Determination of structural principles underlying three different modes of lymphocytic choriomeningitis virus escape from CTL recognition

Lymphocytic choriomeningitis virus infection of H-2(b) mice generates a strong CD8(+) CTL response mainly directed toward three immunodominant epitopes, one of which, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. This CTL response acts as a selective agent for the emergence of viral escape variants. These variants generate altered peptide ligands (APLs) that, when presented by class I MHC molecules, antagonize CTL recognition and ultimately allow the virus to evade the cellular immune response. The emergence of APLs of the gp33 epitope is particularly advantageous for LCMV, as it allows viral escape in the context of both H-2D(b) and H-2K(b) MHC class I molecules. We have determined crystal structures of three different APLs of gp33 in complex with both H-2D(b) and H-2K(b). Comparison between these APL/MHC structures and those of the index gp33 peptide/MHC reveals the structural basis for three different strategies used by LCMV viral escape mutations: 1) conformational changes in peptide and MHC residues that are potential TCR contacts, 2) impairment of APL binding to the MHC peptide binding cleft, and 3) introduction of subtle changes at the TCR/pMHC interface, such as the removal of a single hydroxyl group.     

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.     

High affinity xenoreactive TCR:MHC interaction recruits CD8 in absence of binding to MHC

The TCR from a xenoreactive murine cytotoxic T lymphocyte clone, AHIII 12.2, recognizes murine H-2D(b) complexed with peptide p1058 (FAPGFFPYL) as well as human HLA-A2.1 complexed with human self-peptide p1049 (ALWGFFPVL). To understand more about T cell biology and cross-reactivity, the ectodomains of the AHIII 12.2 TCR have been produced in E. coli as inclusion bodies and the protein folded to its native conformation. Flow cytometric and surface plasmon resonance analyses indicate that human p1049/A2 has a significantly greater affinity for the murine AHIII 12.2 TCR than does murine p1058/D(b). Yet, T cell binding and cytolytic activity are independent of CD8 when stimulated with human p1049/A2 as demonstrated with anti-CD8 Abs that block CD8 association with MHC. Even in the absence of direct CD8 binding, stimulation of AHIII 12.2 T cells with "CD8-independent" p1049/A2 produces p56(lck) activation and calcium flux. Confocal fluorescence microscopy and fluorescence resonance energy transfer flow cytometry demonstrate CD8 is recruited to the site of TCR:peptide MHC binding. Taken together, these results indicate that there exists another mechanism for recruitment of CD8 during high affinity TCR:peptide MHC engagement.     

How a single T cell receptor recognizes both self and foreign MHC

alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.     

Protective Efficacy of Cross-Reactive CD8+ T Cells Recognising Mutant Viral Epitopes Depends on Peptide-MHC-I Structural Interactions and T Cell Activation Threshold

Emergence of a new influenza strain leads to a rapid global spread of the virus due to minimal antibody immunity. Pre-existing CD8⁺ T-cell immunity directed towards conserved internal viral regions can greatly ameliorate the disease. However, mutational escape within the T cell epitopes is a substantial issue for virus control and vaccine design. Although mutations can result in a loss of T cell recognition, some variants generate cross-reactive T cell responses. In this study, we used reverse genetics to modify the influenza NP_336–374 peptide at a partially-solvent exposed residue (N->A, NPN3A mutation) to assess the availability, effectiveness and mechanism underlying influenza-specific cross-reactive T cell responses. The engineered virus induced a diminished CD8⁺ T cell response and selected a narrowed T cell receptor (TCR) repertoire within two Vβ regions (Vβ8.3 and Vβ9). This can be partially explained by the H-2D^bNPN3A structure that showed a loss of several contacts between the NPN3A peptide and H-2D^b, including a contact with His155, a position known to play an important role in mediating TCR-pMHC-I interactions. Despite these differences, common cross-reactive TCRs were detected in both the naïve and immune NPN3A-specific TCR repertoires. However, while the NPN3A epitope primes memory T-cells that give an equivalent recall response to the mutant or wild-type (wt) virus, both are markedly lower than wt->wt challenge. Such decreased CD8⁺ responses elicited after heterologous challenge resulted in delayed viral clearance from the infected lung. Furthermore, mice first exposed to the wt virus give a poor, low avidity response following secondary infection with the mutant. Thus, the protective efficacy of cross-reactive CD8⁺ T cells recognising mutant viral epitopes depend on peptide-MHC-I structural interactions and functional avidity. Our study does not support vaccine strategies that include immunization against commonly selected cross-reactive variants with mutations at partially-solvent exposed residues that have characteristics comparable to NPN3A.     

