Manuarii Manuel

High diversity of the immune repertoire in humanized NOD.SCID.γc−/− 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.γc−/− 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‐β chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR‐β 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.γc−/− 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.

Targeting regulatory T cells

Cancers express tumor-associated antigens that should elicit immune response to antagonize the tumor growth, but spontaneous immune rejection of established cancer is rare, suggesting an immunosuppressive environment hindering host antitumor immunity. Among the specific and active tumor-mediated mechanisms, CD4+CD25high T regulatory cells (Treg) are important mediators of active immune evasion in cancer. In this review, we will discuss Treg subpopulations and the mechanisms of their suppressive functions. Treg depletion improves endogenous antitumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We will also discuss specific strategies for devising effective cancer immunotherapy targeting Treg.

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.

Half of the T-cell repertoire combinatorial diversity is genetically determined in humans and humanized mice

In humanized mice, the T‐cell repertoire is derived from genetically identical human progenitors in distinct animals. Thus, careful comparison of the T‐cell repertoires of humanized mice with those of humans may reveal the contribution of genetic determinism on T‐cell repertoire formation. Here, we performed a comprehensive assessment of the distribution of V‐J combinations of the human β chain of the T‐cell receptor (hTRBV) in NOD.SCID.γc−/− (NSG) humanized mice. We observed that numerous V‐J combinations were equally distributed in the thymus and in the periphery of humanized mice compared with human references. A global analysis of the data, comparing repertoire perturbation indices in humanized NSG mice and unrelated human PBMCs, reveals that 50% of the hTRBV families significantly overlapped. Using multivariate ranking and bootstrap analyses, we found that 18% of all possible V‐J combinations contributed close to 50% of the expressed diversity, with significant over‐representation of BV5‐J1.1+1.2 and BV6‐J1.1+1.2 rearrangements. Finally, comparison of CD3− and CD3+ thymocyte repertoires indicated that the observed V‐J combination overlap was already present before TCR‐MHC selection in the thymus. Altogether, our results show that half of the T‐cell repertoire combinatorial diversity in humans is genetically determined.

ImmunTraCkeR® as a reliable TCR repertoire profiling tool to understand immune response and to explore immunotherapy biomarkers

Meeting abstracts Over the last decade, the host immunity has emerged as a critical determinant of cancer development and of response to therapy. Great progress has been made in the development of immunotherapies, which leverage the patient’s immune system to reach better clinical outcomes.

T cell receptor diversity evaluation to predict patient response to Ipilimumab in metastatic melanoma

Background Ipilimumab blocks the immunologic checkpoint cytotoxic T lymphocyte antigen-4 (CTLA-4) and improves overall survival in patients with metastatic melanoma. Since only a subset of patients achieves benefit from ipilimumab, biomarkers that can predict patient outcome are needed. CTLA-4 blockade has been shown to affect the peripheral T cell receptor (TCR) pool, but whether diversity of the peripheral TCR repertoire is relevant as a biomarker for ipilimumab remains unknown. Methods In this pilot study, we analyzed the baseline (pretreatment) peripheral blood TCR repertoire in 12 patients with metastatic melanoma treated with ipilimumab at 3mg/kg (investigator assessed clinical benefit, n = 4; non-clinical benefit, n = 8). TCR diversity was evaluated using a multiN-plex polymerase chain reaction (PCR) assay which measures TCR combinatorial diversity between V and J genes from genomic DNA. The diversity of the TCR pool was studied through richness (observed V-J rearrangements) and clonality (evenness of the repertoire based on frequencies of specific V-J rearrangements). The Wilcoxon rank sum test was used to compare results between patients with clinical benefit and those without. A receiver operating characteristic (ROC) curve was used to determine an appropriate threshold for dichotomized analysis (i.e. low vs. high diversity richness/clonality). Association with benefit in the low vs. high groups was assessed through a Fisher’s exact test. Overall survival was studied through log-rank analysis. Results There was a significant difference in diversity richness (p = 0.033) and in clonality (p = 0.028) between patients with and without clinical benefit. Dichotomized analysis showed that none of the patients with low diversity richness (n = 0/5, p = 0.081) nor low clonality (n = 0/7, p = 0.01) achieved clinical benefit. 4 out of the 5 patients with a high clonality had clinical benefit. There was no significant difference in overall survival between patients with low vs. high diversity richness (p = 0.218) and clonality (p = 0.26). Conclusions In a small group of melanoma patients treated with ipilimumab, TCR diversity in the peripheral blood at baseline was associated with patient outcomes. Further investigation is ongoing in larger cohorts of patients treated with novel immunologic checkpoint antibodies to explore these preliminary findings and determine whether TCR diversity can be used as a predictive biomarker in cancer immunotherapy.

