The immune system must be able to mount a response against pathogens and transformed cells while remaining tolerant to healthy host tissue. A key process for ensuring this self-tolerance is the negative selection of self-reactive thymocytes. Expression of Programmed Death-1 (PD-1), a co-inhibitory member of the CD28 family associated with dampened peripheral immune responses,was found to be upregulated in 20-40% of thymocytes undergoing negative selection in the HYcd4model of thymic development. Although analysis of gene and protein expression directly ex vivo indicates that PD-1- and PD-1+ thymocytes are equally apoptotic, PD-1+ thymocytes appear to be protected from apoptosis in an in vitro stimulation assay. Analysis of HYcd4PD-1-/- mice indicates that thymocytes receive a higher intensity signal in the absence of PD-1. Future work utilizing HYcd4PD-1-/- mice will increase our understanding of the role of PD-1 in thymic negative selection. / Immunology

No description available.

The administration of soluble antigenic peptides is known to be effective at inducing tolerance in naïve antigen-reactive CD4+ T cells. This observation forms the basis of antigen-based therapy, which offers the potential to specifically target the auto-reactive CD4+ T cells involved in driving autoimmune disease pathogenesis, whilst leaving the rest of the immune system intact. The prophylactic administration of soluble autoantigen-derived peptides has proven to be effective at inhibiting disease induction in various experimental models of autoimmune disease. However, the clinical requirement is to switch off the activated antigen-experienced CD4+ T cells that are present during an ongoing immune response. The effect of soluble peptide administration of antigenexperienced CD4+ T cells is poorly understood, and several clinical trials using peptides in multiple sclerosis patients had to be halted due to the exacerbation of disease. This thesis characterises the effect of soluble peptide administration on pathogenic antigen-experienced CD4+ T cells, using experimental autoimmune encephalomyelitis (EAE) as a model of autoimmune disease of the central nervous system. Using traceable myelin-reactive T cells from Tg4 mice, it was determined that soluble peptide administration induces substantial expansion of antigen-experienced CD4+ T cells. Despite the increase in number, these cells were no longer able to induce EAE. Production of effector cytokine was significantly decreased in peptide treated antigen-reactive CD4+ T cells, and this correlated with high level expression of the co-inhibitory molecule PD-1. The induction of tolerance in both naïve and antigen-experienced CD4+ T cells was found to be dependent upon PD-1 expression, whereby peptide treatment of naïve and antigen-experienced CD4+ T cells that were deficient in PD-1, did not inhibit disease induction. This thesis identifies a novel mechanism of peptide-induced tolerance in CD4+ T cells, and demonstrates that soluble peptide administration can induce tolerance in antigen-experienced T cells.

Cancer is the second most common cause of death in the United States behind heart disease. While current treatments such as surgery, chemotherapy, and radiation therapy are effective and widely used, medicine is moving towards more targeted and personalized therapies. Immunotherapy is one such treatment that utilizes the patient’s own immune system to target and eliminate tumor cells. It allows for the patient’s adaptive immune system to bypass the self-tolerance mechanisms used by the cancerous cells and be activated against the cancer. Two such self-tolerant mechanisms that are co-opted by tumor cells are the interactions between CTLA-4 and T lymphocytes and the interactions between PD-1 and PD-L1. Blocking these interactions allows for the recruitment of CTLs to the site of the tumor and subsequent attack. CTLA-4 and PD-1 are inhibitory costimulators that play a role in the suppression of the adaptive immune system. The interaction of these receptors with their respective ligands leads to self-tolerance, and is a common mechanism used as a protective measure against autoimmune reactions. Monoclonal antibodies against these two receptors and ligand have been tested in clinical trials and have shown efficacy against ovarian cancers, non-small cell lung carcinomas, colon cancers, and melanomas. By targeting the inhibitory signals, these monoclonal antibodies expose cancer cells as being “non-self” thus prompting the immune system to attack. Now, studies are focusing on combination therapies, which combine chemotherapeutics or other monoclonal antibodies with PD-1 and CTLA-4 inhibitors to enhance the effectiveness of the drug. However, drawbacks and side effects to the therapy range from fatigue and nausea to development of autoimmune diseases. It brings forward that future studies will need a panel of predictive biomarkers to identify the best candidates for the immunotherapy. While there are many obstacles, such as a lower than expected efficacy of the immunotherapy, the progress made has important implications in the development of personalized medicine.

