Reduced Frequencies and Activation of Regulatory T Cells After the Treatment of HIV-1-Infected Individuals with the CCR5 Antagonist Maraviroc Are Associated with a Reduction in Viral Loads Rather Than a Direct Effect of the Drug on Regulatory T Cells

Abstract

Regulatory T cells (Tregs) play an important role in the pathogenesis of HIV-1 infection and they frequently express the chemokine receptor CCR5. We therefore investigated whether antiretroviral treatment with the CCR5 antagonist Maraviroc affected Tregs in chronically HIV-1-infected individuals. HIV-1-infected patients with high viral loads had elevated frequencies of activated Tregs in the peripheral blood compared with healthy controls. In patients successfully treated with antiretroviral drugs (undetectable viral loads), the frequency and the activation status of Tregs were comparable with healthy controls without any specific effect related to the treatment with Maraviroc. These results indicate that the control of viral replication in general rather than a direct binding of Maraviroc to CCR5-positive Tregs influences Treg responses in successfully treated chronically HIV-1-infected individuals.

Introduction

Recent reports have shown that Tregs expand and become activated during chronic HIV-1 infection in the lymph nodes, gut-associated lymphoid tissue, and the blood, despite general loss of CD4⁺ T cell numbers (19). Two contradicting hypothesizes have been postulated based on several findings from studies of HIV-1-infected patients: (i) Tregs have a detrimental role in HIV-1 infection by suppressing antiviral immune responses and thereby contribute to disease progression (1,7,21,29) or (ii) Tregs have a beneficial role during infection by limiting the generalized immune activation, controlling the pool of HIV-1 target cells, and limiting immunopathology (3,14,16,19). Most likely both Treg functions play a role in HIV-1 pathogenesis, which is why the Treg responses in HIV-1-infected patients need to be carefully analyzed in further studies. A factor that could complicate above-mentioned studies in the future is that the new HIV-1 entry inhibitor, Maraviroc, which antagonizes the HIV-1 coreceptor CCR5 expressed on CD4⁺ T cells, might directly influence Treg responses. Unfortunately, only limited literature is available on this topic. For this reason, we set out to investigate the long-term effect of Maraviroc treatment on Treg frequencies and activation in the blood of chronically HIV-1-infected patients.

Materials and Methods

Sampling strategy and processing

Blood samples from age- and gender-matched HIV-1-positive and -negative individuals were collected at the University Hospital Essen, Essen, Germany. The HIV-1-positive individuals enrolled in the study were chosen based on their HIV-1 RNA copy number per mL blood and the type of antiretroviral therapy (ART) (ART with or without Maraviroc) they received. The different groups enrolled were the following: HVL-patients with high viral loads (HVLs), despite conventional ART (n = 9; males 86%); lower40-patients on conventional ART, with viral loads below 40 viral RNA copies/mL (n = 13; males 85%); MVC-patients on conventional ART plus additional Maraviroc, with viral loads below 40 viral RNA copies/mL (n = 7; males 71%); and HC-HIV-1-negative healthy controls (n = 3; males 100%). Peripheral blood mononuclear cells (PMBCs) were isolated by Ficoll purification. PBMCs were counted and stained with surface antibodies for anti-CD3 (SK7), anti-CD4 (RPA-T4), anti-CD19 (HIB19), anti-CD25 (M-A251), anti-CD127 (A019D5), anti-CD38 (HIT2), anti-CCR5 (HEK/1/85a), and anti-TNFRII (22235; R&D). The antibodies used for cell staining were purchased from BD, Biolegend, or eBioscience unless otherwise noted. Intracellular Foxp3 staining was performed using anti-Foxp3 (PCH101) according to the manufacturer's recommendations using the eBioscience Foxp3 fixation kit. Dead cell exclusion was performed using a singlet gating strategy and Live/Dead aqua (Invitrogen) stain before fixation. The flow cytometric data were acquired on an LSR II and analyzed using FlowJo.

Statistical analysis

Statistical analysis was performed using Prism (GraphPad Software 5). For Figure 1A, a paired t-test was used. The analysis for Table 1 and Figure 1B–E was done using a one-way ANOVA with Tukey's multiple comparison test. A p-value of <0.05 was considered significant.

FIG. 1.

