ADVANCES IN
Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California
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ADVANCES IN
Immunology EDITED BY FRANK J. DIXON The Scripps Research Institute La Jolla, California
ASSOCIATE EDITORS
FREDERICK ALT K. FRANKAUSTEN TADAMITSU KISHIMOTO FRITZMELCHERS JONATHAN W. UHR
VOLUME 57
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ADVANCES IN IMMUNOLOGY, VOL. 57
Molecular Basis of Fc Receptor Function MARK D. HULEll AND P. MARK HOGARTH The Austin Research Insiiitih, Heidelbeg 3084, AustmIm
1. Introduction
Cell membrane receptors specific for the Fc portion of immunoglobulin (FcR) play an important role in immunity and resistance to infection, providing a system that couples antibody-antigen interaction with cellular effector mechanisms. Distinct cell membrane FcRs have been described for all classes of immunoglobulin, including IgG (FcyR), IgE (FceR),IgD (FcsR), IgM (FcpR), and IgA (FcaR). Of these receptors only the leukocyte FcyR and FceR have been extensively characterized. The FcyRs comprise a multimembered family of structurally homologous but distinct receptors and are expressed on the vast majority of leukocytes. The diversity ofthese receptors is reflected in the wide variety of biological responses mediated upon their binding of IgG-antigen complexes, including phagocytosis, endocytosis, antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators, and regulation of B-cell function (reviewed in Unkeless et al., 1988; Mellman et al., 1988; Kinet, 1989; Ravetch and Anderson, 1991; Van de Winkel and Anderson, 1991; Ravetch and Kinet, 1991; Van de Winkel and Capel, 1993). In contrast, the FceRs comprise only two members which are structurally unrelated to each other. The highaffinity receptor for IgE is closely related to the FcyR. This receptor is exclusively expressed on mast cells, basophils, Langerhans cells, and eosinophils, and is responsible for triggering the IgE-mediated allergic response (reviewed in Metzger et aZ., 1986; Kinet, 1990; Ravetch and Kinet, 1991). The distinct low-affinity receptor for IgE has a much wider cellular distribution and plays a role in B-cell development and IgE-dependent cytotoxicity against parasites (reviewed in $Dieselbeg, 1984; Conrad, 1990; Delespesse et aZ., 1992). In addition to the membrane-bound FcRs, soluble FcRs or immunoglobulin binding factors ( IBFs) have also been described; however, these are not discussed here (for reviews see Fridman and Sautes, 1990; Fridman et al., 1992,1993). This review focuses on studies of the murine and human leukocyte FcyR and FcsRI, with particular reference to the structural characterization of these receptors, the molecular nature of their interaction with 1
Copyright Q 1904 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
MARK D. HULETT AND P. MARKHOGARTH
Ig, and their mechanisms of signal transduction. In addition we also review aspects of FcpR, FcaR, the poly Ig receptor, the receptor for the transport of Ig in neonatal gut (FcRn), and receptors for IgD. The FcR nomenclature used throughout this review follows that proposed by Ravetch and Kinet (1991), unless stated otherwise.' II. Characterization of FcR
A. FcyR Significant advances have been made in recent years in the characterization of the receptors for the Fc portion of IgG (FcyRs) at the protein, transcript, and gene levels. Three distinct classes of mouse and human FcyR are currently recognized: FcyRI, FcyRII, and FcyRIII. These classes can be distinguished on the basis of a number of serological and biochemical criteria, including specificity and affinity for immunoglobulin, cell distribution, molecular size, and recognition with monoclonal antibodies (mAb). The cell-surface FcyRs are all integral membrane glycoproteins, with the exception of the glycosyl-phosphatidylinositol (GP1)anchored hFcyRIIIb isoform. Molecular cloning and sequence analysis of the cDNAs encoding mouse and human FcyRI (Allen and Seed, 1989; Sears et al., 1990), FcyRII (Ravetch et al., 1986; Lewis et al., 1986; Hibbs et al., 1986,1988; Hogarth et al., 1987; Stuart et al., 1987,1989; Stengelin et al., 1988; Brooks et al., 1989), and FcyRIII (Simmons and Seed, 1988; Ravetch and Perussia, 1989; Peltz et al., 1989) have indicated that they are all structurally related, containing conserved extracellular ligand-binding regions of Ig-like domains and as such belong to the Ig superfamily. FcyRI contains three Ig-like domains, whereas FcyRII and FcyRIII contain two Ig-like domains. The homology in the extracellular regions of the FcyR contrasts to the pronounced sequence differences observed in the transmembrane and cytoplasmic tail domains of these receptors. The divergence in the cytoplasmic regions of the FcyR suggests these domains are involved in triggering unique intracellular signals and, combined with selective cellular expression, presumably accounts for the diverse functions of the different FcyR classes. In the mouse, single genes encode each of the three FcyR classes (Qiu et al., 1990; Kulczycki et al., 1990; Hogarth et al., 1991; Osman et al., 1992); whereas in the human, multiple genes have been described for each class: three FcyRI genes, three FcyRII genes, and The prefixes m, h, and rt will be used to denote mouse, human, and rat, respectively.
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
3
two FcyRIII genes, which encode multiple forms of these receptors (Ravetch and Perussia, 1989; Qiu et al., 1990; Van de Winkel et al., 1991; Ernst et al., 1992). The genes encoding the mouse and human FcyR are all located in the same region of chromosome 1 in both species (Sammartino et al., 1988; Grundy et al., 1989; Peltz et al., 1989; Qiu et al., 1990; Ernst et al., 1992; De Wit et al., 1993) with the exception of the mouse FcyRI gene on chromosome 3 (Osman et al., 1992), and have clearly arisen by duplication and divergence of a common ancestral FcyR gene. The following sections describe the properties of the three mouse and human FcyR classes, which are summarized in Tables I and I1 for FcyRI, Tables IV and V for FcyRII, and Tables VI and VII for FcyRIII, aspects of which have been reviewed in Unkeless et al. (1981,1988),Anderson and Looney (1986), Mellman et al. (1988), Van de Winkel and Anderson (1991),Ravetch and Anderson (1991),Ravetch and Kinet (1991), and Van de Winkel and Cape1 (1993). 1. FcyRI a. Biochemical and Molecular Structure
Human FcyRI (CD64)is classically defined as a 72-kDa glycoprotein (Anderson et al., 1982,1986; Frey and Engelhardt, 1987; Dougherty et al., 1987; Peltz et al., 1988a), which following removal of N-linked carbohydrate has a protein core of 55 kDa (Peltz et al. 1988a) (Table I). In contrast, reports of the molecular mass of mouse FcyRI have been conflicting and include the description of a 70-kDa protein (Lane et al., 1980; Lane and Cooper, 1982), a 100-kDa protein of two 50kDa subunits (Fernandez-Botran and Sukuki, 1986; Hirata and Suzuki, 1987; Kagami et al., 1989), and a 57-kDa protein (Loube et al., 1978). However, a recent study has definitively determined the molecular weight of mFcyRI to be similar to that described for its human homologue, identifying the receptor as a 70-kDa phosphoprotein on mFcyRI-transfected Chinese hamster ovary cells and on the myeloid cell line, J774 (Quilliam et al., 1993). The cDNA cloning of human and mouse FcyRI has demonstrated that the above forms of the receptor are structurally unique among the FcyR, containing an extracellular region of three Ig-like domains, in contrast to the two domain structures of FcyRII and FcyRIII (Table 11). The third extracellular domain is distinct, whereas the first two domains are homologous to the extracellular domains of FcyRII and FcyRIII, suggesting that the unique IgG binding characteristics of FcyRI are conferred by domain three. Indeed, this has subsequently been demonstrated (see below and Hulett et al., 1991).
4
MARK D. HULETT AND P. MARKHOGARTH TABLE I CHARACTERISTICS OF FcyRI Genes Human Characteristic
hFcyRIA
Isofoms
hFcyRIa
Alleles Chromosome localization Ig-like domains Receptor Associated subunits Molecular mass (kDa) Apparent Protein backbone Affinity for IgG' (&)
-
Specificity hIgG mIgG Cellular distribution
Regulation of expression
hFcyRIB
Mouse mFcyRI
hFcyRIC
hFcyRIb 1, hFcyRIc mFcyRI hFwRIb2
-
-
lq21.1
3
3 TM y chain FcsRI
2 TM, S
2 S
-
3 TM ?
72 40 108-109
ND 22 ND, 4 M-'
ND 24 ND
72 42 107-108 M -
ND ND
3>1>4>>>2 2a>>>l, 2b, 3 Monocytes Macrophages Neutrophils
ND
IFN-y
lq21.1
lq21.1
M-1
2*,
3>1>4>>>2 ND 2a=3>>>1,2b ND Monocytes Macrophages Neutrophilsb Eosinophils' IFN? t , IL-10 t c ND G-CSF t , IL-4 J.
0 7
-
t
Note. TM, transmembrane; S, soluble; ND, not determined. Monomeric I&. * Expression inducible with 1FN-y. Expression upregulated on monocytes with IL-10 or IFN-y, downregulated with IL-4. Expression also upregulated on neutrophils with G-CSF.
In the human, three distinct hFcyRI cDNA clones were initially isolated using a ligand-mediated expression cloning procedure, designated p90, p135, and p98/X2 (Allen and Seed, 1989).All three clones encode integral membrane glycoproteins with an extracellular region of 292 amino acids comprising three Ig-like domains, a single membrane spanning domain of 21 amino acids, and an intracytoplasmic domain of either 61 (p90 and p135) or 31 amino acids (p98/X2). The p90 and p135 cDNA clones are identical in their coding regions, with the exception of two nucleotide substitutions that result in two amino acid differences in the first extracellular domain, suggesting these two clones represent different polymorphic forms of the receptor. These
5
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION TABLE I1 FcyRI GENES AND TRANSCRIPTS Name h F q RIA
hFcy RIB
hFcy RIC
Gene structure' L1 L2 D1
D2
I
I
D
Transcripts
D3
I
I
L1 L2 D1 I
I
I D2 I
hFcy RIbl [L11L2 D1 h F q RIb2
I
TM/C hFcy RIc ~ - ~ L l l L 2D1
I
TM/C mFcy RI p - - [ L l I L 2 I D1
I D2 I D3 I TM/C 1
-1
I
L1 L2 D1 I
hFcy RIa L11 L2 I D l
TM/C
D2
I
D3
n
I
D2
D3
I TM/C
I TM/C I
1
I
stoD in D3
stop in D3
D2
I A3 I TM/C]
stop
L1 L2 D1 mFcy RI
I
I
D2 I
D3
I
Exons shown as boxes, translated regions shaded, untranslated regions open. L, leader peptide; D, extracellular domain; TM, transmembrane; C, cytoplasmictail coding regions; PA, polyadenylation site. Alternate splicing of D3 exon.
amino acid substitutions do not affect the IgG binding characteristics of the receptors (Allen and Seed, 1989). The sequence of the p98/X2 cDNA clone diverges markedly from the other two cDNAs at its 3' end, becoming a complex pattern of repeats of upstream sequences, which encodes a divergent cytoplasmic tail. The authenticity of this clone has therefore been questioned on the basis of the suggestion that it may have resulted from a cloning artifact (Ravetch and Kinet, 1991). The subsequent cloning of the hFcyRI genes supports this conclusion, as the gene sequences do not account for such a transcript (Ernst et al., 1992). Three hFcyRI genes (designated hFcyRIA, B, and C) have been isolated (Van de Winkel et al., 1991; Ernst et aZ., 1992). The genes demonstrate a high degree of similarity, all containing an identical introdexon structure comprising six exons; two exons encoding the 5'-untranslated region (UTR) and leader sequence, one exon for each Ig-like domain, and a single exon for the transmembrane, cytoplasmic tail and 3' UTR (Table 11). Each gene spans 9.4 kb, and maps to chromosome lq21.1 (Ernst et al., 1992; Osman et al., 1992; De Wit et al., 1993; Dietzsch et al., 1993). Of the three genes, only hFcyRIA encodes an integral membrane receptor with three Ig-like domains
6
MARK D. HULETT AND P. MARKHOGARTH
(FcyRIa, the 72-kDa form), as both the hFcyRIB and IC genes contain translation termination codons in the exon encoding the third extracellular domain. Transcripts derived from the hFcyRIB and IC genes containing these stop codons have been described (hFcyRbl and hFcyRIc, respectively), and these may code for soluble receptors; however, the existence of such receptor proteins has not yet been demonstrated. In addition, an alternatively spliced product from the hFcyRIB gene has been reported (hFcyRIb2), in which the third extracellular exon is spliced out to produce a transcript encoding a twodomain integral membrane receptor (Ernst et al., 1992; Porges et al., 1992). Such a receptor would be expected to bind IgG, however, with low affinity, based on the observation that the first two domains of mouse FcyRI function as a low-affinity receptor (Hulett et al., 1991). Indeed, upon transfection into COS cells hFcyRIb2 binds only IgG complexes and not monomeric IgG (Porges et al., 1992). However, the expression of hFcyRIb2 on the surface of hematopoeitic cells has not been demonstrated. The transcript from the hFcyRIA gene resembles that of the hFcyRI cDNA clones p135 and p90 described previously (Allen and Seed, 1989), with the exception of two amino acid substitutions in the first extracellular domain and a single substitution in the cytoplasmic tail (Ernst et al. 1992). Recently, hFcyRIa has been shown to associate with homodimers of the y-subunit of the high-affinity receptor for IgE, FceRI (Ernst et al., 1993; Masuda and ROOS, 1993). This association was observed in monocytes and the myelomonocytic cell line U937 and could be reconstituted by cotransfection of the hFcyRIa and FceRI-y-subunit cDNAs into COS cells. It should be noted that the cell-surface expression of hFcyRIa is not dependent on association with FceRIy (Allen and Seed, 1989; Ernst et al., 1993). This finding adds FcyRI to the growing list of leukocyte FcR that are known to associate with FceRIy homodimers. The association of y with FceRI and FcyRIII has been well characterized, and more recent studies have now suggested not only hFcyRIa, but hFcaRI (L. Pfefferkorn, personal communication) and possibly some forms hFcyRII (Masuda and Roos, 1993)also associate with the y-subunit (see below). A single mouse FcyRI cDNA has been isolated and encodes a receptor homologous to hFcyRIa, comprising an extracellular region of 273 amino acids containing three Ig-like domains, a single transmembrane region of 23 amino acids, and a cytoplasmic tail of 84 amino acids (Sears et al., 1990).Comparison of the predicted amino acid sequences of mouse and human FcyRIa reveals an overall 75% amino acid identity in the extracellular regions and transmembrane domain, yet diver-
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
7
gence is seen in the cytoplasmic tails which are only 25% identical, with the mouse FcyRI tail containing an additional 23 amino acids (Sears et al., 1990). In contrast to the existence of multiple hFcyRI genes, only a single mouse FcyRI gene has been isolated, the structure of which is conserved with that of the human FcyRI genes, comprising six exons of identical organization spanning 9 kb (Table 11) (Osman et al., 1992). The mFcyRI gene has been mapped to chromosome 3 and is located in a conserved linkage group which contains the genes for CD1, LFAS, CD2, and the ATPase gene, which is syntenic with the region spanning the centromere and the proximal long- and shortarm regions of human chromosome 1 (Osman et al., 1992; Oakey et al., 1992; Dietzsch et al., 1993).
b. Ligand Affinity and Specificity In addition to the unique structure of FcyRI, this class of FcyR is also functionally distinct as it is the only FcyR that binds monomeric IgG with high affinity and as such is referred to as the high-affinity FcyR. Scatchard analysis of the direct binding of monomeric IgG has indicated that human FcyRI displays an equilibrium affinity constant (K,) of -z 108-109 M-' (Anderson and Abraham, 1980; Anderson, 1982; Fries et al., 1982; Kurlander and Batker, 1982; Cohen et al., 1983; Perussia et al., 1983; AlIen and Seed, 1989). Human FcyRI exhibits a specificity for hIgGl and hIgG3, binding monomeric forms of these isotypes. The receptor also binds hIgG4, but with a lower affinity and does not bind hIgG2 (Anderson and Abraham, 1980;Woof et al., 1986). The specificity of hFcyRI for mouse IgG is distinctive among the human FcyR, binding only the IgG2a and IgG3 isotypes (Anderson, 1982; Perussia et al., 1983; Jones et al., 1985; Van de Winkel et al., 1987; Ceuppens et al., 1988). The binding of aggregated IgG by FcyRI is reported to be of a similar affinity to monomeric IgG (Cosio et al., 1981; Kurlander and Batker, 1982; Woof et al., 1986). This raises the question of how FcyRI is able to distinguish IgG-coated particles in vivo as presumably due to the high serum levels of monomeric IgG, the receptor would be expected to be continually saturated with ligand. The reported upregulation of the receptor by IFN-y at sites of inflammation may therefore be crucial in the function of FcyRI (see below). The mouse homologue of hFcyRI exhibits many of the unique IgG binding characteristics of hFcyRI. The affinity of mFcyRI for monomeric IgG is also high; however, it is somewhat lower than that of hFcyRI, with a K, in the order of 107-108M-' (Unkeless and Eisen, 1975; Sears et aE., 1990; Hulett et al., 1991). Mouse FcyRI is unique as
8
MARK D. HULETT AND P. MARKHOGARTH
it is the only FcyR that binds a single mIgG class-mIgG2a-making it distinctive from even its human homologue which also binds mIgG3 (Haeffner-Cavaillon et al., 1979b; Sears et al., 1990; Hulett et al., 1991). However, the binding of hIgG subclasses by mFcyRI is similar to that by hFcyRI, with hIgGl and IgG3 binding preferentially over hIgG4 and no binding of hIgG2 (Haeffner-Cavaillon et al., 1979a). c. Cell Distribution and Monoclonal Antibodies Human FcyRI is constitutively expressed on monocytes and macrophages and can be selectively induced on neutrophils and eosinophils with IFN-y, which also upregulates expression on monocytes and macrophages (Guyre et al., 1983; Perussia, 1987; Shen et al., 1987; Pan et al., 1990; Hartnell et al., 1992). Indeed, IFN-.)Iresponse elements have recently been identified in the promoter of the hFcyRIB gene (Pearse et al., 1991; Benench et al., 1992). In a similar manner to IFNy , IL-10 also enhances hFcyRI expression on monocytes (Te Velde et al., 1992). However, in contrast to both IFN-y and IL-10, IL-4 has been shown to downregulate monocyte FcyRI expression (Te Velde et al., 1990). It has also been reported that G-CSF can upregulate hFcyRI expression on neutrophils (Repp et al., 1991). A number of mAb to hFcyRI have been reported, including 32.2 (Anderson et al., 1986), FR51 (Frey and Engelhardt, 1987), 10.1 (Dougherty et al., 1987), 197.1,22,62, and 44.1 (Guyre et al., 1989) (Table 111). All of the mAb have been shown to recognize epitopes distinct from the IgG binding site, with the exception of the FR51 and 10.1 mAb which block IgG binding to hFcyRI, although 10.1 blocks binding only partially (Frey and Engelhardt, 1987; Dougherty et al., 1987). The nonblocking mAb, 22, 32.2, 44.1, and 62, are specific for epitopes distinct from the ligand binding site, where mAb 22 and 44.1 define one epitope and 32.2 and 62 define a second. Determination of the cellular distribution of mFcyRI has been complicated by the lack of anti-mFcyRI mAb, combined with the overlapping expression of other FcyR classes which also bind mIgG2a (originally thought to bind only FcyRI). However, despite these problems, mFcyRI has been identified umambiguously on macrophages and monocytes (Unkeless and Eisen, 1975; Walker, 1976; Unkeless et al., 1979). IFN-y has also been demonstrated to upregulate the expression of mFcyRI on macrophages (Sivo et al., 1993; and N. Osman, personal communication).
d . Polymorphism A functional polymorphism of hFcyRI has been described with the identification of some members of a Belgium family which appear to
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
9
TABLE 111 FcR MONOCLONAL ANTIBODIES Epitope in relation to binding site Receptor hFcyRI mFcyRI hFcyRII
Blocking Fr51, 10.1
Nonblocking 32, 197, 22,44,62
-
-
CIKM5', 8.2c
mFcyRIII hFcsRIa
IV-3, KuFc79, 41H16"sb 2E14 KB61b, ATIOb 7.3@, 8.7b,8.26 2.4G2, Ly-17.2 3G8,4F7, VEPI3,1D3 GRM-ld, B73.1e, CLB-GRANII" Leulla, Leullb 2.4G2 15A5, 12E7,6F7,4B4
hFcaRI
My43
mFcyRII hFcyRIII
-
BW20912
2237, llB4,5D5,8C8 29C9,39D5,3B4 A3, A59, A62, A77
Specific for hFcyRIIa HR isoform. Preferential binding to B-cell FcyRII. Nonblocking onIy in Fab form. Specific for hFcyRIIIb NA-2 isoform. Specific for hFcyRIIIb NA-1 isoform.
lack hFcyRI on their blood monocytes. This was demonstrated as monocytes from these individuals did not bind mIgC2a or mIgG3 in an FcyRI-dependent anti-CD3-induced T-cell proliferation assay. Furthermore, their monocytes did not bind the anti-hFcyRI mAb 32.2 and 44.1, which detect two distinct epitopes of the receptor (Ceuppens et al., 1988; Ceuppens and van Vaecjk, 1989).In addition, stimulation of both monocytes and neutrophils from these individuals with IFNy , which strongly upregulates FcyRI expression (see above), did not induce FcyRI on these cells (Ceuppens et al., 1988).The absence of hFcyRI on the cells of these individuals did not, however, appear to alter their immune function or increase their susceptibility to infection. This suggests that the functions performed by FcyRI can be compensated for by the other FcyR classes, raising the question as to the functional importance of FcyRI. The molecular basis of this polymorphism has yet to be determined. Comparison of cDNA and genomic sequences also suggests genetic polymorphisms of hFcyRIa, specifically two amino acid substitutions in the first extracellular domain and one in the cytoplasmic tail (see above) (Allen and Seed, 1989; Ernst et al., 1992). A polymorphism has also recently been described for mFcyRI, whereby the nonobese diabetic (NOD) mouse strain was found to
10
MARK D. HULETT AND P. MARKHOGARTH
express a form of FcyRI containing 17 amino acid substitutions and a premature stop codon at position 337 which results in a deletion of 73 amino acids in the cytoplasmic tail (Prins et al., 1993).This mutant form of FcyRI demonstrated a 73% reduction in the turnover of cellsurface receptor-antibody complexes. Interestingly, the mutant FcyRI allele was shown to be tightly linked to a diabetic phenotype, suggesting that defective FcyRI function may play a role in susceptibility to the disease. 2. FcyRll a. Biochemical and Molecular Structure Human FcyRII (CD32)has been characterized as a 40-kDa glycoprotein (Cohen et al., 1983; Rosenfeld et al., 1985; Looney et al., 1986a; Van de Winkel et al., 1989; Ierino et al., 1993), with a putative protein core of 36 kDa, determined following treatment with endoglycosidaseF (Van de Winkel et al., 1989). Mouse FcyRII is more heterogeneous in size, with a molecular weight ranging from 40 to 60 kDa (Mellman and Unkeless, 1980; Hibbs et al., 1985; Holmes et al., 1985) (Table IV). The cDNA cloning of mouse and human FcyRII has demonstrated that this class of FcyR comprises multiple isoforms in both species. The receptor isoforms are all integral membrane glycoproteins, with the exception of a single putative soluble receptor in both the human and mouse, and contain extracellular regions of two Ig-like domains. The extracellular and transmembrane domains are highly conserved, yet their intracytoplasmic regions differ considerably, suggesting that the different isoforms of FcyRII are likely to transduce different signals to mediate different cellular responses (see Section IV). The cloning of human FcyRII cDNAs predicted the presence of multiple receptor isoforms (Stuart et al., 1987,1989; Hibbs et al., 1988; Stengelin et al., 1988, Brooks et al., 1989; Seki, 1989; Warmerdam et al., 1990; Rappaport et al., 1993), and the source of the heterogeneity was subsequently demonstrated at the genomic level with the cloning of three distinct genes, FcyRIIA, IIB, and IIC (formally hFcyRIIa, IIb, and IIa’, respectively), encoding a total of six transcripts (Table V) (Qiu et al., 1990; Warmerdam et al., 1993). The three genes are similar in structure, each comprising eight exons; two exons encode the 5’ UTR and leader sequence, one exon for each of the Ig-like domains and the transmembrane region, and three exons encode the cytoplasmic domain and 3’ UTR (Qiu et al., 1990). The existence of multiple exons encoding the transmembrane, cytoplasmic tail and the 3’ UTR regions of the human FcyRII genes (and the mouse FcyRII
TABLE IV
CHARACTERISTICS OF FcyRII Genes Human Characteristic
hFcyRIIA
Isoforms Alleles Chromosome localization Ig-like domains Receptor topology Associated subunitsb Molecular mass (kDa) Apparent Protein backbone Affinity for I@ (&) Specificity hIgG
hFcyRIIa1, hFcyRIIa2 HR, LR" 1q23-24
mIgG Cellular distributionf
Regulation of expression
hFcyRIIB
Mouse mFcvRII
hFcyRIIC
hFcyRIIb1, b2, b3
hFcyRIIc
1q23-24 2 TM
1q23-24 2 TM ?
mFcyRIIbl, b2, b3 Ly-17.1, Ly-17.2 1 2 TM, TM, S ?
40 31
40 29,27
4 0 7 M-1
4 0 7 M-1
40 31 ND
40-60 33,29 4 0 7 M-'
LR 3>1 = 2>>>4 HR 3>1>>2>4 LR 2a=2b>>l HR2a=lb=1 Monocytes Macrophages Neutrophils Platelets Langerhans cells IL-4 1
3>1>4>2d
ND
3>1>2>>4
2a= 2b>l
ND
1=2a=2b>>>3"
Monocytes (IIbl, IIb2) Macrophages B cells (IIbl, IIb2)
Monocytes Macrophages Neutorphils B cells
Monocytes, mast cells Macrophages, platelets Neutrophils, B cells
ND
ND
ND
2
TM, S ?
?
Notes. TM, transmembrane; S, soluble; ND, not determined. a HR, high responder; LR, low responder. F c E R I reported ~ to associate with FcyRII, but isoform unknown. Monomeric I&. Only determined for hFcyRIIbl isoform. mFcyRII also binds mIgE with low affinity. 'Cellular distribution not completed for all FcyRII isoforms and all cell types.
-
12
MARK D. HULETT AND P. MARKHOGARTH TABLE V FmRII GENESAND TRANSCRIPTS
Name
Transcripts
Gene structurp h F q RIIal
L1 L2 D1
D2
TM Clb
h F q RIIA
I L11 L2 I D1 I D2
ITM IC2 I C31 hFcy RIIa2 L11 L2 I D1 I D2 I C21 C3 h F q RIIbl I L l I L 2 I D l I D2ITMIC11C21C31 h F q RIIb2 I L ~ ~ L ~ I D ~I TI MDI c z~ I c ~ ] h F q RIIb3 [ L l l D1 I D2l TM I C2 I C31
1
L1
L2 D1
D2
TM C1 C2 C3 p~
h F q RIIB
I
-I
hFcy RIIc L l ] L2 I D1 I D2 ITM lC2l ~ 3 1
h F q RIIC
mFcv RIIbl
L1 L2 L3
m F q RII
n
LA D1
~m
D2
TM C1 C2 C3
Exom shown as boxes, translated regions shaded, untranslated regions open. alternate spicing indicated. L, leader peptide: D, extra-
cellular domain, TM, transmembrane;C, cytoplasmic tail coding regions;PA, polyadenylation site. C1 exon is cryptic (always spliced out in hFcyRIIA and hFcyRIIC).
gene, see below) is unique to this FcyR class as the FcyRI and FcyRII genes in both species contain a single exon encoding this region. The human FcyRII genes have been located to band q23-24 on chromosome 1(Sammartino et al., 1988; Grundy et al., 1989; Qiu et al., 1990) and are linked with the human FcyRIII genes on a 200-kb genomic fragment (Peltz et al., 1989). The hFcyRIIA gene (formally hFcyRIIa) encodes three transcripts, two of which arise through the use of alternate polyadenylation sites, producing either a 1.4- or 2.4-kb mRNA (Stuart et al., 1987; Hibbs et al., 1988; Stengelin et al., 1988, Brooks et al., 1989; Qiu et al., 1990; McKenzie et al., 1992), which encode identical integral membrane receptors (hFcyRIIal), and a third transcript (hFcyRIIa2) encoding a putative soluble hFcyRIIa product that is believed to arise from alternate splicing of the transmembrane region encoding exon ( Warmerdam et al., 1990; Rappaport et al., 1993; Astier et al., 1994).The predicted hFcyRIIal receptor contains an extracellular region of two Ig-like domains spanning 178 amino acids, a single transmembrane domain
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
13
of 29 amino acids, and a 76-amino acid cytoplasmic tail. The soluble hFcyRIIa2 receptor would be identical to the hFcyRIIa1 receptor, but lacking the 29-amino acid transmembrane region. The hFcyRIIB gene (formerly hFcyRIIb) encodes three distinct transcripts (bl, b2, and b3), which arise by alternate splicing (Brooks et al., 1989; Qiu et al., 1990). The b l and b2 isoforms are produced as a result of alternate splicing of the first cytoplasmic tail encoding exon (Brooks et al., 1989; Qiu et al., 1990; Hogarth et al., 1991). The hFcyRIIb3 transcript arises through alternate splicing of the second exon encoding the leader peptide cleavage site. The predicted hFcyRIIb1 receptor comprises a two Ig-like domain extracellular region of 179 amino acids, a single transmembrane domain of 23 amino acids, and a cytoplasmic tail of64 amino acids. The hFcyRIIb2 receptor is identical to hFcyRIIbl except for the deletion of 19 amino acids from the cytoplasmic tail as a result of alternate splicing of the first cytoplasmic tail encoding exon. The mature form of the hFcyRIIb3 receptor, if expressed, would be identical to hFcyRIIb1, differing only by a 7 amino acid deletion in the leader sequence corresponding to the second exon (Brooks et al., 1989). A single transcript has been reported from the hFcyRIIC gene (formally hFcyRIIa’) (Brooks et d.,1989),which predicts a receptor almost identical to hFcyRIIa, with an extracellular region of 178 amino acids, a transmembrane domain of 29 amino acids, and a cytoplasmic tail of 75 amino acids. The protein products predicted from the cDNAs of the three hFcyRII genes are all closely related, displacing an overall 85% amino acid identity in their extracellular and transmembrane regions (>95% if only the extracellular regions are compared) and only diverge in their leader sequences and cytoplasmic tail regions. The hFcyRIIa and hFcyRIIc receptors differ only in their leader sequences; the leader sequence of hFcyRIIc is homologous to that of hFcyRIIb, whereas the leader sequence ofhFcyRIIa is related to that of hFcyRII1. The hFcyRIIb receptors differ markedly from both FcyRIIa and IIc in their cytoplasmic tail regions, where after the first 10-12 residues, the hFcyRIIb sequence diverges from that ofthe FcyRIIalIIc receptors (Brooks et al., 1989; Qiu et aZ., 1990). Allelic forms of hFcyRIIa have been described which further increase the diversity of hFcyRII. These allelic variants were identified on the basis of a functional polymorphism for the binding of mIgGl or human IgG2 and termed the high-responder (HR) and lowresponder (LR) isoforms of FcyRIIa. The molecular basis of the polymorphism has been defined through recent cDNA cloning studies
14
MARK D. HULETT AND P. MARKHOGARTH
(Clark et at., 1989; Warmerdam et al., 1990; Tate et al., 1992) (see below). A recent report has suggested that hFcyRII, like hFcyRI and hFcyRIII, associates with the y-subunit ofFcER1; however, this association is not required for celI-surface expression (Masuda and ROOS, 1993). In contrast to hFcyRII, only two distinct murine FcyRII cDNAs encoding integral membrane proteins have been cloned, FcyRIIbl and FcyRIIb2 (formally FcyRIIPl and FcyRIIP2) (Table V) (Ravetch et al., 1986; Lewis et al., 1986; Hogarth et al., 1987). These cDNAs encode identical receptors, with the exception of a 47-amino acid inframe deletion in the cytoplasmic domain of FcyRIIb2. Both receptors comprise an extracellular region of two Ig-like domains spanning 180 amino acids, a single transmembrane domain of 26 amino acids, and cytoplasmic tails of either 94 (mFcyRIIb1) or 47 (mFcyRIIb2) amino acids. A third mFcyRII cDNA has also recently been isolated (designated mFcyRIIb3, but distinct from hFcyRIIb3) and encodes a molecule identical to mFcyRIIb2; however, it lacks the transmembrane region and thus encodes a soluble form of mFcyRII (Tartour et al., 1993). The cloning of a single mouse FcyRII gene indicated that the b l and b2 isoforms arise by differential splicing of the 141-bp first cytoplasmic tail encoding exon (corresponding to the 47 amino acid insertion) in an analogous manner to the two human FcyRIIB gene products: hFcyRIIb1 and b2. The b3 isoform arises by splicing of the transmembrane and first cytoplasmic tail encoding exons (Qiu et al., 1990; Kulczycki et al., 1990; Hogarth et al., 1991).The mFcyRII gene comprises 10 exons spanning 18 kb; 4 exons encode the 5' UTR and leader sequence, single exons encode each of the two Ig-like domains and the transmembrane region, and 3 exons encode the cytoplasmic tail and 3' UTR (see Table V). The mFcyRII gene maps to the Ly-17 locus on chromosome 1 (Davidson et al., 1983; Holmes et al., 1985; Hibbs et al., 1985) and is linked to mFcyRIII on a 160-kb genomic fragment (Kulczycki et al., 1990). The mouse FcyRIIbl, IIb2, and IIb3 receptors demonstrate an overall 60% amino acid identity with the hFcyRII receptors in their extracellular regions. mFcyRIIb1 and IIb2 are clearly most closely related to hFcyRIIbl and b2; the FcyRIIbl receptors exhibit an overall 59% amino acid identity across their entire lengths, and the FcyRIIb2 receptors display 57% identity (Brooks et al., 1989). Two allelic forms have been described for mouse FqRII, identified originally with mouse monoclonal alloantibodies and known as the Ly-17 polymorphism. The molecular basis of the polymorphism has been defined as a two residue difference in the second extracellular domain of mFcyRII (see below).
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
15
The original mFcyRII clone (mFcyRIIP) was isolated at the same time as another distinct yet homologous clone that exhibited 95% identity in its extracellular region, yet contained divergent leader peptide, transmembrane, and cytoplasmic tail regions (Ravetch et al., 1986). The receptor encoded by this cDNA bound the anti-mFcyRII mAb 2.4G2 and exhibited identical ligand binding characteristics to mFcyRII and as such was designated an FcyRII isoform, FcyRIIa (Weinshank et al., 1988).This receptor has subsequently been reclassified as mFcyRIII, based on its homology to hFcyRIII in cDNA sequence and gene structure, and is discussed below.. The isolation and determination of the introdexon structure of the human and mouse FcyRII and FcyRIII genes has suggested that these two receptor classes have arisen by two evolutionary pathways from an ancestral FcR gene, producing the low-affinity FcyR receptors (Qiu et al., 1990; Kulcyzcki et al., 1990). Mouse FcyRII and FcyRIII have been proposed as the prototype receptors for these two classes (Qiu et al., 1990). As described, the mouse FcyRIII gene structure differs from the FcyRII gene as it contains a single exon encoding the transmembrane, cytoplasmic tail, and 3’ UTR, in contrast to the four exons encoding these regions in the FcyRII gene (Kulcyzckiet al., 1990).The FcyRII evolutionary stream includes only the FcyRII genes, while the FcyRIII stream also includes the FcERI a-chain gene on the basis of similar introdexon organization (Qiu et al., 1990; Ye et al., 1992).The FcyRI gene is structurally unique as it contains an additional exon encoding a third extracellular domain, although the transmembrane and cytoplasmic tail regions are encoded by one exon, thus the gene structure most closely resembles that of FcyRIII (Allen and Seed, 1989; Sears et al., 1990; Qiu et al., 1990; Osman et al., 1992; Ernst et al., 1992). It has been proposed that the FcyRIIA gene has arisen through a recombination event between the mouse FcyRII and FcyRIII primordial genes, whereby the 5‘ end of the gene has been derived from FcyRIII and the 3’ end from FcyRII (Qiu et al., 1990). Based on the gene structures and cDNA sequences of the mouse and human low-affinity FcyR, an order of human FcyRII gene evolution has been suggested, with the order of homology to mouse FcyRII as IIB > IIC > IIA (Qiu et al., 1990).An alternative theory for the evolution of the human FcyRII genes has also been proposed, which suggests that the human FcyRIIC gene was generated by an unequal crossover event between FcyRIIA and FcyRIIB (Warmerdam et al., 1993).This theory implies the order of human FcyRII gene evolution as FcyRIIB > FcyRIIA > FcyRIIC, which is in contrast to that proposed above (Qiu et d.,1990).However, these findings clearly demonstrate that the multiple human FcyRII genes have arisen via the pro-
16
MARK D. HULETT AND P. MARKHOGARTH
cesses of gene duplication, divergence, and recombination, from a primordial FcyRII gene resembling the single mouse FcyRII gene.
b. Ligand Afinity and Spect$city In contrast to FcyRI, FcyRII demonstrate a significantly lower affinity for ligand and, together with FcyRIII, comprise the low-affinity FcyRs (Table IV). Human FcyRII binds monomeric IgG poorly (K,< lo7A4-') and essentially only interacts with IgG complexes (Cohen et al., 1983; Kurlander et al., 1984; Rosenfeld et al., 1985; Rosenfeld and Anderson, 1989; Van de Winkel et al., 1989). However, it has been reported that the affinity of hFcyRII for IgG can be influenced by proteases, which increase affinity for IgG (Van de Winkel et al., 1989,199013;Tax and Van de Winkel, 1990).Early determinations of the specificity of hFcyRII for different subclasses of IgG produced some conflicting results, which was due in part to the coexpression of FcyRII with other FcyR and also the unrecognized heterogeneity of this receptor class, now known to comprise a number of different isoforms some of which exhibit functional polymorphisms (see below). Experiments performed prior to the cloning of hFcyRII suggested that this class of FcyR preferentially bound hIgGl and hIgG3 and to a lesser extent hIgG2 and hIgG4 (Dickler, 1976; Karas et al., 1982; Anderson and Looney, 1986). Human FcyRII was also shown to bind mouse isotypes IgGl and IgG2b (Abo et al., 1984; Tax et al., 1984; Jones et al., 1985; Looney et al., 1986a).The subsequent cDNA cloning of hFcyRII has enabled examination of the specificity of individual isoforms using transfection systems; however, data on the binding of all the isoforms are still incomplete. The hFcyRIIaLRand hFcyRIIaHRisoforms have been shown to display distinct specificities for both human and mouse IgG isotypes (Warmerdam et al., 1990,1991; Tate et al., 1992). Examination of human IgG isotype binding demonstrates that both the HR and LR isoforms bind hIgG3, hIgG1, but not hIgG4. However, these isoforms differ markedly in their binding of hIgG2, with hFcyRIIam exhibiting strong binding, in contrast to hFcyRIIaHRwhich binds hIgG2 weakly (Warmerdam et al., 1991). Examination of mouse IgG isotype binding indicates that both the HR and LR isoforms bind mIgG2a and mIgG2b, whereas only hFcyRIIaHRbinds mIgGl strongly (Warmerdam et al., 1990; Tate et al., 1992).The molecular basis of the differential binding of mIgGl and hIgG2 by these two isoforms has been determined (see below). The specificity of hFcyRIIb1 has also been defined recently, with the avidity of binding of hIgG istoypes following the order
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
17
hIgG3 = h I g G l > > hIgG2 > hIgG4 and mouse isotypes IgG2a = mIgG2b > mIgG1 (Warmerdam et al., 1992). The hFcyRIIb2 and b3 isoforms have not yet been examined; however, as they contain identical extracellular regions to hFcyRIIbl it is likely that they have similar IgG binding specificities. Interestingly, the binding of mIgG1 and hIgG2 to hFcyRIIbl was shown to be temperature sensitive, with the binding of complexes of these isotypes increasing significantly upon raising the temperature from 4 to 37°C (Warmerdam et al., 1992). Mouse FcyRII, like hFcyRII, binds monomeric IgG with a low affinity that is essentially undetectable using direct binding methods (K,< 10 M - I ) (Unkeless et al., 1988; Mellman et al., 1988; Hulett et al., 1991). A recent study has suggested that the specificity of this receptor for IgG is not absolute, providing evidence to demonstrate that mFcyRII can bind mIgE with low affinity (Takizawa et al., 1992). The specificity of mFcyRII for mouse IgG subclasses is broad, as the receptor binds mIgG1,2b, and 2a; however, mFcyRII does not bind mIgG3 (Unkeless, 1977,1979; Heusser et al., 1977; Haeffner-Cavaillon et al., 1979a; Teillaud et al., 1985; Lopez et al., 1985; Hulett et al., 1991). It has been proposed that a distinct mouse receptor exists specific for IgG3, but has not been characterized either biochemically or by molecular cloning (Diamond and Yelton, 1981). Mouse FcyRII binds hIgG subclasses with a specificity similar to hFcyRII, binding hIgGl and hIgG3 preferentially and hIgG2 and IgG4 less well (Haeffner-Cavaillon et al., 197913).
Cell Distribution and Monoclonal Antibodies Human FcyRII is the most widely distributed class of hFcyR, being expressed on almost all leukocytes, including monocytes, macrophages, neutrophils, basophils, eosinophils, Langerhans cells, platelets, B cells, and some T-cell subclasses, but is absent on NK cells (Vaughn et al., 1985; Looney et al., 1986a7b,1988;Valent et al., 1989; Anselmino et al., 1989; Schmitt et al., 1990; Sandor and Lynch, 1992; Mantzioris et al., 1993). Human FcyRII has also been demonstrated on nonimmune cells including placental trophoblasts (Stuart et al., 1989) and placental endothelial cells (Sedmak et aZ., 1991). The specific cell-type expression of the different hFcyRII isoforms is not welldefined; however, using the polymerase chain reaction (PCR), transcripts of the hFcyRIIA and hFcyRIIC genes have been detected in monocytes, macrophages, and neutrophils, whereas hFcyRIIB gene mRNA has been detected in monocytes, macrophages, and B cells (Brooks et al., 1989). A more recent study using both Northern blot and PCR analysis has further defined the cellular distribution of the individual transcripts of the three hFcyRII genes (Cassel et al., 1993). c.
