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T-Cell Dysfunction in HIV-1–Infected Patients With Impaired Recovery of CD4 Cells Despite Suppression of Viral Replication

Erikstrup, Christian MD, PhD*†; Kronborg, Gitte MD, DMSc; Lohse, Nicolai MD, PhD§; Ostrowski, Sisse Rye MD, PhD; Gerstoft, Jan MD, DMSc; Ullum, Henrik MD, PhD

JAIDS Journal of Acquired Immune Deficiency Syndromes: March 1st, 2010 - Volume 53 - Issue 3 - p 303-310
doi: 10.1097/QAI.0b013e3181ca3f7c
Basic Science

Introduction: CD4 T-cell recovery is impeded in some HIV-infected patients despite successful combination antiretroviral therapy (cART) with suppressed HIV RNA. We hypothesized that T-cell dysfunction would be increased in these patients.

Methods: In the Danish HIV Cohort Study, we identified HIV-1-infected patients initiating cART with a CD4 cell count <100 cells per microliter, followed by HIV RNA<50 copies per milliliter for 3 years. Patients with a CD4 count <200 cells per microliter after 3 years were identified as cases; 42 patients with a CD4 count ≥200 cells per microliter were selected as controls. Six-color flow cytometry was performed on whole blood. Cytokine levels in supernatants from whole blood stimulations were assessed.

Results: The case and control groups comprised 18 and 35 patients, respectively. Cases were older than controls (median: 54/46 years). The fraction of CD28+ cells was decreased among cases in the CD4+ and CD8+ T-cell subsets (P = 0.0014/P = 0.0349) and in the corresponding naive subsets (P = 0.0011/P < 0.0001). Cases had higher expression of human leukocyte antigen (HLA)-DR on naive CD4 and CD8 T cells (P = 0.0007/P = 0.0028). The production of interleukin (IL)-10 and IL-2 to phytohemagglutinin was decreased in cases (P < 0.0001/P = 0.019).

Conclusions: Patients with impaired CD4 recovery shared a dysregulated T-cell phenotype with low CD28, high HLA-DR expression, and low IL-2 and IL-10 production.

From the *Department of Clinical Immunology, Aarhus University Hospital, Skejby, Aarhus, Denmark; †Department of Clinical Immunology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; ‡Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark; §Department of Clinical Epidemiology, Aarhus University Hospital and The Danish HIV Cohort Study, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; and ‖Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.

Received for publication April 16, 2009; accepted November 9, 2009.

Supported by grants from Fonden Til Lægevidenskabens Fremme, Direktør Leo Nielsens Fond, and the Danish AIDS Foundation.

Presented in part as an abstract and poster at CROI 2008 (abstract number 452), The 15th Conference on Retroviruses and Opportunistic Infections, February 3-6, 2008, Boston, MA.

The authors have no commercial or other association that might pose a conflict of interest.

Correspondence to: Christian Erikstrup, MD, PhD, Department of Clinical Immunology, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, DK-8200 Aarhus N, Denmark (e-mail:

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Treatment of HIV infection with combination antiretroviral therapy (cART) resulting in suppression of viral replication with low or undetectable circulating levels of HIV RNA is usually followed by a substantial CD4 cell count increase as first described more than a decade ago.1,2 However, in few patients, the CD4 cell count recovery is impaired despite no evidence of viral failure.3 These patients share an increased risk of disease progression.3-5 Impaired CD4 recovery has been linked to high age, low nadir CD4 count, and genetic constitution.6-9

