Lack of disease progression in a minority of HIV-infected individuals remains a mystery. Nonprogressors maintain normal CD4 T cell counts without combination antiretroviral therapy (cART) and can be divided into 2 groups with and without viral replication. Long-term nonprogressors (LTNPs) do not progress for years despite ongoing viral replication. In contrast, elite and viremic controllers (VCs) limit viral replication to undetectable or low levels [elite controllers (ECs)/VCs, <50/2000 copies per milliliter, respectively] and also maintain normal CD4 T cell counts.1 Both LTNP and controllers have proven to be infected with replication-competent virus,2 suggesting the host immune system to be involved in nonprogression. Because viral load is an important predictor for the progression of HIV infection, it is likely that different mechanisms may be responsible for maintaining CD4 T cell counts in LTNPs and controllers. Thus, although the lack of progression in controllers may be partly explained by control of viral replication, there is no good explanation for nonprogression in LTNPs, and the ability to maintain normal CD4 T cell counts despite ongoing viral replication is of particular interest.
The discovery of T cell subsets with immunoregulatory properties, including Regulatory T cells (Tregs), Th17 cells, and Tc17 cells, has provided new insight into the regulation of inflammation and immune activation. Tregs are antiinflammatory T cells that are able to suppress immune activation and inflammation.3,4 Tregs may downregulate chronic immune activation in HIV infection making Tregs a key element in understanding the interaction between the host immune system and HIV.5 However, understanding the role of Tregs has proven to be complex as recent studies have suggested Tregs to consist of 3 phenotypically and functionally distinct subpopulations according to their expression of Foxp3 and CD45RA.6,7 Further, the redistribution of Tregs between blood and lymphoid tissue during HIV infection has been suggested, possibly complicating the interpretation of Treg findings in blood (Gaardbo JC, Hartling HJ, Ronit A, et al., unpublished data, 2013).8–10 Importantly, Tregs are related to proinflammatory CD4+Th17 cells as they share a reciprocal maturation pathway and function together in opposing ways to control the inflammatory response upon infection. Although Tregs inhibit autoimmunity, Th17 cells play a role in autoimmune responses.11 The number and function of Th17 cells have been suggested to be altered in HIV infection.12–15 Finally, Tc17 cells, that is, the CD8 T cell pendent to Th17 cells are described as having similar properties, and the depletion of Tc17 cells in HIV infection was recently shown.16 Thus, profound alterations in the T cell subsets that normally regulate the inflammatory responses have been found in HIV infection.
This study aimed to evaluate the immunoregulatory cells in nonprogressing HIV infection. Tregs, Treg subpopulations, CD161+Th17 cells, and CD3+CD8+CD161highTc17 cells were measured in the peripheral blood of HIV-infected LTNPs, controllers, progressors, and HIV-negative individuals. To assess the redistribution of Tregs, Foxp3+ cells were determined in lymphoid tissue.
A total of 64 HIV-infected patients and 34 healthy controls were included. All the patients had CD4 T cell counts within the normal range (reference interval, 390–1200 cells per microliter in our clinic). The patients were divided into 3 groups: (1) 14 LTNPs with HIV infection for a minimum of 10 years, HIV RNA >5000 copies per milliliter and no decay in CD4 T cell counts for a minimum of 2 consecutive years before inclusion, (2) 25 controllers including 5 ECs and 20 VCs with HIV infection for a minimum of 2 years and HIV RNA always <50 per 2000 copies per milliliter, respectively, and (3) 25 progressors with a minimum rate of CD4 T cell decay of 50 cells per microliter per year for a minimum of 2 consecutive years before inclusion, and HIV RNA >5000 copies per milliliter. For comparison, 34 HIV-negative healthy controls matched for age, gender, and ethnicity were included (Table 1). Study participants also took part in 2 other studies.16,17 Exclusion criteria were coinfection with hepatitis B or C virus, acute or chronic ongoing infections other than HIV, malignancy, immunosuppressive treatment, and pregnancy. All the patients were enrolled from the Department of Infectious Diseases, Rigshospitalet or Hvidovre Hospital, University of Copenhagen, Denmark. Healthy controls were recruited among hospital staff. Informed consent was obtained from all the participants after providing written and oral information. The study was performed in accordance with the ethical guidelines of the 1975 Declaration of Helsinki and approved by the Local Ethical Committee (H-2-2009-089) and the Danish Data Protection Agency.