18
MARK D. HULETT AND P. MARKHOGARTH
FcyRIIA was shown to be expressed in megakaryocytic cells, with both the a1 and a2 transcripts present in comparable amounts. In contrast, B cells express FcyRIIbl, b2, and FcyRIIc mRNA, but not FcyRIIal or a2. Myelomonocytic cells were shown to contain transcripts from all three hFcyRII genes, i.e., FcyRIIal, b l , b2, and c, with FcyRIIal the predominant mRNA species (Cassel et al., 1993). These findings clearly demonstrate that the FcyRIIA, B, and C genes products are differentially expressed in hematopoietic cells. It should be noted that substantial quantities of soluble FcyRIIa2 are present in and secreted from platelets, Langerhans cells, and megakaryocytic cell lines (Rappaport et al., 1993; Cassel et al., 1993; Astier et al., 1994).The levels of hFcyRII expression can be influenced by a number ofcytokines. IFN-y and IL-3 have been shown to upregulate the expression of hFcyRII on eosinophils (Hartnel et al., 1992). In contrast to hFcyRI and hFcyRII1, hFcyRII levels on monocytes and neutrophils appear not to be upregulated by any cytokine; however, IL-4 has been reported to downregulate its expression (Te Velde et al., 1990). In addition, GM-CSF has been shown to enhance cytotoxicityby hFcyRII on eosinophils; however, this enhanced function appears to be a result of increased receptor affinity and not due to increased hFcyRII expression (Graziano et al., 1989; Valerius et al., 1990; Koenderman et al., 1993). The distribution of cellular expression of mFcyRII is similar to its human counterpart, displaying a broad range of expression on hematopoietic cells, including monocytes, macrophages, neutrophils, mast cells, eosinophils, platelets, B cells, and some T cells (Unkeless et al., 1988; Mellman et al., 1988; Ravetch and Anderson, 1991). The mFcyRIIb1 and mFcyRIIb2 isoforms exhibit tissue-specific expression, with mFcyRIIb2 found predominantly in monocytes and macrophages, whereas mFcyRIIb1 is preferentially expressed in B lymphocytes (Ravetch et al., 1986; Lewis et al., 1986; Hibbs et al., 1986). The mFcyRIIb3 transcript has been identified in macrophages, and a soluble product possibly encoded by this mRNA has been detected in macrophage supernatents (Tartour et al., 1993). Numerous mAb recognizing hFcyRII have been described and include IV-3 (Looney et al., 1986a),CIKM5 (Pilkington et al., 1986),KuFc79 (Vaughn et al., 1985), 41H16 (Gosselin et al., 1990), 2E1 (Farace et al., 1988), KB61 (Pulford et al., 1986), AT10 (Greenman et al., 1991), 7.30, 8.2, 8.26, and 8.7 (Ierino et al., 1993) (see Table 111).All of these mAb exhibit the capacity to block Fc binding to hFcyRII, with the exception of mAb 8.2 and CIKM5 which have been shown to bind to an epitope distinct from the Ig binding site; however, these mAb can block IgG
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
19
binding to FcyRII via their Fc portions (Van de Winkle et al., 1987; Ierino et al., 1993). Differences in the reactivity of a number of the hFcyRII mAb are also demonstrated in the ability of some to preferentially bind B cells, which include mAb 2EI (Farace et al., 1988), KB61 (Pulford et al., 1986), 41H16 (Gosselin et al., 1990), AT10 (Greenman et al., 1991), 8.7, and 7.30 (Ierino et al., 1993). The 41H16 mAb is unique in that it is able to discriminate between hFcyRIIaHRand hFcyRIIaLR,selectively binding the HR isoform (see below) (Gosselin et al., 1990).The epitopes of a number of the hFcyRII mAb have been mapped in detail using a combination of crossblocking studies and reactivity with chimeric FcRs. The findings reveal that IV-3, 8.26, 8.7, and 7.30 (blocking mAb) have epitopes located in the second extracellular domain of hFcyRII, whereas CIKM5 and 8.2 (nonblocking mAb) have epitopes that seem to comprise regions from both the first and second extracellular domains. This suggests that the second extracellular domain of hFcyRII is involved in the binding of IgG (Ierino et al., 1993). Two mAb have been described that bind mFcyRII, a rat mAb 2.4G2 (Unkeless et al., 1979) and a mouse mAb anti-Ly-17.2 (Hibbs et al., 1985) that specifically detects the Ly-17.2 polymorphic form of mFcyRII (Holmes et al., 1985; Hibbs et al., 1985).Both mAb can block the binding of IgG to the receptor. The 2.4G2 mAb also recognizes mFcyRII1. d. Polymorphism A number of polymorphisms have been identified in hFcyRII. A genetic polymorphism defined as the high-responder/low-responder polymorphism has been identified in hFcyRIIa. This polymorphism was originally observed in anti-CD3 T-cell mitogenesis assays used to examine the interaction of mIgGl and human monocytes. Monocytes of different individuals were found to stimulate mIgGl anti-CD3 mAb T-cell proliferation in such assays either strongly or weakly, and these individuals were termed high responders (HR)or low responders (LR) (Tax et al., 1983). The distribution of high- and low-responder individuals was shown to be distinct in different races, with the finding that Caucasians are 70% HR, 30% LR, whereas in asians the ratio is reversed with 15% HR, 85% LR (Abo et al., 1984). The involvement of hFcyRII in this polymorphism was directly demonstrated with the finding that the anti-hFcyRII mAb IV-3 could block T-cell proliferation in the assay (Looney et al., 1986b) and was not due to differences in FcR numbers on the monocytes of high- and low-responder individuals (Anderson et al., 1987).This implied that there was an intrinsic differ-
20
MARK D. HULETT AND P. MARKHOGARTH
ence in FcyRII on the monocytes of HR and LR individuals, and, indeed, a structural difference in hFcyRII between HR and LR individuals was suggested, as different isoelectric focusing patterns were observed between receptors from these individuals (Anderson et al., 1987). Complementary DNA cloning of hFcyRII from the peripheral blood mononuclear cells of HR and LR individuals enabled the molecular basis of the polymorphism to be determined. The polymorphism has been defined as a two-residue difference in the extracellular region of the FcyRIIa isoform, with a glutamine or tryptophan at position 27 in the first extracellular domain and an arginine or histidine at position 131in the second extracellular domain (Clark et al., 1989;Warmerdam et al., 1990; Tate et al., 1992). Transfection experiments examining the IgG binding capacity of the various alleles have indicated that residue 131is responsible for the functional polymorphism, with arginine critical for the binding of mIgGl and found in HR isoforms, whereas histidine is present in LR isoforms. The tryptophan or glutamine at position 27 has no effect on the binding of mIgGl, and both residues have been described in HR and LR alleles (Warmerdam et al., 1990; Tate et al., 1992).Residue 131has also been shown as crucial for the binding of human IgG2; however, the amino acid required for binding is the reverse of that observed for mIgG1, with histidine (LR form) and not arginine (HR) promoting strong binding of hIgG2 (see above) (Warmerdam et al., 1991; Parren et al., 1992). The observation that residue 131is important for the binding of both mIgGl and hIgG2 supports the finding that the second extracellular domain is the IgG binding domain of hFcyRII (Hulett et al., 1993; see Section 111). It should be noted that hFcyRIIaLRdoes have the capacity to bind mIgG1, demonstrated if high concentrations of mIgGl anti-CD3 mAb are used in the T-cell proliferation assay (Tax et al., 1984; Most et al., 1992) or with immune complexes sensitized with increasing concentrations of mIgGl (Tate et al., 1992). The HR/LR polymorphism has also been described on other cell types including alveolar macrophages (Kindt et al., 1991), neutrophils (Gosselin et al., 1990),and platelets (Looney et al., 1988; Gosselin et al., 1990), but not B cells (Gosselin et al., 1990). As detailed above, the 41H16 mAb can discriminate between the HR and LR isoforms, specifically recognizing only the HR form (Gosselin et al., 1990). A polymorphism has also been described in hFcyRIIb1, whereby T y P 5in the cytoplasmic tail is substituted with an aspartic acid (Warmerdam et al., 1993). T y P 5has been proposed to form part of a signaling motif required for receptor internalization (Van den Herik-Oudijk et
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
21
al., 1994), and as such this polymorphism may result in defective receptor function. Differences in the levels of expression of hFcyRII on platelets (presumably hFcyRIIa) between individuals have also been described. The quantitative differences in FcyRIIa expression, detected using the mAb IV-3, were shown to correlate with platelet activation in response to IgG immune complexes and as such may result in increased susceptibility to immune complex disease (Rosenfeld et al., 1987). A polymorphic receptor for murine IgG2b has been described on human monocytes and EBV-transformed B lymphocytes (Holtrop et al., 1991). The molecular basis of this polymorphism has yet to be determined; however, the receptor appears to be distinct from FcyRII or FceRII (Holtrop et al., 1993a,b). A genetic polymorphism of mouse FcyRII has also been identified, defined by the Ly-17 alloantigen system, and is comprised of two alleles, Ly-lT and L ~ - l 7 that ~ , encode two polymorphic forms of mFcyRI1, designated the Ly-17.1 and Ly-17.2 antigens, respectively (Shen and Boyse, 1980; Davidson et al., 1983; Hibbs et al., 1985). The molecular basis of the polymorphism has been defined using direct sequencing of PCR-amplified mFcyRII extracellular region sequences, derived from the genomic DNA of Ly-17.1 or Ly-17.2 inbred mouse strains. The two alleles were found to differ genetically in only two codons, encoding amino acids 116 and 161; where Pro'16 and Gln161are found in the Ly-17.1 form and Leu116and LeulG1in the Ly17.2 form (Lah et al., 1990). Both these substitutions are situated in the second extracellular domain of the receptor; as antibodies specific for Ly-17.2 antigen have been demonstrated to completely inhibit the binding of IgG to mFcyRII, this suggests that residues 116 and/or 161 are closely associated with the ligand binding site and provides further evidence implicating domain 2 of FcyRII in the binding of IgG. 3. FcyRZZI a. Biochemical and Molecular Structure Human FcyRIII (CD16) is heterogeneous in size with a molecular weight ranging from 50 to 80 kDa (Fleit et al., 1982; Kulczycki, 1984; Lanier et at., 1988) (Table VI). This heterogeneity is due to extensive N-linked glycosylation of two distinct isoforms, hFcyRIIIa and hFcyRIIIb. In addition, two polymorphic forms of hFcyRIIIb have been described which also differ in N-linked glycosylation. Following deglycosylation, the hFcyRIIIa form has a molecular weight of 3334 kDa, and hFcyRIIIb has two distinct sizes of 29 and 33 kDa, which correspond to the different polymorphic forms (Selvaraj et al., 1989;
22
MARK D. HULE'lT AND P. MARKHOGARTH
TABLE VI CHARACTERISTICS OF FcyRIII Genes Human Characteristic Isoforms Alleles Chromosome localization Ig-like domains Receptor topology Associated subunits Receptor formsc Molecular mass (kDa) Apparent Protein Backbone Affinity for IgG
hFcyRIIIA hFcyRIIIa"
-
1~23-24
2
TM
hFcyRIIIb" NAUNA2 1~23-24
-
2 GPI-anchored
2 TM
y-chain, FcsRI (-chain TCR1CD3 a y e , (UrL
Mouse mFcyRIII
hFcyRIIIB
mFcyRIII
1
y-chain, FcsRI p-chain, F C E R I ~ aYz> a h b
4 2
50-80 33
50-80
29
40-60 33
z x 107 M - 1
4 0 7 M-1
4 0 7 M-1
(Ka)
Specificity hIgG mIgG Cellular distribution Regulation of expression
1=3>>>2=4 ND 3>2a>2b>>l 3>2a>2b>> 1 Macrophages Neutrophils NK cells, y6 T cells Eosinophilse monocytes (subpopulati on) TGF-p t (monocytes) TNF-a .1 (neutrophils) IFN-7, GM-CSF, GIL-4 .1 CSF t
3= 1>2>>4 1= 2a = 2b>>>3d Macrophages NK cells yS T cells IFNy
1
Note. TM, transmembrane; S, soluble; ND, not determined; GPI, glycosylphosphatidylinositol. Soluble hFcyRIIIalb generated by proteolytic cleavage of membrane isofoms. Demonstrated for mFcyRIIIa in mast cells, also for hFcyRIIIa in transfection reconstitution experiments. ay2 form expressed in macrophages q 5 ; ay2,a52 expressed in NK ceIls. mFcyRIII also binds mIgE with low affinity. IFNy induces expression hFcyRIIIb on eosinophils.
Kindt et al., 1991). Mouse FcyRIII is also heterogeneous, with amolecular weight ranging from 40 to 80 kDa (Mellman et aZ.,1988). FcyRIII are structurally similar to FcyRII, containing extracellular regions of two Ig-like domains; however, FcyRIII exhibit unique characteristics in that they differ in their forms ofmembrane anchoring. The hFcyRIIIb isoform is the only membrane FcR that is not an integral
23
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
membrane protein and instead is attached to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) moiety (Kurosaki and Ravetch, 1989; Lanier et al., 1989a; Hibbs et al., 1989). The hFcyRIIIa isoform and mouse FcyRIII are integral membrane proteins, yet are also distinct, requiring association with additional subunits for efficient cell-surface expression (Fleit et al., 1982; Kulczycki, 1984; Lanier et al., 1988, 1989b; Kurosaki and Ravetch, 1989; Ra et al., 1989a; Hibbs et al., 1989; Anderson et al., 199Oc; Letourneur et al., 1991) (see below). As described for hFcyRI and hFcyRII, FcyRIII in humans exists in multiple isoforms where two distinct genes FcyRIIIA and FcyRIIIB (formerly FcyRIII-2 and FcyRIII-1, respectively) have been identified, each producing a single transcript that encodes the hFcyRIIIa and FcyRIIIb isoforms, respectively (Simmons and Seed, 1988; Scallon et al., 1989; Ravetch and Perussia, 1989; Peltz et al., 1989). The two hFcyRIII genes are identical in structure comprising five exons, spanning approximately 8 kb: two exons encoding the 5’ UTR and leader sequence, one exon for each of the Ig-like domains, and a single exon encoding the transmembrane, cytoplasmic tail and 3’ UTR (see Table VII) (Ravetch and Perussia, 1989; Qiu et al., 1990).The human FcyRIII genes have been mapped to the q23-24 region of chromosome 1(Peltz et al., 1989; Qiu et al., 1990) and, as detailed above, are linked to the hFcyRII genes.
TABLE VII FcyRIII GENES AND TRANSCRIPTS Name
Gene structurea L1
~FcyRIIIA
L2 D1
Transcripts D2
hFcy RIIIa
TM/C
f lI L l I L 2 1 D1 I D2 ITMK I --c
;heb
I I I 1 < L1
Fey RIIIB
D2
hFcy RIIIb
TM/C
Ll(L2 D1
D2 ITMK Aerb
L1 iFcy RIII
L2 D1
L2 D1
D2
mFcy RIII
TM/C ----c
ILlIL2
1 Dl 1
D2 lTM/C]
a Exons shown as boxes, translated regions shaded, untranslated regions open. L, leader peptide;D, extracellulardomain; ‘M, transmembrane;C, cytoplasmic tail coding regions; PA, polyadenylation site. b Crucial residue in determinationof membrane anchoring form; Phe directs TM, Ser directs GPI.
24
MARK D. HULE'lT AND P. MARKHOGARTH
The transcripts of the two hFcyRIII genes exhibit 10 nucleotide differences in their coding regions which result in only 6 amino acid differences in the hFcyRIIIa and hFcyRIIIb isoforms. Both transcripts encode receptors with extracellular regions of 191 amino acids comprising two Ig-like domains, single transmembrane domains of 21 amino acids, and cytoplasmic tails of 25 (hFcyRIIIa) or 4 amino acids (hFcyRIIIb). The different length cytoplasmic tails result from a single nucleotide change in the cytoplasmic tail coding exons of the two genes, generating an earlier stop codon in the hFcyRIIIB gene. However, a critical amino acid difference between the two forms is observed at position 203, which results in alternate membrane-anchored receptors. Human FcyRIIIb contains Se?03 which specifies a GPIlinked molecule, whereas hFcyRIIa contains Phe203which disrupts the signal for the formation of a GPI anchor, thus preserving the transmembrane and cytoplasmic tail and producing a transmembrane molecule (Kurosaki and Ravetch, 1989; Lanier et al., 1989a; Hibbs et al., 1989). The hFcyRIIIa transmembrane form is the homologue of the single mouse FcyRIII (see below) (Ravetch and Perussia, 1989). The transmembrane form of hFcyRIII requires coexpression of associated molecules for efficient cell-surface expression. In macrophages, hFcyRIIIa is associated with homodimers of the y-subunit of the highaffinity receptor for IgE (FceRI) (Kurosaki and Ravetch, 1989; Ra et al., 1989a; Lanier et al., 1989a; Hibbs et al., 1989) and in NK cells is associated with homo- and/or heterodimers of the FceRI y-subunit and the ( subunit of the T-cell receptor (TCR)-CD3 complex (Lanier et al., 198913; Anderson et al., 199Oc; Letourneur et al., 1991). These accessory chains form disulfide-linked dimeric complexes which noncovalently associate with the transmembrane region of hFcyRIIIa to enable cell-surface expression (Lanier et al., 1991) and are also important in the signaling of the receptor (see Section IV). The y- and (-chains, together with the 7-chain of the TCR-CD3 complex, are all closely related and form a family of these small subunits (Orloff et al., 1990). cDNA and genomic clones have been described in the human for both the y-subunit (Kuster et al., 1990) and (-subunit (Wiessman et al., 1988). The genes for the y- and (-subunits map to the q23-24 region of human chromosome 1, which also contains the hFcyRII, hFcyRII1, and hFceRI a-chain genes (Weissman et al., 1988; Le Conait et al., 1990). Human FcyRIIIa has also been shown to have the capacity to associate with the p-subunit of FceRI. This was demonstrated upon immunoprecipitation of the reconstituted complex, formed by cotransfection of hFcyRIIIa with the FcsRI y- and FceRI p-subunits into the mouse
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
25
mastocytoma cell line P815 (Kurosakiet al., 1992).Given the similarity in the transmembrane regions of the a-chains of FcyRIII and FcsRI, and that the transmembrane regions appear to be the principal sites where the subunits interact, it is not surprising that the FcyRIIIal y complex associates with p-chains (see below). In addition to the association of hFcyRIIIaly with p, it is also possible that FcyRIIIaly may associate with other p-like molecules such as CD20, CD37, or CD53, in NK cells and macrophages (Stamenkovic and Seed, 1988; Classon et al., 1989; Angelisova et al., 1990). Soluble forms of both hFcyRIIIa and hFcyRIIIb have also been described. These molecules are derived from the membrane receptors following proteolytic cleavage from the cell surface. The release of the GPI-anchored hFcyRIIIb isoform from neutrophils has been shown to involve serine proteases, whereas the transmembrane hFcyRIIIa isoform is released upon cleavage by metalloproteases (Harrison et al., 1991). The soluble form of hFcyRIIIb from neutrophils can be detected at high concentrations in normal human sera (Khayat et al., 1987; Huizinga et al., 1988, 1990d) and as such may have important biological role(s). Two allelic forms of hFcyRIIIb have been identified and designated NA-1 and NA-2 (Tetteroo et al., 1988; Huizinga et al., 198913, 1990a; Trounstine et al., 1990; Salmon et al., 1990; Kindt et al., 1991). The molecular basis of this polymorphism has been determined (see below). In contrast to hFcyRII1, only a single isoform of mouse FcyRIII has been described. Mouse FcyRIII is an integral membrane glycoprotein, comprising an extracellular region of 184 amino acids containing two Ig-like domains, a single transmembrane region of 21 amino acids, and a cytoplasmic tail of 26 amino acids (Ravetch et al., 1986). The extracellular region of mFcyRIII is highly conserved with mFcyRII, exhibiting 95% amino acid identity. This conservation contributed to the early classification of mFcyRIII as an isoform of mFcyRIImFcyRIIa (see above). However, mFcyRII1 diverges markedly from mFcyRII in the leader peptide, transmembrane, and cytoplasmic tail regions; these regions of mFcyRIII display homology to hFcyRII1 and specifically the transmembrane form hFcyRIIIa (Ravetch and Perussia, 1989). Mouse FcyRIII is encoded by a single gene that is structurally related to the hFcyRII1 genes comprising five exons spanning 9 kb, including two exons encoding the 5' UTR and leader sequence, a single exon for each of the Ig-like domains, and a single exon encoding the transmembrane, cytoplasmic regions and 3' UTR (see Table VII). The mFcyRIII gene is linked to the mFcyRII gene on a 160-kb geno-
26
MARK D. HULETT AND P. MARKHOCARTH
mic fragment that maps to the Ly-17 locus of chromosome 1(Kulczycki et al., 1990; Qiu et al., 1990). Mouse FcyRIII also associates with accessory chains which are required for efficient cell-surface expression (Ra et al., 1989a; Kurosaki and Ravetch, 1989) and signaling of the receptor (Amigorena et al., 199213; Bonnerot et al., 1992) (see Section IV). However, in contrast to hFcyRII1, mFcyRIII has only been found to associate with the FcsRI y-subunit, as the mCD3lTCR (-subunit does not promote cellsurface expression of mFcyRIII (Kurosaki and Ravetch, 1989; Ra et aZ., 1989b). Interestingly, mouse FcyRIII will associate with the hCD3/TCR (-subunit (Kurosaki and Ravetch, 1989; Ra et al., 1989b). The mouse y-subunit cDNA has been cloned, and, as for human y, the gene mapped to chromosome 1 in the same region in the lowaffinity FcyR genes (Ra et al., 1989b; Huppi et al., 1989).As described for hFcyRIIIa, mFcyRII1 has also been shown to associate with the p-chain of FcsRI in mast cells. This was demonstrated by immunoprecipitation of an endogenously expressed FcyRIIIalylp complex from the mouse mast cell line, MC9, and by reconstitution of the complex by transfection into COS-7 cells (Kurosaki et al., 1992). Rat FcyRIII cDNAs have also been cloned and form a family of multiple isoforms, in contrast to the single mouse FcyRIII. Several distinct isoforms have been isolated, all encoding transmembrane receptors that require subunit association for expression (Zeger et al., 1990; Farber and Sears, 1991). As described for mouse FcyRIII, only the FcsRI y-subunit and not the endogenous rat CD3lTCR (-subunit (in contrast to hCD3ITCR () promotes efficient expression of rat FcyRIII (Farber and Sears, 1991; Farber et al., 1993). The existence of multiple rat FcyRIII genes has been suggested with an organization similar to the mouse and human FcyRIII genes (Farber and Sears, 1991). Examination of the transmembrane region of hFcyRIIIa, mFcyRIII, and the rat FcyRIII isoforms reveals a conserved stretch of eight amino acids (LFAVDTGL) including a negatively charged aspartic acid residue. This region is unique to these FcyR and the a-chain of the tetrameric FcsRI, all of which associate with accessory subunits. The yand (-subunits also have highly conserved transmembrane regions, suggesting that the interaction between these heterologous proteins involves their transmembrane regions. This possibility has been suggested by examination of the association of hFcyRIIIa and the FcsRI y- and CD3 (-subunits, where truncation of the cytoplasmic tails of these molecules did not effect cell-surface expression of hFcyRIIIa (Lanier et al., 1991). A molecular model of the interaction between
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
27
the conserved transmembrane regions of these FcRs and the associated subunits has been proposed (Farber and Sears, 1991).
b. Ligand Affinity and Specificity Human and mouse FcyRIII comprise a second class of low-affinity FcyR. The two hFcyRIII isoforms have been reported to display slightly different affinities for monomeric IgG, the GPI-anchored form (hFcyRIIIb) has an affinity of K , < lo7 M-' (Fleit et al., 1982; Kulczycki, 1984; Anderson and Looney, 1986; Simmons and Seed, 1988), whereas the transmembrane form (hFcyRIIIa) has a higher affinity of K , = 2 x 107M-' (Vance et al., 1992).As described for hFcyRI1, data on the IgG isotype binding specificity of the hFcyRIII isoforms is incomplete. However, the specificity of the GPI-anchored form of hFcyRIII for hIgG subclasses has been determined using dimeric complexes, and the receptor preferentially binds hIgG3 and hIgG1, but not hIgG2 or hIgG4 complexes (Kulczycki, 1984; Huizinga et al., 1989a). The specificity of the transmembrane form of hFcyRIII for hIgG subclasses has not been determined. Reports on the specificity of binding of mouse IgG isotypes by hFcyRIII are conflicting; however, it appears that both the transmembrane and GPI-anchored forms bind mIgG2a, mIgG3, and to a lesser extent mIgG1, but not IgG2b (Kipps et al., 1985; Anasetti et al., 1987; Simmons and Seed, 1988; Braakman et al., 1993). Human FcyRIIIb has been shown to have the unique ability to interact with lectins, probably via its high mannose containing oligosaccharides. This has been demonstrated as the phagocytosis ofconcanavalin A-treated erythrocytes and nonopsonized Escherichia coli by human neutrophils and can be inhibited with the anti-hFcyRIII mAb 3G8, aggregated IgG, and monosaccharides such as D-mannose (Salmon et al., 1987; Kimberley et al., 1989).Thus hFcyRIIIb appears to be able to bind ligands other than IgG through lectin-carbohydrate interactions. These findings also suggests that oligosaccharides may contribute to the integrity of the IgG binding site on hFcyRIIIb. Mouse FcyRIII exhibits identical ligand binding characteristics to mFcyRI1, displaying a similar low affinity for monomeric IgG (K,< lo7 M - ' ) and the same specificity for both mouse and human IgG isotypes, preferentially binding mouse IgG1, 2a, and 2b and human IgGl and 3 (Unkeless, 1977, 1979; Heusser et al., 1977; HaeffnerCavaillon et al., 1979a; Teillaud et al., 1985; Lopez et al., 1985). A recent study has demonstrated that mFcyRIII also binds mIgE with low affinity, as described for mFcyRII (Takizawa et al., 1992). The
28
MARK D. HULETT AND P. MARKHOGARTH
similar interaction of mFcyRIII and mFcyRII with Ig is not surprising as the extracellular ligand binding regions of these receptors are highly conserved with 95% amino acid identity. c. Cell Distribution and Monoclonal Antibodies The expression of the two different hFcyRII1 isoforms is cell specific (Table VI). The GPI-anchored FcyRIIIb is expressed exclusively on neutrophils, whereas the transmembrane FcyRIIIa is expressed on macrophages and NK cells (Simmons and Seed, 1988; Selvaraj et al., 1988,1989; Peltz et al., 1989; Scallon et al., 189; Ravetch and Perussia, 1989; Hibbs et al., 1989; Lanier et al., 1989a; Edberg et al., 1989; Perussia and Ravetch, 1991). The latter form has also been demonstrated on a small population of freshly isolated monocytes (Passlick et al., 1989; Anderson et at., 1990b) and on some T cells (Braakman et al., 1993). Human FcyRIII has also been observed on mesangial cells of the kidney (Sedmak et al., 1990) and on placental trophoblasts (Sedmak et al., 1991). Immunohistochemical staining of normal lymphoid and nonlymphoid tissues with anti-hFcyRII1 mAb has demonstrated strong staining of mantle zone cells and interfollicular zone cells (Tuijnman et al., 1993). The expression of hFcyRIII has been shown to be influenced by cytokines; the transmembrane form on monocytes was found to be upregulated by TNF-P (Welch et al., 1990; Phillips et at., 1991), and the GPI-anchored form on neutrophils can be upregulated by IFN-y, GM-CSF, and G-CSF (Buckle and Hogg, 1989) and also downregulated by TNF-a, which has no effect on the transmembrane form (Mendel et al., 1988). It has also been reported that the GPI-anchored form can be induced on eosinophils by IFN-y (Hartnell et at., 1992).In addition, IL-4 downregulates monorgte hFcyRII1, as for the other FcyR classes (Te Velde et al., 1990). Mouse FcyRIII has a similar cell distribution to hFcyRIIIa and has been described on macrophages, NK cells, subpopulations of T cells, and mast cells (Ravetch et al., 1986; Weinshank et al., 1988; Katz et al., 1990; Benhamou et al., 199Oa). mFcyRIII has also been described on early fetal thymocytes (Rodewald et al., 1993). The expression of mFcyRIII on macrophages is also modulated by IFN-y (Weinshank et al., 1988). Several anti-hFcyRIII mAb have been described and include 3G8 and 4F7 (Fleit et al., 1982), VEPl3 (Rumpold et al., 1982), Leu l l a and Leu l l b (Lanier et al., 1983, 1985), B73.1 (Perussia et al., 1984), GRM-1 (Phillips and Babcock, 1983), CLB GranII (Werner et al., 1988), 1D3 (Tetteroo et al., 1988), and BW209/2 (Huizinga et at., 1990d) (see Table 111).Most of the mAb block the binding of IgG to
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
29
the receptor; however, mAb BW209/2 binds to hFcyRIII when it is occupied with IgG (Huizinga et al., 1990d).A number ofthe hFcyRIII mAb show differential reactivity with the NAUNA2 polymorphic forms, with mAb B73.1 and CLB GranII being specific for the NA1 form, and mAb GRM-1 for the NA-2 form (Huinzinga et al., 1989b; Trounstine et al., 1990; Salmon et al., 1990) (see below). The only mAb demonstrated to bind mFcyRIII is the mAb 2.4G2, which also detects mFcyRII (Unkeless et al., 1979). d. Polymorphism A genetically determined structual polymorphism has been described for hFcyRIIIb, identified by biochemical analysis and reactivity with mAb (Tetteroo et al., 1988; Huizinga et al., 1989b, 199Oa; Trounstine et al., 1990; Salmon et at., 1990; Kindt et al., 1991). The polymorphism has been designated the neutrophil antigen (NA) system and comprises two allelic forms, hFcyRIIIbNA-'and hFcyRIIIbNA-',which exhibit phenotypic frequencies in Caucasians of 37% and 63%, respectively (Lalezari, 1984). The polymorphism is apparent in the different molecular masses observed for the two allotypes following deglycosylation; the NA-1 form has a mass of 29 kDa and the NA-2 form a mass of 33 kDa (Ory et al., 1989; Huizinga et al., 1990a). The two allotypic forms are also distinguishable with mAb as described above. The molecular basis of this polymorphism has been determined and arises from a four-amino acid difference between the two forms, which results in the loss of two N-linked glycosylation sites in the NA-1 form, which has four sites in contrast to the six sites of NA-2 (Ravetch and Perussia, 1989; Ory et al., 1989). In addition, a Taq-1 restriction length fragment polymorphism is also associated with the two hFcyRIIIb alleles (Ravetch and Perussia, 1989; Ory et al., 1989). Individuals who do not express hFcyRIIIb have also been identified. These include a patient with systemic lupus erythematosus (SLE) (Clark et al., 1990) and two healthy individuals who exhibited no sign of increased susceptibility to infection or elevated levels of circulating immune complexes (Huizinga et al., 1990b). The defect in each case appears to be as a result of a disorganized or absent hFcrRIIIB gene. These findings raise the question of the functional significance of hFcyRIIIb and again suggest that the loss of a single class of FcyR can be compensated by the other FcyR. No polymorphism has been described for mFcyRII1; however, based on the high amino acid identity with mFcyRII (which exhibits the Ly-17 polymorphism), and as the mFcyRIII cDNA was isolated
30
MARK D. HULETT AND P. MARKHOGARTH
from an Ly-17.2 mouse strain, it would be interesting to determine if the mFcyRIII sequence is also different in Ly-17.1 strains. B. FcsR Two distinct classes of receptors for the Fc portion of IgE, FcsRI, and FceRII have been defined on the basis of differential affinity for IgE, reactivity with mAb, cell distribution, biological function, and molecular cloning. The isolation of cDNA clones for these receptors has indicated that FcsRI and FcERIIare structurally unrelated, FcsRI belonging to the Ig superfamily and closely related to the leukocyte FcyR, whereas FcsRII belongs to a family of animal lectins. As this review focuses on the Ig superfamily FcR, only FceRI is discussed in detail (summarized in Tables VIII and IX); however, FceRII is described briefly for completeness (for reviews see Spiegelberg, 1984; Metzger et al., 1986, Kinet, 1990; Ravetch and Kinet, 1991; Conrad, 1990; Metzger, 1992a; Delespesse et al., 1992).
TABLE VIII CHARACTERISTICS OF FcsRI Characteristic Affinity for IgE' (K,) Specificity IgE Associated subunitsb Receptor forms Ig-like domains Receptor topology Molecular mass (kDa) Apparent Protein backbone Chromosomal localization Cellular distribution
Human FcsRI
10'oMul-' hIgE, rtIgE, mIgE a,p, y .BY29
ayzc
2 (a) TM
45-65,32,7-9 26.4,25.9,7-8 lq23, llq13,lq23
Rat FcsRI
Mouse FcERI
10lOM-l 10'0M-1 rtIgE, mIgE only rtIgE, mIgE only Q, P, Y a, Y
cvavz
cvarz
TM
TM
45-65,32,7-9 25.2,27, 7.8 ND
45-65,32,7-9 25.8,25.9,7.8 lq, 19,lq
2 (4
Mast Cells Mast Cells Basophils Basophils Langerhans Cells Eosinophils Monocytes (activated)
2 (4
Mast Cells Basophils
Note. TM, transmembrane; ND, not determined. Affinity of receptors for their species specific ligand. a-chain, ligand binding subunit. hFceRI a-chain only requires y-subunit for expression, thus may also exist in ay2 form.
31
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
TABLE IX FceRI GENESAND TRANSCRIPTS Name rtFceRIa
mFc,RIa
Gene structure'
Ll
L2 D1
D2
TM/C
L1
L2 D1
D2
TM/C
L1
L2 D1
D2
TM/C
hFcERIa
- [LIIL~ -
Transcripts
rFc, RIa
[ L i I u 1 DI I DZ ITMK) mFcE RIa
I
DI
I DZ ITM/C\
hFc, RIa
I L ~ I IDI ~ I DZ ITME]
Exons shown as boxes, translated regions shaded, untranslated regions open. L, leader peptide: D, extracellulardomain: TM, transmembrane; C, cytoplasmic tail coding regions; PA, polyadenylation site.
1. FcsRl
a. Biochemical and Molecular Structure FcsRI, also known as the high-affinity IgE receptor, has been characterized at a molecular level in three different species, mouse, rat and human, and has been defined structurally as a tetrameric complex of three distinct polypeptides, comprising an @-subunit(the IgE binding chain) that is homologous to the FcyR, a @subunit, and a dimer of two y-subunits. The early biochemical characterization focused on rat FcsRI of the rat basophilic leukemia cell line RBL-2H3 (Kulczycki et al., 1974). Initial attempts to purify the receptor identified a single polypeptide with an apparent molecular weight of 50-60 kDa (Conrad and Froese, 1976; Kulczycki et al., 1976; Kanellopolous et al., 1979). However, other studies suggested that the receptor was composed of more than one polypeptide (Holowka et al., 1980). This was subsequently found to be correct, as the polypeptide isolated initially (the a-subunit) was found to be associated with a single p-chain of 33 kDa (Holowka et al., 1980; Holowka and Metzger, 1982; Perez-Montfort et al., 1983), and two disulfide-linked y-subunits each of 7-9 kDa (Perez-Montfort et al., 1983; Alcaraz et al., 1984). The initial failure to detect these additional subunits was due to the sensitivity ofthe noncovalent associ-
32
MARK D. HULETT AND P. MARKHOGARTH
ation between the subunits to mild detergents, with purification of the intact tetrameric complex requiring protective phospholipid or submicellular concentrations of detergent (Rivnay et al., 1982; Kinet et al., 1985). Several mAb were also raised against rat FcsRI and allowed further characterization of the membrane topology of the receptor subunits (Basciano et al., 1986). Based on this biochemical data it was suggested that FceRI was a tetrameric complex comprising noncovalently associated subunits: the a-subunit, a highly glycosylated polypeptide expressed on the outer surface of the cell, and two non-glycosylated intramembrane components, the p-subunit, and a dimer of two disulfide-linked y-subunits (Metzger et al., 1983, 1986). The cDNAs for each of the subunits of FcsRI in the rat, mouse, and human have been cloned, which has enabled their molecular structures to be determined and a model for the topology of the FceRI receptor complex to be proposed (Blank et al., 1989). a-Subunit. cDNA clones for the a-subunit have been isolated from all three species (Kinet et al., 1987; Kochan et al., 1988; Shimizu et al., 1988, Liu and Robertson, 1988; Ra et al., 1989b) and encode integral membrane glycoproteins with extracellular regions of two Ig-like domains spanning 180 (human) or 181 (rat, mouse) amino acids; a single transmembrane region of 21 amino acids; and cytoplasmic tails of 31 (human), 25 (mouse), or 20 (rat) amino acids. A single transcript has been identified in each species. Three additional rat FcsRI a-chain cDNA clones have been reported but have substantial differences from the cloned FceRI a-chain gene and as such are likely to be cloning artifacts (Liu and Robertson, 1988). The predicted human, mouse, and rat a-chain protein products exhibit substantial sequence identity (38%), but are the least conserved of the three FcsRI subunits (Ra et al., 198913).The leader peptide and the cytoplasmic domains are the least conserved, exhibiting 17 and 16% amino acid identity between the species, respectively. However, the extracellular and transmembrane regions are more highly conserved, displaying 42 and 62% amino acid identity, respectively, when sequences are compared between these species (Ra et al., 1989b). The high degree of sequence divergence of the predicted a-chain cytoplasmic tails suggest that the cytoplasmic tail ofthis subunit is not involved in a crucial receptor function. In contrast, the high level of amino acid identity observed in the transmembrane region across the species, which all contain a conserved consecutive 8-amino acid motif (LFAVDTGL), suggests this region performs a specific function (Kinet and Metzger, 1990). The finding that this motif is also conserved in the transmembrane region of mFcyRII1, hFcyRII1, and rat FcyRIII, which like the FcsRI a-chain
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
33
are all receptors that require association with the y-subunit for cellsurface expression (see above), provides strong evidence to suggest this region interacts with the y-subunit (Farber and Sears, 1991). Indeed, as described for hFcyRIIIa, recent mutagenesis experiments indicate this is also the case for the FceRI a-chain (see below) (VarinBlank and Metzger 1990). The predicted FcsRI a-chains are homologous to all the FcyR described above, but are most closely related to the FcyRIII subclass (Ra et al., 1989b; Ravetch and Kinet, 1991) (Table IX). This is demonstrated on comparison of mouse FceRIa with mouse FcyRIII, which exhibit an overall 33% amino acid identity across their entire sequence, with 35 and 48% identity in their extracellular and transmembrane regions, respectively. Furthermore, of the 95 conserved residues in the extracellular regions of the rat-mouse-human FceRI a-chains, 61 are also found in both human and mouse FcyRIII, suggesting the unique 34 residues of the a-chains may be specific for IgE (Ravetch and Kinet, 1991). This high degree of conservation suggests that the FceRI a-chain and FcyRIII genes probably arose from a common ancestor by gene duplication. Indeed, the cloning of the rat and mouse and human FceRI a-chain genes has demonstrated that they share a high degree of structural conservation with each other and the mouse and human FcyRIII genes-containing five exons, two of which encode the 5' UTR and leader sequence, one exon for each of Ig-like domains, and a single exon encoding the transmembrane, cytoplasmic tail and 3' UTR (Tepler et al., 1989; Ye et al., 1992; Pang et al., 1993). The human FceRI a-chain gene has been mapped to the same region on chromosome 1 as the low-affinity hFcyR genes-band lq23 (Le Conait et al., 1990). It has also been demonstrated that the mouse FceRI a-chain gene is linked to the mouse low-affinity FcyR genes on chromosome 1 (Huppi et al., 1988). P-Subunit. cDNA clones of the @subunit of FceRI have been isolated from the rat (Kinet et al., 1988), mouse (Ra et al., 1989b), and human (Kuster et al., 1992). Two mRNA species of both mouse and rat p FceRI (1.75 and 2.7 kb) which arise by alternate polyadenylation have been observed. Two transcripts of human FceRIP have also been described, detected as a doublet around 3.9 kb (Kuster et al., 1990). The predicted rat, mouse, and human p-subunits are 243, 235, and 244 amino acids in length, respectively, and are highly homologous, displaying 69% amino acid identity. Based on hydrophobicity plots and studies with mAbs, it has been proposed that the FceRI P-subunit comprises four membrane spanning regions with both the N- and Ctermini in the cytoplasm (Kinet et al., 1988; Ra et al., 1989b). The
34
MARK D. HULETT AND P. MARKHOGARTH
human FcsRI p-subunit gene has been isolated and appears to be a single copy gene comprising seven exons spanning 10 kb (Kuster et al., 1990). The 5’UTR and part of the N-terminal cytoplasmic tail are encoded by exon 1, the first transmembrane region is encoded by exons 2 and 3, transmembrane 2 by exons 3 and 4, transmembrane 3 by exon 5, transmembrane 4 by exon 6, and the C-terminal cytoplasmic tail and 3’UTRs in exon 7. The mouse and rat FceRI p-subunit genes have not been isolated; however, the mouse gene is believed to be encoded by a single gene that maps to chromosome 19, linked to the Ly-l locus (Huppi et al., 1989). The human FceRI p-gene has been mapped to chromosome l l q 1 3 (Sandford et al., 1993). y Subunit. cDNA clones have been isolated for the FcsRI ysubunit in the rat (Blank et al., 1989), mouse (Ra et al., 1989b) and human (Kuster et al., 1990).The y-subunit in all three species is highly conserved, the predicted polypeptide products exhibiting 86% amino acid identity (Kuster et al., 1990).FcsRIy is a small integral membrane protein, with a single transmembrane spanning region of 21 amino acids, a small extracellular region of only 5 residues, and a larger cytoplasmic tail of 42 amino acids. The y-subunit has been demonstrated to exist in dimeric form through the formation of a disulfide bond between the N-terminal cysteine residue (Varin-Blank and Metzger, 1990) and exhibits homology to the (-and 7-chains of the TCR/ CD3 complex, with which it forms a family of disulfide-linked dimers (Orloff et al., 1990). As described above, the y-subunit of FcERI also associates with mouse and rat FcyRIII, human FqRIIIa, FcyRI and FcyRII. In all these receptors (with the exception of FcyRI and FcyRII) the y-subunit is essential for efficient cell-surface expression and also plays a crucial role in signal transduction. The human ysubunit gene has been isolated and mapped to the chromosome 1923, the same region that contains the FceRI a-chain gene and the lowaffinity FcyR genes (Kuster et al., 1990). The mouse y-subunit gene has also been mapped to a region containing the FceRI a-chain, FcyRII and FcyRIII genes on chromosome 1 (Huppi et al., 1989). This close linkage of the genes encoding the FceRI a- and y-subunits, and the low-affinityIgG receptors, suggests the possibility ofcoordinate regulation of these FcR genes. The cDNA cloning of each of the subunits of FceRI in the rat, mouse, and human has enabled cotransfection studies to be performed to assess the requirements for efficient cell-surface expression of the receptor. Initial experiments with the rat FceRI a-chain demonstrated that this subunit could not be expressed on the cell surface following its transfection in isolation into COS-7 (Kinet et al., 1987; Shimizu et
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
35
al., 1988). Subsequently, this has been shown for both the human and mouse FceRI a-chains; however, it should be noted that cell-surface expression does occur, but with an extremely low efficiency making detection difficult (Ra et al., 198913). Efficient cell-surface expression of the a-chain has been shown to require the coexpression of the yand/or /+subunits, and these requirements are different for the rodent and human receptors. In the rat and mouse systems, both the y- and p-subunits are required for efficient cell-surface expression of the asubunit, in contrast to the human a-subunit which requires coexpression of only the y-subunit (which can be of rat, mouse, or human origin) (Miller et al., 1989; Ra et al., 1989b; Kuster et al., 1990). The cotransfection the mouse, rat, or human @chain with hFceRIa and hFceRIy does not increase expression efficiency (Kuster et al., 1990). These findings raise the possibility that hFceRI can exist as an a ( ~ ) ~ complex in vivo and may therefore have the capacity to mediate a distinct intracellular signal. Recent mutagenesis experiments have been performed on the rat FceRI subunits to assess the roles of different regions of the subunits in association and cell-surface expression (Varin-Blank and Metzger, 1990). Truncation of the cytoplasmic tails of any or all of the subunits had little effect on cell-surface expression of the receptor. However, even minor changes in the transmembrane regions resulted in reduced expression levels. These experiments suggest that the transmembrane regions are critical for optimal expression of rat FceRI, and a model to describe the molecular interaction between the transmembrane regions of the subunits has been proposed (Varin-Blank and Metzger, 1990). An interesting finding from this study was that the human achain, when coexpressed with a rat y-chain lacking a cytoplasmic tail, was not expressed on the cell surface, again suggesting that the human and rodent receptors assemble differently. As described above, hFcyRIIIa has been shown to associate with both FceRI y- and the TCR/CD3 (-subunits, and based on the high homology of these subunits and the conserved nature of the hFcyRIIIa and hFceR1 a-chain transmembrane regions, it might be expected that the (-subunit could also associate with the hFceRI a-chain. Indeed, it has been demonstrated recently that the 6-subunit is able to substitute for the y-subunit in the assembly and functional expression of rat FceRI, in a Xenopus oocyte expression system (Howard et al., 1990). However, such an association would not be expected to occur in vivo, as the (-chain appears not to be coexpressed with the FcERI a-chain. Hamawy et a1 (1992) have also indicated that other molecules may be closely associated with the FceRI on the cell surface. A monoclonal
36
MARK D. HULE'IT AND P. MARKHOGARTH
antibody (BD6) that detects a 40-kDa molecule on the surface of RBL2H3 cells blocks IgE binding. This molecule can be chemically crosslinked to the FcsRI complex indicating its likely proximity to the receptor. It is also of interest that a number of novel proteins can be coprecipitated with the FceRI y-subunit (Schoneich et al., 1992).
b. Ligand Affinityand Specificity FcsRI of rat, mouse, and human all bind monomeric IgE with an affinity of approximately 10" M-' (Kulczycki and Metzger, 1974; Ishizaka et al., 1985; Miller et al., 1989). Although FcsRI of each species specifically binds IgE, the specificity for IgE from different species varies. Human FcsRI binds human, rat, and mouse IgE, although rodent IgE binds less well. In contrast, mouse and rat FcsRI only bind rodent IgE, not hIgE (Conrad et al., 1983). Of interest is the recent finding that FcsRI on rat RBL cells can bind EBP, a P-galactoside binding lectin shown to be identical to Mac-2 (Frigeri et al., 1993). c. Cell Distribution and Monoclonal Antibodies FceRI traditionally has been thought to have a unique cell distribution, being exclusively expressed on mast cells and basophils (Metzger et al., 1986; Metzger, 1988; Kinet and Metzger, 1990; Ravetch and Kinet, 1991). However, more recently it has become apparent that FceRI has a broader distribution, as is also found on Langerhans cells (Bieber et al., 1992; Wang et al., 1992), eosinophils (Abdelillah et al., 1994), and activated monocytes (Maurer et al., 1994). Numerous mAb detecting the rat FcsRI a-chain have been described (Basciano et al., 1986). Recently, a number of anti-human FcsRI achain mAb have also been produced (see Table 111)(Riske et d.,1991; and P. M. Hogarth, unpublished). These mAb have been divided into inhibitory and noninhibitory classes on the basis of their capacity to block the binding of IgE to hFcsRI. The inhibitory mAb included 15A5, 12E7,6F7, and 4B4. MAb 15A5 was specifically mapped to the region comprising amino acids 100-115 in the second extracellular domain, and mAb 12E7,6F7, and 4B4 recognized epitopes that were identical to or overlapping that detected by 15A5. The noninhibitory mAb included 2237,1lB4,5D5,8C8,29C9, and 39D5, of which 2237, 5D5, and 8C8 were shown to have epitopes in the first extracellular domain, as all competed with l l B4 which was shown to recognize the peptide corresponding to residues 18-23 of domain 1 (Riske et al., 1991). MAb 29C9 recognized an epitope that was proposed to comprise regions from both domains, since it was able to block the binding of both 15A5 and 2237. The epitope of mAb 39D5 was not mapped.
