
==== Front
eBioMedicine
EBioMedicine
eBioMedicine
2352-3964
Elsevier

S2352-3964(24)00310-4
10.1016/j.ebiom.2024.105274
105274
Articles
Altered memory CCR6+ Th17-polarised T-cell function and biology in people with HIV under successful antiretroviral therapy and HIV elite controllers
Yero Alexis a
Goulet Jean-Philippe b
Shi Tao a
Costiniuk Cecilia T. c
Routy Jean-Pierre c
Tremblay Cecile de
Mboumba Bouassa Ralph-Sydney a
Alexandrova Yulia a
Pagliuzza Amélie d
Chomont Nicolas de
Ancuta Petronela de
Jenabian Mohammad-Ali jenabian.mohammad-ali@uqam.ca
ae∗
a Department of Biological Sciences and CERMO-FC Research Centre, Université du Québec à Montréal (UQAM), Montreal, QC, Canada
b CellCarta, Montreal, QC, Canada
c Chronic Viral Illness Service and Research Institute of the McGill University Health Centre, Montreal, QC, Canada
d Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CR-CHUM), Montreal, QC, Canada
e Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada
∗ Corresponding author. Department of Biological Sciences, Université du Québec à Montréal (UQAM), 141 Avenue President Kennedy, Room SB3385, Montreal, QC, H2X 1Y4, Canada. jenabian.mohammad-ali@uqam.ca
22 8 2024
9 2024
22 8 2024
107 10527430 1 2024
17 7 2024
27 7 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Background

Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17 cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied.

Methods

mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and in vitro functional assays.

Findings

Decreased Th17 cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production in vitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67.

Interpretation

These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17 cells.

Funding

This study was funded by the 10.13039/501100000024 Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Keywords

Th17 cells
CCR6
HIV infection
Transcriptomics
HIV persistence
DNA damage
DNA repair
==== Body
pmc Research in context

Evidence before this study

Th17-polarised cells are key players of mucosal immunity that are rapidly depleted during HIV infection. The impact of viral suppression by antiretroviral therapy (ART) on the recovery of Th17 cells remains controversial, with some reports showing no Th7 restoration following ART, while others suggest Th17 cell recovery mostly when ART is initiated early following HIV infection. Importantly, even in those individuals where Th17 cell frequencies/numbers are restored, profound functional impairments persist. Moreover, long-lived Th17 cells carry replication-competent virus and are known as HIV viral reservoirs during ART. Furthermore, the dynamics of Th17 cells and functions under natural control of HIV replication in elite controllers (EC) have been studied less, and contrasting findings on their Th17 dynamics and functions have also been reported. However, to date, the molecular mechanisms that account for these observations have not been thoroughly investigated. Thus, we performed a comprehensive genome-wide transcriptional analysis, along with in vitro functional assays, in order to assess the dynamics, biology, and functions of these cells in virally suppressed people with HIV (PWH) compared to uninfected controls.

Added value of this study

Our study uncovered novel molecular mechanisms behind three major aspects of impaired Th17-polarised T-cell biology during HIV infection, including (1) altered differentiation and functions; (2) increased HIV susceptibility and viral persistence; and (3) increased activation and exhaustion/senescence in line with elevated DNA damage and DNA modifications. Accordingly, Th17 cell frequencies remained lower in ST and EC compared to uninfected controls, along with their functional impairment in the production of Th17 cell-specific cytokines. These observations are supported by their increased expression of NR1D1/REV-ERBa and lower expression of Semaphorin 4D, which are respectively a repressor and inducer of Th17 cell differentiation and functions. We also found a marked downregulation in genes associated with host restriction factors and inhibitors of HIV infection in Th17-polarised cells from both ST and EC individuals, suggesting higher susceptibility/permissibility to HIV infection. Moreover, Th17 cells from ST and EC contain total and integrated HIV DNA, in line with the dysregulated expression of known cellular factors associated with viral latency and persistence. Finally, Th17 cells from PWH display peculiar features related to increased DNA damage and modifications, which support their increased immune exhaustion and senescence, and reduced proliferation.

Implications of all the available evidence

We uncovered potential molecular paths that could explain the Th17 cell dysfunction and viral persistence in PWH despite suppressed viral replication. The potential development of therapeutic strategies targeting these mechanisms may restore Th17 frequencies and functions and potentially promote immunological remission at the mucosal level in PWH.

