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Structure of the Complex Between Human T-cell Receptor, Viral Peptide and HLA-A2
Abstract
Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.
... For adaptive immune recognition, peptides (also referred to as epitopes in this context) are presented by MHC molecules on cell surfaces and then recognized by TCRs. Although a TCR binds to an epitope and the corresponding MHC molecule partner simultaneously, the core binding regions of the complex are between the complementarity Thresholds (Å) 30 10 20 Thresholds (Å) 30 TEIM Next, an interaction extractor, mainly consisting of 2D CNNs, is used to extract the pairwise residue interaction information. Alongside, an epitope feature vector generated by an autoencoder is fed into an interaction extractor for global epitope information. ...
... For adaptive immune recognition, peptides (also referred to as epitopes in this context) are presented by MHC molecules on cell surfaces and then recognized by TCRs. Although a TCR binds to an epitope and the corresponding MHC molecule partner simultaneously, the core binding regions of the complex are between the complementarity Thresholds (Å) 30 10 20 Thresholds (Å) 30 TEIM Next, an interaction extractor, mainly consisting of 2D CNNs, is used to extract the pairwise residue interaction information. Alongside, an epitope feature vector generated by an autoencoder is fed into an interaction extractor for global epitope information. ...
... available mutation data of a well-studied TCR-epitope sample, A6 TCR and Tax epitope presented by HLA-A:0201 (named as A6-Tax) 30 . For the wild-type A6-Tax complex (in which the sequences of the CDR3β and the epitope are CASRPGLAGGRPEQYF and LLFGYPVYV, respectively), TEIM-Res can accurately capture the two contact regions, corresponding to the interactions of residue Y5 and residues P6V7Y8 of the epitope with residues of CDR3β ( Fig. 3a and Extended Data Fig. 3). ...
Computational modelling of the interactions between T-cell receptors (TCRs) and epitopes is of great importance for immunotherapy and antigen discovery. However, current TCR–epitope interaction prediction tools are still in a relatively primitive stage and have limited capacity in deciphering the underlying binding mechanisms, for example, characterizing the pairwise residue interactions between TCRs and epitopes. Here we designed a new deep-learning-based framework for modelling TCR–epitope interactions, called TCR–Epitope Interaction Modelling at Residue Level (TEIM-Res), which took the sequences of TCRs and epitopes as input and predicted both pairwise residue distances and contact sites involved in the interactions. To tackle the current bottleneck of data deficiency, we applied a few-shot learning strategy by incorporating sequence-level binding information into residue-level interaction prediction. The validation experiments and analyses indicated its good prediction performance and the effectiveness of its design. We demonstrated three potential applications: revealing the subtle conformation changes of mutant TCR–epitope pairs, uncovering the key contacts based on epitope-specific TCR pools, and mining the intrinsic binding rules and patterns. In summary, our model can serve as a useful tool for comprehensively characterizing TCR–epitope interactions and understanding the molecular basis of binding mechanisms.
... The eight exons include exons 2 and 3 that encode the variable alpha 1 (α1) and alpha 2 (α2) domains, and exon 4 which encodes the constant alpha 3 (α3) domain. Exons 2 and 3 are responsible for forming the foreign peptide binding groove and with exon 4 make up the constant T-cell receptor binding region of HLA-A (Bjorkman et al. 1987a, b;Garboczi et al. 1996). Exon 5 encodes the transmembrane region, and exons 6 and 7 encode the cytoplasmic tail; exon 8 terminates the translation of the gene. ...
... We find seven of the eleven conserved residues in the α1 and α2 domains described by Garboczi et al. 1996 at the HLA-A antigen recognition site that bind to TCR, with three conserved residues in Variable Area #1 (residues 58, 68, 72); and four in Variable Area #2 (residues 155, 159, 166, 170). In contrast, we observe NSM at Asn 66 with codon AAT, that mutates to Lys AAA and is in Variable Area #1. ...
... . We locate the five polymorphic residues in the HLA-A foreign peptide binding that interface with TCR as described by Garboczi et al. 1996, with one in Variable Area #1 (Residue 65) and three in Variable Area #2 (residues 151, 163, 167) (Sf1 : Table S17). However, we find the fifth residue 69, GCC (ala4) displays an invariant Ala (Supplementary file 3 (.PDF 3.9 MB)) and the other four residues show variability indices less than 6 (Sf1 : Table S17). ...
The structure and function of human leucocyte antigen (HLA-A) is well known and is an extremely variable protein. From the public HLA-A database, we chose 26 high frequency HLA-A alleles (45% of sequenced alleles). Using five arbitrary references from these alleles, we analyzed synonymous mutations at the third codon position (sSNP3) and non-synonymous mutations (NSM). Both mutation types showed non-random locations of 29 sSNP3 codons and 71 NSM codons in the five reference lists. Most sSNP3 codons show identical mutation types with many mutations resulting from cytosine deamination. We proposed 23 ancestral parents of sSNP3 in five reference sequences using conserved parents in five unidirectional codons and 18 majority parents in reciprocal codons. These 23 proposed ancestral parents show exclusive codon usage of G3 or C3 parents located on both DNA strands that mutate to A3 or T3 variants mostly (76%) by cytosine deamination The sSNP3 and NSM show clear separation of the two variant types with most sSNP3 located in conserved areas in exons 2, 3 and 4, compared to most NSM appearing in two Variable Areas with no sSNP3 in the latter parts of exons 2 (α1) and 3 (α2). The Variable Areas contain NSM (polymorphic) residues at the center of the groove that bind the foreign peptide. We find distinctly different mutation patterns in NSM codons from those of sSNP3. Namely, G-C to A-T mutation frequency was much smaller, suggesting that evolutional pressures of deamination and other mechanisms applied to the two areas are significantly different.
... The T cell receptor is classified as a member of the immunoglobulin (Ig) gene superfamily as its domain structure is strikingly similar to that of Ig. As with Ig, TcR are encoded by a family of gene segments that rearrange to generate the diverse TcR repertoire, estimated to contain 10^"^ different sequences (Garboczi et al, 1996). The Vlike domains are encoded by V-, D-and J-region gene segments in the P chain, and Vand J-region segments in the a chain. ...
... The first two variable loops (CDRl and CDR2) of the human a-and (B-chain are encoded by the 42 Va and 46 V|3 genes, while the third variable loop (CDR3) is encoded following the joining of V genes to one of a number of J-region and, in the case of Vf3, D-and J-region gene segments. The joining reactions also contribute to the generation of further diversity by removing nucleotides and by introducing non-germline ('N') bases at each junction (Garboczi et al, 1996). ...
... Most models for the interaction between TcR and peptide-HLA complex have proposed that the relatively less diverse CDRl and CDR2 loops of both the a and P chains interact with helices of the less diverse HLA molecule, while the highly diverse CDR3 loops contact the (diverse) bound peptides (Bentley and Mariuzza, 1996). A study of the structure of the complex between human TcR, viral peptide and HLA-A2 (Garboczi et al, 1996), however, has shown that the TcR is oriented diagonally over the peptide-HLA complex, with CDRl and CDR3 from both Va and Vp contacting the peptide and CDR2 loops contacting the HLA molecule ( Fig. 1.4.2). The diagonal binding observed is thought to result from properties shared by all classical class I and class II HLA molecules and probably all aP TcR, suggesting that this might represent a general mode of binding (Garboczi etal, 1996 After interaction of the TcR with bound peptide-HLA, signal transduction is accomplished through a complex of proteins stably associated with the TcR known as the CD3 complex ( Fig. 1.4.1). ...
p>The aim of this study was to investigate pathological tissue from cases of enteropathy- associated T cell lymphoma (EATL), previously shown to over-express p53 protein, for the presence of p53 mutations with the view of correlating over-expression of the protein with the presence of genetic alterations. Single-stranded conformation polymorphism analysis was used to screen a series of formalin-fixed, paraffin-embedded EATL tissue samples and histologically uninvolved tissue Irom the same patients for the presence of p53 mutations in exons 5-8. DNA sequence analysis was performed on cases showing PCR fragments with mobility shifts to confirm the presence of mutations. Immunohistochemistry was used to assess expression of p53 and related cell cycle regulatory proteins in the same cases. Mobility shifts were detected in 10/29 (34.5%) tumour (T) samples and in 3/20 (15%) samples of adjacent (A) uninvolved tissue. Only 2 samples (1 T and I A sample) showing mobility shifts showed no over-expression of p53. DNA sequence analysis identified the presence of mutations in 3/29 (10%) T samples and in 2/20 (10%) A samples. Additional silent mutations were also detected in 2 T samples from different cases. The DNA sequencing results suggest that p53 over-expression is associated with p53 mutations in a small proportion of EATL cases. An alternative mechanism may be responsible for the stabilisation of p53 protein in the remaining cases. The detection of multiple mutations in samples 6om some individual patients may indicate that mutations arise in more than one T cell clone within enteropathic bowel.</p
... Unfortunately, at present such an understanding remains incomplete 37,40 . Our data show that cross-reactive (multi-speci c) TCRs share sequence features with PIT-matched TCRs, namely shorter and more hydrophobic TRA junctions and diverse TRB chains, reminiscent of a previous report in mouse 41 TCRs that predominantly utilize germline-like TRA CDR3 regions [43][44][45][46][47] . Less is known about such TRA-centric, MHC class II-restricted TCRs, such as those we have observed. ...
... Less is known about such TRA-centric, MHC class II-restricted TCRs, such as those we have observed. One of the V genes enriched in PIT-matched TRA chains ( Figure S5), TRAV12-2, is also a feature of multiple MHC class I-restricted TCRs 43,44,46,47 . The other V gene enriched in PIT-matched TRA chains was TRAV21-2, which is associated with celiac disease, while paired with TRBV9 chains 48 . ...
