The ultimate goal of antitumor vaccines is to develop memory CD8+ cytotoxic T lymphocytes (CTLs), which are critical mediators of antitumor immunity. Previous work in our lab demonstrated that the ovalbumin (OVA)-specific CD4+ T cell-based (OVA-TEXO) vaccine generated using OVA-pulsed dendritic cell (DCOVA)-released exosomes (EXOOVA) stimulates CTL responses via interleukin (IL)-2 and costimulatory CD80 signaling. To assess the potential involvement of other costimulatory pathways and to define the key constituent of costimulation for memory CTL development, we first immunized wild-type (WT) C57BL/6 and gene-knockout mice with WT CD4+ OVA-TEXO cells or OVA-TEXO cells with various molecular deficiencies. We then assessed OVA-specific primary and recall CTL responses using PE-H-2Kb/OVA257–264 tetramer and FITC-anti-CD8 antibody staining by flow cytometry. We also examined antitumor immunity against the OVA-expressing B16 melanoma cell line BL6-10OVA. We demonstrate that CD4+ OVA-TEXO cells form immunological synapses with cognate CD8+ T cells in vitro. By assessment of the pattern of ex vivo interactions between OTI CD8+ T cells and OVA-TEXO or (Kb-/-)TEXO cells lacking peptide/major histocompatibitity complex (pMHC)-I expression, we provide the first visible evidence on the critical role of exosomal pMHC-I in targeting OVA-TEXO to cognate CD8+ T cells using two-photon microscopy. By assessing primary and recall CTL responses in mice immunized with OVA-TEXO cells or with OVA-TEXO cells lacking the costimulatory molecules CD40L, 4-1BBL or OX40L, we demonstrated that these costimulatory signals are dispensable for CTL priming by OVA-TEXO cells. Interestingly, CD40L, but not 4-1BBL or OX40L, plays a crucial role in the development of functional memory CTLs against BL6-10OVA tumors. Overall, this work suggests that a novel CD4+ T cell-based vaccine that is capable of stimulating long-term functional CTL memory via CD40L signaling may represent a novel, efficient approach to antitumor vaccination. Breast cancer is the most common cancer among women in the western world. Approximately 20-30% of invasive breast carcinomas are proto-oncogene human epidermal growth factor receptor (HER)-2 positive and associated with increased metastatic potential and poor prognosis. The survival benefit of anti-HER2 driven therapies demonstrated in clinical trials indicates that HER2 is one of the most promising molecules for targeted therapy to date. Above results prompt us to assess whether CD4+ T-cell-based vaccine can stimulate efficient HER2-specific CD8+ CTL responses and antitumor immunity in transgenic mice with HER2-specific self-immune tolerance. We prepared HER2-specific HER2-TEXO using ConA-stimulated CD4+ T cells with uptake of exosomes released from HER2-expressing AdVHER2-transfected DCs. We found that HER2-TEXO vaccine is capable of inducing HER2-specific CTL responses and protective immunity against transgene HLA-A2/HER2-expressing B16 melanoma BL6-10HLA-A2/HER2 in 2/8 double transgenic HLA-A2/HER2 mice with HER2-specific self-immune tolerance. The remaining 6/8 mice had significantly prolonged survival. Therefore, the novel T cell-based HER2-TEXO vaccine may provide a new therapeutic alternative for women with HER2+ breast cancer. In contrast to CD4+CD25+ regulatory T cells (Tregs), mechanisms of CD8+CD25+ Treg-mediated immunosuppression are not well understood. In this study, we purified polyclonal CD8+CD25+ Tregs from C57BL/6 mouse splenocytes and expanded them in culture medium containing CD3/CD28 microbeads. By using these amplified CD8+CD25+ Tregs, we demonstrated that CD8+CD25+ Tregs inhibit naive CD4+ T-cell proliferation and induce naive T-cell anergy by up-regulating T-cell anergy-associated early growth response 2 (EGR2), and by decreasing T-cell proliferation and IL-2-secretion upon stimulation. They also impact the expression of perforin on effector CTLs and directly induce perforin-mediated CTL apoptosis. CD8+CD25+ Tregs, when pulsed with OVA323-339 peptide, exert an enhanced inhibition. Interestingly, CD8+CD25+ Tregs, when pulsed with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide, become capable of inhibiting MOG35-55-induced experimental autoimmune encephalomyelitis (EAE). Two-photon microscopic observations suggest that OVA323-339-pulsed (armed) CD8+CD25+ Tregs reduce the interactions between DCs and cognate CD4+ T cells ex vivo by increasing velocities of T cells in mouse