engleski

Multi-color phenotyping of FOXP3 and IL-7 receptor-alpha levels on human CD4+CD25high regulatory T cells

Regulatory T cells (Tregs) are key suppressor cells in maintenance of peripheral tolerance. Murine Tregs are largely CD4+CD25+, which facilitates their identification. In humans though, CD25 is found on activated T cells as well. The difference between activated and regulatory T cells appears to be in the CD25 expression level, former being CD25int (intermediate) and latter CD25hi (high), but there’s no consensus yet as to where the boundary lies. Master regulator of Tregs is FOXP3 transcription factor, a lack of which manifests as severe autoimmune disorder both in humans (IPEX and XLAAD syndromes) and in mice (scurfy mice). Moreover, Foxp3 gene transfer converts naive resting CD4 T cells into functional Tregs. However, FOXP3+ cells in humans were found not only in CD4+CD25hi, but also in CD25int and CD25- T cells, although in lesser amounts. Therefore, additional markers for human Treg identification are required. One of the candidates is CD127 (IL-7 receptor-alpha), expressed on IL-7-dependent T cells (mostly non-regulatory) and considered absent on IL-2-dependent Tregs. We addressed this issue using multi-color flow cytometry for phenotyping human peripheral blood mononuclear cells with CD4, CD25, CD127 and FOXP3 monoclonal antibodies. For determining more accurately the extent of CD25 expression, we used fluorescence minus-one controls. Curiously, among the putative Tregs (CD4+CD25hi) we found both CD127- and CD127+ cells. Moreover, both subsets expressed FOXP3. However, FOXP3 mean fluorescence intensity (corresponding to protein level/ cell) was twice as high on CD127- than on CD127+ cells. Therefore, a quest for Treg markers in humans is far from over, and requires detailed multi-color phenotyping.

regulatory T cells; CD127; FOXP3

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