Mechanisms leading to the observed immune dysregulation in HIV-1 infection are not well understood. HIV-specific IL-10-positive CD8+ T cells are increased in advanced HIV disease. We have previously reported that Gag-specific IL-10-positive CD8+ T cells suppressed cytolysis. In this study we describe the suppressive effect of Nef-specific IL-10-positive CD8+ T cells. Interestingly, simultaneous removal of both Gag-and Nef-specific IL-10-positive CD8+ T cells led to higher HIV-specific cytolysis compared with the removal of Nef-specific IL-10-positive CD8+ T cells alone. We also examined the level of programmed cell death-1 (PD-1) as a measure of immune dysfunction in association with IL-10-positive suppressor CD8+ T cells. The level of PD-1 expression on CD107-positive effector CD8+ T cells was significantly increased when IL-10-positive suppressor CD8+ T cells were present (p< 0.05). Our results suggest that IL-10-positive suppressor CD8+ T cells contribute to the immune dysfunction observed in advanced HIV infection and that the concomitant presence of multiple IL-10-positive CD8+ T cell populations may have an additive suppressive effect.