The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands.

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.

CD25+ regulatory T cells isolated from HIV-infected individuals suppress the cytolytic and nonlytic antiviral activity of HIV-specific CD8+ T cells in vitro.

HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.

Suppression of HIV-specific T cell activity by lymph node CD25+ regulatory T cells from HIV-infected individuals.

CD25(+) CD4(+) FoxP3(+) regulatory T (Treg) cells isolated from the peripheral blood of asymptomatic HIV-infected individuals have been demonstrated to significantly suppress HIV-specific immune responses in vitro. CD25(+) Treg cell suppressor activity in the peripheral blood seems to diminish with progression of HIV disease, and it has been suggested that loss of Treg cells contributes to aberrant immune activation and disease progression. However, phenotypic studies suggest that Treg cells may migrate to, and be maintained or even expanded in, tissue sites of HIV replication. Currently, it is not known whether tissue-associated Treg cells maintain suppressive activity in the context of HIV infection, particularly in individuals with advanced disease. The present study demonstrates that CD25(+) Treg cells isolated from lymph nodes and peripheral blood of HIV(+) subjects, even those with high viral loads and/or low CD4(+) T cell counts, maintain potent suppressive activity against HIV-specific cytolytic T cell function. This activity was better in lymph node as compared with peripheral blood, particularly in patients with high levels of plasma viremia. In addition, the expression of certain CD25(+) Treg-associated markers on CD4(+) T cells isolated from lymph nodes differed significantly from those on CD4(+) T cell subsets isolated from the peripheral blood. These data suggest that CD25(+) Treg cell-mediated suppression of HIV-specific responses continues throughout the course of HIV disease and, because of their particularly potent suppression of HIV-specific CTL activity in lymphoid tissue, may considerably impact the ability to control HIV replication in vivo.

Correlates of preserved CD4(+) T cell homeostasis during natural, nonpathogenic simian immunodeficiency virus infection of sooty mangabeys: implications for AIDS pathogenesis.

In contrast to HIV-infected humans, naturally SIV-infected sooty mangabeys (SMs) very rarely progress to AIDS. Although the mechanisms underlying this disease resistance are unknown, a consistent feature of natural SIV infection is the absence of the generalized immune activation associated with HIV infection. To define the correlates of preserved CD4(+) T cell counts in SMs, we conducted a cross-sectional immunological study of 110 naturally SIV-infected SMs. The nonpathogenic nature of the infection was confirmed by an average CD4(+) T cell count of 1,076 +/- 589/mm(3) despite chronic infection with a highly replicating virus. No correlation was found between CD4(+) T cell counts and either age (used as a surrogate marker for length of infection) or viremia. The strongest correlates of preserved CD4(+) T cell counts were a low percentage of circulating effector T cells (CD28(-)CD95(+) and/or IL-7R/CD127(-)) and a high percentage of CD4(+)CD25(+) T cells. These findings support the hypothesis that the level of immune activation is a key determinant of CD4(+) T cell counts in SIV-infected SMs. Interestingly, we identified 14 animals with CD4(+) T cell counts of <500/mm(3), of which two show severe and persistent CD4(+) T cell depletion (<50/mm(3)). Thus, significant CD4(+) T cell depletion does occasionally follow SIV infection of SMs even in the context of generally low levels of immune activation, lending support to the hypothesis of multifactorial control of CD4(+) T cell homeostasis in this model of infection. The absence of AIDS in these "CD4(low)" naturally SIV-infected SMs defines a protective role of the reduced immune activation even in the context of a significant CD4(+) T cell depletion.

Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection.

In this study, we demonstrate that the in vitro interactions between a CD56(neg)/CD16(pos) (CD56(neg)) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1-infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK-DC activation and maturation as well as a defect in the NK cell-mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56(neg) NK cell subset, largely accounts for the highly defective NK cell-mediated lysis of autologous iDCs. Moreover, mDCs generated from HIV-1 viremic but not aviremic patients are substantially impaired in their ability to secrete interleukin (IL)-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of interferon-gamma.

Identification of NKG2A and NKp80 as specific natural killer cell markers in rhesus and pigtailed monkeys.

