Helios + Regulatory T cell frequencies are correlated with control of viral replication and recovery of absolute CD4 T cells counts in early HIV-1 infection

The acute phase of HIV infection is characterized by massive depletion of CD4 T cells, high viral plasma levels and pronounced systemic immune activation. Regulatory T cells (Tregs) have the potential to control systemic immune activation but also to suppress antigen specific T and B cell response. The co-expression of FoxP3 and Helios transcription factors, has been described for identification of highly suppressive Tregs. The aim of this study was to characterize the phenotype of classic Tregs during early HIV infection, and to assess the correlations between the frequencies and phenotype of Tregs with the plasma viral load, CD4 counts, immune activation and the frequency of antibodies reactive to HIV-1 proteins, measured by an immunochromatographic test. The relative frequency of classic Tregs cells in peripheral blood correlated positively with HIV viral load and immune activation of CD8 T cells, and inversely with absolute CD4 counts and development of anti-HIV antibodies in subjects with early HIV infection. However, the expression of Helios in classic Tregs was inversely correlated with viral replication and immune activation, and positively with recovery of CD4 T cell counts and appearance of antibodies reactive to HIV-1 proteins. These results raise the hypothesis that classic Tregs are inefficient at controlling systemic immune activation in subjects with early HIV infection and may be associated with delayed production of antibodies against HIV proteins, delaying the control of viral replication. Conversely, Helios expressing Tregs might contribute to control of viral replication by mechanisms involving the limitation of systemic immune activation.


Background
Regulatory T cells (Tregs) are a subset of CD4 T cells with potential to suppress the immune response, preventing excessive immune activation. Immune suppression by Tregs is mediated by soluble factors or by mechanisms requiring cell-to-cell contact [1]. To date several cells with regulatory properties have been described, but the classic Tregs are the most extensively studied.
Classic Tregs have been characterized by high expression of the activation marker CD25, presence of the transcription factor Foxp3 or expression of low levels of the alpha chain of Interleukin-7 (IL-7) receptor, CD127 [2]. In addition, classic Tregs can be classified as natural or induced Tregs [3,4]. Natural Tregs emerge from the thymus with regulatory properties while induced Tregs are generated in the periphery following T cell activation [5,6]. The co-expression of the transcription factors Helios and FoxP3 identifies highly suppressive Tregs, initially identified as natural Tregs [7,8].
The role of Tregs preventing the development of exacerbated reactions during the course of an immune response has been highlighted in autoimmune diseases [9], transplantation [10], tumors [11] and infectious diseases [7], including HIV infection [12]. In HIV infection Tregs can be beneficial by controlling chronic immune activation, but also unfavorable or even harmful by preventing development of humoral and cellular anti-HIV immune responses [12][13][14]. A relative increase of classic Tregs in peripheral blood has been observed in treatment-naive patients with chronic progressive HIV infection, compared with healthy uninfected individuals [15,16]. In addition, it has been suggested that virologic control induces a decrease in classic Tregs [17]. Little information, however, is available on the role of classic Tregs during early HIV infection, particularly in subtype C infections, the most prevalent HIV subtype in Sub-Saharan Africa and globally [18].
The early phase of HIV infection is critical as it determines the fate of the progressive infection [19][20][21] and the extent of virus transmission at population level [22,23]. The early phase starts with an exponential rise in HIV viremia, reaching a peak when acquired cellular and humoral immune responses against viral proteins emerge [23].
The aim of this study was to characterize the phenotype of Tregs in individuals with early HIV infection, and to assess how staging of the infection, based on plasma viral load, immune activation, absolute CD4 T cell counts and the frequency of antibodies reactive to HIV-1 proteins, correlates with the relative abundance and phenotype of Tregs. We report opposite correlations between total classic Tregs and those expressing Helios, during early HIV-infection, with the plasma viral load, T cell activation, development of anti-HIV antibodies and absolute counts of CD4 T cells.

