Proline substitution independently enhances H‐2Db complex stabilization and TCR recognition of melanoma‐associated peptides

The immunogenicity of H‐2D^b (D^b) restricted epitopes can be significantly increased by substituting peptide position 3 to a proline (p3P). The p3P modification enhances MHC stability without altering the conformation of the modified epitope allowing for T‐cell cross‐reactivity with the native peptide. The present study reveals how specific interactions between p3P and the highly conserved MHC heavy chain residue Y159 increase the stability of D^b in complex with an optimized version of the melanoma‐associated epitope gp100_25–33. Furthermore, the p3P modification directly increased the affinity of the D^b/gp100_25–33‐specific T‐cell receptor (TCR) pMel. Surprisingly, the enhanced TCR binding was independent from the observed increased stability of the optimized D^b/gp100_25–33 complex and from the interactions formed between p3P and Y159, indicating a direct effect of the p3P modification on TCR recognition.     

A novel T‐cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice

We used a newly generated T‐cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10‐mer) or SSVWNATTA (9‐mer) in complex with class I MHC antigen H‐2D^b, were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8⁺ T cells can detect 10‐ and 9‐mer length variants similarly. This result varies from the idea that a given T‐cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10‐mer was presented on the surface of uninfected and infected CD8α⁺CD11c⁺ dendritic cells, which suggests the use of direct and cross‐presentation pathways. In contrast, CD11b⁺CD11c^? cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell‐type specific mechanisms of antigen presentation in vivo.     

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2D^b using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind D^b. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to D^b. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the D^b pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited D^b-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all D^b-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, D^b-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to D^b, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.     

MHC I tetramer staining tends to overestimate the number of functionally relevant self-reactive CD8 T cells in the preimmune repertoire

Previous studies that used peptide-MHC (pMHC) tetramers (tet) to identify self-specific T cells have questioned the effectiveness of thymic-negative selection. Here, we used pMHCI tet to enumerate CD8 T cells specific for the immunodominant gp33 epitope of lymphocytic choriomeningitis virus glycoprotein (GP) in mice transgenically engineered to express high levels of GP as a self-antigen in the thymus. In GP-transgenic mice (GP⁺), monoclonal P14 TCR⁺ CD8 T cells that express a GP-specific TCR could not be detected by gp33/D^b-tet staining, indicative of their complete intrathymic deletion. By contrast, in the same GP⁺ mice, substantial numbers of polyclonal CD8 T cells identifiable by gp33/D^b-tet were present. The gp33-tet staining profiles of polyclonal T cells from GP⁺ and GP-negative (GP⁻) mice were overlapping, but mean fluorescence intensities were ∼15% lower in cells from GP⁺ mice. Remarkably, the gp33-tet⁺ T cells in GP⁺ mice failed to clonally expand after lymphocytic choriomeningitis virus infection, whereas those of GP⁻ mice did so. In Nur77^GFP-reporter mice, dose-dependent responses to gp33 peptide-induced TCR stimulation revealed that gp33-tet⁺ T cells with high ligand sensitivity are lacking in GP⁺ mice. Hence, pMHCI tet staining identifies self-specific CD8 T cells but tends to overestimate the number of truly self-reactive cells.     

Immunization with glycosylated Kb-binding peptides generates carbohydrate-specific,unrestricted cytotoxic T cells

Cytotoxic T cells (CTL) recognize target proteins as short peptides presented by major histocompatibility complex (MHC) class I restriction elements. However, there is also evidence for peptide-independent T cell receptor (TCR) recognition of target proteins and non-protein structures. How such T cell responses are generated is presently unclear. We generated carbohydrate (CHO)-specific, MHC-unrestricted CTL responses by coupling di- and trisaccharides to K^b- or D^b-binding peptides for direct immunization in mice. Four peptides and three CHO have been analyzed with the CHO either in terminal or central positions on the carrier peptide. With two of these glycopeptides, with galabiose (Galα1-4Gal; Gal₂) bound to a homocysteine (via an ethylene spacer arm) in position 4 or 6 in a vesicular stomatitis virus nucleoprotein-derived peptide (RGYVYQGL binding to K^b), CTL were generated which preferentially killed target cells treated with glycopeptide compared to those treated with the core peptide. Polyclonal CTL were also found to kill target cells expressing the same Gal₂ epitope in a glycolipid. By fractionation of CTL, preliminary data indicate that glycopeptide-specific K^b-restricted CTL and unrestricted CHO-specific CTL belong to different T cell populations with regard to TCR expression. The results demonstrate that hapten-specific unrestricted CTL responses can be generated with MHC class I-binding carrier peptides. Different models that might explain the generation of such responses are discussed.     

