed to identify any such class. I genes(125). Perhaps morethan 36 class I genes are present in the BALB/c mouseand one or moreof the unclonedgenes falls in this category. Third, restriction enzymesite polymorphisms occur with considerably greater frequencyin the K andDregions than in the Qa-2,3and Tla regions (125). This observation is in completeaccordancewith the extensive serological polymorphism of the K and Dmoleculesas contrasted with the limited serological polymorphismof Qa-2,3 and TL molecules. Hence, the extensive polymorphismsexhibited by the K and D genes also are seen in their flanking sequences, whereasthe morelimited polymorphisms of the Qa-2,3 and Tla genesalso are reflected by a lack of polymorphism in their flanking sequences.Hence,these differences mayreflect the differential operationof mechanisms for generating polymorphisms (see below). Finally, the results obtainedusing 13 single- or low-copyprobesto analyzethe numberof class I bandsin four different inbred strains suggest that modestexpansionand contraction of the class I gene family occurs, presumablyby homologous unequalcrossing-over (125). Theseobservations are in accord with those discussed earlier on the gene homologiesin the cluster 1 of BALB/c DNA andthe loss of the L genein certain strains. An elegant approachhas been used to study the organization of human class I genes (83). Treatmentof humancell lines with gamma rays leads the randomdeletion of various chromosomalsegments throughout the humangenome,including the loss of various portions of the humanMHC or HLAcomplex. The corresponding loss of one or more humanclass I genescan be followedby Sbuthernblot analyseswith class I probes. These studies havepermittedthe correlation of particular bandsdetected on the Southernblot with certain HLAalleles. Thesestudies should reveal the location of the as yet unidentified humanclass I genesequivalentto those of the mouseQa-2,3and Tin regions. Mechanisms for the Generation of Polymorphism COMPLEX ALLOTYPES,

SPECIES-ASSOCIATED

RESIDUES,

NO "K-

NESS"OR"D-NESS" Three observations madewith the initial N-terminal K

I

S

D,L

Oa2,5

Tla

Figure 8 Locations of the 13 claso I gene clusters in the MHC.The order of the clusters in a region is not known.Clusters 5 and 12 mapeither to the Qa-2,3 or Tin regions. Arrowspoint to the locations of the probes used for mapping. Open boxes indicate expressed genes by DNA-mediatedgene transfer, whereas dark boxes represent nonexpressed genes. (From 125.)

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MHC GENES 547 protein sequencedata on transplantation antigens suggestedthat the individual membersof the class I multigenefamily could be rapidly expanded andcontractedor correctedagainst oneanother(101, 102). First, the alleles of the K(and D) locus differ fromone anotherby multipleresidues. These multiplysubstituted alleles arc denotedcomplexallotypes (102), and they represent a contrast to the typical simple allotypes of the giobins, which differ generallyby single aminoacid substitutions. Thus,a special genetic mechanism appears necessaryto accountfor the extensive allelic ditferences. Second,the class I polypeptides of humanand mouseare distinguishedfromoneanotherby species-associatedresidues at various positions (102). Thus, the multiple mouse(and human)class I genesmust evolve parallel by a processtermedcoincidentalevolution(38). Onceagain, coincidental evolution amongmultiple genes requires the operation of mechanismsfor gene correction (38). Finally, the N-terminalregions of the alleles andthe Dalleles exhibitedno "K-ness"or "D-hess";that is, in a pool of sequencesfor KandD alleles, no features permittedthe Kalleles to be distinguished from their D counterparts (101, 102) (see also 81). These observationssuggestthat there is a rapid exchangeof informationbetween Kand Dgenesso that they fail to evolveindependentcharacteristics. The mechanismof gene conversion--the correction of one sequenceagainst a second--is also suggestedby data on the mutanttransplantation antigens. MUTANT TRANSPLANTATION ANTIGENS Mutant transplantation antigens weredetected by carrying out reciprocal skin grafts among siblings

in an inbredstrain of mice(80). Occasionally oneanimalwill reject the graft fromhis siblings. Subsequentcharacterization of these mutantsoften reveals that there is an aminoacid sequencevariation in one transplantation bantigen that leads to the subsequentgraft rejection. Mutantsof the K transplantation antigen appearto be remarkablein several contexts (Figure 9 ~/). First, they are very frequent, occurringat a frequencyof approximately5 XlO-~/cell/generation. Second,there are generally two or more aminoacid substitutions that are relatively closely spacedto one another in each mutantantigen. It appears improbablethat one isolated mutation wouldalwaysbe followedby a second closely linked mutation. Third, the sameindependentlyderivedvariants are seen repeatedly. Finally, virtually all of the Kb mutationsoccur at positions wherethe mutantresidues are observedin other class I molecules.For example,the L° polypeptideappears to contain mostof the substituted aminoacids foundin the Kb series of mutants(23, 87). Presumably one or moreclass I genescontainingthese residues also exist in the b haplotypemouse.Thesedata suggest that the Kb gene can undergoa single geneconversionevent with one of the other class I genes leading to variants containing multipleaminoacid substitutions (23, 56).

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I H-2 Kb[

91 ~


bml brn5 brn4. bm 5

18~ I

275 I

\~26/ -~,~ ’li I1 ....

I I

brn 6 bin7 bm8 bin9 bm I0 broil bml6

"II

II

145 150 152 155 156 160 165 HIS Ly5 TRP~LU ~LNALk GLY~LUALAGLUARGLEUAR~ALATYRLEU~LUGLYTHR

b K Kbm-I

ARG

ALA~TYR TYR

GLU-

Figure 9 (,4) Schematicrepresentation of aminoacid substitutions in 11 independently isolated Kb mutants.The top illustration showsthe presumptiveexon organization of the Kb mRNA. Substituted residues in the mutantsare indicated by vertical bars. (Adaptedfrom 80.) (B) Sequencecomparisonof the ~, Kbmm,and Ld genes from codon position 145 to 165. Horizontallines indicate identity to the Kb protein or DNA sequences.(Adaptedfrom120.) The class I gene encoding the mutant Kbml transplantation antigen has recently been isolated (97, 119). This mutant class I polypeptide differs positions 152, 155, and 156 from the wild-type Kb antigen. DNAsequence analyses of the Kb, Kbin1, and Ld genes demonstrate that the Kbin1 mutant has seven nucleotide substitutions arising over a stretch of 13 nucleotides, which lead to the three amino acid substitutions (Figure 9B). All seven these substitutions are seen in the Ld bml gene, which is identical to the K gene, over a stretch of 52 nucleotides encompassing this region. This observation raises the possibility that the mutant Kbin1 gene arose by gene conversion against a b haplotype class I gene identical in sequence to the L~ gene in this region. These data also may be explained by a double recombinational event, which appears less likely. Since this mutant sequence is not

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MHC GENES 549 locatedin the single class I genecloselylinkedto Kb (119), the putativegene conversionof the Kb genepresumablyoccurswith class I geneslocated 0.3 or morecMaway(Figure 1). Thus, gene conversion apparently mayoccur over extensivedistances(41, 42).


GENE CONVERSION

AND GENE EXPANSION

AND CONTRACTION

CREATE POLYMORPHISMS Gcnc correction mechanisms such as gene expansionand contraction or geneconversionare required to explain complex allotypes, species-associatedresidues, the lack of "D-hesS"and "Khess," and the patterns of substitutions seen in the mutanttransplantation antigens (see 38 for a discussionof these issues). Bothmechanisms appear to play a fundamentalrole in generatingthe complexallotypes of immunoglobulin Ca genes(4, 81a, 96). Theanalyses of class I genenumberswith low- andsingle-copyprobes clearly indicate that duplication and deletion of class I genes occurs, presumablyby homologous unequalcrossing over. Furthermore,the mutantbmlgene appearsto havearisen by a geneconversion event. Since manymutantclass I polypeptideshaveproperties similar to those of bml, it is logical to concludethat they also arose by gene conversion and that this process is common throughout the class I gene family. Geneconversionalso appears to accountfor interesting aminoacid sequencerdationships in humanclass I molecules(65). Apparentlygene conversioncannotoccur amongall class I geneswith the sameefficiency. For example,the Db gene showsa 10-fold lower mutation rate than the Kb gene (45). The important point is that both gene expansionand contraction and genecorrection mechanisms play a fundamentalrole in generating the extensive polymorphism of certain class I genes. Anintriguing question is whetherone or both of these genecorrection mechanisms also give rise to the extensive flanking region polymorphisms of the K and D gene dusters, or alternatively, whetherthese polymorphisms reflect the operation of yet a third unknowngenetic mechanism.Onemust ask why certain regions and subregions [K, D, I-A (see below)] appear to exhibit extensive polymorphisms, whereasothers [Qa, Tla, I-E, S (see below)] not.

Expressionof Class I GenesAfter GeneTransfer APPROACH Theunequivocalidentification of serologically defined class I genes requires gene transfer experimentsin whichthe cloned gene is expressedin a foreign cell so that its gene product can be identified by serologic techniques.ClassI geneshavebeensuccessfullytransferred into mouseL cells lacking the thymidine kinase gene by co-transfer with a herpes virus thymidinekinase gene with the calcium phosphatetransfer technique(5, 23, 29, 63, 74, 105). Underappropriateselective conditions,

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cells expressingthe herpes thymidinekinase genewill grow(1 in 104-105 L cells), and about 90%of the thymidinekinase-positive cells expressthe newlyintroducedclass I geneproducton the cell surface. Mouse L cells are derived from C3Hmice of the k haplotype, whichappear to express only two transplantation antigens, Kk and Dk. Monoclonalantibodies can readily distinguisheachof the BALB/c (d haplotype)serologicallydefinexlclass I products from their C3Hcounterparts. The new(BALB/c)class I molecule can be identified by radioimmunoassay, by the fluorescence-activated cell sorter, by two-dimensionalgel electrophoresis, and by peptide map analysis. Data obtained by these meanscan then be correlated with the results obtained by DNA sequenceanalysis and the mappingof the gene by restriction enzymesite polymorphisms. IDENTIFICATION

OF SEROLOGICALLY

DEFINED

CLASS

I

GENES

Thegenetransfer techniquehas beenusedto identify all of the serologically defined class I genes in the BALB/c mouseexcept for the Qa-1gene (28). Thereis no monoclonalantibodyto the Qa-1antigen, and identification of class I geneproductsin mouseL cells after genetransfer is difficult with heterogeneousalloantisera. Six serologically defined genes have been locatedin their correspondingcosmidclusters andare identified in Figures 5 and 8. TheKd, Dd, Ld, Qa-2,3, and two Tla genes havebeen identified. In each case the mappingby restriction enzymesite polymorphisms correlates with the mapping by serologicalidentification after genetransfer (125). TheKb gene isolated from the C57BL/6mousealso has been identified by gene transfer experiments(74). Theb gene resides i n aclass I gene cluster containingtwoclass I genes,whichis similar in organizationto the BALB/c Kd gene cluster (27, 112, 125). Genomicclones for humanclass I moleculesalso have been transferred into mouseL cells to study expressionof HLA antigens (5, 62, 63). These studies haverevealed that humanclass I genes can be expressedin mouse fibroblasts and that these products associate efficiently with mouse$2microglobulinand in somecases induce conformationalchanges of mouse $2-microgiobulin(62). Four humanclass I genes encodingHLA-A2, HLAA3, HLA-B7,and HLA-CW3 antigens were identified with monoclonal antibodies (5; and F. Lemonnier,personal communication). NOVEL CLASSI GENEPRODUCTS Six of the 36 class I genes isolated from BALB/c DNA appear to encode serologically defined antigens (28). To determinewhetherany of the remainingclass I genes are expressed, a radioimmunoassay wasdevelopedfor cell-surface fl2-microgiobulin (28). MouseL cells express relatively constant amountsof the Kk and Dk molecules on their cell surface. Since eachknownclass I moleculeis associated with ~82-microglobulin,the amountof/~2-microglobulinassociated with

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MHC GENES 551 endogenousclass I gene products on the cell surface does not vary. When foreign class I genes are transferred into mouse L cells, the amountof surface ~2-rnicroglobulin generally increases, because foreign class I gene products also must associate with/32-rnicroglobulin to be expressed on the cell surface, and excess/~2-microglobulin appears to be synthesized. Thus, each of the 36 class I genes was independently transferred into mouseL cells and I0 novel gene products were identified by the/~2-nficroglobulin immunoassay(Figure 8). This assay wouldnot identify class I gene products expressed in low levels or not associated with ~2-microglobulin.All of these genes mapto the Qa-2,3 and Tla regions. Indeed, the two additional class I genes in the K and L gene clusters do not appear to be expressed by this gene transformation assay. One of the transformed L cells expressing a novel gene product was used to immunizeC3Hmice, the strain from which the L cells were derived (28). A specific alloantiserum to the novel gene product was raised. Analysis by two-dimensional gel electrophoresis after immunoprecipitation with this antiserum established that the novel gene product was 45,000 daltons and was associated with ~2-microglobulin. Thus, the novel gene product has the typical features of a class I molecule. Furthermore,this novel gene product appears to be present on spleen cells but not on cells from other tissues in the mouse. Alloantisera to these novel gene products should be useful in defining their tissue distribution, their developmentalregulation, and ultimately their functions.

Functionof Class I Genes Cytotoxic or killer T cells carry out an immunosurveillance to eliminate host cells that have been infected with virus (129). The T-cell receptor must recognize both the foreign viral antigen and a self transplantation antigen. The transplantation antigen is termed a restricting element, which permits the T cell to detect foreign antigens in the context of self. Aew~ordingly, the transplantation antigen plays an important role in permitting the cytotoxic T-cell system to distinguish self from nonself. DNA-mediated gene transfer permits the functional analysis of individual class I genesin two ways.First, the restricting elements (K, D or L) that permit different viruses to eliminated by cytotoxic T cells can be determined. Second, in vitro mutagenesis either by the shuffling of exons betweendifferent class I genes or by mutation of individual nucleotides can be used to perturb T-cell recognition and killing functions. Cellular assays such as the one depicted in Figure 10 have been employed to analyze cytotoxic T cells raised in response to a particular viral infection against mouseL cells expressing various class I genes (74, 82). Cytotoxic T cells raised in a mouseof a particular haplotype can only employrestrict-

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ing elements from the same haplotype. Thus, cytotoxic T cells raised in BALB/c mice against the LCMvirus can kill LCMvirus-infected mouse L cells only if they have been transformed with the appropriate BALB/c transplantation antigen. A summaryof the results of T-cell killing studies from several laboratories is presented in Table 2. Several stalking results emerge. First, a particular virus often uses a particular transplantation antigen as restriction element (e.g. the LCMvirus infection in the BALB/c d mouse employs the L element and the influenza infection in the C57BL/10 mouse employs the Kb element). Second, cytotoxic T cells raised in mutant d haplotype animal lacking the L gene (BALB/cdin2) do not employ the Ld gene as a restricting element (82). Thus, the dm2 mice appear to capable of employing another class I molecule as restriction element to L- cell transformants

~

LCMV i.p.

LCMV ~ 2 days ¯

7 doys spleen

trypsinize wash Slcr- labelling wash

suspension

cytotoxicity assay

Figure 10 A schematic ofthe procedures used in the testing of LCM virus-specific H-2 restriction withL-cell transformants.L cells transformedwiththe Ld geneare infected with LCM virus and then are labeled with 5~Cr.CytotoxicT cells isolated fromLCM virus-infected BALB/c mice are then used to showby 5~Crrelease that the Ld moleculeon the surface of the transformedL cells can serve as a restricting elementfor LCM virus.

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553

Table2 Productsof transfectedH-2class I genesact as Iestriction element for anti-viralcytotoxicTcells Transfected gene bH-2K dH-2L


d H-2L

Virus Influenza Lymphocytic choriomeningitis virus Vesicular stomatitisvirus

References 74 82 25

achieve T-cell recognition and killing of LCMvirus-infected cells. Therefore, the systemis flexible. Third, cloned T cells behaveessentially the same way as their heterogeneous counterparts. Cloned T cells will be essential reagents in the in vitro mutagenesis experiments described bdow. Since a transplantation antigen is required to evoke a cytotoxic T-cell response against a particular virus, this element maybe mutated to determine which portions of the molecule are important for the recognition and effector functions of T-cell killing. Preliminary exon shuffling experiments between the Ld gene and gene 27.1 have been carried out (I. Stroynowski, personal communication).The ~ element can no l onger serve as a restricting element if both the a 1 and a2 exons are exchanged with homologous 27.1 exons. Thesedata suggest that the T-cell receptor recognizes antigenic determinants on the crl and/or or2 domains. Moreextensive in vitro mutagenesis and exon shuffling experiments for class I genes are being carried out nowin a numberof different laboratories. Structure and Expression of the ~2-Microglobulin Gene The exon-intron organization of the/~2-microglobulin gene, which appears to be a single-copy genein contrast to the class I multigenefamily, has been determined by DNAsequencing (84). This gene consists of four exons, with most of the coding region present in one exon (amino acids 3-95) (Figure 3.4). Fromthe comparison in Figure 3.4, it is obvious that the B2-microglobulin gene has a very similar exon-intron structure to the antibody, class I, and class II genes. This homologyalso is reflected at the aminoacid (88) and DNAsequence level (73, 84) and indicates an evolutionary relationship between these genes (see below). The gene for/~2-microglobulin is located in a region on chromosome2 encoding several non-MHC-linkedtransplantation antigens and several immuneresponse genes (75). Although/~2-microglobulin and class I genes are located on different chromosomes, they are coordinately regulated. Both genes are not expressed in teratocarcinoma stem ceils, which are presumablyequivalent to early embryoniccells, whereasboth are expressed in differentiated cell fines

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derived from the teratocarcinomastem cells (19, 77). Theregulation these genesappearsto be controlled transcriptionally. Analyses of t Haplotype Mice with Class I cDNAProbes Proximalto the MHC on chromosome 17 is a large genetic~region termed the t complex(7, 8, 103). Some25%of wild micecarry a mutationin this region that results in early embryonicdeath whenit is present on both chromosomes. Thesemutations fall into eight eomplementation groups(7, 8). A chromosome 17 that carries one of these mutations has been termed a t chromosome and its DNA is said to be of t haplotype origin. All t haplotypes showseveral unusual properties. First, males transmit the t chromosometo their progeny with a muchhigher frequency than the normalchromosome 17. Second, genetie recombinationbetweenthe t and the normalchromosome 17 is suppressedover a region of 20 cMthat starts fromthe centromereand includes the H-2complex(7, 8, 103). Avariety of biochemical, serological, and genetic data have suggested that the t complexmaybe evolutionarily related to the MHC complex(2). To analyze the t complex,a ehromosomal walk from the H-2 complexto the t complex maybe feasible (110). Asa first approach,class I eDNA clones have been usedto analyzeclass I genesby Southernblot analysesin different t haplotype micethat expressantigenicallydistinct class I molecules(100, 104). Surprisingly, restriction enzymesthat revealed a considerable polymorphismbetweenthe class I genesof different normalinbred strains of mice failed to showa similar kind of polymorphism betweendifferent t haplotypes. This finding has beeninterpreted to indicate a close evolutionary relationship of all t haplotypes.Thesignificanceof these observations,as well as the possibility of an evolutionaryrelationshipbetweenthe t complex and the MHC, remainsfor future studies to elucidate. CLASS II

GENES AND POLYPEPTIDES

Class H Polypeptides The I region of the MHC encodes the elass II molecules and has been divided by recombinationalanalyses into five distinct subregionsdenoted I-A, I-B, I-J, I-E, and I-C (78) (Figure 11). Twoof these subregions, and I-E, contain genes for the class II molecules, whichhave beenwell characterized by serological and biochemicalmethods.The two class II molecules encodedin the I-A and I-E subregions are both heterodimers composed of alpha and beta chains (Figure 11). Thealpha chains range molecularweight from30,000-33,000and the beta chains range in molecular weightfrom27,000-29,000.Thedifference in molecularweightbetween the t~ and ~ chains is primarily dueto differences in glycosylation.Thea

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MHCGENES chain contains two carbohydrate units, whereasonly one is boundto B chains. TheA~, A~, and E~genesare containedwithin the I-A subregion, whereasthe Ea geneis located within the I-E subregion(Figure 11). Athird subregion,I-J, appears to encodepolypeptidesthat are subunits of the suppressorfactors secreted by suppressorT cells. Althoughthe I-J polypeptideshavebeencharacterized in functional assays, their biochemical characterization is only beginning(53, 117, 120). Thecharacterization of suppressorfactors andtheir geneshas beenof vital interest becauseit represents a uniqueopportunity to study one form of the elusive T-cell rer, eptor. TheI-B andI-C subregionshavebeendefinedexclusivelyon the basis of their ability to modulatecertain immune responsesin mice, and someimmunologistsquestion whetherthese subregions exist (48). Thestructures of the class II polypeptideshavebeendeterminedprimarily fromthe analysis of humaneDNA clones (3, 32, 51, 57, 58, 60, 64, 113, 118, 123) and morerecently fromthe analysis of mouseand humangenomic clones (9, 50, 61, 71, 73). Onlyvery limited aminoacid sequenceinformation is availablefor mouseclass II molecules(116), whereasmoreextensive sequenceinformation has been obtained for humanclass II polypeptides (98). Thesedata suggest that the mouseI-A and I-E moleculesare homologousto the humanDCand DRmolecules, respectively (12) (Figure 1). counterpart to the third humanclass II molecule, SB(40), has not been found in the mouse.The three humanclass II moleculeseach have a and /5 chains denoted DCa,DC0, DRy, DR0, SBa, and SBO.Each class II polypeptideis composed of twoexternal domains,each about 90 aminoacid residues in length, ~1 and ~2, or fl 1 and ~2, a transmembrane region of about 30 residues, and a very short cytoplasmicregion of about 10--15 residues (Figure 2). Three of the four external domainshave centrally placed disulfide bridges (~2,/~1, and/~2). Thus,the class I and class moleculesappearto be very similar in overall structure and domainorganization (Figure 2). Theclass II moleculesserve as restricting elementsthat permitregulatory T ceils (helper, suppressor,amplifier)to viewantigenin the contextof self on the surface of other T cells, macrophages, or B cells (49, 79). Class genesappearto control the proliferationof regulatoryT cells as well as the effector reactionsearried out by these T cells, suchas the amplificationof other T-cell subsets and the promotionof B-cell differentiation. I A A~ A~ E~,

B

J

E

J?