Human islet antigen reactive CD4 + memory T cells (IAR T cells) from peripheral blood have been studied extensively for their role in the pathogenesis of autoimmune type 1 diabetes (T1D). However, IAR T cells are rare, and it remains poorly understood how they affect T1D progression in the pancreas. Using single cell RNA-sequencing coupled with a multiplexed activation induced marker (AIM) enrichment assay, we identified paired TCR alpha/beta ( TRA/TRB ) T cell receptors (TCRs) in IAR T cells from the blood of healthy, at-risk, new onset, and established T1D donors. Using TCR sequences as barcodes, we measured infiltration of IAR T cells from blood into pancreas of organ donors with and without T1D. We detected extensive TCR sharing between IAR T cells from peripheral blood and pancreatic infiltrating T cells (PIT), with perfectly matched or single mismatched TRA junctions and J gene regions, comprising ~ 34% of unique IAR TCRs. PIT-matching IAR T cells had public TRA chains that showed increased use of germline-encoded residues in epitope engagement and a propensity for cross-reactivity. The link with T cells in the pancreas implicates autoreactive IAR T cells with shared TRA junctions and increased levels in blood with the prediabetic and new onset phases of T1D progression.
... First, the results provided by the molecular dynamics of this study indicated that the P1 position of the peptide played more of a key role in the stabilization of the peptide by the N-terminus than previously assumed, since classically, P2 and P9 were the residues of the most important peptides for anchoring to the MHC [42]. This prominent role of P1 is evident in Figure 2, where significantly more important contact scores were observed in P1 than in P2 in the three peptides that remained anchored in their N-terminus part (ASTN1 neo, AKAP6 WT, and AKAP6 neo). ...
... This prominent role of P1 is evident in Figure 2, where significantly more important contact scores were observed in P1 than in P2 in the three peptides that remained anchored in their N-terminus part (ASTN1 neo, AKAP6 WT, and AKAP6 neo). The two neoantigens followed the classically reported amino acid sequence pattern for class I epitopes for binding to MHC-I: X-(L/I)-X(6−7)-(V/L), where L/I and V/L represent the residues, whose side chain anchors the peptide to the MHC [42,43]. In the case of AKAP6, this was generated by a mutation in a non-anchor position (P6), which caused the mutant peptide to be released more quickly from the MHC through the C-terminus. ...
Personalized cancer vaccines based on neoantigens are a new and promising treatment for cancer; however, there are still multiple unresolved challenges to using this type of immunotherapy. Among these, the effective identification of immunogenic neoantigens stands out, since the in silico tools used generate a significant portion of false positives. Inclusion of molecular simulation techniques can refine the results these tools produce. In this work, we explored docking and molecular dynamics to study the association between the stability of peptide–HLA complexes and their immunogenicity, using as a proof of concept two HLA-A2-restricted neoantigens that were already evaluated in vitro. The results obtained were in accordance with the in vitro immunogenicity, since the immunogenic neoantigen ASTN1 remained bound at both ends to the HLA-A2 molecule. Additionally, molecular dynamic simulation suggests that position 1 of the peptide has a more relevant role in stabilizing the N-terminus than previously proposed. Likewise, the mutations may have a “delocalized” effect on the peptide–HLA interaction, which means that the mutated amino acid influences the intensity of the interactions of distant amino acids of the peptide with the HLA. These findings allow us to propose the inclusion of molecular simulation techniques to improve the identification of neoantigens for cancer vaccines.
... The majority of T cells express a and b isoforms to form a heterodimer i.e., ab T cells or more generally referred to as just T cells. The a/b TCRs bind to antigenic peptides presented in molecular grooves on the surface of MHC I or MHC II molecules present on APCs (17)(18)(19)(20). Only a small proportion of T cells express TCRg and TCRd isoforms known as gd T cells. ...
... The a chain is generated by VJ recombination while the b chain is generated by VDJ recombination. Theoretically, a TCR repertoire consists of 2x10 19 unique TCRab pairs generated by the recombination process along with non-templated addition or deletion of nucleotides between spliced gene segments. Therefore, each T cell repertoire is shaped by both genetically determined biases, as well as immune exposures. ...
Antigen-specific T cells play a central role in the adaptive immune response and come in a wide range of phenotypes. T cell receptors (TCRs) mediate the antigen-specificities found in T cells. Importantly, high-throughput TCR sequencing provides a fingerprint which allows tracking of specific T cells and their clonal expansion in response to particular antigens. As a result, many studies have leveraged TCR sequencing in an attempt to elucidate the role of antigen-specific T cells in various contexts. Here, we discuss the published approaches to studying antigen-specific T cells and their specific TCR repertoire. Further, we discuss how these methods have been applied to study the TCR repertoire in various diseases in order to characterize the antigen-specific T cells involved in the immune control of disease.
... First, the results provided by the molecular dynamics of this study indicate that the P1 position of the peptide plays a key role in the stabilization of the peptide by the N-terminus than previously assumed since classically P2 and P9 were the residues of the most important peptides for anchoring to the MHC (42). This prominent role of P1 is evident in Figure 2, where significantly more important contact scores are observed in P1 than in P2 in the three peptides that remain anchored in their N-terminal part (ASTN1 neo, AKAP6 WT, and AKAP6 neo). ...
... This prominent role of P1 is evident in Figure 2, where significantly more important contact scores are observed in P1 than in P2 in the three peptides that remain anchored in their N-terminal part (ASTN1 neo, AKAP6 WT, and AKAP6 neo). The two neoantigens follow the classically reported amino acid sequence pattern for class I epitopes for binding to MHC-I: X-(L/I)-X(6−7)-(V/L), where L/I and V /L represent the residues whose side chain anchors the peptide to the MHC (42,43). In the case of AKAP6, this is generated by a mutation in a non-anchor position (P6), which causes the mutant peptide to be released more quickly from the MHC through the C-terminus. ...
Personalized cancer vaccines are presented as a new and promising treatment alternative for cancer, especially in those cases where effective treatments do not yet exist. However, multiple challenges remain to be resolved so that this type of immunotherapy can be used in the clinical setting. Among these, the effective identification of immunogenic peptides stands out, since the in-silico tools currently used generate a significant portion of false positives. This is where molecular simulation techniques can play an important role when it comes to refining the results produced by these tools. In the present work, we explore the use of molecular simulation techniques such as docking and molecular dynamics to study the relationship between stability of peptide-HLA complexes and their immunogenicity using two HLA-A2-restricted neoantigens that have already been evaluated in vitro. The results obtained agreed with the in vitro immunogenicity of the immunogenic neoantigen ASTN1 the only one that remains bound at both ends to the HLA-A2 molecule. Additionally, molecular dynamics indicates that position 1 of the peptide has a more important role in stabilizing the N-terminal part than previously assumed. Likewise, the results suggest that the mutations may have a "delocalized" effect on the peptide-HLA interaction, that is, they may modulate the intensity of the interactions of other amino acids in the peptide. These results highlight the suitability of this type of in silico strategy to identify peptides that form stable complexes with HLA proteins that are highly immunogenic for CD8+ T cells.
... TCRα or TCRβ-pET22b constructs were used to transform the E. coli BL21 (DE3) pLysS-derived Rosetta strain, under chloramphenicol and ampicillin selection, for protein expression in inclusion bodies 61 . In brief, 40 ml of overnight starter culture in LB medium with chloramphenicol and ampicillin was divided between 4 l of low-salt LB (5 g NaCl per litre) in four 2-l Erlenmeyer flasks and grown for about 2-3 h to an optical density at 600 nm of 0.4-0.6, ...
... Chemical refolding was conducted essentially as previously published 61 , but with minor modifications. A 25 mg quantity of each inclusion body's purified TCR α-and β-chains was diluted in 500 ml of denaturing refolding mix (5 M urea, 0.4 M l-arginine, 100 mM Tris-HCl, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione) with stirring overnight at 4 °C. ...
Human leucocyte antigen B*27 (HLA-B*27) is strongly associated with inflammatory diseases of the spine and pelvis (for example, ankylosing spondylitis (AS)) and the eye (that is, acute anterior uveitis (AAU))¹. How HLA-B*27 facilitates disease remains unknown, but one possible mechanism could involve presentation of pathogenic peptides to CD8⁺ T cells. Here we isolated orphan T cell receptors (TCRs) expressing a disease-associated public β-chain variable region–complementary-determining region 3β (BV9–CDR3β) motif2–4 from blood and synovial fluid T cells from individuals with AS and from the eye in individuals with AAU. These TCRs showed consistent α-chain variable region (AV21) chain pairing and were clonally expanded in the joint and eye. We used HLA-B*27:05 yeast display peptide libraries to identify shared self-peptides and microbial peptides that activated the AS- and AAU-derived TCRs. Structural analysis revealed that TCR cross-reactivity for peptide–MHC was rooted in a shared binding motif present in both self-antigens and microbial antigens that engages the BV9–CDR3β TCRs. These findings support the hypothesis that microbial antigens and self-antigens could play a pathogenic role in HLA-B*27-associated disease.
... Unfortunately, the experimental solving of the 3D structure of Tax remains elusive. To date, the only published structures concerning HTLV-1 Tax are that of short peptides in complex with HLA molecules [13][14][15][16][17][18][19][20][21][22][23] or structures of the last eight residues of the C-terminal extremity of Tax, forming a PDZ-binding motif, in complex with PDZ proteins [24][25][26][27]. ...
Human T-cell Leukemia Virus type 1 (HTLV-1) is a human retrovirus responsible for leukaemia in 5 to 10% of infected individuals. Among the viral proteins, Tax has been described as directly involved in virus-induced leukemogenesis. Tax is therefore an interesting therapeutic target. However, its 3D structure is still unknown and this hampers the development of drug-design-based therapeutic strategies. Several algorithms are available that can be used to predict the structure of proteins, particularly with the recent appearance of artificial intelligence (AI)-driven pipelines. Here, we review how the structure of Tax is predicted by several algorithms using distinct modelling strategies. We discuss the consequences for the understanding of Tax structure/function relationship, and more generally for the use of structure models for modular and/or flexible proteins, which are frequent in retroviruses.
... MHC I-neoepitope interactions have also been examined through structural approaches. The visualization of peptide-MHC complexes (37,38) and, later, their complexes with T cell receptors (39,40) transformed immunology. A pair of analyses used the collective wisdom of such studies to identify features associated with immunogenicity (41,42). ...