Inflammation-associated nitrotyrosination affects TCR recognition through reduced stability and alteration of the molecular surface of the MHC complex

Nitrotyrosination of proteins, a hallmark of inflammation, may result in the production of MHC-restricted neoantigens that can be recognized by T cells and bypass the constraints of immunological self-tolerance. Here we biochemically and structurally assessed how nitrotyrosination of the lymphocytic choriomeningitis virus (LCMV)-associated immunodominant MHC class I-restricted epitopes gp33 and gp34 alters T cell recognition in the context of both H-2D(b) and H-2K(b). Comparative analysis of the crystal structures of H-2K(b)/gp34 and H-2K(b)/NY-gp34 demonstrated that nitrotyrosination of p3Y in gp34 abrogates a hydrogen bond interaction formed with the H-2K(b) residue E152. As a consequence the conformation of the TCR-interacting E152 was profoundly altered in H-2K(b)/NY-gp34 when compared to H-2K(b)/gp34, thereby modifying the surface of the nitrotyrosinated MHC complex. Furthermore, nitrotyrosination of gp34 resulted in structural over-packing, straining the overall conformation and considerably reducing the stability of the H-2K(b)/NY-gp34 MHC complex when compared to H-2K(b)/gp34. Our structural analysis also indicates that nitrotyrosination of the main TCR-interacting residue p4Y in gp33 abrogates recognition of H-2D(b)/gp33-NY complexes by H-2D(b)/gp33-specific T cells through sterical hindrance. In conclusion, this study provides the first structural and biochemical evidence for how MHC class I-restricted nitrotyrosinated neoantigens may enable viral escape and break immune tolerance.     

The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2(b) and H-2(d) haplotypes. In contrast, there is a dearth of structural data regarding H-2(k)-encoded molecules. Therefore, the structures of H-2K(k) complexed to an octameric peptide from influenza A virus (HA(259-266)) and to a nonameric peptide from SV40 (SV40(560-568)) have been determined by x-ray crystallography at 2.5 and 3.0 A resolutions, respectively. The structure of the H-2K(k)-HA(259-266) complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2K(k) groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2K(k) peptide-binding groove. As a result, the Calpha atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2K(b)-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2K(k), the backbone of the SV40(560-568) nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2L(d). This structural convergence occurs despite the totally different architectures of the H-2L(d) and H-2K(k) peptide-binding grooves. Therefore, these two H-2K(k)-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.     

Structural and biological basis of CTL escape in coronavirus-infected mice

Cytotoxic T lymphocyte escape occurs in many human infections, as well as mice infected with the JHM strain of mouse hepatitis virus, which exhibit CTL escape variants with mutations in a single epitope from the spike glycoprotein (S510). In all CTL epitopes prone to escape, only a subset of all potential variants is generally detected, even though many of the changes that are not selected would result in evasion of the T cell response. It is postulated that these unselected mutations significantly impair virus fitness. To define more precisely the basis for this preferential selection, we combine x-ray crystallographic studies of the MHC class I (D(b))/S510 complexes with viral reverse genetics to identify a prominent TCR contact residue (tryptophan at position 4) prone to escape mutations. The data show that a mutation that is commonly detected in chronically infected mice (tryptophan to arginine) potently disrupts the topology of the complex, explaining its selection. However, other mutations at this residue, which also abrogate the CTL response, are never selected in vivo even though they do not compromise virus fitness in acutely infected animals or induce a significant de novo CTL response. Thus, while structural analyses of the S510/D(b) complex provide a strong basis for why some CTL escape variants are selected, our results also show that factors other than effects on virus fitness limit the diversification of CD8 T cell epitopes.     

In vivo selection of a lymphocytic choriomeningitis virus variant that affects recognition of the GP33-43 epitope by H-2Db but not H-2Kb

