HIV infection is associated with a progressive depletion of CD4 T lymphocytes and defective HIV-specific T-cell responses. Persistent immune activation plays a central role in driving CD4 T-cell depletion and progression to AIDS [1,2]. High levels of immune activation occur early in primary HIV infection (PHI) and may be related to activation of innate and adaptive immune responses, microbial translocation, activation by HIV viral proteins and reactivation of other viruses (e.g. cytomegalovirus, hepatitis viruses) . During PHI, immune activation set point was demonstrated to predict the subsequent CD4 T-cell decrease independently of viral load . Thus, the early control of immune activation could be a goal in clinical management of PHI patients.
CD4+CD25+ natural regulatory T cells (nTregs) are able to suppress antigen-specific T-cell responses against a variety of pathogens and may also control inappropriate or exaggerated immune activation, thus, limiting immune-mediated tissue damage . Thus, nTregs may influence the outcome of various infections . In HIV/SIV infection, Tregs, capable of suppressing HIV/SIV-specific immune responses, are detected in peripheral blood and lymphoid tissues and may contribute to immune deficiency [7–9]. Whether these cells are harmful by suppressing HIV-specific immune responses and/or beneficial through a decrease in immune activation remains debatable [8,10–14] and may depend on the stage of the disease. Indeed, our previous data suggested that nTregs may be efficient in controlling residual immune activation in patients with antiretroviral-mediated viral suppression but failed to control immune activation associated with viral rebound following treatment interruption .
We previously demonstrated that HIV-specific nTregs were present in PHI and exhibited suppressive activity against HIV-specific CD4 T-cell responses . In PHI, however, only sparse data are available regarding the relationship between the proportion of nTregs and immune activation, and no longitudinal study has definitely elucidated the impact of nTregs on generalized immune activation in the post-PHI phase in humans. In addition, studies on regulatory T cells in HIV infection were mainly focused on natural CD4+CD25highFoxP3+ Tregs. However, other T-cell subsets including CD8+FoxP3+ regulatory T cells [17,18] and ‘double negative’ CD3+CD4−CD8− T cells (double negative T cells) [19,20] have also been shown to possess the ability to down-regulate specific immune responses in viral infections and/or transplantation. Neither the presence nor the role of double negative T cells have been studied yet in HIV infection.
In the present study, we investigated whether nTregs and double negative T cells in PHI may participate in the control of generalized T-cell activation and, therefore, influence the immunologic and virologic outcome of HIV infection. To assess the impact of these regulatory T-cell subsets on immune activation in PHI, we analyzed the relationship between the activation level of peripheral CD4 and CD8 T cells and the frequency and/or the number of peripheral regulatory T-cell subsets.
This is a prospective multicenter study conducted in four clinical sites in France. To be enrolled, individuals must have shown evidence of acute HIV infection as defined by a negative or weakly positive ELISA, and a known contamination date by one of these criteria: less than three bands on HIV Western Blot, a positive p24 antigenaemia, and/or a detectable plasma HIV-RNA (patients coincluded in the CO6-PRIMO ANRS cohort). Individuals were offered to participate in this study before initiation of combination antiretroviral therapy (ART). Some of the patients started ART during the follow-up, based on clinical symptoms, CD4 cell counts (below 500 cells/μl according to French recommendations) and the decision of both physicians and patients. Written informed consent was provided by study participants according to French ethical laws. The ethical committee of Ile de France II approved the study. Blood from patients was collected at day 0 of enrollment (baseline), day 15, month 1 (M1), month 3 (M3) and month 6 (M6). Blood was also collected from healthy volunteers (n = 3).