E~

C

Figure11Geneticmapof the I region(seetext).

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DNA sequences the E~H - (73), Structure of ofClass GenesEk~(71), and A~(M. Malissen, personal communication) geneshavre beende’~erminedand ~he exon-intronorganization of these genesis illustrated in Figure12. Onceagainthere is a striking correlation betweenthe organizationof exonsandstructural domainsof the class II genes and molecules. Both gene~contain separate exons for the leader peptide and the a 1 and a2 domains.However,the E~gene employs a single exon to encodethe transmembrane and the cytoplasmicdomains, whereasthe A#gene appears to contain an intervening sequencein the cytoplasmicportion separating the exons encodingthe transmembrane and part of the cytoplasmicdomains.The Eogene has a fourth intron located in the 3’ untranslatedregion(71). Thus,the structures of the class II a and B genesappear to differ in several regards. Thegenefor B2-microglobulin showsan exon-intron organization very similar to that of the E~gene (Figure 3.4). For both genes, the exonencodingthe leader peptide is separated by a long intervening sequencefromthe rest of the codingsequence [2.8 kb for fl2-microglobulin(77) and about 2.3 kb for Eo (71)] and intervening DNA sequenceis found in the beginningof the 3’ untranslated region. TheA~genedisplays a structure moresimilar to a class I genein that transmembrane and at least part of the cytoplasmicregions are separated by an intron. Thesedata suggest that the class II ~ genes are more closely related to B2-microglobulin,whereasclass II ~ genes are more closely related to class I genes. ThehumanDRogene has been sequenced and has the sameexon-intron organization as the E~gene(50, 61). Comparison of the aminoacid sequencesencodedby the A~ and Ek genes has revealed an overall homologyof 50%betweenthe two proteins (9). Quite surprisingly, the most conservedregion is the transmembranedomain, whichexhibits 78%homology,perhaps indicating structurally and functionallyimportantinteractions of these parts of the moleculeswith the fl chains or other transmembrane proteins. ~’1

L

,81

,’,2 TM/CY/5’UT 3’UT

,82

TM

Figure 12 Schematicrepresentation of the exon-intronorganizationof the E~ and A~genes. Blackboxesshowexons; 3’ untranslated regions (3’ UT)are hatched; TMdenotes transmembrane; andCYindicates cytoplasmic.Theexonsencodingthe leader peptide and part of the cytoplasmic region and the 3’ untranslated region have not yet been identified by DNA sequencingfor the Aa gene. (Adaptedfrom70; and M. Malissen,personal communication.)

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MHC GENES 557


Homology of Class L Class IL /~2-Microglobulin,Antibody, and Thy 1 Genes Thereare several similarities between class I, class II, 82-microglobulin, and antibodygenes(Figures2 and3A).First, all genesexhibit a precise correlation betweenexonsand the protein domainsthey encode.Second,the sizes of the external domainsandthe central placementandsize of the disulfide bridges are similar. Third, RNA splicing alwaysoccurs betweenthe first and second baseof the junctional codonsaccording to the GT/AG rule for both genefamilies. In other genesandgenefamilies, RNA splicing also can occurafter the secondand third base of the junctional codons(99). Fourth, X-ray data (P. Bjorkman,J. Strominger,and D. Wiley, personal communication) suggestthat the class I and antibodymoleculeshavea twofoldaxis of symmetry and, accordingly,probablyexhibit paired domains(Figure 2). Fifth, the DNA sequencesof the class I a3 exons, the class II 02 and 82 exons, and 82-microglobulinare homologous to the exon sequencesencoding antibodyconstantregions(9, 11, 50, 57, 58, 71, 73, 84, 109)(Figure A lowlevel of homology also has beenobservedbetweenthe class I a 1 and the class II 81 domains(58). Sixth, the class I al and~2 andthe class a 1 exonsdo not showsignificant sequencehomology to one another nor to the antibodygenes. However,their sizes are similar to those of the other class I, class II, and antibody exons. Furthermore,the class I a2 exon encodesa centrally placeddisulfide bridge. Theseobservationssuggestthat the completeclass I, class II, andantibodygenesshareda common ancestor andmarkedchangesoccurredafter divergenceof the genesto fulfill different functions. Finally, homology to the T-cell differentiation antigen, Thy 1, also is observedat the protein level (122). Becauseof their sequenceand structural relationships,it is attractive to postulatethat all of these genes ~lass I, class II, 82-microglobulin, immunoglobulin,and Thy 1--must have descendedfrom a common ancestor and are therefore membersof a supergenefamily. Twoqualifications mustbe raised. First, it is impossible to distinguish betweendivergent and convergentevolution. Accordingly, the membrane-proximal domainsof these molecules mayhave arisen from distinct genes that convergedtowardone another becauseof common functional constraints. If this hypothesisis correct, onewouldhaveto arguethat convergentevolution also generated moleculeswith external domainsof similar size and with similar disulfide bridge placement.This possibility appearsunlikely, but cannotbe formallyexcluded.Second,perhapsonly the membraneproximal domains evolved from a commonancestor, whereas the remainingportions of these genescould haveindependentorigins. Once again, the sharedsize andcentrally placeddisulfide bridgesof the external domainsmakethis an unattractive but formally possible hypothesis.

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Linkage Relationship of Class H Genes A continuous stretch of about 230 kb of DNA has been isolated from the I region by first screening a BALB/c spermcosmidlibrary with a human DR~eDNAprobe and then by using single-copy probes for chromosomal walking(110). Theclonedregion appearsto contain genesencodingall four serologieally defined class II polypeptides.Theclass II genes havebeen identified through the use of synthetic DNA probes whosesequenceswere generatedfrom protein data, mouseB cell-specific eDNA probes, and humaneDNA probes that cross-hybridized to the mouseclass II genes. These data are depicted in Figure 13. This 230-kbregion of DNA includes the right-hand boundaryof the I region, because the structural gene for the complementcomponentC4 mappinginto the S region of the MHC could be identified (M. Steinmetz, L. Fors, A. 0rn, unpublishedresults). TheC4genelies about 90 kb distal to the E~geneand wasidentified with a synthetic oligonueleotide probe specific for the N-terminal portion of the C4 a subunit. Theleft-hand boundaryof the I region cannot be defined at present becausethe cosmid clones do not extend into the K region. Five class II genes, extending over a stretch of DNA encompassing. approximately90 kb, have beenidentified. TheE~(73), A,, (20), and (M. Malissen,personal communication) genes havebeenidentified by direct DNA sequenceanalysis, the EOgene has beenidentified by hybridization with a specific oligonucleotide probe (110), and the EO2gene has been identified by cross-hybridization to a humanDCachain eDNA clone and the mouseEo gene (110). Identification of the Et~ gene waseonfumed the use of restriction enzyme site polymorphisms to localize a serologically defined EOrecombinantto the middle of the EOgene (110). Whetherthe E02gene is functional or represents a pseudogeneis unknown. The A0and Eo geneshavethe same5’ to 3’ orientation, whereasthe E,, geneshowsan opposite orientation. The orientation of the A~gene has not yet been determined. Thelocation of the A~genebetweenthe AOandEOgenesis different from the gene order of A~-Ao-E0proposed from peptide mapanalyses of the A~polypeptide (95). Strain A.TL,whichis an intra-H-2 recombinantdedyed from a cross between B10.S (A~), and A.AL(A~) was found bear an A,, chain whosepeptide fragments appeared to be a composite of As and Ak peptides. If this interpretation of the data is correct, the Aa ggne wott~d mapproximalto the Aagene. However,other investigators could not find a difference betweenthe A~polypeptides isolated from A.TLand B10.A(1R)mice (15, 72). Until the recombination point in the A.TLmice has been mappedat the DNA level this will remain con-


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troversial. However,the gene cloning data unequivocally suggest that the class II gene Order in BALB/cmice is 5’-A~-Aa-E~-Eo-3’. The region corresponding to that containing the five class II genes in BALB/cDNA(d haplotype) also has been isolated from the DNAof AKR mice (k ha..plotype) by screening an AKR cosmid library (M. Steinmetz, Fors, A. Orn, unpublished results). Twoimportant points arise from comparisonOf the Iregions from the k and d haplotypes (Figure 14). First, the same five class II genes are present in the k haplotype DNA.Moreover, their organization is virtually identical in both haplotypes without any major insertions, ddetions, or rearrangements. Second, a striking boundary of polymorphismproximal to the E~ gene is observed whenthe restriction maps of d and k haplotype DNAare compared. Although the two DNAs are almost identical to one another distal to this boundary, they show extensive restriction enzymesite polymorphismproximal to this point. Interestingly, this polymorphismbreakpoint correlates with a hot spot of recombination in the I region (see below). This difference in restriction enzyme site polymorphisms has been noted previously by Southern blot analyses of various mouse strains (110) and correlates nicely with the serological polymorphismsof the corresponding genes (47). Only two alleles each are knownfor the Eo, C4, and Sip genes, whereas the Aa and E# genes appear to be as polymorphic as the class I genes K and D. The important point is that the sequence conservation or restriction enzymesite polymorphismsare not confined to coding sequences but cover large regions of flanking DNA.This observation obviously has important implications for evolutionary mechanismsthat attempt to explain the generation of the extensive polymorphismof certain MHC alleles (see earlier discussion). Southern blot analyses have been carded out on mouseDNAwith t~ and fl probes under conditions that favor extensive cross-hybridization in an V//A HIGHLY BALB/c

0

~ CONSERVED

~?._ t?~

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Figure 14 A polymorphie breakpoint i~ the I region. Cloned regions of the I region BALB/e(d haplotype) and AKR(k haplotype) mice are compared over a stretch of of DNA.The region to the left ofthe breakpoint exhibits extensive restriction enzymesit~

lx~lyraorphisms, whereasthe regionto Nefight doesnot. Thepolymorphism breakl~int locateddoseto the recombinational hotspot at the 3’ endof the E~gene.(From M.Steinmetz L. ForsandA.~rn, unpublished results.)

Annual Reviews


MHC GENES 561 attempt to define the total numberof a and fl genes (110). Thesedata suggest that there are two a genes and four to six ~ genes in the mouse genome.Thus,the total numberof class II genesthat can cross-react with the available class II DNA probes will be less than 10, althoughthe existence of more distantly related class II genes in the mousecannot be excluded.This striking paucityof class II genesstands in sharpcontrast to the exquisite specificity of immune reponsivenessregulatedby the class II genes. Thesedata suggest that the specificity of immune responsivenessis likely to be governedby the repertoire of T-cell receptors. Correlation of the Molecular with the Genetic Mapof the I Region Tensingle-copyDNA probeswere isolated fromvarious portions of the 230 kb of clonedDNA in the I region (110) (see Figure 13). Theseprobes used to search for restriction enzymesite polymorphisms in various inbred, eongenic, and recombinantcongenicstrains of mice. Thesepolymorphisms werethen correlated with the serological polymorphisms, whichdefine the various subregionsof the I region. In this mannerthe molecularmapof the I region (Figure 13) could be correlated with the genetic mapproduced recombinationalanalysis of serological polymorphisms (Figure 11). These analysesmapped eachof the four serologicallydefinedclass II genesto the appropriatesubregions(Figure 15). Thus,the A0, the A~, and at least the 5’ half of the E0genes are all encodedin the I-A subregion, whereasthe E~gene is encodedin the I-E subregion. The genetic and molecularmaps are consistent in the locations of these genes. Aninconsistencyexists betweenthe genetic and molecularmapsfor the I-B and I-J subregions. The analysis of nine recombinantcongenicmice defining the right-handand left-hand boundariesof the I-A and I-E subregions, respectively, haveled to the conclusionthat these twosubregionsare separated by no morethan 3.4 kb of DNA (110) (Figure 15). To complicate mattersfurther, the Yhalf of the Eogeneresides in at least 2.4 kb of this 3.4-kb region. Thus, about 1 kb of DNAremains for the I-B and I-J subregions. Indeed, it is possible that the I-A and I-E subre~ionsjoin directly to oneanother. Twoobservationsexcludethe possibility that the BALB/c mousehas deleted or rearranged the I-B and I-J subregions in comparisonto other mice. First, extensiveSouthernblotting data indicate that the organization of BALB/cDNA around the 3.4-kb region is no different fromthat foundin three other mousestrains (b, k, s haplotypes), whichhavebeenused to define the I-B and I-J subregions (110). Second, the corresponding regions in the cloned k and d haplotype DNAsare co-linear (M.Steinmetz,L. Fors, A. t)rn, unpublishedresults) (Figure


Annual Reviews 562 HOODET AL

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MHC GENES 563


These observations pose a paradox for explaining the location of the genes of the I-B and I-J subregions. Others have suggested that the I-B subregion can be explained by gene complementation between the I-A and I-E subregions (48). Our data supported the notion that the I-B subregion does not exist. However,it is moredifficult to dismiss the I-J subregion because I-J polypeptides have recently been identified by monoclonal antibodies (T. Tada, personal communication).

The Paradox of the 1-J Subregion Howcan the expression of the I-J polypeptides be encodedby 3.4 kb or less of DNA? There are two possible categories of explanations (110). First, the J gene or genes may be encoded outside that segment of DNAresiding between the I-A and I-E subregions. For example, the interpretation of recombinantevents in the recombinant congenic mice could be incorrect in that these mice mayhave undergone multiple recombinational rather than the usually assumedsingle recombinational events within the MHC.Alternatively, this 3.4-kb segment of DNAmaycontain a regulatory element that controls the expression of a battery of polymorphicJ genes located elsewhere in the mousegenome. A second category of hypotheses suggests that at least part of the J polypeptidesare encodedin this 3.4-kb region. The I-J gene might be identical with the Ea gene and appropriate post-translational modification(e.g. glycosylation, ere) could generate the serological determinants of the J polypeptide. Alternatively, the J polypeptides maybe encoded by someEa exons, whichare then linked to distinct I-J exons by alternative patterns of RNA splicing muchas alternative splicing exists for ~membrane and /x~retea mRNA’s of antibody heavy chain genes. Finally, the J gene may be transcribed off the DNAstrand opposite that which encodes the E a gene. The possibility that the J gene is encodedentirely in the remaining 1 kb of DNAin this region appears remote since most eukaryotic genes require far more DNA. Evidence has already been obtained that argues against the second category of explanations (52). RNAisolated from a numberof suppressor T-cell lines does not show hybridization to DNAsequences covering this 3.4-kb region. These RNAsalso fail to hybridize with extensive regions of DNA upstream and downstreamfrom the 3.4-kb region. These data suggest that the structural gene for I-J is not located betweenthe I-A and I-E subregions. The location of the J gene is a question of considerable importance because this polypeptide is expressed alone or in conjunction with another polypeptide in two distinct kinds of T-suppressor factors. These T-suppressor factors have been viewed by manyas the easiest way to approach the structure and molecular biology of the T-cell receptor. Hence, isolation of the I-J gene could be of considerable significance as an approach to the cloning of the T-cell receptor.

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Nonlinearity of Recombination Nine recombinanthaplotypes for the I-A and I-E subregions have been examinedby mappingof restriction enzymesite polymorphisms and in all strains the point of crossing-overfalls withina region of 8 kb of DNA (110) (Figure 15). Indeed, these recombinationsmayall have occurred at precisely the samepoint. Theseobservationssuggestthat recombination in the eukaryotiegenome is not randomlyscattered throughoutbut, at least in this case, is highly localized to a discrete region. Theoccurrenceof hot spots of recombinationhas obvious implications for any attempts to translate centimorgansof DNA from the genetic or recombinationalmapsinto kilobases of DNA from molecular maps. Such a conversion must assumethat recombinationis randomlyscattered throughout the mousegenome.Since the Aa, E~, and C4genes havebeen linked by molecularcloning, recombination frequenciesfor this region can be comparedto physical distances: As and Ea are 0.1 cMapart by recombinationalmappingand are separated by 85 kb of DNA.The E~ and C4 genes separated by 0.11 cMare 90 kb fromone another. Earlier calculations based on the assumptionof random recombinationsuggested that 1 eMshould equal 2000kb. Thusthe actual distances are shorter by about a factor of twoto three than the theoretical distances. It will be interesting to examineother chromosomal regions of the eukaryotic genomewheremultiple recombinations have been mapped to determinewhetherthe occurrenceof recombinational hot spots is specific to the I region or whetherthis is a muchmore general phenomenon.

Expression of Class H Genes Fourinbred strains of mice(b, f, q, s haplotypes)do not express the I-E moleculeon the cell surface. Twoof these (b and s) do not express the E~polypeptidebut have cytoplasmicEOchains, and two (f and q) neither expressE,, nor E~. BySouthernblot analyseswithspecific probes,it appears that the failure to expressthese genesis not dueto a completedeletion of these genesin the respective haplotypes(11). Moredetailed analysesof the structure and transcription of the Ea genein these inbred strains has revealed that at least two different mechanismsare responsible (70). transcription of the E~geneis observedin miceof the b and s haplotypes, presumablybecausea small deletion occurredin the promotorregion of the gene. Obviouslydefective RNAtranscripts of various sizes are found in mice of the f and q haplotypes. Noone has succeededunequivocallyin expressingclass II genesin mouse L ceils after DNA-mediated gene transfer. This mightbe due to the presence of a weakpromotorregulating class II genetranscription, inappropriate splicing, or instability of mRNA’s. Furthermore,the expressionof a

Annual Reviews MHC GENES 565 third chain, called the invadant (Ii) chain, might be necessary for the transport of class II a and fl chains to the cell surface (55). The powerful approachesof molecular biology will certainly lead to class II gene expression within the relatively near future. The long-termgoal is to transfer class II genes into biologically rdevant cells such as macrophages,B cells, and T cells so that their functions can be assessed and perturbed by in vitro mutagenesis.


