T-cell recognition of glycolipids presented by CD1 proteins

The most well-known molecular paradigm of antigen recognition by T cells involves partial digestion of proteins to generate small peptides, which bind to major histocompatibility complex (MHC) proteins. Recent studies of CD1, an MHC class I homolog encoded outside the MHC, have revealed that it presents diverse glycolipids to T cells. The molecular mechanism for lipid antigen recognition involves insertion of the lipid portion of antigens into a hydrophobic groove to form CD1-lipid complexes, which contact T-cell receptors (TCRs). Here, we examine the known antigen structures presented by CD1, the majority of which have sugar moieties that are capable of interacting with TCRs. Recognition of carbohydrate epitopes is precise, and lipid-reactive T cells alter systemic immune responses in models of infectious and autoimmune disease. These findings provide a previously unrecognized mechanism by which the cellular immune system can recognize alterations in many types of carbohydrate structures.     

Viral Escape Mutant Epitope Maintains TCR Affinity for Antigen yet Curtails CD8 T Cell Responses

T cells have the remarkable ability to recognize antigen with great specificity and in turn mount an appropriate and robust immune response. Critical to this process is the initial T cell antigen recognition and subsequent signal transduction events. This antigen recognition can be modulated at the site of TCR interaction with peptide:major histocompatibility (pMHC) or peptide interaction with the MHC molecule. Both events could have a range of effects on T cell fate. Though responses to antigens that bind sub-optimally to TCR, known as altered peptide ligands (APL), have been studied extensively, the impact of disrupting antigen binding to MHC has been highlighted to a lesser extent and is usually considered to result in complete loss of epitope recognition. Here we present a model of viral evasion from CD8 T cell immuno-surveillance by a lymphocytic choriomeningitis virus (LCMV) escape mutant with an epitope for which TCR affinity for pMHC remains high but where the antigenic peptide binds sub optimally to MHC. Despite high TCR affinity for variant epitope, levels of interferon regulatory factor-4 (IRF4) are not sustained in response to the variant indicating differences in perceived TCR signal strength. The CD8+ T cell response to the variant epitope is characterized by early proliferation and up-regulation of activation markers. Interestingly, this response is not maintained and is characterized by a lack in IL-2 and IFNγ production, increased apoptosis and an abrogated glycolytic response. We show that disrupting the stability of peptide in MHC can effectively disrupt TCR signal strength despite unchanged affinity for TCR and can significantly impact the CD8+ T cell response to a viral escape mutant.     

Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.     

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

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

Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with alpha beta TCRs

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.     

A flexible docking approach for prediction of T cell receptor–peptide–MHC complexes

T cell receptors (TCRs) are immune proteins that specifically bind to antigenic molecules, which are often foreign peptides presented by major histocompatibility complex proteins (pMHCs), playing a key role in the cellular immune response. To advance our understanding and modeling of this dynamic immunological event, we assembled a protein–protein docking benchmark consisting of 20 structures of crystallized TCR/pMHC complexes for which unbound structures exist for both TCR and pMHC. We used our benchmark to compare predictive performance using several flexible and rigid backbone TCR/pMHC docking protocols. Our flexible TCR docking algorithm, TCRFlexDock, improved predictive success over the fixed backbone protocol, leading to near‐native predictions for 80% of the TCR/pMHC cases among the top 10 models, and 100% of the cases in the top 30 models. We then applied TCRFlexDock to predict the two distinct docking modes recently described for a single TCR bound to two different antigens, and tested several protein modeling scoring functions for prediction of TCR/pMHC binding affinities. This algorithm and benchmark should enable future efforts to predict, and design of uncharacterized TCR/pMHC complexes.     

The CD8 T cell coreceptor exhibits disproportionate biological activity at extremely low binding affinities

T lymphocytes recognize peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells. Recognition specificity is determined by the alphabeta T cell receptor (TCR). The T lymphocyte surface glycoproteins CD8 and CD4 enhance T cell antigen recognition by binding to MHC class I and class II molecules, respectively. Biophysical measurements have determined that equilibrium binding of the TCR with natural agonist peptide-MHC (pMHC) complexes occurs with KD values of 1-50 microm. The pMHCI/CD8 and pMHCII/CD4 interactions are significantly weaker than this (KD >100 microm), and the relative roles of TCR/pMHC and pMHC/coreceptor affinity in T cell activation remain controversial. Here, we engineer mutations in the MHCI heavy chain and beta2-microglobulin that further reduce or abolish the pMHCI/CD8 interaction to probe the significance of pMHC/coreceptor affinity in T cell activation. We demonstrate that the pMHCI/CD8 coreceptor interaction retains the vast majority of its biological activity at affinities that are reduced by over 15-fold (KD > 2 mm). In contrast to previous reports, we observe that the weak interaction between HLA A68 and CD8, which falls within this spectrum of reduced affinities, retains substantial functional activity. These findings are discussed in the context of current concepts of coreceptor dependence and the mechanism by which TCR coreceptors facilitate T cell activation.     

