Recently, our laboratory reported that secondary CD8+ T cell-mediated antitumor responses were impaired following successful initial antitumor responses using various immunotherapeutic approaches. Although immunotherapy stimulated significant increases in CD8+ T cell numbers, the number of CD4+ T cells remained unchanged. The current investigation revealed a marked differential expansion of CD4+ T cell subsets. Successful immunotherapy surprisingly resulted in an expansion of CD4+Foxp3+ regulatory T (Treg) cells concurrent with a reduction of conventional CD4+ T (Tconv) cells, despite the marked antitumor responses. Following immunotherapy, we observed differential up-regulation of PD-1 on the surface of CD4+Foxp3+ Treg cells and CD4+Foxp3- Tconv cells. Interestingly, it was the ligand for PD-1, B7-H1 (PDL-1), that correlated with Tconv cell loss after treatment. Furthermore, IFN-γ knockout (IFN-γ-/-) and IFN-γ receptor knockout (IFN-γR-/-) animals lost up-regulation of surface B7-H1 even though PD-1 expression of Tconv cells was not changed, and this correlated with CD4+ Tconv cell increases. These results suggest that subset-specific expansion may contribute to marked shifts in the composition of the T cell compartment, potentially influencing the effectiveness of some immunotherapeutic approaches that rely on IFN- +.