THE FUTURE Molecular genetics has given us stalking insights into the structures and organization of genes encoded by the MHC.The tools of molecular biology, when employedin conjunction with the well-developed assays of cellular immunology,provide a unique opportunity to dissect the functions of these multigene families involved in self-nonsdf recognition phenomena.It also will be interesting to determine whetheror not additional multigenefamilies fall into the supergenefamily, whichincludes the antibody, class I, and class II genes. Future experiments will showthe extent to which strategies of regulation and information expansion are shared by these homologousgene families. ACKNOWLEDGMENTS Wethank R. Flavell, B. Mach, H. McDevitt, and S. Nathenson for sending preprints of their workprior to publication, B. Larsh for her careful preparation of the manuscript, and E. Kraig and J. Parnes for thoughtful comments. M.S. was supported by a Senior Lievre Fellowship from the California Division of the American Cancer Society and B.M. was supported by a CNRSand DGRSTFellowship from the French Government. The work of L.H. and M.S. was supported by grants from the National Institutes of Health. LiteratureCited 1. Alper,C. A.1980TheRoleof the Major Histocompatibility Complexin lm. munology, ed. M.E. Doff,pp. 173-220. NewYork: Garland STPM 2. Artzt, K., Bennett,D. 1975. Nature 256:545-47 A.J., Roux-Dos3. Auffray,C., Korman, seto, M.,Bono,R., Strominger, J. L. 1982.Proc. NatL.4cad. Sc~USA79: 6337-41 4. Baltimore,D. 1981.Cell 24:592-94 5. Barbosa,J. A., Kamarck, M.E., Biro, P. A., Weissman, S. M.,Ruddle,F. H.

1982. Proc. NatLAcad.ScL USA79: 6327-31 6. Battey,J., Max,E. E., McBride, O.W., Swan,D., P. 1982.Proc.Natl. Acaa~ ScLLeder, USA79:5956-60 7. Bennett,D.1975.Cell 6:441-54 8. Bennett, D. 1980. HarveyLectures 74:1-21 9. Benoist,C. O., Mathis,D.J., Kanter, M.R., Williams, V.E., McDevitt, H.O. 1983. Proc. Natl. Acad.Sc£ USA.In press 10.Biro,P. A., Pereira, D., Sood,A.K., DeMartinville, B., Francke,N., Weiss-

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man,S. M. 1981. ImmunoglobulinIdiotypes, ICN-UCLASymposium on Molecular and Cell Biology, ed. C. Janeway, E. E. Sercarz, H. Wigzell, 20:315-26. NewYork: Academic 11. BrSl~Sg~re, F., Abastado, J. P., Kvist,$., Rask,L., Lalanne,J. L., et al. 1981. Nature 292:78-81 12. Bono,M.R., Stominger,J. L. 1982.Nature 299:836-38 13. Cami,B., Br~g~g~re,F., Abastado,J. P., Kourilsky, P. 1981. Nature 291: 673-75 14. Coligan,J. E., Kindt,T. J., Uehara,H., Martinko, J., Nathenson,S. G. 1981. Nature 291:35-39 15. Cook,R. G., Capra,J. D., Bednarczyk, J. L., U’hr,J. W.,Vitetta,E. S. 1979.J. lmmunol. 123:2799-803 16. Cosman,D., Khoury,G., Jay, G. 1982. Nature 295:73-76 17. Cosman,D., Kress, M., Khoury, G., Jay, G. 1982.Proc. Natl. dcad.Sc~USA 79:4947-51 18. Crabtree, G. R., Kant,J. A. 1982. Cell 31:159-66 19. Croc¢, C. M., Linnenbach, A., Huebner,K., Parnes, J. R., Margulies, D.H., et al. 1981.Proc.NatLAcad. US.478:5754-58 20. Davis, M. M., Nielsen, E., Cohen,D., Steinmetz, M., Hood, L. 1983. Proc. NatLAcad. ScL USA.In press 21. D6mant,P., Ivb~nyi, D., OudshoornSnoek, M., Calafat, J., Roos, M. H. 1981. Immunol.Rev. 60:5-22 22. Early, P., Rogers, J., Davis, M., Calame,K., Bond,M., et al. 1980. Cell 20:313-19 23. Evans, G. A., Margulies, D. H., Camerini-Otero, R. D., Ozato,K., Seidman,J. G. 1982. Proc. Natl. .4ca~ ScL USA79:1994-98 24. Flaherty, L. 1980. TheRole of the Major Histocompatibility Complexin Immunology,ed. M. E. Doff, pp. 33-57. NewYork: Garland STPM 25. Forman,L, Goodenow, R. S., Hood,L., Ciavarra, R. 1983. J. Exp. MecLIn press 26. Goding, J. W. 1981. J. ImmunoL 126:1644-46 27. Golden,L., Mellor,A. L., Weiss,E. H., Bullman,H., Bud,H., et aL Submitted for publication 28. Goodenow, R. $., McMillan,M., Nicolson, M., Sher, B. T., Ealde, K., et al. 1982. Nature300:231-37 29. Goodenow, R. S., McMillan,M., ~rn, A., Nicolson,M., Davidson,N., et al. 1982. Science 215:677-79

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AnmRev. ImmunoL1983 1:569-607 Copyright©1983b.v AnnualReviewsIn~ All rights reserved


GENETICS, EXPRESSION, AND FUNCTION OF IDIOTYPES Klaus Rajewsky and Toshitada Takemori Institute for Genetics,Universityof Cologne, Cologne, Federal Republicof Germany

1. INTRODUCTION In this article we speak about the networkof idiotypes in the immune system. Westart by defining the vocabularyweare goingto use. Wethen discussthe structural basis of idiotypicdeterminants (i.e. antigenicdeterminants on the antibodyvariable region; also called idiotopes) and of their complementary anti-idiotypic antibodybindingsites (anti-idiotopes). Idiotopes and anti-idiotopes are encodedby antibody V, D, and J genes. The idiotypic repertoire thus reflects the repertoire of these genesas they are expressed, selected, and somatically modifiedin the immune system. The idiotypicrepertoire, its geneticbasis andits selectionin ontogeny,is a main themeof the present view.This relates directly to the functional idiotypic network:Whichrules do idiotypie interactions in the immune systemfollow andwhatis their physiologicalrole? Wewill see that idiotypic interactions seemto be involvedin the growthcontrol of lymphocyte clones with defined receptorspecificity andmaythus be essential for the generationof a diverse systemof interacting ceils. Theyalso stabilize the expressionof a given receptorrepertoire in active immune responses.Idiotypie interactions occur within the compartment of B lymphocytes,but they also involveT cells. We discuss the crucial issue of whetheror not recognitionof B-cell idiotypes is oneof the principles by whichT cells are selected in the immune system. Thisarticle cannotdeal with all facets of idiotypic research. Werestrict ourselves largely to the discussionof experimentalworkin the mouseand, in particular, wedo not discussin anydetail the role of idiotypic interactions in regulatory T-cell circuits controlling immune responses. Detailed modelsof immunologiccontrol through cell interactions have emerged fromrecent workin this fidd. Werefer the disappointedreader to a few 569 0732-0582/83/0410-0569502.00

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reviews on this and other matters that are not discussed here (9, 22, 57, 73a, 78, 95, 184, 198, 204).


2. VOCABULARYAND STRUCTURAL PRINCIPLES OF IDIOTYPIC INTERACTIONS BETWEENANTIBODIES Anidiotype (129, 155) is a set of idiotypic determinants or idiotopes expressed on the V region of a particular antibody or the V regions of a set of related antibodies. Idiotypes and idiotopes are defined serologically, i.e. by anti-idiotypic and anti-idiotope antibodies, respectively. Anti-idiotypic antibodies recognize, in general, a set of idiotopes and are therefore polyclonal. Anti-idiotope antibodies recognize individual idiotopes and are therefore, in general, monoclonalreagents. The idiotype of a given antibody distinguishes the latter from most other antibodies, but, as is discussed in more detail below, idiotypic crossreactions are frequently observed. In Figure 1 we depict schematically the interaction of two complementary antibodies, antibody 1 (abl) and antibody 2 (ab2). Wemayconsider ab2 as an anti-idiotope, raised against abl and binding with its hapten binding site (paratope) to an idiotope of ab 1. However,this distinction into anti-idiotope and idiotope-beadng antibody is merely operational: we might as well have immunized an animal with ab2 and obtained, as an antiidiotope, abl. In this situation the anti-idiotope wouldbind with a structure outside of the hapten binding cleft (paratope) to the idiotope-bearing ab2, and the idiotope recognized wouldbe the paratope of ab2 itself. The scheme in Figure 1 indicates that idiotypic interactions between antibodies occur through complementarystructures in antibody V regions and must involve aminoacid residues on protrusions and clefts of the interacting V regions. Becauseof the selectivity of idiotypic recognition, the interacting complementary structures must be largely determined by the hypervariable regions

pten1

~’~1~hapten2

Figure1 Schematic drawingof twointeracting antibodies.Complementary structures are meantto be in the Vregions.

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of the V domains, an expectation verified by a large body of experimental work(see Section 3). It is satisfying to see that in the available threedimensional models of antibody V domains, obtained by X-ray diffraction techniques and indirect methods, the hypervadable regions are indeed partly arranged as pockets and partly exposed on the surface of the V domain(reviewed in 75, 159). If this were not the case, idiotypic interactions of the exquisite specificity observed wouldbe impossible.1 The structural requirements of idiotypic interactions as depicted in Figure 1 are no morethan a restatement of the "sticky end" modelof antibody V domains developed by Hoffmann (88). The matter has recently been discussed in detail by Jerne et al (101), and wewouldlike to point out, along with the latter discussion, a fewspecific features of idiotypic interactions as they can be deduced from the schemein Figure 1. If we had raised ab2 as anti-idiotope against abl, 2 we would find that the anti-idiotope would not competewith hapten (hapten 1) for binding to its target (abl). In classical nomenclature we would call the idiotope recognized by the anti-idiotope non-binding-site related. If, on the other hand, ab 1 were the aati-idiotope it wouldcompetewith the hapten (hapten 2) for binding to its target (ab2); here the idiotope recognized wouldbe binding-site-related and, in fact, could be the binding site itself. In the original formulation of the network hypothesis (97), the structure on abl recognizing ab2 is the internal image of hapten 2. In line with these considerations, binding-site-related (28) and nonbinding-site-related idiotopes (114) have been described in a variety experimental systems. It is clear, however,that a hapten-inhibitable antiidiotope must not necessarily be a true internal image of the corresponding hapten, but may recognize an idiotope that is associated with the hapten binding site for structural and/or genetic reasons. Genetic associations even of nonbinding-site-related idiotopes with certain binding specificities have indeed been observed and are discussed below (Section 4.3). On the other hand, there is also goodevidence for the existence of true internal images of ligands from the word outside the immunesystem within the immune systemitself, at the level of antibodyidiotopes. Stalking examplesof this are anti-idiotypic antibodies against various hormone-specific antibodies. SubtThearrangement of the hypervadable loops in antibodyVdomainshas presumably been selectedin evolutionnot onlyto permitefficient idiotypieinteractionsof antibodies.The interactionof antibodieswithexternalantigenssuchas proteinsmayoftennot be restricted to a cleft in the Vregion,as suggested for smallhaptensbythe availablethree-dimensional models,but mayalso involveresiduesat the Vregionsurfaceas in the interactionof the V regionof proteinKolwithits ownFcpiece(90, 138). 2Inthe nomenclature of Jerneet al (101),ab2in Figure1 would be an anti-idiotope (against abl) of the ~ type(ab2a),whereas abl wouldbe anti-idiotope(against ab2) of the~ ty (ab2fl).

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sets of such anti-idiotypic antibodieshavebeenshownto be able to mediate hormone-specific effects (183, 193). Likewise,a selected anti-idiotypic antiserum against antibodies with specificity for tobaccomosaicvirus (TMV) coat protein wasable to induce anti-TMV antibodies in animalsof several species (132). Thereis also evidencefor internal imagesof constant region allotypes at the level of antibodyidiotypes in the rabbit (101, 178). As shownbelow, the idiotypic repertoire in any given animalis genetically restricted so that not any possible three-dimensionaldeterminantis expressedat the idiotopelevel (see Section4.3). Internal imagesmaytherefore exist for certain antigenic determinantsbut not for others. Let us reiterate that idiotope-bearingand anti-idiotopeantibody(idiotype and anti-idiotype) are operational terms by whichwelabel the partners in an idiotypic interaction (Figure 1) on the basis of an ad hoe criterion (mostlyconsideringas "anti-idiotope" the antibodyraised by immunization with the "idiotope-bearing"antibody). Whenone considers the functional role of idiotypic interactions in the immune system,oneshouldbear in mind that the anti-idiotope maycontrol the expressionof the idiotope-bearing antibodyas well as vice versa. As is shownbelow, this fundamentalpoint has not beensufficiently taken into considerationin the interpretation of regulatory experiments. 3. STRUCTURAL

LOCALIZATION

OF IDIOTOPES

Canwestructurally localize idiotopes within theV region? Thisquestion isofimportance notonly fortheunderstanding ofidiotypic interactions (see above), buta/soforthegeneticist whowants touseidiotopes asmarkers ofthegenes thatencode antibody V regions (Section 4.3). Intheabsence ofdirect information based uponX-ray analysis, oneisleftwithattempts tolocalize idiotopcs onsubunits oftheantibody molecule andtocorrelate amino acidsequences withidiotopc expression. Asonemight expect, a complex picture emerges fromthiskindofanalysis. Chain reassociation experiments haveshown thatidiotypic determinants usually require both heavy (H)andlight (L)chains forfull expression, although incertain cases some reactivity ofisolated chains with theanti-idiotypic antibody isfound (e.g. 19a,30,33a, 34,74,79,90a,134, 180a, 190a, 213;seealso 57,89). Anti-idiotypic reagents specific fordeterminants oneither L orH chain can bcselectively induced byimmunizing withhybrid antibody molecules in whicheither theH or theL chains arcderived frompooled "normal" immunogiobulin (I90,220). Suchrcagcnts arcparticularly useful forstudyingthegenetic basis ofV gcncexpression. Inspection ofamino acidsequences inidiotypically related mycloma andhybddoma antibodies support thenotion thatidiotopes arcphcnotypic markers of complementarity-

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IDIOTYPES 573 determining regions (CDR)(33, 34). In the case of a(1-3)-dextran (4-hydroxy-3-nitrophenyl)acetyl (NP)-binding k-chain-bearing murine antibodies, attempts have beenmadeto correlate aminoacid sequencesof the V, D, and J segmentsof the H chain with the expression of certain idiotypic determinants.[k-Chainbearingmurineantibodiesare particularly useful for this purposesince ), chains showlittle variability in the mouse (207).] In the case of ¢(1-3)-dextran antibodies, wherethe aminoacid sequencesof a large numberof closely related antibodies havebeendetermined(181), it appearedthat the expressionof a certain idiotypic determinant wasdependenton two positions in the D segment,and that of another was dependent on two residues in the second CDR(CDR2in V~(38). However,as the authors noted, the situation is muchmore complexthan wouldappearat first glance. Table1 showsthe reaction of three monoclonal anti-idiotope antibodieswith a series of monoclonal anti-dextranantibodies of definedprimarystructure. Inspectionof the data reveals that the interaction of idiotype and anti-idiotope (whichin all cases also dependson the presenceof the ~ light chain) canin no casebe attributed to a single stretch of aminoacids, and sequencesin V, D, and J seemto be involvedin various combinations.Table1 also contains sequencesand idiotypic characteristics of a series of closely related anti-NPantibodies together with a non-NPbindingsomaticvariant (the sourcesof these antibodies are given in Table 1). Althoughthe D region is again clearly of importancefor idiotope expression, the Vri region is also involved. (Comparethe expression of idiotope Ac146in antibodies B1-8, B1-8.V3,B1-48, and B1-8.V1;and of idiotope Ac38in antibodies B1-8, B1-48, G100.24,and B1-8.V3.For the contributionOf VHto idiotope Ac38,see also Section4.3.) Thus,the situation is again morecomplexthan originally anticipated (172). That amino acid residuesin both DandVHcan contributeto the constructionof a single idiotopeis also stronglysuggestedby the workof Potter andhis collaborators on the idiotypic propertiesof a series of interrelated galactan-binding antibodies (160). Oneof the idiotopes they analyzedappearedto be influenced by amino acid residues in D and CDR2,and three-dimensional modelsgenerated by a computerprogramindicate that the residues in questionare exposedon the surface of the V domain,in reasonabledistance from each other. Other idiotypic determinants in the samesystem also appear to involve the participation of morethan a single CDR. Despitethe complexstructure of idiotopes, very smallchangesin primary sequencecan cause drastic changesin idiotypic specificity. Anexampleof this is antibodyB1-8.V3in Tablel, wherea single aminoacid exchangein Dleads to the loss of oneof the idiotopes, the modificationof the second, and, in fact, to the loss of six other idiotopes of the parent molecule.The variant antibody fully retains NP-bindingspecificity (A. Radbruch,S. Zaiss, K. Rajewsky,K. Bcyreuther, to be published).

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Table 1 Idiotopes and primary structure regions

of c~(1-3)-dextran

bV region segments


a Antibody J558 Hdex9 Hdex36 Hdex2 Hdex25 Hdexl2 Hdex24 MIOC104E

B1-8 B1-48 G100.16 G100.24 B1-8.V3 B1-8.V1

V H 1 3 1 1 1 1 1 1

186-2 186-2 186-2 186-2 186-2 186-2/102

cIdiotopes

D

JH

RY RY RY NY SY GN SS YD

1 1 2 1 1 4 2 1

YD. YYGSS -L LG Y.. - .... --TH ..... R-....

and NP-binding antibody

2 1 4 2 2 2

EB3-72

EB3-16

AB3

+++ ++ ++ ++ ++ ++ ++ -

+++ + + -

+++ ++ +++ ++ -

Ac38

Ac146

NP-bdg

+ + + +

+ dND eCR -

+ + + + + -

acffl-3)-dextran (upper panel) and NP-binding(lower panel) myelomaand hybridomaproteins. bAminoacid sequences of heavy-chain variable regions in abbreviated form (the L chains are germline-encoded ~.1 chains in all cases). TheHsegment si i dentical i n most of t he dextran-binding (VHI) and the NP-binding molecules (V186-2). VH3differs from VH1in 4 positions in CDR2. VH186-2/102 differs from VH186-2in 10 positions, most of them in CDR2.The sequences of the Dsegmentare given in the one-letter code. The JHsegmentis also indicated. The sequencedata on dextran binding moleculesare taken from Schilling et al and Clevinger et al (38,181). Thesequences of the NP-binding molecules are from Bothwell et al (23) (B1-8); A. Bothwell, M. Paskind, Baltimore, personal communication(B1-48); H. Sakano, K. Karjalainen, personal communication (G100.16and G100.24); S. Zaiss, K. Beyreuther, personal communication(B1-8.V3); and Dildrop et al (49) (B1--8.V1). CEB3-72,EB3-16,and AB3are idiotopes of J558. The typing results are from Clevinger (37). Ac38and Ac146are idiotopes of B1-8. NP-bdg, NP-binding. The typing of B1-8.V3was done by A. Radbruch. dNot determined. eAffinity of anti-idiotope 10× lower than on B1-8.

