Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

The avidity of tumor‐specific T cells amplified by a plasmacytoid dendritic cell‐based assay can predict the clinical evolution of melanoma patients

The advent of immune checkpoint blockers and targeted therapies has changed the outcome of melanoma. However, many patients experience relapses, emphasizing the need for predictive and prognostic biomarkers. We developed a strategy based on plasmacytoid dendritic cells (pDCs) loaded with melanoma tumor antigens that allows eliciting highly efficient antitumor T‐cell responses. We used it to investigate antitumor T‐cell functionality in peripheral blood mononuclear cells and tumor‐infiltrating lymphocytes from melanoma patients. The pDCs elicited tumor‐specific T cells in different proportions and displaying diverse functional features, dependent upon the stage of the disease, but independent of the histological parameters at diagnosis. Strikingly, the avidity of the MelA‐specific T cells triggered by the pDCs was found to predict patient relapse time and overall survival. Our findings highlighted unexplored aspects of antitumor T‐cell responsiveness in melanoma, and revealed for the first time the structural avidity of tumor‐specific T cells as a crucial feature for predicting clinical evolution.     

Variability in T cell receptor V beta gene usage in human peripheral blood lymphocytes. Studies of identical twins, siblings, and insulin-dependent diabetes mellitus patients.

Recent studies focused on the diversity and molecular organization of the human TCR-beta complex have begun to establish the genetic basis for the potential repertoire of V beta specificities in T cells. The scope and variability of the actual repertoire derived from this potential repertoire, however, remains to be clarified. In this study, V beta usage by human peripheral T cells derived from serial samples of the same individual, identical twins, and the members of three nuclear families that include four members with insulin-dependent diabetes mellitus (IDDM) was assessed by both quantitative polymerase chain reaction and Northern blotting with V beta subfamily-specific probes. Samples taken from the same individual over a period of 21 months and analyzed in separate experiments indicated stability in the peripheral repertoire, whereas the similarity in peripheral V beta usage in a pair of identical twins suggested a strong role for genetics in shaping the peripheral T cell repertoire. In contrast, V beta usage in siblings and in unrelated individuals was observed to differ substantially. In particular, peripheral expression of V beta 3 and V beta 20 differed by more than sixfold among members of two different families. Segregation analysis of TCR and HLA haplotypes in these families suggested that variation in V beta 20 expression was TCR haplotype specific. Subsequent nucleotide sequence analysis of the V beta 20 gene segment in multiple members of these families revealed the presence of a null allele for V beta 20 expression. No consistent significant differences in V beta usage were observed in IDDM patients relative to their siblings or between identical twins discordant for IDDM. These results suggest that the repertoire of peripheral T cell specificities present in different individuals in human populations varies dramatically because of the effects of multiple factors, including TCR germ-line polymorphism.     

Regulatory T cells constrain the TCR repertoire of antigen‐stimulated conventional CD4 T cells

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non‐self‐antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T‐cell repertoire was used. The response of EF4.1 mice to an I‐Ab‐associated epitope of the F‐MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg‐depleted EF4.1 mice were immunized, and the extent of the Vα2‐bearing, antigen‐specific TCR repertoire was characterized by high‐throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre‐immune repertoire. Injection of anti‐CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non‐self‐antigens.     

Longitudinal analysis of peripheral blood T cell receptor diversity in patients with systemic lupus erythematosus by next-generation sequencing

