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Basic and Translational Science

HIV-Specific CD8+ T Cell–Mediated Viral Suppression Correlates With the Expression of CD57

Jensen, Sanne S. MSc*,†,‡; Tingstedt, Jeanette Linnea MSc*; Larsen, Tine Kochendorf MSc*; Brandt, Lea PhD*; Gerstoft, Jan MD, DMSc§; Kronborg, Gitte MD, DMSc; Pedersen, Court MD, DMSc; Fomsgaard, Anders MD, DMSc*,‡; Karlsson, Ingrid PhD*

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: January 1, 2016 - Volume 71 - Issue 1 - p 8-16
doi: 10.1097/QAI.0000000000000837



The HIV-1–specific cellular immune response has been correlated with control of infection, but the natural CD8+ T-cell response is not sufficient to eradicate the virus or prevent progression to AIDS.1–4 Antiretroviral therapy (ART) suppresses viral replication and to some extent restores the immune capacity and life expectancy of HIV-1–infected individuals. CD8+ T-cell responses, measured as the ability to proliferate and generate a multifunctional response, have been comprehensively analyzed in viremic individuals, individuals on ART, and HIV-controllers5–9 to gain knowledge to support the development of a potential therapeutic and/or prophylactic HIV-1 vaccine. Although the definition of what constitutes an effective HIV-1 T-cell response remains a topic of debate,10–12 a new ex vivo assay enables the characterization of the cytotoxic capacity of CD8+ T cells to suppress HIV-1 replication.13

CD8+ T cells can be divided into distinct subsets that define major steps in the differentiation of memory T cells.14,15 These distinct subsets display specific transcriptional programs and express different surface markers and intracellular molecules for homing, stimulation, survival, and effector function.16 The programed death-1 (PD-1), 2B4, and certain other markers have previously been identified as expressed on the surface of exhausted CD8+ T cells.17 PD-1 expression has been linked to a proapoptotic CD8+ T cell phenotype.18 An increasing body of evidence demonstrates a role for PD-1 in regulating virus-specific T-cell responses in vivo19,20 and ex vivo.21,22 Recent studies on the expression of 2B4 on CD8+ T cells demonstrate an increase in 2B4 expression during HIV-1 disease progression, and functional studies of 2B4+ CD8+ T cells suggest that these cells may play a role in inhibiting constrained HIV-1 epitopes.23 The CD160 marker is upregulated on HIV-1–specific CD8+ T cells during chronic infection, and when coexpressed with PD-1, it can define a functional exhausted subset.24 CD57 is a maturation marker in CD8+ T cells from HIV-1–positive individuals.25 The percentage of CD8+ T cells expressing CD57 was found to increase with differentiation, reaching maximum levels in the CD27lowCD45ROlow population.18

This study examines the cytotoxic capacity of CD8+ T cells from 44 HIV-1–positive individuals to suppress HIV-1 replication in CD4+ T cells in an ex vivo assay. We compare the actual cytotoxic capacity of the CD8+ T cells to the expression of various inhibitory and maturation markers (PD-1, CD57, CD160, and 2B4) and to HIV-1–specific cytokine responses.


Study Participants and Informed Consent

Forty-four HIV-1–positive individuals followed at the University Hospitals of Copenhagen and Odense in Denmark were recruited for the study. The study included 32 individuals treated with ART and 12 ART naive individuals. Participants on ART were virologically suppressed for at least 2 years before sampling. Table 1 outlines the clinical characteristics of the study participants.

Clinical Characteristics of Study Participants

The study was approved by the National Committee for Health Research Ethics of the Danish Ministry of Health (H-3-2011-031 and H-3-2012-104). Study participants provided written informed consent.

The plasma viral loads were quantified using the COBAS Ampliprep/COBAS TaqManHIV-1 Test, version 2.0 (Roche Diagnostics, Copenhagen, Denmark). The absolute CD4+ T-cell counts were determined using the FACS count system (BD Bioscience, San Jose, CA) according to the manufacturer's protocol. Whole blood or peripheral blood mononuclear cells (PBMCs) were used for analysis. PBMCs were obtained by density-gradient centrifugation and were cryopreserved until the analysis was performed.