Study participants
A total of 37 individuals, 14 with early HIV infection (< than 3 months), 8 with HIV chronic infection (> 9 months) and 15 uninfected controls were enrolled in this study. All subjects were part of a large cohort study (RV363) established for assessment of HIV incidence and associated risk factors, and willingness to participate in future HIV vaccine trials. The HIV incidence study enrolled participants in Maputo, Mozambique, at Centro de Investigação e Treino em Saúde da Polana Caniço (CISPOC) between December 2013 and November 2014. A total of 500 uninfected individuals between 18 and 35 years, at high risk for HIV infection, were enrolled for assessment of HIV incidence every three months, over a period of two years. A smaller group of HIVinfected individuals, as part of the chronic infection group, were also included in the study, as a community masking purposes. Pregnant women were excluded from the study. Peripheral blood samples were collected from HIV early infected participants, those whom the rapid test shift from negative to positive in a period of 3 months (median) and masking group individuals for assessment of virologic and immunologic parameters. For the Tregs sub-study, peripheral blood mononuclear cells (PBMC) were available for 14 out of 19 individuals who seroconverted during the HIV incidence study. In addition, PBMC from 3 seroconverted individuals and 5 chronically infected individuals, were evaluated 9 months after the first PBMC collection and at least 2 years after infection, respectively. Randomly selected individuals who did not seroconvert and were negative for HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and syphilis were considered as control subjects (uninfected). Immunophenotyping of PBMCs was done on 22 HIV infected individuals and 15 uninfected controls.

HIV diagnosis, CD4 counts and viral load testing
The HIV-1 diagnosis was performed on fresh venous whole blood samples, following the Mozambican national algorithm for HIV testing, which consists of two sequential rapid immunochromatographic tests for detection of anti-HIV-1/2 antibodies. Screening was first performed using the Alere Determine™ HIV-1/2 (Alere Medical Co., Japan) rapid test. Non-reactive specimens were classified as negative. Reactive specimens were confirmed by a second rapid test, the Uni-Gold™ HIV (Trinity Biotech PLC, Ireland). Indeterminate results, reactive for Determine but non-reactive for UniGold, were resolved by a fourth-generation ELISA, Genscreen Ultra HIV Ag-Ab (BioRad, France). The antibody reactivity pattern for HIV-1 (p31, gp160, p24 and gp41) proteins was analyzed in all seroconverted individuals, using the Geenius HIV 1/2 Confirmatory Assay (BioRad, France) at seroconversion visit and at time of PBMC collections ( Fig. 1a and b). CD4 counts were performed on whole blood cells as previously described [24]. Acquisition was performed on the four-color flow cytometer FACS CALIBUR (BD, USA). Plasma HIV-1 viral loads were determined using the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, v2 (Roche, USA).

Peripheral blood mononuclear cells immunophenotyping
Peripheral blood mononuclear cells (PBMC) were isolated from freshly obtained heparin anti-coagulated blood, using Ficoll-Paque Plus (GE Healthcare, Sweden) and Leucosep tubes (Greiner Bio-One, German), and stored in liquid nitrogen, in a freezing media (10% dimethyl sulfoxide (DMSO) + 90% fetal calf serum (FCS)). After thawing in a water bath at 37°C, PBMC were washed twice in complete RPMI medium supplemented with 20% of FCS followed by 10% of FCS (R20 and R10, respectively) and viable cells were counted using the Nucleocounter NC-100 (Chemometec, Denmark). Prior to cell staining, PBMC were allowed to recover overnight in R10 medium at 37°C in an atmosphere of 7.5% CO 2 . Subsequently, PBMC were counted and washed in phosphate buffered saline (PBS). Cell viability was assessed by adding 50 μl of the viability dye (fixable viability stain (FVS) 510 (BD, USA)) to one million PBMC. Finally, cells were washed again and stained for cell surface markers. Three eight-color panels were prepared per subject, each containing 500,000 cells. One panel was used to stain cell surface markers only and two other panels were used for combined staining of surface markers and transcription factors, FoxP3 and Helios, all following the instructions from the manufacturer. The FoxP3 buffer set from BD was used to fix and permeabilize PBMC for FoxP3 and Helios staining following manufacturer recommendations.

Statistical analysis
Statistical analyses were performed using the software GraphPad Prism version 6.0 h (San Diego, CA). The Mann-Whitney test was used to test the heterogeneity among different groups. The analysis of correlations between two variables was performed by the Spearman Rank correlation. Difference or correlation with p-values less than 0.05 were considered statistically significant.

