Paired TCR?? analysis of virus-specific CD8(+) T cells exposes diversity in a previously defined 'narrow' repertoire.
ABSTRACT: T-cell receptor (TCR) usage has an important role in determining the outcome of CD8(+) cytotoxic T-lymphocyte responses to viruses and other pathogens. However, the characterization of TCR usage from which such conclusions are drawn is based on exclusive analysis of either the TCR? chain or, more commonly, the TCR? chain. Here, we have used a multiplexed reverse transcription-PCR protocol to analyse the CDR3 regions of both TCR? and ? chains from single naive or immune epitope-specific cells to provide a comprehensive picture of epitope-specific TCR usage and selection into the immune response. Analysis of TCR repertoires specific for three influenza-derived epitopes (D(b)NP(366), D(b)PA(224) and D(b)PB1-F2(62)) showed preferential usage of particular TCR?? proteins in the immune repertoire relative to the naive repertoire, in some cases, resulting in a complete shift in TRBV preference or CDR3 length, and restricted repertoire diversity. The NP(366)-specific TCR?? repertoire, previously defined as clonally restricted based on TCR? analysis, was similarly diverse as the PA(224)- and PB1-F2(62)-specific repertoires. Intriguingly, preferred TCR characteristics (variable gene usage, CDR3 length and junctional gene usage) appeared to be able to confer specificity either independently or in concert with one another, depending on the epitope specificity. These data have implications for established correlations between the nature of the TCR repertoire and response outcomes after infection, and suggest that analysis of a subset of cells or a single TCR chain does not accurately depict the nature of the antigen-specific TCR?? repertoire.
Project description:TCR repertoire diversity has been convincingly shown to facilitate responsiveness of CD8+ T cell populations to mutant virus peptides, thereby safeguarding against viral escape. However, the impact of repertoire diversity on the functionality of the CD8+ T cell response to cognate peptide-MHC class I complex (pMHC) recognition remains unclear. Here, we have compared TCRbeta chain repertoires of three influenza A epitope-specific CD8+ T cell responses in C57BL/6 (B6) mice: D(b)NP(366-374), D(b)PA(224-233), and a recently described epitope derived from the +1 reading frame of the influenza viral polymerase B subunit (residues 62-70) (D(b)PB1-F2(62)). Corresponding to the relative antigenicity of the respective pMHCs, and irrespective of the location of prominent residues, the D(b)PA(224)- and D(b)PB1-F2(62)-specific repertoires were similarly diverse, whereas the D(b)NP(366) population was substantially narrower. Importantly, parallel analysis of response magnitude, cytotoxicity, TCR avidity, and cytokine production for the three epitope-specific responses revealed no obvious functional advantage conferred by increased T cell repertoire diversity. Thus, whereas a diverse repertoire may be important for recognition of epitope variants, its effect on the response to cognate pMHC recognition appears minimal.
Project description:Ecology is typically thought of as the study of interactions organisms have with each other and their environment and is focused on the distribution and abundance of organisms both within and between environments. On a molecular level, the capacity to probe analogous questions in the field of T-cell immunology is imperative as we acquire substantial datasets both on epitope-specific T-cell populations through high-resolution analyses of T-cell receptor (TCR) use and on global T-cell populations analyzed via high-throughput DNA sequencing. Here, we present the innovative application of existing statistical measures (used typically in the field of ecology), together with unique statistical analyses, to comprehensively assess how the naïve epitope-specific CD8(+) cytotoxic T lymphocyte (CTL) repertoire translates to that found following an influenza-virus-specific immune response. Such interrogation of our extensive, cumulated TCR CDR3? sequence datasets, derived from both naïve and immune CD8(+) T-cell populations specific for four different influenza-derived epitopes (D(b)NP(366), influenza nucleoprotein amino acid residues 366-374; D(b)PA(224), influenza acid polymerase amino acid residues 224-233; D(b)PB1-F2(62), influenza polymerase B 1 reading frame 2 amino acid residues 62-70; K(b)NS2(114), and influenza nonstructural protein 2 amino acid residues 114-121), demonstrates that epitope-specific TCR use in an antiviral immune response is the consequence of a complex interplay between the intrinsic characteristics of the naïve cytotoxic T lymphocyte precursor pool and extrinsic (likely antigen driven) influences, the contribution of which varies in an epitope-specific fashion.