IntroductionT cells play an important role in the pathogenesis of systemic lupus erythematosus (SLE). Clonal expansion of T cells correlating with disease activity has been observed in peripheral blood (PB) of SLE subjects. Recently, next-generation sequencing (NGS) of the T cell receptor (TCR) β loci has emerged as a sensitive way to measure the T cell repertoire. In this study, we utilized NGS to assess whether changes in T cell repertoire diversity in PB of SLE patients correlate with or predict changes in disease activity.MethodsTotal RNA was isolated from the PB of 11 SLE patients. Each subject had three samples, collected at periods of clinical quiescence and at a flare. Twelve age-matched healthy controls (HC) were used for reference. NGS was used to profile the complementarity-determining region 3 (CDR3) of the rearranged TCR β loci.ResultsRelative to the HC, SLE patients (at quiescence) demonstrated a 2.2-fold reduction in repertoire diversity in a given PB volume (P <0.0002), a more uneven distribution of the repertoire (Gini coefficient, HC vs SLE, P = 0.015), and a trend toward increased percentage of expanded clones in the repertoire (clone size >1.0 %, HC vs SLE, P = 0.078). No significant correlation between the overall repertoire diversity and clinical disease activity was observed for most SLE patients with only two of eleven SLE patients showing a decreasing trend in repertoire diversity approaching the flare time point. We did not observe any overlap of CDR3 amino acid sequences or a preferential Vβ or Jβ gene usage among the top 100 expanded clones from all SLE patients. In both HC and SLE, the majority of the expanded clones were remarkably stable over time (HC = 5.5 ±0.5 months, SLE = 7.2 ±2.4 months).ConclusionsA significant decrease in T cell repertoire diversity was observed in PB of SLE patients compared to HC. However, in most SLE patients, repertoire diversity did not change significantly with increases in disease activity to a flare. Thus, without a priori knowledge of disease-specific clones, monitoring TCR repertoire in PB from SLE patients is not likely to be useful to predict changes in disease activity.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0655-9) contains supplementary material, which is available to authorized users.     

Cancer of the gastrointestinal tract results in a restricted T-cell repertoire dependent upon tumor differentiation

We investigated the influence of tumor tissue differentiation on the diversity of TCR repertoire. CDR3 spectratypes of CD4⁺ and CD8⁺ T cell subsets were analyzed from 27 patients with gastrointestinal tract tumors exhibiting varying degrees of differentiation. A CDR3 spectratype complexity scoring system was used to quantify the diversity of TCR repertoire. Each patient was matched with an age-matched healthy group to control for age variability. Results show that the complexity scores (TCR repertoire diversity) have a significant correlation with the degree of tumor differentiation, which provides useful information for understanding immune response in cancer patients.     

T cell repertoire development in humans with SCID after nonablative allogeneic marrow transplantation

Transplantation of HLA-identical or haploidentical T cell-depleted allogeneic bone marrow (BM) into SCID infants results in thymus-dependent T cell development in the recipients. Immunoscope analysis of the TCR V beta repertoire was performed on 15 SCID patients given BM transplants. Before and within the first 100 days after bone marrow transplantation (BMT), patients' PBMC displayed an oligoclonal or skewed T cell repertoire, low TCR excision circles (TREC) values, and a predominance of CD45RO(+) T cells. In contrast, the presence of high numbers of CD45RA(+) cells in the circulation of SCID patients >100 days post-BMT correlated with active T cell output by the thymus as revealed by high TREC values and a polyclonal T cell repertoire demonstrated by a Gaussian distribution of V beta-specific peaks. Ten years after BMT, we observed a decrease of the normal polyclonal T cell repertoire and an increase of a more skewed T cell repertoire. A decline of TREC levels and a decrease in the number of CD45RA(+) cells beyond 10 years after BMT was concomitant with the detection of oligoclonal CD3(+)CD8(+)CD45RO(+) cells. The switch from a polyclonal to a more skewed repertoire, observed in the CD3(+)CD8(+)CD45RO(+) T cell subset, is a phenomenon that occurs normally with decreased thymic output during aging, but not as rapidly as in this patient population. We conclude that a normal T cell repertoire develops in SCID patients as a result of thymic output and the repertoire remains highly diverse for the first 10 years after BMT. The TCR diversity positively correlates in these patients with TREC levels.     

Limited T Cell Receptor Repertoire Diversity in Tuberculosis Patients Correlates with Clinical Severity

Background

The importance of CD4⁺ and CD8⁺ T cells in protection against tuberculosis (TB) is well known, however, the association between changes to the T cell repertoire and disease presentation has never been analyzed. Characterization of T-cells in TB patients in previous study only analyzed the TCR β chain and omitted analysis of the Vα family even though α chain also contribute to antigen recognition. Furthermore, limited information is available regarding the heterogeneity compartment and overall function of the T cells in TB patients as well as the common TCR structural features of Mtb antigen specific T cells among the vast numbers of TB patients.