Thedatain Table1 also showthat the absenceof a binding-site-related idiotope[Ac146(174)] is not necessarilyaccompanied by a changein hapten bindingspecificity. Clearly,therefore,the corresponding anti-idiotope andthe hapteninteract with the target antibodyin different ways.The anti-idiotypemaybind to a larger surfacearea than the haptenor to an idiotopethat is, for structuraland/orgeneticreasons,in its close vicinity. Anidiotope of the latter kind could, at the level of the total antibody repertoire,be mostlyassociatedwithantibodiesof the samehaptenspecificity--as indeedhas beenobservedin the case of idiotope Ac146--although the corresponding anti-idiotopedoesnot carrythe "internalimage"of the hapten.

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To concludethis section, idiotypic determinantsappear to be complex structures to whichoften both Hand L chains and, within one chain, more than a single CDRmaycontribute. Theymaybe distant from, or closely associatedwith, a hapten-binding site of the antibody,but evenan idiotope invariablyassociatedwith antibodiesof a givenhaptenspecificity andrecognized by a hapten-inhibitable anti-idiotope does not necessarily coincide with the haptenbinding site. 4.

THE IDIOTYPIC

REPERTOIRE

4.1 What are the Questions? Idiotypesare serological markersof antibodyVregions, andthus in principle allowthe analysis of the expressionandselection of antibodyVregions in the immune system. In viewof the diversity of V regions, any analysis of the repertoire remains a priori fragmentary. However,a numberof questionscan be askedthat allowstraightforwardanswers.(a) Is the idiotypie repertoire generatedat the B-cell level restricted andcan wecorrelate it with the genetic informationavailable in the antibodystructural genes? (b) AreB cells selected accordingto their receptoridiotype whenthey leave the bone marrowand enter the peripheral immunesystem?(c) Howare cells selected in antigen-driven immune responsesand, specifically, are idiotypicinteractionsinvolvedin this selection?(d) If idiotypieinteractions are involvedin B-cell selection at any stage, whatis its mechanism? Thesequestions lead to the field of regulatory T cells, whichare known to be able to specifically recognizeandexpressidiotypic markers.Wedis- " cuss later the problemsof T-cell idiotypes and their regulatory function. 4.2 Analyzing the Idiotypic Repertoire of B-Cell Receptors Theclassical wayof studyingidiotype expressionin the immune systemis the idiotypic analysis of antibodies producedin antigen-driven immune responses.This approachis limitedin severalways.First, it is restricted to antibodies of a given antigen-bindingspecificity, and thus does not allow one to study the representation of a given idiotypic markerin the total population of antibodies. This limitation can be overcomeby inducing idiotypic responsesby immunization with anti-idiotypic antibodies, an approach pioneered by the groups of Urbain (205) and Cazenave(35). analysis of suchresponsesis complicatedby the fact that an anti-idiotypic antibody will induce the production of two types of complementary antibodies, namely(a) antibodies carrying the idiotype defined by the antiidiotype used for immunization,and (b) anti-anti-idiotypes. Theusual serological assays used for idiotype titration do not distinguish between these twotypes of molecules.This has led to confusionin the literature for

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the following reason: If any complementary antibody produced in response to immunizationwith anti-idiotypc is considered to express the corresponding idiotypc, then the idiotypc will be found expressed in any immune system, since each system is sufficiently diverse to allow the induction of complementaryantibodies. Several ways arc open to distinguish between idiotype and anti-anti-idiotypc produced in response to anti-idiotype. One is to identify idiotypc by a variety of anti-idiotypic reagents in addition to the one used for immunization (18, 217). Another one is to identify the idiotype by an unusual structural property such as, for example, expression of k chains in the case of the mouse. Since in mice k chains are expressed in less than 5% of total immunoglobulin, anti-anti-idiotypes would bc expected to express r light chains. The latter approachhas been successfully used (170, 171, 199), and its validity has been recently verified in an independent (third) way that is generally applicable (19), namely, by showing that the idiotypic molecules were indeed encoded by V genes similar to those encoding the original idiotypic antibody (M. Siekevitz, R. Dildrop, K. Bcyrcuthcr, K. Rajcwsky, to be published). Methods of this kind are essential for the identification of idiotypic molecules producedin response to anti-idiotope. One should bear in mind, however, that they all give a minimumestimate of the idiotypic response and might classify an unknown fraction of idiotypic moleculesas anti-anti-idiotopes. The mainproblem of analyzing idiotypic repertoires a.t the level of induced serum antibodies is that the cells producing these antibodies have already been subject to complexregulation, be it before or after immunization. Onewouldtherefore like to see howthe idiotypic patterns determined at the level of serum antibodies compare to the frequencies of B cells expressing the corresponding idiotypic determinants at the various stages of B-cell ontogeny. Several methodsare available for this purpose. One is the limiting dilution assay for mitoge~-reactive B cells (4, 5), in whichthe frequency of mitogen-reactive B cells expressing a #oven antibody V region can be determined. The assay is a priori limited to mitogen-reactive cells --~ the case of bacterial lipopolysacchadde (LPS), roughly every third cell in the spleen of a young mouse(4). Since LPS-reactive cells can generated from bone marrowor fetal liver pre-B cells in vitro (66, 141), frequency determinations of, for example, idiotype-expressing cells in the population of newly generated B lymphocytes are possible (147). During ontogeny, certain fractions of the newly generated (LPS-reactive) B cells becomeresponsive to T-cell help (77, 120, 189) and (or?) enter the pool long-lived, recirculating B cells (64, 191, 192). As T-cell lines delivering help to B cells in vitro in a polyclonal or antigen-specific fashion have recently becomeavailable (6, 105, 117, 162), frequency determinations B cells responsive to T-cell help in an in vitro systemappear feasible, but

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they havenot yet been carried out. However,the morecomplicatedtechnique developedby Klinmanand his colleagues (119), in whichlimiting numbersof B cells are injected into irradiated hosts andthe T-cell dependent responsesof single cell clonesin splenicfragmentsculturedin vitro are recorded,is thoughtto allow frequencydeterminationsin just that B-cell population.Thereis thus a variety of methodsavailable for the determinations of V-regionrepertoires in B cells, eachmethoddeterminingthe repertoire expressedin cells at a particular stage of differentiation.

4.3 The GeneticBasis of the Idiotypic Repertoire Classicalworkon the expressionof idiotypesat the level of inducedantibodies (reviewedin 57) has revealed two types of idiotypic markers,private (minor) idiotypes, expressedirregularly in someindividuals of a given species or strain, and recurrent (major, cross-reactive) idiotypes, which regularly appear in certain immune responsesin the members of a certain speciesor strain or evenseveral species(102b,200a).In the case of strainspecific recurrentidiotypes,geneticanalysis has shown that idiotypeexpression is almost invariably controlled by antibodystructural genesonly, in most eases by the Vn(reviewed in 208), in someinstances by both the VI~and the VLlocus (51, 91, 107, 149). The interpretation of these resuits wasthat idiotypes are markersof antibodyVgenes, that strain-specific idiotypes are due to V-genepolymorphism, and that the predominant control by the VHlocus reflects moreextensive polymorphism of VHthan V~. genes. (Wenowknowthat D and J region polymorphism must also be taken into account.) Byusing recombinantmousestrains it waspossible to construct roughgenetic mapsin whichregions controlling various idiotypic markers were separated from each other by recombination breakpoints. Thesesubjects have been extensively reviewed(56, 137, 208). The modem recombinantDNA techniques have madeit possible to study the genetic basis.ofidiotypeexpressionat the molecularlevel. Suchstudies havelargely confirmedthe classical workand, in addition, dramatically improvedour understandingof the problem.The molecularanalysis of several immune responses dominatedby recurrent idiotypes showedthat each of these responsesis basedon the expressionof a single or very few Vnanda single or very few VLgenesin the germline. This is the case in the responseof miceto arsonate [dominatedby the Arsidiotype (148)], a(1-3)-dextran J558 and MOPC104E idiotype (17, 182)], phosphorylcholine(PC) [domimated by the T15 idiotype (41, 133)], and NP[dominated by the b idiotype (137, 93)]. In these four systems,the antibodyresponseis known to consist of oneor a fewantibodyfamilies, each of whichis characterized by the expression of a single Vngene in combinationwith several D and JI~ segmentsand of one or a few V~and Ji~ genes(23, 24, 33a, 38, 48, 72,

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78, 173, 174, 181, 187) [for the molecularbiology of antibodystructural genes, see Honjo(88a) in this volume].Themembers of each familyare thus closely related to eachother, but the families are verylarge, as in addition to combinatorialdiversity, somaticmutationsin the rearranged variable region genes occur, sometimesat high frequency(11, 23, 24, 48, 72, 161, 181,187, 207; for further families ofidiotypicallyrelated antibodiessee 78). Thus,at least in responsesto certain epitopes, the Y-regionrepertoire is controlled and limited by a few germlinegenes, despite extensive somatic mutation--amodernversion of the "germlinetheory" of antibodydiversity. Asimple interpretation of the phenomenon of recurrent and private idiotypes in antigen-inducedimmuneresponses emerges:recurrent idiotypes are markers of those germline ¥ genes from whichthe antibodies in the correspondingimmune responseare derived and of part of their somatically mutatedprogeny.Private idiotypes are characteristic for somaticmutants. Wehavetested this propositionwhichhas beenwidelyentertained [78] with a pair of monoclonalanti-NP antibodies derived from C57BL/6mice. AntibodyB 1-8 expresses a germline-encodedV region; antibody $43 uses the sameV-regiongenesbut carries somaticpoint mutationsboth in VII and YL(23, 24,173). Non-cross-reactingidiotopes on antibody B1-8and $43, respectively, were defined by monoclonalanti-idiotope antibodies. As expected, the B 1-8 idiotopes are regularly expressedin anti-NPresponsesof C57BL/6mice (111, 174, 175). In contrast, the $43 idiotopes are only occasionallydetectable, and eventhen at very low levels (G. Wildner,T. Takemori,K. Rajewsky,to be published). Theresult is consistent with the viewthat recurrent idiotopes are markersof germlineV(and possibly D) genesand that idiotopes specific for somaticvariants are irregularly expressedand thus private. This does not, of course, necessarily implythat all private idiotypic markersmust be the result of somaticmutation, nor that all germlineV genesare regularly expressedin the immune system;we return to these pointslater. Thefindingof extensivesomaticmutationin B cells engagedin a specific immune responsealso providesa potential explanationfor a finding that has puzzledimmunologists since its original description (156), namely,that idiotypic cross-reactivityof antibodiesdirected at different determinantsof the immunizing antigen (64a, 87b, 102a, 108, 125, 143, 170, 199, 214a,217). Onemightspeculate that in a given B-cell clone, activated in responseto oneof the antigenic sites of the immunizing antigen, somaticvariants arise, someof whichexpress specificity to another determinant on the same antigen. Suchvariants wouldthen be further stimulated by the immunogen and maybe selected for higher affinity by subsequentsomaticmutation.The mutantsmaystill share idiotypic specificity with the antibodyexpressedby the original ("wild type") clone, and the idiotypie networkmightthus also

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IDIOTYPES 579 contribute to their selection (97). This picture has recently emergedfrom the elegant experimentsby W.Gerhardand his colleagues in whichmonoclonal antibodieswith specificity for influenzahemagglutinin wereisolated from individual immunizedmice and were comparedin terms of primary structure andepitope andidiotypic specificity. Theevidencesuggestsstepwisemutationin single B-cell clones, resulting in the productionof antibodies with different, fine specificities (194; W.Gerhard,M.Weigert,personal communication). These considerations add an important aspect to the problemof the generationof the antibodyrepertoire. If somaticB-cell mutantswith altered bindingspecificity constitute a substantial part of the repertoire of functional B cells they might play an important role in responsesfor which suitable V, D, and J genes have not been selected in the germline, e.g. responses against variants of common pathogens. Wecan only say at this point that somaticmutationis not extensive enoughto obscurethe clear pattern of germlinedeterminationin responseslike those to NP,Ars, and PC. Geneticdeterminationandrestriction of the idiotope repertoire can also be demonstratedwhenidiotope expressionis studied in the total antibody populationrather than in populations defined by a given antigen-binding specificity. In the initial experimentsof this type, rabbits wereimmunized with anti-idiotypic antibodies (ab2 in the designationof the authors) produce antibodies complementaryto ab2 (ab3). The population of ab3 containedlittle or no antibodieswith specificity for the antigen, whichwas recognized by the idiotypic antibody (abl) against whichab2 has been raised; however,uponimmunizationwith that antigen, the ab2-sensitized animalsproducedantigen-specificantibodies(ab 1’) that wereidiotypieally indistinguishable from abl (35, 205). Theseexperimentswere later reproducedin other systems(18, 20, 131, 132, 170, 171, 199, 217). It wasalso shownthat in certain experimentalsystemsthe response to ab2 wasdominatedby ab 1, as definedby its antigen-binding specificity (178). Thus,a way appearedopento determinethe idiotypic repertoire in the functionally active B-cell population.Suchexperimentsrequired, however,the differentiation in the ab3populationof idiotype-bearingantibodiesfromanti-antiidiotypes. When this wasdone(as outlinedin Section4.2), twoclear results emerged.In general, the populationcontains a large component of idiotypebearing antibodies that do not share antigen bindingspecificity with the idiotype-bearingantibodiesusedfor the inductionof ab2 (18, 170, 171,199, 217). Togetherwith the workon idiotype sharing betweenantibodies of differentfine speeiticity, the structural data discussedin Section3, andother work(52a, 60), this clearly demonstratesthat the distributions of idiotopes and antigen-bindingspecificities in antibody-Vregions are overlappingbut

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not identical. This important point is exemplified by the data in Figure 2, where the expression of two idiotopes of the germline-eneoded anti-NP antibody B 1-8 in the total antibody population is analyzed, upon immunization with the corresponding cross-linked anti-idiotopes. Clearly, one of the two idiotopes (Ac38) is mainly expressed in non-NPbinding antibodies, whereas another (Ac146) is largely restricted to antibodies carrying binding sites. The appearance ofidiotope Ac38in non-NPbinding antibodies is not due only to somatic mutation or to different waysof V-D-Jjoining. The structural analysis of a somatic variant of antibody B1-8, in whichpart of the VrI region is encodedby a different germ line Vri gene (30, 49) and of anti-idiotypically induced Ac38+ monoclonalantibodies (R. Dildrop, M. Siekevitz, K. Rajewsky,K. Beyreuther, to be published), demonstrates that a series of closely related Vri genes, only one of which is involved in encoding NP-specitic antibodies, encodes Vn regions of Ac38+ molecules. This is direct evidence for idiotype sharing between products of V genes in the germline expressedin antibodies differing in antigen-bindingspecificity. A second result can also be read from the data in Figure 2, namely, that also in this approachidiotope expressionturns out to be strictly genetically ~l-bearing

antibodies(~ug/ml)

1.0 10 100

1.0 10 100

1.0 10 100

÷ A. Ac38

÷ B. Ac146

÷ C. A6-24

1.0 10

C57BL/6 BALB/c CB.20 D. NPb

Figure2 Analysisof idiotopeexpressionby immunization witha cross-linkedmonoelonal anti-idiotopeantibody.Thisantibody(Ae38)detectsan idiotope(Ae38)on the germlineencodedNP-binding antibodyB1-8.Thelatter antibodyalso carries idiotopesAc146and A6-24,definedbymonoclonal anti-idiotopes.Alltheseidiotopesare recurrentlyexpressed in anti-NPresponsesof micecarryingthe Ighb haplotype.Idiotope-bearingandNP-binding molecules in the sera of miceimmunized withcross-linkedantibodyAc38weredetectedon plates coatedwitheitheranti-idiotope(left threepanels)or NP-bovine serumalbumin (right panel),witha radioactive monoclonal anti-k1 antibody [the anti-idiotopes carry~¢chains,the idiotope-bearing molecules carryk chains(174)].Absorption oninsolubilizedantibodyAc38 removed> 98%of all idiotypie and NP-binding molecules,absorptionon NF-Sepharose + and 17%Ac38 + moleculesin strain C57BL/6 removed83%Ac146 (Ighb/b). Strain CB.20 b carries the Igh locusonthe BALB/c background. BALB/c is Igh~/°. Thedata are takenfrom Takemori et al (199),wherefurtherdetails canbe found.

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IDIOTYPF-,8 5 81 controlled, again by genesin the Igh linkage group. Thus,Ighb/b but not Igha/a mice express the Ac146and A6-24idiotopes, and although both + antibodies, it is only in Ighb/b micethat strains of micecan produceAc38 this idiotope is associated in part with NPbinding specificity. Since in + NP-bindingantibodies of the IgG1class all (Ighb X Igha)Fl mice, Ac38 C~ 95%)carry the b allotype (199), it wouldappear that indeed the D, and JH genes in the Igha haplotype are unable to encode an anti-NP antibody with the Ac38idiotope. TheF1 experiment,also carried out in anotherexperimentalsystem(53), argues against the possibility that lack of idiotope expressionis due to anti-idiotypic (network)control. Astrong argument against this possibility has also beenmadeby Siekevitzet al (186) for the Ars idiotype, whosestrain-specific expressioncould be correlated with the presence or absenceof the correspondingstructural Vri gene. Strains lackingthis genedo not expressthe Arsidiotype, althoughthey are undercertain conditionsable to produceidiotypically cross-reactiveantibodies (136)~presumably products of related, but non-identical, V genes. Asimilar situation appearsto emergein the case of the M460 idiotype(131). Asis discussedin Section6.1, lack of idiotypeexpressionhas beenascribed to anti-idiotypic control in certain other systems. To summarizethis section, recurrent idiotypes have turned out to be encodedby single, or small groupsof, germlineVaand VLgenes and some of their somaticallymutatedprogeny.Theidiotypic repertoire is thus in part controlled andrestricted by the Vgenesin the germline,anddifferent strains in a speciesmaydiffer in their repertoirebecauseof Vgenepolymorphism. The systemgenerates somatic mutants, and since this process appears to be based on randommutation, idiotypes characteristic for such mutantsare probablyusually nonrecurrent,i.e. private. Thetotal repertoire of idiotopes in the immune systemis the sumof the repertoire of recurrent andof private idiotypes. Wewantto stress again specifically that although certain immune responseslike those to PC, NP,Ars, ere, are dominatedby recurrent idiotypes (and somaticmutationmayin these cases mainlyserve to improvethe specificity of the response), other responsesand cellular interactions maydependentirely on somatic mutants whosegeneration mighttherefore be of vital importancefor the system. Finally, the data discussedin this section showthat idiotopes can be sharedbetweenantibodies of different antigen-binding specificity andvice versa, andit providesa clue of howidiotypicallyrelated antibodieswithdifferent epitopespecificity might arise in immune responses. 4. 4 ldiotypic Repertoire at the Level of B-Cell Precursors Animportant approachto studying the sdeetion of Vregions in the immunesystemis to analyze the V-regionrepertoire expressedin newlygener-

