Cédric Rébé

Stat3 and Gfi-1 Transcription Factors Control Th17 Cell Immunosuppressive Activity via the Regulation of Ectonucleotidase Expression

Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

In situ immune response after neoadjuvant chemotherapy for breast cancer predicts survival.

Accumulating preclinical evidence suggests that anticancer immune responses contribute to the success of chemotherapy. However, the predictive value of tumour‐infiltrating lymphocytes after neoadjuvant chemotherapy for breast cancer remains unknown. We hypothesized that the nature of the immune infiltrate following neoadjuvant chemotherapy would predict patient survival. In a series of 111 consecutive HER2‐ and a series of 51 non‐HER2‐overexpressing breast cancer patients treated by neoadjuvant chemotherapy, we studied by immunohistochemistry tumour infiltration by FOXP3 and CD8 T lymphocytes before and after chemotherapy. Kaplan‐Meier analysis and Cox modelling were used to assess relapse‐free survival (RFS) and overall survival (OS). A predictive scoring system using American Joint Committee on Cancer (AJCC) pathological staging and immunological markers was created. Association of high CD8 and low FOXP3 cell infiltrates after chemotherapy was significantly associated with improved RFS (p = 0.02) and OS (p = 0.002), and outperformed classical predictive factors in multivariate analysis. A combined score associating CD8/FOXP3 ratio and pathological AJCC staging isolated a subgroup of patients with a long‐term overall survival of 100%. Importantly, this score also identified patients with a favourable prognosis in an independent cohort of HER2‐negative breast cancer patients. These results suggest that immunological CD8 and FOXP3 cell infiltrate after treatment is an independent predictive factor of survival in breast cancer patients treated with neoadjuvant chemotherapy and provides new insights into the role of the immune milieu and cancer. Copyright

STAT3 activation: A key factor in tumor immunoescape

Cancer growth is controlled by cancer cells (cell intrinsic phenomenon), but also by the immune cells in the tumor microenvironment (cell extrinsic phenomenon). Thus cancer progression is mediated by the activation of transcription programs responsible for cancer cell proliferation, but also induced proliferation/activation of immunosuppressive cells such as Th17, Treg or myeloid derived suppressor cells (MDSCs). One of the key transcription factors involved in these pathways is the signal transducer and activator of transcription 3 (STAT3). In this review we will focus on STAT3 activation in immune cells, and how it impacts on tumor progression.

Dacarbazine-Mediated Upregulation of NKG2D Ligands on Tumor Cells Activates NK and CD8 T Cells and Restrains Melanoma Growth

Dacarbazine (DTIC) is a cytotoxic drug widely used for melanoma treatment. However, the putative contribution of anticancer immune responses in the efficacy of DTIC has not been evaluated. By testing how DTIC affects host immune responses to cancer in a mouse model of melanoma, we unexpectedly found that both natural killer (NK) and CD8(+) T cells were indispensable for DTIC therapeutic effect. Although DTIC did not directly affect immune cells, it triggered the upregulation of NKG2D ligands on tumor cells, leading to NK cell activation and IFNγ secretion in mice and humans. NK cell-derived IFNγ subsequently favored upregulation of major histocompatibility complex class I molecules on tumor cells, rendering them sensitive to cytotoxic CD8(+) T cells. Accordingly, DTIC markedly enhanced cytotoxic T lymphocyte antigen 4 inhibition efficacy in vivo in an NK-dependent manner. These results underscore the immunogenic properties of DTIC and provide a rationale to combine DTIC with immunotherapeutic agents that relieve immunosuppression in vivo.

An atypical caspase-independent death pathway for an immunogenic cancer cell line

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.

Tumor exosome-mediated MDSC activation.

