Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment

The human immunodeficiency virus (HIV)-mediated immune response may be beneficial or harmful, depending on the balance between expansion of HIV-specific T cells and the level of generalized immune activation. The current study utilizes multicolor cytokine flow cytometry to study HIV-specific T cells and T-cell activation in 179 chronically infected individuals at various stages of HIV disease, including those with low-level viremia in the absence of therapy ("controllers"), low-level drug-resistant viremia in the presence of therapy (partial controllers on antiretroviral therapy [PCAT]), and high-level viremia ("noncontrollers"). Compared to noncontrollers, controllers exhibited higher frequencies of HIV-specific interleukin-2-positive gamma interferon-positive (IL-2(+) IFN-gamma(+)) CD4(+) T cells. The presence of HIV-specific CD4(+) IL-2(+) T cells was associated with low levels of proliferating T cells within the less-differentiated T-cell subpopulations (defined by CD45RA, CCR7, CD27, and CD28). Despite prior history of progressive disease, PCAT patients exhibited many immunologic characteristics seen in controllers, including high frequencies of IL-2(+) IFN-gamma(+) CD4(+) T cells. Measures of immune activation were lower in all CD8(+) T-cell subsets in controllers and PCAT compared to noncontrollers. Thus, control of HIV replication is associated with high levels of HIV-specific IL-2(+) and IFN-gamma(+) CD4(+) T cells and low levels of T-cell activation. This immunologic state is one where the host responds to HIV by expanding but not exhausting HIV-specific T cells while maintaining a relatively quiescent immune system. Despite a history of advanced HIV disease, a subset of individuals with multidrug-resistant HIV exhibit an immunologic profile comparable to that of controllers, suggesting that functional immunity can be reconstituted with partially suppressive highly active antiretroviral therapy.     

Increased frequency of regulatory T cells accompanies increased immune activation in rectal mucosae of HIV-positive noncontrollers

Gut-associated lymphoid tissue (GALT) is a major site of HIV replication and CD4(+) T cell depletion. Furthermore, microbial translocation facilitated by mucosal damage likely contributes to the generalized immune activation observed in HIV infection. Regulatory T cells (Treg) help maintain homeostasis and suppress harmful immune activation during infection; however, in the case of persistent viral infections such as HIV, their role is less clear. Although a number of studies have examined Treg in blood during chronic infection, few have explored Treg in the gastrointestinal mucosa. For this study, paired blood and rectal biopsy samples were obtained from 12 HIV noncontrollers (viral load of >10,000 copies/ml plasma), 10 HIV controllers (viral load of <500 copies/ml plasma for more than 5 years), and 12 HIV seronegative control subjects. Noncontrollers had significantly higher percentages of Treg in rectal mononuclear cells (RMNC), but not in blood, compared to seronegative subjects (P = 0.001) or HIV controllers (P = 0.002). Mucosal Treg positively correlated with viral load (P = 0.01) and expression of immune activation markers by CD4(+) (P = 0.01) and CD8(+) (P = 0.07) T cells. Suppression assays indicated that mucosal and peripheral Treg of noncontrollers and controllers maintained their capacity to suppress non-Treg proliferation to a similar extent as Treg from seronegative subjects. Together, these findings reveal that rather than experiencing depletion, mucosal Treg frequency is enhanced during chronic HIV infection and is positively correlated with viral load and immune activation. Moreover, mucosal Treg maintain their suppressive ability during chronic HIV infection, potentially contributing to diminished HIV-specific T cell responses and viral persistence.     

