HIV-specific cytotoxic cell frequencies measured directly ex vivo by the Lysispot assay can be higher or lower than the frequencies of IFN-gamma-secreting cells: anti-HIV cytotoxicity is not generally impaired relative to other chronic virus responses

CD8(+) T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8(+) T cells, as measured by IFN-gamma secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN-gamma-secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN-gamma-secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-gamma. These results indicate that measurement of IFN-gamma production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN-gamma ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of "preterminal differentiation" of HIV-specific CD8(+) T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.     

Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection

Unlike HIV-1-infected people, most HIV-2-infected subjects maintain a healthy CD4+ T cell count and a strong HIV-specific CD4+ T cell response. To define the cellular immunological correlates of good prognosis in HIV-2 infection, we conducted a cross-sectional study of HIV Gag-specific T cell function in HIV-1- and HIV-2-infected Gambians. Using cytokine flow cytometry and lymphoproliferation assays, we show that HIV-specific CD4+ T cells from HIV-2-infected individuals maintained proliferative capacity, were not terminally differentiated (CD57-), and more frequently produced IFN-gamma or IL-2 than CD4+ T cells from HIV-1-infected donors. Polyfunctional (IFN-gamma+/IL-2+) HIV-specific CD4+ T cells were found exclusively in HIV-2+ donors. The disparity in CD4+ T cell responses between asymptomatic HIV-1- and HIV-2-infected subjects was not associated with differences in the proliferative capacity of HIV-specific CD8+ T cells. This study demonstrates that HIV-2-infected donors have a well-preserved and functionally heterogeneous HIV-specific memory CD4+ T cell response that is associated with delayed disease progression in the majority of infected people.     

Antiretroviral treatment induces a shift to type-2 cytokine responses in HIV-1 infected pregnant women

We report on a cross-sectional study on proliferation and cytokine production (IFN-gamma, IL-12, IL-5 and TNF-alpha) by peripheral blood mononuclear cells (PBMC), activated or not with phytohemagglutinin (PHA) in HIV-1-infected pregnant women, untreated or treated with zidovudine. We compared the results with healthy women, either pregnant or not, and with HIV-1-infected, non-pregnant women. The most significant results indicate that basal IL-5 production in HIV-1-infected pregnant women was higher than in the rest of the groups, being even higher in the zidovudine-treated than in the untreated group. IL-5 and TNF-alpha production by PHA-activated PBMC was also higher in HIV-1 pregnant women than in controls and infected non-pregnant women. IFN-gamma production was much higher in healthy women than in the other groups. Finally, the IFN-gamma/IL-5 (Th1-type/Th2-type-cytokine) ratio was lower in HIV-infected than in uninfected groups. Zidovudine treatment reduced basal IL-12 and increased PHA-stimulated IL-5 production. Our results indicate that both HIV-1 infection and pregnancy favored a Th2-type response by T cells. Interestingly, zidovudine-treated pregnant women had a significantly higher Th2-type response than untreated ones.     

Cross-reactive cytotoxic T lymphocytes against human immunodeficiency virus type 1 protease and gamma interferon-inducible protein 30

The gamma interferon (IFN-gamma)-inducible protein 30 (IP-30) signal peptide -11 to -3 (LLDVPTAAV) is a prominent self peptide expressed with the class I human histocompatibility leukocyte antigen A2 (HLA-A2). Stimulation of peripheral blood mononuclear cells (PBMC) from HLA-A2 human immunodeficiency virus type 1 (HIV-1)-infected individuals with an HLA-A2-restricted HIV protease (PR) peptide 76-84 (LVGPTPVNI) activated cytotoxic T lymphocytes (CTL) against the IP-30 signal peptide. Since HIV-1 PR 76-84 stimulated CD8+ T cells from these individuals to secrete IFN-gamma, we tested whether the activation of IP-30-specific CTL in vitro resulted from T-cell cross-reactivity or from up-regulation of IP-30 by IFN-gamma. Neither high levels of exogenous IFN-gamma nor incubation of PBMC with other HIV peptides triggering substantial IFN-gamma release activated IP-30-specific CTL. Although the IP-30 signal peptide did not stimulate IFN-gamma release from freshly isolated PBMC, it activated CTL in vitro against itself and HIV PR 76-84. Peptide-stimulated IFN-gamma release, cold target inhibition, and HLA-A2/immunoglobulin dimer-mediated binding and depletion of effector cells all indicated that in vitro stimulation with HIV PR 76-84 or the IP-30 signal peptide activated a comparable population of cross-reactive effector cells. Neither IP-30 nor HIV PR 76-84 activated CTL against themselves following in vitro stimulation of PBMC from non-HIV-infected HLA-A2 individuals. Peptide titrations indicated higher-avidity T-cell interactions with HIV PR 76-84 than with the IP-30 signal peptide. These data indicate that HIV PR 76-84 is a heteroclitic variant of the IP-30 signal peptide -11 to -3, which has implications for immune memory and autoimmunity.     

