A short term assay for evaluating the functional potential of isolated human regulatory T cells (89.14)

Abstract

Abstract Naturally occurring human regulatory T cells (Tregs) are able to actively suppress auto reactive immune responses. Phenotypically, they are characterized as CD4+, CD25++, CD127- and FoxP3+. Determining whether Tregs can suppress the antigen-specific cytokine expression of autologous PBMC would provide useful insight into the functional abilities of these cells. Long term assays such as proliferation are routinely used to analyze the functional capability of Tregs, but for practical reasons, a short term intracellular cytokine staining (ICS) assay would seem to be a more valuable tool for assessing the potency of Treg. In this study, we have demonstrated that highly purified and in vitro expanded Treg are capable of suppressing T-cell specific cytokine responses in 7 hr ICS assays. Treg were purified from healthy donor PBMC by sorting the CD4+CD25++CD127dim/- cells with a BD FACSAria(tm) flow cytometer. The functional ability of the highly purified Treg was evaluated either after activation for approximately 20 hours, or after in vitro expansion for 12 to 14 days. Activation of PBMC with SEB and/or CD3/CD28 results in the expression of inflammatory cytokines (IFNγ, and TNFα) by CD4+ and CD8+ T cells. The data from this study indicate that the addition of CD4+CD25++ cultured Treg to the ICS assay of autologous PBMC results in suppression (i. e. reduced frequency of) cytokine expressing T cells. There was no correlation between the frequency of FoxP3+ Treg and % suppression of cytokine+ effector cells. Since the intracellular T-cell cytokine expression assay has been validated extensively, this short-term assay could reduce the time needed to qualify Treg for immunotherapy.