Since its implementation, antiretroviral therapy (ART) has dramatically decreased AIDS death rate and improved the quality life of people living with HIV.1 However, if treatment is interrupted, plasma viral load (VL) rapidly increases2 because of the persistence of the virus in cellular reservoirs that cannot be cleared by ART.3,4 Long-lived and proliferating latently infected resting CD4+ T cells are the principal barriers to HIV eradication.5,6 Cellular immune responses play a central role in the control of viral replication.7 HIV-specific cytotoxic T lymphocytes (CTLs) are crucial to virus clearance, and there is a high interest in understanding these cells to harness for HIV eradication. An association between CTL responses with virus control has been widely documented.8–11 We have reported that during primary infection, the effectiveness of the HIV-specific CTL function was directly related to the magnitude of the anti-Gag response,12 which showed higher degranulating capacity plus higher production of IFN−γ, and to a differentiation profile enriched in terminal and effector memory (EM) phenotype.13 Immunoregulatory molecules, such as programmed cell death protein 1 (PD-1), have been of special interest because of their role in T-cell exhaustion and impairment of HIV-specific immunity.14,15 PD-1 expression on HIV-specific CTLs has been linked to disease progression parameters,13 higher T-cell activation levels,16,17 lower proliferative capacity of CTLs,18 and exhausted phenotype.19–21 Likewise, PD-1+CD4+ T cells have been described as a preferential HIV reservoir during effective ART.22 Consequently, PD-1 has been proposed as a marker of viral persistence in the CD4+ T-cell compartment.23,24 Moreover, evidence revealed the profound benefits of early ART on virological and immunological parameters.25–28 ART initiation during chronic infection (ie, longer antigen persistence) results in a dysfunctional HIV-specific CTL response26,29 and a larger reservoir size, which also shows greater complexity in its composition.30 However, it is still unknown how the early immune responses shape persistence of HIV on ART. Here, we aimed to determine the relationship between the quality of the early immune response (functionality and phenotype) and HIV persistence on ART.
Detailed methods are described as supplementary material. Briefly, 29 subjects were enrolled during the acute/early HIV infection following the inclusion criteria described by our group.12,13,31,32 The study was reviewed by the Comité de Ética Humana institutional review board (Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina). All participants provided written informed consent. All subjects started ART within 12 months after infection. For certain analyses, subjects were further divided into 2 subgroups defined as starting ART within 4 or between 4 and 12 months of the presumed date of infection (early-ART and delayed-ART, respectively). Plasma and peripheral blood mononuclear cells were collected at enrollment (baseline sample) and every 3 months up to 2 years post-ART initiation. Plasma VL was determined by branched-DNA method (bDNA, VERSANT HIV-1 RNA 3.0 Assay). CD4+ T-cell count was determined by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). Immune activation, functionality, and phenotype of T cells were assayed by flow cytometry in samples obtained before ART, as reported previously.12,13,33,34 Representative flow cytometry plots are shown in Figure S1, Supplemental Digital Content, https://links.lww.com/QAI/B227. Cell-associated HIV DNA and unspliced RNA (US-RNA) were quantified on CD4+ T cells purified from samples on ART by real-time polymerase chain reaction as described elsewhere.35,36 Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). Data were analyzed using nonparametric statistics. Correlations were determined using Spearman's rank test.
For this study, 29 HIV-infected subjects were enrolled during seroconversion (see Table S1, Supplemental Digital Content, https://links.lww.com/QAI/B227). Baseline samples (median: 60 days after presumed date of infection) were obtained during the later Fiebig stages (V and VI).37 Median baseline VL was 53,303 RNA copies per milliliter (IQ25–75 = 13,412-500,000 copies per milliliter), median CD4+ T-cell count was 399 cells/μL (292–603), and median %CD38+/HLA-DR+ CD8+ and CD4+ T cells were 21.90% (11.75%–67.70%) and 2.50% (1.05%–7.02%), respectively.
