The immune system is a complex network of interacting cells and tissues that is designed to protect the body from pathogens and other foreign substances. T cells are a major component of the immune system and consist of two distinct lineages distinguished by the expression of αβ or γδ T cell receptors (TCR). The Tec family kinase, Itk is an important mediator of signaling downstream of the TCR. Past studies on Itk has focused on how Itk regulates development, activation and differentiation of conventional αβ T cells and more recently how Itk regulates the development of innate-like αβ T cells. However, very little is known about the influence of Itk on γδ T cells. My studies show a previously unknown role for Itk in the development and function of γδ T cells. We report in the absence of Itk, γδ T cells were responsible for the spontaneously elevated levels of serum IgE and Itk-/- mice γδ T cells produced high levels of TH2 cytokines. Furthermore, there was an increase in γδ T cells specifically in the Vγ1.1+Vδ6.3+ (V6) subset that represents the dominant population of γδ NKT cells in Itk-/- mice. In addition, the V6 subset had increased expression of PLZF, a transcription factor normally required for αβ iNKT cell development. We further show that V6 cells develop and mature similar to αβ iNKT cells. Similar to defects previously seen in the terminal differentiation of Itk-/- αβ iNKT cell, V6 cells also had impaired maturation in the thymus in the absence of Itk. This data demonstrates a previously unknown role of Itk for the terminal maturation of V6 cells that has been shown to be the cell population that led to spontaneous dermatitis in mice. Given that drug companies have targeted Itk as a potential allergy drug due to Itk’s role in TH2 development and function, our data suggests that further studies on Itk are warranted.

A HAM/TSP é uma doença de caráter neurológico que acomete aproximadamente 5% dos infectados pelo vírus HTLV, afetando-os principalmente em sua capacidade locomotora. Não são conhecidos até o momento os fatores que fazem o indivíduo infectado desenvolver ou não as patologias associadas ao vírus. Nesse contexto, estudamos a participação das células natural killer T (NKT) visando contribuir na elucidação da patogênese da paraparasia espática tropical HAM/TSP. As células natural killer T (NKT) são um subtipo de linfócitos T, com capacidade efetora e auxiliar, e está associado à doenças infecciosas, alergias e a doenças autoimunes, e a hipótese deste trabalho é que elas participem da progressão da HAM/TSP. Após realizarmos testes para avaliação da ativação das NKT (PD-1), bem como avaliar sua capacidade de secretar IFN- y e Granzima B concluimos que as mesmas não possuem potencial de ativação na periferia e pressupomos que estas podem estar no sítio acometido pela patologia, o SNC. Neste contexto, nosso estudo acrescenta informações quanto à patogenia da HAM/TSP e contribui com conhecimento para a futura elucidação da doença / The HAM/TSP is a neurological disease that affects approximately 5% of HTLV infected individuals, especially in their locomotor capacity. They are not known yet the factors that make the infected individual to develop or not the pathologies associated with virus. In this context, we study the participation of natural killer T cells (NKT), trying to contribute to clarify the HAM/TSP pathogenesis. NKT cells are a subtype of lymphocytes with effector and helper capacities, and are associated with infectious, allergies and autoimmune diseases. Our hypothesis is that they participate in the progression of HAM/TSP. After evaluate the activation of NKT through PD-1 marker, as well evaluate assess their ability to secrete IFN-y and Granzyme B, we conclude that the NKT cells lack periphery in activation potential, and presuppose that they are present at the central nervous system, the site affected by the disease. In this context, our study adds information on the pathogenesis of HAM / TSP and contributes knowledge for future elucidation of disease

