Immune signatures for HIV-1 and HIV-2 induced CD4⁺T cell dysregulation in an Indian cohort

An estimated total of 36.7 million [34.0 million–39.8 million] individuals worldwide, are people living with human immunodeficiency virus (PLHIV) [1].Of these, around 2.1 million [1.71 million–2.64 million] are estimated to live in India [2].The HIV epidemic in India largely involves sexual transmission of HIV-1 Clade C viruses [3]. Furthermore, HIV-2 single infection and dual (HIV-1 along with HIV-2) infection is clearly present and persistent in the HIV infected population in India [4, 5]. Both HIV-1 and HIV-2 share the same modes of transmission, namely sexual contact, blood-borne exposure (blood transfusion, shared needles) and perinatal transmission. Also, both infections are associated with a progressive decline of CD4⁺T cells and loss of immune function, which manifests clinically as an increased susceptibility to opportunistic infections. However, the pathogenesis associated with these two viruses is reported to be disparate. HIV-2 infection has been associated with a significantly longer asymptomatic stage, lower plasma viral load, slower decline in CD4⁺T-cell count, and lower mortality rate attributable to AIDS compared to HIV-1 [6‐8]. In addition, a well-preserved and polyfunctional HIV-specific memory CD4⁺T cell response has been shown to be a hall mark of HIV-2 infection [9, 10].

Thus delineating unique immune signatures associated with HIV-2 pathogenesis may provide therapeutically useful insight into the management of HIV-1 infection and AIDS. The CD4⁺T cell compartment is critical in disease progression as it serves both, as a target and also as a marker of disease progression. Also, CD4⁺ T cell help is an important component of immune responses, both cellular and humoral. In this study, we examined the dynamics of the CD4⁺ T cell compartment in terms of various important subsets, based on homeostatic markers, focussing on chronic HIV-1 and HIV-2 infected individuals at various stages of disease progression.

To address homeostatic turnover, CD4⁺T cell subsets have been defined with respect to expression of Interleukin-2 (IL-2) and Interleukin-7 (IL-7) receptors [11]. The receptors for IL-2 and IL-7 share the common γ-chain in combination with unique high-affinity receptors: IL-2Rα (CD25) or IL-2Rβ (CD122) in the case of IL-2 and IL-7Rα (CD127) in that for IL-7 [12]. IL-2 and IL-7 function through their receptors as master regulators of T cell development, naïve and memory T cell homeostasis, proliferation, and differentiation [12‐14]. CD4⁺ T cells can be categorized into three subsets on the basis of expression of IL2Rα (CD25) and IL7R (CD127): CD127⁺CD25^low/−, CD127⁻CD25⁻, and CD25^highCD127^low as previously reported [11]. This report characterized these phenotypes for the state of differentiation (i.e., naive vs. central memory vs. effector memory) using CD45RA and CD62L in HIV-1 and seronegative individuals. It was observed that CD127⁺CD25^low/− subset corresponded to both CD45RA⁺CD62L⁺ naive cells and CD45RA⁻ CD62L⁺ central memory cells. The CD127⁻ CD25⁻ subset consisted of mainly CD45RA⁻CD62L⁻ effector memory cells. The CD25^highCD127^low phenotype of Regulatory T cells (Treg) has been defined by various study groups [15, 16] and was validated to correspond to CD4⁺CD25⁺Foxp3⁺ Treg cells also by us (Additional file 1: Figure S1). As, the dynamics of the CD4⁺T cell compartment may differ based on the presence or absence of active viral replication, we have evaluated the relative proportions of three subsets of CD4⁺ T cells that are defined based on expression of CD127 and CD25 under anti-retroviral therapy (ART) naïve and therapy receiving conditions in both HIV-1 and HIV-2 infected individuals. Furthermore, some HIV-1 infected individuals were followed longitudinally to assess the effect of long term ART on the dynamics of CD4⁺T cell compartment.

Systemic chronic immune activation is considered as the driving force of CD4⁺T cell depletion and resulting disease progression following HIV infection [17, 18], making it an important cellular parameter to evaluate in the context of HIV-1 and HIV-2 infection. Also, cytotoxic T cells (CTL) expressing the effector molecule Granzyme B (GrzB) are central in conferring host cell immunity against viral pathogens through recognition and killing of infected cells [19‐22]. Thus, this study was designed, to examine levels of activation as well as Granzyme B expression in the both CD4⁺T and CD8⁺ T cells in chronic HIV infection and to delineate any unique immune signatures specific to either HIV-1 or HIV-2 infection.