lymph nodes. Therefore, redirecting antigen-specificity to nonspecific CD8+CD25+ Tregs can be achieved for enhanced immunosuppression through their arming with the antigen-specific pMHC-II complexes. This approach may have great impact on improvement of endogenous polyclonal Treg-mediated immunotherapy for autoimmune diseases. Taken together, our studies demonstrate that nonspecific polyclonal CD4+ T cells and CD8+CD25+ Tregs, when armed with HER2 and MOG antigen-specific pMHC-I and -II complexes, become capable of stimulating enhanced HER2-specific CTL responses and antitumor immunity in double transgenic HLA-A2/HER2 mice and inducing enhanced MOG-specific immunosuppression in MOG-induced EAE mice, respectively. Therefore, redirecting antigen specificity to nonspecific CD4+ T and CD8+CD25+ Tregs by pMHC complex arming may have great impact in development of novel T cell-based vaccines for treatment of cancer and autoimmune diseases.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21302号 / 医博第4391号 / 新制||医||1030(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 生田 宏一, 教授 椛島 健治, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

MHCI molecules are constitutively expressed in all nucleated cells and play a key role in antigen presentation to CD8+ T lymphocytes. One of the tumor immune evasion strategies is MHCI expression downregulation. This leads to an impaired recognition of tumor antigens by CD8+ T lymphocytes that are unable to start the immune response. Since the MHCI expression downregulation occurs in up to 90 % of some tumors it is neccesary to have a clinical relevant tumor model without a MHCI surface expression that would be used for testing of immunotherapeutic approaches. This thesis describes a production of new model cell lines of TC-1 tumor cells with irreversibly downregulated MHCI. That was achieved by an inactivation of B2m, which is a part of MHCI, by gene editing using CRISR/Cas9. The B2m inactivation was confirmed by flow cytometry, western blot and sanger sequencing of single alleles. The inactivation slowed down the cell growth for both in vitro and in vivo. The cell metastatic activity was not affected. The tumors established by cells without the B2m expression are not sensitive to DNA vaccine against HPV16 E7 oncoprotein by a pBSC/PADRE.E7GGG vaccine. The main effector function against these tumors possess the NK1.1+ cells. In a therapeutic vaccination experiment it was repeatedly achieved of...

Les cellules dendritiques (DC) tiennent une place centrale dans l'initiation des réponses immunitaires et dans le contrôle du développement des tumeurs. La sous-population cDC1 suscite aujourd'hui en grand intérêt de par ses fonctions d'activation de réponses cytotoxiques par présentation croisée d'Ag aux lymphocytes T (LT) CD8+ ainsi que son implication dans l'immunité antitumorale et la réponse aux immunothérapies chez la souris. Le rôle des cDC1 chez l'Homme est cependant peu décrit. Les cDC1 murines et humaines sont aussi connues pour produire de larges quantités d'interféron (IFN) de type III (IFN-III), aussi appelés IFN-λ. Tout comme les IFN-I avec lesquels ils partagent la même voie de signalisation, les IFN-III ont un rôle antiviral bien décrit. Des modèles murins ont également suggéré un rôle antitumoral, mais ces IFN n'ont jamais été étudiés dans un contexte de cancer chez l'Homme. Il est donc crucial de comprendre les mécanismes expliquant l'impact pronostique positif des cDC1 ainsi que le rôle des IFN-III dans l'immunité antitumorale, en particulier pour le développement de nouvelles approches thérapeutiques. Nous avons démontré pour la première fois l'infiltration des tumeurs humaines de sein et d'ovaire par diverses sous-populations de DC. Les cDC1 sont particulièrement enrichies par rapport au sang des patientes et forment de nombreuses interactions avec les LT CD8+ dans les tumeurs. Une approche de bio-informatique a permis de révéler que les cDC1 représentent l'unique population de DC associée à une meilleure survie des patients dans la majorité des cancers du TCGA. De façon intéressante, la signature de réponses aux IFN-I et III est enrichie dans les tumeurs de sein fortement infiltrées par les cDC1 mais pas par les autres sous-populations. L'expression des gènes codant pour l'IFN-λ1 ou le récepteur aux IFN-III est également associée à une meilleure survie sans rechute dans le cancer du sein. De plus, nous avons démontré la capacité des cDC1 à produire