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The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8–12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.

SignificanceCD4+ regulatory T (Treg) cells expressing CD25 and the transcription factor forkhead box P3 (FOXP3) play indispensable roles for immunological self-tolerance and homeostasis. Because human FOXP3+CD25+CD4+ T cells are heterogeneous in function and differentiation status, their analysis and manipulation for treating immunological diseases remains a challenge. Here we show that CD15s (sialyl Lewis x) is specifically expressed by activated, terminally differentiated, and most suppressive FOXP3high Treg cells, allowing their separation from nonsuppressive FOXP3+CD4+ T cells secreting inflammatory cytokines. Removal of CD15s+CD4+ T cells from human blood is indeed sufficient to enhance in vitro antitumor and antiviral antigen responses. CD15s is therefore useful for phenotypic as well as functional analysis of human Treg subpopulations and for targeting them to control immune responses.

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