Impact of highly active antiretroviral therapy initiation on CD4+ T-cell repopulation in duodenal and rectal mucosa
Hayes, Timothy L.a; Asmuth, David M.b; Critchfield, J. Williama; Knight, Thomas H.b; McLaughlin, Bridget E.a; Yotter, Tammyb; McConnell, Delandy H.a; Garcia, Juan Carlosc; Pollard, Richard B.b; Shacklett, Barbara L.a,b
aDepartment of Medical Microbiology and Immunology
bDivision of Infectious Diseases
cDivision of Gastroenterology, Department of Internal Medicine, School of Medicine, University of California, Davis, California, USA.
Correspondence to Barbara L. Shacklett, PhD, Department of Medical Microbiology and Immunology, 3146 Tupper Hall, 1 Shields Avenue, Davis, CA 95616, USA. Tel: +1 530 752 6785; fax: +1 530 752 8692; e-mail: firstname.lastname@example.org
Received 9 August, 2012
Revised 31 October, 2012
Accepted 15 November, 2012
Objective: The objective of this study was to assess the effects of HAART initiation on CD4+ T-cell repopulation and T-cell immune activation in rectal and duodenal mucosa.
Design: The effects of HAART on the gastrointestinal tract remain controversial, and studies have reached different conclusions regarding its effectiveness at restoring mucosal CD4+ T cells depending upon time of initiation, duration of treatment and gastrointestinal tract region studied.
Methods: We obtained blood, rectal biopsies and duodenal biopsies from 14 chronically infected individuals at baseline and at 4–9 months post-HAART initiation. We examined CD4+ T-cell frequencies in blood, rectum and duodenum at both time points, and performed a detailed assessment of CD4+ T-cell phenotype, immune activation marker expression and HIV-specific CD8+ T-cell responses in blood and rectal mucosa.
Results: CD4+ T-cell percentages increased significantly in blood, rectal and duodenal mucosa after 4–9 months of HAART (P = 0.02, 0.0005, 0.0002), but remained lower than in uninfected controls. HIV-specific CD8+ T-cell responses in blood and rectal mucosa declined following HAART initiation (P = 0.0015, 0.021). CD8+ T-cell coexpression of CD38 and HLA-DR in blood and mucosa, as well as plasma sCD14, declined significantly. CD28 expression on blood and mucosal CD8+ T cells increased, whereas programmed death receptor-1 expression on blood HIV-specific CD4+ and CD8+ T cells decreased.
Conclusion: Within the first months of HAART, limited CD4+ T-cell reconstitution occurs in small and large intestinal mucosa. Nevertheless, decreased immune activation and increased CD28 expression suggest rapid immunological benefits of HAART despite incomplete CD4+ T-cell reconstitution.
Primary HIV-1 infection is associated with rapid and massive depletion of mucosal CD4+ T cells in the gastrointestinal tract, home to the majority of the body's lymphocytes . CD4+ T-cell loss occurs more rapidly in the gut than in peripheral blood [2–5]. With recent advances in HAART, many HIV-infected individuals are able to maintain plasma viraemia at levels undetectable by most assays. This viral suppression is also associated with significant reconstitution of blood CD4+ T cells. However, CD4 recovery is often incomplete, failing to reach levels observed in uninfected individuals. The extent of recovery may vary widely depending on factors such as age and extent of immunodeficiency at the time of HAART initiation [6,7]. In lymph nodes, immune activation, contributing to collagen deposition and lymphoid tissue fibrosis, may severely limit CD4+ T-cell reconstitution . Similar mechanisms likely impact CD4+ T-cell reconstitution in the gut lamina propria . However, the kinetics and extent of HAART-induced CD4+ T-cell reconstitution in the gut are less well understood. To date, most studies have focused on a single portion of the gastrointestinal tract, such as jejunum, ileum, or colon [2–5,10–16]. Sheth et al. observed near complete recovery of CD4+ T cells in sigmoid colon following long-term HAART , while Chun et al. saw incomplete recovery in terminal ileum. These discrepancies may be related to differences between patient groups and treatment protocols, and/or to differences between small and large intestine. To our knowledge, no studies have longitudinally tracked changes in mucosal memory/effector T-cell subsets, immune activation and antigen-specific T-cell responses during the weeks and months immediately following HAART initiation.
