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γδ T cells contribute to the immune response against many cancers, notably through their powerful effector functions that lead to the elimination of tumor cells and the recruitment of other immune cells. However, their presence in the tumor microenvironment has been associated with poor prognosis in breast, colon, and pancreatic cancer, suggesting that γδ T cells may also display pro-tumor activities. Here, we identified in blood from healthy donors a subpopulation of Vδ1T cells that represents around 20% of the whole Vδ1 population, expresses CD73, and displays immunosuppressive phenotype and functions (i.e., production of immunosuppressive molecules, such as IL-10, adenosine, and the chemotactic factor IL-8, and inhibition of αβ T cell proliferation). We then found that in human breast tumors, γδ T cells were present particularly in late stage breast cancer samples, and that ∼20% of tumor-infiltrating γδ T cells expressed CD73. Taken together, these results suggest that regulatory γδ T cells are present in the breast cancer microenvironment and may display immunosuppressive functions through the production of immunosuppressive molecules, such as IL-10, IL-8, and adenosine, thus promoting tumor growth.

The mammalian targets of rapamycin (mTOR) inhibitors (sirolimus [SRL] and everolimus [EVR]) are used after transplantation for their immunosuppressive activity. Regulatory T-cells (Tregs) play a crucial role in immune tolerance. mTOR inhibitors appear to preserve Tregs, unlike Tacrolimus (Tac).The aim of this study was to evaluate the number and function of Tregs in liver transplant recipients before and after conversion from Tac to mTOR inhibitors.Fifteen patients with stable graft function where converted to SRL (n=5) or EVR (n=10). Tregs (CD4Patients of both groups displayed a sustained rise in Treg levels after introduction of mTOR inhibitors (Treg levels at 3 months: 6.45±0.38% of CD4 T-cells, vs. baseline level of 3.61±0.37%, P0.001; mean fold increase 2.04±0.73). In SRL group, 3-month Treg levels were 6.01±0.53 vs. 3.79±0.39; P=0.037, while in EVR group they were 6.63±0.67 vs. 3.54±0.51; P=0.001. By contrast, no statistical change was observed in an unconverted Tac control group. Tregs also preserved their functional ability to suppress activated T-cells.These results suggest that mTOR inhibitors induce a significant increase in Tregs while maintaining suppressive activity after LT.

Liver transplantation remains the only treatment for terminal liver diseases. However, immunosuppressive drugs required for allograft acceptance are toxic and may be responsible for severe side effects. Modulating the immune system to induce tolerance is a promising approach to reduce immunosuppressive regimen. More particularly, promoting natural CD4+ CD25+ FoxP3+ Tregs could be crucial in achieving tolerance. Contrary to calcineurin inhibitors, reports indicate that mTOR inhibitors may have a positive impact on Tregs. Here we present the first randomized prospective clinical study where Tregs levels from liver transplanted patients receiving either tacrolimus or everolimus were monitored for 6 months, starting from the day of transplantation. A total of 30 patients from four centers were monitored. Blood samples were obtained at day 0, day 14, one month, three months and six months post-transplantation. Flow-cytometry immunophenotyping of Tregs (CD4+ CD25+ CD127- FoxP3+) and functional assays with Tregs were performed to assess their immunosuppressive capacity. Levels of Tregs were significantly reduced after one month of standard tacrolimus-based immunosuppressive regimen (p

Summary Background Immune response failure against hepatitis C virus (HCV) has been associated with an increased regulatory T cell (Treg) activity. After liver transplantation (LT), 80% of patients experience an accelerated progression of hepatitis C recurrence. The aim of this work was to assess the involvement of Tregs, T helper (Th) 1, 2 and 17 cells in recurrent hepatitis C. Methods Peripheral blood cells obtained before and one month after LT from 22 recipients were analysed. Forty-four key molecules related to Treg, Th1, 2 and 17 responses, were evaluated using qRT-PCR. Liver recipients were classified in two groups according to graft fibrosis evaluated by the METAVIR score on the biopsy performed one year after LT (mild: F ≤ 1, n = 13; severe: F > 1, n = 9). Patients developing a severe recurrence were compared with patients with a mild recurrence. Results mRNA levels of Treg markers obtained one month after LT were significantly increased in patients with a severe disease course when compared to patients with a mild recurrence. Markers of the Th1 response were elevated in the same group. No differences in the markers determined before LT were observed. Conclusion These findings suggest that Treg, induced by a multifactorial process, which could include a strong Th1 response itself, may play a role in suppressing the early antiviral response, leading to a severe recurrence of hepatitis C.