A proportion of somatic mutations in tumors create neoepitopes that can prime T cell responses that target the MHC I–neoepitope complexes on tumor cells, mediating tumor control or rejection. Despite the compelling centrality of neoepitopes to cancer immunity, we know remarkably little about what constitutes a neoepitope that can mediate tumor control in vivo and what distinguishes such a neoepitope from the vast majority of similar candidate neoepitopes that are inefficacious in vivo. Studies in mice as well as clinical trials have begun to reveal the unexpected paradoxes in this area. Because cancer neoepitopes straddle that ambiguous ground between self and non-self, some rules that are fundamental to immunology of frankly non-self antigens, such as viral or model antigens, do not appear to apply to neoepitopes. Because neoepitopes are so similar to self-epitopes, with only small changes that render them non-self, immune response to them is regulated at least partially the way immune response to self is regulated. Therefore, neoepitopes are viewed and understood here through the clarifying lens of negative thymic selection. Here, the emergent questions in the biology and clinical applications of neoepitopes are discussed critically and a mechanistic and testable framework that explains the complexity and translational potential of these wonderful antigens is proposed.
... Peptides presented in class I and class II MHC follow a sequence pattern that contains anchor position and, thus, is in the interior of the peptide binding groove and away from TCR [26]. The remaining residues interact with TCR and mediate epitope specificity. ...
Animal models are essential in medical research for testing drugs and vaccines. These models differ from humans in various respects, so their results are not directly translatable in humans. To address this issue, humanized mice engrafted with functional human cells or tissue can be helpful. We propose using humanized mice that support the engraftment of human hematopoietic stem cells (HSCs) without irradiation to evaluate vaccines that influence patient immunity. For infectious diseases, several types of antigens and adjuvants have been developed and evaluated for vaccination. Peptide vaccines are generally used for their capability to fight cancer and infectious diseases. Evaluation of adjuvants is necessary as they induce inflammation, which is effective for an enhanced immune response but causes adverse effects in some individuals. A trial can be done on humanized mice to check the immunogenicity of a particular adjuvant and peptide combination. Messenger RNA has also emerged as a potential vaccine against viruses. These vaccines need to be tested with human immune cells because they work by producing a particular peptide of the pathogen. Humanized mice with human HSCs that can produce both myeloid and lymphoid cells show a similar immune response that these vaccines will produce in a patient.
... CD3δ, CD3ε, and CD3γ chains each comprise an ECD, a CP, a TM domain, and a cytoplasmic tail containing one ITAM. CD3ζ has a short nine-amino acid ECD and a long cytoplasmic tail with three ITAMs [35][36][37][38][39]. In addition, T cells also express TCR coreceptor CD4 or CD8, and this transmembrane glycoprotein binds to the MHC class II or class I molecule [40,41]. ...
... Based on the TCR3d database of experimentally determined TCR-pMHC structures 58 , there are >40 structures containing TCRs that possess the TRAV12-2 germline gene and that bind HLA-A2, collectively representing at least 10 unique human TCRs. Several of these, including the TCR A6-Tax-HLA-A2 complex (PDB code 1AO7) 59 and TCR DMF5-MART-1-HLA-A2 complex (3QDG) 60 , have α chain interactions with MHC, as well as with peptide backbone, that are highly their predictability of binding to a dominant nucleocapsid LLL epitope. a Summary of LLL tetramer + CD8 + T cells isolated from uninfected, COVID-19 convalescent donors, and in vitro expanded donors by cell sorting and scTCR-seq. ...
The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8⁺ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8⁺ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8⁺ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8⁺ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8⁺ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8⁺ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR–LLL–HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8⁺ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8⁺ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8⁺ T cell responses with a restricted TCR repertoire.
... In αβ T cells, the six complementaritydetermining region (CDR) loops of the TCR are responsible for scanning the pMHC surface and determining the appropriate immunological response. Since the first crystal structures of a TCR-pMHC complex were solved in 1996 [1,2], general "rules of engagement" between TCR and pMHC have been identified and reliably reproduced in subsequent structures [3,4]. First, the peptide is presented in the "groove" formed by the two α-helices of either the class I or class II MHC molecule [5,6]. ...
T cells are critically important components of the adaptive immune system primarily responsible for identifying and responding to pathogenic challenges. This recognition of pathogens is driven by the interaction between membrane-bound T cell receptors (TCRs) and antigenic peptides presented on major histocompatibility complex (MHC) molecules. The formation of the TCR-peptide-MHC complex (TCR-pMHC) involves interactions among germline-encoded and hypervariable amino acids. Germline-encoded and hypervariable regions can form contacts critical for complex formation, but only interactions between germline-encoded contacts are likely to be shared across many of all the possible productive TCR-pMHC complexes. Despite this, experimental investigation of these interactions have focused on only a small fraction of the possible interaction space. To address this, we analyzed every possible germline-encoded TCR-MHC contact in humans, thereby generating the first comprehensive characterization of these largely antigen-independent interactions. Our computational analysis suggests that germline-encoded TCR-MHC interactions that are conserved at the sequence level are rare due to the high amino acid diversity of the TCR CDR1 and CDR2 loops, and that such conservation is unlikely to dominate the dynamic protein-protein binding interface. Instead, we propose that binding properties such as the docking orientation are defined by regions of biophysical compatibility between these loops and the MHC surface.
... ;https://doi.org/10.1101https://doi.org/10. /2023 1998) (Garboczi et al., 1996a) (Garboczi et al., 1996b). Antigen-specific T-cell clones were generated as described previously (Braun and Zielinski, 2014;Zielinski et al., 2012). ...
Adoptive T-cell therapy has become a powerful weapon for cancer treatment. The efficacy of antitumor immunity is associated with the metabolic state of cytotoxic T cells, which is highly sensitive to the tumor microenvironment. It is therefore of considerable interest to bypass immunosuppressive signals in the tumor microenvironment and to identify factors that augment cytotoxic effector functions and ultimately tumor killing. Whether ionic signals serve as aberrant immune signals and influence the adaptive human antitumor immune response is still largely unexplored. We therefore investigated the effect of sodium on the phenotype, function and metabolic regulation of human CD8+ T cells using transcriptomic, metabolomic, high-dimensional flow cytometric and functional assays. We demonstrate a significant enrichment of sodium in solid tumors from patients with breast cancer, which leaves a transcriptomic imprint on intratumoral immune cells. Sodium chloride (NaCl) enhanced the activation state and effector functions of human CD8+ memory T cells. These functional alterations were associated with enhanced metabolic fitness, particularly increases in glycolysis, oxidative phosphorylation and overall nutrient uptake. These NaCl-induced effects translated into increased tumor cell killing in vitro and in a tumor mouse model in vivo. We therefore propose NaCl as a positive regulator of acute antitumor immunity that could be harnessed for ex vivo conditioning of adoptively transferred T cells, such as CAR T-cells.
... Crystallographic studies, spanning over 25 years, have shed light on the structural basis of TCR-pMHC recognition and its relation to the T-cell immune response [14][15][16] . These studies, using soluble ectodomain proteins, have shown that TCRs use three complementarity-determining regions (CDRs) on each chain to make contacts with the pMHC molecule. ...
The recognition of antigenic peptide-MHC (pMHC) molecules by T-cell receptors (TCR) initiates the T-cell mediated immune response. Structural characterization is key for understanding the specificity of TCR-pMHC interactions and informing the development of therapeutics. Despite the rapid rise of single particle cryoelectron microscopy (cryoEM), x-ray crystallography has remained the preferred method for structure determination of TCR-pMHC complexes. Here, we report cryoEM structures of two distinct full-length α/β TCR-CD3 complexes bound to their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230–239). We also determined cryoEM structures of pMHCs containing MAGEA4 (230–239) peptide and the closely related MAGEA8 (232–241) peptide in the absence of TCR, which provided a structural explanation for the MAGEA4 preference displayed by the TCRs. These findings provide insights into the TCR recognition of a clinically relevant cancer antigen and demonstrate the utility of cryoEM for high-resolution structural analysis of TCR-pMHC interactions.
... Phosphorylation by kinase activity is counteracted by receptor protein tyrosine phosphatases. CD3, cluster of differentiation 3. onto pMHC in a diagonal fashion relative to the long axis of the peptide-binding groove [9,10], and interactions with the peptide are predominantly mediated by the hypervariable CDR3s, while the CDR1s and CDR2s are mainly involved in contacts to the MHC [7]. A salient feature of TCRab complexes is their ability to combine cross-reactivity with high specificity and sensitivity for their agonist pMHC ligands in interactions of medium to low three-dimensional (solution) affinity (K d z 1e10 mM) with typical maximal half-lives of only several seconds. ...
Immunoreceptors, also named non-catalytic tyrosine-phosphorylated receptors, are a large class of leukocyte cell-surface proteins critically involved in innate and adaptive immune responses. Their most characteristic defining feature is a shared signal transduction machinery where binding events of cell surface-anchored ligands to the small extracellular receptor domains are translated into phosphorylation of conserved tyrosine-containing cytosolic sequence motifs initiating downstream signal transduction cascades. Despite their central importance to immunology, the molecular mechanism of how ligand binding activates the receptors and results in robust intracellular signaling has remained enigmatic. Recent breakthroughs in our understanding of the architecture and triggering mechanism of immunoreceptors come from cryogenic electron microscopy studies of the B cell and T cell antigen receptors.
... MHC class I (MHCI) a chain (HLA-A2) and b 2 -microglobulin (b2m) recombinant proteins were generated by overexpression in Escherichia coli and purification of inclusion bodies following a previously described protocol (28). To generate individual peptide:MHC monomer complexes, we set up a refolding reaction in an L-argininerich, glutathione redox refolding buffer with protease inhibitors in the presence of a high concentration of the HLA-A2restricted UV labile peptide H-KLLT(1051)ILTI-OH (Mimotopes), where 1051 denotes a 3-amino-3 (2-nitro-phenyl)-propionic acid residue. ...
The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.
... The peptides presented by HLA class I are generally short, although they can still accommodate larger peptides. HLA class I peptides generally follow the X-(L/I)-X(6-7)-(V/L) pattern, where L/I and V/L represent residues whose side chains anchor the peptide to the pHLA, while the other side is attached to the pHLA on the TCR [45]. As for the peptides presented by HLA class II, they are generally more varied in terms of length and sequence but still have anchoring sites. ...