CD8 T-Cells from Most HIV-Infected Patients Lack Ex Vivo HIV-Suppressive Capacity during Acute and Early Infection

The strong CD8+ T-cell-mediated HIV-1-suppressive capacity found in a minority of HIV-infected patients in chronic infection is associated with spontaneous control of viremia. However, it is still unclear whether such capacities were also present earlier in the CD8+ T cells from non controller patients and then lost as a consequence of uncontrolled viral replication. We studied 50 patients with primary HIV-1-infection to determine whether strong CD8+ T-cell-mediated HIV suppression is more often observed at that time. Despite high frequencies of polyfunctional HIV-specific CD8+ T-cells and a strong CD4+ T-helper response, CD8+ T-cells from 48 patients lacked strong HIV-suppressive capacities ex vivo. This indicates that the superior HIV-suppressive capacity of CD8+ T-cells from HIV controllers is not a general characteristic of the HIV-specific CD8+ T cell response in primary HIV infection.     

Maintenance of large numbers of virus-specific CD8+ T cells in HIV-infected progressors and long-term nonprogressors

The virus-specific CD8+ T cell responses of 21 HIV-infected patients were studied including a unique cohort of long-term nonprogressors with low levels of plasma viral RNA and strong proliferative responses to HIV Ags. HIV-specific CD8+ T cell responses were studied by a combination of standard cytotoxic T cell (CTL) assays, MHC tetramers, and TCR repertoire analysis. The frequencies of CD8+ T cells specific to the majority of HIV gene products were measured by flow cytometric detection of intracellular IFN-gamma in response to HIV-vaccinia recombinant-infected autologous B cells. Very high frequencies (0.8-18.0%) of circulating CD8+ T cells were found to be HIV specific. High frequencies of HIV-specific CD8+ T cells were not limited to long-term nonprogressors with restriction of plasma virus. No correlation was found between the frequency of HIV-specific CD8+ T cells and levels of plasma viremia. In each case, the vast majority of cells (up to 17.2%) responded to gag-pol. Repertoire analysis showed these large numbers of Ag-specific cells were scattered throughout the repertoire and in the majority of cases not contained within large monoclonal expansions. These data demonstrate that high numbers of HIV-specific CD8+ T cells exist even in patients with high-level viremia and progressive disease. Further, they suggest that other qualitative parameters of the CD8+ T cell response may differentiate some patients with very low levels of plasma virus and nonprogressive disease.     

Cell surface markers of regulatory T cells are not associated with increased forkhead box p3 expression in blood CD4+ T cells from HIV-infected patients responding to antiretroviral therapy

Regulatory T (Treg) cells may attenuate host immune responses to pathogens, including HIV and opportunistic pathogens in HIV-infected patients. Treated and untreated progressive HIV disease represent a range of immunological scenarios with potentially different roles for Treg cells. A cell surface marker to determine Treg cell numbers would assist in identifying situations where Treg cells are important. Here we show that levels of Foxp3 mRNA are increased in CD4+ T cells from HIV-infected patients responding to antiretroviral therapy. However, the proportion of peripheral blood CD4+ and CD8+ T cells expressing CD25, neuropilin-1, glucocorticoid-induced TNF receptor and lymphocyte activation gene-3 did not differ as a result of treated or untreated HIV infection when compared with HIV-seronegative controls. Hence, none of the putative Treg cell surface markers identified T-cell populations in peripheral blood that mirrored the effects of HIV infection and antiretroviral therapy on Foxp3 expression.     

Expansion and contraction of HIV-specific CD4 T cells with short bursts of viremia, but physical loss of the majority of these cells with sustained viral replication