On the basis of earlier studies, we predicted that initiation of suppressive HAART in chronically HIV-infected individuals would lead to measurable reductions in lymphocyte activation and begin to elicit mucosal CD4+ T-cell reconstitution in both small and large intestine. To address these issues, we sampled rectal and duodenal mucosa from 14 chronically infected individuals prior to initiation of HAART, then at a second time point 4–9 months postinitiation. We also performed a detailed analysis of HIV-specific CD8+ T-cell responses and expression of T-cell activation and phenotypic markers in blood and rectal mucosa at both time points. Our data revealed incomplete CD4+ T-cell reconstitution; nevertheless, significant increases in CD4+ T-cell percentages were detected in blood, rectal and duodenal mucosa, along with decreased expression of immune activation markers and a decline in HIV-specific T-cell response magnitudes in blood and mucosa.
Materials and methods
Individuals and HAART
Fourteen HIV-seropositive individuals, all infected for a minimum of 1 year and naive to antiretroviral therapy (ART), or who had been briefly exposed to ART (less than 30 days) in the past were enrolled. The only exclusion criteria were safety parameters related to upper endoscopy with biopsies (subjects with Grade II anemia or abnormal coagulation parameters were excluded). After baseline clinical parameters were established, the individuals were prescribed a HAART regimen. The 14 HIV-positive individuals in this research study were also participating in a separate clinical trial comparing the effects of a raltegravir (RGV)-based HAART regimen versus a nonnucleoside reverse transcriptase inhibitor (NNRTI)-based HAART regimen on gut immune reconstitution in HAART-naive individuals (Clinical Trial Registry Number NCT00661960). The primary findings from the clinical trial have been reported elsewhere . Written informed consent for phlebotomy, rectal and duodenal biopsy was obtained through study protocols approved by the Institutional Review Board, School of Medicine, University of California, Davis and the UC Davis CTSC Clinical Research Center (CCRC).
Samples were obtained from gastrointestinal mucosa and peripheral blood at baseline or 4–9 months after initiation of HAART. Blood was collected by sterile venipuncture using EDTA as an anticoagulant. Rectal biopsies were obtained via flexible sigmoidoscopy at 10–15 cm from the anal verge as previously described . Duodenal biopsies were obtained via upper endoscopy. Rectal and duodenal biopsies and blood were immediately transported to the laboratory and processed on the day of collection.
Peripheral blood mononuclear cells (PBMCs) were isolated from blood using Ficoll-Paque (Pfizer-Pharmacia, New York, New York, USA). Isolation of mononuclear cells from rectal and duodenal biopsies was performed as previously described . Following isolation, PBMC, duodenal mononuclear cells (DMCs) and rectal mononuclear cells (RMCs) were either stained the same day for cell surface phenotypic analysis or incubated overnight in complete medium [RPMI-1640 supplemented with foetal calf serum (15%), penicillin (100 U/ml), streptomycin (100 μg/ml) and glutamine (2 mmol/l)]. DMCs and RMCs were treated with Piperacillin-Tazobactam (0.5 mg/ml) (Zosyn; Wyeth Pharmaceuticals, Philadelphia, Pennsylvania, USA) to inhibit overgrowth of intestinal bacteria.
For immunophenotypic analysis, freshly isolated PBMCs and RMCs were labelled with fluorochrome-conjugated antibodies. Cells were stained with Aqua Amine Reactive Dye (Invitrogen Molecular Probes, Eugene, Oregon, USA) to discriminate dead cells and the following fluorescently conjugated mAbs: CD3 clone UCHT1 (Pacific Blue), CD8 clone SK1 (QDot 605), CD38 clone HIT2 (phycoerythrin [PE]-Cy5), C-C chemokine receptor type 7 (CCR7) clone 3D12 (PE-Cy7), human leukocyte antigen–DR subregion clone Tu-39 (fluorescein isothiocyanate), C-X-C chemokine receptor type 4 clone 12G5 (allophycocyanin [APC]) and C-C chemokine receptor type 5 clone 2D7 (PE) (BD Biosciences, San Jose, California, USA); CD45RA clone 2H4 (electron coupled dye) (Beckman Coulter, Fullerton, California, USA); CD4 clone S3.5 (APC-Cy5.5), (Invitrogen Caltag, Burlingame, California, USA). In some experiments, CD28 clone CD28.2 (PE-Cy7) (eBiosciences, San Diego, California, USA) was included. In each experiment, fluorescence minus one controls (FMOs) were included to facilitate gating . Stained cells were washed, fixed in 1% formaldehyde and stored at 4°C until data acquisition (within 24 h).