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ated B cells, whichappear constantly in large numbersin the bonemarrow (154), i.e. the "available" repertoire, and to compareit to the V-region repertoire expressedat later stages of B-cell ontogenyand in immune responses-the "functional" repertoire. Unfortunately,studies of this type are still in their infancy.Theearliest analysesof frequenciesof idiotypically defined B cells showedthat T-dependent(188) and T-independent(TI-2) (42) T15positive precursorcells occurin spleencells at a frequencyof 1-2 X10-5. Theseresults werelater confirmedby Fung& K/Shier(71 a). Clearly, B cells in the spleenmayalreadybe selected fromthe "available"repertoire. This notion is compatible with subsequent results of Kllnmanand his colleagues (189), whichshowthat T15-positive, T-dependentprecursor cells couldbe detectedin the spleenonly whenthe micewereat least 7 days old. In fact, at the level of B-cellprecursorsdefinedby their antigenbinding specificity, the late appearanceof certain specificities has beenobserved repeatedly(reviewedin 120a).In line with this result, Fung&K/Shler(69) recently found only T-independent(TI-1) T15+ responses in B cells from very younganimals; T-dependentresponseswere observedonly later. Frequencies of T15+ precursors in newlyarising B cells have not yet been determined.In other systems, frequencies of LPS-reactive, idiotypically defined B precursor cells were measured.Eichmannet al (60) determined + LPS-reactivesplenic B cells to be 4 X 10-4. [ASA the frequencyof ASA is a recurrent idiotype in the responseof A/J miceto groupAstreptococcal carbohydrate(54).] In a recent stqdy, Nishikawaet al (147) measured frequencyof LPS-reactivesplenic B cells expressingidiotopes of the germ line-encodedantibodyB 1-8 (23, 24, 173; see also Table1 andFigure2). One of the idiotopes (Ac38,see Section4.3) known to be presenton the products of several VHgenes in combinationwith the Vxl domainoccurred at a frequencyof"d0-a; another one, restricted to antibodies with an NPbinding site (Ac146,Section 4.3), wasfoundat a frequencyof -5. Identical frequencies wereobservedin LPS-reactivecells from spleens of newborn and adult animals and in cells generatedfrom bonemarrowpre-Bcells in vitro, in agreementwith similar data of Jui et al (106a). Overall, the frequenciesmeasuredfor recurrent idiotypes or idiotopes in the various assaysystemsrangefrom10-3 - 10-5 (see also 10). Thereis the indication that in newlyarising B cells, the frequency dependson the numberof genes involved in the expression of a given idiotypic marker (147). However, to date, there is not a single analysis availablein whichfor a given idiotypic markerfrequencydeterminationshavebeencarried out at the various stages of B-cell ontogeny.LPS-reactivecells mayrepresent immatureB cells (70, 139), and it is thus not surprising to find similar precursor frequencies in such cells, be they isolated fromthe spleen or generatedin vitro frompre-Bcells. It will be essential for an understanding

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IDIOTYPES 583 of B-cell selection in generalandthe idiotypic networkin particular to see howthese frequenciescompareto those in B-cell populationsat later stages of ontogeny,e.g. B edls responsiveto specific T-cell help and/orlong-lived, recirculating B cells (64, 69, 71, 191, 192). Below(Section5.3) wegive examplethat strikingly showsthat idiotypic frequencies in LPS-reactive spleencells may,in certain instances,not at all reflect whatis expressedin the immuneresponse generated by the sameanimal’s B-cell population. Otherinformationthat is lacking are the frequenciesof precursor cells expressing,at the various stages of ontogeny,idiotypic markerscharacteristic for somaticmutantsand, in the samecontext, of cells expressingantiidiotypesagainst recurrentidiotypicmarkers.Thelatter point has to be seen in connectionwith the so far unresolvedquestion of whetheror not the germlinedirectly encodesa networkof idiotypes (see Section4.3). In addition, the frequencyof anti-idiotypesmaybe of importancefor the activation of idiotype-bearingcells in the system.

4.5 ImmunoglobulinIdiotypes on T Cells After the initial findingsthat anti-idiotypieantisera couldspecificallyinduce idiotype-bearing T helper (TH)cells (62) and that alloantibodies sharedidiotypes with alloreactive T cells (14), a bulk of experimentalevidence has further demonstratedthe expressionof recurrent antibody idiotypesin functional T cells andantigen-binding material(factors) of T-cell origin. Earlyliterature on this subject has beenreviewed(15, 135, 169), and wequote here only morerecent workon idiotype or idiotope expressionin TH (59, 104, 144, 194a) and T suppressor(Ts) (81, 132a, 185, 193a, cells, in T cells mediatingdelayed-typehypersensitivity(DTH)(7, 96, 196, 201, 218), andcytotoxicreactions(68) in antigen-binding materialof T-cell origin (46, 47, 127) including suppressor(73, 86, 150) and helper factors (145). However,with respect to the molecularnature of the T-cell receptor for antigen, idiotypic cross-reactivity of this kind doesnot havemuchsignificance, in particular if one remembers the discussion of "internal images" and the idiotypic cross-reactions betweenanti-hormoneantibodies and hormonereceptors (Section 2). It was only after the demonstrationof the ~ontrol of both T- andB-cell idiotypesby the Igh locus that the hypothesis could be advancedthat antibodyV genes[and specifically VHgenes, since neither VI. determinantsnor control of idiotype expression by VLgenes couldbe identified in T cells (46, 57, 196a)]participate in encodingT-cell receptors for antigen (15, 169). Despitethe availability of specific antiidiotypic reagents andof clonedT-cell lines, little progresshas beenmade over the last years with respect to the elucidationof the structure of T-cell receptors. In addition, although recent immunogenetic data indicate that

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genes also linked to the Igh complexmayencodeconstant regions of T-cell receptors (158, 202), the available evidencefrommolecularbiology argues against the notion that the sameVII genesthat encodeantibodyV regions are expressedin T cells: regular Vngenerearrangement has so far not been observedin suchcells (128, 130). Wedo not wishto go into this complicated matter further in this context, in particular since the molecularbasis of T-cell recognitionof antigen has not, to date, been resolved. However,wewant to maketwo points, which lead directly into the topic of the next sections, namely,that of idiotypic regulation. First, since T cells expressidiotypes [and, consequently,also anti-idiotypicspecificities (3, 27, 36, 61, 76, 86, 116, 157,163, 197, 276)], they are by definition partners in the idiotypic network.Second,if T-cell idiotypes are not encodedby antibody V genes, wehave to explain how idiotypic polymorphism developsin parallel in the B- and T-cell compartment. If wedo not want to invoke a complicatedmechanism of adaptation andrectification of separatesets of Vgenesin evolution,wehaveto consider the possibility that T cells are selected in the immune systemin ontogeny such that their receptor idiotypically resemblesthat of B-cell receptors. Sucha mechanism wouldimplya functional idiotypic networkin the most fundamentalsense. It wouldbe expectedto lead to a situation wherethe idiotypic (and anti-idiotypic) repertoires expressedin the twosets of cells wouldbe similar, but not quite identical, in keepingwith substantial experimental evidence (13, 15, 76, 102, 126, 146, 163). Wecomeback to this matter in Section6. 4. 6 Co-Existence of Lymphocytes with Complementary Receptors." The ldiotypic Network Lymphoeytes expressing complementaryreceptors co-exist in the immune system.At the level of B cells, anti-idiotypic antibodiescan be inducedin animals knownto possess B-cell precursors expressing the corresponding idiotype (reviewedin 57). In addition, animalsoften produce,in the course of an immune response,anti-idiotypic antibodiesagainst idiotypic determinants of their ownantibodies, althoughmostlyin low concentration(12, 39, 67, 77a, 94, 110, 121, 140, 177, 197a,200). At the level oft cells, idiotypebearingand anti-idiotypic T helper and suppressorcells can be inducedin animalsthat expresssimilar idiotypes andanti-idiotypesat the B-cell level (see Sections5 and6). Thereis thus no questionthat self-recognitionat the level of receptor idiotopes is possible within the immune system,quite as Ramseier and Lindenmann (171a) pointed out 10 years ago. Whatare the physiological consequencesof this situation? Wecan look at the problemfrom the standpoint of classical immunology and ask how the systemis able to establish self-tolerance since it allowsthe expression

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IDIOTYPES 585 of idiotypesin immune responses,undisturbedby anti-idiotypic cells. Aswe shall see, regulatory mechanisms havebeenidentified that potentially resolve this difficulty. Fromthe standpointof Jerne’s hypothesis(97, 98), the co-existenceof idiotypesandanti-idiotypesis an essential feature of the immune system. Cells with complementary binding sites interact with each other and an equilibriumof idiotypes and anti-idiotypes is established. Immune responsesare seen as disturbancesof this equilibriumand attempts of the systemto re-equilibrate. In addition, idiotypic interactions mightbe essential for the generationof the functionalreceptorrepertoire, in the sense of both promotingsomaticdiversification and restricting diversity. Quantitative modelsof idiotypic networkshavebeendeveloped(2, 87, 88, 176), and the essential points of three of themhavebeen summarized by Jerne (99). In the sections belowwediscuss someof the experimentalevidence that supports the notion of a functional idiotypic network. 5. IDIOTYPIC REGULATION OF THE RECEPTOR REPERTOIRE BY ANTIBODIES 5.1 Effect of .4nti-Idiotype on Idiotype Expression In this andthe next section weconsideronly experimentsthat approachthe physiologicalsituation in that antibodiesare applied in native formandin low doses. Underthese conditions, anti-idiotypic antibodies are extraordinarily potent regulators of idiotype expression,in the sense of enhancementor suppression. In general, thoughnot without exception (18, 61, 164a, 165, 203), anti-idiotype, whengiven in native form, did not directly inducesynthesis of idiotypic molecules.Rather,its effect appearedto be a long-lastingmodificationof the functionalidiotypicrepertoire. Theclassical workin this area, largely basedon xenogeneicand allogeneic conventional anti-idiotypic antibodies, has beenextensivelyreviewed(22, 57). Weconcentrate the present discussion on morerecent workand on the mechanisms throughwhichanti-idiotypes exert their regulatory function. Themonoclonalantibody technique (122) has madeit possible to study antibody-mediated idiotypic regulation at the level of individual idiotopes and with isogeneic monoclonalidiotype-bearingand anti-idiotope antibodies. Experimentsof this type, carded out in our laboratory, showedthat anti-idiotopes of mouseorigin injected into the mousewereas ettieient in terms of idiotypic regulation as werexenogeneic(guineapig) anti-idiotypic antibodies injected into the mouse(54, 55, 62): 10-100ng of anti-idiotope enhanced,whereas10/~g suppressedthe expressionof the target idiotope in immune responses induced4-8 weekslater (111, 112, 175). Theinteresting observationthat the class of a xenogeneicanti-idiotype determinesits regulatory function in the mouse[guinea pig IgG1 enhanced

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whereasIgG2suppressed idiotype expression (54, 55, 62)] could not reproducedwith the monoclonalmudneanti-idiotopes. In recent experiments(C. Miiller, K. Rajewsky,to be published), two families of such antibodies were constructed and employed.The membersof each family carry the sameV region on different heavy-chainconstant regions [spontaneousclass switchvariants (167)]. It wasshownthat anti-idiotopes of the IgG1, IgG2a, and IgG2bclass all enhancedidiotope expression at nanogram doses and were suppressive at microgramdoses. In the isologous situation, the regulatoryfunction of anti-idiotopeantibodiesof these three classes is thus determinedonly by the antibody dose. Onewonders,however, whetherthe nature of the target idiotope mayalso play a role, since in earlier experimentswehad failed to induceenhancement with a particular anti-idiotope andsuspectedclass-specific regulation(175). Theexperimentsdescribed abovewererestricted to the analysis of idiotype expressionat the antibodylevd. Anti-idiotypes havealso been successfullyusedto induceidiotypiceffeetor cells in cellular reactions, suchas T cells mediatingDTH (7, 96, 196, 201). Theclass of the anti-idiotope may play a role in these cases. Astriking effect of anti-idiotypic antibodiesis also seen whensuchantibodiesare either injected into newborn animals(1, 8, 71, 87a, 109,115, 116, 124, 182, 209) or enter into the newborn’scirculation fromthe mother, either through the placenta or the milk (210). As a consequence,longlasting suppressionof idiotype expressionis generallyobserved,althougha case of enhancement has also beenreported (87a, see also Section6.1). Very smalldosesof anti-idiotypeare requiredfor suppression(1, 8, 71, 109, 115, 116, 182, 209). In our ownexperimentswehavefound that 1/.tg of monoclonal anti-idiotope injected into the newbornwassufficient for essentially complete idiotope suppression over several months (T. Takemori, K. Rajewsky,submittedfor publication).

5.2 Effect of Idiotype on Idiotype Expression Experimentshave also been done in whichthe effect of injection of an antibody with a given idiotype on the expression of that sameidiotype (instead of complementary binding sites, as in the experimentsdescribed above)wasassessed. All experimentsin whichsoluble "idiotypic" antibody wasapplied had in principle the sameresult. Urbainand co-workersimmunized a pregnant female rabbit with anti-idiotype to producecomplementary antibodies. In the offspring of the immunizedanimal, the correspondingidiotype was recurrently expressed in the immuneresponse, in contrast to the controls (214). Similarly, Rubinsteinet al (179) injected small doseof an anti-levan antibodywhoseidiotype is only rarely expressed in the anti-levan response into newbornmice. Whenthe animalshad grown

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IDIOTYPES 587 up, the sameidiotype wasdominantin their anti-levan antibodies (179). Enhancement of idiotype expression was also seen whenmonoclonalantibodiescarrying a recurrent idiotype wereinjected into adult mice. In one study, the induction of idiotype-expressingplaque-formingcells wasreported, without deliberate addition of antigen to the system(92; however see also 85a). In an experimentdone in our laboratory it wasshownthat injection of 10 #g of antibody B1-8led to enhancedexpression of B1-8 idiotopes in an anti-NP response induced6-8 weekslater. Ananalogous result wasobtainedby Ortiz-Ortiz et al (153) in the Ars system,but much higher dosesof idiotype wereused in this case. Formally,these results can be describedas idiotypic memory:the immune systemsees the structure of an antibody variable region and reproducesit later on in an immuneresponse. Idiotypic antibodyhas also beeninjected into animalsafter it hadbeen chemicallycoupledto syngeneicthymocytesor spleen cells (50, 195). contrast to the aboveexperiments,this resulted in suppressionof idiotype productioneither at the level of antibodies or of cells mediatingDTH. 5.3 Mechanisms of ldiotypic Regulation by Antibodies Since enhancement ofidiotype expressionby idiotype occursundera variety of experimentalconditions, morethan a single mechanism might mediate the effect, suchas inductionof idiotype-specitie T-cell help (179) and/or enhancinganti-idiotypic antibody(112) or elimination of suppression anti-idiotype (153). Thesuppressioninducedby cell-boundidiotype is due to the inductionof anti-idiotypic suppressorT-cell circuits (50, 195). Howdoesanti-idiotype exert its effect on idiotype expression?Enhancing doses sensitize idiotype-expressing Tr~cells (16, 62, 103), T edls mediating DTH(7, 196, 201), and B cells whosefrequencyis found enhancedin the population of LPS-reactive spleen cells (60; T. Takemori,K. Rajewsky, unpublishedresults); it is not clear, however,whetheror not the antiidiotypic antibodysensitizes B cells directly. Alternatively,anti-idiotypic Ta ceils (see section 6.1), whichmightalso be inducedby anti-idiotypie antibody,possiblyvia idiotypicTri cells, couldbe responsiblefor this effect (84). Amplification of idiotype-bearingB cells couldalso be dueto interaction with idiotypic Ta ceils via bridginganti-idiotypic antibody(61). It difficult to distinguishexperimentally betweenthe latter twopossibilities. However, the role of T cells in anti-idiotype-indueed B-cell sensitization is indirectly supportedby the finding that T-cell populationssensitized by anti-idiotype preferentially collaboratewith idiotype-bearingB cells (84). In the case of idiotype suppressionby anti-idiotypic antibody, wefind again that both B and T calls are involvedin this phenomenon. Suppressor T cells that specifically preventedidiotypeexpressionin the B-cell response

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were first demonstratedin the ASAidiotypic system(55) and subsequently identified in a variety of cases of adult and neonatal suppressionof both B-cell and DTH responses(71, 113, 116, 157, 218). In someinstances, the suppressorcells wereshownto expressanti-idiotypic specificity by virtue of their ability to bind idiotype (116, 157). However, the situation is more complicatedin that the T-suppressorpathwayincludes interacting T-cell subsets, such that, for example,an idiotypie suppressorinducercell could activate an anti-idiotypic suppressoreffector, thus mimicking the effect of the latter (150, 197, 211,219).Anidiotype-expressingcell wouldof course appear as the natural target of anti-idiotypic antibodies. Wedo not go further into these complicatedmatters (whichinclude the problemof the target of suppressoreffeetor cells) in the contextof the presentdiscussion. This particular subject has recently beenreviewed,and werefer the reader to this literature (9, 73a, 78, 95, 184,198). As in the case of enhancement,idiotype suppressioncan also be demonstrated within the B-cell population itself. In adult mice injected with microgram amountsof anti-idiotype, evidencefor transient nonresponsiveness of idiotypicB cells wasobtained(85) despite the fact that the frequencies of idiotypic LPS-reactiveB-cell precursorsin such animalsare usually only slightly reduced(60; T. Takemori,K. Rajewsky,submittedfor publication). This phenomenon mayrepresent a classical ease of B-cell tolerance or "anergy"[see Nossal(149a)in this volume].Alongthe lines of the work of Nossaland his colleagues,Nishikawa et al (147)haverecently shownthat anti-idiotypic antibody in microgramconcentrationspreventedthe maturation of pre-Bcells into idiotype-expressingLPS-reactiveB cells in vitro. This result suggests that if a given antibody is producedin the immune systemin such concentrations, the generation in the bone marrowof functional B cells expressingcomplementary (anti-idiotypic) receptors may shut off. Theworkof Iverson(92a) and of Rowleyand his colleagues(179a) together with the inability of Sakato&Eisen (180) to generate anti-Tl5 antibody in mice in which microgramamountsof T15+ molecules were circulating (133)supportthis notion(see also section 5.4). Long-lasting unresponsivenessin the B-cell compartmentwasobserved in systems of neonatal suppression. Whennewbornmice were suppressed + precursorB cells could not for the T15idiotype by anti-T 15 antibody,T15 be detected in the micefor several months(1, 40, 70). Thepossibility that this effect wasmediatedby T cells has beenshownto be unlikely in other cases where suppression of the MOPC104E and T15 idiotypes was achievedby injecting anti-idiotype into newborn(T-cell deficient) BALB/ cnU/numice(115, 209). Onthe other hand, there is also someevidencefor the existenceof idiotypespecific suppressorTcells in animalsinjected with anti-idiotypie antibodyat birth (71, 116). Theeffect of neonatallyapplied

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589

anti-idiotype on both T and B cells is exemplified by an experiment we depict in Figure 3. Micewere injected at birth with 100/~gof anti-idiotope antibody Ac38, recognizing an idiotope recurrently expressed in the primary anti-NP response (see Section 4.3). At 13 weeksof age, splenic T and B cells were isolated from the animals and were injected, together with T and B cells from normal controls, in various combinations into irradiated syngeneie hosts. The hosts were then immunized with NP coupled to chicken gammaglobulin, and the expression of the target idiotope in the humoral anti-NP response was recorded (T. Takemod, K. Rajewsky, submitted for publication). As a result, the B cells from the suppressedanimals mounted a normal anti-NP response but were unable to express idiotope Ac38 even though they were stimulated in the presence of normal T calls. This is particularly remarkable, since in limiting dilution analysis of LPSreactive precursor cells, the frequencyof Ae38+ precursors was identical to a

100 ¯

8 ¯ ¯

¯

Bcell" cell

N N

¯

N N

N S

¯

N S S S

¯

S

N

S S

S N

Figure3 Effectof neonatalinjectionof a monodonal anti-idiotopeantibodyonT andB cells in the adult mouse.Antibody Ac38(100/zg),whichdetectsa recurrentidiotope(Ac38) the anti-NPresponse(174;legendto Figure2), wasinjected into newborn C57BL/6 mice. At week13, T andB cells fromthe spleensof the injected animals(S) andfromcontrol spleens(N)weretransferedin variouscombinations (bottomof Figure)into irradiatedhosts (10Tcells of eachtypeper host). Thehosts wereimmunized withNPchickengamma globulin andthe sera weretitrated 12dayslater for total IgG1anti-NPantibody(whichwasthe same in all groups;data notshown) andfor anti-NPantibodies expressing the Ae38idiotopeeither togetherwitha secondidiotope(Ac146, left panel)or the Ac38idiotopealone(fight panel). Antibody titers in individualsera are indicated.Fordetails of titration methods seeKelsoe et al (112).Dataare fromT. Takemori, K. Rajewsky, submittedfor publication.

