ADVANCES IN
Immunology
VOLUME 23
CONTRIBUTORS TO THIS VOLUME Bo DUPONT A. HANSEN KIMISHIGEISHIZAKA T. P. KING DONAL...
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ADVANCES IN
Immunology
VOLUME 23
CONTRIBUTORS TO THIS VOLUME Bo DUPONT A. HANSEN KIMISHIGEISHIZAKA T. P. KING DONALD M. MARCUS GERALD A. SCHWARTING EDMOND J. YUNIS JOHN
ADVANCES IN
Immunology E D I T E D BY
HENRY G. KUNKEL
FRANK J. DIXON
The Rockefeller University
Scrippr Clinic and Research Foundation
N e w York, N e w York
La Jolla, California
VOLUME 23
1976
ACADEMIC PRESS New York
Sun Francisco
A Subsidiary o f Harcourf Broce Jovanovich, Publishers
London
COPYRIGHT 0 1976, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS, INC.
111 Fifth Avenue, New York,New York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London N W l
LIBRARY OF CONGRESS CATALOG
CARD
NUMBER:6 1 - 17057
ISBN 0-12-022423-2 PRINTED IN THE UNITED STATES OF AMERICA
CONTENTS LIST OF CONTRIBUTORS . PREFACE
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1 3 12 20
Cellular Events in the IgE Antibody Response
KIMISHIGE ISHIZAKA
I. Introduction
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11. Immunoglobulin E Antibody Formation in Viuo and in Vitro 111. Immunological Factors Essential for IgE Antibody Responses . . IV. Cellular Basis of IgE Antibody Responses .
V. Regulation of IgE Antibody Responses VI. Discussion and Summary . . . . . . . . . References
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Chemical and Biological Properties of Some Atopic Allergens
T. P. KING I. 11. 111. IV. V. VI.
Introduction . . . . . . . . . Allergen Assay . . . . . . . . Chemical and Biological Properties of Some Allergens General Observations on Allergens . . . . Uses of Purified Allergens . . . . . . . . . . . . Concluding Remarks . References . . . . . . . . .
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Human Mixed-lymphocyte Culture Reaction: Genetics, Specificity, and Biological Implications
Bo DUPONT,JOHNA. HANSEN,AND EDMONDJ. YUNIS I. Introduction: Major Histocompatibility System in Man . . 11. Serology of Human Leukocyte Alloantigens (HLA-A,B,C) . 111. Cell-Mediated Allogeneic Reactions in Vitro . . . . IV. Measurement of Antigenic Differences in Mixed-Lymphocyte C u h r e Reaction . , . . . . . . . . V. Single-Locus Concept for Mixed-Lymphocyte Culture Stimulation (HLA-D Locus) . . . . . . . . . . V
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VI Mixed-Lymphocyte Culture ( HLA-D ) Specificities Defined by HLA-D-Homozygous Typing Cells . . . . . . . VII . Genetic Control of Immune Response Related to Histocompatibility VIII . Mixed-Lymphocyte Culture As a Histocompatibility Test for Clinical Transplantation . . . . . . . . . IX Genetic Mapping of the HLA Complex on Chromosome C-6 . X . Conclusions . . . . . . . . . . . . References . . . . . . . . . . . .
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203 204 229 233 233
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lmmunochemical Properties of Glycolipids a n d Phospholipids
DONALDM . MARCUSAND GERALDA. SCHWARTING
I. I1. 111. IV
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Introduction . . Glycolipids . . Phospholipids . . Concluding Remarks References . .
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CONTENTS OF PREVIOUS VOLUMES.
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LIST
OF CONTRl BUTORS
Nurnlxm in parentheses indicate the pages on which the authors’ contributions begin.
Bo DUPONT,Tissue Typing Laboratory, Sloan-Kettering Institute for Cancer Research, New York, New York (107)
A. HANSEN, Tissue Typing Laboratory, Sloan-Kettering Institute for Cancer Research, New York, New York (107)
JOHN
KIMISHICE ISHIZAKA, Department of Medicine, The Johns Hopkins University School of Medicine at the Good Samaritan Hospital, Baltimore, Maryland (1) T. P. KING, The Rockefeller University, New York, New York (77)
DONALD M. MARCUS,Departments of Medicine, Microbiology and Zmmunology, Albert Einstein College of Medicine, Bronx, New York (203) GERALD A. SCHWARTING, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York (203) EDMOND J. YUNIS, Department of Pathology and Laboratory Medicine, University of Minnesota Hospitals, Minneapolis, Minnesota ( 107)
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PREFACE
The familiar and somewhat tiresome debate over the relative merits of fundamental versus applied research has if anything intensified in the last few years. This has occurred largely as a result of the greatly increased competition for funds that exists today. It has been fostered to a considerable degree by the “somewhat snobbish attitude of many academics to applied research.” The distinction is purely arbitrary; scientific knowledge is a continuum in which every component part can and does feed back on every other. Nowhere is this more clearly apparent than in the field of immunology, as exemplified by the articles in Volume 23. The first paper is by Dr. Kimishige Ishizaka, the individual primarily responsible for the basic work on IgE antibodies and their role in reaginic hypersensitivity. The initial definitive work was carried out in the human system, and the extension to the cellular regulation of IgE antibodies, the main topic of the review, was continued in various experimental animals. The important role of both helper and suppressor T cells in this regulation is quite apparent. I t is still uncertain whether the same cells are involved as those defined for the major immunoglobulin classes. Promising approaches to therapy derived from the animal-model work are discussed. The work of Dr. T. P. King, author of the second article, has centered on the chemistry of the allergens, a subject which has advanced markedly in the last few years, largely through his efforts. Ragweed pollen allergens have received the most attention, and antigen E, the dominant antigen involved in hypersensitivity, has been isolated and characterized in considerable detail. It consists of two non-identical polypeptide chains with molecular weights of approximately 26,000 and 13,000. Additional ragweed allergens have been isolated, but their significance relative to antigen E remains to be defined. Many other types of allergens have been isolated as well. Of special interest is the current active work on the chemical modification of these isolated proteins for possible therapeutic immunization. The third article is written by Drs. Dupont, Hansen, and Yunis, and deals primarily with the new and exciting developments in MLC typing in human histocompatibility studies. These workers have played a major role in placing this system on a firm scientific basis. The use of homozygous cells from specific individuals has made it possible to delineate a t least six different distinct MLC antigens, and there are clearly more. Some of these can also be recognized by B-cell-specific alloantisera and clearly relate to the Ia antigens of the murine system. I t is of special iX
X
PREFACE
interest that certain disease associations, as well as the genes involved in certain of the complement components, appear more closely linked to the MLC genes than to the other components of the HLA system. The last paper covers the somewhat neglected area of the immunology of lipids and glycolipids. The authors, Drs. Marcus and Schwarting, have had wide experience in this field and their contributions have played a major role in current recognition of the significance of these antigens. Suddenly, with the great expansion of interest in cell membranes, the glycolipids have assumed a particular importance and their study by immunological procedures as specific moieties of the cell membrane is receiving great emphasis. Much remains to be learned about the many different types of lipid antigens and their cross reactions, but this review provides the many interested investigators with an up-to-date treatment of the subject. HENRYG. KUNKEL FRANKJ. DIXON
ADVANCES IN
Immunology
VOLUME 23
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Cellular Events in the IgE Antibody Response’ KlMlSHlGE ISHIZAKA Department of Medicine, The Johns Hopkinr University School of Medicine ot the Good Samaritan Hospital, Baltimore, Marylond
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I. Introduction . . . . . . . 11. Immunoglobulin E Antibody Fomiation in Vioo and in Vitro . A. Kinetics of IgE Antibody Responses in Various Animal Species B. Helminth Infection and IgE Responses . . . C. Distribution of IgE-Forming Cells . . . D. Immunoglobulin E Antibody Response in Vitm . 111. Immunological Factors Essential for IgE Antibody Responses . A. Genetic Control of IgE Responses . . . B. Adjuvant for IgE Antibody Response . . . . C. Nature and Dose of Antigen . . . . . N.Cellular Basis of IgE Antibody Responses . A. Requirement for T and B Lymphocytes . . . . B. Type B Lymphocytes in IgE Antibody Response . C. Generation of a Helper Function for IgE Antibody Response D. Mechanisms of T Cell-B Cell Collaboration . . . V. Regulation of IgE Antibody Responses . A. Suppression by Humoral Antibodies . . . . . . , B. Unresponsiveness in IgE-B Cells C. Regulation by T Cells D. Experimental Model for Immunotherapy . . . . . VI. Discussion and Summary References . . .
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I. Introduction
Since the discovery of IgE in the serum of hay fever patients (45), much progress has been made in the field of reaginic hypersensitivity. It is now established that reaginic hypersensitivity reactions in atopic diseases are mediated by IgE antibody [reviewed by Ishizaka and Ishizaka (%)I. Meanwhile, homocytotropic antibodies, which are similar to human IgE antibodies, were detected in experimental animals. Mota (109) and Binaghi et al. (11) first described production of rat “reaginic” antibodies after immunization with antigen plus Bordetella pertussis vaccine. Subsequently, antibodies that were capable of sensitizing homologous skin ‘Supported by research grants AI-11202 from the U.S. Public Health Service, GB-41443 from National Science Foundation, and a grant from John A. Hartford Foundation. This is publication No. 223 from the O’Neill Laboratories at the Good Samaritan Hospital.
1
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KIMISHIGE ISHIZAKA
were found in rabbit (46, 91, 189), dog (133), mouse (110, 112, la), monkey (47), guinea pig (88), pig ( 7 ) , and cattle (39). The physicochemical properties of the reaginic antibodies in experimental animals are similar to those of human IgE, and their molecular sizes are distinct from those of immunoglobulins of the other isotypes. It was also found in each species that the antigenic structure of the immunoglobulin class to which the reaginic antibody belongs was different from IgG, IgA, and IgM. More recently, Bazin et al. ( 9 ) reported that the inbred Lou/WST rat strain presented a high incidence of spontaneous ileocecal immunocytoma, which secreted monoclonal immunoglobulins, and that nearly one-third of them represented a unique isotype to which reaginic antibody belonged. From the biological viewpoint, human IgE and reaginic antibodies in experimental animals share common characteristics. Once skin sites of homologous species are passively sensitized with the antibody, sensitization persists for 2 to 3 weeks. This property and the molecular size of reaginic antibodies are distinct from those of another type of skinsensitizing antibodies that belong to a subclass of IgG. A crucial role of IgE antibody in atopic diseases suggested that prevention or suppression IgE antibody formation is beneficial for atopic individuals. Identification of IgE antibodies in experimental animals provided an important tool for studying this problem. Fortunately, the scope of our knowledge on the mechanisms of antibody response has considerably broadened in the past decade [reviewed by Katz and Benacerraf (64)]. It is firmly established that collaboration of two distinct types of lymphocytes, i.e., bone marrow-derived precursors of antibody-forming cells ( B cells) and thymus-derived lymphocytes ( T
4
8 1970
1 2 4
8 1971
1 2 4 1972
FIG.1. Titers of IgE and IgC antibody in the serum of ragweed-sensitive patient. Both IgE ( A )and IgC ( 0 )antibody titers are expressed by units. The IgE antibody unit corresponds to the minimal concentration of the antibody required to give a positive Prausnitz-Kiistner reaction. [From Ishizaka and Ishizaka ( 4 4 ) .I
CELLULAR EVENTS I N THE IGE ANTIBODY RESPONSE
3
cells), is essential for the induction of antibody responses to most protein antigens. This principle obtained with IgM and IgG antibody responses has been proved to be the case also in IgE antibody formation. From the immunological viewpoint, however, it became clear that IgE antibody responses in experimental systems have certain characteristic features that are not easily demonstrated in IgG antibody response. The purpose of the present review is to analyze the cellular events involved in the IgE antibody response in different experimental systems in comparison with the IgG antibody response. It is hoped that elucidation of the mechanisms for induction and suppression of IgE antibody response will provide a clue to future therapy for atopic diseases. II. Immunoglobulin E Antibody Formation in Vivo a n d in Vitro
A. KINETICS OF IGE ANTIBODYRESPONSESIN VARIOUSANIMAL SPECIES Many years ago, Sherman et al. (142) followed reaginic antibody titers in hay fever patients by recording Prausnitz-Kiistner reactions and showed that antibody titers persisted in the sera of ragweed-sensitive individuals. The results were recently confirmed by quantitative measurement of IgE antibody by a radioimmunoassay (RAST technique), which was developed by Wide et al. (185). Application of this method to measure serum IgE antibody in untreated ragweed-sensitive patients revealed that the antibody level persisted and that most patients showed secondary IgE antiragweed antibody responses after the ragweed season (Fig. 1 ) (44). Because the catabolic rate of IgE is very fast, with an average halflife of 2 to 3 days (178), persistence of IgE antibody titers in the sera of atopic patients indicates that IgE antibody is being formed continuously. Such a pattern of antibody formation, however, is not characteristic only for IgE. Titration of IgG antiragweed antibody in the sera of the same untreated patients by double antibody radioimmunoassay showed that IgG antibody formation also persisted, and the antibody titer definitely increased after the ragweed season. As shown in Fig. 1, the time course of IgG antibody produced to ragweed antigen E paralleled that of IgE antibody. Several investigators injected allergen into non-atopic individuals in the course of their studies of hyposensitization treatment. Some normal individuals who received parenteral injections of alum-precipitated allergen developed IgE antibody against the allergen. The IgE antibody in the sera of these individuals disappeared within 2 to 3 months; however, many of them showed secondary IgE antibody responses after the
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pollen season (99). De Weck (20) has shown a similar pattern of IgE antibody response in patients with hypersensitivity to penicillin. In many patients, the IgE antibody was detected when they had clinical symptoms but disappeared within several weeks after the administration of penicillin. Obviously, these patients will show secondary IgE antibody responses after reexposure to the drug. Although the physicochemical properties and biological function are similar for IgE antibodies from various animal species, the kinetics of IgE antibody responses are different depending on the species and strains of the animals. Immunization of rats with usual protein antigens, such as ovalbumin (OA) (109) or human IgG (11) together with pertussis vaccine or aluminum hydroxide gel (alum) as adjuvants resulted in the formation of IgE antibody, but the antibody response was transient in nature. Maximum IgE antibody titer was reached at 10 to 14 days after the immunization and rapidly declined thereafter. A booster injection of the same antigen 4 to 5 weeks after the primary immunization did not elicit secondary IgE antibody response. Even when a secondary response was observed, maximum IgE antibody titer after a booster injection was lower than the maximum titer after primary immunization. As will be described later, the dose of antigen and nature of adjuvant employed for the primary immunization appear to be important factors in obtaining a secondary antibody response, By using a purified antigen from Ascaris suum extract (Asc-1), Strejan et al. (151) have shown a definite secondary IgE antibody response after a booster injection. More recently, Jarrett et al. (60) immunized Hooded Lister strain rats with 1 to 10 pg OA or keyhole limpet hemocyanin (KLH) together with 10'O pertussis vaccine and then gave a booster injection of homologous antigen without adjuvant at 30 days after primary immunization. This immunization schedule gave a definite secondary IgE antibody response. Other strains of rats, e.g., Sprague-Dawley, Wister, and Lewis, however, failed to show secondary IgE antibody response after a booster injection of antigen without adjuvant. A unique system for obtaining an IgE antibody response in the rat was described by Tada et al. (155). Their immunization schedule was based on previous observations of Strejan and Campbell ( 148), who found that two closely spaced injections of A. mum extract ( Asc) were effective in obtaining a high titer of reaginic antibody in the rat. Tada et al., injected 1 mg of dinitrophenyl derivatives of A. suum extract ( DNP-Asc) together with 10'O Bordetella pertussis vaccine into footpads of Wistar rats, followed by an intramuscular injection of 0.5 mg of DNP-Asc on day 5. In most animals, IgE antibody to homologous antigen was detected after the second injection. The IgE antibody titer reached a
CELLULAR EVENTS I N THE ICE ANTIBODY RESPONSE
5
maximum at 3 days after the second injection and declined thereafter. I n their experiment, neither the first injection of antigen with pertussis vaccine nor the injection of antigen alone induced IgE antibody response. An average maximum antibody titer, which was determined by the homologous passive cutaneous anaphylaxis (PCA) reaction, was on the order of 1:80, and the antibody became undetectable at about 4 weeks. I t was also found that a booster injection of the same antigen 4 weeks after the immunization failed to give a secondary IgE antibody response. Their immunization regimen is unique in that a large dose of antigen was used to obtain an IgE antibody response and that a single injection of antigen with pertussis vaccine failed to elicit the antibody response. As will be discussed later, usually a small dose of antigen is favorable for the IgE antibody response, and a single injection of an adequate dose of antigen with either pertussis vaccine or alum gives primary IgE antibody response. Subsequently, Tada et al. ( 162) succeeded in sustaining the IgE antibody response by irradiation of rats with sublethal doses (200400 R ) of X-ray, 1 day before or 1 day after the initial injection of DNP-Asc with pertussis vaccine. In the irradiated rats, IgG antibody was undetectable, but serum IgE antibody titer was higher than that obtained in nonirradiated animals, and the titer was maintained more than 3 weeks. This immunization schedule was frequently used by Tada and his associates when they wished to analyze the mechanisms involved in the IgE antibody response. Unfortunately, irradiation abolished rather than sustained IgE antibody responses in some other strains such as Sprague-Dawley and Lewis (see Section V,C,1). Rabbit IgE antibody was first described by Zvaifler and Becker (189) who had immunized animals with a relatively high dose of DNP-bovine y-globulin ( BGG ) included in complete Freunds adjuvant ( CFA) . The antibody did not persist for long, and these animals failed to show secondary IgE antibody responses after a booster immunization. Subsequently, it became clear that immunization with a relatively small dose of antigen precipitated with alum (132) gave a primary IgE antibody response and that the animals immunized by this procedure frequently gave secondary IgE antibody responses after a booster immunization. In our experience, more than one-half of rabbits immunized with DNP-Asc showed secondary antihapten IgE antibody responses in which maximal antibody titers were higher than primary responses (48 ). Mota and Peixoto (112) detected reaginic antibody in the mouse, after they had immunized outbred mice with a relatively high dose (50-100 p g ) of antigen included in CFA, alum, or with pertussis vaccine. The IgE antibody response was transient in nature, and antibody became
6
KIMISHIGE ISHIZAKA
undetectable in the sera within 3 weeks after the immunization. Similar results were obtained by Revoltella and Ovary (131) in several inbred strains of mice using DNP-KLH as antigen. Thus, the kinetics of reaginic antibody formation in the mouse were believed to be different from that observed in hay fever patients. Such a difference was overcome in a model developed by Levine and Vaz (go), who immunized several inbred strains of mice with 0.1-1.0 pg of protein antigens absorbed to alum. Repeated immunization at 4-week intervals resulted in a secondary response with a high titer of reaginic antibodies. Subsequently, Vaz et al. (173) succeeded in obtaining a persistent reaginic antibody response by injecting alum-absorbed OA (0.1 pg) into SW-55 strain mice. The reaginic antibody titer persisted for several months without booster injections. So far, the IgE antibody response in this system is the best model for reaginic antibody formation in humans. A persistent IgE antibody response has now been achieved with several different combinations of antigens and inbred strains of mice. For example, a minute dose of OA (0.05-0.2 pg) adsorbed to alum produced a persistent antibody response in DBA/1 and (C57B1/6 x DBA/2)F, mice (176). Immunization of these strains with 1-2 pg DNP-KLH absorbed to 1-2 mg of alum gave a persistent anti-DNP antibody response (120). An injection of alumabsorbed ragweed antigen E into the A/ J strain gave a similar pattern of IgE antibody response ( 5 2 ) .
B. HELMINTH INFECTION AND ICE RESPONSFS It has been known for a long time that an intracutaneous injection of an extract of Ascaris lumbricoides into normal individuals frequently elicits a positive erythema wheal reaction, suggesting that IgE antibody is formed following Ascaris infection. Johansson et al. as well as others reported that total IgE levels in sera increased in most individuals infected with any one of a variety of helminths including A. lumbricoides (61), Capillaria phillipinensis ( 183), and Ancylostoma ( 6). Infected individuals’ other serum immunoglobulins, such as IgG, IgM, IgA, and IgD, were usually in the normal range or were elevated only slightly emphasizing the strong relationship of helminth infections with IgE. The IgE antibody formation following helminth infection was established in experimental animals such as the rat, mouse, and rabbit (108, 115, 118, 137, 190). Nematodes, cestodes, trematodes, as well as arthropods all share this immunogenic characteristic ( 117). A typical example was shown in the rat by Ogilvie (115), who demonstrated IgE antibody formation after infection with Nippostrongylus brasiliensis larvae. The IgE antibody against worm extract became detectable 3 4 weeks after the infection, and antibody persisted for a longer period of time than that
CELLULAR EVENTS I N THE IGE ANTIBODY RESPONSE
7
obtained by an artifical immunization with protein antigen included in an appropriate adjuvant. Furthermore, the animals showed a definite secondary IgE antibody response upon reinfection (116, 188). Recently, Jarrett and Bazin (58) determined total IgE levels in rats infected with N . brasiliensis. Their results showed that total IgE levels in the sera of normal Hooded Lister rats were less than 0.35 pg/ml, but these levels increased to 250-500 pg/ml at 12 days after infection. Recently, we studied the relationship between total IgE and IgE antibody against worm antigen, following the infection of Sprague-Dawley rats with N . brasiliensis larvae ( 5 8 ) . The results showed that total IgE level began to increase about 10 days after the infection and reached a maximum on the fourteenth day. On the other hand, IgE antibody against worm antigen became detectable at 3 to 4 weeks after the infection, when total IgE level had already begun to decline (Fig. 2 ) . It is apparent that the kinetics TOTAL Ig E Nl/ml
100
PCA
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I I I
10 -
-80
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-20
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5
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9
WEEKS
t
INF
t
INF
FIG.2. Total IgE (0) and IgE antibody in the serum of a rat infected with Nippostrongylus brasiliensis. The IgE antibody titer ( A ) was determined by PCA reactions using an extract of worm as antigen. [From Ishizaka et al. ( 5 6 ) . ]
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KIMISHIGE ISHIZAKA
of the IgE antibody formation did not parallel the total IgE synthesis. Lack of correlation between total IgE and IgE antibody was confirmed in Hooded Lister rats (57). Another interesting finding in parasite infection is that infection of rats with N . brasiliensis or Fasciola hepatica causes nonspecific potentiation of unrelated IgE antibody responses to antigens such as OA and KLH (12, 59, 126, 127). Orr and Blair (126) first described this phenomenon following the infection of OA-primed animals with N . brasiliensis. Bloch et al. (12) found that augmentation of the antibody response after parasite infection was directed only to IgE antibodies: Neither the IgGl nor IgG2 antibody response was altered following the infection. There are some requirements for obtaining the potentiation. First of all, rats have to be primed in such a way as to produce IgE antibody prior to the infection. Second, there should be an appropriate interval between the priming immunization with antigen and infection. In Sprague-Dawley rats, which were employed by Orr and Blair (127), an interval of 1week to 10 days was optimal for the potentiation. Neither the infection prior to the immunization nor late infection after the primary IgE antibody response gave potentiation. This interval, however, did not appear to be critical when Hooded Lister rats were used in the experiments. Jarrett and Bazin (58) immunized these rats with OA together with pertussis vaccine and infected them 20 days after the priming for successful potentiation. The difference among the strains may be related to the fact that the primary IgE antibody response to OA in Hooded Lister rats was more persistent than that observed in the other strains. It is also known that the Hooded Lister strain show a secondary IgE antibody response to OA without adjuvant, whereas Sprague-Dawley rats fail to respond to a booster injection. In both strains, potentiation of the IgE antibody response was observed at 12 to 14 days after the infection when the total IgE increase was maximum. These results suggest that potentiation is due to nonspecific stimulus on B cells that have been programmed for IgE antibody production by previous immunization. This idea is supported by the finding of Jarrett et al. (59), who demonstrated that IgE antibodies against both OA and KLH were potentiated following parasitic infection if the rat had been primed with both antigens. The potentiation of an IgE antibody response after N . brasiliensis infection was observed in the mouse as well (82). In this species, however, infection with parasites 5 to 14 days prior to primary immunization was mogt effective for potentiation, whereas the infection after the immunization was ineffective. The reasons for these differences between rats and mice are unknown at the present time.
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CELLULAR EVENTS I N THE IGE ANTTBODY RESPONSE
C. DISTRIBUTION OF IGE-FORMING CELLS Th IgE-forming cells were first detected in primate lymphoid tissues by using a fluorescent antibody technique ( 154). In nonatopic individuals, recurrently infected tonsils and adenoids removed by surgery possessed a large number of plasma cells that stained with anti-IgE. Some germinal centers in these tissues also stained. Bronchial and peritoneal lymph nodes contained IgE-forming plasma cells as well as germinal centers. By contrast, IgE-forming cells were scarce in spleen and subcutaneous lymph nodes. The IgE-forming cells were detected in respiratory and gastrointestinal mucosa. In nasal mucosa, some of the plasma cells under epithelial cells stained with anti-IgE. Immunoglobulin E-forming cells were found in the bronchial mucosa especially around the mucous serous glands. In the stomach, small intestine, colon, and rectum, IgE-forming cells were observed in the lamina propria, especially around the crypts of Lieberkuhn. Lymphoid cells in bone marrow, lung tissues, and peripheral blood from nonatopic individuals did not stain with anti-IgE. The distribution of plasma cells and germinal centers that stained with anti-IgE is summarized in Table I, which also shows the distribution of IgE-forming cells in monkey tissues. It would appear that the IgE-forming cells predominate in the respiratory and gastrointestinal mucosa and in the regional lymph nodes. TABLE I DISTRIBUTION OF IGE-FORMING CELLSI N LYMPHOID TISSUES Monkeya
Humana
Lymphoid tissues Tonsil Adenoid Bronchial and peritoneal Subcutaneous lymph node Spleen Respiratory mucosa Gastrointestinal mucosa Lung Blood Bone marrow
Plasma cells
Germinal center
Plasma cclls
+ + - +++ + - + + +-+++ + - + + ++ ++ (+) 5 +-+ +-++ +-+ + + - -+ + -
Parentheses indicate negative in some cases; nd, not determined. Plus in Peyer’s patches.