Intracellular cytokine staining (ICS) was performed on PBMC and RMC rested overnight at 37°C and 5% CO2 as described previously . Briefly, cells were stimulated with HIV-1 consensus B Gag overlapping peptides (JPT Peptide Technologies, Berlin, Germany) at 3.5 μg/ml for each peptide and with the costimulatory antibodies CD28 (2.5 μg/ml) and CD49d (5 μg/ml), in the presence of 5 μg/ml brefeldin A (Sigma-Aldrich, St. Louis, Missouri, USA) and 1 μmol/l monensin (GolgiStop; BD Biosciences). PE-Cy5-labelled CD107a antibody clone H4A3 (PE-Cy5) (BD Biosciences) was also added, and the cells were incubated for 5 h at 37° and 5% CO2. A negative control well (cells stimulated with costimulatory antibodies and dimethyl sulfoxide alone) and a positive control well (CEF peptides: immunodominant peptides from cytomegalovirus, Epstein–Barr virus and influenza virus, JPT Peptide Technologies) were run in parallel for each sample.
Following stimulation, cells were stained for surface antigens CD4, CD8 and programmed death receptor-1 (PD-1) clone J105 (PE) (eBiosciences) and for viability (Aqua Amine Reactive Dye, AARD; Invitrogen). Cells were fixed and then permeabilized using FACS Perm 2 (BD Biosciences). For intracellular staining, cells were incubated with antibodies against CD3, IFNγ clone B27 (PE-Cy7), interleukin (IL)-2 clone 5344.111 (APC) and tumour necrosis factor-alpha (TNF-α) clone Mab11 (Alexa 700) (all from BD Biosciences). Cells were then washed and stored at 4°C in 1% formaldehyde until acquisition. Flow cytometry data were acquired on an LSRII (BD Immunocytometry Systems, San Jose, California, USA) and analysed using FlowJo Software, V.8 (TreeStar, Ashland, Oregon, USA). For experiments measuring four functional responses, individual responses were evaluated alone and also processed through Boolean combinations to partition responding cells into one of 16 possible specific response categories. Cytometry data were biexponentially transformed in order to include all events. SPICE software (Mario Roederer, Vaccine Research Center, NIAID/NIH, Bethesda, Maryland, USA) was used to graph response data .
Plasma viral loads
Plasma was separated from EDTA-anticoagulated blood. The quantification of HIV RNA copy number was performed by reverse transcriptase PCR using the Amplicor HIV-1 Monitor Standard and UltraSensitive kits (Roche Diagnostic, Branchburg, New Jersey, USA).
Soluble CD14 assay
Soluble CD14 levels in plasma samples were quantified by ELISA with the Quantikine Human sCD14 Immunoassay (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer's protocol. Samples were assayed in duplicate.
Mean values of CD4+ T-cell count, gut CD4+ T-cell percentages, HIV viral load, T-cell differentiation status, costimulatory molecule expression and immune activation levels were compared before and after HAART initiation. Statistical analyses were performed using paired Student's t-test or Mann–Whitney tests, when appropriate, and Wilcoxon's signed rank test. P values were two-tailed and were considered significant when less than 0.05. GraphPad Prism (GraphPad Software, San Diego, California, USA) and XLStat software (Addinsoft SARL, Paris, France) were used for statistical analyses.
Baseline patient characteristics
The study participants included three women and 11 men, with a median age of 38 years (Table 1). HAART-naive patients had median absolute CD4+ T-cell counts of 328 cells per microlitre and a median viral load of 29 000 RNA copies per millilitre plasma. Peripheral blood and rectal mucosa CD4+ T-cell data from 10 seronegative individuals enrolled in previous studies were used as historical controls along with data from two HIV-negative volunteers enrolled in the present study to provide reference values. Seronegatives included six women and four men with an average age of 41 years; whenever possible, these individuals were recruited from the same risk groups as HIV-positive individuals.
