Increased T cell turnover, elevated proportions of activated T cells, and augmented levels of pro-inflammatory cytokines and chemokines are hallmarks of chronic immune activation in HIV infection with persistent high viremia (1–3). In the primary HIV infection (PHI), there is prolific viral replication, high levels of immune activation, and induction of HIV-specific CD4 and CD8 T-cell cytotoxic responses (4–6). The rate of CD4 T-cell loss and the subsequent course of infection are determined by the “set point” of CD8 T-cell activation established within the first 6 months following infection (7). While the initial immune response partially controls HIV replication in PHI, it, however, does not eradicate the infection and does not prevent the consequent gradual loss of CD4 T cells and CD8 T-cell functional impairment. In patients treated by ART in chronic HIV infection (CHI) there is rapid decline of activated T cells; however, they rarely reach a normal steady state compared to uninfected individuals (8, 9). Conventional regulatory T cells (Tregs) are known to be capable of controlling inappropriate or exaggerated immune activation mediated either through cellular contact via CTLA-4 (10) or through secretion of immunosuppressive cytokines such as IL-10 and TGF-β (11). Although they are competent in controlling low residual T-cell activation in ART-treated patients (12), it was found that they are not sufficient in terms of numbers and/or activity to dampen the exaggerated immune activation that is associated with high levels of HIV replication during PHI (13). Instead, IL-10-producing Foxp3⁻ type I Tregs (Tr1) and double negative (DN) T cells were shown to play a beneficial role in controlling T-cell activation (13, 14). Moreover, in SIV infection, it had been observed that natural hosts had higher proportions of DN T cells than found in pathogenic hosts that were less frequently infected and exhibited polyfunctionality, indicating their critical role in providing help during SIV infection (15, 16).

Double negative T cells are a subclass of T cells with more than 70% of them devoid of CD4 and CD8 (17). They constitute 1–5% of T cells in peripheral blood and lymphoid organs and can express either αβ or γδ T cell receptor. In humans, six Vγ genes (Vγ2,3,4,5,8,9) can combine with three other commonly used Vδ genes (Vδ1,2,3) to create different combinations that allow their preferential homing to specific anatomical localizations. In healthy individuals, Vδ2⁺ cells predominate in peripheral blood, whereas Vδ1⁺ cells and Vδ3⁺ cells are localized in the liver and gut epithelia. Vδ1⁺ cells are also found to be present in thymus, spleen, and dermis (18, 19). Vδ2⁺ cells mainly respond to mycobacterial antigens and tumors. They are also activated by phosphoantigens, such as 4-hydroxy-3-methyl-but-2-enyl pyrophosphate or isopentenyl pyrophosphate (IPP), that get accumulated in virus-infected and cancer cells due to alterations in the mevalonate pathway. Vδ1⁺ and Vδ3⁺ cells participate in defense against viral and fungal infections as well as hematological malignancies (20). In HIV infection, expansion of Vδ1⁺ cells with concomitant depletion of Vδ2⁺ cells in peripheral blood results in an inverted Vδ1⁺/Vδ2⁺ ratio compared to healthy individuals (21, 22). Although not entirely clear, indirect mechanisms involving CCR5/α4β7 signaling as well as direct infection of γδ T cells have been reported to be plausible explanations for Vδ2⁺ cell loss in HIV infection (23–25). Generalized immune activation during UT HIV infection was reported to induce transient expression of CD4 on Vδ2⁺ cells, which enables HIV infection of γδ T cells in vivo (25).

As we had previously found that DN T cells including mainly γδ T cells may play a role in controlling high levels of T-cell activation in PHI (13), we put forward the hypothesis that γδ T cells might be involved in the control of immune activation in PHI. Therefore, the primary objective of this study was to characterize phenotype and function of γδ T cells and to investigate their role as potential Tregs in primary HIV infection. We also aimed at analyzing potential changes in the profile of γδ T cells with the stage of infection (primary vs chronic), and their alterations by ART. We thus performed a cross-sectional study including HIV-infected patients in primary and chronic phase with or without antiretroviral treatment.

