HYPOTHESIS

T-Cell Allorecognition of HLA Class II Annemarie Termijtelen

INTRODUCTION The most important biologic function of the major histocompatibility complex (MHC) is its role in the regulation of the immune response: immunocompetent T cells can only recognize foreign antigen in the context of self-MHC. The recently solved three-dimensional structure of the HLA-A2 molecule has greatly improved insight into the molecular basis of antigen recognition [ 1]. The polymorphic residues of the HLA-A2 molecule are clustered in and around a groove. This groove was identified as the binding site for processed antigen [2]. Based on sequence homology and functional studies it was proposed that this model applies to both MHC class I and class II molecules [3]. Although the precise structure of processed antigens is not known, they can be replaced in functional assays by synthetic peptides. Such synthetic peptides were shown in functional studies measuring T-cell proliferation [4,5] and in biochemical studies measuring direct binding [6,7] to be able to bind to some but not all MHC molecules. Thus, the MHC restriction phenomenon is believed to result from the allele-specific binding of different peptides in the groove [8,9]. In this concept the MHC-restricted T-cell receptor may even interact with a conserved rather than a polymorphic part of the MHC molecule [10]. The majority of MHC molecules may be occupied by peptides of endogenous origin [10]. All self-peptides can potentially be anchored in the MHC groove. If foreign antigen appears in high enough concentrations, it may compete with the self-peptides for binding to the MHC molecules and induce an immune response [11]. Occupied by many different endogenous and foreign peptides, the MHC molecules will provide a large variety of potentially stimulatory entities when encountered by allogeneic T cells. The fact that allogeneic T cells proliferate abundantly in a mixed lymphocyte culture (MLC) without prior in vivo immunization [12] may thus be explained by the relatively high number of different stimulating MHC-peptide complexes on the stimulator cells [ 13,14]. Since HLA-

From the Department of lmmunohaematology and Blood Bank, Building I, E3-Q, University Hospital Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Address reprint requests to Dr. Annemarie Termijtelen, Department of Immunohaematology and Blood Bank, Building 1, E3-Q, University Hospital Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Received August 11, 1989; accepted November 20, 1989.

Human Immunology 28, 1-10 (1990) © American Society for Histocompatibility and Immunogenetics, 1990

1 0198-8859/90/$3.50

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A. Termijtelen identical siblings are nonstimulatory in the MLC, the majority of MHC-bound peptides then have to be nonpolymorphic. The function of alloreactivity in very early days may have been to prevent loss of individuality. Primitive invertebrates like sponges, corals, and sea anemones, living a sedentary life, must have developed a system to differentiate between self and nonself to prevent the fusion of tissue. On this assumption Klein postulated that when invertebrates became mobile, the need for alloreactivity was lost, but "the function of the prehistocompatibility antigens was adapted to protect the individual from neoplasms and exogenous pathogens" [15]. The similarity between alloreactivity and antigen-specific T-cell responses has been demonstrated in a number of studies. For example, Ashwell and Schwartz [16] showed that of 62 mouse T-cell clones specific for cytochrome c of sheep insulin, 60% were also alloreactive. In human beings HLA-restricted, Candida albicans-specific T-cell clones [17], Mycobacterium leprae-specific T-cell clones [18], and a tetanus toxoid-specific T-cell clone [19] were described that showed alloreactivity with unrelated HLA antigens. The fact that such dual recognition can result from cross-reactive recognition by the same T-cell receptor was shown in T-cell receptor-gene transfection experiments [20]. It is not known whether the recognition of alloantigen is dependent on peptide bound to the MHC molecule or if alloreactive T cells recognize MHC molecules irrespective of the presence of peptide. If peptides are involved, they may be derived from degraded MHC molecules. In addition, there is a large pool of nonMHC peptides available. The majority are nonpolymorphic, but polymorphic antigens like minor histocompatibility antigens or viral antigens can potentially bind to MHC molecules as well. The present review is meant to summarize and discuss the data available for the T-cell allorecognition of HLA class II. It is shown that the majority of T-cell reactions correlate with the presence of polymorphic sequences on the MHC molecule. The most simple explanation for such reactions would be that the T cells recognize the polymorphic part of the class II molecule, irrespective of the presence of a specific peptide. In exceptional instances, however, the T-cell recognition does not correlate with polymorphic sites on the MHC molecule itself. In these cases the involvement of peptide is extremely likely.

