Although active replication of HIV-1 can be effectively suppressed with antiretroviral combination therapy, HIV-1 persists life long in the human body by establishing a latent, transcriptionally silent infection in CD4 T cells.1,2 Central memory and effector memory CD4 T cells may represent the predominant reservoir for these latently infected cells3; however, naive CD4 T cells, a long-lasting, antigen-inexperienced quiescent T-cell population, also contribute to the latently infected cell pool.4 Current efforts focus on mobilizing this reservoir by pharmaceutical agents that can reactivate HIV-1 replication in latently infected cells5; however, reactivation of HIV-1 replication itself may not necessarily eliminate these cells, and the combinations of immune-mediated and pharmaceutical interventions may ultimately be necessary to reduce viral persistence.
HIV-1–specific CD8 T cells represent an important component of the natural immune response to HIV-1 and can kill HIV-1–infected cells by major histocompatibility complex class I–restricted cytolysis. Such cells are observed in the vast majority of HIV-1–infected patients, but their functional properties and antiviral effectiveness vary. The most powerful HIV-1–specific CD8 T cells can be detected in elite controllers, a small group of HIV-1–infected patients who, in the absence of antiretroviral therapy, maintain undetectable levels of HIV-1 replication.6–8 HIV-1–specific CD8 T cells from elite controllers can effectively inhibit active HIV-1 replication in autologous CD4 T cells in vitro9–11 and can also facilitate elimination of autologous latently infected CD4 T cells after viral reactivation with pharmaceutical agents.12
Such superior antiviral effects of HIV-1–specific CD8 T cells from the elite controllers have mainly been attributed to specific functional characteristics of these cells. For instance, previous work demonstrated a “polyfunctional” profile of HIV-1–specific T cells from controllers with improved antigen-specific proliferation and cytokine secretion,6,8 optimized affinity/avidity to the target epitope,13,14 and enhanced intracellular expression of the cytotoxic molecules perforin and granzyme B.7,15 However, killing of HIV-1–infected CD4 T cells by CD8 T cells involves a bidirectional cellular interaction that, in addition to CD8 T-cell functionality, critically depends on the susceptibility of CD4 T cells to CD8 T-cell–mediated killing. Previous studies have indeed shown that leukocellular subsets differ substantially with regard to their responsiveness to cytotoxic effects of HIV-1–specific CD8 T cells.16 Whether such differences influence the cellular composition and structure of the latent HIV-1 reservoir in persons with durable suppression of active HIV-1 replication is not known.
To better understand the host factors that shape the size and cellular composition of the latent HIV-1 reservoir, we here systematically investigated the susceptibility of CD4 T-cell subsets to cytotoxic effects of HIV-1–specific CD8 T cells in individuals with spontaneous viral control or highly active antiretroviral therapy (HAART)–mediated suppression of viral replication. Our results show that the responsiveness of CD4 T cells to CD8 T-cell–mediated killing is significantly elevated in elite controllers and inversely correlated to the size of the viral reservoir, particularly in naive CD4 T cells. Together these data suggest that cell-intrinsic functional characteristics in host CD4 T cells modulate the dynamics of viral persistence in these cells.
PATIENTS AND METHODS
Peripheral blood mononuclear cell (PBMC) samples from HIV-infected individuals and HIV-1–negative persons were used for this study according to protocols approved by the Institutional Review Board of Massachusetts General Hospital. All study participants gave written informed consent. Demographic and clinical characteristics of the study cohorts are summarized in Table 1.
Generation of CD8 T-Cell Clones
Cytotoxic T-cell clones targeting the HLA-B57–restricted epitopes KF11 (KAFSPEVIPMF) and TW10 (TSTLQEQIGW), the HLA-A2–restricted epitope SL9 (SLYNTVATL), and the HLA-B8–restricted epitope EI8 (EIYKRWII) (all epitopes in HIV-1 Gag) were generated by limiting dilution as previously described.17
For phenotypic characterization of CD4+ T cells, cells were stained with a viability dye and antibodies directed against CD4 and other surface markers, as indicated. After 20 minutes, cells were washed, fixed in paraformaldehyde, and acquired on an LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were analyzed using FlowJo software (TreeStar, Ashland, OR).