Staining of CD8+ T-Cell Markers

Whole blood or PBMCs were stained. First, differences in marker expression on CD8+ T cells between whole blood and thawed PBMCs were established. Differences were observed in the expression levels of all examined markers (CD45RO, CD27, CCR7, CD57, CD160, PD-1, and 2B4), with the result that PBMC staining could not be used for this analysis (results not shown). For this reason, we only report marker expression data from whole blood. Six individuals (study ID: 3, 16, 65, 98, 118, and 119) were only used for the whole blood phenotypic analysis, because of limited sample material, and those were not tested in the intracellular cytokine staining assay and the HIV-1–specific ex vivo viral suppression assay. For these experiments, 100 μL of whole blood was incubated for 20 minutes at room temperature in the dark and in the presence of LIVE/DEAD Fixable Dead Cell Stain Kit marker (Life Technologies, Naerum, Denmark), anti-CD3 PerCP (BD Biosciences, San Jose, CA), anti-CD8 Qdot605 (Life Technologies), anti-2B4 FITC (BioLegend, London, United Kingdom), anti-CD45RO APC-H7 (BD Biosciences), anti-PD1 PECy7 (eBioscience, San Diego, CA), anti-CD57 APC (BD Biosciences), anti-CD27 AlexaFluor700 (eBioscience), anti-CCR7 PerCP/Cy5.5 (BioLegend), and anti-CD160 PE (BioLegend) antibodies. The antibodies were pretitrated to determine the optimal staining concentrations.

Whole blood was lysed for 20 minutes at room temperature using BD FACS Lysing Solution (BD Biosciences). The cells were fixed using BD Stabilizing Fixative (BD Biosciences). The fluorescence levels were measured using BD LSRII and analyzed using FlowJo (Treestar version 8.8.7; Ashland, OR). For detailed gating strategies, see Supplemental Digital Content 1 ( The surface markers CD27, CD45RO, and CCR7 were used to define memory populations. We define central memory (CM) cells as CD45RO+CD27+CCR7+, transitional memory (TM) cells as CD45RO+CD27+CCR7-, effector memory (EM) as CD45RO+CD27CCR7-, effector cells as CD45ROCD27CCR7-, and naive CD8+ T cells as CD45ROCD27+CCR7+.

Intracellular Cytokine Staining Assay

The number of cells available limited the intracellular cytokine staining assay used to test 32, as described previously.5 In brief, CD107a and intracellular interferon-γ (IFNγ), interleukin (IL)-2, tumor necrosis factor–α (TNFα), and macrophage inflammatory protein-1β (MIP-1β) were analyzed by stimulating 0.5 × 106 cells per milliliter PBMCs with HIV-1 antigen peptide pools (8 μg/mL of each peptide) for 6 hours at 37°C and 5% CO2. Peptide pools contained overlapping 15-mers covering the full-length HIV-1 consensus sequence (subtype B, National Institutes of Health AIDS Research and Reference Reagent Program, Germantown, MD). A total of 757 peptides were divided into 5 pools (Gag, Pol, Env, Nef, and TRVVV). Staphylococcus enterotoxin B (Sigma-Aldrich, St. Louis, MO) (1 μg/mL) was used as a positive control stimulation. Nonstimulated samples served as negative controls. Human cytomegalovirus was used as virus-specific control (8 μg/mL of each peptide) (National Institutes of Health AIDS Research and Reference Reagent Program). Brefeldin-A (12.5 μg/mL) and anti-CD107a PE (BD Bioscience) were added before stimulation. After stimulation, cells were incubated with EDTA and stained for 20 minutes using LIVE/DEAD Fixable Dead Cell Stain Kit marker (Life Technologies), and then permeabilized using the Cytofix/Cytoperm Kit (BD Bioscience) according to the manufacturer's protocol. Cells were stained in the dark with anti-CD3 PerCP (BD Bioscience), anti-CD4 Qdot605 (Thermo Fisher Scientific, Waltham, MA), anti-CD8 APC-H7 (BD Bioscience), anti-MIP-1β FITC (R&D systems, Minneapolis, MN), anti-IL-2 APC (BD Bioscience), anti-TNFα AlexaFlour 700 (BD Bioscience), and anti-IFNγ Pe-Cy7 (BD Bioscience). The fluorescence levels were acquired using the BD LSRII and analyzed using FlowJo (Treestar version 8.8.7). For detailed gating strategies, see Supplemental Digital Content 2 ( Samples were considered positive if the following criteria were met: percentages of CD107a-positive or cytokine-positive cells greater than 2 times background (nonstimulated cells); percentages of CD107a-positive or cytokine-positive cells greater than 0.05% after background subtraction; and more than 10 positive cells.5