Introduction

The discovery of Th17 cells as a new functionally distinct subset of CD4+ T-cells opened the path toward a new understanding of immunological processes beyond the Th1/Th2 theory.1, 2, 3 Th17 cells are key players in mucosal immunity and are unique in their ability to produce the hallmark cytokines IL-17A, IL-17F, IL-21, IL-22, and IL-26, which exert effects at the interface between innate and adaptive immunity,4,5 mainly for maintaining mucosal immunity at barrier surfaces, protecting against extracellular pathogen infections. Previous studies demonstrated that human Th17 cell differentiation requires, in addition to transforming growth factor beta (TGF-β), a combination of the cytokines IL-1β, IL-6, IL-21, and IL-23, which are essential to Th17 specification/polarization.6,7 Th17 cell development and functions are influenced by the master regulator transcription factor retinoic acid receptor-related orphan receptor (ROR)γt in mice (RORC2 in humans), along with interferon regulatory factor 4 (IRF4), basic leucine zipper transcription factor (BATF), and signal transducer and activator of transcription 3 (STAT3).4,5 Extracellular markers such as C–C motif chemokine receptor 6 (CCR6), CD26, and C-type lectin CD161 are used to phenotypically identify Th17-polarised cells.4,5

The expression of CCR6 by CD4+ T-cells is well-known to identify cells with Th17 cell features, while higher expression of CCR6 is linked to greater Th17 cell-specific genes and cytokine production.4,8 Within the CCR6+ CD4 T-cells fraction exist four distinct sub-populations that share Th17 cells polarizing markers and stable Th17-lineage commitment, including CCR6+CCR4+CXCR3- (Th17 cells), CCR6+CCR4−CXCR3+ (Th1/Th17 cells), CCR6+CCR4−CXCR3- (double negative, CCR6+DN), and CCR6+CCR4+CXCR3+ (double positive, CCR6+DP) cells.9 Th1/Th17 cells produce IFN-γ and, similar to traditional Th17 cells, express IL-17A and other markers such as IL23R, IL-1R, CD26, CD161, and granulocyte-macrophage colony-stimulating factor (GM-CSF).4,10,11 CCR6+DP cells produce IL-17A levels similar to conventional Th17 cells and also generate IFN-γ.9 In contrast, CCR6+DN cells appear to represent an early stage in Th17 cell differentiation, as they express IL-17F, STAT3, and homing markers associated with migration toward lymph nodes, including CCR7 and CD62L.9 The presence of various cell populations exhibiting characteristics of Th17 cells and other effector cells or regulatory T-cells (Tregs) underscores the remarkable instability and adaptability of Th17 cells.

Th17 cells are located at the portal sites of HIV entry and are among the first HIV targets, notably in mucosal tissues.4,12 Because of their heightened susceptibility to HIV infection, Th17 cells are rapidly depleted in the initial stages of the infection in the blood, gut, and genital mucosa.4,12 This depletion persists throughout the chronic infection, as documented by our group and other studies.13, 14, 15, 16 Depletion of Th17 cells has significant consequences, including the breakdown of mucosal barrier integrity, microbial translocation, higher immune activation, accelerated disease progression, and the development of non-AIDS comorbidities.17, 18, 19, 20 Importantly, Th17 cells and immunosuppressive Tregs share developmental pathways, while changes in fate decisions between Th17 cells and Tregs influence pro-inflammatory versus anti-inflammatory responses.5

The impact of the suppression of HIV replication by antiretroviral therapy (ART) or natural viral control on the recovery of Th17 cell frequencies and functions remains a subject of debate. Different studies have yielded conflicting results, with some indicating that ART fails to normalize Th17 cell frequencies,4,21 while others have shown the restoration of this cell subset.22, 23, 24, 25 Importantly, even when their frequencies are normalized, Th17 cells' functions are not fully recovered.22,25 In addition, specific subsets of Th17 cells with stem-cell-like characteristics represent significant HIV reservoirs, underscoring their critical role in HIV persistence under ART.9,26, 27, 28 However, the molecular mechanisms underlying Th17 cells’ elevated susceptibility to HIV infection and persistent alterations under ART are understudied. HIV elite controllers (EC), a minority group of untreated people with HIV (PWH) comprising less than 1% of subjects, constitute a distinctive subgroup within long-term non-progressors (LTNP) individuals. ECs sustain an undetectable plasma viral load (VL) in the absence of ART and maintain higher CD4 T-cell counts for an extended duration, likely throughout their entire lives.29, 30, 31 Th17 cell frequencies in EC tend to be comparable to those found in uninfected individuals by some groups32, 33, 34; however, we recently reported in the same study cohort as the current study a decrease in frequencies of blood memory CCR6+ CD4 T-cells in EC associated with the duration of the infection.16

