Rôles immunomodulateurs des MDSC Tie-2+ et de la radiochimiothérapie dans les cancers bronchiques non à petites cellules

Monoclonal antibodies targeting immune checkpoints (ICPs) have revolutionized the management of cancers. However, these immunostimulatory therapies are effective in a small proportion of patients.One of the major obstacles to their effectiveness is a tumor microenvironment hostile to the effectors of antitumor immunity, especially T cells. A better understanding of anti-tumor immunity and mechanisms driving resistance to ICP blockade are required to sensitize patients to these promising therapies. Here, we studied a subpopulation of immunosuppressive myeloid cells (MDSC) which is likely to play a major role in this resistance. In the second part of this work, we analyzed the effect of concomitant chemoradiation (RTCT) on the tumor immune microenvironment and wondered how it could counteract with resistance to ICP blockade.First, we showed in a cohort of Non-Small Cell Lung Cancer patients (NSCLC) the expansion of monocytic MDSC populations (M-MDSC) overexpressing Tie-2, a tyrosine kinase receptor which binds the pro-angiogenic factor angiopoietin-2 (Ang-2). A positive correlation has been measured between percentages of Tie2+ M-MDSC and Ang-2 serum level. Furthermore, the presence of a strong Tie2+ M-MDSC/Ang-2 signature was associated with a decrease or loss of T cells responses against NY-ESO1, Telomerase and WT-1, whereas anti-viruses T cell responses were preserved. Indeed, we have demonstrated that Ang-2 increased Tie2+ M-MDSC suppressive activity. In addition, a high Ang-2/ Tie-2+ M-MDSC signature was associated with suppressive cytokines such as VEGF and TGF-β and the accumulation of circulating regulatory T cells. Our results could therefore explain the poor prognosis associated with a strong Ang-2/ Tie-2+ M-MDSC signature in our cohort. Thus, Tie-2 could be used as a phenotypic marker to study suppressive M-MDSC. So, our study open new prospects for the evaluation of Ang-2/ Tie-2+ M-MDSC as a mechanism involved in ICP blockade resistance in NSCLC.In the second part of this project, we used a transcriptomic and immunological approach in TC1 and CT26 mouse tumor models to analyze the effects of chemotherapy (CT), radiotherapy (RT) or concomitant chemoradiation (RTCT) on the immune cells of mice tumor microenvironment (MET). Transcriptomic data demonstrated the superiority of RTCT in stimulating pathways associated with adaptive anti-tumor immunity, dendritic cell activation and antigen cross-presentation within the MET. The strong pro-inflammatory signature induced by RTCT resulted in massive infiltration of cytotoxic CD8 TILs, NKs and also MDSCs. Furthermore, we have shown in anti-tumor T cells an overexpression of CTLA-4, PD-1 and TIM-3 receptors, which are involved in T lymphocytes activation/exhaustion. Combination of RTCT with CTLA-4/PD-1 double blockade (IO combo) conferred a strong anti-tumor protection which was higher than those observed with CT or RT + IO combo. Indeed, complete responses were observed in more than 80% of mice treated with RTCT+ IO combo, whereas only 37% and 0% of complete responses were measured in CT+ IO combo and RT+ IO combo groups respectively. In addition, RTCT+ IO combo triggered a long-term anti-tumor memory response which protected mice more than 100 days after the end of the therapy. Thus, our results demonstrate that RTCT triggers a highly inflammatory MET, suitable for the action of immune checkpoint blockade and encourage the clinical evaluation of RTCT+ IO combo.To conclude, our results provide two potential strategies to overcome primary resistance to ICPs blockade in NSCLC: Tie-2+M-MDSC/Ang-2 axis targeting and the use of chemoradiation in order to turn a hostile MET into an inflammatory MET, suitable for immune checkpoint blockade effectiveness.Les anticorps ciblant les immune checkpoints (ICP) ont révolutionné la prise en charge des cancers. Cependant, ces thérapies ne sont efficaces que chez une faible proportion de patients. Un des obstacles à leur efficacité est un microenvironnement tumoral hostile aux lymphocytes T. Afin de sensibiliser une majorité de patients à ces nouvelles immunothérapies, une meilleure compréhension de la régulation de l’immunité anti-tumorale et des mécanismes de résistance est nécessaire. Dans ce projet de thèse, nous avons étudié une sous-population de cellules myéloïdes immunosuppressives (MDSC) susceptible de jouer un rôle majeur dans cette résistance. Dans la seconde partie de ce travail, nous avons analysé l’effet de la combinaison radiochimiothérapie sur le microenvironnement immunitaire tumoral et étudié comment elle contribue à contrecarrer les mécanismes de résistance aux anti-ICP.Dans ce travail, nous avons mis en évidence dans une cohorte de patients atteints de cancers bronchiques, l’expansion de populations de MDSC monocytaires (M-MDSC) surexprimant Tie-2, un récepteur à tyrosine kinase qui lie le facteur pro-angiogénique angiopoiétine-2 (Ang-2). Une corrélation positive a été observée entre le pourcentage de M-MDSC-Tie2+ et le taux sérique d’Ang-2. La présence d’une forte signature MDSC-Tie2+/Ang-2 était associée à une diminution voire une perte des réponses T dirigées contre des antigènes tumoraux (NY-ESO-1, télomérase et WT-1). En effet, nous avons montré que l’Ang-2 augmentait l’activité suppressive des MDSC via le récepteur Tie-2. De plus, un environnement riche en Ang-2/MDSC-Tie-2+ favorise l’accumulation de cytokines immunosuppressives telles que le VEGF et le TGF-b. Ces résultats pourraient donc expliquer le mauvais pronostic associé à la forte signature Ang-2/MDSC Tie-2+. Ainsi Tie-2 pourrait être un marqueur phénotypique caractéristique des MDSC suppressives. Ces résultats ouvrent des perspectives pour évaluer l’axe Ang-2/MDSC Tie-2+ comme mécanisme impliqué dans la résistance aux anti-ICP.Dans la seconde partie, nous avons utilisé dans deux modèles de tumeurs murines (TC1 et CT26), une approche transcriptomique et immunologique pour analyser les effets de la chimiothérapie (CT), de la radiothérapie (RT) ou de la combinaison des deux : la radiochimiothérapie (RTCT) sur les cellules immunitaires du micro environnement tumoral (MET). Les données transcriptomiques ont monté la supériorité de la RTCT à stimuler au sein du MET les voies associées à l’immunité antitumorale à médiation cellulaire, l’activation des cellules dendritiques et la présentation croisée de l’antigène. La forte expression d’une signature pro inflammatoire induite par la RTCT s’est traduite par une infiltration massive du MET par des TILs CD8+ cytotoxiques, des NK mais aussi des MDSC. Nous avons montré sur les TIL CD8+ une surexpression de CTLA-4, PD-1, et TIM-3, des récepteurs impliqués dans l’activation/épuisement des lymphocytes T. La combinaison de la RTCT avec le double blocage de CTLA-4/ PD1a conféré une protection tumorale largement supérieure à celle observée avec la CT ou la RT, puisque des réponses complètes ont été observées chez plus de 80% des souris du groupe RTCT+ anti-CTLA-4/ PD1 versus 37% et 0% dans le groupe CT+ CTLA-4/ PD1 et RT + CTLA-4/ PD1 respectivement. La combinaison RTCT+ anti-CTLA-4/ PD1 a induit une réponse mémoire T antitumorale protectrice, plus de 100 jours après le traitement. Ainsi nos résultats démontrent que la RTCT crée un MET inflammatoire très favorable à l’action des anti-ICP et constituent un argument solide pour son évaluation en clinique.En conclusion nous avons mis en évidence deux stratégies potentielles permettant de lutter contre la résistance aux thérapies ciblant les immune checkpoints dans les cancers bronchiques: le ciblage de l’axe MDSC Tie-2+/Ang-2 et l’utilisation de la RTCT afin de créer un microenvironnement immunitaire propice à l’action des immunothérapies