Cell isolation and flow cytometric analysis
Fresh peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation (Isopaque-Ficoll, PAA, Austria) within 2–4 h after blood sampling. After washings, cells were stained using multicolor panels and analyzed by flow cytometry (LSRII, Becton Dickinson). The following monoclonal antibodies (mAbs) conjugated to Phycoerythrin Texas Red (ECD), peridinin chlorophyll protein–cyanin 5.5 (PerCP–Cy5.5), Alexa Fluor 488, Alexa Fluor 700, allophycocyanin (APC), allophycocyanin–Hilite7 (APC–H7), phycoerythrin–cyanin 7 (PE–C7), phycoerythrin (PE), fluorescein isothiocyanate (FITC), allophycocyanin–eFluor 780, or V450 were used: anti-CD4–PerCP–Cy5.5, anti-CD4–APC–H7, anti-CD8–Alexa Fluor 488, anti-CD8–Alexa Fluor 700, anti-CD25–APC, anti-CD25–PE–Cy7, anti-HLA–DR–PerCP–Cy5.5 anti-CD38–APC, anti-CD152(CTLA-4)–PE and anti-perforin–FITC (BD Biosciences); anti-CD8–APC–eFluor 780, anti-CD45RA–V450, anti-CD127–PE–Cy7, anti-CD39–PE–Cy7 and anti-FoxP3–Alexa Fluor 700, (eBiosciences, San Diego, California, USA); anti-CD3–ECD and anti-γδ T-cell receptor–PE (Beckman Coulter, Brea, California, USA); and anti-Ki-67–FITC (Dako, Glostrup, Denmark). For intracellular staining of FoxP3, CTLA-4 and Ki-67, cells were fixed and permeabilized using FoxP3 Staining Buffer Set (eBioscience) according to the manufacturer's recommendations. Analyses were performed using FlowJo software (TreeStar, Ashland, Oregon, USA).
Intracellular cytokine production was assessed by flow cytometry on fresh PBMC stimulated with plate-bound anti-CD3 and soluble anti-CD28 mAbs (1 μg/ml of each; Invitrogen, Carlsbad, California, USA) for 24 h at 37°C. After 2 h of culture, brefeldin A (5 μg/ml; Sigma, St Louis, Missouri, USA) was added. Anti-CD3–ECD (Beckman Coulter), anti-CD4–APC–H7, anti-CD8–PE-Cy7, anti-IL-4–FITC, anti-IFN-γ–Alexa Fluor 700, anti-IL-17–Alexa Fluor 700 (BD Biosciences), anti-IL-10–eFluor 450 (eBiosciences) and anti-TGFβ–PE (IQ Products) mAbs were used for immunostaining.
Data were described by medians and interquartile ranges (IQR) for continuous variables. Nonparametric tests were used to avoid the impact of potential outlier values in a small study. All patients at baseline and only untreated patients at M6 were considered for the analyses. Comparisons between treated and untreated patients were performed using the Mann–Whitney test. The Wilcoxon-matched pairs test was used to estimate the changes in the level of T-cell activation, HIV-RNA levels and CD4 T-cell count throughout the follow-up. The Spearman's nonparametric correlation was used to estimate the association of two continuous variables of interest. P values below 0.05 were considered statistically significant.