Chronic infection with the HIV results in poor HIV-specific CD4 T cell proliferation, but more recent analyses using intracellular cytokine staining demonstrated that IFN-gamma-producing, HIV-specific CD4 T cells can be detected for years in HIV-infected subjects. Because it is not known whether the majority of HIV-specific T cells are lost or become dysfunctional, we examined the kinetics of the T cell response over an extended period of time using a panel of 10 HLA-DR tetramers loaded with HIV p24 peptides. Tetramer+ CD4 T cells were present at a relatively high frequency during acute infection, but the size of these populations substantially contracted following suppression of viral replication. Short-term cessation of antiretroviral therapy resulted in a burst of viral replication and concomitant expansion of tetramer+ CD4 T cells, and these populations again contracted following reinitiation of therapy. The kinetics with which these cell populations contracted were characteristic of effector T cells, a conclusion that was supported by their phenotypic (CCR7-CD45RA-) and functional properties (IFN-gamma+). Continued high-level viremia resulted in the physical loss of the majority of tetramer+ CD4 T cells, and the decline of HIV p24-specific CD4 T cells occurred more rapidly and was more substantial than the reduction of total CD4 T cell numbers. We conclude that the population of HIV p24-specific CD4 T cells is initially responsive to changes in the levels of viral Ags, but that the majority of these cells are lost in a setting of chronic viremia.     

HIV-specific CD4 T cell responses to different viral proteins have discordant associations with viral load and clinical outcome

A successful prophylactic vaccine is characterized by long-lived immunity, which is critically dependent on CD4 T cell-mediated helper signals. Indeed, most licensed vaccines induce antigen-specific CD4 T cell responses, in addition to high-affinity antibodies. However, despite the important role of CD4 T cells in vaccine design and natural infection, few studies have characterized HIV-specific CD4 T cells due to their preferential susceptibility to HIV infection. To establish at the population level the impact of HIV-specific CD4 T cells on viral control and define the specificity of HIV-specific CD4 T cell peptide targeting, we conducted a comprehensive analysis of these responses to the entire HIV proteome in 93 subjects at different stages of HIV infection. We show that HIV-specific CD4 T cell responses were detectable in 92% of individuals and that the breadth of these responses showed a significant inverse correlation with the viral load (P = 0.009, R = -0.31). In particular, CD4 T cell responses targeting Gag were robustly associated with lower levels of viremia (P = 0.0002, R = -0.45). Importantly, differences in the immunodominance profile of HIV-specific CD4 T cell responses distinguished HIV controllers from progressors. Furthermore, Gag/Env ratios were a potent marker of viral control, with a high frequency and magnitude of Gag responses and low proportion of Env responses associated with effective immune control. At the epitope level, targeting of three distinct Gag peptides was linked to spontaneous HIV control (P = 0.60 to 0.85). Inclusion of these immunogenic proteins and peptides in future HIV vaccines may act as a critical cornerstone for enhancing protective T cell responses.     

HIV-specific CD8+ T cells exhibit markedly reduced levels of Bcl-2 and Bcl-xL

Human immunodeficiency virus-specific CD8(+) T cells are highly sensitive to spontaneous and CD95/Fas-induced apoptosis, and this sensitivity may impair their ability to control HIV infection. To elucidate the mechanism behind this sensitivity, in this study we examined the levels of antiapoptotic molecules Bcl-2 and Bcl-x(L) in HIV-specific CD8(+) T cells from HIV-infected individuals. Bcl-2 expression was markedly decreased in HIV-specific CD8(+) T cells compared with CMV-specific and total CD8(+) T cells from HIV-infected individuals as well as total CD8(+) T cells from healthy donors. CD8(+) T cell Bcl-2 levels inversely correlated with spontaneous and CD95/Fas-induced apoptosis of CD8(+) T cells from HIV-infected individuals. HIV-specific CD8(+) T cells also had significantly lower levels of Bcl-x(L) compared with CMV-specific CD8(+) T cells. Finally, IL-15 induces both Bcl-2 and Bcl-x(L) expression in HIV-specific and total CD8(+) T cells, and this correlated with apoptosis inhibition and increased survival in both short- and long-term cultures. Our data indicate that reduced Bcl-2 and Bcl-x(L) may play an important role in the increased sensitivity to apoptosis of HIV-specific CD8(+) T cells and suggest a possible mechanism by which IL-15 increases their survival.     