de l'IFN-III sans aucune réactivation ex vivo. Ce résultat indique clairement que dans un contexte de réponse immunitaire antitumorale chez l'Homme, la synthèse d'IFN-III est une spécificité des cDC1 comparées aux autres sous-populations. La présence d'IFN-III dans les surnageants tumoraux a été confirmée au niveau protéique et démontrée comme étant fortement corrélée avec l'IL-12p40, les CXCR3-L, le CX3CL1 et le TNF-α. Ces données soulèvent alors l'hypothèse de l'association entre l'IFN-III, produit dans le microenvironnement tumoral par les cDC1, et la présence de cytokines et chimiokines impliquées dans le recrutement et l'activation de lymphocytes cytotoxiques tels que les LT CD8+ ou cellules NK. Notre étude apporte des informations détaillées quant à la nature des différentes sous-populations de DC infiltrant les tumeurs humaines de sein et d'ovaire et démontrent pour la première fois la production d'IFN-III par les cDC1. L'association de ces cellules et des IFN qu'elles produisent avec une meilleure survie des patientes confirme l'intérêt de développer de nouvelles immunothérapies ciblant les cDC1, en particulier dans le cancer du sein / Dendritic cells (DCs) represent a promising target for the development of new immunotherapies because of their central role in the initiation and the control of immunity. The rare cDC1 population is under considerable scrutiny because their murine counterparts called CD8α+ DCs are essential for cross-presentation to CD8+ T cells, antitumor immunity and response to immunotherapies. In contrast, the role of human cDC1 in cancer has not been investigated as extensively as in mice. They were identified in several tumors and transcriptomic analyses revealed their association with a favorable patient outcome. They also represent a major source of type III interferon (IFN-III), also called IFN-λ, playing a crucial role in viral infections, similarly to IFN-I that share the same signaling pathway. Its antitumor activity was also reported in mouse models, therefore raising questions regarding the use of IFN-IIl in clinical oncology. We believe that understanding the underlying mechanisms of cDC1 favorable prognostic impact and the role of IFN-III in antitumor immunity will be central to design new therapeutic approaches. Here, we demonstrated the infiltration of human primary breast and ovarian tumors by several DC populations and the enrichment of cDC1 compared with patient blood. We also showed for the first time close contacts between cDC1 and T cells in breast tumors. An in silico approach using MCPcouter on the TCGA data sets revealed that cDC1 represented the only DC subset associated with a prolonged overall survival in the majority of solid tumors. Interestingly, type I/III signature was strongly enriched in tumors highly infiltrated only with cDC1. Furthermore, we observed by feature intracytoplasmic flow cytometry analysis a spontaneous production of IFN-λ1 that is restricted to cDC1 in the absence of any ex vivo stimulation in one third of tumors. This result clearly indicates that IFN-λ1 production is a distinct of cDC1 compared with other DC subsets, even in a human tumor context. Notably, a high expression level of genes coding for IFN-III or its receptor was correlated with an increased relapse-free survival in breast cancer. We confirmed the presence of the IFN-λ1 protein in more than 50% of tumors and observed its abundancy compared with other IFN subtypes. IFN-λ1 was strongly correlated with IL-12p40, CXCL9, CXCL10, CXCL11, CX3CL1 and TNF-α. These results raised the hypothesis that IFN-λ1, produced by cDC1 in the TME, could be associated with the production of cytokines and chemokines involved in the recruitment and activation of cytotoxic lymphocytes (NK cells and CD8+ T cells). Our study provides detailed information about the DC compartment infiltrating human breast and ovarian tumors, revealing their potential implication in the antitumor immunity. By focusing on the pathways associated with each DC subset, our findings shed new light on the link between DC population called cDC1 and IFN-I/III signature in tumors. Our clear demonstration of IFN-III production by cDC1 and of its positive impact on the prognosis of cancer patients provides valuable evidences to support the development of new therapeutic strategies targeting cDC1 to amplify the response to immunotherapies, especially in breast cancer