Twenty-five patients diagnosed early during PHI [median (IQR) of estimated time postinfection: 42 (30–51) days] were prospectively enrolled in the study. Patients’ clinical characteristics at baseline and at M6 are depicted in Table 1. Twelve patients remained untreated during the study period. Ten patients were treated with cART just after baseline sampling; one patient was treated between M3 and M6. Treated patients had significant lower CD4 cell counts and higher plasma HIV-RNA levels at baseline (P = 0.0003 and P = 0.03; respectively). CD8 and CD4 T-cell activation levels were determined by the proportion of cells that expressed the CD38, CD25, HLA-DR and/or Ki-67 activation markers at all time points of follow-up. Treated and untreated patients did not differ for CD4 and CD8 T-cell activation at baseline. As expected, at baseline, the proportion of CD8 T cells coexpressing CD38 and HLA-DR and of CD8 T cells expressing Ki-67 positively correlated with viral load (r = 0.72, P = 0.0001 and r = 0.80, P < 0.0001, respectively). The proportion of HLA-DR+CD4+ T cells and Ki-67+CD4+ T cells also positively correlated with viral load (r = 0.60, P = 0.002 and r = 0.53, P = 0.006, respectively). During the 6 months of follow-up (Fig. 1), the proportion of double positive HLA-DR/CD38 among CD8 T cells decreased from a median (IQR) of 25.5% (12.5–60.8) to 10.1% (5.8–12.1) in untreated patients (P = 0.0005). The proportion of Ki-67-expressing CD8 T cells (Fig. 1) and of Ki-67-expressing CD4 T cells (not shown) also significantly decreased. In contrast, HIV-RNA plasma levels did not significantly decrease between baseline and M6, as previously reported in the French ANRS primo cohort . CD8 T-cell activation remained stable between M3 and M6. At 6 months, the proportion of CD8 T cells that expressed CD38, the density of CD38 expression and the proportion of Ki-67+CD8+ T cells were positively correlated to viral load (r = 0.67, P = 0.017; r = 0.85, P = 0.0004 and r = 0.84, P = 0.0006, respectively).
Double negative T cells but not natural regulatory T cells negatively correlated with CD8 T-cell activation level
We put forward the hypothesis that regulatory T cells including nTregs and/or double negative T cells could impact the level of immune activation during PHI. We investigated the relationship between CD4 and CD8 T-cell activation levels and the proportion or absolute numbers of CD3+CD4+CD25+CD127lowFoxP3+nTregs and of CD3+CD4−CD8− double negative T cells during the follow-up (Fig. 2). Longitudinal data of these regulatory T-cell subsets are depicted in Supplemental Digital Content Table 1, https://links.lww.com/QAD/A187. We did not observe any significant variation over the time in the frequencies and absolute numbers of neither nTregs nor double negative T cells.
As illustrated in Fig. 2a, neither CD4 nor CD8 T-cell activation at baseline was associated with the frequencies or absolute numbers of nTregs. In contrast, we found a strong negative relationship between the proportion of double negative T cells and the level of CD8 T cells that coexpressed CD38 and HLA-DR at baseline (r = −0.56, P = 0.005; Fig. 2b) as well as with the proportion of Ki-67-expressing CD8 T cells (r = −0.58, P = 0.002). In addition, the proportion of double negative T cells but not that of nTregs negatively correlated with HIV-RNA plasma levels at baseline (r = −0.42, P = 0.034; Fig. 2). No relationship was found between the proportion of CD4 T cells expressing CD25, HLA-DR and/or Ki-67 and the proportion or the number of double negative T cells at baseline. The absolute numbers of double negative T cells were not related to CD8 and CD4 T-cell activation or to HIV-RNA plasma levels.
We next performed similar analyses in untreated patients at M6. We found that the proportion of double negative T cells still negatively correlated with the proportion of CD38+HLA-DR+ CD8 T lymphocytes (r = −0.59, P = 0.041) but not anymore with viral load (Supplemental Digital Content Fig. 1, https://links.lww.com/QAD/A187). We also observed a trend to a negative relationship between the proportion of double negative T cells and CD4 T-cell activation (CD25+HLA-DR+ CD4 T cells) at M6 (r = −0.57, P = 0.055); in addition, the absolute numbers of double negative T cells were negatively correlated with the proportion of CD25+HLA-DR+ activated CD4 T cells (r = −0.62, P = 0.033). Therefore, double negative T cells, but not nTregs, were found to be strongly negatively correlated with the level of CD8 T-cell activation during early PHI and at month 6 of follow-up.