This is the first study evaluating concurrently the dynamics of the T cell compartment using homeostatic markers such as CD25 (IL-2Rα) and CD127 (IL-7R), level of immune activation and circulating cytotoxic T cell levels in HIV-2 infected individuals. Furthermore, the dynamics, in terms of these markers of the CD4⁺T cell compartment has been addressed separately in the absence of viremia due to effective ART in individuals infected with HIV-1 or HIV-2 in the Indian population for the first time.

Progressive decline of CD4⁺T cells following HIV infection is a hallmark of disease progression resulting in HIV associated immune dysfunction [26]. In this study, both ART-naïve HIV-1 and HIV-2 infected individuals showed apparent dysregulation (altered CD25 and CD127 expression) in CD4⁺T cell subsets and this dysregulation was found to be associated with disease progression. These results suggest an altered cytokine network with respect to IL-2 and IL-7 homeostasis present in HIV infected individuals. Also, the increased frequency of Tregs (CD25^highCD127^low) and effector memory (CD127⁻CD25⁻) subsets seen in both infections within this study may reflect an increased level of immune activation present in these individuals as consequence of viremia in the absence of ART. The significantly lower frequency of the naive/memory (CD127⁺CD25^low/−) T cell subset is congruent with loss of central memory CCR5 expressing CD4⁺T cells as they represent the primary target of HIV infection. Our results with respect to increased frequency of Tregs in ART-naïve HIV infected individuals compared to seronegative individuals are in agreement with some previous reports describing HIV-1 clade B and HIV-2 clade A infection [24, 27‐29]. However, our result with respect to increased frequency of Tregs in ART-naïve HIV-1 infected individuals are not in agreement with some reports [11, 27] that used PBMC staining and were unable to detect differences in frequency of Tregs defined as CD4⁺CD25^bright [27] and CD25^highCD127^low [11] compared to seronegative individuals.

Effective Antiretroviral therapy (ART) blunts viral replication and should result in an increase of the CD4⁺T cell count in peripheral blood, a phenomenon known as immune reconstitution [30]. However the data available so far is limited and conflicting with respect to observed differences in the frequency of Tregs in HIV-1 infection followed by extensive ART [30‐34]. No study has assessed the effect of ART on CD4⁺T subset defined on the basis of expression of CD25 and CD127 in HIV-1 and HIV-2 infection. Analysing the effect of ART on these CD4⁺T cell subsets revealed that ART-receiving HIV infected individuals showed similar frequencies of circulating CD4⁺T cell subsets to those in ART-naïve HIV infected individuals and were higher than seronegative individuals. Also longitudinal assessment of these CD4⁺T cell subsets in ART- receiving HIV-1 infected individuals revealed that they were maintained at high levels over an extended period of ART in the absence of detectable viral replication. These results suggest that ART was not able to rectify a disruption which was occurring in immune homeostasis with respect to IL-2Rα (CD25) and IL-7R (CD127) expressing CD4⁺T cells in both HIV-1 and HIV-2 infection. Though, we recognise that this data would be stronger if it included baseline frequency value of these subsets, which was not available, before the initiation of ART.

Median fluorescent intensity is ‘per-cell’ basis expression of a marker on a cell and alteration in MFI’s could also indicate the functional state for that marker. This paradigm has been supported by studies reporting CD25 expression and functionality of Treg cells [35]. Thus, we evaluated the MFI of both CD127 as well as CD25 in CD4⁺T cell subsets to ascertain if these correlated with disease progression as was found to be the case for frequencies of the subsets that are defined by them. The MFI of CD127 within naive/memory (CD127⁺CD25^low/−)T cell subset and MFI of CD25 within Tregs (CD25^highCD127^low) in ART-naïve HIV infected individuals showed no association with disease progression which suggests that frequency of CD4⁺ T cell subsets defined by their expression are better predictors of disease progression compared to their per cell expression on these subsets.

Though there were no differences in the frequency of CD4⁺T cell subsets in ART- receiving HIV infected individuals compared to ART-naïve HIV infected individuals, we observed reduced per cell expression of CD25 and CD127 in ART- receiving HIV infected individuals compared to ART-naïve HIV infected individuals. This suggests that ART was able to alter the expression of IL-2Rα and IL-7R following successful virological control. While this is indicative of possible or impending immune restoration, it is noteworthy that it was the frequency of CD4⁺ T cell subsets and not MFI of CD25 or CD127 within these subsets that continued to show stronger correlation with disease progression (absolute CD4 count) in HIV infected individuals undergoing virologically effective ART.

Overall, we showed that dysregulation of CD4⁺T cell subsets occurred in infected individuals irrespective of the presence or absence of ART suggesting that the relative disruption in cytokine networks with respect to IL-2 and IL-7 homeostasis caused by HIV infection is persistent in infected individuals and may not be rectifiable through ART alone. From a clinical management point of view in India, our work provides the rationale for exploring new therapeutic strategies for both HIV-1 and HIV-2 infection aimed at directly correcting the HIV-induced immune dysfunction. Also both IL-2 and IL-7 are being investigated as therapies to improve the immune system function in HIV-infected individuals [36‐39].