To provide insights into the molecular mechanisms of Th17 cell alteration during HIV-1 infection under suppressive ART or natural viral suppression in ECs, we performed a comprehensive genome-wide transcriptional analysis, along with in vitro functional assays, in memory CD4+ T-cells expressing the Th17 marker CCR6 from successfully ART-Treated PWH and EC compared to uninfected participants.

Methods

Study population

Sixty-five study participants were enrolled in three groups including: successfully ART-treated PWH (ST, n = 23); 18 elite controllers (EC, n = 18) who were ART-naïve, with CD4+ T-cell count >500 cell/μl and undetectable plasma VL; and 24 uninfected healthy donors (HD). The information on the individuals included in our study is summarized in Supplementary Figure S1 and Supplementary Table S1, while the clinical characteristics are detailed in Supplementary Table S2A–D. Our study cohort was mainly dominated by Men Who Have Sex With Men (MSM), self-reported by study participants. For the transcriptomics study, cells were purified and FACS-sorted from fresh leukapheresis specimens collected at the McGill University Health Centre (Montreal, QC, Canada) from a total of 14 individuals, including 5 HDs, 6 STs, and 3 ECs. Gene expression validation by RT-PCR was done on FACS-sorted cells from frozen leukapheresis in 20 individuals, including 9 HDs, 7 STs, and 4 ECs. Total and integrated HIV DNA was assessed in FACS-sorted cells from frozen leukapheresis in 11 individuals, including 7 STs and 4 ECs. For RT-PCR and viral reservoirs assessment were needed at least 100–150 million of frozen PBMC to obtain around 0.5–1.5 million of sorted non-Tregs memory CCR6+ T-cells, which was only possible in preserved samples from leukapheresis. Flow cytometry analysis and in vitro cytokine production assays were performed on frozen PBMCs of 61 individuals from the Montreal Primary and Slow Progressors HIV Infection cohorts, including 20 HDs, 23 STs, and 18 ECs. In all cases, study participants were negative for other active sexually transmitted infections (STI), including syphilis, gonorrhoea, Chlamydia, and hepatic viral infections (HCV, HBV).

Ethical considerations

The Université du Québec à Montréal (UQAM) Ethical Review Board approved this study (#2014-452), which adhered to the Helsinki Declaration. Prior to blood collection, all study participants signed a written informed consent form for research-exclusive biobanking.

Flow cytometry analysis

Multiparameter flow cytometry analysis was performed on frozen PBMCs. The appropriate concentration of fluorochrome-conjugated antibodies was used for immunological staining in four distinct panels of 14 colours each. To remove dead cells, the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Cat. L34957, Invitrogen, Oregon, USA) was used. Extracellular staining was done for 1 h at 4 °C in PBS + 2% fetal bovine serum (FBS). Following extracellular staining, cells were fixed and permeabilized for 40 min at 4 °C with the Transcription Factor Buffer Set (Cat. 562574, AB_2869424, BD Bioscience, New Jersey, USA). Cells were then stained intracellularly for FoxP3, H2A.X Variant Histone (H2AX), Poly [ADP-Ribose] Polymerase (PARP), Ki-67, and CTLA-4 for 1 h at 4 °C in a Perm/Wash solution (Cat. 562574, AB_2869424, BD Bioscience, New Jersey, USA). Data was collected using a 3-laser BD Fortessa X-20 cytometer and analyzed using FlowJo v10.9.0 (Oregon, USA). The antibodies used for immune phenotyping are listed in Supplementary Table S3.