Characterization of subjects with early HIV infection.
An overview of characteristics of the study participants is shown in Table 1. In total, 14 early infected individuals were included in this sub-study, of whom seven were female. The median age was 21 years and none of the participants had received any HIV antiretroviral treatment at time of PBMC collection. The time between the last HIV negative test and first positive rapid test were 90 days (median) while the PBMC collections were performed 95 days (median) after infection. The median viral load at time of PBMC collection was 4.42 Log10 copies/mL. As expected, the viral load was inversely correlated with the frequency of antibody reactive bands to HIV-1 proteins (r = −0.63; p = 0.02) as shown in Fig. 1c. For some participants, the number of reactive bands increased from between the seroconversion visit and the time of PBMC collection ( Fig. 1a and b).

CD8 T cell activation correlates with the frequency of reactive bands to HIV-1 proteins in peripheral blood of patients with early infection
It is well known that HIV infection and disease progression is associated with systemic immune activation. Tcell activation starts at the acute phase of HIV infection and persists during the chronic phase, even after initiation of highly effective antiretroviral therapy [25]. Both CCR5 and CCR3 participate in T cell activation and migration during the inflammatory process, and are expressed predominantly on memory Th1 cells [26].
The activation profile of CD4 T cell (CD3 + CD4 + ), measured by analyzing the co-expression of HLA-DR and CD38, was significantly higher in the HIV early infected group as compared to controls (p = 0.02). The proportions of both CD4 + CD38 + HLADR + and CD4 + HLADR High tended to correlate inversely with the frequency reactive bands of HIV proteins (r = −0.45; p = 0.12 and r = −0.66; p = 0.01, respectively) and with the absolute CD4 T cell counts (r = −0.51; p = 0.07 and r = −0.4; p = 0.17, respectively). However, no significant correlation was found between the CD4 T cell activation and viral load (=0.34; p = 0.26). We also found a positive correlation between the percentage of recent CD4 thymic emigrants, defined as CD3 + CD4 + CD31 + CD45RA + , and the frequency of reactivity to HIV-1 proteins (r = 0.6; p = 0.04), but With regard the expression of chemokine receptors on CD4 T cells, we did not find significant differences of CD4 + CCR5 + (p = 0.24) and CD4 + CXCR3 + T cells (p = 0.97) expression levels between the two groups. In addition, the proportion of CD4 + CCR5 + (p = 0.24) and CD4 + CXCR3 + T cells did not correlate significantly with viral load (r = −0.16; p = 0.59 and r = −0.16; p = 0.59, respectively) or with the frequency of antibody response against HIV proteins (r = 0.15; p = 0.60 and r = 0.15; p = 0.60, respectively).
Proportions of classic CD4 + CD25 High FoxP3 + Treg cells correlates with viral load, reactivity to HIV-1 proteins, and CD4 counts in HIV patients with early infection The absolute counts and percentages of classic Tregs was analyzed. We observed a decrease of absolute numbers of classic CD4 + CD25 High FoxP3 + Tregs in HIV early infected patients compared with control group (p = 0.02) but not with those with chronic infection. No difference was observed in proportion when compared with control group (p = 0.60) ( Fig. 3a and b). However, the proportion of classic Tregs, in the HIV early infected group correlated positively with viral load (r = 0.8; p = 0.002), the proportion of activated CD8 + CD38 + HLADR + (r = 0.68; p = 0.03) cells, and absolute numbers of CD4 T cells (r = −0.9; p = 0.0004) (Fig. 3c-e). Despite the absence of significant correlation between the frequency of Tregs and the frequency of bands reactive to HIV proteins (r = −0.53; p = 0.1), at time of PBMC collection, we found a significant correlation with the number of bands at seroconversion visit (r = −0.67; p = 0.03) ( Fig. 3f and g).
Increased expression of CCR5 and CXCR3 on classic CD4 + CD25 High FoxP3 + Tregs in HIV patients with a recent infection Chemokine receptor expression on Tregs can predict their function and their potential to accumulate in nonlymphoid tissues [27]. It has been shown that CXCR3 + and CCR5 + Tregs are recruited to sites with ongoing Th1 inflammation [28,29]. Furthermore, increased expression levels of CCR5 and integrin β7 are associated with higher susceptibility to HIV infection and poor immunological response [30][31][32].
We observed an increased proportion of CCR5 (p = 0.01) and CXCR3 (p = 0.03) on classic Tregs in patients with a recent HIV infection as compared to controls (Fig. 4). However, the increased expression of CCR5 and CXCR3 on classic Tregs did not correlate with viral load, nor with reactivity to HIV-1 proteins or with T cell activation.
The proportion and absolute number of classic CD4 + CD25 High FoxP3 + Tregs expressing β7 integrin did not differ between HIV patients and controls (p = 0.45 and p = 0.22 respectively). Their expression did not correlate with viral load, nor with frequency of antibodies reactive to HIV-1 proteins, T cell activation or with absolute numbers of CD4 T cells.