Which part of the HLA class II molecule is involved in the induction of alloproliferation? Alloreactivity can be investigated in vitro with T-cell clones derived from bulk MLCs. The proliferative T-cell clones recognize the sensitizing DR molecule or molecules. Depending on the cell combination used, a considerable number of DQ- and DP-specific clones may also be obtained. Since amino acid sequences are known for many HLA class II molecules, it is possible to investigate which part of the molecule is associated with recognition by the T cells. Mainly data on the first, rather than the second, domain of the class II molecules will be considered here, for the following reasons: (1) less sequence data are available for the second domain, (2) little polymorphism is shown in the second domain, and most importantly, (3) the first domain is the most likely part of the molecule to interact with the T-cell receptor. As exemplified in Figure I(A), the first domain of the DR B chain encompasses three hypervariable regions: two on the/3 sheets forming the bottom of the groove and one on the o~ helix overlying the B-sheet platform. In the case of HLA-DR, the ~ chain is nonpolymorphic. If T-cell clones are, for instance, reactive to the DRB1 chain of HLA-DRw13 (DRw1331), stimulator cells can be selected that share different hypervariable regions (HV) with this

F-Cell Allorecognition of HLA Class II

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FIGURE 1 (A) The hypothetical model of an MHC class II molecule based on the structure of HLA-A2 [1,3]. The picture represents the surface of the molecule as seen by the T cell, i.e., the first domains of the HLA-DR ~ and/3 chains, both composed of ~plated sheets, forming the bottom of a groove, and an ~ helix that may directly interact with the T-cell receptor. The three hypervariable regions as identified on HLA-DR have been indicated. Not visible are the underlying second domains of DR~ and DR~ (B) HLADR sequences, using HLA-DR1 as reference sequence. The horizontal bars indicate the location of the three hypervariable regions and the sequential numbers of the amino acids. Underlined are HV regions shared with the DRy1 chain of DRw13. The reaction patterns of the DRw13~1-directed T-cell clones C6 and C50 have been indicated at the right-hand side. For further details see the text. (C) The same legend as to (B). Underlined are HV regions shared with the DRy1 chain of DR3,Dw3. The reaction patterns of the DR3,Dw331-directed T-cell clones c1656, C1606, and C1671 have been indicated at the right-hand side. For further details see the text. (D) The same legend as to (B). The T-cell reaction patterns [30] have been indicated at the right-hand side. Patterns 1, 2, and 3 represent alloreactive T-cell clones specific for Dw24, Dw25, and Dw26, respectively. Pattern 4 represents T-cell clones recognizing Dw25 and Dw26, and pattern 5 represents antigen-specific T-cell clones, restricted by Dw24 and Dw26. The DR sequences have been taken from the following references: D I ~ I : DR1 [21], DRwl 3,Dw18 [18], DRw13,Dw19 [23], DR3Dw3 [21,22], DRwll,Dw5 [24], DR4,Dwl0 [25], DRw14,Dw9 [22], DRw11~vM [21], and DR3,RSH [26]. DRy3:DRw52,Dw24 [22], Dw52,Dw25 [22], and DRw52,Dw26 [30].