The proportion of double negative T cells at baseline predicted the level of T-cell activation at month 6
We next assessed the impact of double negative T cells at baseline on T-cell activation and viral set point after 6 months of follow-up. As shown in Fig. 3, the proportion of double negative T cells at baseline was negatively related to the proportion of activated CD8 T cells at M6 whether assessed by the expression of HLA-DR (r = −0.76, P = 0.004) or by the coexpression of HLA-DR and CD38 (r = −0.62, P = 0.031) on CD8 T cells. The proportion of double negative T cells at baseline also negatively correlated with the proportion of HLA-DR-expressing CD4 T cells (r = −0.72, P = 0.008) and of CD25+HLA-DR+ CD4 T cells (r = −0.78, P = 0.003) at M6. We also found that the absolute numbers of double negative T cells at baseline negatively correlated with T-cell activation at M6 (r = −0.60, P = 0.035 for HLA-DR+ CD8 T cells and r = −0.64, P = 0.024 for HLA-DR+CD25+ CD4 T cells). In contrast, the proportion or the absolute number of double negative T cells at baseline did not influence the viral set point. Thus, the proportion of double negative T cells at baseline was found to be predictive of T-cell activation level but not of viral load at M6.
Double negative T cells display an anti-inflammatory cytokine profile
In order to gain insight into the mechanisms by which double negative T cells could exert immunomodulatory properties, we then characterized their phenotypic and functional characteristics. We assessed the expression of regulatory T-cell markers (CD25, CD127, FoxP3, CTLA-4) as well as activation and proliferation markers (CD38, HLA-DR and Ki-67). As shown in Supplemental Digital Content Table 2, https://links.lww.com/QAD/A187, double negative T cells did not express CD25 and FoxP3 but exhibited slight expression of CTLA-4 (median 5.3%). Double negative T-cell activation level was positively correlated with activation of CD4 and CD8 T cells (data not shown). Interestingly, a median of 34% of double negative T cells expressed high levels of perforin and a median of 53% expressed the γδ T-cell receptor.
We then studied the cytokine profile of double negative T cells. Fresh PBMCs were stimulated for 24 h with anti-CD3/anti-CD28 mAbs and assessed for IL-4, IL-10, IL-17, TGF-β1 and IFN-γ production. As illustrated in Fig. 4, in contrast to CD4 T cells, double negative T cells from PHI patients and controls displayed an anti-inflammatory cytokine profile expressing IL-10 and/or TGF-β1⋅ Expression of TGF-β1 was detected in 30–40% of stimulated double negative T cells (vs. 2–8% of unstimulated cells) from PHI patients. In healthy donors, the proportion of TGF-β1 expressing double negative T cells tended to be lower increasing from 2–4% in unstimulated cells to 8–10% in stimulated conditions. In patients, expression of IL-10 was found in 3–30% of stimulated double negative T cells (vs. 1–3% of unstimulated cells). In healthy donors, the proportion of IL-10 expressing double negative T cells was less than 5% even in stimulated conditions. Double negative T cells from healthy donors and PHI patients did not upregulate IL-4, IFN-γ nor IL-17 in the same experimental conditions.
Our data showed that, in contrast to nTregs, double negative T cells strongly negatively correlated with CD4 and CD8 T-cell activation. Interestingly, the level of double negative T cells at baseline predicted the immune activation set point reached by M6 . In our cohort, levels of viral load and T-cell activation remained stable between M3 and M6 in untreated patients indicating that the viral and immunologic set points were reached by M3.
The role of nTregs in controlling harmful T-cell activation was still questionable. We previously investigated the role of nTregs in the pathogenesis of HIV infection by studying their suppressive effect on HIV-specific T-cell responses in acute and chronic infection [7,15,16]. Although nTregs were found to negatively correlate with residual immune activation in patients with ART-mediated viral suppression, they were incapable of downmodulating immune activation resulting from ART interruption . We previously demonstrated that nTregs from PHI patients were functional on the basis of suppressive activity on antigen-specific CD4 T-cell proliferation . In the present study, nTregs defined as CD3+CD4+CD25+CD127lowFoxp3+ were found to express CTLA-4 and CD39 (data not shown), markers associated with a suppressive function. As previous reports  suggested a reduction in CD25 expression in nTregs from HIV-infected patients, we performed the same analyses after excluding CD25 from the Treg phenotype and still found a lack of relationship between frequency of Tregs and T-cell activation. Data from a small cross–sectional study in eight patients also suggested that CD4+CD25+ CD127low FoxP3+ cells were not effective in reducing pathogenic immune activation . In most studies of acute FIV or SIV infection, nTregs were not likely to play a protective role. Indeed, CD4+CD25+ Treg cell depletion did not significantly alter viral load, CD4 cell count or immune activation in the acute phase of FIV infection . Furthermore, following acute SIV infection, the frequency of nTregs was found to increase in Rhesus Macaques but not in Sooty Mangabeys despite a lower activation in the Sooty Mangabeys nonpathogenic model . Taken together, data from previous and present studies strongly suggest that nTregs are not able to decrease T-cell activation in patients with untreated PHI.