One year after inclusion, a follow-up registration was performed to clarify if the study participants still fulfilled inclusion criteria: All the ECs still fulfilled inclusion criteria 1 year after inclusion in the study, and none of them had initiated cART. In the group of VCs, 2/20 (10%) had progressed and had initiated cART according to European AIDS Clinical Society guidelines prescribing treatment if CD4 T cell count <350 cells per microliter. The median CD4 T cell count was unchanged (640 vs. 650 cells per microliter, P = 0.346). None of the VCs displayed HIV RNA >2000 copies per milliliter. In the LTNP group, 8/14 (57%) had initiated cART (5 due to patient request, 2 due to planned pregnancy, and 1 due to unspecific complaints). None of the LTNP initiated cART due to CD4 T cell count <350 cells per microliter. The median CD4 T cell count was unchanged (530 vs. 550 cells per microliter, P = 0.496). In the progressor group, 10/25 (40%) participants initiated cART during follow-up (8 due to patient request, 8 according to European AIDS Clinical Society guidelines with CD4 T cell count <350 cells per microliter).
Ethylenediamine tetraacetic acid–stabilized blood was used to obtain a full blood count and for flow cytometry. Plasma HIV RNA was measured with a polymerase chain reaction quantitative kit (COBAS AmpliPrep/COBAS TaqMan 48 System; Roche, Basel, Switzerland) according to the manufacturer's instructions. The detection threshold for HIV RNA was 20 copies per milliliter. Heparinized blood was used to obtain peripheral blood mononuclear cells (PBMCs). PBMCs were isolated by means of density gradient centrifugation (Histopaque; Sigma, St Louis, MO), 400 g for 25 minutes. Freshly isolated PBMCs were used for the analysis of Tregs and Treg subpopulations by means of flow cytometry.
In all the participants, CD161+Th17 cells (CD4+CD161+) and CD3+CD8+CD161highTc17 cells were determined. The gating strategy is shown in Figure 1A, B. In brief, 100 μL of ethylenediaminetetraacetic acid blood was incubated with fluorescent dye–conjugated monoclonal antibodies at room temperature for 20 minutes according to the manufacturer's instructions. Erythrocytes were lysed with 2 mL of Lysing Solution [Becton Dickinson (BD), Franklin Lakes, NJ] at room temperature for 20 minutes, and samples were washed and resuspended in Facs flow (BD). Freshly isolated PBMCs were used for the determination of Tregs (CD4+CD25+CD127lowFoxp3+), resting Tregs (CD4+CD25+CD127lowFoxp3lowCD45RA+), activated Tregs (CD4 +CD25+CD127lowFoxp3highCD45RA−), and nonsuppressive Tregs (CD4+CD25+CD127lowFoxp3lowCD45RA−). The gating strategy is shown in Figure 1C. PBMCs were incubated with relevant surface marker antibodies for 20 minutes followed by fixation and permeabilization (Human Foxp3 Buffer Set; BD), and incubation with antibodies against intracellular Foxp3 for 30 minutes. Monoclonal antibodies used to determine lymphocyte subsets were isotype control immunoglobulin G (IgG)1/IgG2a Phycoerythrin (PE), IgG1 peridinin chlorophyll proteins–cyanine (PerCP–Cy5.5), IgG1/IgM fluorescein isothiocyanate (FITC), IgG1/IgG2b allophycocyanin (APC), IgG1 PE–Cy7, IgG1 APC-H7, CD161–PE, Foxp3–PE, CD8–PerCP–Cy5.5, CD25–PerCP–Cy5.5, CD3–FITC, CD127–FITC, CD45RA–APC, and CD4–APC-H7, all purchased from BD. Six-color acquisition was performed using an FACSCanto, and data were processed using FACSDiva software (BD). For each sample, a minimum of 50,000 cells were acquired.