Résumé : Le carcinome hépatocellulaire (HCC) est la troisième cause commune de décès de cancer et affecte plus les hommes que les femmes. Le HCC résulte d’une dérégulation des voies de signalisation impliquées dans l’initiation de l’inflammation menant ainsi à des répercussions désastreuses. De part la complexité de ce type de cancer, les traitements qui existent à ce jour ne sont pas très prometteurs et ont un faible pourcentage de « rémission ». L’immunothérapie soulève beaucoup d’espoir quant à l’orientation vers un traitement efficace plausible. En effet, plusieurs suppresseurs de tumeur se retrouvent réprimés, parmi lesquels le SOCS1. C’est dans cette optique que nos recherches se sont orientées en mettant la lumière sur le SOCS1 «suppresseur de signalisation des cytokines 1 (SOCS1) » qui est réprimé au niveau du HCC et dont la restauration pourrait contribuer à un pronostic favorable à la rémission. La protéine SOCS1 a beaucoup attisé la curiosité des chercheurs de part son rôle suppresseur de tumeur. Pour comprendre les mécanismes d’action de SOCS1 et son implication dans la neutralisation de la tumeur, nous avons généré trois types stables de la lignée cellulaire du carcinome hépatocellulaire de souris Hepa1-6, une portant un vecteur vide, l’autre exprimant le type sauvage du gène SOCS1 (SOCS1-WT; Hepa-S) et une portant une mutation au niveau du domaine SH2 (SOCS1-R105K; Hepa-R). Le mutant ne peut plus inhiber la signalisation des cytokines. Lors de l'implantation sous-cutanée des cellules Hepa1-6 modifiées, chez des souris C57BL/6 et NOD.scid.gamma (NSG). Nous avons observé que les cellules Hepa1-6 exprimant le vecteur de contrôle (Hepa-V) formaient de grosses tumeurs tandis que les cellules Hepa-S formaient de petites tumeurs chez les deux types de souris. Les cellules Hepa-R quant à elles, formaient de grosses tumeurs seulement chez des souris immunodéficientes (NSG) mais montraient une croissance nettement retardée lorsqu’elles étaient greffées aux souris (C57BL/6) immunocompétentes. Partant de ce constat intrigant, nous avons postulé que SOCS1 favorise l'immunogénicité des cellules tumorales par son domaine SOCS Box. Par conséquent, les cellules Hepa-R offrent une occasion unique de démêler le potentiel pro-immunogène de SOCS1, et ceci dans le but d'élucider les fonctions immunogènes de SOCS1 dans le cancer du foie. Jusqu'à présent aucune précédente recherche ne s’est aventurée à chercher l’implication de SOCS1 dans l’augmentation de l’immunogénicité. / Abstract : Hepatocellular carcinoma (HCC) is the third common cause of cancer deaths and affects men more than women. HCC results from a disregulation that affects many factors including inflammation, leading to disastrous repercusions. Due to the complexity of this type of cancer, existing treatments nowadays are not promising and have a low remission percentage. Immunotherapy raises great hope regarding the direction towards a plausible effective treatment. It is in this context that our research has focused on shedding light on the SOCS1 "signaling suppressor of cytokine 1 '(SOCS1)" which was found to be suppressed in HCC and whose restoration could contribute to a favorable prognosis. The SOCS1 many fueled the curiosity of researchers due to its suppressor role in tumor. To understand the underlying mechanisms, we generated three types of stable mice cell line hepatocellular carcinoma Hepa1-6, one carrying an empty vector, the other expressing the wild-type gene SOCS1 (SOCS1-WT; hepatic S) and carrying a mutation in the SH2 domain (SOCS1-R105K; Hepa-R). These mutants cannot inhibit JAK-STAT cytokine signaling. Upon subcutaneous implantation of the modified Hepa1-6 cells in C57BL/ 6 and NOD.scid.gamma (NSG) mice, the Hepa1-6 cells expressing the control (Hepa-V) formed large tumors while the Hepa-S cells formed small tumors in both types of mice. Hepa-R cells in turn, formed large tumors only in immunodeficient mice (NSG), but showed a markedly delayed growth when transplanted into (C57BL / 6) immunocompetent mice. Based on this intriguing finding, we speculate that SOCS1 could promote immunogenicity of tumor cells, which was masked by the ability of SOCS1 to inhibit signaling of growth factors. Therefore, Hepa-R cells provide a unique opportunity to unravel the pro-immunogenic potential of SOCS1, and this in order to elucidate the immunogenic functions of SOCS1 in liver cancer. So far no previous research studied the potentiel of SOCS1 in increasing immunogenicity.

