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<p>Linker for Activation of T cells, LAT, is a transmembrane adaptor protein that is vital for integrating TCR-mediated signals that modulate T cell development, activation, and proliferation. Upon engagement of the T cell receptor, LAT is phosphorylated and associates with Grb2, Gads, and PLC&#947;1 through its four distal tyrosine residues. Mutation of tyrosine 136 abolishes LAT binding to PLC&#947;1. This results in impaired TCR-mediated calcium mobilization and Erk activation. LATY136F knock-in mice have a severe but incomplete block in T cell development. Yet, CD4+ &#945;&#946; T cells undergo uncontrolled expansion in the periphery, resulting in a severe autoimmune syndrome characterized by Th2 skewing and resultant B cell autoreactivity. Here, we further studied the role of LAT-PLC&#947;1 signaling in T cell lineage commitment, cytokine production, and autoimmunity.</p><p>First, we investigated the importance of the LAT-PLC&#947;1 interaction in &#947;&#948; T cells by crossing LATY136F mice with TCR&#946;-deficient mice. Our data showed that the LATY136F mutation had no major effect on the homeostasis of epithelial &#947;&#948; T cells, which could be found in the skin and small intestine. Interestingly, a population of CD4+ &#947;&#948; T cells in the spleen and lymph nodes underwent continuous expansion and produced elevated amounts of IL4, resulting in an autoimmune syndrome similar to that caused by &#945;&#946; T cells in LATY136F mice. Development of these hyperproliferative &#947;&#948; T cells was not dependent on expression of MHC class II or CD4, and their proliferation could be partially suppressed by regulatory T cells. Our data indicated that a unique subset of CD4+ &#947;&#948; T cells could hyperproliferate in LATY136F mice and suggested that LAT-PLC&#947;1 signaling may function differently in various subsets of &#947;&#948; T cells. </p><p>In addition to examining &#947;&#948; and &#945;&#946; T cell development, we also were interested in further exploring the role of LAT in cytokine production. While our previous data have demonstrated that T cells in LATY136F mice are Th2 skewed, producing large amounts of IL4, we investigated other cytokines that may be important for autoimmunity and found that these CD4+ &#945;&#946; T cells could also produce the proinflammatory cytokine IL6. Analysis of whole cell lysates from CD4+ &#945;&#946; LATY136F T cells demonstrated that NF&#954;B, AKT, and p38 were constitutively phosphorylated, and inhibition of these pathways resulted in reduced IL6 production. By crossing LATY136F mice with IL6 deficient mice, we demonstrated that early T cell survival was diminished in the absence of IL6. We further showed that this reduced CD4+ T cell pool was not due to further blocks in development, or an increase in FoxP3+ regulatory T cells. Finally, we demonstrated that over time, CD4+ T cells do hyperproliferate, yet B cell class switching and autoreactivity remains low. Our data uncovered a novel role for LAT-PLC&#947;1 signaling in regulating IL6 production by T cells during autoimmunity. </p><p>Finally, we wanted to further examine IL4 production and T helper cell differentiation in LATY136F mice. We examined IL4 production using KN2 reporter mice, where huCD2 marks T cells that have recently produced IL4 protein. We demonstrated that only a small proportion of the LATY136F T cells were actively secreting IL4. This subset of T cells were Tfh cells that expressed BCL6 and localized to B cell-rich germinal centers within the spleen. Most studies to date have examined Tfh cells in infection models, and have demonstrated that Tfh cells have very low expression of GATA3. Our results revealed in a spontaneous T cell-mediated autoimmune model system, that Tfh cells express both high levels of BCL6 and GATA3. Additionally, using an inducible deletion system, where normal development occurs, we showed that Tfh cells differentiation is the result of aberrant LAT signaling, rather than autoreactive TCRs with high affinity for self-peptide-MHC. LATY136F Tfh cells did require B cells for their development. Together, these results displayed a novel role for tonic LAT-PLC&#947;1 signaling in modulating Tfh cell differentiation and BCL6 expression.</p> / Dissertation