Production of Cytokines
To determine the production of cytokines, 0.4 mL of peripheral blood was cultured in 1.6 mL of RPMI 1640 (Sigma) and phytohemagglutinin (PHA; 1 μg/μL). Cultures were incubated at 37°C for 24 hours followed by the harvest of supernatants. The supernatants were stored at −80°C until use. Cultures were performed in 66 participants (controls = 17, ECs = 5, VCs = 20, LTNPs = 5, and progressors = 19). Interleukin-10 (IL-10), transforming growth factor beta (TGF-β), and IL-17 were measured in the supernatants by the Flourokine Human MulitAnalyte Profiling Base Kit assay (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions and analyzed on the Luminex 100platform (Luminex Corp, Austin, TX). The samples were measured in duplicates.
Tonsil biopsies were performed in those participants with preserved tonsils willing to donate a biopsy (n = 32). Clinical characteristics of the patients with a tonsil biopsy are presented in Table 1. After applying local anesthesia, a biopsy was obtained using Weils forceps and knife from the easiest accessible tonsil. Hemostasis was secured by compression and ice application. All the biopsies were performed by the same experienced otorhinolaryngologist (K.S.). Biopsies were fixed in neutral buffered formalin (4%) overnight, followed by paraffin embedding. Serial 2-μm sections were cut and processed for hematoxylin eosin staining and immunolabeling. Affinity-purified monoclonal mouse antihuman Foxp3 236 A/E7 was purchased from eBioscience (14-4777; San Diego, CA). Heat-induced epitope retrieval was performed by Dako PT-link (Glostrup, Denmark). Thereafter, the sections were incubated for 20 minutes with a dilution 1:40 of Foxp3 at room temperature, followed by staining using Dako EnVision Flex High pH Link detection system (EnVision FLEX + Mouse, EnVision FLEX HRP, EnVision FLEX DAB + Chromogen, EnVision Substrate Buffer) for Dako Autostainer Link Instruments. The sections were counterstained using EnVision Flex Hematoxylin and mounted by Pertex mounting media (Leica Microsystems, Wetzlar, Germany).
Scoring of Tregs was performed by counting the number of Foxp3+ cells in 3 interfollicular hotspot areas at ×40 magnification and calculating the average (Fig. 2A). Areas of high Foxp3+Treg density were selected as hotspots. In the mucosal epithelium and in the germinal centers, only occasional Foxp3+ cells were encountered (data not shown). Immunolabeling was always nuclear and strong in intensity.18 The pathologist was blinded to clinical characteristics.
Differences between groups were analyzed using the Kruskal–Wallis test followed by the Mann–Whitney U test to compare ranks between groups. Values for IL-10 and TGF-β are presented with and without adjustment for the absolute numbers of Tregs. Associations were examined by Spearman rank order correlations. Two-tailed P values <0.05 were considered significant. Results are given as median and interquartile ranges. Data were analyzed using Graphpad Prism 5.0 (GraphPad Software, La Jolla, CA).
Unaltered Numbers of Foxp3+ Cells in Tonsil Tissue in Nonprogressors and Progressors
The numbers of Foxp3+ cells in lymphoid tissue in tonsils in controls, VCs, LTNPs, and progressors were 133 cells per interfollicular hotspot (104–208), 135 cells per interfollicular hotspot (4–171), 108 cells per interfollicular hotspot (48–137), and 120 cells per interfollicular hotspot (107–152), respectively. There was no overall difference between groups (P = 0.704; Fig. 2B).
Elevated Activated Tregs in LTNPs and ECs
In peripheral blood, percentages of Tregs in CD4 T cells in controls, ECs, VCs, LTNPs, and progressors were 5.0% (3.9–6.5), 5.2% (4.1–6.3), 4.7% (3.4–5.9), 5.5% (4.9–8.0), and 5.4% (3.7–6.9), respectively. There was no overall difference between groups (P = 0.3057; Fig. 3A).
Activated Tregs in controls, ECs, VCs, LTNPs, progressors, and controls were 29.0% (21.3–35.6), 43.6% (37.7–53.5), 30.1% (23.3–39.4), 41.5% (24.3–50.5), and 24.6% (18.2–35.6) of Tregs, respectively. Activated Tregs in LTNPs and ECs were elevated compared with that in controls (P = 0.001 and P = 0.003) and with that in progressors (P = 0.005 and P = 0.005). Activated Tregs in ECs were elevated compared with that in VCs (P = 0.032), and that in VCs were elevated compared with that in progressors (P = 0.023; Fig. 3B).