The development of autoimmune diseases is frequently linked to exposure to environmental factors such as chemicals, drugs or infections. In the experimental model of metal-induced autoimmunity, administration of subtoxic doses of mercury (a common environmental pollutant) to genetically susceptible mice induces an autoimmune syndrome with rapid anti-nucleolar antibody production and immune system activation. Regulatory components of the innate immune system such as the costimulatory molecules PD-1, CTLA-4 and their ligands PD-L1, PD-L2, B7-1 and B7-2 can also modulate the autoimmune process. We examined the interplay among environmental chemicals and these costimulatory molecules in the regulation of autoimmunity. Additionally, we studied PD-1, CTLA-4 and its ligands in a tolerance model where pre-administration of a low dose of mercury in the steady state renders animals tolerant to metal-induced autoimmunity. Overall, PD-1/CTLA-4 blockade by blocking antibodies enhanced the manifestations of metal-induced autoimmunity. Although we expected that the blockade of both PD-1 ligands would mimic blocking the receptor, blocking the ligands resulted in the opposite effect when co-injected with mercury, reducing the manifestations of metal-induced autoimmunity. Individual PD-1 ligands differed in their ability to enhance the mercury treatment, with PD-L1 being the major regulator in the model. Likewise, we showed that PD-L1 is essential to keep the recall response to mercury at check. Our data suggest that these effects could be mediated by the modification of cytokine profiles, B cells and T cell subpopulations numbers. / Microbiology and Immunology

La réponse immunitaires est régulée par des molécules de co-signalisation, qui, en apportant des signaux inhibiteurs ou activateurs, modulent les fonctions lymphocytaires. Nous avons focalisé notre étude sur deux co-effecteurs et membres de la famille CD28/B7 : PD-1 (Programmed Death 1) et BTLA (B and T Lymphocyte Attenuator), tous deux décrits, à l’image de CTLA-4, comme inhibitrices de l’activation lymphocytaire T. Au cours de ma thèse, nous avons pu évaluer l’expression et le rôle de PD-1 et BTLA sur les lymphocytes B normaux et pathologiques. Dans une première partie, nos travaux ont montré que ces molécules sont exprimées à la surface des sous populations B du sang périphérique et des tissus et qu’elles sont également finement régulées au cours de l’activation B. Par la suite nous avons démontré pour la première fois que BTLA et PD-1, à l’image des cellules T, sont recrutées, après activation, au niveau du récepteur de la cellule B et qu’elles exercent un rôle inhibiteur sur l’activation de ces mêmes cellules. Dans une seconde partie, grâce au développement d’un outil en cytométrie multicouleurs, nous avons analysé, dans une cohorte de 72 lymphomes B, l’infiltrat immunitaire ainsi que l’expression de PD-1, BTLA et leurs ligands. Ce travail, par son analyse de la physiologie B d’une part et de pathologies tumorales B d’autre part, donne un nouvel éclairage sur les rôles complexes des co-récepteurs BTLA et PD-1. / The immune response is regulated by co-signaling molecules, which, by providing signals inhibitors or activators, modulate lymphocyte functions. We focused our study on two co-effectors and CD28/B7 family members: PD-1 (Programmed Death 1) and BTLA (B and T Lymphocyte Attenuator), both described, like CTLA-4, as inhibitors of T lymphocyte activation. This work allowed us to evaluate the expression and the role of BTLA and PD-1 on normal and tumoral B lymphocytes. In the first part, our work has shown that these molecules are expressed on B cell subsets of peripheral blood and tissues and are also finely regulated during the B cell activation. Subsequently, we have demonstrated for the first time that BTLA and PD-1, like T cells, are recruited after activation at the B cell receptor, and they exert an inhibitory role on the activation of these cells. In the second part, through the development of a multicolor cytometry tool, we analyzed the immune infiltrate and the expression of PD-1, BTLA and their ligands in a cohort of 72 B-cell lymphomas. Taken together, these results, by his analysis of the physiology of B cell from B cell malignancies, give a new insight into the complex roles of BTLA and PD-1 co-receptors.