Células tumorais desenvolvem diversas estratégias para escapar da identificação e eliminação pelo sistema imune. Dessa forma, a investigação dos mecanismos envolvidos na comunicação celular no microambiente tumoral e na desregulação local do sistema imune é crítica para uma melhor compreensão da progressão da doença e para o desenvolvimento de alternativas terapêuticas mais eficazes. Nós aqui demonstramos que SIGIRR/IL-1R8, um importante regulador negativo de receptores de Interleucina-1 (ILRs) e receptores do tipo Toll (TLRs), apresenta expressão aumentada em uma linhagem celular epitelial mamária transformada pela superexpressão do oncogene HER2 e em tumores primários de mama, e promove o crescimento tumoral e metástase através da modulação da inflamação associada ao câncer e da atenuação da resposta imune antitumoral. Observamos que IL-1R8 tem sua expressão correlacionada com HER2 em tecidos mamários e sua alta expressão é fator de pior prognóstico em câncer de mama de baixo grau. Notavelmente, níveis aumentados de IL-1R8 foram observados especialmente nos subtipos HER2+ e Luminais de tumores de mama, e sua expressão aumentada em células epiteliais de mama transformadas por HER2 diminui a ativação da via de NF-κB e a expressão de diferentes citocinas pro-inflamatórias (IL-6, IL-8, TNF, CSF2, CSF3 e IFN-β1). Meio condicionado de células transformadas por HER2, mas não de variantes celulares com o gene IL-1R8 silenciado, induz a polarização de macrófagos para o fenótipo M2 e inibe a ativação de células NK. Em um modelo murino transgênico de tumorigênese espontânea mediada por HER2, MMTV-neu, verificamos que a deficiência de IL-1R8 (IL-1R8-/-neu) retardou o aparecimento de tumores e reduziu a incidência, a carga tumoral e a disseminação metastática. Contudo, não foram observadas diferenças significativas no crescimento tumoral quando animais IL-1R8-/-neu receberam medula óssea de animais IL-1R8+/+, confirmando um papel importante da expressão de IL-1R8 em células não hematopoiéticas na tumorigênese da mama. Tumores IL-1R8+/+neu apresentaram maiores níveis de citocinas pró-inflamatórias como IL-1β e VEGF, e menores níveis da citocina imunomodulatória IFN-γ. Além disso, tumores que expressavam IL-1R8 apresentaram menor infiltrado de células NK maduras, células dendríticas (DCs) e linfócitos T-CD8+ e um maior infiltrado de macrófagos M2 e linfócitos T-CD4+. Coletivamente, esses resultados indicam que a expressão de IL-1R8 em tumores de mama pode representar um novo mecanismo de escape da resposta imune e suportam IL-1R8 como potencial alvo terapêutico. / Tumor cells develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. Identification of new players that regulate the cellular crosstalk within the tumor microenvironment and promote local immune dysregulation is critical to understand disease progression and to improve therapeutic strategies. Here, we demonstrate that SIGIRR/IL-1R8, a negative regulator of IL-1R and TLRs, is up-regulated in a HER2-transformed epithelial mammary cell line and in primary breast tumors and promotes tumor growth and metastasis by modulating cancer-related inflammation and impairing anti-tumor immunity. IL-1R8 expression is correlated with HER2 in mammary tissue, and higher tumor IL-1R8 expression is a poor prognostic factor in lower grade breast tumors. Notably, higher levels of IL-1R8 expression were observed in HER2+ and Luminal breast tumor subtypes and IL-1R8 up-regulation in HER2-transformed mammary epithelial cells inhibited NF-κB activation and the expression of pro-inflammatory cytokines (IL-6, IL-8, TNFα, CSF2, CSF3, IFN-β1). Conditioned medium from HER2-transformed cells, but not from IL-1R8 knockdown variants, induced M2-macrophage polarization and inhibited natural-killer (NK) cell activation. IL-1R8 