Chemokine receptor CCR5 correlates with functional CD8⁺ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation

Abstract

C-C chemokine receptor 5 (CCR5) plays an essential role in HIV pathogenesis as the major coreceptor on CD4⁺ T cells used by HIV, yet the function of CCR5 on CD8 T cells is not well understood. Furthermore, the immunologic effects of the CCR5 inhibitor maraviroc (MVC), despite approval for clinical use, have not yet been well evaluated for their potential effects on cytotoxic T-cell responses. In this study, we characterized the development and function of CCR5⁺CD8⁺ T cells in rhesus macaques with or without Simian immunodeficiency virus (SIV) infection. We also investigated the effects of the CCR5 antagonist MVC on functional CCR5⁺CD8⁺ T-cell responses in vitro. The data show that CCR5⁺CD8⁺ T cells have an effector memory phenotype and increase with age in systemic and mucosal lymphoid tissues as a heterogeneous population of polyfunctional CD8 T cells. In addition, CCR5 is highly expressed on SIV gag-specific (CM9⁺) CD8⁺ T cells in SIV-infected macaques, yet CCR5⁺CD8⁺ T cells are significantly reduced in mucosal lymphoid tissues with disease progression. Furthermore, in vitro MVC treatment reduced activation and cytokine secretion of CD8⁺ T cells via a CCR5-independent pathway. These findings suggest that surface CCR5 protein plays an important role in differentiation and activation of CD8⁺ T cells. Although MVC may be helpful in reducing chronic inflammation and activation, it may also inhibit virus-specific CD8⁺ T-cell responses. Thus optimal use of CCR5 antagonists either alone or in combination with other drugs should be defined by further investigation.—Wang, X., Russell-Lodrigue, K. E., Ratterree, M. S., Veazey, R. S., Xu, H. Chemokine receptor CCR5 correlates with functional CD8⁺ T cells in SIV-infected macaques and the potential effects of maraviroc on T-cell activation.

C-C chemokine receptor 5 (CCR5) is expressed on a number of cells, including T lymphocytes, macrophages, and dendritic cells (1), and is a receptor for the β-chemokines chemokine ligand (CCL)-3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES), and is the major coreceptor for HIV-1 entry (2–4). CCR5 is critical for the transmission and spread of CCR5-tropic HIV-1, which are the predominant transmitted strains in newly infected individuals (5–7). Envelope glycoprotein GP120 on the surface of HIV binds to cellular CD4 receptors, leading to a conformational change that exposes the V3 loop. The subsequent interaction between the exposed V3 loop of envelope glycoprotein GP120 and coreceptors on the host cells (such as CD4 and CCR5) helps HIV entrance (8). Mutation in the CCR5 gene leads to a 32-bp deletion (Δ32) in the CCR5 protein, and the resulting lack of CCR5 on the surface of cells (homozygotes for Δ32) is protective against HIV acquisition (9–11), and individuals with heterozygous expression of CCR5 have delayed HIV disease progression (12, 13). An understanding of HIV entry thus provides therapeutic targets to develop CCR5 antagonists against HIV infection. A series of clinical trials demonstrated that the CCR5 antagonist maraviroc (MVC) could efficiently suppress HIV infection (8, 14, 15), reduce immune activation (16–21), and even prolong organ survival against Graft-versus-host disease (22). However, possible limitations have also been reported (23–25).

In addition to usage as an HIV coreceptor, CCR5 plays a crucial role in the differentiation, activation, and recruitment of CD8⁺ T cells to inflamed tissues (26–28). CCR5 deficiency results in decreased recruitment of memory T cells and impaired viral containment during early immune responses (29–31). It is also reported that CCR5 signaling results in decreased IFN-γ production and cytotoxic T lymphocyte (CTL) activity in response to viral infections in which CCR5 expression normally increases on activated CD8⁺ T cells (26, 32–35). CCR5 is also responsible for the migration and localization of Ag-specific CD8⁺ T cells to inflamed tissues and secondary lymphoid tissues (33). In addition, West Nile virus and tick-borne encephalitis infection up-regulate CCR5, which correlates with animal survival (36–40). Although these findings indicate that CCR5 plays functional roles in host immunity, the effects of CCR5 blockade on CD8⁺ T-cell responses are not well studied. To determine the effects of CCR5 blockade on T-cell function, here we characterized CCR5 expression on CD8⁺ T cells in rhesus macaques with or without Simian immunodeficiency virus (SIV) infection and investigated the effects of MVC on T-cell responses in vitro.