Selection of T-cell receptors with a recurrent CDR3β peptide-contact motif within the repertoire of alloreactive CD8(+) T cells

Peptide/MHC complexes recognized by alloreactive T lymphocytes (TLs) have been identified, but their contribution to in vivo allo‐rejection is not known. We previously characterized the peptide pBM1, highly represented among endogenous H‐2K^b (K^b)‐associated peptides and critically required to induce full activation of H‐2^k monoclonal CD8⁺ TLs expressing the cognate TCR‐BM3.3. Here, we asked whether a pBM1/K^b‐specific TL subset could be detected within a polyclonal TL population rejecting allogeneic cells in vivo. We show that the proportion of pBM1/K^b‐binding CD8⁺ TLs increased from <0.04% in naïve mice to 3% of activated CD44⁺ CD8⁺ TLs in H‐2^k mice rejecting K^b‐expressing cells. Among these, TCR‐Vβ2 usage was greatly enriched, and 75% of them shared a TCR‐Vβ2 CDR3β motif with the prototype TCR‐BM3.3. Fewer than 5% of K^b‐reactive CD44⁺ CD8⁺ TLs not binding pBM1/K^b displayed this CDR3β motif. We found that the recurrent CDR3β motif of pBM1/K^b‐binding TLs was assembled from distinct V/D/J recombination events, suggesting that it is recruited upon immunization for its optimal TCR‐peptide/MHC fit. Thus, a CDR3β motif generated by a process akin to “convergent recombination” accounts for a sizable fraction of the alloreactive anti‐K^b TCR repertoire.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Major histocompatibility complex recognition by immune receptors: Differences among T cell receptor versus antibody interactions with the VSV8/H-2Kb complex

The surface residues of the VSV8/K^b complex important for recognition by N15 and N26 αβ T cell receptors (TCR) were mapped by mutational analysis and compared to each other and with epitopes of well-characterized K^b specific monoclonal antibodies (mAb). Three features of immune receptor recognition emerge. First, the footprints of the two TCR on VSV8/K^b are similar with more than 80% overlap between sites. Given that only 8 of 14 surface exposed VSV8/K^b residues identified as critical for TCR interaction are in common, the chemical basis of the N15 and N26 interactions is nevertheless distinct. Second, the cognate peptide is a major focus of TCR recognition: mutation at any of the three exposed side chains (at p1, p4 or p6) abrogates interaction of both TCR as measured by functional T cell activation. Third, in contrast to TCR, mAb bind to discrete segments on the periphery of the α1 and/or α2 helices without orientational restriction. These findings suggest that unlike soluble antibodies, surface membrane receptor-ligand interactions on opposing cells (i.e. TCR-peptide/MHC, CD8-MHC) limit the orientational freedom of the TCR in the immune recognition process.     

Major histocompatibility complex class I-restricted presentation of influenza virus nucleoprotein peptide by B lymphoma cells harboring an antibody gene antigenized with the virus peptide

We analyzed the capacity of B cells to process and present a peptide from the variable region of an endogenous immunoglobulin heavy (H) chain to a major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocyte (CTL) clone. The H-chain gene was engineered to express 14-amino acid peptide from the sequence of the influenza virus nucleoprotein (NP) antigen in the third complementarity-determining region (CDR3). This NP peptide is presented in association with the D^b allele in H?2^b mice. We demonstrate that B lymphoma cells (H-2^b) harboring the antigenized H-chain gene process and present the NP peptide in association with the D^b molecule and are lysed by a CTL clone specific for that peptide in an MHC-restricted way. In contrast, the soluble antigenized antibody failed to mediate lysis of H?2^b target cells. The endogenously processed immunoglobulin CDR3 peptide could be eluted from surface D^b molecules in transfected cells. This study formally demonstrates that peptides from the hypervariable loops of endogenous immunoglobulin are processed through the endogenous degradative pathway and are presented to CD8⁺ T cells in the context of MHC class I molecules. The implication of these findings for processing and presentation of endogenous immunoglobulin peptides in B cells and network regulation by idiopeptides is discussed.     

Availability of autoantigenic epitopes controls phenotype,severity, and penetrance in TCR Tg autoimmune gastritis