Polyfunctionality and phenotype of the immune response were studied at baseline. As expected, monofunctional HIV-specific CTLs dominated the early response (87.7% of total response, Fig. 1A). Of these, more than 85% were degranulating cells (CD107A/B+). CTL memory profile showed the following distribution: terminal effector (TE) cells comprised the highest proportion (median = 55.9%; IQ25–75 = 44.2%–73.1%) followed by naïve cells (19.8%; 15.9%–34.9%), EM (10.6%; 5.9%–15.9%), and central memory (CM) cells (1.7%; 0.7%–4.9%; Fig. 1B). Phenotype distribution of HIV-specific CTLs mirrored that of the bulk compartment (Fig. 1C). Median proportion of bulk PD-1+CD4+ and CD8+ T cells was 18.2% (IQ25–75 = 9.6%–27.1%) and 22.6% (13.5%–34.2%), respectively (Fig. 1D). Within the HIV-specific T cells, a slight increase in the percentage of PD-1 was recorded in both compartments (CD4+ = 20.1%; 11.0%–31.6% and CD8+ = 25.3%; 17.6%–43.4%; Fig. 1E). For certain analysis, PD-1+ events were subdivided into PD-1Low and PD-1High according to the intensity (based on mean fluorescence intensity) of PD-1 expression. Most CD4+ and CD8+ T cells, within both bulk and HIV-specific compartments, showed a PD-1Low phenotype. By contrast, a smaller proportion of T cells displayed a high intensity of this molecule (Figs. 1D, E).
Total HIV DNA and US-RNA were quantified on ART samples. When stated, the subjects enrolled were segregated into 2 subgroups whether they started ART within 4 months of presumed date of infection or after it (early-ART or delayed-ART groups, respectively). HIV DNA and US-RNA diminished after subjects started ART in both subgroups (Figs. 1F, G). Of note, DNA decayed more rapidly in early-ART group reaching a plateau by 30 weeks after ART (median HIV DNA 73 copies/106 CE). Also, US-RNA diminished in the early-ART group (median US-RNA 14 copies/106 CE), but this was not observed in the delayed-ART group. At 1 year after ART, HIV DNA was significantly higher in the delayed-ART versus the early-ART subgroup (P = 0.0420, Fig. 1H). A similar trend was recorded for the US-RNA, although it was not statistically significant (Fig. 1I).
Next, the relationship between the immune response that arises early after infection and viral persistence after ART initiation was assessed. No correlations were found between the baseline magnitude of the HIV-specific CTL response (either evaluated by ELISPOT or flow cytometry) and the reservoir size on ART. A negative correlation between the percentages of bulk naïve CTLs and US-RNA levels was observed (r = −0.6569, P = 0.0017; Fig. 2A). There was also a strong positive correlation between HIV DNA on ART and the percentages of bulk TE CTLs measured at baseline (r = 0.6048, P = 0.0078; Fig. 2B) and HIV-specific EM and TE CTLs (r = 0.5989, P = 0.0203 and r = 0.6033, P = 0.0118; respectively; Figs. 2C, D). A higher proportion of baseline bulk PD-1High CTLs directly correlated with higher levels of HIV DNA at 1 year after ART (r = 0.5804, P = 0.0092; Fig. 2E). The same result was observed in the CD4+ compartment (r = 0.5962, P = 0.0090; Fig. 2F). Within the HIV-specific cells, a strong positive correlation was observed between the proportion of HIV-specific PD-1High CTLs measured at baseline and the HIV DNA on ART (r = 0.7156, P = 0.0037; Fig. 2G). Also, a significantly correlation between both parameters was observed at the CD4+ compartment (r = 0.7276, P = 0.0043; Fig. 2H). Taken together, these results suggest an association between the immune response evaluated early after infection and HIV persistence on ART.
