HIV infection is associated with persistent, aberrant immune activation that supports viral replication and contributes to the progressive immune dysfunction associated with HIV disease progression. Chronic immune activation, whether induced by persistent foreign antigens or loss of self-tolerance, activates and expands host-mediated immunosuppressive mechanisms that serve to prevent immune-mediated damage to the host. In addition to limiting autoimmunity, several of these mechanisms suppress immune responses directed against foreign antigens and affect the ability of the host to eliminate or control certain tumors and bacterial, parasitic and viral infections. Two of these mechanisms in particular, suppressor CD25+ CD4+FoxP3+ regulatory T (Treg) cells and the negative regulatory surface molecule programmed death (PD)-1, play a role in suppressing virus-specific T cell responses in chronic viral infection and/or vaccination. [unreadable] [unreadable] We have previously demonstrated that CD25+ Treg cells isolated from peripheral blood (PB) of asymptomatic HIV+ subjects inhibit numerous HIV-specific CD4+ and CD8+ T cell immune responses in vitro, including proliferation, cytokine and chemokine secretion, and the ability of CD8+ T cells to kill HIV-expressing target cells. CD25+Treg cells have been found to accumulate in lymphoid tissue of viremic individuals and we demonstrated that these cells are capable of suppressing HIV-specific cytolytic (CTL) activity, but not proliferation, in individuals at all stages of disease. We now find that the mechanisms by which Treg cells suppress HIV-specific proliferation and CTL activity differ, and that HIV disease progression is associated with dysfunction of or reduced sensitivity to mechanisms involved in suppressing proliferation, but not CTL activity. [unreadable] [unreadable] We are also conducting studies investigating the role of Programmed Death (PD)-1 and its ligands PD-L1 and PD-L2, negative regulatory members of the B7 superfamily. Persistent antigen exposure has been shown to upregulate PD-1 and its engagement suppresses T cell receptor (TCR)-mediated signaling events. In vitro neutralization of PD-1 ligands has been reported to rescue the function of exhausted HIV-specific T cells. The ability to restore the quality (broad functionality), and not just the quantity, of HIV-specific T cells has been a primary goal of immune-based therapies for the treatment of HIV disease. However, controversy exists regarding whether blocking PD-1 activation rescues functionality of exhausted HIV-specific T cells or simply allows for increased expansion of existing, functional HIV-specific T cells. Also, the differential roles of PD-L1 and PD-L2 have not yet been determined in the context of HIV disease.[unreadable] [unreadable] We observed that neutralization of PD-L1, but not PD-L2, significantly enhances HIV-specific T cell proliferative responses. In contrast, PD-L2, but not PD-L1, appeared to play a modest role in apoptosis of PD-1+ T cells. Neutralization of neither PD-L1 nor PD-L2 rescued cytokine production following a short stimulation of PBMCs with HIV peptides; however, enhancing effects of PD-1 ligand neutralization on cytokine production were observed under conditions that rely on expansion of HIV-specific T cells and re-stimulation. These data point to potentially differential roles/mechanisms of endogenous PD-L1 and PD-L2 in the suppression of HIV-specific T cell proliferation and survival, respectively. Furthermore, these data support the concept that blocking the PD-1 axis enhances expansion of pre-existing functional HIV-specific T cells. In addition, we observed that both PD-1 and PD-1 ligand expression was greater in LT than in PB and that PD-1 ligand expression in the LT, but not the PB, correlated with markers of HIV disease progression. It remains to be established whether the quality of HIV-specific T cell function can be enhanced by blocking PD-1 and PD-1 ligand interactions in the LT.[unreadable] [unreadable] Finally, we have investigated the role of the PD-1 axis in the context of TCR-independent, cytokine-driven T cell activation. Of the numerous cytokines tested, only the common gamma chain (gC) cytokines, IL-2, IL-7, IL-15 and IL-21 were found to directly upregulate PD-1 and PD-L1 on purified T cells as well as inducing PD-L1 and PD-L2 on on monocytes in the context of total PBMCs. In support of these observations, significant increases the frequency of PD-1+ T cells and PD-L1+ and PD-L2+ monocytes were observed in PBMCs of HIV+ individuals following administration of IL-2 in vivo. Engagement of PD-1 failed to inhibit gC cytokine-driven T cell proliferation, survival or signaling events; however, the PD-1 axis induced by gC cytokines did act to suppress proliferation and function of T cells upon triggering the TCR (antigen). These gC cytokines play an important role supporting T cell expansion, survival and function and are currently being used as immunotherapy or vaccine adjuvants. Our data suggest that under conditions of high gC cytokine levels, up-regulation of the PD-1 axis could impair, at least transiently, antigen-specific responses.