Systemic immune activation is a feature of chronic HIV infection and determines the rate of disease progression [17, 18, 40]. HIV-2 infection has been reported to be associated with slower disease progression as compared to HIV-1.However the data available with respect to immune activation associated with infection is limited, conflicting and has reported on either CD4 or CD8 compartment in isolation [41‐44]. The present study explored the level of immune activation in ART-naïve HIV-1 and HIV-2 infection in CD4⁺T and CD8⁺T cells.

Our findings demonstrate that in ART-naïve HIV-2 infected individuals, the level of immune activation in both CD4⁺T and CD8⁺ T cell compartment was lower compared to ART-naïve HIV-1 infected individuals and higher than seronegative individuals. While lower levels of immune activation could possibly be explained, in part due to lower viral load as has been reported in previously [41, 45], it is important to note that in our study, the levels of circulating Tregs were observed to be similar to those in ART-naïve HIV-1 infected individuals. Furthermore, these levels correlated inversely with absolute CD4 counts. Thus, these results suggest that the activation induced Treg response following both HIV-1 and HIV-2 infection while similar in magnitude may be more effective in ART-naïve HIV-2 infected individuals. This would explain the lower levels of immune activation as well as higher absolute CD4 counts observed and hence contribute to better disease progression. Interestingly, we also observed that the level of immune activation in CD4⁺T and CD8⁺T cell compartment was not associated with disease progression (absolute CD4 count) in ART-naïve HIV-2 infected study group in contrast to ART-naïve HIV-1 infected individuals. This data indicates that the observed lower levels of immune activation in ART-naïve HIV-2 infected individuals were not sufficiently high enough to drive persistent immune activation driven CD4⁺T cell depletion as observed in ART naïve HIV-1 infected individuals.

Release of Granzyme B, the principle Granzyme in CD8⁺cytotoxic T lymphocyte (CTL) and accompanying cytolytic destruction of target cells is believed to be one of the key immunological mechanisms for destruction of virally infected cells including those infected by HIV [46]. Also, with respect to HIV-1 and SIV infection, studies have delineated a role for CD4⁺ T cytotoxic responses in the prevention of acquisition, peak and set point viremia, control of rebounding virus in the absence SIV specific CD8⁺T cells and in effective CD8⁺T cell mediated responses against infected cells [47‐50].

We report on the level of Granzyme-B expressing circulating CD4⁺T and CD8⁺T cells in ART-naïve HIV-2 infected individuals which, as in the case of ART-naïve HIV-1 infected individuals was observed to be higher compared to seronegative controls. These cytotoxic CD4⁺T and CD8⁺T cell may be assumed to include the HIV-specific cytotoxic T cells and indeed, correlation analysis between Granzyme-B expressing circulating CD4⁺T and CD8⁺T cells with absolute CD4 count showed a significant, though opposing correlation in HIV-1 infected individuals, suggesting that Granzyme-B expressing circulating CD4⁺T cells may be playing a protective role against disease progression in HIV-1 infection. Also, as expected, these CD4⁺T cell Granzyme-B responses correlated negatively with Treg frequencies in ART-naïve HIV-1 infected individuals. Intriguingly, no such correlation of Granzyme-B producing T cells with Treg frequency (or absolute CD4 count) and thus disease progression was observed in ART- naïve HIV-2 infected individuals. Taken together with the lack of correlation of activation levels and absolute CD4 counts in ART-naïve HIV-2 infected individuals, this data suggests a disparate pathogenesis for HIV-2 where possibly lower viral load and efficient Treg activity are associated with disease progression rather than activation and cytotoxic function of T cells. A future study, focusing on virus specific cytotoxic T cell responses would definitely provide a clearer picture.

The study reports for the first time on levels of circulating Granzyme-B expressing CD4⁺T and CD8⁺T cells in chronic HIV-2 infection. Our data also highlights a possibly distinct pathogenesis mechanism operating in HIV-2 infected individuals who, despite having similar duration of infection have possibly lower viral load and efficient Treg activity. This would contribute to less activation driven CD4⁺T cell depletion and thus better disease progression. Furthermore, the observed disruption in cytokine networks with respect to IL-2 and IL-7 homeostasis caused by HIV infection is persistent in infected individuals and may not be rectifiable through ART alone. Our work also provides the rationale for exploring novel immunomodulatory therapeutic strategies for both HIV-1 and HIV-2 infection aimed at directly correcting the HIV-induced immune dysfunction.