T-cell epitope prediction with combinatorial peptide libraries.

T cell receptors (TCR) recognize antigenic peptides in complex with the major histocompatibility complex (MHC) molecules and this trimolecular interaction initiates antigen-specific signaling pathways in the responding T lymphocytes. For the study of autoimmune diseases and vaccine development, it is important to identify peptides (epitopes) that can stimulate a given TCR. The use of combinatorial peptide libraries has recently been introduced as a powerful tool for this purpose. A combinatorial library of n-mer peptides is a set of complex mixtures each characterized by one position fixed to be a specified amino acid and all other positions randomized. A given TCR can be fingerprinted by screening a variety of combinatorial libraries using a proliferation assay. Here, we present statistical models for elucidating the recognition profile of a TCR using combinatorial library proliferation assay data and known MHC binding data.     

T cell receptor recognition via cooperative conformational plasticity

Although T cell receptor cross-reactivity is a fundamental property of the immune system and is implicated in numerous autoimmune pathologies, the molecular mechanisms by which T cell receptors can recognize and respond to diverse ligands are incompletely understood. In the current study we examined the response of the human T cell lymphotropic virus-1 (HTLV-1) Tax-specific T cell receptor (TCR) A6 to a panel of structurally distinct haptens coupled to the Tax 11-19 peptide with a lysine substitution at position 5 (Tax5K, LLFG[K-hapten]PVYV). The A6 TCR could cross-reactively recognize one of these haptenated peptides, Tax-5K-4-(3-Indolyl)-butyric acid (IBA), presented by HLA-A*0201. The crystal structures of Tax5K-IBA/HLA-A2 free and in complex with A6 reveal that binding is mediated by a mechanism of cooperative conformational plasticity involving conformational changes on both sides of the protein-protein interface, including the TCR complementarity determining region (CDR) loops, Valpha/Vbeta domain orientation, and the hapten-modified peptide. Our findings illustrate the complex role that protein dynamics can play in TCR cross-reactivity and highlight that T cell receptor recognition of ligand can be achieved through diverse and complex molecular mechanisms that can occur simultaneously in the interface, not limited to molecular mimicry and CDR loop shifts.     

Posttranslational modification of gluten shapes TCR usage in celiac disease

Posttranslational modification of Ag is implicated in several autoimmune diseases. In celiac disease, a cereal gluten-induced enteropathy with several autoimmune features, T cell recognition of the gluten Ag is heavily dependent on the posttranslational conversion of Gln to Glu residues. Evidence suggests that the enhanced recognition of deamidated gluten peptides results from improved peptide binding to the MHC and TCR interaction with the peptide-MHC complex. In this study, we report that there is a biased usage of TCR Vβ6.7 chain among TCRs reactive to the immunodominant DQ2-α-II gliadin epitope. We isolated Vβ6.7 and DQ2-αII tetramer-positive CD4(+) T cells from peripheral blood of gluten-challenged celiac patients and sequenced the TCRs of a large number of single T cells. TCR sequence analysis revealed in vivo clonal expansion, convergent recombination, semipublic response, and the notable conservation of a non-germline-encoded Arg residue in the CDR3β loop. Functional testing of a prototype DQ2-α-II-reactive TCR by analysis of TCR transfectants and soluble single-chain TCRs indicate that the deamidated residue in the DQ2-α-II peptide poses constraints on the TCR structure in which the conserved Arg residue is a critical element. The findings have implications for understanding T cell responses to posttranslationally modified Ags.     

A new twist in TCR diversity revealed by a forbidden alphabeta TCR

We report crystal structures of a negatively selected T cell receptor (TCR) that recognizes two I-A^u-restricted myelin basic protein peptides and one of its peptide/major histocompatibility complex (pMHC) ligands. Unusual complementarity-determining region (CDR) structural features revealed by our analyses identify a previously unrecognized mechanism by which the highly variable CDR3 regions define ligand specificity. In addition to the pMHC contact residues contributed by CDR3, the CDR3 residues buried deep within the Vα/Vβ interface exert indirect effects on recognition by influencing the Vα/Vβ interdomain angle. This phenomenon represents an additional mechanism for increasing the potential diversity of the TCR repertoire. Both the direct and indirect effects exerted by CDR residues can impact global TCR/MHC docking. Analysis of the available TCR structures in light of these results highlights the significance of the Vα/Vβ interdomain angle in determining specificity and indicates that TCR/pMHC interface features do not distinguish autoimmune from non-autoimmune class II-restricted TCRs.     