β‐arrestin‐1 mediates the TCR‐triggered re‐routing of distal receptors to the immunological synapse by a PKC‐mediated mechanism

T‐cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen‐presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β‐arrestin‐1 as a ligand of non‐phosphorylated resting TCRs. Using dominant‐negative and knockdown approaches we demonstrate that β‐arrestin‐1 is required for the internalization and downregulation of non‐engaged bystander TCRs. Furthermore, TCR triggering provokes the β‐arrestin‐1‐mediated downregulation of the G‐protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β‐arrestin‐1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR‐triggered PKC‐mediated phosphorylation of β‐arrestin‐1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal.     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.     

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

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

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

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

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

The Third Way: Progress on pathways of antigen processing and presentation by CD1

CD1 proteins are a third family of antigen presenting molecules that bind bacterial and autologous lipid antigens for presentation to T cells. With the solution of the crystal structures of several complexes of CD1 molecules with lipids, a greater appreciation has been gained of the adaptability of CD1 in binding lipid antigens with diverse structural features. Biochemical studies of the interactions between the TCR and CD1-lipid complexes have revealed striking contrasts with TCR that bind to peptides presented by MHC-encoded class I and class II molecules. The sphingolipid activating proteins (SAP) have recently been found to facilitate the transfer of lipid antigens onto CD1 molecules. This helps to provide an explanation as to how the thermodynamic barrier, caused by loading hydrophobic lipid antigens in a hydrophilic environment, can be overcome. Mechanisms of CD1 endosomal trafficking are being delineated, including the means by which adaptor proteins induce the localization of some types of CD1 molecules to lysosomes, where they bind antigens. Unlike MHC class I and class II proteins, specialized molecules that function solely in chaperoning CD1 molecules, or in facilitating their antigen loading, have not been found. This suggests that the CD1 antigen presenting system, which diverged early in vertebrate evolution from MHC antigen presenting molecules, is a simpler system with a character closer to the primordial antigen presenting function.     

Display, engineering, and applications of antigen-specific T cell receptors

The use of T cell receptors (TCRs) as potential therapeutic agents provides an opportunity to target a greatly expanded array of antigens, compared to those now targeted with monoclonal antibodies. With the advent of new display technologies and TCR formats for in vitro engineering, it should be possible to generate high-affinity TCRs against virtually any peptide antigen that is shown to bind to a major histocompatibility complex (MHC) molecule (e.g. peptides derived from viral antigens or from self proteins that are associated with the transformed phenotype). What remains, however, are challenges associated with effective targeting of very low numbers of cell surface antigens (pepMHC), fewer than the case for conventional monoclonal antibody-based therapies. This hurdle might be overcome with the attachment of more effective payloads for soluble TCR approaches, or by using TCR gene transfer into T cells that can then be adoptively transferred into patients. There is considerable work to be done on the physiological aspects of either approach, including pharmacokinetic studies in the case of soluble TCRs, and T cell trafficking, persistence, and autoreactivity studies in the case of adoptively transferred T cells. As with the field of monoclonal antibodies, it will take time to explore these issues, but the potential benefits of TCR-based therapies make these challenges worth the effort.     

以T细胞受体为基础的免疫疗法研究进展

T细胞是机体抗肿瘤免疫的核心,以T细胞功能调控为基础的免疫检查点疗法已经在多种肿瘤的临床治疗中取得了重大突破,以基因工程化T细胞为基础的过继性免疫细胞疗法在血液瘤治疗中取得了重要进展,免疫治疗已经对肿瘤的临床治疗产生了深刻变革,成为肿瘤临床治疗策略的重要组成部分。T细胞受体（T cell receptor,TCR）赋予了T细胞识别肿瘤抗原的特异性,能够识别由主要组织相容性复合体（major histocompatibility complex,MHC）呈递的包括胞内抗原在内的广泛肿瘤抗原,具有高度的抗原敏感性,因而具有广泛的抗肿瘤应用前景。2022年第一款TCR药物的上市开启了TCR药物开发的新纪元,多项TCR药物临床研究表现出潜在的肿瘤治疗价值。本文综述了以TCR为基础的免疫治疗策略研究进展,包括T细胞受体工程化T细胞（T cell receptor-engineered T cell,TCR-T）和TCR蛋白药物,以及基于TCR信号的其他免疫细胞疗法,以期为以TCR为基础的免疫治疗策略开发提供参考。     

Targeting hepatitis B virus-infected cells with a T-cell receptor-like antibody

Virus-specific CD8 T cells are activated when their T-cell receptors (TCRs) recognize the specific viral peptide/major histocompatibility complex (MHC) class I (pMHC) complexes present on the surface of infected cells. Antibodies able to recognize the specific pMHC can mimic TCR specificity and both represent a valuable biological tool to visualize pMHC complexes on infected cells and serve as a delivery system for highly targeted therapies. To evaluate these possibilities, we created a monoclonal antibody able to specifically recognize a hepatitis B virus (HBV) envelope epitope (Env at positions 183 to 91 [Env183-91]) presented by the HLA-A201 molecule, and we tested its ability to recognize HBV-infected hepatocytes and to deliver a cargo to a specific target. We demonstrate that this antibody detects and visualizes the processed product of HBV proteins produced in naturally HBV-infected cells, is not inhibited by soluble HBV proteins present in patient sera, and mediates the intracellular delivery of a fluorescent molecule to target cells. Additionally, compared to CD8 T cells specific for the same HBV epitope, the TCR-like antibody has both a superior sensitivity and a specificity focused on distinct amino acids within the epitope. These data demonstrate that a T-cell receptor-like antibody can be used to determine the quantitative relationship between HBV replication and specific antigen presentation to CD8 T cells and serves as a novel therapeutic delivery platform for personalized health care for HBV-infected patients.     