deficiency in a transgenic mouse model of breast tumorigenesis (MMTV-neu) significantly delayed tumor onset and reduced tumor incidence, burden and metastasis. No significant differences in tumor growth were observed when IL-1R8-/-neu mice were transplanted with bone marrow from IL-1R8+/+ animals, confirming an important role for IL-1R8 expression in non-hematopoietic cells during breast tumorigenesis. IL-1R8+/+neu mammary tumors presented higher levels of pro-inflammatory cytokines such as IL-1β and VEGF, but lower levels of IFN-γ. Besides, a lower infiltrate of mature NK cells, dendritic cells (DCs) and CD8+ T cells but higher infiltrate of M2-macrophages and CD4+ T cells were present in IL-1R8 expressing tumors. Collectively, our results support IL-1R8 expression as a novel tumor immune escape mechanism in breast cancer and putative target for immunotherapy.

Células tumorais desenvolvem diversas estratégias para escapar da identificação e eliminação pelo sistema imune. Dessa forma, a investigação dos mecanismos envolvidos na comunicação celular no microambiente tumoral e na desregulação local do sistema imune é crítica para uma melhor compreensão da progressão da doença e para o desenvolvimento de alternativas terapêuticas mais eficazes. Nós aqui demonstramos que SIGIRR/IL-1R8, um importante regulador negativo de receptores de Interleucina-1 (ILRs) e receptores do tipo Toll (TLRs), apresenta expressão aumentada em uma linhagem celular epitelial mamária transformada pela superexpressão do oncogene HER2 e em tumores primários de mama, e promove o crescimento tumoral e metástase através da modulação da inflamação associada ao câncer e da atenuação da resposta imune antitumoral. Observamos que IL-1R8 tem sua expressão correlacionada com HER2 em tecidos mamários e sua alta expressão é fator de pior prognóstico em câncer de mama de baixo grau. Notavelmente, níveis aumentados de IL-1R8 foram observados especialmente nos subtipos HER2+ e Luminais de tumores de mama, e sua expressão aumentada em células epiteliais de mama transformadas por HER2 diminui a ativação da via de NF-κB e a expressão de diferentes citocinas pro-inflamatórias (IL-6, IL-8, TNF, CSF2, CSF3 e IFN-β1). Meio condicionado de células transformadas por HER2, mas não de variantes celulares com o gene IL-1R8 silenciado, induz a polarização de macrófagos para o fenótipo M2 e inibe a ativação de células NK. Em um modelo murino transgênico de tumorigênese espontânea mediada por HER2, MMTV-neu, verificamos que a deficiência de IL-1R8 (IL-1R8-/-neu) retardou o aparecimento de tumores e reduziu a incidência, a carga tumoral e a disseminação metastática. Contudo, não foram observadas diferenças significativas no crescimento tumoral quando animais IL-1R8-/-neu receberam medula óssea de animais IL-1R8+/+, confirmando um papel importante da expressão de IL-1R8 em células não hematopoiéticas na tumorigênese da mama. Tumores IL-1R8+/+neu apresentaram maiores níveis de citocinas pró-inflamatórias como IL-1β e VEGF, e menores níveis da citocina imunomodulatória IFN-γ. Além disso, tumores que expressavam IL-1R8 apresentaram menor infiltrado de células NK maduras, células dendríticas (DCs) e linfócitos T-CD8+ e um maior infiltrado de macrófagos M2 e linfócitos T-CD4+. Coletivamente, esses resultados indicam que a expressão de IL-1R8 em tumores de mama pode representar um novo mecanismo de escape da resposta imune e suportam IL-1R8 como potencial alvo terapêutico. / Tumor cells develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. Identification of new players that regulate the cellular crosstalk within the tumor microenvironment and promote local immune dysregulation is critical to understand disease progression and to improve therapeutic strategies. Here, we demonstrate that SIGIRR/IL-1R8, a