Resting Tregs were lower in LTNPs and ECs compared with that in controls (P = 0.001 and P = 0.024), progressors (P = 0.004 and P = 0.024), and VCs (P = 0.047 and P = 0.072; Fig. 3C), whereas no overall difference was found between groups in nonsuppressive Tregs (P = 0.419; Fig. 3D).
Elevated CD161+Th17 Cells in ECs
Percentages of CD161+Th17 cells of CD4 T cells in controls, ECs, VCs, LTNPs, and progressors were 25.3% (18.2–30.7), 34.4% (26.1–37.8), 24.9% (21.3–33.8), 19.1% (16.4–29.5), and 22.4% (17.8–26.5), respectively. CD161+Th17 cells in ECs were elevated compared with that in LTNPs, progressors, and controls (P = 0.037, 0.019, and 0.068, respectively). In VCs, LTNPs, and progressors, the percentages of CD161+Th17 cells were comparable with that in controls (all P values >0.05; Fig. 4A).
CD3+CD8+CD161highTc17 Cells Are Preserved in ECs but Not in Other Groups of HIV-Infected Patients
CD3+CD8+CD161highTc17 cells in controls, ECs, VCs, LTNPs, progressors were 3.3% (2.1–6.9), 4.9% (1.0–6.8), 1.0% (0.4–1.7), 0.4% (0.1–0.9), and 0.6% (0.3–1.7), respectively. In ECs, CD3+CD8+CD161highTc17 cells were comparable with that in controls (P > 0.05) and elevated compared with that in VCs, LTNPs, and progressors (P = 0.041, 0.044, and 0.037, respectively). CD3+CD8+CD161highTc17 cells in LTNPs and VCs were lower compared with controls (P values <0.001; Fig. 4B).
Impaired IL-10 Production in All HIV-Infected Patients Except for ECs
IL-10 production in controls, ECs, VCs, LTNPs, and progressors was 541 pg/mL (420–670), 491 pg/mL (310–909), 269 pg/mL (162–386), 154 pg/mL (133–233), and 216 pg/mL (139–280), respectively. IL-10 production in ECs was similar to that in controls (P = 0.8930), whereas the production of IL-10 in VCs, LTNPs, and progressors was lower compared with that in controls (P values <0.001). Also, the production of IL-10 in ECs was elevated compared with that in VCs, LTNPs, and progressors (P = 0.083, 0.052, and 0.024, respectively, Fig. 4C).
When adjusted for the absolute numbers of Tregs in the culture, IL-10 production in controls, ECs, VCs, LTNPs, and progressors was 15.1 pg per Treg (8.3–19.7), 15.6 pg per Treg (7.5–17.8), 9.5 pg per Treg (6.3–11.0), 5.8 pg per Treg (3.0–8.9), and 5.8 pg per Treg (4.3–15.1), respectively. In the adjusted values for IL-10 production, ECs were similar to controls (P > 0.05), whereas VCs, LTNPs, and progressors were lower than controls (P = 0.047, 0.007, and 0.017, respectively).
TGF-β production in controls, ECs, VCs, LTNPs, and progressors were 4786 pg/mL (4304–5842), 4902 pg/mL (4068–5899), 4290 pg/mL (2746–6435), 5173 pg/mL (3207–6960), and 4326 pg/mL (3026–5989), respectively. No overall difference was found (P = 0.8046). Likewise, no overall difference was found when adjusting for the absolute numbers of Tregs (P = 0.673, Fig. 4D).
No overall difference was found in IL-17 production in controls, ECs, VCs, LTNPs, and progressors (P = 0.811, Fig. 4E).