MATERIALS AND METHODS

Ethics statement

All animals in this study were housed at the Tulane National Primate Research Center in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International standards. All studies were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee under protocol number P0049. Animal housing and studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals (AAALAC 000594; National Institutes of Health (NIH), Bethesda, MD, USA) and with the recommendations of the Weatherall Report, “The use of non-human primates in research.” All clinical procedures were carried out under the direction of a laboratory animal veterinarian. All procedures were performed under anesthesia using ketamine, and all efforts were made to minimize stress, improve housing conditions, and to provide enrichment opportunities (i.e., objects to manipulate in cage, varied food supplements, foraging and task-oriented feeding methods, interaction with caregivers and research staff).

Animals and virus

A total of 68 adult rhesus macaques (Macaca mulatta) that were either negative for SIV (n = 40) or infected by SIV (n = 28), yet negative for type D retrovirus and Simian T-cell leukemia virus type 1 infection, were used in this study. The SIV naive animals included newborn (n = 5), infants/neonates (age: 0–6 mo, n = 12), or juvenile (age: 6 mo–3 yr, n = 8), and the rest were adults (>3 yr, n = 15). Of the 28 SIVmac251-infected animals, 6 were in acute stage (d 7–21) and 22 were in chronic stage (3 mo and later post-SIV infection, including 3 Mamu A*01⁺ macaques). Blood and lymph node biopsies were collected from animals. To examine cells from tissues (such as spleen, intestine, etc.), macaques were euthanized for tissue collection.

Phenotyping blood and tissue mononuclear cells

Mononuclear cells from peripheral blood, lymph node, spleen and intestinal tissues were isolated and processed as previously described (41). Cells were stained with CD3 (SP34), CCR5 (3A9), CD8 (SK1), CD4 (L200), CD45RA (5H9), CCR7 (TG8/CCR7; BioLegend, San Diego, CA, USA), CD28 (CD28.2), CD69 (FN50), human leukocyte antigen–DR isotype (HLA-DR; L243), IL-2 (MQ1-17H12), IL-17 (CZ8-23G1; Miltenyi Biotech, Bergisch Gladbach, Germany), TNF-α (MAB11), IFN-γ (B27), and CM9 tetramer (Thermo Fisher Scientific, Waltham, MA, USA). All antibodies and reagents were purchased from BD Biosciences (San Jose, CA, USA) unless otherwise noted. Stained samples were resuspended in BD Stabilizing Fixative (BD Biosciences) and acquired on a BD FacsVerse flow cytometer [Becton Dickinson (BD), San Diego, CA, USA]. Data were analyzed with FlowJo software (BD Biosciences).

Cell stimulation and intracellular cytokine staining

To evaluate the effects of MVC on surface CCR5 expression, activation, and cytokine secretion of CD8⁺ T cells, peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood and resuspended in Roswell Park Memorial Institute (RPMI) medium 1640–10% fetal calf serum. PBMCs were then plated in 96-well plates (5 × 10⁵/well) treated with or without MVC (0, 1, 10, and 50 μM) at 37°C in a 5% CO₂ for 5 and 24 h, when early activation markers and CCR5 expression were analyzed. To detect SIV gag-specific CD8⁺ T-cell responses, PBMCs were stimulated by a pool of 15-mer gag peptides (5 μg/ml each peptide), medium (negative control), or phorbol-12-myristate-13-acetate (PMA; 5 ng/ml; MilliporeSigma, Burlington, MA, USA) plus ionomycin (50 μg/ml) (positive control) with or without MVC treatment (10 μM; NIH) for 6 h. The cultures also contained brefeldin A (MilliporeSigma) and 1 μg/ml of anti-CD49d and anti-CD28 costimulatory molecules (BD Biosciences). Cultured cells were stained with monoclonal antibodies specific for surface molecules (CD3, CD4, CD8, CCR5, and Live/Dead cell-staining kit). After fixation and permeabilizion with Cytofix/Cytoperm solution (BD Biosciences), cells were stained with antibodies specific for IFN-γ and TNF-α and then washed by Perm/Wash buffer (BD Biosciences). Finally, labeled cells were fixed in 1.5% paraformaldehyde, acquired with a FacsVerse cytometer, and data were analyzed using FlowJo software. The background level of cytokine staining varied within different samples and different cytokine patterns but was typically <0.05% of total CD8⁺ T cells (median, 0.01%). Only samples in which the percentage of cytokine-stained cells was at least twice that of background were considered positive.

