The introduction of combination antiretroviral therapy (cART) has improved the prognosis of HIV-infected patients with access to treatment enormously. However, there is evidence of excess comorbidity and mortality among otherwise successfully treated HIV-infected patients due to particularly cardiovascular disease and non–AIDS-defining cancers.1–4 Residual immune activation and dysfunction even after long-term treatment have been suggested as contributory factors of this problem. Several studies have examined the immunological effects of cART, but the long-term effects on immune reconstitution are not clear-cut.
cART effectively induces a decrease in viral load accompanied by an increase in total CD4 count, which is used as a surrogate marker of immune reconstitution. CD4 count in successfully treated patients can continue to increase 8–10 years after treatment initiation.5,6 naive CD4 cells and recent thymic emigrants (RTEs) are depleted in chronic HIV disease and are suggested markers of CD4 recovery because thymic output, which is associated with age and the amount of thymic tissue, correlates with CD4 T cell restoration.7,8
Resting, antigen-experienced T cells are thought to undergo a linear differentiation through several phenotypically distinct subsets: central memory (TCM) and effector memory T cells (TEM), intermediately differentiated T cells (TID), and terminally differentiated T cells (TTD).9 TCM have the capacity to home to secondary lymphoid organs, and TEM display effector functions at the site of inflamed tissue.10 TCM and TEM CD4 cells have been shown to serve as a long-lived reservoir of HIV that prevents eradication of the virus because of the long half-life of these cells.11,12
T cells react to chronic viral infections with distinct phenotypical and functional profiles, that is, HIV-specific T cells primarily display a TID phenotype and play a role in viral control.9,13–16 Progressive disease causes an increase in the magnitude of the HIV-specific CD8 response and a disturbance in the quality of both CD4 and CD8 HIV-specific responses. cART decreases the amount of circulating HIV-specific CD8 cells but whether cART corrects the qualitative displacement remains controversial.13,15,17–19
Immune activation is an important part of the immune response to any acute viral infection, but in HIV infection, immune activation continues and drives disease progression and high levels of cellular immune activation as measured by CD8+CD38+HLA-DR+ T cells that are associated with poorer disease outcome in cART-treated patients.20,21 Although cART effectively reduces, and even in some cases normalizes, these levels,22,23 residual immune activation can persist through years of successful treatment.21,24
The present study investigated T cell subsets in patients with long-term viremic suppression after 12 years of continuous cART to (1) determine the degree of quantitative immune reconstitution and phenotypic T-cell imbalance, particularly excess activation, and (2) examine the relationship between residual viremia and immune activation and reconstitution in patients with long-term viremic suppression.
MATERIALS AND METHODS
The study was conducted at the Department of Infectious Diseases and the Department of Clinical Immunology at Rigshospitalet (Copenhagen, Denmark). The study population comprises a cohort of patients, who were included in between September 1997 and August 1998 on the basis of having reproducible plasma HIV RNA levels <200 copies/mL after starting combination antiretroviral treatment. Several articles based on data from the cohort have previously been published.25–28
One hundred one patients entered the study in 1997 to 1998—at follow up in 2009, 17 of those had died and 13 were lost to follow-up, leaving 71 patients who participated in the present study. Blood samples were obtained in connection with the patients' routine visits to the outpatient clinic, and background data were obtained from the patients' charts and the Danish HIV Cohort.29 All patients gave written informed consent, and the study was approved by the local ethics committee (journal number H-C-2008-077). As treatment interruptions have never been part of the Danish treatment guidelines, the patients received cART continually since the inclusion, although the drug combinations changed over the years because of drug development, side effects, etc. The control group consisted of 16 gender- and age-matched healthy volunteers from the Danish Blood Donor Corps.
T-cell subsets were analyzed in EDTA anticoagulated fresh whole blood by 6-color flowcytometry using a BD FACSCanto II flowcytometer and antibodies stained with fluorescein isothiocyanate (FITC)-CD45, FITC-CD27, FITC CD45RO, FITC-CD31, phycoerythrin (PE)-CD14, PE-CD28, PE-CD38, PE-CD62L, allophycocyanin (APC)-CD45RA, PE-Cy7-CD27, PE-Cy7-HLA-DR, PE-Cy7-CD127, peridinin–chlorophyll–protein complex (PerCP)-CD8, and APC-Cy7-CD4, all from BD Pharmingen (Franklin Lakes, NJ) except PE-Cy7-CD127 from eBioscience (San Diego, CA).