CD8 T cells drive the protective immune response to lymphocytic choriomeningitis virus (LCMV) infection and are thus a determining force in the selection of viral variants. To examine how escape mutations affect the presentation and recognition of overlapping T-cell epitopes, we isolated an LCMV variant that is not recognized by T-cell receptor (TCR)-transgenic H-2Db-restricted LCMV GP33-41-specific cytotoxic T lymphocytes (CTL). The variant virus carried a single-amino-acid substitution (valine to alanine) at position 35 of the viral glycoprotein. This region of the LCMV glycoprotein encodes both the Db-restricted GP33-43 epitope and a second epitope (GP34-42) presented by the Kb molecule. We determined that the V-to-A CTL escape mutant failed to induce a Db GP33-43-specific CTL response and that Db-restricted GP33-43-specific CTL induced by the wild-type LCMV strain were unable to kill target cells infected with the variant LCMV strain. In contrast, the Kb-restricted response was much less affected. We found that the V-to-A substitution severely impaired peptide binding to Db but not to Kb molecules. Strikingly, the V-to-A mutation did not change any of the anchor residues, and the dramatic effect on binding was therefore unexpected. The strong decrease in Db binding explains why the variant virus escapes the Db GP33-43-specific response but still elicits the Kb-restricted response. These findings also illustrate that mutations within regions encoding overlapping T-cell epitopes can differentially affect the presentation and recognition of individual epitopes.     

Thymic selection and peripheral activation of CD8 T cells by the same class I MHC/peptide complex

Thymic selection is controlled by the interaction between TCR and MHC/peptide. Strength and quality of the signal determine whether thymocytes are selected or deleted. The factors that contribute to this signal remain poorly defined. Here we show that fetal thymic organ cultures (FTOCs) derived from OT-I transgenic mice (the OT-I TCR is restricted by K(b)-SIINFEKL) on a K(b)D(b-/-) background support positive selection, but only when provided with soluble H-2K(b)-SIINFEKL complexes. Selection of CD8 T cells is independent of the valency of the ligand or its capability to coengage CD8 molecules. Both CD8alphaalpha and CD8alphabeta T cells are selected by H-2K(b)-SIINFEKL, but only CD8alphabeta cells are capable of releasing IFN-gamma in response to the same ligand. The alpha(4)beta(7) integrin is up-regulated on postselection thymocytes from FTOCs. After adoptive transfer, FTOC-derived OT-I CD8 T cells divide in response to the agonist peptide SIINFEKL. These results establish that CD8 T cells responsive to their nominal peptide-Ag can be generated in FTOC supplemented with soluble MHC class I molecules equipped with the same peptide.     

The mode of ligand recognition by two peptide:MHC class I-specific monoclonal antibodies.

The Ig superfamily members TCR and B cell receptor (BCR) share high structural and amino acid homology, yet interact with Ags in a distinct manner. The overall shape of the TCR ligand is rather constant, with the variation coming from the MHC polymorphism and the peptide heterogeneity. Consequently, the TCR alpha- and beta-chains form a relatively flat ligand-binding site that interacts with the peptide:MHC (pep:MHC) ligand in a fixed diagonal orientation relative to the MHC alpha-helices, with the alpha- and beta-chains of the TCR contacting the N and C termini of the pep:MHC complex, respectively. By contrast, the shape of BCR ligands varies dramatically, and the BCR exhibits much greater variability of the Ag-binding site. The mAbs 25-D1.16 (D1) and 22-C5.9 (C5), specific for the OVA-8:H-2Kb complex, allowed us to directly compare how TCR and BCR approach the same ligand. To that effect, we mapped D1 and C5 footprints over the OVA-8:H-2Kb complex. Using peptide variants and mutant MHC molecules, we show that the D1 and C5 contacts with the OVA-8:Kb complex C terminus overlap with the TCR beta-chain footprint, but that this footprint also extends to the regions of the molecule not contacted by the TCR. These studies suggest that D1 and C5 exhibit a hybrid mode of pep:MHC recognition, in part similar to that of the TCR beta-chain and in part similar to the conventional anti-MHC Ab.     

Conversion of tyrosine to the inflammation-associated analog 3'-nitrotyrosine at either TCR- or MHC-contact positions can profoundly affect recognition of the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein 33 by CD8 T cells

Immunohistochemical detection of increased levels of protein-associated nitrotyrosine has become widely used as a surrogate marker of in situ inflammation. However, the potential consequences of protein-associated nitrotyrosine formation in terms of cellular immune recognition has received surprisingly little attention. Using a well-defined I-E(K)-restricted epitope of pigeon cytochrome c, we previously demonstrated that conversion of a single tyrosine residue to nitrotyrosine can have a profound effect on recognition by CD4 T cells. In this study, we used the MHC class I-restricted epitope of lymphocytic choriomeningitis virus glycoprotein (gp33) to demonstrate that conversion of tyrosine to nitrotyrosine can also profoundly affect recognition of MHC class I-restricted epitopes. Conversion of the Y4 residue of the gp33 epitope to nitrotyrosine completely abrogated recognition by gp33-specific T cells from P14 TCR-transgenic mice. In contrast, CD8(+) T cells specific for "nitrated gp33" (NY-gp33) can be readily elicited in C57BL/6 mice after immunization with NY-gp33 peptide. Interestingly, T-T hybridomas specific for NY-gp33 peptide were found to fall into two distinct subsets, being specific for NY-gp33 presented in the context of either H-2D(b) or H-2K(b). This latter result is surprising in light of previous structural studies showing that Y4 comprises a critical TCR-contact residue when presented by H-2D(b) but that the same residue points downward into the peptide-binding groove of the MHC when presented by H-2K(b). Together, these results indicate that nitrotyrosine formation can impact T cell recognition both directly, through alteration of TCR-contact residues, or indirectly, through alterations in MHC-contact positions.     