negative regulator of IL-1R and TLRs, is up-regulated in a HER2-transformed epithelial mammary cell line and in primary breast tumors and promotes tumor growth and metastasis by modulating cancer-related inflammation and impairing anti-tumor immunity. IL-1R8 expression is correlated with HER2 in mammary tissue, and higher tumor IL-1R8 expression is a poor prognostic factor in lower grade breast tumors. Notably, higher levels of IL-1R8 expression were observed in HER2+ and Luminal breast tumor subtypes and IL-1R8 up-regulation in HER2-transformed mammary epithelial cells inhibited NF-κB activation and the expression of pro-inflammatory cytokines (IL-6, IL-8, TNFα, CSF2, CSF3, IFN-β1). Conditioned medium from HER2-transformed cells, but not from IL-1R8 knockdown variants, induced M2-macrophage polarization and inhibited natural-killer (NK) cell activation. IL-1R8 deficiency in a transgenic mouse model of breast tumorigenesis (MMTV-neu) significantly delayed tumor onset and reduced tumor incidence, burden and metastasis. No significant differences in tumor growth were observed when IL-1R8-/-neu mice were transplanted with bone marrow from IL-1R8+/+ animals, confirming an important role for IL-1R8 expression in non-hematopoietic cells during breast tumorigenesis. IL-1R8+/+neu mammary tumors presented higher levels of pro-inflammatory cytokines such as IL-1β and VEGF, but lower levels of IFN-γ. Besides, a lower infiltrate of mature NK cells, dendritic cells (DCs) and CD8+ T cells but higher infiltrate of M2-macrophages and CD4+ T cells were present in IL-1R8 expressing tumors. Collectively, our results support IL-1R8 expression as a novel tumor immune escape mechanism in breast cancer and putative target for immunotherapy.

L’axe adénosinergique est au cœur de divers processus pathophysiologiques. L’enzyme CD73 joue un rôle pivot dans la génération de l’adénosine en catalysant la déphosphorylation de l’adénosine monophosphate. L’adénosine contribue à un éventail large de processus biologiques et pathologiques, principalement via l’activation de récepteurs transmembranaires. L’adénosine est principalement reconnue pour son activité régulatrice des cellules immunitaires et CD73 pour son rôle dans l’accumulation de l’adénosine dans le microenvironnement tumoral. En effet, en altérant la réponse immunitaire anti-tumorale via l’inhibition des fonctions effectrices de divers types de cellules immunes, CD73 et l’adénosine sont fréquemment associés à la progression tumorale et s’inscrivent comme cibles thérapeutiques intéressantes. Les rôles de CD73 et l’adénosine dans d’autres processus immunitaires physiologiques ne sont pas tous aussi bien compris, notamment concernant les processus d’immunisations. En utilisant un modèle murin d’immunisation contre le pneumocoque, cette thèse démontre un rôle positif, mais non essentiel, de CD73 et de l’adénosine dans la commutation isotypique des lymphocytes B et la génération d’une immunité protectrice contre l’infection au S. pneumoniae. Cette découverte est pertinente au développement de stratégies thérapeutiques afin d’augmenter l’efficacité d’immunisation dépendante des cellules B, plus particulièrement chez les populations à risque en bas âge. Ensuite, alors que la modulation de l’axe adénosinergique, notamment via l’inhibition de CD73, est une avenue thérapeutique étudiée dans divers contextes de tumeurs solides, ce potentiel thérapeutique demeure largement inexploré dans des modèles de néoplasmes sanguins. En utilisant un modèle de souris transgénique de leucémie spontanée, cette thèse démontre un rôle pro-tumorigénique, avec un biais sexuel, de CD73 dans la leucémie lymphoïde chronique des lymphocytes B (LLC), via l’altération de l’immunité anti-tumorale. Enfin, alors que les rôles immunosuppressifs de CD73 et