Immunomodulatory roles of Tie-2+ MDSCs and concomitant chemoradiation in Non-Small Cell Lung Cancers

Les anticorps ciblant les immune checkpoints (ICP) ont révolutionné la prise en charge des cancers. Cependant, ces thérapies ne sont efficaces que chez une faible proportion de patients. Un des obstacles à leur efficacité est un microenvironnement tumoral hostile aux lymphocytes T. Afin de sensibiliser une majorité de patients à ces nouvelles immunothérapies, une meilleure compréhension de la régulation de l’immunité anti-tumorale et des mécanismes de résistance est nécessaire. Dans ce projet de thèse, nous avons étudié une sous-population de cellules myéloïdes immunosuppressives (MDSC) susceptible de jouer un rôle majeur dans cette résistance. Dans la seconde partie de ce travail, nous avons analysé l’effet de la combinaison radiochimiothérapie sur le microenvironnement immunitaire tumoral et étudié comment elle contribue à contrecarrer les mécanismes de résistance aux anti-ICP.Dans ce travail, nous avons mis en évidence dans une cohorte de patients atteints de cancers bronchiques, l’expansion de populations de MDSC monocytaires (M-MDSC) surexprimant Tie-2, un récepteur à tyrosine kinase qui lie le facteur pro-angiogénique angiopoiétine-2 (Ang-2). Une corrélation positive a été observée entre le pourcentage de M-MDSC-Tie2+ et le taux sérique d’Ang-2. La présence d’une forte signature MDSC-Tie2+/Ang-2 était associée à une diminution voire une perte des réponses T dirigées contre des antigènes tumoraux (NY-ESO-1, télomérase et WT-1). En effet, nous avons montré que l’Ang-2 augmentait l’activité suppressive des MDSC via le récepteur Tie-2. De plus, un environnement riche en Ang-2/MDSC-Tie-2+ favorise l’accumulation de cytokines immunosuppressives telles que le VEGF et le TGF-b. Ces résultats pourraient donc expliquer le mauvais pronostic associé à la forte signature Ang-2/MDSC Tie-2+. Ainsi Tie-2 pourrait être un marqueur phénotypique caractéristique des MDSC suppressives. Ces résultats ouvrent des perspectives pour évaluer l’axe Ang-2/MDSC Tie-2+ comme mécanisme impliqué dans la résistance aux anti-ICP.Dans la seconde partie, nous avons utilisé dans deux modèles de tumeurs murines (TC1 et CT26), une approche transcriptomique et immunologique pour analyser les effets de la chimiothérapie (CT), de la radiothérapie (RT) ou de la combinaison des deux : la radiochimiothérapie (RTCT) sur les cellules immunitaires du micro environnement tumoral (MET). Les données transcriptomiques ont monté la supériorité de la RTCT à stimuler au sein du MET les voies associées à l’immunité antitumorale à médiation cellulaire, l’activation des cellules dendritiques et la présentation croisée de l’antigène. La forte expression d’une signature pro inflammatoire induite par la RTCT s’est traduite par une infiltration massive du MET par des TILs CD8+ cytotoxiques, des NK mais aussi des MDSC. Nous avons montré sur les TIL CD8+ une surexpression de CTLA-4, PD-1, et TIM-3, des récepteurs impliqués dans l’activation/épuisement des lymphocytes T. La combinaison de la RTCT avec le double blocage de CTLA-4/ PD1a conféré une protection tumorale largement supérieure à celle observée avec la CT ou la RT, puisque des réponses complètes ont été observées chez plus de 80% des souris du groupe RTCT+ anti-CTLA-4/ PD1 versus 37% et 0% dans le groupe CT+ CTLA-4/ PD1 et RT + CTLA-4/ PD1 respectivement. La combinaison RTCT+ anti-CTLA-4/ PD1 a induit une réponse mémoire T antitumorale protectrice, plus de 100 jours après le traitement. Ainsi nos résultats démontrent que la RTCT crée un MET inflammatoire très favorable à l’action des anti-ICP et constituent un argument solide pour son évaluation en clinique.En conclusion nous avons mis en évidence deux stratégies potentielles permettant de lutter contre la résistance aux thérapies ciblant les immune checkpoints dans les cancers bronchiques: le ciblage de l’axe MDSC Tie-2+/Ang-2 et l’utilisation de la RTCT afin de créer un microenvironnement immunitaire propice à l’action des immunothérapies.Monoclonal antibodies targeting immune checkpoints (ICPs) have revolutionized the management of cancers. However, these immunostimulatory therapies are effective in a small proportion of patients.One of the major obstacles to their effectiveness is a tumor microenvironment hostile to the effectors of antitumor immunity, especially T cells. A better understanding of anti-tumor immunity and mechanisms driving resistance to ICP blockade are required to sensitize patients to these promising therapies. Here, we studied a subpopulation of immunosuppressive myeloid cells (MDSC) which is likely to play a major role in this resistance. In the second part of this work, we analyzed the effect of concomitant chemoradiation (RTCT) on the tumor immune microenvironment and wondered how it could counteract with resistance to ICP blockade.First, we showed in a cohort of Non-Small Cell Lung Cancer patients (NSCLC) the expansion of monocytic MDSC populations (M-MDSC) overexpressing Tie-2, a tyrosine kinase receptor which binds the pro-angiogenic factor angiopoietin-2 (Ang-2). A positive correlation has been measured between percentages of Tie2+ M-MDSC and Ang-2 serum level. Furthermore, the presence of a strong Tie2+ M-MDSC/Ang-2 signature was associated with a decrease or loss of T cells responses against NY-ESO1, Telomerase and WT-1, whereas anti-viruses T cell responses were preserved. Indeed, we have demonstrated that Ang-2 increased Tie2+ M-MDSC suppressive activity. In addition, a high Ang-2/ Tie-2+ M-MDSC signature was associated with suppressive cytokines such as VEGF and TGF-β and the accumulation of circulating regulatory T cells. Our results could therefore explain the poor prognosis associated with a strong Ang-2/ Tie-2+ M-MDSC signature in our cohort. Thus, Tie-2 could be used as a phenotypic marker to study suppressive M-MDSC. So, our study open new prospects for the evaluation of Ang-2/ Tie-2+ M-MDSC as a mechanism involved in ICP blockade resistance in NSCLC.In the second part of this project, we used a transcriptomic and immunological approach in TC1 and CT26 mouse tumor models to analyze the effects of chemotherapy (CT), radiotherapy (RT) or concomitant chemoradiation (RTCT) on the immune cells of mice tumor microenvironment (MET). Transcriptomic data demonstrated the superiority of RTCT in stimulating pathways associated with adaptive anti-tumor immunity, dendritic cell activation and antigen cross-presentation within the MET. The strong pro-inflammatory signature induced by RTCT resulted in massive infiltration of cytotoxic CD8 TILs, NKs and also MDSCs. Furthermore, we have shown in anti-tumor T cells an overexpression of CTLA-4, PD-1 and TIM-3 receptors, which are involved in T lymphocytes activation/exhaustion. Combination of RTCT with CTLA-4/PD-1 double blockade (IO combo) conferred a strong anti-tumor protection which was higher than those observed with CT or RT + IO combo. Indeed, complete responses were observed in more than 80% of mice treated with RTCT+ IO combo, whereas only 37% and 0% of complete responses were measured in CT+ IO combo and RT+ IO combo groups respectively. In addition, RTCT+ IO combo triggered a long-term anti-tumor memory response which protected mice more than 100 days after the end of the therapy. Thus, our results demonstrate that RTCT triggers a highly inflammatory MET, suitable for the action of immune checkpoint blockade and encourage the clinical evaluation of RTCT+ IO combo.To conclude, our results provide two potential strategies to overcome primary resistance to ICPs blockade in NSCLC: Tie-2+M-MDSC/Ang-2 axis targeting and the use of chemoradiation in order to turn a hostile MET into an inflammatory MET, suitable for immune checkpoint blockade effectiveness