Germinal center
++ (+)
-
+
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KIMISHIGE ISHIZAKA
Morphologically, most of the cells stained by anti-IgE had the characteristics of plasma cell with an eccentric nucleus, abundant cytoplasm, and a clearly defined Golgi apparatus. The others appeared to be lymphoid cells that are normally present in the germinal centers. The IgEforming cells were different from plasma cells forming other immunoglobulins such as IgA or IgG, and germinal centers stained by anti-IgE were distinct from those stained by either anti-IgA or anti-IgG. The distribution of IgE-forming cells in primate lymphoid tissues suggests that spleen and peripheral lymph nodes are not the source of IgE. This idea was supported by the finding of Mota (111) that splenectomy of rats prior to immunization with OA did not affect the IgE antibody level in their sera. Recently, we studied IgE-forming cells in spleens and mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis. Smears of spleen and mesenteric lymph node cells were treated with rabbit anti-rat IgE antibody and then with fluoresceinated antirabbit IgG (56). Both mesenteric lymph nodes and spleen contained IgEforming plasma cells, however, the number of IgE-forming cells in the spleen was significantly less than that observed in the mesenteric lymph nodes. After these rats were reinfected with the parasite to induce secondary IgE antibody responses, suspensions of their spleen cells, mesenteric lymph nodes, and parathymic lymph node cells were injected intracutaneously into Sprague-Dawley rats for passive sensitization. On the basis of the number of mononuclear cells, parathymic lymph nodes had the highest sensitizing activity, and mesenteric lymph node cells gave a lower PCA titer, whereas spleen cells failed to give the PCA reaction. In the rabbit, however, IgE-antibody forming cells were detected in the spleen. As described, an anti-DNP IgE antibody response was obtained by intraperitoneal injections of a minute dose of DNP-Asc included in alum. In order to see the distribution of IgE antibody-forming cells in these animals, cell suspensions were prepared from their spleens, mesenteric lymph nodes, popliteal lymph nodes and thymuses, and serial dilutions of the cell suspensions were injected intracutaneously into outbred normal rabbits. Challenge of the recipients with DNP-human serum albumin (HSA) at 48 hours after sensitization showed that spleen cells gave the highest PCA titers and mesenteric lymph node cells gave twoto four-fold lower titers, Neither thymus cells nor popliteal lymph node cells were capable of sensitizing homologous skin for a positive PCA reaction. Because the same cells killed by freezing and thawing failed to sensitize rabbit skin, it appears that IgE-forming cells released the antibody in the skin tissues. Kind and Macedo-Sobrinho ( 7 1 ) performed similar experiments using mouse lymphoid tissues. They injected cell suspensions of spleen, bone marrow, and lymph nodes from immunized
CELLULAR EVENTS I N THE ICE ANTIBODY RESPONSE
11
animals into rat skin to exclude possible proliferation of immunocompetent cells in the recipients. Their data showed that spleen cells gave the highest PCA titer when the same number of viable cells were used for passive sensitization. Taken collectively, it appears that distributions of IgE antibodyforming cells are different depending on the method and route of immunization. Thus, preferential distribution of IgE-forming cells in the respiratory and gastrointestinal tracts in the primate might result from frequent exposure of the lymphoid tissues to small doses of environmental antigens, which may be favorable for IgE synthesis. D. IMMUNOGLOBULIN E ANTIBODY RFSPONSEin Vitro In view of recent progress in tissue culture techniques and successful antibody formation in vitro (95, l05), attempts were made to form IgE antibody in cell culture. So far, the primary IgE antibody response has not been obtained in vitro; however, a secondary antihapten IgE antibody response was observed using mesenteric lymph node cells of rabbits that had been properly immunized for IgE antibody response (48).An experimental design for the culture system is shown in Fig. 3. Rabbits were immunized with 10 pg DNP-Asc included in 10 mg alum and boosted with the same dose of alum-absorbed antigen 4 weeks after the primary immunization. Animals giving a PCA titer of 1:160 or more against DNPHSA were sacrificed 2 weeks after the booster injection to obtain mesenteric lymph nodes. The cells were suspended in minimum essential medium, enriched with 20%fetal calf serum and 2 mM L-glutamine, and incubated for 24 hours at 37°C with homologous antigen. After being washed to remove free antigen, 1 ml of the cell suspension containing 1-2 x lo7 nucleated cells was cultured for 6 days by the method of Marbrook (95). By this procedure, primed mesenteric lymph node cells formed anti-DNP IgE antibody together with the IgG and IgM antibodies specific for the DNP group, whereas unstimulated cultures of the same cells failed to produce the antibody. Kinetic studies of antibody formation showed that both IgE and IgG antibodies became detectable after 3 to 4 days in culture, and their concentration in the culture fluid increased exponentially. It was also found that an optimal concentration of antigen for maximum antibody formation was comparable for the three immunoglobulin classes. Because the rabbits used in the experiments were outbred, the limitation of this system was that lymphocytes from two different animals could not be mixed in the culture. Nevertheless, this system was useful for studying cell requirements for a secondary IgE antibody response, and for analyzing the mechanisms involved in T cell-R cell collaboration.
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KIMISHIGE ISHIZAKA
Ill.
Immunological Factors Essential for IgE Antibody Responses
A. GENETICCONTROL OF IGE RESPONSES Immunization of inbred strain of mice with several protein antigens and a hapten-protein conjugate by Levine and Vaz (90) revealed that the reaginic antibody response is controlled by genes at a single autosoma1 locus, closely linked to the H-2 system on the 1X linkage group, which controls immune responses per se. They observed marked differences among the strains in the production of both IgGl and IgE antibodies, if minute doses (0.1 pg) of the immunogens were used for immunization. When large doses of the immunogens (100 pg) were used, such a strain difference became less apparent. As the result of systematic experiments in many inbred strains with three different antigens, i.e., benzyl penicilloyl-BGG ( BPO-RGG ), OA, and chicken ovomucoid in low doses, they found that the responsiveness of a given strain was antigen-specific. One strain was a good responder to one antigen but a poor responder to another antigen. When good and poor responders to a given antigen were grouped, responsiveness correlated with H-2 geno-
CELLULAR EVENTS IN THE IGE ANTIBODY RESPONSE
13
types. For example, strains A/He, CBA/J, C E / J and C3H, whose H-2 genotypes is either a or k, were good responders to BGG, BPO-BGG, and ovomucoid, but poor responders to OA. On the other hand, DBAI1 and C57BL/6 having type q or b were good responders to OA but poor responders to ovomucoid. Subsequent breeding experiments and immunization of strain pairs congenic at the H-2 locus established the genetic control. The experiments have shown that the F1 generation between A/He (H-2") and C57BL/6J ( H-2b) were good responders to ovomucoid. The backcross (F1 x poor responder) yielded approximately 50% responders, indicating that the immune response is controlled by a single gene. The results of the experiments by Levine and Vaz (90) as well as Vaz et al. (175, 177) are in agreement with previous studies by McDevitt and Chinitz (101) who established that the H-2-linked Ir-I gene controls the immune response to synthetic branched polypeptide (TG ) -AL. Comparisons between the two series of experiments indicated that responsiveness to ovomucoid correlated with that to (TG)-AL, whereas responsiveness to OA correlated with that to ( HG ) -AL. The experiments of Levine and Vaz (90) clearly showed that strain difference was related to carrier protein when mice were immunized with hapten-protein conjugates. As will be discussed later, both T and B cells are required for an IgE antibody response. Correlation of the immune response with carrier protein suggested that the genetic control observed in their experiments is on T cells. The mechanisms by which allele products of the 17-1 locus influence immune responsiveness are unknown; however, evidence has accumulated that they are expressed on T lymphocytes ( 100). Although the genes linked to the H-2 locus control a major portion of the responsiveness of the strains, there are some variations that cannot be due to the presence of a particular combination of H-2 alloantigen specificities. Thus, among H-2" mice, strain AKR/J responded better to both ovomucoid and OA than the other H-2rcstrains. Similarly, (C57BL/6 X DBA/B)F, (BDF1) mice gave better responses to OA than both parental strains and genetically related strains such as B10.D2. Vaz et d. (174) suggested that a portion of the heightened responsiveness of BDFl mice to OA might be due to genes present in the DBA/2 background. An entirely different kind of genetic factor found in mice uniquely controls the immune response of IgE antibody (87). When a large number of mouse strains were immunized with different antigens in various doses, all strains produced IgG antibody to one or another antigen. The IgE antibody responses were high in some strains but low or absent in others. For example, the SJL strain showed a poor IgE antibody response
14
KIMISHIGE ISHIZAKA
to six different antigens, but a reasonably high titer of IgG antibody to some of these antigens. In this situation, there was neither dose effect nor antigen specificity and the effect was on IgE antibody alone. Breeding experiments showed that the genetic control is by more than one locus and not linked to H-2. Thus, it appears that two different genetic controls operate in determining whether or not a given mouse strain will produce IgE antibody to a given antigen. Genetic control of the IgE antibody response was found in humans as well. In the typical case of pollen allergy, the individual became sensitized to extremely low doses of allergens within the pollen. Even if all allergens were extracted completely from pollen in the mucosa of the respiratory tract, a dose of a major allergen in ragweed pollen (antigen E ) or in grass pollen (Group I antigen) would be less than 1 pglpollen season for an individual in Baltimore (96). It is quite conceivable that the antibody response to such a minute dose of antigen is induced only in high responders. Indeed, most individuals who are not allergic to these pollen antigens lack both IgE and IgG antibodies, but almost all allergic individuals have antibodies of both immunoglobulin classes. Levine et al. (89) performed family studies in order to demonstrate Ir genes for ragweed antigen. Their results showed that members of a family having the HL-A1, HL-A8 (18) haplotype had intense immediate skin reactivity to antigen E, whereas none of the subjects having the other HLA haplotypes had immediate skin reactivity. Data for seven families, in which ragweed hay fever occurred in more than one member showed that 22 out of 26 of the members having the hay fever-associated haplotypes had ragweed hay fever and skin reactivity to antigen E. By contrast, non of the 11 members who had the other haplotypes had clinical hay fever. In the seven families, however, the hay fever-associated haplotype was different from one family to another. It is not known whether the antibody response to antigen E is controlled by only one or several different Zr genes. Nevertheless, the authors have shown that sensitivity to the other allergens has no relationship to the ragweed hay fever-associated haplotype, and suggested that the HLA-linked control of immune responsiveness to antigen E has antigenic specificity. As expected from their studies, population studies that were carried out by Marsh et al. (97) did not show a clear-cut relationship between a single HLA haplotype and the sensitivity to ragweed antigen. Genetic studies of Marsh et al. (97), however, showed a relationship between HLA haplotype and the sensitivity to a minor ragweed allergen, Ra-5, which has a relatively simple structure comprised of only 43 amino acids. Their approach was to examine skin sensitivity of ragweed-sensitive individuals to both antigen E and Ra-5, and to classify the patients into Ra-5-sensitive and Ra-5-insensitivegroups.
CELLULAR EVENTS IN THE I G E ANTIBODY RESPONSE
15
All patients were sentitive to antigen E, indicating that the patients received enough allergen to produce IgE antibody against antigen E. Statistical analysis of the two groups with respect to HLA types indicated that the control of IgE responsiveness to Ra-5 is determined by a single Zr gene that is intimately associated with genes controlling the expression of HL-A7 Creg antigens. Marsh et al. (98) studied the possibility that IgE synthesis may be genetically controlled. Statistically, allergic persons have high IgE levels compared with nonallergic individuals ( 3 2 ) , but some nonallergic persons have atypically high and some allergic individuals have atypically low IgE levels. The authors set up a cutoff point between high and low IgE at the level at which the combined percentages of such atypical subjects were minimized. This cutoff point was 95 & 5 international units ( I U ) / ml which was in excellent agreement with a value of 91 f 5 IU/ml calculated by replotting data of Gleich et al. (32). Thus, they analyzed three types of mating in 28 families: ( A ) low x low giving at least one high IgE, ( B ) low x high giving at least one high IgE, and ( C ) high x high. If one assumes recessive inheritance of high IgE, types A, B, and C would be RrX Rr, Rr X r r , and rr x rr, respectively. As predicted, all siblings of type C families had high IgE levels, and the incidences of high IgE level children in types A and B families were close to, but slightly higher than 1 standard deviation ( S D ) above the theoretically predicted values. From these studies they speculated that the inheritance of a high serum IgE level could be a simple Mendelian recessive trait and that there is no linkage between HLA haplotype and IgE level. An interesting observation in their studies is an implication that hay fever patients with high IgE levels have allergies to multiple allergens, whereas those with low IgE levels are usually sensitive to few allergens. Thus, in most allergic families, a gene regulating serum IgE level appears to mask the role played by hypothetical Zr genes linked to an HLA haplotype in controlling the expression of a specific IgE antibody response to different allergens. On might speculate that a genetic factor capable of controlling total IgE synthesis in humans may correspond to a factor in mice that uniquely controls the immune response of IgE antibody. It appears that IgE antibody responsiveness to low doses of specific allergens may be controlled by two distinct genetic factors in mice and humans.
B. ADJUVANT FOR IGE ANTIBODYRESPONSE The nature and dose of adjuvant are critical factors in the IgE antibody response to protein antigens. Injections of various doses of soluble antigen without adjuvant into rodents failed to give an IgE antibody response. In many different animal species, such as rabbits, guinea pigs, mice, and
16
KIMISHIGE ISHIZAKA
rats, alum is a better adjuvant than CFA for the IgE antibody response. Revoltella and Ovary (132) as well as others (147) have reported that the percentage of rabbits that produce reaginic antibodies is greater with alum than with CFA. It was also found that immunization of rabbits with protein antigens included in CFA failed to establish a memory for the IgE antibody response. Yet, repeated immunization with an adequate dose of alum-absorbed antigen elicited secondary IgE antibody responses in many of the animals immunized. In the rat, it is difficult to obtain a secondary IgE antibody response whichever adjuvant is employed; however, a primary IgE antibody response obtained with alum is higher than that obtained with CFA as an adjuvant. In certain inbred strains of mice, both alum and CFA are effective for inciting an IgE antibody response. It is generally observed, however, that the minimum dose of an appropriate antigen required for giving an IgE antibody response is less if alum is employed as an adjuvant. Using DNP conjugates of three different carriers, i.e., KLH, OA, and BGG, Hamaoka et al. ( 3 8 ) confirmed that alum-absorbed antigen favored IgE rather than IgG responses, whereas the reverse was true for antigen included in CFA. Another important adjuvant used for IgE antibody response is Bordetella pertussis vaccine. This adjuvant is effective in the rat (109) and mouse (112) but essentially useless in the rabbit. It is known that pertussis vaccine induces severe inflammation in the lung and lymphocytosis in peripheral blood (15). It is also known that the sensitivity of the rat and mouse to histamine increases after the administration of pertussis vaccine (113). Attempts were, therefore, made to isolate active substance with an adjuvant effect from the vaccine. Clausen et al. (16) obtained a saline extract from the B . pertussis organisms at an alkaline pH and showed that the extract exerted an adjuvant effect for IgE antibody production in the mouse. In their experiment, the active component was not distinguishable from the histamine-sensitizing factor. They have also shown that endotoxin from B. pertussis was not effective in stimulating IgE antibody response. Tada et al. (157) studied the effect of “lymphocytosis promoting factor,” which was obtained from a culture filtrate of B. pertussis, and showed that less than 1 pg of this component had a definite adjuvant effect on IgE antibody production in the rat. Because this substance induced lymphocytosis in the peripheral blood and depleted small lymphocytes in thymus-dependent areas of lymphoid tissues, the authors speculated that treatment with lymphocytosis-promoting factor caused a depletion of a certain subpopulation of T cells from the lymphoid tissue that regulate the IgE antibody response. Recent studies by Lehrer et al. (85) suggested that both histamine-sensitizing activity
CELLULAR EVENTS IN THE ICE ANTIBODY RESPONSE
17
and lymphocytosis-promoting activity were associated with the same molecule. Adjuvant other than alum and pertussis vaccine gave inconsistent results for IgE antibody responses. Clausen et al. (15) have reported that endotoxin ( lipolysaccharide, LPS ) from Salmonella minesota was not as effective as pertussis vaccine in stimulating an IgE antibody response in the mouse. Newberger et al. (114), however, showed that administration of LPS with DNP-Asc into irradiated recipient mice that received DNP-Asc-primed spleen cells enhanced antibody responses in both IgE and IgG classes. It appeared that CFA, LPS, and Poly A:U enhanced antibody responses of both IgE and IgG classes in the mouse and did not preferentially affect the IgE antibody response. The requirement of adjuvant for IgE antibody response in the mouse is limited to the primary response. If the mouse was primed with an appropriate antigen absorbed to alum for IgE antibody response, a booster injection of homologous antigen without adjuvant gave a secondary IgE antibody response (120). Within 2 weeks after the booster injection, the IgE antibody level declined to the same level as that observed before the booster injection. As described before, secondary IgE antibody responses were not observed in most rat strains. However, in the Hooded Lister strain, a secondary IgE antibody response was easily obtained by injecting antigen without adjuvant (60). The antibody titer increased within 4 days after the booster injection and rapidly declined by the seventh day. Recently, we primed the same strain with DNP-OA either with pertussis vaccine or with alum for primary antihapten antibody responses and gave a booster injection of homologous antigen without adjuvant. The magnitude of the primary IgE antibody response was higher with alum than with pertussis vaccine. By contrast, the secondary antihapten IgE antibody response was readily observed when pertussis vaccine was employed for the primary immunization. When alum was employed for priming, essentially no secondary IgE antibody response was observed. I t would appear that pertussis vaccine is a better adjuvant than alum to establish immunological memory for an IgE antibody response in the rat.
C. NATUREAND DOSEOF ANTIGEN In all animal experiments, IgE antibody was formed against T-dependent antigens. Attempts to produce IgE antibody in mice by the immunization with T-independent antigens were unsuccessful. TO date, immunization of mice with Salmonella bacilli ( 143), pneumococcus polysaccharide SIII, or the DNP-derivative of an acidic copolymer of D-tyro-
18
KIMISHIGE ISHIZAKA
sine, glutamine, and lysine ( DNP-D-TGluL) failed to induce IgE antibody responses ( 121 ). Although some atopic individuals were sensitive to some T-independent antigens such as dextran (63), there was no evidence that the T-independent antigens were actually immunogens. It is quite possible that such T-independent antigens were in the form of complexes with a carrier protein in the natural state and that the complex was immunogenic with respect to the IgE antibody response. This possibility is conceivable in view of the work by Paul et al. (128), who demonstrated an anti-SIII antibody response in the rabbit to immunization with SIIIBGG conjugates. In this system, BGG served as a carrier to which helper cells were directed. Because hay fever patients have IgE antibodies against a variety of allergens, and helminth infection induces IgE antibody responses in many animal species, pollen allergens as well as Ascaris extract were frequently used to induce IgE antibody responses in experimental animals. It has been shown that both ragweed antigen E and extract of Ascaris suum produced IgE antibody responses in rabbits (48, 76, 150), rats ( 139, 149), and mice ( 5 2 ) . Strejan et al. (151) compared the immunogenicity of Asc with other protein antigens, such as OA, KLH, and bovine serum albumin (BSA), for the IgE antibody response in the rat, and found that Asc was superior to the other antigens. Purified Ascaris antigen ( Asc-1) (42) was a strong immunogen for an IgE antibody response in the rat, whereas BSA and BGG were unable to produce reaginic antibody. Under the same immunization regimen, OA and KLH were less immunogenic than Ascaris antigen but better than BSA for the formation of IgE antibody. It should be noted that all of these protein antigens were capable of producing large amounts of IgG antibody. Potent antigens for IgE antibody formation are useful as carrier proteins for the formation of antihapten IgE antibody. Strejan and Marsh ( 152) compared various DNP-coupled protein antigens for their ability to induce anti-DNP IgE antibody in the rat, and found that DNP coupled to Ascaris extract was a potent immunogen and superior to DNP conjugates with KLH or BGG. Similar results were obtained in the rabbit as well. Both DNP-Asc and DNP coupled to ragweed Fraction D (Rag), in which antigen E is the major immunogen, are superior to DNP-BGG or DNP-KLH for the formation of antihapten IgE antibody (76). As both Asc and ragweed antigen ( Fraction D and purified antigen E ) themselves are excellent immunogens for IgE antibody response in the rabbit, one may generalize that allergens serve as excellent carriers for producing antihapten IgE antibody responses. The peculiar properties of allergens stimulating IgE antibody formation suggest that they have physicochemical properties in common; however,
CELLULAR EVENTS I N THE IGE ANTIBODY RESPONSE
19
no common structure for allergen has been established. On the other hand, an extensive study on IgE antibody formation in inbred strains of mice suggests that the common immunological property of potent allergens is high immunogenicity. Levine and Vaz (90) showed that almost all inbred strains of mice gave a transient IgE antibody response to protein antigens or hapten-protein conjugates when they were immunized with 50 to 100 pg antigen included in alum, but a booster injection of the same dose of antigen failed to give a secondary IgE antibody response. If the same strains were immunized with a low dose (0.1-1 p g ) of antigen in responder mice to OA, such as Sw-55 or DBA/1, the IgE antibody responses, and IgE antibody titers increased upon booster injections. In high responder mice to OA, such as Sw-55 or DBA/l, the IgE antibody response to a minute dose of the antigen was comparable to or higher than that obtained by immunization with allergens such as Asc or ragweed antigen. In these animals, DNP-OA and DNP-KLH induced a higher antihapten IgE antibody response than did DNP-Asc. Thus, IgE antibody responses of high responder strains of mice to certain antigens or haptencarrier conjugates were similar to those of individual outbred animals to potent allergens. Another important factor in the IgE antibody response is the dose of antigen used for immunization. Strejan et a2. (151) immunized rats with different doses of alum-absorbed purified Asc-1 every 3 to 4 weeks and showed that the secondary IgE antibody response was obtained by a l-pg dose rather than by a 10-pg dose. Similarly, Jarrett et al. (60) reported that 1-10 pg of OA with pertussis vaccine was optimal for establishing memory for the IgE antibody response. Rats immunized with 100 pg to 1 mg OA gave the antibody response, but secondary IgE antibody responses of these animals were lower than those observed in rats primed with 0.1-1 pg OA. The dose dependence of IgE antibody formation was also shown in the rabbit by Revoltella and Ovary (132). They found that a low dose of antigen in alum favored reagin production. As already described, Levine and Vaz (90) showed that in high-responder strain mice a low dose of antigen gave higher and longer-lasting IgE antibody responses than did a high dose. It is known that a high dose of antigen is required for producing antibody responses in poor responder mice. One might speculate that the failure of poor responder mice to give persistent IgE antibody responses may be owing to the fact that optimal conditions for the IgE antibody response, i.e., a low dose of antigen with a relatively high dose of adjuvant cannot induce an immune response in a low responder. The dose of antigen is particularly important for the induction of an antihapten antibody response ( 51). When DBA/ 1mice were immunized
20
KIMISHICE ISHIZAKA
with DNP-OA included in alum, a minimum immunogenic dose (0.05 pg) of the antigen gave a persistent antihapten IgE antibody response. If the antigen dose was increased from 0.2 to 1 p g , the anti-DNP antibody titer reached a maximum soon afterward and then declined between 2 to 3 weeks. An interesting observation was that the anticarrier (OA) IgE antibody response was persistent even with 0.2-1 p g of DNP-OA (51). The reasons for the discrepancy between the time course of an antihapten antibody response and an anticarrier antibody response are unknown. Because carrier-specific helper cells must be common to both anti-DNP and anti-OA antibody responses, the decline of antihapten antibody titers may not simply be ascribed to some possible changes in helper cell population in the course of the response. IV. Cellular Basis of IgE Antibody Responses
A. REQUIREMENTFOR T
AND
B LYMPHOCYTES
It has been established that the collaboration of two distinct types of lymphocytes, i.e., bone marrow or bursa-derived ( B ) lymphocytes and thymus-derived ( T ) lymphocytes, is essential for induction of an antibody response by mice to certain antigens, such as sheep erythrocytes and proteins (14, 104). An analogous cooperation of two lymphocyte cell lines was demonstrated in the mouse (106) as well as in the guinea pig and rabbit (69) in the formation of antihapten antibodies. In the haptenspecific antibody response, T cells are usually primed with the determinants present on the carrier, whereas B cells are precursors of antibodyforming cells and have the same specificity as the antibody formed by their progeny. Naturally, a question arose as to whether T cells are required for the IgE antibody response. As will be described below, several attempts were made to establish the requirement of T and B cells for the IgE antibody response. Okumura and Tada (122) reported that rats thymectomized within 24 hours of birth failed to produce IgG and IgE antibodies upon subsequent immunization with DNP-Asc. Supplementation of the neonatally thymectomized rats with normal thymocytes restored the ability to produce IgE antibody to the antigen. Similarly, Michael and Bernstein (103) reported that congenitally athymic (nulnu) mice were unable to produce IgE antibody against OA, but with supplementation by thymocytes from nu/ mice the nude mice were able to produce the antibody. In the antihapten IgG and IgM antibody responses, Mitchison (106) and Katz et al. (69) have shown that priming of mice or guinea pigs with free carrier enhanced the primary antihapten antibody response to
+
CELLULAR EVENTS I N THE ICE ANTIBODY RESPONSE
21
hapten-homologous carrier conjugate. This phenomenon, i.e., carrier effect, is due to priming of carrier-specific T cells. The same principle was reproduced in the IgE antibody response. For example, immunization of rats with Asc extract and pertussis vaccine on day 0, followed by an intramuscular iiijectioii of DNP-Asc on day 5, resulted in the formation of IgE antibody to DNP-Asc (156). Similarly, priming of rabbits with ASC extract included in alum 4 weeks prior to the immunization with DNP-Asc induced both IgE and IgG anti-DNP antibody responses (75). Under the experimental condition employed, a single injection of DNPAsc in alum was insufficient to induce an antihapten IgE antibody response. Although IgE antibody responses in the rabbit and rat were inconsistent, an enhancing effect of carrier priming was demonstrated in DBA/1 mice that were primed with a subimmunogenic dose (0.02-0.05 p g ) of OA included in CFA (51).The animals did not have a detectable amount of either IgE or IgG anti-OA antibody; but priming definitely enhanced the anti-DNP antibody response of both IgE and IgG classes to a subsequent immunization 2 weeks later with DNP-OA. Thus, a carrier effect on the IgE antibody response has been demonstrated in three animal species. However, the dose of carrier, the adjuvant vehicle employed for priming, and/ or the intervals between carrier-priming and immunization with hapten-carrier conjugate were critical for the demonstration of a carrier effect in the IgE antibody response. Priming with an immunogenic dose of OA, which enhanced the antihapten IgG antibody response in the mouse, suppressed the IgE antibody response to DNP-OA. Evidence was obtained for the participation of carrier-specific helper cells in the secondary IgE antibody response by rabbit mesenteric lymph node cells in uitro (82). In this experiment, rabbits were primed with DNP-Asc in alum and some of the animals received supplemental immunization of partially purified ragweed pollen extract ( Rag) [Fraction D by King et al. (72)]. Four weeks after the priming immunization, all animals received a booster injection of DNP-Asc in alum and were sacrificed 2 weeks later. Mesenteric lymph node cells were stimulated by either DNP-Asc or DNP-Rag for the antibody response. As shown in Table 11, the lymph node cells of rabbits that did not receive a supplemental immunization formed anti-DNP IgE antibody upon stimulation with the homologous antigen ( DNP-Asc) but failed to form the antibody upon stimulation with DNP-Rag. On the other hand, lymph node cells from the animals that received a supplemental immunization of alumabsorbed Rag formed both IgE and IgG anti-DNP antibodies upon stimulation with either DNP-Asc or DNP-Rag. Comparisolls between the two groups indicated that the DNP-specific B cells raised by the
22
KIMISHIGE ISHIZAKA
TABLE I1 HELPER FUNCTION OF CARRIER-SPECIFIC CELLS A G A I N S T PRIMARY A N D SECONDARY CARRIERS FOR IGE A N D IGE ANTIBODY RESPONSES Anti-DNPb Supplemental immunization0 None Ragweed Ag in alum Ragweed Ag in CFA
Antigen in vitro None IINP-ASC DNP-Rag None IINP-Asc DNP-Rag None DNP-ASC DNP-Rag
IgG bglml) 0.29 39.0 0.35 0.32 18.5 23.5 0.40
IgE (PCA)
40.0
venom, but apamin was not active. A detailed inspection of thc allergenic activity data showed that the relative activities of phospholipasc to hyaluronidase or melittin are different in different individuals. This variability indicates that allergcnicity is dependent on the nature of the allergens as well as on the individual responses to the allergen. Other
92
T. P. KING
reports have also indicated that phospholipase is the major bee venom allergen (Hoffman and Shipman, 1975; Ilea et al., 1975; Sobotka et al., 1975) and that melittin is a weak allergen (Mackler, 1972). Chemical modifications of bee venom phospholipase (King et al., 1976) showed that its allergenic determinants depend on the charge, the conformation, and the amino acid sequence of the molecule, just as has been indicated in the preceding sections on ragweed and ryegrass pollen allergens. The chemical modifications of phospholipase include succinylation of 8 of its 11 amino groups, cyanogen bromide cleavage of its three methionyl bonds, or reduction and carboxymethylation of its four disulfide bonds. All the derivatives showed reduced allergenic activities. The reduced and carboxymethylated enzyme showed a large decrease in its allergenic activity, about that of the native enzyme. The relative activity of the succinylated enzyme compared to that of the native enzyme covered a range of 0.003 to 0.7 in 5 allergic persons tested; the activity of the cyanogen bromide-cleaved enzyme also showed a wide range, The widely differing decreases in activities of these two modified enzymes in different persons tested is indicative of the fact that the test subjects recognize different antigenic determinants of the native enzyme; that is, the specificities of their IgEs differ so that they will show different activities with different modified enzymes. Melittin is immunogenic, but apamin is not. Melittin with a formula weight of 2840 is strongly associated in solution to form a complex with a molecular weight of about 12,000, but apamin with a formula weight of 2038 does not associate. This association property may be of importance as to why one is an immunogen and the other is not. IV. General Observations on Allergens
Under natural conditions of exposure, especially inhalants, atopic persons become sensitized on repeated challenges with extremely small doses of allergens. As an example, the estimated annual dosage of ragweed antigen E for a person is in the microgram range (Marsh, 1975). Prior to the development of RAST, the presence of allergen-specific IgEs could be detected only by biological tests because of their very low concentrations. The very high sensitivity of biological tests, coupled with the very low concentrations of allergens required for sensitization and of the specific IgEs formed, led a number of workers to consider the possibility of allergens being different from antigens. For example, Berrens (1974) has suggested that the allergenic activity of a molecule is dependent primarily on the proportion of N-glycosidic protein-sugar residues and their resulting decomposition products, and Stanworth
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(1973) has stressed the importance of dimeric structure for protein allergens. However, chemical characterizations of allergens and immunological studies in the past decade have provided solid indic at'lolls that allergens are not different from antigens. Some of these studies will be reviewed in this section. The inhalants, foods, or insect venoms are all multiallergen systems as indicated in the preceding section. Each source has one or more major allergens to which the majority (>go%) of allergic persons are highly reactive and several minor allergens to which a smaller group (10-50%) of persons is also highly reactive. People show varying responses to the different allergens. This individual variation indicates that the allergenicity depends both on the chemical nature of allergen and on the response of the host. The major allergens from the inhalants and foods are all acidic proteins in the molecular weight range of 20,000 to 40,000 daltons with PIS in the range of 4 to 6. The minor allergens can be acidic or basic proteins (polypeptides), with molecular weights as large as 65,000 daltons for serum albumin and as small as 2800 for the bee venom allergen, melittin. The major allergens from bee venom, hyaluronidase and phospholipase, are exceptions, being basic proteins. However, the route of sensitization to bee venom is clearly different from that for inhalants or foods. All the known allergens from the inhalants and foods are globular proteins, as indicated by their chromatographic properties on porous gels and by their diffusion coefficients. Some allergens contain carbohydrates and some do not. Some allergens are proteins of a single polypeptide chain (ragweed antigens Ra3, 4, and 5; codfish allergen M; serum albumins) and some are proteins of two nonidentical polypeptide chains (ragweed antigen E ) or of two identical polypeptide chains (p-lactoglobulin). Some allergens (ragweed antigen E and mite allergen) in their native state are strongly resistant to proteolytic digestion, but others like grass and tree pollen allergens are readily digested. The amino acid compositions of most of the allergens described in Section I11 are known and they do not show any unusual chemical features. The complete amino acid sequences of several allergens are known: ragweed antigen Ra5, codfish allergen M, p-lactoglobulin, bovine serum albumin, and bee venom phospholipase and melittin. Simple inspection of these amino acid sequences does not reveal any obvious unusual structural features. Melittin is an exception, having an unusual distribution of the hydrophobic and hydrophilic amino acid residues. We cannot state unequivocally that allergens do not contain special features, since we do not know what combinations of amino acid residues participate in forming the antigenic determinants.
