engleski

Detection of regulatory T cells in human blood

Regulatory T (T_reg) cells have the capacity to inhibit potentially harmful immune responses in different disorders such as autoimmune diseases. Many distinct types of T_reg cells have been described according to their surface phenotype or by their ability to secrete immunoregulatory cytokines, such as interleukin 10 (IL-10) and transforming growth factor beta (TGF-beta). So-called natural CD4+ T_reg cells express high levels of CD25 accompanied by the expression of CD45RO, HLA-DR and intracellular CTLA-4. Although these cells express CD25, which is usually considered as an activation marker, they lack the expression of other activation markers such as CD69. The aim of this study was to develop an approach to detect T_reg cells in human peripheral blood. For that purpose, we first identified natural T_reg cells using four-color flow cytometry. In healthy adults, 2, 6 ± 1, 5 % of CD4+CD25^high natural T_reg cells among all CD4+ cells were detected. As observed earlier, CD4+CD25^high were CD69-HLA-DR+ CD45RO+CTLA-4+. In addition, we established a method for detection of IL-10 and TGF-beta in different subsets of T cells. Peripheral blood mononuclear cells were cultured alone or in the presence of phorbol esther and ionomycin for 6 to 72 hours. Maximal percentage of cytokine positive T cells was observed after 24 hours of culture in CD3+CD4+ helper T cells and in CD3+CD8+ cytotoxic T cells, although cytotoxic T cells produced more IL-10 and TGF-beta. These results indicate that in addition to phenotypic analysis by 4-color flow cytometry, intracellular cytokine staining and subsequent detection of cytokine positive cells by flow cytometry are useful methods for the detection of different types of cells that secrete IL-10 and TGF-beta. Further studies are underway to determine the role of T_reg cells in allergic patients undergoing immunotherapy.

asthma; children; T-lymphocytes; immunotherapy

nije evidentirano