Cutting edge: culture with high doses of viral peptide induces previously unprimed CD8(+) T cells to produce cytokine

Culturing naive T cells with 50 microM selected HIV-1 envelope peptides for 6 days in the presence of IL-2 drives the emergence of a substantial CD8(+) population that secretes IFN-gamma following short-term stimulation with 1 microM peptide. This response is H-2K(b) restricted, epitope specific, and requires the continuing presence of peptide. The same effect was found for known H-2D(b)-restricted peptides from two influenza virus proteins. The great majority of these influenza-specific CD8(+)IFN-gamma(+) T cells neither stained with the cognate tetramer nor expressed the TCR Vbeta bias that is characteristic of the CD8(+) set expanded in vivo during an infection. Thus, multipoint binding of low affinity TCRs on naive CD8(+) T cells can drive peptide-specific cytokine production. However, at least for two influenza-derived epitopes, the avidity of the TCR-MHC peptide interaction appears to be insufficient to stabilize a tetrameric complex of MHC class I glycoprotein plus peptide on the lymphocyte surface.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.     

Effect of decreasing the affinity of the class II-associated invariant chain peptide on the MHC class II peptide repertoire in the presence or absence of H-2M

The class II-associated invariant chain peptide (CLIP) region of the invariant chain (Ii) directly influences MHC class II presentation by occupying the MHC class II peptide-binding groove, thereby preventing premature loading of peptides. Different MHC class II alleles exhibit distinct affinities for CLIP, and a low affinity interaction has been associated with decreased dependence upon H-2M and increased susceptibility to rheumatoid arthritis, suggesting that decreased CLIP affinity alters the MHC class II-bound peptide repertoire, thereby promoting autoimmunity. To examine the role of CLIP affinity in determining the MHC class II peptide repertoire, we generated transgenic mice expressing either wild-type human Ii or human Ii containing a CLIP region of low affinity for MHC class II. Our data indicate that although degradation intermediates of Ii containing a CLIP region with decreased affinity for MHC class II do not remain associated with I-A(b), this does not substantially alter the peptide repertoire bound by MHC class II or increase autoimmune susceptibility in the mice. This implies that the affinity of the CLIP:MHC class II interaction is not a strong contributory factor in determining the probability of developing autoimmunity. In contrast, in the absence of H-2M, MHC class II peptide repertoire diversity is enhanced by decreasing the affinity of CLIP for MHC class II, although MHC class II cell surface expression is reduced. Thus, we show clearly, in vivo, the critical chaperone function of H-2M, which preserves MHC class II molecules for high affinity peptide binding upon dissociation of Ii degradation intermediates.     

Recognition of an MHC class I-restricted antigenic peptide can be modulated by para-substitution of its buried tyrosine residues in a TCR-specific manner

Conformational dependence of TCR contact residues of the H-2Kb molecule on the two buried tyrosine side chains of the vesicular stomatitis virus (VSV)-8 peptide was investigated by systematic substitutions of the tyrosines with phenylalanine, p-fluorophenylalanine (pFF), or p-bromophenylalanine (pBrF). The results of peptide competition CTL assays revealed that all of the peptide variants, except for the pBrF analogues, had near-native binding to the H-2Kb molecule. Epitope-mapped anti-H-2Kb mAbs detected conformational differences among H-2Kb molecules stabilized with these VSV-8 variants on RMA-S cells. Selective recognition of the VSV-8 analogues was displayed by a panel of three H-2Kb-restricted, anti-VSV-8 TCRs. Thus, these substitutions result in an antigenically significant conformational change of the MHC molecular surface structure at both C and D pockets, and the effect of this change on cognate T cell recognition is dependent on the TCR structure. Our results confirm that the structure of buried peptide side chains can determine the surface conformation of the MHC molecule and demonstrate that even a very subtle structural nuance of the buried side chain can be incorporated into the surface conformation of the MHC molecule. The ability of buried residues to modulate this molecular surface augments the number of residues on the MHC-peptide complex that can be recognized as "foreign" by the CD8+ T cell repertoire and allows for a higher level of antigenic discrimination. This may be an important mechanism to expand the total number of TCR specificities that can respond to a single peptide determinant.     