During acute HIV infection, direct killing of cells by HIV prevails, but this cannot account for the loss during chronic infection,10 and viral production (including complete and noncomplete replication cycles) is presumably low in the discordant responding patients with suppressed plasma HIV RNA. The normal immune reconstitution after initiation of cART consists of an initial steep increase in numbers of CD4 T cells primarily due to diminished apoptosis and redistribution of memory CD4 T cells from lymphoid tissues and a subsequent plateau phase with a slower but often persistent increase due to production of new naive CD4 T cells.11,12 The redistribution of memory T cells from their sequestered localizations has been linked to a decrease in immune activation.11 Immune activation and associated dysregulation and apoptosis of T cells has repeatedly been suggested to be a main feature of HIV pathogenesis.13-16 The cause of HIV-related immune activation is incompletely understood.17 However, persistent triggering of the innate and adaptive immune system by the virus, increased production of proinflammatory cytokines, increased proliferation and turnover of T cells seem to play a role.17-19 Also the deterioration of the intestinal mucosa with the depletion of effector cells and subsequent inflow of products from microorganisms may contribute.17 The extent of the depletion of gut-associated lymphoid tissue (GALT) and the disruption of the lymphoid tissue architecture have been proposed to impact the extent of immune reconstitution.20,21

The importance of chronic immune activation in the development of AIDS is indicated in a study with persistent activation of CD4 cells in mice constitutively expressing CD70.22 The activation led to depletion of naive CD4 cells and the mice died of Pneumocystis jiroveci infection. Similarly, extreme acute lymphopenia was described among humans stimulated by anti-CD28.23 Simian immunodeficiency virus (SIV)-infected sooty mangabeys is another example. In contrast to SIV-infected rhesus macaques and HIV-infected humans, the sooty mangabeys preserve their CD4 subset and remain free of AIDS despite heavy viral replication but with low levels of immune activation.24 We hypothesized that T-cell activation and dysfunction would be linked to impaired CD4 cell count recovery during cART.

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In the Danish HIV Cohort Study,25 we identified patients followed at the departments of infectious diseases at Rigshospitalet or Hvidovre Hospital, Copenhagen, Denmark, who initiated cART with a CD4 cell count <100 cells per microliter followed by undetectable HIV RNA (<50 copies/mL) for at least 3 years. Twenty-two patients had a CD4 count of <200 cells per microliter after 3 years of viral suppression and were characterized as cases. Fourty-two patients who had achieved a CD4 count ≥200 cells per microliter after 3 years of viral suppression were randomly selected as controls. Individuals were subsequently included in the study at routine visits: 18 cases and 35 controls.

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Flow Cytometry

Whole blood samples were stained according to the manufacturer's recommendations. The monoclonal antibodies (Mabs) used were conjugated to fluorescein-isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), PE-Cy7, allophycocyanin (APC), or APC-Cy7. Three profiles were run for each sample: (1): CD14 (clone:MøP9), CD16, CD45 (2D1), human leukocyte antigen(HLA)-DR (L243), toll-like receptor (TLR)2, TLR4; (2): CD4 (SK3), CD8 (SK1), CD45RA (HI100), CD62L (SK11), CD28 (C28.2), CCR7 (3D12); (3): CD4, CD8, CD45RA, CD45RO (UCHL-1), HLA-DR, CD38 (HB7). All Mabs were from BD Biosciences (Franklin Lakes, NJ), except for TLR2 and TLR4 Mabs, which were from eBioscience (San Diego, CA). The stained samples were subjected to 6-color flow cytometry (FacsCanto; BD Biosciences). The subsequent computer analyses were performed with BD FacsDiva v4.1.2. IgG1 antibodies conjugated with FITC, PE, PerCP, PE-Cy7, APC, and APC-Cy7 were used as negative controls for marker setting. Samples from 2 patients in the case group and 2 in the control group were lost due to technical issues (FacsCanto was defunct on day of analysis).

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Whole Blood Stimulations

Heparinized whole blood was mixed with RPMI1640 (1:4) and stimulated with endotoxin (lipopolysaccharide, LPS, 1 μg/mL), phytohaemagglutinin (PHA 20 μg/mL), or control medium (unstimulated) and incubated at 37°C, 5% CO2. Supernatants were harvested after 24 hours and kept at −80°C until analysis. The choice of dose for PHA was based on previous experience with 24 hours of stimulation.26 Whole blood stimulation was not performed for one patient (control); insufficient sample material.