4

A. Termijtelen chain. Figure l(B) shows that the DR/31 chain of DRw 13,Dw18 is identical to the DR/31 chain of DRw13,Dw19 with one exception at position 86. In addition, both DRw1331 chains share HV1 with DR3,Dw3 and DRwl 1,Dw5; HV3 with DR4,Dwl0; HV1 and HV2 with DRw14,Dw9 (except residue 37); and HV1 and HV3 with D R w l l J V M . By testing alloreactive T-cell clones sensitized to a DRw 13,Dw 19 haplotype, we found that 14 out of 15 of the DR31-specific clones responded to DRw 13 (i.e., Dw 18 and Dw 19)-positive stimulator cells only. The pattern of reactivity of these clones is represented by C6 [Figure I(B)]. One clone, C50, was identified which reacted with all stimulator cells that were either DRw13,Dw18; DRw13,Dw19, or DR4,Dwl0, and in addition with JVM [27]. These stimulator cells shared HV3 with the original priming antigen, DRw13,Dw19, although they differed at HV1 and/or HV2. Thus, I out of 15 DRw1331-specific clones was reactive to a structure determined by HV3 of the molecule leading to cross-reactivity with other than the original sensitizing DR molecule [Figure I(B)]. In a similar analysis we found that two out of six DR3,Dw3-specific T-cell clones (represented by C1656) were dependent on the presence of HV1 + HV2 + HV3 on the stimulating molecule. These clones responded only when stimulated with DR3,Dw3 stimulator cells [Figure 1(C)]; three out of six responded in addition to DR3,RSH, which shares HV1 and HV3 with DR3,Dw3 (represented by C1606). One clone, C1671, responded to DR3,Dw3, to DR3,RSH, and to all the DRw52,Dw24-positive donors in the panel. Dw24 represents an allele of the DRw5233 chain and shares HV3 and the majority of HV2 with the DR331 chain. Since RSH is Dw24-positive, it could not be determined conclusively whether C1671 was reactive to the/31 and the/33 chain of RSH or to the/33 chain only. Thus, the reactivity pattern of C 1671 was determined by HV3, although the additional influence of HV2 could not be excluded. Seyfried et al. similarly described a clone that recognized all DR1-, DR4,Dw14-, DRwI4,Dw16-, and DRwl0-positive cells. These DR types differ by HV1 and HV2 but share their HV3 region [28]. The specificity of all clones mentioned so far was determined by one or more HV regions, always including HV3. An exception to this rule would be the cytotoxic T-cell clones reactive to DR4 haplotypes described by Reinsmoen et al. [29]. Although some of the T-cell clones were specific for the HV3-defined HLAD specificity Dw4, some clones recognized all DR4-positive target cells. The "DR4-ness" of a DR molecule is defined by HV1 and/or HV2 but not by HV3 [25]. Although the hypervariable regions may represent functional elements, the schematic subdivision of HLA-DR into three HV regions is artificial. As may be clear from Figure I(A), it is not unlikely that one antigenic epitope can be composed of HV3 on the a helix in combination with the underlying, interhelical parts of the/3 sheets. Gorski and coworkers showed that the DRw52/33 antigenic epitopes are composed ofpolymorphic clusters of amino acids from the interhelical part of the/3 sheets combined with amino acids in the ~ helix. One central cluster was composed of residues in HV2 and HV3 [including residues 26, 28, and 74; Figure I(A and D)]. A second cluster, including residues 30, 37, 51, 57, and 60, created a restriction element for tetanus toxin-specific T-cell clones [30]. In conclusion, these data show that for the majority of HLA-DR-reactive Tcell clones, the stimulation could be ascribed to a dluster ofpolymorphic residues on the MHC molecule. Such clusters could be composed of one or more hypervariable regions but could also be formed by apparently randomly distributed residues that combine in the three-dimensional structure. To explain this type of reaction patterns, the T cell may directly interact with the polymorphic part of

T-Cell Allorecognition of HLA Class 1I A

5 B

C

FIGURE 2 Schematic representation of various models for the recognition of MHC by alloreactive T cells. MHC: cross-section through the MHC molecule composed of an ce and a/3 chain. 1, first domain; 2, second domain; P, peptide anchored in the MHC groove; TcR, allospecific T-cell receptor. (A) The T-cell receptor recognizes a polymorphic entity on the MHC molecule. The involvement of peptide is here not a prerequisite. (B) The Tcell recognizes a structure defined by the combination of MHC and peptide. The peptide may originate from different endogenous or foreign sources. (C) The T cell recognizes degraded MHC presented by an intact MHC molecule.

the M H C molecule without the involvement of peptide [exemplified in Figure 2(A)]. W h a t is the role o f M H C - b o u n d p e p t i d e s in a l l o p r o l i f e r a t i o n ? A second type of T-cell clone appears to recognize entities that cannot be translated to a cluster of polymorphic residues on the stimulating class II molecule. Lang and coworkers [31] described a T-cell clone responding to DR3 and DR4,Dw14 although no specific amino acid sequence could be identified that correlated with these responses. A similar reaction pattern was observed with our T-cell clone C1443 [27] that could be stimulated by DRw13 ( D w l 8 and Dw19)- and DR4,Dw4-positive cells. These responses again were not related to specific amino acid sequences on either the first or the second domain of the stimulating D R molecules. In the latter study two findings favor the involvement of a peptide: First, C1443 was "broadly reactive" in such a way that no specific polymorphic cluster of the stimulating class II molecules could be responsible for the stimulation. We hypothesized that a yet unidentified polymorphic peptide (for example, from a frequently occurring viral antigen or a minor histocompatibility antigen) was bound in the groove of DRw13 and DR4,Dw4 in such a manner that once bound, the T-cell receptor could not discriminate between Dw18-, Dw19-, and Dw4-positive stimulator cells. The fact that M H C - b o u n d peptide can indeed "camouflage" the specificity of a class II molecule and lead to broad T-cell recognition is also known from antigen-specific T-cell clones. Some clones, sensitized to synthetic peptides, can give very broad recognition patterns that do not correlate with amino acid sequences of the antigen-presenting class II molecules. These "promiscuous" T cells were postulated to recognize peptide in combination with a monomorphic part of the M H C molecule, for example, DR~x [33,34].