HIV-1–Specific Ex Vivo Viral Suppression Assay

We performed the HIV-1 suppression assay of samples from 44 HIV-1–positive individuals as previously described.13 Briefly, CD4+ T cells were isolated by positive selection on an LS column and CD8+ T cells by negative selection according to the manufacturer's protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). After 3 days of stimulation, CD4+ T cells were infected and centrifugal inoculated (1 hour, 1200 g) with HIV-1 BaL. The virus was pretitrated to the optimal infection concentration. After infection, cells were set up as triplicates as follows in a 96-well plate: CD4+ noninfected; CD4+ plus CD8+ T cells (1:1); infected CD4+ T cells; and infected CD4+ T cells plus CD8+ T cells (1:1). CD4+ and CD8+ T-cell concentrations were set to 1 × 106 cells per milliliter.

HIV-1 Antigen ELISA

Supernatants were collected 3, 7, 10, and 14 days after infection, and the p24 concentration was determined by enzyme-linked immunosorbent assay (ELISA) as an indication of the viral suppression by CD8+ T cells. In brief, supernatants were diluted 1:4000 in dilution buffer (SSI Diagnostica, Hillerød, Denmark) and incubated overnight at 4°C. HIV-1 antigen-positive control (in house) was used at a concentration of 25–6400 pg/mL. Biotinylated HIV-1 antigen detection antibody no. 26 (SSI in-house) was added and incubated for 3 hours. Avidin-peroxidase (ThermoFisher Scientific, Waltham, MA) was diluted 20,000× and incubated for 1 hour. Tetramethylbenzidine ready-to-use substrate (Kem-En-Tec Diagnostics, Taastrup, Denmark) was added. The colorigenic reaction was stopped with 0.2 M H2SO4, and the absorbance was measured at 450–620 nm.

The suppressive activity (suppression factor) of CD8+ T cells was calculated from the ELISA results as follows:

Individuals with CD8+ T cells effective at suppressing HIV-1 infection are defined as suppressors, and individuals with CD8+ T cells with a lower suppressive effect are defined as nonsuppressors. The cutoff (>0.532) was defined as the mean level of suppression by CD8+ T cells from 8 HIV-negative individuals plus 3 SDs.

Statistical Analysis

Data analysis was performed using GraphPad Prism 6 software version 6.0c (GraphPad Software Inc., La Jolla, CA), and the results are presented as the medians and interquartile ranges in the figures. All data were tested for normality. The Mann–Whitney 2-tailed test or unpaired t test with Welch's correlation was used to test for differences between groups. Correlations were tested using Spearman or Pearson 2-tailed tests. Fisher exact test was used to test for differences between the number of ART-treated and ART naive in the suppressor and the nonsuppressor group. Polyfunctional data were analyzed in PESTLE version 1.7 and SPICE version 5.35 (provided by Mario Roederer, ImmunoTechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, MD).


HIV-1 Replication was Effectively Suppressed by CD8+ T cells From 14 of 44 Individuals Independent of ART, CD4+ T-Cell Count, or Plasma Viral Load

We tested the HIV-1–specific viral suppression of CD8+ T cells from 44 HIV-1–positive individuals, of which 32 were receiving ART and 12 were ART naive. In total, CD8+ T cells from 14 individuals effectively suppressed HIV-1 replication ex vivo (defined as suppressors), with 8 of those individuals on ART and 6 ART naive (P = 0.1524, data not shown). The suppression factor for individuals on ART did not correlate with the duration of therapy (P = 0.7181, data not shown). Moreover, viral suppression did not correlate with CD4+ T-cell count (P = 0.5242), viral load (P = 0.4348), or the CD4+ T-cell count at the time of ART initiation (P = 0.4767, data not shown).