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Cytokine Measurements

Plasma TNF-α was measured by the enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). Supernatant cytokine levels of IL-10, TNF-α, IFN-γ, IL-2, IL-5, IL-6, and IL-8 and plasma cytokine levels of IL-10, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 were measured by multiplexed assays (Fluorokine MAP Multiplex kits, Human cytokine panel A, R&D Systems) on the Luminex platform (Luminex Corporation, Austin, TX).

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Patients were included at routine visits, and blood for this study was drawn in conjunction with routine blood samples. The Scientific Ethical Committee of Copenhagen and Frederiksberg Municipalities approved the study (KF 01-175/04); oral and written informed consent was obtained from each participant.

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Parameters were compared between case and control groups by T test or the Mann-Whitney U test. T tests were supplemented with analysis of covariance allowing for age adjustments. Results are reported as means/geometric means with 95% confidence intervals (CIs) or medians with interquartile range.

Correlation between CD4 T-cell subsets was assessed by univariate regression analysis, and results are presented as regression coefficients (RC) with CI and R2.

Parameters were log10 transformed when appropriate to approach normality. Normality of parameters and residuals was checked graphically. A P value of 0.05 was considered significant.

For parameters only detectable in a fraction of samples, parameters were categorized as above or below the limit of detection and groups were compared by logistic regression.

The analyses of lymphocyte and monocyte subsets were only performed with fractions and not with absolute cell counts as the recruitment into the case group was dependent on a low CD4 cell count. For the markers HLA-DR, CD38, TLR2, and TLR4, the expression in the form of mean fluorescence intensity (MFI) on subsets was compared between groups.

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At the time of inclusion, the CD4 cell count was approximately 3-fold lower among cases, whereas cases and controls did not differ in time on cART (Table 1). However, patients in the case group were older than patients in the control group (Table 1) and, thus, all analyses were run unadjusted and adjusted for age. The cohort only included 4 females who were all in the control group. All analyses were additionally performed with male participants only. This did not change conclusions; only results from analyses including females are reported.



Patients were of white ethnicity except for 4 patients who were of African or Asian (Thai) origin (Table 1). The 5 patients who were hepatitis B coinfected were all white males. Similarly, 5 patients were hepatitis C coinfected, 4 white males, and 1 Asian male. None were dually infected with hepatitis B and C. When the hepatitis B and C cases were combined, there was a trend toward a higher prevalence of hepatitis among cases (Fisher exact test: P = 0.07). All subsequent analyses were additionally performed with adjustments for hepatitis B and C infection; hepatitis status did not change conclusions of the age-adjusted analyses.

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CD4+ T Lymphocytes

Results from flow cytometric analyses appear in Table 2. In the CD4-gated lymphocyte subset, the fraction of naive (CD45RA+ and CD62L+) cells was decreased in the case group in the unadjusted analysis (unadjusted/adjusted: P = 0.0057/P = 0.06). CD28 expression was reduced on CD4 T cells (P = 0.0014/P = 0.0071) and, similarly, the fraction of CD28 expressing naive CD4 T cells was lower among cases (P = 0.0011/P = 0.0075; Fig. 1A). In unadjusted, but not in the adjusted analysis, the fraction of memory (CD45RA− CD45RO+) cells in the CD4 T-cell subset was increased in the case group (P = 0.0418/P = 0.21). The expression of the activation marker HLA-DR was increased in the case group on phenotypically naive (CD45RA+ CD45RO−) CD4 T cells (P = 0.0007/P = 0.0345; Fig. 1B). However, we did not detect any difference between groups in the expression of the activation marker CD38 on the naive CD4 T cells. Similar results applied to memory CD4 T cells: hence, HLA-DR expression was increased on memory (CD45RA- CD45RO+) CD4 T cells in the case group (P = 0.0041/P = 0.0345), whereas no difference between groups was detected in CD38 expression. The expression of HLA-DR on CD4 cells correlated negatively with CD28 expression (P = 0.0019, RC: HLA-DR MFI 23 units lower per percentage point increase in CD4 cell CD28 expression, CI: −37 to −8.9, R2: 0.17). Similarly, on the naive CD4 T-cell subset, the expression of HLA-DR correlated negatively with CD28 expression (P = 0.0004, RC: HLA-DR MFI 0.986 fold higher per percentage point increase in naive CD4 cell CD28 expression, CI: 0.978 to 0.993, R2: 0.22). There was no correlation between CD38 and CD28 expression on CD4 cells (P = 0.78, R2 = 0.00). CCR7 expression did not add to the information already obtained from CD45RA and CD62L.