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The dominant antigenic determinants of the major allergens of ragweed and ryegrass pollens, codfish, and bee venom are dependent on both the primary structure and the conformation of the molecule, a property in common with other globular protein antigens. This was shown by the loss of their allergenic activities on denaturation, chemical modification, or proteolysis. Attempts to isolate haptenic peptides from proteolytic digests of modified ragweed antigen E or rye Group I antigen were unsuccessful. However, there are reports claiming the isolation of low molecular weight haptenic materials from timothy pollen ( Malley and Harris, 1967; Malley et nl., 1973) and from ragweed pollen ( Attalah and Sehon, 1969). Studies on the immunogenicities of the major allergens of ragweed and ryegrass pollens showed that the pollen allergens are no more potent than other protein antigens in eliciting IgG and IgE responses in mice ( Chang and Marsh, 1974). In addition to the “natural” allergens described in Section 111, many other diverse proteins are known to stimulate IgE responses in atopic or normal man, for example, bovine pancreatic ribonuclease ( Salvaggio et d.,1964), diphtheria and tetanus toxoids (Kuhns, 1962), insulins (Berson and Yallow, 1963), keyhole limpet hemocyanin ( Salvaggio et al., 1969), and the laundry detergent additives, proteases and amylases from Bacillus subtilis (Pepys et al., 1969). The biological functions of some of these protein allergens are known, and they are quite varied including enzymes, hormones, and blood ‘transport proteins. The widely different biological functions as well as origins of these protein allergens also argue for the unlikeliness that they may have certain structural features particularly suited for the induction of IgE production, such as the presence of hydrophobic surfaces of the molecule for attachment to immunocompetent cells. Most allergens are absorbed on inhalation or ingestion, and the allergens have to be absorbed through the respiratory or gastrointestinal tracts. These are the very sites where the IgE-forming plasma cells are known to be predominantly located (Tada and Ishizaka, 1970). Obviously there will be an upper size limit as to the solutes that can be rapidly absorbed through the mucosal membranes. The size of albumin molecule of about 65,000 daltons may well represent the upper limit of permeability of mucosal membrane, as serum albumins are the largest known allergens. There is one known exception to this size limit, namely keyhole limpet hemocyanin, an associating protein with estimated molecular weight of about 1 X lo6 daltons at pH 8.6 (Barte and Campbell, 1959). Intranasal immunization of normal or atopic persons with this protein as an aerosol led to the production of specific IgE ( Salvaggio et nl., 1969). Hemocyanins are known to be composed of associating subunits in the
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size range of about 30,000 daltons (Pickett et al., 1966), so it is possible that the dissociated subunits are being absorbed. Studies have shown that atopic persons do not differ from normal persons by having greater permeability of the nasal mucosal membrane ( Kontou-Karakitsos et al., 1975). The immunogenic potency of a protein is dependent on its molecular complexity, its degree of foreignness to the host ( Landsteiner, 1936; Crumpton, 1974), and the genetic makeup of the immunized host (Benacerraf and McDevitt, 1972). Complex proteins with a large number of antigenic determinants are more likely to meet the genetic requirements of the immunized host than small proteins or peptides with fewer antigenic determinants. For example, keyhole limpet hemocyanin is a larger and more foreign protein to man than bovine pancreatic ribonuclease, and hemocyanin induced specific IgE formation in atopic and normal persons more frequently and readily than ribonuclease did (Salvaggio et al., 1964,1969); with either immunogen a greater percentage of atopic persons developed specific IgE than the percentage of normal persons. This combination of permeability and immunogenicity requirements may very well be the explanation as to why proteins of molecular weights of 20,000 to 40,000 are better allergens than proteins of other sizes. They are not too large, so that they cannot be readily absorbed through the mucosal membranes of atopic individuals. Yet they are not too small so that they can be sufficiently complex to be good immunogens. Studies on the effects of inbred strain and H-2 type, antigen, and antigen dose on immune responsiveness in the mouse (Levine and Vaz, 1970; Vaz and Levine, 1970) have provided several findings applicable to human atopy. They suggest the following: ( a ) Atopic persons differ from normal persons in their ease to respond immunologically to minute doses of antigen; ( 1 ) ) low-dose immunization favors persistent and boosterable IgE production or IgG production, and the reverse is true for high-dose immunization; and ( c ) both IgE and IgG responses to specific antigens are under the control of histocompatibility linked immune response genes. The last area has prompted a number of studies to establish the association of a specific HL-A type and IgE and IgG responses to a specific allergen. To date such studies have been carried out with the following purified allergens: ragweed antigen E (Levine et al., 1972; Blumenthal et al., 1974; Bias and Marsh, 1975), ragweed antigen Ra5 (Marsh et al., 1973) and rye Group I antigen ( Marsh, 1974). The presence of an IgE-regulating gene in man has been postulated (Hamburger et al., 1973; Marsh et al., 1974), and this gene in some way regulates the serum level of IgE. The IgE-regulating gene is believed to
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play an important role together with the immune response gene in the expression of atopic allergy in man ( Marsh et al., 1974). V. Uses of Purified Allergens
One important use for purified allergens is in the standardization of allergen extracts. These extracts are important reagents for the diagnosis and treatment of a number of allergic disorders. Since the beginning of clinical allergy practice, extracts have been standardized on the basis of their protein nitrogen contents, and they are not sbandardized on the basis of their biological activities. It is not uncommon to have two commercial extracts having the same protein nitrogen content but differing markedly in their biological activities (Baer et al., 1970). The difficulty in standardizing allergen extracts on the basis of their biological activities lies in the variability of individual responses to allergens. This problem can be partly overcome by doing RAST or RAST inhibition with a reference pool of reaginic sera. In cases where the major allergens( s ) are known and well-characterized, a simpler approach will be to standardize the extracts on the basis of their allergen contents. The allergen contents can be established by a number of simple methods independent of human reaginic sera. Such methods may include various forms of simple or electroimmunodiffusion using animal antisera specific for the allergens or, in cases where known, enzyme assays. Two other related uses of purified #allergensare as reagents for studying the immunological mechanism of immunotherapy and for the development of chemically modified derivatives better sui'ted for immunotherapy. Immunotherapy is one well-accepted form of treatment of allergic diseases since its iniitial description by Noon in 1911. The effectiveness of properly manlaged immunotherapy for hay fever has been reported in several studies, but symptom relief is not complete for most patients (Norman and Lichtenstein, 1971; Lichtenstein et al., 1974). On immunotherapy there is usually an initial rise in the serum level of allergenspecific IgE followed by a slow decrease, but the serum level of specific IgG continues to rise during therapy, usually reaching a plateau. The best clinical improvement has been associated with high doses of allergen extracts, high serum levels of allergen-specific IgG, and lowered levels of specific IgE, but it is not clear whether the clinical improvement is a consequence of changes in IgG, IgE, or both. Another serological change for hay fever patients is that immunotherapy prevents the seasonal rise in specific IgE following each pollinating season (reviewed by Irons et al., 1975). The immunological mechanism of these changes in IgG and IgE levels on immunotherapy is under study in animal models by several
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laboratories. Some workers have suggested these changes to be a form of feedback suppression of IgE antibody production by IgG antibody (Tada and Okumura, 1971). Others have indicated these changes to be a form of immunological tolerance specific for the IgE class antibody (Ishizaka and Ishizaka, 1973; Gleich and Yunginger, 1974). Whatever the mechanism may be, the results of immunotherapy indicate that the best symptom relief is obtained on treatment with high doses of allergens. The only difficulty is that many persons cannot tolerate large doses of allergens because of allergic reactions. Investigators have sought to circumvent this problem by using adjuvants to give slow release of allergens into tissues and to enhance their immunogenicity. The repository treatment using allergen extracts incorporated as a mineral oil emulsion (Loveless, 1957) was used, but this practice was discontinued because of undesirable side effects, Alum-precipitated allergen extract is also used, but the amount that can be used safely is not significantly greater than that for the aqueous allergen extract ( Norman et al., 1972). For ragweed pollen extract, the mean total amount of pollen proteins to be injected into a person after a season’s treatment is on the order of 1.5 mg (or 25,000 PNU) ; less commonly, the total dose may be as high as 15 mg of pollen proteins for some individuals ( Melam et al., 1971; Yunginger and Gleich, 1973). Another approach, which has been tried (Stull et al., 1940; Fuchs and Strauss, 1959; Naterman, 1957, 1965), is to use chemically modified allergens. Marsh et al. (1970b) had proposed the term allergoids for modified allergens having reduced allergenic activities but retaining the immunogenic properties of the native molecule. The desired immunogenic property of the modified allergen may involve not only the induction of allergen-specific IgG antibody production but also IgE class-specific tolerance. However, this may not be the only important property, as it is indicated in the preceding paragraph that immunotherapy may be a form of IgE class-specific tolerance. The term allergoid was presumably chosen by analogy to the reduction of toxicity of a toxin to yield immunogenic tosoid. I t is appropriate to note here the difference in the toxic actions of allergens and toxins. The action of a toxin such as diphtheria or cholera depends on two welldefined reactive sites, one having enzymatic activity necessary for toxicity and the other for interaction with cell receptor (Gill et al., 1973; van Heyningen, 1974). Chemical modification of the single enzymatically active site of a toxin is sufficient for its conversion to toxoid. An allergen with molecular weight of about 40,000 daltons will have at least four or five antigenic determinants, by analogy to other known protein antigens, and its toxic action depends on the interaction of at least two of these antigenic determinants with cell-bound IgE ( Ishizaka, 1973 ) . Therefore,
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for the conversion of an allergen to an allergoid, chemical modifications of different combinations of antigenic determinants of an allergen are required so that the resulting allergoid is a mixture of derivatives. In the idealized case, each derivabive retains one of the determinants of the native allergen, and the derivatives together retain all the specificities of the native allergen. Marsh prepared allergoids of rye Group I antigen by formaldehyde treatment under conditions similar to those used for preparing bacterial toxoids (Marsh et al., 1970b). The product so prepared was a mixture of components of different molecular sizes, and the major components (about 70%) were dimers and trimers of the rye Group I antigen (Marsh, 1971) , The chemical reactions involved mainly inter- and intramolecular cross-linking of the €-amino groups of the protein through methylene bridges. The allergenic activity of the resulting allergoid was 10'-109 times less than that of the native antigen, and the large range of activity decrease is a reflection of the variability of allergic individuals in recognizing different antigenic determinants. The rye Group Iallergoid was immunogenic in normal persons in inducing IgG production specific for the native allergen. Following the pilot studies, allergoids of whole ragweed and grass pollen extracts were also prepared, and these allergoids were 102-104 times less allergenic than the unmodified extracts. Controlled studies on immunotherapies with the whole ragweed extract and the ragweed allergoid indicated that the allergoid was somewhat more effective clinically that the extract. The average dose of ragweed allergoid used was about 40 times larger than that of the extract, and the higher dose of allergoid was more effective than the limited dose of extract in inducing allergen-specific IgG production. However, complete symptom relief was still not attained ( Norman et al., 1975). Patterson and co-workers ( 1973a,b) have modified ragweed antigen E by treatment with glutaraldehyde. The resulting cross-linked preparation was separated into high and low molecular weight fractions with apparent molecular weight ranges of 0.2 to 4 million and of 4 to 20 million daltons, respectively. There was a greater reduction of the allergenic activity of the high molecular weight fraction than of the low molecular weight fraction in accord with their molecular size differences. Both fractions were as immunogenic in rabbits as the native antigen in inducing specific IgG antibody production. Interestingly, they differed in their induction of IgE antibody production: The native antigen produced a tnansient IgE response; the low molecular weight fraction gave an IgE response weaker than the native antigen, but the high molecular weight fradion gave a stronger and longer lasting IgE response than that of the native antigen ( Patterson and Suszko, 1974 ).
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The allergenicity of a molecule can also be reduced by attaching to it large molecular weight groups. The bulky large groups can prevent or reduce the accessibility of antigenic determinants for combination with antibodies, as illustrated with the following two derivatives of ragweed antigen E. Antigen E attached to 2-5 molecules of devtran of molecular weight of about 20,000 was prepared by reductive coupling with periodate-oxidized dextran (King et al., 1975). Also, antigen E attached to about 8 molecules of methovypolyethylene glycol, of molecular weight of about 2,000, was prepared by reaction with a monochlorotriazine derivative of methoxypolyethylene glycol ( T. P. King, unpublished work; Abuchowski et al., 1974). On a molar basis, the allergenic activity of the dextran and the methoxJplyethylene glycol derivatives of antigen E were, respectively, about one-eighth and one-fiftieth of the native antigen. The methoxypolyethylene glycol antigen E did not form immune precipitates with sheep anttiantigen E sera, whereas the dextran-antigen E still did. The polyethylene glycol-antigen E has all the antigenic determinants of antigen E as shown by its inhibition of hemagglutination of antigen E-coated cells with anti-E sera, and, on a molar basis, its inhibitory activity was one-fortieth of the native antigen. Rabbits immunized wicth the polyethylene glycol derivative gave a lower antibody titer (about one-sixth ) than those with the native antigen. Although the polyethylene glycoliantigen E was less immunogenic than antigen E, it was just as effective as antigen E in priming the rabbiits for a secondlary response. The immunogenic property of the polyethylene glycol-antigen E for IgE response has yet to be studied. The modifications described in this paragraph differ from the formaldehyde and glutanaldehyde treatments in one significant aspect, namely that the dextran, or the polyethylene glycol, derivative is more homogeneous in size than are the formaldehydeor glutaraldehyde-treated allergens and that it does not contain large molecular weight aggregates. This chemical difference of these derivatives may be of importance for their capacities to alter IgE responses in man or animal, and this is indicated by the work of Patterson and Suszko ( 1974 ) on the immunogenic property of glutaraldehyde-treated antigen E. If the clinical effcct of immunotherapy is a result of depression of the IgE antibody response, then denatured or extensively modified allergens can be useful therapeutic reagents. This suggestion results from the following observations: As indicated in Section 111, the urea-denatured antigen E and its dissociated polypeptide chains are essentially inactive as allergens, that is, they have less than 0.0001 of the originla1 activity. Studies in mice indicated that denatured and native antigen E do not share common B-cell determinants, but they do share common T-cell determinants (Ishizaka et al., 1974, 1975). Mice immunized with the
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denatured antigen E did not produce IgG or IgE specific for the native antigen, but the denatured antigen E primed the animals for a secondary response to the native antigen E. More interestingly, repeated injections of the denatured antigen E into antigen E-primed mice depressed the ongoing IgE antibody response, whereas injections of the same dose of native antigen E showed little or no depression. The animals treated with the denatured antigen showed only a small rise in their IgG antibody specific for the native antigen E, whereas those treated with the native antigen E showed a very large rise. These findings on the treatment of mice with the denatured antigen E are believed to result from a suppression of the helper T-cell function as well as from decreases of antigen E-specific IgG and IgE B-cell populations ( Takatsu et al., 1976). These observations on the denatured antigen E are of general applicability and they may prove to be of practical importance. Another example on the use of chemically modified protein antigen to depress an ongoing IgE response specific for the native antigen in the mouse model has been reported (Bach and Brashler, 1975). The protein antigen studied was ovalbumin, and chemical modifications of the lysyl, the arginyl, and tryptophan$ residues were carried out. I t was found that the primary structural requirement for efficacy of the modified antigen was reduced antigenicity to prevent anaphylaxis. Since the antigenicity of globular proteins depends primarily on the conformation of the molecule, the findings can be taken to indicate that the more altered the antigen is conformationally, the better it is for suppression of the IgE response. VI. Concluding Remarks
Many different proteins from animal, plant, or microbial sources can be allergens in man. The known allergens are usually in the molecular size range of 20,000 to 40,000 daltons, and they do not have any distinguishing chemical features. The purified allergens are useful reagents as they can help us to understand better the heightened responsiveness of susceptible persons to minute doses of immunizing allergens for IgE and IgG productions. Continued chemical and immunological studies of the purified allergens will indicate the types of chemical modification required to prepare derivatives useful for altering the immune responsiveness of allergic man. The findings will be of general applicability, as most environmental allergens are proteins. ACKNOWEDGMENTS I would like to thank Dr. David Marsh for sending me a copy of his review article on allergens prior to its publication. My own studies on allergens have been supported in part by a grant from the National Institutes of Health (AI-08445).
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Ishizaka, K., Okudaira, H., and King, T. P. (1975). J. Zmmunol. 114, 110. Johnson, P., and Marsh, D. G. (1965). Eur. Polym. J. 1, 63. Johnson, P., and Marsh, D. G. (1966a). Immunochemistry 3, 91. Johnson, P., and Marsh, D. G. (1966b). Immunochemistry 3, 101. Kabat, E. A., Turino, C. M., Tarrow, A. B., and Maurer, P. H. (1957). J. Clin. Inoest. 36, 1160. Katz, D. H., and Benacerraf, B. (1972). Adu. Zmmrcnol. 15, 1. Kawai, T., Marsh, D. G., Lichtenstein, L. M., and Norman, P. S. (1972). J. Allergy Clin. Immunol. 50, 117. King, T. P. (1972). Biochemistry 11, 367. King, T. P., and Norman, P. S. (1962). Biochemistry 1, 709. King, T. P., Norman, P. S., and Connell, J. T. (1964). Biochemistry 3, 458. King, T. P., Norman, P. S., and Lichtenstein, L. M. (1967a). Biochemistry 6, 1992. King, T. P., Norman, P. S., and Lichtenstein, L. M. (1967b). Ann. Allergy 25, 541. King, T. P., Norman, P. S., and Tao, N. (1974). Immunochemistry 11, 83. King, T. P., Kochoumian, L., Ishizaka, K., Lichtenstein, L. M., and Norman, P. S. (1975). Arch. Biochem. Biophys. 169, 464. King, T. P., Sobotka, A. K., Kochoumian, L., and Lichtenstein, L. M. (1976). Arch. Biochem. Biophys. 172, 661. Kontou-Karakitsos, K., Salvaggio, J. E., and Mathews, K. P. (1975). J . Allergy Clin. Zmmunol. 55, 241. Kuhns, W . J. (1962). J. Zmmunol. 89, 652. Landsteiner, K. ( 1936). “The Specificity of Serological Reactions” (rev. ed. Dover Publications, New York, 1962 ). Leskowitz, S., and Lowell, F. C. (1961). J . Allergy 32, 152. Levine, B. B. (1965). J . Zmmunol. 94, 111. Levine, B. B. (1966). N . Engl. J. Med. 275, 1115. Levine, B. B., and Vaz, N. M. (1970). Int. Arch. Allergy Appl. Immunol. 39, 156. Levine, B. B., Stember, R. H., and Jotino, M. (1972). Science 178, 1201. Levy, D. A. ( 1975). Int. Arch. Allergy Appl. Immunol. 49,219. Lichtenstein, L. M. (1974). Allergol., Proc. Int. Congr., 8th, 1973 pp. 294-305. Lichtenstein, L. M., King, T. P., and Osler, A. G. (1966). J . Allergy 38, 174. Lichtenstein, L. M., Marsh, D. G., and Campbell, D. H. (1969). J . Allergy 44, 307. Lichtenstein, L. M., Roebber, M., and Goodfriend, L. (1973). J . Allergy Clin. Immunol. 51, 285. Lichtenstein, L. M., Norman, P. S., and Ishizaka, K. (1974). Allergol., Proc. Int. Congr., 8th, 1973 pp. 61-74. Loveless, M. H. ( 1957). J. Immunol. 79, 68. LZwenstein, H., Weeke, B., and Nielsen, L. (1974). Acta Allergol. 29, 418. McKenzie, H. A., ed. ( 1970). “Milk Proteins: Chemistry and Molecular Biology,” Vol. 1. Academic Press, New York. McKenzie, H. A., ed. ( 1971). “Milk Proteins: Chemistry and Molecular Biology,” Vol. 2. Academic Prew, New York. Mackler, B. F. (1972). Clin. Allergy 2, 317. Malley, A,, and Harris, R. L., Jr. ( 1967). 1. Zmmunol. 99, 825. Malley, A., Crossley, G., Baecher, L., Wilson, B. J., Perlman, F., and Burger, D. ( 1973). I n “Mechanisms in Allergy” ( L . Goodfriend, A. H. Sehon, and R. P. Orange, eds.), pp. 83-96. Dekker, New York. Marsh, D. G. ( 1971). Int. Arch. Allergy Appl. Zmmunol. 41, 199. Marsh, D. G. (1974). Allergol., Proc. Znt. Congr., 8th, 1973 pp. 381-393.
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Marsh, D. G. (1975). In “The Antigens” (M. Sela, ed.), Vol. 3, pp. 271459. Academic Press, New York. Marsh, D. G., and Haddad, 2. H. (1968). Fed. Proc., Fed. Am. SOC. Erp. Biol. 27, 368. Marsh, D. G., Haddad, Z. H., and Campbell, D. H. (1970a). J . Allergy 46, 107. Marsh, D. G., Lichtenstein, L. M., and Campbell, D. H. (1970b). Immunology 18, 705. Marsh, D. G., Bias, W. B., Hsu, S. H., and Goodfriend, L. (1973). Science 179, 691. Marsh, D. G., Bias, W. B., and Ishizaka, K. (1974). Proc. Natl. Acad. Sci. U.S.A. 71, 3588. Melam, H., Pruzansky, J., Patterson, R., and Zinger, S. (1971). J . Allergy 47, 262. Miller, H., and Campbell, D. H. (1950). 1. Allergy 21, 522. Miyamoto, T. (1974). Allergol., Proc. Int. Congr., 8th, 1973 pp. 403-410. Mole, L. E., Goodfriend, L. Lapkoff, C. B., Kehoe, J. M., and Capra, J. D. (1975). Biochemistry 14,1216. Nakashima, S., Sugimura, K., Sawada, T., and Mazaki, M. (1974). J. Biochem. (Tokyo) 76, 349. Naterman, H. L. (1957). J. Allergy 28,76. Naterman, H. L. ( 1965). J . Allergy 36,226. Noon, L. (1911). Lancet 1, 1572. Norman, P. S. (1971). In “Immunological Diseases” ( M . Samter, ed.), 2nd ed., pp. 775-783. Little, Brown, Boston, Massachusetts. Norman, P. S., and Lichtenstein, L. M. (1971). In “Immunological Diseases” (M. Samter, ed.), 2nd ed., pp. 840-858. Little, Brown, Boston, Massachusetts. Norman, P. S., Winkenwerder, W. L., and Lichtenstein, L. M. (1971). J. Allergy 47, 273. Norman, P. S., Winkenwerder, W. L., and Lichtenstein, L. M. (1972). J. Allergy Clin. Immunol. 50, 31. Norman, P. S., Lichtenstein, L. M., and Ishizaka, K. (1973). J. Allergy Clin. Immunol. 52, 210. Norman, P. S., Marsh, D. G., Lichtenstein, L. M., and Ishizaka, K. (1975). J. Allergy Clin. Immunol. 55, 78. Ohman, J. L., Jr., Lowell, F. C., and Bloch, K. J. ( 1973). J . Allergy Clin. I m m u d . 52, 231. Ohman, J. L., Jr., Lowell, F. C., and Bloch, K. J. (1974). J . Immunol. 113, 1668. Ohman, J. L., Jr., Lowell, F. C., Bloch, K. J., and Kendall, S. ( 1975). J . Allergy Clin. Immunol. 55, 77 (abstr). Osler, A. G., Lichtenstein, L. M., and Levy, D. (1968). Ado. Immunol. 8, 183. Parish, W. E. (1967). Nature (London) 215,738. Patterson, R., and Suszko, I. M. (1974). J . Immunol. 112, 1855. Patterson, R. Suszko, I. M., and McIntyre, F. C. (1973a). J. Immunol. 110, 1402. Patterson, R., Suszko, I. M., Pruzansky, J. J., and Zeiss, C. R. ( 1973b). J . Immunol. 110, 1413. Pepys, J., Hargreaves, F. E., Longbottom, J. L., and Faux, J. (1969). Lancet 1, 1181. Pickett, S. M., Riggs, A. F., and Larimer, J. L. (1966). Science 151, 1005. Ponterius, G., Brandt, R., Hultbn, E., and Yman, L. (1973). Int. Arch. Allergy Appl. Immunol. 44, 679. Prausnitz, C., and Kustner, H. ( 1921). Zentralbl. Bakteriol., Pumsitenk. Infektionskr., Abt. 86, 160. Ricci, M., Romagnani, S., and Bilotti, G. (1974). Allergol., Proc. Int. Congr., 8th, 1973 pp. 411-416.