Virus suppression and CD4+ T-cell reconstitution
The initial median plasma virus load was 29 000 RNA copies/ml, with a range of 974–552 000 copies/ml (Table 1). HAART significantly reduced median plasma virus load to 108 copies/ml (Fig. 1a), with no detectable virus in six individuals. Median pre-HAART mucosal CD4+ T-cell percentages, presented here as proportion of CD3+ cells expressing CD4 but not CD8, were 12.3% in rectal mucosa and 5.6% in duodenal mucosa. In blood, rectal and duodenal mucosa, significant increases were observed in total CD4+ T-cell percentages after HAART, although in all three cases, posttherapy levels were still significantly lower than CD4+ T-cell percentages in uninfected controls (Fig. 1b). It is important to note that the percentage of CD4+ T cells in duodenal mucosa was significantly lower than in rectal mucosa; this was true for healthy control individuals as well as for HIV-positive individuals pre and post-HAART.
Using linear regression analysis, we tested for significant correlations between baseline CD4 cell count, baseline viral load and immune reconstitution in blood and gut. There were no significant relationships between either baseline CD4 cell count or viral load and CD4 cell reconstitution in blood, rectal or duodenal mucosa.
Given that the time of evaluation post-HAART varied from 4 to 9 months, we used regression analysis to check for any significant relationships or trends between time of evaluation post-HAART and CD4+ T-cell reconstitution in blood and rectal mucosa. No significant relationships were detected between time of evaluation and any of the following: change in blood CD4+ T-cell count, blood CD4+ T cells as a percentage of CD3+ T cells or rectal mucosa CD4+ T cells as a percentage of CD3+ T cells.
Changes in T-cell memory/effector phenotype after HAART initiation
To examine the effect of HAART initiation on T-cell differentiation profiles, blood and rectal CD4+ and CD8+ T cells were analysed by flow cytometry for expression of maturation markers CCR7 and CD45RA (Fig. 2) . Cells expressing both CCR7 and CD45RA were considered naive, cells positive for CCR7 but negative for CD45RA were considered central memory (TCM), cells expressing neither antigen were designated as effector memory (TEM) and cells expressing CD45RA but not CCR7 were considered terminally differentiated effectors (TEMRA) [23,24]. Changes in these subsets were monitored longitudinally in each patient before and after HAART. Few significant differences in memory/effector phenotype were observed between pre and post-HAART T-cell subsets. After HAART initiation, a significant increase was observed in the blood CD4+ TCM population along with a concomitant decrease in the peripheral CD4+ naive T-cell subset. However, significant early HAART effects were not apparent in other CD4+ or CD8+ T-cell memory subsets in either blood or rectal mucosa.
Expression of T-cell costimulatory markers
Costimulatory molecules provide a vital ‘second signal’ for activation of T cells  when they encounter antigen-presenting cells. CD28 ligation provides an important second signal to naive T cells , and memory CD8+ T cells require CD28 costimulation for maximal responsiveness in vivo. Furthermore, T cells from HIV-infected individuals with impaired CD4+ T-cell recovery during HAART may express decreased levels of CD28 . Changes in expression of CD28 on peripheral and mucosal T cells were monitored before and after HAART initiation (Fig. 3a). No significant changes were observed on CD4+ T cells in either compartment. However, a significant increase in the proportion of CD8+ T cells expressing CD28 was measured after HAART in both blood and rectal mucosa.
PD-1, another member of the CD28 family of receptors, has been implicated in T-cell exhaustion in the context of chronic viral infection  and impaired HIV-specific T-cell responses [30–32], due in part to increased spontaneous apoptosis of HIV-specific T cells . We assessed the expression of PD-1 by HIV-specific CD8+ T cells by determining the median fluorescence intensity (MFI) of PD-1 staining on the population of cells that produced interferon-gamma (IFNγ) in response to HIV Gag peptide stimulation (Fig. 3b). In peripheral blood, PD-1 MFI was found to decrease following HAART initiation in both HIV-specific CD4+ and CD8+ T cells (Fig. 3b). Due to the small number of HIV peptide responsive cells in rectal mucosa, particularly after HAART initiation, this comparison was not feasible on rectal HIV-specific CD8+ T cells.