Flowcytometric data were processed with BD FACS Diva software version 5.0.3. The cell definitions used are given in Table 1.9,16,30,31 All analyses were conducted using a lymphocyte gate with <2% CD14+ monocytes, and further distinction of T cells were made by CD4 and CD8 gating. The absolute concentrations of cells were calculated by multiplying the cytometrically derived proportions with the lymphocyte concentrations obtained by standard hematological analysis.
Ultrasensitive HIV RNA Measurements
Quantification of HIV RNA was performed at the AIDS laboratory, Rigshospitalet, on EDTA plasma by an ultrasensitive method based on a modified Amplicor assay (Cobas Amplicor HIV-1 monitor test, version 1.5 ultrasensitive assay; Roche Diagnostics, Branchburg, NJ) to reach a lower level of detection (LLD) of 2.5 copies/mL as used in several other studies.32–35 Plasma was kept at −80°C until analysis. The virus was pelleted from 2 mL of plasma after 2 hours of centrifugation at 23,600 × g at 4°C. Half the normal amount of quantification standard was used, and the RNA pellet was resuspended in 50 μL of diluent. The entire volume of resuspended RNA was assayed by reverse transcriptase polymerase chain reaction according to the manufacturer's instructions. Sixty-four samples were tested with both the above-mentioned method and the local standard TaqMan polymerase chain reaction with LLD = 20 copies/mL and correlated well (Spearman rho = 0.569, P < 0.001).
Statistical analyses were conducted using SPSS 11.5, GraphPad Prism 5.03 and Microsoft Office Excel 2007. For flow cytometric data, means were compared using Mann–Whitney test. Correlation analyses were performed using Spearman rho. All data are presented as medians with interquartile range unless otherwise stated. HIV RNA measurements of <2.5 copies/mL were set as 2.4 copies/mL. P values less than 0.05 were considered significant.
The cohort consisted primarily of white, male HIV-infected patients with a median age of 55 years and a median baseline CD4 count of 0.18 × 109/L. The patients had been diagnosed with HIV for a median of 230 months (range, 143–328 months) and had received cART for a median of 150 months (range, 143–175 months). Nineteen patients had experienced AIDS-defining events, and six patients had chronic hepatitis.
At the date of sampling, 45 patients had HIV RNA <2.5 copies/mL, 17 patients had HIV RNA between 2.5 and 20 copies/mL, 8 patients had HIV RNA between 20 and 128 copies/mL, and 1 patient had 8888 copies/mL. HIV RNA has been measured regularly over the years (between 31 and 68 times) and was below 40 copies/mL, which was the highest applied LLD in the study period, in a median of 86.7% of measurements. Only 7 patients did not experience any blips >40 copies/mL during the entire follow-up.
The control group consisted of 88% male participants with a median age of 56 years. As control subjects were blood donors, they were known to be hepatitis seronegative. The HIV-infected patients had lower concentrations of CD4 T cells and higher concentrations of CD8 T cells than the controls (Figs. 1A, B). When stratified according to baseline CD4 count, the HIV-infected patients showed similar total CD4 gain with ongoing increase even after 12 years of treatment. The flattening slopes of CD4 increase were similar between groups and displayed a good fit to a logarithmic curve (Fig. 1C).
Naive Cells, Naive CD127+ Cells, and RTEs
Two different definitions of naive cells were used to examine both naive cells in the CD27, CD28 maturation pathway, and the expression of CD127 [interleukin (IL)-7 receptor]. HIV-infected patients had lower concentrations of naive CD4 T cells and higher concentrations of naive CD8 T cells than controls (Figs. 2A, D). There were no differences in proportions. Proportions of naive CD4 cells in HIV-infected patients did not correlate with viremia measured by ultrasensitive assay on the same day. Naive CD127+ cells showed a pattern similar to naive cells defined by CD27/CD28 (Figs. 2B, E).
