Cell Repertoire Diversity Research Articles

Abstract 5017: T-cell repertoire turnover induced by anti-CTLA-4 antibody treatment in cancer patients

Abstract While treatment with anti-CTLA-4 antibody can induce clinical responses in advanced cancer patients, its effects on the breadth of the T cell response is unknown. We used a next-generation sequencing-based method to assess T cell repertoire diversity in 46 patients with metastatic castration resistant prostate cancer or metastatic melanoma. Peripheral blood mononuclear cells were obtained from patients prior to and during treatment with anti-CTLA-4 antibody. mRNA was amplified using locus-specific primer sets for T cell receptor (TCR) beta, and the amplified product was sequenced. Sequence reads were used to quantitate absolute TCR frequencies using standardized clonotype determination algorithms with normalization by spiked reference TCR sequences. Following clonotype quantitation, repertoire differences between serial samples were assessed by the Morisita index, a statistical measure of population dispersion. 97 paired samples were assessed, of which 46 (47%) had increases and 22 (23%) had decreases in TCR diversity by more than 2-fold. By comparison, none of 9 untreated sample pairs underwent more than a 2-fold change in diversity (P = 0.005, Fisher's exact test, two tailed). TCR repertoire differences between monthly samples were markedly higher than for time-matched controls. After the first treatment, median Morisita index between samples was 0.197 for treated samples versus 0.039 for untreated (P = 0.0005, Mann-Whitney U test). The median number of clones that significantly changed in abundance was 421 for treated versus 45 for controls. In patients with multiple time points, this rapid clonotype evolution continued through treatment. Whereas the number of clonotypes that increased with treatment was not associated with clinical outcome, improved overall survival was associated with maintenance of high frequency clones present at baseline. In contrast the highest frequency clonotypes fell with treatment in patients with shorter overall survival. Stably maintained clonotypes included T cells possessing high avidity TCR such as CMV-reactive T cells. Together, these results suggest that CTLA-4 blockade induces T-cell repertoire evolution and diversification. Moreover, improved clinical outcomes are associated with less clonotype loss, consistent with the maintenance of high frequency TCR clonotypes during treatment. These clones may represent the presence of pre-existing high avidity T cells that may be relevant in the anti-tumor response. Citation Format: Lawrence H. Fong, Edward Cha, Mark Klinger, Yafei Hou, Craig Cummings, Antoni Ribas, Malek Faham. T-cell repertoire turnover induced by anti-CTLA-4 antibody treatment in cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5017. doi:10.1158/1538-7445.AM2014-5017

Abstract 2937: Localization of sipuleucel-T- expanded T cell clones into tumors of treated prostate cancer patients

Abstract Sipuleucel-T is an FDA-approved autologous cellular therapy demonstrated to prolong overall survival in subjects with asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer (mCRPC). Sipuleucel-T is generated by culturing patients' own peripheral blood mononuclear cells (PBMC) with a fusion protein of prostatic acid phosphatase and granulocyte macrophage colony stimulating factor. While antigen presenting cells take up this antigen and can prime antigen specific T cells in vivo, the majority of the cells in the sipuleucel-T product are in fact T cells. Moreover, antigen-specific T cells in the product become activated during production of sipuleucel-T, particularly for the 2nd and 3rd infusions. To examine whether T cells contained in sipuleucel-T might be homing to the site of the tumor, we used a next-generation sequencing-based method to assess T cell repertoire diversity in the blood, sipuleucel-T product, and resected tissues of patients participating in a neoadjuvant study of sipuleucel-T treatment (P07-1; NCT00715104). In this trial, patients with localized prostate cancer received sipuleucel-T prior to undergoing radical prostatectomy (RP). DNA extracted from the different samples was amplified using a multiplex PCR assay to amplify the CDR3 region of the T cell receptor spanning the variable region formed by the junction of the V, D and J segments and their associated non-templated insertions. Sequence reads were used to quantitate absolute TCR frequencies using standardized clonotype determination algorithms. We found that a subset of T cell clones was enriched in the generation of sipuleucel-T. We also found that T cell clones were expanded in patient blood after each successive infusion. When we examined the T cell clonotypes in the prostate tissues resected after sipuleucel-T treatment, we found that there were T cell clones in the prostate in common with the sipuleucel-T product. Interestingly a number of the clones present in the pre-treatment blood were reduced or lost in the blood by the second and third infusion but were present in the post-treatment prostate tissue, consistent with trafficking of the T cell clones from the peripheral circulation. These results indicate that ex vivo production of sipuleucel-T induces the expansion of certain T cell clones that are ultimately present in the prostates of patients treated prior to RP. Sipuleucel-T may therefore also function as an adoptive T cell therapy. Citation Format: Lawrence Fong, Todd DeVries, Jason Cham, Li Zhang, Mark Frohlich, James Trager, Nadeem Sheikh. Localization of sipuleucel-T- expanded T cell clones into tumors of treated prostate cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2937. doi:10.1158/1538-7445.AM2014-2937