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
37
These findings strongly suggest that the second extracellular domain of the hFcsR1 a-chain is important in the binding of IgE, which supports the findings of others using direct binding site mapping approaches (Hulett et al., 1993; Robertson, 1993; Mallimaci, 1993).
d. Polymorphism
No polymorphisms have been reported for FcsRI in the rat, mouse, or human.
2. FcsRZZ a. Biochemical and Molecular Structure FcsRII (CD23)is a single-chain type I1 integral membrane glycoprotein with an apparent molecular mass of 45-50 kDa (Conrad, 1990; Delepesse, 1991,1992). Complementary DNA cloning studies suggest FcsRII contains a large C-terminal extracellular region of 277 amino acids, a single transmembrane domain of 20 amino acids, and a short N-terminal intracytoplasmic tail of 23 residues (Kikutani et al., 1986a; Ikuta et al., 1987; Ludin et al., 1987).The extracellular region has been
proposed to comprise a C-terminal C-type (Ca2+-dependent)lectin domain, homologous to a family of proteins including the adhesion proteins termed “selectins” and the asialoglycoprotein receptor (Wong et al., 1991). Located on the C-terminal side of the lectin domain is an “inverted RGD” sequence (Arg-Gly-Asp) which has been proposed to have a role in cell adhesion-in a manner similar to the RGD sequence of the integrins. On the N-terminal side of the lectin domain is a repetitive region containing five heptadic repeats of hydrophobic leucineholeucine residues, predicted to form an a-helical “stalk” region which mediates trimer formation (Beavil et al., 1992; Dierks et al., 1993). A single N-linked glycosylation site is situated on the transmembrane side of the stalk region. A single gene has been isolated for human FcsRII and mapped to chromosome 19 (Suter et al., 1987; Wendel-Hansen et al., 1990). The gene comprises 11 exons spanning 13 kb. The mouse FcsRII gene structure is almost identical to the human, with the exception that it contains an additional exon encoding a fourth repeat region (3 in the human) (Richards et al., 1991). Two transcripts (designated A and B) are encoded by the human FcsRII gene, differing only in their 5’ untranslated and intracytoplasmic tail encoding regions. These transcripts are derived from the use of different promoters which control different first exons (Yokota et al., 1988). Mouse FceRII exhibits 52% amino acid identity with human FcsRII (Delepesse et al., 1992). Similarly, in the mouse two isoforms of FcsRII have been described that
38
MARK D. HULETT AND P. MARKHOGARTH
arise by the same mechanism as that for the human forms (Richards et al., 1991). Soluble forms of human FceRII have been described and arise by proteolytic cleavage of the membrane form. Initially a 37- or 33-kDa fragment is released following cleavage at amino acid 82 in the "stalk" region. Additional soluble forms are derived from these by further proteolysis steps, producing fragments of 29, 25 and 16 kDa. All of these soluble fragments retain the capacity to bind IgE (Letellier et al., 1989, 1990).
b. Ligand AfJinity and Speci3city FcsRII binds monomeric IgE with an affinity of K , < lo7 M-' and is referred to as the low-affinity IgE receptor (Conrad, 1990; Delespesse et al., 1991, 1992). FceRII also binds CR2-a membrane protein found on B cells, follicular dendritic cells, T cells, and basophils (Aubry et al., 1992; Pochon et al., 1992).As such CR2 is referred to as a counter-receptor for FceRII. The IgE and CR2 binding functions of FceRII reside entirely in the lectin domain (reviewed in Sutton and Gould, 1993). c. Cell Distribution and Monoclonal Antibodies In the human, FceRII is expressed on a diverse range of hematopoietic cells including T and B cells, monocytes, eosinophils, platelets, follicular dendritic cells, Langerhans cells, and epithelial cells of the bone marrow and thymus (Conrad, 1990; Delepesse et al., 1991,1992). Expression of the two FceRII forms is regulated in a tissue-specific manner. The FceRIIa form is expressed only on antigen-activated B cells; however, following differentiation into Ig secreting plasma cells expression is lost (Kikutani et al., 1986b; Snapper et al., 1991). FcsRIIb is expressed on all the cell types outlined above following induction with IL-4 (Delespesse et al., 1991, 1992). Mouse FcsRII has been described on B cells, monocytes, and eosinophils (Delepesse et al., 1992). C. FcaR FcaRs have been described on hematopoietic cells in both the human and mouse. The FcaR on human myeloid cells has been most extensively characterized and is a member of the Ig superfamily, structurally related to the FcyR and FceRI. The existence of distinct lymphocyte FcaR has also been suggested; however, lymphocyte FcaR are far less well-defined and remain controversial. This section of the review, therefore, focuses primarily on human FcaRI.
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
39
1. FcaRl
a. Biochemical and Molecular Structure Human FccuRI (CD89) is a heavily glycosylated protein of 5575 kDa (Albrechtsen et al., 1988; Monteiro et al., 1990, 1992; Mazengera and Kerr, 1990)(Table X). The observed molecular heterogeneity has been shown to arise from variable glycosylation of a single protein product. The removal of N-linked oligosaccharides by treatment with N-glycanase reveals two molecular species of 32 and 36 kDa (Monteiro et al., 1990, 1992). The 32-kDa form has been proposed to represent the protein core, with the 36-kDa form resulting from incomplete deglycosylation rather than being the product of an alternatively spliced transcript (Monteiro et al., 1992). The mouse homologue of human FccuRI has not been defined. A human FcaRI cDNA clone has been isolated and the predicted amino acid sequence indicates an integral membrane glycoprotein of 287 amino acids comprising an extracellular region of 206 amino acids containing six potential N-linked glycosylation sites, a single transmembrane region of 19 amino acids, and a cytoplasmic tail of 41 amino acids (Maliszewski et al., 1990).The extracellular region of this FcaR is homologous to that of the other Ig superfamily FcRs, FcyRII, FcyRIII, FcsRI a-chain, and the first two domains of FcyRI. However, it is
TABLE X CH~RACTERISTICS OF FcaR Characteristics Chromosome localization Ig-like domain Receptor topology Associated subunit Receptor forms Molecular mass (kDa) Apparent Protein backbone Affinity for IgA Specificity Cellular distribution Regulation of expression
Human FcaRI'
19
2
TM FcERIy-chain a,4
55-756
32
5 x 107M-* Monomeric and polymeric IgAl and IgA2 Monocytes, macrophages, neutrophils, eosinophils PMA t (neutrophils), Ca2+ionophore t (eosinophils)
Human FcaRI only FcaR cloned; mouse FcaRI form not reported. Distinct FcaR on human and murine lymphocytes but not described at biochemical/molecular level. Cell type dependent; eosinophils 70-100 kDa.
40
MARK D. HULETT AND P. MARKHOGARTH
more distantly related to these receptors than the FcyR and FccRI are to each other, suggesting FcaRI diverged from a common ancestor early in the evolution of the Ig superfamily FcR (Maliszewski et al., 1990). The gene encoding hFcaRI has recently been mapped to chromosome 19q3.4 (Kremer et al., 1992) and as such is not linked to the other Ig superfamily FcR, which are all found on chromosome 1q23-24 (with the exception of mFcyRI, see above). Genomic clones encoding hFcaRI have not as yet been isolated. Transfection experiments have demonstrated that hFcaRI does not require accessory subunits for cell-surface expression (Maliszewski et al., 1990). However, recent studies suggest that it does associate with the y-subunit of FcERI (L. Pfefferkorn, personal communication), also known to associate with hFcyRI, FcyRII, and FcyRIII (see above). Interestingly, examination of the amino acid sequence of hFcaRI transmembrane region does reveal some homology to the 8-amino acid motif in the transmembrane region of human and mouse FcyRIII and FcERIa-chain, believed to be crucial for interaction with the y-subunit, including the presence of a charged residue (Arg230)(Maliszewski et al., 1990; Farber and Sears, 1991). As described above, FcaR have also been postulated to occur on subpopulations of human and murine T and B cells (reviewed in Mestecky and McGhee, 1987; McGhee et al., 1989; Kerr, 1990; also Lum et al., 1979; Gupta et al., 1979; Sjoberg, 1980a; Lynch and Sandor, 1990; Millet et al., 1989; Roa et al., 1992; Aicher et al., 1992). The lymphocyte FcaR appear to be structurally distinct from FcaRI; however, their biochemical nature and molecular structures have not yet been determined.
b. Ligand Specificity and Affinity Human FcaRI is specific for IgA, binding both monomeric and polymeric forms of IgAl and IgA2 (Albrechsten et al., 1988; Monteiro et al., 1990; Stewert and Kerr, 1990). The receptor binds IgA with high affinity, binding monomeric IgA with an affinity of K , = 5 x lo' M-' (Mazengera and Kerr, 1990). It should be noted that the binding of IgA by hFcaRI is species specific, binding only human IgA and not mouse IgA (M. Kerr, personal communication). c. Cell Distribution and Monoclonal Antibodies Human FcaRI is expressed on monocytes, macrophages, neutrophils, and eosinophils (Gauldie et al., 1983; Chevailler et al., 1989; Fanger et al., 1983; Maliszewski et al., 1985; Abu-Ghazaleh et al., 1989; Monteiro et al., 1990,1992,1993; Shen et al., 1989; Mazengera and Kerr, 1990; Monteiro et al., 1993). FcaRI has also recently been described on human mesangial cells (Gomez-Guerrero et al., 1993).
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
41
There is some evidence to suggest that hFcaRI expression is differentially regulated on myeloid cells, as PMA enhances FcaRI expression on the monocyte cell lines U937 and PLB985 but not on eosinophils, whereas Ca2+ ionophores enhance FcaRI expression on eosinophils but not monocyte cell lines (Monteiro et al., 1993). In support of this, FcaR expression is elevated on eosinophils but not neutrophils of allergic individuals (Monteiro et al., 1993). In addition, the treatment of neutrophils with GM-CSF or G-CSF has been shown to reduce the number of IgA binding sites, while increasing the affinity of the remaining receptors (Weisbart et al., 1988); however, it is not known whether this is due to the induction ofa high-affinity FcaR (i-e.,FcaRI) or the modification of a preexisting low-affinity receptor. A number of monoclonal antibodies have been described that specifically bind hFcaRI, including My43 (Shen et d.,1989) (used in the expression cloning of the receptor), A3, A59, A62, and A77 (Monteiro et al., 1990). Only My43 has been shown to inhibit the binding of IgA to hFcaRI, the remaining mAb appearing to bind determinants located outside the IgA binding site. As mentioned, novel FcaR apparently distinct from FcaRI have been postulated to exist on subpopulations of human and mouse T and B cells; however, no mAb have been described that recognize these proposed receptors. d. Polymorphisms No polymorphisms have been described to date which identify additional isoforms of hFcaRI to the original cDNA clone. However, molecular heterogeneity is apparent which arises by differential glycosylation, as on eosinophils FcaRI appears to have a molecular mass of 70-100 kDa (Monteiro et al., 1992), in contrast to FcaR on other cell types which exhibit a molecular mass of 55-75 kDa (Albrechtsen et al., 1988; Monteiro et al., 1990, 1992; Mazengera and Kerr, 1990). Deglycosylation of FcaRI on these cells in all cases produces a core protein of 32 kDa, which, combined with the apparent existence of only a single FcaRI gene (Maliszewski et al., 1990), suggests that multiple receptor isoforms with different numbers of glycosylation sites are not an explanation for the observed heterogeneity.
D. OTHERFCR 1. FcpR In contrast to the FcyR, FcsR, and FcaR, receptors for the Fc portion of IgM are not well-characterized; however, recent studies have begun to define the biochemical nature of these receptors.
42
MARK D. HULETT AND P. MARKHOGARTH
The existence of IgM binding molecules has been well-documented on subpopulations of human and murine B and T cells using EA rosetting and immunoflourescence techniques (Moretta et al., 1975, 1977; Lamon et al., 1976; Pichler and Knapp, 1977; Ferrarini et al., 1977; Burns et al., 1979; Rudders et al., 1980; Mathur et al., 1988a,b; Lydyard and Fanger, 1982; Anderson et al., 1981) (Table XI). Human NK cells have also been reported to express FcpR (Pricop et al., 1991, 1993).Although these approaches have clearly demonstrated IgM binding function of these cell types, it is only recently that FcpR have been biochemically defined. An FcpR of 58 kDa has been isolated from human B-cell lineages (Ohno et al., 1990)and a similar but apparently distinct molecule of 60 kDa has been isolated from human T cells following their short-term culture (Nakamura et al., 1993). The B-cell FcpR is an 0-glycosylated protein linked to the membrane by a GPI anchor and is inducible following cell activation. In contrast, the Tcell FcpR is resistant to phospholipase C treatment, suggesting it may be an integral membrane protein, and is downregulated following cell activation. The question of whether these FcpR are different forms of the one receptor or entirely different FcpR remains to be determined. FcpR on mouse B and T cells have not yet been isolated. The affinity ofthe described FcpRs appears to be quite low, their detection necessitating the use of IgM complexes (Pichler and Knapp, 1977; Mathur et al., 1988a; Lydyard et al., 1982).The function of FcpR on lymphocytes is interesting, and it is tempting to speculate that these receptors play a similar role in immune regulation as the FcyR and FcsR receptors on lymphocytes. Clearly much further work is needed to understand the biology of FcpB.
TABLE XI CHARACTERISTICS OF FcpR Characteristic
Humana
Mr Cell distribution Receptor topology Specificity Regulation expression*
58 B cell GPl IgM
a
t
No FcpR isolated from murine cells.
* Following cell activation. ND, not determined.
60
T cell ? IgM
t
ND NK cell ND IgM ND'
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
43
2. FcsR Receptors for IgD have been described on murine and human T cells and T-cell clones. The expression of these IgD-R is induced upon exposure to IgD complexes, 11-2,Il-4,or T-cell activating agents (Coico et al., 1985, 1987, 1988, 1990). In mice, IgD-R are expressed only on CD4+ T cells and cloned CD4+ T cells (Coico et al., 1985, 1987),whereas in humans they are expressed on both CD4+ and CD8+ T-cell subsets and T-cell lines (Coico et al., 1990; Tamma and Coico, 1991). However, IgD-R on murine and human T cells have not been biochemically characterized and have only been functionally detected on the surface of T cells by rosetting with IgD-coated erythrocytes. The presence of IgD receptors on human B cells have also reported (Sjoberg 1980b; Rudders and Anderson, 1982). Murine T-cell IgD-R have been shown to recognize N-glycans of murine IgD, as the interaction of these receptors with IgD is inhibited by N-acetlyglucosamine, N-acetylgalactosamine, and galactose. As such these receptors have been suggested to be lectin-like molecules (Amin et al., 1991). In contrast to receptors for the other Ig isotypes, the binding of mIgD to murine IgD-R is not specific to the Fc portion of IgD, as both Fab and Fc fragments can block the binding of IgD. The interactive region of murine IgD has been localized to the first and third constant regions of the heavy-chain domains (Tamma et al., 1991). Recent studies have suggested that the IgD-R on human T cells is also a lectin that binds N-glycans. However, in contrast to the murine IgD-R, the hIgD-R appears to interact with both hIgD and hIgAl (G. Thorbecke, personal communication). Both of these Ig isotypes contain Gal 1-3Gal NAc-rich 0-linked glycans. It should also be noted that murine and human IgD do not exhibit cross-species inhibition as assessed by EA rosetting. Clearly, extensive further studies are required to understand the molecular nature of murine and human IgD-R. 3. Polymeric IgR The polymeric IgA/IgM receptor (poly-IgR) is expressed on the basolateral surface of glandular epithelial cells and is responsible for the transcytosis of these polymeric Igs into external secretions (Table XII). Proteolytic cleavage of the extracellular polymeric Ig binding portion of the receptor produces secretory component (SC).The receptor binds polymeric Ig basolaterally and endocytosis of the receptor-ligand complex is followed by transcytosis to the apical cell surface and proteolytic cleavage of the receptor to release polymeric Ig into the apical medium in association with SC (Mostovet al., 1984; Brandtzaeg, 1985).
44
MARK D. HULETT AND P. MARKHOCARTH
TABLE XI1 CHAFIACTERISTICS OF POLY-IGR Characteristic
Rabbit
Isoforms Molecular mass (LDA) Ig-like domains Receptor topology Specificity
A0 70 3 T M ~sc , PkA, PIgM
Cellular distribution
Glandular epithelial cells NDd
Chromosome localization
Rat B‘ 90-95
5
TM, SC PI&, PI@ Glandular epithelial cells ND
Human
A 120 5 TM, SC PkAC
Glandular epithelial cells ND
Glandular epithelial cells lq31-41
Two forms encoded by differentiallyspliced transcripts,the “A, B” nomenclature is not standard and is used here for the sake of comparison. TM (transmembrane) and SC (secretory component) arise by cleavage of receptor from cell surface in association with polymeric Ig following transcytosis. Polymeric IgM binds weakly. ND, not determined.
*
The poly-IgR has been characterized at a biochemical and molecular level in three species, the rabbit, rat, and human. The receptor has been defined as a membrane glycoprotein of 100 kDa in the human (Brandtzaeg, 1985), 120 kDa in the rat (Banting et al., 1989), and two isoforms of 70 and 90-95 kDa in the rabbit (Mostov et al., 1984) (which are the products of alternatively spliced mRNAs of a single gene, see below). Molecular cloning of cDNAs encoding the poly-IgR in the rabbit (Mostov et al., 1984), rat (Banting et al., 1989), and human (Eiffert et al., 1984, 1989; Krajci et al., 1989, 1991, 1992) indicate the receptor is a member of the Ig superfamily and is structurally conserved in all three species, The receptor is an integral membrane molecule comprising an extracellular region of 5 Ig-like domains, a single transmembrane region, and a cytoplasmic tail. The 5 Ig-like domains are highly conserved and exhibit a significant degree ofhomology with the variable domain of Ig and thus are only distantly related to the leukocyte Ig superfamily FcR which contain Ig-like domains of the C2 set (Williams et al., 1989). Comparison of the predicted amino acid sequences of the rabbit, rat, and human poly-IgR reveals an overall 41% amino acid identity. The extracellular regions exhibit only 36% identity; however, the transmembrane and cytoplasmic tail regions are highly homologous displaying 74 and 60% identity across the three species, respectively (Mostov et al., 1984; Banting et al., 1989; Krajci et al., 1989).The significant conservation of these regions
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
4s
suggests they are important for receptor function. Indeed, the high degree of sequence homology of the cytoplasmic tails presumably reflects conservation of intracellular signals required for correct targeting/sorting/transcytosis (see below). A single mRNA species has been observed of 2.8 kb in the human (Krajci et al., 1989)and 3.5 kb in the rat (Banting et al., 1989),whereas two distinct related mRNAs of 2.6 and 2.8 kb are present in the rabbit which arise by differential splicing (Mostov et al., 1984). The gene encoding the human poly-IgR has been isolated and comprises 11 exons spanning 19 kb (Krajci et al., 1992b). Two exons encode the signal peptide (exons 2 and 3), single exons encode three of the five Ig-like domains (domains 1, 3, and 4 encoded by exons 3,5, and 6, respectively), whereas domain 2 and 3 are encoded by the same exon (exon 4). Exons 8 to 11encode the cytoplasmic tail region, with exon 8 also encoding the transmembrane region. As described above, the rabbit poly-IgR exists in both high- and low-molecular-weight forms which are encoded by two distinct differentially spliced mRNA transcripts (Mostov et al., 1984). The sequence of these transcripts indicates that it is the region encoding domains 2 and 3 which is alternatively spliced, which corresponds precisely to exon 4 of the human gene (Krajci et al., 1992b). The 4 exons encoding the cytoplasmic domains seem to correlate with the regions defining the structural determinants proposed to be responsible for the intracellular sorting of the poly-IgR in the rabbit (Apodaca et al., 1991). These regions include a 14-amino acid segment (residues 655-668 in the rabbit) proposed to direct the receptor to the basolateral surface (Casanova et al., 1991); the corresponding region in the human is exons 8 and 9, exon 9 containing Ser655-the phosphorylation of which appears crucial for receptor transcytmis (Hirt et d,1993). Residues 670-707 of the rabbit poly-IgR encode a region believed to be involved in the protection of receptor from lysosomal degradation (Breitfeld et al., 1990), and the corresponding human region is also found in exon 9. The 30 C-terminal residues of the rabbit poly-IgR have been shown to be responsible for the rapid basolateral endocytosis of the receptor (Breitfeld et al., 1990), and the corresponding human region is found in exon 11. The human poly-IgR gene has been mapped to chromosome lq31-41 by direct and genetic approaches (Davidson et al., 1988; Krajci et al., 1991, 1992a). A recent study has demonstrated that the human poly-IgR mRNA is upregulated in a time- and concentration-dependent manner by IFN-y (Krajci et al., 1993). Other proinflammatory cytokines which increase the epithelial expression of the human poly-IgR include TNF-
46
MARK D. HULETT AND P. MARKHOGARTH
a and IL-4 (Sollid et al., 1987; Kvale et al., 1988; Phillips et al., 1990).
In contrast to the human and rabbit poly-IgRs, which both bind polyIgA and poly-IgM, the rat receptor appears to bind only poly-IgA well and not poly-IgM (Underdown et al., 1992). 4. FcRn
FcRn is a receptor for IgG on intestinal epithelial cells which mediates the transfer of maternal Ig from milk to the bloodstream of newborn mice and rats (Table XIII). The receptor has been defined at a molecular level in both the rat and the mouse and is a heterodimer of an integral membrane glycoprotein similar to MHC class I antigens (IgG binding a-subunit) and @2-microglobulin(Simister and Mostov, 1989). The FcRn a-chain has been described as a 45- to 53- or 50-kDa glycoprotein in the rat (Simister and Mostov, 1989)and mouse (Ahouse et al., 1993),respectively. The p2m component has an apparent molecular mass of 14 kDa in both species. The association of the FcRn achain with p2m has been directly demonstrated in the rat using crosslinking studies of the receptor on brush border epithelial cells (Simister and Mostov, 1989) and is also suggested in the mouse as neonate1 mice homozygous for a targeted disruption of the p2m gene lack the ability to bind IgG through FcRn (Ziljstra et al., 1990). A human form of FcRn has also recently been suggested, as microvilli membranes from the small bowel of fetal intestine exhibit pH-dependent binding of IgG with a dissociation constant of 2 x lo7 M - l , which is similar to that of the rodent FcRn (see below) (Israel et al., 1993). cDNA cloning of the rat (Simister and Mostov, 1989) and mouse TABLE XI11 CHARACTERISTICS OF FcRn Characteristic
Rat
Mouse ~~
Molecular mass (kDa) Ig-like domains Receptor topology Associated subunits Cellular distribution Specificityb Affinity Chromosomal localization
45-53 3
50 3
TM /32ma Intestinal epithelial cells, fetal yolk sac IgG lo8M-'
TM P2m" Intestinal epithelial cells, fetal yolk sac
ND
p2m mouse/rat, 14 kDa. Selectively binds I& at pH 6.4; releases bound Ig at pH 7.4.
I& lo8 M-' ch 7
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
47
(Ahouse et al., 1993) FcRn a-chains indicates they are highly conserved integral membrane proteins with a predicted structure comprising an extracellular region of 3 Ig-like domains homologous to class I MHC antigens (therefore only distantly related to the leukocyte FcR and the poly-Ig receptor), a single transmembrane region, and a cytoplasmic tail. The rat and mouse FcRn exhibit 91% overall amino acid identity, with 84, 88, and 100% identity in the al, a2, and a3 domains, respectively, 91% in the transmembrane regions, and 98% in the cytoplasmic tail regions. Additional support for the similar structures of rat FcRn (and by analogy mouse FcRn) with MHC class I is based on the similarity in the circular dichroism spectra of rat FcRn and HLA-B40 (Gastinel et al., 1992). Comparison of the amino acid sequence of rat FcRn with MHC class I antigens reveals significant homology, the highest homology being in the a3 domains (35-37%), followed by a1 (27-30%), and a2 (22-29%) (Simister and Mostov, 1989). Mouse FcRn also exhibits similar homology with MHC class I, with an average of 27% identity in a l , 23% in a2, and 34% in a3 (Ahouse et al., 1993). The gene encoding mouse FcRn has been mapped to the proximal region of chromosome 7 and is therefore not encoded in the same region as the majority of MHC class I antigens which are found on chromosome 17 (Ahouse et al., 1993).This finding, together with the results of amino acid sequence comparisons, supports a divergence of FcRn from MHC class I early in the mammalian lineage. The binding of IgG by rat and mouse FcRn is of high affinity ( K , = lo7 - lo8 M-I) and is pH dependent with Ig being bound at pH 6.5 in the acidic environment of the gut and released at pH 7.4 in the neutral environment of the bloodstream (Simister and Rees, 1985; Hobbs and Jackson, 1985; Raghavan et al., 1993). A secreted form of rat FcRn has been cocrystallized with its ligand, and although no structural data have been reported, the stoichiometry of the interaction has been determined, with two FcRn molecules binding per Fc portion (Gastinel et al., 1992; Huber et al., 1993). Studies of the tissue distribution of FcRn by Northern blot analysis of mRNA has demonstrated that the receptor is expressed in epithelial cells of the neonate1 rat and mouse small intestine (but not adult intestine) (Simister and Mostov, 1989; Ahouse et al., 1993) and in the yolk sac (Ahouse et al., 1993; Roberts et al., 1990). A single 2.2-kb FcRn a-chain mRNA is present in these tissues of the mouse (Ahouse et al., 1993) and in two mRNAs of 1.7 and 3.1 kb in the rat, which possibly arise by the use of alternate polyadenylation sites (Simister and Mostov, 1989).
48
MARK D. HULETT AND P. MARKHOGARTH
111. Molecular Basis of the FcR-19 Interactions
The main focus of studies on the molecular nature of the FcR-Ig interaction has been the identification of the regions in the Fc portion of Ig involved in binding to FcRs, and little attention has been given to the determination of the sites on FcRs responsible for binding Ig (aspects reviewed in Metzger, 1988; Burton and Woof, 1992; Sutton and Gould, 1993).As a consequence, a distinct bias exists in the understanding of the FcR-Ig interaction. However, recent studies performed by ourselves and others examine this interaction from the receptor side, and significant advances are being made into understanding the molecular basis of the interaction of FcRs with Ig. The current. state of understanding of the FcyR-IgG and FceRI-IgE interactions is presented below and summarized in Table XIV and Fig. 1. A. FcyRI The site(s) of interaction on mouse or human FcyRI with IgG are not well-characterized at present. However, by generating chimeric mFcyRIlmFcyRI1 receptors we have been able to define the Ig binding roles of the extracellular domains of mFcyRI (Hulett et al., 1991). The extracellular region of mFcyRI can be divided into two main regions with distinct Ig bindingroles: (i)the first two domains (homologous to the two domains of the other leukocyte Ig superfamily FcR) responsible for the direct binding of IgG and (ii) the unique domain 3, which confers the distinctive specificity and affinity of the receptor (Table XIV). The first two domains of mFcyRI can bind IgG in their own right. However, the removal of domain 3 converts the Ig binding function of mFcyRI to an “FcyRII-like” receptor, domains 1and 2 of mFcyRI in the absence of domain 3 lose the ability to specifically bind mIgG2a with high affinity and instead exhibit a low affinity and broad specificity for mIgGl, 2a, and 2b, characteristic of mFcyRII and I11 (Hulett et al., 1991). This finding demonstrates that the first two domains of mFcyRI represent an IgG binding motif conserved with the lowaffinity FcyR and that domain 3 of mFcyRI is modifying the binding of IgG by domains 1 and 2. Consistent with this finding is that the two-domain fork of hFcyRIb, which lacks domain 3, also exhibits lowaffinity IgG binding (Porges et al., 1992). In addition, it has been claimed that preliminary studies on hFcyRI, whereby point mutations were introduced into domain 3, also indicate that this domain is important in conferring high-affinity binding, although no data were presented (Allen and Seed, 1989).Based on these observations, it would
TABLE XIV
SUMMARYOF FcR-Ig INTERACTIVEREGION^ Receptor-ligand interactionb D1
FcyRI - IgG
Stability ?
I
D2
I
1
F9RII - IgG
Ig binding regions
Receptor binding regions
,,
AffinitY/S#city
D2
.I
’ ,111-114
\
/
0,
Binding domain
D1
D3
I
Stability/AfEnity ?
1!2%134 1 5 p 1 6 1 5
Binding domain
F3RIII - IgG \
FcERI- IgE
I
D1
Stahility/Minity ?
?
CE2
D2 87-12.W
129-137
Binding domain*
154-161
/
/ \
Binding domain ?
,
Stability ?
I=
CE3
,, =
Binding domain
I
CE4
1 0
/ \
Minity&nd site
Schematic diagram of FcR extracellular domains and Ig Fc portion constant domains. Identified binding regions shaded and key residues indicated. Based on studies of mFcyRI, hFcyRII, rat FcyRIII and hFc,RI. Demonstrated directly for hlgGln and inferred for other IgG subclasses. Site located in hinge proximal region. Direct binding role demonstrated for 15p161 region, secondary or indirect binding contribution by 111-114 and 130-135 regions. Direct binding role for all three regions, three subregions of 87-128, ie., &104,10&115, and 111-125 imphcted in binding (see text). f For binding of rat IgE by rat Fc,RI, domain 1appears to be the crucial domain.
*
A
*
*
*
6
*
*
*
C
*
F G L T A N S - D T H L L O G Q S L T L T L E S - P P G S S P S V Q C R S P R G - - - K 86
110
100
90
120
E W L V L Q T P H L E F Q E G E T I M L R C H S W K D - V K V T F F Q N - G - K S Q
D W L L L Q T P Q L V F L E G E T I T L R C H S W R N K L & N R I S F F H N - E - K S V G W L L L Q A P R W V F ' E E D P I H L R C H S W K N T A L H K V T Y L Q N - 2 - K D R G W L L L Q A P R W V T K P P 3 P I H L R C H S W K N T A L H K V T Y L Q N - D - K D R D W L L L Q T P Q R V F ; E G S T I T L R C H S W R N K L L N R I S F F H N - E - K S V
-
--
- -- ------
-
D W L L L Q T P Q L V F L E ' G S R I T L . R C H G W K S I Q L A R 1 S F L Q N - G - Q F V
D W L L L Q T P Q L V F E E G E T I T L R C H S W K N K Q L T K V L L F Q N - G - K P V D W L L L Q A S R R V L T E G E P L A L R C H G W K N K L V T N V V F Y R N - G - K S F G W L L L Q V S S R V F T E G E P L A L R C H A W K D K L V Y N V L Y Y R N - G - K A F
D W L L L Q A S A E V V M E G O P L F L R C H G W R N W D V Y K V I Y Y K D - G E A L K
C *
*
E
G
F
*
*
*
*
*
*
*
N I Q G - G K T L S V S Q L E L Q D S G T W T C T V L Q - N Q K K V Q F K I D I V V L 130
K R K K R
@ Y Y Y Y
S H F F H
140
150
160
@ L D @ T F S I P Q A N H S H S G D Y H C T G [ N ~ Y T ~ S K P V T I T V Q H Y S S N F S I P K A N H S H S G D Y Y C K G S L G R T L H Q S K P V T I T V H H N S D F ~ I P K A T L K D S G S Y F C R G L ~ G S ~ N V S S E T V N H H N S D F H I P K A T L K D S G S Y F C R G L V G S K N V S S E T V N I T I H Y K S N F S I P K A N H S H S G D Y Y C K G S L G S T Q H Q S K P V T I T V
--------- --
-
170
Q I T Q
G T Q D
P I T Q G L A V S T G L A V S T P A T
S F H P Y N V S Y S I S N A N H S H S G D Y Y C K A Y L G R T E H V S K P V T I T V Q G
R Y Y Y Q S S N F S I P K A N H S H S G N Y Y C K A Y L G R T M H V S K P V T I T V Q G
Q F S - S D S E V A I L K T N L S H S G I Y H C S G T - G R H R Y T S A G V S I T V K E L K F F H W N S N L T I L K T N I S H N G T Y H C S G M - G K H R Y T S A G I S V T V K E L Y W Y E N - H N I S I T N A T V E D S G T Y Y C T G I K V W Q L D Y E S E P L N I T V I
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
51
be expected that the hFcyRIbl and c l transcripts, which contain stop codons in their third extracellular domain coding regions, would encode functional soluble low-affinity FcyRs. However, protein products of these transcripts have not as yet been identified. It should be noted that the modifying effect on Ig binding by domain 3 was found to be specific to mFcyRI, as the linking of domain 3 to domains 1 and 2 of mFcyRII did not produce the specific high-affinity binding of mIgGSa, this receptor retaining the specificity and affinity of mFcyRII (Hulett et al., 1991). Furthermore, the binding of mIgG2a by mFcyR1 appears to be a specialized interaction between domains 2 and 3, as replacement of domain 1 of mFcyR1 with domain 1 of mFcyRII does not alter the specificity of IgG binding or have a major influence on the high-affinity binding of mIgG2a, in contrast to the replacement of both domains 1 and 2 of mFcyRI with domains 1 and 2 of mFcyRII (M. Hulett, unpublished observations). Based on these findings it is tempting to speculate that domain 2 of mFcyRI is the key domain involved in the direct binding of IgG, especially in the light of the observation that it is the homologous second extracellular domain of both human FcyRII and FceRI a-chain that is responsible for the binding of IgG and IgE, respectively (see below). The definition of the Ig binding roles of the extracellular domains of mFcyRI should now enable the fine specificity of the molecular interaction with IgG to be determined. In contrast to the limited information available on the FcyRI binding site for IgG, significant advances have been made into identifying the binding site(s) on IgG for hFcyRI (Burton et al., 1988; Burton and Woof, 1992). Early studies using proteolytic fragments of IgG suggested the C y 3 domain of IgG was crucial for the interaction with human FcyRI (Okafor et al., 1974; Ciccimarra et al., 1975); however, this was subsequently shown to be incorrect with purified IgG fragFIG.1. Alignment of Ig superfamily FcR second extracellular domain amino acid sequences. The positions of the putative p strands are overlined and the core hydrophobic residues are indicated by asterisks and are based on comparison with the solved structure of CD4 domain 2 (Ryu et al., 1990; Wang et al., 1990; Hogarth et al., 1992). Regions implicated in the binding of Ig using chimeric receptor studies are boxed. Specific residues implicated in Ig binding through mutagenesis studies are circled. Polymorphic residues also suggested to play a binding role are underlined. Amino acid differences between rat FcyRIIIA and rat FcyRIIIH or hFcyRIIIa and FcyRIIIb are indicated by lines between the two sequences. Three subregions of the 87-127 IgE binding region of hFceRI have been implicated in binding and are underlined. See text for sequence details. The numbering is based on that for hFcyRIIa, with every 10th residue indicated.
52
MARK D. HULETT AND P. MARKHOGARTH
ments and myeloma proteins containing deleted domains (Woof et al., 1984). It was suggested that the Cy2 domain had an important role in the binding of IgG to FcyRI in experiments where removal of N-linked carbohydrate from Cy2 resuIted in a significant loss in affinity for FcyRI (Leatherbarrow et aZ., 1985, Walker et al., 1989).The role of Cy2 in the binding of IgG to hFcyR1 was demonstrated directly in experiments that examined the capacity of anti-human IgG mAb to inhibit the binding of IgG to FcyRI, as only mAb that recognized the N-terminal portion of Cy2 blocked IgG binding to FcyRI (Partridge et al., 1988). These same mAb could not bind to IgG that was bound to FcyRI. In support of this important binding role of Cy2, recent experiments using chimeric immunoglobulins generated between hIgG1 and mIgE, where Cy2 andlor Cy3 were exchanged with the homologous Cs3 and Cs4, demonstrated that mutant immunoglobulins lacking Cy2 did not bind to FcyRI, whereas those containing Cy2 bound to FcyRI (Shopes et al., 1990). However, Cy3 does seem to play a role in the binding of IgG to FcyRI, as calculations of the relative contributions of each domain to binding reveal that Cy3 contributes 25% of the overall drop in free energy on binding, Cy2 contributing 73%. This contribution of Cy3 to the binding of IgG by FcyRI has been proposed as a stabilizing role on the Fc structure (Shopes et al., 1990). A similar study using chimeric hIgG2 and hIgG3 molecules supports the above findings, as IgG2 (which does not bind to FcyRI) substituted with Cy3 of IgG3 (which binds to FcyRI) did not bind to hFcyRI, whereas IgG3 containing Cy3 from IgG2 did bind to FcyRI (Canfield and Morrison, 1991). The Cy2 domain of hIgGl has also been shown as the principal domain involved in the binding of this isotype to hFcyRI. Using a similar chimeric approach as that described above, but with hIgGl and hIgG2, it was demonstrated that only those chimeric molecules containing Cy2 of IgGl were able to bind to hFcyRI (Chappel et al., 1991). In order to identify the binding site in Cy2 for hFcyRI, experiments were performed where a range of IgGs of different isotypes from different species was tested for their ability to bind to hFcyRI (Woof et al., 1986). Examination of the amino acid sequence of the Cy2 domain of these IgGs enabled the identification to be made of sites potentially involved in the binding to FcyRI. The region Leuw to S e P 9 (Leu-Leu-Gly-Gly-Pro-Ser) in the N-terminal region of Cy2, which forms part of the hinge proximal region, was proposed as crucial for interaction with FcyRI, being present in all IgG isotypes that bound to hFcyRI with high affinity, i.e., hIgG1, hIgG3, mIgG2a, rat IgC2b, and rabbit IgG (Woof et al., 1986) (Table XV). Mouse IgG2b and
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
53
TABLE XV
COMPARISON IgG CH2HINGEPROXIMAL REGIONS IgG" hIgGl hIgG3 rIgGb mIgG2a mIgG2b hIgG2 mIgGl a
Amino acid sequence alignmen9
P P P P P P T
A A P A A A V
P P P P P P P
E L L G G P S V
E E N N P
€3-
L L L L A
L G G P L G G P L G G P E G G P - A G P -- V S
S S S S S S
V V V V V V
h, human; m, mouse; r, rabbit. Corresponding to residues 230 to 240 of hIgG1.
hIgG4, which bind weakly to FcyRI, differ in this region, with mIgG2b containing Glu at position 235 and hIgG4 containing Phe at position 234. The importance of the 234-239 region in the binding of IgG to FcyR has been confirmed using site-directed mutagenesis. Mutagenesis of this region in hIgG3 has demonstrated that substitutions between residues 234 and 237 reduce the binding to hFcyRI. Residue when substituted for Glu had a >100-fold decreased binding affinity, whereas replacement of Leu234,GlyU6,and GlyZ3' with Ala had less effect, with affinities reduced 4-, 4-, and 30-fold, respectively. Using the reverse approach, the weak binding of mIgG2b was converted to high affinity (comparable to hIgGl), following replacement of G ~ with Leu (Duncan et al., 1988; Lund et al., 1991). Similarly, point mutations in this region of hIgGl have been shown to either significantly reduce or abolish its hFcyRI binding activity (Chappel et al., 1991). An independent study examining the 234-237 region by sitedirected mutagenesis supported the above findings and confirmed the importance of this region in the binding of IgG to hFcyRI (Canfield and Morrison, 1991). Replacement of in hIgG3 with Glu also resulted in a >lOO-fold reduction in affinity for hFcyRI. In addition, hIgG4, which binds weakly to hFcyRI and differs from the high-affinity IgG isotypes containing a Phe at position 234, was converted to a highaffinity binding immunoglobulin (3-fold lower than hIgG3 for FcyRI) upon substitution of Phem with Leu. In the reciprocal experiment, replacement of Leu2%in IgG3 with Phe produced a molecule with a low affinity for hFcyRI equivalent to that of hIgG4 (Canfield and Morrison, 1991). The inability to impart full high-affinity binding to hIgG4 by replacing PheZa with Leu led to the proposal that other residues in C72 may be involved in the binding to FcyRI. Indeed, a
u
~
54
MARK D. HULETT AND P. MARKHOGARTH
second region of Cy2 comprising a hinge proximal bend which lies in close proximity to the 234 to 237 region has also been implicated in the binding of IgG by hFcyRI (Canfield and Morrison, 1991). This was demonstrated as substitution of Pro331situated in this loop region (Pro is found in this position in the high-affinity binding hIgGl and IgG3 isotypes in contrast to Ser in hIgG4) and was found to reduce the affinity for hFcyRI 10-fold. It has also been proposed that another bend region lying close to the hinge proximal region may be an important contributor to binding, as aglycosylation of Cy2, which lowers the affinity for hFcyRI, appears to result in structural alterations in this region as assessed by NMR methods, which may in turn effect the structure of the lower hinge region (Matsuda et al., 1990).It should be also noted that the binding of IgG to hFcyRI has been shown to involve only one heavy chain, as the valency of hFcyRI for IgG was determined to be one (O’Grady et al., 1986), and monomeric mIgG2a2b and mIgG2a bind equally well to mFcyRI (Koolwijk et al., 1989).