Concentrations of CD4 RTEs did not differ between groups (Fig. 2C), but concentrations of CD8 RTEs were higher in HIV-infected patients than controls (Fig. 2F). Both CD4 and CD8 RTE proportions were similar between groups. Proportions of CD4 RTEs tended toward a negative correlation with age but did not reach significance (Spearman rho = −0.107, P = 0.325) and showed no correlation with viremia on the sample data (data not shown). Patients who had experienced viremic blips above 40 copies/mL during the last 2 years before sampling (n = 39) did not have lower proportions of naive CD4 cells or CD4 RTEs than those with persistent viremic suppression (n = 32) (data not shown).
Central and Effector Memory and Intermediately, Late, and Terminally Differentiated T Cells
In general, only small differences were observed in the CD4 compartment (Figs. 3A–E), and all proportions were similar. Greater differences were seen within the CD8 maturation pathway (Figs. 3F–J), where significant differences were seen for all levels of differentiation, especially within the CD8 TID cells, where not only concentrations but also proportions differed significantly between HIV-infected patients and controls. No differences were found between HIV-infected patients and controls in CD4 TCM and TEM subsets in either concentrations or proportions (Figs. 3A, B). HIV-infected patients had higher concentrations CD8 TCM and TEM than controls (Figs. 3F, G), but there were no differences in proportions.
CD4 TID concentrations were similar between groups whether measured as concentrations or proportions (Fig. 3C), but CD8 TID concentrations (Fig. 3H) were higher compared with controls, and the HIV-infected patients also had higher proportions of CD8 TID cells than controls (P = 0.036), making CD8 TID the only sign of a skewing of the maturation pathway that cannot be explained by higher total CD4 or CD8 counts. CD4 TLD concentrations were lower in HIV-infected patients than controls, but the numbers were exceedingly small and based on few events that might not represent a true difference. CD8 TLD concentrations were higher in HIV-infected patients (Figs. 3D, I). Proportions were similar in both CD4 and CD8 cells. CD4 TTD showed no differences between groups in concentrations (Fig. 3E) or proportions. CD8 TTD concentrations were higher in patients than controls (Fig. 3J), but the proportions were similar.
HIV-infected patients did not show signs of excess activation within the CD4 compartment in either concentrations or proportions (Fig. 4A), but activated CD8 cells were more abundant in HIV-infected patients than controls (Fig. 4B) and constituted a larger percentage of CD8 cells (P = 0.047). CD8 activation did not significantly correlate to viremia measured by ultrasensitive assay on the same day (Fig. 4C), and no correlation was found between viremia and CD8 activation when looking exclusively at the 26 patients with detectable viremia (>2.5 copies/mL) (Spearman rho = 0.306, P = 0.128, data not shown).
The 39 of 71 patients in the study who had experienced one or more viremic blips >40 copies/mL within 2 years before inclusion did not show higher proportions of activated CD8 cells (P = 0.428) than the rest nor did the 7 patients who never experienced any blips during cART treatment differ in activated CD8 proportions from the rest of the study subjects. The patients' percentage of HIV RNA measurements >40 copies/mL during the 12 years of follow-up (frequency of blips) did not correlate to CD8 activation.
The principal findings of the study were as follows: (1) HIV-infected patients differ from controls in having lower absolute and relative CD4 T-cell counts and higher absolute and relative CD8 T-cell counts even after 12 years of successful cART; (2) HIV-infected patients had lower concentrations of naive CD4 cells and naive CD4 cells expressing CD127 than controls but proportions were similar; (3) HIV-infected patients had higher concentrations of CD8+ T cells than controls in all of the examined subsets, but only a higher proportion of CD8+ cells in the TID and activated subset; and (4) residual viremia at the time of sampling or throughout the years did not correlate to the proportions of naive CD4, CD4 RTEs, or activated CD8 T cells.
