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The Journal of Immunology, 2002, 169: 3146-3154.
Copyright © 2002 by The American Association of Immunologists

MHC Allele-Specific Molecular Features Determine Peptide/HLA-A2 Conformations That Are Recognized by HLA-A2-Restricted T Cell Receptors1

Zichun Wang*, Richard Turner*, Brian M. Baker{dagger} and William E. Biddison2,*

* Molecular Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and {dagger} Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The structures of {alpha}{beta} TCRs bound to complexes of class I MHC molecules and peptide show that the TCRs make multiple contacts with the {alpha}1 and {alpha}2 helixes of the MHC. Previously we have shown that the A6 TCR in complex with the HLA-A2/Tax peptide has 15 contact sites on HLA-A2. Single amino acid mutagenesis of these contact sites demonstrated that mutation of only three amino acids clustered on the {alpha}1 helix (R65, K66, A69) disrupted recognition by the A6 TCR. In the present study we have asked whether TCRs that recognize four other peptides presented by HLA-A2 interact with the MHC in identical, similar, or different patterns as the A6 TCR. Mutants K66A and Q155A had the highest frequency of negative effects on lysis. A subset of peptide-specific CTL also selectively recognized mutants K66A or Q155A in the absence of exogenous cognate peptides, indicating that these mutations affected the presentation of endogenous peptide/HLA-A2 complexes. These findings suggest that most HLA-A2-restricted TCRs recognize surfaces on the HLA-A2/peptide complex that are dependent upon the side chains of K66 and Q155 in the central portion of the peptide binding groove. Crystallographic structures of several peptide/HLA-A2 structures have shown that the side chains of these critical amino acids that make contact with the A6 TCR also contact the bound peptide. Collectively, our results indicate that the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs as well as influence the binding and presentation of the bound peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Differentiation of T cells and execution of T cell effector functions are triggered by interactions between the TCR and MHC/peptide complexes (1, 2, 3). The specificity of {alpha}/{beta} TCR Ag recognition is determined by independent interactions of the TCR CDR loops with the bound peptide and the MHC molecule (1, 2, 3). Structural studies have shown that TCRs bind peptide/MHC complexes in a generally conserved binding mode, with the receptor positioned diagonally over the peptide/MHC, but with variations in the diagonal orientation of as much as 30o (4, 5, 6, 7, 8, 9, 10, 11, 12). Approximately one-third of the surface area contacted by the TCR is contributed by the peptide, and two-thirds are contributed by the MHC molecule. Compared with Ag-Ab interactions, the interface between the TCR and peptide/MHC is poorly packed, with cavities, channels, and buried water molecules (6, 8, 12, 13). This poor shape complementarity between TCRs and peptide/MHC complexes is reflected in the weak to moderate affinity of the TCR for its ligand (14).

Previously, we have shown that the human A6 {alpha}{beta} TCR is present on a CD8+ CTL clone that recognizes the human T cell leukemia virus type 1 (HTLV-I)3 Tax 11–19 peptide presented by HLA-A2 (15). We have studied the interaction of the A6 TCR with the Tax peptide/HLA-A2 complex functionally, structurally, biochemically, and biophysically (5, 7, 8, 13, 16, 17, 18, 19, 20). Briefly, our studies showed that the A6 TCR binds the HLA-A2 molecule by contacting six amino acids on the {alpha}1 helix and nine amino acids on the {alpha}2 helix (5). To investigate their relative contributions to binding, alanine scanning mutagenesis was performed on these contact amino acids. The results showed that only three amino acids (R65, K66, A69), clustered on the {alpha}1 helix, were critical for recognition by the A6 TCR and that mutation of at least one of these amino acids affected recognition by 201 other Tax-specific CTL lines (19). Thus, the area around amino acids R65, K66, and A69 appears to provide a critical focus for all Tax-specific TCRs that were examined.