B. FcyRII Of the three classes of FcyR, FcyRII has the most reported information on the molecular basis of its interaction with IgG (Table XIV, Fig. 1). A contribution to the understanding of how FcyRII binds IgG has been made through the identification and characterization of a functional polymorphism of hFcyRII, the high-responder/low-responder polymorphism. This polymorphism has been described extensively above and identifies residue 131 in the second extracellular domain of hFcyRIIa as important in the binding of IgG. The nature of residue in this position is crucial for the binding of both mIgGl (Warmerdam et al., 1990; Tate et al., 1992) and hIgG2 (Warmerdam et al., 1991). The presence of Arg directs the strong binding of mIgGl, yet results in weak binding of hIgG2; whereas the presence of His promotes the strong binding of hIgG2 and weak binding of mIgG1. These findings indicate that position 131 of hFcyRIIa is probably contributing to the binding site of both mIgGl and hIgG2 and suggest that domain 2 has an important role in the binding of IgG. It should be noted that this polymorphism does not affect the binding of other mouse and human IgG isotypes (Warmerdam et al., 1991), suggesting the existence of additional regions important in the binding of IgG by hFcyRII (see below). The mouse Ly-17 polymorphism of mFcyRII (Shen and Boyse, 1980; Hibbs et al., 1985; Holmes et al., 1985) also implicates the second extracellular domain of this FcyR class in the binding of IgG. The
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
55
polymorphism, described above, has been defined at the molecular level as two allelic variants that differ only in residues 116 and 161, where Pro'16 and Glnl6I are found in the Ly-17.1 form and Leu''6 and Leu'" in the Ly-17.2 form (Lah et al., 1990). MAb specific for the Ly17.2 form inhibit the binding of IgG to the receptor, implying that residues 116 and/or 161are involved in binding themselves, or closely situated to residues crucial in the interaction of FcyRII with IgG. W e have used a chimeric receptor strategy, involving the exchange of homologous segments of hFcyRII and the structurally similar hFcsRI a-chain, to definitively demonstrate that domain 2 of hFcyRII is responsible for the direct binding of IgG (Hulett et al., 1993). A chimeric receptor comprising domain 1 of the hFcsRI a-chain and domain 2 of hFcyRIIa was found to bind only IgG immune complexes, whereas the reciprocal receptor containing domain 1of hFcyRIIa and domain 2 of the hFceRI a-chain did not exhibit any IgG binding and instead bound only IgE complexes. In addition, we have also demonstrated that domain 2 of hFcyRIIa contains the epitopes recognized by anti-hFcyRII mAb which block the binding of IgG to hFcyRII, providing further supporting evidence that domain 2 is the principle domain involved in the binding of IgG (Ierino et al., 1993). Although domain 1 of hFcyRII appears not to be directly involved in the binding of IgG, it does play an important structural role. This is suggested as replacement of hFcyRIIa domain 1 with domain 1 of hFcsRIa reduced the capacity to bind IgG, as shown by the failure of this receptor to bind dimeric human or mouse IgG1, which bind to wild-type hFcyRII (M. D. Hulett, unpublished observations). These data imply that the role of domain 1 in Ig binding is likely to be an influence on receptor conformation, stabilizing the structure of domain 2 to enable efficient IgG binding by hFcyRII. However, the possibility remains that a direct interaction of domain 1with IgG can occur following initial binding to domain 2. The systematic examination of hFcyRII domain 2, again using a chimeric hFcyRII/hFcsRI a-chain strategy, has enabled the localization of the IgG binding region to an 8-amino acid segment contained within residues Asn'% to Ser''l (Table XIV). Site-directed mutagenesis on this region identified residues Ilel5' and Gly'56 as crucial in the binding of both mIgGl and hIgGl by hFcyRII as replacement of these residues with alanine resulted in almost complete loss of binding. The importance of this region in the binding of IgG was further supported with the finding that replacement of Leu159,PhelGO,and SerlG1with Ala substantially increased the affinity of these mutant receptors for mIgGl and/or hIgGl (Hulett et al., 1994) (Fig. 1).
56
MARK D. HULETT AND P. MARKHOGARTH
We have generated a three-dimensional model of hFcyRII domain 2 based on the previously described related structure of CD4 domain 2 (Hogarth et al., 1992; Hulett et aE., 1994) (Fig. 2). The model represents an Ig-like domain of the “truncated C2 set,” comprising two anti-parallel 6 sheets of three and four6 strands, respectively (Williams and Barclay, 1988).The putative eight-residue binding region lies in the F-G loop of domain 2 at the interface with domain 1. The spacial location of the Ile”‘ and Gly’= in the hFcyRII domain 2 model suggests these residues contribute to a possible hydrophobic cleft between the F-G and B-C loops (Fig. 2). Based on these findings, this hydrophobic pocket is postulated to be a critical structure for the binding of IgG by hFcyRII. The similar Ig binding specificity of the FcyR, combined with their high amino acid sequence identity, makes it tempting to speculate that the F-G loop of domain 2 may be a conserved binding region in this class of FcR. With this in mind, it is significant to note that comparison of the F-G loop sequences of the human and mouse FcyR reveals that the putative crucial IgG interactive residues of hFcyRII, i.e., Ile15’ and G1y1%, are the only conserved residues, as Gly’= is found in all FcyR and a hydrophobic residue is present at position 155 in all the low-affinity FcyR (Fig. 1). In light of these observations, it is interesting to note that the F-G loop sequences of the two hFcyRIII isoforms differ only in the nature of the hydrophobic residue at position 155, where FcyRIIIA contains a phenylalanine and hFcyRIIIB a valine (Fig. 1). These hFcyRII1 isoforms exhibit distinct affinities for IgG as hFcyRIIIA has a K , = 2 x lo7M - l whereas hFcyRIIIB a K , < lo7M-‘. (Simmons and Seed, 1988; Vance et al., 1992). Thus, based on the proposed importance of the F-G loop in the binding of Ig, these findings suggest that the hydrophobic residue at position 155 may indeed be playing a crucial role in the binding of IgG by hFcyRIII. Other residues implicated in the binding of IgG by FcyRII through the polymorphism studies are situated in loop regions in close proximity to the identified 154-161 binding region. Residue 131 lies in the C’-E loop, and the human equivalents of the mouse Ly-17 mFcyRII polymorphism, i.e., and Led5’, are located in the adjacent B-C and F-G loops, respectively. These findings suggest that the C’-E and B-C loops of FcyRII also contribute to the binding of IgG. Indeed, site-directed mutagenesis on both of these regions has identified a number of residues which when replaced with Ala substantially effect the binding of IgG. These include Lys113,Pro114,and Leu”‘ of the BC loop and Phe12’, Arg/His13’, and Prola of the C’-E loop (M. D. Hulett, unpublished observations) (Fig. 1).However, based on our
FIG.2. Ribbon diagram of hFcyRII second extracellular domain model. The model is oriented with the C’-E and B-C face at the front and adjoins domain 1at the top of the page and the transmembrane region at the bottom (for details see Hogarth et al., 1992).The B-C, C’-E, and F-G loops are shown in dark blue, and the disulfide bond between Cys’“ and Cys’” in the B and F strands, respectively, in yellow. The F-G loop residues IleIss and Gly’” identified as crucial in the binding of IgG by hFcyRII (see text for details) are shown in magenta. t
MOLECULAR BASIS OF Fc RECEPTOR FUNCTION
57
findings from the chimeric receptor studies, the F-G loop of hFcyRII appears to be the major interactive region for mIgG1 and hIgG1. This is clearly evident as the substitution of the 154-161 region of hFcyRII with the corresponding region of hFceRIa totally eliminates IgG binding, whereas insertion of this region into FceRI imparts IgG binding to FceRI (Hulett et al., 1994). This effect on IgG binding was not observed for any other regions of FcyRII domain 2. Thus, although both the C’-E and B-C loops are involved in the binding of IgG, their roles appear to be secondary to that of the F-G loop region. In summary, these findings suggest that the IgG interactive site on hFcyRII is at the interface of domains 1 and 2, with loop regions at the membrane-distal end of domain 2 playing the crucial binding roles. It should be noted that the affinity of hFcyRII for IgG immune complexes has been demonstrated to increase following treatment with proteolytic enzymes such as pronase and elastase (van de Winkel et al., 1989); however, the molecular basis of this observation has yet to be determined. The identification of the FcyRII binding site(s) in IgG has proved more difficult for this receptor compared with FcyRI, due to the low affinity of FcyRII for monomeric IgG. However, significant advances have been made into localizing the region in the Fc portion of IgG important for interaction with hFcyRII (Burton and Woof, 1992). As for the binding of hIgGl and hIgG3 by hFcyRI, aglycosylation of the Cy2 domain of these isotypes resulted in a dramatic loss in their capacity to bind hFcyRII, suggesting Cy2 is important in the binding of IgG by hFcyRII (Walker et al., 1989). Studies have been performed to identify the key domains in the Fc portion of IgG responsible for binding to hFcyRII (Shopes et al., 1990). The experiments were performed using the same chimeric hIgGl/mIgE molecules described in the analogous experiments for hFcyRI (see above). Results indicated that only those chimeric Ig containing both Cy2 and Cy3 were able to mediate rosette formation with K562 cells (FcyRI-, FcyRII+), suggesting both these domains are required for the binding of hIgGl to hFcyRII (Shopes et al., 1990). These findings are therefore in contrast to the requirement of hFcyRI seemingly for only the Cy2 domain for the binding of hIgGl (Weetall et al., 1990). The importance of the Cy 2 domain of hIgG3 in the interaction with h F q R I I has been demonstrated using the panel of hIgG3 mutants in the 234-237 region, as described above for binding to hFcyRI (Lund et al., 1991). These mutant hIgG3 molecules were assessed for their ability to form rosettes with the FcyRII expressing B-cell line Daudi. The number of rosettes formed with hIgG3 substituted with Leu2%to Ala, to Ala,
to Glu, Gly236to Ala, and GlyZ3’to Ala was reduced in each case compared with wild-type hIgG3, suggesting this region is important in the binding of hIgG3 to hFcyRII. Human IgG3 containing replaced with Ala exhibited the lowest binding capacity to hFcyRII, forming lo9 clones. Randomization of six residues results in a library diverse in sequences but constrained in structure. The remodeling of a single combining site to bind a variety of antigens might best be achieved by focusing on structural diversity, i.e., randomizing stretches which are longer than can be completely surveyed and display a greater degree of structural freedom. This hypothesis is testable using a competetive selection scheme. Barbas et al. (1993d) utilized three HCDR3 libraries of length 5, 10, and 16. This report focused on developing a strategy for the iterative selection of catalytic antibodies which utilized metal cofactors. Fabs were selected to bind a variety of metal ions by immobilizing the metal with an iminodiacetic acid support. Success of the selection strategy could be verified with some of the metals, such as Cu2+and Zn2+,by knowledge of side chains which have been shown to be important in chelating these metals. The Cu2+and Zn2+selected clones were abundant in His, Cys, Met, and Asp as would be predicted. Binding to free Cu2+was verified by fluorescence quench experiments. The selective panning was also applied to the selection of Fab which could bind the surface of the metal oxide magnetite. This experiment produced four clones which were similar to other loop sequences which had previously been reported to bind magnetite. No clones from the five residue-length HCDR3 library were observed following analysis of 49 selected clones. In another report (Barbas et al., 1993a),libraries of HCDR3, HCDR3 and LCDR3, and LCDR3 alone were examined using a competetive selection scheme for the selection of anti-hapten Fab. In this report, selection for binding three different haptens resulted in 18 antihapten antibodies; additional clones were available but were not sequenced. The specificities of a number of these were examined and are shown in Fig. 22. Various degrees of specificity were observed with binding constants in the range 1-3 x 1O'M-l as determined by surface plasmon resonance. Again no clones from the five-residue HCDR3 were se-
255
ANTIBODIES FROM COMBINATORIAL LIBRARIES
100
' s v)
8
80 60
40
20 0
P2
F22
s2
s4
s10
C15
om rn ""-8 Tetanus toxoid
1
Bovine serum albumin FIG.22. The specificity of Fabs selected from semisynthetic libraries by panning against various haptens. F22 and P2 are Fabs selected with conjugate 1; S2, S4, and S10 selected with conjugate 2; and C15 selected with conjugate 3. From Barbas et al., 1993a).
lected with the competitive selection scheme. Additionally none were selected from the LCDR3 only randomized libraries where a maximum of six residues were randomized. The selected clones were derived mostly from HCDR3/LCDR3 libraries. Furthermore, all clones selected were derived from HCDRS libraries wherein Asp-101 was fixed. These results support the notion that structurally diverse libraries are a requirement for remodeling a given framework. Extrapolation of these results may be used to rationalize the utilization of shorter HCDRS lengths in mice as compared with humans. Structural diversity lacking in HCDR3 of the mouse may be compensated by the use of a far greater number of V segments. Synthetic libraries can be utilized as probes for molecular recognition of antigens from which it is difficult to prepare antibodies for reasons of tolerance, toxicity, or reactivity. To this end, S. M. Barbas et al. (1994) have utilized the aforementioned libraries to study the recognition of double-stranded DNA. Two Fabs were isolated which bound double- and single-stranded DNA with moderate to high affin-
256
DENNIS R. BURTON AND CARLOS F. BARBAS 111
ity. The Fabs did not bind the negatively charged polyelectrolyte dextran sulfate or lipid A. The ability of one of the Fabs to perform as a naturally occurring DNA binding protein was investigated in electrophoretic mobility shift assays. The ability to form nucleoprotein complexes was clearly demonstrated (Fig. 23). The two Fabs isolated contained HCDR3s of 10 and 16 residues in length. Interestingly, although the CDRs differed in length, the amino- and carboxy-ends of the HCDR3 were virtually identical (Fig. 24). The clone with the 16-residue HCDR3 demonstrated a clear preference for poly(dGdC).poly(dGdC),whereas the other clone bound different oligonucleotides with similar affinity (Fig. 25). This observation and the fact that the two clones differ only in the central portion of HCDR3 suggest this region is crucial for the sequence preference displayed by one of the clones and may provide the basis for the design and selection of antibodies capable of sequence-specific recognition. In unpublished studies, these synthetic libraries have further been utilized in the selection of Fabs which bind a number of protein antigens and in one case neutralize HIV. Fabs with catalytic activity may also be selected from the aforementioned synthetic antibody libraries. The direct covalent selection of Fabs from phage display libraries was proposed in the original Fab display report of Barbas et al. (1991). The strategy proposed was to use mechanism-based inhibitors or affinity labels to select for appropriately positioned functionalities within the combining site of an antibody. These functionalities, amino acid side chains with the appropriate chemical characteristics and geometries, would then catalyze a chemical reaction with the appropriate substrate. To this end, Janda et al. (1994) have utilized an pyridyl disulfide affinity label to trap an appropriately positioned thiol in the active site of an synthetic antibody. Phage which covalently bind to the support via a disulfide bond are then selectivly released by reduction of the disulfide following elution with acid of noncovalently attached phage (Fig. 26). Sequencing of 10 of the selected Fabs revealed 2 with unpaired cysteines. One of these was examined for its ability to hydrolyze a thiol ester substrate designed to place the electrophilic center of the carbonyl in the position of original active disulfide ofthe affinity label. The Fab was shown FIG.23. (a) Interaction between the synthetic Fab SD1 and DNA in an electrophoretic mobility shift assay (EMSA). Lanes 1-6 contain 0.08 pmol 32P-labeleddoublestranded probe (5'-AAT-GTA-TGC-GCG-CGC-GCT-TTA-GGC-GCC-CC-3') with 0.2 pmol Fab SD1 (lanes 2, 3, and 4). Lanes 3 and 6 included a preincubation with an anti-Fab reagent (a-Fab IgG). Lanes 4 and 5 included 0.2 pmol of a control Fab reactingwith HIV-1 surface glycoprotein gp120 (HIV Fab). The thick arrow indicates the
257
ANTIBODIES FROM COMBINATORIAL LIBRARIES
a
- - - +
HIVFab a-FablgG SD1 Fab
- - + + - -
- + +
+
+
-0
as-
--+
-
1 2 3 4 5 6
-FP
b 0 0 0 0
0
0
0 0
08RszO88s$
1
2 3 4 5 6 7 8 9 10 11
0
0
0 0 0 0 8 8 8 0 0 0 0 oV)6lV)roV)OIV)r
12 13 14 15 16 17 18 19 20 21 22
position of the specific Fab SDl nucleoprotein complex and the a,arrow indicates the position of the supershifted Fab SD1 nucleoprotein complex. 0 marks the origin of migration and FP the position of the free probe. (b) Specificity of binding of Fab SD1 to DNA. Lanes 1-22 contain 0.08 pmol %P-labeled probe and 0.2 pmol Fab SDl in lanes 2-11 and 13-22, respectively. In addition increasing concentrations of competitor oligonucleotide are included as shown. Numbers refer to molar excess of competitor oligonucleotide relative to the probe. Arrows indicate the position of the Fab nucleoprotein complex. From S. M. Barbas et al. (1994).
258
DENNIS R. BURTON AND CARLOS F. BARBAS I11
Library E
GxxxxxxxxxxxxxDx
QQM;GsRN
Library F
GxxxxxxxDx
QQyI;Gspw
Library ~1 O I E
GxxxxxxxxxxxxxDx
QQYxxxxxxT
Fab SD1
GRAYGGWMSLDN
QwGGsRN
Fab SD2
GRGWSGSLDI
QQyt;Gspw
FIG.24. Amino acid sequences of the variable domain CDR3 regions of the heavy chains (HC) and light chains (LC) from the DNA binding antibodies SD1 and SD2 and the semisynthetic libraries from which they were selected. x refers to a position which was randomized. From S. M. Barbas et al. (1994).
to be catalytic in its hydrolysis of the ester and was futher characterized to proceed through a covalent intermediate. Thus, the catalytic activity had been directly selected to proceed through the mechanism dictated by the design of the affinity label/substrate pair. This is the first report of the selection of a catalytic protein, in this case a Fab, from a random protein library and will likely have a major impact on the field of catalytic antibodies. Garrard and Henner (1993) have constructed a synthetic antibody library with the introduction of limited diversity over four CDRs of a humanized anti-HER-2 Fab. The doping strategy was designed to incorporate mostly amino acids which are naturally found in the CDRs. The library of Fab fragments was selected to bind rsCD4, insulin-like growth factor 1 (IGF-1), and tissue plasminogen activator. A single Fab was isolated which bound IGF-1 with an affinity of 3 X lo5M-’. Two other reports, Hoogenboom and Winter (1992) and Akamatsu et al. (1993), have utilized combinations of genomic V segments with synthetic CDR3 segments as had been previously suggested (Barbas et al., 1992a).The report of Hoogenboom and Winter utilized a defined collection of 49 V, genes and a single light chain. Both groups constructed scFv libraries as opposed to Fab as discussed above. The former report combined a HCDR3 segment randomized over five amino acids with V, genes and selected the library to bind phOX-BSA, 3-iodo-4-hydroxy-5-nitrophenylacetate-BSA (NIP-BSA), BSA, turkey egg white lysozyme, TNF-a, and human thyroglobulin. Several scFv’s were shown to bind phOX and NIP while only a single clone was found to bind weakly to one of the protein antigens, TNF-a. The affinities of the phOX and NIP binding scFv’s were reported to be 105-106 M - 1 . Akamatsu et al. introduced,a biased set of amino acids over portions
259
ANTIBODIES FROM COMBINATORIAL LIBRARIES
a 3
2 In 0 Q
a 1
0
-1
0
1
log Fab conc. (Ug/ml)
.
-
poly(dA).poly(dT) poly(dAdT).poly(dAdT) poly(dG).poly(dC) poly(dGdC).poly(dGdC)
2
260
DENNIS R. BURTON AND CARLOS F. BARBAS 111
FIG.26. Scheme illustrating the selection of Fab fragments containing an unpaired cysteine from a phage display library. From Janda et al. (1994).Nonbinders are eluted in detergent and noncovalent binders in acid before covalent binders (via a disulfide linkage) are eluted with dithiothreitol (DTT). Panning steps are repeated as in Fig. 7.
of both HCDRS and LCDRS to a set of genomic V genes. The library was selected to bind ConA. Six scFv's were characterized to bind ConA with affinities of 5 X 104-105M-'. The ConA scFv's demonstrated good specificity in binding the target antigen. Targeted CDR mutagenesis can also be utilized to make very modest specificity changes in an antibody. An example of altering the reactivity of an antibody to its anti-Ids has been reported by Glaser et al. (1992). In this report LCDR 1 and 2 were targeted for mutagenesis using a minimal doping scheme called codon-based mutagenesis. This restricted doping scheme employs oligonucleotide synthesis using two columns and the opening, mixing an exchange of resin between the columns during synthesis. This synthesis scheme was necessitated by the use of screening rather than selection in the examination of the library. The system utilized for library screening has been discussed (vida supra, Huse et al., 1992).Screening required that a larger number of positive clones would be represented in the library as only 1000clones would be screened for reactivity. The goal was to moderate the binding of a number of anti-Ids which had been previously characterized as reacting with the LCDRs and the experiment was successful in this regard.
C. COMBINING DESIGNAND SELECTION IN SYNTHETIC HUMAN ANTIBODIES An alternative to a random search of binding sites for the desired specificity is a directed search that incorporates information known to be relevant to a given binding problem. For anti-receptor antibodies
ANTIBODIES FROM COMBINATORIAL LIBRARIES
261
information relevant to the binding of the ligand to the receptor may be utilized to construct specialized libraries directed to bind a given receptor or receptor family. The minimal binding sequences of a number of ligands for their receptors have been characterized. Barbas et al. (1993~) described a methodology which could allow for the direct design and selection of human antibodies reactive with human receptors. The overall strategy is outlined in Fig. 27. A minimal sequence known to be important for the binding of the ligand to the receptor is transplanted into an antibody CDR. Since the correct conformational display of the sequence will, in almost all cases, be critical for interaction with the receptor, the transplanted sequence is flanked by randomized segments which form the elements of diversity within the library. The randomized elements also allow for the selection of additional contact residues. As a demonstration of the validity of this approach a member of the Asp-Gly-Arg (RGD)binding integrin receptor family, aVp3was chosen as a target receptor. The simple tripeptide RGD formed the basis for the construction of the library. In this case, the RGD sequence was placed near the apex of the extended hairpin loop of the HCDR3 of a human anti-HIV Fab. The HCDR3 had a moderately long sequence, 18 residues. Residues at the N- and C-terminal of the CDR were conserved to retain the stem of the loop. On each flank of the RGD were three randomized residues. Following selection of the 3 X lo7 member library for binding to avp3 5 Fab clones were characterized. The selected flanking regions showed some homology with known integrin binding peptides; however, some elements were quite unique. The affinities of the Fabs were astonishingly high, 10" it4-' (Table VI). Specificity characterization revealed the Fabs also bound aI&3 but not avp5, two integrins highly related to a,p3. These three integrins can share the same high-affinity ligand vitronectin and all bind RGD containing peptides. Furthermore, aVp3and aVp5bind with the same affinity to some RGD containing peptides. Fabs were shown to compete with RGD peptides for the integrin ligand binding site as designed. Further functional characterization demonstrated the Fabs to be potent in adhesion assays and subsequently (Smith et al., 1994) in inhibiting platelet aggregation (Fig. 28). Collectively, antagonism of these integrins could have potential in the treatment of osteoporosis and as anti-metastatic and anti-thrombotic agents. Synthetic antibodies can also be utilized to derive novel minimal ligands for receptors. Smith et al. (1994) demonstrate that randomization of the RGDX sequence within the context of the optimized Fab-9 (Barbas et al., 1993c) architecture and reselection for aII& binding leads to the identification of a number of novel non-RGD ligand sequences within the HCDR3. This experiment led to the sug-
262
DENNIS R. BURTON AND CARLOS F. BARBAS I11
IQ
Receptor
L
/ptitnize
Minimal Ligand
Ligand in an Antibody
FIG.27. The design and selection of human anti-receptor antibodies is aided by a knowledge of residues within the ligand that are involved in binding to the receptor. The first step involves the characterization of this interaction by mapping regions of the ligand involved in binding or by selecting peptides from linear or constrained peptide libraries to bind the receptor. The minimal ligand is then transplanted into a semisynthetic antibody library. The random sequence is provided to optimize the conformational display of the transplanted sequence which will be dependent on constaints imposed by the antibody. Random sequence also allows for selection ofadditional contact residues. Selection horn a vast library of variants provides the optimal antireceptor antibody. For integrins which bind RGD containing peptides, the simple RGD sequence is transplanted. For this case we have chosen the heavy-chain CDR3 for insertion of the sequence and optimization. The sequence of the starting a n t i - e l 2 0 antibody HCDR3 was VGPYSWDDSPQDNYYMDV. Following transplantation into a synthetic version of HCDRB the library consisted of variants where the HCDR3 sequences are VGCXXXRGDXXXCYYMDV,where X represents a mixture ofall 20 amino acids. Following selection for binding to the integrin a& the sequence of the HCDR3 of the highest A n i t y antibody, Fab-9, was VGCSFGRGDIRNCYYMDV. From Barbas et al. (1993~).
gestion of a new minimal sequence consensus for a I I b p 3 , namely, R/K-X-D, where Xis virtually any amino acid except proline. Several of these novel non-RGD Fabs demonstrated the capacity to discriminate between avp3and a I I b p 3 with a maximal difference of 100-fold preference (affinity) for aIIbp3.Prospects for the use of these antibodies as lead compounds were examined through the characterization of HCDR3 peptides. Peptides were shown to maintain receptor specificity albeit at reduced affinity.
D. IMPROVING THE AFFINITYOF HUMAN ANTIBODIES The systems and mutagenesis strategies described for producing new antibodies can also be utilized to improve the properties of ex-
ANTIBODIES FROM COMBINATORIAL LIBRARIES
263
TABLE VI AND AMINOACID SEQUENCES OF RGD CONTAINING SYNTHETIC INHIBITION CONSTANTS HUMAN ANTIBODIES ICa (Molar) Antibody No. 4
7
8 9 10
d 3 3
2.5 X 2.0 x 2.0 x 1.0 x 2.5 x
10-l0 10-10 10-10 10-lo
%BP3
2.5 X 5.0 x 1Olo 3.5 x 10-lo 1.0 x 10-lo 2.5 x 10-lo
4
5
5X NI NI NI NI
lo-'
Sequence TQG-RGD-WRS TYG-RGD-TRN PIP-RGD-WRE SFG-RGD-IRN TWG-RGD-ERN
Note. The ability of the recombinant antibodies to block vitronectin binding to avb3 and avb5 and fibrinogen binding to was determined with a purified receptor binding assay. The sequences flanking the RGD motif of each antibody are shown. NI, no inhibition at concentrations of up to 5 x I@'M. From Barbas et al. (1993~).
isting antibodies. Several strategies have been applied to improve the affinity of antibody fragments. For all of these studies it is important to consider the affinity range of the antibody to be improved. It is expected, and indeed observed, that greater relative improvements are more readily obtained with low-affinity antibodies as starting points. High-affinity antibodies require more modest adjustments of an already tight antigedantibody interface and are therefore less readily improved.
20xl09M JOxlO@M
Fab-9 FIG.28. The synthetic human antibody fragment Fab-9 blocks platelet aggregation. Human platelets (1 x 108) were mixed with 100 pg/ml of purified fibrinogen and 2.0 mM Ca2' in Tyrodes buffer. These were placed in a glass aggregation tube. The indicated concentration of Fab was added and the platelets were stimulated with 20 p M ADP. Aggregation was measured as light transmission through the platelet suspension using an aggregometer. From Smith et al. (1994).
264
DENNIS R. BURTON AND CARLOS F. BARBAS I11
Gram et al. (1992) have utilized error-prone PCR conditions to construct libraries of point mutations. This strategy is interesting since it mimics the random point mutations generated in natural somatic mutation. This approach was utilized to improve a Iow-affinity mouse scFv selected from a naive library to bind progesterone. The monovalent display system pComb3 allowed for the selection of an scFv with 30-fold improved affinity. The final scFv had an affinity of ca. lo6M-’. Hawkins et al. (1992) have utilized a similar strategy to improve a mouse anti-NIP scFv. In this case the multivalent display system fdtet-DOG1 was utilized. In order to select for higher affinity variants from this multivalent display system, the antigen was biotinylated and the selection performed in solution. Following several rounds of selection a clone was isolated which was improved fourfold in NIP binding to a final affinity of lo8M’. In a subsequent report, Hawkins et al. (1993) have utilized the same strategy and vector with a highaffinity mouse anti-lysozyme scFv the structure of which had been previously determined. The library was selected in two different fashions. The first experiment entailed 13rounds ofbiotin capture panning. The second experiment was performed in a competitive selection strategy where biotin-captured lysozyme was used with nonlabeled lysozyme as a competitor for five rounds of selection. The second selection was performed to bias the selection for the capture of clones with slower off-rates. These two experiments yielded two clones which were improved threefold in affinity. The two sets of mutations found in these clones were combined by site-directed mutagenesis to yield an scFv with an affinity of 2 x lo9M-‘,a fivefold overall improvement in affinity. One of the potential problems with random mutagenesis of the entire gene is that many of the mutations occur outside the CDRs and thus might more easily generate an antigenic antibody. Chain shuffling as proposed by Huse et al. (1989)may be used with great success when starting with a low-affinity clone (Marks et al., 1992). Starting with low-affinity clone and shuffling against a library of light chains allowed for the selection of a clone with 20-fold improved affinity. This clone was then combined with a V, library and selected for an additional improvement of 18fold. The final clone had an affinity of 109M-’ for phOX. This approach may be of lesser utility for the improvement of the high-affinity antibodies (Barbas et al., 1993b) as the sequence changes are too great. A final approach termed “CDR walking” (C. F. Barbas et aZ., 1994) may prove to be a general method for the improvement of antibody affinity. This approach is a variant of the synthetic antibody approach with one important difference. In this case library completeness is stressed over structural diversity and randomization is limited to six
ANTIBODIES FROM COMBINATORIAL LIBRARIES
265
residues or less. The approach may be applied in a parallel fashion where libraries are constructed in different CDRs. Following selection, the improved CDRs are then assembled to give the best antibodies, assuming additivity. The approach may also be applied in a sequential fashion. C. F. Barbas et al. (1994) applied the sequential approach for the improvement of a human anti-HIV-1 gp120 Fab. The Fab chosen (designated here as HIV-4 has an identical sequence to Fab b12 discussed above) had already been shown to potently neutralize HIV1.In this case a library of HCDRl variants with five residues randomized was selected by four rounds ofpanning against rgpl20. The collection of clones which resulted were then used in the construction of a HCDRS library where four additional residues were randomized. The resulting library was selected by an additional six rounds of panning. A clone was isolated with an 8-fold improvement of affinity for a final affinity of 1.4 x 1O’M-l. The epitope recognized by this antibody is the CDCbinding site on gp120. As this functional feature, which is not formed by a linear sequence, is retained by all variants of HIV-1, it was proposed that a f h i t y should also be increased for divergent isolates. This was tested by determining the affinities of several clones 15
EL g 5
h
5-
!2
3B4 HIV4 0
.:I :;; 5
0
2 4 6 8 1 0 1 2 1 4 K. (MN) [wl] (x 10’) CDRl CDRS
383
.-
3B9
384 HIV-4
-
NFTVH NFTLM NFTVH NYTLl NFV I H
EWGW QWNW PWRW PWNW P Y SW
MAA A6AA A4AA A6AA
FIG.29. The affinity increases of evolving Fabs for binding the divergent envelope proteins gp120 IIIB (LAI)andgpl20 MN are well-correlated.Affinities were determined using the surface plasmon resonance technique. The sequences of evolved clones are ranked as compared to the parent and changes in the amino acid sequence from the parent are shown as A M . The complete VH sequence of HIV-4 is given in Fig. 15 as Fab b4 and is identical with Fab b12. From C. F. Barbas et al. (1994).
266
DENNIS R. BURTON AND CARLOS F. BARBAS Ill
for gp120 derived from MN and IIIB strains. These proteins differ in over 80 amino acids and are highly divergent. As shown in Fig. 29 the affinity increases for both gpl20’s are well correlated. Functional improvement was assayed in virus neutralization studies with laboratory isolates and a 54-fold improvement was determined for the highest affinity clone. Furthermore, neutralization studies with primary clinical isolates demonstrated that the improved Fab acquired the ability to neutralize additional variants not neutralized by the parent. VI. Conclusions
Since its conception 5 years ago, the combinatorial approach has allowed unprecedented access to the human antibody response. The cloning of antibodies from preimmune, immune, and memory compartments ofthe human immune system has been demonstrated. Combinatorial antibodies have been shown to provide an accurate functional reflection of the natural response as demonstrated by the ability of cloned antibodies to compete with serum antibodies for binding antigens. Combinatorial antibodies also provide a useful (if somewhat incomplete) guide to the molecular biology of the response. The ability to select large numbers of antibodies against the same antigen and even the same epitope has led to a greater understanding ofthe molecular and structural biology ofthe immune response. Examination of natural libraries has highlighted the functional significance of HCDR3 and its prominent role in recognition. This observation coupled with the observation of light-chain plasticity characterizes an immune response which weights HCDR3 diversity over other elements. The ability to select antibodies to a multiplicity of antigens from synthetic libraries which differ only in the HCDR3 supports this notion. Furthermore competitive selections with synthetic libraries demonstrate that limited structural diversity within the rest of the binding site can be compensated for by increased structural diversity within the HCDR3. This observation may be important in rationalizing the differences between murine and human repertoires as reflected in HCDR3 length and the number of expressed V genes. These studies complement the recent increased appreciation of the role of HCDR3 gained through structural studies (Wilson and Stanfield, 1993). With its ability to provide large numbers of human antibodies directed against a single antigen, the combinatorial approach allows for the rapid assessment of immunodominant as well as neutralizing epitopes in the context ofthe human response. This information should be utilized in the future to guide the design of more effective vaccines. Antibodies neutralize viruses by mechanisms which in most cases
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are not yet defined. Mechanistic investigation of anti-viral antibodies should allow for the elucidation of novel pathways which might be targeted by small-molecule pharmaceuticals. Thus we suggest that combinatorial antibodies will play a significant role in the design of vaccines and new anti-viral agents. Antibodies may also provide a way of determining receptor function in vivo and serve as templates for the design of small molecules. One hundred years ago von Behring suggested, “Considering that antitoxin is an inanimate chemical substance, the possibility cannot be discounted that it may, at a later date, be able to be produced without the aid of an animal body,” (as translated in Gronski et al., 1991). Indeed this has now been realized. Our new-found ability to generate human antibodies and to evolve their specificities and affinities ex vivo promises increased use of this class of molecules in the service of human health. ACKNOWLEDGMENTS We acknowledge the enthusiasm and support of Richard Lemer for our efforts in the combinatorial antibody area. We are grateful to Gregg Silverman for a critical review of the manuscript and to members of our laboratories for valuable comments and suggestions including Shana Barbas, James Binley, Henrik Ditzel, Paul Parren, Jonathan Rosenblum, Pietro Sanna, and Anthony Williamson. We thank Joanne Marshall for help in preparing the manuscript. Some of our work described is supported by NIH (A133292 and A135165) and by Johnson and Johnson. C.F.B. is a Scholar of the American Foundation for AIDS Research and the recipient of an Investigator Award from the Cancer Research Institute.
REFERENCES Akamatsu, Y., Cole, M.S., Tso, J. Y., and Tsurushita, N. (1993). Construction o f a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments. J. Immunol. 151,4651-4659. Amadori, A. (1993). AIDS and autoimmunity. I n “The Molecular Pathology of Autoimmune Diseases” (C. A. Bona, K. A. Siminovitch, M. Zanetti, and A. N.Theofilopoulos, eds.), pp. 727-750. Hanvood Academic Publishers, Chur, Switzerland. Aulitzky, W. E., Schulz, T. F., Tilig, H., Niederwieser, D., Larcher, K., Ostberg, L., Scriba, M., Martindale, J., Stern, A. C., Grass, P., Mach, M., Dierich, M. P., and Huber, C. (1991). Human monoclonal antibodies neutralizing cytomegalovirus (CMV) for prophylaxis of CMV disease:Report of a phase I trial in bone marrow transplant recipients. J. Infect. Dis. 163, 1344-1347. Balachandran, N., Bacchetti, S., and Rawls, W. E. (1982). Protection against lethal challenge of Balb/c mice by passive transfer of monoclonal antibodies to five glycoproteins of herpes simplex virus type 2. Infect. Imrnun. 37,1131-1137. Balfour, H. H. (1988).Varicella zoster virus infection immunocompromised hosts. Am. J . Med. 85,68-73. Barbas 111, C. F. (1993). Recent advances in phage display. Cum. Opinion Biotechnol. 4,526-530. Barbas 111, C. F. (1994). The combinatorial approach to human antibodies: The pharmacology of monoclonal antibodies. Handb. Erp. Phannacol. 13, in press.
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Barbas 111, C. F., and Lerner, R A. (1991). Combinatorial immunoglobulin libraries on the surface of phage (Phabs): Rapid selection of antigen-specific Fabs. In “Methods: A Companion to Methods in Enzymology” (R. A. Lerner and D. R. Burton, eds.), Vol. 2, pp. 119-124. Academic Press, Orlando. Barbas 111, C. F., Kang, A. S., Lerner, R. A., and Benkovic, S . J. (1991). Assembly of combinatorial antibody libraries on phage surfaces: The gene 111 site. Proc. Natl. A d . Sci. U.S.A.88, 7978-7982. Barbas 111, C. F., Bain, J. D., Hoekstra, D. M., and Lerner, R. A. (1992a).Semisynthetic combinatorial antibody libraries: A chemical solution to the diversity problem. Proc. Natl. Acad. Sci. U S A . 89,4457-4461. Barbas 111, C. F., Bjorling, E.,Chiodi, F., Dunlop, N., Cababa, D., Jones, T. M., Zebedee, S. L., Persson, M. A. A,, Nara, P. L., Norrby, E.,andBurton, D. R. (199213).Recombinant human Fab fragments neutralize human type 1immunodeficiencyvirus in vitro. Proc. Natl. A d . Sci. U.S.A. 89, 9339-9343. Barbas 111, C. F., Crowe, J. E., Jr., Cababa, D., Jones, T. M., Zebedee, S. L., Murphy, B. R., Chanock, R. M., and Burton, D. R. (1992~).Human monoclonal Fab fragments derived from a combinatorial library bind to respiratory syncytial virus F glycoprotein and neutralize infectivity. Proc. Natl. Acad. Sci U.S.A.89, 10,164-10,168. Barbas 111, C. F., Amberg, W., Simoncsits, A., Jones, T. M., and Lemer, R. A. (1993a). Selection of human anti-hapten antibodies from semisynthetic libraries. Gene 137, 57-62. Barbas 111, C. F., Collet, T. A., Amberg, W., Roben, P., Binley, J. M., Hoekstra, D., Cababa, D., Jones, T. M., Williamson, R. A., Pilkington, G. R., Haigwood, N. L., Satterthwait, A. C., Sanz, I., and Burton, D. R. (1993b).Molecular profile ofan antibody response to HIV-1 as probed by combinatorial libraries. J . Mol. Biol. 230,812-823. Barbas 111, C. F., Languino, L. R., and Smith, J. W. (1993~).High-affinity self-reactive human antibodies by design and selection: Targetingthe integrin ligand binding site. Proc. Natl. Acad. Sci. U.S.A.90, 10,003-10,007. Barbas 111, C. F., Rosenblum, J. S., and Lemer, R. A. (1993d). Direct selection of antibodies which coordinate metals from semisynthetic combinatorial libraries. Proc. Natl. Acad. Sci. U.S.A. 14,6385-6389. Barbas 111, C. F., Hu, D., Dunlop, N., Sawyers, L., Cababa, D., Hendry, R. M., Nara, P. L., and Burton, D. R. (1994a).In vitro evolution of a neutralizing human antibody to HIV-1 to enhance affinity and broaden strain cross-reactivity. Proc. Natl. Acad. Sci. U.S.A., 91, 3809-3813. Barbas, S. M., Ghazal, P., Barbas 111, C. F., and Burton, D. R. (1994b). Recognition of DNA by synthetic antibodies. J. Am.Chem. SOC. 116,2161-2162. Bass, S., Greene, R., and Wells, J. A. (1990).Hormone phage: An enrichment method for variant proteins with altered binding properties. Proteins Struct. Funct. Genet. 8,309-314. Beasley, R. P., Hwang, L. Y.,Stevens, C. E., Lin, C. C., Hsieh, F. J., Wang, K. Y.,Sun, T. S., and Szmuness,W. (1983).Efficacy of hepatitis B immune globulin for prevention of perinatal transmission of the hepatitis B virus carrier state: Final report of a randomized double-blind, placebo-controlled trial. Hepatology 3, 135-141. Bebbington, C. R. (1991). Expression of antibody genes in nonlymphoid mammalian cells. In “Methods: A Companion to Methods in Enzymology” (R. A. Lerner and D. R. Burton, eds.), Vol. 2, pp. 136-145, Academic Press, Orlando. Behring, E. A. (1893). “Die Gesehichte der Diphtherie,” p. 186. Thieme, Leipzig. Behring, E. A. (1894).“Das neue Diphtherieheilmittel,” p. 40. 0. Hering, Berlin. Bender, E., Woof, J. M., Atkin, M. D., Barker, M. D., Bebbington, C. R., and Burton, D. R. (1993). Recombinant human antibodies: Linkage of an Fab fragment from a
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combinatorial library to an Fc fragment for expression in mammalian cell culture. Hum. Antibod. Hybridomas 4,74-79. Bender, E., Pilkington, G. R., and Burton, D. R. (1994). Human monoclonal Fab fragments from a combinatorial library prepared from an individual with a low serum titer to a virus. Hum. Antibod. Hybridomas, in press. Berberian, L., Goodglick, L., Kipps, T. J., and Braun, J. (1993). Immunoglobulin VH3 gene products: Natural ligands for HIV gp120. Science 261, 1588-1591. Better, M., Chang, C. P., Robinson, R. R. et al. (1988).Escherichia co2i secretion of an active chimeric antibody fragment. Science 240, 1041-1043. Bigazzi, P. E. (1993).Autoimmunity in Hashimoto’s disease. In “The Molecular Pathology of Autoimmune Diseases” (C. A. Bona, K. A. Siminovitch, M. Zanetti, and A. N. Theofilopoulos, eds.), pp. 493-510. Hanvood Academic Publishers, Chur, Switzerland. Binley, J. M., Ditzel, H. J., Hendry, M., Sawyer, L., Dunlop, N., Nara, P. L., Barbas 111, C. F., and Burton, D. R. (1994). Generation of neutralizing human antibodies reactive with conformational epitopes of gp41, the transmembrane glycoprotein of HIV-1. Submitted for publication. Bird, R. E., and Walker, B. W. (1991). Single chain antibody variable regions. Trends Biotechnol. 9, 132-137. Boulianne, G. L., Hozumi, N., and Schulman, M. J. (1984). Production of functional chimaeric mouse/human antibody region domains. Nature (London)312,643-646. Britt, W . J., Vugler, L., Buffiloski, E. J.,and Stephens, E. B. (1990).Cell surface expression of human cytomegalovirus (HCMV) gp55-116(gB): Use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response. J . Virol. 64, 1079-1085. Burioni, R., Williamson, R. A., Sanna, P. P., Bloom, F. E., and Burton, D. R. (1994). Recombinant human Fab to glycoprotein D neutralizes infectivity and prevents cellto-cell transmission of herpes simplex virus type-1 and -2 in uitro. Proc. Natl. Acad. Sci. U.S.A.91,355-359. Burton, D. R. (1990). The conformation of antibodies. In “Fc receptors and the action of antibodies” (H. Metzger, ed.), pp. 31-54. American Society of Microbiology,Washington, DC. Burton, D. R. (1991). Human and mouse monoclonal antibodies by repertoire cloning. Trends Biotechnol. 9, 169-175. Burton, D. R. (1992).Human monoclonal antibodies: Achievement and potential. Hospital Practice. 27, 67-74. Burton, D. R. (1993a). Human monoclonal antibodies to viruses from combinatorial libraries. In “Vaccines 93,” pp. 1-5. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Burton, D. R. (1993b). Monoclonal antibodies from combinatorial libraries. Acc. Chem. Res. 26,405-411. Burton, D. R.,and Barbas 111, C. F. (1992). Antibodies from libraries. Nature (London) 359,782-783. Burton, D. R., and Barbas 111, C. F. (1993a). Human antibodies from combinatorial libraries. In “Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man” (M. Clark, ed.), pp. 65-82, Academic Titles, Nottingham, England. Burton, D. R., and Barbas 111, C. F. (1993b).Monoclonal Fab fragments from combinatorial libraries displayed on the surface of phage. Zmmunomethods 3, 1-9. Burton, D. R., and Woof, J. M. (1992).Human antibody effector function. Adu. Zmmunol. 51, 1-86.