Impact of ageing on the response and repertoire of influenza virus-specific CD4 T cells.

BackgroundAgeing has been shown to reduce CD8 T cell repertoire diversity and immune responses against influenza virus infection in mice. In contrast, less is known about the impact of ageing on CD4 T cell repertoire diversity and immune response to influenza virus infection.ResultsThe CD4 T cell response was followed after infection of young and aged C57BL/6 mice with influenza virus using a tetramer specific for an immunodominant MHC class II epitope of the influenza virus nucleoprotein. The appearance of virus-specific CD4 T cells in the lung airways of aged mice was delayed compared to young mice, but the overall peak number and cytokine secretion profile of responding CD4 T cells was not greatly perturbed. In addition, the T cell repertoire of responding cells, determined using T cell receptor Vβ analysis, failed to show the profound effect of age we previously described for CD8 T cells. The reduced impact of age on influenza-specific CD4 T cells was consistent with a reduced effect of age on the overall CD4 compared with the CD8 T cell repertoire in specific pathogen free mice. Aged mice that were thymectomized as young adults showed an enhanced loss of the epitope-specific CD4 T cell response after influenza virus infection compared with age-matched sham-thymectomized mice, suggesting that a reduced repertoire can contribute to impaired responsiveness.ConclusionsThe diversity of the CD4 T cell repertoire and response to influenza virus is not as profoundly impaired by ageing in C57BL/6 mice as previously shown for CD8 T cells. However, adult thymectomy enhanced the impact of ageing on the response. Understanding the impact of ageing on CD4 T cell responses to influenza virus infection is an important prerequisite for developing better vaccines for the elderly.

Genetic and environmental determinants of human NK cell diversity revealed by mass cytometry.

Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.

IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5′RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.

TGF-β Cytokine Signaling Promotes CD8+ T Cell Development and Low-Affinity CD4+ T Cell Homeostasis by Regulation of Interleukin-7 Receptor α Expression

Interleukin-7 receptor α chain (IL-7Rα) is induced upon T cell positive selection and controls thymic CD8-lineage specification and peripheral naive T cell homeostasis. How IL-7Rα expression is regulated in developing thymocytes is unclear. Here, we show that transforming growth factor β (TGF-β) signaling promoted IL-7Rα expression and CD8+ T cell differentiation. In addition, TGF-β signaling was required for high IL-7Rα expression in CD4+ T cells bearing low-affinity T cell receptors, and the abrogation of TGF-β receptor expression led to failed maintenance of peripheral CD4+ T cells. Compromised IL-7Rα expression in TGF-β-receptor-deficient T cells was associated with increased expression of the Il7ra transcriptional repressor, Gfi-1. IL-7Rα transgenesis or T-cell-specific ablation of Gfi-1 restored IL-7Rα expression and largely ameliorated the development and homeostasis defects of TGF-β-receptor-deficient T cells. These findings reveal functions for TGF-β signaling in controlling IL-7Rα expression and in promoting T cell repertoire diversification.

Effect of anti-CTLA-4 antibody treatment on T-cell repertoire evolution in treated cancer patients.