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that of control animals(data not shown).Thus,as discussedin Section4.4, the repertoire of LPS-reactiveprecursors mightnot be the sameas that expressedin the mature, functionally active B-cell population. Alternatively, the idiotypic B-cell responsecouldbe suppressedby anti-idiotypic B cells whosefrequencyin the suppressedanimalsremainsto be determined. Mostinteresting in this context are recent data of Okumura et al (151), who ascribea critical role in idiotypic Controlto a B-cellpopulationexpressing the Lyt-1 surface antigen. Comingbackto the experimentin Figure3, the T cells of the suppressedanimalswerealso affected in that they did not allow the expressionof the target idiotope Ac38in a normalB-cell population, althoughthey helpedthe B cells to mounta normalanti-NPresponse. Mixingexperimentshaveso far failed to establish whetherthis deficiency is dueto lack of idiotype-specifichelp, active suppression,or both. Interestingly, although nonresponsiveness in the B-cell compartment comprisesall antibodies expressingidiotype Ac38,the T cells seemto see only a subset of these antibodies, namelythose co-expressinga secondidiotope (Ac 146). This not only controls for the purity of the B-cell populationin the experiment,it also suggeststhat the idiotypic determinantsseen by T andB cells maynot be the same. Wecomeback to this point in Section 6.2. Toconcludethis section, smalldoses of antibodies, appliedto the animal in native form, profoundlyaffect the equilibrium of the corresponding idiotypes and anti-idiotypes in both the T- and the B-cell compartments of the immunesystem. 5.4 Interpretation of the Regulatory Effects of ldiotypic and Anti-ldiotypic Antibodies Theinterpretation of the results described abovehas been madedifficult because of a problem of nomenclature, namely, the terms idiotype and anti-idiotype. Certainly, any idiotype arising in the immune systemhas to deal with the problemof anti-idiotypes; however,whenweinject an "antiidiotype"into the mouse,it is incorrect to interpret this experimentsolely in the sense of control of the corresponding"idiotype," as has usually been done. Asdiscussed in detail in section 2, idiotype and anti-idiotype are operational terms for functionally equivalentcomplementary bindingsites, and anyidiotype is equally well an anti-idiotype and vice versa. Whenweinterpret the data on this basis, a surprisingly simple pattern emerges.At low doses (nanogramrange), antibodies enhancethe expression of complementary binding sites in the system. This enhancement is not at the level of effector functions, suchas high-rate antibodyproduction,but presumablyit involves mainlythe recruitmentof T and B cells expressing complementary bindingsites. It is attractive to think that the enhancement phenomenon reflects a fundamentaldriving force in the immune system in the senseof the networkhypothesis.Lowlevel antibodyproduction(natural

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IDIOTYPES 591 antibody) could accompany and promoteinteractions betweendiverse sets of cells with complementary receptors (61). Oncethe concentrationof certain antibodybindingsites is abovea certain threshold (microgramrange), for examplewhenthe system engagesin active immune response,a different set of mechanisms is set in motion.Now the systemis designedto lock itself into the expressionof a givenantibody repertoire. The"suppression"experimentsdiscussed abovereveal someof these mechanisms: microgram quantities of antibodies ("anti-idiotypes" in this case) shut off, in the bonemarrow,the productionof cells with complementary("idiotypic") receptors and suppressor T cells suppressing the activation of suchcells in the peripheryare induced.Thesystemstabilizes the expressionof those bindingsites (in this case the "anti-idiotype") whichit has committed itself. This is strikingly demonstrated by the finding that idiotype can facilitate the expressionof the sameidiotype["idiotypic memory,"Section 5.2; the problemof stabilizing immuneresponses has beendiscussedfrommanydifferent angles (e.g. 82, 83, 88)]. Theentire set of data discussed above can thus be interpreted in a coherentwaythat makessense in terms of the physiologyof the systemand ascribes the idiotypic networka physiologicalregulatoryrole. Afinal remarkabout idiotypic control of antigen-specific antibodyresponses: As discussedin Section 4.3, such responsesconsist of antibody families in whichsets of idiotopes are expressedon the members in various combinations.Ananti-idiotypic responseagainst the morecommon of these idiotopes would have as its commondenominator, because of its heterogeneity,the bindingsites for these idiotopes. Becauseof this asymmetry it couldunidirectionallyregulate the expressionof the idiotypic antibodyfamily, with the implication, however,that regulation could extendto members of the familythat do not possess the antigen-bindingspecificity of the initial immune response. Theabl-ab2-ab3 experimentsof Urbain, Cazenaveand others (204; section 4.3) can be consideredto exemplifythis and can be interpreted in this waywithoutcomplicatedgenetic assumptions (168). Idiotypie control wouldbe strictly specific for the antigen-induced responseif the activatedcells wereparticularlysusceptibleto the regulatory signals (36), anda priori so in caseswherethe anti-idiotypic responsewould see the antigen-binding site as the common denominatorin the antigeninducedantibodypopulation, and thus consist largdy of "internal images" 000, 101). 5.5 Antibody Idiotypes and Growth Receptom on Pre-B and B Cells A potentially importantissue has not beenmentionedso far, namely,that of idiotypic relationshipof antibodiesandgrowthreceptorsexpressedin the B-cell lineage. Coutinhoandhis collaborators (44) havefoundthat certain

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anti-idiotypic antibodies against recurrent idiotypes can bind to a large fraction of B andpre-Bcells andcan drive these cells into proliferation. A similar phenomenon was subsequentlydescribed for antibody M460(164), whoseidiotype is recurrently expressedin BALB/c anti-dinitrophenyl responses(21, 51, 52, 131). Theantibodiesare thoughtto interact with a set of growthreceptors expressedin different combinations on different subsets of pre-B and B cells, and a theory was developed according to which antibodyidiotypes and B-cell growthreceptors havebeenselected in evolution for efficient interaction, providinga mechanism for self-stimulation within the B-cell system (43). This mechanism wouldbe operating already at the pre-Bcell level, that is before the cells expresssurface Ig [it is interesting in this connection,however,that pre-B cells maycarry small amountsof free/~ chains on the surface (158a, 206)], and maternalantibodies mightthus influence B-cell development in the organismfight fromthe beginning(10). Thestudies in this field are still in their infancyin terms molecularanalysis. However,together with other evidence (163a, 165a, 183, 193), they point in a direction that mayturn out to be of physiological importancein a general way,namely,the possible involvementof antibody idiotypesin interactionswith surfacereceptorsother than the antigenreceptors of the immunesystem. 6.

T CELLS AND IDIOTYPE

REPERTOIRE

6. ] Silent Clones and Idiotype Dominance Thediscussionin the previoussections has shownthat antibodyidiotypes, be they expressedon circulating or cell-boundantibodies, can specifically induceT-cell activities. It is thus not surprising that in antigen-driven immune responses[in whichin addition to antigen-specificantibodies spontaneous auto-anti-idiotypes often appear and have beenascribed a regulatory role (39, 67, 77a, 110, 121, 153, 197a, 200; see also Section 4.6)] idiotype-beadngand anti-idiotypic Tn and Ts cells have beenobservedin a variety of experimentalsystems, including antibodyand DTH responses. Theinduction of such cells is thus not restricted to manipulationswith anti-idiotypic antibodies. Basedon sophisticated workwith techniquesof cellular immunology, idiotypic interactions of regulatory T cells havebeen incorporatedinto schemesof T cell circuits regulating immune responses. Thediscussion of these matters is beyondthe scopeof the present review andwerefer the reader to the literature (9, 73a, 78, 95, 184, 198). However,since the immunesystem continuously lives with antibody idiotypes, starting in ontogenywith immunoglobulins of maternal origin, idiotype-dependentmodulationof T-cell activities must be expected to occurthroughoutlife. Thisleads us directly to the issue of silent clonesand

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IDIOTYPES 593 ofidiotype dominancein the B-cell compartment. There is evidence,mainly fromthe workof Urbain, Cazenaveand Bonaand their colleagues, that the bone marrowproducesclones of cells not expressedas recurrent or dominant idiotypes in immune responses,either becauseof suppressionby T cells or becauseof the lack of T-cell hdp (21, 106, 179, 204). Alongthese lines, several groupshavepresented data suggestingthe dependenceof dominant expressionof recurrentidiotypeson idiotype-specificTHcells (3, 25, 36, 85, 216).It seemsclear that classical, cartier (i.e. antigen)-specifichelpercells are sufficient to drive B cells expressingantibodiesof the IgMandIgGclass (and also a "dominant"idiotype) into proliferation and antibody production in vitro (31), but this doesnot excludethe possibilitythat anti-idiotypie TI~ cells often participate in the preselection and/or expansionof certain B-call clones that dominatecertain immune responses. Preferential expansion by anti-idiotypic helpers couldbe responsiblefor the dominance of an idiotype in the anti-lysozymeresponse, being established only late in the response(142). Clearly, such evidencehas to be weighedagainst the classical conceptof selection in the immune responseof ceils expressingantibodies with high affmity for antigen (63). Such selection .(in which idiotype-specific T cells could also be involved)mayfinally explain many eases of idiotype dominance,in particular since the correspondingantibody Vregions (often specific for bacterial antigens) mighthave beenalready selected in evolution(29, 65, 215). Theessential role of antigen in driving immune responsesis evident fromthe fact that the expressionof idiotypes in antigen-inducedresponses is usually, although not always (156), restricted to antigen-bindingmoleculesin the serum(e.g. 54, 148, 170, 217). 6.2 Antibody Idiotypes and the T-Cell Repertoire Theconcept that the existence of silent clones and idiotype dominance reflects idiotype-specitic T-cell control represents a complicationfor the discussionof the geneticdetermination of the idiotypic repertoirein B cells (Section 4.3). Thus,if a givenidiotype is not expressedin the B-cell compartment,wemightconsiderthis to be unrelatedto the absenceor presence of certain antibodystructural genesanddue to suppressionby T cells. Let us try to clarify the situation. Almostwithoutexception,idiotypeexpression is exclusivelyunderthe control of the Igh locus (Section 4.3). Therefore, if a given idiotype is expressedin a givenstrain, but not in another,then the idiotype-negativestrain either lacks the structural informationfor that idiotype (as exemplifiedin several cases, see Section4.3) or a regulatory elementrequired for its expression. That elementmustalso be located in the Igh locus. Clear candidatesfor such hypothetical regulatory elements are genesencodinganti-idiotypes. If suchgeneswereexpressedin T cells,

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these cells would,in principle, be able to interact with idiotype-bearingB cells, either in the senseof suppression(generatingsilent clones)or enhancement(generatingidiotypic dominance). However, this type of strain-specific indirect control of idiotype expressionvia regulatory T cells wouM again be due to polymorphism of genesin the lgh locus. If, indeed, the repertoire of antibody V-regionstructural genes were sharedbetweenT- andB-cell receptors, the extensiveidiotypic interactions betweenthe two types of lymphoeyteswouldnot be surprising. In particular, idiotypic polymorphism wouldbe expectedto be expressedequally at the level of T and B cells, as is indeedfound(Section 4.5). However, as discussedabove,evidencefrommolecularbiology arguesagainst the notion that the V, D, and J genesexpressedin antibodies are also expressedin T cells. Furthermore, there is evidencethat idiotypic control of B-cell activities by regulatoryTcells andwithinregulatoryT-cell circuits is restricted by the Igh locus,suggestinga selectionof T cells for the recognitionof B-cell idiotypes expressedin the samesystem(see 9, 26, 106, 198, 219). These considerationslead to the far-reachinghypothesisthat the T-cell repertoire mightbe selected in ontogenynot only for the recognitionof self-histocompatibility (MHC) antigens(118, 222), but also for (self) B-cell idiotypes. its extremeform, this hypothesisexplains idiotype sharing betweenT and B cells by idiotypicinteractions betweenthese cells leadingto the selection of a T-cell receptor repertoire complementary to that of B-cell receptors. Since the latter contains complementary receptors in itself, and since, in addition, anti-idiotypic T cells selected for B-cell idiotypes mayin turn expandidiotypic T cells, a situation wouldresult in whichnot only efficient idiotypic T-Bcell interactions wouldbe guaranteed,but also the receptor repertoire of T cells wouldbe idiotypically (and anti-idiotypically)similar, althoughnot identical, to that of the B cells. Theexperimentalresults in Figure3 can easily be interpreted in this sense. Thereis suggestiveevidencealso in other systemsthat T and B cells see similar, but not identical, idiotopes on antibodies (76, 80, 102, 163). One mightspeculate that "regulatory" idiotopes in the sense of Paul & Bona (159a)mightbe those that T ceils are able to see. Apriori, a B call appears to be an ideal target to be seen by T cells: It expressesclass I andclass II MHC antigens on its surface, as well as a unique structure, the immunoglobulin idiotype. Furthermore,a close structural similarity of immunoglobulin domainsand the domainsof class I and class II antigens has recently emerged(130a, 152, 221). This has led to the speculation that domainsas well as MHC antigens mightrepresent restriction elementsfor T cells (25, 26, 95, 106), and that Vregions could havebeenselected evolutionfor structural similarity with MHC antigens (45). Thelatter could relate to the findingthat helper andsuppressorT cells see different idiotypic

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IDIOTYPES 595 determinantsin certain systems(80, 184), and to the issue of silent clones and idiotype dominance(Section 6.1). An answerto the question as to what extent the T-cell repertoire is determinedby idiotypic interactions with B ceils will require the analysis of T-cells that haveditfercntiated in the absenceof B-ceil idiotypes.Experimentsof Bottomlyand her co-workerssuggest that T cells from Ig-suppressedmicestill contain THcells specific for conventionalantigens (25); but of course the suppressedmiceare not free of the antibody idiotypes present on the suppressingantibody. Bottomlyand Mosier(27) have also suggested that anti-idiotypic THcells are absent from mice lacking the correspondingidiotype in the B cell compartment. This result is controversial at present (32, 166). Further experimentsalong these lines appear urgently needed.Theissue is whetheror not idiotypic interactions are the natural wayfor T cells to communicate with each other and with B cells. 7.

CONCLUSIONS

Westarted our discussionwith the principles of idiotypic interactions and the structural andgenetic basis of idiotypic dctcrminants.Thereis clear evidencethat the germlinecontrols and limits the availableidiotypic repertoire in newlyarising B cells. Thecells in the immune system arc continuouslyexposedto idiotypic interactions. A pre-B cell arising in the bone marrowmayalready be positively selected by antibodyidiotypcs throughinteraction with growth receptorson its surface. When the cell expressesits Ig receptor, it becomes subject to idiotypic control of several types. Thereis cvidcnccthat cells arising in the bone marrowand expressing receptors complementaryto idiotypes present in the circulation in microgramamountsare turned off. Onthe other hand, nanogram doses of idiotypes in circulation lead to the expansionof cells with complementary receptors. Idiotypes maythus play a crucial role in establishing the functional repertoire in the system,by recruiting B cells that are otherwiseshort-lived into the pool of recirculating, long-livedcells and/orthat of B cells responsiveto specific T-cell help (these two compartments could bc overlappingor identical). In the idiotypicselectionof B cells, regulatoryT cells can be specifically involved,they themselvesbeing targets of regulation by antibodyidiotypes. Theinterplay of T and B cells via idiotypic interactions will lead to the selection of sets of complementary cells whosereceptor repertoire might well differ fromthat of the newlygeneratedcells. Idiotypc sharing between T andB cells andthe control of idiotypcs by the Igh locus couldthus either indicate the involvementof Vngenesin the receptors of both cell types or the adaptationof the T-cell repertoire to B-cell idiotypesin ontogcnyand/

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or evolution, similar to its adaptation to the (structurally related) MH antigens. There is experimental evidence that the receptor repertoire of T cells is idiotypically related but not identical to that of antibodies, and hints that T cells might adapt to the recognition of antibody idiotypes in ontogeny. If it will turn out that idiotypically selected cells represent an importan part of the functional repertoire, then immuneresponses might indeed, quite in the sense of the original network hypothesis (97), depend on the interference of antigen with interactions of complementarycells and reflect release from suppression. Experimentaldata pointing in this direction have recently been presented (58, 123). Oncethe system expresses a given repertoire of binding sites in an immuneresponse, the expression of these binding sites is stabilized via idiotypic control ("idiotypic memory"). A functional network of idiotypes maynot only exist at the level of T-B (or T-T) cell interactions but also in the B-cell compartmentitself. Here could either have been selected in evolution or reflect an interplay of germ line encoded with somatically mutated V regions (168). The question whether the germline encodes an idiotypic network of antibody V regions can now be directly approached by the tools of molecular biology. ACKNOWLEDGMENTS

Weare grateful to K. Beyreuther, M. Cramer, A. Radbruch, M. Siekevitz, F. Smith, and H. Tesch for valuable criticism and advice, and to E. Sieg: round, C. Tomas, and W. Muntjewerf for typing the manuscript. Wealso thank K. Karjalainen for samples of monoclonal antibodies, and him and H. Sakano, A. Bothwell, M. Paskind, D. Baltimore, S. Zaiss, and K. Beyreutber for letting us have unpublished sequence information. Wewish to acknowledgespecifically the contributions of M. Reth to our work and thinking. The work from our laboratory was supported by the Deutsche Forschungsgemeinschaft through SFB 74. LiteratureCited 1. Accolla, R. S., Gearhart, P. J., Sigal,N. H., Cancro,M. P., Klinman,N. R. 1977.Idiotype-spccificneonatalsuppressionof phosphorylcholine-responsire B cells. Eur.Z ImmunoL 7:876-81 2. Adam,G., Weiler, E. 1976. Lymphocytepopulationdynamicsduring ontogeneti¢ generation of diversity.In TheGeneration of Antibody Diversity:A NewLook,ed. A. J. Cunningham, pp. 1-20. NewYork: Academic 3. Adorini,L., Harvey,M.,Sercarz,E. E. 1979.Thefine specificityof regulatory T cells. IV. Idiotypiccomplementarity

andantigen-bridging interactionsin the anti-lysozyme response.Eur. J. Im. munol.9:906--9