l’adénosine sont bien décrits, leurs activités pro-tumorigéniques qui s’étendent au-delà de l’immunité anti-tumorale sont peu connues. En accord avec la littérature, cette thèse explore plusieurs hypothèses selon lesquelles CD73 module l’activité métabolique mitochondriale des cellules cancéreuses. Les résultats présentés dans cette thèse suggèrent un rôle pro-tumorigénique à l’enzyme CD73, indépendant de la signalisation adénosinergique et de l’inhibition de l’immunité anti-tumorale, qui favorise la flexibilité métabolique et plus particulièrement la respiration mitochondriale des cellules cancéreuses, via la voie de récupération de la biosynthèse du nicotinamide (NAD+). En résumé, cette thèse apporte plusieurs précisions quant aux rôles biologiques de l’enzyme CD73 qui sont pertinents à l’immunisation dépendante des lymphocytes B, à la pathogénèse de la LLC ainsi qu’à la régulation de l’activité métabolique des cellules cancéreuses. Cette thèse offre de nouvelles pistes de réflexion quant au potentiel thérapeutique que renferme l’axe adénosinergique et plus particulièrement CD73, en approfondissant nos connaissances quant à l’éventail de ses fonctions. / The adenosinergic axis is central to a plethora of pathophysiological processes. The enzyme CD73 is key to the generation of adenosine by catalyzing the dephosphorylation of adenosine monophosphate. Adenosine’s contribution to biological and pathological processes is mainly carried through the activation of transmembrane receptors. Adenosine is mostly appreciated for its regulatory activity on a variety of immunes cells whereas CD73 is often referred to the enzyme responsible for adenosine accumulation within tumor microenvironment. Thus, by hindering antitumoral immune responses, CD73 and adenosine are frequently associated with cancer progression and targeting these offers great therapeutic potential in clinic. CD73 and adenosine’s role in other immune physiological processes are not fully understood, notably regarding immunization processes. Using a murine model of pneumococcal immunization, this thesis herein demonstrates a positive, but non-essential, role for CD73 and adenosine in B cells’ isotype class switching required to protective immunity against S. pneumoniae. This finding is particularly relevant to the development of novel strategies aimed at enhancing B cell-dependent immunization in high-risk populations such as young infants. While targeting the adenosinergic axis, particularly CD73, was extensively proven efficient in restoring antitumor immunity in many solid tumor contexts, its therapeutic potential in blood neoplastic malignancies remain largely unexplored. Using a transgenic mouse model of spontaneous leukemia, this thesis identifies a sex-oriented pro-tumorigenic role for CD73 in favoring B cells chronic lymphocytic leukemia (CLL) progression, through the inhibition of antitumor immunity. Finally, while immunosuppression by CD73 and adenosine is well described in cancer, other immune-independent pro-tumorigenic roles of CD73 are poorly understood. In accordance with literature, this thesis explores various hypotheses by which CD73 regulates cancer cells’ mitochondrial metabolic activity. Results presented herein suggest an immune- and adenosine signaling-independent pro-tumorigenic function for CD73 in favoring cancer cells’ metabolic flexibility and more particularly mitochondrial respiration through the nicotinamide (NAD+) salvage biosynthesis pathway. In sum, this thesis brings many insights into CD73’s biological functions relevant to B cells-dependent immunization, in CLL pathogenesis and in cancer cells’ metabolic activity. By expanding our knowledge of the extend of CD73’s biological functions, this thesis further discusses novel potential therapeutic opportunities.