International audience; Background: Immunosuppressive-drugs are needed after solid organ transplantation to prevent allograft rejection but induce severe side effects. Understanding the alloimmune response is critical to modulate it and to achieve graft operational tolerance. The role of regulatory T cells and tolerogenic dendritic cells (Tol-DCs) is undoubtedly essential in tolerance induction. Tacrolimus is considered as the cornerstone of immunosuppression in solid organ transplantation. mTOR inhibitor such as rapamycin are thought to induce tolerance and are used as anticancer drugs in several cancers. The aim of this study was to better understand the effect of these immunosuppressive drugs on the differentiation, maturation and function of human monocyte derived dendritic cells (DCs).Material and methods: DCs were differentiated from monocytes of healthy donors with either rapamycin (Rapa-DCs) or tacrolimus (Tac-DCs). The phenotype was evaluated by flow cytometry analysis. The production of pro- and anti-inflammatory cytokines was assessed by ELISA. The mRNA expression level of IDO and PD-L1 was assessed by RTqPCR. Mixed leukocytes reactions were performed to analyse suppressive activity of DCs.Results: Rapa-DC were characterised by a lower expression of the co-stimulatory molecules and CD83 than control-DCs (CTR-DC) (p < 0.05). In contrast, tacrolimus had no effect on the expression of surface markers compared to CTR-DCs. Rapamycin reduced both IL-12 and IL-10 secretions (p < 0.05). Rapa-DCs had a suppressive effect on CD4+ allogenic T cells compared to CTR-DCs (p < 0.05). However, neither Rapa-DCs nor Tac-DCs favoured the emergence of a CD4+CD25highFoxp3+ population compared to CTR-DCs. Surprisingly, Rapa-DCs had a reduced expression of IDO and PD-L1 compared to Tac-DCs and CTR-DCs.Conclusion: Rapa-DCs exhibit an incomplete phenotypic tolerogenic profile. To our knowledge this is the first paper showing a reduction of expression of pro-tolerogenic enzyme IDO in DCs. Tacrolimus does not change the phenotypical or functional characteristics of moDCs.

Hepatitis C viral infection persists and becomes chronic in a majority of affected individuals. Numerous factors have been described to explain how the virus manages to escape the host immune system. One important escape mechanism is the increase in regulatory T cells induced by the virus. In this review, we will focus on the status of regulatory T cells throughout the natural history of hepatitis C infection and after liver transplantation. The molecular mechanisms involved in increasing the number of regulatory T cells are also discussed, as are data regarding the impact of regulatory T-cells on hepatic fibrosis in the context of hepatitis C viral infection.

International audience; Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immuno-suppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9-Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epi-topes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.