Here, we studied double negative T cells and report on a strong negative correlation between their frequency and the proportion of CD8 T cells coexpressing HLA-DR and CD38 at baseline and M6. This suggests a role for double negative T cells in the control of immune activation, although we cannot exclude that double negative T cells relocate to lymphoid organs in patients with high T-cell activation. Concomitant HLA-DR and CD38 expression on CD8 T cells, referred to as generalized immune activation, was demonstrated to be a strong predictor of disease progression . Also noteworthy is that the proportion of double negative T cells at baseline predicted the level of CD8 T-cell activation at M6 whether assessed by the coexpression of HLA-DR and CD38 or the expression of HLA-DR alone. In contrast, double negative T cells at baseline did not impact CD38 expression on CD8 T cells at M6. Although double negative T cells were not related to CD4 T-cell activation at baseline, their proportion was found to predict the level of HLA-DR-expressing CD4 T cells at M6. Thus, double negative T cells seem to principally impact the frequency of T cells that express HLA-DR, which, it has been suggested, reflects the capacity of cells to proliferate , rather than CD38. Expression of CD38 on CD8 T cells is known to be closely associated with HIV plasma viral load . Accordingly, we did not find any relationship between double negative T cells, whether measured at baseline or at M6, and plasma viral load at M6.
Double negative T cells detected in PHI patients could originate from CD4 T cells that had downmodulated the membrane CD4 protein following activation. However, several findings here argue against this hypothesis including the higher double negative T-cell frequencies observed in patients with lower T-cell activation and the proportion and number of double negative T cells that remained stable within the first months of PHI (data not shown). Although double negative T cells were found to be target cells of HIV , mature double negative T cells were reported to be highly resistant to apoptosis . Thus, in the context of PHI in which immune activation is particularly high, double negative T cells could possibly resist activation-induced cell death.
Zhang et al. were the first to characterize the suppressive function of antigen-specific double negative regulatory T cells and their role in the control of allograft rejection in mice. They showed that double negative T cells can specifically downmodulate proliferative T-cell responses against alloantigens allowing significant prolongation of skin graft survival. Double negative T cells were demonstrated to inhibit proliferation and cytotoxicity of CD8 T cells carrying the same TCR specificity. Fischer et al. first characterized in humans, double negative T cells with similar functional properties to those described in mice. Interestingly, an inverse relationship between frequency of double negative T cells and graft-versus-host disease severity has been reported in patients receiving haematopoietic stem-cell transplantation , suggesting that double negative T cells participate in peripheral tolerance when expanded after allogeneic stimulation.