In the present study we have asked whether TCRs that are specific for other peptides presented by HLA-A2 also focus on this same area of the HLA-A2 molecule. We have analyzed CTL lines specific for four other peptides presented by HLA-A2 for the capacity to recognize their specific peptides presented by the same panel of HLA-A2 mutants that were used for the Tax peptide-specific TCR study (19). Our results suggest that for most HLA-A2-restricted TCRs, only a select few amino acids (positions 66 and 155 on the {alpha}1 and {alpha}2 helixes) are critical for peptide/HLA-A2 recognition. Furthermore, by making contacts to both peptide and TCR, these critical amino acids may directly influence both peptide presentation as well as TCR binding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation and expression of HLA-A2 mutant transfectants

A panel of HLA-A2 mutants (Table IGo) was created as previously described (19) and transfected into Hmy2.C1R cells (21). In this mutant panel each of the HLA-A2 amino acids contacted by the Tax-specific A6 TCR was replaced with alanine (or glycine in cases of alanine in the wild-type) by site-directed mutagenesis. Transfectants were assayed for cell surface expression using the anti-HLA-A2 Abs BB7.2 and MA2.1 (22, 23, 24, 25) with a FACSCalibur (BD Biosciences, Mountain View, CA) as previously described (19). All mutant transfectants showed cell surface expression of HLA-A2 at similar levels as wild-type HLA-A2 as detected by one or both HLA-A2-specific Abs (19).


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  		Table I. Summary of the effect of HLA-A2 mutants on recognition by the Tax-specific A6 TCR

 
Generation and assay of HLA-A2-restricted peptide-specific CTL

The peptides recognized by our panels of HLA-A2-restricted CTL are listed in Table IIGo. A panel of 14 influenza virus matrix peptide M158–66-specific CTL lines was generated in limiting dilution culture from the PBL of two normal HLA-A*0201 donors, as previously described (26). A panel of nine melanoma Ag recognized by T cells-1 (MART-1) 27–35-specific CTL lines was generated from the PBL of two HLA-A*0201 patients with metastatic melanoma, as previously described (27). One melanoma gp100209–217-specific CTL population (JH) was obtained from the tumor-infiltrating lymphocytes of metastatic melanoma lesions from an HLA-A*0201 melanoma patient (28). These melanoma Ag-specific CTL lines were gifts from Dr. F. Marincola (National Institutes of Health, Bethesda, MD). A panel of 12 CTL lines specific for the human CMV (HCMV) matrix protein pp65495–503 was generated in limiting dilution culture from the PBL of two normal HLA-A*0201 donors, exactly as we previously described for the generation of M1-specific CTL (26). The HTLV-I Tax-specific A6 TCR-bearing clone, RS56, was isolated from the PBL of an HLA-A*0201 patient with HTLV-I-associated myelopathy/tropical spastic paraparesis (15). All these CTL populations were shown to lyse HLA-A2+ targets pulsed with their cognate peptide and did not lyse HLA-A2+ targets pulsed with other HLA-A2-restricted peptides. Cytotoxicity was quantified by a time-resolved fluorometric assay using HLA-A2 wild-type and mutant-transfected Hmy2.C1R cells as target cells, as described previously (7).


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  		Table II. Peptides used in these experiments

 
Peptide binding to cell surface HLA-A2 molecules

A modification of the acid strip procedure reported by Storkus et al. (34) was used as previously described (35). HLA-A2 wild-type and mutant transfectants were incubated with 20 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO) for 2 h at 37°C to block the transport of newly synthesized class I molecules to the cell surface. The cells were washed in PBS and resuspended in 0.13 M citric acid/PBS (pH 3.0), 0.5% human serum albumin, and 10 µg/ml human {beta}2-microglobulin for 2 min on ice. The cells were washed with PBS, 0.5% human serum albumin, and 5 µg/ml {beta}2-microglobulin, and resuspended in the same buffer plus 5 µg/ml peptide and 2 µg/ml brefeldin A. The cells were incubated for 3 h at room temperature, and then cell surface HLA-A2 expression was quantified by indirect immunofluorescence with BB7.2 and was analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of HLA-A2 mutations on recognition of influenza matrix peptide M158–66/HLA-A2

Fourteen CTL lines specific for influenza virus M158–66/HLA-A2 were isolated from the PBL of two HLA-A2+ donors. These CTL lines were assayed on our panel of HLA-A2 mutants at A6 TCR contacts pulsed with varying concentrations of the M1 peptide (Table IGo). Thirteen of 14 of these CTL lines displayed the pattern exhibited by CTL line 2; only K66A ({alpha}1 helix) and Q155A ({alpha}2 helix) showed a >100-fold reduction in recognition of the M1 peptide relative to wild-type HLA-A2.1 (Fig. 1Go, A and B). The remaining {alpha}1 or {alpha}2 helix mutants showed a <10-fold reduction in M1 recognition. CTL line 35 was unique in that it recognized K66A and Q155A with a <10-fold reduction in M1 presentation relative to HLA-A2. (Fig. 1Go, C and D). The results with all the mutants are summarized in Table IIIGo.