Differential peptide dynamics is linked to major histocompatibility complex polymorphism

Peptide presentation by major histocompatibility complex (MHC) molecules is of central importance for immune responses, which are triggered through recognition of peptide-loaded MHC molecules (pMHC) by cellular ligands such as T-cell receptors (TCR). However, a unifying link between structural features of pMHC and cellular responses has not been established. Instead, pMHC/TCR binding studies suggest conformational and/or flexibility changes of the binding partners as a possible cause of differential T-cell stimulation, but information on real-time dynamics is lacking. We therefore probed the real-time dynamics of a MHC-bound nonapeptide (m9), by combining time-resolved fluorescence depolarization and molecular dynamics simulations. Here we show that the nanosecond dynamics of this peptide presented by two human MHC class I subtypes (HLA-B*2705 and HLA-B*2709) with differential autoimmune disease association varies dramatically, despite virtually identical crystal structures. The peptide dynamics is linked to the single, buried polymorphic residue 116 in the peptide binding groove. Pronounced peptide flexibility is seen only for the non-disease-associated subtype HLA-B*2709, suggesting an entropic control of peptide recognition. Thermodynamic data obtained for two additional peptides support this hypothesis.     

Cross-reactivity in T-cell antigen recognition

The molecular interactions between the T-cell receptor (TCR) and peptide-MHC (pMHC) have been elucidated in recent years. Nevertheless, the fact that binding of only slightly different ligands by a TCR, or ligation of the same pMHC at different developmental stages of the T cell, can have opposing consequences, continues to pose intellectual challenges. Kinetic proofreading models, which have at their core the dissociation rates of pMHC from the TCR, are best suited to account for these observations. However, T cells can be triggered by peptides with often minimal homology to the primary immunogenic peptide. This cross-reactivity of the TCR is manifest at several levels, from positive selection of immature thymocytes to homeostasis and antigen-cross- reactive immune responses of mature peripheral T cells. The implications of the high cross-reactivity of T-cell antigen recognition for self-tolerance and T-cell memory are discussed.     

A reliable and safe T cell repertoire based on low-affinity T cell receptors

Antigens are presented to T cells as short peptides bound to MHC molecules on the surface of body cells. The binding between MHC/peptides and T cell receptors (TCRs) has a low affinity and is highly degenerate. Nevertheless, TCR-MHC/peptide recognition results in T cell activation of high specificity. Moreover, the immune system is able to mount a cellular response when only a small fraction of the MHC molecules on an antigen-presenting cell is occupied by foreign peptides, while autoimmunity remains relatively rare. We consider how to reconcile these seemingly contradictory facts using a quantitative model of TCR signalling and T cell activation. Taking into account the statistics of TCR recognition and antigen presentation, we show that thymic selection can produce a working T cell repertoire which will produce safe and effective responses, that is, recognizes foreign antigen presented at physiological levels while tolerating self. We introduce "activation curves" as a useful tool to study the repertoire's statistical activation properties.     

The study of high-affinity TCRs reveals duality in T cell recognition of antigen: specificity and degeneracy

