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Decellularized extracellular matrices have been a growing area of interest in the biomedical engineering fields of tissue engineering and regenerative medicine.As these materials move toward clinical applications, the immune response to these materials will be a driving force toward their success in clinical approaches. Fully digested decellularized matrix constructs derived from porcine liver, muscle and lung were created to test the adaptive immune response. Hydrogel characterization ensured that the materials had relatively similar stiffness levels to reduce variability, and in vitro studies were conducted. Each individual construct as well as a gelatin control were plated with a co-culture of macrophages and T-cells to measure T-cell proliferation. In addition standard markers of inflammation through qPCR were measured in the macrophage group. Constructs were then placed into animals for 3 and 7 days in addition to a second group that received constructs for 21 days before secondary constructs were placed. These groups were then sacrificed following 3, 7 and 14 days to measure the residual and memory-like response of the constructs. Our results showed that t-cell proliferation was increased with decellularized constructs, particularly in tissue with higher DNA content. In vivo, animals with secondary treatments showed extended inflammatory response, driven by Th1 and Th17 polarization suggesting a memory-like response due to recognition of peptides in the constructs from secondary placements.

Flagelinas de bactérias gram-negativas ativam o sistema imune inato resultando em efeitos adjuvantes para antígenos co-administrados. No entanto, não existem relatos sobre as propriedades adjuvantes de flagelinas de bactérias gram-positivas. O objetivo do projeto foi estudar os efeitos imunomoduladores da flagelina Hag de B. subtílis. Inicialmente foi purificada a Hag e subseqüentemente avaliada sua propriedade imunológica quando administrada junto com a ovalmbumina (OVA) pelas vias intra-nasal (i.n.) ou subcutânea (s.c.) em camundongos Balb/C e C57BL/6. Os resultados demonstraram as propriedades imunomoduladoras da Hag nas duas linhagens em relação à indução de anticorpos antígeno-específicos. Não foi possível demonstrar a ativação de linfócitos TCD4+ e CD8+ antígenos-específicos em animais Balb/C imunizados com OVA e Hag tanto pela via s.c. como pela i.n.. Resultados mais promissores do efeito adjuvante frente a células T foram encontrados em animais C57BL/6. Os resultados abrem perspectivas para o estudo do potencial da flagelina Hag de B. subtilis como adjuvante vacinal. / Flagellin of gram negatives bacteria actives the innate immune system resulting in adjuvants effects against coadministrated antigens. However, there is not information about immunological properties of flagellins produced by gram positive bacteria. This study aimed the evaluation of immunomodulator effects of Hag flagellin from B. subtilis. Vaccine formulations based in purified Hag and Ovalbumine protein (OVA) were administrated intranasal (i.n.) and subcutaneously (s.c.) in Balb/C and C57BL/6 mice and then the population of B and T lymphocytes were monitored for production of antibodies and citocines and/or surface markers expression. Results showed the immunomodulatory properties of Hag in both mice lineages regarding the induction of antigen specific antibodies when immunized with OVA and Hag by i.n. and s.c. via. Most promising results concerning the adjuvant effects observed on T cells activation were found in C57BL/6 mice. The results obtained open future perspectives for the study of the potential use of B. subtilis Hag flagellin as adjuvant in vaccines.

Flagelinas de bactérias gram-negativas ativam o sistema imune inato resultando em efeitos adjuvantes para antígenos co-administrados. No entanto, não existem relatos sobre as propriedades adjuvantes de flagelinas de bactérias gram-positivas. O objetivo do projeto foi estudar os efeitos imunomoduladores da flagelina Hag de B. subtílis. Inicialmente foi purificada a Hag e subseqüentemente avaliada sua propriedade imunológica quando administrada junto com a ovalmbumina (OVA) pelas vias intra-nasal (i.n.) ou subcutânea (s.c.) em camundongos Balb/C e C57BL/6. Os resultados demonstraram as propriedades imunomoduladoras da Hag nas duas linhagens em relação à indução de anticorpos antígeno-específicos. Não foi possível demonstrar a ativação de linfócitos TCD4+ e CD8+ antígenos-específicos em animais Balb/C imunizados com OVA e Hag tanto pela via s.c. como pela i.n.. Resultados mais promissores do efeito adjuvante frente a células T foram encontrados em animais C57BL/6. Os resultados abrem perspectivas para o estudo do potencial da flagelina Hag de B. subtilis como adjuvante vacinal. / Flagellin of gram negatives bacteria actives the innate immune system resulting in adjuvants effects against coadministrated antigens. However, there is not information about immunological properties of flagellins produced by gram positive bacteria. This study aimed the evaluation of immunomodulator effects of Hag flagellin from B. subtilis. Vaccine formulations based in purified Hag and Ovalbumine protein (OVA) were administrated intranasal (i.n.) and subcutaneously (s.c.) in Balb/C and C57BL/6 mice and then the population of B and T lymphocytes were monitored for production of antibodies and citocines and/or surface markers expression. Results showed the immunomodulatory properties of Hag in both mice lineages regarding the induction of antigen specific antibodies when immunized with OVA and Hag by i.n. and s.c. via. Most promising results concerning the adjuvant effects observed on T cells activation were found in C57BL/6 mice. The results obtained open future perspectives for the study of the potential use of B. subtilis Hag flagellin as adjuvant in vaccines.