Influence of Maraviroc treatment on Treg frequencies, activation, and CCR5 expression in chronically HIV-1-infected patients. (A) Representative example for the expression of CCR5 on Treg (CD3⁺CD4⁺CD25^hiCD127^loFoxp3⁺) and Tcon (CD3⁺CD4⁺CD25^loCD127^hi) from a patient with HVLs, despite ART. Statistical analysis for all patients evaluated in this study revealed significantly higher expression of CCR5 on Tregs compared with Tcon: p < 0.0001 (t-test). (B–E) Comparison of different HIV-1-infected patient groups: HVL-patients with HVLs, despite conventional ART (n = 9); lower40-patients on conventional ART with viral loads below 40 viral RNA copies/mL (n = 13); MVC-patients on conventional ART plus additional Maraviroc with viral loads below 40 viral RNA copies/mL (n = 7); HC-HIV-1-negative healthy controls (n = 3). PBMCs were stained ex vivo for FACS analysis and (B) the numbers per million live cells of total CD3⁺CD4⁺ T cells, (C) the frequency of CD127^loCD25^hiFoxp3⁺ Tregs of CD3⁺CD4⁺ T cells, (D) the frequency of activated (CD38⁺TNFRII⁺) Tregs, and (E) the frequency of Tregs expressing CCR5 was determined for patients of the different groups. Statistical analysis was done using a one-way ANOVA with Tukey's multiple comparison test. ART, antiretroviral therapy; HVL, high viral load; MVC, Maraviroc; ns, not significant; *<0.05; **<0.005; ***<0.0005.

Table 1.

Summary of Demographical, Immunological, and Virological Patient Parameters Included in the Study

	Age (years)	Nadir (cells/μL)	CD3⁺CD4⁺ (cells/μL)	VL before therapy (HIV-1 RNA copies/mL)	Time from diagnosis (months)
HVL
Mean	43	110	212	844,818	63
SD	13	89	180	1,929,192	65
Geometric mean	42	60	123	196,352	36
Lower40
Mean	48	302	670	99,667	212
SD	11	251	226	27,934	83
Geometric mean	47	196	637	97,135	195
MVC
Mean	57	245	683	72,204	239
SD	8	282	221	89,674	58
Geometric mean	56	142	650	44,346	232
p
Lower40 vs. HVL	ns	ns	^***	ns	^***
Lower40 vs. MVC	ns	ns	ns	ns	ns
HVL vs. MVC	^*	ns	^***	ns	^***

Statistical analysis was done using a one-way ANOVA with Tukey's multiple comparison test.

HVL, high viral load; MVC, Maraviroc; ns, not significant; SD, standard deviation; VL, viral load.

< 0.05; ^***< 0.0005.

Results and Discussion

CCR5 is expressed on activated conventional CD4⁺ T cells (Tcon) (17), but an even higher expression was found on Foxp3⁺ Tregs (Fig. 1A). Up to 50% of this T-cell population expressed CCR5 in HIV-1-infected individuals. This high proportion of CCR5-expressing Tregs might render them susceptible to direct effects of Maraviroc and could potentially influence their activation or proliferation in HIV-1-infected patients on ART.

Indeed, in a current study by Pozo-Balado et al., Maraviroc treatment of HIV-1-infected patients resulted in a short- and long-term reduction of Treg frequencies (24). However, the reduced Treg frequencies also correlated with decreased viral loads in the Maraviroc-treated patients, making it difficult to interpret whether or not Maraviroc had a direct effect on Tregs or if viral loads were mainly influencing the Treg response.

In an attempt to determine the long-term effects of Maraviroc treatment on Treg frequencies and activation, we investigated three different groups of chronically HIV-1-infected individuals, which were distinguished by their viral loads. One group consists of therapy failures with HVLs (HIV-1 RNA copies 2,221–130,600 per mL blood), despite conventional ART (HVL), and two groups of ART responders with HIV-1 RNA below 40 copies per mL blood (with Maraviroc [MVC] or without MVC treatment [lower40]). Demographical, immunological, and virological important parameters were compared between the different groups (Table 1). No differences between all three groups were found for the nadir and the viral loads before treatment initiation. Patients with HVLs were slightly younger than patients of the MVC group. Furthermore, the time of diagnosis was more recent for HVL patients. A comparison between the two successfully treated (viral loads below 40 viral RNA copies/mL) and virologically suppressed patient groups showed no differences.

The lowest number of CD3⁺CD4⁺ T cells was found in the HLV group (Fig. 1B, Table 1), characteristic for an HIV-1-induced CD4⁺ T-cell depletion. Interestingly, this group of patients showed significantly higher frequencies of CD127^loCD25^hiFoxp3⁺ Tregs compared with ART-treated patients with undetectable viral loads (Fig. 1C). The finding that patients with HVLs have a higher frequency of Tregs and low CD4⁺ T-cell counts is in line with the current literature (18,20,25). Both, viral loads and CD4 depletion might influence Treg numbers in HIV-1-infected patients. Treg numbers in humans have been described to increase with age (13,27). Considering that HVL patients were slightly younger than MVC patients suggests that the increase of Treg frequencies in this patient group depends on the ongoing HIV-1 infection with high viral loads and immune activation rather than the age of the patients. No difference in the frequency of Tregs was found between patients who successfully received ART or ART plus Maraviroc (Fig. 1C). This result indicates that the control of HIV-1 replication in general rather than the choice of a specific treatment protocol determined the frequency of Tregs in HIV-1-infected patients.