GB Virus C Infection Is Associated with Altered Lymphocyte Subset Distribution and Reduced T Cell Activation and Proliferation in HIV-Infected Individuals

GBV-C infection is associated with prolonged survival and with reduced T cell activation in HIV-infected subjects not receiving combination antiretroviral therapy (cART). The relationship between GBV-C and T cell activation in HIV-infected subjects was examined. HIV-infected subjects on cART with non-detectable HIV viral load (VL) or cART naïve subjects were studied. GBV-C VL and HIV VL were determined. Cell surface markers of activation (CD38⁺/HLA-DR⁺), proliferation (Ki-67+), and HIV entry co-receptor expression (CCR5+ and CXCR4+) on total CD4+ and CD8+ T cells, and on naïve, central memory (CM), effector memory (EM), and effector CD4+ and CD8+ subpopulations were measured by flow cytometry. In subjects with suppressed HIV VL, GBV-C was consistently associated with reduced activation in naïve, CM, EM, and effector CD4+ cells. GBV-C was associated with reduced CD4+ and CD8+ T cell surface expression of activation and proliferation markers, independent of HIV VL classification. GBV-C was also associated with higher proportions of naïve CD4+ and CD8+ T cells, and with lower proportions of EM CD4+ and CD8+ T cells. In conclusion, GBV-C infection was associated with reduced activation of CD4+ and CD8+ T cells in both HIV viremic and HIV RNA suppressed patients. Those with GBV-C infection demonstrated an increased proportion of naive T cells and a reduction in T cell activation and proliferation independent of HIV VL classification, including those with suppressed HIV VL on cART. Since HIV pathogenesis is thought to be accelerated by T cell activation, these results may contribute to prolonged survival among HIV infected individuals co-infected with GBV-C. Furthermore, since cART therapy does not reduce T cell activation to levels seen in HIV-uninfected people, GBV-C infection may be beneficial for HIV-related diseases in those effectively treated with anti-HIV therapy.     

HIV-specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-gamma, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.     

Immune activation, CD4+ T cell counts, and viremia exhibit oscillatory patterns over time in patients with highly resistant HIV infection

The rates of immunologic and clinical progression are lower in patients with drug-resistant HIV compared to wild-type HIV. This difference is not fully explained by viral load. It has been argued that reductions in T cell activation and/or viral fitness might result in preserved target cells and an altered relationship between the level of viremia and the rate of CD4+ T cell loss. We tested this hypothesis over time in a cohort of patients with highly resistant HIV. Fifty-four antiretroviral-treated patients with multi-drug resistant HIV and detectable plasma HIV RNA were followed longitudinally. CD4+ T cell counts and HIV RNA levels were measured every 4 weeks and T cell activation (CD38/HLA-DR) was measured every 16 weeks. We found that the levels of CD4+ T cell activation over time were a strong independent predictor of CD4+ T cell counts while CD8+ T cell activation was more strongly associated with viremia. Using spectral analysis, we found strong evidence for oscillatory (or cyclic) behavior in CD4+ T cell counts, HIV RNA levels, and T cell activation. Each of the cell populations exhibited an oscillatory behavior with similar frequencies. Collectively, these data suggest that there may be a mechanistic link between T cell activation, CD4+ T cell counts, and viremia and lends support for the hypothesis of altered predator-prey dynamics as a possible explanation of the stability of CD4+ T cell counts in the presence of sustained multi-drug resistant viremia.     

Programmed death 1 expression on HIV-specific CD4+ T cells is driven by viral replication and associated with T cell dysfunction