La reconnaissance des cellules tumorales par le système immunitaire, appelée immunosurveillance, permet le contrôle de lacroissance des tumeurs voir dans certains cas leur élimination. Cependant, de nombreuses études d’exploration du système immunitaire dans le contrôle de la réponse antitumorale ont mis en évidence des mécanismes complexes d’échappement à cette immunosurveillance,avec par exemple pour les lymphocytes TCD8+ (LT CD8+) principal acteur de cette réponse, un défaut de migration, de reconnaissance des cellules tumorales ou d’activation au sein de la tumeur. Cette inhibition de fonction des LTCD8+, peut être liée à un phénomène appelé exhaustion, lié à l’expression à leur surface de molécules de costimulation inhibitrices telles que PD1, TIM-3, Lag3, CTLA-4. L’interaction de ces récepteurs avec leurs ligands engendre une perte de contrôle de la réponse antitumorale, favorisant alors la progression tumorale. Afin de lever ce phénomène d’exhaustion induit sur les LT CD8+ et restaurer la réponse antitumorale, plusieurs stratégies de traitements, visant à inhiber ces « checkpoints inhibiteurs » ont étédéveloppées. L’effet clé de la ciclosporine-A(CsA) repose sur la modulation de l’activité des lymphocytes T, ce qui explique son rôle dans la prévention du rejet de greffe. Cependant, il reste à déterminer si la CsA exerce d'autres effets sur le système immunitaire.Les évidences scientifiques montrent un effet paradoxal de la faible dosage de cyclosporine-A (fd-CsA). Ces résultats nous ont permis d’émettre l’hypothèse d’un rôle de la fd-CsA dans la modulation de la réponse antitumorale des LT CD8+, Nous avons pu observer à l’inverse, qu’à faible dose de ciclosporine-A, soit une dose équivalente entre 10 et 30 ng/mL, l’expression de PD1 était significativement diminuée à la surface des LT CD8+ activé. En revanche, à cette faible dose, aucun effet significatif sur la diminution d’expression du marqueur d’activation CD69 n’a été observé. Des expériences effectuées in vivo dans le modèle murin de mélanome B16F10 et MCA nous ont par ailleurs permis de montrer une réduction de la croissance tumorale chez les souris traitées par fd-CsA par rapport aux souris non traitées. En utilisant un modèle murin de fibrosarcome MCA, nous avons montré que nous restaurions in vivo une réponse immunitaire antitumorale et qu’un traitement de ces souris par fd-CsA en combinaison avec l’anti PDL-1 permettait une guérison après traitement, alors que l’anti PDL-1 seul n’avait pas d’effet. Cet effet novateur de la fd-CsA permet donc de visualiser de nouvelles stratégies thérapeutiques dans la réponse antitumorale qui pourraient bénéficier aux futurs patientsdiagnostiqués d’un cancer. / The recognition of tumor cells by the immune system, called immunosurveillance, allows the control of tumor growth or in some cases their elimination. However, numerous studies of the immune system in the control of the antitumor response have revealed complex mechanisms by means of which this immunosurveillance can be evaded. Examples of this occurrence are CD8+ T lymphocytes, the main element of this response are defective migration, tumor cell non-recognition or non-activation within the tumor. This inhibition of CD8+ T cell function may be related to a phenomenon called exhaustion, which may be a result of the expression on their surface of inhibitory costimulatory molecules such as PD1, TIM-3, Lag3, CTLA-4. The interaction of these receptors with their ligands causes a loss ofcontrol of the antitumor response, thus promoting tumor progression. To overcome this phenomenon of exhaustion induced in CD8+ T cells and restore the antitumor response, several treatment strategies aimed at inhibiting these "inhibitory checkpoints" have been developed. The key effect of cyclosporin-A (CsA) is modulation of T-cell activity, which explains its role in the prevention of transplantrejection. However, it remains to be determined whether CsA has other effects on the immune system. Preliminary results have allowed us to demonstrate the paradoxical effect of cyclosporin-A (CsA) in the antitumor response. Thus, these unexpected results have allowed us to hypothesize a role for Ld-CsA in modulating the antitumor response of CD8+ Tcells. We observed that Ld-CsA at equivalent dose between 10 and 30 ng/mL, significantly decreased PD1 expression at the activated CD8+ T cell surface. Furthermore, at this lowdose, no significant effect was observed on the expression of the CD69 activation marker. We have also shown that Ld-CsA increases the vaccine response in vivo. In vivo experiments with the murine B16F10 melanoma model and MCA fibrosarcoma have also shown areduction in tumor growth in mice treated with d-CsA compared to untreated mice. More recently, using a mouse model of MCA-OVAfibrosarcoma, we have shown that we can restore the in-vivo antitumor immune response and that the treatment of these mice by Ld-CsAin combination with the anti PDL-1, allowed tumor regression, whereas anti PDL-1 alone had no effect. This novel effect of Ld-CsA allows us therefore to visualize new therapeutic strategies for the antitumor response which may benefit future patients diagnosed with cancer.