O objetivo deste estudo foi analisar a expressão e o padrão de distribuição de linfócitos citotóxicos (LCs) e linfócitos T regulatórios (LTregs) no tecido gengival de pacientes com doença periodontal através de análise imunoistoquímica. Foram selecionados 30 pacientes (10 por grupo) com diagnóstico de periodontite agressiva (PA), periodontite crônica (PC) e gengiva clinicamente saudável (controle); dos quais foi colhida uma amostra de tecido gengival. A distribuição das células (epitélio e córion) foi identificada usando os imunomarcadores CD56, CD57, Granzima B e Perforina (LCs); CD4, CD25, FOXP3 (LTregs). A imunoexpressão foi avaliada, utilizando representação de imagem por meio de um sistema computadorizado, constituído por microscópio de luz, adaptado a uma câmera de alta resolução. Contagens independentes de 10 campos separados para cada caso foram feitas. Two-way ANOVA e posterior teste de Fisher foram utilizados para observar diferenças entre os diagnósticos e os marcadores; e teste t de Student para observar diferenças entre epitélio e córion (p<0.05). Os resultados indicaram que pacientes com PA e PC apresentaram um número significantemente maior de células CD56+ e CD57+, em relação ao grupo controle, porém sem diferenças entre si; um número significantemente maior de células CD56+ e CD57+ foi observado em relação às células Granzima B+ e Perforina+ em todos os pacientes. Em relação aos LTregs, o número de células CD25+ e FOXP3+, foi significativamente diferente entre PA, PC e controle, aparecendo em maior número na PC. Células CD4+ foram observadas em número similar em pacientes com PA e PC, diferindo significantemente do grupo controle; em pacientes com PA e PC, foi observado um número significantemente maior de CD4+, em relação às células CD25+ e FOXP3+. Pacientes com PA e PC tem maior número de LCs no tecido gengival em relação ao grupo controle sugerindo a participação destas células na patogênese da PA e PC. Pacientes com PA apresentaram menor número de LTregs no tecido gengival em comparação aos pacientes com PC, sugerindo que estas células podem estar envolvidas no mecanismo de regulação do processo inflamatório e reabsorção óssea. / This project aims to observe the expression and distribution of cytotoxic lymphocytes (LCs) and regulatory T lymphocytes (LTregs) in gingival tissue from periodontal disease affected patients through immunohistochemical analysis. 30 patients (10 per group) diagnosed with aggressive periodontitis (PA), chronic periodontitis (PC) and clinically healthy gingiva (control) were selected; from which a sample of gingival tissue was collected. The distribution of cells (epithelium and chorion) was identified using the immunomarkers CD56, CD57, Granzyme B, Perforin (LCs); CD4, CD25, FOXP3 (LTregs). The immunoexpression was assessed using image representation by a computer system, comprising a light microscope adapted to a high resolution camera. Independent counts of 10 separate fields for each case were done. Two-way ANOVA and posterior Fisher´s Test were used to observe differences between diagnostics and immunomarkers; and unpaired Student t test to observe differences between epithelium and chorium (p<0.05). The results indicates that patients with PA and PC presented a significantly higher number of CD56+ and CD57+ cells, in relation to control, but without differences between each other; a significantly higher number of CD56+ and CD57+ cells was observed in relation to Granzyme B and Perforine cells in all patients. Related to the LTregs, the number of CD25+ and FOXP3+ cells was significantly different between PA, PC and control, appearing in greater number in PC. CD4+ cells were observed in similar number in patients with PA and PC, it was observed a significantly higher number of CD4+, in relation to CD25+ and FOXP3+ cells. Patients with PA and PC have a greater number of LCs in gingival tissue in relation to control group - suggesting the participation of this cells in the pathogenesis of PA and PC. Patients with PA presented less LTregs in gingival tissue when compared to PC patients, suggesting that this cells may be involved in the regulatory mechanism of the inflammatory process and bone resorption.