Ex Vivo Cytotoxicity Assay
Susceptibilities to CD8 T-cell–mediated killing were investigated using a previously described cytotoxicity assay.16 Target CD4 T cells were pulsed with the indicated epitopic peptides at 10 μg/mL for 1 hour at 37°C. CD4 T cells were then stained with fluorescently conjugated antibodies against CD4, CCR7, and CD45RA (all antibodies from BD Biosciences) and fluorescein isothiocyanate–labeled Annexin V at room temperature for 15 minutes. HLA-matched effector CD8 T-cell clones were added and cocultured with target cells at the E:T ratios of 1:1 for 60 minutes, followed by surface staining with allophycocyanin-labeled Annexin V. Unpulsed and peptide-pulsed target cells without exposure to CD8 T effector cells and CD8 T effector cells mixed with unpulsed target cells were used as controls. Cells were acquired on an LSRII flow cytometer (BD Biosciences), using DiVA software. Data were analyzed using FlowJo software (TreeStar). To calculate the susceptibility of cells to epitope-specific cytotoxicity, the proportions of Annexin V–positive cells were normalized to proportions of Annexin V–positive cells before co-incubation with effector cells and the background from negative controls was subtracted.
Analysis of HIV-1–Specific CD8 T Cells
ELISPOT assays were performed as described previously to quantify the magnitude of HIV-1–specific CD8 T cells.17,18 PBMCs were plated in 96-well polyvinylidene plates that had been precoated with 0.5 μg/mL of an antihuman interferon-γ monoclonal antibody (Mabtech, Cincinnati, OH). PBMCs were added at a concentration of 50,000–100,000 cells per well in a volume of 100 μL. Plates were incubated overnight at 37°C, 5% CO2, and developed on the following day. To calculate the number of specific T cells, the number of spots in the negative control wells was subtracted from the counted number of spots in each well. Responses were considered positive if there were >50 spot-forming cells/106 PBMCs and at least 3 times the mean number of spot-forming cells of the 3 control wells.
Quantification of HIV-1 DNA
Total, naive, and memory CD4 T cells were isolated by immunomagnetic selection using commercial kits according to the manufacturer's protocol (Miltenyi, San Diego, CA). Purity of cells was confirmed by flow cytometry and was >95%. Isolated CD4 T cells were digested as previously described3 to extract cell lysates. We amplified total HIV-1 DNA with primers and probes previously described.19 As a standard curve, we amplified serial dilutions of chronically infected 293 T cells (kindly provided by Dr Bushman, University of Pennsylvania). Proviral HIV-1 DNA copy numbers were calculated relative to the CCR5 gene copy number previously quantified with the same standards.
Pearson correlation coefficient was calculated to analyze correlations. Differences between nominal data were tested for statistical significance by 2-tailed Student t test, Mann–Whitney U test, or paired Wilcoxon test as appropriate.
Higher Susceptibility of CD4 T Cells From Elite Controllers to Cytotoxic Effects of CD8 T Cells
To analyze the susceptibility of target cells to HIV-1–specific CD8 T-cell killing, we pulsed CD4 T cells from HIV-1–negative individuals, HAART-treated persons, and elite controllers with antigenic peptides corresponding to HLA-B8-, HLA-B57–, and HLA-A2–restricted immunodominant CD8 T-cell epitopes in HIV-1 Gag (B8-EI8, B57-TW10, B57-KF11, and A2-SL9), followed by coculture with HIV-1–specific CD8 T-cell clones targeting these epitopes. Antigen-specific killing of target cells was then detected in CD4 T cells by flow cytometric detection of Annexin V, as described in a previously published protocol.16 An example for the flow cytometric assessment of CD4 T-cell susceptibility to cytotoxic effects of CD8 T cells is demonstrated in Figure 1A, and the demographic and clinical characteristics of the 3 different study cohorts are summarized in Table 1.