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Human Mixed-Lymphocyte Culture Reaction: Genetics, Specificity, and Biological Implications BO DUPONT AND JOHN A. HANSEN Tissue Typing laboratory, Sloan-Kettering lnrtitute far Cancer Research, New York, New Yark
AND EDMOND J. YUNlS Department of Pathology and laboratory Medicine, University o f Minnesota Hospitals, Minneapolis, Minnesota
I. Introduction: Major Histocompatibility System in Man . . . . 11. Serology of Human Leukocyte Alloantigens (HLA-A,B,C) . . . A. Introduction . . . . . . . . . . . , B. One Genetic System of Several Closely Linked Loci . . . . C. Cross-reactivity . . . . . . . . . . . D. Genetic Linkage Disequilibrium . . . . . . . . 111. Cell-Mediated Allogeneic Reactions in Vitro. . . . . . . A. Mixed-Lymphocyte Culture Reaction , , . . . . . B. Induction of Cytotoxic Effector Cells in Mixed-Lymphocyte Culture C. Induction of Immunological Memory Cells in Mixed-Lymphocyte . . . . . . . . . . . . . Culture IV. Measurement of Antigenic Differences in Mixed-Lymphocyte Culture Reaction . . . . . . . . . . . . . . V. Single-Locus Concept for Mixed-Lymphocyte Culture Stimulation (HLA-D LOCUS) . . . . . . . . . . . . A. Family Studies . . . . . . . . . . . . B. Mixed-Lymphocyte Cultures between Unrelated Individuals . . VI. Mixed-Lymphocyte Culture ( HLA-D ) Specificities Defined by HLA-D. . . . . . . . . Homozygous Typing Cells . A. Families with Shared Parental Histocompatibility Haplotypes . . R . Identification of HLA-D-Homozygous Typing Cells . . . . C. Sources of HLA-D-Homozygous Typing Cells . . . . . D. Definition of Typing Responses . . . . . . . . E. Characterization of HLA-D Specificities . . . . . . F. Complexity of the HLA-D Locus: Cross-reacting Specificities versus Multiple Subloci . . . . . . . . . . . G . Family Studies with HLA-D-Homozygous Typing Cells . . . H. HLA-D Typing of Families with Recombinations within the HLA . . . . . . . . . . . . Complex . I. Population Studies with HLA-D-Homozygous Typing Cells . . J. Serological Identification of Alloantigens with Restricted Tissue . . . . . . . . . . . . Distribution K. Role of Lymphocyte Subpopulations in Mixed-Lymphocyte Culture . . . . . . . . . . . . . Reaction 107
108 110 110 114 116 117 119 119 120 123 124 130 130 132 135 136 138 140 142 146 148 154 154 162 164 168
108
DUPONT, HANSEN, AND YUNIS
VII. Genetic Control of Immune Response Related to Histocompatibility VIII. Mixed-Lymphocyte Culture As a Histocompatibility Test for Clinical . . . . . . . . . . . . Transplantation IX. Genetic Mapping of the HLA Complex on Chromosome C-6 . . . X. Conclusions . . . . . . . . . . . . . References . . . . . . . . . . . . .
169 177 183 185 187
I. Introduction: Maior Histocompatibility System in M a n
In all vertebrates studied thus far, the major histocompatibility complex ( M H C ) has been described as a genetic system of closely linked genes. The MHC controls alloantigens that are the predominant determinants in allograft or transplantation reactions. In addition to the control of individual diversity, this system also controls determinants of tissue diversity. Interest in the MHC goes beyond transplantation to include a number of biologically important functions. For example, genes linked to the MHC control immune responses to some specific antigens and production of some components of the complement system. The MHC in mouse ( H-2) controls certain factors influencing resistance or susceptibility to virus infections, autoimmune disease, and neoplastic disease. The MHC in man (HLA) is at present defined by at least four distinct loci (Fig. 1) : HLA-A, B, C, and D. Three of these loci code for alloantigens readily detectable by serological methods (HLA-A, B, and C ) . The fourth locus ( HLA-D ) controls lymphocyte responses in the in uitro mixed-lymphocyte culture reaction ( MLR) , Three important characteristics are shared by the genes or gene products of each locus: ( 1 ) each locus represents an allelic system with considerable polymorphism; ( 2) the determinants of each locus demonstrate cross-reactivity; and ( 3) genetic linkage disequilibrium exists among the alleles of the different loci. In addition to these four HLA loci, several different genetic traits have been linked to HLA, although the actual mapping of bhe genes in relation to HLA is still largely unknown (Fig. 1 ) . This review will summarize the major achievements in the clarification of the genetic control and polymorphism of the serologically deteotable HLA-A, B, and C determinants. The emphasis will, however, be placed on the recent developments in the identification of the HLA-D locus and HLA-D determinants. The mixture of lymphocytes from two different individuals usually results in a blastogenic response with cell proliferation. During the MLR, a series of events, in addition to cell proliferation, takes place, including the production and secretion of biologically active molecules called lymphokines, which mediate certain immunological readions [e.g., production of migration inhibitory factor ( MIF), lymphocytotoxic factors,
109
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION Location of Cantrornara
n
-8
=0.0074
--
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.
Lorua Symbol‘
’OM3
= 0.0017-
-e
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FACIOR I C-2 DEFICIENCY C-4 DEFlCllNCY I-CELL ALLOANTIGENS
SIRONG MLR GvH REACIIONS1
1
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DISEASE ASSOCIATIONS1
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IMMUNE RESPONSE GENES (lr)
FIG. 1. Schematic diagram of the major histocompatibility complex (HLA) in man and associated genetic traits. The four presently recognized HLA loci are designated by the letters A, B, C, and D following the Sixth International Histocompatibility Workshop 1975. The A locus was previously known as LA or first locus; B locus as four or second locus; C locus as AJ or third locus; and D locus as MLR-S, MLC locus, LD-I, or LAD. The bars indicate associated genetic traits. Open bars indicate that no definite position for a locus can be determined. A filled bar underneath a region of HLA indicates that there is strong evidence for localizing the trait within this region. The arrows indicate a specific localization of a trait based on study of at least one recombinant family. ( e ) Recombination fraction between two loci; ( PGMI ) phosphoglucomutase-3; ( Chido) erythrocyte antigen, Chido; ( Factor B ) glycine-rich &glycoprotein (GBC), Bf, complement C3 proactivator; ( C2) serum complement C2 deficiency; ( C 4 ) serum complement C4 deficiency; (CML) cell-mediated lympholysis; ( MLR ) mixed-lymphocyte culture reaction; ( GvH ) graft-versus-host reactions.
and mitogenic factors]. Following 6-7 days of in uitro mixed-lymphocyte culture, specific cytotoxic “killer” lymphocytes may appear, and after 10-14 days of in uitro culture, small lymphocytes with specific memory for secondary responses, both proliferative and cytotoxic, appear. The MLR is of special interest, since this in uitro test reflects the individual’s capacity to identify foreign cells and to initiate specific immunological reactions against the foreign cells. The study of genetic control of human MLR has advanced rapidly during the last few years as a result of the development of new methods for identifying HLA-D specificities. Using
1
110
DUPONT, HANSEN,
AND YUNIS
lymphocytes from HLA-D-homozygous individuals as stimulators in MLR, it is possible to identify the HLA-D specificities in the popul at'ion. As a result of these studies, six to eight different provisional groups of HLA-D specificities have been defined, accounting for approximately 50%of the determinants in random Caucasians. The study of MLR has ramifications in cellular immunology, immunogenetics, and clinical transplantation. Although many immunological traits and allogeneic phenomena are clearly associated with HLA, the precise region or determinant involved in a specific effect is sometimes difficult to define. The MLR typing now allows direct analysis of the HLA-D region, with the possibility of looking at HLA-D determinants either as markers or as factors in many different immune reactions and in susceptibility to disease. The biological significance of the determinants controlling the MLR in man is largely unknown as is also the case for the other presently known HLA determinants. The conclusions that can be made from the study of genetic control of immune response and from clinical transplantations emphasize, however, the significance of the HLA-D segment of the HLA complex. II. Serology of Human leukocyte Alloantigens (HLA-A,
B, C)
Several authors have furnished extensive reviews of the major histocompatibility system in man: Amos (1969), Amos and Yunis (1973), Amos and Ward ( 1975), Bodmer ( 1973), Ceppellini (1971), Ceppellini and van Rood ( 1974), Dausset ( 1971, 1973), Kissmeyer-Nielsen and Thorsby ( 1970), McDevitt and Bodmer ( 1974), Morris ( 1974), Terasaki and Singal (1969), Thorsby (1974), and Walford et al. (1970). The early history of the detection of antibodies has been reviewed by Walford et al. (1969), Killman (1960), and van Rood (1962). For reviews of the mouse histocompatibility system, consult Stimpfling ( 1971), Shreffler and David (1975), and Klein ( 1975). Reviews focusing on human mixed-lymphocyte culture have been published by SGrensen ( 1973), Bach and Bach (1974), Eijsvoogel (1974), and Thorsby (1974). The following section summarizes the developments in HLA serology and includes information obtained during the Sixth International Histocompatibility Workshop, Aarhus, Denmark, 1975. In Table I are given the presently recognized HLA specificities (World Health Organization Terminology Committee, 1975; Kissmeyer-Nielsen, 1975). A. INTRODUCTION Knowledge of the serology and inheritance of human leukocyte alloantigens (HLA-A, B, and C ) has resulted from the studies of many dif-
TABLE I LISTINGO F CUBRENTLT ItECOGNIZED HLA
A locus N em
Previous
c locus
B locus New
Previous
sPECIFICITIi,S
Sew
Previous
(1975)" 1) locus
New
Previous
HLA-I>Wl
L1) 101-Pf, J, Lad 27a, L1) SVIII, L1) iV5a LI) 102-L1)7a, Pi, S , Ld V L1) 10:3-L1>8~. L1) X I LI) 104-LI) \\l.;a, li, L, L1) X l r l l l LI) lI).?-Ll) I V LI) 106-L1>-pin, L1) SIL' Ll>ll'a, LI) XI1 L 1Lae
~
HLA-A1 HLA-A2 HLL4-A3 HLA-A9 HLA-A10 HLA-A11 HL.4-ABX HLA-A29
HL-A1 HL-A2 HL-A:3 HL-A!) HL-A10 HL-A11 WJ28 W29
HLA-B.5 HL.4-J%7 HLA-BX HLA-Bl2 HLA-Bl3 HLA-B14 HLA-B1X HLA-B27
HL-A,; HL-.47 HL-AH HL-Al2 HL-A1:3 W14 \?'I8 W27
H1.A-AWBB - .--
W'L3
HLA-AW24 HLA-AW24 HLA-AWB6 HLA-AWS1 HLA-AWS2
W'24 W2.i W26 L$rS1 W32
HT, 4.- . A-W 3 3 -
U'l9.6
HLA-AW34 HLA-AWX HLA-AW43
Malay 2 No* BK
HLh-BW1.i HL.4-BIV16 HLA-BW17 HLA4-B\V21 HLA-BW22 HL&B\V3S HL.4-B\V37 I-ILA-BW3H HLA-BbV3'3 HLA-B\\'40 HLA-B\V41 HLA-BW42
W15 LV16 W17 W21 lV22 W5 TY W16.1 W16.2 tV 10 Sahell 11i\-A4
HLA-CW1 HLA-CLV2 HLA-C\V:I HLA-CW4 HL.4-CW5
TI-AJ Ti-Sa532, 170 T3-UPS T4-315 TS
HL.4-I>W2 HL.4-I)jVS HLA-I)W4 HLA-I)\V5 HLA4-I)\V6 LI) 107 LI) 10%
a The genetic region containing the genetic determinants of the rnajor histocompatil~ilitycomplex in man is called HL.4 following the Sixth International Histocompatibility IVorksIrop 1'375. The locus previously called LA (or first) is now designated the letlrr A ; four locus (or second) is now designated 13, and the AJ locus (or third) is now designated C. The genetic locus controlling hlLR is now designated the letter 1) (previously MLIt-S, LI)-l, hlLC locus). T h e alleles of the A and B loci are nuinliered jointly (for historical reasons). The alleles of the C and I) loci are nuinbered separately for each locus starting in sequence from 1. Individual alleles of each locus are designated a numI)er following the locus symbol. Provisionally identified specificities will in addition carry the letter \V, inserted between the locus letter and the specificity number for the allele. An HLA ohenotvoe will lie written as HLA-.41, 2. B7. 12, CLVl, C\V2, I)\Vl, lllV2, and the genotype could be HLA-A2, B12, CW1, I>Wl)Al, B7: CW2, l)\V2 No locus symhol has been designated to the alloantigen systenr coding for antigens n i t h restricted tissue distrihution (Ia equiralents?).
8 5 2 E
3
?
w
4
m
2 m
3
5
! c1
112
DUPONT, HANSEN, AM, WNIS
ferent investigators over the last 18 years. The development of this body of knowledge is an unprecedented example of international collaboration, taking place during six histocompatibility workshops: Histocompatibility Testing, Duke University, North Carolina 1964 (Russell et al., 1965); Histocompatibility Testing, Leiden, The Netherlands, 1965 ( Balner et al., 1965); Histocompatibility Testing, Torino, Italy, 1967 ( Curtoni et al., 1967) ; Histocompatibility Testing, Los Angeles, California, 1970 ( Terasaki, 1970) ; Histocompatibility Testing, Evian, France, 1972 ( Dausset and Colombani, 1973); and Histocompatibility Testing, Aarhus, Denmark, 1975 ( Kissmeyer-Nielsen, 1975). In the beginning of this collaboration, two developments, among many, contributed most to the rapid progress in the field: ( 1 ) the miniaturization and standardization of the lymphocytotoxic technique (Terasaki and McClelland, 1964) and (2) the introduction of rabbit complement for lymphocytotoxic reactions ( Walford et aZ., 1965). The characterization of a human leukocyte alloantigen, Mac, was furnished by Dausset (1958). Following this, van Rood and van Leeuwen (1963) described the first leukocyte antigen system, which consisted of two alleles, 4a and 4b, controlled by one locus. Van Rood and van Leeuwen also introduced the use of two-by-two comparison analysis to identify similar patterns of serological reactions of leukocyte alloantibodies. This has been an essential tool in the subsequent identification of HLA specificities. A second group of leukocyte antigens, labeled LA 1 to 4, was identified by Payne et al. (1964). These antigens belonged to the same allelic system and were shown by family studies to be mutually exclusive. Dausset et al. (1965) studied ten identifiable antigens in the population and postulated a unified concept of human leukocyte antigens, describing a single complex genetic system, which was then termed Hu-1, and later changed to HL-A (Nomenclature Committee, 1968). Assumption of the control of human leukocyte antigens by a single genetic system of closely linked allelic groups was proven in family studies (Dausset et al., 1967; Ceppellini et al., 1967; Amos, 1967). According to this concept, one genetic system controls a large number of alleles. The segregation of paternal and maternal leukocyte antigens can be identified by serological reactions of the cells from family members, and only six different genotypes can be found in a given family. The paternal haplotypes were usually designated a and b, with the genotype ab; the maternal haplotypes were designated c and d, with the genotype cd. Four genotypes could thus occur in the family among the children: ac,ad, bc, bd (Fig. 2 ) .
113
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION
a b
I c!l
1
a c
Cornbina tion
Pdrent-Child
0 Haplotype Diff.
none
c d
b
b
I
L
a d
b c
b d
I Haplotype D i f f .
2 Haplo type
Diff.
ab
or
cd
ab
or
cd
vs
ad
b
or
cd
vs
bc
ab
or
cd
vs
bd
d C
VS
ad
ac
vs
bc
dc
vs
bd
ad
vs
bd
ad
vs
bc
bc
vs
bd
d
vs
ac
none
FIG.2. Segregation of HLA haplotypes in a family. Letters a, b, c, and d denote HLA haplotypes; a/b, c/d, a/c, a/d, b/c, and b / d denote HLA genotypes. The probability for genotypically identical siblings in each subsequent mating is 0.25. Parent-child combinations will always share one HLA haplotype. The MLC between HLA-identical siblings ( 0 haplotype difference) will cause no response. The MLC between parent-child and some sibling combinations will cause one-haplotype response; MLC between other sibling combinations will cause two-haplotype response. If the parents share a HLA-D specificity (e.g., the D specificity of the a and c haplotype is cross-reacting ), the disparity in MLR combinations involving this shared specificity (e.g., a/d vs c / d ) will not represent full one-haplotype stimulation. The concept of a single genetic system in control of human leukocyte antigens received further support when Bach and Amos (1967) observed that the in uitm MLR between leukocytes from family members corresponded with the results obtained by serological identification of the leukocyte antigens. klixed-lymphocyte cultures between cells from siblings genotypically identical for the serologically defined leukocyte antigens failed to stimulate. It was also demonstrated that the genes controlling the production of these leukocyte antigens were codominant, since one paternal and one maternal haplotype was expressed in each child.
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B. ONE GENETICSYSTEMOF SEVERAL CLOSELY LINKEDLOCI Based on population and family studies (Ceppellini et al., 1967; Dausset et al., 1970), it was concluded that the HLA specificities belong genetically to the same system but are probably the product of at least two different genetic loci, or regions, now known as the HLA-A locus (previously LA, first locus), and the HLA-B locus (previously four or second locus). It was also concluded that the two loci, each with multiple alleles, were closely linked on the same chromosome. This concept was proven when crossing over was found to take place between the two segregant series ( Kissmeyer-Nielsen et d., 1969; Ward et al., 1969). From analysis of the haplotypes of Scandinavians, it was estimated that the recombinant fraction between the HLA-A and the HLA-B loci was 0.0056 (Svejgaard et al., 1970). In a later report, the same investigators analyzed information on 1362 parental meiotic divisions; in 11 cases, recombinations had occurred and the recombinant fraction was calculated to be 0,0081 & 0.0024, with the maternal recombination frequency slightly higher than the paternal one (Svejgaard et al., 1971). Combined data obtained from six European laboratories have recently been analyzed, and the recombination fraction between the HLA-A and HLA-B locus has been determined to be 0.00874 0.00136. These data consisted of 4614 parental meiotic divisions, with forty recombinations (Belvedere et al., 1975). Within a family, the alleles of one locus cannot be transmitted with the same gamete, and cannot be brought together by crossover (recombinations). By contrast, the alleles of the A and B loci are inherited together as a genetic unit through the gamete, and may be changed by crossover. The combination of the two alleles, transmitted together within the same chromosome, is called a haplotype, and a complete genofype is formed b y two haplotypes. Mattiuz ei al. (1970) used computer analysis of the HLA specificities in families and populations. First, they demonstrated that HLA specificities could be distributed into two nonoverlaping series, corresponding to the two HLA loci, HLA-A and HLA-B. Second, they calculated the haplotype frequencies from the phenotype frequency in the population. This was performed by using gene frequencies found in the population with a correction factor-the delta value-compensating for the nonrandom association between different alleles on the two loci. Third, they studied the zygotic assortment of the four parental haplotypes in individual sibships. The observed random segregation of the parental haplotypes suggested that genetic linkage disequilibrium between the HLA-A and the HLA-B locus was not based on selection at
*
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the level of gametic assortment. These findings were confirmed during the Fourth International Histocompatibility Workshop 1970 ( Allen et al., 1970). It was concluded that the alleles from each of the two series did not occur with more than two specificities in the phenotypes (very few triplets were observed) and that the phenotype frequencies for the two antigen series fitted the Hardy-Weinberg distribution. One additional locus controlling HLA antigens, now considered to be well established, is the HLA-C locus ( AJ locus or third locus). Sandberg et al. (1970) described an HLA antiserum that reacted with about 10% of Scandinavians; the specificity detected by this antiserum did not seem to fit with any of the known antigens of the HLA-A or HLA-B locus. Thorsby et aZ. (1971) further defined two additional antisera that seemed to belong to the same series. These sera described specificities that were clearly shown to segregate with the HLA complex. A number of antigens that segregate as true alleles and fit the Hardy-Weinberg distribution have been assigned to this series (Svejgaard et nl., 1973). Formal proof of a separate third locus has come from the observation of recombinations between the HLA-A locus and the HLA-C locus, and between the HLA-C locus and the HLA-B locus (Mayr et al., 1973; Low et d., 1974; H. E. Hansen et al., 197s). Only two families, however, have been described with a recombination between the HLA-C locus and the HLA-I3 locus (Low et al., 1974; H. E. Hansen et al., 1975), but several families have been found with rccoinbination between the HLA-A locus and the HLA-C locus (Pierres et al., 1975). The HLA-C locus is thus presumably closely linked to the HLA-B locus, but the exact position of the C locus within the HLA complex is still uncertain. Only five specificitics are at present defined for the HLA-C locus (CW1-CW5) ( World Health Organization Tcmninology Committee, 1975; KissmeyerNielsen, 1975). The existence of these three different series of antigens has also been demonstrated by the phenomenon of determinants capping on the cell membrane. Because the detcrminants of HLA-A, 13, and C were shown to be present on the cell surface as separate molecules, they had to be controllcd by different cistrons (Bcrnoco et d.,1973; Solheim et al., 1973). Two important factors have complicated the definition of polymorphism at the three HLA loci: ( 1 ) serological cross-reactivity between different HLA antigens, and ( 2 ) the linkage disequilibrium between the determinants at different loci. The present concept of three HLA loci controlling serologically defined HLA antigens could well be an oversimplification. Most, if not all, HLA antisera are multispecific, and definition of the specificities is only obtained by careful control of
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methodology in terms of length of incubation, temperature of incubation, quality and quantity of complement, dilution of the reagents (antisera), and/or addition of antihuman globulin, Therefore, the antisera against the HLA antigens are only operationally monospecific. It is possible that the HLA antigens of the different loci have considerable homology at the molecular level, which could explain the cross-reactivity observed among them. Such homologies have recently been suggested from studies of tyrosine residues within different HLA antigens (C. CunninghamRundles et al., 1975). C. CROSS-REACTIVITY Leukocyte antibodies may be mono- or multispecific. In general, it may be assumed that a leukocyte antibody is monospecific when it can be exhaustively absorbed by the immunizing cells and by cells of several other individuals possessing the same specificity. There may be common determinant parts between allelic products of one locus, but each allele must have individual or unique determinant. Monospecific antibodies may be more or less cross-reactive among the different alleles of one locus or they may be directed toward a unique antigenic determinant. Antibodies reacting against a panel of different lymphocytes can define at least two different types of specificities: ( 1 ) narrow HLA specificities, which are defined when the antibody reacts with one known specificity, and can only be absorbed by cells giving positive reactions for that specificity and ( 2 ) cross-reacting groups ( CREG), which are complex and defined by antibodies reacting with cells expressing specificities for two or more different alleles. These antisera can be completely absorbed by cells of any one of these specificities. The shared specificities are said to be included in the products of each of the different alleles in the cross-reacting group. Analogous to the H-2 system of the mouse, this phenomenon has been explained on the basis of private (narrow) or public (broad) specificities. The crossreacting antibodies are of at least two different types: ( I ) antibodies defined by antigens of relatively restricted specificity, for example, one specificity of the HLA-A locus was originally designated as A9 but was later found to include two cross-reacting specificities, AW23 and AW24, and ( 2 ) the broad reacting antibodies, termed supertypic by Ceppellini (1971), such as anti-4a (W4) and anti-4b ( W 6 ) . These broad antibodies include several specificities of the HLA-B locus: 4a includes HLA-B5, 12, 13, W35, W17, W21, 27; and 4h includes B7, 8, W40, 14, 15, 16 and 22 (van Rood, 1973). The cytotoxicity negative and absorption positive ( CYNAP ) phenomenon was introduced by Ceppellini et al. (1965) and is illustrated in the following example. One serum
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( B C ) was shown to cross-react with HLA-AS, A l l , and A1 and could be absorbed by cells phcnotyped as A3, All , or Al, although at some dilutions it was cytotoxic only for A3 (Yunis et al., 1970). This may be due to different avidity of some antibodies to some restricted antigenic specificities, and could explain the many examples of the CYNAP phenomenon. The foregoing description is oversimplified, since there are many paradoxes that cannot at present be explained. For instance, many sera fail to differentiate HLA-B13 and W40, and yet HLA-B13 is included in W4 and W40 in W6 supertypic specificities. Most women, responding to the conceptus, recognize more readily the supertypic specificities dctected by CREG than the type-specific HLA antigens, and therefore the shared portion or the molecule must be highly immunogenic (Nielscn and Svejgaard, 1972; Ceppellini, 1971). The knowledge of CREG has practical applications, since matching for traiisplantation finds its greatest limitation in the nonavailability of fully matched donors. Matching at cross-reactive groups may be important for kidney allograft survival (Dausset et al., 1975).