Enhancer role of STAT5 in CD2 activation of IFN-gamma gene expression

IFN-gamma is an important immunoregulatory protein with tightly controlled expression in activated T and NK cells. Three potential STAT binding regions have been recognized within the IFN-gamma promoter: 1) an IL-12-mediated STAT4 binding site at -236 bp; 2) a newly identified IL-2-induced STAT5 binding element at -3.6 kb; and 3) CD2-mediated STAT1 and STAT4 binding to an intronic element in mucosal T cells. However, functional activation of these sites remains unclear. In this study we demonstrate CD2-mediated activation of the newly characterized -3.6-kb IFN-gamma STAT5 binding region. CD2 signaling of human PBMC results in activation of the -3.6-kb IFN-gamma promoter, whereas mutation of the -3.6-kb STAT5 site attenuates promoter activity. Functional activation is accompanied by STAT5A but little STAT5B nucleoprotein binding to the IFN-gamma STAT5 site, as determined by competition and supershift assays. STAT5 activation via CD2 occurs independent of IL-2. Western and FACS analysis shows increased phospho-STAT5 following CD2 signaling. AG490, a tyrosine kinase inhibitor affecting Jak proteins, inhibits CD2-mediated IFN-gamma mRNA expression, secretion, and nucleoprotein binding to the IFN-gamma STAT5 site in a dose-dependent fashion. This report is the first to describe CD2-mediated activation of STAT5 and supports STAT5 involvement in regulation of IFN-gamma expression.     

Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Sustained impairment of IFN-gamma secretion in suppressed HIV-infected patients despite mature NK cell recovery: evidence for a defective reconstitution of innate immunity

The impairment of NK cell functions in the course of HIV infection contributes to a decreased resistance against HIV and other pathogens. We analyzed the proportion of mature and immature NK cell subsets, and measured subsets of IFN-gamma and TNF-alpha-producing NK and T cells in viremic or therapy-suppressed HIV-infected subjects, and noninfected control donors. Viremic HIV(+) individuals had significantly lower proportions of mature CD3(-)/CD161(+)/CD56(+) NK cells and of IFN-gamma-producing NK cells compared with noninfected donors, independent of CD4(+) T cell counts. HIV-infected subjects with undetectable viral load recovered mature CD3(-)/CD161(+)/CD56(+) NK cells and cytotoxicity against tumor (K562) and HSV-infected target cells to percentages comparable with those of uninfected individuals, but their NK cells remained impaired in their ability to produce IFN-gamma. In parallel to these ex vivo findings, in vitro NK cell differentiation of CD34-positive cord blood precursors in the presence of R5 or X4 HIV-1 resulted in the production of NK cells with a normal mature phenotype, but lacking the ability to produce IFN-gamma, whereas coculture of uninfected PBMC with HIV failed to affect mature NK cell properties or IFN-gamma secretion. Altogether, our findings support the hypothesis that mature NK cell phenotype may be uncoupled from some mature functions following highly active antiretroviral therapy-mediated suppression of HIV-1, and indicate that relevant innate immune functions of NK cell subsets may remain altered despite effective viral suppression following antiretroviral treatment.     

Preferential apoptosis of HIV-1-specific CD4+ T cells

In contrast to other viral infections such as CMV, circulating frequencies of HIV-1-specific CD4+ T cells in peripheral blood are quantitatively diminished in the majority of HIV-1-infected individuals. One mechanism for this quantitative defect is preferential infection of HIV-1-specific CD4+ T cells, although <10% of HIV-1-specific CD4+ T cells are infected. Apoptosis has been proposed as an important contributor to the pathogenesis of CD4+ T cell depletion in HIV/AIDS. We show here that, within HIV-1-infected individuals, a greater proportion of ex vivo HIV-1-specific CD4+ T cells undergo apoptosis compared with CMV-specific CD4+ T cells (45 vs 7.4%, respectively, p < 0.05, in chronic progressors). The degree of apoptosis within HIV-1-specific CD4+ T cells correlates with viral load and disease progression, and highly active antiretroviral therapy abrogates these differences. The data support a mechanism for apoptosis in these cells similar to that found in activation-induced apoptosis through the TCR, resulting in oxygen-free radical production, mitochondrial damage, and caspase-9 activation. That HIV-1 proteins can also directly enhance activation-induced apoptosis supports a mechanism for a preferential induction of apoptosis of HIV-1-specific CD4+ T cells, which contributes to a loss of immunological control of HIV-1 replication.     