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  		FIGURE 1. Presentation of the M1 peptide by wild-type and {alpha}1 helix (A and C) and {alpha}2 helix (B and D) mutants to M1-specific CTL lines 2 (A and B) and 35 (C and D). E:T cell ratio, 2.5:1.

 

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  		Table III. Summary of effects of HLA-A2 mutants on recognition by Tax, M1, MART-1, pp65, and gp100 peptide-specific CTL lines

 
Effects of HLA-A2 mutations on recognition of MART-127–35/HLA-A2

A panel of nine CTL lines specific for the MART-1 peptide was assayed on the same panel of HLA-A2 mutants. K66A produced a marked reduction (>100-fold) in recognition of MART-1 by six of nine CTL lines tested (e.g., B10–88; Fig. 2GoA), but had no significant effect on recognition by two other CTL lines (e.g., B10–49; Fig. 2BGo). K68A had moderate effects (one to two orders of magnitude more MART-1 peptide required vs A2.1) on all CTL lines (e.g., B10–88; Fig. 2GoB). For the {alpha}2 helix mutants, Q155A produced marked negative effects on MART-1 recognition by seven of nine CTL lines (e.g., B10–177; Fig. 2GoC), while having little or no effect on recognition by the other two CTL lines (e.g., B1–35; Fig. 2GoD). The results for all mutants are summarized in Table IIIGo.



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  		FIGURE 2. Presentation of the MART-1 peptide by wild-type and {alpha}1 helix (A and B) and {alpha}2 helix (C and D) mutants to MART-1-specific CTL lines B10–88 (A), B10–49 (B), B10–177 (C), and B1–35 (D). E:T cell ratio, 2.5:1.

 
Effect of HLA-A2 mutations on recognition of HCMV matrix peptide pp65495–503/HLA-A2

Twelve CTL lines specific for pp65 were assayed on the same panel of mutants as described above. Of the {alpha}1 helix mutants, K66A produced a >100-fold reduction in the amount of pp65 required to sensitize target cells for lysis by seven of 12 CTL lines tested (e.g., 4.74; Fig. 3GoA), but had no effect on the other five CTL lines (e.g., 4.65; Fig. 3GoB). None of the {alpha}2 helix mutants had negative effects on the majority of pp65-specific CTL lines (e.g., 4.65; Fig. 3GoC). The results for all mutants are summarized in Table IIIGo.



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  		FIGURE 3. Presentation of the pp65 peptide by wild-type and {alpha}1 helix (A and B) and {alpha}2 helix (C) mutants to pp65-specific CTL lines 4.74 (A) and 4.65 (B and C). E:T cell ratio, 2.5:1.

 
Effect of HLA-A2 mutations on recognition of melanoma gp100209–217/HLA-A2

We have also analyzed one peptide-specific polyclonal CTL population that was not derived under limiting dilution culture conditions and is specific for melanoma peptide gp100209–217. The results at the limiting peptide concentration of 1 nM are shown in Fig. 4Go. Only mutants K66A and Q155A had clear negative effects on recognition by this CTL population.



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  		FIGURE 4. Recognition of HLA-A2 mutants by the gp100-specific CTL population. All targets were pulsed with 1 nM gp100209–217. No lysis by this CTL population was observed on any target in the absence of peptide (data not shown). E:T cell ratio, 5:1.