A HAM/TSP é uma doença de caráter neurológico que acomete aproximadamente 5% dos infectados pelo vírus HTLV, afetando-os principalmente em sua capacidade locomotora. Não são conhecidos até o momento os fatores que fazem o indivíduo infectado desenvolver ou não as patologias associadas ao vírus. Nesse contexto, estudamos a participação das células natural killer T (NKT) visando contribuir na elucidação da patogênese da paraparasia espática tropical HAM/TSP. As células natural killer T (NKT) são um subtipo de linfócitos T, com capacidade efetora e auxiliar, e está associado à doenças infecciosas, alergias e a doenças autoimunes, e a hipótese deste trabalho é que elas participem da progressão da HAM/TSP. Após realizarmos testes para avaliação da ativação das NKT (PD-1), bem como avaliar sua capacidade de secretar IFN- y e Granzima B concluimos que as mesmas não possuem potencial de ativação na periferia e pressupomos que estas podem estar no sítio acometido pela patologia, o SNC. Neste contexto, nosso estudo acrescenta informações quanto à patogenia da HAM/TSP e contribui com conhecimento para a futura elucidação da doença / The HAM/TSP is a neurological disease that affects approximately 5% of HTLV infected individuals, especially in their locomotor capacity. They are not known yet the factors that make the infected individual to develop or not the pathologies associated with virus. In this context, we study the participation of natural killer T cells (NKT), trying to contribute to clarify the HAM/TSP pathogenesis. NKT cells are a subtype of lymphocytes with effector and helper capacities, and are associated with infectious, allergies and autoimmune diseases. Our hypothesis is that they participate in the progression of HAM/TSP. After evaluate the activation of NKT through PD-1 marker, as well evaluate assess their ability to secrete IFN-y and Granzyme B, we conclude that the NKT cells lack periphery in activation potential, and presuppose that they are present at the central nervous system, the site affected by the disease. In this context, our study adds information on the pathogenesis of HAM / TSP and contributes knowledge for future elucidation of disease

Les cellules invariantes « natural killer » T (iNKT) constituent une population particulière de LT non conventionnelle qui exprime un récepteur TCRαβ semi-invariant composé de la chaine Vα14-Jα18 associée aux chaines Vβ8, -7 ou -2 chez la souris et dont le développement a lieu dans le thymus. Ainsi, les cellules iNKT sont capables de reconnaitre via leur TCR des antigènes de type glycolipidique présentés par une molécule de classe I non polymorphique : le CD1d. Ces cellules sont connues pour être impliquées dans diverses réponses immunes grâce à leur capacité à produire rapidement des cytokines immunorégulatrices. De la même façon que les LTc SP CD4+, il existe trois phénotypes de cellules iNKT : Th1, -2 et -17 permettant de distinguer trois sous-populations de cellules iNKT. La sous-population iNKT1 dite iNKT conventionnelle exprime des récepteurs appartenant au lignage NK. Cette sous-population est localisée préférentiellement dans le foie, le thymus et la rate et produit majoritairement de l’IFN-. La sous-population iNKT2, qui reste jusqu’à aujourd’hui insuffisamment décrite, se localise préférentiellement dans les poumons et produit majoritairement de l’IL-4 et de l’IL-13. La sous-population iNKT17 a été caractérisée et mise en évidence au sein de notre laboratoire comme une sous-population de cellules iNKT exprimant le facteur de transcription RORt et capable de sécréter de l’IL-17 en réponse à l’IL-1 et l’IL-23 et se localisant principalement dans les ganglions périphériques et la peau. A ce jour, seul le développement des cellules iNKT conventionnelles est bien connu tandis que celui des cellules iNKT17 demeurent ignorés. Ainsi, ayant remarqué une faible proportion des cellules iNKT17 dans le thymus de la souris C57BL/6 comparées aux autres sous-populations de cellules iNKT, nous nous sommes intéressés dans un premier temps à expliquer les causes de cette faible distribution de cette sous-population, ainsi qu’à définir la séquence d’acquisition de ses marqueurs lors de son développement thymique et de sa migration en périphérie. Les résultats montrent que ces cellules n’ont aucun défaut de prolifération, ni de réponse aux cytokines de l’homéostasie permettant d’expliquer leur plus faible nombre. En revanche, nous avons constaté une absence d’accumulation thymique de ces cellules qui ont la capacité de migrer en périphérie, accompagnée d’une sensibilité plus accrue à la mort par apoptose et une diminution de l’expression des facteurs de survie comme le Bcl-2 pouvant ainsi expliquer leur nombre