We examined TCR:MHC/peptide interactions and in vivo epitope availability to explore the Th1‐ or Th2‐like phenotype of autoimmune disease in two TCR Tg mouse models of autoimmune gastritis (AIG). The TCR of strains A23 and A51 recognize distinct IA^d‐restricted peptides from the gastric parietal cell H/K‐ATPase. Both peptides form extremely stable MHC/peptide (MHC/p) complexes. All A23 animals develop a Th1‐like aggressive, inflammatory AIG early in life, while A51 mice develop indolent Th2‐like AIG at 6–8 wk with incomplete penetrance. A51 T cells were more sensitive than A23 to low doses of soluble antigen and to MHC/p complexes. Staining with IA^d/peptide tetramers was only detectable on previously activated T cells from A51. Thus, despite inducing a milder AIG, the A51 TCR displays a higher avidity for its cognate IA^d/peptide. Nonetheless, in vivo proliferation of adoptively transferred A51 CFSE‐labeled T cells in the gastric lymph node was relatively poor compared with A23 T cells. Also, DC from WT gastric lymph node, presenting processed antigen available in vivo, stimulated proliferation of A23 T cells better than A51. Thus, the autoimmune potential of these TCR in their respective Tg lines is strongly influenced by the availability of the peptide epitope, rather than by differential avidity for their respective MHC/p complexes.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Characterization of the human CD4+ T‐cell repertoire specific for major histocompatibility class I‐restricted antigens

While CD4⁺ T lymphocytes usually recognize antigens in the context of major histocompatibility (MHC) class II alleles, occurrence of MHC class‐I restricted CD4⁺ T cells has been reported sporadically. Taking advantage of a highly sensitive MHC tetramer‐based enrichment approach allowing detection and isolation of scarce Ag‐specific T cells, we performed a systematic comparative analysis of HLA‐A*0201‐restricted CD4⁺ and CD8⁺ T‐cell lines directed against several immunodominant viral or tumoral antigens. CD4⁺ T cells directed against every peptide‐MHC class I complexes tested were detected in all donors. These cells yielded strong cytotoxic and T helper 1 cytokine responses when incubated with HLA‐A2⁺ target cells carrying the relevant epitopes. HLA‐A2‐restricted CD4⁺ T cells were seldom expanded in immune HLA‐A2⁺ donors, suggesting that they are not usually engaged in in vivo immune responses against the corresponding peptide‐MHC class I complexes. However, these T cells expressed TCR of very high affinity and were expanded following ex vivo stimulation by relevant tumor cells. Therefore, we describe a versatile and efficient strategy for generation of MHC class‐I restricted T helper cells and high affinity TCR that could be used for adoptive T‐cell transfer‐ or TCR gene transfer‐based immunotherapies.     

Going Pro to enhance T‐cell immunogenicity: Easy as π?

MHC class I molecules bind intracellular oligopeptides and present them on the cell surface for CD8⁺ T‐cell activation and recognition. Strong peptide/MHC class I (pMHC) interactions typically induce the best CD8⁺ T‐cell responses; however, many immunotherapeutic tumor‐specific peptides bind MHC with low affinity. To overcome this, immunologists can carefully alter peptides for enhanced MHC affinity but often at the cost of decreased T‐cell recognition. A new report published in this issue of the European Journal of Immunology [Eur. J. Immunol. 2013. 43:3051–3060] shows that the substitution of proline at the third residue (p3P) of a common tumor peptide increases pMHC affinity and complex stability while enhancing T‐cell receptor recognition. X‐ray crystallography indicates that stability is generated through newly introduced CH‐π bonding between p3P and a conserved residue (Y159) in the MHC heavy chain. This finding highlights a previously unappreciated role for CH‐π bonding in MHC peptide binding, and importantly, arms immunologists with a novel and possibly general approach for increasing pMHC stability without compromising T‐cell recognition.     

Elongated peptides,not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein

The peptides recognized by an H-2D^b-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2D^b binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2D^b was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2D^b with an efficacy similar to that of the nonapeptide corresponding to the H-2D^b motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2D^b cleft. Because the carboxy-terminal part is thus larger than predicted this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.     

Efficient help for autoreactive B‐cell activation requires CD4+ T‐cell recognition of an agonist peptide at the effector stage

T‐cell recognition of peptide/MHC complexes is flexible and can lead to differential activation, but how interactions with agonist (full activation) or partial agonist (suboptimal activation) peptides can shape immune responses in vivo is not well characterized. We investigated the effect of stimulation by agonist or partial agonist ligands during initial CD4⁺ T‐cell priming, and subsequent T‐B‐cell cognate interactions, on antibody production by anti‐chromatin B cells. We found that autoantibody production required TCR recognition of an agonist peptide at the effector stage of B‐cell activation. However, interaction with a weak agonist ligand at this effector stage failed to promote efficient autoantibody production, even if the CD4⁺ T cells were fully primed by an agonist peptide. These studies suggest that the reactivity of the TCR for a target self‐peptide during CD4⁺ T‐B‐cell interaction can be a critical determinant in restraining anti‐chromatin autoantibody production.     