In this study, we found that the dynamics in γδ T cells were distinct in different clinical stages of HIV infection—primary vs chronic. First, we observed that the γδ T cell frequency was similar between PHI patients and HD; nevertheless, UT-CHI patients exhibited a higher proportion of γδ T cells. This observation was in contrast with one study that showed no difference in the proportion and absolute count of γδ T cells between healthy controls, acute HIV infection, and slow and fast progressors (26). However, earlier studies have reported an increase in γδ T cells in HIV infection as well as an inverted Vδ1⁺/Vδ2⁺ ratio, a consequence of expansion of Vδ1⁺ cells and/or depletion of Vδ2⁺ cells (21, 22, 27). In accordance with them, we also found that there was skewed Vδ2⁻/Vδ2⁺ ratio, which positively correlated with γδ T cell numbers in HIV-infected patients including PHI and CHI with or without ART. Second, the proportion of γδ T cells negatively correlated with CD8 T-cell activation in PHI, which was in contrast with UT-CHI where we observed a positive correlation. We had previously reported that in PHI patients, the proportion of DN T cells negatively correlated with the proportion of CD38⁺HLA-DR⁺CD8 T cells within the first weeks of acute infection (13). In addition, the frequency of DN T cells at baseline predicted the level of CD8 T-cell activation at month 6 [i.e., the immune set point (7)]. Fifty percent of DN T cells expressed the γδ TCR. Earlier studies have demonstrated that γδ T cells are able to suppress innate and adaptive immune responses (28–30). Third, we found that Vδ2⁺ γδ T cells comprised a significantly lower proportion of terminally differentiated cells in PHI compared to UT-CHI. Furthermore in PHI patients, Vδ2⁺ γδ T cells displayed an anti-inflammatory cytokine profile whereas in UT-CHI patients Vδ2⁺ cells exhibited pro-inflammatory cytokine characteristics. Taken together, our findings suggest a potential role of γδ T cells as immune regulators in PHI.

Alterations in the phenotype and function of the γδ T cell compartment have been observed in several viral infections (31). In CMV infection, it has been shown that Vδ2⁺ and Vδ2⁻ cells display distinct evolution and phenotypic pattern. As opposed to Vδ2⁺ subset, the Vδ2⁻ subset was found to express higher levels of CD38 in patients with active CMV infection (32). During primary CMV infection, Roux et al. reported that activated Vδ2⁻ cells directly correlated with CD8 T-cell activation (32). In our study, we observed that there was a positive association between activated γδ T cells and activated CD8 T cells in UT-CHI but not in PHI clearly indicating that during CHI, immune activation encompasses all T-cell subsets. We also found that although level of CD38 expression on Vδ2⁺ cells was similar between PHI and UT-CHI, the %Ki-67⁺Vδ2⁺ cells was significantly higher in PHI compared to UT-CHI indicating active proliferation of these cells during acute infection. A positive correlation of %CD38⁺Vδ2⁺ cells and %Ki-67⁺Vδ2⁺ cells supported this observation. It was recently demonstrated that immune activation can render Vδ2⁺ cells susceptible to HIV infection and serve as latent reservoir for HIV (25). We found that there was a high ratio of Vδ2⁻/Vδ2⁺ cells in all groups of HIV-infected patients compared to HD. Noteworthy, the absolute numbers of Vδ2⁺ cells remained unperturbed between different groups of HIV patients despite strong activation and cellular proliferation of Vδ2⁺ cells in PHI. We were therefore intrigued by the nature of Vδ2⁺ cells, which motivated us to analyze them further. It is well known that as cells proceed toward terminal differentiation they lose their proliferative potential and effector functions and progress to exhaustion. We observed that such events happened to occur in the Vδ2⁻ subset as in viremic patients, the number of Vδ2⁻ cells positively correlated with the proportion of terminally differentiated Vδ2⁻ cells. In contrast, no such correlation was observed for Vδ2⁺ cells suggesting that Vδ2⁺ subset of γδ T cells could be involved in immune regulation in PHI. Both mucosal and circulating Vδ1⁺ cells in CHI have been shown to have high frequency of T_EMRA cells compared to PHI (27). Furthermore, in CMV infection, it was observed that the Vδ2⁻ subset exhibited predominance of terminally differentiated cells compared to Vδ2⁺ subset (32, 33). It is suggested that differentiation phenotype described in γδ T-cell subsets could be due to the type of stimulus or pathogen they react to (phosphoantigens for Vδ2⁺ γδ T cells) (32). Therefore, to specifically activate Vδ2⁺ cells and investigate their functional capacity, we used IPP as the stimulus. In addition, we used mAbs against CD3 and CD28 to stimulate both Vδ2⁺ and Vδ2⁻ cells and to assess and compare their cytokine production in PHI and CHI. We found that Vδ2⁺ cells were able to produce both the anti-inflammatory cytokine TGF-β as well as the pro-inflammatory cytokine IFN-γ. Interestingly, there was a high %TGF-β⁺ but low frequency of IFN-γ producers among Vδ2⁺ cells in PHI patients compared to UT-CHI patients. In viremic patients, the %TGF-β⁺Vδ2⁺ cells negatively correlated with the %Vδ2⁺ T_EMRA cells indicating that higher was the terminal differentiation of Vδ2⁺ cells the lower was their ability to produce TGF-β. In addition, we observed that the ratio of TGF-β⁺/IFN-γ⁺Vδ2⁺ cells was significantly higher in PHI compared to UT-CHI indicating the conversion of Vδ2⁺ cells from an anti-inflammatory cytokine profile in PHI to pro-inflammatory profile in UT-CHI.

