Clonal restriction and predominance of regulatory T cells in coronary thrombi of patients with acute coronary syndromes.

AIMS: Regulatory T cells (Treg) exert anti-inflammatory and atheroprotective effects in experimental atherosclerosis. Treg can be induced against specific antigens using immunization strategies associated with clonal restriction. No data exist on Treg in combination with clonal restriction of T cells in patients with acute coronary syndromes (ACS). METHODS AND RESULTS: Among T cell subsets characterized by flow cytometry, Treg (CD4(+) CD25(+) CD127(low)) were twice as frequent in coronary thrombi compared with peripheral blood. Treg prevailed among T cell subsets identified in coronary thrombi. To evaluate clonal restriction, genomic DNA was extracted from coronary thrombi and peripheral blood in order to evaluate T cell receptor (TCR) ß chain diversity by means of Multi-N-plex PCR using a primer specific for all TCR ß V gene segments and another primer specific for TCR ß J gene segments. T cell receptor diversity was reduced in thrombi compared with peripheral blood (intra-individual comparisons in 16 patients) with 8 gene rearrangements in the TCR common in at least 6 out of 16 analysed coronary thrombi. Compared with age-matched healthy controls (n = 16), TCR diversity was also reduced in peripheral blood of patients with ACS; these findings were independent of peripheral T cell numbers. CONCLUSION: We provide novel evidence for a perturbed T cell compartment characterized by clonal restriction in peripheral blood and coronary thrombi from patients with ACS. Our findings warrant further studies on Treg as novel therapeutic targets aimed at enhancing this anti-inflammatory component of adaptive immunity in human atherothrombosis.

Decreased T-cell repertoire diversity in sepsis: a preliminary study.

OBJECTIVE: Septic syndromes are the leading causes of mortality in intensive care units. In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality, although the exact role of lymphocyte dysfunctions is not well established. The objective of this study was to investigate T-cell receptor diversity, an important feature of T-cell response, in patients with septic shock. DESIGN: Preliminary prospective observational study. SETTING: Adult intensive care units in a university hospital. SUBJECTS: Patients with septic shock (n = 41) sampled twice after the onset of shock (early after inclusion [day 1] and at the end of the first week [day 7]). MEASUREMENTS AND MAIN RESULTS: Using a novel molecular biology technique, the combinatorial diversity of human T-cell receptor ß-chain (TRB locus) was measured in peripheral blood. Patients with septic shock presented with a marked decreased T-cell receptor diversity after the onset of shock in comparison with normal values. Importantly, in paired samples, a very steep recovery slope of T-cell receptor diversity, never described in other clinical situations, was observed between day 1 and day 7 (p < 0.0001, Wilcoxon's paired test). Decreased T-cell receptor diversity was associated with mortality (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence interval 1.96-53.32), and the development of nosocomial infections (p < 0.05, Mann-Whitney U test). CONCLUSION: Our results show for the first time that septic patients present with a marked decreased T-cell receptor diversity that returned rapidly toward normal values over time. This opens novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis. After confirmation in larger cohorts of these preliminary results, T-cell receptor diversity measurements may become a crucial tool to monitor immune functions in ICU patients.

Numerical modelling of the V-J combinations of the T cell receptor TRA/TRD locus.

T-Cell antigen Receptor (TR) repertoire is generated through rearrangements of V and J genes encoding alpha and beta chains. The quantification and frequency for every V-J combination during ontogeny and development of the immune system remain to be precisely established. We have addressed this issue by building a model able to account for Valpha-Jalpha gene rearrangements during thymus development of mice. So we developed a numerical model on the whole TRA/TRD locus, based on experimental data, to estimate how Valpha and Jalpha genes become accessible to rearrangements. The progressive opening of the locus to V-J gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. Furthermore, the possibility of successive secondary V-J rearrangements was included in the modelling. The model points out some unbalanced V-J associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the J genes, depending on their location in the locus. The model shows that 3 to 4 successive rearrangements are sufficient to explain the use of all the V and J genes of the locus. Finally, the model provides information on both the kinetics of rearrangements and frequencies of each V-J associations. The model accounts for the essential features of the observed rearrangements on the TRA/TRD locus and may provide a reference for the repertoire of the V-J combinatorial diversity.