CD8+ T Cell–Mediated HIV-1 Suppression Correlated With the Expression of CD57

We aimed to test whether the capacity to suppress HIV-1 replication ex vivo correlated with a specific CD8+ T-cell phenotype. The correlations between the suppression factor for each individual (both suppressors and nonsuppressors) and the frequency of effector, EM, CM, TM, and naive CD8+ T cells were all insignificant (data not shown). Moreover, no correlation was observed between the expression of the inhibitory markers 2B4, CD160, and PD-1 on the effector and memory subsets (data not shown). However, both frequency and the intensity mean fluorescence intensity (MFI) of the maturation marker CD57 on CD8+ T cells correlated with the suppression factor (P = 0.0047 and P = 0.0189, respectively) (Figs. 1A, B).

Viral suppression of HIV-1 in an autologous ex vivo assay correlates with CD57 mean fluorescence intensity (MFI) on effector CD8+ T cells. A, The suppression factor correlates with the frequency of total CD8+ T cells expressing CD57. B, The suppression factor correlates with CD57 MFI on total CD8+ T cells. C, The suppression factor of CD8+ T cells effectively suppressing HIV-1 infection (suppressors) correlates with CD57 MFI on effector CD8+ T cells.

To further determine whether the correlation between viral suppression and CD57 expression was determined by a specific CD8+ T-cell subset, we investigated the correlation of the suppression factor with the expression of CD57 on effector, EM, CM, TM, and naive CD8+ T cells. When focusing only on suppressors, a group of ART-treated and ART naive individuals, a correlation was found between the intensity of CD57 on effector cells and the suppression factor (P = 0.0178, Fig. 1C). No correlation was found between the expressions of CD57 on EM, CM, TM, or naive CD8+ T cells. Moreover, the frequency of effector and EM CD8+ T cells expressing CD57 and the duration of ART correlated (Figs. 2A, B, P = 0.0260 and P = 0.0472, respectively). In addition, the intensity of CD57 on effector CD8+ T cells correlated with the duration of ART (Fig. 2C, P = 0.0028).

The frequency of CD8+ T effector cells and EM cells expressing CD57 is correlated with the length of ART. A and C, The frequency of effector CD8+ T cells and the MFI correlated with the number of years that individuals received ART. B and D, The frequency of EM CD8+ T cells and the MFI was also correlated with the number of years of ART.

Phenotype of CD8+ T Cells Did Not Differ Between Suppressors and Nonsuppressors

To define whether individuals who effectively suppress HIV-1–replication have a distinct distribution of memory CD8+ T-cell subsets, we compared their distributions to those of nonsuppressors (see Supplemental Digital Content 3, No difference was found in the frequency of effector, EM, CM, TM, and naive within CD8+ T cells (see Supplemental Digital Content 3, Likewise, the expression of the inhibitory markers 2B4, CD160, and PD-1 on total CD8+ T cells or on memory CD8+ T-cell subsets was compared between suppressors and nonsuppressors (see Supplemental Digital Content 4A–D, The only significant differences seen were a slightly higher frequency of PD-1 on naive cells (P = 0.0034; see Supplemental Digital Content 4B, and coexpression of PD-1 and 2B4 on total CD8+ T cells (P = 0.0363) from suppressors (Fig. 3). For EM, we did see a significant higher coexpression of PD-1+2B4+CD160+ in suppressors compared with nonsuppressors (P = 0.0203, data not shown). Although the expression of maturation marker CD57 correlated with HIV-1–specific viral suppression, the CD8+ T cells from suppressors did not have a higher expression of CD57 compared with nonsuppressors (see Supplemental Digital Content 4D,

The frequency of CD8+ T cells coexpressing PD-1 and 2B4 was significantly higher in suppressors compared with nonsuppressors. We examined the frequency of CD8+ T-cell coexpression of PD-1 and 2B4 and found that suppressors (open circles) exhibit significantly higher coexpression of PD-1 and 2B4 compared with the nonsuppressors (open squares).