Os mastócitos (MC) são células teciduais, ricas em mediadoras inflamatórias, envolvidos na resposta alérgica, com papel imunomodulador cada vez mais reconhecido. Células Dendríticas (DCs), por sua vez, são sabidamente necessárias para a resposta imune, sendo as células apresentadoras de antígenos mais eficientes, capazes de responder prontamente frente à sinais de perigo. Neste trabalho, demonstramos que o contanto celular de DCs com MCs (iDC-MC) induz a geração de DCs com imunofenotipo tolerogênico. As iDC-MC exibiram menor expressão de HLA-DR e maior expressão de PD-L1, assim, não foram capazes de manter uma proliferação alogeneica. Ainda, os linfócitos expostos as iDC-MC induziram maior expressão de FoxP3+, IL-10 e TGF-β, capazes de suprimir a proliferação de linfócitos T naïve estimulados com mitógeno. Além disso, o contato resultou na maior produção de IDO, fenômeno este, bloqueado quando MCs foram tratados com anti-PD-1 ou iDCs tratadas com PD-L1 ou PD-L2, mas a produção se manteve inalterada após o tratamento das iDCs com anti-histamínicos. / Mast cells (MC) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between iDCs and MC bends DCs towards tolerance induction. These MC-exposed DCs decreased HLA-DR but increased PD-L1 expression and stimulated T lymphocytes to express FoxP3+, to secrete TGF-β and IL-10, and to suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, contact with MC induced DCs to express higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs.

Os mastócitos (MC) são células teciduais, ricas em mediadoras inflamatórias, envolvidos na resposta alérgica, com papel imunomodulador cada vez mais reconhecido. Células Dendríticas (DCs), por sua vez, são sabidamente necessárias para a resposta imune, sendo as células apresentadoras de antígenos mais eficientes, capazes de responder prontamente frente à sinais de perigo. Neste trabalho, demonstramos que o contanto celular de DCs com MCs (iDC-MC) induz a geração de DCs com imunofenotipo tolerogênico. As iDC-MC exibiram menor expressão de HLA-DR e maior expressão de PD-L1, assim, não foram capazes de manter uma proliferação alogeneica. Ainda, os linfócitos expostos as iDC-MC induziram maior expressão de FoxP3+, IL-10 e TGF-β, capazes de suprimir a proliferação de linfócitos T naïve estimulados com mitógeno. Além disso, o contato resultou na maior produção de IDO, fenômeno este, bloqueado quando MCs foram tratados com anti-PD-1 ou iDCs tratadas com PD-L1 ou PD-L2, mas a produção se manteve inalterada após o tratamento das iDCs com anti-histamínicos. / Mast cells (MC) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between iDCs and MC bends DCs towards tolerance induction. These MC-exposed DCs decreased HLA-DR but increased PD-L1 expression and stimulated T lymphocytes to express FoxP3+, to secrete TGF-β and IL-10, and to suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, contact with MC induced DCs to express higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs.

Les thérapies anticancéreuses nécessitent aujourd’hui une approche plus ciblée. L’approche immunologique est devenue dans cette optique un enjeu d’importance notamment grâce à sa capacité d’atteindre de manière plus spécifique des cibles tumorales. Récemment, mon équipe a identifié des peptides antigéniques issus de la préprocalcitonine (ppCT), qui étaient capables d’induire une réponse T cytotoxique spontanée chez un patient atteint de cancer bronchique non à petites cellules bénéficiant d’une longue survie et correspondent donc à des candidats potentiels dans des approches vaccinales. Dans ma thèse, je fournis une preuve de concept préclinique pour un vaccin fondé sur la ppCT à usage thérapeutique contre le cancer du poumon. Cette approche est optimisée en la combinant avec un anticorps monoclonal anti-PD- 1. En plus, j'ai identifiée de nouveaux épitopes issus de cet antigène et j'ai construit un lentivirus codant la ppCT pour des perspectives thérapeutiques. / Cancer therapies today require a more targeted approach to optimize the anti-tumor response. In this context, the immunological approach has become an important issue, mostly through the identification of specific tumor-associated antigens (TAA). Recently, my team identified new tumor antigens derived from the preprocalcitonin (ppCT) which were able to induce a spontaneous cytotoxic T-cell response with long term survival in patients with non-small cell lung cancer and therefore correspond to be potential candidates for vaccine approaches. In my thesis, I provide a preclinical proof of concept for the ppCT peptide vaccine for the therapeutic use against lung cancer which is optimized by combining the treatment with the monoclonal antibody anti-PD-1, nivolumab. In addition, I identified new epitopes from this Ag and constructed a lentivirus encoding ppCT for further therapeutic perspectives.