réduit. Les analyses de leur développement aux stades précoces ont montré un biais préétabli de leur faible nombre observable dès le stade CD44-. L’étude de leur ontogénique a permis de montrer une cinétique d’acquisition séquentielle des marqueurs CCR6 et CD138 permettant d’établir pour la première fois un modèle de maturation thymique de cette sous-population iNKT17 qui était encore inconnue. Ainsi, au stade précoce HSAhigh, les cellules iNKT RORt+ observé correspondent à des précurseurs communs des cellules iNKT et non pas à des précurseurs des cellules iNKT17 montrant que la différenciation de ces cellules ne se fait pas au stade de sélection positive et que leur faible nombre dépend des signaux que ces cellules reçoivent lors de leur engagement vers le lignage “Th17 like“. / Invariant natural killer cells T (iNKT) constitute a particular population of unconventional LT which expresses a semi-invariant TCRαβ receptor composed of the Vα14-Jα18 chain associated with the Vβ8, -7 or -2 chains in mice and which develops in the thymus. Thus, iNKT cells are able to recognize glycolipid antigens via their TCR presented by a non-polymorphic class I molecule: CD1d. These cells are known to be involved in various immune responses because of their ability to rapidly produce cytokines. However, like conventional SP T CD4+ lymphocytes, iNKT cells can differentiate into three phenotypes: Th1, -2 and -17. The iNKT1 subset also named conventional iNKT cells expresses receptors belonging to the NK lineage, is mainly located in the liver, thymus and spleen and produces mainly IFN-. The iNKT2 subset which until now remains insufficiently described, is localized preferentially in the lungs and produces mainly IL-4 and IL-13. The iNKT17 subset has been characterized in our laboratory as a subset of iNKT cells expressing the RORt transcription factor and capable of secreting IL-17 in response to IL-1 and IL-23 and located mainly in the peripheral lymph nodes and the skin. To date, only the development of conventional iNKT cells is well known while that of iNKT17 cells remains unknown. Thus, having noticed the low distribution of the iNKT17 cells present in the thymus of the C57BL/6 mouse compared to other iNKT cell subset, we were initially interested in explaining the causes of this poor distribution of this subset, as well as to define the acquisition sequence of its markers during its thymic development and peripheral migration. The results show that these cells have no defect of proliferation or response to cytokines of homeostasis that can explain their lower number in the thymus. In contrast, we found a lack of thymic accumulation of these cells that have the ability to migrate peripherally, accompanied by increased sensitivity to death by apoptosis and decreased expression of survival factors such as Bcl-2 which can explain their reduced number. Analyzes of their development at early stages showed a pre-established bias of their low number from the CD44- stage. The study of their ontogeny has shown a sequential acquisition kinetics of CCR6 and CD138 markers to establish for the first time a model of thymic maturation of this iNKT subset which was still unknown.

Metastatic breast cancer treatment has seen few advances in recent years, yet treatment resistance continues to rise, causing disease recurrence. A pilot study was performed to determine the efficacy of ex vivo expansion and reprogramming of tumor-reactive immune cells from experimental metastatic tumor-sensitized mice. Also, phenotypic changes in tumors due to metastasis or tumor microenvironment influences were characterized. Metastatic neu+ mouse mammary carcinoma (mMMC) and its distant relapsing neu-antigen-negative variant (mANV) were investigated in FVBN202 mice. Tumor-reactive central memory CD8+ T cells and activated NK/NKT cells were successfully reprogrammed and expanded during 6-day expansion from mMMC- and/or mANV-sensitized mice, resulting in tumor-specific cytotoxicity. mMMC exhibited a flexible neu-expression pattern and acquired stem-like, tumorigenic phenotype following metastasis while mANV remained stable except decreased tumorigenicity. Myeloid-derived suppressor cell (MDSC) levels were not increased. Adoptive cellular therapy (ACT) with reprogrammed tumor-reactive immune cells may prove effective prophylaxis against metastatic or recurrent breast cancer.