Methodology/Principal Findings

CDR3 spectratypes of CD4⁺ and CD8⁺ T cells were analyzed from 86 patients with TB exhibiting differing degrees of disease severity, and CDR3 spectratype complexity scoring system was used to characterize TCR repertoire diversity. TB patients with history of other chronic disease and other bacterial or viral infections were excluded for the study to decrease the likely contribution of TCRs specific to non-TB antigens as far as possible. Each patient was age-matched with a healthy donor group to control for age variability. Results showed that healthy controls had a normally diversified TCR repertoire while TB patients represented with restricted TCR repertoire. Patients with mild disease had the highest diversity of TCR repertoire while severely infected patients had the lowest, which suggest TCR repertoire diversity inversely correlates with disease severity. In addition, TB patients showed preferred usage of certain TCR types and have a bias in the usage of variable (V) and joining (J) gene segments and N nucleotide insertions.

Conclusions/Significance

Results from this study promote a better knowledge about the public characteristics of T cells among TB patients and provides new insight into the TCR repertoire associated with clinic presentation in TB patients.     

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.     

A TCRβ Repertoire Signature Can Predict Experimental Cerebral Malaria

Cerebral Malaria (CM) is associated with a pathogenic T cell response. Mice infected by P. berghei ANKA clone 1.49 (PbA) developing CM (CM⁺) present an altered PBL TCR repertoire, partly due to recurrently expanded T cell clones, as compared to non-infected and CM^- infected mice. To analyse the relationship between repertoire alteration and CM, we performed a kinetic analysis of the TRBV repertoire during the course of the infection until CM-related death in PbA-infected mice. The repertoires of PBL, splenocytes and brain lymphocytes were compared between infected and non-infected mice using a high-throughput CDR3 spectratyping method. We observed a modification of the whole TCR repertoire in the spleen and blood of infected mice, from the fifth and the sixth day post-infection, respectively, while only three TRBV were significantly perturbed in the brain of infected mice. Using multivariate analysis and statistical modelling, we identified a unique TCRβ signature discriminating CM⁺ from CTR mice, enriched during the course of the infection in the spleen and the blood and predicting CM onset. These results highlight a dynamic modification and compartmentalization of the TCR diversity during the course of PbA infection, and provide a novel method to identify disease-associated TCRβ signature as diagnostic and prognostic biomarkers.     

Large TCR diversity of virus-specific CD8 T cells provides the mechanistic basis for massive TCR renewal after antigen exposure

Ex vivo analysis of virus-specific CD8 T cell populations by anchored PCR has shown that the CD8 TCR repertoire was less oligoclonal (seven to nine clonotypes per individual epitope) than previously thought. In the current study, TCR diversity was investigated by assessing both the overall TCR β-chain variable regions usage as well as the CDR3 regions in ex vivo-isolated CMV- and EBV-specific CD8 T cells from 27 healthy donors. The average number of clonotypes specific to most single viral epitopes comprised between 14 and 77. Changes in the CD8 TCR repertoire were also longitudinally assessed under conditions of HIV-1 chronic infection (i.e., in patients with suppressed virus replication and after treatment interruption and Ag re-exposure). The results showed that a large renewal (≤ 80%) of the TRB repertoire occurred after Ag re-exposure and was eventually associated with an increased T cell recognition functional avidity. These results demonstrate that the global CD8 TCR repertoire is much more diverse (≤ 9-fold) than previously estimated and provide the mechanistic basis for supporting massive repertoire renewal during chronic virus infection and Ag re-exposure.     

The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state.     

