ARTICLE ABSTRACTAdoptive immunotherapy with antitumor T cells is a promising novel approach for the treatment of cancer. However, T-cell therapy may be limited by the cotransfer of regulatory T cells (Treg). Here, we explored this hypothesis by using 2 cell surface markers, CD44 and CD137, to isolate antitumor CD4 T cells while excluding Tregs. In a murine model of B-cell lymphoma, only CD137negCD44hi CD4 T cells infiltrated tumor sites and provided protection. Conversely, the population of CD137posCD44hi CD4 T cells consisted primarily of activated Tregs. Notably, this CD137pos Treg population persisted following adoptive transfer and maintained expression of FoxP3 as well as CD137. Moreover, in vitro these CD137pos cells suppressed the proliferation of effector cells in a contact-dependent manner, and in vivo adding the CD137posCD44hi CD4 cells to CD137negCD44hi CD4 cells suppressed the antitumor immune response. Thus, CD137 expression on CD4 T cells defined a population of activated Tregs that greatly limited antitumor immune responses. Consistent with observations in the murine model, human lymphoma biopsies also contained a population of CD137pos CD4 T cells that were predominantly CD25posFoxP3pos Tregs. In conclusion, our findings identify 2 surface markers that can be used to facilitate the enrichment of antitumor CD4 T cells while depleting an inhibitory Treg population. Cancer Res; 72(5); 1239–47. ©2012 AACR.