Vaccines are an upcoming medical intervention for breast cancer. By targeting the tumor antigen, cancer vaccines can be designed to train the immune system to recognize tumor cells. Therefore, along with technological advances, the vaccine design process is now starting to be carried out with more rational methods such as designing epitope-based peptide vaccines using immunoinformatics methods. Immunoinformatics methods can assist vaccine design in terms of antigenicity and safety. Common protocols used to design epitope-based peptide vaccines include tumor antigen identification, protein structure analysis, T cell epitope prediction, epitope characterization, and evaluation of protein‒epitope interactions. Tumor antigen can be divided into two types: tumor associated antigen and tumor specific antigen. We will discuss the identification of tumor antigens using high-throughput technologies. Protein structure analysis comprises the physiochemical, hydrochemical, and antigenicity of the protein. T cell epitope prediction models are widely available with various prediction parameters as well as filtering tools for the prediction results. Epitope characterization such as allergenicity and toxicity can be done in silico as well using allergenicity and toxicity predictors. Evaluation of protein‒epitope interactions can also be carried out in silico with molecular simulation. We will also discuss current and future developments of breast cancer vaccines using an immunoinformatics approach. Finally, although prediction models have high accuracy, the opposite can happen after being tested in vitro and in vivo. Therefore, further studies are needed to ensure the effectiveness of the vaccine to be developed. Although epitope-based peptide vaccines have the disadvantage of low immunogenicity, the addition of adjuvants can be a solution.
... The mIg is associated with the CD79AB signaling dimer (also known as Igαβ) (5, 6) ( Fig. 1A) to form the complete receptor complex. The TCR is an octameric complex consisting of either a TCRαβ or γδ subunit for antigen binding, which is reminiscent of a membrane-tethered antibody Fab (7)(8)(9), and three signaling dimers, namely CD3δε, CD3γε, and ζζ (10-15) (Fig. 1B). The TCR, like most other modular activating receptors, assembles its components through basic and acidic residues in the TM domains of the antigen-binding and signaling subunits, respectively (10-17) ( Fig. 1 B inset). ...
The B cell and T cell antigen receptors (BCR and TCR) share a common architecture in which variable dimeric antigen-binding modules assemble with invariant dimeric signaling modules to form functional receptor complexes. In the TCR, a highly conserved T cell receptor αβ (TCRαβ) transmembrane (TM) interface forms a rigid structure around which its three dimeric signaling modules assemble through well-characterized polar interactions. Noting that the key features stabilizing this TCRαβ TM interface also appear with high evolutionary conservation in the TM sequences of the membrane immunoglobulin (mIg) heavy chains that form the BCR’s homodimeric antigen-binding module, we asked whether the BCR contained an analogous TM structure. Using an unbiased biochemical and computational modeling approach, we found that the mouse IgM BCR forms a core TM structure that is remarkably similar to that of the TCR. This structure is reinforced by a network of interhelical hydrogen bonds, and our model is nearly identical to the arrangement observed in the just-released cryo-electron microscopy (cryo-EM) structures of intact human BCRs. Our biochemical analysis shows that the integrity of this TM structure is vital for stable assembly with the BCR signaling module CD79AB in the B cell endoplasmic reticulum, and molecular dynamics simulations indicate that BCRs of all five isotypes can form comparable structures. These results demonstrate that, despite their many differences in composition, complexity, and ligand type, TCRs and BCRs rely on a common core TM structure that has been shaped by evolution for optimal receptor assembly and stability in the cell membrane.
... Human virus-specific CD8 pos T cells express a heterodimeric alpha(α)/beta(β) T-Cell Receptor (TCR) that specifically recognizes a viral-peptide in the context of a human leukocyte antigen (HLA)-class-I molecule. The TCR-β chains have highly variable gene and their variability is based on the different V-genes [4,5]. The sequence at the recombined V-D-J and V-J regions encodes for the CDR3 region, which is highly variable and greatly determines the specificity of the TCR. ...
Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino-acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2FLY -specific public TCR as model and modified the highly-variable position 5 of the CDR3β sequence with all 20 amino-acids. Our results demonstrate that amino-acids at this particular position in the CDR3β region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance. This article is protected by copyright. All rights reserved.
... The first two milestone papers published in 1996 as well as subsequent studies that succeeded in elucidating crystal structures of αβTCR:pMHC complexes have advanced our understanding of how TCRs see pMHC (19,20). In general, αβTCR heterodimers dock on pMHC complexes diagonally, and TCR α and β chains are placed above the MHC class I α2 and α1 helices, respectively (21). ...
Complementarity-determining regions (CDRs) of αβ T-cell receptors (TCRs) sense peptide-bound MHC (pMHC) complexes via chemical interactions, thereby mediating antigen specificity and MHC restriction. Flexible finger-like movement of CDR loops contributes to the establishment of optimal interactions with pMHCs. In contrast, peptide ligands captured in MHC molecules are considered more static because of the rigid hydrogen-bond network that stabilizes peptide ligands in the antigen-binding groove of MHC molecules. An array of crystal structures delineating pMHC complexes in TCR-docked and TCR-undocked forms is now available, which enables us to assess TCR engagement-induced conformational changes in peptide ligands. In this short review, we overview conformational changes in MHC class I-bound peptide ligands upon TCR docking, followed by those for CD1-bound glycolipid ligands. Finally, we analyze the co-crystal structure of the TCR:lipopeptide-bound MHC class I complex that we recently reported. We argue that TCR engagement-induced conformational changes markedly occur in lipopeptide ligands, which are essential for exposure of a primary T-cell epitope to TCRs. These conformational changes are affected by amino acid residues, such as glycine, that do not interact directly with TCRs. Thus, ligand recognition by specific TCRs involve not only T-cell epitopes but also non-epitopic amino acid residues. In the light of their critical function, we propose to refer to these residues as non-epitopic residues affecting ligand plasticity and antigenicity (NR-PA).
... Crystallographic studies, spanning over 25 years, have shed light on the structural basis of TCR-pMHC recognition and its relation to the T-cell immune response [14][15][16] . These studies, using soluble ectodomain proteins, have shown that TCRs use three complementarity determining regions (CDRs) on each chain to make contacts with the pMHC molecule. ...
The recognition of antigenic peptide-MHC (pMHC) molecules by T-cell receptors (TCR) initiates the T-cell mediated immune response. Structural characterization is key for understanding the specificity of TCR-pMHC interactions and informing the development of therapeutics. Despite the rapid rise of single particle cryoelectron microscopy (cryoEM), x-ray crystallography has remained the preferred method for structure determination of TCR-pMHC complexes. Here, we report cryoEM structures of two distinct full-length a/b TCR-CD3 complexes bound to their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230-239). We also determined cryoEM structures of pMHCs containing MAGEA4 (230-239) peptide and the closely related MAGEA8 (232-241) peptide in the absence of TCR, which provided a structural explanation for the MAGEA4 preference displayed by the TCRs. These findings provide insights into the TCR recognition of a clinically relevant cancer antigen and demonstrate the utility of cryoEM for high-resolution structural analysis of TCR-pMHC interactions.
... Our high-resolution reconstruction allowed the molecular contacts underpinning the assembly of the TCR to be visualized at a high level of detail. The structures of the extracellular IgSF domains of the TCR-ab and CD3 heterodimers are well characterized by NMR and X-ray crystallography (Arnett et al., 2004;Garboczi et al., 1996;Garcia et al., 1996;Kjer-Nielsen et al., 2004;Sun et al., 2001). How these parts of each subunit are linked to The 59 tilt between the gp100/HLA-A2 and TCR extracellular domains and the plasma membrane is denoted. ...
The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαβ/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.
... The TCR CDR loops are known to undergo structural rearrangements to recognize peptides and lipids presented by classical (29, 39) and nonclassical MHC molecules (40), respectively. Likewise, there is documented evidence for conformational changes in peptide antigens during pMHC-TCR interactions (32,41,42). A particularly noteworthy study (43) of a human autoreactive TCR targeting a Multiple Sclerosis-relevant pMHCII identified structural rearrangements in both the MHCII molecule and the peptide in response to TCR engagement. ...
We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-A g7 -restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-A g7 complex, both in isolation as well as bound to the 4.1-TCR. We find that HIP promiscuity of the 4.1-TCR is dictated, on the one hand, by an amino acid sequence pattern that ensures I-A g7 binding and, on the other hand, by the presence of three acidic residues at positions P5, P7 and P8 that favor an optimal engagement by the 4.1-TCR’s complementary determining regions. Surprisingly, comparison of the TCR-bound and unbound HIP/I-A g7 structures reveals that 4.1-TCR binding triggers several novel and unique structural motions in both the I-A g7 molecule and the peptide that are essential for docking. This observation indicates that the type 1 diabetes-associated I-A g7 molecule is structurally malleable and that this plasticity allows the recognition of multiple peptides by individual TCRs that would otherwise be unable to do so.
Development of T cell receptors (TCRs) as immunotherapeutics is hindered by inherent TCR cross-reactivity. Engineering more specific TCRs has proven challenging, as unlike antibodies, improving TCR affinity does not usually improve specificity. Although various protein design approaches have been explored to surmount this, mutations in TCR binding interfaces risk broadening specificity or introducing new reactivities. Here we explored if TCR specificity could alternatively be tuned through framework mutations distant from the interface. Studying the 868 TCR specific for the HIV SL9 epitope presented by HLA-A2, we used deep mutational scanning to identify a framework mutation above the mobile CDR3β loop. This glycine to proline mutation had no discernable impact on binding affinity or functional avidity towards the SL9 epitope but weakened recognition of SL9 escape variants and led to fewer responses in a SL9-derived positional scanning library. In contrast, an interfacial mutation near the tip of CDR3α that also did not impact affinity or functional avidity towards SL9 weakened specificity. Simulations indicated that the specificity-enhancing mutation functions by reducing the range of loop motions, limiting the ability of the TCR to adjust to different ligands. Although our results are likely to be TCR dependent, using framework engineering to control TCR loop motions may be a viable strategy for improving the specificity of TCR-based immunotherapies.
Major Histocompatibility Complex (MHC) I and II and the αβ T-cell antigen receptor (TCRαβ) govern fundamental traits of adaptive immunity. They form a membrane-borne ligand-receptor system weighing host proteome integrity to detect contamination by nonself proteins. MHC-I and -II exhibit the “MHC-fold”, which is able to bind a large assortment of short peptides as proxies for self and nonself proteins. The ensuing varying surfaces are mandatory ligands for Ig-like TCRαβ highly mutable binding sites. Conserved molecular signatures guide TCRαβ ligand binding sites to focus on the MHC-fold (MHC-restriction) while leaving many opportunities for its most hypervariable determinants to contact the peptide. This riveting molecular strategy affords many options for binding energy compatible with specific recognition and signalling aimed to eradicated microbial pathogens and cancer cells. While the molecular foundations of αβ T-cell adaptive immunity are largely understood, uncertainty persists on how peptide-MHC binding induces the TCRαβ signals that instruct cell-fate decisions. Solving this mystery is another milestone for understanding αβ T-cells’ self/nonself discrimination. Recent developments revealing the innermost links between TCRαβ structural dynamics and signalling modality should help dissipate this long-sought-after enigma.