Overall, we observed that the susceptibility of CD4 T cells to HIV-1–specific CD8 T-cell–mediated killing was substantially higher in elite controllers, compared with CD4 T cells from HAART-treated persons or HIV-1–negative individuals (Fig. 1B). These differences were most significant after exposure to CD8 T-cell clones restricted by the protective HLA class I allele HLA-B57. Susceptibilities to the HLA-A2– or HLA-B8–restricted CD8 T cells were not statistically significantly different between elite controllers and HAART-treated persons, although there was a trend for higher levels of susceptibility in elite controllers (Fig. 1B). Because spontaneous cell death rates can influence the susceptibility of CD4 T cells to CD8 T-cell–mediated killing, we simultaneously analyzed Annexin V expression in CD4 T cells from the study subjects in the absence of CD8 T effector cells; however, these did not substantially differ among the different study cohorts (Fig. 1C). Because the level of cellular activation may influence the susceptibility to CD8 T-cell–mediated killing, we analyzed the expression of activation surface markers, including HLA class I, HLA-DR, and CD38 on CD4 T cells from the different study cohorts. In line with previous reports, expression of these cell surface markers was slightly higher in HAART-treated persons compared with elite controllers and HIV-1–negative persons, but there was no correlation between these markers and corresponding levels of susceptibility to CD8 T-cell killing, neither within elite controllers nor HAART-treated patients or HIV-1–negative persons (data not shown); this suggests that possible differences between the levels of HLA class I–mediated cytotoxic T-cell epitope presentation in the different CD4 T–cell subsets were not responsible for the observed effects. Overall, these experiments indicate elevated susceptibilities of CD4 T cells from elite controllers to CD8 T-cell–mediated killing, specifically in the context of restriction by the protective HLA class I allele B57.
Cell Subset–Specific Differences in Susceptibility to CD8 T-Cell Killing
CD4 T cells consist of distinct subsets, which may differ with regard to their susceptibility to CD8 T-cell–mediated killing. To investigate this, we selectively analyzed the susceptibilities of CCR7+CD45RA+ naive, CCR7+CD45RA− central memory, CCR7−CD45RA− effector memory, and CCR7−CD45RA+ terminally differentiated CD4 T cells to cytotoxic effects of the described 4 HIV-1–specific CD8 T-cell clones. Overall, we observed that the susceptibility to HIV-1–specific CD8 T-cell–mediated killing was highest in effector memory CD4 T cells, followed by central memory, terminally differentiated, and naive CD4 T cells; this hierarchy was seen across all analyzed patient populations (Fig. 2A). Interestingly, we observed that among naive CD4 T cells, susceptibility to CD8 T-cell killing was highest in elite controllers and significantly exceeded corresponding levels in HIV-1–negative persons and HAART-treated individuals. Again, this difference was most strongly detectable in the context of epitope restriction by HLA-B57, but a similar trend was also seen for the epitopes restricted by HLA-A2 and HLA-B8 (Fig. 2B). Similar but less pronounced effects were observed for central memory and terminally differentiated CD4 T cells from elite controllers, which both had higher susceptibilities to CD8 T-cell killing compared with the 2 alternative study cohorts; again, such differences were more obvious in the context of restriction by the protective HLA class I allele B57 (Figs. 2C–E). Differences between the study cohorts were least pronounced for the effector memory CD4 T cells, but a trend for higher susceptibility to HLA-B57–restricted CD8 T cells remained also visible within this subset (Fig. 2D). To test whether increased susceptibilities of CD4 T-cell subsets to CD8 T-cell–mediated killing were related to an altered activation status of the cells, we analyzed the phenotypic expression of HLA class I, HLA-DR, and CD38 in the different T-cell subsets. Yet, none of these markers correlated with susceptibility to CD8 T-cell–mediated killing of the respective CD4 T-cell subsets (Figs. 2B–E). These data indicate that naive CD4 T cells and to a lesser extent terminally differentiated, central memory, and effector memory CD4 T cells from elite controllers have higher susceptibilities to cytotoxic effects of CD8 T cells, particularly in the context of restriction by the protective HLA class I allele B57.