The evolution of vertebrate antigen receptors: a phylogenetic approach

Classical T cells, those with alpha beta T-cell receptors (TCRs), are an important component of the dominant paradigm for self-nonself immune recognition in vertebrates. alpha beta T cells recognize foreign peptide antigens when they are bound to MHC molecules on the surfaces of antigen-presenting cells. gamma delta T cells bear a similar receptor, and it is often assumed that these T cells also require specialized antigen-presenting molecules for immune recognition, which we term "indirect antigen recognition." B-cell receptors, or immunoglobulins, bind directly to antigens without the help of a specialized antigen-presenting molecule. Phylogenetically, it has been assumed that T-cell receptors and the genes that encode them are a monophyletic group, and that "indirect" antigen recognition evolved before the split into two types of TCR. Recently, however, it has been proposed that gamma delta-TCRs bind directly to antigens, as do immunoglobulins (Ig's). This calls into question the null hypothesis that indirect antigen recognition is a common characteristic of TCRs and, by extension, the hypothesis that all TCR gene sequences form a monophyletic group. To determine whether alternative explanations for antigen recognition and other historical relationships among TCR genes might be possible, we performed phylogenetic analyses on amino acid sequences of the constant and variable regions which encode the basic subunits of TCR and Ig molecules. We used both maximum-parsimony and genetic distance-based methods and could find no strong support for the hypothesis of TCR monophyly. Analyses of the constant region suggest that TCR gamma or delta sequences are the most ancient, implying that the ancestral immune cell was like a modern gamma delta T cell. From this gamma delta-like ancestor arose alpha beta T cells and B cells, implying that indirect antigen recognition is indeed a derived property of alpha beta-TCRs. Analyses of the variable regions are complicated by strong selection on antigen-binding sequences, but imply that direct antigen binding is the ancestral condition.     

Fine specificity of TCR complementarity-determining region residues and lipid antigen hydrophilic moieties in the recognition of a CD1-lipid complex

alphabeta TCR can recognize peptides presented by MHC molecules or lipids and glycolipids presented by CD1 proteins. Whereas the structural basis for peptide/MHC recognition is now clearly understood, it is not known how the TCR can interact with such disparate molecules as lipids. Recently, we demonstrated that the alphabeta TCR confers specificity for both the lipid Ag and CD1 isoform restriction, indicating that the TCR is likely to recognize a lipid/CD1 complex. We hypothesized that lipids may bind to CD1 via their hydrophobic alkyl and acyl chains, exposing the hydrophilic sugar, phosphate, and other polar functions for interaction with the TCR complementarity-determining regions (CDRs). To test this model, we mutated the residues in the CDR3 region of the DN1 TCR beta-chain that were predicted to project between the CD1b alpha helices in a model of the TCR/CD1 complex. In addition, we tested the requirement for the negatively charged and polar functions of mycolic acid for Ag recognition. Our findings indicate that the CDR loops of the TCR form the Ag recognition domain of CD1-restricted TCRs and suggest that the hydrophilic domains of a lipid Ag can form a combinatorial epitope recognized by the TCR.     

Antigen specificity of semi-invariant CD1d-restricted T cell receptors: the best of both worlds?

T lymphocytes are characterized by the use of structurally diverse TCR. The discovery of subsets of canonical T cells that have structurally homogeneous TCR presents an enigma: What antigens do these T cells recognize, and how does their antigen specificity relate to their functions? One subset of canonical T cells is restricted by CD1d, a non-classical antigen presenting molecule that presents lipids and glycolipids. Canonical CD1d-restricted T cells have semi-invariant TCR consisting of an invariantly rearranged TCR alpha chain, paired with diversely rearranged TCR beta chains. Most respond strongly to the unusual glycolipid alpha-galactosylceramide (alpha-GalCer), and can also respond to cellular antigens presented by CD1d. Mounting evidence indicates that alpha-GalCer responsive T cells are heterogeneous in their reactivities to cellular antigens, suggesting that an individual semi-invariant TCR may be capable of recognizing more than one ligand. Recent crystal structures of CD1b molecules with three different bound lipids indicate that the antigenic features of lipids may be localized over a smaller area than those of peptides, and that the positioning of the polar head group can vary substantially. A model that explains how CD1d-restricted T cells could possess both conserved and heterogeneous antigen specificities, is that different lipid antigens may interact with distinct areas of a TCR due to differences in the positioning of the polar head group. Hence, canonical CD1d-restricted TCR could recognize conserved antigens via the invariant TCR alpha chain, and have diverse antigen specificities that are conferred by their individual TCR beta chains.