Quantitative and qualitative changes in V-J alpha rearrangements during mouse thymocytes differentiation: implication for a limited T cell receptor alpha chain repertoire.

Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR alpha repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3' side than 5' end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.

High diversity of the immune repertoire in humanized NOD.SCID.gamma c-/- mice.

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.gammac(-/-) immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V-J rearrangements at the TCR-beta chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR-beta chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.gammac(-/-) mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA-restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen-specific immune responses.

Peripheral Blood TCR Repertoire Profiling May Facilitate Patient Stratification for Immunotherapy against Melanoma.

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.

Analysis of the TCR alpha-chain rearrangement profile in human T lymphocytes.

The size of the available human alphabeta T cell repertoire is difficult to determine and is open to debate. Empirical analysis of TCR beta-chain diversity reveals approximately 10(6) different beta chains in peripheral blood. Due in part to locus complexity, comparable information for TCR alpha is lacking. Rather, current estimates for human TCR alpha diversity, and hence, total repertoire diversity, are based on theoretical analyses that assume equal probabilities of rearrangement between any V alpha gene and J alpha gene. Here, we report on a systematic locus-wide rearrangement analysis of the TCR alpha-chain in human T cells. We first demonstrate that the V-J alpha recombination in the thymus is not random but depends on the reciprocal V alpha and J alpha position within the locus. Characterization of the frequency of gene usage combined with identification of five previously unrecognized pseudogenes enables us to empirically estimate the human TCR alpha combinatorial repertoire. The number of V-J alpha combinations achieved is approximately 44-56% of the total combinatorial possibilities, significantly lower than theoretical estimates. We also demonstrate that TCR alpha-chain diversity in peripheral T lymphocytes mimics the same general patterns of rearrangement as observed in the thymus, and these patterns appear conserved among different individuals. This unexpected observation indicates that, unlike the TCR beta locus, the human TCR alpha-chain repertoire is primarily predetermined by genetic recombination and its size is restricted by limits on the combinatorial repertoire rather than post-thymic selection.

Role of the T cell receptor alpha chain in the development and phenotype of naturally arising CD4+CD25+ T cells.

The T cell receptor alpha chain repertoire and the possible influence of the alpha chain on the development and phenotype of naturally arising mouse CD4+CD25+ T cells have not been extensively analysed. We used all available Valpha-specific monoclonal antibodies and a sensitive multiplex genomic DNA PCR assay to study the Valpha repertoire of CD4+CD25+ T cells in normal mice. To address whether CD4+CD25+ T cells express two TCR alpha chains, we have carried out four-colour flow cytometry using combinations of the available anti-Valpha reagents in mice where one allele of the TCRA locus had been inactivated. Results indicate that the Valpha repertoire of CD4+CD25+ T cells is as diverse as their CD25- partners. In addition, CD4+CD25+ T cells develop normally in Tcralpha+/- mice and we show for the first time that despite expressing only one TCRalpha chain, they retain their characteristic CD4(low), CD3(low), TCRbeta(low), CD5(high), CD45RB(low) and cytoplasmic CD152(high) phenotype.

IMGT/GeneInfo: T cell receptor gamma TRG and delta TRD genes in database give access to all TR potential V(D)J recombinations.