 
Recognition of HLA-A2 mutants in the absence of exogenous cognate peptide

For each of the peptide-specific CTL line/target cell combinations tested, a negative control of CTL with target cells pulsed without exogenous cognate peptide was always included. This control was always negative for wild-type HLA-A2 without exogenous peptide, because that condition was one of the selection criteria used in screening the CTL lines that grew out of limiting dilution cultures. However, in a subset of peptide-specific CTL/mutant HLA-A2 combinations, the mutant targets were strongly lysed in the absence of exogenous cognate peptide. The most dramatic example of recognition without exogenous cognate peptide was found in our panel of HCMV pp65-specific CTL lines: 10 of the 12 CTL lines from two different donors selectively lysed Q155A without exogenous peptide (one representative line from each donor is shown in Fig. 5Go, A and B). The other two pp65-specific CTL lines did not recognize any mutant without peptide (e.g., 4.65; Fig. 3GoC). Similarly, two MART-1-specific CTL lines, B10–98 and B10–143, selectively recognized K66A in the absence of exogenous peptide (B10.98; Fig. 5GoC; B10–143 had the same pattern (not shown), but expressed different V{beta} genes (27)). No mutant targets were recognized by any of the M1-specific CTL lines or the gp100-specific polyclonal CTL population. These results suggest that 1) a subset of peptide-specific TCRs can recognize endogenous peptide(s) when presented by K66A or Q155A, but not when these endogenous peptides are presented by wild-type HLA-A2 or any of the other HLA-A2 mutants; and/or 2) the K66A and Q155A mutants change the composition and/or conformation of the endogenous peptides that they bind, and a subset of peptide-specific TCRs exists that can recognize these different peptides or different conformations.



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  		FIGURE 5. Recognition of HLA-A2 mutants Q155A and K66A in the absence of exogenous peptide. The pp65-specific CTL lines IIG9 (A) and 4.74 (B) and the MART-1-specific CTL line B10–98 (C) were assayed on the panel of mutants in the absence of exogenous peptides or in the presence of increasing concentrations of their cognate peptides (D). E:T cell ratio, 2.5:1.

 
If the highly selective recognition of K66A and Q155A in the absence of exogenous peptide was due to recognition of endogenous peptides differentially presented by these mutant molecules, it may be possible to block this recognition with a high concentration of a noncognate HLA-A2 binding peptide. However, lysis of K66A by MART-1-specific B10–98 and lysis of Q155A by pp65-specific IIG9 were not blocked by addition of 1000 nM Tax peptide (data not shown). Although addition of the cognate peptide produced a slight increase in lysis (Fig. 5GoD), these results together cannot distinguish between the possibilities that these peptide-specific TCRs cannot discriminate between bound endogenous peptide(s) and the noncognate peptide, or that peptide recognition is not necessary for lysis. To more directly examine the role of endogenous peptide(s), wild-type HLA-A2 and mutant K66A and Q155A cell surface molecules were acid stripped of endogenous peptides (34) and reconstituted with HLA-A2 binding peptides. Flow cytometric analysis of cells reconstituted with no peptide revealed a 1–2 log reduction in cell surface expression of HLA-A2 (histograms are shown in Fig. 6GoA; mean fluorescence intensity values for each curve are plotted in Fig. 6GoB). Reconstitution with individual HLA-A2 binding peptides resulted in partial reconstitution of cell surface expression, but not to levels before acid stripping. Q155A displayed poor reconstitution with all HLA-A2 binding peptides. Despite the significant reduction in cell surface expression of HLA-A2 following acid stripping, both K66A (Fig. 7GoA) and Q155A (Fig. 7GoB) showed little reduction in lysis in the absence of reconstituting peptide, and little effect on lysis was observed upon addition of the various HLA-A2-binding peptides. Thus, peptide-specific TCR recognition of these mutant molecules is either peptide independent or involves peptide(s) that are not removed by the acid-stripping procedure.



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  		FIGURE 6. Flow cytometric analysis of HLA-A2, K66A, and Q155A before and after acid stripping and reconstitution without exogenous peptide or with HLA-A2 binding peptides. Cells were stained with BB7.2 before (No Rx) and after acid stripping and reconstitution with no peptide or with the indicated peptide. Flow cytometric histograms are shown in A, and mean fluorescence intensity values are plotted in B.

 


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  		FIGURE 7. Lysis of target cells before and after acid stripping and reconstitution. The MART-1-specific CTL line B10–98 was assayed on K66A (A), and the pp65-specific CTL line IIG9 was assayed on Q155A (B) before (No Rx) and after acid stripping and reconstitution without exogenous peptide or with the indicated peptides. E:T cell ratio, 2.5:1.