TCRs exhibit a high degree of Ag specificity, even though their affinity for the peptide/MHC ligand is in the micromolar range. To explore how Ag specificity is achieved, we studied murine T cells expressing high-affinity TCRs engineered by in vitro evolution for binding to hemoglobin peptide/class II complex (Hb/I-Ek). These TCRs were shown previously to maintain Ag specificity, despite having up to 800-fold higher affinity. We compared the response of the high-affinity TCRs and the low-affinity 3.L2 TCR toward a comprehensive set of peptides containing single substitutions at each TCR contact residue. This specificity analysis revealed that the increase in affinity resulted in a dramatic increase in the number of stimulatory peptides. The apparent discrepancy between observed degeneracy in the recognition of single amino acid-substituted Hb peptides and overall Ag specificity of the high-affinity TCRs was examined by generating chimeric peptides between the stimulatory Hb and nonstimulatory moth cytochrome c peptides. These experiments showed that MHC anchor residues significantly affected TCR recognition of peptide. The high-affinity TCRs allowed us to estimate the affinity, in the millimolar range, of immunologically relevant interactions of the TCR with peptide/MHC ligands that were previously unmeasurable because of their weak nature. Thus, through the study of high-affinity TCRs, we demonstrated that a TCR is more tolerant of single TCR contact residue substitutions than other peptide changes, revealing that recognition of Ag by T cells can exhibit both specificity and degeneracy.     

Peptide Modulation of Class I Major Histocompatibility Complex Protein Molecular Flexibility and the Implications for Immune Recognition

T cells use the αβ T cell receptor (TCR) to recognize antigenic peptides presented by class I major histocompatibility complex proteins (pMHCs) on the surfaces of antigen-presenting cells. Flexibility in both TCRs and peptides plays an important role in antigen recognition and discrimination. Less clear is the role of flexibility in the MHC protein; although recent observations have indicated that mobility in the MHC can impact TCR recognition in a peptide-dependent fashion, the extent of this behavior is unknown. Here, using hydrogen/deuterium exchange, fluorescence anisotropy, and structural analyses, we show that the flexibility of the peptide binding groove of the class I MHC protein HLA-A*0201 varies significantly with different peptides. The variations extend throughout the binding groove, impacting regions contacted by TCRs as well as other activating and inhibitory receptors of the immune system. Our results are consistent with statistical mechanical models of protein structure and dynamics, in which the binding of different peptides alters the populations and exchange kinetics of substates in the MHC conformational ensemble. Altered MHC flexibility will influence receptor engagement, impacting conformational adaptations, entropic penalties associated with receptor recognition, and the populations of binding-competent states. Our results highlight a previously unrecognized aspect of the “altered self” mechanism of immune recognition and have implications for specificity, cross-reactivity, and antigenicity in cellular immunity.     

A single autoimmune T cell receptor recognizes more than a million different peptides

The T cell receptor (TCR) orchestrates immune responses by binding to foreign peptides presented at the cell surface in the context of major histocompatibility complex (MHC) molecules. Effective immunity requires that all possible foreign peptide-MHC molecules are recognized or risks leaving holes in immune coverage that pathogens could quickly evolve to exploit. It is unclear how a limited pool of <10(8) human TCRs can successfully provide immunity to the vast array of possible different peptides that could be produced from 20 proteogenic amino acids and presented by self-MHC molecules (>10(15) distinct peptide-MHCs). One possibility is that T cell immunity incorporates an extremely high level of receptor degeneracy, enabling each TCR to recognize multiple peptides. However, the extent of such TCR degeneracy has never been fully quantified. Here, we perform a comprehensive experimental and mathematical analysis to reveal that a single patient-derived autoimmune CD8(+) T cell clone of pathogenic relevance in human type I diabetes recognizes >one million distinct decamer peptides in the context of a single MHC class I molecule. A large number of peptides that acted as substantially better agonists than the wild-type "index" preproinsulin-derived peptide (ALWGPDPAAA) were identified. The RQFGPDFPTI peptide (sampled from >10(8) peptides) was >100-fold more potent than the index peptide despite differing from this sequence at 7 of 10 positions. Quantification of this previously unappreciated high level of CD8(+) T cell cross-reactivity represents an important step toward understanding the system requirements for adaptive immunity and highlights the enormous potential of TCR degeneracy to be the causative factor in autoimmune disease.     

Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.     