Viral Reservoirs in Naive CD4 T Cells Correlate With Susceptibility to CD8 T-Cell–Mediated Killing
We subsequently analyzed whether altered susceptibility of CD4 T cells to CD8 T-cell–mediated killing influences the size and composition of the HIV-1 reservoir in CD4 T cells from elite controllers and HAART-treated persons. For this purpose, we isolated CD4 T cells from our study subjects and determined their amount of cell-associated HIV-1 DNA. We observed that in total, unselected CD4 T cells, naive CD4 T cells, and memory CD4 T cells, viral reservoirs were substantially lower in elite controllers compared with HAART-treated persons (Fig. 3A). In addition, within elite controllers, amounts of cell-associated HIV-1 DNA in total CD4 T cells were significantly lower in carriers of HLA-B57 compared with elite controllers expressing alternative HLA class I alleles (Fig. 3B). To investigate mechanisms that may influence the size of the viral reservoir, we calculated correlations between quantities of cell-associated HIV-1 DNA and corresponding susceptibilities of CD4 T cells to CD8 T-cell–mediated killing (Fig. 4A) or numbers of HIV-1–specific CD8 T cells determined by interferon-γ ELISPOTS (Figs. 4B, C) in elite controllers. Overall, we observed that the amount of cell-associated HIV-1 DNA in bulk CD4 T cells was related neither to the susceptibility to CD8 T-cell–mediated killing nor to the magnitude of total or gag-specific, HIV-1–specific CD8 T cells. Yet, within isolated naive CD4 T cells, a statistically significant inverse correlation was observed between reservoir sizes and respective susceptibilities to CD8 T-cell–mediated killing; a trend for such an inverse correlation was also noted in isolated memory CD4 T cells. No correlation was observed between reservoir sizes in naive or memory CD4 T cells and corresponding frequencies of total or Gag-specific, HIV-1–specific CD8 T cells. These data suggest that susceptibilities to cytotoxic effects of CD8 T cells influence the size of the latent viral reservoir in naive and, to a lesser degree, in memory CD4 T cells.
HIV-1 persists for the entirety of a person's life, even when active viral replication is effectively suppressed with currently existing pharmaceutical agents. Moreover, lifelong viral persistence is also observed in the rare group of patients who are able to suppress HIV-1 to undetectable levels in the absence of antiretroviral therapy. HIV-1–specific cytotoxic T cells, an essential component of the adaptive immune response against HIV-1, seem to play a critical role for limiting active viral replication in elite controllers,7,20 and recent studies suggest that they can also influence the size of the latently infected CD4 T-cell pool.21,22 In contrast, the impact of functional characteristics of CD4 T cells on the size and composition of the latent viral reservoir is less certain. Here, we used a recently described assay to measure the susceptibility of CD4 T cells to HIV-1–specific CD8 T-cell–mediated killing, a functional parameter in CD4 T cells that has not systematically been assessed in previous studies. In comparison with viral inhibition assays, in which the abilities of HIV-1–specific CD8 T cells to block active viral replication are evaluated,23 our assay measures the responsiveness of directly ex vivo isolated, peptide-pulsed CD4 T-cell to CD8 T-cell–mediated killing without being biased by different susceptibilities of CD4 T cells to HIV-1 infection,24–26 differences in cell-intrinsic antigen presentation or processing,27 or artifacts resulting from in vitro culture. Using this experimental system, we show that the susceptibility of CD4 T cells from elite controllers to CD8 T-cell–mediated killing is significantly enhanced in comparison with alternative patient populations and that this difference is associated with a lower HIV-1 reservoir size in corresponding CD4 T-cell populations. Moreover, we observed that within elite controllers, higher susceptibility to CD8 T-cell–mediated killing in combination with lower viral reservoirs were more visible in carriers of the protective HLA class I allele HLA-B57, as compared with individuals expressing alternative HLA class I alleles. Together, our findings indicate that the susceptibility of CD4 T cells to CD8 T-cell–mediated killing may represent an important factor for shaping and structuring the size and cellular composition of the latent HIV-1 reservoir and likely needs to be considered in future clinical strategies that aim at reducing HIV-1 persistence.