Zusammenfassung Neueste Daten deuten daraufhin, dass maligne Tumoren der immunologischen Überwachung über eine Herunterregulierung der T-Zell-Aktivierung mittels eines PD-1 (programmed death 1)/ PDL-1/PDL-2 Signals ausweichen. Dies führt offensichtlich zu einer herabgesetzten Immunantwort und kann somit das Tumorwachstum fördern. Das Oberflächenmolekül PD-1, welches auf T- und B-Zellen, myeloischen Zellen und auf vielen menschlichen Karzinomen exprimiert wird, gehört zu der CD28 Familie, und PDL-1 (B7H1) und PDL-2 (B7DC) wurden als Liganden zu PD-1 beschrieben. Der CD28/B7 Signalweg gehört zu der Gruppe kostimulatorischer Signale, die beim zustande kommen einer T-Zell-gerichteten Immunantwort als zweites kostimulatorisches Signal notwendig sind. In dieser Arbeit wurde die molekulare Expression von PD-1, PDL-1 und PDL-2, von Zytokinen und T-Zell-Subpopulationen im Tumorgewebe von 81 Patienten analysiert, die sich einer kurativen oder palliativen Operation (gemäß UICC- Stadien I-IV) eines primären kolorektalen Karzinoms unterzogen hatten. Es zeigte sich auf Protein- als auch auf molekularer Ebene erstmals, dass PDL-1 und PDL-2 im Tumorgewebe fortgeschrittener Tumorstadien (UICC III/IV) signifikant überexprimiert wurden, PD-1 dagegen erniedrigt exprimiert war. PD-1 wurde dagegen deutlich auf infiltrierenden CD4+ Zellen bei Patienten fortgeschrittener Tumorstadien (UICC III/IV) detektiert, wohingegen PDL-1 auf CD4+ Zellen in frühen Stadien gefunden wurde. Im Vergleich zu den frühen Stadien (UICC I/II) wurde eine grössere Anzahl an T-Zellen mit regulatorischem Charakter in den Tumorstadien III/IV beobachtet. Letzteres würde dem Tumor im Bezug auf sein fortschreitendes Wachstum von Vorteil sein, da regulatorische T-Zellen T-Effektorzellen inhibieren können. Im Tumorgewebe fand sich zudem eine verminderte Expression an IFN-gamma, welches unter anderem T-Effektorzellen aktiviert und damit eine Immunantwort verstärkt. Das Zytokin IL-10, welches mit regulatorischen T-Zellen, aber auch mit T-Helfer (Th)2-Zellen und Tumorgewebe assoziiert ist und antiinflammatorische Eigenschaften besitzt, wurde in höheren Stadien verstärkt nachgewiesen. Dadurch verschafft es dem Tumor einen Überlebensvorteil. Die Beobachtungen in dieser Arbeit zeigen, dass PDL-1 und PDL-2 eine Schlüsselrolle während der Tumorprogression zukommen. Regulatorische T-Zellen sind offensichtlich in den Prozeß der Immunantwort gegen den Tumor eingebunden. Die deutliche PDL-1- und PDL-2-Expression auf T-Zellen insbesondere in frühen Tumorstadien lässt darauf schließen, dass die PD-1/PDL-1- bzw. PD-1/PDL-2-Interaktion inhibitorische Signale zwischen Tumorzellen und den T-Zellen vermittelt. In fortgeschrittenen Stadien (UICC III/IV) waren diese kostimulatorischen Signale auf den T-Zellen nur vermindert vorzufinden. Dies hat zur Folge, dass der Tumor sein Wachstum ungehindert fortsetzen kann, da die anti-Tumor-T-Zell-Antwort, die den Tumor normalerweise in seiner Expansion beeinträchtigt, gestört ist. Es wird somit festgestellt, dass eine Blockade der untersuchten Oberflächenmoleküle PDL-1 oder PDL-2 auf Tumorzellen eine wertvolle Option in der Immuntherapie des humanen kolorektalen Karzinoms darstellen könnte. Angesichts der diskutierten Tatsachen im Hinblick auf das Verhalten der regulatorischen T-Zellen in höheren Tumorstadien könnte sich eventuell eine