 
Comparison of effects of HLA-A2 mutations on recognition by five peptide-specific CTL lines: general effects

Table IIIGo compares the effects of the HLA-A2 mutants on recognition of the Tax-specific A6 TCR and the major effects of the mutants on recognition by the majority of the CTL lines from the panel of M1-, MART-1-, and pp65-specific CTL lines and the gp100-specific CTL population. For each peptide tested, very few mutants had any significant negative effects (defined as a >100-fold reduction in the amount of peptide required to achieve lysis relative to wild-type A2.1). These results suggested three general possibilities: 1) the amino acids at those positions are not contacted by these TCRs; 2) the amino acids at those positions are contacted, but the interaction does not contribute significantly to the stability of the TCR-peptide/MHC interaction; or 3) the amino acids at those positions are contacted, but the TCRs have enough flexibility to be able to accommodate the alanine or glycine substitutions. Furthermore, in general, none of the mutations results in global destabilizing effects within the TCR-peptide/MHC interface, since every mutant was lysed by at least one CTL line.

Among those few mutants that did show strong negative effects, K66 was the most widely shared element. Mutation of K66 to alanine had a negative effect on recognition by the majority of CTL lines specific for each of the five peptides examined. The next most widely shared element was Q155, in which the Q155A mutant produced marked reduction in recognition of M1, MART-1, and gp100. The Q155A mutant was lysed in the absence of added peptide by 10 of 12 pp65-specific CTL lines tested, also suggesting a critical role for position 155 in these CTL.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our previous study on HLA-A2 recognition by Tax peptide-specific T cells identified a critical TCR focal point on the {alpha}1 helix of HLA-A2 consisting of lysine 66, arginine 65, and alanine 69 (19). Here we have extended that study to include HLA-A2-restricted T cells of different peptide specificity. The results demonstrate that lysine 66 of the Tax/HLA-A2 focal point is a common requirement for most HLA-A2-restricted TCRs. A second site critical for many HLA-A2-restricted TCRs, not identified in our previous study, is glutamine 155 on the {alpha}2 helix.

Lysine 66 and glutamine 155 are situated near the center of the {alpha}1 and {alpha}2 helixes, respectively, and structures of TCRs bound to class I peptide/MHC complexes (5, 7) indicate that both positions are contacted by the TCR. Alterations at these positions might thus be expected to affect TCR recognition by changing the number and type of contacts made as well as changing the electrostatic surface potential and overall MHC topology. However, K66 and Q155 frequently make multiple contacts with the bound peptide. In four nonamer peptides crystallized with HLA-A2, K66 makes multiple contacts with all four peptides, including a potential hydrogen bond with the peptide backbone, and Q155 makes contacts with three of the four peptides (36). K66 is on the rim of the B pocket, and Q155 is on the rim of the D pocket, where they can contact peptide main chain polar atoms adjacent to a side chain that extends into these pockets (37). Because of the potential influence of these peptide-MHC interactions, it is likely that in addition to directly altering TCR contacts, changing the electrostatic surface potential, and overall MHC topology, the mutations result in changes in peptide conformation or mobility that cannot be accommodated by most peptide-specific TCRs. Thus, the generalized effects of changes at these critical amino acids are probably due to the fact that they can be directly contacted by TCRs, and they can influence the presentation of the bound peptides. The extent to which these different mechanisms influence our findings cannot yet be ascertained and may, in fact, differ with different TCR/peptide combinations. In this regard it is instructive to note that the K66A mutation results in an ~20-fold weakening of TCR binding affinity with the A6 TCR (from ~1 to 20 µM) (19). This value is in the range of weak agonists for the A6 TCR (18), yet Tax/HLA-A2 targets with the K66A mutation are not lysed by the vast majority of Tax/HLA-A2-specific CTL lines (19). As indicated above, K66 makes a number of contacts to the Tax peptide in the Tax/HLA-A2 crystal structures (5, 7, 36).

In the crystallographic structure of the M1 peptide with HLA-A2, the side chain of Q155 forms the rim of a pocket that retains the ring of phenylalanine 5 of the M1 peptide in a position away from where a TCR would be predicted to sit (36); the contacts between Q155 and the Tax peptide in the Tax/HLA-A2 structure appear much less substantive (36). This may provide a structural explanation for why the Q155A mutation has such a dramatic effect with CTL specific for the M1 peptide presented by HLA-A2 and not Tax (Table IIIGo). Again, though, as Q155 is also in a position to contact M1-specific TCRs, the extent to which contacts to peptide and contacts to TCR differentially influence TCR recognition cannot yet be determined.