HIV-1–Specific CD8+ T cells From Individuals With the Ex Vivo Capacity to Suppress Viral Replication Expressed Higher Levels of CD107a and TNFα

To examine whether the cytokine expression by HIV-1–specific CD8+ T cells correlated with the ex vivo suppressive capacity of HIV-1 replication, we evaluated the CD107a and cytokine expression after stimulation with overlapping peptides covering the entire HIV-1 proteome. Suppressors expressed significantly higher levels of CD107a and TNFα compared with the nonsuppressors (P = 0.0044 and P = 0.0275, respectively, Fig. 4A). No differences were observed in the expression of IFNγ, IL-2, and MIP-1β between suppressors and nonsuppressors. The suppression factor was correlated with the expression levels of CD107a, TNFα, IFNγ, IL-2, and MIP-1β. A negative correlation was found between CD107a and the suppression factor, only for suppressors (P = 0.0268, data not shown). The ability of the CD8+ T cells to express multiple cytokines (ie, polyfunctionality) was also tested in the suppressors and the nonsuppressors, but no difference was observed (data not shown).

The total CD107a expression and TNFα response are elevated in suppressors compared with nonsuppressors. A, We examined the total CD107a and cytokine responses (IFNγ, IL-2, MIP-1β, and TNFα) in suppressors (open circles) and nonsuppressors (open squares). The CD107a expression and the TNFα response were significantly elevated in suppressors compared with the nonsuppressor group. B, No difference between suppressors and nonsuppressors was observed in the total responses to the 5 peptide pools (Gag, Pol, Env, Nef, and TRVVV).

We tested the total CD107a and cytokine responses toward 5 peptide pools corresponding to the HIV-1 proteins Gag, Pol, Env, Nef and the accessory proteins Tat, Rev, Vif, Vpr, and Vpu (TRVVV). The ability to suppress HIV-1 replication did not correlate with the specificity of CD8+ T cells to a certain HIV-1 protein. No significant difference was observed in the responses to any of the proteins between the suppressors and the nonsuppressors (Fig. 4B), and no correlation was found between the suppression factor and the specificity of the CD8+ T cells (data not shown).


This study presents a comprehensive analysis of viral suppression in correlation with the cytokine expression profiles and phenotypic profiles of CD8+ T cells from 44 HIV-1–positive individuals. The ex vivo viral suppression assay allowed us to measure the capacity of CD8+ T cells to suppress HIV-1–specific superinfection of autologous CD4+ T cells through cytotoxic activity.4 This assay is likely the most relevant ex vivo/in vitro method to measure the cytotoxic capacity of CD8+ T cells.

A positive correlation was demonstrated between the viral suppression factor and the frequency of CD8+ T cells expressing CD57, and also with the intensity of CD57 expression. The correlation coefficient is weak, but the small number of individuals included in the study could explain this. The positive potential of the maturation marker CD57 has gained a great deal of attention in recent years in connection with different types of cytotoxic cells.18,25–28 A comprehensive study of T and natural killer cells showed that CD57 could be used as a surrogate marker for the simultaneous expression of granzyme A, granzyme B, and perforin.26 We also demonstrated a correlation between long-term ART (>2 years) and the CD57 expression on total CD8+ T cells. The suppressor group consisted of both ART naive and ART-treated individuals, which taken together with the small number of studied individuals could explain the fact that no difference was observed between the frequencies of CD8+ T cells expressing CD57 in the suppressor group compared to the nonsuppressor group. Age has previously been shown to be an important determinant of CD57 expression;29 however, it does not significantly confound the correlation between CD57 expression and the viral suppression factor demonstrated here. This could be explained by the limitation in the relative few study individuals examined.

A decrease in inhibitory markers on CD8+ T cells during ART has been reported30,31 and is expected to be a measure of the restoration of CD8+ T-cell function. We hypothesized that a lower expression of inhibitory markers would correlate with viral suppression. However, we observed an increase in the coexpression of PD-1 and 2B4 in the suppressor group compared with the nonsuppressor group, independent of treatment. The simultaneous expression of several coinhibitory markers in the regulation of CD8+ T cells has been described in several studies, and the coexpression of PD-1 and 2B4 could not be linked either to less functional CD8+ T cells or to increased viremia.31–33 Thus, the coexpression of PD-1 and 2B4 may describe mature effective cytotoxic CD8+ T cells.