Project description:Pathogen-specific responses are characterized by preferred profiles of peptide+class I MHC (pMHCI) glycoprotein-specific T-cell receptor (TCR) Variable (V)-region use. How TCRV-region bias impacts TCR?? heterodimer selection and resultant diversity is unclear. The D(b)PA(224)-specific TCR repertoire in influenza A virus-infected C57BL/6J (B6) mice exhibits a preferred TCRV-region bias toward the TRBV29 gene segment and an optimal complementarity determining region (CDR3) ?-length of 6 aa. Despite these restrictions, D(b)PA(224)-specific BV29(+) T cells use a wide array of unique CDR3? sequences. Structural characterization of a single, TRBV29(+)D(b)P(A224)-specific TCR??-pMHCI complex demonstrated that CDR3? amino acid side chains made specific peptide interactions, but the CDR3? main chain exclusively contacted peptides. Thus, length but not amino acid sequence was key for recognition and flexibility in V?-region use. In support of this hypothesis, retrovirus expression of the D(b)PA(224)-specific TCRV?-chain was used to constrain pairing within a naive/immune epitope-specific repertoire. The retrogenic TCRV? paired with a diversity of CDR3?s in the context of a preferred TCRV? spectrum. Overall, these data provide an explanation for the combination of TCRV region bias and diversity within selected repertoires, even as they maintain exquisite pMHCI specificity.
Project description:Earlier studies of influenza-specific CD8(+) T cell immunodominance hierarchies indicated that expression of the H2K(k) MHC class I allele greatly diminishes responses to the H2D(b)-restriced D(b)PA(224) epitope (acid polymerase, residues 224-233 complexed with H2D(b)). The results suggested that the presence of H2K(k) during thymic differentiation led to the deletion of a prominent V?7(+) subset of D(b)PA(224)-specific TCRs. The more recent definition of D(b)PA(224)-specific TCR CDR3? repertoires in H2(b) mice provides a new baseline for looking again at this possible H2K(k) effect on D(b)PA(224)-specific TCR selection. We found that immune responses to several H2D(b)- and H2K(b)-restricted influenza epitopes were indeed diminished in H2(bxk) F(1) versus homozygous mice. In the case of D(b)PA(224), lower numbers of naive precursors were part of the explanation, though a similar decrease in those specific for the D(b)NP(366) epitope did not affect response magnitude. Changes in precursor frequency were not associated with any major loss of TCR diversity and could not fully account for the diminished D(b)PA(224)-specific response. Further functional and phenotypic characterization of influenza-specific CD8(+) T cells suggested that the expansion and differentiation of the D(b)PA(224)-specific set is impaired in the H2(bxk) F(1) environment. Thus, the D(b)PA(224) response in H2(bxk) F(1) mice is modulated by factors that affect the generation of naive epitope-specific precursors and the expansion and differentiation of these T cells during infection, rather than clonal deletion of a prominent V?7(+) subset. Such findings illustrate the difficulties of predicting and defining the effects of MHC class I diversification on epitope-specific responses.
Project description:The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCR?-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2?(+/-) TCR?(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of J? usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.
Project description:The ?? T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3?, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.
Project description:A diverse T cell receptor (TCR) repertoire is essential for controlling viral infections. However, information about TCR repertoires to defined viral antigens is limited. We performed a comprehensive analysis of CD8+ TCR repertoires for two dominant viral epitopes: pp65495-503 (NLV) of cytomegalovirus and M158-66 (GIL) of influenza A virus. The highly individualized repertoires (87-5,533 ? or ? clonotypes per subject) comprised thousands of unique TCR? and TCR? sequences and dozens of distinct complementary determining region (CDR)3? and CDR3? motifs. However, diversity is effectively restricted by preferential V-J combinations, CDR3 lengths, and CDR3?/CDR3? pairings. Structures of two GIL-specific TCRs bound to GIL-HLA-A2 provided a potential explanation for the lower diversity of GIL-specific versus NLV-specific repertoires. These anti-viral TCRs occupied up to 3.4% of the CD8+ TCR? repertoire, ensuring broad T cell responses to single epitopes. Our portrait of two anti-viral TCR repertoires may inform the development of predictors of immune protection.