Nadir CD4 cell counts did not predict CD4 counts neither among cases (P = 0.78, RC: nadir CD4 cell count 0.23 cells per microliter higher with CD4 cell count 1 cell per microliter higher, CI: −1.52 to 1.97, R2: 0.00) nor among controls (P = 0.17, RC: 2.54, CI: −1.18 to 6.26, R2: 0.03). Similarly, CD28 expression on CD4 cells or the fraction of naive cells in the CD4+ subset were not predicted by nadir CD4 cell count among cases or controls (data not shown).

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CD8+ T Lymphocytes

The total number of CD8 T cells was lower among cases (P = 0.006/P = 0.02; Table 2). The fraction of naive cells in the CD8 T-cell subset was lower among cases (P = 0.007/P = 0.049). The expression of CD28 on CD8-gated T cells was only lower among cases in unadjusted analysis (P = 0.0349/P = 0.09). However, CD28 was diminished on naive CD8 cells among cases (P < 0.0001/P = 0.0004, Fig. 1A). The expression of HLA-DR on naive CD8 T cells was increased in the case group (P = 0.0028/P = 0.0248; Fig. 1B) with no differences in CD38 expression between groups. No difference in HLA-DR or CD38 expression on memory T cells between groups was found. As for CD4 T cells, the expression of HLA-DR correlated negatively with CD28 expression on CD8 cells (P = 0.042, RC: HLA-DR MFI 32 units lower per percentage point increase in CD8 cell CD28 expression, CI: −62 to −1.8, R2: 0.06) and on naive CD8 T cells (P = 0.014, HLA-DR MFI 28 units lower per percentage point increase in CD4 cell CD28 expression, CI: −50 to −5.8, R2: 0.10).

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The total numbers of monocytes did not differ between groups and neither did the fraction of CD14+ CD16+ positive cells in the monocyte subset differ between groups (Table 2). No difference between TLR2 or TLR4 expression on monocytes or on the CD14+ CD16+ monocyte subset was found between groups.

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Cytokine Production From LPS and PHA Stimulated Whole Blood

The production of IL-10 to PHA was markedly attenuated in cases in univariate and age-adjusted analysis (P < 0.0001/P < 0.0001, Table 3). The production of IL-10 to endotoxin was also decreased in the case group (P = 0.0192/P = 0.0439). The production of IL-2 to PHA was also markedly lower in the case group (P = 0.0019/P < 0.0001). Adjusting for the leukocyte subsets best correlating with the respective cytokines did not change conclusions.



To test if the balance between proinflammatory and anti-inflammatory potential differed between patients with impaired CD4 cell recovery and controls, we compared the relationship between IL-10 production with PHA stimulation, primarily from lymphocytes, and TNF-α production to endotoxin, primarily from monocytes (Fig. 2). In regression analysis, we found that there was an interaction between group and TNF-α production (P = 0.013). Thus, the slope of the IL-10/TNF-α association differs between groups. Similar findings applied when TNF-α to endotoxin was substituted with TNF-α to PHA or plasma TNF-α.



For all cytokines, the production from unstimulated whole blood was undetectable for several patients and cytokine production did not differ between the case and control groups (data not shown).