Fluorescence-activated cell sorting (FACS) of memory CCR6+ CD4+ T-cells

Total CD4+ T cells were first enriched from freshly isolated PBMCs obtained from fresh leukapheresis samples by negative magnetic selection using the EasySep Human CD4+ T-cell Enrichment Kit (Cat. 17952, STEMCELL, Vancouver, Canada) with a purity higher than 95%. Cells were then stained extracellularly with anti-human CD3-Alexa Fluor 700/CD4-FITC/CD25-PE/CD127-PE-Cy7/CD45RA-BV-650/CCR6-BV-786 antibodies all from BD Biosciences (New Jersey, USA), and LIVE/DEAD™ FixableBlue Dead Cell Stain Kit (Cat. L34955, Invitrogen, Oregon, USA) to exclude dead cells for 1 h at 4 °C. To exclude the Tregs, the sorting gates for non-Tregs memory CCR6+ T-cells were set on CD4+CD25lowCD45RA−CCR6+ as described in Fig. 1a, which were sorted using a BD FACSAria.Fig. 1 Differential gene expression in memory CCR6+CD4+ T-cells from PWH under successful ART and elite controllers compared to HIV-uninfected individuals. a) Before cell-sorting, total CD4+ T-cells were first isolated from PBMCs of HIV− healthy donors (HD, n = 5), successfully ART-Treated HIV+ individuals (ST, n = 6), and HIV elite controllers (EC, n = 3) by negative selection using magnetic beads. Gating strategy used for the FACS-sorting of memory CD4+CD25−CD45RA−CCR6+ T-cells. Total RNA from FACS-sorted cells was used for genome-wide transcriptomics analysis. The differential expression analysis was performed using a linear model with moderated t-statistics as implemented in the Bioconductor package limma (version 3.58.1). b) Heatmap showing differential gene expression in sorted non-Tregs memory CCR6+ cells in the 3 study groups related to different T-helpers (Th1, Th2, and Th17) and Tregs cells. c) Representation of the number of commonly and differentially expressed genes when comparing EC versus HD, EC versus ST, and ST versus HD individuals. d) Principal component analysis plots. Scores plot of PC1 versus PC2 from PCA are shown for mRNA-seq results. Each dot represents one sample. Colours are assigned based on the known of specimen assignment. e) Volcano plots showing differentially expressed genes when comparing EC versus HD, EC versus ST, and ST versus HD individuals. f) Top-modulated 50 genes in ST and EC individuals compared to HD subjects. Highlighted genes correspond to genes that were previously associated with (1) Th17 cell differentiation, stability, and functions; (2) susceptibility to HIV infection; (3) HIV latency and persistence; and (4) DNA replication, DNA damage repair, cell cycle and cell proliferation.

Transcriptomics mRNA-seq analysis

FACS-sorted cells from 14 individuals were used for RNA extraction. Total RNA was extracted using the RNeasy Plus mini kit (Cat. 74134, Qiagen, Germantown, Maryland, USA). Genome-wide transcriptional profiling was performed by Genome Québec (Montreal, Québec, Canada) using the Illumina RNA-Sequencing technology (NovaSeq6000 S4 PE 100bp 25M reads). Briefly, the paired-end sequencing reads were aligned to coding and non-coding transcripts from Homo Sapiens database GRCh 38 revision 96 and quantified using Kallisto software version 0.46.0.35 The entire RNA-Sequencing data set and the technical information requested by Minimum Information About a Microarray Experiment (MIAME) are available at the Gene Expression Omnibus database under accession GSE252771.

The principal component analysis (PCA) was applied to the global gene expression matrix, including all the 16,541 detected in samples. A filter on variation was applied, where the lower 10% of genes based on variance were excluded from the calculations. The gene expression values were scaled and centered. Rotation is based on the resulting eigenvectors, as implemented in R package PCATools version 2.16.36 Two significant principal components were identified using the Elbow method.36

The top 50 genes were selected for a representation that allowed the readability of gene labels and highlighted relevant genes. No GO-term annotation was performed per se. Pathway analysis was performed using the method Gene set Variation Analysis (GSVA), after which we obtained an expression value associated with a given gene set. This expression value is then applied to the same linear model as differential gene expression analysis.

