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The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.

Resting CD4(+) T cells containing integrated HIV provirus constitute one of the long-lived cellular reservoirs of HIV in vivo. This cellular reservoir of HIV had been thought to be quiescent with regard to virus replication based on the premise that HIV production in T cells is inexorably linked to cellular activation as determined by classical activation markers. The transition of T cells within this HIV reservoir from a resting state to an activated HIV-producing state is believed to be associated with a shorten life span due to susceptibility to activation-associated apoptosis. Evidence is mounting, however, that HIV production may occur in T cells that have not undergone classic T cell activation. HIV encodes several proteins, including envelope and Nef, which trigger a variety of signaling pathways associated with cellular activation, thereby facilitating HIV replication in nondividing cells. The present study demonstrates that production of infectious virus from resting CD4(+) T cells isolated from HIV-infected individuals can be induced following exposure of these cells to HIV-1 recombinant (oligomeric gp140) envelope protein. Envelope-mediated induction of HIV expression occurs in the presence of reverse transcriptase inhibitors and is not associated with markers of classic T cell activation, proliferation, or apoptosis. The ability of HIV envelope to induce virus replication in HIV-infected resting CD4(+) T cells without triggering apoptosis provides a mechanism for the virus itself to directly participate in the maintenance of HIV production from this cellular reservoir.

The role of exogenous stimulation of CD40 by CD40 ligand (CD40L) in dendritic cell (DC) maturation, CC-chemokine production, and CCR5 receptor expression was examined using a soluble trimeric CD40L agonist protein (CD40LT). Stimulation of monocyte-derived DCs with CD40LT enhanced the production of the CC-chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES and diminished surface expression of CCR5. Based on these findings, the functional role of CD40LT stimulation on the ability of DCs to replicate and transmit HIV viral infection was studied. The addition of CD40LT to cocultures of naive CD4+ T cells and autologous DCs (T/DC) infected with the macrophage-tropic isolate, HIVBaL, caused a striking reduction in reverse transcriptase (RT) activity after 10 and 14 days of culture. The addition of a mixture of Abs against CC-chemokines abrogated the decrease in RT activity, demonstrating that the inhibitory effect mediated by CD40LT was CC-chemokine-dependent. In contrast, the presence of CD40LT in T/DC cocultures infected with the T cell-tropic isolate, HIV IIIB, caused an increase in RT activity that was CC-chemokine-independent. Of note, CD40LT stimulation also inhibited RT activity in cultures containing macrophage-tropic virus (HIVBaL)-infected DC only. However, in contrast to the results seen in the T/DC cocultures, CD40LT stimulation inhibited RT activity in cultures of DCs alone in a CC-chemokine-independent manner. Together, these results show that CD40LT stimulation of DCs suppresses HIV replication and transmission to CD4+ T cells by two potentially different mechanisms.

A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria. Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria. The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV. Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF). Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted. Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha. IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production. In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production. Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells.

Replication of HIV is regulated by virus-encoded regulatory proteins, as well as by a variety of cellular factors including cytokines. In the present study, we have investigated the autocrine/paracrine effects of endogenous cytokines on HIV replication in primary PBMCs of healthy HIV seronegative individuals. Addition of rIL-2 to cultures between 0 and 72 h after isolation of PBMCs allowed the replication of primary HIV isolates and laboratory-adapted HIV strains to levels comparable with or greater than those obtained in parallel cultures of autologous PHA-blasts. In this regard, both major cellular targets of HIV infection, CD4+ T lymphocytes and mononuclear phagocytes, were maintained for several weeks in IL-2-stimulated PBMC cultures and virion production was observed in both cell lineages. The kinetics of secretion of several cytokines (such as TNF-alpha, IL-1 beta, IL-6, and IFN-gamma), as well as expression of cellular activation markers, paralleled HIV replication in IL-2-stimulated PBMCs. Endogenous pro-inflammatory cytokines and IFN-gamma played a major role in the regulation of HIV replication in IL-2-stimulated PBMCs, as determined by the ability of several anti-cytokine Abs or antagonists to suppress HIV production; this was not the case in parallel cultures of autologous PHA-blasts. Thus, IL-2-stimulated PBMCs may represent a more physiologic in vitro system than PHA-blasts for the study of HIV infection and replication, and should prove useful in investigating the role of cytokines and other host factors in the regulation of HIV production.