International audience; Vγ9Vδ2 T cells contribute to the immune response against many tumor types through their direct cytotoxic activity and capacity to regulate the biological functions of other immune cells, such as dendritic cells and IFN-γ-producing CD8+ T cells. However, their presence in the tumor microenvironment has also been associated with poor prognosis in breast, colon and pancreatic cancers. Additionally, recent studies demonstrated that cytokines can confer some plasticity to Vγ9Vδ2 T cells and promote their differentiation into cells with regulatory functions. Here, we demonstrated that activation of Vγ9Vδ2 T cells isolated from healthy donors and cultured in the presence of IL-21 favors the emergence of a subpopulation of Vγ9Vδ2 T cells that express the ectonucleotidase CD73 and inhibits T cell proliferation in a CD73/adenosine-dependent manner. This subpopulation produces IL-10 and IL-8 and displays lower effector functions and cytotoxic activity than CD73-negative Vγ9Vδ2 T cells. We also showed, in a syngeneic mouse tumor model, the existence of a tumor-infiltrating γδ T cell subpopulation that produces IL-10 and strongly expresses CD73. Moreover, maturation, IL-12 production and induction of antigen-specific T cell proliferation are impaired in DC co-cultured with IL-21-amplified Vγ9Vδ2 T cells. Altogether, these data indicate that IL-21 promotes Vγ9Vδ2 T cell regulatory functions by favoring the development of an immunosuppressive CD73+ subpopulation. Thus, when present in the tumor microenvironment, IL-21 might negatively impact γδ T cell anti-tumor functions.

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca2+) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca2+ mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca2+ mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4+ T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca2+ mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4+ T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.

Comment in :- RE: Effect of Nasopharyngeal Carcinoma-Derived Exosomes on Human Regulatory T Cells. [J Natl Cancer Inst. 2015]- Response. [J Natl Cancer Inst. 2015]; International audience; BACKGROUND: Regulatory T cells (Treg) and tumor-exosomes are thought to play a role in preventing the rejection of malignant cells in patients bearing nasopharyngeal carcinoma (NPC).METHODS: Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients' plasma (Patient-Exo), and CCL20 were tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model (n = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA, and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). The Student's t test was used for group comparisons. All statistical tests were two-sided.RESULTS: CCL20 allowed the intratumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34 fold increase, P < .001), which was statistically significantly inhibited (P < .001) by an anti-CCL20 blocking mAb. NPC-Exo also recruited conventional CD4(+)CD25(-) T cells and mediated their conversion into inhibitory CD4(+)CD25(high) cells. Moreover, NPC-Exo enhanced (P = .0048) the expansion of human Treg, inducing the generation of Tim3(Low) Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75 (P < .001). These results were consistent with a stronger suppression of responder cells' proliferation and the secretion of immunosuppressive cytokines (IL10, TGFB1).CONCLUSION: Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.

Background Galectin-9 is a mammalian lectin which possesses immunosuppressive properties. Excessive production of galectin-9 has been reported in two types of human virus-associated diseases chronic hepatitis C and nasopharyngeal carcinoma associated to the Epstein-Barr virus. The objective of this study was to produce new monoclonal antibodies targeting galectin-9 in order to improve its detection in clinical samples, especially on tissue sections analysed by immunohistochemistry. Methods Hybridomas were produced through immunization of mice with the recombinant c-terminus part of galectin-9 (residues 191 to 355 of the long isoform) and semi-solid fusion of spleen cells with Sp2/0 cells. Monoclonal antibodies were characterized using ELISA, epitope mapping, western blot and immunohistochemistry. Results We selected seven hybridomas producing antibodies reacting with our recombinant c-terminus galectin-9 in ELISA. Five of them reacted with the epitope “TPAIPPMMYPHPA” (common to all isoforms, residues 210 to 222 of the long isoform) and stained all three isoforms of galectin-9 analysed by western blot. One of them, 1G3,demonstrated very good sensitivity and specificity when used for immunohistochemistry. Using 1G3, we could confirm the intense and constant expression of galectin-9 by Epstein-Barr virus positive malignant cells from nasopharyngeal carcinomas. In most samples, specific staining was detected in both cytoplasm and nuclei. Galectin-9 was also detected in liver biopsies from patients infected by the human hepatitis C or B viruses with expression not only in inflammatory leucocytes and Kupffer cells, but also in hepatocytes. In contrast, galectin-9 was virtually absent in non-infected liver specimens. Conclusion The 1G3 monoclonal antibody will be a powerful tool to assess galectin-9 expression and distribution especially in diseases related to oncogenic viruses.