Differentially expressed genes (DEG) were identified based on nominal p-values (p < 0.05), and fold-change (FC, cutoff 1.3).9 Statistical analyses were performed using R version 4.0.4. Differential expression analysis was performed using the voom method35 limma Bioconductor R package (version 3.44.3) on the log2-counts per million (logCPM) transformed transcript-level and gene-level data. Gene set enrichment analysis (GSEA) was performed using the GSVA method (package version 1.36.3)37 on the logCPM data using a Gaussian cumulative distribution function using MSigDB v7.4.38 GSVA analysis was done at once on all the canonical (C2) pathways and then selected for their relationship with (1) Th17 cell differentiation, stability, and functions; (2) susceptibility to HIV infection; (3) HIV latency and persistence; and (4) DNA replication, DNA damage repair, cell cycle, and cell proliferation. Some C2 pathways might be redundant since several databases were interrogated. The colours in all heatmaps correspond to z-score for which each gene was scaled across all samples to better compare relative expression between genes.

Quantification of RNA expression by RT-PCR

Total DNA and RNA were co-extracted in parallel from each FACs-sorted population using the AllPrep DNA/RNA Mini Kit (Cat. 80204, Qiagen, Hilden, Germany) following the manufacturer's recommendations. cDNA was then synthesized using M-MLV reverse transcriptase (Cat. 28025013, Invitrogen, Oregon, USA) on total RNA according to the manufacturer's protocol. The specific primers and housekeeping gene ACTB are described in Supplementary Table S4. Reactions were carried out in duplicate in a LightCycler 480 Instrument II (Roche, Switzerland) with a LightCycler 480 SYBR Green I Master (Cat. 04707516001, Roche, Germany). Cycling conditions: 5 min at 95 °C; 40 amplification cycles of 15 s at 95 °C, 20 s at 59 °C, and 20 s at 72 °C; 1 cycle of 5 s at 95 °C and 30 s at 65 °C; and finally, 10 s at 40 °C. To ensure that no contamination occurred, we created a negative control with no cDNA. The negative control resulted in no amplification or a crossover point (Cp) value of 35 or above. Each gene's expression was measured in relation to the housekeeping gene ACTB. RT-PCR validation was performed for 1) the genes included within the Top 50 DEG; 2) genes that were not within the top 50 DEGs, but were significant and with high fold-change; 3) genes that were part of a significant C2 pathway and previously validated in the literature; and, 4) other genes which were previously validated in the literature and relevant to our study.

Th17 cell-specific cytokine production following in vitro stimulation

One million PBMCs were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA, Cat. P8129, SIGMA-ALDRICH, Darmstadt, Germany) at 100 ng/ml and ionomycin (Cat. I9657, Millipore-SIGMA, Darmstadt, Germany) (1 μg/ml), followed by another 4 h in the presence of brefeldin A (Cat. 555029, AB_2869014) and monensin (Cat. 554724, AB_2869012) (both from BD Bioscience). After stimulation, cells were washed and stained for flow cytometry analysis as we previously described.15 Antibodies used for in vitro analysis are listed in Supplementary Table S3. Th17 cell-specific cytokine production by memory CCR6+ CD4+ T-cells was calculated as the delta between the stimulated and unstimulated conditions.

Total and integrated HIV DNA quantification

Cell lysates from FACS-sorted memory CCR6+ CD4+ T-cells were used to measure total and integrated HIV DNA by real-time PCR as described previously.39 Briefly, cell pellets of sorted cells were resuspended in a lysis buffer (10 mM Tris–HCl pH 8.0, 50 nM KCl, 400 μg/ml Proteinase K, Invitrogen) and digested for 12–16 h. Proteinase K was inactivated, and cell lysates were directly used in all pre-amplification reactions. In all PCR reactions, primers specific to the human CD3 gene were used to quantify the exact number of cells present in the reaction tube. Total HIV DNA or integrated HIV DNA genomes (using alu oligonucleotides) together with the CD3 gene were pre-amplified for 12 cycles. The second rounds of PCR were carried out in real-time on the RotorGene Q instrument (Qiagen) with the Rotor Gene Probe Master Mix (Qiagen) following the manufacturer's instructions. In all experiments, serial dilutions of digested ACH2 cells (containing a single copy of integrated HIV DNA per cell) were used as standards for quantification.