3020 Background: CTL-associated antigen 4 (CTLA-4) is an immune checkpoint expressed by T cells. While treatment with anti-CTLA-4 antibody can induce clinical responses in advanced cancer patients, its effects on the breadth of the T cell response is unknown. Methods: We used a sequencing-based method, LymphoSIGHT, to assess T cell repertoire diversity in 46 patients with metastatic castration resistant prostate cancer or metastatic melanoma. Peripheral blood mononuclear cells were obtained from patients prior to and during treatment with anti-CTLA-4 antibody. mRNA was amplified using locus-specific primer sets for T cell receptor (TCR) beta, and the amplified product was sequenced. Sequence reads were used to quantitate absolute TCR frequencies using standardized clonotype determination algorithms with normalization by spiked reference TCR sequences. Following clonotype quantitation, repertoire differences between serial samples were assessed by the Morisita index, a statistical measure of population dispersion. Results: 97 paired samples were assessed, of which 46 (47%) had increases and 22 (23%) had decreases in TCR diversity by more than 2-fold. By comparison, none of 9 untreated sample pairs underwent more than a 2-fold change in diversity (P = 0.005, Fisher’s exact test, two tailed). TCR repertoire differences between monthly samples were markedly higher than for time-matched controls. After the first treatment, median Morisita index between samples was 0.197 for treated samples versus 0.039 for untreated (P = 0.0005, Mann-Whitney U test). The median number of clones that significantly changed in abundance was 421 for treated versus 45 for controls. In patients with multiple time points, this rapid clonotype evolution continued through treatment. Despite this global turnover in repertoire, a subset of high frequency clones, including CMV-specific T cells, remained relatively constant over the course of the study. Conclusions: CTLA-4 blockade increases the global rate of T cell clonotype turnover and influences TCR diversity. This evolution of the TCR repertoire may reflect a mechanism by which CTLA-4 blockade enhances tumor-specific T cells over time.

Quantifying the unanticipated diversity of the human NK cell repertoire (P1456)

Abstract Like their T and B lymphocyte counterparts, natural killer (NK) cells exist in small and specialized subpopulations. However, while T and B cell repertoire diversity is generated primarily through DNA rearrangement to produce a single antigen-specific receptor, the diversity of the NK cell repertoire is determined by the expression of a spectrum of activating and inhibitory receptors. To evaluate the human NK cell repertoire on a single-cell basis, we simultaneously measured more than 25 NK cell receptors in the peripheral blood of 22 healthy individuals via mass cytometry. We used Boolean gating to classify cellular phenotypes. Based on the Simpson index, an ecological measure designed to quantify population diversity, we found that total NK cell repertoire diversity was approximately normally distributed across individuals. Using rarefaction curves and non-parametric species estimators, we calculate the expansiveness of the NK cell repertoire at a minimum of 100,000 unique phenotypes. Furthermore, within an individual, the ex vivo addition of the homeostatic cytokine IL-15 biased the repertoire toward highly common and uncommon phenotypes, increasing the total population richness but decreasing the overall diversity. These data show, for the first time, that the receptor-based NK cell repertoire is exquisitely diverse. They further suggest an optimal baseline diversity that is highly sensitive to optimization by exogenous factors.

Evolution of T Cell Repertoire Diversity After Reduced-Intensity Conditioning and Double Umbilical Cord Blood Transplantation with or without Exposure to FT1050 (16,16-dimethyl Prostaglandin E2)

Evolution of T Cell Repertoire Diversity After Reduced-Intensity Conditioning and Double Umbilical Cord Blood Transplantation with or without Exposure to FT1050 (16,16-dimethyl Prostaglandin E2)

Fractal Organization of the Human T Cell Repertoire in Health and after Stem Cell Transplantation

T cell repertoire diversity is generated in part by recombination of variable (V), diversity (D), and joining (J) segments in the T cell receptor β (TCR) locus. T cell clonal frequency distribution determined by high-throughput sequencing of TCR β in 10 stem cell transplantation (SCT) donors revealed a fractal, self-similar frequency distribution of unique TCR bearing clones with respect to V, D, and J segment usage in the T cell repertoire of these individuals. Further, ranking of T cell clones by frequency of gene segment usage in the observed sequences revealed an ordered distribution of dominant clones conforming to a power law, with a fractal dimension of 1.6 and 1.8 in TCR β DJ and VDJ containing clones in healthy stem cell donors. This self-similar distribution was perturbed in the recipients after SCT, with patients demonstrating a lower level of complexity in their TCR repertoire at day 100 followed by a modest improvement by 1 year post-SCT. A large shift was observed in the frequency distribution of the dominant T cell clones compared to the donor, with fewer than one third of the VDJ-containing clones shared in the top 4 ranks. In conclusion, the normal T cell repertoire is highly ordered with a TCR gene segment usage that results in a fractal self-similar motif of pattern repetition across levels of organization. Fractal analysis of high-throughput TCR β sequencing data provides a comprehensive measure of immune reconstitution after SCT.