Option damit eröffnen, die regulatorischen T-Zellen mittels eines spezifischen Antikörpers gegen PDL-1 und/oder PDL-2 zu beeinflussen, um somit die hemmenden Auswirkungen gegenüber der anti-Tumor-Immunantwort zu verhindern oder zu revidieren. / Summary Recent data have suggested that malignant tumors are able to escape from the immunological surveillance on a downregulation of the T cells via a PD-1 (programmed death 1) / PDL-1/PDL-2 signal. This obviously leads to a reduced immune response and thus may promote tumor growth. The surface molecule PD-1, which is expressed on T and B cells, myeloid cells and on many human carcinoma, belongs to the CD28 family, and PDL-1 (B7H1) and PDL-2 (B7DC) are ligands to PD-1. The CD28/B7 signal belongs to the group of costimulatory pathways, which is needed for a T-cell-directed immune response as a second costimulatory signal. In this work, the molecular expression of PD-1, PDL-1 and PDL-2, cytokines and T-cell-subpopulations was analyzed in tumor tissue of 81 patients, which have undergone a curative or palliative surgery (according UICC- stages I-IV) of a primary colorectal cancer. For the first time it was shown on the protein and molecular level, that PDL-1 and PDL-2 were significantly overexpressed in tumor tissue of advanced tumor stages (UICC III/IV), the expression of PD-1, on the other hand was decreased. PD-1 was much more detected on infiltrating CD4+ cells in patients of advanced tumor stages (UICC III/IV), while PDL-1 was found on CD4+ cells in the early stages. In comparison to the early stages (UICC I/II), a larger number of T cells with regulatory character in the tumor stage III/IV was observed. The latter would be an advantage for the tumor in relation to its progressive growth, because regulatory T cells are able to inhibit T-effector-cells. In tumor tissue the expression of IFN-gamma was also reduced, which among others activate T-effector-cells and thus reinforced an immune response. The cytokine IL-10, which is associated with regulatory T cells, but also with T-helper(Th)2-cells and tumor tissue, has antiinflammatory properties and was demonstrated enhanced at higher stages. Thus it gives the tumor a survival advantage. The observations in this study show that PDL-1 and PDL-2 plays a key role during tumor progression. Regulatory T cells are obviously involved in the process of immune response against the tumor. The significant PDL-1- and PDL-2-Expression on T-cells, particularly in the early stages of tumor suggests that the PD-1/PDL-1- or PD-1/PDL-2-interaction mediated inhibitory signals between tumor cells and the T-cell. In advanced stages (UICC III/IV) the costimulatory signals on T-cells were decreased. This means that the tumor's growth can continue unhindered, because the anti-tumor-T-cell-response which the tumor usually affected is disrupted in its expansion. It is thus found that a blockade of the investigated surface molecules PDL-1 and PDL-2 in tumor cells could be a valuable option in the immunotherapy of human colorectal cancer. Given the facts discussed in relation to the behavior of regulatory T cells in advanced tumor stages could possibly be an option to influence the regulatory T cells with a specific antibody against PDL-1 and / or PDL-2, thus to prevent or revise the inhibitory effects against the anti-tumor-immunresponse.