RESUMO
The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.
Assuntos
Antígenos de Neoplasias , Neoplasias , Proteômica , Receptores de Antígenos de Linfócitos T , Antígenos de Neoplasias/metabolismo , Epitopos , Imunoterapia , Linfócitos do Interstício Tumoral , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
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 recognized 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 focused 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.
Assuntos
COVID-19 , SARS-CoV-2 , Linfócitos T CD8-Positivos , Epitopos de Linfócito T , Antígenos HLA-A , Antígenos de Histocompatibilidade Classe I , HumanosRESUMO
Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.
Assuntos
Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Sistemas CRISPR-Cas , Estudo de Associação Genômica Ampla , Humanos , Imunoterapia/métodos , Ativação Linfocitária/imunologia , CamundongosRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
In the thymus, high-affinity, self-reactive thymocytes are eliminated from the pool of developing T cells, generating central tolerance. Here, we investigate how developing T cells measure self-antigen affinity. We show that very few CD4 or CD8 coreceptor molecules are coupled with the signal-initiating kinase, Lck. To initiate signaling, an antigen-engaged T cell receptor (TCR) scans multiple coreceptor molecules to find one that is coupled to Lck; this is the first and rate-limiting step in a kinetic proofreading chain of events that eventually leads to TCR triggering and negative selection. MHCII-restricted TCRs require a shorter antigen dwell time (0.2 s) to initiate negative selection compared to MHCI-restricted TCRs (0.9 s) because more CD4 coreceptors are Lck-loaded compared to CD8. We generated a model (Lck come&stay/signal duration) that accurately predicts the observed differences in antigen dwell-time thresholds used by MHCI- and MHCII-restricted thymocytes to initiate negative selection and generate self-tolerance.
Assuntos
Autoantígenos/imunologia , Tolerância Imunológica , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Cinética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Cadeias de Markov , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/imunologiaRESUMO
Studies of individual T cell antigen receptors (TCRs) have shed some light on structural features that underlie self-reactivity. However, the general rules that can be used to predict whether TCRs are self-reactive have not been fully elucidated. Here we found that the interfacial hydrophobicity of amino acids at positions 6 and 7 of the complementarity-determining region CDR3ß robustly promoted the development of self-reactive TCRs. This property was found irrespective of the member of the ß-chain variable region (Vß) family present in the TCR or the length of the CDR3ß. An index based on these findings distinguished Vß2(+), Vß6(+) and Vß8.2(+) regulatory T cells from conventional T cells and also distinguished CD4(+) T cells selected by the major histocompatibility complex (MHC) class II molecule I-A(g7) (associated with the development of type 1 diabetes in NOD mice) from those selected by a non-autoimmunity-promoting MHC class II molecule I-A(b). Our results provide a means for distinguishing normal T cell repertoires versus autoimmunity-prone T cell repertoires.
Assuntos
Autoimunidade , Regiões Determinantes de Complementaridade/genética , Diabetes Mellitus Tipo 1/imunologia , Subpopulações de Linfócitos T/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Diferenciação Celular , Tolerância Central , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
The structural characteristics of the engagement of major histocompatibility complex (MHC) class II-restricted self antigens by autoreactive T cell antigen receptors (TCRs) is established, but how autoimmune TCRs interact with complexes of self peptide and MHC class I has been unclear. Here we examined how CD8(+) T cells kill human islet beta cells in type 1 diabetes via recognition of a human leukocyte antigen HLA-A*0201-restricted glucose-sensitive preproinsulin peptide by the autoreactive TCR 1E6. Rigid 'lock-and-key' binding underpinned the 1E6-HLA-A*0201-peptide interaction, whereby 1E6 docked similarly to most MHC class I-restricted TCRs. However, this interaction was extraordinarily weak because of limited contacts with MHC class I. TCR binding was highly peptide centric, dominated by two residues of the complementarity-determining region 3 (CDR3) loops that acted as an 'aromatic-cap' over the complex of peptide and MHC class I (pMHCI). Thus, highly focused peptide-centric interactions associated with suboptimal TCR-pMHCI binding affinities might lead to thymic escape and potential CD8(+) T cell-mediated autoreactivity.
Assuntos
Apoptose , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Secretoras de Insulina/imunologia , Linfócitos T CD8-Positivos/química , Antígenos de Histocompatibilidade/imunologia , Humanos , Células Secretoras de Insulina/patologia , Modelos Moleculares , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.