Beside the frequency of Tregs, we also wanted to study their activation status and CCR5 expression levels under therapy. CD38 and TNFRII are the most reliable markers for Treg activation in vivo (4,5,22,26), so we stained for these molecules on gated Tregs (CD3⁺CD4⁺CD127^loCD25^hiFoxp3⁺ cells) from HIV-1-infected patients. Similar results as before were obtained, in that individuals with HVLs had significantly higher percentages of activated Tregs than successfully treated HIV-1⁺ patients. Maraviroc treatment did not affect the activation status of Tregs compared with patients receiving ART without Maraviroc (Fig. 1D).

We finally investigated the frequency of CCR5⁺ Tregs in the different patient groups. No significant differences were found between the groups (Fig. 1E), indicating that the activation status as well as the treatment with Maraviroc did not influence CCR5 expression on Tregs. These results indicate that antiretroviral treatment with Maraviroc reduced viral loads, which were associated with reduced frequencies of Tregs, but did not result in direct activation of Tregs in HIV-1-infected patients.

Our findings were in line with several other studies indicating that ART significantly decreases or even normalizes Treg frequencies to levels similar to that of healthy donors (2,10,18,25,28). Furthermore, patients with HVLs, due to irresponsiveness to ART (28), treatment interruption (30), or no treatment (10), exhibited higher Treg frequencies compared with healthy controls or virologically suppressed individuals. Hence, it was recently concluded by Chevalier and Weiss (6) that HIV-1 replication drives Treg expansion. In a recent study published by Pozo-Balado et al., (24) it was found that Maraviroc containing ART was able to reduce Treg frequencies in the short-term and even reduce Treg frequencies to levels observed in healthy controls in the long-term after treatment initiation. In their study, neither a short-term reduction (comparison between day 0, day 8, and day 30 after treatment initiation) nor a long-term normalization (comparison between 48 weeks after treatment initiation and healthy individuals) of Treg frequencies was found in patients successfully treated with ART without Maraviroc. This was especially surprising for the long-term effect of ART on Treg frequencies as they were still increased in patients with low viral loads compared with Treg frequencies in healthy individuals. This finding is in contrast to the results presented here and many other studies cited above. Interestingly, Pozo-Balado et al. found that in individual patients (follow-up analysis of ART-treated patients), ART was able to reduce the Treg frequencies significantly; however, the Treg frequencies did not normalize to levels observed in healthy individuals (24). This result is similar to what was reported for a few smaller studies that did not observe normalization of Treg frequencies after successful HIV therapy (9,12,15).

Up till now, this and the mentioned study by Pozo-Balado et al. (24) are the only two studies investigating the effect of Maraviroc treatment on Treg frequencies in HIV-1-infected patients. Some of the observed discrepancy between this study and the study by Pozo-Balado et al. (24) could result from the different study design and the gating strategy for Tregs. In the study by Pozo-Balado et al., (24) treatment naïve HIV-1-infected patients were treated with a high-dose Maraviroc monotherapy for 8 days, followed by a combination regime, including Maraviroc, at a lower dose. The observation period for this study was 48 weeks post-treatment initiation. In our study, chronically HIV-1-infected patients on conventional ART were additionally treated with Maraviroc and analyzed at different time points ranging from 1 to 91 months (median 24 month) post-treatment initiation. Furthermore, Pozo-Balado et al. (24) defined Tregs by the expression of CD4 and Foxp3, as well as high expression of CD25 (CD4⁺CD25^hiFoxp3⁺), whereas the definition of Tregs used in this study (CD4⁺CD25^hiCD127^loFoxp3⁺) is more stringent and most likely analyzed a purer Treg population. It will be interesting to see further studies comparing Treg frequencies in HIV-1-infected patients on cART with and without Maraviroc, and the potential effects of this CCR5 inhibitor on the function of CCR5-expressing Tregs in HIV-1-infected patients.

Most cross-sectional studies also report that peripheral Treg frequencies were lower in ART-treated patients compared with untreated chronically HIV-1-infected patients (8,10,11,28). Interestingly, the Treg frequency remained higher in patients who did not respond to treatment (11,23). Thus, ART that successfully decreases viral loads also results in reduced HIV-1-induced Treg expansion independent of the specific drug used for therapy. This suggests that the Treg frequency in HIV-1-infected patients is mainly influenced by viral replication and CD4 depletion. This is a widely accepted conclusion in the field. According to our data, Maraviroc does not have a direct long-term effect on blood Treg frequencies and activation in chronically HIV-1-infected subjects, but rather mediates a secondary effect on Tregs through suppression of viral replication and thereby immune activation.

Footnotes

Acknowledgment

This work was supported by a grant from the DFG (TRR60 Project B4).

All individuals included in this study provided written informed consent.

Author Disclosure Statement

No competing financial interests exist.

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