BACKGROUND: Adaptative immune repertoire diversity in vertebrate species is generated by recombination of variable (V), diversity (D) and joining (J) genes in the immunoglobulin (IG) loci of B lymphocytes and in the T cell receptor (TR) loci of T lymphocytes. These V-J and V-D-J gene rearrangements at the DNA level involve recombination signal sequences (RSS). Whereas many data exist, they are scattered in non specialized resources with different nomenclatures (eg. flat files) and are difficult to extract. DESCRIPTION: IMGT/GeneInfo is an online information system that provides, through a user-friendly interface, exhaustive information resulting from the complex mechanisms of T cell receptor V-J and V-D-J recombinations. T cells comprise two populations which express the alphabeta and gammadelta TR, respectively. The first version of the system dealt with the Homo sapiens and Mus musculus TRA and TRB loci whose gene rearrangements allow the synthesis of the alphabeta TR chains. In this paper, we present the second version of IMGT/GeneInfo where we complete the database for the Homo sapiens and Mus musculus TRG and TRD loci along with the introduction of a quality control procedure for existing and new data. We also include new functionalities to the four loci analysis, giving, to date, a very informative tool which allows to work on V(D)J genes of all TR loci in both human and mouse species. IMGT/GeneInfo provides more than 59,000 rearrangement combinations with a full gene description which is freely available at http://imgt.cines.fr/GeneInfo. CONCLUSION: IMGT/GeneInfo allows all TR information sequences to be in the same spot, and are now available within two computer-mouse clicks. This is useful for biologists and bioinformaticians for the study of T lymphocyte V(D)J gene rearrangements and their applications in immune response analysis.

IMGT/GeneInfo: enhancing V(D)J recombination database accessibility.

IMGT/GeneInfo is a user-friendly online information system that provides information on data resulting from the complex mechanisms of immunoglobulin (IG) and T cell receptor (TR) V(D)J recombinations. For the first time, it is possible to visualize all the rearrangement parameters on a single page. IMGT/GeneInfo is part of the international ImMunoGeneTics information system (IMGT), a high-quality integrated knowledge resource specializing in IG, TR, major histocompatibility complex (MHC), and related proteins of the immune system of human and other vertebrate species. The IMGT/GeneInfo system was developed by the TIMC and ICH laboratories (with the collaboration of LIGM), and is the first example of an external system being incorporated into IMGT. In this paper, we report the first part of this work. IMGT/GeneInfo_TR deals with the human and mouse TRA/TRD and TRB loci of the TR. Data handling and visualization are complementary to the current data and tools in IMGT, and will subsequently allow the modelling of V(D)J gene use, and thus, to predict non-standard recombination profiles which may eventually be found in conditions such as leukaemias or lymphomas. Access to IMGT/GeneInfo is free and can be found at http://imgt.cines.fr/GeneInfo.

Peripheral T cell receptor diversity is associated with clinical outcomes following ipilimumab treatment in metastatic melanoma.

BACKGROUND: Ipilimumab improves overall survival in a subset of patients with metastatic melanoma. Peripheral blood T cell receptor (TCR) repertoire diversity has been associated with favorable outcomes in patients with cancer, but its relevance as a biomarker for ipilimumab outcomes remains unknown. FINDINGS: In this pilot study, we analyzed the pre-treatment peripheral blood TCR repertoire in 12 patients with metastatic melanoma who received ipilimumab at 3 mg/kg (clinical benefit, n = 4; no clinical benefit, n = 8). TCR diversity was evaluated using a polymerase chain reaction assay which measures TCR combinatorial diversity between V and J genes from genomic DNA. TCR repertoire diversity was studied through richness (observed V-J rearrangements) and evenness (similarity between the frequencies of specific V-J rearrangements). The Wilcoxon rank sum test was used to compare patients with clinical benefit and those without. Association with benefit in a dichotomized analysis was assessed through a Fisher's exact test. Overall survival was studied through log-rank analysis. There was a significant difference in richness (p = 0.033) and evenness (p = 0.028) between patients with and without clinical benefit. Dichotomized analysis showed that none of the patients with low richness (n = 0/5, p = 0.081) nor low evenness (n = 0/7, p = 0.01) achieved clinical benefit. There were no significant differences in overall survival. CONCLUSIONS: In this small group of patients, baseline TCR diversity in the peripheral blood was associated with clinical outcomes. Further investigation is ongoing in larger cohorts of patients to explore these preliminary findings and determine whether TCR diversity can be used as a predictive biomarker in cancer immunotherapy.