The proportion of Helios expressing Tregs correlates with HIV-1 viral loads and CD4 T cell absolute counts
It has been shown that co-expression of FoxP3 and Helios identifies Tregs that do not produce inflammatory cytokines [33]. In addition, it has also been shown that deletion of Helios contributes to progressive systemic immune activation [34]. We analyzed the proportion of classic CD4 + CD25 High FoxP3 + Tregs expressing Helios (Fig. 5a) and found an inverse correlation between Tregs expressing Helios and viral load in early infection (r = −0.63; p = 0.04) (Fig. 5c). This correlation was preserved, when including chronically infected subjects with detectable viral loads (r = −0.62; p = 0.01) (Fig. 5d). In addition, the proportion of Tregs expressing Helios showed a trend to correlate positively with the recovery of CD4 T cells absolute counts, in early infected group (r = 0.6; p = 0.056). Furthermore, when adding data from progressive and chronically infected individuals with detectable viral loads, the correlation between Helios expressing Tregs and absolute CD4 T cell counts became significant (r = 0.56; p = 0.03) (Fig. 5e). We also found tendencies to correlations, between proportions of Tregs expressing Helios in early infected patients with a b c d e f g  (11) and chronically (eight) infected patients, were isolated and manipulated as described in methods section. These PBMC were stained with the following combination of monoclonal antibodies: CD31 FITC / FoxP3 PE / CD45RA PerCP / CD25 PE-CY7 / Helios APC / CD3 APC-H7 / CD4 V450 / FVS510. Data acquisition was performed on a flow cytometer (FACS CANTO II). Viral loads and frequency of antibodies reactive to HIV-1 proteins was determined using plasma samples as described on methods section. Absolute frequency of CD4 T cells was determined using a four-color flow cytometer (FACS Calibur), and the TruCount method, as described previously described [24].