D. GENETICLINKAGE DISEQUILIBRIUM Antigens of the HLA-A and HLA-R loci can be in linkage disequilibrium. This means that some alleles of the two loci can be found together on the same haplotype with a inuch higher or lower frequency than expected from the genc frequency of the alleles in the population. This association is measured by a constant ( A ) that is defined as the difference between the observed haplotype frequency and that expected from the product of the gene frequencies (Mattiuz et al., 1970). The delta values for the different combinations of alleles vary from population to population. For instance, in European Caucasoids, the highest delta value is found in the HLA-A1, B8 haplotype; in Japanese, HLA-All-B22; and in Asian Indians, HLA-A1-Bwl7 ( Dausset and Colombani, 1973). The reason for these associations is unknown. Chance inbreeding in genetic isolatcs, natural selection, and genetic drift, however, may be factors in the occurrencc of this phenomenon. Two important basic mechanisms may I,c involved: ( I) the selective advantage derived from maintaining closely linked irninune response genes within the HLA complex, and ( 2 ) control of recombination frequency within the HLA complex by other genes. Dunn and Gluecksohn-Waelsch ( 1953) observed that different strains of mice had different recombination frcquency within the H-2 complex. Bennet et al. (1972) have recently noted that the suppression of recombinations within the H-2 complex was controlled by genes at another
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locus, the T locus, within the ninth linkage group of chromosome 17 in the mouse. Similarly, it is possible that a T locus equivalent, which would control the recombination frequency between the different loci of the HLA complex, may exist in man (Amos and Ward, 1975). In summary, HLA is a system of several closely linked codominant genes. Three different loci are assumed to exist for the serologically defined HLA antigens, namely HLA-A, B, and C. Only the HLA-A and HLA-B loci, however, fulfill formal genetic requirements, since they control genetically the expression of different mutually exclusive alleles, and since several proven recombinations between the determinants of the two loci have been described. The HLA-C locus has tentatively been assigned five specificities identified by lymphocytotoxic antibodies, but for many individuals the HLA-C determinants cannot yet be defined due to lack of specific antibodies for identification of other possible specificities, Independent capping of cell-surface determinants, however, confirms the existence of all three loci. The exact mapping of the HLA-C locus cannot be made at present, since only two recombinant families have separated the HLA-C determinant from the HLA-B determinant. It is characteristic of the HLA antigens that the identification of different specificities by antisera is complicated by the serological crossreactivity that exists between some antigens. These serological reactions also demonstrate inclusions between different specificities which may represent public and private determinants. Genetic linkage disequilibrium between the alleles of the different loci further complicates the evaluation of the reactions of the HLA antisera. The pattern of reactions obtained with an antiserum when tested against a panel of lymphocytes may indicate that one HLA specificity is included in another specificity. This phenomenon would, however, also be observed if the two specificities defined by the antiserum were in strong positive linkage disequilibrium. A selected panel that minimizes the influence of genetically determined associations between HLA antigens of different loci and family studies make it possible to discriminate antibodies identifying HLA inclusions from antibodies directed against multiple determinants controlled by closely linked genes. Very little is presently known about the biological significance of the serologically defined HLA antigens, These antigens occur as cell surface antigens on all tissue cells and are, thus, different from the alloantigens that have restricted tissue distribution, as described in Section VI,J. The most direct proof that HLA antigens are important in transplantation comes from the observation that allografts are rejected in an accelerated manner when performed in the presence of alloantibody specific
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to antigen(s) of the donor (detected in cross-match testings). As will be discussed later, the reports of longer allograft survival in HLA-matched unrelated donor-recipient combinations could be explained by assuming that the HLA-A and B antigens themselves elicit the allograft immunity. Another possibility is that there are loci independent from but closely linked to HLA-A and B that are responsible for allograft immunity. Ill. Cell-Mediated Allogeneic Reactions
in Vifro
A. MIXED-LYMHPOCYTE CULTURE REAC~ION The MLR is an in vitro test of lymphocyte recognition and proliferation. Bain et al. (1963) were the first to show that the mixture of lyniphocytes from different individuals in vitro resulted in the production of blastlike cells, the appearance of some cells in mitosis; and the appearance of cells labeled with thymidine- 'H in radioautograph smears. The control and specificity of this reaction and its relation to certain allogeneic determinants is still an area of intense investigation. The very early studies, however, established that the strength of the MLR was related to certain genetic differences. Bain et nl. (1964) showed that there was no reaction in mixed-lymphocyte cultures between three pairs of monozygotic twins, but reactions between four pairs of dizygotic twins were variable. Two pairs of dizygotic twins showed rcactions comparable to those seen bctwcen unrelated individuals, and two pairs showed no reaction. Hirschhorn et al. (1963) confirmed that the mixture of lymphocytes from unrelated individuals led to cell enlargement and division. Bach and Hirschhorn (1964) suggcsted that the degree of transformation in mixed-lymphocyte culture might prove to be useful as a quantitative measure of histocompatibility. Although the reactions between the majority of normal unrelated persons were found to be positive, there was great variation between sibling pairs, ranging from a large numbcr of blast cells in some combinations to no reaction in others ( Bain and Lowenstein, 1964; Chalniers et al., 1966). These studies suggested that some genetic factor( s ) was segregating in families and that this factor( s ) determined certain antigenic differences on leukocytes that could be detected by the MLR. Important innovations in mixed-lymphocvtc culture methodology have included: ( I) the use of isotopically labeled thymidine to determine the synthetic ratc of DNA in reactive cultures (Bain et a]., 1964); ( 2 ) the development of microculture systems (Hartznian et al., 1971); ( 3 ) the development of multisamplc harvesting machines ( Hartzmm et al., 1972); and ( 4 ) the one-way mixed-lymphocyte culture technique. The
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one-way mixed-lymphocyte culture technique makes possible the measurement of proliferation of a single responder in a given combination. In the two-way mixed-lymphocyte culture, lymphocytes from two diffcrent donors are allowed to respond against each other so that the final reactivity measured reflects the total proliferation of both populations. A unidirectional response is obtained by metabolically inhibiting the proliferative capacity of one of the cells in the mixture while leaving its stimulating capacity intact. Stimulation in the mixed-lymphocyte culture requires a metabolically active and viable lymphocyte. Heat-inactivated cells, freeze-thawed cells, or disrupted cells are not able to stimulate allogeneic lymphocytes, even though the serologically detected HLA antigens may still be present (Hardy and Ling, 1969; Schellekens and Eijsvoogel, 1970). The one-way reaction can be achieved by pretreatment of aliquots of the designated stimulating cells with X-rays (Kasakura and Lowenstein, 1965) or mitomycin C (Bach and Voynow, 1966). By different mechanisms these agents block lymphocyte proliferation by inhibition of DNA synthesis, Measurement of DNA synthetic rate has become the standard method to measure lymphocyte proliferation in MLR. Deoxyribonucleic acid synthesis in unstimulated lymphocytes cultured for 3 to 6 days is relatively low and the rate of DNA synthesis in stimulated cultures can be readily determined by measuring radioactive labeled thymidine-14C or -3H incorporation. To make quantitative comparisons between weak and strong stimulation, pulse labeling with thymidine is performed during the log phase of proliferation (Sgrensen et al., 1969). Depending on the culture conditions, the log phase of proliferation for stimulated lymphocytes cultured in a microsystem is 96-144 hours (DuBois et al., 1974; Bondevik et al., 1974; Jorgensen and Lamm, 1974). Different aspects of MLR methodology have recently been analyzed in a cooperative study between several laboratories and reported by Thorsby et al. (1974b). In addition to the proliferative response in the mixed-lymphocyte culture, other cell-mediated allogeneic reactions may occur, The induction of cytotoxic effector cells and immunological memory, as shown by accelerated responses in secondary culture, will be briefly summarized.
B. INDUCTION OF CYTOTOXIC EFFECTOR CELLS IN MIXED-LYMPHOCYTE CULTURE Following stimulation by allogeneic cells or tumor cells, lymphocytes proliferate and acquire a capacity for specific cytotoxicity against the stimulating cells (Cerottini and Brunner, 1974). Some degree of proliferative response requiring several days of incubation is generally necessary before a cytotoxic effect of cells activated in vitro can be de-
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tected. This cell-mediated cytotoxicity is independent of antibody or complement. The first in oitro system showing some specific sensitization against transplantation antigens was developed by Ginsburg and associates, utilizing the xenogeneic combination of rat lymphocytes sensitizcd on mouse cell monolayers (Ginsburg, 1968; Berke et al., 1969). They showed that sensitized rat cells had their greatest cytotoxicity when tested against mouse cells syngeneic with the immunizing monolayer. Hiiyry and Defcndi ( 1970) used the one-way mixed-lymphocyte culture to sensitize mouse lymphocytes against allogeneic mouse lymphocytes and showed cytotoxicity by chromium-51 release against lymphoid cell lines isogeneic with thc sensitizing cells. Solliday and Bach (1970) showed that human cells could also generate cytotoxicity in mixedlymphocyte culturc and that cytotoxicity was greatest against the same lymphoid cell linc that was used to sensitize. However the necessity of using lymphoid cell lines that were more sensitive to the cytotoxic effect of sensitized lymphocytcs than normal cells severely restricted the possibilities for studying the specificity of the cytotoxic reaction. Lightbody et al. (1971) and associates (Miggiano et al., 1972) introduced an innovation that made it possible to use normal lymphocytes as targets in the cytoxicity reaction. Normal lymphocytcs treated with phytohemagglutinin ( PHA ) produce blasts that function as targets just as well as lymphoid cell lines in the chromium-51 release assay. Target lysis of PHA blasts is maximum for the PHA blasts syngeneic with the cells used to sensitize in mixed-lymphocyte culture. Usually no cytotoxicity occurs against PHA blasts syngeneic with the sensitized effector cells, This test for cell-mediated lympholysis, following mixed lymphocyte culture and using PHA blasts as target, is called the CML test. During the mixed-lymphocyte culture cytotoxic lymphoid cells are produced when the responder and stimulator cells differ at the HLA chromosomal region (Eijsvoogel et al., 1972b, 1973a,b,c; Miggiano et al., 1972; Trinchieri et al., 1973; Bonnard et al., 1973; Mawas et al., 1973; Alter and Bach, 1974). Studying the CML test in families, Miggiano et al. (1972) showed that a greater cytotoxicity could be achieved between two-haplotype different siblings as compared to one-haplotype different siblings. Eijsvoogel et al. (1972b, 1973c) studied families with recombination within the HLA region. When the CML test was performed between combinations differing at one or two HLA haplotypes, clear cytotoxicity always resulted, Cytotoxicity did not occur, however, between MLR-positive combinations that were seroidentical. In three combinations that were HLAD-identical but serologically incompatible, no proliferation occurred and
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no cytotoxicity could be detected. The conclusion from these studies was that disparity at HLA-D is necessary to generate effector cells, but measurable cytotoxicity in CML depends on disparities at the serologically defined HLA-A or B loci or at some other closely linked determinants that are responsible for the specificity of effector and target cell interaction ( Eijsvoogel, 1973a,b). More recently, it has been suggested that the HLA-D determinants are not esssential for the generation of cytotoxic cells (Long et al., 1975). Studies with CML both in recombinant families and between unrelated individuals, matched or mismatched for various serologically defined HLA antigens, indicate that HLA-A antigens tend to be weaker immunogens in CML than most HLA-B antigens (Eijsvoogel et al., 1973a,b). The HLA-A antigen A2, however, has been shown to be as strong a target as the HLA-B antigens (Trinchieri et al., 1973; Eijsvoogel, 1974; Grunnet et al., 1974). The specificity of the killer cells generated is directed toward the stimulator cell, but another cell may be a target cell if it shares determinants with the stimulator cell. These determinants are either HLA-A or HLA-B, or products of closely linked genes. In the mouse, it has been demonstrated that cytotoxic cells can be generated in MLC combinations where the responder and the stimulator cells differ at the H-2K locus alone. Conversely, Sprrensen, using congeneic strains carrying recombinant alleles of the H-2 complex, found that the specificity of the effector phase is determined by serologically detectable antigens or by products of genes that map together with H2-D or H-2K and that incompatibility at MLC is not an absolute requirement for sensitization to occur (Sgrensen and Hawkes, 1973; Sorensen, 1973). More recently, Fcstenstein and Dkmant ( 1975) have presented evidence for the existence of a mouse CML-stimulating locus, ECS, mapping in thc I-B and I-C subrcgions of thc H-2 complex. In man, the HLA antigens A, B, and C may be important as target determinants. Whether or not these antigens are the exclusive target determinants has been studied by testing the CML between HLA-identical individuals. Bach et al. (1973) investigated 5 HLA-A, B-identical unrelated individuals and found that one combination exhibited positive CML. Schapira and Jeannet (1974) found several positive CML reactions in HLA-identical unrelated individuals. The CML effectors educated between HLA-A, B-identical unrelated individuals cxhibited a higher mean chromium release than combinations involving HLA-identical siblings (Grunnet et al., 1974; Grunnet and Kristensen, 1975). Kristensen and Grunnet (1975) used two CML effectors educated against different HLA-A, B, C antigens on two different stimulators and found
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that they gave a similar pattcrn of cytotoxicity against a panel of target cells. The “CML determinant” prcmmed to be the target for cytotoxicity was found to be in association with HLA-B8, both in population and family studies. This study suggests the possibility of a separate CML locus within HLA and also suggests :in approach for using hlLC-activatcd effector cells in “CML typing.”
c. INDUCTION OF IN
IMMUiXOLOGICAL MEhfonY CELLS MIXED-LYMPHOCYTE CULTURE
Following the first 6-7 days of mixed-lymphocyte culture, mouse cells revert to small nondividing cells with loss of cytotoxic effect against the primary target cells (Andersson and Hayry, 1973, 1974). When primed mouse cells are rechallenged between 14-21 days of culture with fresh primary target cells, they regain their cytotoxic effect within 24 hours, and the rate of secondary proliferation is accelerated (Hayry and Andersson, 1973; Cerottini et al., 1974; MacDonald et al., 1974; Wagner et al., 1972). Similarly, the proliferative response of human lymphocytes primed in oitro and restimulated on day 14 is accelerated (Fradelizi and Dausset, 1975; Sheehy et al., 1975a). Studying an HLA-B/D recombinant family, Mawas et al. (1975a) have shown that disparity for HLA-D alone is sufficient for the secondary response of primed cells, and disparity for HLA-A, B, or C alone does not initiate secondary proliferation. An accelerated secondary response can occur against a new cell donor if the donor shares an HLA-D determinant with the stimulator in primary culture (Fradelizi et al., 1975; Sheehy et al., 1975a). If a haploidentical cell donor is used to stimulate in primary MLC, the primed cells will identify the same HLA-D determinant in secondary culture by an accelerated response. The use of primed lymphocytes as typing cells against unrelated individuals has been proposed as a new approach to HLA-D typing ( Sheehy et al., 1975a,b; Fradelizi et al., 1975). In addition to the acceleration of proliferation following restimulation, lymphocytes primed in vitro show an increased efficiency as effector cells when rechallenged with the same target cell (Wagner et al., 1972; Andersson and Hayry, 1973; Hiiyry and Andersson, 1974; Cerottini et al., 1974; MacDonald et al., 1974). The reinduction of specific effector cells was found to result from activation by HLA-D products, which need not be identical to those present on the primary cells (Charmot et al., 1975a,b). Even nonspecific niitogens were shown to be capable of reinducing specific effector cells. However, HLA-A, B, or C differences alone could not restimulate primed effector cells. Mawas et al. (1975b,c) have studied two families with HLA-B/D recombinations using the more sensitive secondary CML test. In one combination, an HLA-D difference
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did not elicit killer cells after secondary challenge, but in the other combination an HLA-D difference did elicit killer cells after secondary challenge. These preliminary observations are consistent with the possibility of a locus between HLA-B and HLA-D that controls a CML determinant. A similar locus ( H D R ) had been previously postulated to map between HLA-B and D, based on skin graft survival in HLA-A,B-identical HLA-D-different siblings (Yunis et al., 1973). In addition, Mawas et al. (1975b,c), using the secondary CML test to prime and rechallenge HLA-AS, B7 heterozygous responders with a homozygous HLA-AS, B7,DW2 stimulator showed that the secondary effector cells were cytotoxic against several unrelated target cells, most of them carrying the HLA-DW2 determinant, These experiments, as well as those reported by Kristensen and Grunnet (1975), using primary CML, suggest that CML is directed toward antigen( s ) in strong linkage disequilibrium with other antigens of the HLA-B, D region. In summary, the in vitro triggering of allogeneic responses and the resulting cellular differentiation might enable us to study the cellular events leading to rejection of a graft or a graft-versus-host reaction. Mixture of allogeneic lymphocytes in vitro results in proliferation in the MLR. The generation of killer lymphocytes is measured in the CML test. Both MLC and CML have immunological specificity, and MLC-primed cells display specific immunological memory ( secondary MLC and CML). The proliferative phase in primary MLC occurs without previous immunization and is initiated by differences a t determinants genetically controlled by the HLA-D locus. The determinants controlling the specific lysis of target cells are not known but are probably genetically controlled by genes different from, but closely linked to HLA-B. IV. Measurement of Antigenic Differences in Mixed-lymphocyte Culture Reaction
Quantitative expression of MLR data for man has been an important feature in the deductive analysis of the genetic factors controlling the mixed-lymphocyte culture reaction. Responses in MLR (one-way) have been expressed using increments or net counts per minute, stimulation ratios ( S R ) , or relative responses ( R R ) . Stimulation ratios are calculated by dividing the counts per minute in stimulated cultures by the counts per minute in autostimulated or nonstimulated cultures:
SR
=
cpm st,imulated culture (test) cpm autostimulated culture (cont,rol)
Relative responses are calculated by dividing the net counts per minute
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in a test combination by the net counts per minute in an average maximally stimulated combination (which is said to define the 100%reference value ) :
RR
=
cpm stimulated - cpm autostimulated cpm reference response - cpm autostimulated
x
100%
The reference value for a given responder is derived from the average response against a panel of lymphocytes from unrelated individuals (usually 5 different donors) or from the response against a standardized pool of stimulator cells containing lymphocytes from three to five HLAdisparate unrelated donors. Osoba and Falk (1974) suggested that a pool of cells from 3 different individuals representing the major cross-reacting groups of serologically defined HLA antigens be used to compose a standardized pool. We have used the cells from 4 different individuals who are HLA heterozygous and who are known not to carry any of the common HLA-D determinants. The calculation of stimulation ratio corrects for interexperimental variation in autostimulated cultures but does not allow for correction in the variation of maximal response capacity. Small variations in counts per minute in unstimulated cultures can have a great influence on the value for SR. The use of relative responses, with a reference value included to stabilize the maximum response, has been shown by Jorgensen et al. (1973) to allow better reproducibility of interexperimental results than the use of stimulation ratios. Studies on the kinetics of lymphocyte responses in weak and strong stimulation indicate that the doubling time of DNA-synthesizing cells is constant and independent of the antigenic strength of the stimulating cells (Wilson et al., 1968; Bach et al., 1969). The number of cells initially responding in MLC seems to be related to the antigenic strength of the stimulating cells. It has been shown through experiments involving sequential elimination that diff erent clones of responder cells react to specific stimulating cells (Salmon et al., 1971; Zoschke and Bach, 1971). The depletion of a specific antigen-reactive clone by broinodeoxyuridine (BUDR) incorporation and UV irradiation inhibits the response of that population of cells to repeated challenge by the same stimulating cell but not the response to other allogeneic cells. Assuming that the lag phase before onset of proliferation is the same for strong and weak combinations, proliferation, which is measured by the DNA synthetic rate, should be proportional to the nuniber of antigen-reactive cells. Empirically, the measurement of proliferation in MLC does reflect differences in antigenic strength and allows the ranking of MLR for combinations representing strong, intermediate weak, and zero stimulation.
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In MLC family studies, it has repeatedly been shown that quantitative differences can be measured between combinations representing HLAseroidentical siblings ( zero haplotype differences, Oh), discrepancy at one haplotype ( 111. differences), or discrepancy at two haplotypes (212 differences ) ( Albertini and Bach, 1968; Sprrensen and Kissmeyer-Nielsen, 1969; Schellekens and Eijsvoogel, 1970; Seigler et al., 1971). The average response for one haplotype-different combinations is approximately half the average response for two haplotype-different combinations ( Schellekens and Eijsvoogel, 1970); Sgrensen, 1970; Mempel et al., 1973a; Dupont et al., 1974a; Jiirgensen and Lamm, 1974). No overlap is seen between the responses of HLA-seroidentical siblings and the responses of family members different for two haplotypes. Sprrensen (1970) found no significant difference in the average responses between two haplotypedifferent family members as compared to the average responses between randomly chosen unrelated individuals. It has been shown by several investigators that the stimulation between HLA-seroidentical sibling pairs is essentially zero, as measured by standard MLC techniques. Thus, it is concluded that the histocompatibility system controlled by the HLA region is the only significant contributor to stimulation in the MLR in vitro (Bach and Amos, 1967; Amos and Bach, 1968; Albertini and Bach, 1968; Eijsvoogel et al., 1970; S@rensen,1970). A quantitative analysis of a large number of MLC tests, accumulated from two different laboratories and representing 983 different one-way combinations, has been evaluated with respect to average stimulation between HLA seroidentical siblings (Oh), family members discrepant for one haplotype ( l h ) , and combinations discrepant for two haplotypes (2h) ( Dupont et al., 1974a; Thomsen et al., 1974). When the stimulation ratios for Oh, lh, and 2h combinations were converted to log values (log SR ) and then plotted against the cumulative percentage of observations in each group, three straight-line curves were generated, indicating that the log-converted stimulation ratios in each group of combinations are normally distributed, From the log-converted SRs, the mean and 95% confidence interval for each group was calculated. These values, retransformed into SRs, are given in Table IIa. The mean SR, derived from ninety-six different combinations between Oh sibling pairs, was 0.95 (0.45-2.0). This range defines the mean SR and 95%confidence limits for MLC identity. The mean for combinations differing by l h was 6.5 (2.0-2l.O), and the mean SR for combinations differing by 2h was 12.0 (3.7-38.0). Although the mean SR for 2h differences is nearly twice the mean for l h differences, there is a large overlap between the two groups. This overlap between I h and 2h groups has been demonstrated in several different studies ( Albertini and Bach,
127
H U M AN MIXED-LYMPHOCYTE CULTURE REACTION
TABLE I1
Q u INTIT.I~IYON O F MLC I)Isr \ n i w a. STIMUI, I T I O N I1 \ T I O S ( S Z 2 ) u . b HLA (haplotype) disparity:c Parameter
Oh
lh
2h
No. of combinations Mean (95 % Confidence range)
96 0.9-5 (0.4.5-2.0)
266 6..5 (2.0-21 , 0)
62 1 12.0 (3.7-38.0)
HLA (haplotype) disparity :c Parameter
No. of combinations Mean (net rpm) Mean (relative response) (9.5 % Confidence range)
Oh 41 92 0 7% (0-3 6 %)
lh
2h
117 7489 66 % (16-1 16 %)
130 924,; 90 % (48-132 %)
Reference response to pooled cells 78 10,826 100 % -
D a t a from Dupont et al. (1974a) and Thornsen et al. (1975a) Table I I a represents 983 different one-way MLC combinations. The mean and 95 % confidence limits are obtained from log converted stimulation ratios. (Oh) IILA-identical sibling conibinations; ( 1 h) one HLA haplotype-different family combinations; (2h) two HLA haplotype-different family or unrelated combinations. Unpublished data, J. A . Hansen and B. Dupont (1973). Table IIb represents 288 different one-way MLC combinations. The reference response is the response to a pool of 4 different unrelated, IILA-heterozygous individuals. The relative response ( K R ) is clalculated as the percentage of the net counts per minute in stimulated cultures relative to the net reference response. a
1968; Schellekens and Eijsvoogel, 1970; SGrensen, 1970). It may be the result of some technical factors causing interexperimental variation or it may be the result of genetic heterogeneity within each group. The assumption that antigenic disparity between combinations within the l h group or 2h group is always of the same strength may not be true. Genetic uniformity in the strength of l h stimulation can be expected only if the antigenic disparity between the four haplotypes inherited within a family is of equal strength. If parents share a haplotype with cross-reacting specificities, then the disparity in l h combinations involving the haplotypes will not represent full l h stimulation, and the response in MLR will be less than the average l h response (Fig. 3 ) . The
128
DUPONT, HANSEN, AND YUNIS
argument that shared specificities on different haplotypes may came less stimulation than expected is also relevant to the occasional observation of weak MLR between some unrelated combinations. Jorgensen and Lamm ( 1974) have evaluated relative responses among fifty-four random unrelated 2h combinations and found a good fit with the normal distribution, The analysis of our own data for the responses in Oh, lh, and 2h combinations is shown in Fig. 3. The average response in Oh combinations, expressed as relative responses, is 0.7%,with a range of 0 to 3.6%(Table IIb). There is no overlap between the Oh responses and the responses in the 2h groups. Although the mode of distribution of l h and 2h combinations approaches normality, there are a number of outliers, particularly in the low range of distribution. These outliers probably represent combinations that share more than the expected number of determinants. Regardless of whether the data are expressed in stimulation ratios or relative responses, the same quantitative distinction between the different groups of responses (Oh, lh, and 2h) can be made. Lack of MLR between randomly selected unrelated individuals is an extremely rare event. If, however, unrelated combinations are selected on the basis of HLA seroidentity, the number of weak MLC responses increases greatly (van Rood and Eijsvoogel, 1970; Kissmeyer-Nielsen et al., 1970; Sgrensen and Nielsen, 1970; Singal et al., 1975). In more recent studies, it has been shown that approximately 10%of HLA seroidentical combinations may show no response or very weak response ( Mempel et al., 1973a,b), Even unrelated combinations,which are selected for identity at only the HLA-B locus, may show some very weak reactions (Mempel et al., 1973b; Thomsen et al., 1974; L'Espkrance et al., 1975). Repeated testing of 5 HLA-B locus-identical individuals, selected as relatively MLR identical from a group of 16 HLA-B locus-identical, unrelated normal blood donors, indicated that the mutual MLR between these 5 unrelated individuals constituted a group of very weak responses that were clearly separable from both Oh- and lh-different combinations ( Thomsen et al., 1974). As shown in Section VI, the degree of stimulation produced by HLA-D-homozygous cells against heterozygous cells that share the same HLA-D specificity belongs to this same weak intermediate range of responses. These weak responses are known as typing responses. In conclusion, MLR can be ranked into four different groups based on antigenic differences between responder and stimulator. A clear separation exists between HLA-seroidentical combinations (Oh) and combinations that differ by one or two haplotypes ( l h and 211). The mean for one haplotype responses is approximately half the mean for two haplotype
HUMAN MIXED-LYMPHOCYTE
CULTURE =(;TION
129
15
Differences
10 5
HLA Identical Siblings
I
0
N=77
o;
' 40
60 ti0 ' 160 Relative Response (%)
o1;
140
FIG.3. Frequency distribution of relative responses of 305 heterozygous responders against one HLA-D homozygous typing cell ( DW2 specificity). Frequency distribution of relative responses for 77 combinations representing HLA-identical siblings, 287 combinations representing one-haplotype stimulation, and 328 combinations representing two-haplotype stimulation.
130
DUPONT, HANSEN, AND YUNIS
responses. Considerable overlapping, however, exists between these two groups. Evaluation of this intermediate group in terms of HLA genetic differences is discussed further in Sections V and VI. V. Single-Locus Concept for Mixed-Lymphocyte Culture Stimulation (HLA-D locus)
A. FAMILY STUDIES The genetic control of stimulation in the MLR was clearly assigned to the major histocompatibility system (HLA) in man by Bach and Amos (1967; Amos and Bach, 1968). They demonstrated that a significant correlation existed between lack of response in MLR and identity between siblings, with respect to the histocompatibility antigens detected by the lymphocytotoxicity test. Of a total of 209 siblings, 29.2% showed no response in reciprocal MLR, whereas 282 unrelated cell combinations all showed significant stimulation. It was concluded that the genetic system controlling MLR is identical with the major histocompatibility locus (HLA). Similar observations had been obtained in the mouse where strains differing at H-2 showed stimulation, whereas H-2-identical cell combinations did not react (Dutton, 1966). Bach and Amos (1967), however, observed one MLR-positive combination of cells from siblings in one family where the siblings were identical for the serologically defined histocompatibility antigens. Also, two combinations between parent and child did not show a positive MLR. Similar observations were subsequently obtained. Albertini and Bach (1968) confirmed the findings by Bach and Amos and further demonstrated the quantitative differences in MLR elicited by one and two HLA-haplotype-different family combinations. These investigators, as well as Bach et al. (1969), presented a few combinations of HLA-seroidentical siblings with positive MLR, and single, one haplotype-different combination with lack of positive MLR, or very weak response. Plate et aZ. (1970) described another family in which one pair of HLA-A, B-seroidentical siblings showed strong positive MLR in mutual MLC testings. Seigler et al. (1971) observed some parent-child combinations with very weak mutual responses in MLR, or with positive MLR in only one direction. The possibility that MLR might be controlled by a separate gene closely linked to the serologically detectable HLA antigens was suggested by Yunis et aZ. (1971), based on studies in recombinant families. Two different phenomena were observed: ( I ) The MLR in a known recombinant family, with recombination occurring between the HLA-A locus and the HLA-B locus, was controlled by genetic factors closely linked
131
CULTURE REACTION
HUMAN MIXED-LYMPHOCYTE
to the HLA-B locus; ( 2 ) MLR in another family with two pairs of HLA seroidentical siblings did not correspond to the results of the HLA serotyping. In this family, the sibling combination with the HLA genotypes a l c was mutually nonresponsive in MLC, whereas the sibling combination with genotypes a l d was mutually responsive. One of the siblings, however, with the a l d genotype was mutually nonresponsive with the two a / c haplotype siblings (Fig. 4).This suggested that the child's d haplotype was a recombinant haplotype with a maternal recombination occurring between the HLA-B locus and the hypothetical locus for the control of MLR (MLR-S), now designated HLA-D. These findings, together with findings from studies of HLA seroidentical unrelated combinations, led to the hypothesis of a separate locus for the genetic control of MLR. This locus was tentatively placed outside the do/b
F
I Responder Cells
Family
d
Stimulating
Ch 1
Chi
7
ac
Ch3
ad
-
-
Ch4
ad
+
+
Yunis & A m a s ,
-
-
Ch2
by
~~
Ch3
-
ac
-
described
Cells
ChZ
1971
.