6

A. Termijtelen

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FIGURE 3 Schematic representation of DR sequences in relation to the pattern of reactivity of the DR/31-specific T-cell clone C1443. The origin of the sequence data is as follows: WT51 [21], PRIESS [32], HHK [22]. The second argument for the involvement of a peptide in the allorecognition by C1443 was the fact that the B-cell line PRIESS was the only one out of nine DR4,Dw4-positive cells that could not stimulate C 1443, even though the DRB 1 sequence of this cell line is identical to the DR4,Dw4/31 sequence of WT51, which was stimulatory (Figure 3). Apparently the clone could discriminate between DR4,Dw4 molecules that are identical by sequence. In addition, the cell line KRA (Dwl9) was the only one out of 20 DRwl3-positive stimulator cells that could not stimulate C 1443 although it was perfectly able to stimulate other DRw1331specific T-cell clones [27]. If a peptide was involved in the allorecognition by C1443, then PRIESS and KRA, although expressing the appropriate DR molecule, might not have the disposal of the relevant peptide. This "non-sequence-defined" reactivity ofC 1443 is not an exception. We found another T-cell clone that showed a similar unexpected pattern of recognition. This clone discriminated between DQwl molecules that were identical for the amino acid sequences of the first domain of DQa and DQ3 (in preparation). Thus, when a T cell discriminates between stimulating molecules that are identical by sequence, we favor the explanation that a polymorphic peptide, bound to the MHC molecule, is involved. Several other lines of evidence show that MHC-bound peptides can be involved in allorecognition. Evidence for the simultaneous recognition of MHC and tissuespecific peptide has been given by Marrack and Kappler [35], who suggested that B-cell-specific peptides can be involved in allorecognition. Bernhard and coworkers showed that HLA-A2-transgenic mice, immunized with human HLAA2, could lyse human HLA-A2-positive target cells but not the murine transfectants. A likely explanation for this finding is that the human HLA-A2 molecule carried a species-specific peptide absent in the transgenic mice [36]. Eckels and coworkers demonstrated that DRl-specific alloreactivity could be modulated by the peptide HA (306-320) of influenza virus hemagglutinin [37]. This peptide specifically bound to DR1 molecules. Competition experiments showed that HA (306-320) could block some but not all DRl-specific T-cell clones. It was postulated that HA (306-320) might differentially displace endogenous peptides. Only the T cells recognizing DR1 in combination with an endogenous peptide replaced by HA (306-320) would then be inhibited. The data from T-cell clones recognizing entities that are not solely defined by the stimulatory MHC molecules support the concept that at least part of the alloreactive T cells recognize MHC in combination with peptide [exemplified in Figure 2(B)]. This peptide could then be tissue-specific or species-specific. In addition, the expression of a certain peptide could vary between individuals, i.e.,