Functional impairment of HIV-specific CD4(+) T cells during chronic HIV infection is closely linked to viral replication and thought to be due to T cell exhaustion. Programmed death 1 (PD-1) has been linked to T cell dysfunction in chronic viral infections, and blockade of the PD-1 pathway restores HIV-specific CD4(+) and CD8(+) T cell function in HIV infection. This study extends those findings by directly examining PD-1 expression on virus-specific CD4(+) T cells. To investigate the role of PD-1 in HIV-associated CD4(+) T cell dysfunction, we measured PD-1 expression on blood and lymph node T cells from HIV-infected subjects with chronic disease. PD-1 expression was significantly higher on IFN-gamma-producing HIV-specific CD4(+) T cells compared with total or CMV-specific CD4(+) T cells in untreated HIV-infected subjects (p = 0.0001 and p < 0.0001, respectively). PD-1 expression on HIV-specific CD4(+) T cells from subjects receiving antiretroviral therapy was significantly reduced (p = 0.007), and there was a direct correlation between PD-1 expression on HIV-specific CD4(+) T cells and plasma viral load (r = 0.71; p = 0.005). PD-1 expression was significantly higher on HIV-specific T cells in the lymph node, the main site of HIV replication, compared with those in the blood (p = 0.0078). Thus, PD-1 expression on HIV-specific CD4(+) T cells is driven by persistent HIV replication, providing a potential target for enhancing the functional capacity of HIV-specific CD4(+) T cells.     

Dynamics of HIV-specific CD8+ T lymphocytes with changes in viral load.The RESTIM and COMET Study Groups

The influence of HIV burden variations on the frequencies of Ag-specific CD8+ T cell responses was evaluated before and during highly active antiretroviral therapy by analyzing the number, diversity, and function of these cells. The frequencies of HLA-A2-restricted CD8+ PBL binding HLA-A2/HIV-epitope tetramers or producing IFN-gamma were below 1%. A panel of 16 CTL epitopes covering 15 HLA class I molecules in 14 patients allowed us to test 3.8 epitopes/patient and to detect 2.2 +/- 1.8 HIV epitope-specific CD8+ subsets per patient with a median frequency of 0.24% (0.11-4. 79%). During the first month of treatment, viral load rapidly decreased and frequencies of HIV-specific CD8 PBL tripled, eight new HIV specificities appeared of 11 undetectable at entry, while CMV-specific CD8+ PBL also appeared. With efficient HIV load control, all HIV specificities decayed involving a reduction of the CD8+CD27+CD11ahigh HIV-specific effector subset. Virus rebounds triggered by scheduled drug interruptions or transient therapeutic failures induced four patterns of epitope-specific CD8+ lymphocyte dynamics, i.e., peaks or disappearance of preexisting specificities, emergence of new specificities, or lack of changes. The HIV load rebounds mobilized both effector/memory HIV- and CMV-specific CD8+ lymphocytes. Therefore, frequencies of virus-specific CD8 T cells appear to be positively correlated to HIV production in most cases during highly active antiretroviral therapy, but an inverse correlation can also be observed with rapid virus changes that might involve redistribution, sequestration, or expansion of these Ag-specific CD8 T cells. Future strategies of therapeutic interruptions should take into account these various HIV-specific cell dynamics during HIV rebounds.     

Comprehensive analysis of frequency and phenotype of T regulatory cells in HIV infection: CD39 expression of FoxP3+ T regulatory cells correlates with progressive disease

There are conflicting data about the frequency and role of regulatory T cells (Tregs) during the course of HIV infection. Peripheral blood of a large cohort of HIV-infected patients (n = 131) at different stages of disease, including 15 long-term nonprogressors and 21 elite controllers, was analyzed to determine the frequency and phenotype of Tregs, defined as CD4(+), CD25(high), CD127(low), FoxP3(high) cells. A significantly increased relative frequency of Tregs within the CD4(+) compartment of HIV(+) patients compared to that of healthy controls (P < 0.0001) was observed. Additionally, the relative frequency of Tregs directly correlated with HIV viral load and inversely with CD4(+) counts. However, the absolute Treg number was reduced in HIV-infected patients versus healthy controls (P < 0.0001), with the exception of elite controllers (P > 0.05). The loss of absolute Treg numbers coincided with rising markers of immune activation (P < 0.0006). The initiation of antiviral therapy significantly increased absolute Treg numbers (P < 0.0031). We find that the expression of CD39, a newly defined ectonucleotidase with immunomodulatory functions on Tregs, correlated with progressive HIV disease, HIV viral load, and immune activation. Of note, when tested in peripheral blood mononuclear cells of healthy volunteers, the in vitro capacity to suppress T-cell proliferation was limited to CD4(+), CD25(high), CD39(+) T cells. Interestingly, Tregs of elite controllers exhibited not only the highest expression of CCR5, CTLA-4, and ICOS but also the lowest level of CD39. The data presented here reconcile the seemingly contradictory results of previous studies looking at Tregs in HIV and highlight the complexity of Treg-mediated immunoregulation during human viral infections.     