+
I
Chd
+ + +
a = AI,B8 b:
A9.612
c
6
A3.BW17
d
= A2,BIZ
FIG. 4. Informative families with indirect evidence of HLA-B,D recombination. Pedigree indicating the serologically defined HLA haplotypes in a family: a, b, c, and d. The lower part of the figure gives the results of the MLC testings between the siblings. An MLR identity is found between u/c ( C h l ) = u / c (Ch2) = a / d (Ch3). Positive MLR is seen between the two HLA-seroidentical siblings u/d (Ch3) and n / d (Ch4). The HLA-B/D locus recombination can, therefore, be assigned to the d haplotype of Ch3. ( - ) MLC identity (or very weak responses); ( + ) strong positive MLR.
132
DUPONT,
HANSEN,
AND W N I S
segment of the chromosome controlling the serologically defined HLA-A and HLA-B antigens (Yunis and Amos, 1971). A series of reports of mixed-lymphocyte cultures in families with recombination between the HLA-A and the HLA-B locus followed. These studies consistently demonstrated that sibling combinations that differ for only one HLA-A antigen did not generate strong stimulation in MLR. In combinations where the siblings differed, however, for a single HLA-B locus antigen, the MLR was strongly positive (Dupont et al., 1971; Gatti et al., 1971; Lebrun et al., 1971; Eijsvoogel et al., 1972a,b; Yunis et al., 1974, 1975). This confirmed that the genetic determinants responsible for strong MLR were located close to the HLA-B locus, or that they were identical to the HLA-B determinants. Several other family studies of MLR demonstrated HLA-seroidentical sibling combinations that were mutually strongly responsive (Eijsvoogel et al., 1972a,b,c; Mempel et al., 1972; Sasportes et al., 1972, 1973; Thorsby et al., 1973b; Keuning et al., 1975b). In other families, it was shown that some siblings differing for one HLA haplotype did not generate positive MLR( Eijsvoogel et al., 1972a,b; Sasportes et al., 1972; Dupont et al., 1972, 1974a). This information further supported the hypothesis that a separate gene, different from the serologically defined HLA determinants, was responsible for MLR. B. MIXED-LYMPHOCYTE CULTURES BETWEEN UNRELATED INDIVIDUALS The MLR between cells from randomly selected unrelated individuals is practically always positive. Even when HLA-seroidentical combinations are selected, it has been shown that most of these pairs develop strong positive responses. Approximately 10%of these combinations, however, are MLR identical or show only weak positive responses (van Rood and Eijsvoogel, 1970; Kissmeyer-Nielsen et al., 1970; Sgrensen and Nielsen, 1970; Eijsvoogel et al., 1970; 1971; Sengar et al., 1971; Mempel et al., 1973a,b; Jorgensen and Kissmeyer-Nielsen, 1973; Segall et al., 1973). This discrepancy demonstrated that typing for the serologically defined HLA-A and HLA-B determinants cannot be used to predict the results obtained in mutual mixed-lymphocyte cultures and that the HLA-A and HLA-B determinants cannot be the major genetic factors controlling MLR. The observation that randomly selected unrelated pairs practically always show positive MLR, whereas 101%of the HLA-seroidentical combinations are clearly weakly responsive or MLR-identical, indicates that HLA-A antigens and/or the HLA-B antigens must have some relation to the specificity of the MLR controlling determinants. This concept is further supported by the observation of some (less than 10%)MLR-identical or weakly responsive MLR combinations among individuals carrying the same HLA-B specificities. The majority of pairs in this group will
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION
133
show strong mutual MLR (Mempel et nl., 1973a,b; Dupont et al., 1973c; L’Espkrance et al., 1975) . A few reports have described MLR identity, or weak MLR, among unrelated HLA-A, B-different combinations, even with differences between the two cells on the HLA-B locus (Dupont et d., 1972; Pentycross et al., 1972; Mempel et ul., 1973a,b). Some of these combinations have later been reevaluated and shown to represent HLA-A, B-heterozygous, but HLA-D locus-homozygous cells (Dupont et al., 1973c, 1974a; Mempel et aZ., 1975). The results of family studies and studies of unrelated combinations can be summarized: The genetic control of strong MLR in man derives from a ringle gene (or genetic region) closely linked to HLA-B. It is located outside the HLA-A, B segment of the HLA complex. This genetic region controls an allelic system, wherein the specificity of determinants to some extent is related to the specificity of the determinant on the HLA-B locus. A schematic representation of the position of the four, presently defined genetic determinants of the msjor histocompatibility complex (HLA) in man is given in Fig. 1. The locus controlling strong MLR has been named HLA-D, following nonieiiclature established by the Sixth international Histocompatibility Workshop, 1975. As shown in the figure, the recombination fraction between the HLA-A and the HLA-€3 locus has been estimated to 0.008 (Svcjgaard et id., 1971; Belvedere et nl., 1975). Thorsby et al. ( 1975a) estimated the recombination fraction between HLA-B/D to be 0.014. The rcwmibination fraction between the HLA-B and HLA-D has recently been estimated to be 0.00’74 by Keuning et al. (1975b). These authors studied 39 familics, including 8 families in which parents did not share an HLA-B, D hapIotype (incIuding the HLA-D allele), 24 families in which the parents shared one haplotype, and 12 sets of HLA seroidentical siblings from 10 families in which parents or grandparents were not available for study ( 3 of which also belong to the second group). Positive MLR was found in two pairs of HLA-seroidentical siblings in two different families (the material represents 167 children). The families selected were, in most instances, studied for possible HLA-D-homozygous offspring from first-cousin marriages. The material contains a high fraction of HLAidentical siblings and a high fraction of mating pairs sharing at least one HLA-A, B haplotype. The studies were also based on the assumption that there was no more than one recombination between the HLA-B locus and thc HLA-D locus per family. Corrections were made to compensate for parental homozygosity, but the influence of genetic linkage disequilibrium, which exists between some HLA-B alleles and HLA-D alleles, was ignored.
134
DUPONT, HANSEN, AND YTJNIS
This indirect approach for determining the recombination fraction between the HLA-B and the HLA-D locus is, however, the first attempt to obtain an estimation of the distance between these two loci. Although the HLA-B determinants are identified readily in the lymphocytotoxicity test, the HLA-D determinants were not characterized by typing but indirectly assumed by evaluating the results of MLC between HLA-A, B-seroidentical siblings and parent-child combinations. Further development of typing for the HLA-D determinant will supply the tools for direct determinantion of the recombination fraction between the HLA-B and the HLA-D locus ( see Section VI, H ) . The genetic control of the mixed-lymphocyte culture reaction in mouse developed in parallel with the studies in man. Rychlikovl et al. (1970, 1971) demonstrated that differences at the K end of the H-2 gene complex in general provoked much stronger MLR than H-2D end differences. Studies of MLR in pairs of congenic strains were subsequently performed with combinations carrying H-2 recombinant haplotypes or with mutants within the H-2 gene complex (see Shreffler and David, 1975, for review). Bach et a2. (1972a,b) and Meo et al. (1973a,b) established that strong MLR was controlled by genes within the I region of the H - 2 gene complex and not by the H-2K region or the H-2D region. It was, however, demonstrated that genetic differences within other areas of the €€-2 gene complex would generate definite but weak MLR ( Abbasi et al., 1973; Widmer et al., 1973a,b; Meo et al., 1973a,b; Plate, 1973). Festenstein et al. (1970, 1974) and Huber et n2. (1973) demonstrated another genetic system ( M locus) separate from H-2 which can provoke moderate MLR. The MLR in the mouse is, thus, controlled by several genes, most of which are located within the H-2 gene complex. Strong MLR is controlled by genes within the I region, whereas weak-to-moderate MLR can be induced by all other regions of the H-2 gene complex, as well as by some non-H-2 genes. It is concluded that strong MLR in man can be caused by disparity on a single genetic locus (or genetic region) HLA-D. It is, however, not known if disparities within the HLA-D locus can cause weak MLR and if weak MLR can be induced by disparities on other genetic factors, other than HLA-D, within the HLA complex. A few family studies have been reported involving children with recombination between the HLA-A and the HLA-B locus, or between the HLA-B and the HLA-D locus, in which the single HLA-A locus disparity or disparity for the entire HLA-A, B segment of the HLA complex induced weak MLR (Eijsvoogel et al., 1972a; Mempel et al., 1973a; Thorsby et al., 1973b; Dupont et al., 1974a). It was postulated that another locus within the HLA-A, B segment of
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION
135
HLA, possibly close to the HLA-A locus, could be responsible for weak MLR. Proof for the existence of such a locus has not been presented. The main difficulties involved in resolving the question of genetic control in MLR are closely related to the problem of quantitation of the responses. The present conclusions are based on the classification of responses into three groups: (1) MLR identity, as seen in most cases of HLA-A, B-seroidentical siblings; ( 2 ) weak MLR, as seen between some HLA-seroidentical unrelated or HLA-B locus-identical individuals; and ( 3 ) strong MLR obtained in combinations differing for one or two HLA haplotypes. The conclusions made in this section are based on the assumption that there is a qualitative difference between strong and weak MLR. Strong MLR can result only from disparities between two cells at the HLA-D locus (or HLA-D region). A few recombinant families demonstrate weak responses in MLR, which may be causcd by disparities at genetic determinants (located close to HLA-A or HLA-B) other than HLA-D, although this has not been proven. In man, MLR has never been shown to result from genetic determinants outside of HLA. VI. Mixed-Lymphocyte Culture (HLA-D) Specificities Defined by HLA-D-Hornozygous Typing Cells
When it was recognized that strong MLR is controlled by a separate gentic locus (HLA-D) different from the genes controlling the serologically defined HLA-A, B, C antigens, attempts were made to develop methodologies for typing the HLA-D determinants. The breakthrough in the development of HLA-D typing was a consequence of the following observations ( I ) The weak MLR obtained from 10%of HLA-seroidentical unrelated individuals indicated a genetic linkage disequilibrium between the serologically defined HLA antigens and the determinants controlling strong MLR; ( 2 ) since weak MLR is observed in 10% of HLA-seroidentical unrelated individuals, it could be assumed that the polymorphism of the strong MLR determinants is relatively restricted and that the strong MLR specificities could be defined not only within families but in populations. Two different approaches were used to identify HLA-D specificities : the study of serological methods for identification of lymphocyte membrane components shared by individuals identical for the HLA-D determinants and the study of MLR, using well-defined HLA-D-homozygous or HLA-D-heterozygous cells as stimulators. The use of HLA-D-homozygous cells as stimulators in typing experiments has been the major advance in the identification and study of HLA-D specificities.
136
DUPONT,
HANSEN, AND WNIS
A. FAMILIES WITH SHARED PARENTAL HISTOCOMPATIBILITY HAPLOTYPES In the early reports of MLR in families, Bach and Amos (1967; Amos and Bach, 1968) mentioned that a few parent-child combinations were nonresponsive in MLR. Seigler et al. (1971) demonstrated that some HLA-seroidentical parent-child combinations, and some family combinations differing for only one HLA-A or B allele, were nonresponsive in MLR or showed very low levels of response. In addition, they described a few combinations with stimulation in only one direction. Nonresponsive HLA-seroidentical parent-child combinations were also observed by Eijsvoogel et al. (1971) and Thompson et al. (1972). These authors presented data from families in which the parents had one HLA-A, B haplotype in common. In such families a child could inherit the same HLA haplotype from each parent and thus be genotypically homozygous. Eijsvoogel et al. (1971) observed that in mixed-lymphocyte cultures some of these HLA-A, B-homozygous cells generated very low response when used as stimulators against cells from parents or siblings who were heterozygous for the shared parental haplotype (e.g., aJ/bvs U J / U , ) . The low levels of MLR obtained in these unusual combinations, however, were clearly different quantitatively from the MLR identity responses obtained from HLA-identical sibling combinations. The first suggestion of a restricted polymorphism in determinants responsible for strong MLR was derived from further studies of outbred families, in which HLA-seroidentical parent-child combinations were mutually nonresponsive in MLR. When parents share one HLA haplotype, as defined by serological typing, there is a 50%chance that a child will be genotypically HLA-seroidentical with at least one of the parents (Fig. 5). Children who are seroidentical with a parent will inherit one haplotype from that parent and the second haplotype, the shared haplotype, from the other parent. The MLR between the seroidentical parent-child combinations will test a l h difference, represented by the two parentally shared haplotypes. Identity response in this combination will indicate that the MLR dkterminants on these two serotypically identical haplotypes aretalsa identical. As illustrated in Fig. 5, the two parental haplotypes HLA-AS, B7 must carry the same HLA-D determinant, because the MLR between the father and child 3 is mutually nonresponsive. These observations indicate that certain HLA-A, B haplotypes are associated with specific HLA-D specificities. The MLR-identical parentchild combinations are most frequently observed in families in which the parents share one of the more common HLA haplotypes, e.g., HLA-A1,
HUMAN MIXED-LYMPHOCYTE CULTURE REACI'ION
A3,B7/A3,#7
'm
'f
Ch I
A3, B7
A2, B12
A l l , B W 4 0 /A3,B7 b am
af i h 2
Ch 3 Ch 1 ChZ Ch3
HLA-D HLA-D HLA-D
137
All,BW40 /A2>#12 b C
Ch
4
Homozygous i f a f = a m Identical w i t h mother i f d f = d m Identical H i t h father i f a f = a m
FIG. 5. Segregation of HLA haplotypes in a family with one shared haplotype. The a,, b, a,, and c denote the HLA haplotypes in the family. The a, = a, for the serologically defined HLA-A, B antigens. Child 1 is HLA-A, B-homozygous. If the a, and a , haplotype carry the same HLA-D determinant, the a,/a, cell is a Dhomozygous typing cell. In other families, the parents may share a HLA-B antigen (e.g., B7), but differ for the HLA-A antigens (e.g., a, = A3, B7 and a m = Al, B7). If these two haplotypes carry the same HLA-D determinant, the a,/a, cell is a D-homozygous typing cell.
B8; A3, B7; or A2, B12 (Mempel et al., 1973a,b; Dupont et al., 1973c; Dausset et al., 1973; Lebrun et aZ., 1973; Sasportes et al., 1973). The association of different HLA-D determinants with these common HLA-A, B, haplotypes suggests that the HLA-D determinants are in linkage disequilibrium with certain HLA antigens and that some HLA-D specificities are associated with common HLA-A, B haplotypes. Therefore, these HLA-D specificities should also be relatively frequent determinants in the population. Additional family studies have demonstrated that the association between HLA-D specificities and the HLA-A, B antigens is strongest between HLA-D and the HLA-B determinants. Some parent-child combinations differing for one HLA-A antigen but having the same HLA-B antigen on the shared parental haplotype, were MLR-identical (Fig. 5 ) (Seigler et al., 1971; Thompson et al., 1972; Dupont et al., 1973c; Dausset et al., 1973). One intriguing puzzle in MLR has been the finding of HLA-heterozygous unrelated combinations with lack of MLR response in one direction, but with positive response in the opposite direction (Dupont et al., 1972, 1973b; Mempel et al., 1973a, b; Pentycross, 1972). Some of these HLA-A, B-heterozygous individuals have been shown to be HLA-D-homozygous (Mempel et al., 1973b; Dupont et al., 1973a b, 1974a; J. A. Hansen et al., 1975). The results between some other of these heterozygous combinations remain unexplained. Nevertheless, the observation that an HLA-A,
138
DUPONT, HANSEN,
AND WNIS
B-heterozygous cell can be HLA-D-homozygous ( demonstrating that one HLA-D determinant may occur together with more than one HLA-B determinant further suggests that polymorphism of the HLA-D system is relatively restricted. In summary, MLR family studies have shown that up to 50%of HLAseroidentical parent-child combinations may be mutually nonresponsive or only very weakly responsive. These nonresponsive combinations are usually seen when the shared HLA haplotype involves serologically identified HLA antigens that are common in the population e.g., Al, B8; A3, B7; A2, B12; A2, BW15; A l l , BW35. The association between certain HLA-D and HLA-B determinants is much stronger than the association between the HLA-D and the HLA-A determinants. The experiments that led to these conclusions are based on the following assumptions: ( I ) that disparities between HLA-D determinants induce strong MLR and ( 2 ) that the weak MLR seen in some combinations is caused either by disparities at other loci or near identity at the HLA-D locus. The frequent observation of either MLR identity or weak MLR between HLA-seroidentical parent-child combinations in the outbred population indicates that the polymorphism of the HLA-D determinants is relatively restricted.
B. IDENTIFICATION OF HLA-D-HOMOZYGOUS TYPINGCELLS Family studies of MLR constitute the basis for identification of HLA-D-homozygous typing cells. Most frequently such homozygous cells are obtained from families in which the parents are known to share one HLA haplotype. This is based on serological typing for the HLA-A and B antigens. An example of such a family is given in Fig. 5 and Table 111. The shared parental haplotype in this family is designated a. The paternal haplotype af and the maternal haplotype a, carry the same HLA-D determinant. The two remaining HLA haplotypes, b and c, are different. The two HLA-seroidentical parent-child combinations ( q b X u,b) and ( GC X q c ) should be mutually nonresponsive in MLR, and the cells of the homozygous a,af child should not stimulate the cells from either parent or from siblings who are heterozygous for the a haplotype. The use of HLA-D-homozygous cells for typing of HLA-D determinants in the population was first formulated by Mempel et al. ( 1973a). The concept was built on the basis of substantial MLR data obtained in both related and unrelated combinations. Mempel et al. divided their data into three different groups of responses representing Oh-, lh-, and 2h-different combinations. They further identified an intermediate group of weak MLR which consisted of some combinations of HLA-seroidentical
139
H U M A N MIXED-LYMPHOCYTE CULTURE MACXION
TABLE I11 MIXED-LYMPHOCYTE CULTURE RE.\CTIONI N A FAMILY WITH A N HLA-I>-IIOMOZYGOUS CHILD(PARENTS SHARET H E HLA HAPLOTYPE A3,B7,DW2)a Stimulating cells* HLA haplotypes Subject
A
Father Mother Child1 Child 2 Child3 Child 4
3 3 3 3
B 7 7
7
7 1 1 W40 11 W40
D
A
DW2 DW2 DW2 DW2 X X
11 2 3 2 3 2
B
D
W40 X 12 Y 7 I>W2 12 Y 7 1lW2 12 Y
Father Mother AS
+ + + +
Child Child Child Child 1 2 3 4
+
-
AS + -
A
+
+
-
+
+ S + A S + A + +
- + + + + + S A
+ + + S
a All family members who are heterozygous for the same haplotype that is homozygous in Child 1 give negative or weak MLIt, which is characteristic of a typing response against homozygous cells. Child 1 is homozygous HLA-DW2. Father-Child 3 and Mother-Child 2 represent seroidentical parent-child combinations. They are mutually nonresponsive in MLR, indicating that the maternal DW2 and paternal DW2 are identical (+) Positive MLR; ( - ) negative or weak MLR; (AS) autostimulated control cultures.
or HLA-B-identical unrelated individuals. Combinations belonging to this intermediate group repeatedly gave responses midway between the zero and one haplotype responses. Mempel et al. also identified 2 unrelated individuals, homozygous for HLA-AS, B7, who were mutually MLR identical. When cells from these 2 individuals were used as stimulators against several unrelated cells heterozygous for the same haplotype, it was observed that some of the responses fell into the intermediate group of weak responses. This weak reaction against a homozygous stimulator cell is called a typing response. It was suggested that HLA-D-homozygous cells could be used for the typing of HLA-D specificities of unrelated cell donors. The authors discussed the possibility that perhaps the HLA-AS, B7 haplotype in these two unrelated families carried the same HLA-D specificity as a result of some common genetic origin, since these two families lived in the same geographically isolated region. These first two typing cells were assumed to be HLA-D-homozygous on the basis of family studies in which the homozygous cells did not stimulate the two parental cells. The two unrelated typing cells appeared to be homozygous for the same HLA-D specificity because of mutual MLR identity. Similar families were studied by Lebrun et al. (1973). In spite of the observation that the HLA-seroidentical parent-child combinations were mutually MLR identical, the HLA homozygous cells generated a weak
140
DUPONT, HANSEN,
AND YUNIS
but definitely positive response against the parental cells. This could not be explained by assuming a single locus was responsible for strong activation in MLR, and these reaction patterns are still not conceivable with our present concept of the genetic control of MLR. It was suggested that stimulation induced by HLA-D-homozygous typing cells in responders heterozygous for this HLA-D specificity is caused by allelic interaction. The problems of defining HLA-D-homozygous typing cells and typing responses will be discussed later in this section. It can, however, be concluded that even typing cells that elicit relatively high typing responses within the family can be useful in the population for identification of HLA-D specificities. Operationally, a typing cell is useful if it gives a clear bimodal distribution of responses.
C. SOURCEOF HLA-D-HOMOZYGOUS TYPINGCELLS Two different approaches have been used to find HLA-D-homozygous typing cells. One approach is based on the concept that some HLA-D specificities are in strong positive linkage disequilibrium with certain common HLA-B antigens. Therefore, some HLA-D-homozygous individuals should be found even in the outbred population by searching for HLA-A, B-homozygous children in families in which the parents share one of the common HLA-A, B haplotypes (Mempel et al., 1973a; Dupont et al., 1973a,b). The second approach, the study of inbred families, is genetically cleaner. In most inbred families studied, the parents have been first cousins (van den Tweel et al., 1973; Jorgensen et al., 1973). In a first-cousin marriage, or incest family, the HLA-homozygous child will be a true HLA homozygote by descent. The segregation of HLA haplotypes in such a family is shown in Fig. 6. I
FIG.6. Pedigree of a family with a first-cousin marriage in generation 111 illustrating the segregation of HLA haplotypes a, b, c, d, n, m, p, and 9 in the family. Child IV.1 is HLA-homozygous ( a / a ) by descent. The probability of an HLAhomozygous child in any first-cousin marriage is 0.0625.
HUMAN MIXED-LYMPHOCYTE CULTURE REACIlON
141
As will be described later, there does not seem to be any special advantage in using HLA-D-homozygous typing cells obtained from either one or the other source. It should be noted that the weak response obtained when typing cells stimulate parental cells is seen with both homozygous cells from inbred families and with homozygous cells from the outbred population (Eijsvoogel et al., 1971; du Toit et al., 1973; JIdrgensen et al., 1973; Thorsby and Piazza, 1975). Proof that a suspected HLA-D-homozygous cell is truly homozygous may be difficult to obtain: Critical family members may not be available for study, and the prcsumed HLA-D-homozygous typing cell may induce weak or even a moderate degree of stimulation in family members heterozygous for the same HLA-D specificity. When testing a family for a possible HLA-D-hamozygous cell, the most informative combinations to study are those of the HLA-seroidentical parent-child. If this combination is mutually nonresponsive, the HLA-homozygous child must be HLA-D-homozygous, assuming that a recombination has not occurred. The HLA-D-homozygous typing cells almost always induce some weak stimulation of cells from heterozygous siblings and parents. This weak response presents the main difficulty in evaluating HLA-D typing experiments. Since the discrimination between weak responses and the group of responses representing lh-different-sibling or child-parent combinations is relatively poorly defined, the limits of a typing response cannot be clearly defined. The identification of a typing cell is, in practice, based on how the cell functions in a typing experiment. If all of the responses against a homozygous cell can be separated into a bimodal distribution representing a group of weak responses, on the one hand, and a group of I h or greater responses on the other, then empirically the cell is functioning as a typing cell. Some HLA-D-homozygous cells that are heterozygous for the serologically defined HLA-A,B antigens have been identified (Dupont et al., 1972, 1973b,c, 1974a; Mempel et al., 1973b, 1975; Suciu-Foca and Dausset, 1975; J. A. Hansen et al., 1975; F u et al., 1975a,b). Such cells are identified from family studies by the same criteria as used for identification of HLA-A, B-homozygous cells. Family members heterozygous for any of the HLA haplotypes of the typing cell should show typing responses when stimulated by the typing cell. When the two parental haplotypes, assumed to have the same HLA-D determinant, are tested against each other in a parent-child combination, the MLR should be nonresponsive. In summary, HLA-D-homozygous typing cells can be obtained from random matings of the outbred population (Mempel et al., 1973a,b; Dupont et al., 1973c) and from inbred populations, primarily from children of first-cousin marriages (van den Tweel, 1973; Jpgensen et al.,
142
DUPONT, HANSEN,
AND YUNIS
1973; Keuning et al., 1975a), but also from geographical isolates (Layrisse et al., 1975). The main source for HLA-D-homozygous typing cells are families in which the parents share one serologically defined HLA haplotype. Homozygosity for the HLA-D determinant can be demonstrated if the HLA-homozygous cell induces typing responses in family members heterozygous for the haplotype of the homozygous cell and if the HLAseroidentical parent-child combinations are mutually nonresponsive. This approach is applicable to outbred and inbred families. If family members of the HLA-homozygous individual are not available for study, the potential HLA-D-homozygous typing cell may be identified by mutual MLC testing against a panel of defined HLA-D-homozygous typing cells. If none of these approaches can answer the question, a potential typing cell may be operationally identified by using it as a stimulating cell against a large responder cell panel. If the potential typing cell gives a bimodal stimulation pattern that shows a discrimination between weak responses and strong responses, the cell may be said to behave as a typing cell. A typing cell that represents a very rare HLA-D specificity will have to be tested against a large number of responders before it can be definitely identified as a typing cell.
D. DEFINITION OF TYPING RESPONSES A typing response is defined as the weak MLC response obtained when a responder cell reacts to an HLA-D-homozygous typing cell that has at least one D specificity identical to that of the responder. As discussed previously (Section IV), it is only possible to discriminate clearly MLC responses obtained between HLA-identical sibling combinations, on the one hand, and family members or unrelated combinations that differ for one or more HLA-D specificities or haplotypes, on the other. The discrimination between identity responses and positive responses in MLR is a quantitative one. Evaluation of response differences in the weak intermediate range, however, remains a major problem in MLR interpretation. To minimize technical variation in experimental results, many attempts have been made to normalize the data, particularly so that comparisons can be made between data obtained in different experiments and in different laboratories. Variation in the results obtained in any mixed-lymphocyte culture experiment may be the result of: ( I ) various technical factors, ( 2 ) the status of the responding cells, (3) the status of the stimulating cells, and ( 4 ) genetic disparity between the responder and stimulator cells. As discussed previously, responder cell function can be normalized by using relative responses; stimulator cell function is normalized by using the methods of Ryder and Thomsen (Thomsen et al., 1975a; Ryder et al., 1975).