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26. Bottomly,K., Maurer,P. H. 1980. Antigen-specifichelperT cells requiredfor dominant production of an idiotype (ThId) are not under immuneresponse (It) gene control. J. Exp. Med~152: 1571-82 27. Bottomly,K., Mosier,D. E. 1979.Mice whoseB cells cannot producethe T 15 idiotype also lack an antigen-specific hdper T cell required for T 15 expression. J. Exp. Med.150:1399-409 28. Brient, B. W.,Nisonoff,A. 1970.Quautitative investigationsof idiotypicantibodies. IV. Inhibition by specific haptens of the reactionof anti-haptenantibodywithits anti-idiotypicantibody.J. Exp. Med. 132:951-62 29. Briles, D. E., Forman,C., Hudak,S., Claliin, J. L. 1982. Anti-phosphorylcholine antibodiesof the T 15 idiotype are optimallyprotective against Steeptococcus pneumoniae. J. Exp. Med. 156:1177-85 30. Briiggemann, M., Radbruch, A., Rajewsky, K. 1982. Immunoglobulin V regionvariants in hybridoma cells. I. Isolation of a variantwithaltered idiotypic and antigen-bindingspecificity. EMBO J. 1:629-34 31. Cammisuli,S., Schreier, M. H. 1981. Individual clones of carder-specific T cells help idiotypicallyandisotypically heterogeneousanti-hapten B-cell responses. Immunology43:581-89 32. Cancro,M. P., Klinman,N. R. 1980. B ceil repertoire diversity in athymic mice..i.. Exp. Meg151:761-66 33. C.apra,J. D., Kehoe,J. M.1975.Hypervariableregions,idiotypy,andthe antibody-combiningsite. Adv. ImmunoL 20:1-40 33a. Capra,J. D., Slaughter,C., Milner,E. C. B., Estess, P., Tucker,P. W.1982. The cross-reactive of A-strain ndce. Immunologyidiotype Today 3:332-39 34. Car~n, D., Weigert, M. 1973. Immunochemical analysis of cross-reacting idiotypes of mousemyelomaproteins withanti-dextranactivity andnorreal anti-dextran antibody.Proc. NatL Acad. ScL USA70:235-39 35. Cazenave,P.-A. 1977. Idiotypic-antiidiotypic regulationof antibodysynthesis in rabbits. Pro~NatLdcad. ScLUSA 74:5122-25 36. Corny,J., Caulfield,M.J. 1981.Stimulation of specificantibody-forming cells in antigen-primed nude mice by the adoptivetransfer of syngeneic anti-idiotypic T cells. J. ImmunoZ 126:2262-66 37. Clevinger,B. 1982.In Idiotypes~"Antigens on the Inside, ed. I. Westen-

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43. Coufiaho,A. 1 cfiminationan, tion of the a~ Immunol. (PaJ 44. Coutinho, A., | 1980. The pob munoglobulin nants on the n their precurso~ munopathoL3 45. Coutinho,A., 1 Ivars, F. 1983. Tautologic?It Geneticsof the andE. MiSller, press 46. trainer, M.,I~ I., Imanishi-K Givol, D., R@ hapten-bindin~ lymphocytes. munoglobulin hydroxy-3-nitr specificrecept~ B and T lymph 9:332-38 47. Cramer, M., R 1,981. T Cell ~ Immunoglobut pression,ed. J E., Wigzell, H Academic 48. Crews, S., (~ Calame,K., H H genesegmen

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IDIOTYPES sponse to phospholTlcholine:Somatic mutationis correlatedwith the class of the antibody. Cell 25:59-66 49. Dildrop, R., Brii~gemann, M., Radbruch, A., RajewsKy,K., Beyreuther, K. 1982. Immunoglobulin V region variants in hybridoma cells. IL Recombination between V genes. EMBO d. 1:635-40 50. Dohi, Y., Nisonoff, A. 1979. Suppression of idiotype and generationof suppressor T cells withidiotype-conjugated thymocytes, d. Exp. Meal150:909-18 51. Dzierzak,E. A., Janeway,C. A. Jr., Rosenstein, R. W., Gottlieb, P. D. 1980. Expressionof an idiotype (Id-460) during in vivoanti-dinitrophenylantibody responses. I. Mapping of genes for Id460expressionto the variableregionof immunoglobulin heavy-chainlocus and to the variable region of immtmoglobufin k-light-chain locus, d. Exp. Med. 152:720-29 52. Dzierzak, E. A., Rosenstein, R. W., Janeway,C. A. Jr., 1981.Expressionof an idiotype(Id-460)duringin vivoantidinitrophenyl antibody responses. II. Transient idiotypic dominance..LExp. Meal 154:1432-41 52~ Dzierzak, E. A., Janeway,C. A. Jr., 1981.Expressionof an idiotype(Id-460) duringin vivo anfi-dinitrophenylantibodyresponses.III. Detectionof Id-460 in normalserumthat does not binddinitrophenyl, d. Exp. Meal154:1442-54 53. Eichmaun,K. 1973. Idiotype expression andthe inheritance of mouseantibodyclones. £ Exp. Med. 137:603-21 54. Eichmmm, K. 1974. Idiotype suppression. I. Influenceof the doseandof the effectorfunctionsof anti-idiotypicantibody on the production of an idiotype. Eur..L ImmunoL 4:296-302 55. Eichmaun,K. 1975. Idiotype suppression. IL Amplification of a suppressorT cell withanti-idiotypicactivity. Eur.~. ImmunoL5:511-17 56. Eichmann,K. 1975. Geneticcontrol of antibodyspecificity in the mouse.Immunogentics2:491-506 57. Eichnmnn, K. 1978. Expression and function of idiotypes on lymphocytes. Adv. ImmunoI26:195-254 K. 1982. Idiotypes.Antigens 58. Eichmmm, on the Inside, ed. I. Westen-Schnurr, pp. 209-21. Basel: Editiones Roche 59. Eichmann,K., Ben-Neriah,Y., Hetzelberger, D., Polke,C., Givol,D., Lonai, P. 1980. Correlated expression of Vn frameworkand Vn idiotypic determinantson T helper cells and onfunctionally undefinedT cells binding groupA

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streptococcal carbohydrate.Eur. L Immuno£10:105-12 60. Eiehmaun,K., Coutinho,A., Melehers, F. 1977. Absolute frequencies of Lipopolysaecharide-reaetive B cells producing A5Aidiotype in unprimed, streptococcal A earbohydrate-primed, anti-A5Aidiotype-sensitized and antiA5Aidiotype-suppressed A/J mice. Exp, Med. 146:1436-49 61. Eiehmaun,K., Falk, I., Rajewsky,K. 1978.Recognitionof idiotypes in lymphocyte interaetions. II. AntigenindependentcooperationbetweenT and B lymphocytes that possesssimilar and complementary idiotypes. Eur. ~. lmmuno~8:853-57 62. Eiehmann,K., Rajewsky,K. 1975. Induction of T and B cell immunityby anti-idiotypic antibody. Eur. ~. ImmunoZ5:661-66 63. Eisen, H. N., Siskind, G.W.1964.Variations in affinities of antibodiesduring the immune response. Biochemistry3: 996-1008 64. Elson, C. J., Jablonska,K. F., Taylor, R. B. 1976.Functional half-life of virgin and primed B lymphocytes.Eur. £ Immunol. 6:634-38 64a. Enghofer, E. M., Glandemans,C. P. L, Bosma, M. L 1979. Immunogiobufins withdifferentspeeificitieshavesimilar idiotypes. MoLImmunoL16: 1103-10 65. Etlinger, H. M., Julius, M.H., Heusser, Chr. H. 1982. Mechanismof clonal dominancein the murine anti-phosphorylchofineresponse.I. Relationbetweenantibody avidity128:1685-91 and elonal dominance. J. Immunol. 66. Fairchild, S. S., Cohen,J. J. 1978.B lymphocyteprecursors. I. Induction of Lipopolysaccharideresponsivenessand surface immunoglobulin expression in vitro, d. ImmunoL121:1227-31 67. Fernandez,C., M/511er,G. 1979. Antigen-inducedstrain-specific autoantiidiotypie antibodies modulatethe immuneresponse to dextran B512.Proc. NatLAcad.Sci. US.476:594zl---47 68. Frischknecht,H., Binz, H., Wigzell,H. 1978.Inductionof specific transplantation immune reactions using anti-idiotypic antibodies. £ Exp. Med. 147: 500-14 69. Fung,J., KShler,H. 1980. Late clonal selection andexpansionof the TEPC-15 germ-line specificity, d.. Exp. Meal 152:1262-73 70. Fung, J., KShler, H. 1980. Mechanism of neonatal idiotype suppression. I.

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merit at birth with idiotypes is assoasymmetryin idiotypic interactions. ciated with the expansionof idiotypeAnn. ImmunoLIn press 169. Rajewsky,K., Eichmaun,K. 1977. Anspecific helper T cells. J. Exp. Med. tigen receptors of T helper cells. Con156:506-21 179a.Rowley,D. A., Griflith, P., Lorbach,I. temp. Top. ImmunobioL7:69-112 170. Rajewsky,K., Reth, M., Takemori,T., 1981. Regulation by complementary Kelsoe,G. 1981.A glimpseinto the inidiotypes. Ig protects the cloneproducnet life of the immune system. In The ing it. J. Exp. Med.153:1377-90 Immune System,ed. C. M.Steinberg, I. 180. Sakato,N., Eisen, H. N. 1975. AntibodLefkovitz,2:1-11. Basel: Karger ies to idiotypes of isologous immtmo171. Rajewsky,K., Takemori,%, geth, M. globulins. J. Exp. Med~141:1411-26 1981.Analysisand regulation ofVgene 180a.Schitf, C., Boyer,C., Milili, M., Fougeexpression by monoclonalantibodies. reau, M. 1981. Structural basis for MOPC 173 idiotypic determinants disIn Monoclonal.~ntibody and T Cell Hybridoma:Perspectiveand Technical tinctively recognizedin syngeneicand vances,ed. G.J. I-I’~immerling, U.Hiimallogenc~cimmunization:contribution merling, J. F. Kearney,pp. 399--409. of DH,JH, and JK regions to an idiNewYork: Elsevier otyperecognizedby allogeneicantisera. ~4ng Immunol. Ins~ Pasteur 132C: 171a. Ramseier, H., Lindenmann,J. 1972. Aliotypic antibodies. Tra~plant. Rev. 113-29 10:57-96 181. Schilling,3., Clevinger, B., Davie,J. M., 172. Reth, M., Bothwell, A. L. M., RaHood,L. 1980. Aminoacid sequenceof homogeneous antibodies to dextran and jewsky,K. 1981. Structural properties DNA rearrangementsin heavy chain Vof the haptenbinding site and of idib otopes in the NP antibody family. In region gene segments. Nature 283: ImmunoglobulinIdiotypes: ICN-UCLd 35-40 Symposia on Molecular and Cellular 182. Schuler, W., Weiler,E., Weiler, I. J. Biology,ed. C. JanewayJr., E. E. Ser1981. Biological and serological comcarz, H. Wigzell,20:169-78.NewYork: parison of syngeneicandallogeneicanAcademic ti-idiotypic antibodies. MolecularImmunology18:1095-105 173. Reth, M., I-~knmerllng, G. J., Rajewsky,K. 1978.Analysisof the reper- 183. Scge, K., Pcterson, P. A. 1978. Useof toire of anti-NPantibodies in C57BL/6 anti-idiotypicantibodiesas cell-surface miceby cell fusion. I. Characterization receptor probes. Proc. NatLAcad.ScL of antibodyfamilies in the primaryand USA75:2443-47 hyperimmuneresponse. Eur. J. Im- 184. Sercarz, E. E., Metzger, D. W.1980. Epitope-specificand idiotype specific munoL8:393-400 cellular interactions in a modelprotein 174. Reth, M., Imanishi-Kari,T., Rajewsky, K. 1979. Analysisof the repertoire of antigen system. Springer Semin. Immunopatho£3:145-70 anti -(4 -hy.droxy-3ultrophenyl)acetyl (NP) antibodies in C57BL/6mice 185. Sherr, D. H., Doff, M.E. 1982.Haptenceil fusion.II. Characterization of idispecificT ceil responsesto 4-hydroxy-3otopes by monoclonalanti-idiotope anmtrophenylacetyl. XIII. Characterizatibodies. Eur. Y.. Immuno£ 9:1004-13 tion of a third T cell L~pulatiunin175. Reth, M., Kelsoe, G., Rajewsky,K. volvedin suppression of in vitro PFC 1981.Idiotypic regulation by isologous responses. J. ImmunoL128:1261-66 monoclonal anti-idiotope antibodies. 186. Siekevitz, M., Gefter, M. L. Brodeur, Nature 290:257-59 P., Riblet, R., Marshak-Rothstein,A. 176. Richter, R. H. 1975. A networktheory 1982.Thegeneticbasis of antibodyproof the immunesystem. Eur. Y.. Imduction: The dominantanti-arsonate muno~5:350-54 idiotype response of the strain mice. 177. Rodkey,L. S. 1974.Studies ofidiotypic Eur. J. Immuno~12:1023-32 antibodies. Productionand characteri- 187. Siekevitz, M., Huang,S. Y., Gefter, M. zationof autoanti-idiotypicantisera. J. L. 1983. Thegenetic basis of antibody Exp. Med~139:712-20 production: Onevariable region heavy 178. Roland,J., Cazenave,P.-A. 1981. Rabchain geneencodesall moleculesbearbits immunized against b6 allotype exing the dominantanti-arsonate idiotyp¢ press s’mdlaranti-b6 idiotopes. Eur.J. in the strain A mouse.Eur. I. ImmunImmunoL11:469-74 ol. In press 179. Rubinstein,L. J., Yeh,M., Bona,C. A. 188. Sigal, N. H., Garhart, P.J., Klinman, 1982. Idiotype-anti-idiotype network. N. R. 1975. The freque~myof phosII. Activationof silent clonesby treatphorylcholine-specific Bcells in conven-

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189. Sigal, N. H., Picard, A. R., Metcalf,E. S., Gearhart, P. J., Klinman,N. R. 1977.Expressionof phosphorylchollnespecific B cells duringmurinedevelopment. J. Exp. Med.146:933-48 190. Sogn,J. A., Yarmush,M.L, Kindt, T. J. 1976.Anidiotypic markerfor the VL region of an homogeneousantibody. .,Inn. ImmunoL(Paris) 127C:397-408 190a.Somm6, G., Semi,J. R., Leclercxl, L., Moreau,J.-L., Mazi6, J.-C., Momier, D., Fougereau, M., Th~ze, J. 1982. Contribution of the H- and L-chains andof the bindingsite to the idiotypic specificities of mouseanti-GATantibodies. MoLImmunoL19:1011-19 191. Sprent, J. 1973. Circulating T and B lymphocytes of the mouse.II. Life span. Cell. lmmunoL7:40-59 192. Strober,S. 1972.Initiation of antibody responsesby different classes of lymphocytes. V. Fundamentalchanges in the physiological characteristicsof virgin thymus-independent ("B") lymphocytes and "B" memory cells..L Exp. Mea~136:851-71 193. Strosberg,D. A., Couraud,P.-O., Shreiher, A. 1981.Immunological studies of hormone receptors: A two-way approach. Immunol. Today2:75-9 193a. Sugimura,K., Kishimoto,T., Maeda, K., Yamamura,Y. 1981. Demonstration of T15idiotype-positive effector and suppressorT cells for phosphorylcholine-specificdelayed-typehypersensitivity response in CBA/N or (CBA/N X BALB/c)Ft male mice. Fur. d. ImmunoL11:455-61 194. Staudt, L. 1982. In Idiotypes-dntigens on the Inside, ed. I Westen-Schnurr, pp. 96-111. Basel: Editiones Roche 194a. Suzan, M., Valstedt, F., Boned,A., Rubin, B. 1982. Genetic chasing of T helper cell idiotype andallotypegenes. Immunogenetics16:229-41 195. Sy, M.-S., Bach, B. A., Brown,A., Nisonoff, A., Benacerraf, B., Greene, M. I. 1979. Antigen- and receptordriven regulatory mechanisms.IL Inductionof suppressorT cells with idiotype-coupled syngeneicspleencells, d. Exp. Med. 150:1229-40 196. Sy, M.-S.,Brown,A. R., Benacerraf,B., Greene,M.I. 1980. Antigen-andreceptor-driven regulatory mechanisms. III. Induction of delayed-type hypersensitivity to azobenzenearsonate with anti-cross-reactiveidiotypicantibodies,d. Exp. Med. 151:896-909 196a. Sy, M.-S., Brown,A., Bach, B. A., Benacerraf,B., Gottlleb, P. D., et al. 1981. Geneticand serological analysis

of the expressionof crossreactiveidiotypic determinants on anti-p-azobenzenarsonate antibodies and p-azobenzenarsonate-specificsuppressorT cell factors. Proe. NatL Acad. ScL USA78: 1143-47 197. Sy, M.-S., Dietz, M. H., Germain,R. N., Benacerraf,B., Greene,M.I. 1980. Antigen- and receptor-driven regulatoT mechanisms.IV. Idiotype-bearing I-J suppressor T cell factors induce second-ordersuppressor T cells which express anti-idiotypicreceptors,d.. Exp. Med. 151:1183-95 197a. Szewczuk,M. R., Campbell, R. J. 1981.Lackof age-associatedauto-antiidiotypic antibodyregulation in mucosM-associated lymphnodes. Eur. J. Immunol. 11:650-56 198. Tada, T., Okumura,K., Hayakawa,K., Suzuki, G., Abe,R., Kumagai,Y. 1981. Immunologicalcircuitry governed by MHCand VH gene products. In Immunoglobulin Idio~ypes: ICN-UCLM Symflosia on Molecular and Cellular Biology, ed. C. JanewayJr., E. E. Serearz, H. Wigzell,20:563-72.NewYork: Academic 199. Takemori,T., Tesch,H., Reth, M., Rajewsky, K. 1982. The immuneresponse against anti-idiotype antibodies.I. Induction of idiotope-bearingantibodies andanalysis of the idiotoperepertoire. Eur. Z ImmunoL12:1040-46 200. Tasiaux, N., Leuwenkroon, R., Bruyns, C., Urbain,ft. 1978.Possibleoccurence and meaningof lymphocytes bearing auto anti-idiotypic receptorsduringthe immune response. Eur. Z ImmunoL 8:464-68 200a. Th~ze,J., Morean,J.-L. 1978.Genetic control of the immune response to the GATterpolymer. ,~nn. ImmunoL (Paris) 129C:721-26 201. Thomas,W.R., Morahan,G., Walker, I. D., Miller,J. F. A. P. 1981.Induction of delayed-typehypersensitivityto azobenzenearsonateby a monoclonalantiidiotype antibody, d. Exp, Med. 153: 743-47 202. Tokuhisa,T., Komatsu,Y., Uchida,Y., Tani~. ehl, M. 1982. Monoclonalalloantlbodiesspecific for the constantre#on of156:888-97 T cell antigenreceptors. ,L ~xp. Med. 203. Trenkner, E., Riblet, R. 1975. Induction of antiphosphorylcholineantibody formationby anti-idiotypie antibodies. d. .Exp. Meal142:1121-32 204. Urbam,J., Wuilmart, C., Cazenave, P.-A. 1981.Idiotypic regulation in immunenetworks. In ContemporaryTop-