Cell line and in vitro cell cultures

The Jurkat-Lat Tat–green fluorescence protein (GFP) (A1) T-cell line, which is infected with retroviruses containing long terminal repeat (LTR)-Tat–internal ribosome entry site–GFP and expresses GFP upon reactivation (42, 43), was used to study the effects of MVC treatment on cell activation. Because Jurkat T cells do not express surface CCR5 (44), it is an appropriate cell model to evaluate whether CCR5 is involved in MVC-mediated cell function. The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum, 1% of Penicillin-Streptomycin, and 1% l-glutamine, and maintained in 37°C with 5% CO₂. To test effects of MVC on cell activation, cells (5 × 10⁵) were incubated with or without MVC (10 μM) for 1 h, then treated by PEP005 (R&D Systems, Minneapolis, MN, USA) for another 23 h as we reported previously (45). After stimulation, cells were analyzed for CD69 and GFP expression by flow cytometry.

Statistics

Graphical presentation and statistical analysis of the data were performed using Prism 4.0 (GraphPad Software, La Jolla, CA, USA). Comparisons between groups were analyzed by a 1-way ANOVA and a nonparametric Mann-Whitney U test. Values of P < 0.05 were considered statistically significant.

RESULTS

Characterization of CCR5⁺CD8⁺ T cells in rhesus macaque

To characterize CCR5⁺CD8⁺ T-cell populations in rhesus macaques, we analyzed phenotype, function (cytokine-producing capacity), and developmental expression from peripheral blood and various lymphoid tissues in infants and adults. Our data showed that all CCR5⁺CD8⁺ T cells are transitional or effector memory cells as indicated by a CD45RA−CCR7−CD28^+/− phenotype (Fig. 1A, B), which is consistent with other reports that CCR5 is not expressed on naive CD8⁺ T cells in humans (33). These CCR5⁺CD8⁺ T cells consist of a heterogeneous population of polyfunctional CD8⁺ T cells that produce multiple cytokines, such as IL-17, IL-2, IFN-γ, and TNF-α, after mitogen stimulation (Fig. 1C). The percentage of CCR5 expression on CD8⁺ T cells dynamically increased with age, from newborn to adult, in the peripheral blood, lymph node, spleen, and mucosal gut-associated lymphoid tissues (Fig. 1D). These findings suggest that multifunctional CCR5⁺CD8⁺ T cells develop with age most likely in response to antigen exposure, inflammation, or maturation of immune responses.

Figure 1.

Characterization of CCR5⁺CD8⁺ T cells in uninfected macaques. A, B) Representative dot plot showing expression of CD45RA, CD28, and CCR7 on CCR5⁺CD8⁺ T cells in blood from adult animals (A); frequencies of naive (CD45RA⁺CCR7⁺CD28⁺), terminally differentiated effector memory (TEMRA; CD45RA⁺CCR7⁻CD28⁻), central memory (CM; CD45RA⁻CCR7⁺CD28⁺), and translational and effector memory (EM; CD45RA⁻CCR7⁻CD28^+/−) subsets in the CCR5⁺CD8⁺ T cells in blood from adult rhesus macaques (n = 10) (B). *P < 0.05, compared with other subsets. C) IFN-γ, TNF-α, IL-17, and IL-2 secretion of CCR5⁺CD8⁺ T cells from adult animals upon mitogen stimulation in vitro. D) Dynamics of CCR5⁺CD8⁺ T cells in systemic and lymphoid tissue compartments with age; newborn (n = 5); neonates (>0–6 mo, n = 12), juveniles (6 mo–3 yr, n = 8), and adults (>3 yr, n = 15). The data are presented as the means ± sem. *P < 0.05, compared with newborns.

