Regulated Compartmentalization of Programmed Cell Death-1 Discriminates CD4 super(+)CD25 super(+) Resting Regulatory T Cells from Activated T Cells

More effective discrimination between CD4 super(+)CD25 super(+) regulatory T cells (Treg) and activated T cells would significantly improve the current level of purification of Treg and their therapeutic application. We observed that similar to 90% of Treg (positive for the nuclear transcription factor Forkhead winged helix protein-3 and able to inhibit naive T cell proliferation) isolated from the spleens or lymph nodes of normal mice did not express significant levels of the inhibitory receptor programmed cell death-1 (PD-1) on their surface, but retained PD-1 intracellularly. An identical phenotype was also identified for human CD4 super(+)CD25 super(high) T cells isolated from peripheral blood of healthy volunteers. By contrast, activated T cells expressed high levels of surface PD-1 that paralleled up-regulation of CD25 during effector cell expansion. This distinction allowed us to isolate CD4 super(+)CD25 super(+)PD-1 super(-) T cells with suppressive activity from mice immunized with mature allogeneic dendritic cells. Although purification was limited to resting Treg because TCR ligation induced up-regulation of surface PD-1, this strategy nevertheless represents a valuable step toward more definitive characterization of Treg and their improved purification for therapeutic assessment.