Rapid assessment of in vitro expanded human regulatory T cell function

Human regulatory T cells (Treg) are able to actively suppress autoreactive immune responses. Phenotypically, they are broadly characterized as CD4+, CD25+, CD127lo/− and FoxP3+. CD45RA can be used to further differentiate the population into naïve (CD45RA+) and induced (CD45RA−) Treg. The functional potential of Treg is routinely determined by assessing their ability to suppress T cell function in 3–5 day proliferation assays. Since Treg are being explored for therapeutic use, a short-term functional assay could serve as a valuable tool for evaluating the potency of Treg. Therefore, an assay designed to measure Treg suppression of activation marker expression by responder T cells in 7 to 20 h has been examined in this report. Using flow cytometry, expression of CD69 and CD154 on T cells, in response to stimulation with CD3/CD28 beads, was used as a measure of activation in the assay. Treg from healthy volunteers were sorted as CD4+CD25+CD127lo/−CD45RA+ cells with a BD FACSAria™ II. The highly purified Treg were then expanded in vitro and their function was assessed in short term activation marker suppression assays using autologous PBMC as responder cells. The data suggest that this short term suppression assay could be a reliable surrogate for assessing Treg functional potential.

► Describes a robust, short term assay to measure function of in vitro expanded Treg.
► Suppression of CD69 and CD154 expression is a reliable surrogate of Treg function.
► The suppression of activation markers is specifically mediated by Treg.
► Assay can be used to identify therapeutic agents to modulate the function of Treg.