Discussion
Tregs regulate immune responses and prevent or downregulate exacerbated immune reactions by an array of mechanisms including suppression of T cell activation [35]. This characteristic of Tregs makes them an attractive target to treat inflammatory disorders but also to improve immunogenicity during vaccination against infectious diseases [7,36,37]. Tregs are expected to down-regulate immune activation after the acute phase of infection. However, this regulatory activity may also have a negative impact on development of effective anti-HIV immune responses. It has been shown that depletion of regulatory T cells improve reactivation of virusspecific CD8+ T cells in chronic viral infections models such as in Friend retrovirus and in lymphocytic choriomeningitis virus (LCMV) infection [38][39][40].
During early stages of HIV infection, a massive depletion of CD4 T cells occurs, mainly from the gut associated lymphoid tissue (GALT), the preferential site of initial replication of the virus [41,42]. Concomitantly, the structure of the GALT is compromised and passive microbial translocation occurs which is believed to drive or contribute to increased non-specific systemic immune activation [42]. We observed an absolute decrease of classic Tregs in patients with HIV early infection, as observed in previous studies [43][44][45]. However, despite the decrease of the CD4 T cell percentage, we did not find a concordant relative decrease of classic Tregs, suggesting that these cells are better preserved, at least during the early phase of HIV infection. We also found a strong but inverse correlation between Tregs percentages and absolute CD4 counts. Interestingly, high levels of T cell activation were seen in patients with higher percentages of classic Tregs. This may be explained by the fact that classic Tregs are effective in suppressing antigen-specific immune activation but not that of bystander immune activation, as observed in previous HIV studies [46].
Since classic Tregs are more effective in controlling antigen-specific immune responses, we evaluated their association with humoral anti-HIV immune responses. At the early stages of HIV infection, HIV-specific CD8 T cell responses have been shown to be critical, but insufficient to control viral replication [23]. The control of the initial viremia occurs at onset of antibody production to HIV proteins, although it has been suggested that these antibodies are less effective in virus neutralizing and mediating virus inhibition by antibody-dependent cellmediated cytotoxicity (ADCC) [47]. Here, for the first time, we suggest an involvement of Tregs delaying antibody response in HIV infection. We observed lower frequencies of antibodies reactive to HIV-1 proteins and lower CD4 counts at higher Tregs proportions. In addition, classic Tregs percentages correlated strongly and positively with HIV plasma viral loads. These observations suggest that classic Tregs contribute to the observed delayed onset of antibody production in HIV infection [48], which result in delayed control of viral replication.
The phenotype of Tregs can also predict their organotropism, their target cells, function and susceptibility to HIV infection [27][28][29]31]. We assessed the frequency of CXCR3 and CCR5 chemokine receptor expression together with the expression of integrin β7 and the transcription factor Helios. We found a relative increase of Tregs expressing CXCR3 and CCR5 in HIV positive patients with early infection. The proportion of both chemokine receptors was at least twice as high as in uninfected controls. In contrast, the relative and absolute abundance of Tregs expressing the integrin β7 remained unchanged. Despite the higher proportions of CD4 cells expressing the HIV co-receptor CCR5, we did not detect any association between the percentage or absolute counts of CCR5 + Tregs on the one hand and the plasma viral load on the other. In previous studies, a higher content of HIV DNA was found in CD25 + FoxP3 + memory CD4 T cells as compared to CD25 − FoxP3 − memory CD4 T cells [30]. However, the sequences of HIV DNA found in memory CD25 + FoxP3+ were not similar to those found in plasma virions [30]. Thus, although Tregs might be preferentially infected by HIV due to expression of the HIV co-receptor CCR5, they probably do not contribute significantly to the overall plasma viral load. Furthermore, it was shown that Tregs are resistant to HIV-1 as consequence of repression of HIV-LTR by FoxP3 [49].
The Helios marker is an ikaros family transcription factor, initially identified as marker of natural Tregs [50,51]. Helios expression confers stability of phenotype, enhanced immunosuppressive function and increased survival in periphery [34,52]. Here we show for the first time a correlation between proportions of Helios expressing Tregs with a positive laboratorial outcome in adults with HIV early infection. We found that the frequency of Helios + Tregs remained preserved in patients with early infection, probably due their fitness compared to Tregs not expressing Helios. Interestingly, at higher expression levels of Helios + Tregs, lower viral loads and higher CD4 T cells counts were observed. These results suggest that these bona fide Tregs in addition to being preserved, might be involved in mechanisms controlling the viral replication, probably limiting the activation of T cells. Studies in mice showed that selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation [34]. However, studies conducted in HIV infected children [51] revealed contradictory results regarding correlation of Helios + Tregs with viral loads and absolute CD4 counts. These discrepancies might be explained mainly due to different strategies for identification of Tregs. Since, here we evaluated Helios + Tregs as CD4 + CD25 High FoxP3 + Helios + rather than memory Tregs identified as CD4 + FoxP3 + Helios + CD45RO + . In addition, the difference in ages and staging of HIV-1 infection may also contribute to the observed discrepancies.
The limitations of our study are the low sample size, evaluation of only four unique HIV-1 epitopes and the consequent lack of precise staging of the acute infection, based on Fiebig classification and absence of functional tests for assessing Tregs and antibodies functionality.

Conclusions
Our results suggest that in early HIV infection, classic Tregs do not contribute to the control of systemic T cell activation but rather may be linked to delayed appearance and possibly maturation of antibodies directed against HIV proteins, delaying the control of viral replication. However, highly suppressive Helios expressing Tregs may have a beneficial effect on mechanisms associated with control of viral replication in the periphery. Nevertheless, anti-inflammatory, antiviral and vaccine therapy trials could consider modulation of classic Tregs to evaluate the mechanistic impact of experimental interventions.