Conformational melding permits a conserved binding geometry in TCR recognition of foreign and self molecular mimics

Molecular mimicry between foreign and self Ags is a mechanism of TCR cross-reactivity and is thought to contribute to the development of autoimmunity. The αβ TCR A6 recognizes the foreign Ag Tax from the human T cell leukemia virus-1 when presented by the class I MHC HLA-A2. In a possible link with the autoimmune disease human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis, A6 also recognizes a self peptide from the neuronal protein HuD in the context of HLA-A2. We found in our study that the complexes of the HuD and Tax epitopes with HLA-A2 are close but imperfect structural mimics and that in contrast with other recent structures of TCRs with self Ags, A6 engages the HuD Ag with the same traditional binding mode used to engage Tax. Although peptide and MHC conformational changes are needed for recognition of HuD but not Tax and the difference of a single hydroxyl triggers an altered TCR loop conformation, TCR affinity toward HuD is still within the range believed to result in negative selection. Probing further, we found that the HuD-HLA-A2 complex is only weakly stable. Overall, these findings help clarify how molecular mimicry can drive self/nonself cross-reactivity and illustrate how low peptide-MHC stability can permit the survival of T cells expressing self-reactive TCRs that nonetheless bind with a traditional binding mode.     

Structure of a human autoimmune TCR bound to a myelin basic protein self-peptide and a multiple sclerosis-associated MHC class II molecule

Multiple sclerosis is mediated by T-cell responses to central nervous system antigens such as myelin basic protein (MBP). To investigate self-peptide/major histocompatibility complex (MHC) recognition and T-cell receptor (TCR) degeneracy, we determined the crystal structure, at 2.8 A resolution, of an autoimmune TCR (3A6) bound to an MBP self-peptide and the multiple sclerosis-associated MHC class II molecule, human leukocyte antigen (HLA)-DR2a. The complex reveals that 3A6 primarily recognizes the N-terminal portion of MBP, in contrast with antimicrobial and alloreactive TCRs, which focus on the peptide center. Moreover, this binding mode, which may be frequent among autoimmune TCRs, is compatible with a wide range of orientation angles of TCR to peptide/MHC. The interface is characterized by a scarcity of hydrogen bonds between TCR and peptide, and TCR-induced conformational changes in MBP/HLA-DR2a, which likely explain the low observed affinity. Degeneracy of 3A6, manifested by recognition of superagonist peptides bearing substitutions at nearly all TCR-contacting positions, results from the few specific interactions between 3A6 and MBP, allowing optimization of interface complementarity through variations in the peptide.     

ATLAS: A database linking binding affinities with structures for wild‐type and mutant TCR‐pMHC complexes

The ATLAS (Altered TCR Ligand Affinities and Structures) database ( https://zlab.umassmed.edu/atlas/web /) is a manually curated repository containing the binding affinities for wild‐type and mutant T cell receptors (TCRs) and their antigens, peptides presented by the major histocompatibility complex (pMHC). The database links experimentally measured binding affinities with the corresponding three dimensional (3D) structures for TCR‐pMHC complexes. The user can browse and search affinities, structures, and experimental details for TCRs, peptides, and MHCs of interest. We expect this database to facilitate the development of next‐generation protein design algorithms targeting TCR‐pMHC interactions. ATLAS can be easily parsed using modeling software that builds protein structures for training and testing. As an example, we provide structural models for all mutant TCRs in ATLAS, built using the Rosetta program. Utilizing these structures, we report a correlation of 0.63 between experimentally measured changes in binding energies and our predicted changes. Proteins 2017; 85:908–916. © 2016 Wiley Periodicals, Inc.     

Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.     

Peptidic termini play a significant role in TCR recognition

TCR recognition of class I MHC is dependent on the composition of the antigenic peptide and the MHC. Single amino acid substitutions in either the MHC or the peptide may dramatically alter recognition. While the major interactions between TCR and the peptide/MHC complex appear to be focused on the complementarity-determining region (CDR)3, it is also clear from the cocrystal structure of class I MHC and TCR that the amino and carboxyl ends of the peptide may play a role through interactions with the CDR1. In this work we show that gp33 variants substituted at the peptidic termini at the putative CDR1 contact regions show improved recognition in B6 mice. The rank order of recognition is different using the P14 transgenic T cells, suggesting that one reason for improved recognition is a change in the TCR repertoire that recognizes the peptide. However, the affinity of the TCR by some of the peptide/MHC complex with increased recognition is improved, as shown by increased tetramer binding to P14 T cells. These substitutions at the termini of the peptide-binding cleft cause localized conformational changes as seen by changes in mAb binding and crystallographic structures. The different peptide structures also show different conformations in the center of the peptide, but these are shown to be energetically similar and thus most likely have no significance with respect to TCR recognition. Therefore, small conformational changes, localized to the CDR1 contact regions, may play a significant role in TCR recognition.     