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION
143
Figure 3 shows the frequency distributions of relative responses obtained when one HLA-D-homozygous typing cell was used as a stimulating cell against a panel of unrelated responders, For comparison, the frequency distribution of relative responses is shown for family combinations of HLA-identical siblings (Oh), Ih-different combinations, and 2h-different family or unrelated combinations, Some of the responses elicited with the typing cell are clearly lower than the responses for lh-different combinations. There is, however, obvious overlap. The problem in evaluating rcsponses of unrelated individuals is the uncertainty in discriminating between 111 stimulators ( i.e., disparity for at least one strong HLA-D determinant) and typing responses. At present, no definition for the upper limit of a typing response that gives statistical limits of confidence is available. From our empirical analysis of the relative responses of each homozygous cell in family studies, and from the frequency distribution of the relative responses of a large donor panel, we have selected an arbitrary limit of 35% for the upper level of a typing response. The definition and reproducibility of typing responses were evaluated in the Joint Report of the collaborative MLC experiments a t the Sixth International Histocompatibility Workshop ( Thorsby and Piazza, 1975) . In the combined analysis of typing experiments, which included a reproducibility experiment, it was clear that the assignment of a positive MLR against a typing cell could be based o n a single testing. The assignment of typing response, however, could not be reproduced in a large proportion of combinations if the experiment was repeated. The number of combinations that gave discordant results between typing versus nontyping responses in repeated experiments varied from 25 to 70%, depending on the method used to define a typing response. Different HLA-D-homozygous cells, independent of the specificity that they represented, had different discriminating capacities. For selected homozygous cells used as typing cells in a standardized procedure in a single laboratory, the reproducibility of typing responses can be quite good (see Fig. 7 ) . Why a weak MLR response results when a heterozygous cell reacts against a typing cell that is homozygous for one of the same HLA-D determinants is unknown. Stimulating cells are pretreated with irradiation of mitomycin C to block DNA synthesis, but it is possible that certain lymphokines may be secreted by the stimulating cell in response to the allogeneic responder cell. This is a postulated reaction that has been called back stimulation. Mitogenic factors or blastogenic factors have been reported in the supernatants of mixed-lymphocyte cultures (Janis et al., 1970). Preliminary studies on the production of blastogenic factor by homozygous
144
DUPONT, HANSEN, AND YUNIS 130 120 110 100
-
-
-
90,
t; 60 80
70
N YI
c
50
-
30 -
J’
40
Dw2
20 A
FIG. 7. Reproducibility testing of responses to two HLA-D-homozygous typing cells. The lymphocytes of 20 unrelated individuals were tested against the DW2 typing cell and 21 were tested against the DW3 typing cell. The correlation coefficient of identity between test 1 and test 2 for each test cell was r = 0.921 (DW2) and r = 0.921 ( DW 3 ) at a significance level of p 0.001.
-X* AW26, BW22, 1)-X i l l , B8, D-Y A2, B13, D-Y) ( A l , B8, D-X
Maternal HLA haplotypes A.3, B17, D-Z * A3, B7, D-Z* AW24, B?, D Z * A3, B7, 1)-Z ilW26, BW22, D-Z* A41,BX, D-Z*
z
SIX
INFORMATIVE
Recombinant haplotypes
A2, B12, D-Q A2, B12, I)-Z A l , B8, D-Z .41, A 3 , 8 7 , D-Q A3, B7, D-Z A428,BW40, D-Q A3, B7, D-X AW24, BW17, D-Q AW26, BWB'L, D-Q A2, B7, D-Q Al, B8, D-Q
KD-Q
C
5
2
References Yunis et al. (1971) Eijsvoogel et al. (1972b) Sasportes el al. (1972) Thorsby et a / . (1973b) Keuning ~t a/. (1975b) Keuning el al. (1975b)
= The HLA-D locus specificities are indicated as X and Y for the paternal specificities and Z and Q for the maternal specificities. The asterisk (*) indicates whether the HLA-B/D recombination has occurred between maternal or paternal haplotypes. If the parents share one HLA-B locus antigen or share the HLA-A, B haplotype, this is indicated by underlining the shared antigens or haplotype. Parentheses indicate that the IILA genotypes of these individuals have been deduced from the family study since the individuals were unavailable for studies.
n
2
158
DUPONT, HANSEN, AND YUNIS
Among the 6 informative families with evidence of recombination, only
1 involved a paternal recombination. In 4 of the families the parents shared one HLA-A, B haplotype, and in the other families the parents shared the HLA-B12 or the B8 determinant, This sharing of parental haplotypes cannot influence the occurrence of the recombination, but it could have implications as to alternative explanations for the results obtained in the family MLC tests. The development of HLA-D typing with homozygous cells has made it possible to evaluate the possible HLA-B/ HLA-D locus recombinants by a direct method. Formal proof of a recombination should be based on the positive identification of one allele on each of the two genetic loci between which the recombination has occurred, and the products of each locus should be identified on the recombinant haplotype. This has only been shown in two HLA-B/D recombinant families. The father, however, was not available for study in one family (Goulmy et al., 1975) (Tablc V I ) . In view of newly acquired knowledge about the determinants of the HLA-D locus, some “atypical” or “abnormal” MLC family studies previously reported as showing unexpected lack of MLR between different family members can now be reinterpreted. This can be illustrated by using as an example the MLC family study previously reported by Dupont et al. (1972, 1973a, 1974a). It was originally concluded that this family demonstrated an HLA-B/ HLA-D locus recombination in one child. This was based on the observation that one pair of HLA-seroidentical, MLC-identical siblings and their 1 h-different sister were MLR identical. The pedigree for key family members of this family ( K J ) is given in Fig. 8. The propositus, 111-2, is a child with severe combined immunodeficiency disease. The parents, 11-3 and 11-4, are unrelated husband and wife. The father 11-4 is DW2-homozygous, in spite of the fact that he is HLA-A, B-heterozygous (Dupont et al., 1973a,b,c, 1974a). Individuals 11-2, 11-3, and 111-1 are DW2-heterozygous, but the paternal grandfather, 1-1, does not carry DW2. The family MLC study is summarized in Table IX. It can be shown that 1-1 is behaving as a D-locus-homozygous typing cell. Grandfather 1-1is HLA-A, B-heterozygous, but his cells are incapable of stimulating the cells of his children or the cells of the patient’s sibling 111-1, who has inherited the A2, BW35 haplotype from 1-1. Since 111-1 has inherited the paternal A3, B7 DW2 haplotype, it is possible to show that the A2, BW35 haplotype carries the same D-locus specificity as the A2, B12 haplotype by testing combinations 11-2 vs 11-3and 11-2 vs 111-3.The MLC identity in these combinations confirms the hypothesis that 1-1 is HLA-D-homozygous. This example illustrates that the observation of MLC identity between HLA-haploidentical siblings does not constitute proof for HLA-B/ HLA-D recombinations.
HUMAN MIXED-LYMPHOCYTE CULTURE REACI'ION FAMILY
159
KJ
I
A2, B W 5 5 . 0 - X A1,BI2,D-X
11-1
~ ~ , m i D-x a, A a, ~ 7 D,w a
(AS,B7,DWD Al,BO,O-Y)
11-2
A2.Bl2,D-X ~ a , ~ owa 7 ,
11-4
Aa,B55,0-X A2,B7 , O W 1 111-1
AS,B7,DWP AlO,DlO, O W 2 Ill?
Ill A3,B7, D W 2 A2,BWSI,D-X
AIO, mia, o m AS,BW55,0-X
FIG. 8. Pedigree of family KJ previously described by Dupont et al. (1972, 1973a, 1974a) and C. Koch et al. (1973). The propositus (111-2) has severe combined immunodeficiency disease. The HLA genotypes are given as HLA-A, B, D specificities. The genotypes of 1-2 are deduced from HLA serotyping of additional family members. Individuals 11-3 and 11-4 are unrelated; 11-4 is HLA-A, B-heterozygous but HLA-D-homozygous for the DW2 specificity (Dupont ct al., 1974a). The DW2 specificity has been identified in 11-1, 11-2, 11-3, and 111-1 in typing experiments which included both 11-4 and other DW2-homozygous typing cells. The D specificity D-X has not been identified in typing experiments, but the family MLR indicates that 1-1 is homozygous for D-X. The segregation of the D-X specificity in the family is deduced from the family MLC testings summarized in Table IX.
Only a few families with HLA recombinations have been studied with HLA-D-homozygous typing cells. Rittner et al. (1975a) reported on 2 families with HLA recombination in which D-locus typing showed that the D specificity segregated with the HLA-B determinants. In both families, however, the recombination occurred between the two maternal haplotypes, both of which had the same HLA-B determinant. Therefore, the precise site of recombination could not be determined. Sasazuki et al. (1975) studied an HLA-A/B recombinant family, but the typing cells used could not identify the recombinant haplotype. Similarly, 5 families studied by Suciu-Foca et al. (1975) were uninformative with respect to the segregation of an identifiable D-locus determinant. Informative studies in HLA-B/ D recombinant families have been demonstrated by Keuning et al. (1975b,c), Mawas et al. (1975b,c), and Dupont et al. ( 1 9 7 5 ~ ) .In the family studied by Mawas et al. (1975a), the recombinant haplotype acquired the DW2 specificity, but the HLA-B specificity could not be determined. In the family studied by Dupont et al. (1975c),
160
DUPONT, HANSEN, AND YUNIS
TABLE I X MIXED-LYMPHOCYTE CULTIJRlC ItEACTION HLA-A, B,
L)
B
1-1 2W35 11-1 2 12 11-2 2 12 11-3 2 W35 111-1 3 7 111-2' 10 18 II-4b 10 18 DW2 cellb 3 7
Second haplotype L)
X X
X X W2 W2 W2
FAMILY KJ
genotypes
First haplotype Responder A
IN
A
B
1)
Stimulating cellsa
1-1 11-1
+
2 12 X A S 2 7 W2 - AS 2 7 W2 2 7 W2 X - n.d. 2 W35 2 W35 X 3 7 W2
W2 3
7 W2
+ + + n.d.
11-2
+ -
AS -
+
+
11-3 111-1 111-2 II-4b
+
+
+- +-
DW2 cellb
+
AS -
n.d. AS
-
-
n.d.
-
-
+ +
+ +
+ +
AS
-
-
AS
-
n.d. -
-
a (n.d.) Not done; (+) positive MLR; ( - ) very weak MLR; (AS) autostimulated control culture. 11-4 and DW2 cell: HLA-DW2-homozygous cells. c Individual 111-2 has severe combined immunodeficiency disease and is, therefore, not included as a responder cell. Immunological reconstitution of the lymphoid T-cell system and partial reconstitution of the B-cell system has resulted following bone marrow transplantation with maternal uncle 11-1 as bone marrow donor. According to this scheme the donor and recipient have inherited unrelated EILA haplotypes, which, however, carry the same HLA-D specificities, DW2 and D-X.
the D specificity carried by the HLA-A2, B12 haplotype of the mother could be defined by two HLA-D-homozygous cells. In the recombinant child, the involved haplotype, A2, B12, no longer carried one of these D specificities and acquired the D-17 specificity (see Table VI). The segregation of D specificities in a presumed recombinant family reported by Keuning et al. (1975b) is also in agreement with the concept of recombination between HLA-B and a separate locus for MLC (HLA-D). Only the family presented in Table VI and the family studied by Goulmy et al. (1975) proves the HLA-B/D recombination. It has previously been suggested that some genetic factors between HLA-A and HLA-B may contribute to at least weak MLR activation (Eijsvoogel et al., 197213; Thorsby et al., 1973b; Dupont et al., 1974a). The contribution of this additional genetic factor( s ) to the MLR activation has not been proven. An additional family suggesting the existence of an MLR-stimulating determinant associated with the HLA-A locus has recently been reported by Johnson et al. (1975). This family study demonstrates a unique finding that an HLA-A/ B recombinant child (11-1,Table X and Fig. 9 ) is strongly responsive to an HLA-B locusidentical sibling. The HLA-D typing in this family indicates that the
161
HUMAN MIXED-LYMPHOCYTE CULTURE REACTION
TABLE X MIXED-LYMPHOCYTE CUI.TURER.EACTION IN FAMILY HuR HLA-A, B genotypes First haplotype
Second haplotype
Stimulating cells*
Re- - sponder A B A B 1-1 1-2 11-1 11-2 11-3 11-4
2 11 2 2 9 2
18 9 W35 28 7 11 18 28 7 11 18 28
1-1
7 D W 2 W35 w35 W35 W35 DW2 W35
AS
+ + + + +
1-2
11-1
11-2
11-3
+ + + + + AS + 2 + AS + + AS 2 + + +
+ + + +
+
AS
-
11-4
DW2 cell'
+ + + +
-
+
2
+ +
AS
The HLA-A/HLA-B recombinant child (11-1) is HLA-B-identical to sibling 11-3. However, the MLIt between 11-1 and 11-3is positive. The l)-lorus typing with I)W2 typing cell identifies the paternal haplotype IILA-AS, 13, 7, 11W2 (in the fathrr 1-1 and rhild 11-3). Recombinant child 1-1 shows a strong positive response to the DW2 typing rrll. (15. Yunis, unpublishcd data.) * (+) Positive MLR; (-) MLC identity; (2) denotes the critical MLR-positive combinations; (AS) autostimulated control culture. & 0 OH
HO
O-CH,
I
0
I F I1 ;
CH-0-C-R CH,-
0--C -R
C. Galactosylglucosyl diglyceride [ I ,2-diacyl-3-o-(a-o -galactopyranosyl( 1+2)-O-p-O -glucopyranosyl)-sn-glycerol]
FIG.3. Structures of some glycosyl glycerides.
dry weight of the chloroplasts. Among microbial organisms, glycosyl glycerides are present in most gram-positive bacteria and mycoplasma (Brundish et al., 1965a,b), in treponemas, and in some gram-negative bacteria (Sastry, 1974). In animals they have been found only in the nervous system (Steim, 1967). Since many of the microbial and animal glycosyl glycerides contain the same sugar sequences as the plant compounds the latter are a useful source of material for chemical and immunological studies. Glycosyl glycerides containing up to five sugars have been described but diglycosyl glycerides are the most abundant compounds, and some
GLYCOLIPIDS AND PHOSPHOLIPIDS
223
TABLE I\' B.\cTI,: I
~ AI L
1) I G LY COSY I, ( ;L Y c I,:RII) E
S ~
(:lc(a,1+2)(;1c (a,1+3)-diglyccridc Glc(P, 1 ~ 6 ) ( ~ 1 c ( ~ , l + : 3 ) - d i ~ l y r e r i d ~ Gal@, l+G)C;al(~,l-t3)-di~lyrcridc Gal(a, 1+2)C;nl(a, 1+3)-diglyrcride hlan(a,l-t3)P\lan(a,l--r9)-diglyceridc Ahbrcviations: (>lC = D-glUCOSe;
(;a1 = u-galactose;
Man
=
D-mannosc.
of the most common disaccharide structures are listed in Table IV. Galactose, glucose, and mannose are the major constituents of microbial glycosyl glycerides, and the mono- and digalactosyl glycerides are the only compounds detected thus far in animals. Microbial glycosyl glycerides contain palmitic, stearic, and oleic acids, fatty acids of plant compounds are highly unsaturated, and bacteria contain branched fatty acids (Sastry, 1974). A comprehensive review of the structures of bacterial glycolipids and glycophospholipids was published recently by Shaw (197s). 2. Immunological Properties Antibodies to glycosyl glycerides are produced in the course of natural (Beckman and Kenny, 1968; Plackett et al., 1969) and experimental infections (Brunner et al., 1973) caused by mycoplasma. They may also be elicited by immunization with whole mycoplasma cell membranes or pure glycosyl glycerides aggregated with a membrane protein from Acholeplasma laidlawii (Razin et al., 1970, 1971b). The immunological specificity of these compounds is determined by their sugar sequence and linkages, and the nonreducing terminal residue is immunodominant. For optimal complement fixation, glycosyl glycerides must be mixed with lecithin and cholesterol (Plackett et al., 1969; Kenny and Newton, 1973). Cross-reactions betwecn glycosyl glycerides of a number of microorganisms are summarized in Table V (Sugiyama et al., 1974; Kenny, 1975). Extensive cross-reactions between galactosyl glyceride and galactosyl ceramide have been demonstrated with rabbit antisera to brain tissue or to pure glycolipids, and with human sera from healthy subjects, and patients with neurological diseases or syphilis ( Dupouey, 1972; Dupouey et al., 1976). Treponema reiteri contains galactosyl diglyceride ( Dupouey et al., 1970), and the cross-reaction between this treponeme and nervous
224
DONALD M. MARCUS AND GERALD A. SCHWARTING
TABLE V CROSS-REACTING GLYCOSYL GLYCERIDES” Source Acholeplasma laidlawii Acholeplasma modicum Mycoplasma granularum
Structures
Glc (a, 1+ 3)-diglyceride Glc (a, 142)Glc (a,l+ 3)-diglyceride
Mycoplasma pneumoniae Mycoplasma neurolyticum Streptococcus MG Mycoplasma pncumoniae Spinach
Gal@, 1+ 6)Gal (P,1 4 3)-diglyceride Gal(a,l-+6)Gal(a,l-+6)Gal(~,I-+3)-diglyceride
Abbreviations: Glc = D-glucose; Gal = D-galactose.
tissue presumably involves both of these galactose-containing glycolipids. Galactosyl glyceride has not been found in Treponemu pallidum ( Dupouey and Betz, 1969). Myc,oplasma pneumoniae causes “primary atypical pneumonia” and tracheobronchitis in man (reviewed by Chanock, 1965; Couch, 1973)) and these illnesses may be accompanied by other clinical syndromes that appear from 4 to 14 days following the onset of respiratory symptoms (reviewed by Murray st al., 1975). These associated conditions include Stevens-Johnson syndrome, autoimmune hemolytic anemia caused by cold agglutinins, aseptic meningitis, meningoencephalitis, Gullain-Barre syndrome, and acute psychosis. The Stevens-Johnson syndrome ( erythema multiforme ) is generally considered to be a hypersensitivity reaction (Fellner and Bystryn, 1971), and it has been suggested that the neurological manifestations noted above may represent a hypersensitivity response to the mycoplasma and/or host tissues modified by the mycoplasma (Taylor et al., 1967; Biberfeld, 1971). The sera of convalescent patients contain antibodies that fix complement with lipid extracts of M . pneumoniae (Kenny and Grayston, 1965), and these antigens have been identified as glycosyl glycerides (Beckman and Kenny, 1968; Plackett et al., 1969). Five compounds have been identified: monogalactosyl, digalactosyl, and trigalactosyl glycerides and additional diglycosyl and tetraglycosyl glycerides (reviewed by Kenny, 1975). The structures of these compounds have not been completely elucidated. Human sera contain antibodies to the digalactosyl and trigalactosyl compounds, and a diglucosyl glyceride isolated from StreptoCOCCUS MG (Kenny and Newton, 1973). Glucose has been detected in
CLYCOLIPIDS AND PHOSPHOLIPIDS
225
crude glycolipid fractions of M . pneumoniae, but a glycolipid containing glucose has not been isolated to date. There is some uncertainty whether the dignlactosyl or trigalactosyl glyceride is the major complement-fixing antigen, but recent evidence favors the latter (Kenny, 1975). The sera of patients with primary atypical pneumonia contain antibodies against human lung, liver, heart, and kidney (Thomas et al., 1943; Thomas, 1964), and Biberfeld ( 1971) demonstrated antibodies to human brain. The antibodies studied by Biberfeld reacted with a lipid extract of brain and could be absorbed by M . pneumoniae. They were present in the sera of 80%of patients with M . pneumoniae infections not associated with neurological symptoms, and in all 7 patients with neurological symptoms, and their role, if any, in the pathogenesis of these disorders is unclear. The brain antigen was not identified, but, in retrospect, it might well have been galactosyl glyceride and/or galactosyl ceramide. Individuals with previous M . pneumoniae infections develop positive skin tests (Mizutani et al., 1971) and in vitro evidence of cell-mediated immunity to extracts of this organism ( Fernald, 1972; Biberfeld, 1972). Patients with immunodeficiency diseases who develop M . pneumoniae infections exhibit minimal or no pulmonary infiltrates (Foy et al., 1973). The latter investigators and Mizutani and associates ( 1971) suggested that the pulmonary pathology in normal persons infected by M . pneumoniae may represent a hypersensitivity reaction. In accord with this suggestion is the observation of Taylor-Robinson et al. (1972) that immunosuppressed mice infected by M . pneumoniae develop much less peribronchial and perivascular lymphocytic cuffing than normal mice. The antigen that elicits cell-mediated immunity in humans and guinea pigs infected with M . pneumoniae appears to be a protein and not a glycolipid ( Mizutani and Mizutani, 1975). The origin of the cold agglutinin anti-I antibodies found in the sera of many patients with this disease is not clear. The cold agglutinins are not adsorbed by M . pneumoniae (Liu et al., 1959; Feizi and Taylor-Robinson, 1967; Biberfeld, 1971) . Mycoplasma pneumoniae produces peroxides that alter the erythrocyte membrane, and it has been suggested that the cold agglutinins represent a response to altered autologous erythrocytes (Feizi et al., 1969). Cold agglutinins with I-like specificity have been obtained by immunization of rabbits with M . pneumoniae (Costea et al., 1971; Lind, 1973), and it is possible that the mycoplasma contains an antigen that cross-reacts with the I antigen but is present in a too small quantity to adsorb the antibody. The serological activities of glycolipids from other mycoplasma (Sugiyama et al., 1974; Kenny, 1975) and streptococcal L forms (Feinman et al., 1973) have recently been summarized.
226
DONALD M. MARCUS AND GERALD A. SCHWARTING
C. LIPOTEICHOIC ACIDS
1. Structure and Distribution Teichoic acids are a group of phosphate-containing polymers that are constituents of the cell walls and membranes of gram-positive bacteria (reviewed by Knox and Wicken, 1973). The cell wall teichoic acids are covalently linked to peptidoglycan and consist of glycerol or ribitol phosphate polymers (Fig. 4 ) that are substituted with D-alanine or sugar residues. The cell membrane lipoteichoic acids are composed of 25-30 glycerol phosphate residues that are covalently linked to membrane glycolipid ( glycosyl glycerides) (reviewed by Wicken and Knox, 1975). The membrane glycolipid is linked to teichoic acid by a phosphodiester bond between a sugar hydroxyl group and the terminal glycerol residue of the teichoic acid (Fig. 5 ) . The hydroxyl groups of glycerol are substituted by D-alanine and glycosyl residues, and the terminal glycerol group of the polymer or one of its substituent sugars may be acylated (Fig. 5). The glycolipid portion of lipoteichoic acids is inserted into the plasma membrane, and the polar glycerophosphate polymer is thought to be intercalated into the peptidoglycan network of the cell wall ( Wicken and Knox, 1975). The glycerophosphate polymers extend to the surface of the cell wall in some instances, as demonstrated by their accessibility to ferritin-labeled antibodies (van Driel et al., 1973; Dickson and Wicken, 1974; Joseph and Shockman, 1975) and by agglutination of intact organisms by antibodies to intact lipoteichoic acids (Shattock, 1949). R
Ala
Ala
R
- I - I H O O O H OH H O O O H I I ! l . l I I I I I I -0-c-c--c -c -c-o-P-o-c-c-c-c-c-o-P I I I I I II I I I I I H
H
H
H
H
H
0
H
H
H
OH
I II
0
H
A. Ribitol teichoic acid
R
I I
l l
R
Ala
I
H 0 H
l l
OH
I II
I
H 0 H
I I
l l
l l
-o-c-c-c-o-P-o-c-c-c-o-P-o-c-c-c-o H
H
H
0
H
H
H
OH
I II
0
I l
H 0 . H
I I
H
l
H
l l
H
B. Glycerol teichoic acid
FIG.4. Schematic structures of teichoic acids. R
= H or glycosyl; Ala = D-alanyl.
227
GLYCOLIPIDS AND PHOSPHOLIPIDS Lactobacillus c a x i
Streptococcus lactis
tz Lactobacillus fermenti
Streptococcus faecalis
Ha*. ,",,, y,l"i GIC
Gal
I
Glc
Gal
I-?
..
I 1-2
1-7
Gal-Glc-
I
-
iatty
~ L K Ic \ t e r
I
+o-~H,-(
q
t
I Glc GlcI
I
IG I L
GlcI I-?
I-?
.....
H
HOH-(
GlC
Glc
Gic-Glc-
I H-0-P-
II
I
?
t
glycerol rc\iduc
OH
FIG. 5. Proposed partial structures of some lipoteichoic acids. (Reproduced from an article by Wicken and Knox, 1975, by permisssion of the authors; copyright 1975 by the American Association for the Advancement of Science.)
2. Immunological and Biological Properties Some lipoteichoic acids are immunogenic in the intact organism, and antisera can be obtained readily by immunization with disintegrated organisms or a high molecular weight micellar lipoteichoic acid-protein complex that can be extracted from bacteria with hot phenol (Wicken and Knox, 1971; Wicken et al., 1973). Most of the antibodies to lipoteichoic acids are directcd against the glycerophosphate backbone or its carbohydrate substituents. The former type of antibodies cross-react widely with a number of lipoteichoic acids (Knox and Wicken, 1973). At present, lipoteichoic acids have been identified as group-specific antigens of bacteria of only two genera, Streptococcus and Lactobacillus. Organisms with group-specific antigens include Group D and N streptococci and Group F lactobacilli (Knox and Wicken, 1973). The immunodominant groups of the lipoteichoic acids are the glucosyl and galactosyl substituents of the glycerophosphate polymer; some of thcse determinants are listed in Table VI (Wicken and Knox, 1975). The few human antibodies studied to date have becn directed against the glycerophosphate backbone (Decker et al., 1972; Markham et al., 1973).
228
DONALD M. MARCUS AND GERALD A. SCHWARTING
TABLE VI STRUCTURES O F GROUP-SPGCIFIC CARBOHYDRATE DETERMINANTS O F LIPOTEICHOIC ACIDS Genus
Group
Determinant
Lactobacillus Lactobacillus Streptococcus Streptococcus Streptococcus
A F
a-~-Gl~co~yl a-o-Galactosyl a-D-GlUCosyl-(1+2)-glUCOSyl a-D-Galactosy 1 P-D-Galactosyl
n N Serotype a
Purified lipoteichoic acid or soluble antigen obtained from culture fluid or saline washings of gram-positive organisms adhere firmly to erythrocytes and sensitize them to hemolysis or hemagglutination by antibodies (Rantz et al., 1956; Gorzynski et al., 1960). Lipoteichoic acids can also exchange between erythrocytes and tissues. The ability of lipoteichoic acids to adhere to cell membranes is dependent on their content of esterlinked fatty acids (Hewett et al., 1970; Matsuno and Slade, 1971; Ofek et d.,1975). It has been suggested that complexes of lipoteichoic acid with streptococcal antigens may bind to host tissues and play a role in the pathogenesis of poststreptococcal diseases such as rheumatic fever and acute glomerulonephritis ( Moskowitz, 1966). Lipoteichoic acids are analogous in some respects to lipopolysaccharides of gram-negative bacteria (Wicken and Knox, 1975) : Both are amphipathic molecules capable of attaching to cell membranes and both can elicit local and generalized Schwartzman reactions. Lipoteichoic acids are not mitogenic for B cells and they do not possess endotoxic properties. Other immunological properties of lipoteichoic acids include their ability to depress the immune response to sheep erythrocytes and enhance the immune response to lipopolysaccharides (Miller and Jackson, 1973, 1974) as well as their crossreactions with cardiolipin (Wicken & al., 1972). The latter property could be responsible for some false positive serological reactions for syphilis.
D. OTHERGLYCOLIPIDS Other glycolipids with immunological properties, which are not discussed in this review, include acylated sugars ( Coulon-Morelec, 1968, 1972; Coulon-Morelec et al., 1967, 1968, 1970; Faure and Coulon-Morelec, 1974; Shaw, 1970) and mycolic acids (reviewed by Lederer et al., 1975). Immunochemical properties of lipopolysaccharides have been reviewed extensively (for a recent review, see Luderitz et al., 1971).