T Cell Repertoire Diversity After Umbilical Cord Transplant Predicts Mortality From Infection

Abstract 4202 BackgroundIn a transplant study run at Fred Hutchinson Cancer Research Center, 34 patients with high risk hematological malignancies underwent a myeloablative procedure and were subsequently transplanted with double umbilical cord blood units. Peripheral blood samples were collected from each patient before myeloablation, and at 28, 56, 100, 180, and 360 days post-transplant. MethodsAt each time point, we used the immunoSEQ platform to perform high-throughput sequencing of rearranged T Cell Receptor (TCR) loci. Using immunoSEQ data, we were able to track the presence and frequency of individual TCR clones in each patient across time-points, as well as measuring the diversity of the TCR repertoire as a whole. We correlated our measure of TCR repertoire diversity with clinical outcomes in this cohort. ResultsThe study produced two primary results. First, using the ability to track clones, the reconstituting TCR repertoire is shown to oscillate wildly with nearly complete turnover of the T cell repertoire occurring at least monthly after CB transplant. The largest T cell clones present in each blood draw drop below detection within weeks, contrasting with control data in which the top clones in healthy patients are not only observed in multiple subsequent time-points, but remain at high frequency. The second result is a test of the hypothesis that diversity of the T cell repertoire is a measure of immunocompetence, as a clinical application of high-throughput sequencing. Of the 34 patients, six died between Day 100 and Day 360 of non-relapse causes. At both Day 56 and Day 100, the diversity of the T cell repertoires of those six patients were far lower than the T cell repertoire diversity values of the remaining patients (P-value = 0.015). ConclusionsWe have demonstrated that the reconstitution of clinical immunity in cord blood transplantation patients is characterized by a highly unstable T cell compartment with very rapid turnover of T cell clones. Despite the transience of individual T cell clones, however, by two months after transplant T cell repertoire diversity as measured by high-throughput TCR sequencing accurately predicts risk of non-relapse mortality. Disclosures:Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Fractal Organization of the Human T Cell Repertoire in Health and Following Stem Cell Transplantation

AbstractAbstract 4193T cell repertoire diversity is generated by recombination of variable (V), diversity (D) and joining (J) segments in the T cell receptor (TCR) locus. Further variability and antigen recognition capacity is introduced by nucleotide insertion (NI) in the recombined sequences resulting in a complex repertoire, the organization of which is poorly understood. We postulate that TCR b D, J and V gene segment usage in an individual would result in a TCR repertoire with a fractal, self-similar frequency distribution of T cell clones with respect to gene segment usage. To determine this, the TCR repertoire of donors and recipients of HLA matched-related and unrelated allogeneic stem cell transplantation (SCT) was evaluated by high-throughput (HT) sequencing of the CDR3 region of TCR b.Ten SCT donor-recipient pairs were selected for HT-TCR b sequencing. cDNA was isolated from T cells obtained from the donors at baseline and recipients at day 100, 1 year post SCT or at the time of graft-versus-host disease (GVHD) diagnosis. HT-TCR β sequencing was performed and analyzed using the ImmunoSEQ analyzer tool (Adaptive Biotechnologies, Seattle, WA). TCR b clone frequencies were used to determine the TCR self-similarity score (SSS = log clone frequency × log scale), evaluating clonal frequency at each gene segment-scale (J, VJ and VJ+NI). This revealed a TCR SSS with a relatively narrow distribution of 1.64 ± 0.1 (mean ± SD) for J, 1.69 ± 0.2 for VJ, and 1.41 ± 0.01 for VJ+NI, which was consistent between all normal stem cell donors analyzed.Relative proportional distribution (RPD) graphs were then generated to allow for the depiction of the TCR b D, J, V distribution for simultaneous comparison at an individual level. Representative data in Figure 1 shows the relatedness across donors at the TCR b J segment level. Relative clonal frequency was determined for each unique TCR clone at the specified segment level and plotted according to frequency-rank. Slope of the resulting linear regression lines from log-log plots of rank-frequency was used to determine self-similarity and fractal dimension. These plots were comparable among donors with resulting slopes of 1.6 ± 0.01 and 1.8 ± 0.1, respectively for TCR J and VJ containing clones (Figure 2). The plots also revealed a hierarchy of T cell clones, with few dominant clones occupying the high ranks and a multitude of clones in the later ranks.For donor-recipient comparisons, we examined the dominant ranking clones which would be most likely to be involved in GVHD and response to infection. The ordered TCR clonal frequency distribution seen in donors was perturbed in recipients following SCT, with recipients demonstrating a lower level of complexity in their TCR repertoire, and a large shift in the frequency distribution of the dominant T cell clones compared to the donor. When dominant clones were compared between donors and recipients, recipients shared only a small proportion of TCR clonotypes with their donors despite full donor T cell chimerism. This difference did not change over time, suggesting an alternate T cell clonal hierarchy and repertoire develops in transplant recipients when compared with their donors.Using simple mathematical analysis, we demonstrate that the TCR b repertoire has a fractal, self-similar pattern with a hierarchy of dominant and minor clones. We note that the complexity of the TCR repertoire is diminished and TCR b hierarchy is altered following SCT. Restoration of this order may serve as a marker for post-SCT immune reconstitution. Further, by demonstrating shifts in TCR clonal dominance, fractal analysis comparing donor and recipient T cell repertoire may allow for more accurate monitoring of immunotherapy of malignancies in general, beyond allogeneic SCT. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