Longitudinal assessment of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon responses during the first year of life in HIV-1-infected infants

Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We evaluated the association between HIV-1-specific gamma interferon (IFN-gamma) release from the cells of infants of 1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and peripartum, respectively (P = 0.09), and in approximately 50% of infants thereafter. Peaks of HIV-specific spot-forming units (SFU) increased significantly with age in all infants, from 251/10(6) peripheral blood mononuclear cells (PBMC) at 1 month of age to 501/10(6) PBMC at 12 months of age (P = 0.03), although when limited to infants who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P = 0.18). Over the first year of life, infants with IFN-gamma responses at 1 month had peak plasma viral loads, rates of decline of viral load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth of IFN-gamma responses at 1 month were not significantly associated with viral containment or mortality. These results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific CD8+-T-cell responses early in life that are not clearly associated with improved clinical outcomes.     

Clonality of HTLV-2 in Natural Infection

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) both cause lifelong persistent infections, but differ in their clinical outcomes. HTLV-1 infection causes a chronic or acute T-lymphocytic malignancy in up to 5% of infected individuals whereas HTLV-2 has not been unequivocally linked to a T-cell malignancy. Virus-driven clonal proliferation of infected cells both in vitro and in vivo has been demonstrated in HTLV-1 infection. However, T-cell clonality in HTLV-2 infection has not been rigorously characterized. In this study we used a high-throughput approach in conjunction with flow cytometric sorting to identify and quantify HTLV-2-infected T-cell clones in 28 individuals with natural infection. We show that while genome-wide integration site preferences in vivo were similar to those found in HTLV-1 infection, expansion of HTLV-2-infected clones did not demonstrate the same significant association with the genomic environment of the integrated provirus. The proviral load in HTLV-2 is almost confined to CD8⁺ T-cells and is composed of a small number of often highly expanded clones. The HTLV-2 load correlated significantly with the degree of dispersion of the clone frequency distribution, which was highly stable over ∼8 years. These results suggest that there are significant differences in the selection forces that control the clonal expansion of virus-infected cells in HTLV-1 and HTLV-2 infection. In addition, our data demonstrate that strong virus-driven proliferation per se does not predispose to malignant transformation in oncoretroviral infections.     

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.     

HIV-1-specific T cell precursors with high proliferative capacity correlate with low viremia and high CD4 counts in untreated individuals

Evidences have recently suggested that the preservation of vaccine-induced memory rather than effector T cells is essential for better outcome and survival following pathogenic SIV challenge in macaques. However, an equivalent demonstration in humans is missing, and the immune correlates of HIV-1 control have been only partially characterized. We focused on the quantification of Ag-specific T cell precursors with high proliferative capacity (PHPC) using a peptide-based cultured IFN-gamma ELISPOT assay (PHPC assay), which has been shown to identify expandable memory T cells. To determine which responses correlate with viral suppression and positive immunologic outcome, PBMC from 32 chronically untreated HIV-1-infected individuals were evaluated in response to peptide pools, representing the complete HIV-1 Gag, Nef, and Rev proteins, by PHPC and IFN-gamma ELISPOT assay, which instead identifies effector T cells with low proliferative capacity. High magnitude of Gag-specific PHPC, but not ELISPOT, responses significantly correlated with low plasma viremia, due to responses directed toward p17 and p15 subunits. Only Gag p17-specific PHPC response significantly correlated with high CD4 counts. Analysis of 20 additional PBMC samples from an independent cohort of chronically untreated HIV-1-infected individuals confirmed the correlation between Gag p17-specific PHPC response and either plasma viremia (inverse correlation) or CD4 counts (direct correlation). Our results indicate that the PHPC assay is quantitatively and qualitatively different from the ELISPOT assay, supporting that different T cell populations are being evaluated. The PHPC assay might be an attractive option for individual patient management and for the design and testing of therapeutic and prophylactic vaccines.     

Differences among HIV-1 variants in their ability to elicit secretion of TNF-alpha

HIV-1 infection of human PBMC has been shown to elicit secretion of several different cytokines. TNF-alpha secretion induced by this virus has been of particular interest because it has been associated with the development of HIV-1 dementia and because TNF-alpha increases viral replication by enhancing NF-kappaB interaction with the viral promoter, the HIV-1 long terminal repeat. Thus, an autocrine pathway is potentially created in which HIV-1 stimulates its own replication. Conflicting reports exist, however, on the ability of HIV-1 to induce TNF-alpha secretion in vitro or in vivo. Using experimental protocols that controlled for potential bacterial endotoxin-induced TNF-alpha secretion, the current study demonstrates significant differences in TNF-alpha-eliciting properties among primary and laboratory obtained HIV-1. The relative TNF-alpha-inducing ability of different variants is conserved when tested using PBMC from different individuals. Elicitation of TNF-alpha secretion was not blocked by exposure of cells to zidovudine, indicating that viral integration was not required to induce secretion. Rather, the interaction between the virus and cell surface is critical for TNF-alpha induction, as Abs against CD4 or CCR5 blocked the induction of TNF-alpha synthesis by PBMC when added before virus exposure. Furthermore, the ability to induce TNF-alpha secretion mapped to a region of the HIV-1 env gene that includes the third hypervariable domain. Differences in the ability of different HIV-1 variants to elicit TNF-alpha may account for individual differences in HIV-1 disease course.