Included in our panel of mutants were Q72A and W167A. It is useful to compare these positions to K66 and Q155, as both Q72 and W167 contact both the A6 and B7 Tax-specific TCRs and the Tax peptide bound to HLA-A2 (5, 7), yet neither of these mutants had a major effect on T cell recognition. These findings indicate that although several HLA-A2 amino acids can make contacts with both the TCR and the bound peptide, not all these interactions have the same functional significance. It is also possible that additional amino acid positions on the surface of HLA-A2 are critical for TCR recognition, but were not identified in our panel of A6 TCR contact mutants.

Both K66 and Q155 are highly conserved in all known HLA-A alleles; position 66 has only lysine or arginine and position 155 is always glutamine (38). It was recently predicted that the naturally selected mature T cell repertoire contains remnants of conserved interactions with MHC residues and that these residues vary from one TCR/MHC pair to another (39). Evidence in favor of this prediction may be obtained by comparison of the data presented in this report on TCR/HLA-A2 with data on recognition of TCR/H-2Kb (40). In that study TCR/H-2Kb recognition was analyzed with panels of two virus-specific CTL and alloreactive CTL clones and a series of H-2Kb mutants that were either selected by an alloreactive H-2Kb-specific CTL clone or by H-2Kb-specific mAbs (40) (no mutations at positions 66 and 155 were included in this panel). The results showed that a predominant recognition pattern of most H-2Kb-specific TCRs existed; the most disruptive mutants were clustered on the {alpha}2 helix around position 167, with a second area of disruption on the {alpha}1 helix around position 82. For known H-2K alleles, position 167 is almost always tryptophan, and position 82 is only leucine or glutamine (41). A similar analysis of CTL recognition of H-2Ld mutants using a panel of alloreactive, viral peptide- and tumor Ag peptide-specific CTL, also found a common recognition pattern (42). The H-2Ld mutants that had the most negative effects for 75% of the clones examined in that study were at positions 69, 72, 76, and 155/157. This common recognition pattern for H-2Ld involves both relatively conserved (72 and 76) and polymorphic (69 and 155) amino acid residues (42). Thus, it appears that most H-2Kb-specific, H-2Ld-specific, and HLA-A2-specific TCRs have allele-specific recognition patterns for elements on the {alpha}1 and {alpha}2 helices of these class I molecules; however these allele-specific recognition patterns are clearly different from each other. Fig. 8Go illustrates the locations of these elements on the surface of HLA-A2, H-2Kb, and H-2Ld (we note that in the structure of H-2Ld with the p29 peptide, position 155 forms numerous interactions with the peptide (43), again highlighting the possibility that mutations here can affect peptide as well as TCR binding).



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  		FIGURE 8. Locations of regions on the surfaces of HLA-A2, H-2Kb, and H-2Ld experimentally determined to provide critical functional requirements for TCRs restricted for each MHC (structures from Refs. 36 48 , and 49 ). Position 155 (outlined on H-2Kb) is the only critical element predicted to be in common for these three MHC molecules (see Discussion). The figure was generated with InsightII (Accelrys, San Diego, CA).

 
Another comparison of importance to our understanding of TCR/MHC interactions is the effect of mutations at position 155 in H-2Kb with that observed here in HLA-A2. The mutant H-2Kb molecule, Kbm1, has an arginine to tyrosine mutation at position 155 (44). Comparison of the crystal structures of H-2Kb and H-2 Kbm1 bound to vesicular stomatitis virus (VSV) and Sendai virus (SEV) peptides indicates that the mutation at position 155 causes a major change in local electrostatic potential and surface topology of the H-2 Kbm1 molecule (45). The mutations in H-2Kbm1 block recognition of the VSV and SEV epitopes by H-2Kb-restricted CTL, and they also alter the peptide conformations and decrease the half-life of the peptide/MHC complexes at 37°C (45); particularly interesting is the loss of an interaction between the terminal (charged) nitrogen on the side chain of R155 and the polar hydroxyl on tyrosine 3 of the VSV peptide (no such interactions are present between R155 of H-2Kb and the SEV peptide). As noted previously, it is difficult to ascertain whether the loss of activity with H-2Kbm1 is due to changes in TCR contacts at position 155 or alterations in the mode of peptide presentation. Jaulin et al. (46) showed that a panel of 21 CTL clones that recognize a Plasmodium berghei peptide presented by H-2Kd was unable or had a diminished capacity to recognize the peptide presented by an H-2Kd mutant with a tyrosine to alanine substitution at position 155. Finally, as noted by Rudolph and colleagues (45), the amino acid at position 155 contacts the TCR in all class I TCR/peptide/MHC crystal structures determined to date. Together, these data suggest that the amino acid at position 155 may provide critical contacts with most TCRs that recognize class I molecules.