Mechanical force is critical for the interaction between an αβ T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR α and β chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the C β FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCR αβ -pMHC agonist and antagonist complexes.
Introduction
The HLA region is the hallmark of balancing selection, argued to be driven by the pressure to present a wide variety of viral epitopes. As such selection on the peptide-binding positions has been proposed to drive HLA population genetics. MHC molecules also directly binds to the T-Cell Receptor and killer cell immunoglobulin-like receptors (KIR).
Methods
We here combine the HLA allele frequencies in over six-million Hematopoietic Stem Cells (HSC) donors with a novel machine-learning-based method to predict allele frequency.
Results
We show for the first time that allele frequency can be predicted from their sequences. This prediction yields a natural measure for selection. The strongest selection is affecting KIR binding regions, followed by the peptide-binding cleft. The selection from the direct interaction with the KIR and TCR is centered on positively charged residues (mainly Arginine), and some positions in the peptide-binding cleft are not associated with the allele frequency, especially Tyrosine residues.
Discussion
These results suggest that the balancing selection for peptide presentation is combined with a positive selection for KIR and TCR binding.
Intrinsically disordered protein regions exist in a collection of dynamic interconverting conformations that lack a stable 3D structure. These regions are structurally heterogeneous, ubiquitous and found across all kingdoms of life. Despite the absence of a defined 3D structure, disordered regions are essential for cellular processes ranging from transcriptional control and cell signalling to subcellular organization. Through their conformational malleability and adaptability, disordered regions extend the repertoire of macromolecular interactions and are readily tunable by their structural and chemical context, making them ideal responders to regulatory cues. Recent work has led to major advances in understanding the link between protein sequence and conformational behaviour in disordered regions, yet the link between sequence and molecular function is less well defined. Here we consider the biochemical and biophysical foundations that underlie how and why disordered regions can engage in productive cellular functions, provide examples of emerging concepts and discuss how protein disorder contributes to intracellular information processing and regulation of cellular function.
T-cell receptors (TCRs) are involved in most human diseases, but linking their sequences with their targets remains an unsolved grand challenge in the field. In this study, we present TAPIR (T-cell receptor and Peptide Interaction Recognizer), a T-cell receptor (TCR) language model that predicts TCR-target interactions, with a focus on novel and rare targets. TAPIR employs deep convolutional neural network (CNN) encoders to process TCR and target sequences across flexible representations (e.g., beta-chain only, unknown MHC allele, etc.) and learns patterns of interactivity via several training tasks. This flexibility allows TAPIR to train on more than 50k either paired (alpha and beta chain) or unpaired TCRs (just alpha or beta chain) from public and proprietary databases against 1933 unique targets. TAPIR demonstrates state-of-the-art performance when predicting TCR interactivity against common benchmark targets and is the first method to demonstrate strong performance when predicting TCR interactivity against novel targets, where no examples are provided in training. TAPIR is also capable of predicting TCR interaction against MHC alleles in the absence of target information. Leveraging these capabilities, we apply TAPIR to cancer patient TCR repertoires and identify and validate a novel and potent anti-cancer T-cell receptor against a shared cancer neoantigen target (PIK3CA H1047L). We further show how TAPIR, when extended with a generative neural network, is capable of directly designing T-cell receptor sequences that interact with a target of interest.
Mechanical force is critical for the interaction between an alpha beta T cell receptor (TCR) and a peptide-bound major histocompatibility complex (pMHC) molecule to initiate productive T-cell activation. However, the underlying mechanism remains unclear. We use all-atom molecular dynamics simulations to examine the A6 TCR bound to HLA-A*02:01 presenting agonist or antagonist peptides under different extensions to simulate the effects of applied load on the complex, elucidating their divergent biological responses. We found that TCR alpha and beta chains move asymmetrically, which impacts the interface with pMHC, in particular the peptide-sensing CDR3 loops. For the wild-type agonist, the complex stabilizes in a load-dependent manner while antagonists destabilize it. Simulations of the C beta FG-loop deletion, which reduces the catch bond response, and simulations with in silico mutant peptides further support the observed behaviors. The present results highlight the combined role of interdomain motion, fluctuating forces, and interfacial contacts in determining the mechanical response and fine peptide discrimination by a TCR, thereby resolving the conundrum of nearly identical crystal structures of TCR-pMHC agonist and antagonist complexes.
Different strategies based on peptides are available for cancer treatment, in particular to counter-act the progression of tumor growth and disease relapse. In the last decade, in the context of therapeutic strategies against cancer, peptide-based vaccines have been evaluated in different tumor models. The peptides selected for cancer vaccine development can be classified in two main type: tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), which are captured, internalized, processed and presented by antigen-presenting cells (APCs) to cell-mediated immunity. Peptides loaded onto MHC class I are recognized by a specific TCR of CD8+ T cells, which are activated to exert their cytotoxic activity against tumor cells presenting the same peptide-MHC-I complex. This process is defined as active immunotherapy as the host’s immune system is either de novo activated or restimulated to mount an effective, tumor-specific immune reaction that may ultimately lead to tu-mor regression. However, while the preclinical data have frequently shown encouraging results, therapeutic cancer vaccines clinical trials, including those based on peptides have not provided satisfactory data to date. The limited efficacy of peptide-based cancer vaccines is the consequence of several factors, including the identification of specific target tumor antigens, the limited immunogenicity of peptides and the highly immunosuppressive tumor microenvironment (TME). An effective cancer vaccine can be developed only by addressing all such different aspects. The present review describes the state of the art for each of such factors.
Formation of major histocompatibility (MHC)-peptide-T cell receptor (TCR) complexes is central to initiation of an adaptive immune response. These complexes form through initial stabilization of the MHC fold via binding of a short peptide, and subsequent interaction of the TCR to form a ternary complex, with contacts made predominantly through the complementarity-determining region (CDR) loops of the TCR. Stimulation of an immune response is central to cancer immunotherapy. This approach depends on identification of the appropriate combinations of MHC molecules, peptides, and TCRs to elicit an antitumor immune response. This prediction is a current challenge in computational biochemistry. In this chapter, we introduce a predictive method that involves generation of multiple peptides and TCR CDR 3 loop conformations, solvation of these conformers in the context of the MHC-peptide-TCR ternary complex, extraction of parameters from the generated complexes, and use of an AI model to evaluate the potential for the assembled ternary complex to support an immune response.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
Cancer is the second biggest cause of death globally, and the use of therapeutic peptides to specifically target and destroy cancer cells has gotten much interest. Cancer peptides or vaccinations are utilized to treat cancer nowadays, apart from chemotherapy, which has significant discomfort, side effects and costly. It is time demanding to identify and predict potential anticancer peptides using computational biology approaches. Thus, 3-D molecular modeling is being used to find possible ACP candidates. In this research, Curcuma longa has predicted peptide sequences were docked on breast cancer receptors and used a molecular docking technique to assess the anticipated peptides' binding affinities to MHC molecules. A similar approach was utilized to simulate the interactions of the chosen peptide with the TCR. Additionally, the Pep10 LIRQHVASNIGIAKSKIREPIV was examined, and our findings indicated interaction with MHC class I and II. However, the maximum binding energy was obtained with TCR at 695.61, giving strength through eight hydrogen bonds. Similarly, the pep20, GAIIGNRKIKLQPHIIIRID, the projected, has the most significant overall binding energy with MHC class I and II but a lower global E total value with TCR, namely −600.97 kj/Mol, and also four hydrogen bonds. This research could lead to the development of novel anticancer drugs based on the anticancer activity of the Curcuma longa medicinal plant.
Autoimmunity is a breakdown of the normal mechanisms that maintain immunologic homeostasis in the immune system response to specific antigens. The pathogenesis of autoimmune disease is multifactorial, including both genetic and environmental factors affecting the onset, maintenance, and progression of diseases. Autoimmune disorders result from the recognition of self-antigen(s) and the subsequent attack on self-tissues, usually by the adaptive immune response. Autoimmune pathology can be caused by T lymphocytes, natural killer cell, and/or antibodies and can be modulated by hormonal effects. One of the unifying themes of many autoimmune disorders is that they are associated with specific alleles and genotypes of the highly polymorphic major histocompatibility complex where, in humans, the human leukocyte antigen loci reside. In addition, a host of other genes can contribute to the onset, course, management, and response to therapy of specific diseases.
We studied the prevalent cytotoxic CD8 T-cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent Human Leukocyte Antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘variants of concern’, was not recognised by the large CD8 T-cell response seen across cohorts of HLA A∗02⁺ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T-cell receptors. Viral escape at prevalent T-cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focussed on a single protein such as SARS-CoV-2 Spike providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T-cell escape in new SARS-CoV-2 variants.
The crystal structure of the Vα domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin
topology set different from those previously described. A switch in a polypeptide strand from one β sheet to the other enables
a pair of Vα homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable
loops face in one direction. On the basis of the observed mode of Vα association, a model of an (αβ)2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (αβ)2 tetramer with the third hypervariable loop of Vα over the amino-terminal portion of the antigenic peptide and the corresponding loop of Vβ over its carboxyl-terminal residues. TCR dimerization that is mediated by the α chain may contribute to the coupling of antigen
recognition to signal transduction during T cell activation.
Most of the polymorphic amino acids of the class I histocompatibility antigen, HLA-A2, are clustered on top of the molecule in a large groove identified as the recognition site for processed foreign antigens. Many residues critical for T-cell recognition of HLA are located in this site, in positions allowing them to serve as ligands to processed antigens. These findings have implications for how the products of the major histocompatibility complex (MHC) recognize foreign antigens.