Consistent with previous phenotypic analyses of double negative T cells in humans [20,29,30], those from PHI patients did not express markers of conventional nTregs such as FoxP3 or CD25. They consisted of both naive CD45RA+ and antigen-experienced CD45RAneg T cells (see Supplemental Digital Content Table 2, https://links.lww.com/QAD/A187). We also found that they expressed high levels of perforin. A slight proportion of double negative T cells exhibited CTLA-4 expression. There is some evidence that double negative T cells mediate direct T-cell suppression through cell-to-cell contact [19,20,30] and, in mice, through a perforin/granzyme-dependent pathway , a mechanism that might be involved in HIV infection. However, we did not find any correlation between perforin expression by double negative T cells and their ability to control immune activation. This is consistent with a recent study suggesting that, in contrast to mice, human double negative T cells do not eliminate effector T cells but rather suppress their proliferation . In addition to cell–cell contact, other mechanisms could be involved in double negative-mediated suppression. A high proportion of double negative T cells were found here to produce the immunosuppressive cytokines TGF-β and IL-10. Among CD3+CD4−CD8− cells, human T cells bearing the γδ TCR were demonstrated to produce these cytokines [32,33]. Approximately half of double negative T cells from patients with PHI expressed the γδ TCR. These cells were described as potential effectors playing a direct role in HIV infection by killing infected cells through cytotoxic NK-like mechanisms and/or by producing chemokines . However, several studies also suggested that γδ T cells exhibited immunoregulatory properties [32,35–38], and that their suppressive function might be mediated through secretion of TGF-β and/or IL-10 [33,38–40]. Although higher TGF-β plasma levels were found in patients with advanced stages of HIV infection [41,42], it was shown to inhibit viral replication of HIV  and hepatitis B virus . TGF-β also inhibited DC-SIGN expression on dendritic cells  and, thus, could indirectly reduce transinfection of T cells caused by DC-SIGN/HIV binding. There is increasing evidence that HIV infection disrupts gastrointestinal mucosal barrier integrity leading to microbial translocation . Increased lipopolysaccharide (LPS) plasma levels were found to correlate with innate and adaptive immune activation . Microbial translocation might, thus, be one of the major mechanisms involved in chronic generalized immune activation during HIV infection. Interestingly, mucosal TGF-β and IL-10 have been shown to prevent LPS-driven epithelial damages in the human colon . Therefore, double negative T cells producing TGF-β and IL-10 may participate in the control of immune activation both by reducing inflammatory responses and by preserving mucosal barrier integrity and, therein, preventing microbial translocation.
Altogether, data from the present study strongly supports a role for double negative T cells in the control of harmful generalized T-cell activation during PHI. Because the frequency of double negative T cells in PHI patients was found to predict the immune activation set point, reported to impact disease progression, this marker should be considered for use in clinical practice. It can be easily measured in hospitals without any additional set up using the regular four-color CD4 protocol. This needs to be evaluated in larger prospective cohort studies. Whether double negative T cells are able to inhibit the development of HIV-associated immunopathology without compromising specific immune responses remains to be determined.
We thank all patients involved in this study and we also thank Nelly Desplanques, Nadia Valin, Laurent Fonquerine (Hôpital Saint Antoine, Paris); Marina Karmochkine, Pascale Kousignian, Jean Derouineau, Martin Buisson, Isabelle Pierre, Dominique Batisse (Hôpital Européen Georges Pompidou, Paris), Gilles Pialoux, Laurence Slama, Thomas Lyavanc, Laura Iordache (Hôpital Tenon, Paris), Christine Katlama and Marc-Antoine Valantin (Hôpital Pitié-Salpétrière, Paris) for including patients in the study.
We thank members of the ANRS PRIMO Cohort study group and especially Christiane Deveau.
L.W designed and supervised the study; G.P, M.F.C, M.M-T, F.B.S, D.S-A and L.W contributed to the experimental design and provided intellectual input; G.P, M.F.C, C.D and A-S.L performed experiments; P.C, P-M.G, M.E.M included patients and reviewed the manuscript; G.P, M.F.C, F.T, L.M and L.W. analyzed data; M.M-T reviewed the manuscript and G.P, M.F.C, D.S-A and L.W wrote the paper.
G.P. and M.F.C. were recipients of fellowships from the Agence Nationale de Recherches sur le SIDA et les Hépatites Virales (ANRS). This work was supported by the ANRS and the Assistance Publique – Hôpitaux de Paris (AP-HP, Paris).
Conflicts of interest
There are no conflicts of interest.
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