Project description:The T cell receptor (TCR) repertoire is an essential component of the CD8 T-cell immune response. Here, we seek to investigate factors that drive selection of TCR repertoires specific to the HLA-A2-restricted immunodominant epitope BRLF1109-117 (YVLDHLIVV) over the course of primary Epstein Barr virus (EBV) infection. Using single-cell paired TCR?? sequencing of tetramer sorted CD8 T cells ex vivo, we show at the clonal level that recognition of the HLA-A2-restricted BRLF1 (YVL-BR, BRLF-1109) epitope is mainly driven by the TCR? chain. For the first time, we identify a CDR3? (complementarity determining region 3 ?) motif, KDTDKL, resulting from an obligate AV8.1-AJ34 pairing that was shared by all four individuals studied. This observation coupled with the fact that this public AV8.1-KDTDKL-AJ34 TCR pairs with multiple different TCR? chains within the same donor (median 4; range: 1-9), suggests that there are some unique structural features of the interaction between the YVL-BR/MHC and the AV8.1-KDTDKL-AJ34 TCR that leads to this high level of selection. Newly developed TCR motif algorithms identified a lysine at position 1 of the CDR3? motif that is highly conserved and likely important for antigen recognition. Crystal structure analysis of the YVL-BR/HLA-A2 complex revealed that the MHC-bound peptide bulges at position 4, exposing a negatively charged aspartic acid that may interact with the positively charged lysine of CDR3?. TCR cloning and site-directed mutagenesis of the CDR3? lysine ablated YVL-BR-tetramer staining and substantially reduced CD69 upregulation on TCR mutant-transduced cells following antigen-specific stimulation. Reduced activation of T cells expressing this CDR3 motif was also observed following exposure to mutated (D4A) peptide. In summary, we show that a highly public TCR repertoire to an immunodominant epitope of a common human virus is almost completely selected on the basis of CDR3? and provide a likely structural basis for the selection. These studies emphasize the importance of examining TCR?, as well as TCR?, in understanding the CD8 T cell receptor repertoire.
Project description:Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCR? of rhesus monkeys. We identified 1.26 million TCR? sequences corresponding to 643,570 unique TCR? sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys.
Project description:BACKGROUND:CD4+ T cells play critical roles in the pathogenesis of IgG4-related disease (IgG4-RD). The aim of this study was to investigate the TCR repertoire of peripheral blood CD4+ T cells in IgG4-RD. METHODS:The peripheral blood was collected from six healthy controls and eight IgG4-RD patients. TCR ?-chain libraries of CD4+ T cells were constructed by 5'-rapid amplification of cDNA ends (5'-RACE) and sequenced by Illumina Miseq platform. The relative similarity of TCR repertoires between samples was evaluated according to the total frequencies of shared clonotypes (metric F), correlation of frequencies of shared clonotypes (metric R), and total number of shared clonotypes (metric D). RESULTS:The clonal expansion and diversity of CD4+ T cell repertoire were comparable between healthy controls and IgG4-RD patients, while the proportion of expanded and coding degenerated clones, as an indicator of antigen-driven clonal expansion, was significantly higher in IgG4-RD patients. There was no significant difference in TRBV and TRBJ gene usage between healthy controls and IgG4-RD patients. The complementarity determining region 3 (CDR3) length distribution was skewed towards longer fragments in IgG4-RD. Visualization of relative similarity of TCR repertoires by multi-dimensional scaling analysis showed that TCR repertoires of IgG4-RD patients were separated from that of healthy controls in F and D metrics. We identified 11 IgG4-RD-specific CDR3 amino acid sequences that were expanded in at least 2 IgG4-RD patients, while not detected in healthy controls. According to TCR clonotype networks constructed by connecting all the CDR3 sequences with a Levenshtein distance of 1, 3 IgG4-RD-specific clusters were identified. We annotated the TCR sequences with known antigen specificity according to McPAS-TCR database and found that the frequencies of TCR sequences associated with each disease or immune function were comparable between healthy controls and IgG4-RD patients. CONCLUSION:According to our study of CD4+ T cells from eight IgG4-RD patients, TCR repertoires of IgG4-RD patients were different from that of healthy controls in the proportion of expanded and coding degenerated clones and CDR3 length distribution. In addition, IgG4-RD-specific TCR sequences and clusters were identified in our study.