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munesystem. Common idiotypic speicsinMolecular Immunology, ed.F. P. Inwan,W.J. Mandy,8:113-48. New citicites betweenidiotypesandantibodYork:Plenum ies raised againstanti-idiotypicantibod205.Urbain, J.,Wilder, M.,Franssen, J.D., ies in rabbits. Z Exp. Med.150:184 Collignon, C.1977. Idiotypic regulation 215. Williams,K, R., Claflin, J. L. 1982. oftheimmune system bytheinduction Clonotypesof anti-phosphocholineanofantibodies against anti-idiotypic antitibodies inducedwith Proteusmorganii bodies. Proc. Natl. Acad. Sc~ USA (Potter). II. Heterogeneity,class and 74:5126-30 idiotypie analysesof the repertoires in 206. Yowler, L. B., Preod’homme,J. L., BALB/c and A/I-Id mice. J. Immunol. Scligmarm, M., Gathings,W.E., Grist, 128:600-7 W.M., et al. 1981. Diversity of im- 216. Woodland,R., Cantor, H. 1978. Idimunoglobulinexpression in leukaemie otype-speeific T helper cells are recells resemblingB-lymphocyte precurquired to reduce idiotype-positive B sors. Nature290:339-41 memory cells to secrete antibody.Eur. Y. lmmunol. 8:600-6 207. Weigert,M,, Riblet, R. 1976, Genetic control of antibody variable regions, 217. Wysocki,L. J., Sato, V. L. 1981. The ColdSpringHarborSyrup. Quant.Biol. strain A anti-p-azophenylarsonatcma41:837-46 jor crossreactive idiotypic family in208. Weil~ert dudes memberswith no reactivity to, M., Riblet, R. 1978. Thegewardp-azophenylarsonatc.Fur. J. Imnetic coatrol of aatibodyvariable regions in the mouse.SpringerSemi~Immunol. 11:832-39 munopathol.1:133-69 218. Yamumoto,H., Nonaka, M., Katz, D. 209. Weiler,E. 1981.In ldiotypes-Antigens H. 1979.Suppressionof hapten-speeitie on the lnside, ed. I. Westen-Sclmurr, delayedtype hypersensitivityresponses in miceby idiotype-specitic suppressor p.p.. 142-49.Basel: EditionesRoche 210.weiler, I. J., Weiler,E., Sprenger,R., T cells after administration of anti-idioCosenza,H. 1977. Idiotype suppression typic antibodies. Z Exp. Med. by maternalinfluence. Eur. Z lmmunol. 150:818-29 7:591-97 219. Yamauchi,K., Chao, N., Murphy,D. 211. Weinberger, J. Z., Get’main, R. N., B., Gershon, R. K. 1982. Molocular Benacerraf,B., Doff, M.E. 1980. Haptcomposition of an antigen-specificLy-1 en-speeifie T cell responses to 4T suppressorinducer factor. Onemolehydroxy-3-nitrophenylacetyl. V. Role culebindsantigenandis I-J-, anotheris of idiotypesin the suppressorpathway. I-J+, does not bindantigen, andimparts an Igh-variable region-linkedrestricZ Exp. Med. 152:161-69 212. Weinuerger,J. Z., Germain,R. N., Ju, tion. J. Exp. Med.155:655-65 $.-T., Greene,M.I., Benacerraf, B., 220. Yarmush,M., Sogn,J. A., Mudgett,M., Doff, M.E. 1979.Hapten-speeifie T cell Kindt, T. J. 1977. The inheritance of responses to 4-hydroxy-3-nitrophenyl antibodyV regionsin the rabbit: Linkacetyl. II. Demonstration of idiotypie .age of an H-chain-specificidiotype to determinantson suppressor T cells. Z unmunoglobulin allotypes. Z Exp. Med. Exp. Meal. 150:761-76 145:916-30 213. Wells, J. V., Fudenberg,H. H., Givol, 221. Yang, C., Kratzin, H., GiSt2, H., D, 1973.Localizationof idiotypicantiThiunes,F. P., Kruse,T., et al. 1982. genie determinantsin the F, region of PrimLrstruktur mensehlicher Hismurine myeloma 315. tokompatibilit~tsantigene der KlasseII Proc. Natl. Acad.protein Sc£ USAMOPC 70:1585-87 2. Mitteilung: Aminos~uresequenz der 214. Wilder, M., Demeur,C., Dewasme, G., N-terminalen179Reste der ~-Kettedes Urbain, J. 1980. Immunoregulatory HLA-Dw2/DR2-Alloantigeus.Hopperole of maternalidiotypes. Ontogeny of Seyler’s Z. Physiol. Cher~363:671-76 immunenetworks. :. Exp. Meal. 152: 222. Zinkernagel, R. M., Doherty, P. C. 1024-35 1974.Restrictionof in vitro T-cell me214a. Wilder, M., Fraussen, J.-D., Colligdiated eytoxieity in lymphocyticehorinon, C., leo, O., M~’iam6,B,, et al. omeningitis within a syngeneic orse1979. Idiotypic regulation of the iramiallogcneic system. Nature 248:701-2




An~Rev, lrnmunoL1983. D609-32 Copyright©1983by AnnualReviewsInc, All rights reserved


EPITOPE-SPECIFIC REGULATION Leonore .4. Herzenberg, Takeshi Tokuhisa, and Kyoko Hayakawa Genetics Department, Stanford California 94305

University

School of Medicine, Stanford,

INTRODUCTION Longbefore lymphocyteshad been identified with certainty as the precursors of antibody forming ceils, immunologists and immunogeneticists were well aware that animals immunizedwith the complex antigens like bovine serum albumin individually produce antibodies to different subsets of the epitopes (determinants) on the antigen. Over the years, manyof the cells and cell interactions that regulate antibody production have been defined in great detail; however,the processes that control the characteristic individuality of antibody responses still remain shrouded in mystery. The epitope-specific regulatory system described here offers a workableexplanation of howthis variation is generated and maintained. Furthermore, as we shall show, the joint operation of the carrier-specific induction mechanismand the epitope-specific effector mechanismthat defines the characteristics of the regulated response provides an explanation for manyof the odd observations encountered in studies of immunologicmemoryand carrier-specific regulation. Current concepts of howcarrier-specific regulation influences antibody responses to the epitopes (immunogenicstructures) on carrier molecules date from the early 1970s, whenMitchison (I) and Rajewskyet al (2) first demonstrated that carrier-primed T cells help hapten-primed B cells give rise to adoptive secondary antibody responses. These studies, which showed that "... the antigen [hapten-carrier conjugate] is recognized by two receptors, one directed to the hapten and the other to a determinanton the carrier protein" (1), in essence formulated the contemporary definition of a dependentantigen, i.e. a macromoleculewith at least one "carrier determi609 0732-0582/83/0410-0609502.00

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nant" recognized and used by T cells to regulate antibody responses to the various epitopes on the antigen. The hapten-carrier bridge mechanismsuggested for carrier-specific helper activity in these early studies is as viable today as it was whenfirst stated (and still remains unproven); however,the simplistic two-cell model (helper T and memoryB) introduced initially has undergone considerable expansion. Twofunctionally distinct carrier-specific regulatory T cells have been added, one which suppresses antibody responses (3-8) and another which contrasuppresses such responses (9). Furthermore, several carrierspecific cells have been identified as part of developmentalcascades leading to the emergenceof the functional cell types. Finally, on a theoretical level, a series of carrier-specific regulatory circuits have been proposed locating the various functional cells in a self-limiting ("feed back") type systemthat controls antibody production by controlling the supply of carrier-specific help (10). This construct is consistent with most of the available data; however, it fails to explain a numberof relevant observations (several of whichpreMate its inception). In particular, becauseit is predicated on the idea that carrierspecific regulatory interactions do not selectively influence antibody production to individual epitopes on an antigen, it tends to trivialize findings that suggest links between cartier-specific and epitope-specifie or other regulatory mechanisms.In essence, it has led to consistent disregard of evidence suggesting that immunizingcarrier-primed animals with a "new" hapten coupled to the priming carrier results in the induction of specific suppression for antibody responses to the hapten (see below). Mitehison (1) and Rajewskyet al (2) overtly chose to ignore this peculiar response failure to simplify consideration of the mechanismsinvolved in carrier-specific help. Thus, although they carefully cite evidence from several studies (including their own) showingthat carder-primed animals often fail to producedetectable levels of antibodies to haptens introduced subsequently on the priming carrier, they put more trust in opposing evidence showingthat significant anti-hapten antibody production can be stimulated by this "carrier/hapten-carrier" immunizationprotocol. Rajewskyet al (2) makethis point quite directly by stating: "Wesuggest that, in general, an animal pretreated with free carrier and receiving a secondary injection of this carrier eomplexedwith a hapten will be found to produce a better anti-hapten response than without the pre-treatment, if the experimental design is aimed at detecting this effect." This "leap in faith" (although largely incorrect) was probably crucial the orderly progress of early studies exploring the mechanismsregulating antibody production. The idea that carrier-priming wouldaugment (rather than suppress) subsequent antibody responses to a hapten presented on the

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EPITOPE-SPECIFICREGULATION 611 primingcartier cleared awayconfusion and allowed rational planning of adoptiveand in vitro experimentsthat followedfromthe newlypublished carrier-specific help studies. However,although this idea wasadvanced originally as a prediction and had beenshownto be invalid undercertain circumstances,it rather rapidly assumed the mantleof truth. Consequently, several years later, we(and mostof our colleagues)werequite surprised the suppressedrather than augmentedanti-hapten antibody responsesthat weobtained followingcarrier/hapten-earrier immunization(11). Theseunexpectedfindings (e.g. see Figure1) engendereda rapid series of experimentsexploring the mechanism of the suppressionand a somewhat moreleisurely search throughthe literature lookingfor precedentsfor our observations. Thus,by the time wediscoveredthat previousinvestigators had ascribed this kind of responsefailure to impairedanti-hapten memory development (14) or to the presenceof anti-carrier antibodies(12, 13), hadalready ruled out these possibilities andrealized that weweredealing with a previously unrecognized"epitope-specific" regulatory systemthat selectively controls antibodyproductionto individualepitopes accordingto the dictates of carrier-specific (and other) regulatoryT cells presentin the immunologicenvironmentwhensuch epitopes are first introduced (11, 15-22). In pursuingour studies of this system,wehavebeenconcernedprimarily with finding out howit works; however,we have also put considerable thought and someexperimentaleffort into determininghowit could have escapednotice for so manyyears. Not surprisingly, the answersto these questions often merge.For example,in attempting to establish adoptive assays for the inducerandetfector cells responsiblefor the suppression,we foundthat quite strong in situ suppressionis diti~cult to transfer to irradiated recipients, particularly whenmeasuredas the ability to suppress a responsemountedby a co-transferredcell population(16). This characteristic tendencyfor help to predominate over suppressionin irradiated recipients put several investigatorsoff the track, includingSarvaset al (14) who in 1974reasonedcorrectly that cartier-specific suppressorT cells induce suppressionfor anti-hapten responsesin carrier/hapten-carrier immunized virgin B ....... > EarlyMemory ....... > Mature Memory .......... > IgGAFC

:

DEVELOPMENT

: : EXPRESSION

Figure 1 Regulation of IgG (memory) responses. Mechanismsthat regulate memoryB-cell developmentregulate the potential for IgG antibody production (22). Mechanismsthat regulate memoryB-cell expression control which and howmanyof the memoryB cells present in a given animal actually give rise to AFC(17).

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animals(see below),but discardedthe idea becauseit "requires postulating that the effect of these suppressorcells is abolishedin cell transfer experiments.’’ Thegeneral tendencyto measureanti-hapten antibody productionexclusively whilestudyingthe mechanisms involvedin carrier-specific regulation also appears to haveplayed a majorrole in "hiding" the epitope-specitic system. Ishizaka & Okudaira(23), for example,demonstratedthat antihapten responses were suppressed while anti-carrier antibody responses proceededto secondarylevels in carder-primedanimalsstimulated with a haptenon the primingcarrier. Discussingthis work(in 1973),these investigators referred to work by Tada & Okumura(24-26) demonstrating the existence of suppressorT cells and suggested"... that immunizationwith 1 to 10 #g of ovalbuminmayresult in the formationof so called [carrierspecific] suppressorT cells that mightsuppresspreferentially the primary anti-DNPresponse" (23). Immuno-historywouldprobablybe quite different had Tada(or others interested in carder-specific suppressorT cells) taken this suggestionseriously enoughto test anti-carrier antibody responsesin suppressionassays routinely. All in all, the attractivesimplicityof the idea that carrier-specificsuppressor T ceils regulate antibodyproductionby depleting carrier-specific help appears to be sutticient to explain the willingness of manylaboratories (including our own)to ignore subtle inconsistencies and leave a fewmosscoveredstones unturned.Perhapsthis has beenmainlyfor the good,since the mechanisms involved in epitope-specific regulation are considerably morecomplexandwould,in fact, havebeendifficult to explorewithoutthe levd of technologyand theoretical understandingreachedwith the last few years. In any event, we nowappear to have reached the time whenreevaluationof past evidenceis essential to present progress. Thesections that follow describe various aspects of our studies of the epitope-specific regulatory system. Webegin with an overviewsection (immediatelybelow)that broadly outlines the systemas a whole, documenting statements with references rather than with evidence. Theremainingseetions, in contrast, discussaspects of the systemin moredetail andinclude muchof the evidence uponwhichour conclusions are based. The workwediscuss here has mainly been conductedin our laboratory at Stanford; however,studies with carder-specific suppressorT cells and soluble factors were conductedin Dr. MasaruTaniguchi’s laboratory in Chiba, Japan. Almostwithout exception, the evidence we report derives from experimentsthat repeat earlier workbut include key controls aimed at distinguishingcarrier-specific fromepitope-specificregulatoryeffects. Thus, weindependentlymeasureboth the anti-hapten and anti-carder antibodyresponsesfollowingvarious antigenic stimulations, andweconfirmthe

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EPITOPE-SPECIFICREGULATION 613 induction of epitope-speeific suppressionfor a given anti-hapten antibody response by a final immunizationwith the hapten conjugated to an unrelated carder molecule.As weshall show,the application of these rather straightforwardtechniquesprovidessurprising insights into the older data andreveals the outlines of a highly flexible central regulatorysystemresponsiblefor controlling all aspectsof antibodyresponses.


EPITOPE-SPECIFIC

REGULATION:

AN OVERVIEW

Theepitope-specific systemapparently provides a common channelthrough whichcarrier-specific, isotype-specific, allotype-speeific, and 1-regiondefined mechanisms exert control over antibody responses(11, 15-21). plays a key role in regulating IgGantibodyresponsesto haptensand native epitopes on commonly used carrier moleculessuch as KLH (keyhole limpet hemocyanin)and CGG(chicken gammaglobulin) (11, 15-18). Furthermore,it is active in regulating IgGresponsesto epitopes on the synthetic ter-polymerTGAL, even in genetic "non-responders"to this antigen (20). Carder-specificinteractions induce it to suppressantibodyproduction(to individual epitopes on the carrier); however,onceinducedto suppress given anti-epitope response, it will suppressthat responseevenwhenthe epitope is presented in immunogenic form on a different carder molecule (II, 15-17). Theeffector mechanism in this systemcontrols memory B-cell expression (as opposedto development;see Figure 1) and appears to be the ultimate arbitor of whichandhowmanysuch B cells will be permittedto differentiate to IgGantibody-formingcells (AFC)in response to a given antigenic stimulus.It is epitopespecific in that it independently regulatesthe amount and affinity of the antibodyresponseproducedto each of the epitopes on a complex antigen(11, 15-17);andit is Igh restricted in that it selectively controls the IgChisotype andallotype expressionin such responses(17-19). Theseproperties suggestthat the overall systemis composed of individually specific elements,each chargedwith the regulation of a subset of B cells committedto produceunique or closely related IgGmolecules(17). Theflexibility of this (compound) regulatorysystemandthe nature of its effects on in situ antibodyresponsesare extraordinary. It is capable of suppressingvirtually the entire primaryand secondaryantibody response to a given epitope. Therefore, it can completelyconceal the presence of normalanti-epitope memory B-cell populations clearly demonstrablein adoptive transfer assays (11, 15, 16). Alternatively, it can support the expression of a subset of memory B ceils and suppress the expression of others, thereby defining the uniquespectrumof an antibodyresponse producedby a given animal(17-19).

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Carrier-specific and other regulatory mechanisms operative in the immunologicalenvironmentwhenan epitope is first introduced determine whichcomponentsof the antibody response will be suppressed and which will be supported(11, 15-17). Prior immunization history, genetic predisposition towardresponsiveness(20) and perinatal immunologic "conditioning" (18) all contribute to this determination.Thus,the epitope-specific systemconstitutes an ideal candidate for a central integrative mechanism capableof resolving conflicting signals fromvarious peripheral regulatory systemsand translating a coherent decision to the antibody-producing apparatus. In keepingwith this role, this systemoffers a uniqueregulatorycapability that providesboth the stability requiredto maintaina responsepattern once inducedand the flexibility to modifythat responsepattern whenstimulatory conditionschangedramatically.In essence, the individual epitope-specific, Igh-restricted elementsin the systemappearto behavesimilarly to bistable electronicbinary("flip-flop") circuits that can be switchedinitially into "on"or "off" positionby a smallelectrical force andthen require a substantially larger force to switchthemto the oppositeposition. Thatis, initial immunization conditions that induce epitope-specific elementsto suppress a givenIgGantibodyresponsewill usuallyfail to do so oncethe systemhas beeninducedto supportthat response; and, similarly, conditionsthat inducethe systemto support a response will be far less effective oncethe system has been induced to suppress the response. Nevertheless, either suppression or support can be reversed by sul~cient stimulation in the oppositedirection (17). The evidenceuponwhichthese conclusions (and hypotheses) are based is presentedin detail in several publicationsthat haveappearedwithin the last few months(16-19). Therefore, in the discussion whichfollows (and in Tables 1 and 2), we summarizethese findings and concentrate more intensively on several recent studies defining the T cells that mediateepitope-specific suppression and demonstrating someof the more complex aspects that exist within the system. CARRIER/HAPTEN-CARRIER IMMUNIZATION INDUCES EPITOPE-SPECIFIC SUPPRESSION FOR IgG ANTI-HAPTEN RESPONSES Theepitope-specific regulatory systemcan be specifically inducedto suppress primaryand secondaryIgG antibody responses to the dinitrophenyl hapten (DNP)without interfering with antibody responses to epitopes the carrier moleculeon whichthe DNPis presented(see Table3). Furthermore,onceso induced,it specifically suppressesantibodyresponsesto DNP presented on unrelated carrier molecules. Themagnitudeof a suppressed

Annual Reviews EPITOPE-SPECIFICREGULATION 615


primary anti-DNP response is usually about 30%of the normal primary response; however,the affinity of a suppressedresponse is about 10-fold lower than normal. Suppressedsecondaryanti-DNPresponses are typically less than10%of normalandhaveaverageaffinities that are at least 100-fold below normal(11, 16). For most of our studies, we induce this suppression by immunizing animals sequentially with a carder molecule such as KLH (keyhole limpet hemocyanin) and then DNPconjugated to" the carrier (i.e. with the Table 1 Induction of epitope-specific tions

suppression occurs under a wide variety of condia Result

Variable tested Epitope

DNP, TNP, NIP

Suppression induced for both epitopes by carrier/hapten-carrier; suppression inducible for KLHepitopes by other protocols

Carrier

KLH, CGG, OVA, TGAL

All prime for suppression induction; 100 ~.g on alum sufficient; somegenetic restrictions (see table 2)

Age

KLHat 8 weeks to >6 months

Suppression equally strong at all ages

Timing

1 to 13 weeks between KLH and DNP-KLH

Suppression equally strong for all intervals between carrier and hapten-carrier

KLH/DNP-KLH then DNP-KLH or DNP~-CGG up to 1 yr later

Suppression equally strong for all intervals between first and second hapten-carrier immunizations

BALB/C, BAB/14, SJL, SJA, C3H, C3H.SW, A/J, (SJL × BALB/C), C57BL/10, C57BL/6

Suppression inducible in all strains

Strains

aSummarized from References 1 through 7. Table 2 Epitope-specific suppression in genetically controlled regulatory conditions Condition I-region controlled responses Impaired response to KLHcarrier (non-MHC) Allotype suppression for Igh-lb (IgG2a) allotype production in (SJL × BALB/C)F1

Immunization

Result

TGAL/TNP-TGAL in C3H(H-2k) and C3H.SW(H-2b) KLHpriming in A/J or C57BL/10

Suppression induction occurs in responder and non-responder strains (20)

DNP-KLHat 8 weeks of age (prior to midlife remission from allotype suppression)

Poor suppression induction by KLH/ DNP-KLH;normal induction by CGG/ DNP-CGG (21) Igh-lb responses to DNPand KLH specifically suppressed during remission (18)

Annual Reviews

616

HERZENBERG ET AL

Table 3 Anti-hapten antibody production is specifically hapten-carrier immunizedmice aImmunizations Second DNP

K -K -K --

-D-K D-K D-C D-C D-K D-K

-----D-K D-K

K ---

D-K D-K D--C

D-C D--C D-C

K


In situ IgG2a b antibody responses

First DNP

Carrier

suppressed in carrier/

Anti-DNP c~tg/ml (Ka) < 3 (< 0.3) 5 (

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