From an analysis of the immunoglobulins of known structure we derive a list of 40 sites crucial for the conserved structure of the variable domains. We show that, with marginal exceptions, the sequences of the T-cell alpha beta receptors contain, at sites homologous to these 40, the same or very similar residues. Thus the V alpha-V beta dimer has a framework structure very close to that of the immunoglobulins. Further comparisons show that parts of the surface of the V alpha-V beta framework are hypervariable. They also show that the loops that form the antigen-binding site are similar in size to those commonly found in the immunoglobulins but have different conformations. Only limited sequence variations occur in the first loop of the antigen-binding site in both V alpha and V beta. This, and their geometrical arrangement, suggest that they mainly interact with the MHC proteins.
Class II and class I histocompatibility molecules allow T cells to recognize 'processed' polypeptide antigens. The two polypeptide chains of class II molecules, alpha and beta, are each composed of two domains (for review see ref. 6); the N-terminal domains of each, alpha 1 and beta 1, are highly polymorphic and appear responsible for binding peptides at what appears to be a single site and for being recognized by MHC-restricted antigen-specific T cells. Recently, the three-dimensional structure of the foreign antigen binding site of a class I histocompatibility antigen has been described. Because a crystal structure of a class II molecule is not available, we have sought evidence in class II molecules for the structural features observed in the class I binding site by comparing the patterns of conserved and polymorphic residues of twenty-six class I and fifty-four class II amino acid sequences. The hypothetical class II foreign-antigen binding site we present is consistent with mutation experiments and provides a structural framework for proposing peptide binding models to help understand recent peptide binding data.
A number of recent studies have suggested that the main functional role of the product of the immune response (Ir) genes is in the process of antigen recognition by the T lymphocyte. The observation in the accompanying report that the interaction of macrophage-associated antigen with immune T lymphocytes requires that both cells share histocompatibility antigens raised the question as to whether the macrophage played a role in the genetic control of the immune response or even if the macrophage were the primary cell in which the product of the Ir gene is expressed. In the current study, parental macrophages were pulsed with an antigen, the response to which is controlled by an Ir gene lacking in that parent; these macrophages were then mixed with T cells derived from the (nonresponder x responder)F(1) and the resultant stimulation was measured. No stimulation was seen when column-purified F(1) lymph node lymphocytes were mixed with antigen-pulsed macrophages from the nonresponder parent. However, when the highly reactive peritoneal exudate lymphocyte population was used as the indicator cells, parental macrophages pulsed with an antigen whose Ir gene they lacked were capable of initiating F(1) T-cell proliferation. The magnitude of stimulation was approximately 1/10 that seen when macrophages from either the responder parent or the F(1) were used. In order to explain this observation, we hypothesize that antigen recognition sites on the T lymphocyte are physically related to a macrophage-binding site and both are linked to the serologically determined histocompatibility antigens. Thus, parental macrophages pulsed with an antigen, whose Ir gene they lack, activate F(1) cells poorly because the recognition sites for the antigen are physically related to the macrophage-binding site of the responder parent while the main contacts between the cells are at the nonresponder binding sites. Experiments performed with alloantisera lend support to this hypothesis. Thus, when parental macrophages are pulsed with any antigen and added to F(1) T cells, an alloantiserum directed against parental histocompatibility antigens reacts with both the lymphocyte and the macrophage and thereby inhibits macrophage-lymphocyte interaction and abolishes antigen-induced lymphocyte transformation. When the alloantisera are directed at determinants present solely on the T lymphocyte, they only inhibit the recognition of antigens controlled by the Ir gene linked to the histocompatibility antigen against which they are directed. We conclude from these studies that antigen recognition by the T lymphocyte is a complex multicellular event involving more than simple antigen binding to a specific lymphocyte receptor.
For many years immunologists have been well served by the viewpoint that the immune system's primary goal is to discriminate between self and non-self. I believe that it is time to change viewpoints and, in this essay, I discuss the possibility that the immune system does not care about self and non-self, that its primary driving force is the need to detect and protect against danger, and that it does not do the job alone, but receives positive and negative communications from an extended network of other bodily tissues.
The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.
The frequencies of human T cell lymphotropic virus type 1 (HTLV-1)-specific CD8+ precursor cytotoxic T lymphocytes (pCTL) were quantitated from lymphocytes obtained from the peripheral blood and cerebrospinal fluid (CSF) of infected individuals with and without HTLV-1-associated neurological disease. An estimate of the pCTL was obtained by separating CD8+ cells, plating these cells in limiting dilution, and testing wells for HTLV-1 specific lysis. Targets consisted of autologous lymphoblastoid cell lines (LCL) infected with vaccinia constructs expressing HTLV-1 gene products or LCL pulsed with HTLV-1 synthetic peptides. In patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), the frequency of HTLV-1 p40X-specific pCTL was at least 40-280-fold higher than in asymptomatic HTLV-1-infected individuals. All HAM/TSP patients (five of five) predominantly recognized HTLV-1 products encoded within the pX region. Lower pCTL to env were demonstrated in three patients, and only one of five HAM/TSP patients had pCTL to gag. A synthetic peptide corresponding to the tax region of HTLV-1 (peptide 11-19, amino acid sequence LLFGYPVYV) was recognized in association with human histocompatibility leukocyte antigen (HLA)-A2 in two HLA-A2 HAM/TSP patients with a high CD8+ pCTL frequency of 1/325 and 1/265, respectively. A second immunodominant region of HTLV-1 tax (peptide 90-55, amino acid sequence VPYKRIEEL) was identified to be restricted by HLA-B14 in two HLA-B14 HAM/TSP patients with a CD8+ pCTL frequency of 1/640 and 1/1,125, respectively. Lymphocytes from the CSF of a patient with HAM/TSP also showed a pCTL frequency against p40X of similar magnitude to that demonstrated from peripheral blood lymphocytes (PBL). The HLA-A2-mediated CSF pCTL activity to the immunodominant tax-specific peptide 11-19 was also comparable to pCTL from PBL. These results indicate that an extremely high pCTL frequency to HTLV-1 tax-encoded peptides may be related to pathogenesis of myeloneuropathy associated with HTLV-1.
Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.
A T cell-mediated immune response is mainly determined by the 3-5 aa residues that protrude upwards from a peptide bound to an MHC molecule. Alterations of these peptide residues can diminish, eliminate or radically alter the signal that the T cell receives through its T cell receptor (TCR). We have used peptide immunizations of normal mice and mice carrying alpha or beta chain TCR transgenes to identify three distinct peptide contact points. One, near the carboxyl terminus of the peptide, involves the beta chain CDR3 region; the second was centrally located and interacted with both the alpha and beta chain CDR3 loops; the third was near the amino terminus of the peptide, and affected V alpha gene usage, but not the structure of CDR3 of either TCR chain. Based on these results, we propose an orientation for the TCR of this cloned line and argue for its generality.
From an analysis of the immunoglobulins of known structure we derive a list of 40 sites crucial for the conserved structure of the variable domains. We show that, with marginal exceptions, the sequences of the T‐cell alpha beta receptors contain, at sites homologous to these 40, the same or very similar residues. Thus the V alpha‐V beta dimer has a framework structure very close to that of the immunoglobulins. Further comparisons show that parts of the surface of the V alpha‐V beta framework are hypervariable. They also show that the loops that form the antigen‐binding site are similar in size to those commonly found in the immunoglobulins but have different conformations. Only limited sequence variations occur in the first loop of the antigen‐binding site in both V alpha and V beta. This, and their geometrical arrangement, suggest that they mainly interact with the MHC proteins.
The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.
Different antibodies adopt substantially different shapes in order to recognize their respective antigens. Local and more global structural rearrangements, in addition to sequence and size diversity of complementarity determining loops, result in antigen-binding sites that can vary considerably in size, shape and charge distribution, even though the antibody framework structure remains invariant. The structures of several free and bound Fabs show that induced-fit mechanisms best describe the antibody-antigen recognition process. This affects the prediction, engineering and design of antibody combining sites.
The structural origins of antibody diversity are well understood as a result of X-ray crystallographic and molecular modelling studies of Fab fragments. A similar understanding of antibody specificity is beginning to emerge from an analysis of structures of hapten, peptide and protein antibody complexes. While the nature of the antibody-antigen interface, and any conformational changes that occur on complex formation, can be described in structural terms, a full explanation of the thermodynamic and mechanistic basis of affinity is less accessible from structure alone. A number of physiochemical studies carried out on wild type and mutant antibodies have raised questions about the nature of the energetics of the interaction, and the possibility of a key role for water molecules has been discussed. The possibility of ‘induced fit’ as a common mechanism for antigen-antibody interactions has been raised, and a molecular basis for hapten and protein cross-reactivity proposed. These recent contributions to the field, as well as providing partial solutions to old problems, have provided exciting new insights.
To test models of T-cell recognition, mice transgenic for T-cell receptor alpha or beta chain have been immunized with variant peptides that force changes in the resulting T-cell response. In particular, charge substitutions on the peptide often elicit reciprocal charges in the junctional (CDR3) sequences of T-cell receptor V alpha or V beta chains, indicating direct T-cell receptor-peptide contact, and allowing derivation of a topology for the T-cell receptor-MHC interaction. At one position on the peptide, variants transformed a homogeneous V beta response into a very heterogeneous one.
Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.
The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to greater than 1.5-A resolution.
We review recent data that increase our understanding of the ternary complex of the T cell receptor (TCR), antigenic peptides, and molecules of the major histocompatibility complex (MHC). Studies using synthetic peptide analogs for T-cell antigens have identified peptide residues that appear to interact with the MHC molecule and/or the TCR. The logical extension of these studies, using a complete replacement set of peptide analogues for a model peptide antigen, has more precisely defined the biochemical character of putative MHC and TCR contact residues, and indicated that the TCR is highly sensitive to subtle changes in peptide conformation. Insight into the binding site for peptide on the TCR has recently come from variant peptide immunization of TCR single-chain transgenic mice. These experiments indicate that residues encoded by the V(D)J junctions of both TCR chains contact peptide directly. TCR-MHC contacts have also been studied, using in vitro-mutagenized MHC molecules, particularly those altered at residues predicted to point "up," toward the TCR. These studies reveal that TCR-MHC contacts appear to be quite flexible, and vary between even closely related TCRs. A measure of the affinity of TCR for peptide/MHC complexes has come from competition experiments using soluble MHC complexed with specific peptides. This affinity, with a KD of 5 x 10(-5) M, is several orders of magnitude lower than that of most antibodies for their protein antigens and suggests that the sequence of events leading to T-cell activation begins with antigen-independent adhesion.