T-Cell Allorecognition of HLA Class II

7

could be polymorphic. The possibility that the peptide is derived from the MHC itself is discussed in the next section. What is the role of degraded MHC in alloproliferation? In the models described above, the peptide recognized by alloreactive T cells was never identified. The only endogenous, well-characterized peptides that are known to be capable of binding to and being presented by MHC molecules are peptides derived from the MHC itself. Parham and coworkers found that synthetic peptides from the ~2 domain of HLA-A2 could inhibit alloreactive cytotoxic T lymphocytes (CTLs). These authors showed that the inhibition was most likely at the level of the T-cell receptor and speculated on the possibility that the CTLs recognized degraded class II molecules as nominal antigen presented by intact class I molecules as restriction elements [38]. More definite proof for the simultaneous recognition of MHC and MHCderived peptide has been given by Maryanski and coworkers. They immunized mice with syngeneic cells transfected with human Cw3. Thus, H-2K-restricted cytotoxic T-cell clones specific for Cw3 were isolated. It could be recognized that a synthetic Cw3 peptide served as target for the CTL clones [39]. The class ll-restricted recognition of MHC class 1I peptides has been demonstrated by De Koster and coworkers. They made T-cell clones from a DR4,5; DPw3,w4-positive responder against a synthetic peptide homologous to the third hypervariable region of HLA-DR331 (DR3~I 67-85) [40]. The T cells gave a conventional peptide-specific, DPw3-restricted proliferative response. In addition, they could be stimulated by DPw3-positive stimulating cells without the addition of the peptide, if the stimulating cells were also DR3-positive. This finding implied that degraded DR3 bound to DPw3 was continuously present on the lymphocytes of the DR3,DPw3-positive individuals. The hypothesis that MHC-derived peptides can be presented by autologous MHC and recognized by alloreactive T cells is clearly proven by these studies [exemplified in Figure 2(C)]. Whether the recognition of degraded MHC is rule or exception in allorecognition still needs to be determined. Using conventional MLC cloning techniques without prior in vivo immunization, the recognition of one MHC antigen restricted by an other MHC antigen (for example an anti-DR2 response restricted by DR1 or DQwl) does not seem to be a frequent event. This suggests that if MHC-derived peptides are recognized in the context of intact MHC, the MHC-derived peptide is usually presented by "its own" MHC molecule (DR2 by DR2, DR3 by DR3, etc). DISCUSSION The reaction patterns of HLA-DR-specific T-cell clones frequently correlate very well with the linear amino acid sequences of the DR molecules. The most simple explanation for this observation is that the anti-DR-reactive T cells recognize "empty" DR molecules or that the MI-IC-bound peptide itself is irrelevant for recognition. Recent investigations have shown that a peptide, bound to class I, is needed to stabilize the molecule [41]. A class II-bound peptide similarly may be needed to induce a stable conformation recognized by alloreactive T cells. Such peptide does not necessarily have to contribute to the specificity of the T-cell epitope. It is experimentally difficult, however, to exclude the possibility that the T-cell receptor recognizes (1) polymorphic DR peptides, specifically bound to their "own" intact DR. molecules or (2) specific conformational changes induced in nonpolymorphic peptides.

8

A. Termijtelen One of the reasons to believe in the involvement of peptides in alloreactivity is the relatively high number o f T cells taking part in the MLC as compared to the number o f T cells reactive to nominal antigen. The majority of M H C molecules are expected to be occupied by endogenous or foreign peptides. If each MHC-peptide complex can be recognized by different T cells, then these complexes would provide a very large variety of potentially stimulatory entities. The high reactivity in MLC may, on the other hand, be explained by the fact that the antigen recognized by alloreactive T cells, namely, the class II molecule irrespective of bound peptide, is presented in the most efficient way possible. Each stimulator cell is loaded with these foreign class II antigens. The concentration of alloantigen per stimulator cell is therefore extremely high as compared to the concentration of nominal antigen, which will be bound to and presented by a very small number of M H C molecules per antigen-presenting cell. Since T cells are selected to recognize self-MHC, it is likely that many T cells carrying receptors with low affinity for the allo-MHC molecules may be induced to proliferate, due to the high concentration of alloantigen. In this model, M H C - b o u n d peptide should only play a minor role in defining the specificity of alloreactivity as measured in vitro. Although for the majority of reactions it will be hard to give the final proof whether peptides are part of the stimulatory entity or not, in some instances the participation of peptide in alloreactivity seems extremely likely. Evidence for the involvement of tissue-specific and species-specific peptides has been presented by several groups. Our own data show that some T cells recognize entities that are not solely defined by the amino acid sequence of the stimulating molecule. Here, the involvement of a polymorphic peptide is postualted. So far a very limited number of studies have shown that such peptides can be of M H C origin.

ACKNOWLEDGMENTS

l wish to thank Saskia de Koster, Rend de Vries, and Jon van Rood for helpful and stimulating discussions, Willeke Schroeijers and Lemke Braun for technical assistence, and Mariette Mulder for typing the manuscript. This work was supported by theJ.A. Cohen Institute for Radiopathology and Radiation Protection.

REFERENCES 1. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC: Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329:506, 1987. 2. Bjorkman PJ, Saper MA, Samraoui B, Bennett WS, Strominger JL, Wiley DC: The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512, 1987. 3. BrownJH,Jardetzky T, Saper MA, Samraoui B, Bjorkman PJ, Wiley DC: A hypothetical model of the foreign antigen binding site of class II histocompatibility molecules. Nature 332:845, 1988. 4. GuilletJG, Lai MZ, Briner TJ, SmithJA, Gefter MA: Interaction of peptide antigens and class II major histocompatibility antigens. Nature 324:260, 1986. 5. Kilgus J, Romagnoli P,Guttinger M, Stuber D, Adorini L, Sinigaglia F: Vaccine T-cell epitope selection by a peptide competition assay. Proc Natl Acad Sci (USA) 86:1629, 1989. 6. Babbit BP, Matsueda G, Haber E, Unanue ER, Allen PM: Binding of immunogenic peptides to Ia histocompatibility molecules. Nature 317: 359, 1985.

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