Patients with metastatic breast cancer leading to CD4+ T cell lymphopaenia have poor outcome.

BACKGROUND: Low lymphocyte count is a prognostic factor in cancer patients including metastatic breast cancer patients (MBC) but the relative role of each lymphocyte subtype is unclear in MBC. METHODS: The impact of lymphocyte subsets was analysed in two prospective MBC patients' cohorts. Cohort A patients (n=103) were included before the first line of chemotherapy and cohort B patients (n=101) were included after at least one line of chemotherapy. Extensive phenotypic analyses were performed on fresh whole blood. Plasma cytokines levels were measured using commercially available Luminex-based multiplex kits. Prognostic value of lymphocyte subsets and circulating cytokines was analysed. RESULTS: In both cohorts, severe lymphopaenia (<0.7 Giga/L) correlated with poor overall survival (OS) (median OS: 6.6 months versus 21.7 months in cohort A and 4.5 versus 9 months in cohort B). CD8(+), CD19(+) and CD56(+) T cell counts had no significant prognostic value for OS. After stratification (≤0.2, [0.20-0.45], >0.45 Giga/L), CD4 lymphopaenia appeared to be correlated with poor OS in both cohorts. Furthermore, severe CD4(+) lymphopaenia (≤0.2 Giga/L) was strongly correlated with poor OS in both cohorts (1.2 months versus 24.9 months in cohort A and 5.7 versus 13.1 months in cohort B). In multivariate analysis, after stratification CD4(+) lymphopaenia appeared to be an independent prognostic factor for OS in both cohorts. CD4(+) lymphopaenia correlated with low plasmatic levels of CCL22 that might directly contribute to CD4(+) lymphopaenia. CONCLUSIONS: CD4(+) lymphopaenia was associated with reduced OS in MBC patients regardless of the chemotherapy line. Decreased levels of plasmatic CCL22 may contribute to CD4(+) lymphopaenia.

Lymphopenia combined with low TCR diversity (divpenia) predicts poor overall survival in metastatic breast cancer patients.

Lymphopenia (< 1Giga/L) detected before initiation of chemotherapy is a predictive factor for death in metastatic solid tumors. Combinatorial T cell repertoire (TCR) diversity was investigated and tested either alone or in combination with lymphopenia as a prognostic factor at diagnosis for overall survival (OS) in metastatic breast cancer (MBC) patients. The combinatorial TCR diversity was measured by semi quantitative multi-N-plex PCR on blood samples before the initiation of the first line chemotherapy in a development (n = 66) and validation (n = 67) MBC patient cohorts. A prognostic score, combining lymphocyte count and TCR diversity was evaluated. Univariate and multivariate analyses of prognostic factors for OS were performed in both cohorts. Lymphopenia and severe restriction of TCR diversity called "divpenia" (diversity ≤ 33%) were independently associated with shorter OS. Lympho-divpenia combining lymphopenia and severe divpenia accurately identified patients with poor OS in both cohorts (7.6 and 10.6 vs 24.5 and 22.9 mo). In multivariate analysis including other prognostic clinical factors, lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (p = 0.005) along with lack of HER2 and hormonal receptors expression (p = 0.011) and anemia (p = 0.009). Lympho-divpenia is a novel prognostic factor that will be used to improve quality of MBC patients' medical care.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRp-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2(125-243), TRP2L(125-376) and TRP2S(291-376). However, only immunization with TRP2L(125-376) induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8(+) epitopes, and strong CD4(+) Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapid screening and evaluation of antigens of varying length and epitope composition.

Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome.

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3(+) T cells are typical Treg based on their CD4(+)CD25(high)CD127(low)FOXP3(+) phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP(+) dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3(+), and CD8(+) T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem.