Tim-3 and PD-1 are powerful immunoinhibitory molecules involved in immune tolerance, autoimmune responses, and antitumor or antiviral immune evasion. A current model for Tim-3 regulation during immune responses suggests a divergent function, such that Tim-3 acts synergistically with TLR signaling pathways in innate immune cells to promote inflammation, yet the same molecule terminates Th1 immunity in adaptive immune cells. To better understand how Tim-3 might be functioning in innate immune responses, we examined the kinetics of Tim-3 expression in human CD14 + M/M 4 in relation to expression of IL-12, a key cytokine in the transition of innate to adaptive immunity. Here, we show that Tim-3 is constitutively expressed on unstimulated peripheral blood CD14 + monocytes but decreases rapidly upon TLR stimulation. Conversely, IL-12 expression is low in these cells but increases rapidly in CD14 + M/M.J, in correlation with the decrease in Tim-3. Blocking Tim-3 signaling or silencing Tim-3 expression led to a significant increase in TLR-mediated IL-12 production, as well as a decrease in activation-induced up-regula-tion of the immunoinhibitor, PD-1; TNF-a production was not altered significantly, but IL-10 production was increased. These results suggest that Tim-3 has a role as a regulator of pro- and anti-inflammatory innate immune responses.

PD-1 (Programmed Cell death protein -1) est un récepteur co-inhibiteur exprimé à la surface de lymphocytes T (LT) activés. Ce récepteur joue un rôle important dans le maintien de la tolérance périphérique tout en protégeant contre les maladies auto-immunes et inflammatoires. Cependant, une expression élevée et permanente de PD-1 et ses ligands PD-L1 et PD-L2 (PD-Ls) perturbe la réponse immunitaire contre les pathogènes et les cellules tumorales. Les inhibiteurs de points de contrôle immunitaires (ICI) ciblant l'axe PD-1/PD-Ls représentent aujourd’hui une avancé majeure dans le traitement de différents types de cancer, tant sur le plan de l’efficacité que de la tolérance. Le nivolumab (nivo) et le pembrolizumab (pembro) sont deux anticorps monoclonaux (AcM) anti-PD-1 qui bloquent l’interaction de PD-1 avec ses ligands. Ces AcM ont montré des résultats prometteurs dans le traitement de multiples types de cancers comme le mélanome, le cancer bronchique non à petites cellules, le carcinome à cellules rénales, etc. Malgré l’importance thérapeutique de nivo et de pembro dans le cancer, aucune étude n’a défini le devenir de PD-1 après la liaison à ces deux AcM. L’objectif de cette étude a été donc d’évaluer l’endocytose de PD-1 suite au liaison à des AcM anti-PD-1 (clone J110, nivo, pembro) et de déterminer s’il y a différence entre nivo et pembro vis à vis leur capacités à induire l'internalisation de PD-1. L’étude de l’endocytose de PD-1 a été réalisé sur des LT humains obtenus à partir du sang périphérique de donneurs sains et activés avec des Ac anti-CD3/anti-CD28 ou concanavaline A afin d’exprimer le récepteur PD-1. L’analyse des données par cytométrie en flux a montré que l’engagement de PD-1 avec l’Ac anti-PD-1 (clone J110) induit son endocytose dans les LT humains et que 50% de la totalité de PD-1 de surface est internalisé dans les premiers 30 minutes suivant l’incubation de cellules à 37°C, suivi d’un taux d’endocytose plus lent (56% en 60min). Notre étude a montré également que la liaison de nivo et de pembro au PD-1 induit son endocytose et que la plupart de récepteur est internalisée dans les 30 min suivant l’incubation de cellules à 37°C. Toutefois, 32 à 50% des récepteurs sont résistants à l’endocytose. L’analyse comparative de nivo et de pembro a révélé une différence statistiquement significative (p=0.03) entre le taux d’internalisation du complexe PD-1/nivo et celui du PD-1/pembro (46% versus 25% en 30min, respectivement). Même à des concentrations élevées de pembro, la liaison de nivo induit une meilleure internalisation de PD-1, ce qui suggère que nivo pourrait être plus efficace. Bien que les ICI comme nivo et pembro sont connus de bloquer l’interaction de PD-1 avec ses ligands, PD-L1 et PD-L2, notre étude a montré pour la première fois que ce deux AcM induisent aussi l’endocytose du récepteur PD-1 dans les LT humains avec des taux différents, et que 32% à 50% de récepteurs PD-1 sont résistants à l’endocytose. Ces résultats pourraient être exploités pour améliorer l’internalisation de PD-1 dans les LT humains et par la suite augmenter les potentiels thérapeutiques de nivolumab et de pembrolizumab dans le traitement du cancer. Mots clés : Récepteur PD-1, Ligands de PD-1, Lymphocytes T, Inhibiteurs de point de contrôle immunitaires, Anticorps anti-PD-1, Nivolumab, Pembrolizumab, Endocytose, Cancer / PD-1 (Programmed Cell death protein -1) is a co-inhibitory receptor expressed on the surface of activated T cells. It plays an important role in maintaining peripheral tolerance and protecting against autoimmune and inflammatory diseases. However, permanent expression of PD-1 and its ligands PD-L1/ PD-L2 (PD-Ls) disrupts the immune response against pathogens and tumor cells. Immune checkpoint blockade (ICB) targeting the PD-1/PD-Ls axis has revolutionized the treatment of many cancers. Nivolumab (nivo) and pembrolizumab (pembro) are two anti-PD-1 monoclonal antibodies (mAb) that block the interaction between PD-1 and its ligands. They have shown promising results in the treatment of multiple types of cancers such as melanoma, non-small cell lung cancer, renal cell carcinoma, etc. Surprisingly, despite the success of anti-PD-1 in cancer immunotherapy, no-one has defined the destiny of surface PD-1 following antibody binding. Therefore, the objective of my master thesis was to define the fate of surface PD-1 following antibody binding and whether different anti-PD-1 Abs in the clinic differ in their ability to induce PD-1 endocytosis. The study of PD-1 endocytosis was performed on human T lymphocytes obtained from peripheral blood of healthy donors and activated with anti-CD3/anti-CD28 Ab or concanavalin A to express PD-1 receptor. Data analysis by flow cytometry showed that following anti-PD-1 Ab binding, 50% of PD-1 becomes endocytosed by 30min. In addition, we found that the PD-1 receptor is internalised upon its engagement with nivo and pembro and that most of the receptor is endocytosed within 30 min. However, 32 to 50% of the receptors are resistant to endocytosis. The comparative analysis of nivo and pembro has revealed a statistically significant difference (p=0.03) between the internalisation rate of the PD-1/nivo complex versus PD-1/pembro (46% versus 25% by 30min, respectively). Even at high concentrations of pembro, nivo induces better internalization of PD-1, suggesting that nivo could be more effective than pembro. Our study showed for the first time that ICB involves not only in the blockade of PD-1/PD-Ls interaction, but also in the endocytosis of PD-1 receptors from the surface of human T-cells, which differs between nivolumab and pembrolizumab. These results could be exploited to increase the therapeutic potential of nivolumab and pembrolizumab in cancer treatment. Keywords: PD-1 receptor, PD-1 ligands, T lymphocytes, Immune checkpoint blockade, Anti-PD1 antibodies, Nivolumab, Pembrolizumab, Endocytosis, Cancer

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19594号 / 医博第4101号 / 新制||医||1014(附属図書館) / 32630 / 京都大学大学院医学研究科医学専攻 / (主査)教授 竹内 理, 教授 河本 宏, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20254号 / 医博第4213号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 川上 浩司, 教授 生田 宏一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21637号 / 医博第4443号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 松田 道行, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Modulação da resposta imune no local do tumor é um mecanismo crítico envolvido com evasão dos tumores. Sinais de PD-1 e PD-L1/PD-L2 podem estar envolvidos com o escape tumoral. Entretanto, pouco se sabe a respeito destas moléculas no desenvolvimento de carcinoma de células escamosas. No presente estudo, nós investigamos a expressão de PD-1 sobre células T das lesões e linfonodos de carcinomas de células escamosas. A carcinogênese de pele foi induzida quimicamente com DMBA/PMA em camundongos. A caracterização da expressão de PD-1, PD-L1 e PD-L2 na lesão e linfonodos foram analisados por citometria de fluxo. A produção IL-10, IL-12, TGF- e IFN- foi determinada por ELISA. Camundongos tratados com DMBA/PMA apresentaram maior taxa de papilomas e progressão para carcinoma de células escamosas. Nós encontramos também que células T CD4+PD-1+ migraram para o local do tumor e que maior nível de CD4+PD-1+, CD8+PD-1+, CD14+PD-1+ e CD4+PD-L1+ foram detectados nos linfonodos quando comparados com o grupo controle. Além disso, as produções de IL-12, IFN- e TGF-, mas não IL-10, foram altamente detectadas nas lesões comparada com do grupo controle. Estes dados indicam que a expressão de PD-1 está regulada positivamente sobre linfócitos infiltrados nos tumores e linfonodos. Este fato e os altos níveis de TGF- e IL-10 podem contribuir para supressão da resposta imune antitumor. / Modulation of immune response in tumor site is a critical mechanism involved with tumor evasion. PD-1 and PD-L1/PD-L2 signals could be involved in tumor escape. However, little is known about the role of these molecules in squamous cell carcinoma development. In the present study, we investigated the expression of PD-1 on T cells from squamous cell carcinoma lesions and lymph nodes. Skin carcinogenesis was chemically induced with DMBA/PMA in mice. Characterization of PD-1, PD-L1 and PD-L2 expression in the lesion and lymph nodes were analysed by flow cytometry. IL-10, IL-12, TGF- and IFN- production was determined by ELISA. DMBA/PMA-treated mice showed higher rate of papillomas and progression to squamous cell carcinoma. We also found that CD4+PD-1+ T cells migrated to the tumor site and that higher level of CD4+PD-1+, CD8+PD-1+, CD14+PD-1+ and CD4+PD-L1+ were detected in the lymph nodes when compared with the control group. Besides, IL-12, IFN- and TGF-, but not IL-10, productions were higher detected in the lesions compared with the control group. These data indicate that PD-1 expression is up-regulated on tumor infiltrating lymphocytes and lymph nodes. This fact and high levels of TGF- and IL-10 may contribute to suppression of anti-tumor immune response.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2017-12-21T18:17:20Z No. of bitstreams: 1 julianamelo.pdf: 1321154 bytes, checksum: 6613c9596412757aeb47ee110faa5b87 (MD5) / Rejected by Adriana Oliveira (adriana.oliveira@ufjf.edu.br), reason: Favor corrigir autor: Sobrenome, Nome on 2017-12-22T12:19:42Z (GMT) / Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2017-12-22T12:28:52Z No. of bitstreams: 1 julianamelo.pdf: 1321154 bytes, checksum: 6613c9596412757aeb47ee110faa5b87 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-12-22T13:02:07Z (GMT) No. of bitstreams: 1 julianamelo.pdf: 1321154 bytes, checksum: 6613c9596412757aeb47ee110faa5b87 (MD5) / Made available in DSpace on 2017-12-22T13:02:07Z (GMT). No. of bitstreams: 1 julianamelo.pdf: 1321154 bytes, checksum: 6613c9596412757aeb47ee110faa5b87 (MD5) Previous issue date: 2017-02-22 / A brucelose é uma doença infectocontagiosa causada por bactérias do gênero Brucella que acometem o homem e uma grande variedade de animais domésticos, resultando em prejuízos econômicos significativos aos sistemas de produção. Em humanos essa infecção pode causar febre ondulante, endocardite, artrite, osteomielite e complicações neurológicas enquanto em animais leva ao aborto e infertilidade. Sabe-se que a resposta imunológica a bactérias intracelulares como a Brucella ocorre essencialmente através da imunidade mediada por células, sendo macrófagos especialmente importantes no combate à infecção. Entretanto, apesar da efetividade da resposta, a B. abortus conta com diversos mecanismos de evasão, o que garante a sua sobrevivência no organismo hospedeiro. Dentre estes mecanismos, a modulação de células apresentadoras de antígenos tem sido apontada como um dos mais relevantes. Recentemente, diversos trabalhos têm evidenciado a importância das NTPDases e da molécula PD-1 na modulação da resposta imune. As NTPDases estão envolidas com a produção de adenosina que apresenta relevante caráter imunomodulador. Já a molécula PD-1 está associada a indução de um perfil anti-inflamatório com diminuição de IL-12 e aumento de IL-10. Neste contexto, o objetivo deste estudo foi determinar se a infecção com B. abortus é capaz de alterar a expressão destas moléculas e assim limitar a ação do sistema imune, favorecendo a sobrevivência do patógeno. Para isso, células RAW 264.7 foram infectadas com B.abortus e a modulação das moléculas CD80, CD86, CD40, MHCII, foi avaliada por citometria de fluxo. Em seguida, a expressão de CD39 também foi determinada por citometria de fluxo e por Western Blot. Por fim analisamos possíveis alterações na expressão da molécula PD-1 induzidas pela bactéria. Como resultados, foi confirmado que a infecção é capaz de inibir a expressão de CD80, CD86 e CD40, embora o mesmo não tenha sido observado com o MHCII. Além disso, a expressão de CD40 se mostrou diminuída mesmo após o estímulo com LPS. De forma surpreendente, foi observada uma diminuição na expressão das moléculas CD39 e PD-1, o que pode ser explicado pela menor ativação celular induzida pela infecção. Assim, os dados obtidos até o momento demonstram que as moléculas CD39 e PD-1 não são utilizadas pela Brucella para modular as APCs, mas são influenciados pela menor ativação celular induzida pelo patógeno. / Brucellosis is an infectious disease caused by bacteria of the genus Brucella that affect humans and a wide variety of domestic animals, resulting in significant economic losses to production systems. In humans the infection can cause undulant fever, endocarditis, arthritis, osteomyelitis and neurological complications while in animals leads to abortion and infertility. It is known that the immune response to intracellular bacteria such as Brucella occurs primarily by cell-mediated immunity, macrophage being especially important in fighting infection. However, despite the effectiveness of the response, B. abortus has several avoidance schemes, which ensures their survival in the host organism. Among these mechanisms, modulation of antigen-presenting cells has been identified as one of the most relevant. Recently, several studies have shown the importance NTPDase and PD-1 molecule to modulate the immune response. The NTPDase are envolidas with the production of adenosine which presents immunomodulatory relevant character. Since PD-1 molecule is associated with induction of an anti-inflammatory profile with decreased IL-12 and IL-10 increase. In this context, the aim of this study was to determine whether infection with B. abortus is able to alter the expression of these molecules and thus limit the action of the immune system, favoring the survival of the pathogen. To this end, RAW 264.7 cells were infected with B.abortus and modulation of CD80 molecules, CD86, CD40, MHCII, was evaluated by flow cytometry. Then the CD39 expression was also determined by flow cytometry and Western blot. Finally, we analyze possible changes in PD-1 molecule expression induced by bacteria. As a result, it was confirmed that the infection is capable of inhibiting expression of CD80, CD86 and CD40, although this has not been observed with MHCII. Additionally, CD40 expression showed decreased even after the stimulation with LPS. Surprisingly, it was observed a decrease in the expression of CD39 and PD-1 molecules, which can be explained by the lower cellular activation induced by the infection. Thus, the data obtained so far show that the PD-1 and CD39 molecules are not used by Brucella Modular APCs but are less influenced by cellular activation induced by the pathogen.

Modulação da resposta imune no local do tumor é um mecanismo crítico envolvido com evasão dos tumores. Sinais de PD-1 e PD-L1/PD-L2 podem estar envolvidos com o escape tumoral. Entretanto, pouco se sabe a respeito destas moléculas no desenvolvimento de carcinoma de células escamosas. No presente estudo, nós investigamos a expressão de PD-1 sobre células T das lesões e linfonodos de carcinomas de células escamosas. A carcinogênese de pele foi induzida quimicamente com DMBA/PMA em camundongos. A caracterização da expressão de PD-1, PD-L1 e PD-L2 na lesão e linfonodos foram analisados por citometria de fluxo. A produção IL-10, IL-12, TGF- e IFN- foi determinada por ELISA. Camundongos tratados com DMBA/PMA apresentaram maior taxa de papilomas e progressão para carcinoma de células escamosas. Nós encontramos também que células T CD4+PD-1+ migraram para o local do tumor e que maior nível de CD4+PD-1+, CD8+PD-1+, CD14+PD-1+ e CD4+PD-L1+ foram detectados nos linfonodos quando comparados com o grupo controle. Além disso, as produções de IL-12, IFN- e TGF-, mas não IL-10, foram altamente detectadas nas lesões comparada com do grupo controle. Estes dados indicam que a expressão de PD-1 está regulada positivamente sobre linfócitos infiltrados nos tumores e linfonodos. Este fato e os altos níveis de TGF- e IL-10 podem contribuir para supressão da resposta imune antitumor. / Modulation of immune response in tumor site is a critical mechanism involved with tumor evasion. PD-1 and PD-L1/PD-L2 signals could be involved in tumor escape. However, little is known about the role of these molecules in squamous cell carcinoma development. In the present study, we investigated the expression of PD-1 on T cells from squamous cell carcinoma lesions and lymph nodes. Skin carcinogenesis was chemically induced with DMBA/PMA in mice. Characterization of PD-1, PD-L1 and PD-L2 expression in the lesion and lymph nodes were analysed by flow cytometry. IL-10, IL-12, TGF- and IFN- production was determined by ELISA. DMBA/PMA-treated mice showed higher rate of papillomas and progression to squamous cell carcinoma. We also found that CD4+PD-1+ T cells migrated to the tumor site and that higher level of CD4+PD-1+, CD8+PD-1+, CD14+PD-1+ and CD4+PD-L1+ were detected in the lymph nodes when compared with the control group. Besides, IL-12, IFN- and TGF-, but not IL-10, productions were higher detected in the lesions compared with the control group. These data indicate that PD-1 expression is up-regulated on tumor infiltrating lymphocytes and lymph nodes. This fact and high levels of TGF- and IL-10 may contribute to suppression of anti-tumor immune response.

La survie des patients atteints de cancer et traités avec des thérapies conventionnelles reste faible dans plusieurs types de tumeurs. Récemment, une nouvelle approche immunothérapeutique a été développée pour cibler le système immunitaire au lieu de la tumeur elle-même, afin de restaurer la fonctionnalité des cellules immunitaires et la destruction des cellules cancéreuses. L’immunothérapie ciblant le récepteur inhibiteur PD-1 occupe une place privilégiée dans les thérapies anticancéreuses en raison de sa haute spécificité et de sa faible toxicité par rapport aux thérapies conventionnelles. Cependant, le taux de réponse reste faible avec seulement 20 à 25% de patients répondant à une immunothérapie anti-PD-1. Il est donc important de comprendre les mécanismes associés à la résistance à ces thérapies et d’identifier des biomarqueurs prédictifs de réponse. L'expression du ligand de PD-1, PD-L1, sur les cellules tumorales, la charge mutationnelle tumorale et l'infiltration tumorale par les lymphocytes ont déjà été décrits, mais de nouveaux biomarqueurs sont nécessaires pour mieux déterminer la sous-population de patients susceptible de bénéficier de ces traitements. Au cours de ce travail, nous avons établi une cohorte de 118 patients atteints d'un cancer du poumon non à petites cellules (CBNPC) traités avec une immunothérapie anti-PD-1/PD-L1, et nous avons étudié l'expression de plusieurs biomarqueurs potentiels, en particulier les cellules T mémoires résidentes dans le tissu (TRM) CD8+CD103+. Ces cellules constituent un candidat potentiel car elles représentent une population privilégiée de lymphocytes T CD8 grâce à l’expression de PD-1 et une forte capacité cytotoxique vis-à-vis des cellules tumorales autologues suite à la neutralisation de l’interaction de PD-1 avec PD-L1. Nous montrons qu’une forte infiltration de tumeurs de CBNPC avec des cellules TRM corrèle à une survie sans progression plus élevée (PFS) et une réponse plus efficace à anti-PD-1 que les tumeurs avec une faible infiltration par des TRM. De plus, les tumeurs qui expriment fortement ICAM-1, un ligand de l’intégrine LFA-1 exprimée sur les lymphocytes T CD8, sont hautement infiltrées par des TRM. Par ailleurs, il est bien connu que le signal TGF-β est crucial pour l’induction de CD103 et la formation de TRM CD8+CD103+. Je me suis donc intéressée à l'activation du TGF-β par les intégrines αV exprimée par les cellules tumorales humaines et murines. À l'aide des modèles in vitro et in vivo, nous montrons que les cellules tumorales exprimant les intégrines αV activent le TGF-β et induisent l'expression de CD103 à la fois par les cellules T CD8+ provenant de cellules mononucléées du sang périphérique (PBMC) et de lymphocytes infiltrant la tumeur (TIL). L’expression plus faible de CD103 par les TIL CD8+ de souris greffées avec des tumeurs déficientes pour l’expression d'αV n'a pas d'effet sur le contrôle de la croissance tumorale. De manière intéressante, nous montrons dans des modèles de tumeurs déficientes pour l’expression d’αV, que le traitement avec des anticorps anti-PD-1 bloquants corrèle avec un meilleur contrôle de la croissance tumorale et une meilleure réponse à l'immunothérapie anti-PD-1 qui sont associés à une infiltration plus forte de TIL et un état d'activation plus élevé des TIL CD8+ exerçant une activité cytotoxique spécifique. De plus, une expression élevée de l'intégrine αV dans les tumeurs corrèle avec une réponse plus faible des patients atteints de CBNPC à une immunothérapie anti-PD-1/PD-L1. Ces données montrent comment trois marqueurs distincts, cellules TRM, ICAM-1 et les intégrines αV, régulent le microenvironnement tumoral et l’immunité T CD8 avec des implications potentielles pour potentialiser les réponses aux immunothérapies. / The survival of cancer patients treated with conventional therapies remains low in multiple cancers. Recently, a new immunotherapeutic approach has been developed to target the immune system instead of the tumor itself, in order to restore immune cell functions in cancer destruction. Immunotherapy targeting the T cell inhibitory receptor PD-1 occupies a privileged place in cancer therapy thanks to its high specificity and low toxicity compared to chemotherapies. However, the response rate remains low with only 20-25% of patients responding to anti-PD-1 immunotherapy. An important issue is therefore to understand the mechanisms associated with resistance to these therapies and to identify the predictive biomarkers of response. The expression of the PD-1 ligand, PD-L1, on tumor cells, tumor mutational burden (TMB) and tumor infiltration by lymphocytes have been described to predict the response to immune checkpoint blockade (ICB). However, new biomarkers are needed to better determine patient subpopulation which could benefit from this treatment. To address this question, we established a cohort of 118 non-small cell lung cancer (NSCLC) patients treated with anti-PD-1/PD-L1 immunotherapy and studied the expression of several potential biomarkers. Tissue-resident memory T (TRM) cells are a potential candidate because they represent a distinct population of CD8+ T cells highly expressing integrin αEβ7 (CD103) and PD-1; and showing strong cytotoxic capacity towards autologous tumor cells upon neutralisation of PD-1/PD-L1 interaction. Results from the present study show that high infiltration of TRM cells in NSCLC tumors correlates with higher progression-free survival (PFS) and a better response to anti-PD-1/PD-L1 immunotherapy. Moreover, tumors with high expression levels of ICAM-1, the ligand of integrin LFA-1 expressed on T cells, show higher TRM infiltration. TGF-β is a cytokine directly involved in CD103 induction on activated tumor-specific T cells. Therefore, I also investigated the role of αV integrins in activating TGF-β and thereby in controlling TRM differentiation and anti-tumor T cell immunity. Using human and mouse models, we show that tumor cells expressing αV integrins activate TGF-β, which can in turn induce expression of CD103 on CD8+ T cells in vitro on peripheral blood mononuclear cells (PBMCs) and in vivo on tumor infiltrating lymphocytes (TIL). However, lower CD103 expression on CD8+ TIL and thus CD103+ TRM cell formation in C57BL/6 mice engrafted with αV-lacking cancer cells had no effect on tumor growth control. Remarkably, αV-deficient tumors responded more effectively to anti-PD-1 immunotherapy than αV-efficient tumors and this response correlates with higher tumor infiltration by activated CD8+ T cells and stronger cytotoxic activity toward autologous cancer cells. Moreover, high expression of αV integrins in NSCLC tumors correlates with worse response to anti-PD-1/PD-L1 immunotherapy. These data show how three distinct markers, TRM cells, ICAM-1, and αV integrins regulate the tumor microenvironment and CD8+ T cell immunity, with potential implications in improving response to ICB immunotherapies.

Les antibiotiques (ATB) inhibent l'efficacité anti-tumorale du blocage de PD1, mais les mécanismes sous-jacents à leurs effets immunosuppresseurs demeurent inconnus. Nous montrons ici que les ATB favorisent l'accumulation des cellules T FoxP3+ et RORct+ a4b7hi dans les ganglions lymphatiques et les lésions tumorales. Les ATB induisent la perte de l'adressine MadCAM-1 iléale provoquant la recirculation des lymphocytes T régulateurs et TH17 α4β7hi de l’iléon vers le microenvironnement tumoral (TME). Cette migration a été visualisée grâce à 2 méthodes, la première consiste en l’ injection directe de carboxyfluoresceine succinimidyl ester (CFSE) dans les ganglions lymphatiques mésentériques des souris porteuses de tumeurs, l’autre utilise des souris transgéniques Kaede contenant une protéine flurorescente photoconvertible. Les hétérodimères d'adressine MAdCAM-1 et d'intégrine α4β7 sont indispensables pour l'efficacité anti-tumorale et dans l'immuno-surveillance induite par les anticorps anti-PD1. L’utilisation de modèles knock-out de MadCAM-1 ou d’ anticorps bloquant son ligand α4β7, compromettent l’efficacité anticancéreuse du blocage du PD1 ceci en mobilisant les cellules entérotropes α4β7hi Treg et TH17 vers le TME et inversement, en réduisant le retour du Treg du TME vers l'intestin. L’inhibition de la voie MadCAM-1 au niveau iléal réduit simultanément les lymphocytes CCR5+ effecteurs et mémoires dans le tissu tumoral. Ces résultats démontrent l’existence d’un lien mécanistique entre la dysbiose intestinale et l'efficacité du traitement anti- tumoral, l’importance de l'axe intestin-tumeur dans l’ immunosurveillance du cancer et ouvre des perspectives d’application clinique pour cibler la voie MAdCAM-1 α4β7+ . / Antibiotics (ATB) inhibit the anticancer efficacy of PD1 blockade but the mechanisms underlying their immunosuppressive effects remains unknown. Here we show that ATB promote the accumulation of enterotropic, ileum egressing FoxP3+ and RORct+ α4β7 hi T cells into tumor draining lymph nodes and tumor lesions. ATB induce the loss of ileal MadCAM-1 adressin provoking the recirculation of α4β7 hi ileal Treg and TH17 cells to the tumor microenvironment (TME), as visualized using direct injection of carboxyfluorescein succinimidyl ester in mesenteric lymph nodes of tumor bearers as well as Kaede transgenic mice harboring a photoconvertible flurorescent protein. MAdCAM-1 addressin and α4β7 integrin heterodimers are indispensable for the anticancer efficacy and the immuno-surveillanc elicited by anti-PD1 antibodies. Gene defects in MadCAM-1 as well as antibodies blocking its ligand α4β7, severely compromise the anticancer effects of PD1 blockade by mobilizing enterotropic α4β7hi Treg and TH17 cells towards the TME and conversely reducing Treg homing from the TME to the gut, concomitantly reducing CCR5+ effector memory tumor infiltrating lymphocytes. These findings demonstrate a mechanistic link between gut dysbiosis and tumor treatment efficacy and unveil the potential clinical relevance of the MAdCAM-1 α4β7+ and the gut-tumor axis in cancer immunosurveillance.