Galectin-9 (gal-9) is a multifunctional β-galactoside-binding lectin which is frequently released in the extra-cellular medium where it can modulate various biological activities (cell adhesion, migration), but it acts mainly as a negative regulator of the immune response. Our team has shown an intense and inappropriate production of gal-9 by malignant epithelial cells in Epstein-Barr virus-associated nasopharyngeal carcinoma. Other groups and ourselves have shown that gal-9 is very abundant in plasma samples from patients chronically infected by hepatitis C or B virus, and particularly in those with hepatocellular carcinomas. The most prominent immunosuppressive effects reported for gal-9 are the induction of apoptosis of CD4+ T-helper 1 (Th1) cells, exhaustion of CD8+ T cells, and stimulation of regulatory T cell activity. However, a recent study (and our own data) demonstrates that gal-9 can also activate and expand a subset of IFNγ; producing Th1 cells and central memory T cells in the surviving population. Despite its critical role in regulating the immune response, our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. Our studies reveal that several proximal components of the TCR-CD3 complex including the Lck kinase were required for the calcium (Ca2+) mobilization induced by gal-9 in Jurkat cells. In contrast, these components were not necessary for the apoptosis pathway triggered by gal-9. These data were confirmed in human CD4+ blood T cells: Lck inhibition with a specific inhibitor abrogates Ca2+ mobilization, but not apoptosis induction in CD4+ T cells. Moreover, this cytosolic Ca2+ release leads to the production of Th1 type cytokines, i.e. IL-2 and IFNγ; also dependent on Lck. These findings support the notion that gal-9 acts on T cells by two distinct pathways: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis which is independent of this complex. More recent data suggest that the CD45 molecule could be a putative receptor for gal-9 on human T cells. Indeed, the J45.01 cell line (CD45-deficient) was not susceptible to gal-9-induced apoptosis. In parallel to this work, we investigated the same cellular events (apoptosis and Ca2+ mobilization) in human B cells. In fact, there are currently very few publications dealing with the effects of gal-9 on B cells. Our preliminary results show that some Burkitt's lymphoma cell lines (BL2, BL41 and Ramos) and CD20+ B cells from blood, were both sensitive to gal-9-induced apoptosis. Besides, gal-9 induced a cytosolic Ca2+ mobilization in BL2 and BL41 cells which is efficiently abrogated by the chemical Lck inhibitor. In contrast, we didn't observe any Ca2+ release in the Ramos cell line, in which the Lck kinase is not functional, as well as in non-lymphoid cells. These preliminary results suggest that gal-9 activates in B cells a signaling pathway somehow similar to the TCR pathway and previously unexplored in B cells. Citation Format: Claire Lhuillier, Clement Barjon, Toshiro Niki, Aurore Gelin, Nadira Delhem, Mitsuomi Hirashima, Ming Wei, Olivier Dellis, Pierre Busson. Exploration of the signaling pathways triggered by exogenous galectin-9 in human T and B cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B177.

Like other human solid tumors, nasopharyngeal carcinoma (NPC) is a tissue and a systemic disease as much as a cell disease. Tumor cell population in NPC is highly heterogeneous. Heavy infiltration by non-malignant leucocytes results at least in part from the production of abundant inflammatory cytokines by the malignant epithelial cells. There is indirect evidence that interactions between stromal and malignant cells contribute to tumor development. Peripheral blood samples collected from NPC patients contain multiple products derived from the tumor, including cytokines, non-cytokine tumor proteins, tumor exosomes and viral nucleic acids. These products represent a potential source of biomarkers for assessment of tumor aggressiveness, indirect exploration of cellular interactions and monitoring of tumor response to therapeutic agents. Most NPC patients are immunocompetent with evidence of active humoral and cellular immune responses against EBV-antigens at the systemic level. Tumor development is facilitated by local immunosuppressive factors which are not fully understood. Local accumulation of regulatory T-cells is probably one important factor. At least two NPC tumor products are suspected to contribute to their expansion, the cytokine CCL20 and the tumor exosomes carrying galectin 9. In the future, new therapeutic modalities will probably aim at breaking immune tolerance or at blocking cellular interactions critical for tumor growth.

