Estudo do papel de células T reguladoras e moléculas co-estimulatórias ma regulação da resposta imune em doenças infecciosas e tumor de cabeça e pescoço / The role of the regulatory T cells and PD-1 molecules in the modulation of the immune response in chronic infections and head and neck tumors

O sistema imune serve como uma barreira contra os patógenos e ao crescimento anormal de células. Para impedir as respostas imunes excessivas ou indiscriminadas que podem comprometer a sobrevivência do organismo, diversos mecanismos regulatórios são ativados visando manter o delicado balanço entre início e término de uma resposta imune. As celular T reguladoras (Treg) parecem desempenhar papel central na regulação da resposta imune em infecções crônicas e durante o desenvolvimento de tumores. Outro mecanismo importante no controle da resposta imune é desempenhado por moléculas co-estimulatórias, dentre as quais estão CTLA-4 e PD-1, todas associadas à função das células T reguladoras. Um aspecto importante é q a sobrevida de tecido tumoral e de transplantes tem sido associada à função das células T reguladoras. Assim, buscamos definir o envolvimento de células T reguladoras e PD-1 na modulação da resposta imune L. braziliensis, ao fungo P. brasiliensis, à doença periodontal e ao tumor de cabeça e pescoço. Baseado nos resultados já publicados e em dados preliminares, as hipóteses são que: (a) a interação do parasita (ou célula tumoral) com o hospedeiro leva à migração de linfócitos T e efetores e células T reguladoras para o local da lesão; (b) a dinâmica do acúmulo dessas células em tais sítios determina a eficiência da eliminação do patógeno ou tumor. No caso das parasitoses, há o desenvolvimento de imunidade concomitante; (c) as células T regulam a resposta imune local de forma contato dependente e modulando a função de APC através da liberação de IL-10 e/ou TGF-β; (d) infecção e progressão tumoral levam à modulação da expressão de PD-1 nos leucócitos e seus ligantes nos órgãos; (e) a interação PD-PDL-1 regula a resposta imune local de forma a favorecer a persistência do patógeno e os mecanismos de escape tumoral.

The immune system serves as a barrier against pathogens and abnormal cellular growth. To avoid tissue and organ damage during immune response several regulatory mechanisms are activated to limit, terminate and attenuate T-cells response. Regulatory T cells (Treg) play a central role in the regulation of the immune response in chronic infections and tumor-specific immunity. Programmed death-1 (PD-1) is a transmembrane protein that acts as a negative regulator in effector T cells, modulating the delicate balance between effective antimicrobial immune defenses and immune-mediated tissue damage. However, recent data suggest that the PD-1:PD-L1 pathway can also block antitumor immune responses even when tumor antigens can be recognized. An important aspect it that the survival of tumor and transplant tissues has been associated with the function or regulatory T cells. Thus, we discuss the role of Treg cells and PD-1 molecules in the modulation of the immune response to L. braziliensis, P. brasiliensis, periodontal disease and head and neck tumors. Based on published results and preliminary data, the hypotheses are that: (a) the interaction of the parasite (or tumoral cells) with the host leads to the migration of effector T lymphocytes and Treg cells to the local; (b) the dynamics of cells accumulation in such sites determinate the elimination efficiency of tumors. In infectious disease, there is the development of concomitant immunity; (c) Treg cells regulate the local immune response, modulating the APC function through the release of IL-10 and/or TGF-β; (d) infection and tumor progression leads to the modulation of PD-1 expression in the leukocytes and their ligands in the tissue; (e) PD-PDL-1 interactions regulate the immune response and may mediate the persistence of pathogen and contribute to immune evasion by cancers.T.

Detection of homodimer formation of CD99 through extracelluar domain using bimolecular fluorescence complementation analysis

Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.

Characterization and epitope mapping of two monoclonal antibodies against human CD99

CD99 plays an critical role in the diapedesis of monocytes, T cell differentiation, and the transport of MHC molecules. Engagement of CD99 by agonistic monoclonal antibodies has been reported to trigger multifactorial events including T cell activation as well as cell-cell adhesion during hematopoietic cell differentiation. In this study, to identify the functional domains participating in the cellular events, we mapped the epitopes of CD99, which are recognized by two agonistic CD99 monoclonal antibodies, DN16 and YG32. Using recombinant fusion proteins of GST with whole or parts of CD99, we found that both antibodies interact with CD99 molecules independently of sugar moieties. DN16 mAb detected a linear epitope located in the amino terminal region of CD99 while YG32 mAb bound another linear epitope in the center of the extracellular domain. To confirm that the identified epitopes of CD99 are actually recognized by the two mAbs, we showed the presence of physical interaction between the mAbs and the fusion proteins or synthetic peptides containing the corresponding epitopes using surface plasmon resonance analyses. The dissociation constants of DN16 and YG32 mAbs for the antigen were calculated as 1.27 X 10(-7) and 7.08 X 10(-9) M, respectively. These studies will help understand the functional domains and the subsequent signaling mechanism of CD99.

Actin binds to the cytoplasmic tail of alpha 1 subunit of integrin in hepatocytes.

alpha 1 beta 1-Integrin is a common receptor for laminin and collagen IV on hepatocytes. The interactions of intracellular domain of integrins with cytoplasmic elements are critical in the initiation and transduction of signals. In order to understand the nature of cytoplasmic components that can interact with cytoplasmic domain of alpha 1 integrin, cytoplasmic extracts of monolayers of rat hepatocytes were subjected to chromatography over an affinity column prepared by coupling a 60-mer synthetic cytoplasmic tail of alpha 1 subunit. SDS-PAGE analysis of the eluate showed the presence of a 47 kDa protein. Dot-Blot assay using radio-iodinated 47 kDa protein showed the binding of the protein to 60-mer C tail in a concentration dependent manner. Immunoblot analysis using specific antibodies showed that the 47 kDa protein is actin.