Identificação de marcadores moleculares para células T reguladoras humanas com perfil CD4+CD25+ por phage display / Peptide phage display for the identification of novel molecular markers on human thymic regulatory CD4+CD25+ T cells

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

There are consistent data in literature indicating that thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed as an independent lineage in the thymus. These cells are known as natural regulatory T cells. Several questions about these cells remain unanswered, such as how they are generated, what is determinant in their regulatory function and which specific molecular markers can be used to identify them. Taking this into consideration, our aim was to identify new potentially important molecules in the development and/or supressive function of natural regulatory T cells, both in the thymus and in CD4+CD25+ thymocytes. For this, the phage display technique was employed, with a peptide phage library and thymic specimens obtained from children who underwent corrective cardiac surgery at the Heart Institute (InCor), in São Paulo. The search for these molecules was separately performed in 3 types of biological material: thymic tissue, thymocytes and CD4+CD25+ thymic cells. In the first stage, the phage peptide-library was incubated with a pool of PBMC (peripheral blood mononuclear cells). After the incubation, phages bound to PBMC were discarded (pre-clearing). In the second stage, unbound phages were incubated with either total thymocytes or CD4+CD25+ thymic cells. The pre-clearing stage was not perfomed in the thymic tissue. The phages obtained with after incubation with the different biological materials were recovered in E. coli culture and used in additional cycles of selection. After three rounds of selection, the recovered phages from the total thymocytes, from thymic tissue and thymocytes CD4+CD25+ were sequenced and their ligands identified. Among the phages selected for validation one ligand of thymic tissue: M2C and one ligand of CD4+CD25+ thymocytes: R2A present similarity to two proteins associated to the metabolism of Vitamin D3, a molecule involved in imunoregulation and toelrance induction in several experimental models. However, there are no data in the literature concerning the possible role of this moelcule in natural regulatory T cells. In the molecular validation of theses phages, although some variability between the diffeterent assays we have verified by ELISA, that the phages present preferential binding to the 1,25 dhydroxyvitamin D3, the active form of Vitamin D3. However, in the functional validation assays, the influence of the Vitamin D3 in the differentiation of these cells could not be consistently confirmed since we could observe an increase in the number of CD4+CD25+ cells cultured with vitamin D in only a few experiments. The ligand-receptor molecules we have defined in this study may have relevant implications in the development of CD4+CD25+ regulatory T cells in the thymus

ASSUNTO(S)

timo regulatory t lymphocytes thymus gland calcitriol receptors imunoregulation linfócitos t reguladores receptores de calcitiol biblioteca de peptídeos peptide library vitamina d3 vitamin d3 biological markers marcadores biológicos imunorregulação

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