Background Bioluminescent tumor cell lines are experimental tools of major importance for cancer investigation, especially imaging of tumors in xenografted animals. Stable expression of exogenous luciferase in tumor cells combined to systemic injection of luciferin provides an excellent signal/background ratio for external optical imaging. Therefore, there is a need to rationalize and speed up the production of luciferase-positive tumor cell lines representative of multiple tumor phenotypes. For this aim we have designed a fusion gene linking the luciferase 2 protein to the c-terminus of a truncated form of the rat CD2 protein (tCD2-luc2). To allow simultaneous assessment of the wild-type luciferase 2 in a context of tCD2 co-expression, we have made a bicistronic construct for concomitant but separate expression of these two proteins (luc2-IRES-tCD2). Both the mono- and bi-cistronic constructs were transduced in lymphoid and epithelial cells using lentiviral vectors. Results The tCD2-luc2 chimera behaves as a type I membrane protein with surface presentation of CD2 epitopes. One of these epitopes reacts with the OX34, a widely spread, high affinity monoclonal antibody. Stably transfected cells are sorted by flow cytometry on the basis of OX34 staining. In vitro and, moreover, in xenografted tumors, the tCD2-luc2 chimera retains a substantial and stable luciferase activity, although not as high as the wild-type luciferase expressed from the luc2-IRES-tCD2 construct. Expression of the tCD2-luc2 chimera does not harm cell and tumor growth. Conclusion Lentiviral transduction of the chimeric tCD2-luc2 fusion gene allows selection of cell clones with stable luciferase expression in less than seven days without antibiotic selection. We believe that it will be helpful to increase the number of tumor cell lines available for in vivo imaging and assessment of novel therapeutic modalities. On a longer term, the tCD2-luc2 chimera has the potential to be expressed from multi-cassette vectors in combination with various inserts of interest.

On the basis of the three-dimensional model of the heme/hemophore TonB-dependent outer membrane receptor HasR, mutants with six-residue deletions in the 11 putative extracellular loops were generated. Although all mutants continued to be active TonB-dependent heme transporters, mutations in three loops abolished hemophore HasA binding both in vivo and in vitro.

Introduction Exosomes are nanovesicles found in large quantities in biological fluids and tumors of patients with nasopharyngeal carcinoma (NPC). These tumor exosomes play an important role in tumor progression due to their immunosuppressive properties. In addition, it has been reported that the frequency and suppressor functions of CD4 + CD25highFOXP3 + CD127low regulatory T cells (Treg) are also higher in NPC patients than healthy donors. Interactions between NPC-derived exosomes and Treg remain unknown. Here we investigated their ability to induce, expand, activate and recruit human Treg. Material and methods Treg recruitment by exosomes derived from NPC cell lines (C15/C17-Exo), exosomes isolated from NPC patients’ plasma (Patient-Exo), and CCL20 was tested in vitro using Boyden chamber assays and in vivo using a xenograft SCID mouse model ( N = 5), both in the presence and absence of anti-CCL20 monoclonal antibodies (mAb). Impact of these NPC exosomes (NPC-Exo) on Treg phenotype and function was determined using adapted assays (FACS, Q-PCR, ELISA and MLR). Experiments were performed in comparison with exosomes derived from plasma of healthy donors (HD-Exo). Results CCL20 allowed the intra-tumoral recruitment of human Treg. NPC-Exo also facilitated Treg recruitment (3.30 ± 0.34-fold increase, P P P = 0.0048) the expansion of human Treg, inducing the generation of Tim3Low Treg with increased expression of CD25 and FOXP3. Finally, NPC-Exo induced an overexpression of cell markers associated with Treg phenotype, properties and recruitment capacity. For example, GZMB mean fold change was 21.45 ± 1.75. These results were consistent with a stronger suppression of responder cells’ proliferation ( P Conclusion Interactions between NPC-Exo and Treg represent a newly defined mechanism that may be involved in regulating peripheral tolerance by tumors and in supporting immune evasion in human NPC.