Here, Charles Janeway argues that the requirement for two signals to initiate the adaptive immune response may reflect the evolutionary history of host defences. Early phases of host defence involve receptors and ligands that may have controlled immune responses prior to the development of clonally-distributed receptors encoded in rearranging genes. The former receptors persist in contemporary vertebrates both to trigger innate or nonclonal responses and to signal to lymphocytes that a particular antigen is associated with a microorganism.
The interaction of antigen-specific T cell receptors (TCRs) with their ligands, peptides bound to molecules of the major histocompatibility complex (MHC), is central to most immune responses, yet little is known about its chemical characteristics. The binding to T cells of a labeled monoclonal antibody to the TCR was inhibited by soluble class II MHC heterodimers complexed to different peptides. Inhibition was both peptide- and TCR-specific and of low affinity, with a KD = 4 x 10(-5) to 6 x 10(-5) M, orders of magnitude weaker than comparable antibody-antigen interactions. This finding is consistent with the scanning nature of T cell recognition and suggests that antigen-independent adhesion precedes TCR engagement.
We present an algorithm for generating images of molecules represented as a set of intersecting opaque spheres. Both perspective and shadows are computed to provide realistic visual cues. Compared to existing programs for generating similar images, our algorithm is both more accurate and several times faster. We present in detail the mathematics used in picture generation, along with examples of the computed images.
Map interpretation remains a critical step in solving the structure of a macromolecule. Errors introduced at this early stage may persist throughout crystallographic refinement and result in an incorrect structure. The normally quoted crystallographic residual is often a poor description for the quality of the model. Strategies and tools are described that help to alleviate this problem. These simplify the model-building process, quantify the goodness of fit of the model on a per-residue basis and locate possible errors in peptide and side-chain conformations.
The antigen-specific receptor of T lymphocytes (TCR) and the Fab moiety of immunoglobulins are expected to fold into similar three-dimensional structures because of their identical protein domain organization, the conservation of key residues and their overall sequence homology. However, T cells mostly appear to recognize short peptide antigens bound to MHC class I or class II presenting molecules. A complete model of the human leucocyte antigen molecule (HLA-A2) reconstructed from the alpha-carbon coordinates was used to investigate the putative organization of a TCR/peptide/HLA-A2 complex. In this article, Jean-Michel Claverie and co-workers show that the respective geometries of a Fab-like TCR structure and of the HLA-A2 antigen binding site suggest a model where the third variable regions of both chains of the TCR mainly interact with the peptide antigen, while the first and/or second less variable regions are in position for making contact with residues pointing up from the alpha 1 and alpha 2 helical regions of the HLA-A2 molecule.
TCRs undergo a series of interactions with ligands during development. We have characterized the interaction of a TCR with its ligand and the attendant co-receptor and co-ligand structures. This characterization has led to the model in which the TCR not only binds to class II MHC, but also binds to CD4 co-receptors and co-ligands such as Mls. We have shown that both cross-linking and conformational change in the TCR are required for optimal T-cell activation. Finally, we have used the observation that a particular self-peptide found abundantly associated with class II MHC in the periphery is essentially lacking from thymic cortical epithelium to argue that positive selection for self-MHC recognition may occur by a novel process in the thymic cortex. A TCR recognizing class II MHC with low affinity could either be multiply cross-linked in the absence of conformational change, which here would be driven by a unique peptide, or could be conformationally changed without cross-linking due to the rarity of the individual high-affinity peptide on thymic cortical epithelial cells. Either proposal leads to a partial signal one delivered via the TCR, which we refer to as signal one-half. This signal one-half would induce the cell to repress its other co-receptor molecule and to undergo maturation events such as up-regulation in TCR expression. Such cells are then rigorously screened for activating interactions with autologous structures, such as Mls. The threshold for clonal deletion is set very low to avoid autoreactivity. By this combination of signaling events, a mature TCR repertoire is generated that has the functional characteristics observed in immune systems.
The four distinct T-cell antigen receptor polypeptides (alpha, beta, gamma, delta) form two different heterodimers (alpha:beta and gamma:delta) that are very similar to immunoglobulins in primary sequence, gene organization and modes of rearrangement. Whereas antibodies have both soluble and membrane forms that can bind to antigens alone, T-cell receptors exist only on cell surfaces and recognize antigen fragments only when they are embedded in major histocompatibility complex (MHC) molecules. Patterns of diversity in T-cell receptor genes together with structural features of immunoglobulin and MHC molecules suggest a model for how this recognition might occur. This view of T-cell recognition has implications for how the receptors might be selected in the thymus and how they (and immunoglobulins) may have arisen during evolution.
We used congenic-resistant mouse strains to answer questions concerning the respective roles of genes coding for major histocompatibility and background genotypes in T (thymus-derived)-B (bone marrow-derived) lymphocyte cooperative responses to hapten-protein conjugates. These studies demonstrate conclusively that the gene or genes present in the H-2 complex control the capacity of antigen-specific T and B cells to effectively interact. These findings led us to postulate that there exists on the B-lymphocyte surface an "acceptor" molecule for the active T-cell product or for the T cell itself.
RECENT experiments1-3 indicate that cooperation between
thymus derived lymphocytes (T cells) and antibody-forming cell
precursors (B cells) is restricted by the H-2 gene complex. Helper
activity in vivo operates only when T cells and B cells share at least
one set of H-2 antigenic specificities. Evidence is presented here that
the interaction of cytotoxic T cells with other somatic cells
budding4-5 lymphocytic choriomeningitis (LCM) virus is
similarly restricted.
A complex between HLA-DR3 and a fragment of invariant chain called CLIP was isolated from a human cell line defective in antigen presentation and its X-ray crystal structure determined. Previous data indicate that this complex is an intermediate in class II histocompatibility maturation, occurring between invariant chain-DR3 and antigenic peptide-DR3 complexes. The structure shows that the CLIP fragment binds to DR3 in a way almost identical to that in which antigenic peptides bind class II histocompatibility glycoproteins. The structure is the substrate for the loading of antigenic peptides by an exchange process catalysed by DM.
Structural similarity between viral T cell epitopes and self-peptides could lead to the induction of an autoaggressive T cell response. Based on the structural requirements for both MHC class II binding and TCR recognition of an immunodominant myelin basic protein (MBP) peptide, criteria for a data base search were developed in which the degeneracy of amino acid side chains required for MHC class II binding and the conservation of those required for T cell activation were considered. A panel of 129 peptides that matched the molecular mimicry motif was tested on seven MBP-specific T cell clones from multiple sclerosis patients. Seven viral and one bacterial peptide efficiently activated three of these clones. Only one peptide could have been identified as a molecular mimic by sequence alignment. The observation that a single T cell receptor can recognize quite distinct but structurally related peptides from multiple pathogens has important implications for understanding the pathogenesis of autoimmunity.
Recognition of class I MHC antigens involves interaction between TCRs of cytotoxic T lymphocytes (CTL) and the two alpha helices of MHC molecules. Using a combined panel of H-2Kb mutants selected by either a CTL clone or MAbs, we have shown evidence that the TCRs of 59 Kb-specific CTL clones shared a common binding pattern on the H-2Kb molecule. Mutations of amino acid residues at the C-terminal regions, but not the N-terminal regions, of the alpha helices abrogated the recognition by the majority of the clones. The data suggests that TCRs predominantly recognize the class I MHC molecule with an orientation that is parallel to the beta-pleated strands and diagonal to the alpha helices.
The ability of MHC molecules to present a broad spectrum of peptide antigens for T cell recognition requires a compromise between high affinity and broad specificity. Three-dimensional atomic structures of several class I and class II MHC molecules reveal a unique structural solution to this problem: Tight binding to the peptide main chain is supplemented by more or less restrictive interactions with peptide side chains. In spite of these contacts, peptide side-chain and conformational variability ensures that the resulting peptide-MHC complex presents an antigenically unique surface to T cell receptors.
Extension of this understanding to other peptide-MHC complexes, including agonist/antagonist peptides and the identification of antigenic peptides within protein sequences, however, requires a detailed analysis of the interactions that determine both peptide-MHC binding affinity and the conformations of bound peptides. While many of these interactions can be modeled by homology with known structures, their specificity can depend sensitively on subtle and long range structural effects. Structurally and immunologically important distinctions are also found between the class I and class II peptide-binding strategies. Taken together, these interactions ultimately determine the ability of an individual to respond successfully to immune challenges.
Complexes of five peptides (from HIV-1, influenza A virus, HTLV-1, and hepatitis B virus proteins) bound to the human class I MHC molecule HLA-A2 have been studied by X-ray crystallography. While the peptide termini and their second and C-terminal anchor side chains are bound similarly in all five cases, the main chain and side chain conformations of each peptide are strikingly different in the center of the binding site, and these differences are accessible to direct TCR recognition. Each of the central peptide residues is seen to point up for some bound peptides, but down or sideways for others. Thus, although fixed at its ends, the structure of an MHC-bound peptide appears to be a highly complex function of its entire sequence, potentially sensitive to even small sequence differences. In contrast, MHC structural variation is relatively limited. These results offer a structural framework for understanding the role of nonanchor peptide side chains in both peptide-MHC binding affinity and TCR recognition.
The crystal structure of the extracellular portion of the beta chain of a murine T cell antigen receptor (TCR), determined
at a resolution of 1.7 angstroms, shows structural homology to immunoglobulins. The structure of the first and second hypervariable
loops suggested that, in general, they adopt more restricted sets of conformations in TCR beta chains than those found in
immunoglobulins; the third hypervariable loop had certain structural characteristics in common with those of immunoglobulin
heavy chain variable domains. The variable and constant domains were in close contact, presumably restricting the flexibility
of the beta chain. This may facilitate signal transduction from the TCR to the associated CD3 molecules in the TCR-CD3 complex.
Recent structural results have shown that antibodies use an induced fit mechanism to recognize and bind their antigens. Here we present the crystallographically determined structure of an Fab directed against an HIV-1 peptide (Fab 50.1) in the unliganded state and compare it with the peptide-bound structure. We perform a detailed analysis of the components that contribute to enhanced antigen binding and recognition.