229
GLYCOLIPIDS AND PHOSPHOLIPIDS
Ill. Phospholipids
A. CARDIOLIPIN 1 . Structure and Distribution The immunological properties of cardiolipin ( diphosphatidylglycerol ) (Fig. 6 ) have been studied extensively because of its role in the serological diagnosis of syphilis. Cardiolipin was first isolated from an alcoholic extract of beef heart by Pangborn (1942), its structure was studied by a number of investigators (reviewed by MacFarlane, 1964), and it was synthesized by de Haas et al. (1966). Cardiolipin is ubiquitous -it is found in mammals, fish, birds, bacteria, protozoa, yeasts, mycobacteria, and treponemas ( MacFarlane, 1964). I t is located principally in the membranes of organelles that display high metabolic activities : mitochondria, bacterial protoplasts, and chloroplasts of photosynthetic bacteria. Bovine heart cardiolipin contains mostly unsaturated fatty acids, approximately 85%linoleic acid ( 18 :2 ) , but synthetic or hydrogenated compounds containing saturated fatty acids have essentially equal immunological activity ( Faure and Morelec-Coulon, 1963; Inoue and Nojima, 1967). 2. lmrnunological Properties Antibodies reactive with cardiolipin occur in sera of patients with syphilis and other diseases caused by spirochetes, leprosy, systemic lupus erythematosus, and transiently in a number of acute viral infections ( Sparling, 1971 ) . Antibodies to cardiolipin have been raised in rabbits immunized with crude lipid extracts mixed with heterologous serum or a foreign protein (Eagle, 1932) or with mitochondria ( Schiefer, 1973a). Liposomes containing cardiolipin, lecithin, and cholesterol, with or without a foreign protein such as human antibody (Fowler and Allen, 1962; Aho et al., 1973) or methylated bovine serum albumin (MBSA) (Inoue and Nojima, 1967; DeSiervo, 1974) are also good immunogens. CardioOH
0
CH,-0-C-R
II
CH,-0-C-R
I
f
I
B
CH,-0-P-0-CH,
I
1
I
CH,-0-P-0-CH,
CH-OH
a
1
0 II
CH-0-C-R
I
CH,-0-C-R
t
OH
FIG.6. Structure of cardiolipin ( diphosphatidylglycerol).
230
DONALD M. MARCUS AND GERALD A. SCHWARTING
lipin-lecithin-MBSA is a good immunogen, but cardiolipin-cholesterolMBSA is very weakly immunogenic. Pure cardiolipin does not react well with antibodies and auxiliary lipids are required for optimal immunological reactivity. Cardiolipinlecithin mixtures are precipitated by antibodies (Osler and Knipp, 1957) and fix complement, but maximum sensitivity in complement fixation is achieved by using cardiolipin-cholesterol-lecithin mixtures ( Maltaner and Maltaner, 1945). The polar head groups of lecithin molecules in these liposomes can be hydrolyzed by phospholipase C. The antigenic activity of liposomes containing cardiolipin is unaffected by hydrolysis of up to 80%of the lecithin, but complete hydrolysis does reduce their activity (Kataoka and Nojima, 1969). Active liposomes cannot be prepared, however, by substituting diglyceride for lecithin. The polar head group of lecithin appears to be important for proper orientation of the molecules when the liposomes are formed, but the structure can be maintained without most of the head groups. Electron microscopic studies of lipid particles revealed that cardiolipin-lecithin-cholesterol liposomes consisted of lamellar structures surrounding a cholesterol core, whereas cardiolipin alone or cardiolipin-cholesterol particles form an irregular network without any lamellar structure ( Kanemasa, 1974). The reactions of human and rabbit antibodies to cardiolipin with molecules related structurally to cardiolipin were examined by Faure and Morelec-Coulon (1963) and Inoue and Nojima (1967, 1969). Derivitization of the free hydroxyl group or removal of one or two fatty acids from cardiolipin markedly decreased its reaction with antibody, and removal of more than two fatty acids essentially abolished its activity. The distance between the two phosphodiester groups is also an important structural feature because synthetic derivatives in which the central glycerol moiety is replaced by longer or shorter methylene chains also exhibit diminished immunological activity ( Inoue and Nojima, 1967). Cross-reactions between anticardiolipin antibodies and phosphatidyl inositol ( P I ) and nucleic acids were reported by Guarnieri (1974) and Guarnieri and Eisner ( 1974). Reciprocal cross-reactions were observed between antisera to cardiolipin and PI and the two antigens; the crossreaction of cardiolipin with anti-PI was stronger than the reciprocal reaction. Guarnieri and Eisner made the interesting observation that DNA and cardiolipin reacted equally with anticardiolipin antibodies and that all of the antibodies to cardiolipin could be absorbed by DNA. These investigators used a microflocculation assay, and it was necessary to mix the DNA with lecithin and cholesterol to detect the reaction. Ribonucleic acid was about 10-203 as effective as DNA in reacting with the cardiolipin antibodies. Guarnieri and Eisner suggested that the basis
CLYCOLIPIDS AND PHOSPHOLIPIDS
231
of the cross-reaction is a structure composed of two phosphodiester groups separated by 3 carbon atoms. They suggested also that the hemiacetal oxygen of the deoxyribose ring might be immunologically equivalent to the hydroxyl group of the central glycerol residue in cardiolipin. The reaction of rabbit anticardiolipin antibodies with mouse tissues was studied by a direct immunofluorescent technique (Kataoka and Nojima, 1968). After fixation of tissue sections with acetone-buffered saline, fluorescent staining was observed in heart, skeletal muscle, kidney, and liver in a distribution suggestive of mitochondria. Rabbit antibodies also bind to intact mitochondria isolated from a variety of tissues (Guarnieri et al., 1971; Schiefer, 1973b). In another study ( Aho et al., 1973), antibodies from rabbits with experimental syphilis or from patients with syphilis or biological false positive serologies reacted with intact mitochondria, but antibodies from rabbits immunized with cardiolipinlecithin-cholesterol liposomes coated with human antibodies did not react. The reason for the discrepancy between this study and those of Guarnieri and Schiefer is not apparent. Guarnieri et al. concluded that the polar head groups of only 9% of cardiolipin molecules of the mitochondrial membrane were accessible to antibodies. This calculation was based on a comparison of the number of cardiolipin molecules in mitochondria with the number of antibody molecules adsorbed. The calculation may not be valid if the binding of one antibody molecule obstructs access of other antibodies to adjacent cardiolipin molecules. Schiefer ( 197317) found that treatment with trypsin and pronase increased the uptake of anticardiolipin antibodies by inner mitochondrial membranes but not by intact mitochondria. The reaction of anticardiolipin antibodies with cardiolipin-containing liposomes was studied by electron spin resonance (Schiefer ct al., 1975). They used spin-labeled derivatives of stearic acid in which nitroxide groups were located near polar head groups of the phosphatides or in the hydrophobic interior of the liposomes. The mobility of the nitroxide group in ithe polar region of liposomes was decreased when the liposomes were 1 exposed to antibodies, but the nitroxidc probc in the hydrophobic region >wasunaffected. The antibody-cardiolipin interaction appears to produce i tightcr packing of the polar head groups of the phosphatides. Anticardiolipin antibodies also blocked the condensing effect of calcium on the 1 liposomes. The precise nature of the antigcnic stimulus that elicits human anti)bodies to cardiolipin is not clear. Cardiolipin is a constituent of many 1 treponemas, including the Rciter strain ( Faure and Pillot, 1960), and ithe immunogenicity of cardiolipin may be enhanced by its presence in a 1 membrane containing foreign antigens. On the other hand, the presence
232
DONALD M. MARCUS AND GERALD A. SCHWARTING
of antibodies to cardiolipin in the sera of normal elderly individuals, drug addicts, and patients with systemic lupus erythematosus, leprosy, and other diseases (reviewed by Sparling, 1971) suggests an autoimmune process. This possibility is supported by the appearance of rheumatoid factors and cryoglobulins in the sera of patients with syphilis. There is no information about the pathogenetic significance of these antibodies. Rabbits producing anticardiolipin antibodies do not develop overt disease and mitochondrial respiratory functions were not impaired by exposure to anticardiolipin antibodies in vitro (Guarnieri et al., 1971). It is unclear, however, whether complement was present during the latter experiment. The extensive cross-reaction between rabbit anticardiolipin antibodies and DNA indicates the need for a careful study of the specificity of human antibodies to these two antigens, particularly when they occur in the same patient, as in systemic lupus erythematosus.
B. PHOSPHATIDYL INOSITOL Rabbit antibodies to PI were produced by rabbits immunized with mitochondria ( Schiefer, 1973a) or liposomes containing PI-lecithincholesterol-MBSA ( Kataoka and Nojima, 1969; Guarnieri, 1974). The general serological properties of these antibodies are similar to anticardiolipin antibodies. Auxiliary lipids are necessary for complement fixation and microflocculation reactions, and hydrolysis of most of the PI in liposomes by phospholipase C does not alter the immunological reactivity of the liposomes. Kataoka and Nojima did not detect the cross-reaction of cardiolipin with anti-PI observed by Guarnieri, but the former group may not have examined many sera or used as sensitive serological techniques as Guarnieri. The flocculation of liposomes containing PI was not inhibited by myoinositol or glycerylphosphoryl inositol ( deacylated PI ). Anti-PI sera fixed complement with intact mitochondria and inner mitochondrial membranes, indicating that the polar head groups of some PI molecules are accessible to antibodies on the membrane (Schiefer, 1973b). Anti-PI antibodies are adsorbed by myelin and synaptosomes (Guarnieri, 1974). The uptake of antibodies by myelin, but not synaptosomes, was increased by performing the incubation with antibodies at 45".
C. SPHINGOMYELIN Antibodies to sphingomyelin have been produced by immunization with conjugates containing deacylated sphingosine or other haptens coupled to carrier proteins. Taketomi and Yamakawa (1966; Taketomi, 1969) coupled N-p-aminobenzyldihydrosphingosylphosphorylcholineto
GLYCOLIPIDS AND PHOSPHOLIPIDS
233
BSA or egg albumin by diazotization. Hapten-specific antibodies were demonstrated by complement fixation with hapten coupled to an unrelated protein and by passive cutaneous anaphylaxis. Teitelbaum et al. (1973) and Arnon and Teitelbaum (1974) used two haptens, dihydrosphingosylphosphorylcholine ( SPC ) and ceramide phosphorylethanolamine ( CPE ), and employed carbodiimides as the coupling agents. Hapten-specific antibodies were elicited by both conjugates, and CPE was more immunogenic than SPC. The antibodies were apparently able to react with sphingomyelin in cell membranes because they lysed sheep erythrocytes, which are rich in sphingomyelin, but not guinea pig erythrocytes, which contain very little sphingomyelin ( Arnon and Teitelbaum, 1974). IV. Concluding Remarks
During the 7 years since the last general review of lipid antigens (Rapport and Graf, 1969), much has been learned about the structure, biosynthesis, and immunological properties of glycolipids, but their biological functions remain elusive. The recurrent suggestions that they may serve to mediate cellular interactions or act as cell membrane receptors or regulatory molecules remain plausible and intriguing, but unproven. Despite the uncertainty about their functions, glycolipids offer unique experimental advantages for studies of the architecture and functional properties of cell membranes. Glycolipids are the only components of cell membranes that are readily isolated and possess a single antigenic determinant. Antisera to these determinants can be used in many ways: to identify cells that are not readily distinguished by morphological differences, such as T and B lymphocytes; to obtain data on the accessibility of specific antigenic determinants to antibodies during different physiological states, such as phases of the cell cycle, and in pathological conditions; to determine the cellular and subcellular distribution of glycolipids; to prepare afFinity columns for fractionation of cells and macromolecules; and to study model membranes containing glycolipid antigens. These studies should clarify the biological role of glycolipids and provide insight into many aspects of cell membrane structure and function.
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SUBJECT INDEX Allergen ( s ) assay of, 78-80 chemical and biological properties foods, 88-91 grass pollens, 85-86 honeybee venom, 91-92 mammalian dander, 87 mite and house dust, 88 ragweed pollen, 80-85 tree pollens, 86-87 general observations, 92-96 purified, use of, 96-100 Allogeneic differences, measurement in mixed-lymphocyte culture reaction, 124-130 Allogeneic reactions cell-mediated induction of cytotoxic effector cells, 120-123 induction of immunological memory cells, 123-124 mixed-lymphocyte culture reactions, 119-I20
major system in man, 108-110 Honeybee venom, nature of allergens, 91-92 House dust, nature of allergens, 88 Human, major histocompatibility system in, 108-110
Immunoglobulin E antibody cellular basis of response cell types, 23-28 helper function generation, 28-36 mechanisms of cell collaboration, 36-45 requirement for T and B lymphocytes, 20-23 factors essential for response adjuvant for, 15-17 genetic control, 12-15 nature and dose of antigen, 17-20 formation distribution of cells, 9-11 helminth infection and, 6-8 kinetics of response, 3-6 response in uitro, 11-12 regulation of responses experimental model, 6 2 4 7 suppression, 45-48 T cells and, 50-62 unresponsiveness of cells, 48-50
Cardiolipin, immunocheniical properties, 229-232
Food( s ) nature of allergens, 88-91
Genetic mapping, HLA complex, 183-185 Glycolipids, immunocheniical properties, 228 Glycosphingolipids, inimunochemical properties, 204-221 Glycosyl glycerides, immunochemical properties, 221-225 Grass pollens, nature of allergens, 85-86
Histocompatibility immune response, genetic control, 169-177
Leukocyte alloantigens serology, 110-113 cross-reactivity, 116-117 genetic linkage disequilibrium, 117119 system of closely linked loci, 114116 Lipoteichoic acids immunochemical properties, 226-228
Mammalian dander, nature of allergens,
87 24 1
242
SUBJECT INDEX
Mite( s), nature of allergens, 88 Mixed-lymphocyte culture allogeneic differences, measurement of, 124-130 as histocompatibility test for clinical transplantation, 177-183 induction of cytotoxic effector cells in, 120-123 induction of immunological memory cells in, 123-124 single locus concept family studies, 130-132 unrelated individuals, 132-135 specificities defined by HLA-Dhomozygous typing cells, 135 characterization of specificities, 146148 complexity of locus, 148-154 definition of typing responses, 142146 families sharing histocompatibility haplotypes, 136138 family studies, 154
identification of cells, 138-140 population studies, 162-164 role of lymphocyte subpopulations, 168-169 serological identification of alloantigens, 164-168 sources of cells, 140-142 typing of families with recombinations, 154-162 Phosphatidyl inositol, imniunochemical properties, 232 Ragweed pollen, nature of allergens, 8085 Sphingomyelin, imniunochemical properties, 232-233 Tree pollens, nature of allergens, 86-87
CONTENTS OF PREVIOUS VOLUMES Volume 1
Antibody Production by Transferred Cells
CHARLES G. COCHRANE AND FRANK J. DIXON
Transplantation Immunity and Tolerance
M. HASEK,A. LENGEROV~, AND T. HRABA
Phagocytosis
DERRICK ROWLEY Immunological Tolerance of Nonliving Antigens
RICHARDT. SMITH
Antigen-Antibody Reactions in Helminth Infections
E. J. L. SOULSBY
Functions of the Complement System
ABRAHAMG. OSLER
Embryological Development of Antigens
REED A. FLICKINGER
In Vifro Studies of the Antibody Response
ABRAM B. STAVITSKY
AUTHOR
INDEX-SUUJECT INDEX
Duration of Immunity in Virus Diseases
J. H. HALE Fate and Biological Action of AntigenAntibody Complexes
WILLIAM 0. WEICLE
Volume 3 In Vifro Studies of the Mechanism of Anaphylaxis
K. FRANKAUSTEN A N D Jorm H. HUMPHREY
Delayed Hypersensitivity to Simple Protein Antigens
P. G. H. GELLA N D B. BENACERRAF
The Role of Humoral Antibody in the Homograft Reaction
The Antigenic Structure of Tumors
CHANDLER A. STETSON
P. A. CORER AUTHOR INDEX-SUB JECT INDEX
Immune Adherence
11. S. NELSON Reaginic Antibodies
D. R. STANWORTH
Volume 2 Immunologic Specificity and Molecular Structure
Nature of Retained Antigen and its Role in Immune Mechanisms
DAN H. CAMPBELL AND JUSTINE S. GARVEY
FREDKARUSH Heterogeneity of 7-Globulins
JOHNL. FAHEY The Immunological Significance of the Thymus
J. F. A. P. MILLER,A. H. E. MAHSHALL,AND R. G. WHITE
Blood Groups in Animals Other Than Man
W. H. STONEA N D M. R. IRWIN Heterophile Antigens and Their Significance in the Host-Parasite Relationship
C. R.
JENKIN
Cellular Genetics of Immune Responses
G. J. V. NOSSAL
AUTHOR INDEX-SUB JECT INDEX
243
244
CONTENTS OF PREVIOUS VOLUMES
Volume 4
Volume 6
Ontogeny and Phylogeny of Adaptive Immunity
Experimental Glomerulonephritis: Immunological Events and Pathogenetic Mechanisms
ROBERT A. GOODAND BEN W. PAPERMASTER
EMIL R. UNANUEAND FRANK J. DIXON
Cellular Reactions in Infection
EMANUEL SUTER AND HANSRUEDY RAMSEIER Ultrastructure of Immunologic Processes JOSEPH
Chemical Suppression of Adaptive Immunity
ANN E. CABRIELSON AND ROBERTA. GOOD
D. FELDMAN Nucleic Acids as Antigens
Cell W a l l Antigens of Gram-Positive Bacteria
MACLYNMCCARTY AND STEPHENI. MORSE Structure and Biological Activity of I mmunoglobulins
SYDNEY COHENAND RODNEYR. PORTER
OTTOJ. PLESCIA AND WERNERBRAUN In Vifro Studies of Immunological Responses of lymphoid Cells
RICHARDW. DUTTON Developmental Aspects of Immunity
STERZLAND ARTHUR M. SILVERSTEIN JAROSLAV
Autoa ntibodies and Disease
H. G . KUNKEL AND E. M. TAN Effect of Bacteria and Bacterial Products on Antibody Response
J. MUNOZ AUTHOR INDEX-SUBJECT INDEX
Anti-antibodies
PHILIPG. H. CELLA N D ANDREWS. KELUS Cong1utin in and I mmunocongIutin ins
P. J. LACHMANN AUTHOR INDEX-SUBJECT INDEX
Volume 5
Volume 7
Natural Antibodies and the Immune Response
Structure and Biological Properties of Immunoglobulins
STEPHENV. BOYDEN Immunological Studies with Synthetic Polypeptides
MICHAEL SELA Experimental Allergic Encephalomyelitis and Autoimmune Disease
PHILIPY. PATERSON The Immunology of Insulin
c. G . POPE
Tissue-Specific Antigens
D. C. DUMONDE
AUTHOR INDEX-SUB J ECT INDEX
SYDNEYCOHENAND CESARMILSTEIN Genetics of Immunoglobulins in the Mouse
MICHAEL POTTERA N D ROSE LIEBERMAN Mimetic Relationships between Group A Streptococci and Mammalian Tissues JOHN
B. ZABRISKIE
lymphocytes and Transplantation Immunity
DAHCY B. WILSONAND R. E. BILLINCHAM
CONTENTS OF PREVIOUS VOLUMES
Human Tissue Transplantation
245
Phylogeny of Immunoglobulins
JOHN P. MERRILL
HOWARDM. GREY
AUTHORINDEX-SUBJECT INDEX
Slow Reacting Substance of Anaphylaxis
ROBERTP. ORANGE AND K. FRANK AUSTEN
Volume 8 Chemistry and Reaction Mechanisms
of Complement
HANS J. MULLER-EBERHARD Regulatory Effect of Antibody on the Immune Response
JONATHANW. UHR AND GORAN MOLLER
Some Relationships among Hemostasis, Fibrinolytic Phenomena, Immunity, and the Inflammatory Response
OSCARD. RATNOFF Antigens of Virus-Induced Tumors
KARL HABEL
The Mechanism of Immunological Paralysis
Genetic and Antigenetic Aspects of Human Histocompatibility Systems
D. BERNARDAMOS
D. W. DRESSER AND N. A. MITCHISON
AUTHORINDEX-SUBJECT INDEX
In Vitro Studies of Human Reaginic Allergy
ABRAHAMG. OSLER, LAWRENCEM. LICHTENSTEIN, A N D DAVIDA. LEVY AUTHORINDEX-SUBJECT INDEX
Volume 11 Electron Microscopy of the Immunoglobulins
N. MICHAELGREEN Volume 9
Genetic Control of Specific Immune Responses
Secretory Immunoglobulins
THOMAS B. TOMASI, JR., JOHN BIENENSTOCK
AND
Immunologic Tissue Injury Mediated b y Neutrophilic leukocytes
CHARLES C . COCHRANE The Structure and Function of Monocytes and Macrophages
ZANVILA. COHN The Immunology and Pathology of NZB Mice
J. B. HOWIE A N D B. J. HELYER
AUTHORINDEX-SUBJECT INDEX Volume 10 Cell Selection b y Antigen in the Immune Response
GREGORYW. SISKINDAND BARUJ RENACERRAF
HUGH 0. MCDEVITT AND BARUJ BENACERRAF The lesions in Cell Membranes Caused b y Complement
H. HUMPHREYAND ROBERTR. DOURMASHKIN
JOHN
Cytotoxic Effects of Lymphoid Cells In Vifro
PETERPERLMANN AND GORANHOLM Transfer Factor
H. S. LAWRENCE Immunological Aspects of Malaria Infection
IVORN. BROWN AUTHORINDEX-SUB JECT INDEX
246
CONTENTS OF PREVIOUS VOLUMES
Volume 12 The Search for Antibodies with Molecular Uniformity
RICHARDM. KRAUSE
Nature and Classification of ImmediateType Allergic Reactions
ELMER L. BECKER AUTHORINDEX-SUBJECT INDEX
Structure and Function of r M Macroglobulins
HENRYMETZCER Transplantation Antigens
R. A. REISFELDAND B. D. KAHAN The Role of Bone Marrow in the Immune Response
NABIH I. ABDOUAND MAXWELLRICHTER Cell Interaction in Antibody Synthesis
D. W. TALMACE, J. RADOVICH,A N D H. HEMMINCSEN The Role of lysosomes in Immune Responses
GERALDWEISSMANNAND PETERDUKOR Molecular Size and Conformation of Immunoglobulins
K E ~ HJ. DORRINCTON AND CHARLES TANFORD
Volume 14 lmmunobiology of Mammalian Reproduction
ALAN E. BEER AND R. E. BILLINCHAM Thyroid Antigens and Autoimmunity
SIDNEYSHULMAN
I mmunolog ica I Aspects of Burkitt's lymphoma GEORGEKLEIN Genetic Aspects of the Complemenl System
CHESTERA. ALPER AND FREDS. ROSEN The Immune System: A Model for Differentiation in Higher Organisms
L. HOODAND J. PRAHL AUTHORINDEX-SUBJECT INDEX
AUTHOR INDEX-SUBJECT INDEX Volume 13
Volume 15
Structure and Function of Human Immunoglobulin E
The Regulatory Influence of Activated T Cells on B Cell Responses
HANSBENNICH AND S. GUNNAR0. JOHANSSON Individual Antigenic Specificity of Immunoglobulins
JOHN E. HOPPERAND ALFRED NISONOFF In Vitro Approaches to the Mechanism of Cell-Mediated Immune Reactions
BARRYR. BLOOM Immunological Phenomena in leprosy and Related Diseases
J. L. TURKAND A. D. M. BRYCESON
to Antigen
DAVIDH. KATZ AND BARUJ BENACERRAF The Regulatory Role of Macrophages in Antigenic Stimulation
E. R. UNANUE Immunological Enhancement: A Study of Blocking Antibodies
JOSEPH D. FELDMAN Genetics and Immunology of Sex-linked Antigens
DAVIDL. GASSERAND WILLYS K. SILVERS
CONTENTS OF PREVIOUS VOLUMES
Current Concepts of Amyloid
Volume 18
EDWARDC. FRANKLIN AND DOROTHEA ZUCKER-FRANKLIN AUTHOR INDEX-SUB JECT INDEX
Genetic Determinants of Immunological Responsiveness
DAVIDL. GASSERA N D WILLYSK. SILVERS Cell-Mediated Cytotoxicity, Allograft Rejection, and Tumor Immunity
Volume 16 Human Immunoglobulins: Classes, Subclasses, Genetic Variants, and Idiotypes
J. B. NATVIGA N D H. G. KUNKEL Immunological Unresponsiveness
WILLIAM0. WEICLE Participation of lymphocytes in Viral Infections
E . FREDERICK WHEELOCKA N D STEPHENT. TOY Immune Complex Diseases in Experimental Animals and Man
c. G.
247
COCHRANEAND D. KOFFLER
The lmmunopathology of Joint Inflammation in Rheumatoid Arthritis
NATIIANJ. ZVAIFLER JECT INDEX AUTHOR INDEX-SUB
JEAN-CHARLES CERO~TINI AND K. THEODORE BRUNNER Antigenic Competition: A Review of Nonspecific Antigen-Induced Suppression
IIUGHF. moss AND DAVIDEIDINCER Effect of Antigen Binding on the Properties of Antibody
HENRYMETZCER lymphocyte-Mediated Cytotoxicity and Blocking Serum Activity to Tumor Antigens
KARL ERIK HELLSTROMAND INCEGERD HELLSTROM
AUTHORINDEX-SUBJECT INDEX Volume 19 Molecular Biology of Cellular Membranes with Applications to Immunology
Volume 17
S. J. SINGER
Antilymphocyte Serum
EUGENE M. LANCE,P. B. MEDAWAR, A N D ROBERTN. TAUB In Vitro Studies of Immunologically Induced Secretion of Mediators from Cells and Related Phenomena
L. BECKER A N D PETER M. HENSON
ELhlEH
Membrane Immunoglobulins and Antigen Receptors on B and T lymphocytes
NOEL L. WARNER Receptors for Immune Complexes on lymphocytes
VICTORNUSSENZWEIC Biological Activities of Immunoglobulins of Different Classes and Subclasses
HANS L. SPIECELBERC Antibody Response to Viral Antigens
KEITH M. COWAN Antibodies to Small Molecules: Biological and Clinical Applications
VINCENTP. BUTLER,JR., SAM M. BEISER
AND
AUTHORINDEX-SUBJECT INDEX
SUBJECTINDEX Volume 20 Hypervariable Regions, Idiotypy, and Antibody-Combining Site
J. DONALD CAPRAAND J. MICHAEL KEHOE
248
CONTENTS OF PREVIOUS VOLUMES
Structure and Function of the J Chain
MARIANELLIOTTKOSHLAND
Thymus-Independent B-Cell Induction and Paralysis
ANTONIO Amino Acid Substitution and the Antigenicity of Globular Proteins MORRIS
REICHLIN
SUBJECT INDEX
The H-2 Major Histocompatibility Complex and the I Immune Response Region: Genetic Variation, Function, and Organization
DONALDc. S H R E F F L E R CHELLAS. DAVID
COUTINHO AND
G O R A N MOLLER
AND
Volume 22 The Role of Antibodies in the Rejection and Enhancement of Organ Allografts CHARLES
Delayed Hypersensitivity in the Mouse
ALFRED J. CROWLE SUBJECT INDEX
B.
CARPENTER,
ANTHONYJ. F. D’APICE, AND ABUL K. ABBAS Biosynthesis of Cornplement HARVEY
R. COLTEN
Volume 21 Graft-versus-Host Reactions: A Review X-Ray Diffraction Studies of Immunoglobulins
STEPHENC. GREBEAND J. WAYNESTREILEIN
ROBERTO J. POLJAK Rabbit Immunoglobulin Allotypes: Structure, Immunology, and Genetics
THOMAS J. KINDT Cyclical Production of Antibody as a Regulatory Mechanism in the Immune Response
WILLIAM0. WIECLE
Cellular Aspects of Immunoglobulin A
MICHAEL E. LAMM Secretory Anti-Influenza Immunity
YA. S. SHVARTSMAN AND M. P. ZYKOV SUBJECTINDEX