Changes in paracrine interleukin-2 requirement, CCR7 expression, frequency, and cytokine secretion of human immunodeficiency virus-specific CD4+ T cells are a consequence of antigen load

Virus-specific CD4+ T-cell responses are thought to be required for the induction and maintenance of many effective CD8+ T-cell and B-cell immune responses in experimental animals and humans. Although the presence of human immunodeficiency virus (HIV)-specific CD4+ T cells has been documented in patients at all stages of HIV infection, many fundamental questions regarding their frequency and function remain. A 10-color, 12-parameter flow cytometric panel was utilized to examine the frequency, memory phenotype (CD27, CCR7, and CD45RA), and cytokine production (interleukin-2 [IL-2], gamma interferon, and tumor necrosis factor alpha) of CD4+ T cells specific for HIV antigens as well as for adenovirus, Epstein-Barr virus (EBV), influenza H1N1 virus, influenza H3N2 virus, cytomegalovirus, varicella-zoster virus (VZV), and tetanus toxoid in normal controls, long-term nonprogressors (LTNP), and HIV-infected patients with progressive disease on or off therapy. The HIV-specific CD4+ T-cell responses in LTNP and patients on therapy were similar in frequency, phenotype, and cytokine production to responses directed against adenovirus, EBV, influenza virus, and VZV. HIV-specific CD4+ T cells from patients off antiretroviral therapy demonstrated a shift towards a CCR7(-) CD45RA(-) phenotype and a reduced percentage of IL-2-producing cells. The alterations in cytokine production during HIV viremia were found to be intrinsic to the HIV-specific CD4+ T cells and caused a requirement for IL-2 supplied exogenously for proliferation to occur. These observations suggest that many previously described changes in HIV-specific CD4+ T-cell function and phenotype are a consequence of high levels of antigen in viremic patients. In addition, defects in function and phenotype of HIV-specific CD4+ T cells are not readily discernible in the context of antiretroviral therapy but rather are similar to responses to other viruses.     

Differential Gag-specific polyfunctional T cell maturation patterns in HIV-1 elite controllers

A small fraction of HIV-infected individuals (<1%), referred to as elite controllers (EC), are able to maintain undetectable viral loads indefinitely without treatment. The role of the maturational phenotype of T cells in the control of HIV infection in these individuals is not well described. We compared the maturational and functional phenotypes of Gag-specific CD4 and CD8 T cells from EC, who maintain undetectable viral loads without treatment; relative controllers (RC), who maintain viral loads of <1,000 copies/ml without treatment; and noncontrollers (NC), who fail to control viral replication. EC maintained higher frequencies of HIV-specific CD4 T cells, less mature polyfunctional Gag-specific CD4 T cells (CD27(+) CD57(-) CD45RO(+)), and Gag-specific polyfunctional CD4 T cells than those observed in NC. In EC, the frequency of polyfunctional Gag-specific CD8 T cells was higher than that observed in RC and NC. RC had a similar functional phenotype to that observed in NC, despite consistently lower viral loads. Finally, we found a direct correlation between the frequency of Gag-specific CD27(+) CD57(-) CD45RO(+) CD4(+) T cells and the frequency of mature HIV-specific CD8 T cells. Altogether, our data suggest that immature Gag-specific interleukin-2 (IL-2)-producing CD4(+) T cells may play an important role in spontaneous control of HIV viremia by effectively supporting HIV-specific CD8 T lymphocytes. This difference appears to differentiate EC from RC.     