Decreased immune activation after HAART initiation
Immune activation is an important prognostic indicator of disease progression in HIV infection [33,34]. To address the effects of HAART initiation on immune activation, the expression of human leukocyte antigen–DR subregion and CD38 was measured on CD4+ and CD8+ T cells in blood and rectal mucosa. In addition, soluble CD14 (sCD14), a marker of activation that is released by monocytes upon activation by lipopolysaccharide , was measured in plasma. Immune activation was significantly diminished in both blood and rectal CD8+ T cells following HAART (Fig. 3c). A similar, but not significant decrease was observed in blood and rectal CD4+ T cells. Plasma sCD14 also decreased after initiation of therapy (Fig. 3c).
For the 10 patients biopsied at baseline and 9 months post-HAART, there was no significant correlation between either baseline viral load or CD4 and baseline CD8 T-cell immune activation or change in blood or rectal CD8 T-cell activation following HAART. There was also no relationship between baseline CD4 or plasma viral load and either baseline rectal CD4 T-cell activation or change in rectal CD4 T-cell activation following HAART. However, there was a trend towards an inverse correlation between baseline CD4 cell count and blood CD4 T-cell activation (P = 0.054, R2 = 0.388), and a similar trend between baseline CD4 cell count and the change in blood CD4 T-cell activation following HAART (P = 0.099, R2 = 0.304). There was also a positive correlation between baseline plasma viral load and blood CD4 T-cell activation (P = 0.017, R2 = 0.530), as well as a trend towards a positive correlation between baseline plasma viral load and reduction in blood CD4 T-cell activation (P = 0.115, R2 = 0.281).
HIV-specific CD8+ T-cell responses
Lower viral antigen levels as a result of HAART lead to decreased frequencies of HIV-specific CD8+ T-cell responses in blood [36,37] and rectal mucosa . To examine the effect of HAART initiation on HIV-specific CD8+ T-cell response magnitude and polyfunctionality, blood and rectal mucosal CD8+ T cells were stimulated with HIV Gag peptide and stained with fluorescent mAbs to measure the production of three cytokines and a marker of degranulation. In both compartments, HIV Gag-specific CD8+ T-cell response magnitudes decreased significantly after beginning HAART: the percentage of Gag-responding CD8+ T cells producing either IFNγ or CD107 was markedly reduced (Fig. 4a). In PBMC, the percentage of Gag-specific cells producing TNFα was also reduced. Using Boolean gating and SPICE software, we then determined the magnitude of the total Gag-specific response, including cells positive for any one, two, three or all four factors, counting each cell only once (Fig. 4b). This analysis revealed that the total Gag-specific CD8+ T-cell response declined in both compartments after HAART, reaching significance in PBMC. We also evaluated response complexity by graphing populations positive for each of the 15 possible combinations of factors (Fig. 4c). This analysis demonstrated that HAART initiation did not lead to an increase in response polyfunctionality within the time frame studied. Instead, minor shifts in functionality were observed in both PBMC and rectal mucosa, with monofunctional responses dominating in rectal mucosa at both time points, and in PBMC following HAART.
Effects of simian immunodeficiency virus/HIV infection on gut mucosal CD4+ T cells have been extensively documented [3,4,38–43]. However, the effects of HAART on CD4+ T-cell recovery in the gut have remained controversial, with studies [10,13–16,38,44,45] reaching varied conclusions depending on the site of the gastrointestinal tract studied, time of sampling, duration of HAART, specific HAART regimens and the stage of HIV infection in which HAART was initiated. These variables make comparisons difficult, particularly as most studies have relied on sampling at a single mucosal site, and several have used cross-sectional rather than longitudinal sampling. It is generally accepted that HAART does not lead to full restoration of mucosal CD4+ T cells, particularly when initiated during chronic infection [9,11,13,15,38,45]; however, one study  found that long-term HAART was associated with rectal mucosal CD4+ T-cell populations that approximated levels found in healthy controls. It should also be noted that when flow cytometry is used to gauge changes in T-cell subset percentages, as was done for this study, it is not possible to differentiate recovery of CD4+ T cells from a decline in CD8+ T cells. Nevertheless, recruitment and/or expansion of CD8+ T cells in the gastrointestinal lamina propria occurs during HIV/simian immunodeficiency virus infection, beginning during the acute phase , contributing to the observed decreased percentages of CD4+ T cells. In the parallel clinical trial in which these individuals were also enrolled, analysis of duodenal tissue samples by immunohistochemistry revealed a significant decline in CD8+ T-cell numbers following HAART initiation. These findings, discussed in detail elsewhere, show that much of the apparent CD4+ T-cell reconstitution observed in mucosal tissue during HAART is in fact due to a normalization (i.e. reduction) of CD8+ T-cell density .
In the present study, effects of HAART on CD4+ T-cell recovery in multiple anatomic sites were measured in individuals initiating therapy. T-cell differentiation, expression of costimulatory molecules, markers of immune activation and HIV-specific CD8+ T-cell responses were analysed before and during HAART in blood and rectal mucosa of 14 individuals. Early effects of HAART were apparent, as significant increases in CD4+ T-cell populations were evident in blood, small intestine (duodenum) and large intestine (rectum). In blood CD4+ T cells, a significant increase in TCM, typically cells with a high proliferative capacity, was observed. Significant decreases in immune activation were measured in CD8+ T cells from blood and rectal mucosa; plasma levels of sCD14, a marker of macrophage activation, also declined with HAART initiation. Despite these changes, CD4+ T-cell populations in all three compartments failed to recover to levels seen in uninfected, healthy controls.
Mucosal HIV Gag-specific CD8+ T-cell responses decreased in magnitude after 4–9 months of therapy. This apparent contraction of the HIV-specific memory T-cell pool was expected on the basis of previous reports showing a rapid decline of HIV-specific T-cell responses in blood following initiation of therapy [36,37], and is also consistent with prior cross-sectional studies from our group [18,21]. However, HAART also led to significant increases in CD28 expression on peripheral and mucosal CD8+ T cells and significant decreases in PD-1 on peripheral HIV-specific T cells, suggesting enhancement in the capacity of the remaining HIV-specific CD8+ T cells to survive and respond to antigenic stimulation [30–32,46].
The present study examined immune reconstitution in blood and both small and large intestine in chronically HIV-infected individuals during the first 4–9 months following HAART initiation. By examining the same individuals longitudinally, HAART-specific effects could be determined for CD4+ vs. CD8+ T-cell subset normalization, T-cell costimulatory molecule expression, differentiation status, immune activation and CD8+ T-cell function. Early effects of HAART included significant CD4 recovery in blood, duodenal and rectal mucosa, decreases in immune activation of T cells and monocytes, changes in T-cell differentiation profiles and costimulatory molecule expression, and decreased HIV-specific CD8+ T-cell responses. These results suggest that initiation of HAART during chronic infection provides modest yet measurable immunologic benefit to mucosal tissues, despite incomplete CD4+ T-cell recovery.
Preliminary data from this study were presented at the Keystone Conference on HIV Vaccines, Banff, Alberta, March 2010 (Poster X5-211), and at AIDS Vaccine 2010, Atlanta, Georgia, USA (Poster P10.08).
D.M.A. and B.L.S. designed the study with input from R.B.P. D.M.A., R.B.P., T.Y., D.H.M. and J.C.G. enrolled patients and procured samples. T.L.H. performed laboratory work with assistance from T.H.K. and D.H.M. T.L.H. analysed data with assistance from J.W.C. and B.E.M. T.L.H. and B.L.S. wrote the article. All authors reviewed the manuscript.
The authors thank the study volunteers for their participation, the CARES clinic, Sacramento, California and the Gastroenterology Laboratory, UC Davis Medical Center.
This research was supported by NIH/NIAID (R01-AI057020 to B.L.S.) and the California Universitywide AIDS Research Program (UARP Center Grant CH05-D-606 to R.B.P.). The study was also supported in part by a research grant from the Investigator-Initiated Studies Program of Merck & Co., Inc. to D.M.A. The opinions expressed in this study are those of the authors and do not necessarily represent those of Merck & Co., Inc. The UC Davis CCRC is supported by Grant Number UL1 RR024146 from the National Center for Research Resources (NCRR), NIH. This investigation was conducted in a facility constructed with support from the Research Facilities Improvement Program (grant C06 RR-12088-01) from the National Center for Research Resources, National Institutes of Health. The LSR-II violet laser was upgraded with funding from the James B. Pendleton Charitable Trust.
Conflicts of interest
There are no conflicts of interest.
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gut; HAART; HIV; immune activation; mucosa; T cells
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