It remains unclear whether HIV-specific CTLs are important in the frequency of cells that remain infected on ART. Here, we determined the relationship between the quality (in terms of immune activation, phenotype, and functionality) of the immune response arising early after infection and HIV persistence in subjects on ART treated within 12 months of infection, in Argentina. Results showed that higher immune activation, dominance of monofunctional CTLs, and a skewed distribution of the memory profile were the hallmarks in this cohort, mirroring previous results of our group.12,13 HIV DNA and US-RNA results corroborated that early ART has an impact on decay rate, size, and composition of the viral reservoir as reported previously.27,38–40
The HIV-specific CTL response and the size of the viral reservoir have been studied in chronic infection,27,41,42 but little is known about what happens during early infection. Here, we showed that a higher percentage of baseline HIV-specific EM and TE CTLs correlated with higher levels of HIV DNA after 1 year on ART. A recent work showed a negative association between baseline granzyme B producing CD8+ T cells and HIV reservoir at 48 weeks after ART, and no associations with IFN-γ producing cells.43 Similarly, we were unable to find associations between CTL functionality in terms of CD107 mobilization or cytokine production, including IFN-γ. Unfortunately, granzyme B production was not evaluated here. Takata et al26 evaluated the HIV immune response on the RV254 Thai cohort members, who were on ART since the earliest stages of acute infection (before seroconversion). Their results indicated that the frequency of fully differentiated effector CTLs at peak viremia was associated with a lower reservoir seeding. These contradictory findings could be related to different timings of sampling during acute infection: while the Thai cohort was enrolled during the earliest stages of acute infection (Fiebig I–IV), the Argentinean cohort was mostly enrolled during Fiebig V and VI (which is closest to the everyday clinical setting in our country). Thus, differentiated CTLs as defined here (only phenotypically) were already experiencing antigen-driven exhaustion, while CTLs in the Thai cohort (defined on functional bases) were still not subjected to this process.
PD-1 molecule has been studied as a marker of exhaustion15,44,45 and associated with disease progression,13 cell activation,17 and HIV persistence.22,46,47 Here, baseline PD-1 expression was evaluated on bulk and HIV-specific CD8+ and CD4+ T cells. Strong correlations were found between higher proportions of baseline bulk and HIV-specific PD-1High CTLs and higher levels of HIV DNA at 1 year after ART. Similar correlations were observed in the CD4 compartment. This result points toward a role of CTLs response in reservoir dynamics. To the best of our knowledge, this is the first report where PD-1 expression evaluated early after infection has been associated with viral persistence on ART. Although we were able to measure higher percentages of fully differentiated cells early after infection, PD-1 results suggest that this vigorous response is at least partially exhausted, thus leading to an ineffective immune response. In other words, the early preservation of a functional T-cell compartment precludes the seeding of the viral reservoir, explaining the direct correlation observed between the baseline memory profile and the viral reservoir on ART. Moreover, Fromentin et al22 found that PD-1, TIGIT, and LAG-3 expression on CD4+ T cells were associated with markers of HIV persistence during ART. Furthermore, SPARTAC trial results, involving treatment interruption analysis, showed that PD-1, Tim-3, and Lag-3 expression, evaluated before ART, could strongly predict time to viral rebound.46 Altogether, results suggest a link between the level of CTL exhaustion early after infection and the viral reservoir dynamics.
Our study has some limitations. First, a small cohort with short follow-up was analyzed. A larger sample size studied for a longer period would permit stronger associations between variables. Although the main focus of our study was to determine the relationship between the baseline immune response and the HIV persistence on ART, it would also be important to investigate the reservoir composition at baseline. Unfortunately, this was not possible due to limited cell numbers. Finally, only polymerase chain reaction–based assays were used to evaluate HIV persistence. Given recent reports regarding the frequency of cells harboring defective genomes,48 it would be of special interest determining the real inducible competent virus in this cohort.
It is accepted that starting early ART limits the size of the HIV reservoir and prevents the irreversible deterioration of the mechanisms involved in the immune response. Data presented here compliment this notion by showing that the quality of the early CTL response shapes the latent reservoir on ART. Thus, it is clear enough that the memory CTL response will play an important role in strategies aimed to achieve sustained viral remission. Further interventions in this field are guaranteed.
The authors specially acknowledge study participants for contributing in this study and for providing blood samples. The authors thank Mr. Sergio Mazzini for assistance during manuscript preparation.
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