Abstract
CD4^+^CD25^+^ regulatory T cells (Treg cells) prevent T cell‐mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS‐ or bead‐sorted CD4^+^CD25^+^ T cells from healthy donors to inhibit anti‐CD3/CD28 activation of CD4^+^CD25^−^ indicator T cells. The data clearly demonstrated classical Treg suppression of CD4^+^CD25^−^ indicator cells by both CD4^+^CD25^+high^ and CD4^+^CD25^+low^ T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO^−^ (naive) or CD45RO^+^ (memory) subpopulations, was independent of the TCR signal strength, required cell–cell contact, and was reversible by interleukin‐2 (IL‐2). Of general interest is that a wider sampling of 27 healthy donors revealed an age‐ but not gender‐dependent loss of suppressive activity in the CD4^+^CD25^+^ population. The presence or absence of suppressive activity in CD4^+^CD25^+^ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4^+^CD25^+^ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4^+^CD25^+^ T cells had significantly higher expression of both CTLA‐4 and GITR than CD4^+^CD25^−^ T cells from the same donors. Moreover, antibody neutralization of CTLA‐4, GITR, IL‐10, or IL‐17 completely reversed Treg‐induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4^+^CD25^+^ T cells declines with age. Published 2003 Wiley‐Liss, Inc.