CD4 T cells represent a highly heterogeneous cell population that includes different subsets with individual developmental programs and functional characteristics,28 which influence the ability of these cells to serve as long-term reservoirs for HIV. In this study, we observed that inverse correlations between the latent viral reservoir size and the corresponding susceptibility to CD8 T-cell–mediated killing were most pronounced in naive CD4 T cells, a quiescent, antigen-inexperienced cell subset with critical functions for replenishing the memory T-cell pool. Although naive HIV-1–infected CD4 T cells in our study were difficult to isolate from terminally differentiated CD4 T memory cells by immunomagnetic selection because of the co-expression of CD45RA, they typically represent a detectable component of the long-lasting viral reservoir that decays very slowly.4,29,30 Our work shows that naive CD4 T cells have comparatively weak susceptibilities to cytotoxic effects of CD8 T cells, which may contribute to long-term viral persistence in these cells. Interestingly, we observed that the susceptibility of naive CD4 T cells to CD8 T-cell–mediated killing was markedly elevated in elite controllers as compared with HAART-treated persons, which seemed to translate into a lower reservoir size in this cell compartment in elite controllers. Together, these findings suggest that the responsiveness of naive CD4 T cells to cytotoxic effects of CD8 T cells represents an important factor for shaping the viral reservoir size in the naive CD4 T-cell compartment.
Although a correlation between cell-associated viral DNA and susceptibility to HIV-1–specific CD8 T cells was also detected in memory CD4 T cells, this association was relatively weak, suggesting that the reservoir size in this cell compartment is influenced by a combination of multiple factors. For instance, previous work suggested that homeostatic and transitional proliferation of memory CD4 T-cell subsets may have a critical role for structuring the latent CD4 T-cell reservoir.3 In addition, susceptibility of memory CD4 T cells to HIV-1 infection varies considerably among different individuals,25,26,31 which may also influence the size of the latently infected CD4 T-cell pool. Notably, despite evidence from previous studies indicating that quantities of Gag-specific CD8 T cells influence the viral reservoir size in memory CD4 T cells,22 our work only showed a moderate correlation between those parameters that was weaker than correlations between reservoir sizes and susceptibility of CD4 T cells to CD8 T-cell–mediated killing. Overall, these findings suggest that the presence of Gag-specific CD8 T cells by itself may not necessarily limit viral reservoirs in CD4 T cells and that the functional responsiveness of CD4 target cells to HIV-1–specific CD8 T cells may be more critical for influencing the size and composition of latently infected CD4 T cells. However, in all these studies, we analyzed the viral reservoir in CD4 T cells by measuring HIV-1 DNA, a large proportion of which is not encoding for replication-competent virus; future studies will therefore be necessary to analyze how HIV-1–specific CD8 T-cell–mediated immune activity may influence the reservoir of cells harboring replication-competent virus.
Despite numerous detailed investigations, the ability of elite controllers to spontaneously control HIV-1 replication remains difficult to explain, likely because of the substantial heterogeneity of patients who develop this specific clinical phenotype.32 Our data show that CD4 T-cell susceptibility to CD8 T-cell killing and viral reservoir size in CD4 T cells vary greatly among elite controllers and tend to be associated with expression of the protective HLA class I allele HLA-B57. This allele restricts specific immunodominant CD8 T-cell epitopes in HIV-1 Gag, and HIV-1–infected CD4 T cells from elite controllers may be more responsive to CD8 T-cell killing when recognized in the context of this HLA allotype. Recently, it was shown that HLA-B57–restricted cytotoxic T cells from elite controllers are highly potent in killing latently infected CD4 T cells after reactivation of active viral replication with histone deacetylase inhibitors.12 These observations have mostly been attributed to superior functional properties of HIV-1–specific CD8 T cells from elite controllers. However, our data suggest that an increased responsiveness of CD4 T cells from elite controllers to CD8 T-cell–mediated killing may also facilitate elimination of latently infected cells in this specific patient population.
The reasons for the elevated susceptibility of CD4 T cells from elite controllers to cytotoxic effects of HIV-1–specific CD8 T cells are unclear at present. Notably, CD4 T cells from these patients were more responsive to HIV-1–specific CD8 T cells irrespectively of their antigenic specificity, suggesting that our findings reflect a general constitutive characteristic of CD4 T cells from elite controllers. Importantly, a considerable proportion of elite controllers paradoxically develops declining CD4 T-cell counts in the absence of detectable viral replication,33,34 and higher sensitivities to apoptosis-inducing processes may in part be responsible for that. As such, increased susceptibility of CD4 T cells from elite controllers to CD8 T-cell–mediated killing may contribute to lowering the reservoir of latently HIV-1–infected cells but in some cases be associated with higher risks for more pronounced CD4 T-cell losses.
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Keywords:© 2014 by Lippincott Williams & Wilkins
HIV-1; viral reservoir; cytotoxic T cells; viral persistence