Unraveling a hotspot for TCR recognition on HLA-A2: evidence against the existence of peptide-independent TCR binding determinants

T cell receptor (TCR) recognition of peptide takes place in the context of the major histocompatibility complex (MHC) molecule, which accounts for approximately two-thirds of the peptide/MHC buried surface. Using the class I MHC HLA-A2 and a large panel of mutants, we have previously shown that surface mutations that disrupt TCR recognition vary with the identity of the peptide. The single exception is Lys66 on the HLA-A2 alpha1 helix, which when mutated to alanine disrupts recognition for 93% of over 250 different T cell clones or lines, independent of which peptide is bound. Thus, Lys66 could serve as a peptide-independent TCR binding determinant. Here, we have examined the role of Lys66 in TCR recognition of HLA-A2 in detail. The structure of a peptide/HLA-A2 molecule with the K66A mutation indicates that although the mutation induces no major structural changes, it results in the exposure of a negatively charged glutamate (Glu63) underneath Lys66. Concurrent replacement of Glu63 with glutamine restores TCR binding and function for T cells specific for five different peptides presented by HLA-A2. Thus, the positive charge on Lys66 does not serve to guide all TCRs onto the HLA-A2 molecule in a manner required for productive signaling. Furthermore, electrostatic calculations indicate that Lys66 does not contribute to the stability of two TCR-peptide/HLA-A2 complexes. Our findings are consistent with the notion that each TCR arrives at a unique solution of how to bind a peptide/MHC, most strongly influenced by the chemical and structural features of the bound peptide. This would not rule out an intrinsic affinity of TCRs for MHC molecules achieved through multiple weak interactions, but for HLA-A2 the collective mutational data place limits on the role of any single MHC amino acid side-chain in driving TCR binding in a peptide-independent fashion.     

T cell recognition of distinct peptide:I-Au conformers in murine experimental autoimmune encephalomyelitis

We have used T cells bearing TCRs that are closely related in sequence as probes to detect conformational variants of peptide-MHC complexes in murine experimental autoimmune encephalomyelitis in H-2(u) mice. The N-terminal epitope of myelin basic protein (MBP) is immunodominant in this model. Our studies have primarily focused on T cell recognition of a position 4 analog of this peptide (MBP1-9[4Y]) complexed with I-A(u). Using site-directed mutagenesis, we have mapped the functionally important complementarity determining region residues of the 1934.4 TCR Valpha domain. One of the resulting mutants (Tyr(95) to alanine in CDR3alpha, Y95A) has interesting properties: relative to the parent wild-type TCR, this mutant poorly recognizes Ag complexes generated by pulsing professional APCs (PL-8 cells) with MBP1-9[4Y] while retaining recognition of MBP1-9[4Y]-pulsed unconventional APCs or insect cell-expressed complexes of I-A(u) containing tethered MBP1-9[4Y]. Insect cell expression of recombinant I-A(u) with covalently tethered class II-associated invariant chain peptide or other peptides which bind relatively weakly, followed by proteolytic cleavage of the peptide linker and replacement by MBP1-9[4Y] in vitro, results in complexes that resemble peptide-pulsed PL-8 cells. Therefore, the distinct conformers can be produced in recombinant form. T cells that can distinguish these two conformers can also be generated by the immunization of H-2(u) mice, indicating that differential recognition of the conformers is observed for responding T cells in vivo. These studies have relevance to understanding the molecular details of T cell recognition in murine experimental autoimmune encephalomyelitis. They are also of particular importance for the effective use of multimeric peptide-MHC complexes to characterize the properties of Ag-specific T cells.     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.