CCR5 expression on virus-specific CD8⁺ T cells following SIV infection

To examine CCR5 expression during SIV infection, we analyzed CCR5 expression on total and SIV-specific CD8⁺ T cells (CTL) in blood and tissues throughout infection, and their responses to MVC treatment ex vivo. Interestingly, the results showed that the majority of SIV gag-specific (CM9⁺) CD8⁺ T cells coexpress CCR5 in blood (2.39 ± 0.11), lymph nodes (2.53 ± 0.23), and intestinal lymphoid tissues (2.72 ± 0.35) of Mamu A*01⁺ SIV-infected macaques in vivo (Fig. 2A). Notably, CCR5 blockade by MVC significantly reduced SIV-specific CD8⁺ T-cell responses against SIV gag, as indicated by lower levels of IFN-γ and TNF-α production after peptide stimulation compared with untreated controls (Fig. 2B, C). Furthermore, CCR5⁺CD8⁺ T cells in the jejunum gradually decreased with disease progression during SIV infection, yet these populations in the blood returned to equivalent levels of uninfected controls at chronic stage (Fig. 2D).

Figure 2.

CCR5 expression on SIV-specific CD8⁺ T cells in SIV-infected adult rhesus macaques. A) Representative dot plots of CCR5 expression on SIV gag-specific CD8⁺ T cells (CM9 clone) in tissues of chronically SIV-infected Mamu A*01⁺ macaques. B, C) Effects of CCR5 antagonist MVC on CD8⁺ T-cell responses against SIV gag stimulation in vitro. PBMCs from chronically SIV-infected macaques (n = 6) were stimulated by pooled SIV gag peptides with or without MVC (10 μM) for a 6-h cell culture, then analyzed by IFN-γ (B) or TNF-α (C) secretion. D) Changes in CCR5⁺CD8⁺ T cells in blood and jejunum lamina propria in Mamu A*01⁻ macaques during SIV infection. Uninfected, n = 15; acute, n = 6, chronic asymptomatic, n = 19. The data are the means ± sem. *P < 0.05, compared with uninfected controls.

Effects of MVC on activation and cytokine secretion of CD8⁺ T cells in vitro

Because all SIV-specific CD8⁺ T cells expressed CCR5 (Fig. 2), we investigated the effects of MVC on activation and stimulation of cytokine production of total CD8⁺ T cells, and CCR5⁻ or CCR5⁺CD8⁺ T cells from uninfected macaques. Although MVC is a CCR5 antagonist, MVC treatment markedly increased surface CCR5 expression on CD8⁺ T cells (Fig. 3A). Furthermore, MVC inhibited IFN-γ, TNF-α, and IL-17 production by PMA/ionomycin-stimulated total CD8⁺ T cells in vitro, compared with non-MVC–treated controls (Fig. 3B–D). To address whether MVC treatment suppressed T-cell function specifically by interacting with surface CCR5 expression, we compared the effects of MVC on CCR5⁻ and CCR5⁺ CD8⁺ T cells by analyzing cell activation and capacity to produce cytokines. The data indicated that both CCR5⁻ and CCR5⁺CD8⁺ T cells had reduced expression of the early activation marker CD69, albeit decreases in HLA-DR were not consistent between these 2 subsets (Fig. 3E, F), yet MVC treatment reduced HLA-DR expression on CCR5⁺CD8⁺ cells, albeit HLA-DR expression was minimally affected by MVC treatment (Fig. 3F). Further results upon mitogen stimulation showed that both CCR5⁻ and CCR5⁺CD8⁺ T cells had markedly reduced proinflammatory cytokine responses (IFN-γ, TNF-α, and IL-17) after MVC treatment, consistent with the decreased cell activation measured by CD69 on both subsets (Fig. 3G–I). In general, these data demonstrate that MVC suppresses activation and responses of all CD8⁺ T cells, which suggests that these effects may be induced in a CCR5-independent manner or possibly from secondary effects that are due to its interactions with other cells.

Figure 3.

Effects of in vitro MVC treatment on CCR5⁺ or CCR5⁻CD8⁺ T cells from SIV naive adult macaques. A) Effects of MVC treatment on CCR5 expression of CD8⁺ T cells. B–D) TNF-α (B), IFN-γ (C), and IL-17 (D) production in PMA plus ionomycin-stimulated total CD8⁺ T cells, with or without MVC cotreatment. E, F) Effects of MVC treatment on activation of CCR5⁺ (opened bar) or CCR5⁻ (closed bar) CD8⁺ T cells, as indicated by the levels of CD69 (E) and HLA-DR (F) expression. G–I) Effects of MVC treatment on cytokine secretion of CCR5⁺ (opened bar) or CCR5⁻ (closed bar) CD8⁺ T cells stimulated by PMA plus ionomycin, as indicated by the levels of TNF-α (G), IFN-γ (H), and IL-17 (I) that were measured. PBMCs were isolated from the peripheral blood of uninfected macaques (n = 10) then stimulated by PMA plus ionomycin in the presence or absence of MVC (10 μM) for 6 h. The data are the means ± sem. *P < 0.05.