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Plasma Cytokine Levels

For the cytokines, TNF-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1, the plasma levels were compared between groups by T tests and by analysis of covariance adjusted for age (Table 3). There were no differences between groups. IL-10 and IL-1β were only detectable in a fraction of samples (47% and 81%, respectively) and, hence, these parameters were dichotomized and compared between groups by logistic regression. IL-10 detection was higher among cases in unadjusted but not in the adjusted analysis (unadjusted: P < 0.046, odds ratio for detectable IL-10, case vs. control group: 3.4; CI: 1.0 to 11; adjusted for age: P = 0.06, odds ratio: 3.6, CI: 0.96 to 13). Plasma TNF-α levels or the interaction between group and plasma TNF-α did not affect plasma IL-10 levels in logistic regression analysis (P = 0.96). IL-1β did not differ between groups (unadjusted: P = 0.11, odds ratio for detectable IL-1β, case vs. control group: 5.9, CI: 0.68 to 51; adjusted: P = 0.10, odds ratio: 6.8, CI: 0.72 to 65).

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T-cell dysfunction with decreased CD28 expression, increased HLA-DR expression, and low IL-10 and IL-2 production were key findings among patients with impaired CD4 recovery in this study.

We found that CD28 expression on peripheral CD4 cells and CD28 expression on naive CD4 and CD8 T cells was lower among cases. In several studies, CD28 was reported to be decreased on T cells from HIV-infected individuals compared with uninfected individuals.27-31 We previously reported that low expression of CD28 on CD4 T cells was linked to immune activation and poor prognosis among untreated HIV-infected patients.32 CD8-positive CD28-negative T cells promoted dendritic cell activation and, hence, may contribute to immune activation.33 Terminally-differentiated T-effector memory cells (TEM) do not express CD28, whereas T central memory cells (TCM) and naive cells are CD28+.34 Thus, the low CD28 T-cell expression among cases may be attributed to high numbers of short-lived terminally-differentiated CD4+ TEM migrating to and partially repopulating depleted lymphoid tissues.17 A notion that may also explain the reduced IL-2 production in cases because IL-2 is primarily produced by CD4+ TCM35 and naive CD4+ T cells.15

An impaired recovery of CD4 T cells was associated with increased immune activation and reduced numbers of regulatory T cells.36 Furthermore, CD4 and CD8 T-cell activation levels (HLA-DR+/CD38+) before cART initiation were negatively correlated with subsequent CD4 cell recovery.37 We found higher levels of HLA-DR on CD4 and CD8 T cells and as expected CD28 expression correlated negatively with HLA-DR expression. Surprisingly, the CD38 expression on CD4+ or CD8+ cells did not differ between groups. Moreover, the total number of CD8 cells was lower among cases. However, taken together, our results show that peripheral CD4 and CD8 T cells from the individuals in the case group share a more activated phenotype. This finding is corroborated by the reduced production of IL-10 among cases. IL-10 is an important anti-inflammatory cytokine that may down regulate immune activation during HIV infection. Thus, we reported that mortality was reduced among treatment-naive HIV-infected individuals carrying a genetic polymorphism associated with high production of IL-10, that is, the G-allele in position -1082 in the promoter to the IL-10 gene.38

In apparent contradiction, cases tended to have higher plasma levels of IL-10 than normal responders. This is, however, in accordance with higher plasma IL-10 levels and a positive correlation to viral load among HIV-infected individuals.38,39 However, as an even stronger correlation between soluble TNF receptor II and HIV RNA was observed, we proposed that plasma IL-10 levels are only increased by HIV because of ongoing immune activation with high levels of circulating TNF-α and subsequent up regulation of IL-10.38

Attention has recently been drawn to the profound depletion of CD4 T cells in GALT described in SIV-infected rhesus macaques40 and human HIV-infected patients.41,42 The GALT depletion has been linked to endotoxin translocation, and it was proposed that endotoxin translocation could be a mediator of immune activation during HIV infection.43 In a recent study, IL-10 in the gut was reported to be crucial to avoid IFN-γ driven inflammation due to endotoxin translocation. Without IL-10, the homeostasis of the gut mucosa was disturbed.44 Interestingly, endotoxin tended to be increased in patients with impaired CD4 recovery.45 Hence, diminished IL-10 production could be associated with endotoxin translocation.

In addition to lower IL-10 production in cases, we found an interesting difference in the relationship between IL-10 production to PHA and TNF-α production to endotoxin. Whereas the full responding patients displayed a positive correlation between TNF-α and IL-10 potential, this was not found among cases. Taken together, our findings suggest that lack of IL-10 enhances immune activation and thereby may impair CD4 recovery.

As for IL-10, the IL-2 production from PHA-stimulated whole blood was decreased among cases. IL-2 is an important growth factor for T cells,46 and HIV-specific CD4 cells producing IL-2 are decreased in HIV progressors but increase with treatment.47 We previously reported that impaired production of cytokines, among them IL-2 and IL-10, was an independent predictor of death among HIV-infected individuals.26 Moreover, studies indicate that IL-2 adjuvance could improve immunological recovery during cART.48 Hence, low production of IL-2 among cases may contribute to the impaired CD4 recovery. Alternatively, it reflects that cases have higher numbers of terminally differentiated CD4+ TEM but fewer TCM and naive cells.

In this study, cases were older than patients with a normal CD4 recovery. Age has previously been associated with an impaired immunological recovery after initiation of cART.49 This is a limitation of the study, however, all group comparisons were supplemented with age adjusted analyses. In these analyses, only CD38 expression on naive CD4 T cells correlated (positively) with age (data not shown). The cases in this study had undetectable HIV RNA and survived for more than 3 years despite a low CD4 cell count. Thus, it should be noted that cases in this study represent a selected subgroup of patients with low CD4 counts. It should also be noted that the CD4 cell count of case patients had not reached a plateau phase; conversely, they did experience immune reconstitution although at a slow rate. According to the case definition, the CD4 cell count after 3 years of full viral suppression was below 200 cells per microliter, and the slightly higher mean CD4 count was due to a subsequent increase from identification until recruitment at routine visits.

Hepatitis B and C infections combined tended to be over-represented among cases. Among a subgroup of cases coinfections may thus be part of the explanation for the exaggerated immune activation. Thus, analyses were performed with adjustments for hepatitis B and C infection (data not shown); this did not change conclusions. It should also be noted that we did not check if earlier treatment with zidovudine was overrepresented among cases.

According to earlier studies and our findings, old age, low nadir CD4 cell counts, high levels of proviral DNA, high levels of immune activation, and possibly coinfections are associated with the risk of an impaired immunological recovery.50 Even though we do not know the cause of these associations, clinicians may still use these as risk factors to identify patients who require closer monitoring. Our findings of lower production of IL-2 and IL-10 point to the existence of an imbalance in the cytokine environment. IL-2 and also IL-7 have been proposed as adjuvances to support the immune reconstitution during cART, but their place in the routine treatment of patients with impaired CD4 cell recovery has not been determined.48,50,51

We did not adjust for multiple comparisons in this study. However, as all findings pointed in the same direction, that is, cases displayed increased immune activation, we considered adjustment for multiple comparisons not to be appropriate.

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Patients with an impaired recovery of CD4 cells shared a dysregulated phenotype including low levels of CD28 expression on CD4 and CD8 T cells, increased expression of the activation marker HLA-DR on T cells, and decreased production of the anti-inflammatory IL-10 and the T-cell growth factor IL-2. Persistent immune activation with an unopposed proinflammatory immune response despite the low rate of viral replication may be the reason for the impaired CD4 recovery among cases in this study.

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We thank Dorthe Petersen from the Department of Infectious Diseases, Hvidovre Hospital, Bente Baadegaard and Lene Pors Jensen from the Department of Infectious Diseases, Rigshospitalet, for their excellent work with logistics and provision of information to the patients. Marie Noan Bruun is thanked for outstanding laboratory assistance.

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HIV; immune reconstitution; CD28; HLA-DR; interleukin-10; interleukin-2

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