The study demonstrated that although some successfully treated HIV-infected patients have normalized their levels of total CD4 and CD8 cells, most still have skewed concentrations of both, which impacts on the concentrations of particularly CD8 subsets. This sign of incomplete immune reconstitution has been reported previously in several studies with shorter observation periods.36–38
However, patients with higher baseline CD4 counts reached higher CD4 counts after treatment, and the present study showed evidence of slowing but continuous CD4 gain up to 12 years following a logarithmic curve unaffected by baseline CD4 count. This indicates that even patients with lower baseline might achieve normalization in time, although the parallel slopes imply that the lowest baseline patients will never catch up to the highest baseline patients, unless/until the slopes reach a plateau somewhere beyond 12 years of treatment. Some studies have demonstrated a plateau effect after 3–5 years39,40 but most have shown continuous gain after up to 10 years in line with the present study.5,6,36,41,42
Naive CD4 cells are depleted in untreated HIV infection and to some degree restored by cART. This study showed an incomplete restoration of the naive CD4 cell subset that seems to account for most of the remaining total CD4 deficit. In studies with shorter follow-up periods, both Robbins et al37 and Vrisekoop et al38 found lower concentrations of naive CD4 cells in cART-treated patients with low baseline CD4 count in accordance with our results, and Lederman et al43 demonstrated lower naive concentrations in HIV patients with CD4 counts below 350 per microliter but not in patients with CD4 counts above 500 per microliter.
The IL7/CD127 (IL7 receptor) pathway is critical for the maturation and differentiation of thymocytes, and untreated HIV infection has been shown to be associated with reduced CD127 expression (partly) reversible by cART.44–47 In the present study, concentrations of CD4 naive CD127+ cells were lower in HIV-infected patients than controls, but the proportions did not differ. The results of the naive cells and the naive CD127+ cells were very similar, and the proportions of these subsets correlated well (Spearman rho = 0.991, P < 0.001, data not shown), suggesting that the 2 combinations of surface markers are in fact 2 ways of quantifying the same subset of naive cells.
CD31 has been suggested as a marker of RTEs primarily on CD4 cells,48–50 and as with CD127 expression, untreated HIV infection reduces thymic output, which can be at least partially restored by cART.51,52 Vrisekoop et al38 recently showed normalization of CD31+ naive cells CD4 cells defined as CD45R0−CD27+CD31+ after long-term cART, and the present study did not find signs of downregulation or depletion of cells expressing CD31 in the CD4 compartment.
Thymic function decreases with age, and HIV-infected children undergoing cART show a more efficient immune reconstitution response than adults.51,53,54 However, the thymus has been shown to contribute to the formation of naive cells in HIV-infected adults on cART.7,8 In the present study, the amount of RTEs had returned to normal levels for age, suggesting that the cause of the low naive CD4 levels is not reduced thymic production but may be found elsewhere, that is, in altered peripheral expansion or premature activation.
Based on their functional capabilities and telomere length resting, antigen-experienced cells are thought to undergo a linear differentiation through several phenotypically distinct subsets, and this study examined the whole line of phenotypic differentiation from central memory cells to terminally differentiated cells based on the markers CD45RA, CD27, CD28, and CCR7.9,15 Only few differences in T cell distribution between HIV-infected patients and controls along this maturation line were found, most pronounced in the CD8 compartment where all subsets' concentrations were higher among HIV patients. Only CD8 cells with TID phenotype existed in higher proportions and concentrations.
T cells show different phenotypes according to their viral specificity, and CD8 TID cells are predominant in HIV-specific CD8 cells, whereas other subtypes are more frequent in other latent infections, such as hepatitis C virus or cytomegalovirus.9,10,13,16,55–58 Thus, it is tempting to speculate that the excess of TID CD8 cells could represent accumulation HIV-specific CD8 T cells; however, we found no differences in proportions among the other CD8 subsets indicating a shift toward senescence in the line of differentiation. Other studies have demonstrated disturbances in T cell maturation resulting in skewed representation of particularly CD8 subsets with predominance of preterminally differentiated cells in untreated and to a lesser degree in cART-treated HIV-infected individuals.15,18,59 Skewed maturation of HIV-specific CD4+ T cells toward intermediately and late differentiated subsets has also been described in patients with progressive disease, but cART seems to be able to correct the skewed representation.19
It has been suggested that even cART-treated HIV-infected patients' immune system share some characteristics with that of the elderly (immunosenescence) in terms of reduced T cell renewal, elevated levels of activated T cells, and progressive enrichment of the late and terminally differentiated phenotypes that are CD28− and CD57+ with shortened telomeres and reduced proliferative potential.60–65 These changes are thought to be brought on by viremia, microbial translocation, bystander activation by CMV, and lack of anti-inflammatory mechanisms, such as regulatory T cells. Although the present study confirmed reduced levels of naive CD4 cells, high CD8 TID levels, and excess CD8 activation, thymic output seemed within normal range and the maturation line did not seem severely displaced.