Effects of MVC on activation of J-Lat cell line in vitro

To further validate whether suppression of T-cell activation is induced by MVC through blockade of surface CCR5, the J-Lat-Tat-GFP cell line, derived from Jurkat T lymphocytes, was used in this study. This cell line bears the integrated HIV LTR-Tat and GFP gene (42, 43, 46) but does not express extracellular CCR5 (44). Resting cells are GFP negative, but cell activation leads to HIV-1Tat/LTR-driven GFP expression. To stimulate HIV/GFP expression, we used PEP005, which is a small molecule activator of protein kinase currently being explored for reactivation of latent HIV reservoirs (45). As confirmed in Fig. 4, J-Lat cells do not express surface CCR5, yet MVC treatment significantly inhibited cell activation and the counterpart reporter signal in cells that were stimulated by PEP005 (10 ng/ml) for 24 h. MVC treatment significantly reduced CD69 (63.8 ± 1.3% vs. 94.2 ± 0.4%) as well as GFP expression (9.01 ± 0.24% vs. 45.6 ± 0.5%) in PEP005-alone stimulated cells. Combined, these findings indicate that MVC treatment inhibits T-cell activation through a CCR5-independent or downstream manner.

Figure 4.

Effects of MVC treatment on activation of J-Lat-Tat-GFP cell line in vitro. A) Representative histogram of surface CCR5 expression on cells (compared with isotype Ab control), CD69 and GFP expression in cells treated by PEP005 alone, or PEP005 plus MVC that compared with untreated controls. B, C) The levels of CD69 (B) and GFP (C) expression in J-Lat-Tat-GFP cells in the presence of PEP005 activators, in combination with or without MVC. Cells (5 × 10⁵/well) were cultured in the presence of PEP005 (10 ng/ml) or PEP005 plus MVC (10 μM) for 24 hr. The early cell activation marker CD69 and the GFP expression in cells (>95% cell survival) were analyzed. These data are representative of at least 3 independent experiments with the means ± sd. *P < 0.05.

DISCUSSION

Chemokine receptor CCR5 plays an essential role in differentiation, activation, and recruitment of T cells and in HIV pathogenesis. As the major HIV coreceptor, CCR5 thus provides a target for treatment and prevention of HIV infection. Currently, MVC is the only CCR5 antagonist currently approved by the U.S. Food and Drug Administration for HIV treatment. Although CCR5 is also expressed on a considerable proportion of CD8⁺ T cells, its potential immunologic effects on CD8⁺ T cells are not well defined. In this study, we examined CCR5 expression on CD8⁺ T cells in uninfected neonatal and adult macaques and in SIV-infected animals, and we specifically evaluated the effects of MVC on CD8⁺ T-cell responses.

It was previously reported that CCR5 is highly expressed on virus-specific CD8⁺ T cells following HIV-1, coronavirus, hepatitis C virus, Epstein-Barr virus, or cytomegalovirus infections (26, 32, 33, 47, 48). In addition, impaired viral control has been demonstrated in cases of CCR5 deficiency (29–31), suggesting CCR5 is a vital functional molecule involved in T-cell responses to viral infections. Consistent with previous reports in humans (33), our data indicated that CCR5⁺CD8⁺ T cells have an effector or transitional memory phenotype, which are able to produce multiple cytokines after stimulation (Fig. 1A–C). Furthermore, these populations increase with age in various systemic and lymphoid tissues of macaques (Fig. 1D), displaying development-associated maturity and differentiation of polyclonal CD8⁺ T cells. It was also reported that type 1 CD8⁺ T cells (Tc1 effector CD8⁺ T cells) predominantly express CCR5 mRNA (and CXCR4 mRNA), but type 2 cells (Tc2 CD8⁺ T cells) do not express CCR5 mRNA (34, 35), suggesting that CCR5 signaling is involved in IFN-γ production and CTL activity but the mechanisms remain unknown. Notably, virtually all SIV-specific CD8⁺ T-cell clones targeting CM9 (a dominant epitope of gag) highly expressed CCR5. Furthermore, SIV infection resulted in significant reductions of jejunal CCR5⁺CD8⁺ T cells (Fig. 2A, D), which may have included other populations of virus-specific CTL clones.