Polyfunctionality, defined by the multiplicity of antigen-specific cytokine production and other effector functions, has previously been demonstrated to be an important trait of T-cell function in HIV-1 pathogenesis.34 The frequency and percentage of HIV-, Gag-, and Nef-specific CD8+ T cells coexpressing CD107a, TNFα, IFNγ, IL-2, and MIP-1β function were inversely correlated with viral load in progressors.2 However, no correlation was observed between a polyfunctional cytokine profile and the ex vivo suppressive function of the CD8+ T cells. In previous studies,5 we have demonstrated decreased CD107a, TNFα, IFNγ, and MIP-1β responses in individuals on ART compared with ART naive individuals.5 Nonetheless, we did observe an elevated expression of the deregulation marker CD107a and TNFα in the suppressor group compared with the nonsuppressor group, independent of ART. Surprisingly, we found that the suppression factor was inversely correlated with CD107a.

No difference was observed between suppressors and nonsuppressors in their responses to the different HIV-1 proteins. Previous studies have shown that despite low antigen levels in their blood,35 HIV-1 controllers exhibit high frequencies of specific CD8+ T-cell responses that preferentially target the viral Gag protein.36–38 enzyme-linked immunospot analysis in HIV-1 controllers and viremic individuals showed that the secretion of IFNγ was correlated with the suppression factor (log p24 decrease).4,39 This could not be confirmed for viremic and ART-treated individuals in this study. These differences could be explained using different assays, as we used pools of overlapping peptides rather than single epitopes for stimulation.

Phenotypic analysis was performed on whole blood, as we found a surprising difference in marker expression when comparing the whole blood and PBMC assays. This deviation between marker expression on CD8+ T cells from separated PBMCs and whole blood was previously observed.40–42 The deviation between marker expression on PBMCs and CD8+ T cells from whole blood and also the treatment status of the studied individuals could partially explain some of the disparity between our results and previous findings. Finally, our study is limited by the fact that we did not study the phenotype of HIV-1–specific CD8+ T cells. The use of a nonautologous virus might lead to some underestimation of the HIV-1–suppressive capacity of the CD8+ T cells.39 However, we tested this possibility in a separate study (data not shown), and the suppression factor measured against the autologous virus correlated with the suppression factor for the nonautologous laboratory strain (BaL) used in this study.

This comprehensive study compares the HIV-1–specific viral suppression of CD8+ T cells ex vivo to the CD8+ T cell phenotype, specificity, and cytokine profile. Nevertheless, the number of suppressors compared with nonsuppressors and the fact that 6 of 14 suppressors are ART naive may affect the results and is a limitation of this study. Further studies should be performed comparing more viremic, ART treated, and HIV-1–controller patients to confirm that ART improves the effect of CD8+ T cells toward a more HIV-1 controller-like cytotoxic effectivity. This analysis extends the findings of Sáez-Cirión A. et al,39 who reported that the suppressive capacity of HIV controllers was significantly stronger compared with viremic and highly active antiretroviral therapy–treated individuals.

In summary, we performed an ex vivo analysis of the capacity of CD8+ T cells to suppress HIV-1 replication and compared this to the phenotypic profile and the HIV-1–specific responses of the degranulation marker CD107a, the cytokines TNFα, IFNγ, and IL-2, and the chemokine MIP-1β. In individuals with effective viral suppression, we observed higher CD107a and TNFα responses. In addition, a strong correlation was found between the expression of CD57 and the CD8+ T cell–mediated suppression of HIV-1 replication. The duration of ART correlated with the expression of CD57 but not with the viral suppression. Although the function of CD8+ T cells is restored during therapy, the intensity and breadth of T-cell responses fade due to the lack of antigen stimulation.4,5,43,44 The consequence of these 2 factors together may be that viral suppression does not change. In this context, it may be beneficial to combine ART with a therapeutic HIV-1 vaccine to induce a potent CD8+ T-cell response. Our results demonstrate that in HIV-1–positive individuals, the best correlation of CD8+ T cell–mediated viral suppression is the expression of CD57.


The authors are grateful to the individuals who participated in this study. The authors acknowledge Lene Pors Jensen, Bente Baadegaard, Philippa Collins at the University Hospital of Copenhagen, and Charlotte Christensen at Odense University Hospital for their help in recruiting individuals into this study and for providing the clinical information. The authors thank Asier Saez-Cirion for training on the HIV-1 suppression assay in his laboratory. The authors are especially grateful to Birgit Knudsen for her technical assistance.


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HIV-1 suppression assay; CD8+ T lymphocytes; phenotype; cytokine; CD57; intracellular cytokine staining; antiretroviral therapy

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