Previous studies that have examined restoration of memory subsets in cART-treated HIV-infected individuals have shorter follow-up, broader definitions of memory cells, or focus solely on CD4 cells. In accordance with our results, Lederman et al43 have described concentrations of TCM and TEM, defined as CD4/CD8+, CD45RA−CCR7+ and CD4/CD8+, CD45RA−CCR7−, respectively, after 2–11 years of cART and found normalization of the CD4 subsets and persistently higher concentrations of the CD8 subsets in patients with total CD4 count above 500 per microliter. Lopez et al59 also found normalization of proportions of 4 CD4 memory subsets defined by CD45RA and CD27 in cART-treated patients compared with controls, but lower proportions of naive CD8 cells and higher proportions of terminally differentiated CD8 cells. Normalization of quantities does not necessarily imply normalization of functional and proliferative capacities and whether cART has potential to correct these qualitative disturbances remains controversial13,17–19,66 and is beyond the scope of this study.
The present study showed persistent CD8 activation after 12 years of continuous cART demonstrated by greater proportions and concentrations of activated T cells but no signs of excess activation within the CD4 subset. Even though CD8 activation persists after long-term cART, it is noteworthy that in some patients proportions do normalize and it is unclear what factors determine this. Several studies have examined activated T cells after cART and the results are contradictory. In all studies, cART reduces immune activation but some studies show persistent residual activation in CD4 and/or CD8 compartments,21,24,37 whereas others demonstrate normalization in one or both compartments.38,43
In this study, no correlation between CD8 activation and low-grade viremia was found, but all patients except one had HIV RNA below 128 copies/mL, and the 2 factors are probably dependent when viral load is substantially higher, that is, over 10,000 copies/mL as shown elsewhere.22,23,59,67 Patients who experienced viremic blips >40 copies/mL within the last 2 years were not prone to higher levels of activated CD8 cells. This is somewhat in contrast to findings in previous articles based on data from the same cohort that found that patients who experienced blips within the first 6 to 24 months of treatment had significantly higher levels of activated CD8 cells.26 This difference suggests that the immune system is less sensitive to viremic blips after several years of immune reconstitution, but undiagnosed viremia between samples and the difference in the assays over the course of time may also be an influencing factor.
The strengths of the study are the long observation period without scheduled treatment interruptions and the possibility to examine several T-cell subsets. The limitations are primarily the cross-sectional design and the lack of qualitative assays that could contribute to describe a potential difference in the cells' functional and proliferative capabilities. Controls were relatively few and only matched on gender and age. Another consideration is that T cell composition and reconstitution in lymphoid tissue (ie, in the gut, where most CD4 T cells reside) may not be reflected in circulating cells.68 It remains to be determined which factors are responsible for the immune imbalance that exists after years of effective viral suppression, and large longitudinal studies are needed to establish whether these discrete changes have any clinical relevance.
In conclusion, the results demonstrate that some degree of T-cell imbalance persists after 12 years of successful cART, namely, in the shape of (1) reduced CD4 counts and elevated CD8 counts, (2) reduced concentrations of naive CD4 cells, and (3) elevated levels of intermediately differentiated and activated CD8 cells. Additionally, the study found no association between low-grade viremia and immune reconstitution and activation.
Author Contributions: Study concept and design: S. R. Ostrowski, T. L. Katzenstein, H. Ullum, J. Gerstoft, F. F. Rönsholt. Conduct of the study: F. F. Rönsholt. Data analysis: F. F. Rönsholt, S. R. Ostrowski, T. L. Katzenstein, J. Gerstoft, H. Ullum. Drafting the manuscript: F. F. Rönsholt. All authors critically reviewed content and approved final version for publication.
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