Investigations of natural killer (NK) cells in simian models of disease have been hampered by a lack of appropriate phenotypic markers and by an inadequate understanding of the regulation of NK cell activities. In the present study, a panel of monoclonal antibodies (mAbs) specific for various human NK receptors was screened for cross-reactivity with NK cells from rhesus macaques and pigtailed macaques. Flow cytometric analyses using anti-human NKG2A and anti-human NKp80 mAbs individually, and particularly in combination with anti-CD16 mAb, allowed for the identification of the entire NK cell population in both species. NK cells in monkeys were generally identified by negative selection of peripheral blood mononuclear cells (PBMCs) for the absence of T-cell, B-cell, and monocyte markers. mAb-mediated ligation of NKp80 induced NK cell cytotoxicity, while in the case of NKG2A it displayed a clear capability to inhibit the lysis of target cells by NK cells from macaques, as well as from humans. This new phenotypic and functional characterization of NKG2A and NKp80 in rhesus and pigtailed macaque NK cells provides a new approach in the analysis of their innate immune system.

Contrasting effects of low-dose IL-2 on vaccine-boosted simian immunodeficiency virus (SIV)-specific CD4+ and CD8+ T cells in macaques chronically infected with SIVmac251.

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

Characterization of CD56-/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals.

Natural killer (NK) cells are an important component of the innate immune response against viral infections. NK cell-mediated cytolytic activity is defective in HIV-infected individuals with high levels of viral replication. In the present study, we examined the phenotypic and functional characteristics of an unusual CD56(-)/CD16(+) (CD56(-)) NK subset that is greatly expanded in HIV-viremic individuals. The higher level of expression of inhibitory NK receptors and the lower level of expression of natural cytotoxicity receptors observed in the CD56(-) NK fraction compared with that of CD56(+) NK cells was associated with extremely poor in vitro cytotoxic function of this subset. In addition, the secretion of certain cytokines known to be important in initiating antiviral immune responses was markedly reduced in the CD56(-), as compared with the CD56(+) NK cell subset. These data suggest that the expansion of this highly dysfunctional CD56(-) NK cell subset in HIV-viremic individuals largely accounts for the impaired function of the total NK cell population.

Neonatal natural killer cells produce chemokines and suppress HIV replication in vitro.

The innate immune system may be critical in the prevention of perinatal HIV infection. Since neonates have limited immunological experience, they may rely more on the ability of the innate immune system to defend against infection than their adult counterparts. To assess the potential of human neonatal natural killer (nNK) cells to suppress HIV infection in a noncytolytic manner, we evaluated their ability to secrete chemokines and inhibit HIV replication in vitro. nNK cells were cocultured with autologous, HIV-infected CD4(+) T cells and their suppressive activity against HIV was compared to nCD8(+) T cells and adult NK cells. We found that nNK cells could suppress HIV replication in autologous CD4(+) T cells infected with a CCR5-utilizing virus, but were unable to suppress replication by a CXCR4-utilizing virus. nNK cell-mediated suppression of HIV replication was comparable to that of nCD8(+) T cells and greater than that of NK cells from adults. Suppression was mediated by soluble factors, and was abrogated by the addition of an excess of anti-CC-chemokine Ab directed at CCR5 ligand chemokines. In contrast, inhibition of HIV replication by autologous nCD8(+) T cells was not fully abrogated with anti-CC-chemokine Abs indicating, as previously reported in HIV-infected adults, that other factors in addition to chemokines play a role in CD8(+) T cell-mediated suppression of HIV replication. Our results show that nNK cells can inhibit HIV replication via a chemokine-mediated mechanism, and support a potential role for the innate immune system in preventing perinatal transmission of HIV in a noncytolytic manner.

CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.

Human immunodeficiency virus (HIV) disease is associated with loss of CD4(+) T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4(+) T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25(+)CD4(+) regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25(+)CD4(+) T cells significantly suppressed cellular proliferation and cytokine production by CD4(+) and CD8(+) T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-beta independent. Individuals with strong HIV-specific CD25(+) T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4(+): CD8(+) T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25(+)CD4(+) T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

Productive HIV infection of resting CD4+ T cells: role of lymphoid tissue microenvironment and effect of immunomodulating agents.

The ability of resting CD4+ T cells to support HIV replication is relevant to understanding how the reservoir of HIV-1-infected resting CD4+ T cells is generated, maintained and, hopefully, how it might be reduced or eliminated. We have utilized a tonsillar histoculture system to demonstrate that HIV, particularly X4 strains, can productively infect phenotypically resting CD4+ T cells in vitro and that this event is largely dependent on the lymphoid tissue microenvironment. Highly purified CD4+ tonsillar T cells that lack expression of both cell surface and nuclear antigens characteristic of classic T cell activation produce X4 HIV-1 mRNA, p24, and infectious virus while maintaining a resting phenotype when cultured in a tonsillar tissue microenvironment; in contrast, comparable purified resting CD4+ tonsillar T cells that have been exposed to X4 HIV do not support HIV replication when cultured in the absence of a lymphoid tissue microenvironment. HIV production from phenotypically resting CD4+ T cells is dramatically inhibited by anti-proinflammatory cytokine agents or immunosuppressive cytokines, but is only modestly suppressed by an inhibitor of the cell cycle. The ability of resting CD4+ T cells to support HIV replication in the microenvironment of the lymphoid tissue has implications in the pathogenesis of HIV disease and may provide an additional avenue for therapeutic intervention.

HIV envelope induces virus expression from resting CD4+ T cells isolated from HIV-infected individuals in the absence of markers of cellular activation or apoptosis.

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 effect of elective cesarean delivery and intrapartum infection on fetal lymphocyte activation and susceptibility to HIV infection.

OBJECTIVE: Mother-to-child transmission of the human immunodeficiency virus may be reduced with elective cesarean delivery before labor. Because immune activation enhances the human immunodeficiency virus infection, we hypothesized that fetal lymphocytes that are obtained at elective cesarean delivery may be less activated, therefore less susceptible to human immunodeficiency virus infection than cells that are obtained after normal spontaneous vaginal delivery at term. A second hypothesis was that intrapartum infection correlates with increased lymphocyte activation and susceptibility to human immunodeficiency virus infection. STUDY DESIGN: Samples were obtained after normal spontaneous vaginal delivery (n = 13), elective cesarean delivery (n = 12), chorioamnionitis (n = 5), and preterm labor (n = 6). Activation markers were measured by flow cytometry, and cord blood mononuclear cells were infected with the human immunodeficiency virus. RESULTS: Cell activation was comparable within the normal spontaneous vaginal delivery and elective cesarean delivery groups; there was no difference in susceptibility to in vitro human immunodeficiency virus infection. Intrapartum infection (chorioamnionitis, preterm labor) was associated with increased cell activation. Chorioamnionitis/preterm labor also tended to increase cord blood mononuclear cell susceptibility to human immunodeficiency virus infection. CONCLUSION: Labor did not activate fetal lymphocytes or alter susceptibility to human immunodeficiency virus infection compared with elective cesarean delivery. Intrapartum infection was associated with cell activation, and there was a trend toward increased susceptibility to human immunodeficiency virus infection. These data suggest that fetal lymphocyte activation correlates with susceptibility to human immunodeficiency virus infection and may account for the increased mother-to-child transmission of the human immunodeficiency virus that has been seen in association with chorioamnionitis and preterm labor.

Interleukin (IL)-4 inhibits phorbol-ester induced HIV-1 expression in chronically infected U1 cells independently from the autocrine effect of endogenous tumour necrosis factor-alpha, IL-1beta, and IL-1 receptor antagonist.

The anti-inflammatory cytokine interleukin 4 (IL-4) has shown both inductive and inhibitory effects on the replication of the human immunodeficiency virus type 1 (HIV-1) in primary CD4+ T cells and mononuclear phagocytes. In this study, IL-4 did not induce virus production, but inhibited phorbol esters (PMA)-stimulated HIV expression in chronically infected promonocytic U1 cells. This effect, however, was not accounted for by a decreased secretion of endogenous TNF-alpha induced by phorbol myristate acetate (PMA). We also observed that PMA upregulated the production of both IL-1beta and of IL-1 receptor antagonist (IL-1ra). IL-4 inhibited the secretion of IL-1beta and strongly increased that of IL-1ra; however, these effects were not responsible of IL-4-mediated inhibition of PMA-induced HIV expression since anti-IL-1ra antibodies did not revert IL-4 mediated suppression. U1 cells were transiently transfected with both wild-type (WT) long terminal repeat (LTR) constructs, or with LTR plasmids containing deletions of either the NF-kappaB or the Sp-1 binding sites. IL-4 inhibited LTR-driven transcription triggered by PMA stimulation of U1 cells, and this effect was dependent upon intact NF-kappaB but not Sp-1 binding sites. Thus, IL-4 may favour a state of microbiological quiescence in infected monocytic cells bypassing the induction of HIV expression mediated by pro-inflammatory cytokines.