O objetivo deste estudo foi analisar a expressão e o padrão de distribuição de linfócitos citotóxicos (LCs) e linfócitos T regulatórios (LTregs) no tecido gengival de pacientes com doença periodontal através de análise imunoistoquímica. Foram selecionados 30 pacientes (10 por grupo) com diagnóstico de periodontite agressiva (PA), periodontite crônica (PC) e gengiva clinicamente saudável (controle); dos quais foi colhida uma amostra de tecido gengival. A distribuição das células (epitélio e córion) foi identificada usando os imunomarcadores CD56, CD57, Granzima B e Perforina (LCs); CD4, CD25, FOXP3 (LTregs). A imunoexpressão foi avaliada, utilizando representação de imagem por meio de um sistema computadorizado, constituído por microscópio de luz, adaptado a uma câmera de alta resolução. Contagens independentes de 10 campos separados para cada caso foram feitas. Two-way ANOVA e posterior teste de Fisher foram utilizados para observar diferenças entre os diagnósticos e os marcadores; e teste t de Student para observar diferenças entre epitélio e córion (p<0.05). Os resultados indicaram que pacientes com PA e PC apresentaram um número significantemente maior de células CD56+ e CD57+, em relação ao grupo controle, porém sem diferenças entre si; um número significantemente maior de células CD56+ e CD57+ foi observado em relação às células Granzima B+ e Perforina+ em todos os pacientes. Em relação aos LTregs, o número de células CD25+ e FOXP3+, foi significativamente diferente entre PA, PC e controle, aparecendo em maior número na PC. Células CD4+ foram observadas em número similar em pacientes com PA e PC, diferindo significantemente do grupo controle; em pacientes com PA e PC, foi observado um número significantemente maior de CD4+, em relação às células CD25+ e FOXP3+. Pacientes com PA e PC tem maior número de LCs no tecido gengival em relação ao grupo controle sugerindo a participação destas células na patogênese da PA e PC. Pacientes com PA apresentaram menor número de LTregs no tecido gengival em comparação aos pacientes com PC, sugerindo que estas células podem estar envolvidas no mecanismo de regulação do processo inflamatório e reabsorção óssea. / This project aims to observe the expression and distribution of cytotoxic lymphocytes (LCs) and regulatory T lymphocytes (LTregs) in gingival tissue from periodontal disease affected patients through immunohistochemical analysis. 30 patients (10 per group) diagnosed with aggressive periodontitis (PA), chronic periodontitis (PC) and clinically healthy gingiva (control) were selected; from which a sample of gingival tissue was collected. The distribution of cells (epithelium and chorion) was identified using the immunomarkers CD56, CD57, Granzyme B, Perforin (LCs); CD4, CD25, FOXP3 (LTregs). The immunoexpression was assessed using image representation by a computer system, comprising a light microscope adapted to a high resolution camera. Independent counts of 10 separate fields for each case were done. Two-way ANOVA and posterior Fisher´s Test were used to observe differences between diagnostics and immunomarkers; and unpaired Student t test to observe differences between epithelium and chorium (p<0.05). The results indicates that patients with PA and PC presented a significantly higher number of CD56+ and CD57+ cells, in relation to control, but without differences between each other; a significantly higher number of CD56+ and CD57+ cells was observed in relation to Granzyme B and Perforine cells in all patients. Related to the LTregs, the number of CD25+ and FOXP3+ cells was significantly different between PA, PC and control, appearing in greater number in PC. CD4+ cells were observed in similar number in patients with PA and PC, it was observed a significantly higher number of CD4+, in relation to CD25+ and FOXP3+ cells. Patients with PA and PC have a greater number of LCs in gingival tissue in relation to control group - suggesting the participation of this cells in the pathogenesis of PA and PC. Patients with PA presented less LTregs in gingival tissue when compared to PC patients, suggesting that this cells may be involved in the regulatory mechanism of the inflammatory process and bone resorption.