Made available in DSpace on 2017-07-21T14:13:10Z (GMT). No. of bitstreams: 1 Stefanie Nolte.pdf: 1509773 bytes, checksum: dbef289d8b9a1e611bef15b9a6d2f33c (MD5) Previous issue date: 2013-02-21 / Introduction: Psoriasis is a skin inflammatory disease characterized by keratinocytes hyperproliferation and their incomplete differentiation in the epidermis. Treatment aims to reduce the psoriatic plaques formation; however, many therapeutic regimens do not produce satisfactory results. Searching for new alternatives, this study evaluated the possible effect of the TTHL compound, isolated from the ethanolic extract of Combretum leprosum, on HaCaT proliferation.Methods: The cellular viability was assessed 24 hours after the treatment with different concentrations of TTHL through MTT and neutral red methods. To evaluate the TTHL anti-proliferative activity, the cells were treated with different concentrations of TTHL, in alternate days (96 hours) and analyzed through MTT and Cyquant methods. The cell cycle was evaluated by flow citometry. The possible toxicity by the topical application of the TTHL (0.3 mg/ear) was assessed in vivo, through skin atrophy, by measuring ear thickness of animals and histology.Results: The MTT assay showed, after 24 hours, that TTHL presents cytotoxic activity in HaCaT cells, at the concentrations of 5, 7, 9 and 10 μM, decreasing, respectively, 53.54, 89.53, 89.69 and 90.30 ± 5.84% the cellular viability compared to the control group. The neutral red assay, in the same concentrations, decreased 39.83%, 48.67%, 47.78% and 50.44 ± 4.42%, respectively. In the cellular proliferation assay, the 5, 7 and 10 μM concentrations decreased 24.27%, 59.25% and 82.30% ± 5.96% the cells number, as showed in the MTT method, and 30.30%, 59.59% e 90.90% ± 6.03% as showed in the Cyquant method. The cell cycle assay showed that, at the concentrations of 5, 7 and 10 μM, the non-viable cells number was higher and statistically significant when compared to the control group. Through the skin atrophy assay was possible to conclude that TTHL does not cause atrophy.Conclusion: TTHL is capable of reducing cell viability. These data suggest that TTHL might be a possible tool for the treatment of hyperproliferative diseases, among them, psoriasis. However, additional studies are needed to verify the action mechanism and triterpene safety. / Introdução: A psoríase é uma doença inflamatória cutânea caracterizada pela hiperproliferação e diferenciação incompleta de queratinócitos na epiderme. O tratamento tem como objetivo reduzir a formação de placas psoriáticas, porém muitos regimes terapêuticos não produzem resultados satisfatórios. Buscando alternativas, este trabalho avaliou o possível efeito do composto TTHL, isolado do extrato etanólico de Combretum leprosum, sobre a proliferação de HaCaT. Métodos: A viabilidade celular foi avaliada 24 horas após o tratamento com diferentes concentrações de TTHL, através do método de MTT e vermelho neutro. Para avaliar a atividade antiproliferativa, as células foram tratadas com diferentes concentrações de TTHL, em dias alternados e analisadas através do método de MTT e Cyquant. O ciclo celular foi avaliado por citometria de fluxo. A possível toxicidade pela aplicação tópica do TTHL foi avaliada in vivo, pelo ensaio de atrofia cutânea, através da medida da espessura da orelha dos animais e histologia. Resultados: No ensaio de MTT, após 24 horas, o TTHL, apresentou atividade citotóxica nas células HaCaT, nas concentrações de 5, 7, 9 e 10 μM, diminuindo, respectivamente em 53,54, 89,53, 89,69 e 90,30 ± 5,84% a viabilidade celular quando comparado ao grupo controle. No ensaio de vermelho neutro, essas mesmas concentrações diminuíram 39,83%, 48,67%, 47,78% e 50,44 ± 4,42%, respectivamente. No ensaio de proliferação celular, nas concentrações de 5, 7 e 10 μM, observou-se diminuição de 24,27%, 59,25% e 82,30% ± 5,96% no número de células, conforme o método de MTT e 30,30%, 59,59% e 90,90% ± 6,03% conforme o método de Cyquant. No ensaio de ciclo celular observou-se que nas concentrações de 5, 7 e 10 μM o número de células não viáveis foi maior e estatisticamente significante quando comparado com o grupo controle. Através do ensaio de atrofia cutânea foi possível concluir que o TTHL não causa atrofia. Conclusão: O TTHL é capaz de diminuir a viabilidade celular. Esses dados sugerem que o TTHL possa ser uma possível ferramenta para o tratamento de doenças hiperproliferativas, dentre elas, a psoríase. Entretanto, estudos adicionais são necessários para verificar o mecanismo de ação e segurança do triterpeno.

Objectives: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. Design: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. Methods: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. Results: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. Conclusion: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.