Positional scanning-synthetic peptide library-based analysis of self- and pathogen-derived peptide cross-reactivity with tumor-reactive Melan-A-specific CTL

Synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) have recently emerged as a useful tool for the analysis of T cell recognition. This includes identification of potentially cross-reactive sequences of self or pathogen origin that could be relevant for the understanding of TCR repertoire selection and maintenance, as well as of the cross-reactive potential of Ag-specific immune responses. In this study, we have analyzed the recognition of sequences retrieved by using a biometric analysis of the data generated by screening a PS-SCL with a tumor-reactive CTL clone specific for an immunodominant peptide from the melanocyte differentiation and tumor-associated Ag Melan-A. We found that 39% of the retrieved peptides were recognized by the CTL clone used for PS-SCL screening. The proportion of peptides recognized was higher among those with both high predicted affinity for the HLA-A2 molecule and high predicted stimulatory score. Interestingly, up to 94% of the retrieved peptides were cross-recognized by other Melan-A-specific CTL. Cross-recognition was at least partially focused, as some peptides were cross-recognized by the majority of CTL. Importantly, stimulation of PBMC from melanoma patients with the most frequently recognized peptides elicited the expansion of heterogeneous CD8(+) T cell populations, one fraction of which cross-recognized Melan-A. Together, these results underline the high predictive value of PS-SCL for the identification of sequences cross-recognized by Ag-specific T cells.     

Skewed T-cell receptor repertoire: more than a marker of malignancy, a tool to dissect the immunopathology of inflammatory diseases

The highly diverse heterodimeric surface T cell receptor (TCR) gives the T lymphocyte its specificity for MHC-bound peptides needed to initiate antigen-recognition. In normal peripheral blood, spleen and lymph nodes, the TCR repertoire of the T lymphocytes is usually polyclonal. However, in malignancies such as leukemias, as well as in lymphoproliferative diseases of mature T cells, the TCR is a reflection of the clonality of the malignant cells and is therefore monoclonal. Several clinical conditions (mainly solid tumors and autoimmune diseases) have been described where the TCR repertoire is restricted. The ability to demonstrate clonal TCR usage provides a useful tool to dissect the immunopathology of inflammatory diseases. In this review we discuss these findings and propose to sub-divide diseases with restricted TCR repertoire into a group of conditions in which there is a known TCR ligand, as opposed to diseases in which the restricted TCR repertoire is the result of impaired T-cell development. This classification sheds light on the pathogenesis of several inflammatory diseases.     

TCR trans-rearrangements: biological significance in antigen recognition vs the role as lymphoma biomarker

V(D)J rearrangements occur within loci of TCR and BCR genes, thus generating the diversity of the AgR repertoire. In addition, interlocus V(D)J rearrangements occur, giving rise to so-called "trans-rearrangements." Such trans-rearrangements increase the diversity of the immune receptor repertoire and can be expressed as functional chimeric TCR proteins on the surface of T cells. Although chimeric receptors are not pathogenic per se, the frequency of AgR trans-rearrangements correlates with the level of genetic instability and thus could be used as a predictive biomarker for lymphoma risk.     

Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.     

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.     

Characterization of Distinct T Cell Receptor Repertoires in Tumor and Distant Non-tumor Tissues from Lung Cancer Patients

T cells and T cell receptors(TCRs) play pivotal roles in adaptive immune responses against tumors.The development of next-generation sequencing technologies has enabled the analysis of the TCRb repertoire usage.Given the scarce investigations on the TCR repertoire in lung cancer tissues,in this study,we analyzed TCRb repertoires in lung cancer tissues and the matched distant non-tumor lung tissues(normal lung tissues) from 15 lung cancer patients.Based on our results,the general distribution of T cell clones was similar between cancer tissues and normal lung tissues;however,the proportion of highly expanded clones was significantly higher in normal lung tissues than in cancer tissues(0.021% ± 0.002% vs.0.016% ± 0.001%,P = 0.0054,Wilcoxon signed rank test).In addition,a significantly higher TCR diversity was observed in cancer tissues than in normal lung tissues(431.37 ± 305.96 vs.166.20 ± 101.58,P = 0.0075,Mann-Whitney U test).Moreover,younger patients had a significantly higher TCR diversity than older patients(640.7 ± 295.3 vs.291.8 ± 233.6,P = 0.036,Mann-Whitney U test),and the higher TCR diversity in tumors was significantly associated with worse cancer outcomes.Thus,we provided a comprehensive comparison of the TCR repertoires between cancer tissues and matched normal lung tissues and demonstrated the presence of distinct T cell immune microenvironments in lung cancer patients.