Les hypersensibilités retardées (HSR) cutanées sont hétérogènes, à la fois par la nature des mécanismes impliqués (allergiques versus non allergiques) mais aussi par les différents degrés de sévérité rencontrés. Seules les HSR allergiques sont dues à la présence de lymphocytes T (LT), mal caractérisés chez l'homme. Le but de ce travail est d'étudier la contribution des LT CD8 cytotoxiques dans le développement et la sévérité des HSR cutanées chez l'homme, à partir de deux pathologies fréquentes : les toxidermies aux béta lactamines et l'eczéma allergique de contact à la para-phénylènediamine (PPD). Tout d'abord, la présence de LT spécifiques de médicament au sein des toxidermies aux béta lactamines a été recherchée in vivo et in vitro. Nous avons montré que les HSR sévères étaient plus souvent d'origine allergique que les HSR bénignes. Nous avons ensuite caractérisé le rôle des LT CD8 dans les HSR allergiques. Dans les toxidermies bénignes à l'amoxicilline, l'étude de la cinétique de recrutement des LT au niveau cutané ainsi que l'analyse des LT spécifiques du sang circulant ont permis de mettre en évidence le rôle essentiel des LT CD8 cytotoxiques dans l'initiation de ces réactions. Ensuite, dans l'eczéma allergique de contact à la PPD, un recrutement épidermique précoce des LT CD8 associés à des marqueurs de cytotoxicité, a été retrouvé, corrélé avec la sévérité des lésions. Ces résultats ont été confortés par ceux obtenus dans un modèle pré-clinique d'HSR allergique à la PPD chez la souris. En conclusion, ce travail montre que les LT CD8 cytotoxiques pourraient être les principales cellules effectrices des HSR cutanées allergiques chez l'homme / Skin delayed hypersensitivity (DHS) are heterogeneous, by the nature of the mechanisms involved (allergic versus non allergic) and also by their different degrees of severity. Only allergic DHS is due to T cells, poorly characterized in humans. The aim of this work is to study the contribution of cytotoxic CD8 T cells in the development and severity of skin DHS in humans, induced by two common diseases: cutaneous adverse drug reactions to beta lactam antibiotics and allergic contact dermatitis to para-phenylenediamine (PPD). First, the presence of drug specific T cells in cutaneous adverse drug reactions to beta lactams was investigated in vivo and in vitro. We showed that severe DHS were more often allergic than benign DHS. Then, we characterized the role of CD8 T cells in allergic DHS. In benign cutaneous adverse drug reactions to amoxicillin, the study of the kinetics of skin T cell recruitment as well as the analysis of circulating specific T cells highlight the essential role of cytotoxic CD8 T cells in the initiation phase of these reactions. In allergic contact dermatitis to PPD, early recruitment of epidermal CD8 T cells associated with cytotoxic markers was found, correlated with the severity of lesions. These results were supported by those obtained in a mouse model of allergic contact dermatitis to PPD. In conclusion, this work showed that cytotoxic CD8 T cells could be the main effector cells of allergic skin DHS in humans

Comparatively, HIV like most viruses is very minute, unadorned organism which cannot reproduce unaided. It remains the most deadly disease which has ever hit the planet since the last three decades. The spread of HIV has been very explosive and mercilessly on human population. It has tainted over 60 million people, with almost half of the human population suffering from AIDS related illnesses and death finally. Recent theoretical and computational breakthroughs in delay differential equations declare that, delay differential equations are proficient in yielding rich and plausible dynamics with reasonable parametric estimates. This paper seeks to unveil the niche of delay differential equation in harmonizing low HIV viral haul and thereby articulating the adopted model, to delve into structured treatment interruptions. Therefore, an ordinary differential equation is schemed to consist of three components such as untainted CD4+ T-cells, tainted CD4+ T-cells (HIV) and CTL. A discrete time delay is ushered to the formulated model in order to account for vital components, such as intracellular delay and HIV latency which were missing in previous works, but have been advocated for future research. It was divested that when the reproductive number was less than unity, the disease free equilibrium of the model was asymptotically stable. Hence the adopted model with or without the delay component articulates less production of virions, as per the decline rate. Therefore CD4+ T-cells in the blood remains constant at 𝛿1/𝛿3, hence declining the virions level in the blood. As per the adopted model, the best STI practice is intimated for compliance. / Mathematical Sciences / Ph.D. (Applied Mathematics)

A histiocitose de células de Langerhans (HCL) é uma neoplasia mieloide inflamatória comumente afetando pacientes pediátricos e apresenta frequentemente mutações ativadoras somáticas em genes da via MAPK, incluindo BRAF e MAP2K1. Vários estudos sugerem que as células lesionais da HCL podem recrutar e modular células inflamatórias e cujas citocinas parecem fornecer sinais recíprocos de sobrevivência celular. Para o presente estudo foram selecionados 15 casos de HCL (10 crianças, 5 adultos), sendo as amostras de tecido avaliadas através de imunoistoquímica utilizando marcadores para macrófagos (CD68 e CD163), células dendríticas maduras (CDm) (CD83 e CD208), linfócitos T regulatórios (LTregs) (CD4, CD25 e FOXP3) e linfócitos citotóxicos (LCs) (CD56, CD57, perforina e granzima B). Além disso, marcadores de células B (CD20), células T (CD3, CD8) e confirmatórios de HCL foram analisados. Todos os casos de HCL foram positivos para S100, CD1a, CD207 e CD4; enquanto que Bcl-2 e Ciclina D1 foram positivos em 13/15 (86,7%) casos. No microambiente imune intralesional, macrófagos M2 (CD68+/CD163+), seguidos por LTregs, foram as populações celulares mais predominantes. Em quantidade significativamente menor, foram observadas CDm, seguidas por escassos LCs. Considerando a população linfoide, linfócitos T CD3+ foram mais numerosos do que linfócitos B CD20+. Dentro dos linfócitos T, linfócitos T CD4+ foram mais numerosos do que linfócitos T CD8+ (p<0,05). Nossos resultados sugerem que a infiltração de células imunes na HCL, provavelmente através de mecanismos pró-tumorais, inflamatórios e/ou imunossupressores mediados por citocinas, pode promover o desenvolvimento e sobrevivência das células lesionais da HCL, fornecendo uma justificativa para a combinação de imunoterapia e terapia gênica (BRAF) na HCL / Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia often affecting children with constitutively somatic activating mutations in MAPK pathway genes including BRAF and MAP2K1. Several studies suggest that LCH cells can recruit and modulate inflammatory cells and whose cytokines appear provide reciprocal survival signals. For the present study, 15 cases of LCH (10 children, 5 adults) were selected, and the tissue samples were evaluated through immunohistochemistry using markers for macrophages (CD68 and CD163), mature dendritic cells (mDC) (CD83 and CD208), regulatory T-cells (Tregs) (CD4, CD25 and FOXP3) and cytotoxic lymphocytes (CLs) (CD56, CD57, perforin and granzime B). Moreover, B-cell (CD20), T-cell (CD3, CD8) and LCH markers were analyzed. All LCH cases were positive for S100, CD1a, CD207 and CD4, while Bcl-2 and Cyclin D1 were positive in 13/15 cases (86.7%). In the immune microenvironment, M2-polarized macrophages (CD68+/CD163+), followed by LTregs, were the predominant cell populations. In a significantly lower amount, mDC were observed, followed by scarce CLs. Moreover, CD3+ Tcells than CD20+ B-cells were more numerous (p>0.05), the former presenting a higher number of CD4+ than CD8+ T-cells (p<0.05). Our results suggest that immune cell infiltration in LCH, probably through cytokine-mediated pro-tumoral, inflammatory and/or immunosupressive mechanisms, can promote LCH cell development and survival, providing a rationale for combining immunotherapy and BRAF-targeted therapy in LCH

Le dysfonctionnement de l'exocytose des granules cytotoxiques est responsable d'une susceptibilité accrue aux pathogènes intracellulaires qui s'accompagne de l'activation continue et anarchique des lymphocytes cytotoxiques et des macrophages. Ce phénomène conduit à la lymphohistiocytose hémophagocytique (HLH), un syndrome auto-inflammatoire fatal en absence d'intervention thérapeutique. Les mutations des gènes codant pour la perforine (PRF-1) ou pour certaines des protéines impliquées dans la biogénèse ou le transport vésiculaire des granules cytotoxiques sont causales des formes familiales ou primaires de la HLH (FHL). La HLH fait également partie des complications secondaires aux infections à herpesviridae et à certains désordres immunologiques importants tels que l'arthrite juvénile idiopathique (SoJIA). Au moment d'entreprendre les travaux présentés dans ce manuscrit, le premier cas de HLH induite par une mutation menant à l'activation constitutive de la composante NLRC4 de l'inflammasome était décrit. L'inflammasome est une structure multimérique composée d'un récepteur cytosolique, de la protéine échafaud ASC et de la Caspase-1. Son activation mène à la maturation des pro-formes de l'IL-1β et l'IL-18 ainsi qu'à leur sécrétion. L'activation constitutive de NLRC4 étant suffisante au déclenchement de la HLH, nous avons tenté de comprendre si cette structure y était essentielle dans le cadre des défauts génétiques de cytotoxicité. Nous avons donc invalidé la protéine ASC ou Caspase-1 dans le modèle murin de HLH déficient pour la perforine (PRF1 -/-). Nous avons également testé l'hypothèse qu'un déficit de cytotoxicité pouvait expliquer le développement de la HLH chez les patients souffrant de SoJIA. Nos résultats montrent que l'inflammasome est nécessaire à la production d'IL-18 lors de la HLH mais qu'il n'est pas essentiel au développement de la maladie dans le cadre des FHL. Par ailleurs, nous montrons que la cytotoxicité des cellules NK semble normale chez les patients atteints de SoJIA, ce qui suggère que les mécanismes immunologiques à l'origine de la HLH dans les FHL et dans les maladies autoinflammatoires comme la SoJIA sont distincts. Dans la seconde partie de ce manuscrit, nous avons étudié sur le rôle de la sérine/thréonine/tyrosine kinase Styk1 dans la régulation des lymphocytes cytotoxiques NK. Ces derniers sont responsables du contrôle immunitaire précoce des pathogènes intracellulaires et contribuent à l'immunosurveillance des cellules tumorales. Suite à leur activation, ils relâchent de très grandes quantités d'IFN-y et de TNF-α, faisant ainsi le lien entre l'immunité innée et adaptative. La reconnaissance des cellules cibles par les lymphocytes NK est gouvernée par l'expression d'un éventail de récepteurs qui transduisent des signaux, activateurs ou inhibiteurs, et dont la balance se traduit par l'activation ou la tolérance. Ces récepteurs sont codés au sein de deux complexes génétiques très denses, le complexe de cytotoxicité naturelle (NCR) et le complexe des récepteurs des leucocytes (LRC). Au moment de commencer ces travaux, nous avions révélé que l'expression de la kinase Styk1 fait partie de la signature transcriptionnelle des lymphocytes NK. Sa fonction dans le système immunitaire demeure toutefois inconnue. Néanmoins, la localisation génétique favorable de Styk1, près du NCR, ainsi que son implication dans la voie PI3K-AKT, en faisaient un candidat potentiel de régulation des lymphocytes NK. Afin de connaître le rôle de Styk1 dans le développement et les fonctions effectrices des lymphocytes NK, nous avons donc généré une souris pour laquelle Styk1 est invalidé. Nos résultats confirment que Styk1 est exprimée de façon spécifique par les cellules NK. Nous avons également détecté une diminution de l'activité constitutive de la voie AKT/mTOR dans les cellules NK, mais le développement, l'homéostasie et la fonction des cellules NK sont cependant normaux dans les souris déficientes en Styk1 / Upon recognition of infected or target cells, CD8+ T and Natural Killer (NK) lymphocytes initiate a polarized degranulation of vesicle storing cytotoxic molecules (perforin: PRF1 and granzyme B). By altering the target cell’s cellular membrane integrity, perforine allows granzyme B to translocate to its cytosol. Genetic anomalies may affect normal cytotoxic functions and severely hamper the control of intracellular pathogens. In this context, the pathogenic signal remains uninterrupted and both cytotoxic lymphocytes and macrophages are continuously stimulated. This auto-inflammatory pathological condition is named hemophagocytic lymphohistiocytosis (HLH) and is fatal without therapeutic intervention. HLH can also occur secondary to infection with viruses from the herpesviridae family, or be concomitant to important immune alterations such as systemic onset juvenile idiopathic arthritis (SoJIA), with no clear etiological cause identified. In 2014, a case of HLH mediated by the constitutive activation of the NLRC4 inflammasome receptor was first described. The inflammasome is a multimeric structure involving a cytosolic receptor, a scaffold protein – ASC – and Caspase-1. In the immune system, the inflammasome is expressed in macrophages and dendritic cells and senses pathogenic (PAMP) and danger (DAMP) associated signals. Once activated, inflammasome’s protein Caspase-1 catalyzes the maturation of pro-IL-1b and pro-IL-18 and leads to their secretion. Since NLRC4 constitutive activation appears to be sufficient for triggering HLH, we aimed to understand if the inflammasome structure was essential to the development of the syndrome. In order to address this question, we invalidated the inflammasome through the abrogation of ASC or Caspase-1 in PRF1 -/- HLH mouse model. We also tested the hypothesis that an altered cytotoxic function could explain the high prevalence of HLH in the proinflammatory context of SoJIA. The results we present here show that the inflammasome is responsible for the elevated levels of IL-18 in the serum of HLH patients. However, the inflammasome is facultative for its development. We also demonstrate that in patients suffering from SoJIA, NK cells show normal cytotoxicity, suggesting that immunological mechanisms involved in FHL and secondary HLH are distinct. In the second part of this manuscript, we aim at understanding the role of Styk1 serine/threonine/tyrosine kinase in NK cells’ regulation. NK cells are in charge of eliminating stressed, virally infected or transformed cells. Upon activation, they secrete large amounts of IFN-γ and TNF-α, thus bridging innate and adaptive immunity. Capabilities for recognition of target cells is endowed by the expression of numerous stochastically expressed activating and inhibitory receptors. The balance between activating and inhibitory signal allows for self-tolerance or activation upon engagement of abnormal cells. Activating and inhibitory receptor are germline encoded in two dense, large complexes, the Natural Killer Complex (NKC) and the Leukocyte Receptor Complex (LRC). At the moment of starting this work, we had recently identified that Styk1 was a signature transcript of NK cells. However, its function in NK cells and more generally in the immune system remains unknown. Nevertheless, its genetic localisation near the NKC and its potential implication in the PI3K-AKT pathway prompt that it may play a role in NK cell development and/or functions. In order to evaluate the role of Styk1 in NK cells’ regulation, we generated a mouse model in which its expression is abrogated. Our data confirms that amongst all immune subsets, Styk1 is specifically expressed by NK cells. Styk1 was also able to discriminate NK cells from other ILCs. In this study, we show that Styk1 invalidation lead to a decrease of activity in the AKT/mTOR pathway. However, NK cells development, homeostasis and function were surprisingly normal in Styk1 -/- mice