Modulation of Determinant Factors to Improve Therapeutic Combinations with Immune Checkpoint Inhibitors

Immune checkpoint inhibitors (ICPi) have shown their superiority over conventional therapies to treat some cancers. ICPi are effective against immunogenic tumors. However, patients with tumors poorly infiltrated with immune cells do not respond to ICPi. Combining ICPi with other anticancer therapies such as chemotherapy, radiation, or vaccines, which can stimulate the immune system and recruit antitumor T cells into the tumor bed, may be a relevant strategy to increase the proportion of responding patients. Such an approach still raises the following questions: What are the immunological features modulated by immunogenic therapies that can be critical to ensure not only immediate but also long-lasting tumor protection? How must the combined treatments be administered to the patients to harness their full potential while limiting adverse immunological events? Here, we address these points by reviewing how immunogenic anticancer therapies can provide novel therapeutic opportunities upon combination with ICPi. We discuss their ability to create a permissive tumor microenvironment through the generation of inflamed tumors and stimulation of memory T cells such as resident (TRM) and stem-cell like (TSCM) cells. We eventually underscore the importance of sequence, dose, and duration of the combined anticancer therapies to design optimal and successful cancer immunotherapy strategies

CIITA-transduced glioblastoma cells uncover a rich repertoire of clinically relevant tumor-associated HLA-II antigens

CD4+ T cell responses are crucial for inducing and maintaining effective anti-cancer immunity, and the identification of human leukocyte antigen class II (HLA-II) cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of the class II major histocompatibility complex transactivator (CIITA) coupled to a detailed and sensitive mass spectrometry based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17 and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared to interferon gamma (IFN&3) treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor-antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but also such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire

PD-1/PD-L1 pathway: an adaptive immune resistance mechanism to immunogenic chemotherapy in colorectal cancer

International audienc

Chemoradiation triggers antitumor Th1 and tissue resident memory-polarized immune responses to improve immune checkpoint inhibitors therapy

BACKGROUND: Multiple synergistic combination approaches with cancer drugs are developed to overcome primary resistance to immunotherapy; however, the mechanistic rationale to combine chemoradiotherapy (CRT) with immune checkpoint inhibitors remains elusive. METHODS: This study described the immunological landscape of tumor microenvironment (TME) exposed to CRT. Tumor samples from patients with rectal cancer (n=43) treated with neoadjuvant CRT or radiotherapy were analyzed by nanostring and immunohistochemistry. Studies in mice were performed using three syngeneic tumors (TC1, CT26 and MC38). Tumor-bearing mice were treated either with platinum-based CRT, radiotherapy or chemotherapy. Anti-CTLA-4 and/or anti-Programmed Cell Death Receptor-1 (PD-1) therapy was used in combination with CRT. The therapy-exposed TME was screened by RNA sequencing and flow cytometry and tumor-infiltrating T lymphocyte functionality was evaluated by interferon (IFN)-γ ELIspot and intracellular cytokine staining. RESULTS: Front-to-front comparison analysis revealed the synergistic effect of CRT to establish a highly inflamed and Th1-polarized immune signature in the TME of patients and mice. In both settings, CRT-exposed TMEs were highly enriched in newly-infiltrated tumor-specific CD8+ T cells as well as tissue resident memory CD103+CD8+ T cells. In mice, CD8 T cells were involved in the antitumor response mediated by CRT and were primed by CRT-activated CD103+ dendritic cells. In the three tumor models, we showed that concurrent combination of CRT with a dual CTLA-4 and PD-1 blockade was required to achieve an optimal antitumor effect and to establish a broad and long-lasting protective antitumor T cell immunity. CONCLUSIONS: Our results highlight the ability of CRT to stimulate strong antitumor T-cell-mediated immunity and tissue resident memory T activation in TME, to foster immune checkpoint inhibitors action. These findings have implications in clinic for the design clinical trials combining chemoradiation with immunotherapy

Rapalogs Efficacy Relies on the Modulation of Antitumor T-cell Immunity

International audienceThe rapalogs everolimus and temsirolimus that inhibit mTOR signaling are used as antiproliferative drugs in several cancers. Here we investigated the influence of rapalogs-mediated immune modulation on their antitumor efficacy. Studies in metastatic renal cell carcinoma patients showed that everolimus promoted high expansion of FoxP3 (+)Helios(+)Ki67(+) regulatory CD4 T cells (Tregs). In these patients, rapalogs strongly enhanced the suppressive functions of Tregs, mainly in a contact-dependent manner. Paradoxically, a concurrent activation of spontaneous tumor-specific Th1 immunity also occurred. Furthermore, a high rate of Eomes(+)CD8(+) T cells was detected in patients after a long-term mTOR inhibition. We found that early changes in the Tregs/antitumor Th1 balance can differentially shape the treatment efficacy. Patients presenting a shift toward decreased Tregs levels and high expansion of antitumor Th1 cells showed better clinical responses. Studies conducted in tumor-bearing mice confirmed the deleterious effect of rapalogs-induced Tregs via a mechanism involving the inhibition of antitumor T-cell immunity. Consequently, the combination of temsirolimus plus CCR4 antagonist, a receptor highly expressed on rapalogs-exposed Tregs, was more effective than monotherapy. Altogether, our results describe for the first time a dual impact of host adaptive antitumor T-cell immunity on the clinical effectiveness of rapalogs and prompt their association with immunotherapies. Cancer Res; 76(14); 4100-12. ©2016 AACR