To the Editor-in-Chief: n nIn a recent issue of The American Journal of Pathology, Xiang et al1 published their results concerning the effects of tumor-derived exosomes on myeloid-derived suppressor cell (MDSC) biology. They compared the biological effects of exosomes derived from in vitro cultured B16 tumor cells (termed C-exo for culture exosome) and exosomes derived from in vivo grown B16 tumor (termed P-exo for primary exosomes). They reported that P-exo induce Toll-like receptor 2 (TLR2)–independent MDSC activation and expansion, whereas C-exo activate and expand MDSC in a TLR2-dependent manner. These data are in contrast with our own recent article demonstrating MDSC activation through TLR2 ligation by heat shock protein 72 (Hsp72) expressed on exosomes from CT26 tumors.2 In their discussion, Xiang et al1 propose a hypothesis to explain this discrepancy between C-exo and P-exo. Here, we provide our point of view about this difference. n nXiang et al1 propose that P-exo harbor a TLR2-independent mechanism of MDSC activation that is different from C-exo. This is probably true. Indeed, in another study from the same group, Xiang et al3 provided evidence that TS/A tumor cell lines produce exosomes containing prostaglandin E2 (PGE2), and it has previously been shown that PGE2 alone is sufficient to induce MDSC expansion and activation in a TLR2-independent manner.4 However, in the models used in our study, we provide evidence that there is no detectable PGE2 in tumor-derived exosomes. Xiang et al1 assert that our work demonstrated that “tumor exosomes trigger MDSC expansion via activation of STAT3.” However, this assumption is false. In our model, we demonstrated that there are indeed two distinct signals: tumor-derived exosomes account for MDSC activation (STAT3 phosphorylation and interleukin 6 secretion), whereas tumor-derived soluble factors [namely, granulocyte-macrophage colony-stimulating factor (GM-CSF)] are responsible for expansion. In the EL4 model, we observed that tumors growing in TLR2−/− mice induced MDSC expansion but not MDSC activation compared with wild-type mice. Thus, we can exclude the in vitro effect hypothesized by Xiang et al,1 because, in this in vivo setting, we observed proliferation dissociated from activation. In the model by Xiang et al, for unknown reasons, C-exo induced both STAT3 activation and expansion of MDSC; however, we never observed proliferation of MDSC cultured with exosomes alone. Moreover, Xiang et al already reported that the expansion and proliferation of MDSC in their model may be related to the presence of PGE2. Indeed, we also observed that PGE2 alone could trigger MDSC expansion and proliferation, so we could postulate that exosomes may have contained PGE2 in the model of Xiang et al, whereas we could not detect PGE2 in exosomes from our own models. These data may explain the discrepancy between our works. n nFurthermore, the preparation of the P-exo raises some methodological concerns. They are prepared from isolated in vivo grown tumors, with less than 5 in vitro passages. Many nontumoral cells, such as myeloid cells or fibroblasts, might be present in the preparation. These cell contaminants could be responsible for the production of PGE2-containing exosomes because it is well known that tumor-infiltrating macrophages may produce PGE25 and exosomes.6 n nXiang et al1 also propose that the difference between C-exo and P-exo is based on the passage number of in vitro cultured cells. We work with cell lines obtained from the American Type Culture Collection and used at passage numbers less than 15 to 20, so we assume the derivation from the parental cell line is minimal. Xiang et al also proposed that mycoplasma contamination could be responsible for the TLR2-dependent activation signal, which would be eliminated by the in vivo passage of the tumor. In our study, we routinely tested for mycoplasma contamination, but Xiang et al suspect that the available tests are insufficient. However, in our study, we demonstrated that exosomes derived from mycoplasma-free Hsp72-silenced CT26 were unable to activate MDSC, whereas mycoplasma-free mock CT26-derived exosomes activated MDSC. Further, in vivo Hsp72-silenced tumors were unable to induce STAT3 activation of MDSC, thus supporting the premise that the Hsp72-TLR2-MyD88 pathway is vital in our model. In our opinion, this clearly excludes concerns about a potential effect of mycoplasma contamination because the Hsp72 status alone would not be a determinant otherwise. n nIn summary, we propose that the discrepancy identified by Xiang et al1 between C-exo and P-exo may actually rely on the presence of tumor exosome–associated PGE2 produced by tumor or contaminant cells, which bypass the Hsp72-TLR2-MyD88 signal.

Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3 Deacetylation

Sirtuin-1 deacetylates proteins and has emerged asxa0axa0critical regulator of different cellular processes, particularly inflammation. Basal SIRT1 activity was previously found to limit Th9 and enhance Th17 differentiation in mice, but the effect of pharmacological SIRT1 activation on Txa0cell differentiation and antitumor responses remains unclear. Here, we find that SIRT1 pharmacological agonists selectively impede mouse and human Th17 cell differentiation. SIRT1xa0activation induces STAT3 deacetylation, thus reducing its ability to translocate into the nucleus, bind to Rorc promoter, and induce its transcription. SIRT1 agonists reduce tumor growth in mice by blocking Th17 cell differentiation. In cancer patients, the SIRT1 agonist metformin reduced the frequency of Th17 cells and STAT3 acetylation levels. Altogether, these data underscore that SIRT1 activation impedes Th17 cell differentiation and thereby limits tumor growth and suggest that SIRT1 activators may directly target IL-17A functions.

Identification of biological markers of liver X receptor (LXR) activation at the cell surface of human monocytes.

Background Liver X receptor (LXR) α and LXR β (NR1H3 and NR1H2) are oxysterol-activated nuclear receptors involved in the control of major metabolic pathways such as cholesterol homeostasis, lipogenesis, inflammation and innate immunity. Synthetic LXR agonists are currently under development and could find applications in various fields such as cardiovascular diseases, cancer, diabetes and neurodegenerative diseases. The clinical development of LXR agonists requires the identification of biological markers for pharmacodynamic studies. In this context, monocytes represent an attractive target to monitor LXR activation. They are easily accessible cells present in peripheral blood; they express LXR α and β and respond to LXR agonist stimulation in vitro. The aim of our study was to identify cell surface markers of LXR agonists on monocytes. For this, we focused on clusters of differentiation (CD) markers because they are well characterized and accessible cell surface molecules allowing easy immuno-phenotyping. Methodology/Principal Findings By using microarray analysis of monocytes treated or not with an LXR agonist in vitro, we selected three CD, i.e. CD82, CD226, CD244 for further analysis by real time PCR and flow cytometry. The three CD were up-regulated by LXR agonist treatment in vitro in a time- and dose- dependent manner and this induction was LXR specific as assessed by a SiRNA or LXR antagonist strategy. By using flow cytometry, we could demonstrate that the expression of these molecules at the cell surface of monocytes was significantly increased after LXR agonist treatment. Conclusions/Significance We have identified three new cell surface markers that could be useful to monitor LXR activation. Future studies will be required to confirm the biological and diagnostic significance of the markers.

Induction of pyroptosis in colon cancer cells by LXRβ.

Liver X receptors (LXRs) have been proposed to have some anticancer properties. We recently identified a new non-genomic role of LXRβ in colon cancer cells. Under LXR agonist treatment, LXRβ induces pyroptosis of these cells in vitro and in vivo, raising the possibility of targeting this isoform in cancer treatment.