Enhanced positive selection of a transgenic TCR by a restriction element that does not permit negative selection

Very little is known about the conformational properties ofthe MHC molecules that are able to signal positive selectionof a given TCR. To try to understand these parameters and todetermine whether these requirements are shared with interactionsduring negative selection and antigen recognition, we have studiedselection and antigen recognition of a transgenic TCR (specificfor lymphocytic choriomeningitls virus glycoprotein and H-2D^b)in the context of two D^b mutants, H-2^bm13 and H-2^bm14. The datashowed that the transgenic TCR was not positively selected bythe H-2^bm14 haplotype but, interestingly, enhanced positiveselection was seen in H-2^bm13 mice. The transgenic TCR couldnot be negatively selected In H-2^bm13animals persistently infectedwith the virus (neonatal virus carrier mice), nor could thetransgenic TCR be activated by H-2^bm13 infected cells in vivoor in vitro. These experiments show that although a TCR maybe selected by a mutant MHC molecule, the corresponding viralantigen cannot be recognized in the context of the mutant MHCmolecule, as Judged by both negative selection and T cell reactivityin vivo and in vitro. The ‘enhanced’ positive selectionoccurring in the context of D^bm13 suggests that a differentconformation of the MHC molecule is able to select the sameTCR and also that various TCR-ligand avidities may permit positiveselection.     

Presentation of a horse cytochrome c peptide by multiple H-2b class I major histocompatibility complex (MHC) molecules to C57BL/6- and bm1-derived cytotoxic T lymphocytes: Presence of a single MHC anchor residue may confer efficient peptide-specific CTL recognition

In this study the immunogenic tryptic fragment from a horse cytochrome c (cyt c) digest recognized by cytotoxic T lymphocytes (CTL), induced by in vitro peptide stimulation from C57BL/6 (B6) and mutant B6.C-H-2^bm1 (bm1) mice is identified. An identical sequence, p40—53, is recognized by CTL from both B6 and bm1 mice. In addition, both B6 and bm1 cloned CTL lines display unusual major histocompatibility complex (MHC) class I-restricted recognition of this peptide in that they respond to it in the context of H-2K^b, H-2D^b, and H-2K^bm1 class I molecules, although the sequence lacks the usual structural K^b and D^b peptide-binding motifs. Truncated analogues which resemble the lengths of naturally processed MHC class I-presented peptides, confer reactivity for B6 and bm1 CTL against EL4 (H-2^b) targets as well as the L cell transfectants, L + K^b, L + D^b, and L + K^bm1. The antigenic peptide with the greatest potency is p41—49, which appears to be generated by angiotensin converting enzyme cleavage of the full-length p40—53 tryptic peptide. The minimum antigenic peptide recognized by both B6 and bm1 CTL, and which targets lysis on each of the transfectants, is the hexamer p43—48 peptide from horse cyt c. Residues Pro⁴⁴ and Thr⁴⁷, which occupy polymorphic positions with respect to other species-variant cyt c molecules, influence recognition of these peptides differently for the B6 and bm1 CTL. The ability of H-2K^b, H-2D^b, and mutant H-2K^bm1 class I molecules to present the same peptide to a single cloned CTL is discussed in the context of current knowledge of peptide anchor residues and side chain-specific binding pockets in the MHC class I peptide-binding site.     

A study of CDR3 loop dynamics reveals distinct mechanisms of peptide recognition by T‐cell receptors exhibiting different levels of cross‐reactivity

T‐cell receptors (TCRs) can productively interact with many different peptides bound within the MHC binding groove. This property varies with the level of cross‐reactivity of TCRs; some TCRs are particularly hyper cross‐reactive while others exhibit greater specificity. To elucidate the mechanism behind these differences, we studied five TCRs in complex with the same class II MHC (1A^b)‐peptide (3K), that are known to exhibit different levels of cross‐reactivity. Although these complexes have similar binding affinities, the interface areas between the TCR and the peptide–MHC (pMHC) differ significantly. We investigated static and dynamic structural features of the TCR–pMHC complexes and of TCRs in a free state, as well as the relationship between binding affinity and interface area. It was found that the TCRs known to exhibit lower levels of cross‐reactivity bound to pMHC using an induced‐fitting mechanism, forming large and tight interfaces rich in specific hydrogen bonds. In contrast, TCRs known to exhibit high levels of cross‐reactivity used a more rigid binding mechanism where non‐specific π‐interactions involving the bulky Trp residue in CDR3β dominated. As entropy loss upon binding in these highly degenerate and rigid TCRs is smaller than that in less degenerate TCRs, they can better tolerate changes in residues distal from the major contacts with MHC‐bound peptide. Hence, our dynamics study revealed that differences in the peptide recognition mechanisms by TCRs appear to correlate with the levels of T‐cell cross‐reactivity.     

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides

The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an αβ TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2D^b MHC molecule. Both peptides were shown to antagonize specifically the T cell cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands.