CD8+ T lymphocyte control of HIV replication in cultured CD4+ cells varies among infected individuals

Production of the human immunodeficiency virus (HIV) by cultured peripheral blood mononuclear cells (PMC) from many seropositive individuals is inhibited by the presence of CD8+ T lymphocytes. In a study of 10 subjects, high levels of virus replication could be detected in cultures of purified CD4+ cells, but not in unseparated PMC. Addition of highly purified, autologous CD8+ cells to the enriched CD4+ cells resulted in a dose-dependent inhibition of HIV growth and revealed that for some individuals, even low numbers of CD8+ cells can prevent replication of the virus. The data also indicated that culturing enriched CD4+ cells could greatly enhance detection of infectious virus in blood specimens and demonstrated that the CD4+ molecule is expressed on infected T cells isolated directly from the peripheral blood.     

CD8+ T cells inhibit HIV replication in naturally infected CD4+ T cells. Evidence for a soluble inhibitor

This study describes the inhibitory effect exerted by activated CD8+ T cells on the replication of HIV in naturally infected CD4+ T cells. Highly purified CD4+ T cells from asymptomatic HIV seropositive individuals were stimulated with anti-TCR mAb-coated beads in the presence of IL-2. HIV was subsequently reproducibly isolated in cell supernatants from all study participants (53 cultures from 42 individuals). Both autologous and allogeneic CD8+ T cells from asymptomatic HIV seropositive and healthy HIV seronegative individuals inhibited the replication of HIV in these cultures in a dose-dependent manner. CD8+ T cells from patients with AIDS showed reduced or no such inhibitory activity. The inhibitory effect was not dependent on direct cell-cell contact: an inhibitory effect was exerted by CD8+ T cells across a semipermeable membrane, and an inhibitory activity was also exerted by the cell-free supernatants from activated CD8+ T cells. These results suggest that activated CD8+ T cells secrete a soluble inhibitor of HIV replication.     

Paucity of HIV DNA methylation in latently infected, resting CD4+ T cells from infected individuals receiving antiretroviral therapy

Maintenance of HIV latency in vitro has been linked to methylation of HIV DNA. However, examinations of the degree of methylation of HIV DNA in the latently infected, resting CD4(+) T cells of infected individuals receiving antiretroviral therapy have been limited. Here, we show that methylation of the HIV 5' long terminal repeat (LTR) in the latent viral reservoir of HIV-infected aviremic individuals receiving therapy is rare, suggesting that other mechanisms are likely involved in the persistence of viral latency.     

Human herpesvirus-6-specific interleukin 10-producing CD4+ T cells suppress the CD4+ T-cell response in infected individuals

Human herpesvirus‐6 (HHV‐6) infection normally persists for the lifetime of the host and may reactivate with immunosuppression. The mechanism behind HHV‐6 latent infection is still not fully understood. In this study, we observed that decreased proliferation of CD4⁺ T cells and PBMCs but not CD8⁺ T cells from HHV‐6‐infected individuals was stimulated with HHV‐6‐infected cell lysates. Moreover, HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals have suppressive activity on naïve CD4⁺ T and CD8⁺ T cells from HHV‐6‐uninfected individuals. However, no increased proportion of CD4⁺ CD25⁺ Treg cells from HHV‐6‐infected individuals contributed to the suppressive activity of the HHV‐6‐stimulated CD4⁺ T cells from HHV‐6‐infected individuals. Transwell experiments, ELISA and anti‐IL‐10 antibody blocking experiment demonstrated that IL‐10 may be the suppressive cytokine required for suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interleukin (IL)‐10 and IL‐4 further implicated the HHV‐6‐speciflc IL‐10‐producing CD4⁺ T cells in the suppressive activity of CD4⁺ T cells from HHV‐6‐infected individuals. Results of intracellular interferon (IFN)‐γ demonstrated a decreased frequency of HHV‐6‐speciflc IFN‐γ‐producing CD4⁺ T, but not CD8⁺ T cells in HHV‐6‐infected individuals, indicating that it was the CD4⁺ Th1 responses in HHV‐6‐infected individuals that were selectively impaired. Our findings indicated that HHV‐6‐specific IL‐10‐producing CD4⁺ T cells from HHV‐6‐infected individuals possess T regulatory type 1 cell activity: immunosuppression, high levels of IL‐10 production, with a few cells expressing IFN‐γ, but none expressing IL‐4. These cells may play an important role in latent HHV‐6 infection.     

Disruption of intestinal CD4+ T cell homeostasis is a key marker of systemic CD4+ T cell activation in HIV-infected individuals

HIV infection is associated with depletion of intestinal CD4(+) T cells, resulting in mucosal immune dysfunction, microbial translocation, chronic immune activation, and progressive immunodeficiency. In this study, we examined HIV-infected individuals with active virus replication (n = 15), treated with antiretroviral therapy (n = 13), and healthy controls (n = 11) and conducted a comparative analysis of T cells derived from blood and four gastrointestinal (GI) sites (terminal ileum, right colon, left colon, and sigmoid colon). As expected, we found that HIV infection is associated with depletion of total CD4(+) T cells as well as CD4(+)CCR5(+) T cells in all GI sites, with higher levels of these cells found in ART-treated individuals than in those with active virus replication. While the levels of both CD4(+) and CD8(+) T cell proliferation were higher in the blood of untreated HIV-infected individuals, only CD4(+) T cell proliferation was significantly increased in the gut of the same patients. We also noted that the levels of CD4(+) T cells and the percentages of CD4(+)Ki67(+) proliferating T cells are inversely correlated in both blood and intestinal tissues, thus suggesting that CD4(+) T cell homeostasis is similarly affected by HIV infection in these distinct anatomic compartments. Importantly, the level of intestinal CD4(+) T cells (both total and Th17 cells) was inversely correlated with the percentage of circulating CD4(+)Ki67(+) T cells. Collectively, these data confirm that the GI tract is a key player in the immunopathogenesis of HIV infection, and they reveal a strong association between the destruction of intestinal CD4(+) T cell homeostasis in the gut and the level of systemic CD4(+) T cell activation.     

CD4+CD25+ regulatory T cells in HIV infection

The immune system faces the difficult task of discerning between foreign, potentially pathogen-derived antigens and self-antigens. Several mechanisms, including deletion of self-reactive T cells in the thymus, have been shown to contribute to the acceptance of self-antigens and the reciprocal reactivity to foreign antigens. Over the last decade it has become increasingly clear that CD4(+)CD25(+) T(Reg) cells are crucial for maintenance of T cell tolerance to self-antigens in the periphery, and to avoid development of autoimmune disorders. Recently, evidence has also emerged that demonstrates that CD4(+)CD25(+) T(Reg) cells can also suppress T cell responses to foreign pathogens, including viruses such as HIV. In this article we review the current knowledge and potential role of CD4(+)CD25(+) T(Reg) cells in HIV infection.     

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.     

CD34+ bone marrow cells are infected with HIV in a subset of seropositive individuals.

Individuals infected with HIV frequently develop cytopenias and suppressed hematopoiesis. The role of direct HIV infection of hematopoietic progenitor cells in this process has not been defined. In this study, purified CD34+ bone marrow progenitor cells from 74 Zairian and American patients were studied by both coculture viral isolation and polymerase chain reaction for evidence of HIV infection. A total of 36.5% of Zairian and 14% of American patients had HIV infection of the CD34+ cell subset, with as many as 1 in 500 CD34+ cells infected. Most of the Zairian patients in this study had advanced HIV infection and markedly decreased CD4/CD8 T lymphocyte ratios (mean 0.160 +/- 0.08), and no laboratory value predicted the presence of infection in the CD34+ subset of a given Zairian individual. In contrast, American patients with CD34+ cell infection had total CD4 cells less than 20/mm3 and a greater decrease of the CD4/CD8 T lymphocyte ratio compared to seropositive Americans without CD34+ cell infection (p = 0.003). Hematopoiesis, studied by methylcellulose colony assays, was depressed in all seropositive patients studied with no significant further suppression when CD34+ cells were infected. Thus, CD34+ bone marrow progenitor cells are infected in vivo in a subset of seropositive individuals and may serve as an additional reservoir of virus in HIV-infected individuals.     

Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4(+) T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8(+) T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-gamma, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8(+)CD127(-) T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4(+) T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8(+)CD127(-) effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8(+) T cells may be useful in the clinical management of HIV-infected individuals.     

CD8+ lymphocytes suppress HIV production by autologous CD4+ cells without eliminating the infected cells from culture

Studies of separated peripheral blood mononuclear cell subsets have indicated that the CD8+ lymphocyte is the primary cell type responsible for suppressing human immunodeficiency virus (HIV) replication by infected CD4+ cells. The effect of this antiviral activity is dose-dependent and does not involve killing of the infected cell. These observations indicate that this response is distinct from the anti-HIV cytotoxic mechanisms also described for human CD8+ cells.     

CD4+CD8+ T cells represent a significant portion of the anti-HIV T cell response to acute HIV infection

Previous studies have revealed that HIV-infected individuals possess circulating CD4(+)CD8(+) double-positive (DP) T cells specific for HIV Ags. In the present study, we analyzed the proliferation and functional profile of circulating DP T cells from 30 acutely HIV-infected individuals and 10 chronically HIV-infected viral controllers. The acutely infected group had DP T cells that showed more proliferative capability and multifunctionality than did both their CD4(+) and CD8(+) T cells. DP T cells were found to exhibit greater proliferation and higher multifunctionality compared with CD4 T cells in the viral controller group. The DP T cell response represented 16% of the total anti-HIV proliferative response and >70% of the anti-HIV multifunctional response in the acutely infected subjects. Proliferating DP T cells of the acutely infected subjects responded to all HIV Ag pools with equal magnitude. Conversely, the multifunctional response was focused on the pool representing Nef, Rev, Tat, VPR, and VPU. Meanwhile, the controllers' DP T cells focused on Gag and the Nef, Rev, Tat, VPR, and VPU pool for both their proliferative and multifunctional responses. Finally, we show that the presence of proliferating DP T cells following all HIV Ag stimulations is well correlated with proliferating CD4 T cells whereas multifunctionality appears to be largely independent of multifunctionality in other T cell compartments. Therefore, DP T cells represent a highly reactive cell population during acute HIV infection, which responds independently from the traditional T cell compartments.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

HIV envelope suppresses CD4+ T cell activation independent of T regulatory cells

We previously demonstrated that HIV envelope glycoprotein (Env), delivered in the form of a vaccine and expressed by dendritic cells or 293T cells, could suppress Ag-stimulated CD4(+) T cell proliferation. The mechanism remains to be identified but is dependent on CD4 and independent of coreceptor binding. Recently, CD4(+) regulatory T (Treg) cells were found to inhibit protective anti-HIV CD4(+) and CD8(+) T cell responses. However, the role of Tregs in HIV remains highly controversial. HIV Env is a potent immune inhibitory molecule that interacts with host CD4(+) cells, including Treg cells. Using an in vitro model, we investigated whether Treg cells are involved in Env-induced suppression of CD4(+) T cell proliferation, and whether Env directly affects the functional activity of Treg cells. Our data shows that exposure of human CD4(+) T cells to Env neither induced a higher frequency nor a more activated phenotype of Treg cells. Depletion of CD25(+) Treg cells from PBMC did not overcome the Env-induced suppression of CD4(+) T cell proliferation, demonstrating that CD25(+)FoxP3(+) Treg cells are not involved in Env-induced suppression of CD4(+) T cell proliferation. In addition, we extend our observation that similar to Env expressed on cells, Env present on virions also suppresses CD4(+) T cell proliferation.     

Age-related changes in mature CD4+ T cells: cell cycle analysis

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.     

CD4+ and CD8+ T Cell Activation Are Associated with HIV DNA in Resting CD4+ T Cells

The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1) in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4⁺ and CD8⁺ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.     

Memory CD8+ T cells in HIV infection

Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent HIV infection in humans. The kinetics and general features of the CTL response are similar to those found during other persisting virus infections in humans. During chronic infection there are commonly between 0.1 and 1.0% of all CD8+ T cells in the blood that are specific for immunodominant virus epitopes, as measured by HLA class I peptide tetramers. These figures are greatly in excess of the numbers found by limiting dilution assays; the discrepancy may arise because in the latter assay, CTLs have to divide many times to be detected and many of the HIV-specific CD8+ T cells circulating in infected persons may be incapable of further division. Many tetramer-positive T cells make interferon-gamma, beta-chemokines and perforin, so are probably functional. It is not known how fast these T cells turn over, but in the absence of antigen they decay in number. Impairment of CTL replacement, because CD4+ T helper cells are depleted by HIV infection, may play a major role in the development of AIDS.