T-cell receptor excision circles and repertoire diversity in children with profound T-cell immunodeficiency

Approximately 40% of patients with profound T-cell immunodeficiency have no identified molecular basis. Early assessment of T-cell impairment is vital for medical intervention, if hematopoietic stem cell transplantation is needed. The dynamics of T-cell receptor excision circles (TRECs) revealing recent thymic output of naïve T cell and T-cell receptor (TCR) repertoire diversity reflecting broader responses to multiple antigens, are both important in resisting infections. The TRECs value and TCR repertoire diversity were evaluated from peripheral blood mononuclear cells in patients with primary severe T cell immunodeficiency, to elucidate the T-cell response. In seven children with <30% of normal phytohemagglutinin (PHA)-stimulated lymphocyte proliferation, including two IL2RG (Try74Gly and Arg226Lys, X-linked) and one RAG2 mutations [(Ser205Tyr) and (del 1366T, frameshift, 484stop); autosomal recessive], lower TRECs value and oligo- and restricted TCR diversity patterns, were associated with increased susceptibility to opportunistic infections, but not inversely correlated to the severity and frequency of infections. Three patients had successful cord blood stem cell transplantation which reconstructed the T cell immunodeficiency, with normalized TRECs value and TCR repertoire diversity at 6 months post-transplant, without clinical events. Low TRECs value and restricted TCR repertoire diversity can help in the early diagnosis of T cell immunodeficiency before irreversible sequelae and in the monitoring of post-transplantation T-cell immune reconstruction.

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.

P3.13 Immune Status Stratification of Metastatic Breast Cancer Patients: Lympho-Divpenia Predicts Overall Survival

ABSTRACT Background Lymphopenia ( 1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor -beta chain (TCR-β), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n = 66) and in a validation series (cohort B, n = 67). A prognostic score, defined NDL (Number & Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results Lymphopenia ( 1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity >33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A + B) (p = 0.005) along with triple negative tumors (p = 0.011) and hemoglobin level (11.5 g/dL) (p = 0.009). Conclusion NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

Lymphopenia combined with low TCR diversity to predict overall survival in metastatic breast cancer patients.