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ADVANCES IN IMMUNOLOGY,VOL. 57
Immune Response against Tumors CLAUDE ROTH, CHRISTOPH ROCHLITZ,’ AND PHlLlPPE KOURILSKY Unit6 de Biologie Mdkulrrirr du Gkre, 0.277 Insem, lnstht Pasteur, 25, rue du Dr. Rou~r,75724 Pans c6dex 15, France
1. Introduction
In the past few years, tumor immunology has made considerable progress and is receiving more and more attention. This increasing interest is justified by several reasons. First of all, the notion of tumorspecific antigens, which initially involved integral cell-surface proteins recognized by antibodies, has been extended to T-cell epitopes presented by MHC molecules and recognized by specific T cells. The field of tumor immunology has then largely shifted toward the analysis and manipulation of T-cell responses. A new conceptual framework has opened up new experimental approaches. Results now obtained in animal models have become so spectacular that expectations have been raised concerning the prospects for the immunotherapy of human cancers. A major transition can be traced back to the mid 1980s, when Townsend et aZ. (1985,1986a)demonstrated that the influenza nucleoprotein is presented to specific cytolytic T cells (CTL) in the form of a small peptide carried by a given class I MHC molecule. It followed that intracellular proteins of the host might also be displayed as processed peptides at the cell surface (Townsend et d.,1985; Kourilsky and Claverie, 1986). The notion that any class I MHC-positive cell could permanently expose on its surface thousands of self-peptides led to a broadening of the concept of immunosurveillance (Kourilsky and Claverie, 1986). It was thus shown that dysregulations in tumor cells might become visible by the immune system, either as peptides derived from unmutated, upregulated self-proteins, or as mutant peptides derived from mutated self-proteins (Kourilsky and Claverie, 1986; Kourilsky et al., 1987,1991).The pioneering work of Boon and his co-workers, started in the mid 1970s, has provided, in 1988-1991, the experimental foundation for the important concept of T-celldefined specific tumor epitopes, the latter being actually derived from Present address: Department Innere Medizin, Abteilung fur Onkologie, Kantosspital Basel am Petergsraben 4, CH-4031 Basel, Switzerland
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mutant self- or upregulated unmutated self-proteins (reviewed in Boon, 1992). A growing body of basic immunology is developing around the fundamental notion that not all potentially presentable self-peptides are actually presented by MHC molecules in a way that triggers T-cell tolerance (Cibotti et al., 1991; Milich et aZ., 1991; reviewed in Sercarz et al., 1993). It is expected that these peptides may serve as a reservoir for some tumor-specific antigens and that understanding the rules that place them in this category will ultimately be useful to tumor immunology. The realization that tumor epitopes can be recognized by T cells opened new avenues for immunointervention. Among these, the transfection of genes which could possibly increase the immunogenicity of tumor cells, such as cytokine genes, was particularly attractive, because immunogenicity could be increased without even knowing the molecular nature of the tumor-specific antigens. The first results obtained in mouse tumor models between 1988 and 1991 were indeed remarkably successful (Bubenik et al., 1988; Fearon et ul., 1990; Gansbacher et uZ., 1990a; Ley et al., 1990,1991; Russel et al., 1991). At the least, they could sustain the claim that, even if natural immune responses are often weakly relevant to tumor control in uiuo, it may be possible to manipulate immune responses in order to make them efficient enough to control tumors. The debate over the relevance of immune responses against tumors was thus muted by the notion that immunointervention might well do the job, provided that the appropriate T-cell responses are activated. In the past few years, the flurry of spectacular results in mouse tumor models has encouraged optimism as to their applicability in man. Dozens of clinical trials are currently under way and dozens are being planned. An understandable trait of tumor immunology is that every finding is almost immediately framed within a therapeutic perspective. This may explain the confusion, commonly encountered in the field, between an antigen and an immunogen-because any antigen is immediately used as an immunogen to try to control tumor growth. In this context, we have attempted to review here a number of aspects related both to the antigenicity and the immunogenicity of tumors. We could not cover the entire field and have selected what we felt are the most relevant trends in today’s research. In doing so, we hope to have reviewed a sufficiently broad area to substantiate the conclusion that the complexity of the field should temper some of the ongoing, naive optimism for human immunotherapy. This and connected fields have been reviewed by others (reviewed in Klein and Boon, 1993), and we apologize in advance for any omission which, in this relatively large survey, we might have unwillingly made.
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It. Tumor Antigens
A. ANTIGENS D E ~ ~ NSEROLOGICALLY ED
It was initially assumed that tumor antigens would be unique to malignancy and not found in normal cells and that they might be specific to the tissue of origin and thus reflect the state of differentiation of the cells within this tissue. Antigens expressed on the cell surface of tumor cells were thought to be the main components detected by the immune system. Specific antisera and, subsequently, monoclonal antibodies were developed to identify them. Tumors often express high levels of differentiation antigens, usually corresponding to the differentiation stage of the cell in which the malignant transformation occurred. An example is provided by the oncofetal antigens, such as a-feto-protein (AFP) on human hepatomas and carcinoembryonic antigen (CEA)on colon carcinomas. These markers were the first human tumor-associated antigens to be discovered. While they are not as tumor specific as originally believed, they still continue to be promising for the purpose of diagnosis and therapy (Mach et al., 1993). On B and T lymphocytes, growth factor receptors, such as receptors for transferrin, interleukin-2, or insulin, also belong to this category of normal differentiation antigens, since they are absent or barely detectable on resting lymphoid cells, but abundant on stimulated and dividing cells. In human melanomas, the gp95/p97 antigen studied by several groups provides a clear example of a tumor-associated antigen shared with normal tissues. The initial biochemical characterization of the p97 antigen from human melanoma cells showed that the parital amino acid sequence of this phosphorylated sialoglycoprotein is structually related to that of transferrin (Brown et al., 1981,1982). The entire gene was then cloned (Rose et al., 1986). Immunization of mice with recombinant vaccinia virus expressing p97 antigen (Hu et al., 1988) induced antibody production and T-cell proliferation. More strikingly, immunized mice rejected melanoma cells expressing p97 while they allowed the outgrowth of p97-negative tumors (Estin et al., 1988). Whether this approach is valid for human therapy is not yet clear, as normal tissues expressing trace amounts of p97 could constitute targets for immune attack. At about the same time, other investigators observed that sera isolated from patients with melanoma bound only to the autologous melanoma, while a mouse monoclonal antibody raised against the purified gp95/p97 antigen could also bind to allogeneic melanomas (Shiku et al., 1976; Real et al., 1984). By immunoprecipitation, this monoclonal antibody was shown to detect a 95-kDa molecule on other melanomas
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and on some normal tissues. It was thus postulated that the patients’ antibodies were able to detect a new epitope on a common molecule, originally identified as a melanoma antigen, and designated gp95 (Dippold et al., 1980) or p97 (Woodbury et al., 1980; Brown et al., 1980). These results were the first to show that a common melanoma antigen bearing specific mutations can behave as an autoimmunogenic epitope that elicits a specific antibody response (Furukawa et al., 1989). An additional example of tumor-associated antigens expressed in normal tissues at a given stage of differentiation is provided by the tyrosinase-related melanosomal antigens and by the cell-surface glycolipids whose expression increases during malignant transformation. The glycoprotein gp75, related to tyrosinase, an enzyme that catalyzes the formation of melanin, was originally identified by autoantibodies in patients with metastatic melanoma (Vijayasaradhi and Houghton, 1991). Using a mouse monoclonal antibody against gp75, it was shown that the passive transfer of this autoantibody into mice bearing B16 melanoma tumor cells induced the rejection of established tumors, but also produced autoimmunity against regenerating melanocytes (Houghton, 1992). Gangliosides have also been identified as target structures for antibodies in cancer patients. In passive immunotherapy, tumor remissions were observed with antibodies directed against gangliosides in melanomas and neuroblastomas (Houghton et al., 1985; Cheung et al., 1989). For active immunotherapy, the main problem is still to induce in vivo a strong enough antibody response, as discussed by Livingston (1991). Various immunization protocols are currently being investigated, using either purified or modified glycolipid antigens (Ritter et al., 1991) or antiidiotypic antibodies against the GD3 ganglioside as the immunogen (Chapman and Houghton, 1991). In mice, the p53 tumor suppressor molecule is another case of a serologically detected tumor antigen. Antibodies reacting against p53 molecules have been found in the sera of animals inoculated with methylcholantrene-induced sarcomas (Deleo et al., 1979). Biochemical analysis of p53 tryptic peptides demonstrated that mutations in p53 might be involved in the individually distinct immunological characteristics of methylcholantrene-induced tumors ( Jay et d., 1979). From these observations, it appears that tumor antigens, recognizable by antibodies, derive either from mutant self-proteins or from unmutated upregulated or modified components (e.g., at the level of carbohydrates) of the cell surface. The concept thus emerged that tumors may express specific epitopes, rather than specific molecules. This became even more evident when T-cell responses against tumor cells were analyzed in detail.
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B. EARLYEVIDENCE FOR TUMOR-SPECIFIC ANTIGENSRECOGNIZED BY T CELLS Evidence for the existence of specific tumor rejection antigens can be traced back to the pioneering work of Gross (1943), Foley (1953), and Prehn and Main (1957),who showed that inbred mice immunized with a syngeneic chemically induced tumor could reject a graft of the same tumor. The antigens were unique for each individual chemically induced tumor, since specific protection was obtained against the' immunizing but not against other tumors, even if induced by the same carcinogen (methycholantren), and in mice of the same strain (Klein et al., 1960). The antigens responsible for tumor rejection were then referred to as tumor-specific transplantation antigens (TSTAs).Several major characteristics could be defined: 1. Each tumor had individually distinct antigens. 2. These antigens were stably expressed during successive passages in vivo and in uitro. They were usually not lost after passage in immunized animals. 3. There was a relationship between the dose of carcinogen used to induce the tumor, the latency period before tumor growth, and the immunogenicity of the tumor. In tumors induced by high doses of carcinogen, the latency period was short and the immunogenicity was generally high (Prehn, 1975; Prehn and Karcher, 1983; Prehn and Prehn, 1987). 4. TSTAs were not restricted to tumors induced by chemical carcinogens but were also found expressed in UV-induced tumors (Kripke, 1981; Ward et al., 1989),mammary tumors induced by mammary tumor virus (Morton et al., 1969),and tumors of spontaneous origin (Carswell et al., 1970). 5. Immunity could be transferred to naive mice with lymphocytes from immunized animals (reviewed in Melief, 1992), strongly implicating T cells.
Extensive biochemical studies on chemically induced murine tumors were undertaken to determine the molecular nature of these antigens. The diversity of the results diminished the hope of identifying common structures that could be used for diagnostic or therapeutic purposes. In some cases, TSTAs were identified as deregulated embryonic antigens (Coggin and Anderson, 1974; Medawar and Hunt, 1983),while in other cases they were characterized as allo- or mutatedMHC molecules (Invernizzi and Parmiani, 1975; Philipps et al., 1985; Stauss et al., 1986a; Lee et al., 1988), as viral antigens derived from
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recombinant murine virus (Roman et al., 1981; Lennox et al., 1981), as antigens linked to the Ig heavy-chain locus (Pravtcheva et al., 1981), or as antigens related to heat shock proteins (Ullrich et al., 1986; Moore et al., 1987). Several hypothesis were proposed to account for this antigenic diversity (Schreiber et al., 1988).First, individual transplantation antigens could result from a direct mutagenic activity of chemical and physical carcinogens in a cellular gene, leading to the expression of a neo T-cell epitope. Alternatively, carcinogens could only lead to clonal amplification of one single cell (Burnet, 1970), expressing a particular antigen, but usually present at insufficient levels on normal cells to be recognized by the immune system. This is observed with malignancies of the B-cell lineage expressing a private idiotope (Lynch et al., 1972). It is now well-accepted that carcinogens can induce the appearance of neoantigens, but it is not always clear whether the latter result from mutated cellular genes or from activated preexisting silent genes (reviewed in Schreiber et al., 1988). The methylcholanthrene-induced tumor MethA has been extensively analyzed (Deleo et d.,1977). In this case, it was not possible to generate tumor-specific CTLs which could select antigen-loss variants, and the immunity involves mainly helper T cells. While initial studies were concerned with serological specificities (Pravtcheva et al., 1981), further work used the transplantation assay to identify tumor rejection antigens. These investigations led to the characterization of several molecules that could be used to specifically inhibit the growth of the MethA tumor in immunized mice. From plasma membrane, or solubilized extracts, two groups of rejection antigens were identified, the 96-kDa (gp96) cell-surface glycoprotein (Srivastava et al., 1986) and the 84- to 86-kDa (p84/86) intracellular antigen (Ullrich et al., 1986). Interestingly, another methylcholanthrene-induced tumor (CMS.5) was shown to contain a tumor rejection activity associated with the 96-kDa fraction of the plasma membrane. The gp96 from these various sources could not be distinguished on the basis of biochemical characteristics. Furthermore, molecular cloning of the cDNA encoding gp96 revealed exactly the same sequence in MethA and CMS.5 tumors and in normal liver (Srivastava and Old, 1988).However, major regions of the gp96 gene still remain to be explored. The other proteins that conferred protection against tumor growth were two polypeptide isoforms of 84 and 86 kDa, displaying considerable homology with each other and with certain stress-induced or heat shock proteins (hsp), namely, the hsp90 family (Ullrich et al., 1986; Moore et al., 1987) or the hspl08 family (Srivastava et al., 1986,1987). The possibility has been raised that these molecules may not be
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immunogenic by themselves but rather serve as immunogenic peptide carriers (Srivastava and Maki, 1991). This was suggested for the gp96 molecules in relation with their capacity to load MHC class I antigens with peptides in the endoplasmic reticulum (Srivastava and Heike, 1991; Li and Srivastava, 1993). The identification of the human homologue of murine gp96 and its homology with hsp.lOO and 108reinforces this hypothesis (Maki et al., 1990). Interestingly, other members of hsp that belong to the hsp.70 family could also contribute, albeit to a lesser extent, to the immunogenicity of various tumors. By interfering with the antigen processing and presentation pathway and facilitating peptide/MHC class I1 interactions, they could enhance tumor antigen presentation and strengthen immune recognition of specific target molecules (Young, 1990; Kaufmann, 1990; De Nagel and Pierce, 1992,1993). Moreover, hsp7O was shown to play a key role in the nuclear transport of various oncogenes and antioncogenes, as mentioned for the T antigen of SV40 (Sawai and Butel, 1989),the adenovirus E1A proteins (White et al., 1988), and the p53 tumor suppressor molecule (Pinhasi-Kimhi et al., 1986). In this situation, stable complexes of mutated p53 and hsp7O (Lam and Calderwood, 1992) could lead to an increased half-life and immunogenicity of this tumor antigen (Davidoff et al., 1992). The antigenicity of the hsp molecules could also be accounted for by the existence of mutated epitopes. At any rate, it is remarkable that these highly conserved proteins should constitute target molecules of immune responses in both healthy and unhealthy individuals (reviewed in Young, 1990; Cohen, 1991; Kaufmann, 1990). In contrast to the MethA tumor, which induces only a relative and dose-dependent protection, the UV-induced tumor 1591 is much more immunogenic and is rejected by normal young mice, even after inoculation of large tumor fragments. Also, in contrast to the MethA tumor, CTL clones could be generated against the UV-induced tumor. This allowed for a characterization of the tumor antigens, by sequential in vitro selection of tumor variants having lost the ability to be lysed by given CTL clones. It was thus demonstrated that a single tumor may express several tumor-specific antigens that can be lost independently of each other (Wortzel et al., 1983,1984; Uytenhove et al., 1983). Two additional main features characterize the 1591 UV-induced tumor. First, there is a hierarchy in the antigenicity of the multiple antigens expressed by the same tumor cell. This results in antigenic changes and loss in vivo and in the appearance of more malignant variants leading to tumor progression (Urban et al., 1984). Second, tumor variants which could not retain a CTL-defined tumor specificity, nor induce protective immunity to itself, could still present a tumor-specific
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antigen defined by T helper cells and cause protective immunity to any variant that expresses a CTL-defined antigen (Van Waes et al., 1986). One antigen of the 1591 tumor has been defined by monoclonal antibodies that recognize a unique specificity on “regressor” tumors. The gene was cloned and found to encode a novel class I molecule, with three antigenic specificities shared with other MHC molecules, namely Lq, Dq and K216 (Stauss et al., 1986a; Lee et al., 1988). While the Lq and Dq specificities were shown to be 100% homologous to the known Lq and Dq specificities at the gene level, the origin of the K216 specificity remains elusive, but it encodes an antigen which is sufficiently immunogenic to induce rejection by normal mice (Stauss et al., 198613). C. TUMOR-SPECIFIC PEPTIDES IDENTIFIED BY T-CELLRECOGNITION Tumor immunology then made considerable progress and this is largely due to the better understanding of the mechanisms of antigen presentation by MHC molecules and of T-cell recognition of peptide-MHC complexes. First, T cells were shown to recognize antigen only in the context of MHC products at the cell surface (Zinkernagel and Doherty, 1974,1979; Bevan, 1975) as small antigenic peptides located in a groove of the MHC molecules (Babbit et al., 1985; Townsend et al., 1985). The three-dimensional structures of human and mouse MHC molecules are now known (Bjorkman et al., 1987; Ejorkman and Parham, 1990)and it appears that class I and class I1 products share very similar structures (Brown et al., 1988,1993). Functional studies demonstrated that both CD4+ helper (TH) and CD8 + cytotoxic T lymphocytes (CTL) recognize short peptides derived from fragmented proteins which are presented by MHC class I1 or class I molecules (Babbit et al., 1985; Maryanski et al., 1986;Townsend et al., 1986a,1989; Buus et al., 1987; Schumacher et al., 1990; Van Bleek and Nathenson, 1990; Falk et al., 1991; Rudensky et al., 1991). Two major pathways of antigen processing and presentation for Tcell recognition have been identified. Antigens of exogenous proteins are usually presented to T cells by professional antigen-presenting cells, such as macrophages, B cells, bone marrow-derived dendritic cells (reviewed in Unanue and Allen, 1987), and other cells that express class I1 molecules (Rock et al., 1984; Lanzavecchia, 1985; Boog et al., 1988; Melief et al., 1988).This “exogenous” presentation pathway involves endocytosis of the foreign protein, followed by its degradation into short peptides in the endocytic compartments (reviewed in Brod-
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sky and Guagliardi, 1991).In general, antigenic peptides bind to class 11 molecules when the invariant chain ( Ii) is removed at low pH in the late endosome (Babbit et al., 1985; Buus et al., 1986,1987; Roche and Cresswell, 1990; Teyton et al., 1990). Class I1 MHC-peptide complexes then reach the cell surface, where they are specifically recognized by the receptor of T lymphocytes expressing the CD4 molecule, which binds to a constant region of class I1 product (Sprent and Webb, 1987). The other processing pathway concerns mostly endogenous proteins, peptides of which are loaded into the peptide presenting groove of MHC class I molecules in the endoplasmic reticulum. In this scheme, proteins originating from either virus infection (Townsend et al., 1985,1986a; Morrison et al., 1988; Yewdell et al., 1988), or osmotic lysis of pinosomes (Moore et al., 1988)or normal cellular components, which are present mostly in the cytoplasm or nucleus, are fragmented into short peptides, ususally eight to nine amino acids length, that are loaded onto MHC class I antigens after transport into the endoplasmic reticulum by MHC-encoded transport molecules (Powis et al., 1991,1992). MHC class I molecules are quite unstable if they are not loaded with a peptide, as shown with mutant cell lines altered in their transporter genes (Powis et al., 1991; Spies et al., 1992).T cells which recognize peptides presented by class I molecules express CD8, which binds to a constant region of class I MHC molecules (reviewed in Parnes, 1989). As a consequence, endogenous proteins are preferentially recognized by CD8+ CTL in the context of MHC class I molecules, whereas exogenous antigens are mainly recognized by CD4' TH cells in the context of MHC class I1 molecules (Morrison et al., 1988). However, there are also T helper cells specific for class I molecules (reviewed in Singer et al., 1987)and CTL specific for class I1 molecules (reviewed in Mills, 1986; Morrison et al., 1986). Likewise, in certain circumstances, exogenously administered peptides can induce MHC class I restricted CTL responses (Carbone and Bevan, 1989; Deres et al., 1989; Aichele et al., 1990; Schultz et al., 1991; Kast et al., 1991), and endogenous proteins can prime class 11-restricted helper T cells, as shown, for example, in a B-cell line transfected with a A light-chain gene (Bogen and Weiss, 1988).Peptides eluted from class I molecules are usually about nine amino acids long (van Bleek and Nathenson, 1990; Rotzchke et al., 1990; Schumacher et al., 1991), while peptides eluted from class I1 molecules are usually longer and heterogeneous in size. The peptide binding pocket of class I molecules, being closed on both sides, is believed to display more stringent peptide length
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requirements than that of class I1 molecules (Falk et al., 1991; Brown et al., 1993). The first antigenic structures to be regularly detected on tumor cells as targets for T-cell immunity were viral antigens on virus-induced murine neoplasms (reviewed in Melief, 1992; Klein, 1991). Subsequently, two additional types oftumor-specific antigens, whose molecular nature had long remained elusive, could be distinguished: the first one comprises antigens derived from structurally abnormal proteins, while the other includes antigens resulting from the abnormal expression of a normal protein (reviewed in van der Bruggen and van den Eynde, 1992). D. TUMOR ANTIGENSON VIRUS-INDUCED TUMORS The first evidence of rejection antigens expressed on tumors induced by oncogenic viruses was based on early work on the polyoma virus (Sjogren et al., 1961; Habel, 1961).These studies demonstrated that, in contrast to the transplantation antigens of chemically induced tumors, polyoma virus-related-specific antigens have identical or crossreactive specificities, even if they are induced in different species (Hellstrom and Sjogren, 1966). For polyoma, for instance, this specific antigen was initially thought to play a key role in maintaining the tumorigenicity of tumor cells, as suggested from the inability to isolate polyoma antigen-negative variants (Sjogren, 1964). However, when these antigens are coexpressed on other tumors, such as Moloney virus-induced lymphoma, the polyoma antigen is lost without affecting the growth potential of the lymphoma. This demonstrated that a given tumor could acquire several rejection antigens independently of each other and that some of the antigens could be lost following passage of tumors in mice. These antigen-loss variants still maintain their malignant phenotype if the disappearing antigen is unrelated to the malignancy of the cell. For polyoma-induced tumors, two antigens have been identified, the middle T antigen (a 56-kDa protein) and the small T antigen (a 22-kDa protein), both of which are localized in the nucleus. In two sets of experimental immunization protocoles, it was observed that middle T antigen can act as a TSTA, since it causes the rejection of transplanted syngeneic polyoma tumors (Lathe et al., 1987; Ramquist et al., 1988). Experimental tumors induced by Gross virus have been shown to stimulate an MHC-restricted CTL response in grafted mice. An epitope was traced down to the gag polyprotein, and it could be demonstrated that mouse L cells transfected with the gag gene and the proper restriction elements (H-2Kb)could vaccinate against the graft (Abas-
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tad0 et al., 1985; Plata et al., 1986,1987).In the case of Friend leukemia virus, the virus-derived FBL-3 tumors were shown to induce a T-cell response against the gag or env viral components in C57B1/6 mice. Both helper T lymphocytes and cytotoxic T cells participated in the rejection of these antigenic tumors (Leclerc and Cantor, 1980; Greenberg et al., 1981). CD8+ cytotoxic T cells recognize a viral gagencoded epitope restricted by H-2Db class I molecules, whereas CD4+ helper T cells recognize a viral env-encoded epitope in the context of I-Ab class I1 molecules (Klarnet et al., 1989). Furthermore, studies using recombinant vaccines demonstrated that both antigens can prime T helper cells, while only the gag nuclear antigen can induce a CTL response (Earl et al., 1986; Klarnet et al., 1989),and both antigens can induce protection against leukemia. Likewise, in an adoptive therapy model, CD4+ and CD8+ populations directed against FBL-3 can eradicate disseminated disease, by a mechanism which involves the induction of tumoricidal macrophages activated by IFN-y (reviewed in Greenberg, 1991). As in the polyoma virus tumor system, the tumor antigens encoded by the genome of the simian virus 40 (SV40)are highly immunogenic. One of these virally encoded proteins, the large T antigen, has been involved in the transformation process leading to malignancy, presumably by disrupting the interaction between transcription factor E2F and the retinoblastoma tumor suppressor gene product (Chellapan et al., 1992). In C57B1/6 mice (H-2bhaplotype), five antigenic sites have been identified so far in large T, on the basis of their recognition by MHC class I-restricted CTLs (Tanaka et al., 1988; Tevethia et al., 1990). Four of the sites have been mapped using synthetic peptides and are H-2Db restricted while one site is H-2Kb restricted (Tanaka et al., 1988; Deckhut et al., 1992). To identify the molecular basis of tumor antigen loss variants, an SV40-transformed B6 mouse kidney cell line (K-0) was cocultivated with SV40 T-antigen site-specific CTL clones (Tanaka and Tevethia, 1988,1990).The resulting epitope-loss variants that were selected for their resistance against CTL clones contained amino acid substitutions which were still presented by MHC molecules but were no longer recognized by the CTL clones (Lill et al., 1992; Deckhut and Tevethia, 1992). These specific amino acid changes did not affect the MHC class I binding motifs, but disrupted the ability to provide a target for specific CTL, which could explain the escape of tumors from immunosurveillance. One could argue against this hypothesis, though, due to the existence of other T cells in the in vivo repertoire which might recognize the mutant tumor cells.
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In other murine or human virus-induced tumors, various T-cell epitopes, which may be potential targets for immune intervention, were defined using CD8+ CTL clones. In the case of human adenovirus early region 1 (AdEQtransformed murine cells, H-2b-restricted CTL clones were found to be directed against a peptide sequence of the viral nuclear oncogene product ElA, presented in the context of the H-2Db class I molecule (Kast et al., 1989; Kast and Melief, 1991). The Ad5-E 1-transformed murine cells, which express this H-2Db-restricted peptide, are, however, incapable of causing tumors in immunocompetent animals, due to efficient surveillance by CD8+ CTLs (reviewed in Melief et al., 1989; Melief and Kast, 1990). No antigen-loss variants were isolated, suggesting that the expression of E1A is essential to maintain the transformed state. Moreover, in this experimental system, CTLs are highly specific, as shown by tumor regression, and longterm memory was induced in immunoincompetent mice after adoptive immunotherapy (Kast et al., 1989). In the case of the human Epstein-Barr virus (EBV), one peptide derived from the nuclear antigen 3 (EBNA-3) was originally characterized by CD8+ CTL clones in the context of HLA-B8 molecules (Burrows et al., 1990).Afterward, additional target antigens for CTL recognition were identified on transformed cells, using recombinant viruses that carry EBV genes. These strategies allowed the identification of EBNA-4- and EBNA-6-derived peptides, presented by HLA-A2 molecules (Gavioli et al., 1992,1993). A hierarchy was established with respect to the immunogenicity of these epitopes (Murray et al., 1992) and for HLA-A2-restrictedCTL peptides, only a few of which appeared to be immunodominant (Gavioli et al., 1993). A main feature of this oncogenic virus is related to its multiple escape mechanisms, which contribute to counteract an effective immune response, as shown by the high frequency of EBV-specific CTL precursors and their remarkable stability. First, it encodes for aprotein sharing many ofthe biological activities of IL-10 (Vieira et al., 1991),which can suppress lymphokine production by the TH-1 cells (Fiorentino et aE., 1989) and which is a potent stimulator for B-cell proliferation (Defrance et al., 1992; Rousset et al., 1992). Second, the emergence of variants having lost the immunodominant A2-restricted CTL epitope (De Campos-Lima et al., 1993) has been observed. This could result in a weaker T-cell response against the infected cells. In the case of papillomaviruses, which cause epithelial proliferative diseases in animals and in man, several epitopes, which induce a CTL response against tumor cells in viuo, have been identified. Recombinant vaccinia viruses expressing the E5, E6, or E7 early region proteins
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of the bovine papillomavirus BPVl were shown to elicit antitumor immunity in animals inoculated with transformed cells (Meneguzzi et al., 1990). In humans, recombinant vaccinia virus expressing E6 or E7, then peptides derived from the HPV type 16 oncogenes E6 and E7, selected for their binding motifs for H-2Kb and H-2Db (Falk et al., 1991), were used to identify a CTL epitope able to protect against a tumor induced by human papillomavirus type 16-transformed cells (Meneguzzi et al., 1991; Feltkamp et al., 1993). Interestingly, in this study, successful antitumor vaccination was achieved with a nonimmunodominant CTL epitope. This result underlines the necessity for identifying peptides which are not necessarily immunodominant (as reviewed by Sercarz et al., 1993),for antigenic determinants presented by MHC class I1 molecules) but which are obvious candidates for inducing an in vivo CTL response. Other virus-induced tumors in humans have been studied and are currently under active investigation. Human T-cell leukemia virus-1, which is, the causative agent of adult T-cell leukemia, does not require additional immunosuppression to be leukemogenic. Hepatitis B virus (HBV)seems to play an important role in the genesis ofprimary hepatomas (Feitelson et al., 1993; reviewed in Buendia, 1992). In this human tumor virus system, target cells have been produced by expression of the viral protein in immortalized human B-cell lines that can stimulate CTLs in vitro (Guilhot et al., 1992; Bertoletti et al., 1993).
E. ANTIGENSDERIVED FROM STRUCTURALLY ABNORMALPROTEINS 1. TurnWhile viral antigens were relatively easy to identify, it has been much more difficult to characterize those tumor rejection antigens which are encoded by the cellular genome. Originally, clonal mouse and tumors were mutagenized with N-methyl-N’-nitrosoguanidine, variants that were no longer tumorigenic in syngeneic animals were isolated (Boon, 1983). Such tum- variants have been obtained in various tumor models, including a teratocarcinoma (Boon and Kellerman, 1977), Lewis lung carcinoma (van Pel et al., 1979),mastocytoma P815 (Uyttenhove et a1., 1980),and radiation-induced or spontaneous leukemia (van Pel and Boon, 1982; van Pel et al., 1983). In the P815 model, the tumor antigens are recognized by CD8+ cytotoxic T lymphocytes, leading to the rejection of the tum- tumors (reviewed in Boon, 1992). Anti-P815 CTL clones were used to define the antigens expressed by the original tum+ tumor (Pl).By transfection of the highly transfectable P1 tum+ cell line (P1 HTR) with the DNA
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of the tum variant, it was found that tum antigens could be expressed on the transfected target cells, thus rendering them sensitive to lysis by specific CTLs (DePlaen et al., 1988).This led to the identification ofthe gene coding for the tum antigen, P91A, and it was demonstrated that a single point mutation is responsible for the antigenicity of the tum- peptide (Lurquin et al., 1989). Using a similar strategy, the gene encoding the tum- antigen specific for the P35B tum- variant was cloned. Again, it appeared to be a mutant differing from the wild-type gene by a point mutation in exon 5 (Szikora et al., 1990). For the P91A epitope, the mutation allowed the peptide to bind to the Ld molecule by generating an agretope. This was not the case for the P35B antigen, since both the wild type and mutated peptides were able to bind to Ddmolecules and to render target cells sensitive to lysis by the specific CTL (Szikora et al., 1990).A third tum- antigen, P198, was identified and shown to be derived from a normal gene bearing a point mutation in one of the exons. Both peptides corresponding to the normal and the mutated sequences could bind to the Kd molecule, but only the tum - P198 mutated antigen was recognized by anti-P198-specific CTLs (Sibille et al., 1990). These results indicated that many cellular genes can mutate to generate tum- variants, which explained their high rate of appearance (about This was the first rigorous demonstration that mutations induced by a mutagenic treatment can generate tumor rejection antigens, specific for each tumor, either by creating an agretope that allows apeptide to bind to MHC class I molecules or by creating a new epitope that competes with the normal peptide for binding to MHC class I molecules (reviewed in Boon, 1992). In mice and in humans, several other tumor antigens that derive from mutated proteins and are recognized by T cells have now been identified. Indeed, there is now mounting evidence that peptides derived from the protein products of altered oncogenes and tumor suppressor genes are associated with the MHC class I or class I1 molecules and thus may provide potential candidates for inducing specific immunity against cancer cells. The protein products of altered oncogenes and tumor suppressor genes (TSG) are interesting potential targets for a T-cell-mediated immune response for at least three reasons. First, alterations in these genes are obligatory events in the carcinogenic process and are thus present in every malignant cell. It has been estimated that, in the development of colorectal cancer, a total of five to eight different “genetic hits” affecting oncogenes and/or tumor suppressor genes have to occur in the same cell to induce the complete transformation from normal mucosa to malignant carcinoma (Reviewed
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in Fearon and Vogelstein, 1990). The emergence of altered peptides derived from the protein products of oncogenes and tumor suppressor genes and potentially presentable in an MHC class I or I1 context thus appears as a general phenomenon in cancer. Second, some of the most common mechanisms of oncogene activation, such as point mutation and chromosomal rearrangement, create protein sequences that do not exist in nontransformed cells (Bishop, 1991). Similarly, the common mechanism of inactivation of a tumor suppressor gene is chromosomal deletion of one allele and point mutation of the second allele, thus creating sequences that are specific of the cancer cells (Marshall, 1991). The induction of a strong immune response against these “foreign” sequences in a therapeutic context should thus not face problems associated with tolerance or autoimmunity. Finally, since the expression of altered oncogene and tumor suppressor gene proteins confers a selective growth advantage to the cancer cell, one escape mechanism in response to immune intervention, namely the downregulation of the expression of the altered gene, might lead to the same therapeutic effect as successful targeting by the Tcell response. Although more than 10tumor suppressor genes and almost 100 oncogenes have been defined (Bishop, 1991), relatively little information is currently available on immune responses directed against these cancer-specific proteins, or on the induction of specific immunity against oncogene and tumor suppressor gene proteins. There is, however, a growing interest in possible immune intervention directed against these “cancer genes”; naturally occurring or induced T-cellmediated immunity against the p53 tumor suppressor gene, the bcr/ abl tyrosine kinase, and the ras-proteins has been reported.
2 . Mutated p53 Proteins Sporadic mutations in the p53 tumor suppressor gene are the most common genetic alterations observed in human cancers (Levine et al., 1991) and approximately 70% of colon cancers, 30 to 50% of breast cancers, 50%of lung cancers, and almost 100% of small-cell carcinomas of the lung harbor these mutations (Hollstein et al., 1991). Germline mutations of the p53 gene have been described in “hereditary cancers” such as in patients with Li-Fraumeni syndrome (Malkin et al., 1990; Srivasta et al., 1990; Santibanez-Koref et al., 1991), in young patients with more than one malignant neoplasm (Malkin et al., 1992), and in adults with sarcoma with a family history of cancer (Toguchida et al., 1992). A computerized scoring system for predicting peptide binding to
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HLA-A2.1 molecules has been applied to the p53 gene (Houbiers et al., 1993). The 393 amino acids of wild-type p53 and the 20 amino acids around each of the 32 published point mutations in colorectal and ovarian cancer were scanned, which resulted in a set of 63 peptides that scored above a predefined level indicating presumptive binding to HLA-A2.1. Peptides were synthesized and about 40% of them actually bound. The human processing defective cell line 174CEM.T2 was then loaded with some of these peptides and utilized as antigenpresenting cells to induce CD8+ CTL clones. These clones were capable of specifically lysing target cells loaded with the respective wildtype or tumor-specific mutant p53 peptides (Houbiers et al., 1993).The authors concluded that p53-specific CTL clones might be potentially useful for cellular immunotherapy of cancer. Yanuck et all. (1993) used another approach to demonstrate the immunogenicity of peptides derived from mutated p53 sequences. Spleen cells of BALB/c (H-2d)mice were pulsed with a 21-amino acid peptide encompassing a point mutation (135Cys to Tyr) in the mutant p53 from a human lung carcinoma and then injected intravenously into syngeneic animals. This immunization protocol generated CD8+ CTL that were capable of specifically killing mouse fibroblasts transfected with the complete mutated human p53 gene, whereas fibroblasts expressing the nonmutated human p53 were not recognized (Yanuck et al., 1993). Since the level of expression of the transfected p53 gene was comparable to that seen in human tumors and leukemias, it was proposed that peptide immunization against mutant p53 could be used for cancer immunotherapy. 3. The bcrlabl Oncogene
More than 95% of patients with chronic myelogenic leukemia (CML) carry the t(9;22)(q34;qll)translocation. This translocation of the c-abl protooncogene (abl) on chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22 produces the Philadelphia chromosome and leads to the formation of a fusion gene termed bcr/abl, which encodes a 210-kDa chimeric protein with abnormal tyrosine kinase activity (Shtivelman et al., 1985; Hermans et al., 1987). In 10% of children and approximately 25% of adults with acute lymphoblastic leukemia (ALL), the abl gene is also translocated to chromosome 22, but to a different region of the bcr gene, and detection of the bcr/abl fusion mRNA in ALL has been associated with poor prognosis (Maurer et al., 1991). The abl and bcr genes are expressed by norma1 cells and thus the encoded proteins are probably nonimmunogenic. However, the join-
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ing region segment of the bcr/abl chimeric protein is composed of unique sequences of abl amino acids joined to bcr amino acids that are expressed only by malignant cells. Synthetic peptides corresponding to the bcr/abl joining region have been used to immunize BALB/c and C57B1/6 mice by repeated subcutaneous injections together with complete Freud's adjuvant (Chen, W. et al., 1992). The immunization protocol elicited peptide-specific, CD4+, class I1 MHC-restricted T cells that recognized only the combined bcr/abl sequences but not bcr or abl sequences alone. The proliferative response of these T-cell clones to the whole bcr/abl protein presented by syngeneic spleen cells demonstrates that processing of the fusion protein and binding of joining-region peptides to MHC molecules also occurs in a less-artificial system (Chen, W. et al., 1992). The bcr/abl protein thus represents a potential tumor-specific antigen related to the transforming event and expressed in the large majority of patients with CML. Further studies will indicate if an immune reaction against bcr/abl dependent on cytotoxic CD8+ T lymphocytes can also be induced. 4 . The ras Oncogenes Mutations of the ras protooncogenes are among the most frequent alterations found in human malignancies. Thus, mutations of the Kiras, N-ras, or Ha-ras genes are found in approximately 90% of pancreatic carcinomas (Almoguera et al., 1988),50% of colorectal carcinomas (Bos et al., 1987a; Forrester and Almoguera, 1987), and 25% of acute myelogenic leukemias (Bos et al., 198713; Farr et al., 1988), but only in less than 5% of breast carcinomas (Rochlitz et al., 1989). Ras oncogenes are activated by point mutations that occur almost exclusively in codons 12, 13, and 61 (Barbacid, 1987). These welldefined and easy to diagnose genetic alterations can be conveniently included in immunization strategies, and this is probably the reason why the ras genes were the first oncogenes whose role as potential targets for immunotherapy of cancer was evaluated. Using a synthetic peptide corresponding to amino acids 5-16 of a mutated ras protein with an exchange of the normal Gly at position 12 by Val, Jung and Schluesener (1991)first demonstrated that specific CD4+ T-cell lines could be derived from the blood of two of four healthy donors analyzed. These T-cell lines were generated by continuous in vitro stimulation of peripheral blood cells and showed no crossreactivity to normal, nonmutated p2lras proteins. Similarly, Peace et al. (1991) showed the presence of class I1 restricted, specific T cells in the repertoire of healthy C57/B16 mice immunized with Freund's
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adjuvant plus mutated ras peptides with an Arg substituted for Gly at position 12 (Peace et al., 1991). Several other mutated ras peptides and mouse strains with different H-2 backgrounds have been evaluated. Some, but not all, are immunogenic in individual strains of mice, presumably reflecting the ability of individual peptides to be efficiently presented by the MHC molecules ofthe host. The same authors have reported preliminary results on the induction of class I MHCrestricted CD8+ CTL, specific for mutated ras peptides, using the B6 leukemia cell line of the C57/B16 (H-zb)mouse (Peace et al., 1992). In line with the results of Jung and Schluesener (1991),Gedde-Dahl et al. (1992a) have shown that the repertoire of a single healthy donor contained T-cell clones specific for six of nine mutated peptides tested. All of these peptides were capable of binding to HLA-DQ molecules and several to HLA-DR molecules (Gedde-Dahl et al., 1992aJ993b). Four T-cell clones were established and their fine specificity determined: two of the clones recognized peptides presented by DR2, and the other two recognized peptides presented by DQ6 (Gedde-Dahl et al., 1993a).An interesting observation was the occurrence of an HLADQ8-restricted clone, specific for a codon 61 Gln to Leu mutation of p21 ras, in a patient with thyroid cancer (Gedde-Dahl et al., 1992b). The authors could not find the corresponding codon 61 mutation in the thyroid carcinoma cells, and they speculate that this might be due to the prior elimination of the mutation-bearing cells during tumor progression. Another interpretation, however, is that the isolation of the clone specific for the mutated codon 61 is accidental and that it belongs to the normal repertoire of the cancer patient, as shown for healthy donors in the studies mentioned above. An encouraging study has involved immunization of C57/B16 mice by intraperitoneal injection of mutant H-ras proteins. A CD8+ CTL activity was demonstrated in the splenocytes of the treated animals against cells carrying Arg instead of Gly in Ha-ras position 12. More importantly, 9110 animals immunized with the mutant protein were protected against subsequent inoculation of lo5 Ha-Balb fibroblasts, a malignant cell line that expresses an activated Ha-ras gene with the same Gly to Arg mutation in position 12. Only 2/10 mice immunized with nonmutated ras protein and 0/10 immunized with ovalbumin were protected against the Ha-Balb cells (Fenton et al., 1993).A drawback of this study was the fact that the same experiments did not work when Ha-ras with Val instead of Arg at position 12 was used to induce immunity. In conclusion, altered oncogenes and tumor suppressor genes are potentially important targets for the immunotherapy of cancer. Not all
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of the mutated oncogenehmor suppressor gene peptides analyzed so far seem to be immunogenic in all circumstances, but strategies to increase immunogenicity might overcome this problem. Clinical studies exploiting the immunogenicity of these cancer-inducing genes are currently under way.
F. ANTIGENSDERIVEDFROM THE OVEREXPRESSION OF NORMALPROTEINS Another mechanism may account for the expression of a tumor antigen on transformed cells. If a gene is silent, or expressed at a low level, in the normal tissue, and if it is activated in tumor cells, the overexpression of the unmutated self-proteins may lead to the production of a set of presentable self-peptides which are potential targets for antitumor CTLs. In the murine mastocytoma P815, comparison of the specificities expressed by tum- variants that are selected either in vitro by coculture with CTL clones or in vivo by passage in syngeneic DBA/2 mice led to the identification of five distinct rejection antigens, named P815 A, B, C, D, and E. Because antigens A and B are usually lost together by tumor cells that escape tumor rejection in vivo, they were identified as dominant antigens that play a significant role in the antitumoral response. Using the same strategy as for molecular cloning of the tumantigens (i-e., transfection of the P1A gene in P1A- B - antigen-loss variants), transfectants were isolated that express both A and B rejection antigens, recognized by CTL clone. This strategy allowed the identification of the P1A gene which encodes both P1A and P1B antigens presented by the Ld restriction element (van den Eynde et al., 1991). The sequence of the P1A gene, which differs completely from the genes coding for Tum- specificities, is identical in tumor cells and in normal mouse tissues. A nonapeptide was synthesized from the coding sequence of P1A and shown to carry specificities for P1A and PlB. Antigen-loss variants of P815 having one mutation were shown to express only one of the two epitopes (Lethe et aZ., 1992). The PlA gene is silent in precursors of most cell lines but is transcribed at a very low level in normal adult tissues. Several hypotheses have been proposed to account for the antitumor immune response, which occurs independently of any observable autoimmune reaction (reviewed in Boon, 1992). P l A might be expressed at an early stage in embryonic tissues before the establishment of natural tolerance. Alternatively, it could be transiently expressed by some cell precursors at a given stage of their differentiation. In any case, normal cells by themselves cannot induce an immune response, and it cannot be ruled
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out that the antitumoral reaction does not eliminate precursor cells that express the antigen, without visible damage to the organism. Similar experiments, using tumorigenic cell lines obtained by transfection with oncogenes, are in agreement with this observation and confirm that activation of a normally silent gene leads to the expression of a potential tumor rejection antigen (Torigoe et al., 1991). The neu/erbB2 oncogene is a transmembrane receptor with a high degree of homology to the epidermal growth factor receptor EGF-R (Schechter et al., 1985). Amplification and/or overexpression of neu/ erbB2 occurs in approximately 20-30% of human ovarian and breast carcinomas and has been associated with poor prognosis in both diseases (Slamon et aZ., 1987,1989; Allred et al., 1992; Gasparini et al., 1992). Preliminary data indicate that tumor-infiltrating lymphocytes isolated from human ovarian carcinoma can recognize peptides corresponding to neu/erbB2 epitopes and’lyse targets pulsed with these peptides (Ioannides et al., 1992). Similarly, CTL have been cloned from the peripheral blood of patients with ovarian and breast cancer that seem to be specific for peptides derived from the neulerbB2 oncogene (Tim Eberlein, Harvard Medical School, personal communication). Thus, the mechanism of activation of the neu/erbB2 oncogene is overexpression of an unaltered protein, and specific T-cell reactions can be raised against it. As above with the P815 antigen, the unresponsiveness against the “self-antigen” in normal cells might result from anergy or suppression of established specific CTL, absent or insufficient antigen processing of the antigen in low expressors, or clonal deletion of neu/erbB2 reactive T cells. One should bear in mind that attempts to induce a specific immune response against malignant cells overexpressing neu/erbB2 do, of course, carry a certain risk of also inducing an autoimmune response. In human tumors, several groups have reported the existence of such specific tumor antigens that are recognized by autologous CTL clones (Hainaut et al., 1990; Knuth et al., 1992). By immunoselection with these CTLs, variants having lost several antigens have been prepared, which allowed the definition of some antigenic specificities in human melanomas (Knuth et al., 1989; van den Eynde et al., 1989; Wolfel et al., 1989; Topalian et al., 1990). Three different epitopes presented by HLA-A2 molecules were identified in a human melanoma tumor. HLA-A2 is the most widespread MHC class I allele in Caucasian populations, and the HLA-A2 molecule is a common restriction element for melanoma-specific effector T cells (Kawakami et al., 1992; Topalian et al., 1989; Slingluff et al., 1993; Viret et al., 1993).Furthermore, it was shown in a study that the antigen expressed
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by melanoma cells and restricted by HLA-A2 molecules is also expressed by HLA-A2 melanocytes derived from normal tissues (Anichini et al., 1993). Biochemical studies have led to the identification of a tyrosinasederived peptide as one of several peptides isolated from an HLAA2 melanoma (Slingluff et al., 1993). Using the previously described strategy which allowed the identification of the P1A gene, T. Boon and his co-workers have prepared a genomic library from DNA of a human melanoma (MZ2-MEL).The library was transfected into a variant which had lost one of the six distinct antigens on the original tumor and was recognized by autologous CTL clones. Transfectants expressing antigen MZ2-E were selected based on their ability to induce the production of tumor necrosis factor by an anti-E CTL clone (Traversari et al., 1992). This led to the isolation of the MAGE-1 gene, which encodes the MZ2-E antigen (van der Bruggen et al., 1991). The sequence of the MAGE-1 gene does not correspond to any previously known protein. It encodes a 30-kDa protein that contains a nonapeptide that binds to the HLA-A1 molecule, and this is recognized by the anti-E CTL clones. While not expressed by normal tissues, except sperm, this gene is expressed by different tumors, such as melanomas (40%),lung cancer, head and neck tumors, sarcomas, and breast tumors. Since about 10% of melanoma patients should express the antigen MZ2-E restricted by the HLA-A1 alIele, vaccination protocols are currently being assessed, using either irradiated cells expressing the MZ2E antigen or genetic constructs expressing the antigen, the HLA-A1 molecule, and/or interleukine, that could improve the antitumor immunity in these melanoma patients. Subsequently, a similar approach has been used to define antigens recognized by T cells in the context of HLA-A2 molecules. At least two antigens have been isolated, a tyrosinase or tyrosinase-associated molecule (Ab) and a molecule termed Aa (Brichard et al., 1993; Wolfel et al., 1993). In one study, another strategy was used to identify human melanoma peptides that are recognized by class I-restricted tumor-infiltrating T lymphocytes (Storkus et al., 1993). Using elution from HLA-A2+ or HLA-A2- melanoma lines by repeated acid treatment, different tumor peptides isolated by fractionation on HPLC were shown to sensitize HLA-A2 nonmelanoma targets for lysis by melanoma-specific CTLs. The relationship between these peptides and the peptide isolated by Slingluff et al. from melanoma cells by affinity chromatography on HLA-A2 molecules still remains to be determined (Slingluff et al., 1993; Storkus et al., 1993). Interestingly, some of these peptides were
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shown to bear the HLA-A2 binding motif, as already predicted from the sequence of self-peptides eluted from MHC molecules (Falk et al., 1991). In other human tumors, there is mounting evidence that autologous CTLs can recognize antigens on tumor cells, such as squamous cell carcinoma of the head and neck (Yasumura et d . ,1993), or renal-cell carcinomas bearing the HLA-A2 restriction element (Schendel et al., 1993)or ovarian tumors bearing the HLA-B5 molecule (Wang et al., 1992). G. THECASEOF MUCIN Several groups, looking for antigens preferentially expressed by malignant cells, have generated tumor-specific monoclonal antibodies that recognize an epitope on the mucin, an epithelial cell glycoprotein, produced by breast and pancreatic carcinomas, but not by the corresponding normal tissues (Girling et al., 1989; Barnd et al., 1989). The MUC-1 antigen, which corresponds to the heavily glycosylated mucin, consists of multiple tandem repeats of a 20-amino acid sequence with abundant 0-linked carbohydrate side chains, the protein core being identical to the one expressed on the normal tissues (Gendler et al., 1988; Burchell et al., 1989; Wreshner et al., 1990; Hareuverni et al., 1990a). The antigenic epitope expressed as the epithelial tumor antigen in a recombinant vaccinia virus was shown to provide a true target molecule for immune responses against tumor cells bearing this antigen (Hareuveni et al., 1990b). Furthermore, the originality of this tumor antigen resides in its capacity to stimulate specific CTLs in an MHC-unrestricted manner, a finding attributed to its highly repetitive nature (Finn, 1992a). In a first attempt, human CTLs were generated from lymph nodes of patients with pancreatic carcinomas (Barnd et al., 1989; Finn, 1992b) and breast carcinomas (Jerome et al., 1991), using allogeneic mucin-producing tumor cell lines as stimulators. These data were then repeated using presenting cells transfected with MUC-1 cDNA as stimulators and targets for mucin-specific CTLs (Jerome and Finn, 1992; Jerome et al., 1993). The results confirmed original observations and demonstrated that both syngeneic- and allogeneic-presenting cells can stimulate mucin-specific CTLs. Furthermore, to determine the precise peptide specificity of TCRs on cloned CTLs, an expression vector that contains only two repeats was constructed, and transfected cells with this vector were shown to be recognized efficiently by specific CTLs (Bu et al., 1993). It was thus postulated that target cells would express a two-repeat molecule that is not extensively glycosylated, leading to a more effective presentation of the CTL epitope. This is in agreement with the observation that a
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major group of T-cell clones requires inhibition of mucin glycosylation for target cell recognition (Jerome et al., 1993). In order to evaluate the immunogenicity of mucin epitopes, peptides derived from the mucin tandem repeat or the whole mucin molecule were assayed for their ability to induce a protective immunity against tumor growth (Ding et al., 1993; Acres et al., 1993). Since mucin is a self-antigen that is preferentially recognized on tumor cells, it should be a good candidate for a tumor-rejection antigen in vaccines, which could be further modified to secrete locally cytokines that stimulate effective helper and cytotoxic T-cell responses. 111. Why Are Tumors Poorly Immunogenic?
A. Low ANTIGENOR MHC CLASS I EXPRESSION Downregulation of MHC class I expression has been found to occur in many animal and human leukemias, lymphomas, and solid tumors, as extensively reviewed elsewhere (Browning and Bodmer, 1992; Moller and Hammerling, 1992; Schrier and Peltenburg, 1993). The notion is generally accepted that loss of MHC expression and the subsequent inability of the cells to present tumor-specific peptides to CTL clones can lead to an escape of the respective tumor cells from immunological control. This notion is supported by the observation that transfection of MHC class I genes into aggressive mouse tumor cell lines with low or no endogenous expression of these genes leads to tumor rejection when mice are transplanted with the transfected cells (Hui et al., 1984; Tanaka et al., 1985). A reciprocal experiment in the murine thymoma AKR showed that tumor growth was promoted by transfection of antisense DNA suppressing endogenous H-2KkMHC expression (Hui et al., 1991). These and similar studies are discussed in more detail in Section IV,A of this review. A variety of mechanisms through which tumor cells can lose MHC expression have been discussed. They include loss of peptide transporter genes in murine RMA-S cells (Franksson et al., 1993) and several human cancers (Restifo et al., 1993),altered binding of regulatory factors to HLA class I enhancer sequences (Blanchet et al., 1992), a frameshift mutation of the P2-microglobulin gene in the class Inegative melanoma cell line SK-MEL-33 (Wang et d . , 1993), among other mechanisms (Chapman and Houghton, 1993). The “immune evasion” theory, however, is not the only possible explanation for MHC loss by tumor cells, and other interpretations
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should be considered. For example, nonimmunological, selective advantages may accompany low class I expression, such as an intrinsic growth advantage for low expressors (Haliotis et al., 1990).Also, MHC class I genes are expressed at different levels in various cells and tissues, so that their level of expression can be taken as a differentiation trait. In a study of Burkitt lymphomas, loss of HLA class I expression was strictly correlated with B-cell differentiation and could thus be considered as a phenotypical trait of the tumor rather than a means to escape immune surveillance (Anderson et al., 1991). Similar findings have been reported in human urothelial tumors (Ottesen et al., 1987; Tomita et al., 1990). Finally, MHC loss might be a consequence of the oncogenic process itself, i.e., of alterations in oncogenes and tumor suppressor genes. This has been shown for the transformation of rodent cells by the adenovirus E1A oncogene, whose expression is responsible for MHC class I downregulation. Similarly, the c-myc and N-myc genes are capable of switching off the expression of MHC class I genes in human melanomas and neuroblastomas, respectively (reviewed in Schrier and Peltenburg, 1993). In several tumors, low MHC expression was found more frequently in metastatic than in primary lesions (Cordon-Cardo et aE., 1991; Pantel et al., 1991), or was associated with bad prognosis, although the evidence on this last point is controversial (Lopez-Nevot et al., 1989; Moller and Hammerling, 1992). These findings can be interpreted as arguments in favor of the “immune evasion” theory but would also fit with m y of the aforementioned explanations for MHC loss. In contrast to complete loss of HLA expression on human tumors, loss of the expression of individual HLA alleles argues in favor of immunoselection of a tumor cell clone (Browning and Bodmer, 1992; Schrier and Peltenburg, 1993) and has been detected in a large percentage of colorectal carcinoma and other epithelial and nonepithelial neoplasms (Momburg et al., 1989; Natali et al., 1989; Smith et al., 1989; Pandolfi et al., 1991; Kaklamanis et al., 1992). Furthermore, there is growing evidence that different MHC alleles either predispose to or protect against the development of certain virally induced tumors (Klitz, 1992). Such MHC associations have been found in Hodgkin’s lymphoma (Bodmer et al., 1989), nasopharyngeal carcinoma (Lu et al., 1990), squamous cell carcinoma of the cervix (Wank and Thomssen, 1991), and rabbit viral papillomas (Han et al., 1992). Similarly, renal transplant recipients treated with long-term immunosuppressive therapy have an increased risk of skin cancer only when they carry certain HLA alleles, while others (HLA-All) seem to partially protect against the development of skin tumors (Bouwes Bavinck et al., 1991; Browning and Bodmer, 1992). It is possible that the ability of a given
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HLA allele to present certain viral immunogenic peptides to stimulate and/or be recognized by CTL clones determines whether a transformed virus-infected cell develops into a tumor or is attacked and removed by cytotoxic cells. Loss of MHC class I expression by tumor cells may be a major obstacle in immune therapy trials designed to activate CTL responses against the tumor. The question of whether MHC class I gene expression remains activatable by IFN-y, TNF-a, or other agents therefore becomes a significant issue. It has also been observed in several mouse models that even modest levels of MHC class I-petide complexes, which do not allow the induction of specific CTL, are nevertheless sufficient to permit specific lysis by activated CTL (Shastri and Gonzalez, 1993). B. LACKOF COSTIMULATORY MOLECULES (B7-CD28/CTLA-4) Maximal stimulation of a T-cell response to a peptide/MHC complex, requires a second, costimulatory signal, usually delivered by antigen-presenting cells, i.e., activated B cells, macrophages, or dendritic cells. A suboptimal proliferative T-cell response can be enhanced by different soluble or membrane-bound molecules, such as nutrients, hormones, trophic factors, interleukins, and cell adhesion molecules (reviewed by Schwartz, 1990). Occupancy of the TCR in the absence of a costimulatory signal induces a state of unresponsiveness in the T cell, characterized by a lack of IL-2 production and proliferation in response to subsequent exposure to antigen, even in the presence of a costimulatory signal. This long-lasting, antigenspecific state of unresponsiveness has been termed anergy (Schwartz, 1990). Although the molecular basis ofT-cell anergy is not fully understood, evidence suggests that one such costimulatory pathway involves the interaction of the T-cell surface antigens CD28 and CTLA-4 with their ligand B7, expressed on APCs (Freeman et al., 1991; Gimmi et al., 1991; Linsley et al., 1991). CD28 is constitutively expressed on 95% of human CD4+ T cells, 50% of CD8+ T cells, and thymocytes coexpressing CD4 and CD8, and its regulates T-cell cytokine production by transcriptional and post-transcriptional mechanisms ( Jenkins and Johnson, 1993). CTLA-4 is a second ligand, with approximately 20fold higher avidity than CD28 for B7, and is virtually undetectable on resting T cells but is expressed on activated CD4+ and CD8+ T-cell subsets at -1/30-50 the levels of CD28 (Linsley et al., 1992). B7, like CD28 and CTLA-4, is a member of the immunoglobulin gene superfamily and is expressed on activated B cells and macrophages and Constitutively on dendritic cells (Schwartz, 1992). The function
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of CTLA-4 on activated T cells may be to promote effective interaction between CD28 and B7. Low-abundance, high-avidity CTLA-4 molecules might facilitate interactions between higher abundance, lower avidity CD28 molecules and the B7 counter receptor (Linsley et al., 1992). Blocking costimulation of the CD28 receptor in the continued presence of a foreign antigen can lead to T-cell unresponsiveness. This has been shown in experiments achieving long-term survival of xenogeneic pancreatic islets when B7 was blocked by the high-affinity inhibitor CTLA-41g (Lenschow et al., 1992). The induction of T-cell activation and proliferation is thus a twostep process. First, cognate recognition of a specific peptidelMHC complex by the T-cell receptor leads to the activation of tyrosine kinases in the T cell and to signal transmission by the cytoplasmic tail of the MHC molecule, which causes increased expression of B7 on the antigen-presenting cell (Nabavi et al., 1992; Baskar et al., 1993). Second, B7 interacts with its natural counterreceptors, CD28lCTLA-4, on the T cell; induces an independent stimulus, probably uia tyrosine phosphorylation of specific substrates, including phopholipase C-1; and triggers calcium-dependent and calcium-independent signals. This subsequently leads to the production of IL-2 and other cytokines and to proliferation of the specific T-cell clone (Freeman et al., 1991; Gimmi et al., 1991; Linsley et al., 1991; Reiser et al., 1992; reviewed in: Jenkins and Johnson, 1993; Linsley and Ledbetter, 1993). In contrast, when peptide antigen is presented by MHC in the absence of €37, T-cell clones are unable to secrete detectable levels of IL-2 or proliferate (Gimmi et al., 1993). In model systems, this anergy can be reversed, even after its full induction, by IL-2 at very high doses, although four or more division cycles are required for complete reversal (Schwartz, 1992). CD28 is not required, however, for all T-cell responses in uivo, since CD28-deficient mice were capable of mounting a CTL response and delayed-type hypersensitivity after being infected with lymphocytic choriomeningitis virus (Shahinian et al., 1993), suggesting that alternative costimulatory pathways may exist, at least in this model. More recently, in agreement with this hypothesis, another surface molecule, that is distinct from B7 and abundantly expressed on activated B.cells, was identified as the predominant ligand for the T-cell activation molecule CTLA.4 (Freeman et al., 1993a,b; Hathcock et al., 1993). This CTLA.4 counterreceptor named B7.2 seems to provide a critical early costimulatory signal for T cells. The importance of the discovery of the B7,CD28/CTLA-4 pathway for tumor immunology lies in the fact that most tumors are derived
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from parenchymal or mesenchymal cells that do not express B7. It was thus speculated that the ability of tumor cells to effectively present tumor-specific antigens (TSA) might be severely impaired and that lack of costimulation could be an important mechanism confering low immunogenicity even to tumor cells expressing MHC and TSA (Schwartz, 1992). Tumor reactive T-cell clones might thus receive inadequate costimulation and enter a state of anergy rather than eradicating the tumor. The introduction of the B7 gene into tumors might result in effective costimulation and tumor immunogenicity in the immunocompetent host. This has been shown in several examples (Chen, L. et al., 1992; Baskar et al., 1993; Harding and Allison, 1993; Townsend and Allison, 1993) and is discussed below (Section IV,B). C. IMMUNE SUPPRESSIVE FACTORS (TGFP AND IL-10) Besides changes in the tumor cells themselves that lead to decreased immunogenicity, as discussed above, it has long been postulated that malignant cells may secrete immunosuppressive factors that could contribute to immune evasion. A variety of immunosuppressive molecules have been found in the culture supernatant of tumor cells, as well as in the sera and effusions of animals and patients with cancer. Although the active molecules have frequently not been purified to homogeneity, they were shown to inhibit a variety of immune reactions: delayed-type hypersensitivity (DTH); macrophage accumulation at sites of inflammation; macrophage chemotaxis, phagocytosis, and cytotoxicity; skin graft rejection; lymphocyte proliferation in response to mitogenic stimuli; antibody synthesis; generation of LAK, TIL, and CTL cells; and lymphokine production (reviewed in Sulitzeanu, 1993). Probably the best characterized among cancer-related immunosuppressive molecules is the transforming growth factor (TGFP). It was originally identified due to its ability to confer a transformed phenotype to normal fibroblasts, but it also exerts a strong inhibitory effect on malignant and nonmalignant epithelial cells (Barnard et al., 1990) and potent immunosuppressive activity. TGFP inhibits the activity of a number of other cytokines, including IL-2, IL-4, IFN-y, and TNFa.It inhibits the proliferation of T and B cells as well as the generation of LAK and CTL. In addition, TGFP blocks NK cytolytic activity and downregulates the expression of the IL-2 receptor and of MHC molecules (Sulitzeanu, 1993).TGFP may thus be responsible for many of the immunosuppressive activities that have been initially attributed to “suppressor” lymphocytes and macrophages (Sulitzeanu, 1993). TGFP was originally identified as an immunosuppressive factor in
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the sera of patients with glioblastoma (reviewed in Siepl et al., 1988), but its mRNA was later detected in a large number of tumors (Derynck et al., 1987). The TGFP protein could be detected by immunocytochemistry or Western blot analysis in cell lines established from colorectal cancer (Coffey et al., 1986), breast carcinoma (Knabbe et al., 1987), endometrial cancer (Boyd and Kaufman, 1990), and other cancers. TGFP was also found in the ascitic effusion of ovarian (Hirte and Clark, 1991) and breast cancer (Sulitzeanu, 1993) patients, where it was at least partially produced by metastatic cancer cells. The transfection of TGFP cDNA into a highly immunogenic UVinduced tumor leads to reduction in immunogenicity and progressive tumor growth in one model (Torre-Amione et al., 1990). In a mouse plasmocytoma, TGFP was secreted in large amounts by the malignant cells and consequently blocked the expression of the surface markers IgM, the CD23 receptor, and the transferrin receptor (Berg and Lynch, 1991). One may speculate that in humans, immunosuppression and increased susceptibility to infection might be caused by similar TGFPassociated mechanisms. An important regulatory cytokine, initially named CSIF (cytokine synthesis inhibitory factor) but now known as IL-10, has been cloned (Moore et al., 1990). In the mouse, CD4 T-cell clones have been classified in three categories, TH-0, TH-1, and TH-2 (reviewed in Mosmann and Coffman, 1989).Even if questions remain on the validity of the classification in wiwo and to its extrapolation to human T cells, it is well-established that IL-10 is secreted by the cells of the mouse TH-2 subset and suppresses the production of IL-2, IFN-.)I,and TNFa by the TH-1 subset (reviewed by Moore et al., 1993).The production of the NK cell stimulatory factor (NKSF or IL-12) by human peripheral mononuclear cells, and consequently the proliferation of NK cells, has also been shown to be blocked by IL-10 (D'Andrea et al., 1993). The inhibition of cytokine synthesis and of several accessory functions of macrophages thus renders IL-10 a potent suppressor of T cells, NK cells, and macrophages. In analogy to the classification of CD4+ helper cells into TH-1 or TH-2 subsets, it has been proposed that murine CD8+ T-cell clones could be subdivided into two groups, the cytotoxic (CTL) and the suppressor (Ts) subsets. As opposed to CTLs, all the Ts clones produce IGlO after stimulation with anti-CD3 mAb and suppress the proliferative activity of both TH-1- and TH-2-type CD4+ clones (Inoue et al., 1993). In a study of 48 human cancer cell lines established from different carcinomas, malignant melanomas, and neuroblastomas, IL-10 was
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found in the supernatant of 15 cell lines, mostly those derived from colorectal cancers (Gastl et al., 1993). In addition, as described for TGFp, IL-10 can be found in the peritoneal fluid and serum of patients with ovarian and other intraperitoneal cancers (Gotlieb et al., 1992), suggesting a role for this cytokine in solid tumor development and antitumor immunity. In addition to TGFp and IL-10, a large number of soluble immunosuppressive factors secreted by tumor cells have been described. These include the lymphocyte blastogenesis-inhibiting factor (LBIF) produced by a macrophage cell line (Fujiwara and Ellner, 1986); a transmembrane retroviral envelope protein, p15E, shown to diminish tumor immunity in vivo (Cianciolo et al., 1985); insulin-like growth factor 1(IGF-1) (Trojan et al., 1993);the suppressive E-receptor (SER), a molecule isolated from malignant effusions of patients with head and neck, ovarian, and lung cancer (Oh et al., 1987); different colonystimulating factors (CSF); and many other molecules (reviewed in Sulitzeanu, 1993). It must be kept in mind, however, that for most of these factors found in sera and effusions, it has not been clearly demonstrated that they are tumor-derived rather than tumor-associated molecules. Many of them have also been detected in the sera of normal patients and they might represent growth regulatory rather than immunosuppressive molecules. Nevertheless, the modulation of “immune suppressive factors” by antisense oligonucleotides, monoclonal antibodies, and other reagents remains an attractive target for future attempts at cancer immunotherapy.
D. MODIFICATION OF THE TUMOR ENVIRONMENT Modifications in the environment of a developing tumor can occur during the entire process of oncogenesis, from the preneoplastic state to the highly metastatic, autonomously growing cancer cell. These changes of extratumoral factors can cause growth-promoting or -inhibiting alterations in the complex interaction between the tumor and its environment. The process involved in tumor spread can be subdivided into the following steps: transformation, growth, angiogenesis, detachment, invasion, intravasation/release, survival in the blood stream, arrest, extravasation/invasion, and growth/angiogenesis (Hart and Saini, 1992). During each ofthese phases, immunological as well as nonimmunological mechanisms can influence the progression of the malignant disease. Since the biology of metastasis and the importance of host-tumor interactions during progression have been extensively reviewed elsewhere (Killion and Fidler, 1989; Hart and Easty, 1990; Liotta et al.,
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1991a,b; Frost and Levin, 1992; Hart and Saini, 1992), only a few important findings are discussed in this section. During progression, tumor cells interact with countless other host cells such as lymphocytes, monocytes, and epithelial and endothelial cells. The regulation of many of these intercellular interactions is mainly dependent on integrins, a class of heterodimeric receptor molecules by which cells attach to extracellular matrices and adhere to other cells. Ligands and counterreceptors of the integrins are cellular adhesion molecules such as JCAM-1 and -2, and VCAM-1, but also molecules like laminin, collagens, fibronectin, or vitronectin (Hynes, 1987,1992). The analysis of ICAM-1 on human melanoma clones demonstrated that resistance to lysis by monocytes was associated with reduced expression of the adhesion molecules, and sensitivity to monocytes could be restored by transfection of the ICAM-1 gene into melanoma cells (Jonjic et al., 1992). On the other hand, increased expression of the cellular adhesion molecules MUC18 and ICAM-1 was correlated with metastasis in other studies (Johnson et aZ., 1989; Lehmann et al., 1989), and human melanoma as well as murine rhabdomyosarcoma cells expressing the integrins VLA-2,5, and 6 show increased adherence to the basement membrane glycoproteins, laminin, and collagen, and consequently display enhanced metastatic properties (Chan et al., 1991; Mortarini et al., 1991). Another study reported that reduced expression of the adhesion molecule cadherin was associated with increased tumor invasion (Takeichi, 1991),and the work of numerous investigators on basement membranes in malignant tissues demonstrates that a dysregulation of laminin expression leads to modifications of the interaction between tumor cells and the matrix and can either increase or decrease metastasis (reviewed in Liotta et al., 1986). An important role in invasion and intravasation is also played by macrophages and monocytes. It has been observed that infiltration of tumors by macrophages could result in an increased release of tumor cells by induction of lysosomes, causing necrosis and consequently an increase in tumor motility (Turner and Weiss, 1980). Similarly, the coculture of rat hepatoma cells with macrophages in vitro increased the invasive potential of the tumor cells in uitro and in vivo (Mukai et at., 1987). Finally, several cytokines such as TNF-a, TGFP, and IL6 can enhance the invasive potential of tumor cells, as measured in different experimental models (Miller, 1993). It has to be emphasized that, in many of the aforementioned studies on tumor invasion, immunological as well as nonimmunological mechanisms might have been responsible for the observed phenomena, and it is not clear whether
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the tumor cells or the tumor environment had undergone the primary alterations. Proteolysis of tissue barriers is another essential component of the invasive/metastatic process and has been linked to several enzymes degrading collagens, proteoglycans, or gelatin. The most extensively studied of these enzymes are the metalloproteinases, of which at least seven are known (Liotta et al., 1991a). These enzymes are produced by tumor cells and have been associated with metastasis in many model systems analyzed. Their function is counteracted by tissue inhibitors of metalloproteinases (TIMP) that are produced by malignant and normal cells, and the balance between the TIMPs and metalloproteinases apparently determines the outcome (Hart and Saini, 1992). A metalloproteinase gene that is specifically expressed in stromal cells of breast carcinomas has been cloned by Basset et al. (1990). The product of this gene, named stromelysin-3 (ST3), could be detected in 30/30 breast carcinomas evaluated and was always found in invasive but never in in situ components of the tumors (Basset et al., 1990). The authors speculated that ST3 might be instrumental in the lytic process leading to cancer invasion and metastasis, while participating at the same time in the host reaction to prevent further spread of the tumor, since ST3 is also associated with desmoplasia, a tissue reaction known to be directed against the most invasive breast cancer lesions (Basset et al., 1990). The formation of new blood vessels is a prerequisite for tumor growth in three dimensions beyond approximately 2 mm ( Folkman et al., 1989). The regulation of angiogenesis is a balance between a variety of angiogenic peptides, such as fibroblast growth factors (FGF), TGFP, and other cytokines. These regulatory factors may be produced by either normal or malignant cells (Mahadevan and Hart, 1990) and represent a further means by which modification of the tumor environment can influence tumor progression and metastasis. In the immunological context, it is important to note that several cytokines that block angiogeneisis do so by affecting the proteolytic activity (Liotta et al., 1991a). Thus, monocytes produce several cytokines, such as TNF-a, TGFP, and IL-1, that can have angiogenic activity (Roberts et al., 1986; reviewed in Miller, 1993). As in the case of adhesion of tumor cells to matrices, proteolysis of membrane barriers, or angiogenesis, other steps involved in the progression of a malignant disease are also subject to regulatory mechanisms imposed by the host and possibly counteracted by the tumor in its attempts to evade “surveillance.” These include factors that regulate survival of tumor cells in the bloodstream, early postarrest events,
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and similar other steps (reviewed by Miller, 1993). These multiple alterations of the tumor environment evoke a number of possible targets for therapeutic interventions. It would be attractive to target the tumor environment rather than the tumor itself because, in contrast to many tumor-directed immunotherapeutical approaches, it is probably unnecessary to modify the entire tissue in order to achieve a beneficial effect, which could also be reached by systemic treatment. IV. Overcoming the Poor Immune Responses Elicited by Tumors
A. INCREASING MHC EXPRESSION ON TUMOR CELLS
1 . Transfection of Allo-MHC into Tumor Cells Attempts to increase the antigenicity and immunogenicity of tumors by introducing foreign genes have been made for more than 20 years. This modification has been called the “xenogenization of tumor cells” (Kobayashi et al., 1969) and can be achieved by the use of viruses (Lindenman and Klein, 1967; Kuzumaki et al., 1978), chemical coupling (Lachmann and Sikora, 1978), enzyme treatment (Currie and Bagshawe, 1969; Bekesi et al., 1971), somatic hybridization (Watkins and Chen, 1969), or transfection of allo-MHC molecules (Itaya et al., 1987). Xenogenized tumors are able to elicit immune responses not only to the “neoantigen,” but also to the TAA of the parental cells, most likely through helper antigen mechanisms (Boone et aE., 1974). Infection of a mixture of four human tumor cell lines by vaccinia virus, in order to potentiate their antigenicity, has been used to produce a lysate which is currently under clinical investigation (Wallack and Sivanandham, 1993). Itaya et al. transfected an allogeneic H-2Ld gene into the Lewis lung carcinoma 3LL/3 derived from the C57B1/6 strain (H-2b).The antigenic expression of the Ld-positive clones was approximately 20-40% of that observed with MethA tumor cells of BALB/c mice (H2-d), and the transfected cells also expressed constant amounts of their native Db antigens (Itaya et al., 1987). The tumorigenicity of these clones was reduced to less than 1/40 of that of the parent tumor cells. The mice also acquired transplantation resistance against challenge with the parental cells after inoculation and regression of viable Ld-positive cells. Surprisingly, the immunogenicity of the Ld-transfected clones was not different from Ld-negative parental cells. The latter was determined by measuring the resistance of C57B1/6 mice against parental 3LL/3 cells after pretreatment with Ld-positive and Ld-negative ceIls rendered nonviable by mitomycin C. The authors speculate that the
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antigenic expression of the allo-MHC molecule might be too strong to enhance the relatively weak TAA, as has been shown for xenogenization by viruses (Yamaguchi et al., 1982).Alternatively, the immunogenicity of the TAA of 3LL may be too low to be recognized by the mice, even after immunogenization by allo-MHC, a mechanism that also has been described in virally xenogenized tumors (Kobayashi et al., 1978). More recently, Plautz et al. (1993) reported on the in uiuo transfer of an H2-KS gene into the colon adenocarcinoma cell line CT26 (H2Kd) and the fibrosarcoma cell line MCA 106 (H2-Kb).This was achieved by surgical exposure of tumors and injection with either retroviral or DNA liposome vectors. In contrast with the above results (Itaya et al., 1987), a cytotoxic T-cell response to H-2Ks and other antigens present on unmodified tumor cells was induced. This immune response attenuated tumor growth in more than 70%ofthe treated animals and resulted in complete cure in approximately 20%. It is important to note that these results were achieved in tumor cell lines that are known to be poorly immunogenic.
2. Transfection of Self-MHC Class ZZ into Tumor Cells The introduction of INF-y and other cytokine genes into tumor cells can upregulate the expression of self-MHC class I and I1 molecules. This aspect is dealt with in more detail in Section IV,C. Only the effects of transfection of the MHC genes themselves are therefore described below. In some murine tumors, introduction of self-MHC class I1 genes into tumor cells leads to the rejection of the transfected cells and can even cause the rejection of nonmodified parental cells. OstrandRosenberg et a2. immunized syngeneic A/J mice with Sal sarcoma cells transfected with self-Ak molecules and showed that the MHC class 11-expressing cells stimulated an improved tumor-specific immune response and that the immunity was cross-reactive with the class 11-negative tumor. Since the transfected MHC molecule could not function as a target molecule for autologous tumor rejection, the increased immunity of Sal/Akcells was probably due to stimulation of a tumor-specific T helper cell population (Ostrand-Rosenberg et al., 1990). In another experimental system, increased immunogenicity after transfection of autologous MHC class I1 molecules was found to be dependent on their cytoplasmic tail. This suggests that enhanced presentation of peptides to CD4+-positive T cells is not the only mechanism by which the transduced MHC molecule enhanced immunogenicity (Baskar et al., 1993).
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3. Transfection of SelfMHC Class I into Tumor Cells Most of the experiments aimed at increasing antitumor immunity by the transfection of tumor cells with MHC genes involved selfMHC class I molecules. Increasing MHC class I expression by gene transfection usually results in increased immunogenicity and decreased tumorigenicity and metastatic capacity in murine models (Hui et al., 1984; Tanaka et al., 1985; Wallich et at., 1985). The decreased tumorigenicity is postulated to be due to enhanced presentation of tumor-specific peptides to CD8+-positive CTLs in viuo. However, increased expression of MHC does not necessarily lead to an increase in the immunogenicity of a tumor. It can also lead to a decrease in NK-mediated cell killing and thus to an increase in the tumorigenicity of the transfected cells (Kame et al., 1986; Glas et al., 1992; Franksson et al., 1993). In addition, several nonimmunological mechanisms are apparently involved in the alterations of tumorigenicity after MHC transfection (Gorelik et al., 1990,1991; Kaufman et al., 1993). a. Zmmunological Effects i. Recognition by CTL Effector Cells The most straightforward consequence of self-MHC class I gene transfection may be to induce or increase the capacity of the transfected tumor cells to present peptides derived from putative tumor-specific antigens. This concept was supported by early experiments in which transfection of H-2Kkgenes into the AKR leukemia led to an increase of the immunogenic properties of these cells and, consequently, to a suppression of growth in vivo (Hui et al., 1984). Similar findings have been reported in several other tumor systems, such as T10 sarcoma, 3LL lung carcinoma, or B16 melanoma transfected with the H-2Kb gene (Wallich et al., 1985; Bahler et al., 1987; Plaksin et al., 1988; Porgador et at., 1989). As described in more detail below (Section IV,A,3,c), in several cases even the metastatic phenotype of the tumor cells could be diminished or completely abrogated by transfection of autologous MHC class I molecules (reviewed in Feldman and Eisenbach, 1991). In some of the tumor models, such as the 3LL lung carcinoma (Plaskin et al., 1988), the AKR leukemia (Hui et al., 1984) and the B16 melanoma (Porgador et al., 1989), preimmunization with MHCpositive cells decreased dramatically the tumorigenicity and/or metastatic spread of the parental MHC-negative cells. Therefore, the very low density of the MHC class I molecule on the parental cells did not confer immunogenic competence per se, but seemed to be sufficient to make these cells susceptible to lysis in uitro by MHC class Irestricted CTLs.
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In contrast, in the T10 sarcoma system, such cross-reactivity was not observed in vitro. CTLs induced by Kb- or Kk-transfected IE7 or IC9 cells did not kill the parental lines, most probably because the parental T10 cells did not express any cell-surface H-2K at all (Wallich et al., 1985). Since an increasing number of human tumors have been found to express very low but detectable levels of MHC class I molecules (see Section III,A, above), antimetastatic immunotherapy with MHC class I-transfected primary tumor cells deserves further investigation. ii. Recognition b y NK Effector Cells In a number of experimental tumor models, the sensitivity of tumor cells to NK cytotoxicity is determined by the level of MHC class I expression on the tumor cells (reviewed in Ljunggren and Karre, 1990; Schrier an& Peltenburg, 1993). Thus, in the murine lymphoma RBL-5, tumor cel(s selected for loss of H-2 expression were less malignant than wild type cells after low-dose injection in syngeneic hosts (Karre et al., 1986). In a P2microglobulin (P2-m)-deficientvariant of the murine lymphoma EL4,which does not express stable MHC class I on the cell surface, a marked reduction in tumorigenicity in syngeneic C57B1/6 mice was also observed when compared to normal EL-4 cells. Transfection of a functional P2-m gene into the variant cells reestablished normal tumorigenicity. In addition, in athymic B6 nude mice, the P2-mtransfected cells were much more tumorigenic than nontransfected variant cells, whereas depletion of NK cells in these mice restored normal tumorigenicity of &-m-deficient cells (Glas et aZ., 1992).Induction of MHC class I expression can thus lead to escape from NK cells in vivo. Similarly, murine RMA-S lymphoma cells, which are defective in the peptide transporter TAP-2 gene, do not express significant levels of MHC class I and are sensitive to NK lysis. Transfection of RMA-S with the missing transporter gene led to a marked increase in tumor outgrowth potential in uivo, despite the fact that the transfected cells had restored antigen presentation and would have been expected to be more immunogenic and thus less tumorigenic (Franksson et al., 1993). The increase in tumorigenicity was shown to be due to a loss of sensitivity to NK lysis. The “missing self” hypothesis (Ljunggren and Eirre, 1990) states that somatic ceIls are permanently scanned for MHC class I expression by surveying NK cells. Cells that have lost MHC will be recognized as targets and killed by NK cells. The authors have proposed two distinct, albeit not mutually exclusive, models to explain this mechanism. According to the “target interference” model, class I MHC molecules prevent the interaction between the tentative NK target and the
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NK cells. The “effector inhibition” model postulates that the delivery of the lytic hit is blocked after interaction of NK cell and target cell and recognition of MHC class I. Thus, if class I MHC molecules are absent, NK cells kill the target. The latter model is supported by the observations that NK cells of normal mice can reject bone marrow from syngeneic mice deficient in &-m expression (Bix et al., 1991) and that normal T cell blasts from MHC class-I-deficient mice can serve as targets for syngeneic NK cells in vitro (Liao et al., 1991). Further support in favor of the “effector inhibition” model was provided by the finding that the Ly-49 molecule, which is expressed on a subset of murine NK cells, can mediate an inhibitory signal upon recognition of certain MHC class I molecules (Karlhofer et al., 1992). In addition, it has been demonstrated that proximal signaling events, such as phospholipase C-mediated hydrolysis of membrane phophoinositides or calcium signaling, are not decreased in NK cells after interaction with MHC class I molecules in transfected target cells. On the contrary, addition of anti-HLA mAb leads to increased lysis of the class I-transfected targets (Kaufman et al., 1993).These results suggest that MHC class I expression on target cells does not block the access of NK cells to their cellular target but that MHC molecules initiate delivery of inhibitory signals in the NK cells once they have bound to their putative receptors. An inverse correlation between NK susceptibility and expression of MHC class I has not, hgwever, been found ubiquitously (Gorelik et al., 1990; Ljunggren and Karre, 1990). Other factors determining NK sensitivity of a tumor must, therefore, be involved. In agreement with this hypothesis, results suggested both MHC-dependent and MHCindependent cytotoxic mechanisms, may influence NK susceptibility, depending on the nature of the target cell (Litwin et al., 1993). The question of whether MHC class I upregulation is systematically beneficial or not thus has no general answer. Opposite results have been reported with regard to tumorigenicity even in H-2-positive and H-2-negative variants ofthe same tumor if different experimental protocols are used (Ljunggren and E r r e , 1990). Most of the available data can be rationalized as follows: the effects of variation in class I expression on tumorigenicity will depend on the dominating immunosurveillance system in the model studied. If NK cells (eliminating class Ideficient cells) play the major role, upregulation of MHC class I will lead to an increase in tumorigenity of the malignant cells. If CTLs (eliminating class I-positive cells that present tumor-specific peptides) dominate, an increase of MHC class I will rather lead to enhanced immunogenicity and thus decreased tumorigenicity (Ljunggren and
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Karre, 1990).What determines which immunosurveillance system dominates is not known. It appears as if domination of NK cells is more prominent in tumors of low inherent immunogenicity, whereas in highly immunogenic tumors, CD8+-positive CTLs play the dominant role (Elliot et al., 1989).
b. Nonimmunological Effects It is generally accepted that the main importance of class I MHC molecules in oncogenesis lies in their key role in eliciting immune responses against the peptides which they present. However, a variety of nonimmunological effects by which MHC molecules may interfere with the development of tumors have also been proposed. It has been reported that the transfection of class I H-2Kb but not of class I1 H-21Akgenes into a BL6 melanoma clone led to a number of alterations in the tumor cells which were potentially irrelavant to their immunological functions. Thus, clones which expressed high levels of H-2Kbdisplayed elevated levels of soybean agglutinin (SBA), Griffonia simplicifolia I-B4 (GSIB4), and peanut agglutinin (PNA) lectin-binding sites (Gorelik et al., 1991). In parallel, these cells had lost the expression of the melanoma-associated antigen (MAA) and became sensitive to natural cell-mediated cytolysis (NCMC) and to lysis by recombinant TNF-a. Natural cytotoxicity (NC) sensitivity of these tumor cells was blocked by anti-TNF-a antibodies, suggesting that expression of the transfected H-2Kb gene resulted in increased sensitivity to TNF-a-mediated cytotoxicity (Gorelik et al., 1990). Since it seemed unlikely that TNF-a could directly recognize class I antigens, it was speculated that transfection of the H-2 gene may have an indirect effect on TNF-a sensitivity. In fact, four H-2Kb-transfected clones that subsequently lost H-2Kbexpression but retained their ability to bind to SBA agarose beads still displayed the modified phenotype described above, namely, an increase in SBA and GS1B4 lectin binding, the loss of MAA, and sensitivity to TNF lysis (Kim et al., 1993). Transfection of BL6 clones with H-2Kbhas also been reported to lead to the inhibition of the production of the endogenous A- and C-type retroviruses found in these melanoma cells and to the concomitant expression of some cellular genes that are usually repressed by these retroviruses (Gorelik et al., 1994). The phenotypic changes in the BL6 melanoma cells were probably induced by the transfection of the H2Kbgene that was later lost or downregulated during the development of the malignant clone. This assumption was based on the observation that ecotropic retrovirus production was lost because of induced stable rearrangements in proviral DNA that were sustained in the absence
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of H-2Kb gene expression (Muller et al., 1994). Electron microscopic analysis of the BL6 melanoma clones expressing transfected or endogenous H-2Kbmolecules revealed that all clones that had lost cell-surface expression of MAA had also lost budding virus particles. This loss of budding particles was associated with DNA rearrangements of proviral DNA in all samples analyzed. The postulated relationship between retrovirus expression and TNF sensitivity is supported by findings indicating that human adenovirus gene products could also control tumor cell sensitivity to TNF lysis (Chen et al., 1987; Gooding et al., 1990). The reversal of TNF resistance in H-2Kb-transfectedBL6 melanoma cells was associated with an increase in p55 TNF receptor expression and enhanced TNF internalization and degradation. TNF-induced activation of phospholipase A2 and release of arachidonic acid metabolites was observed and might have been responsible for the reversion of a block in transduction of the lytic signal present in TNF-resistant parental BL6 cells (Kim et al., 1993).
c. Effects on the Metastatic Phenotype The emergence of a metastatic phenotype is a multistep process. The tumor cell must express a variety of genes and acquire a number of specific capabilities to successfully go through the selective process that finally leads to metastasis. These include the expression of proteolytic enzymes allowing penetration through intercellular matrices and blood capillaries, the matrix-independent growth in serum, synthesis of angiogenic factors controlling metastatic spread, and many others (Hart and Easty, 1990; Liotta et al., 1991b). At each of these steps, interaction with the immune system is possible, and a number of experimental data suggest that MHC class I molecules may play an important role in the suppression of metastatic spread by immune mechanisms (Feldman and Eisenbach, 1991). The first experiments evaluating the association between metastasis and MHC expression were carried out more than 10 years ago and demonstrated a clear dependence of the metastatic properties of a tumor cell on the differential expression of H-2 genes. While some genes (H-2Kband H-2Kk)caused abolition of the metastatic phenotype, others (H-2Db)increased the metastatic competence of the cells (De Baetselier et al., 1980; Katzav et al., 1984). Later, the abrogation of metastatic properties of tumor cells by the transfection of H-2 genes was also demonstrated (Wallich et al., 1985). The expression of H2Db but not H-2Kb was found to be associated with the suppression of metastases in an allogeneic mouse model of metastasis (Lewis lung
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carcinoma), while local proliferation was unhampered by MHC expression (Isakov et al., 1983). Metastatic cells, therefore, appeared more susceptible to allograft rejection than locally proliferating cells. This might be explained by the fact that a metastasizing cell is individually exposed to CTLs, while the primary tumor grows in a bulk where individual cells are less exposed to cytotoxic cells and can survive despite the presentation ofallo-antigens. Similar studies in other allogeneic systems have confirmed the inverse correlation between metastatic phenotype and expression of allo-MHC (Gelber et al., 1989). In another syngeneic model, metastasis was inversely correlated with the level of expression of H-2Kb (Eisenbach et al., 1983,1984). The introduction of the H-2Kb gene into highly metastatic cells led to either a complete loss or a significant reduction of their metastatic competence (Plaskin et al., 1988). The suppression of the metastatic phenotype was associated with H-2Kb-controlled immunogenicity, since the H-2Kb-transfectedcells generated spontanous metastases in immune-suppressed animals, and CTLs specific for the transfected but not the parental cells appeared in immunized immunocompetent mice. The nontransfected, parental cells expressed low amounts of H-2Kb, and it proved possible to protect animals against injection of large numbers of the parental cells by preimmunization with irradiated or mitomycin C-treated H-2Kbtransfectants. In addition, the treatment of established lung metastases from parental cells with H-2Kb-expressing transfectants resulted in cure of some animals and significant tumor reduction in many others (Plaksin et al., 1988). In the B16 melanoma cell line, MHC class I expression does not appear to be an important factor in the control of the metastatic phenotype, since cells of both high and low metastatic competence express virtually no MHC molecules. Metastasis seems instead to be controlled by different levels of collagenase activity, fibrinolytic activity, and adhesiveness to lung cells (Feldman and Eisenbach, 1991). Nonetheless, transfection of syngeneic H-2Kbmolecules into cells of high metastatic competence leads to a very significant fall in the number of metastases formed and an induction of CTLs restricted by H-2Kb. These CTLs manifest high cytotoxicity not only against Kb-transfected targets but also against parental cells expressing very low amounts of Kb (Porgador et al., 1989). It was later shown that the c-fos oncogene, whose expression can be induced by IFN-7, is involved in the control of MHC expression and is differentially expressed in low versus highly metastatic clones (Kushtai et al., 1988). Accordingly, transfection of cells with c-fos
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turned on MHC expression and consequently reduced the metastatic phenotype (Kushtai et al., 1990). It may be concluded that downregulation of MHC class I expression seems to be an important mechanism by which potentially metastatic cells evade immune surveillance during the sequential steps of the metastatic process. This notion is also suworted by the fact that. in humans, metastatic tumor cells frequently express lower levels of MHC class I than their parental primary tumor cells (Cordon-Cardo et al., 1991; Pantel et al., 1991). It has to be kept in mind, however, that the loss of MHC expression was found to lead to increased tumorigenicity in several primary tumor models, as discussed above (Section IV,A). It is conceivable that in appropriate circumstances similar effects might also play a role in metastasis and thus lead to stronger metastatic competence in MHC nonexpressors, as has been observed with T10 cells transfected with the H-2Dk gene (Gopas et al., 1989). B. INTRODUCTION OF THE COSTIMULATORY MOLECULEB7 INTO TUMORS The poorly immunogenic, B7-negative, murine melanoma cell line K1735 expresses both MHC class I and class I1 molecules and stimulates a specific, albeit ineffective, immune response in uiuo. In an attempt to provide the tumor cells with costimulatory activity, K1735 was transfected with the B7 gene (Chen, L. et al., 1992; Townsend and Allison, 1993). Expression of B7 on transfected K1735 cells led to a CD8+ T-cell-mediated immune response in syngeneic mice inoculated with these cells. Tumor rejection was independent of CD4+ helper cells and immunized mice rejected transfected as well as nontransfected K1735 cells when challenged 25 days after vaccination (Townsend and Allison, 1993). In the study by Chen et al., cotransfection with B7 and a strong viral tumor rejection antigen, the E7 gene of human papilloma virus 16, was necessary to achieve successful vaccination in the same murine melanoma model (Chen, L. et al., 1992). Further work is needed to explain this discrepancy. It is interesting to note, however, that, in the experiments of Chen et al., even mice with established micrometastases of E7+B7- K1735 cells could be cured by injection of E7+B7+cells, suggesting that CTL precursors directed against the E7 antigen were not anergic, or that anergy could be reversed by the double-transfectants. In both cases, tumor growth of B7-transfected cells was unaltered in nude mice and in immunocompetent mice deprived of CD8+ cells. Since depletion of CD4+ cells did not impair the animals’ capacity to reject the tumor, it was suggested that, following B7-mediated costimu-
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lation, tumor-specific CD8' CTL clones could make enough IL-2 on their own to become fully activated and expand (Schwartz, 1992). In the two studies on K1735 and in a similar model of B7-transfected P815 mastocytoma cells, it was shown that costimulation is important during the inductive but not the effector phase of the response, since tumor-specific CTL effectors were equally effective in lysing B7transfected and nontransfected cells in uitro or in viuo (Chen L. et al., 1992; Harding and Allison, 1993; Townsend and Allison, 1993). Baskar et al., transfected the very weakly immunogenic murine sarcoma SaI with the B7 gene and achieved complete protection against high doses ofparental tumor cells in mice immunized with the transfectants. Interestingly, CD4+ effector cells were responsible for the observed tumor rejection, suggesting that, in the sarcoma model, the immunogenic tumor-specific antigens were presented by MHC class I1 rather than class I molecules (Baskar et al., 1993). Taken together, these studies demonstrate that under appropriate conditions, coexpression of B7 can stimulate both CD4+ and CD8+ T cells, thereby enhancing the tumor-specific response in both T-cell compartments.
C. ENGINEERING OF TUMOR CELLSWITH CYTOKINE GENES In murine tumor models, it is now generally accepted that the failure of an antitumor immune response is often due not to the absence of tumor specific antigens, but rather to defects in immune regulation. One goal, then, is to overcome these defects by modifying the local immunological environment of the tumor cell in such a way as to enhance the presentation of tumor-specific antigens and/or the activation of tumor-specific lymphocytes. The first successful attempts to stimulate specific effector cells in the vicinity of the tumor were performed in murine tumor models, using malignant cells engineered with the IL-2 gene (Bubenik et al., 1988,1990; Fearon et al., 1990; Gansbacher et al., 1990a; Ley et al., 1990,1991; Russel et al., 1991). In all the experimental systems, the expression of IL-2 by the weakly immunogenic tumor cells resulted in growth inhibition of the modified tumor cells. Furthermore, in several cases, a systemic immune response against the parental tumor was observed, as a consequence ofthe activation of specific T lymphocytes, leading to an immune memory against a challenge with the parental tumor cells (Fearon et aE., 1990; Ley et al., 1991; Gansbacher et al., 1990a). A parallel approach was tried with tumor cells producing IL4, another helper lymphokine that induces a local inflammatory re-
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sponse. All the animals inoculated with the IL-4-producing cells developed an antitumor response, leading to the rejection of genetically modified tumor cells (Tepper et al., 1989;Li et al., 1990; Blankenstein et al., 1990; Golumbek et aZ., 1991).Local immune reactions, evaluated by histological analysis, revealed the recruitment of activated macrophages and eosinophils at the site of tumor rejection (Tepper et al., 1992). In the latter study, the antitumor effect was not mediated by T cells and was also present in nude mice, but subsequent experiments using a renal cell carcinoma transduced with IL-4 revealed a systemic immune response generated against the parental tumor. Similar experiments were performed using IL-7-transfected plasmocytoma 5558. Tumor rejection and generation of a systemic immune response was reported only in syngeneic but not in nude mice (Hock et al., 1991; McBride et al., 1992).In this experimental system, the antitumor effect was shown to be dependent on CD4 T cells, while in other tumor models, the rejection of modified tumor cells involved mainly CD8+ and, to alesser extent, CD4+ T lymphocytes (Aokiet al., 1992).Discrepancies between the effector cell populations required for tumor rejection in the different systems may be due to heterogenous levels of class I or class I1 molecules expressed on the target tumor cells. For example, unlike most tumor models, the 5558 plasmocytoma is a class II-positive cell line that could present tumor antigens to class IIrestricted CD4' T lymphocytes. Other cytokines, such as IFN-y and GM-CSF, might locally enhance the level of antigen presentation by the tumor cells and/or antigenpresenting cells in their vicinity. In various systems, it was shown that local secretion of IFN-y by the tumor cells can protect the mice against tumor growth and induce a long-lasting specific immunity mediated by CD8+ T cells (Watanabe et al., 1989; Cansbacher et al., 1990b; Restifo et al., 1992). It was postulated that loss of tumorigenicity in animals inoculated with IFN-y-secreting tumors is due to enhanced class I expression and antigen presentation (Restifo et al., 1992). However, MHC class I expression, while necessary, was shown to be insufficient by itself to inhibit tumorigenicity, supporting the notion that additional factors are needed (Esumi et al., 1991). Indeed, IFN-y is also an activator of CTL (Hayashi et al., 1985) and NK cells (Weigert et al., 1983), and it promotes the tumoricidal activity of macrophages (Pace et aZ., 1983). More recently, the beneficial effect of another cytokine, GM-CFS, which is required for the differentiation of hematopoietic progenitors and the maturation of specialized antigenpresenting cells (Steinman, 1991),was tested in the highly tumorigenic B16 melanoma model. Transfected tumor cells producing GM-CSF are rejected in syngeneic animals by stimulating specific cytotoxic T
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cells (Dranoff et al., 1993). It was hypothesized that dendritic cells, as they exhibit increased antigen presentation and accessory functions, could be potent stimulators of helper and cytotoxic activities, thus reversing the T-cell anergy to tumor cells (Nonacs et al., 1992).Further experiments in induced tumor models as well as in spontaneous tumors will be required to compare the beneficial effect of these various cytokines in inducing tumor growth inhibition and rejection of preestablished tumors. Interestingly, it was shown that cytokines may not have to be secreted by the tumor cells themselves to deliver their beneficial effect. Using IL-2-secreting allogeneic or even xenogeneic cells as vectors, protection against tumor growth was obtained in two different experimental tumor systems (Roth et al., 1992,1994).In this situation, stimulation of nonspecific (NK) and/or specific (CTL)effector cells was shown to depend on the tumor model. While CTL were shown to be responsible for immune rejection of P815 tumor cells, NK cells were the major effector cells involved in the protective effect against Lewis tumor growth (Roth et al., 1992). Finally, in the mouse models, it appears that the immune reaction against tumor cells can be described as a two-step reaction. In the initial phase, nonspecific effector cells, such as macrophages, activated eosinophils, or neutrophils, and NK cells are recruited in the local inflammatory reaction observed (Cavallo et al., 1992; Hock et al., 1993a,b). It appears that the immune rejection of IL-4-producing transfectants involves two waves of effector cell types. In the early phase (within 18hr), macrophages and activated eosinophils are associated with the local inflammatory reaction observed and play a dominant role in the tumoricidal effect initiated by IL-4, while, in the later phase, T lymphocytes can be stimulated, depending on the inherent immunogenicity of the tumor cell line, which can then lead to systemic immunity (Tepper et al., 1992). In general, then, it appears that the first nonspecific reaction enables the secondary specific immune reaction to take place, by slowing tumor growth and enhancing the level of tumor antigen presentation by the tumor and/or specialized antigenpresenting cells, through MHC class I and class I1 molecules. In agreement with this hypothesis, it has been repeatedly observed that tumors start growing in T-cell-deficient mice only after a latency period that corresponds to the first wave of non-T effectors that slow down tumor growth (Hock et al., 1993a). In immunocompetent animals, this gives T cells the time to proliferate and differentiate into effector cytolytic cells. In the latter phase, CD8+ tumor-specific T cells can arise, leading to long-term immune protection against the parental tumor growth (Colombo et al., 1992).
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D. ESCAPE MECHANISMS In order to evaluate the potential of the above immune manipulations for future therapeutic procedures, it is important to consider some of the known immune escape mechanisms. Initially, some tumor cells enter a prolonged, quiescent state, termed tumor dormancy,” a situation in which tumor cells are present but tumor progression is not clinically apparent. While observed in various human tumors, such as lymphomas, leukemias, carcinomas, and melanomas (Stewart et al., 1991), this phenomenon has been mainly documented in the experimental model of antibody-induced dormancy (Yefenof et al., 1993a,b). In this model, the interaction of antibodies (antiidiotypes) with surface IgM (idiotypes) on BCLl tumor cells is sufficient to stop proliferation of the growing malignant cells. Strikingly, these dormant cells, whose growth was arrested, retained their malignant phenotype, since their injection into a naive animal resulted in the progressive growth of BCLl tumor cells in the adoptive recipient (George et al., 1987). Furthermore, a sporadic loss of dormancy has been observed in immune mice, and the rate of dormancy loss suggests that a single mutation event plays a critical role in the escape process (Yefenof et al., 1993b). This phenomenon is of great interest in dissecting the cellular and molecular mechanisms leading to the induction, maintenance, and termination of the dormant state in various cancers. Tumor escape may also be associated with suppressive effects mediated by CD4+ T cells. In a highly immunogenic UV-induced tumor, in vivo treatment with anti-CD4 antibodies can prevent or slow tumor growth. Suppression of CTL responses can in fact be mediated by CD4’ T cells through immunosuppressive factors such as interleukin10 secreted by the TH-2 subset, or through antibodies or other B-cell products, whose secretion requires T-cell help (Schreiber et al., Fortyfifth Annual Symposium on Fundamental Cancer Research, October 1992, Houston, Texas; Monach et al., 1993). Furthermore, escape from the immune response could result from the selection of tumor variants that are no longer recognized by the specific CTLs. This has been shown in SV40-induced tumors in mice (Lill et al., 1992). Five antigenic sites were identified on the 94-kDa large T antigen of SV40 tumor virus (Tanaka et al., 1988; Deckhut et al., 1992), and CTL-resistant variants were selected by in vitro cocultivation of an SV40-transformedmouse kidney cell line and CTL clones recognizing the various epitopes. Identification of variants lacking sites I, I1 and I11 coding sequences revealed that point mutations ‘I
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were responsible for the loss of the relevant CTL recognition epitope. These results reinforce the view that minimal genetic changes can generate new epitopes that still bind to MHC class I molecule but have lost the CTL recognition sites from tumor antigens, allowing transformed cells to escape immunosurveillance. Finally, an important observation has been made recently. In tumorbearing mice as well as in a number of cancer patients (Mizoguchi et al., 1992; Finke et al., 1993; Nakagoni et al., 1993) CD8' T cells which infiltrate the tumors show a defect in signal transduction, associated with alterations in the structure of the CD3 complex and more particularly of its 5 chain. This might be a major cause for immune defects in tumor-bearing hosts. Whether or not such modifications of the CD3 complex are induced by some diffusable immunosuppressive factors produced by the tumor cells remains opened to investigation. V. Conclusion
We have reviewed here a number of aspects dealing with immune responses directed against and stimulated by tumor cells. As mentioned in the Introduction, the antigenic and immunogenic properties of tumors are intimately related, inasmuch as everything learned about tumor cells is very rapidly applied to fight them. We have certainly not covered the entire field. In particular, there are many other ways to try and overcome the poor immunogenicity of tumor cells than described here, especially with respect to modifications of antigenpresenting cells (as opposed to the tumor cells) and to the use of various gene therapy protocols. The results obtained in animal models are indeed extremely encouraging, but the growing enthusiasm about the applicability of in vivo or ex vivo immunotherapy protocols in humans should be tempered for a variety of reasons. One is that there are very large gaps in our basic knowledge and much remains to be learned about the antigenicity and the immunogenicity of tumor cells. A second reason is that oncologists will in general agree that there are no really accurate animal models for human cancers, and that work performed in the mouse, while necessary and informative, may not yield results directly transposable to man. In this context, it is worthwhile mentionning that preclinical work performed with spontaneous tumors in domestic animals might be extremely useful in approaching the high complexity of the situations which must be faced in human beings. A third reason pertains to the clinical trials themselves: first, a very large number of trials are going to be necessary in order to evaluate the large number of new
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procedures (and of combinations of them) which are being suggested for immune therapy. Second, many such trials will be performed first with patients with advanced diseases, where they are less likely to be efficient. Confidence will have to be gradually gained in a selected number of procedures to permit more extensive and, perhaps, more significant trials to be performed. Nevertheless, it is a fair assessment of the current situation that the impressive progess made in immunology and basic tumor immunology is likely to prove useful in the relatively near future.
ACKNOWLEDGMENTS The authors thank many colleagues who have sent us preprints and unpublished information. We acknowledge Drs. J.-P. Abastado, R. Cohen, J. Even, and D. Ojcius for fruitful discussions. We are especially indebted to Dr. D. Ojcius for critical reading of the review and to Ms. V. Caput for editorial assistance in the preparation of the manuscript.
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ADVANCES IN IMMUNOLOGY, VOL. 57
Formation of the Chicken B-Cell Repertoire: Ontogenesis, Regulation of Ig Gene Rearrangement, and Diversification by Gene Conversion CLAUDE-AGNES INSERM U373, In&t
REYNAUD, BARBARA BERTOCCI, AURIEL DAHAN, AND JEAN-CLAUDE WEILL Nockor, 156 we de Vaugimd, 75730 Pans Codex 05, Fmnco
1. Introduction
Past years have seen the description of immune systems from several species, for which the mechanism of B-cell repertoire formation differs fundamentally from what has been established for the mouse. In the mouse, rearrangement of Ig genes takes place continuously in the bone marrow during the lifelong differentiation of B cells from uncommitted progenitors. It provides the opportunity for random assortment of V-encoding elements (V, D, and J) and for the linked junctional diversification processes in each newly formed B cell (Tonegawa, 1983; Alt et al., 1987). In chickens, sheep, and rabbits, Ig gene rearrangement is not the key event for Ig diversity: postrearrangement diversification processes taking place during an early phase of B-cell amplification generate the B-cell repertoire in these three species. The molecular mechanisms differ however among them: in the chicken, gene conversion diversifies a unique rearranged gene at both heavy- and light-chain loci by recombination with a pool a pseudogene elements (Reynaud et al., 1985,1987,1989);in the sheep, several functional light-chain V genes undergo extensive modification by untemplated somatic mutations (Reynaud et al., 1991a); for the rabbit heavy chain, a major rearranged gene undergoes gene conversion, with possibly extensive somatic mutation of the D region (Becker and Knight, 1990; discussed by Knight and colleagues in the previous volume). These postrearrangement diversification processes take place in primary lymphoid organs in which considerable B-cell proliferation occurs and allow modifications to accumulate as further cell division proceeds: the bursa of Fabricius has been known for a long time to be the primary site of B-cell formation in the chicken; in sheep, ileal Peyer’s patches fulfill a similar function (Reynolds and Morris, 1983), the bone marrow having no B lymphopoietic activity in these two species. The question turns out to be more complex in the rabbit; 353
Copyright 8 1994 by Academic Press, Inc
All rights of reproduction In any form reserved
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whether gut-associated lymphoid tissues (i.e., appendix and sacculus rotondus) are sites where such B-cell diversification takes place, as originally proposed by Archer et al. (1963),and whether this is superimposed to a mouse-type bone marrow lymphopoiesis are still open issues. Among these three species, the chicken immune system is so far the model for which the more complete description has been achieved since the initial description of the bursa-derived B-cell lineage (Cooper et al., 1965).We will summarize the actual knowledge of the formation of the B-cell repertoire, including data on early B-cell commitment and regulation of rearrangement obtained with chicken substrates in transgenic mice. II. Organization of Ig Genes in the Chicken
A. LIGHTAND HEAVY-CHAIN LOCI Both heavy- and light-chain loci share a similar gene organization with unique functional V and J elements, indicative of a striking coevolution (Fig. 1). For the light-chain locus, which is a A isotype, the unique Vhl gene is 1.8 kb upstream of a single J-C unit. This V gene has all the characteristics (promoter with octamer sequence, leader peptide, leader intron, recombination signals) of mouse V elements. Upstream of Vhl are 25 V pseudogenes clustered in 19 kb of DNA with alternate polarities (16 with the same polarity as Vhl and 9 with a reverse orientation) (Reynaud et al., 1985,1987). Most of these pseudogenes (but not all) lack recombination signal sequences. The homology with the VX1 gene never extends farther 5' than approximately 40 bp in the leader intron. None of them have upstream regulatory elements, i.e., transcription signals and leader sequence, some of them being even truncated genes with only part of the V coding sequence. For the heavy chain, the unique vH1 and J H elements are 15 kb apart, and JH is approximately 15 kb upstream of the C p gene. In between vH1 and JH are 16 D elements (Reynaud et d., 1989,1991b). A pool of VHpseudogenes, larger than that for the light-chain locus, has been analyzed but not completely sequenced; it covers 60-80 kb of DNA, with an average spacing of V elements of 0.8 kb (i.e., a total of 80 to 100 pseudogenes). The alternance of polarities is quasisystematic. Like that for Vh pseudogenes, homology with vH1 is restricted in 5' to ca. 80 bp in the leader intron, with no leader sequence nor
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FIG.1. Organization of the chicken light- and heavy-chain loci. The chicken light-chain locus contains a single functional V gene (VAl), a single J-C unit, and a cluster of 25 pseudogenes (Reynaud et al., 1985,1987).A twenty-sixth JIVA element, located between JIV7 and JIVS, has been reported by Kondo et al. (1993) in the chicken H-B15 strain. The heavy-chain locus contains single Cp, JH, and functional VH ( v H 1 ) elements, a cluster of 16 D, and a group of pseudogenes (80-100 JIV, in 60-80 kb of DNA) (Reynaud et d., 1989,199113).Horizontal arrows indicate transcriptional polarities.
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CLAUDE-AGNl?S REYNAUD ET AL.
transcription signals. In 3’, they have no recombination signals, but surprisingly a “D-like” segment fused to the V element; this D-like sequence is even terminated in some pseudogenes by a few nucleotide homology with JH,which has functional relevance to the diversification process (see below). The presence of leader intronic sequence in some of these pseudogenes argues against a processed gene origin for these fused V-D structures. B. DELEMENTS D elements are the only functional V-encoding elements present in multiple copies in the chicken genome. Sixteen D elements exist, with 15 of them being extremely homologous (some even in several identical copies) and 1 being rather different (Dx) (Fig. 2); this last one is however poorly functional, since it has a low rearrangement frequency and is selectively counterselected during B-cell expansion in the bursa (see below) (Reynaud et al., 1991b). All D elements encode an exact number of amino acids in reading frame 1: mostly 10, but also 9 or 8. The amino acid composition of the three reading frames is spectacularly different: Gly-Ser-Ala-Tyr-Cys, i.e., hydrophilic and aromatic residues, in reading frame 1; hydrophobic amino acids (Leu, Val) in reading frame 2; and one or two stop codons in reading frame 3 (Table I). This amino acid composition resembles that of the mouse Dfl16 and Dsp families, and a bias for usage of reading frame 1 is observed in the chicken as in the mouse (cf. section 1II.C). The germline-encoded D elements are only minor contributors to the overall heavy-chain repertoire, which results mainly from gene conversion (cf. 1I.A). What is thus the function of this pool of genomic D elements? Its main contribution could be the formation of D-D junctions: they amount to 25% among D-J alleles and are still maintained around 15% in “functional” VDJ sequences, indicating that such large CDR3 domains are not detrimental for the chicken Ig molecules.’ This indicates also that no specific length matching is required between heavy and light chains in the chicken, the heavy-chain CDRS varying between 15 and 30 amino acids, whereas light-chain CDRS length heterogeneity is only moderate (McCormack et aE., 1989a; Considering a homology-mediatedjunction leading to 50% D-D joining in reading frame 1 and selection of D reading frame 1 in functional VDJ sequences (Reynaud et al., 1991b), the 25%D-D junctions among V-D-J alleles are expected to result after selection in 12.5%V-D-D-J sequences with both D’s in reading frame 1 (see section 1II.C and Table 111).
D6
GGT AGT GGT TAC TGT GGT AGT GGT GCT TAT
D1 D2 D3 D4 D5 D7 D8 D9 D10 D11 D12 D13 D14 D15
GG GGT GGT GGT GGT CGT CGT GGT GGT GGT GGT GGT GGT GGT
FAGT GCT TAC GGT AGT TGT TGT
_ _ _ _ _ _A-_ _ - -
( )
GGT GCT TAT
GCT ( ) ( ) GGT CCT TAT AGT GCT TAC TGT TGT AGT GGT GCT TAT
AGT AGT AGC AGT AGT AGT AGT AGT AGT AGT AGT
GCT TAC TGT ( ) GCT TAC TGT GGT GCT TAC TGT ( ) GCT TAC TGT ( ) GGT TAC TGT GGT GGT TAC TGT GGT GCT TAC TGT ( ) GGT TAC TGT GGT GGT TAC TGT GGT GGT TAC TGT GGT GGT TAC TGT GGT
TGG GAT AGT GGT TGG TAT TGG GAT AGT GCT TGG GGT TGG GAT AGT GCT AGT GCT TGG AGT AGT GGT
GCT GAT GCT TAT GCT GAT GCT GAT GCT TAT GCT TAT GCT GAT GCT TAT GCT TAT GCT TAT GCT GAT
Dx
FIG.2. Genomic D sequences. Genomic D sequences are shown, including recombination signal sequences (Reynaud et al., 1991b). D6 has been chosen arbitrarily to be compared to, with dashes for nucleotide identity and brackets and mows for, respectively, gaps and insertions introduced to maximize homology. The D coding part is written in a triplet mode in reading frame 1 and indicated in full to facilitate comparison. Heptamer-nonamer signal sequences are boxed.
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TABLE I THETHREEREADINGFRAMES OF THE CHICKEN D ELEMENTS HAVEA MARKEDLY DIFFERENT AMINOACID COMPOSITION’ ~~
Frame 1
I
D1 D2 D3 D5 D6 D9/12/13 D10 D14 D15 D4/8/11 D7
GSAYGC-GAY GSA-CC-GPY GSAYCCSGAY GSAYCGSGAY GSGYCGSGAY GSGYCGSAAY GSGYCGWGAY GSGYCGWSAY GSGYCGSGAD GSAYC-h”3 GSAYC-WAD
consensus
G S F F D
Frame 2
Frame 3 L*CLRLFFCL *CL-w-SL *CLLL*WcL *CLLw*WcL *wLIw*WcL *W*CCL *wwJLGcL *wLLWLECL *W*Wc* *CLL-LGC* *W-LVC*
a The amino acids encoded by the three reading frames of the D elements are shown in the one-letter code with asterisks for stop codons, dashes for gaps, and arrows for insertions introduced as in Fig. 2. Frames 1, 2, and 3 refer to sequences beginning at the first, second, and third nucleotide of the D’s, respectively. A consensus sequence is shown for the reading frame 1 (Xstanding for a nonconsewed residue).
Reynaud et al., 1991b). Surprisingly, this D-D joining requires rearrangement between two signals with the same 12-bp spacing, a mechanism that would be expected from model systems to be extremely inefficient. 111. Generation of the Chicken B-Cell Repertoire by Gene Conversion
A. A HYPERCONVERSION MECHANISMDIVERSI~~ES THE UNIQUE FUNCTIONAL VL AND VH GENES The initial observation that most chicken B cells harbor the same Vh gene rearrangement made the question of the origin of the Ig repertoire in this species an obvious one. Light-chain cDNA sequences isolated from splenic cells displayed striking blocks of homologies with the first three pseudogenes analyzed, which led us to propose a gene conversion mechanism between V elements (Reynaud et al., 1985), analogous to the recombination models proposed 25 years ago to explain the generation of diversity, models that turned out to be irrelevant to the mouse immune system (Smithies, 1967; Edelman and Gally, 1967).
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The systematic analysis of rearranged genes during bursal development together with the complete sequence of the V-encoding elements of the locus confirmed that a gene conversion-like mechanism was responsible for generating diversity in this species (Reynaud et al., 1987). The recombination process transfers in a nonreciprocal manner blocks of sequence from the pseudogene pool acting as donor into the unique functional gene acting as acceptor. Ongoing diversification of the V h l gene was also demonstrated by Thompson and Neiman (1987), using restriction sites located in the V sequence. The number of converted segments increases with time, from an average of one to three at Day 18 of embryonic development to four to six at 3 weeks after hatching (Fig. 3); this figure represents an underestimate since conversion tracks may superimpose on each other. Bursa1 cells divide every 8-10 hr during the embryonic period, and a high division rate is probably maintained after hatching. A frequency of one gene conversion event per V sequence every 10-20 cell divisions can thus be estimated (Reynaud et al., 1987).
I
I I I
I
I
I
I
1
I I
I I
I
I
I
I I
I
I
I
I
I
I
I
I
I
I
FIG.3. Diversification of bursal VX1 sequences through multiple gene conversion events. Six rearranged bursal V h l sequences isolated from either Day 18 embryos or a 3-week-old chicken are compared to the original VX1 sequence (Reynaud et al., 1987). The various parts of the variable region are delineated: leader (L), framework (FR), complementarity-determining regions (CDR), and J segment ( J). Domains in white indicate regions of nonmodified VX1 sequence; domains in gray represent approximate borders of gene-converted segments, with the name of the putative pseudogene donor (several numbers in a box refer to multiple equivalent donors). Dots represent single untemplated nucleotide modifications.
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CLAUDE-AGNES AEYNAUD ET AL.
The length and position of converted segments vary widely. Length can be from 10 to more than 100 bp (a 250-bp event was reported) (Reynaud et al., 1987; McCormack and Thompson, 1990). No precise borders are observed for the conversion tracks that affect all parts of the V sequence including the D region. D reading frame 1 is already largely selected among bursa1 VDJ sequences when incidence of gene conversion can be monitored (cf. section 111.C);the D-like sequence is fused to the $VH elements in reading frame 1 and therefore does not modify the overall reading frame of the Ig molecule when it is transferred by gene conversion. Due to the particular structure of several $VH and $Vh elements that end in 3’ with a few nucleotide homology with the J H and Jh segments, gene conversion is moreover able to extend over the V-J (V-D-J) junction. This particular feature of the gene conversion process probably dictates the particular Ig gene configuration of chicken B cells: only one allele is rearranged for both heavy- and light-chain loci, a configuration which avoids the presence of abortive rearrangements for the other allele that would be susceptible to being put back in frame by gene conversion and would result in allelic inclusion of the cell. This point, originally raised by M. Cohn, is discussed at length in a Forum in Immunology (Langman and Cohn, 1993). Differential pseudogene usage has been observed (Reynaud et aE., 1987; McCormack and Thompson, 1990): proximal genes were used more frequently, and the pseudogenes with higher homology to V h l were similarly favored. This homology-biased exchange may vary with further diversification of the V h l gene, possibly restricting pseudogene usage. In the DT40 cell line which undergoes gene conversion in culture (cf section IIB), the very particular CDRl structure of the Vh1 gene (with $VS sequence) biases further gene conversion toward the use of the homologous $Vl8 gene (Buerstedde et al., 1990; Kim et al., 1990). Such sequential pseudogene usage has been described during the gene conversion process leading to surface antigen variation in the trypanosome (Roth et aZ., 1986). The third type of pseudogene usage preference concerns their orientation, the ones with an inverted polarity compared to V h l being more frequent donors (McCormack and Thompson, 1990). By studying cell lines with restriction enzyme polymorphism in the $V cluster, Carlson et aZ. (1990) have brought evidence that the mechanism used was indeed intrachromosomal gene conversion, i.e., a mechanism operating in cis with nonreciprocal exchange of DNA. McCormack and Thompson (1990) observed furthermore that, on the average, a longer stretch of homology flanks gene conversion tracks
CHICKEN B-CELL REPERTOIRE FORMATION
36 1
in 5’ than in 3‘ (referred to V coding sequence), which led them to propose a polarity in the sequence transfer mechanism with initiation on the 5’ side. Untemplated mutations are also observed (8 among 214 mutations collected from bursal sequences before polymerase chain reaction times (Reynaud et aZ., 1987)) frequently located at the border of a conversion event. Such mutations might be intrinsincally linked to the recombination process. The occurrence of further somatic mutation in peripheral B cells has been suggested by Parvari et al. (1990). However, a systematic study of somatic mutations of IgG versus IgM molecules during the course of an immune response has not been performed so far. B. DT40: AN ALV-INDUCED CELLLINEUNDERGOING IG GENE CONVERSION CULTURE Several transformed cell lines from bursal origin have been tested for their capacity to undergo gene conversion of their Ig gene in culture. Bursa1 cells immortalized in uitro by the REV-T virus (reticuloendotheliovirus) have no gene conversion activity in culture (Barth and Humphries, 1988). Avian leukosis virus (ALV)-induced tumors clonal for the virus integration site have heterogeneous diversified lightchain sequences, indicating that gene conversion was active during tumor cell proliferation in the animal, irrespective of the anatomical localization of the tumor; however, among those that have been established as cell lines in uitro, only one (DT40) has shown ongoing gene conversion activity in culture (Thompson and Neiman, 1987; Buerstedde et al., 1990; Kim et al., 1990). There are however major differences in the gene conversion activity of this cell line compared to that of normal bursal cells. The rate of gene conversion appears to be much slower, with small changes of one or two nucleotides being the most frequent event. It is unclear so far whether this represents a trend specific to the DT40 toward recombination between regions of high homology (i.e., exchanging few nucleotides) or whether such small changes are an integral part of the normal process that is wiped out by longer conversion tracks occurring at higher frequency in the chicken bursa. Spontaneous IgM-negative variants of the DT40 cell line were analyzed to characterize possible errors in the gene conversion mechanism; such variants occurred at a low frequency (less than 1%of the total population) and were due to a frameshift mutation in the V h l sequence, thus indicating a low error rate in the gene conversion process. Such negative variants could give rise upon culture to IgM-
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positive subclones, the frameshift mutation being corrected by a superimposed gene conversion (Buerstedde et al., 1990). It is unlikely that such a correction process plays a major role in the bursa, the lifespan of B cells with nonfunctional IgM being probably too short for them to be rescued. A high frequency of gene targeting is obtained upon transfection of the DT40 cell line (Buerstedde and Takeda, 1991). This feature is however shared by other chicken B-cell lines, whether gene conversion active or not, and is not specific for the Ig loci; it might thus reflect some general recombination activity that can be dissociated from the gene conversion process. This observation allows nevertheless specific genes involved in the gene conversion machinery to be sought by knocking them out on both alleles in the DT40 cell line: such experiments have already suggested that the high RAG-2 gene expression observed in the bursa was unnecessary for the maintenance of the gene conversion activity (see below) (Takeda et al., 1992). C. RECOMBINATION MODELSFOR IG GENECONVERSION IN THE CHICKEN: Is THERE FORMATION OF HOLLIDAY JUNCTIONS? Models of gene conversion involve the formation of DNA single- or double-strand breaks as initiator sites ofrecombination, strand displacement, gap repair, and resolution of crossed DNA strands (Holliday junctions). The closest related model is the yeast-mating type switch: this event consists of a programmed gene conversion whereby sequences at the MAT (mating-type specific) locus are replaced using either of two possible donors, which can occur as frequently as once per generation. The whole process is induced by a double-strand break created by the site specific HO endonuclease that results in an exchange of ca. 700 bp. The directionality of the exchange is ensured through the silencing of the donor genes that prevents recognition and cutting by the HO endonuclease (reviewed by Haber, 1992). No such DNA breaks have been reported so far for chicken V genes in the developing bursa (Thompson, 1992),but we are still investigating the question using more sensitive techniques. Heptamer sequences specific for the rearrangement process exist in two locations within the V h l gene (and in the VH1 and half of the D elements as well). Although it was tempting to propose that they could be recognized by some lymphoid-specific, heptamer-nicking activity (Reynaud et al., 1987), it was nevertheless difficult to envision how so few entry sites could generate gene conversion tracks with such diverse end points. This proposal was raised again by Carlson et al. (1991), who observed high RAG-2 gene expression in the bursa and proposed that RAG-2 might represent this heptamer-nicking activity. Correlation of
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RAG-2 expression with gene conversion was however questioned due to the observation that disruption of both RAG-2 alleles in the DT40 cell line did not modify its gene conversion activity (Takeda et al., 1992). The role of RAG-2 in the bursa and the existence of specific DNA breaks promoting gene conversion in the V sequence are thus open issues. McCormack and Thompson (1990)proposed a model of recombination to account for their observation ofpolarity in the conversion tracks, which involves strand displacement following the direction of transcription that would be resolved by strand dissociation. In their model, the authors exclude the formation of Holliday junctions, arguing that no crossing over associated with their resolution has been reported. Such structures however would result in nonfunctional V sequences that would be rapidly eliminated in the bursa; crossing over in a gene conversion involving a pseudogene donor with the same orientation as VA1 would result in a fused 5’ ($V-Vhl-J) 3‘ sequence with excision of the DNA sequence in between (Fig. 4a); when involving a pseudogene donor with an opposite polarity, crossing over then results in an inversion that would produce again similar nonfunctional hybrid $V-Vhl structures (Fig. 4b). Only in the DT40 cell line, whose proliferation does not depend upon a functional Ig molecule, would it be possible to observe them. It is nevertheless anticipated that associated crossing overs are rare events in such intrachromosomal gene conversions, and this is also the case in the mating-type switch (Klar and Strathern, 1984; Ray et al., 1988). Kondo et al. (1993)have described circular DNA structures isolated from the bursa. Two circular DNA with an “abnormal” V gene configuration were reported, both containing a hybrid V gene formed by the 5’ end of the VA1 gene and the 3‘ end of a pseudogene having the same polarity as VX1. Such structures represent strikingly the DNA product expected from a crossing over associated with a gene conversion event (cf. Fig. 4a), although obviously other explanations exist for their formation. Whether such rare structures reflect the normal pathway of gene conversion, i.e., resolution of Holliday junctions, with few percent of associated crossing over due to topological constraints or whether they are accidental, atypical structures remains to be determined. If a systematic link could be established between the presence of such hybrid V genes in circular DNA and the gene conversion activity of a cell, their detection could represent a “gene conversion assay,” analogous to the gene rearrangement assay constituted by the detection of the circular DNA region excised upon rearrangement (McCormack et al., 1989a). When considering an activity or a signal that would activate the
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L Vhl J
wV6
nrft t
b
ww
L Vhl J
t
wEIl-L Vhl J
t
t
6@ I I Vh1 J
-BEG+
Q ~
L Vhl lwV6
FIG.4. Bursa1 circular DNAs with hybrid V genes: Possible products of the Resolution of HoIliday Junctions? The DNA products expected from a crossing-over associated with the resolution of a Holliday junction are depicted, the gene conversion event initiating the strand exchange not being represented. Two possible cases exist, depending upon the orientation of the pseudogene participating in the gene conversion: (a) when a pseudogene with the same orientation as the VAl gene is involved, excision of a circular DNA molecule occurs; (b)with a +V in the reverse orientation, an inversion occurs. Circular DNAs containing hybrid 5' VA1/3' +V sequences as described in (a) have been isolated from bursa1 cells by Kondo et al. (1993).
gene conversion program in chicken B cells, it is striking that the same question pertains both to gene conversion of V genes during chicken B cell development and to somatic mutation of V genes during the secondary immune response. Both" are basic, nonspecific mechanisms, involved in the evolution of genes, that become specifically activated at a high rate at a precise developmental stage and are targeted exclusively to both VH and VL genes without affecting other genomic sites.