This study was designed to study the immunoregulatory T cells in nonprogressing HIV-infected individuals. Although the number of total Tregs was identical in all the groups of HIV-infected patients in both blood and lymphoid tissue, LTNPs and ECs presented with an elevated percentage of activated Tregs and lower percentage of resting Tregs in the blood. Also, ECs displayed elevated CD161+Th17 cells. Interestingly, ECs had preserved CD3+CD8+CD161highTc17 cells, whereas CD3+CD8+CD161highTc17 cells were depleted in all other HIV-infected patients. Finally, the IL-10 response upon stimulation with PHA was preserved in ECs only. Altogether, this suggests immunoregulatory mechanisms to be involved in the nonprogression of HIV infection.
LTNPs and controllers are rare populations of HIV-infected patients characterized by nonprogression with and without viral replication, respectively. The definition of the populations suffers from a lack of consensus making it difficult to compare results across studies. In particular, some define LTNPs without including viral load. Thus, some LTNPs may fulfill controller criteria with low or undetectable viral replication.19–22 Further, the duration of infection is of importance as even 7 years of HIV infection with stable CD4 T cell counts does not distinguish properly between LTNPs and normal progressors.23 Finally, all controllers do not have delayed progression, and some even progress normally to AIDS by clinical events or CD4 T cell count.23 In this study, LTNPs were defined as HIV-infected individuals with stable CD4 T cell counts within the reference interval of >10 years without cART and viremia >5000 copies per milliliter. ECs and VCs were defined as HIV-infected individuals with stable CD4 T cell counts within the reference interval for a minimum of 2 years and viremia never >50/2000 copies per milliliter, respectively, whereas progressors were defined by declining CD4 T cell counts to avoid the inclusion of possible LTNP. The majority of the patients still fulfilled inclusion criteria at 1-year follow-up.
The impact of Tregs in HIV infection is controversial, and conflicting results are reported. There seems to be consensus that an overall depletion of the total number of Tregs during HIV infection takes place. However, Tregs seem to be spared compared with other CD4 T cells resulting in a relative increase in treated and untreated patients.15,24–27 To date, it is unknown whether Tregs are beneficial or harmful in HIV infection. The impact of HIV-specific CD8 + cells on disease progression is well accepted,1 and only recently, the small subset of HIV-specific Tregs was identified.28 If high-frequency HIV-specific Tregs have a beneficial impact on disease progression while total Tregs do not, it may partly explain why the total Treg count cannot predict disease progression. Tregs in LTNPs is not previously described, whereas controllers are reported to have lower percentages than that of progressors, although conflicting results exist.29–35 In this study, no difference in total Tregs was found comparing HIV-infected individuals and healthy controls. The similar percentages may be explained by the fact that all the patients had normal CD4 T cell counts as evidence suggests increased Treg percentages to be preferentially found in patients with low CD4 T cell counts (Gaardbo JC, Hartling HJ, Ronit A, et al., unpublished data, 2013).36,37 In contrast, a great variation was found in Treg subpopulations in different groups of HIV-infected patients. Thus, both LTNPs and ECs displayed high percentages of activated Tregs and low percentages of resting Tregs, whereas VCs were closer to and progressors similar to healthy controls. These findings suggest the overall Treg percentage to be of minor importance, whereas a high percentage of activated Tregs may be involved in nonprogression. We therefore hypothesize that the ability to convert resting Tregs to activated Tregs is beneficial in the setting of untreated HIV infection. This is supported by a previous study reporting the suppressive activity of Tregs in ECs to be preserved and disrupted in progressors,33 and a recent study showing reduced percentages of activated Tregs in HIV-infected progressors.38
HIV binds to resting CD4 T cells and upregulates L-selectin causing homing from blood to lymphoid tissue at enhanced rates.39–41 Also, the redistribution of Tregs between blood and lymphatic tissue in untreated HIV-infected patients has been described,8,9 indicating that conclusions drawn from the studies of Tregs in peripheral blood should be interpreted with caution. In this study, the difference in the number of Foxp3+ cells in tonsil tissue was not found between patients and controls, suggesting a preferential redistribution of Tregs not to be of importance for disease progression. This is in accordance with a recent study showing similar Foxp3+ cells in lamina propria in colon biopsies.10 Further, a small study of 5 nonprogressors showed the depletion of Foxp3+ cells in tonsil tissue,42 whereas another study found increased frequencies in the gut from progressors.8 However, a limitation in this study is the use of Foxp3 as the sole marker of Tregs in lymphoid tissue. Activated T cells may express Foxp3, possibly imposing a bias in the setting of untreated HIV infection with a high level of immune activation. Thus, we cannot rule out that some Foxp3+ cells in the tonsils are activated T cells and not Tregs. However, when examining peripheral blood using Foxp3 alone to define Tregs compared with CD4+CD25+CD127lowFoxp3+, the frequency of Tregs was overestimated by approximately one-third in both healthy controls and in HIV-infected patients (see Table S1, Supplemental Digital Content,https://links.lww.com/QAI/A453), suggesting the potential bias to be of limited impact. Altogether, the small sample sizes, the use of Foxp3 alone and lack of biopsies from ECs complicate firm conclusions concerning the impact of Tregs in lymphoid tissue. Also, it remains unknown if the lymphoid tissue in the tonsils and the gut is comparable. With the findings of great variation in Treg subpopulations in mind, the relevance of the overall Treg percentages might be questioned. Finally, the functionality of Tregs is of importance. In this study, the production of the Treg-associated cytokine IL-10 was measured, and only in ECs was the production intact, further linking Tregs to the control of HIV infection.