Relaxed DM requirements during class II peptide loading and CD4+ T cell maturation in BALB/c mice

Current ideas about DM actions have been strongly influenced by studies of mutant strains expressing the H-2(b) haplotype. To evaluate DM contributions to class II activities in BALB/c mice, we generated a novel mutation at the DMa locus via embryonic stem cell technology. Unlike long-lived A(b)/class II-associated invariant chain-derived peptide (CLIP) complexes, mature A(d) and E(d) molecules are loosely occupied by class II-associated invariant chain-derived peptide and are SDS unstable. BALB/c DM mutants weakly express BP107 conformational epitopes and toxic shock syndrome toxin-1 superantigen-binding capabilities, consistent with partial occupancy by wild-type ligands. Near normal numbers of mature CD4(+) T cells fail to undergo superantigen-mediated negative selection, as judged by TCR Vbeta usage. Ag presentation assays reveal consistent differences for A(d)- and E(d)-restricted T cells. Indeed, the mutation leads to decreased peptide capture by A(d) molecules, and in striking contrast causes enhanced peptide loading by E(d) molecules. Thus, DM requirements differ for class II structural variants coexpressed under physiological conditions in the intact animal.     

CD8+ T cell activation is governed by TCR-peptide/MHC affinity, not dissociation rate

Binding of peptide/MHC (pMHC) complexes by TCR initiates T cell activation. Despite long interest, the exact relationship between the biochemistry of TCR/pMHC interaction (particularly TCR affinity or ligand off-rate) and T cell responses remains unresolved, because the number of complexes examined in each independent system has been too small to draw a definitive conclusion. To test the current models of T cell activation, we have analyzed the interactions between the mouse P14 TCR and a set of altered peptides based on the lymphocytic choriomeningitis virus epitope gp33-41 sequence bound to mouse class I MHC D(b). pMHC binding, TCR-binding characteristics, CD8+ T cell cytotoxicity, and IFN-gamma production were measured for the peptides. We found affinity correlated well with both cytotoxicity and IFN-gamma production. In contrast, no correlation was observed between any kinetic parameter of TCR-pMHC interaction and cytotoxicity or IFN-gamma production. This study strongly argues for an affinity threshold model of T cell activation.     

A structural difference limited to one residue of the antigenic peptide can profoundly alter the biological outcome of the TCR-peptide/MHC class I interaction

The vesicular stomatitis virus (VSV) octapeptide RGYVYQGL binds to H-2K(b) and triggers a cytotoxic T cell response in mice. A variant peptide, RGYVYEGL (E6) with a glutamic acid for glutamine replacement at position 6 of the VSV peptide, elicits a T cell response with features that are quite different from those elicited by the wild-type VSV peptide. The differences found in the nature of the T cells responding to the E6 peptide include changes in both the V beta elements and the sequences of the complementarity-determining region 3 loops of their TCRs. Further experiments found that the E6 peptide can act as an antagonist for VSV-specific T cell hybridomas. To determine whether these differences in V beta usage, complementarity-determining region 3 sequences, and the switch from agonism to antagonism are caused by a conformational change on the MHC, the peptide, or both, we determined the crystal structure of the variant E6 peptide bound to H-2K(b). This structure shows that the only significant structural difference between H-2K(b)/E6 and the previously determined H-2K(b)/VSV is limited to the side chain of position 6 of the peptide, with no differences in the MHC molecule. Thus, a minor conformational change in the peptide can profoundly alter the biological outcome of the TCR-peptide/MHC interaction.     

Crystal structure of a non-canonical high affinity peptide complexed with MHC class I: a novel use of alternative anchors

The crystal structure of a non-standard peptide, YEA9, in complex with H-2Kb, at 1.5 A resolution demonstrates how YEA9 peptide can bind with surprisingly high affinity through insertion of alternative, long, non-canonical anchors into the B and E pockets. The use of "alternative pockets" represents a new mode of high affinity peptide binding, that should be considered when predicting peptide epitopes for MHC class I. These novel interactions encountered in this non-canonical high affinity peptide-MHC complex should help predict additional binding peptides from primary protein sequences and aid in the design of alternative approaches for peptide-based vaccines.