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How the specific recognition of the V sequence is achieved remains the key question in both of these processes. IV. Chicken B-Cell Development
A. N o ONGOING IG GENEREARRANGEMENT IS OCCURRING IN THE CHICKEN BURSA The bursa of Fabricius is the primary organ of B-cell formation in the chicken; bursectomy in the embryo results in a severe agammaglobulinemia, a discovery that allowed the B (bursa-derived)-cell lineage to be identified (Glick et al., 1956; Mueller et al., 1959; Cooper et al., 1965,1969; Warner et al., 1969). The stem cells (prebursal stem cells) that give rise to B lymphocytes colonize the bursa from the general circulation between Days 8 and 14 of embryonic development (Moore and Owen, 1967; Le Douarin et al., 1975; Houssaint et al., 1976).When they reach the bursal epithelium, they induce the formation of lymphoid follicles and, as bursal stem cells, differentiate into B lymphocytes. The bursa is composed of lo4 follicles, colonized by a few prebursal stem cells (two to three on average), these lymphoid follicles reaching a size of 2-5 x lo5cells at maximal bursal size. B cells start to migrate out of the bursa at Day 18 of embryonic development, and install the peripheral lymphoid compartment as postbursal stem cells. Three weeks after hatching, removal of the bursa has no incidence on the further development of the B-cell repertoire and by 6 months of age, the bursa has completely involuted (reviewed by Pink, 1986). The terms prebursal, bursal, and postbursal stem cells were coined by Toivanen and Toivanen (1973);they refer not simply to their successive localization during the development of the animal, but rather to the fact that they either need or do not need to migrate in the bursa to restore the B-cell compartment of a depleted host in adoptive cell transfer experiments. However, despite this terminology, it is still totally unclear for the moment whether there exists a cell population with true “stem cell” characteristics. It remains equally possible that every chicken B cell at both bursal and postbursal stages is endowed with a large proliferative capacity, the B-cell population ensuring as a whole its long-term maintenance. When first described, B-cell development in the bursa appeared as a typical instructive model, wherein a multipotent progenitor would be driven into the lymphoid pathway through the induction of a specific microenvironment. One would thus expect Ig gene rearrangements to
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be constantly produced in each newly formed B cell. This turned out to be doubly wrong; first, stem cells migrating into the bursa are already committed to the B-cell lineage (as discussed in the following section); second, Ig gene rearrangement is a unique event, occurring during a short time and does not take place continuously during the bursal phase of B-cell development. The study by Pink and his colleagues established at first that the bursal stem cell, responsible for restoring the immune capacity of a B-cell-depleted host, was a surface IgM-positive cell (Pink et at., 1985). Accordingly, long-lasting allotype suppression could be achieved by a single anti-IgM anti-allotype injection at Day 13 of development, a situation in striking contrast to the one in the mouse (Ratcliffe and Ivanyi, 1981). Study of Ig gene configuration in individual lymphoid follicles established that rearrangement is not an ongoing event in the bursa; using a restriction enzyme polymorphism at the light-chain locus, individual follicles were shown to be oligoclonal for rearrangement, the number of rearrangement events (two to three on average) corresponding to the number of progenitors that colonized each follicle (Weill et al., 1986). A similar conclusion was reached from the analysis of IgM allotype distribution in bursal follicles (Ratcliffe et al., 1986). IgMcommitted stem cells are thus formed at the very beginning of the colonization process, or even slightly before. This last point was confirmed using systematic polymerase chain reaction (PCR) analysis of Ig gene rearrangement during early development (cf. section 111,B). More recently, the issue of ongoing rearrangement in the bursa was addressed by McCormack et al. (1989a), who monitored by PCR the formation of the circular DNA excised during light-chain rearrangement. This elegant “rearrangement assay,” which is of general relevance for rearrangement of V elements with the same polarity, failed to detect such circular structures at late embryonic and posthatching stages, in agreement with previous conclusions. Analysis of Ig gene rearrangement in the total bursa and in individual follicles has revealed an Ig gene configuration particular to chicken B cells: only one allele is rearranged for both heavy- and light-chain loci; the other allele remains in germline configuration for the light chain and mostly DJ for the heavy chain (with even 5% of cells with a heavy-chain locus in germline configuration). About 1-2% cells were estimated to have both alleles rearranged. A concordant, but slightly higher value was reported from analysis of chicken B-cell lines (6% of cell lines with both light-chain alleles rearranged) (Reynaud et al., 1985,1989;Weill et al., 1986; McCormack et al., 1989b). We proposed
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that this particular feature is caused by rearrangement being attempted only once on a chromosome, the vast majority of cells with unproductive rearrangements being simply discarded (Reynaud et al., 1987). Such a restricted rearrangement would be achieved through a short window of time during which access to the genes would be allowed. A possible regulation for this phenomenon is described in section 1II.D. The most striking feature of B-cell development in the chicken is thus the existence of a unique “wave” of Ig gene rearrangement in B-cell progenitors. The entire B-cell compartment is thus derived from the 2-3 x lo4 productively rearranged bursal stem cells that develop within bursal follicles. Contrary to previous expectations, the main function of the bursa therefore appears to be the expansion and the diversification of the B-cell population (Weill and Reynaud, 1987; Pink and Lassila, 1987). B. EARLY B-CELLCOMMITMENT IN THE DEVELOPING CHICKEN EMBRYO As discussed in the previous section, committed B-cell progenitors have a unique phenotype in the chicken, having their Ig genes in a rearranged configuration and presenting a surface IgM molecule. This unique property allows us to ask where and when these progenitors emerge during embryonic development by following at the singlecell level the rearrangement pattern of embryonic cells. This can be done at all stages of development, taking advantage of the easy access to the embryo; the simplicity of chicken Ig loci with single VH,V,, and J elements; and the use of the PCR technique allowing the detection of one rearrangement event in lo5 cells (Reynaud et al., 1992a). Based on these and on previous results (Moore and Owen, 1967; Dieterlen-Lihvre and Martin, 1981; Lassila et al., 1978,1982;Ratcliffe et al., 1986; Houssaint et al., 1991),one can recapitulate the development of the B-cell system in the early chicken embryo (Fig. 5).Hematopoietic precursors can first be detected in the intraembryonic paraaortic region at Day 3-4. These progenitors then migrate to the yolk sac at Day 5-6 at which stage the specific B-cell progenitors start to perform D to JH rearrangement, thus segregating from the other hematopoietic lineages (chicken T cells as opposed to mouse T cells do not show any DJH rearrangement). This DJH rearrangement could be triggered either by the yolk sac environment or by the removal of a differentiation block as cells migrate out from intraembryonic sites. DJH rearrangement goes on as B-cell progenitors circulate back to the embryo and seed the various lymphoid organs. One or two days later, whether
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I "
\ DJ+ day 10
t J
FIG.5. A proposed scheme for the emergence of B-cell progenitors in the chicken embryo (Reynaud et al., 1992a). Hematopoietic progenitors are first detected within the embryo in the paraaortic region (Lassila et al., 1978; Dieterlen-LiBvre and Martin, 1981); they further migrate through the yolk sac, where DJH-committed progenitors start to segregate from the other cell lineages and seed via the general circulation the various lymphoid organs. These progenitor populations regress thereafter in spleen and bone marrow (marked with a t symbol) and only expand in the bursa ( t t t ) (cf. Table 11). The first day of detection of DJH rearrangement is indicated for each compartment.
in the blood or in a lymphoid organ, they start to rearrange their heavyand light-chain V genes, this event being most probably part of an intrinsic cellular program rather than being induced in any particular site. Once in spleen or in bone marrow the B-cell progenitors, whether they have rearranged their Ig genes productively or not, cease to divide and slowly disappear. To support this view we have shown that there is neither selection for functional sequences in the spleen nor B-cell proliferation (as monitored by the ratio of rearranged cells to light chain circular DNA excision product) (Reynaud et al., 1991b71992a). When put in quantitative terms, the picture indeed favors a role of the general circulation in distributing DJH progenitors to the various organs (Table 11);approximately 1000 DJH progenitors are present at Day 8 in the yolk sac, and then DJHprogenitors dominate in the blood at Day 10 (125,000-250,000 cells). This population remains very high in blood at Day 13 as well as in the spleen and bone marrow
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TABLE I1 NUMBER OF DJH-COMMITTED PROGENITORS IN VARIOUS EMBRYONIC COMPARTMENTS“ Days of Development: Yolk sac
Blood
Spleen Bone marrow Bursa Thymus
Number of DJH Progenitors Day 8
1000-2000
Day 10
Day 13
Day 17
125,OOO-250,000 2,000-5,000 10-20 50-200 100
250.000-500,000 500,000 100,OOO 30,000 400-1,000
500-1,OOO 20,000-40,OOO 2,000-10,000 > 1-2 x 106 1,6bO-4,000
a The total number of DJH-committedprogenitors in various compartments at four developmental stages was estimated from a quantitative PCR analysis reported to the size of each organ at each stage (Reynaud et al., 1992a). Figures underlined represent the major DJH progenitor compartment(s) at a given stage.
(100,000-500,000 cells), while it starts to accumulate in the bursa. At Day 17 these populations have declined in blood, spleen, and bone marrow but expand strikingly in the bursa. Our data do not disagree with previous reports showing commitment of B-cell progenitors in nonbursal sites, these cell populations being able moreover to restore a B-cell-depleted animal in cell transferexperiments (Ratcliffe et al., 1986; Houssaint et al., 1989,1991).We estimate however that such sites represent dead ends rather than functional intermediates for further bursal development. A large excess of cells (1-2 x lo6)is thus engaged in the B-lymphoid lineage, compared to the approximately 2-3 X 104 productively rearranged committed B-cell progenitors that are needed to generate the B-cell compartment in the bursa. IG SEQUENCES ARE SELECTED DURING B-CELL C. FUNCTIONAL PROLIFERATION I N THE BURSA After bursal colonization by B-cell progenitors, there is a defined period (Day 10-18) during which selection for “functional” Ig sequences takes place, before and independent of the occurrence of gene conversion; this selection bears on both in-frame sequences and on the D reading frame and can be easily monitored on the total bursal population since only one Ig allele is rearranged per cell (McCormack et al., 1989b; Reynaud et al., 1991b). Heavy- and light-chain gene rearrangement in the bursa, analyzed at the time of DJ progenitor colonization (Day 10-12), represents the
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outcome of the joining process with characteristics similar to those described for the mouse fetal repertoire: absence of N additions, biased joining at both V-D and D- J junctions due to terminal homology, and favoring the D reading frame 1; 60% of VD and 50% of DJ result in D reading frame 1, which makes about 113 of “functional” VDJ sequences (i.e., in frame with a D in reading frame l), as opposed to 119 expected from random joining (Table 111).The proportion of the three reading frames on the DJ allele does not change with time, which suggests that the selection for the D reading frame is produced on the assembled VDJ structure presented at the cell surface (Reynaud et al., 1991b). For the light chain, the joining appears to be roughly random (McCormack et al., 1989b). A total of 80-90% of Ig sequences is already in frame (with a D in reading frame 1)at Day 15, and this amounts to more than 95% at Day 18 of development (Table 111). What is thus the structure signaling for cell proliferation and selecting for “functional” Ig molecules? It has been proposed that the germline-encoded specificity (i.e., the VHl-VA1 pair) is recognizing some bursal determinant that would induce further development (Reynaud et al., 1989; McCormack et al., 1989b; see also the discussion in Langman and Cohn, 1993). Alternatively, the mere presence of an IgM molecule at the cell surface could be signaling for proliferation and would result in selection of in-frame sequences with a D region ensuring the proper folding of the Ig molecule, i.e., in reading frame 1 (cf. Table 1). The hypothesis of the recognition of a bursal ligand by the germline VH1-VAl specificity has been further developed to propose that abolition of this germline recognition by gene conversion would signal the arrest of proliferation and allow the mutated cell to leave the bursa (McCormack et al., 1989b). It is however difficult to envision, since light (and heavy)-chain sequences can accumulate six to eight gene conversion tracks, that only the very last event abolishes the binding of the bursal ligand, all the previous events having maintained the germline-encoded specific recognition. Is there also a role for external antigens in selection of B cells as further diversification proceeds in the developing bursa? This question was raised, since antigen uptake by specialized bursal epithelial cells has been described (Toivanen et al., 1987). Moreover, bursal duct ligation, isolating the bursa from contact with gut-associated antigens, was proposed to result in reduced B-cell diversification. We do not think however that, since the initial description of B-cell development in the bursa as an antigen-independent process (Lydyard et al., 1976), any function other than a nonspecific mitogenic signal has been estab-
TABLE 111 SELECTIONOF HEAVY-CHAIN REARRANGEMENTS IN BURSALVERSUS NONBWRSAL SITES"
DJ VDJ bursa Day 13 VDJ bursa Day 15 VDJ bursa Day 18 VDJ spleen Day 15
Number of Sequences
In-frame Sequences
48 28 12 45 22
78% 92% 96%
50%
D Reading Frame Frame 1
Frame 2
Frame 3
D-D Junctions
50% 67% 92% 95% 59%
31% 30% 8% 5% 27%
19% 3% 0% 0% 13%
25% 21% 17% 15% 18%
Heavychain rearranged sequences have been analyzed concerning the overall reading frame, the frame ofthe D elements, and the incidence of D-D junctions, at Days 13, 15, and 18 of development in the bursa and at Day 15 in the spleen. The reading frame of the D is referred to J for DJ sequences and to V for VDJ sequences (taken from Reynaud et al., 1991b).
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CLAUDE-AGNES REYNAUD ET AL.
lished for antigens present in the bursa (Ekino, 1993).A similar question on the role of gut-associated antigens in diversification of B cells in sheep ileal Peyer’s patches has a very clear answer: the rate of Ig diversification is unchanged in genn-free animals or in segments of the ileum isolated from the intestinal tract (analogous to bursa1 duct ligation) (Reynaud et aE., 199210; Reynaud et al., in preparation); however, cell proliferation is impaired after some weeks without external antigen contact.
D. ALLELICEXCLUSION IN CHICKEN B CELLS:A SILENCER/ ANTISILENCERREGULATION Regulation of rearrangement at the Ig locus operates in the chicken as well as in the mouse: Ig genes are rearranged in B cells only, and allelic exclusion is performed for both heavy- and light-chain gene rearrangement. The main difference between the two systems comes from the short time during which rearrangement takes place, with two main consequences: first, the concommitant rearrangement of both heavy- and light-chain genes (no heavy-, then light-chain regulation, possibly mediated by a pre-B receptor), which makes chicken rearrangement closer to a “stochastic” process (Benatar et al., 1992; Reynaud et al., 1992a); second, the fact that only one allele is rearranged, no “second trial” being allowed in case of abortive joining (this precludes any regulatory function of the assembled Ig molecule in the process of allelic exclusion). The chicken light-chain locus with its natural compaction is thus an ideal substrate for investigating regulatory elements giving access to the rearrangement enzymes. Rearrangement of such a transgene with 11.5 kb of DNA is observed in mouse B cells (Bucchini et al., 1987). Further deletions/mutations of this rearrangement substrate have defined four regions involved in the regulation of rearrangement (Lauster et al., 1993) (Fig. 6). Positive regulatory elements map to promoter and enhancer elements, the promoter region necessary for efficient rearrangement being larger than the sole octamer motif. Such a role of enhancer elements in the control of Ig and TCR rearrangement has also been shown in the mouse, by both transgenic and knock-out experiments (Ferrier et al., 1990; reviewed by Chen and Alt, 1993). A negative regulatory element is present in the V-J intervening sequence, the region excised upon light-chain rearrangement, this element showing strong transcriptional silencing activity in a CAT assay in uitro. One or two sites flank this region that have no positive effect on rearrangement by themselves, but only in conjunction with the V-J intervening sequence; if these sites are mutated in the context of
CHICKEN B-CELL REPERTOIRE FORMATION
IH 1H
B PROGENITOR
V
“UO“
V
V
C
J
C
J
J” Q
B CELL
J
373
C
Antisilencer binding gives access to the recombinasemachinery
Silent allele V J
C
Transcription
FIG.6. A silencerlantisilencer regulation of chicken light-chain gene rearrangement. Four DNA elements regulating rearrangement of the chicken light-chain locus in transgenic mice have been described (Lauster et al., 1993): two positive regulatory elements, the promoter and the enhancer regions (the enhancer is located 3’ of CA, as described by Hagman et al. (1990)for the mouse A locus); one negative control element, corresponding to a strong transcriptional silencer, located in the V-J intervening sequence excised upon rearrangement (“Uo segment”); one (or two) putative elements located on one (orboth) side(s)ofthe Uo segment, antagonizing the effect ofthe silencer. It is proposed that the antisilencer factors would be present transiently in chicken Bcell progenitors, removing the silencer and allowing rearrangement to be performed on one allele (see Section 111,D). In the mature B cell, the remaining silencing element maintains the unrearranged allele in a silent configuration,the other allele being actively transcribed. Binding of the comesponding promoter (P), enhancer (E), silencer (S),and antisilencer (AS) factors is represented.
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CLAUDE-AGNES REYNAUD ET AL.
the normal V-J negative element, rearrangement is abolished in such a transgenic construct; on the contrary, these mutations have no negative effect if the V-J intervening sequence is replaced by a “neutral” segment of DNA (Lauster et al., 1993). We suggest that they act as antisilencer elements that would function by counteracting the silencing effect of the V-J segment (Fig. 6). We have proposed, to explain the particular configuration of Ig genes in chicken B cells with one allele rearranged and one allele in germline (or DJ) configuration, that a short window oftime allows rearrangement to be attempted only once on a chromosome; a transient antisilencer expression would give access to the Ig gene during a short time, after which silencer activity would dominate to ensure the exclusion of the other allele, making it unnecessary to coordinate the shut-off of the RAG genes with the expression of a functional Ig. Indeed, not only is RAG-2 expressed at a high level in the bursa (Carlson et al., 1991), but we also found RAG-1 transcription (at a lower level, but still detectable by Northern blot analysis (Reynaud et al., 1992a)), which might reflect some “leakiness” in the regulation of its expression, once Ig rearrangement is performed. Silencing activity through the V-J excision segment might also have relevance for gene conversion, maintaining the unrearranged allele in a silent inaccessible configuration, despite the presence of transcription factors and the small distance between promoter and enhancer regulatory elements in the chicken Ig locus in its germline configuration. Such a regulation is observed on a chicken transgene in mouse B cells, suggesting that it has been conserved between these two species: since activtors of rearrangement largely coincide with transcription factors, a dominant negative regulation exerted by the region excised upon rearrangement would be crucial to maintain an allele unrearranged in a recombination-active cell (e.g., a VDJ/DJ mouse pre-B cell proceeding to light rearrangement). V. Concluding Remarks
The chicken B-cell immune system is attractive because of its apparent simplicity: one can easily follow and count the different actors of the play. A few million cells are enrolled at the very beginning of embryonic development to build up the system. Thereafter, 20,000 to 30,000 B-cell progenitors having rearranged productively their unique V, and VL genes and having colonized the 10,000 bursa1 follicles produce the B-cell lineage. During B-cell expansion in the bursa, gene conversion generates a
CHICKEN B-CELL REPERTOIRE FORMATION
37s
diversified B-cell repertoire. After a few months, the bursa involutes completely, the animal maintaining lifelong immunity with this established peripheral B-cell compartment. Strikingly, the chicken model appears today less and less evolutionarily distant from mammalian immune systems. Rabbits use gene conversion to generate B-cell diversity and this process may well occur during a short period of development in gut-associated lymphoid tissues (GALT). Sheep, and probably ruminants in general, use GALT that are only present during early development to generate their Bcell repertoire. Surprisingly in this species, the molecular mechanism used is an antigen-independent hypermutation process. In this new outlook, bone marrow lymphopoiesis involving ongoing Ig gene rearrangement seems mainly a characteristic of rodents and men. Many questions remain unsolved, and they appear to be common to the various B-cell systems which have been studied. These questions concern the control of Ig gene rearrangement and therefore of allelic exclusion, the processes of selection occurring during the different stages of B-cell formation, and the homeostasis between the different B-cell subpopulations and the relative contribution of de novo B-cell production versus expansion of preexisting clones as the animal ages.
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origin of lymphoid stem cells studied in chick yolk sac-embryo chimeras. Nature (London)272,353-354. Lassila, O., Martin, C., Dieterlen-LiBvre, F., Gilmour, D., Eskola, J., and Toivanen, P. (1982).Migration of prebursal stem cells from the early chicken embryo to the yolk sac. Scand. J . Immunol. 16,265-268. Lauster, R., Reynaud, C.-A., Mirtensson, L., Peter, A,, Bucchini, D., Jami, J., and Weill, J.-C. (1993).Promoter, enhancer and silencer elements regulate rearrangement of an immunoglobulin transgene. EMBO J . 12,4615-4623. Le Douarin, N. M., Houssaint, E., Jotereau, F. V., and Belo, M. (1975). Origin of hemopoietic stem cells in embryonic bursa of Fabricius and bone marrow studied through interspecific chimeras. Proc. Natl. Acad. Sci. U.S.A. 72,2701-2705. Lydyard, P. M., Grossi, C. E., and Cooper, M. D. (1976). Ontogeny of B cells in the chicken. I. Sequential development of clonal diversity in the Bursa.]. Erp. Med. 144, 79-97. McCormack, W. T., and Thompson, C. B. (1990). Chicken IgL variable region gene conversions display pseudogene donor preference and 5’ to 3’ polarity. Genes Deu. 4,548-558. McCormack, W. T., Tjoelker, L. W., Carlson, L. M., Petryniak, J. B., Barth, C. F., Humphries, E. H., and Thompson, C. B. (1989a).Chicken Ig, gene rearrangement involves deletion of a circular episome and addition of single nonrandom nucleotides to both coding segments. Cell (Cambridge, Mass.) 56, 785-791. McCormack, W. T., Tjoelker, L. W., Barth, C . F., Carlson, L. M., Petryniak, B., Humphries, E. H., and Thompson, C. B. (1989b).Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development. Genes Deu. 3,838-847. Moore, M. A., and Owen, J. J. T. (1967).Chromosome marker studies in the irradiated chick embryo. Nature (London)215,1081-1082. Mueller, A. P., Wolfe, H. R.,and Meyer, J. (1959).Precipitin production in chickens. XXI. Antibody production in bursectomized chickens and in chickens injected with 19-nortestosterone on the fifth day of incubation.]. Immunol. 83,507-510. Parvari, R.,Ziv, E., Lantner, F., Heller, D. K., and Schechter, I. (1990).Somatic diversification of chicken immunoglobulin light chains by point mutations. Proc. Natl. Acad. Sci. U S A . 87,3072-3076. Pink, J. R. L. (1986).Counting components of the chicken’s B cell system. Immunol. Reu. 91, 115-128. Pink, J. R. L., and Lassila, 0.(1987).B-cell commitment and diversification in the bursa of Fabricius. Curt-. Topics Microbiol. Immunol. 135,57-64. Pink, J. R. L., Ratcliffe, M. J. H., and Vainio, 0. (1985). Immunoglobulin-bearing stem cells for clones of B (bursa-derived) lymphocytes. Eur. 1. Immunol. 15, 617620. Ratcliffe, M. J. H., and Ivanyi, J. (1981).Allotype suppression in the chicken. IV. Deletion of B cells and lack of suppressor cells during chronic suppression. Eur.]. Immunol. 11,306-310. Ratcliffe, M. J. H., Lassila, O., Pink, J. R. L., and Vainio, 0. (1986). Avian B cell precursors: Surface immunoglobulin expression is an early, possibly bursaindependent event. Eur. ]. Immunol. 16,129-133. Ray, A,, Siddiqi, I., Kolodkin A. L., and Stahl, F. W. (1988).Intra-chromosomal gene conversion induced by a DNA double-strand break in Saccharomyces cereoisiae. 1.Mol. Biol. 201,247-260. Reynaud, C. A., Anquez, V., Dahan, A., and Weill, J. C. (1985).A single rearrangement
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event generates most of the chicken immunoglobulin light chain diversity. Cell (Cambridge, Mass.)40,283-291. Reynaud, C. A., Anquez, V., Grimal, H., and Weill, J. C. (1987). A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell (Cambridge, Mass.) 48,379-388. Reynaud, C. A., Dahan, A., Anquez, V., and Weill, J. C. (1989).Somatic hyperconversion diversifies the single V, gene of the chicken with a high incidence in the D region. Cell (Cambridge, Mass.) 59, 171-183. Reynaud, C. A., Mackay, C. R., Miiller, R. G., and Weill, J. C. (1991a).Somatic generation of diversity in a mammalian primary lymphoid organ: The sheep ileal Peyer’s patches. Cell (Cambridge, Mass.) 64,995-1005. Reynaud, C. A., Anquez, V., and Weill, J. C. (1991b). The chicken D locus and its contribution to the immunoglobulin heavy chain repertoire. Eur. J. Zmmunol. 21, 2661-2670. Reynaud, C. A., Imhof, B. A., Anquez, V., and Weill, J. C. (1992a). Emergence of committed B lymphoid progenitors in the developing chicken embryo. EMBOJ. 12, 4349-4358. Reynaud, C. A., Hein, W. R., Imhof, B. A., and Weill, J. C. (1992b).Diversity is generated with diversity. In “Progress in Immunology,” Vol. VIII, pp. 121-128. Springer-Verlag, Heidelberg/Berlin. Reynolds, J. D., and Morris, B. (1983).The evolution and involution of Peyer’s patches in fetal and postnatal sheep. Eur. J. Zmmunol. 13,627-635. Roth, C. W., Longacre, S., Raibaud, A., Baltz, T., and Eisen, H. (1986). The use of incomplete genes for the construction of a Trypanosoma equiperdum. EMBO J . 5, 1065-1070. Smithies, 0. (1967). The genetic basis of antibody variability. Cold Spring Harbor Symp. Quant. Biol. 32, 161-166. Takeda, S., Masteller, E. L., Thompson, C. B., and Buerstedde, J. M. (1992). RAG-2 expression is not essential for chicken immunoglobulin gene conversion. Proc. Natl. Acad. Sci. U.S.A. 89,4023-4027. Thompson, C. B. (1992). Creation of immunoglobulin diversity by intrachromosomal gene cohversion. Trends Genet. 8,416-422. Thompson, C. B., and Neiman, P. (1987).Somaticdiversification ofthe chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell (Cambridge, Mass.)48,369-378. Toivanen, P., and Toivanen, A. (1973).Bursa1 and post-bursa1 stem cells in the chicken: Functional characteristics. Eur. J. Immunol. 3,585-595. Toivanen, P., Naukkarinen, A., and Vainio, 0.(1987).What is the function of the bursa of Fabricius? In “Avian Immunology: Basis and Practice” (A. Toivanen and P. Toivanen, eds.), Vol. 1, pp. 79-99. CRC Press, Boca Raton, FL. Tonegawa, S. (1983). Somatic generation of antibody diversity. Nature (London)302, 575-581. Warner, J., Uhr, W., Thorbecke, G. J., and Ovary, Z. (1969).Immunoglobulins, antibodies and the bursa of Fabricius: Induction of agammaglobulinemia and the loss of all antibody-forming capacity by hormonal buresectomy. J. Zmmunol. 103, 1317-1330. Weill, J.-C., and Reynaud, C.-A. (1987). The chicken B cell compartment. Science 238, 1094-1098. Weill, J. C., Reynaud, C. A., Lassila, O., and Pink, J. R. L. (1986). Rearrangement of chicken immunoglobulin genes is not an ongoing process in the embryonic bursa of Fabricius. Proc. Natl. Acad. Sci. U.S.A. 83,3336-3340.
INDEX
A
phage display antibody fragments, 195-198 vector systems, 199-203 to primary biliary cirrhosis selfantigens from human donors, 244-245 primate-derived, 231-232 principles of combinatorial approach, 192-195,266-267 selection strategies, 206-207,261-263 staphylococcal protein A, 229 study of responses, 232-242 synthetic repertoire approach, 250, 252-260 to thyroid disease self-antigens from human donors, 242-244 viruses, 209-228 cytomegalovirus, 220-221 hepatitis B virus, 220 herpes simplex virus type 1,222-227 herpes simplex virus type 2, 222-227 human immunodeficiency virus type 1,211-217 measles, 227-228 respiratory syncytial virus, 217-219 varicella Zoster virus, 221-222 whole antibody molecules, 208 Antibodies, monoclonal, see Monoclonal antibodies Antigen receptor, homology motif, signal transduction role, 81-83 Antigens Fas, 129-131 MHC, tumor immune response to increased expression of allo-MHC, 312-313
Airway hypersensitivity, guinea pig, interleukin-5 role, 173-174 Alleles, exclusion in chicken B cell development, 372-374 Allergens, antibodies from combinatorial libraries, 228 Amino acid sequences, antibodies from combinatorial libraries analysis, 233-238 RGD, 261-263 Antibodies, human, from combinatorial libraries affinity, strategy for improvement, 263-266 allergens, 228 amino acid sequences analysis, 233-238 RGD, 261-263 bacteria, 228 cloning strategies, 203-206 design, 261-263 Epstein-Ban virus-transformed cell line-derived, 229-230 expression of antibody fragments, 207-208 to Graves' ophthalmopathy selfantigens from human donors, 245 to human immunodeficiency virus type 1 self-antigens from human donors, 246-250 HuSCID mice-derived, 230-231 hybridomas, 229-230 immune donors, 208-242 naive repertoire approach, 250-252 overview, 191-192 379
380
INDEX
MHC class I self-, 314-320 metastatic phenotype affected by, 318-320 nonimmunological effects on tumor cells, 317-318 recognition by cytotoxic T lymphocyte effector cells, 314-315 recognition by natural killer effector cells, 315-317 tumor immunogenicity affected by low level of expression, 303-305 MHC class 11, self-, tumor immune response to increased expression, 313 non-self, antibodies to allergens, 228 bacteria, 228 Epstein-Ban-virus-transformed cell lines, 229-230 HuSCID mice, 230-231 hybridomas, 229-230 overview, 208-209 primates, 231-232 staphylococcal protein A, 229 study of responses, 232-242 viruses, 209-228 self-, from human donors, antibodies to Graves’ ophthalmopathy, 245 human immunodeficiency virus type 1,246-250 primary biliary cirrhosis, 244-245 thyroid disease, 242-244 staphylococcal protein A, antibodies to, 229 tumor, see Tumor antigens tumor immunogenicity affected by low level of expression, 303-305 tumor-specific transplantation, characteristics, 285 Apoptosis, Fas-mediated, 129, 133-134, 140 Asthma, interleukin-5 role, 174-176 Autoimmune diseases, Fas role, 138, 140 Avian leukosis virus, in chicken B cell DT40 induction, 361-362
B Bacteria, antibodies from combinatorial libraries, 228 Basophils, interleukin-5-mediated activity, 156 B cells, chicken, repertoire formation in, 353-375 development allelic exclusion, 372-374 antisilencer regulation, 372-374 bursa role, 364-367 embryonic, 367-369,374 immunoglobulin sequence selection, 369-372 silencer regulation, 372-374 gene conversiop avian leukosis virus induction of DT40 cell line, 361-362 DT40,361-362 Holliday junction, possible formation, 362-365 hyperconversion mechanism, 358-361 recombination models, 362-365 immunoglobulin gene organization amino acid composition, 356, 358 D elements, 356-358 heavy chain loci, 354-356 light chain loci, 354-356 B cells, interleukin-5 effects regulation of development, 154-155 signaling in X chromosome-linked immunodeficient mice, 171-172
C Cell death, Fas-mediated, 129, 133-134, 140 Chicken B cells, repertoire formation in, see B cells Cirrhosis, primary biliary, and antibodies to self-antigens from human donors, 244-245 Cloning, antibodies from combinatorial libraries, 203-206 Combinatorial libraries, human antibodies from, see Antibodies, human, from combinatorial libraries
INDEX
Cytokine genes, in tumor cell engineering, 321-323 Cytomegalovirus, human, antibodies from combinatorial libraries, 220-221 Cytotoxic T lymphocytes Fas expression in, 138 self-MHC class I recognition by effector cells, 314-315
D Death factor, Fas as, 129-140 Diseases autoimmune, Fas role in, 138, 140 human, interleukin-5 role in asthma, 174-176 graft rejection, 177 helminth infections, 176-177 tumors, 177-178 DNA, complementary, interleukin-5, organization, 148-149
E Embryo, chicken, B cell, early commitment in, 367-369,374 Eosinophilia, interleukin-5 role asthmatic patients, 174-176 experimental, ,173 guinea pig, 173-174 mRNA expression in patients, 158-159 parasite infection association, 172 tumors associated with, 177-178 Eosinophils, interleukin-5-mediated production, 156 Epstein-Barr virus antibodies from transformed cell lines, 229-230 tumor antigen induction, 292 Experimental eosinophilia, interleukin-5 role, 173
F Fas, 129-140 antigen, 129-131
38 1
apoptosis role, 129, 133-134, 140 in oitro, 133-134 in oioo, 133-134 autoimmune diseases associated with, 138,140 expression, 130-132 gene, mutation in Zpr mice, 132-133 loss of function mutation, 139 physiologic roles, 137-139 signal transduction role, 134-135,139 T cell development role, 137 Fas ligand, 130, 135-139 characteristics, 135-137 expression in cytotoxic T lymphocytes, 138 Fc receptors characteristics, 1 FcaR, characteristics, 38-39 FcaRI biochemical structure, 39-40 cell distribution, 40-41 characteristics, 39 ligand properties, 40 molecular structure, 39-40 monoclonal antibodies, 9, 40-41 polymorphisms, 41 FcyR biological function, 69-70 characteristics, 2-3 signal transduction role mechanisms, 72-77 phosphorylation, 74-77 second messenger interactions, 72-74 structural factors, 82, 84-89 FcyRI biochemical structure, 3-7 biological function, 69 cell distribution, 8 characteristics, 2-4 gene structure, 4-5,7 immunoglobulin interactions, molecular basis, 48-54 ligand properties, 7-8 molecular structure, 3-7 monoclonal antibodies, 8-9 polymorphisms, 8-10 signal transduction role phosphorylation, 76-77 structural factors, 84
382 FcyRII biochemical structure, 10-16 biological function, 70 cell distribution, 17-19 characteristics, 2-3, 11 gene structure, 11-13,15-16 immunoglobulin interactions, molecular basis, 49-50,54-59 ligand properties, 16-17 molecular structure, 10-16 monoclonal antibodies, 9, 17-19 polymorphisms, 19-21 signal transduction role phosphorylation, 75-77 structural factors, 82, 84-87 transcripts, 13 FcyRIII biochemical structure, 21-27 biological function, 70 cell distribution, 28-29 characteristics, 2-3, 22 gene structure, 22-26 immunoglobulin interactions, molecular basis, 49-50,59-61 ligand properties, 27-28 molecular structure, 21-27 monoclonal antibodies, 9,28-29 polymorphisms, 29-30 signal transduction role phosphorylation, 7 6 7 7 structural factors, 84, 87-88 transcripts, 23 FcsR, characteristics, 43 FccR, characteristics, 30 FceRI biochemical structure, 31-36 biological hnction, 70-71 cell distribution, 36-37 characteristics, 30 gene structure, 31-34 immunoglobulin interactions, molecular basis, 49-50,61-68 ligand properties, 36 molecular structure, 31-36 monoclonal antibodies, 9,36-37 polymorphisms, 37 signal transduction role mechanisms, 77-81 phosphorylation in, 79-81 second messenger interactions, 77-79
INDEX
structural factors, 89-90 a-subunit, 32-33 @subunit, 33-34 y-subunit, 34-36 transcripts, 31 FceRII biochemical structure, 37-38 cell distribution, 38 characteristics, 30 gene structure, 37 ligand properties, 38 molecular structure, 37-38 monoclonal antibodies, 38 FcpR, characteristics, 41-42 FcRn, characteristics, 46-47 function biological, 69-71 FwR, 69-70 FcERI, 70-71 signal transduction, mechanism, 71-81 signal transduction, structural basis, 81-90 genes, see Genes immunoglobulin interactions, molecular basis of FcyRI, 48-54 Fc/RII, 49-50,54-59 FcyRIII, 49-50,59-61 FcERI, 49-50,61-68 overview, 48-50 polymeric IgR, characteristics, 43-46 research directions, 91 signal transduction, mechanism, 71-81 phosphorylation role, 74-77,7931 protein kinase role, 74-77, 79-81 second messenger interactions, 72-74,77-79 signal transduction, structural basis, 81-90 antigen receptor homology motif role, 81-83 F q R role, 82,84-89
G Genes, see also Oncogenes chicken B cell, conversion DT40,361-362 Holliday junction, possible formation of, 362-365
INDEX
hyperconversion mechanism, 358-361 recombination models, 362-365 cytokine, in tumor cell engineering, 321-323 Fas, mutation in Zpr mice, 132-133 Fc receptor FcyRI characteristics, 4 structure, 5, 7 FcyRII characteristics, 11 structure, 12-13, 15-16 FcyRIII characteristics, 22 structure, 23-26 FcERI a-chain, 32-33 structure, 31-34 FcERII, structure, 37 immunoglobulin, see Immunoglobulin genes interleukin-5, organization, 148-149 interleukin-5Rq structure, 162-163 V, chicken B cell diversification, 358-361 recombination models, 362-365 Graft rejection, interleukin-5 role, 177 Graves’ ophthalmopathy, antibodies to self-antigens from human donors, 245
H Helminth infections, interleukin-5 role, 176-177 Hepatitis B virus, antibodies from combinatorial libraries, 220 Herpes simplex virus type 1, antibodies from combinatorial libraries, 222-227 Herpes simplex virus type 2, antibodies from combinatorial libraries, 222-227 Hodgkin’s disease, interleukin-5 mRNA expression in patients, 158 Human antibodies, from combinatorial libraries, see Antibodies, human, from combinatorial libraries
383
Human cytomegalovirus, antibodies from combinatorial libraries, 220-221 Human diseases, see Diseases Human immunodeficiency virus type 1, antibodies from combinatorial libraries, 211-217 to self-antigens from human donors, 246-250 Hybridomas, antibodies from, 229-230
Immune response, against tumors, see Tumors, immune response against Immune suppressive factors, tumor immunogenicity affected by, 307-309 Immunodeficient mouse HuSCID-derived antibodies, 230-231 interleukin-5 production in, 171-172 Immunoglobulin genes, in chicken B cells conversion allelic exclusion, 372, 374 avian leukosis virus induction of DT40 cell line, 361-362 rearrangement in bursa, 365-367 recombination models, 362-365 organization D elements, 356-358 heavy chain loci, 354-356 light chain loci, 354-356 Immunoglobulins Fc receptors for, see Fc receptors interleukin-5-regulated production, 151-154 IgA, 152-153 IgE, 153 IgG, 153 sequence selection in chicken B cell development, 369-372 Infections, interleukin-5 role helminth, 176-177 parasite, 172-173 Inflammation, interleukin-5-associated, in asthmatic patients, 174-176 Interleukin-5 animal models of production airway hypersensitivity, 173-174 eosinophilia in guinea pigs, 173-174 experimental eosinophilia, 173
384
INDEX
parasite infection, 172-173 transgenic mouse, 169-170 tumor rejection, 174 Major histocompatibility complex, see X chromosome-linked Antigens immunodeficient mouse, Measles virus, antibodies from 171-172 combinatorial libraries, 227-228 characteristics, 145-146 MHC, see Antigens functional properties Monoclonal antibodies, Fc receptor FcaRI, 9 , 4 0 4 1 basophil regulation, 156 B cell developmentregulation, 154-155 FwRI, 8-9 eosinophil production, 156 FwRII, 9, 17-19 immunoglobulin production F q R I l l , 9, 29-30 regulation, 151-154 FcERI, 9,36-37 interleukin-2 receptor induction, 155 FcsRII, 38 future perspectives, 178-179 Mouse HuSCID, antibodies derived from, histological background, 146-148 in human disease 230-231 asthma, 174-176 immunodeficient, interleukin-5 graft rejection, 177 production, 171-172 lpr, Fas gene mutation in, 132-133 helminth infections, 176-177 tumors, 177-178 transgenic, interleukin-5 production messenger RNA expression, 156-159 models, 169-170 molecular structure X chromosome-linked biological activity, 149 immunodeficient, interleukin-5 cDNA organization, 148-149 production models, 171-172 Mucin, tumor antigen recognition of, gene organization, 148-149 302-303 polypeptides, 149-151 Mutations Interleukin-10, tumor immunogenicity Fas reduced by, 307-309 genes in lpr mice, 132-133 Interleukin-2 receptor, induction by interleukin-5, 155 loss of function, 139 Interleukin-5 receptor p53 protein-derived tumor antigens, expression 295-296 aberrations in mice, 170-172 analysis, 168-169 function, 165-166 IL-5Ra chain N soluble forms, 163-164 structure, 160-164 Natural killer cells, self-MHC class I IL-5Ra gene structure,. 162-163 recognition in tumor cells, 315-317 IL-5RP chain structure, 164-165 signaling pathway, 166-168 signal transduction, 146 structure, 159-165 0
L Zpr mouse, Fas gene mutation in,
132-133 Lymphocytes, T, see T cells
Oncogenes, tumor antigens derived from abl, 296-297 bcr, 296-297 ras, 297-299 Ophthalmopathy, antibodies to selfantigens from human donors, 245
385
INDEX
P Papillomavirus, tumor antigen induction, 292-293 Parasite infection, interleukin-5 production in animal models, 172-173 Peptides, tumor-specific, T cell recognition, 288-290 Phosphorylation, in Fc receptormediated signal transduction FwR, 74-77 FcsRI, 79-81 Polypeptides, interleukin-5, molecular structure, 149-151 Primary biliary cirrhosis, antibodies to self-antigens from human donors, 244-245 Primates, antibodies derived from, 231-232 Protein kinases, in Fc receptor-mediated signal transduction F v R , 74-77 FcsRI, 79-81 Proteins staphylococcal protein A, antibodies from combinatorial libraries, 229 tumor antigens derived from abl oncogenes, 296-297 bcr oncogenes, 296-297 mutated ~53,295-296 overexpression role, 299-302 ras oncogenes, 297-299 tum-, 293-295
R Respiratory syncytial virus, antibodies from combinatorial libraries, 217-219 RNA messenger, interleukin-5 expression, 156-159
S Second messengers, in Fc receptormediated signal transduction FwR, 72-74 FcsRI, 77-79
Signal transduction Fas-mediated, 134-135, 139 Fc receptor-mediated antigen receptor homology motif, 81-83 FcyR, mechanisms of activity, 72-77 FcyR, structural factors, 82, 84-89 FcERI, 77-81,89-90 mechanism, 71-81 phosphorylation, 74-77,79-81 protein kinase involvement, 74-77, 79-81 second messenger interactions, 72-74,77-79 structural factors, 81-90 Staphylococcal protein A, antibodies from combinatorial libraries, 229
T T cells Fas effects development role, 137 expression in cytotoxic T lymphocytes, 138 self-MHC class I recognition by cytotoxic T lymphocyte effector cells, 314-315 tumor antigen interactions costimulatory pathway-determined immogenicity, 288-290 early evidence of recognition, 285-288 tumor-specific peptide recognition, 288-290 Thyroid disease, antibodies to selfantigens from human donors, 242-244 Transforming growth factor+, tumor immunogenicity affected by, 307-309 Tumor antigens B7, costimulatory effects on immunogenicity, 305-307 CD28, costimulatory effects on immunogenicity, 305-307 costimulatory pathway, 305-307 CTLA-4, costimulatory effects on immunogenicity, 305-307 mucin recognition, 302-303
386
INDEX
protein-derived abl oncogenes, 296-297 bcr oncogenes, 296-297 mutated p53,295-296 overexpression resulting in, 299-302 ras oncogenes, 297-299 tum-, 293-295 serological detection, 283-284 T cell recognition early evidence, 285-288 tumor-specific peptides, 288-290 virus-induced tumors, 290-293 Tumors, immune response against, 281-326 antigens, see Tumor antigens B7-transfected cells, 320-321 cytokine genes in engineering of tumor cells, 321-323 escape mechanisms, 324-325 immunogenicity, factors contributing to low level antigen expression, low, 303-305 costimulatory molecules, lack of, 305-307 immune suppressive factors, 307-309 interleukin-10, 307-309 MHC class I expression, low, 303-305 transforming growth factor-@, 307-309 tumor environment modification, 309-312 MHC expression increased by transfection
allO-MHC, 312-313 self-MHC class I, 314-320 self-MHC class 11,313 tumor antigens, see Tumor antigens Tumors, interleukin-5 effects, 177-178 rejection, 174 Tumor-specific transplantation antigens, characteristics, 285
V Varicella Zoster virus, antibodies from combinatorial libraries, 221-222 Viruses antibodies, from combinatorial libraries cytomegalovirus, human, 220-221 hepatitis B virus, 220 herpes simplex virus type 1, 222-227 herpes simplex virus type 2, 222-227 human immunodeficiency virus type 1,211-217 measles, 227-228 respiratory syncytial virus, 217-219 varicella Zoster virus, 221-222 avian leukosis, in chicken B cell DT40 induction, 361-362 tumor antigen induction by, 290-293 Epstein-Barr virus, 292 papillomavirus, 292-293