Tregs interact with Th17 cells. Elevated percentages of CD161+Th17 cells were found in ECs only. In this study CD161 was used to identify Th17 cells. CD161 has proven to be expressed selectively on Th17 precursors,43 but CD161 may not be a sufficient Th17 cell marker as either CCR6 or IL-17 should ideally be included. However, the results are in accordance with earlier studies reporting a maintained balance between Th17 cells and Tregs in controllers.29,44 Further, elevated percentages of Th17 cells in a group of nonprogressors where most participants met the LTNP criteria have been reported,45 suggesting high percentages of Th17 cells to be involved in the nonprogression of HIV infection. In this study, the percentage of Th17 cells differed between groups, whereas the production of IL-17 after stimulation with PHA was similar. However, measurement of cytokines was not performed on supernatants from purified Th17 cells, which may explain this discrepancy. In future studies, the number of IL-17 copies per cell in purified Th17 cells should be determined. Lately, it has become evident that also a subset of CD8 T cells exhibits proinflammatory properties, complicating the interpretation of the impact of Th17 cells on HIV infection. These Tc17 cells express a number of markers including CD161high, CCR6 and IL-17,46–49 and CD3+CD8+CD161high, however, harbor all of the IL-17 producing CD8 T cells.46,47 Our group has recently shown that CD3+CD8+CD161highTc17 cells are depleted in HIV infection.16 Here, we report ECs to have preserved the percentages of CD3+CD8+CD161highTc17 cells unlike all other groups of HIV-infected patients where CD3+CD8+CD161highTc17 cells were depleted. This is consistent with the finding of 2 studies demonstrating a depletion of Tc17 cells in simian immunodeficiency virus infection along with a partial restoration of Tc17 cells after the initiation of cART,50,51 suggesting the depletion of Tc17 cells to be a possible contributor to immune dysfunction in HIV-infected patients.
In conclusion, immuno-Tregs seem to be of importance for the nonprogression in HIV infection, both regarding patients with and without ongoing viral replication. In particular, a large proportion of activated Tregs may be involved in preserved CD4 T cell counts in both ECs and LTNPs. Thus, the selective activation of resting Tregs may serve as a possible future therapeutic strategy, although further functional studies are warranted. Further, the depletion of CD3+CD8+CD161highTc17 cells may be involved in immune dysfunction in HIV infection as only ECs presented with preserved percentages, whereas all other HIV-infected patients displayed severe depletion of CD3+CD8+CD161highTc17 cells. Indeed, further studies are warranted to clarify the role of Tc17 cells in HIV infection.
The authors gratefully acknowledge the participants who volunteered and made this project possible. The authors thank Professor Niels Obel, DMSc, and the Danish HIV Cohort study (DHCS) for identifying patients. They also thank The Novo Nordisk Foundation, The Lundbeck Foundation, The Augustinus Foundation, The Danish AIDS Foundation, Rigshospitalet Research Council, and University of Copenhagen for financial support.
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