Summary In the thymus, high affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck. Coreceptor scanning is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (~0.2s) to initiate negative selection compared to MHCI restricted TCRs (~0.9s) because more CD4 coreceptors are Lck-loaded compared to CD8. Based on experimental data and mathematical analysis, we generated a model (Lck come&stay/signal duration) that accurately predicts the experimentally observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.

The structural characteristics of autoreactive-T cell receptor (TCR) engagement of major histocompatability (MHC) class II-restricted self-antigens is established, but how autoimmune-TCRs interact with self-MHC class I has been unclear. We examined how CD8+ T cells kill human islet β-cells, in Type-1 diabetes, via autoreactive-TCR (1E6) recognition of an HLA-A*0201-restricted glucose-sensitive preproinsulin peptide. Rigid ‘lock-and-key’ binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHCI-restricted TCRs. However, this interaction was extraordinarily weak, due to limited contacts with MHCI. TCR binding was highly peptide-centric, dominated by two CDR3-loop-encoded residues, acting as an ‘aromatic-cap’, over the peptide MHCI (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8+ T cell-mediated autoreactivity.

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease.

Key Points• MHCI-restricted TCRs exhibit an explicit preference for a single MHCI-peptide length.• Effective CD8 ϩ T-cell immunity can only be achieved by length-matched Ag-specific T-cell clonotypes.␣␤-TCRs expressed at the CD8 ؉ T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8 ؉ T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8 ؉ T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8 ؉ T-cell clonotypes. (Blood. 2013;121(7):1112-1123)

Despite the ∼1018 αβ T cell receptor (TCR) structures that can be randomly manufactured by the human thymus, some surface more frequently than others. The pinnacles of this distortion are public TCRs, which exhibit amino acid-identical structures across different individuals. Public TCRs are thought to result from both recombinatorial bias and antigen-driven selection, but the mechanisms that underlie inter-individual TCR sharing are still largely theoretical. To examine this phenomenon at the atomic level, we solved the co-complex structure of one of the most widespread and numerically frequent public TCRs in the human population. The archetypal AS01 public TCR recognizes an immunodominant BMLF1 peptide, derived from the ubiquitous Epstein-Barr virus, bound to HLA-A*0201. The AS01 TCR was observed to dock in a diagonal fashion, grasping the solvent exposed peptide crest with two sets of complementarity-determining region (CDR) loops, and was fastened to the peptide and HLA-A*0201 platform with residue sets found only within TCR genes biased in the public response. Computer simulations of a random V(D)J recombination process demonstrated that both TCRα and TCRβ amino acid sequences could be manufactured easily, thereby explaining the prevalence of this receptor across different individuals. Interestingly, the AS01 TCR was encoded largely by germline DNA, indicating that the TCR loci already comprise gene segments that specifically recognize this ancient pathogen. Such pattern recognition receptor-like traits within the αβ TCR system further blur the boundaries between the adaptive and innate immune systems.

Background: The molecular principles governing T-cell specificity are poorly understood.Results: High affinity binding of a melanoma-specific T-cell receptor (TCR) is mediated through new MHC contacts and distinct thermodynamics.Conclusion: A novel thermodynamic mechanism upholds TCR-peptide specificity.Significance: TCRs can maintain peptide specificity using a mechanism that may enable widespread, safe enhancement of TCR binding affinity in therapeutic applications.

MHC anchor residue-modified “heteroclitic” peptides have been used in many cancer vaccine trials and often induce greater immune responses than the wild-type peptide. The best-studied system to date is the decamer MART-1/Melan-A26–35 peptide, EAAGIGILTV, where the natural alanine at position 2 has been modified to leucine to improve human leukocyte antigen (HLA)-A*0201 anchoring. The resulting ELAGIGILTV peptide has been used in many studies. We recently showed that T cells primed with the ELAGIGILTV peptide can fail to recognize the natural tumor-expressed peptide efficiently, thereby providing a potential molecular reason for why clinical trials of this peptide have been unsuccessful. Here, we solved the structure of a TCR in complex with HLA-A*0201-EAAGIGILTV peptide and compared it with its heteroclitic counterpart, HLA-A*0201-ELAGIGILTV. The data demonstrate that a suboptimal anchor residue at position 2 enables the TCR to “pull” the peptide away from the MHC binding groove, facilitating extra contacts with both the peptide and MHC surface. These data explain how a TCR can distinguish between two epitopes that differ by only a single MHC anchor residue and demonstrate how weak MHC anchoring can enable an induced-fit interaction with the TCR. Our findings constitute a novel demonstration of the extreme sensitivity of the TCR to minor alterations in peptide conformation.

Background: TCR recognition of bipartite ligands composed of self (MHC) and non-self (peptide) maintains T-cell specificity.Results: Mutation of residues in the cognate peptide override TCR mutations that enhance MHC binding.Conclusion: TCR-pMHC binding affinity requires specific TCR-peptide interactions.Significance: Stabilization of TCR-pMHC engagement by TCR-peptide interactions maintains T-cell specificity and prevents recognition of self-pMHC in the periphery.