CD28 Costimulation Is Essential for Human T Regulatory Expansion and Function

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs (aAPC) we found that CD28, but not ICOS, OX40, 4-1BB, CD27, or CD40-L 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 xeno-GVHD 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 less than 3 weeks. 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 to expand Tregs differ from T effector cells, and furthermore, they extend findings from mouse Tregs to demonstratethat human post-thymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.