One intriguing aspect of our study is the identification of a subset of CTL lines that exhibited the unusual property of highly selective recognition of HLA-A2 mutants in the absence of exogenous peptide. Ten of the 12 pp65-specific CTL lines selectively lysed Q155A targets with no added peptide, and two of the MART-1 specific lines selectively recognized K66A with no added peptide. It is possible that these peptide-specific TCRs cross-react with an endogenous peptide(s) that is bound by both wild-type and mutant HLA-A2 molecules, but can only achieve sufficient affinity to trigger lysis when it binds the mutant HLA-A2 molecule. The failure of noncognate peptide or acid stripping to eliminate recognition could mean that the endogenous peptide was very tightly bound and is recognized with high affinity, or that recognition was largely peptide independent. TCR affinity ceilings or windows have been proposed (47); according to these models, binding tighter than a certain threshold would result in diminished activity. It is possible that peptide-specific TCRs have been selected for specific unfavorable interactions within the interface that act to limit binding affinity to maintain it below such a threshold. In these cross-reactive cases, perhaps the mutations have replaced an unfavorable interaction with a favorable (or neutral) one that allows binding to proceed with an affinity tight enough for activity despite the presence of a suboptimal peptide. However, recent work with a TCR engineered for very high affinity that still results in peptide-dependent T cell activity questions the existence of affinity thresholds (48). An important question is why is this cross-reactivity observed with mutations that generally have a negative impact on T cell recognition, e.g., positions 66 and 155? One possible explanation is that these mutants were specifically selected for recognition from our panel of mutants because both these amino acids contact both the TCRs and the bound peptides in the critical central region of the peptide binding groove and significantly affect interactions with the CDR3 loops of the TCRs and alter the conformations of the bound peptides that are contacted by these loops.

Positions that have the dual capacity to directly affect both peptide as well as TCR binding may not be limited to class I MHC molecules. Amino acids in the third hypervariable region (HVR3, positions 67–74) of class II MHC {beta}-chains form contacts to peptide (49) and, in the case of the structure with the mouse TCR D10, also contact the TCR (11). Furthermore, Doherty et al. (50) demonstrated that, at least in human DR{beta} alleles, mutations in this region can negatively affect peptide binding. Thus, the capacity for MHC amino acids to influence both peptide and TCR binding may be a general consequence of the need for a TCR to recognize a surface contributed to by both peptide and MHC. Finally, our observation that changes at these critical dual contact positions result in alloreactivity is also not unique to class I molecules, as substitutions in the HVR3 region are responsible for the alloreactivity of certain mouse class II alleles with the D10 TCR (51, 52).

In conclusion, we have identified two important positions on the surface of HLA-A2 that are critical for recognition by TCRs specific for five different peptides. These positions, K66 and Q155, may simultaneously affect both TCR binding as well as peptide presentation. Based on these observations, future structural studies can now be conducted that will provide further insights into the mechanisms of TCR interactions with peptide/MHC complexes.


   Acknowledgments
 
We thank Drs. Jay A. Berzofsky and Eleanor S. Metcalf for helpful discussions.


   Footnotes
 
1 This work was supported by University of Notre Dame College of Science and the Department of Chemistry and Biochemistry (to B.M.B.). Back

2 Address correspondence and reprint requests to Dr. William E. Biddison, National Institutes of Health, Building 10/Room 5B-16, Bethesda, MD 20892. E-mail address: web{at}ninds.nih.gov Back

3 Abbreviations used in this paper: HTLV, human T cell leukemia virus type 1; HCMV, human CMV; VSV, vesicular stomatitis virus; MART-1, melanoma Ag recognized by T cells-1; SEV, Sendai virus. Back

Received for publication May 1, 2002. Accepted for publication July 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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