To address whether CCR5 blockade could influence CD8⁺ T-cell function, we performed a series of experiments. First, we compared SIV-specific CD8⁺ T-cell responses stimulated by SIV gag peptides in the presence or absence of MVC treatment from macaques chronically infected with SIV. We then compared levels of activation and functional capacity of both CCR5⁻ and CCR5⁺CD8⁺ T cells obtained from uninfected animals with or without MVC treatment after in vitro stimulation. Surprisingly, MVC treatment significantly increased surface CCR5 expression on T cells (Fig. 3A). Although the mechanisms of this up-regulation were not examined, this may be a compensatory response of T cells following CCR5 engagement. After MVC treatment, cytokine production was markedly reduced by CD8⁺ T cells stimulated by either SIV antigens or mitogens, concomitant with decreased activation measured by CD69 and HLA-DR expression, in which penetrated intracellular MVC might interfere with cell activation–associated signals by unknown mechanisms. Furthermore, this was independent of CCR5 expression as it was observed in both CCR5⁻ and CCR5⁺CD8+ T cells (Figs. 2B and 3). In fact, and consistent with other reports (49), we found that MVC treatment also reduced activation and cytokine secretion by both CD4⁺ (unpublished data) and CD8⁺ T cells. These data suggest that MVC could inhibit T-cell activation via a CCR5-independent pathway. These findings prompted further experiments using J-Lat-Tat-GFP T cells as CCR5 is not expressed on this cell line (Fig. 4A). PEP005 (ingenol-3-angelate), an activator of PKC, induces nuclear translocation of PKC-δ and is under exploration as a candidate drug for HIV latency reactivation (50–52). Our recent study indicated PEP005 could effectively induce maximal cell reactivation and GFP expression in vitro (45). As indicated in Figs. 3E and 4B, MVC treatment was still able to suppress activation of Jurkat T cells, despite the lack of surface CCR5 expression, as shown by significantly reduced levels of CD69 and GFP (Fig. 4C), suggesting that MVC may inhibit T-cell activation by CCR5-independent pathway(s), yet the mechanisms of this remain unknown. Although MVC intensification may have the potential for blocking new infections and reduction of chronic activation in HIV therapy, conceivably, its effects on inhibition of cell activation could also promote establishment of HIV latency in therapy. Therefore, MVC treatment may also interfere with “shock and kill” latency reactivation strategies, as suggested by the effects of MVC treatment on attenuating the reactivation of latently infected cells after stimulation by the potent PKC activator PEP005. Inconsistent with our results, it is reported that MVC could be used as an HIV latent reversing agent, yet the contradictory data revealed the following: MVC induces NF-κB activity, but CCR5 inhibitor blocks cell activation; the effects of MVC and bryostatin-1 combination on cell activation is antagonistic (53, 54), probably the survival of cells (excluding dead cells) should be considered when luciferase/proliferation is analyzed in MVC treatment. Although CCR5 blockade may prevent HIV infection of new cells, it may also reduce activation, cytokine responses, and migration of effector T cells into inflamed tissues. Thus, continued research into the effects of CCR5 inhibitors on HIV patients is needed, especially when considering strategies based on latency reactivation and cellular immunity for an HIV cure.

Glossary

CCL

chemokine ligand

CCR5

C-C chemokine receptor 5

CTL

cytotoxic T lymphocyte

GFP

green fluorescence protein

HLA-DR

human leukocyte antigen–DR isotype

LTR

long terminal repeat

MVC

maraviroc

PBMC

peripheral blood mononuclear cell

PMA

phorbol-12-myristate-13-acetate

SIV

Simian immunodeficiency virus

Tat

transactivator

AUTHOR CONTRIBUTIONS

X. Wang performed experiments and analyzed data; K. E. Russell-Lodrigue and M. S. Ratterree assisted with manuscript preparation; R. S. Veazey edited the manuscript; and H. Xu designed the research and wrote the manuscript.