Assuntos
Antígenos CD8/imunologia , Peptídeos/agonistas , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos/química , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Cinética , Ligantes , Ativação Linfocitária , Modelos Imunológicos , MutaçãoRESUMO
Here, we report an update of the VDJdb database with a substantial increase in the number of T-cell receptor (TCR) sequences and their cognate antigens. The update further provides a new database infrastructure featuring two additional analysis modes that facilitate database querying and real-world data analysis. The increased yield of TCR specificity identification methods and the overall increase in the number of studies in the field has allowed us to expand the database more than 5-fold. Furthermore, several new analysis methods are included. For example, batch annotation of TCR repertoire sequencing samples allows for annotating large datasets on-line. Using recently developed bioinformatic methods for TCR motif mining, we have built a reduced set of high-quality TCR motifs that can be used for both training TCR specificity predictors and matching against TCRs of interest. These additions enhance the versatility of the VDJdb in the task of exploring T-cell antigen specificities. The database is available at https://vdjdb.cdr3.net.
Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Motivos de Nucleotídeos , Receptores de Antígenos de Linfócitos T/genética , Recombinação V(D)J , Sequência de Aminoácidos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Matrizes de Pontuação de Posição Específica , Receptores de Antígenos de Linfócitos T/química , Análise de Sequência de DNA , Software , NavegadorRESUMO
Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule--mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7- CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule-induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.
Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos CD/imunologia , Imunoterapia/métodos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/imunologia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Anticorpos Biespecíficos/imunologia , Humanos , Receptor B1 de Leucócitos Semelhante a Imunoglobulina/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Células Tumorais CultivadasRESUMO
CD4+ T-cells recognize peptide antigens, in the context of human leukocyte antigen (HLA) class II molecules (HLA-II), which through peptide-flanking residues (PFRs) can extend beyond the limits of the HLA binding. The role of the PFRs during antigen recognition is not fully understood; however, recent studies have indicated that these regions can influence T-cell receptor (TCR) affinity and pHLA-II stability. Here, using various biochemical approaches including peptide sensitivity ELISA and ELISpot assays, peptide-binding assays and HLA-II tetramer staining, we focused on CD4+ T-cell responses against a tumor antigen, 5T4 oncofetal trophoblast glycoprotein (5T4), which have been associated with improved control of colorectal cancer. Despite their weak TCR-binding affinity, we found that anti-5T4 CD4+ T-cells are polyfunctional and that their PFRs are essential for TCR recognition of the core bound nonamer. The high-resolution (1.95 Å) crystal structure of HLA-DR1 presenting the immunodominant 20-mer peptide 5T4111-130, combined with molecular dynamic simulations, revealed how PFRs explore the HLA-proximal space to contribute to antigen reactivity. These findings advance our understanding of what constitutes an HLA-II epitope and indicate that PFRs can tune weak affinity TCR-pHLA-II interactions.
Assuntos
Epitopos/imunologia , Antígeno HLA-DR1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cristalografia por Raios X , Epitopos/química , Epitopos/metabolismo , Antígeno HLA-DR1/química , Antígeno HLA-DR1/imunologia , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Simulação de Dinâmica Molecular , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.
Assuntos
Epitopos Imunodominantes/metabolismo , Imunoterapia Adotiva/métodos , Antígeno MART-1/metabolismo , Melanoma/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Aminoácidos , Apresentação de Antígeno , Sítios de Ligação , Células Cultivadas , Células Clonais , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Ativação Linfocitária , Antígeno MART-1/química , Melanoma/terapia , Peptídeos/química , Ligação Proteica , Conformação Proteica , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/transplanteRESUMO
There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/administração & dosagem , Sistema Respiratório/imunologia , Aerossóis , Sequência de Aminoácidos , Animais , Antígenos Virais/química , Epitopos/química , Epitopos/genética , Feminino , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Endogamia , Vírus da Influenza A/patogenicidade , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/transmissão , Masculino , Modelos Animais , Modelos Moleculares , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/veterinária , Sus scrofa/genética , Sus scrofa/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Vacinação/métodos , Vacinação/veterináriaRESUMO
Adoptive transfer of T cells genetically modified to express a cancer-specific T-cell receptor (TCR) has shown significant therapeutic potential for both hematological and solid tumors. However, a major issue of transducing T cells with a transgenic TCR is the preexisting expression of TCRs in the recipient cells. These endogenous TCRs compete with the transgenic TCR for surface expression and allow mixed dimer formation. Mixed dimers, formed by mispairing between the endogenous and transgenic TCRs, may harbor autoreactive specificities. To circumvent these problems, we designed a system where the endogenous TCR-ß is knocked out from the recipient cells using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) technology, simultaneously with transduction with a cancer-reactive receptor of choice. This TCR replacement strategy resulted in markedly increased surface expression of transgenic αß and γδ TCRs, which in turn translated to a stronger, and more polyfunctional, response of engineered T cells to their target cancer cell lines. Additionally, the TCR-plus-CRISPR-modified T cells were up to a thousandfold more sensitive to antigen than standard TCR-transduced T cells or conventional model proxy systems used for studying TCR activity. Finally, transduction with a pan-cancer-reactive γδ TCR used in conjunction with CRISPR/Cas9 knockout of the endogenous αß TCR resulted in more efficient redirection of CD4+ and CD8+ T cells against a panel of established blood cancers and primary, patient-derived B-cell acute lymphoblastic leukemia blasts compared with standard TCR transfer. Our results suggest that TCR transfer combined with genome editing could lead to new, improved generations of cancer immunotherapies.
