Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion

Expression of the IL-2 receptor alpha chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T-R), although cells with regulatory properties are also found in the CD4(+)CD25(-) subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of T-R differentiation, we have evaluated the requirements for CD25 expression by peripheral T-R. We first show that in vivo depletion of CD25(+) cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25(+) T-R. Consistently, Foxp3-expressing T-R encompassed in the CD25(-) cell population convert to CD25(+) after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on T-R is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25(-) cells constitute a peripheral reservoir of differentiated T-R, recruited to the CD25(+) pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T-R takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T-R differentiation from naive CD4 T cells, defined as CD25(-).