Induced fit of Fab 50.1 to its peptide antigen involves a substantial rearrangement of the third complementarity determining region loop of the heavy chain (H3), as well as a large rotation of the variable heavy (VH) chain relative to the variable light (VL) chain. Analysis of other Fab structures suggests that the extent of the surface area buried at the VL-VH interface correlates with the ability to alter antibody quaternary structure by reorientation of the VL-VH domains.
Fab 50.1 exhibits the largest conformational changes yet observed in a single antibody. These can be attributed to the flexibility of the variable region. Comparisons of new data with previous examples lend to the general conclusion that a small VL-VH interface, due in part to a short H3 loop, permits substantial alterations to the antigen-binding pocket. This has major implications for the prediction, engineering and design of antibody-combining sites.
The recent determination of the structure of a class II MHC molecule complexed to a specific peptide reveals both similarities and differences with peptide binding by class I MHC.
The structures of the various regions of an antibody molecule are analysed and correlated with biological function. The structural features which relate to potential applications are detailed.
An influenza virus peptide binds to HLA-DR1 in an extended conformation with a pronounced twist. Thirty-five per cent of the peptide surface is accessible to solvent and potentially available for interaction with the antigen receptor on T cells. Pockets in the peptide-binding site accommodate five of the thirteen side chains of the bound peptide, and explain the peptide specificity of HLA-DR1. Twelve hydrogen bonds between conserved HLA-DR1 residues and the main chain of the peptide provide a universal mode of peptide binding, distinct from the strategy used by class I histocompatibility proteins.
We have generated mice lacking H2-M complexes, critical facilitators of peptide loading onto major histo-compatibility complex class II molecules. Ab molecules in these mice matured into stable complexes and were efficiently expressed at the cell surface. Most carried a single peptide derived from the class II-associated invariant chain; the diverse array of peptides normally displayed by class II molecules was absent. Cells from mutant mice presented both whole proteins and short peptides very poorly. Surprisingly, positive selection of CD4+ T cells was quite efficient, yielding a large and broad repertoire. Peripheral T cells reacted strongly to splenocytes from syngeneic wild-type mice, no doubt reflecting the unique peptide complement carried by class II molecules in mutant animals.
Although the thymus produces many immature thymocytes, few of these cells mature. Positive selection has been thought to limit thymocyte development. In thymuses expressing a single MHC/peptide combination, however, surprisingly large numbers of thymocytes are selected to mature. Many of these react with the selecting MHC, bound to other self-peptides. Therefore, the number of thymocytes that mature is limited by the fact that positively selected cells die because they react too well with MHC bound to self-peptides that are not identical to those involved in positive selection. T cells that mature in thymuses expressing a single MHC/peptide ligand react frequently with foreign MHC, suggesting that the repertoire of alpha beta receptors may be more biased toward reaction with MHC than was previously thought.
The human β T cell receptor (TCR) locus, comprising a complex family of genes, has been sequenced. The locus contains two
types of coding elements—TCR elements (65 variable gene segments and two clusters of diversity, joining, and constant segments)
and eight trypsinogen genes—that constitute 4.6 percent of the DNA. Genome-wide interspersed repeats and locus-specific repeats
span 30 and 47 percent, respectively, of the 685-kilobase sequence. A comparison of the germline variable elements with their
approximately 300 complementary DNA counterparts reveals marked differential patterns of variable gene expression, the importance
of exonuclease activity in generating TCR diversity, and the predominant tendency for only functional variable elements to
be present in complementary DNA libraries.
Recent crystallographic studies of T cell antigen receptor (TCR) fragments from the alpha and beta chains have now confirmed the expected structural similarity to corresponding immunoglobulin domains. Although the three-dimensional structure of a complete TCR alpha beta heterodimer has not yet been determined, these results support the view that the extracellular region should resemble an immunoglobulin Fab fragment with the antigen-binding site formed from peptide loops homologous to immunoglobulin complementarity-determining regions (CDR). These preliminary results suggest that CDR1 and CDR2 may be less variable in structure than their immunoglobulin counterparts, consistent with the idea that they may interact preferentially with the less polymorphic regions of the molecules of the major histocompatibility complex. The region on the variable beta domain responsible for superantigen recognition is analyzed in detail. The implications for T cell activation from the interactions observed between domains of the alpha and beta chains are also discussed in terms of possible dimerization and allosteric mechanisms.
We have developed a method that utilizes site-specific mutation data, sequence analysis, immunological data and free-energy minimization, to determine structural features of the ternary complex formed by the T-cell receptor (TcR) and the class I major histocompatibility complex (MHC) molecule bound by peptide. The analysis focuses on the mouse Kd MHC system, for which a large set of clones with sequenced T-cell receptors is available for specific peptides. The general philosophy is to reduce the uncertainties and computation time in a free-energy minimization procedure by identifying and imposing experimental constraints. In addition to assessing compatibility with various kinds of immunological data, we are particularly interested in differentiating the structural features peculiar to this particular system from generic features, and in ascertaining the robustness of the structure; i.e. determining, in so far as possible, the variations in the structure that leave its compatibility with experiment unaltered from those that do not. This last is equivalent to recognizing that certain features of the model are presented with a reasonable degree of confidence, while others remain highly tentative. The central conclusion in the former category is a placement of the TcR on the Kd peptide complex, which has its beta 2, beta 3 and alpha 3 loops (i.e. the second and third complementarity-determining region of the TcR beta chain, and the third complementarity-determining region of the alpha chain) covering the peptide; the alpha 1 and alpha 2 loops covering the MHC alpha 1 helix; the alpha 2 loop interacting with residues on the MHC beta sheet; and the beta 1 and (part of) the beta 2 loops covering the alpha 2 MHC helix. More specifically, our findings include the following. (1) A highly conserved histidine residue in the first complementarity-determining region of the TcR beta chain (beta:CDR1) points outward and interacts with highly conserved side-chains on the MHC alpha 2 helix. (2) The amino-terminal portion of the beta 2 loop interacts with the carboxyl portion of the peptide. A particularly important interaction is K4 of the loop interacting with E8 of the peptide. (3) Charged side-chains of the 11-residue TcR alpha 2 loop interact with conserved charged side-chains at positions 44, 58, 61 and 68 on the MHC. (4) The TcR beta 3 loop interacts with the amino-terminal part of the peptide, up through position 4. (5) the TcR alpha 3 loop interacts with the central portion of the peptide and stacks against the beta 2 loop. (6) Because of the interaction between the beta 2 loop and the peptide, and stacking of beta 2 on alpha 3, alpha 3 gene and V beta gene selection can be correlated. (7) Using the topology of the recently solved TcR alpha chain we predict that the alpha 2 loop interacts with the loop on the MHC beta sheet floor, which encompasses residues 42 to 44.
The central event in the cellular immune response to invading microorganisms is the specific recognition of foreign peptides
bound to major histocompatibility complex (MHC) molecules by the αβ T cell receptor (TCR). The x-ray structure of the complete
extracellular fragment of a glycosylated αβ TCR was determined at 2.5 angstroms, and its orientation bound to a class I MHC-peptide
(pMHC) complex was elucidated from crystals of the TCR-pMHC complex. The TCR resembles an antibody in the variable Vα and
Vβ domains but deviates in the constant Cα domain and in the interdomain pairing of Cα with Cβ. Four of seven possible asparagine-linked
glycosylation sites have ordered carbohydrate moieties, one of which lies in the Cα-Cβ interface. The TCR combining site is
relatively flat except for a deep hydrophobic cavity between the hypervariable CDR3s (complementarity-determining regions)
of the α and β chains. The 2C TCR covers the class I MHC H-2Kb binding groove so that the Vα CDRs 1 and 2 are positioned over the amino-terminal region of the bound dEV8 peptide, the Vβ
chain CDRs 1 and 2 are over the carboxyl-terminal region of the peptide, and the Vα and Vβ CDR3s straddle the peptide between
the helices around the central position of the peptide.
Antibody specificity is determined by structural v-genes that code for the amino acid sequences of the variable regions of antibody polypeptide chains. The present hypothesis proposes that the germ-cells of an animal carry a set of v-genes determining the combining sites of antibodies directed against a complete set of a certain class of histocompatibility antigens of the species to which this animal belongs. The evolutionary development of this set of v-genes in phylogeny is traced back to the requirements for cell to cell recognition in all metazoa. The hypothesis leads to a distinction between two populations of antigen-sensitive cells. One population consists of cells forming antibodies against foreign antigens; these lymphocytes have arisen as mutants in clones descending from lymphocytic stem cells which expressed v-genes belonging to the subset (subset S) coding for antibody against histocompatibility antigens that the individual happens to possess. The other population consists of allograft rejecting lymphocytes that express v-genes of the remaining subset (subset A) coding for antibody against histocompatibility antigens of the species that the individual does not possess. The primary lymphoid organs are viewed as mutant-breeding organs. In these organs (e. g. in the thymus), the proliferation of lymphocytes expressing the v-genes of subset S and the subsequent suppression of the cells of these “forbidden” clones, leads to the selection of mutant cells expressing v-genes that have been modified by spontaneous random somatic mutation. This process generates self-tolerance as well as a diverse population of antigen-sensitive cells that reflects antibody diversity. The proliferation in the primary lymphoid organs of lymphocytes expressing v-genes of subset A generates the antigen-sensitive cell population that is responsible for allo-aggression.
The determination of macromolecular structure by crystallography involves fitting atomic models to the observed diffraction data. The traditional measure of the quality of this fit, and presumably the accuracy of the model, is the R value. Despite stereochemical restraints, it is possible to overfit or 'misfit' the diffraction data: an incorrect model can be refined to fairly good R values as several recent examples have shown. Here I propose a reliable and unbiased indicator of the accuracy of such models. By analogy with the cross-validation method of testing statistical models I define a statistical quantity (R(free) (T) that measures the agreement between observed and computed structure factor amplitudes for a 'test' set of reflections that is omitted in the modelling and refinement process. As examples show, there is a high correlation between R(free) (T) and the accuracy of the atomic model phases. This is useful because experimental phase information is usually inaccurate, incomplete or unavailable. I expect that R(free) (T) will provide a measure of the information content of recently proposed models of thermal motion and disorder, time-averaging and bulk solvent.
- P. Matzinger
Fundamental Immunology
- S. M. Hedrick
- F. J. Eidelman