Assuntos
Antineoplásicos/metabolismo , Sistemas CRISPR-Cas/genética , Genes Codificadores dos Receptores de Linfócitos T/genética , Linfócitos T/metabolismo , Transgenes , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Técnicas de Inativação de Genes , Células HEK293 , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Reprodutibilidade dos TestesRESUMO
Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígeno HLA-A2/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Coloração e Rotulagem/métodos , Citomegalovirus/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Ativação Linfocitária/imunologia , Melanoma/imunologia , Orthomyxoviridae/imunologia , Ligação Proteica/imunologia , Inibidores de Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA/imunologia , Células Tumorais CultivadasRESUMO
The ability to decode antigen specificities encapsulated in the sequences of rearranged T-cell receptor (TCR) genes is critical for our understanding of the adaptive immune system and promises significant advances in the field of translational medicine. Recent developments in high-throughput sequencing methods (immune repertoire sequencing technology, or RepSeq) and single-cell RNA sequencing technology have allowed us to obtain huge numbers of TCR sequences from donor samples and link them to T-cell phenotypes. However, our ability to annotate these TCR sequences still lags behind, owing to the enormous diversity of the TCR repertoire and the scarcity of available data on T-cell specificities. In this paper, we present VDJdb, a database that stores and aggregates the results of published T-cell specificity assays and provides a universal platform that couples antigen specificities with TCR sequences. We demonstrate that VDJdb is a versatile instrument for the annotation of TCR repertoire data, enabling a concatenated view of antigen-specific TCR sequence motifs. VDJdb can be accessed at https://vdjdb.cdr3.net and https://github.com/antigenomics/vdjdb-db.
Assuntos
Antígenos/química , Bases de Dados de Proteínas , Anotação de Sequência Molecular , Receptores de Antígenos de Linfócitos T/química , Software , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Antígenos/metabolismo , Sítios de Ligação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Internet , Macaca mulatta , Complexo Principal de Histocompatibilidade/genética , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Modelos Moleculares , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The repertoire of human αß T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8+ T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8+ T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8+ T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Regiões Determinantes de Complementaridade/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Vacinas Virais/imunologia , Febre Amarela/imunologia , Vírus da Febre Amarela/fisiologia , Imunidade Adaptativa/genética , Linhagem Celular , Seleção Clonal Mediada por Antígeno , Células Clonais , Citotoxicidade Imunológica , Epitopos de Linfócito T/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária , Especificidade do Receptor de Antígeno de Linfócitos T , Proteínas Virais/metabolismo , Febre Amarela/genéticaRESUMO
Antigen recognition by the T-cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. Here, we used single-molecule localization microscopy to quantify the organization of TCR-CD3 complexes into nanoscale clusters and to distinguish between triggered and nontriggered TCR-CD3 complexes. We found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Moreover, both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. Thus, TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex, and the formation of dense signaling-competent clusters is a process of antigen discrimination.
Assuntos
Antígenos/imunologia , Complexo CD3/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Imunidade Adaptativa/genética , Animais , Antígenos/genética , Humanos , Camundongos , Peptídeos/imunologia , Fosforilação/imunologia , Transdução de Sinais , Imagem Individual de MoléculaRESUMO
T-cell cross-reactivity is essential for effective immune surveillance but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focused antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase. The ILA1 TCR contacted its pMHC with a broad peptide binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding, the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity with important implications for pathogen surveillance, autoimmunity, and transplant rejection.