10562 Background: Lymphopenia (&lt;1 Giga/L) before initiation of chemotherapy is a predictive factor for toxicity and death in metastatic phase for cancer patients. Combinatorial diversity of T Cell Receptor beta chain (TCR-ß), as a measure of T cell repertoire diversity, was investigated and tested either alone or in combination with lymphopenia as a prognostic factor for overall survival (OS) in first line treatment of metastatic breast cancer (MBC) patients. Methods: Using semi quantitative multiplex PCR, the V-D-J combinatorial diversity of the TCR was measured on cryo-preserved blood samples from 2 cohorts of MBC patients before the initiation of the first line chemotherapy: in an experimental cohort (cohort A, n=66) and in a validation series (cohort B, n=67). A prognostic score, defined NDL (Number &amp; Diversity of Lymphocytes) combining lymphocyte count and TCR diversity was delineated. Univariate and multivariate analysis of prognostic factors for OS were performed in both cohorts. Results: Lymphopenia (&lt;1 Giga/L) was associated with shorter OS for cohort B while TCR diversity ≤33% (called divpenia) was associated with a reduced OS in cohort A. The combination of lymphopenia with low TCR diversity (called lympho-divpenia) was associated with poor OS compared to patients with either lymphocyte count ≥1 Giga/L or diversity &gt;33% or both, in cohort A (median OS: 7.6 vs 24.5 months, p.value =0.0006) and cohort B (median OS 10.6 vs 22.9 months, p.value =0.0035). In a multivariate analysis, including all significant clinical factors from the univariate analysis (PS, liver metastasis, hemoglobin) lympho-divpenia was found to be an independent prognostic factor in the pooled cohort (A+B) (p=0.005) along with triple negative tumors (p=0.011) and hemoglobin level (11.5 g/dL) (p=0.009). Conclusions: NDL score combining lymphopenia and reduced TCR diversity seems to be a strong prognostic factor for OS and could be use to improve care quality of MBC patients.

The NLRP3 Inflammasome Promotes Age-Related Thymic Demise and Immunosenescence

The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naive Tcells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in 'lipotoxic danger signals' such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, Tcell progenitors and maintenance of Tcell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates Tcell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the re-establishment of a diverse Tcell repertoire in middle-aged or elderly patients undergoing HSCT.

Regulation of memory CD4 T cell repertoire diversity upon peptide and protein vaccination (160.18)

Abstract Diverse antigen-specific memory TCR repertoires are essential for protection against pathogens. Subunit vaccines that combine peptide or protein antigen with TLR agonists are very potent in inducing T cell immune responses but their capacity to elicit stable and diverse memory CD4 T cell repertoire has not been evaluated. Here, we tracked the development of antigen-specific memory CD4 T cells after peptide or protein vaccination. Both vaccination regimens induced equally diverse effector CD4 TCR repertoires but a skewing toward high affinity clonotypes was observed in the memory TCR repertoire elicited by peptide vaccines. This skewing did not occur after protein vaccination leaving the antigen-specific TCR repertoire essentially intact in the transition from effector to memory. Clear understanding of the factors regulating the TCR repertoire diversity after peptide and protein vaccination contributes to our knowledge of T-cell mediated immune responses and has practical relevance to the design of vaccines against infections requiring robust T cell immunity.

Investigating the involvement of Foxo1 in T cell repertoire diversity (160.8)

Abstract Advanced age is associated with increased susceptibility to and mortality from infectious diseases, especially influenza and pneumonia; and vaccination responses in the elderly are diminished. Declining resistance to infection in aging individuals is correlated with a progressive decline in the size and repertoire of the T cell pool. A better understanding of the mechanisms underlying age-related deterioration of T cell immunity could inform therapies that enhance general immune function and vaccination effectiveness in this vulnerable population. The forkhead transcription factor, Foxo1, has recently been identified as a major regulator of naïve T cell homeostasis. Moreover, a Foxo1 homolog, DAF-16, promotes longevity in C. elegans. Therefore, these studies documented the repertoire diversity of splenic CD4+ and CD8+ T cells in mice selectively deficient for Foxo1 in the T cell compartment (Foxo1 cKO mice). Our studies indicate that T cell-specific Foxo1 deficiency impacts TCR diversity in both the CD4+ and CD8+ splenic T cell populations of young mice. These observations suggest the intriguing possibility that in mice, Foxo1 may participate in aging-related changes in T cell diversity.

The Different Extent of B and T Cell Immune Reconstitution after Hematopoietic Stem Cell Transplantation and Enzyme Replacement Therapies in SCID Patients with Adenosine Deaminase Deficiency

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle(+) lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA-treated patients, it was accompanied by a significant and progressive decrease in circulating CD19(+) lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle(+) cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.