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.     

Composition and Function of T Cell Subpopulations Are Slow to Change Despite Effective Antiretroviral Treatment of HIV Disease

The ability to reconstitute a normal immune system with antiretroviral therapy in the setting of HIV infection remains uncertain. This study aimed to characterize quantitative and qualitative aspects of various T cell subpopulations that do not improve despite effective ART. CD4∶CD8 ratio was evaluated in HIV-infected subjects with viral loads >10,000 copies/µl (“non-controllers”, n = 42), those with undetectable viral loads on ART (“ART-suppressed”, n = 53), and HIV-uninfected subjects (n = 22). In addition, T cell phenotype and function were examined in 25 non-controllers, 18 ART-suppressed, and 7 HIV-uninfected subjects. CD4∶CD8 ratio in non-controllers, ART-suppressed, and HIV-uninfected subjects was 0.25, 0.48, and 1.95 respectively (P<0.0001 for all comparisons). The increased ratio in ART-suppressed compared to non-controllers was driven by an increase of CD4+ T cells, with no change in the expanded CD8+ T cell population. Expansion of differentiated (CD28−CD27−CD45RA+/−CCR7−) T cell subpopulations persisted despite ART and minimal changes were noted in naïve T cell frequencies over time. Increased number of CD8+CD28− T cells and increased CD8+ CMV-specific T cell responses were associated with a decreased CD4∶CD8 ratio. Measures of T cell function demonstrated persistence of high frequencies of CD8+ T cells producing IFN–γ. Lastly, though all CD8+ subpopulations demonstrated significantly lower Ki67 expression in ART-suppressed subjects, CD4+ T cell subpopulations did not consistently show this decrease, thus demonstrating different proliferative responses in the setting of T cell depletion. In summary, this study demonstrated that CD4∶CD8 ratios remained significantly decreased and naïve T cell numbers were slow to increase despite long-term viral suppression on ART. In addition, there is a evidence of differential regulation of the CD4+ and CD8+ T cell subpopulations, suggesting independent homeostatic regulation of the two compartments.     

T cell immune discriminants of HIV reservoir size in a pediatric cohort of perinatally infected individuals

The size of the latent HIV reservoir is associated with the timing of therapeutic interventions and overall health of the immune system. Here, we demonstrate that T cell phenotypic signatures associate with viral reservoir size in a cohort of HIV vertically infected children and young adults under durable viral control, and who initiated anti-retroviral therapy (ART) <2 years old. Flow cytometry was used to measure expression of immune activation (IA), immune checkpoint (ICP) markers, and intracellular cytokine production after stimulation with GAG peptides in CD4 and CD8 T cells from cross-sectional peripheral blood samples. We also evaluated the expression of 96 genes in sort-purified total CD4 and CD8 T cells along with HIV-specific CD4 and CD8 T cells using a multiplexed RT-PCR approach. As a measure of HIV reservoir, total HIV-DNA quantification by real-time PCR was performed. Poisson regression modeling for predicting reservoir size using phenotypic markers revealed a signature that featured frequencies of PD-1+CD4 T cells, TIGIT+CD4 T cells and HIV-specific (CD40L+) CD4 T cells as important predictors and it also shows that time of ART initiation strongly affects their association with HIV-DNA. Further, gene expression analysis showed that the frequencies of PD-1+CD4 T cells associated with a CD4 T cell molecular profile skewed toward an exhausted Th1 profile. Our data provide a link between immune checkpoint molecules and HIV persistence in a pediatric cohort as has been demonstrated in adults. Frequencies of PD-1+ and TIGIT+CD4 T cells along with the frequency of HIV-specific CD4 T cells could be associated with the mechanism of viral persistence and may provide insight into potential targets for therapeutic intervention.