Abstract 2574: Interleukin-7 (CYT107) treatment in lymphopenic 1st line metastatic breast carcinoma patients treated with chemotherapy regimen (Capecitabine) favors the restoration of T-cell subsets number

Introduction: We previously showed that T ( Methods: Lymphocytes, NK, monocytes and DC subpopulations quantification and functional competence after reactivation (PMA/ionomycin, TLR7/8-Ligand) were assessed by multiparametric flow-cytometry (surface markers/ intracytoplasmic cytokines) on fresh whole blood (D0, D21, D57, D78, EOS). Impact of CYT107 treatment before (D0-D21) or during (D57-D78) chemotherapy on immune parameters was assessed. Results: At inclusion, 20 patients presented strong quantitative alteration of all lymphocytes subsets compared to a healthy donor women cohort (n=30). Inflammatory monocytes (infl-Mo) and DC subsets were quantitatively reduced. CD4 CD45R0 + T (IFNγ/IL-2), Tγδ cells and NK (IFNγ) cytokine productions were altered independently of quantitative alterations highlighting the importance to assess both. Pre-chemotherapy, CYT107 significantly increased CD3 T cell absolute number (median relative evolution: CYT107+128.5%[39;606cells/µL] vs placebo +1.6%[-34;85cells/µL], p=0.002) involving both CD4 (p=0.002) or CD8 (p=0.006) T cells and naive (CD4:p=0.005 and CD8:p -3 ) and memory subsets (CD4:p -3 and CD8:p=0.008). CYT107 pre-chemotherapy induced a Treg number increase (p=0.017) remaining however in the normal range. In contrast, B cells, infl-Mo, DC and NK absolute numbers were not significantly modified. During chemotherapy (D57-D78, n=11), T cell absolute number increases in CYT107 group vs placebo (median relative evolution: CYT107 +61.5%[-11.1;229.7cells/µL] vs placebo -0.5%[-30.7;73.5cells/µL], p=0.083), CD4 and CD8 subsets being affected and in particular CD8 naive(median relative evolution: CYT107 +75.8%[39.0;314.6cells/µL] vs placebo -0.6%[-18.7;48.4cells/µL], p=0.022) and memory subsets (median relative evolution: CYT107 +72.1%[-21.9;282.7cells/µL] vs placebo -8.6%[-43.3;32.8cells/µL], p=0.055). Of importance, no significant modulation of immune cells functional competence was observed upon CYT107 treatment. Conclusion: CYT107 administered before chemotherapy, increases all T cell subsets absolute number but no other compartments without affecting their functional responses. Interestingly, during chemotherapy, CYT107 induces an increase of CD8 memory cells, known to play a role in long lasting defense against tumors. Citation Format: Christine Menetrier-Caux, Isabelle Ray-Coquard, Claire Cropet, Estelle Verronese, Thomas Bachelot, Olivier Tredan, Gwenaelle Garin, Pierre Heudel, Axelle N9Kodia, Ana Delgado, Christine Bardin-Dit-Courageot, Chantal Rigal, Gilles Clapisson, Sylvie Chabaud, David Perol, Paul Rebattu, Therese Croughs, Nicolas Pasqual, Manuarii Manuel, Michel Morre, Jean-Yves Blay, Christophe Caux. Interleukin-7 (CYT107) treatment in lymphopenic 1 st line metastatic breast carcinoma patients treated with chemotherapy regimen (Capecitabine) favors the restoration of T-cell subsets number. [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 2574. doi:10.1158/1538-7445.AM2014-2574