Both Vδ1⁺ and Vδ2⁺ cells have been shown to be able to produce TGF-β upon in vitro stimulation with anti-human TCR Vδ1 and CD3/CD28, respectively (29, 34). Vδ2⁺ cells can also suppress T cell proliferation and IL-2 production via interaction of CD86 with CTLA-4 on activated CD4 T cells (30). It is known that IFN-γ along with other inflammatory cytokines contributes to the establishment of chronic immune activation during HIV infection (35). On the other hand, TGF-β has been shown to regulate T cell responses by suppressing their proliferation and IL-2 production (36). We previously showed that systemic immune activation in primary HIV infection is primarily driven by the virus and innate immune responses. Microbial translocation probably does not occur at the time of early PHI (37, 38) despite a loss of intestinal mucosal integrity (39, 40). Our findings in this study suggest that, γδ T cells, particularly Vδ2⁺ cells, might play a role in the control of excessive T-cell activation in PHI through production of TGF-β. In contrast, in UT-CHI, the regulatory capacity of Vδ2⁺ cells is overridden by the presence of viral replication, pro-inflammatory cytokines and other soluble factors that are driving systemic immune activation. The fact that we observed a negative correlation of γδ T cell frequency with CD8 T-cell activation in PHI, but found no such association with either the Vδ2⁺ or Vδ2⁻ subset when assessed separately (data not shown), suggests that probably both subsets work in partnership as immune regulators in PHI. It has been demonstrated that Vδ1⁺ cells can also produce TGF-β (34). We therefore cannot rule out the possibility that Vδ2⁻ cells producing TGF-β may also play a role in the control of immune activation in PHI.

Although ART results in a dramatic decrease in HIV viremia and systemic immune activation, the level of residual immune activation does not necessarily reach that of HIV-uninfected individuals, especially in patients treated late in the chronic phase of infection (8, 9). In our study, we found that in ART-CHI patients, the proportion of activated as well as terminally differentiated Vδ2⁺ cells was reduced to the level of HD. It has been shown that despite the reconstitution of TCR repertoire, the numbers and functions of Vδ2⁺ cells are not totally restored even after prolonged ART (41). Consistent with these reports we found that although the frequency of γδ T cells was reinstated in ART-CHI patients compared to UT-CHI patients, the ratio of Vδ2⁻/Vδ2⁺ cells remained impaired. IFN-γ levels in serum are shown to decline in HIV-infected patients after initiation of ART (35). We also found that the proportion of IFN-γ⁺Vδ2⁺ cells was reduced in ART-CHI compared to UT-CHI patients. In contrast, the frequency of TGF-β producing Vδ2⁺ cells did not recover in ART-CHI patients. Moreover, the ratio of TGF-β⁺/IFN-γ⁺Vδ2⁺ cells was similar between UT-CHI and ART-CHI patients indicating that there was a lasting damage to the regulatory capacity of Vδ2⁺ cells in CHI patients despite effective ART.

There were some limitations in our study. The cross-sectional nature of this study provided valuable insight into the phenotypic characteristics and functional capacities of γδ T cells in different stages of infection—primary, UT chronic and ART-treated chronic. However, a longitudinal design would have allowed individual follow up on the evolution of γδ T cell features during the course of infection. The benefit of a cross-sectional design was that we have analyzed patients who have initiated ART during the chronic phase. A longitudinal design would have been limited to the analysis of patients who had been treated early during chronic infection, the current situation for about 50% of the patients in the real life in Europe (42). Our study focused on analysis of γδ T cells in peripheral blood. We had no access to tissue samples, particularly from the gastro-intestinal tract. Analyses of tissues would have allowed a better understanding of the dynamic changes in the distribution of γδ T cell subsets and their functions.

In summary, we report on the potential role of γδ T cells in the control of immune activation in PHI. We showed that the proportion of γδ T cells negatively correlated with CD8 T-cell activation in PHI patients, and that the Vδ2⁺ subset was probably the predominant player mediating its effect via production of the anti-inflammatory cytokine, TGF-β. Furthermore, we found that there was a sustainable loss in the immune regulatory capacity of Vδ2⁺ γδ T cells in CHI as there was no recovery observed in their function even in ART-treated patients with median duration of suppressed viral load of more than 5 years. Thus based on our data, we believe that therapies aiming to restore the functional properties of Vδ2⁺ γδ T-cell subset in CHI should be considered.

This study was carried out in accordance with the recommendations of “French ethical laws” with written informed consent from all patients. All patients gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by “The ethical committee of Ile de France IV.”

LW designed and supervised the study. NB and LW contributed to experimental design and wrote the manuscript. NB analyzed the data. NB, ML-G, and CDi performed experiments. P-MG, LC, DB, and CDu included patients. CJ collected and analyzed clinical data. CP implemented and managed the project. P-MG and DS-A reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.