Statistical analysis

GraphPad Prism V9 (California, USA) was used for statistical analysis. The Kolmogorov–Smirnov test was first used to determine the distribution of variables. The Kruskal–Wallis test was then used to determine significant differences between more than two research groups. For unpaired variables, the nonparametric Mann–Whitney U rank test was performed. The differential expression analysis was performed using a linear model with moderated t-statistics as implemented in the Bioconductor package limma (version 3.58.1). The correlation between variables was determined using the Spearman correlation coefficient test. Data is presented in all graphs as a median with a 95% confidence interval (CI).

Role of funders

The funding institutions played no role in the study design, experimental workflow, data collection, data analysis and interpretation, or manuscript writing.

Results

Memory CCR6+ CD4+ sub-populations are dysregulated in PWH under successful ART and HIV elite controllers and contain HIV reservoirs

A significant decrease in total memory CCR6+ CD4+ T-cells in ECs was observed compared to STs (median difference: −2.68%, 95% CI: −5.62% to −0.76%) and HDs (median difference: −4.72, 95% CI: −7.01% to −2.59%) (Fig. 2a and b), which is consistent with our previous published data,16 whereas Th1/Th17 (CCR6+CCR4−CXCR3+) cell frequencies were lower in EC compared to STs (median difference: −6.75%, 95% CI: −9.71% to −0.68%) and HDs (median difference: −6.60%, 95% CI: −10.40% to 0.40%), and Th17 (CCR6+CCR4+CXCR3-) cell frequencies were significantly lower in both STs (median difference: −10.75%, 95% CI: −16.30% to −2.90%) and ECs (median difference: −9.10%, 95% CI: −17.0% to −1.50%) compared to HD individuals (Fig. 2c and d). Lower frequencies in EC compared to HDs were observed for CCR6+DP (CCR6+CCR4+CXCR3+) (Fig. 2e), while increased frequencies of CCR6+DN (CCR6+CCR4−CXCR3-) (Fig. 2f) were found in STs and ECs compared to HD individuals. In addition, memory CCR6+ T-cells in ST individuals expressed lower levels of Th17 cell markers CD26 and CD161 (Fig. 2g) than cells from HDs (median difference: −8.75%, 95% CI: −20.40% to −0.20%). Importantly, total and integrated HIV DNA were detected in FACS-sorted memory CCR6+ CD4+ T-cells from all STs and ECs, with a trend for their higher levels in STs versus ECs (Fig. 2h). Altogether, we found profound dysregulations in ST and EC participants, along with memory CCR6+ T-cell contribution in HIV viral persistence despite their natural or ART-induced suppression of plasma VL.Fig. 2 Decreased frequencies of memory CCR6+CD4+T-cell sub-populations in PWH under successful ART and elite controllers compared to HIV-uninfected controls. a) Gating strategy in flow cytometry to determine non-Treg (FoxP3-) memory CCR6+ sub-populations. b) Frequencies of non-Tregs memory CCR6+ T-cells within total CD4 T-cells determined by flow cytometry. Percentages determined in flow cytometry of CCR4−CXCR3+ (c), CCR4+CXCR3− (d), CCR4+CXCR3+ (e), CCR4−CXCR3− (f), and CD26+CD161+ (g) within memory CCR6+ cells. Quantification of total HIV DNA and integrated HIV DNA (h) in memory CCR6+ CD4+ T-cells from successfully ART-Treated HIV+ individuals (ST) (n = 7) and HIV elite controllers (EC) (n = 4). Horizontal lines refer to the median. After the Kruskal–Wallis analysis, the differences among the three study groups were determined by a nonparametric Mann–Whitney rank test for unpaired variables. Sample size in flow cytometry analysis: HD (n = 20), ST (n = 23), and ED (n = 18).

Reduced in vitro Th17-specific cytokine secretion capacity of memory CCR6+ CD4+ T-cells in PWH under successful ART and elite controllers compared to uninfected controls

Upon in vitro stimulation of PBMCs with PMA/Ionomycin (Fig. 3a and b), significantly lower production of Th17 cell-specific cytokines IL-17A, IL-17F, and IL-22 were observed in ST (for IL-17A: median difference: −3.79%, 95% CI: −4.88% to −0.18%; for IL-17F: median difference: −1.29%, 95% CI: −1.60% to −0.060%; for IL-22: median difference: −1.17%, 95% CI: −2.39% to −0.18%) and EC (for IL-17A: median difference: −4.55%, 95% CI: −5.13% to −0.09%; for IL-17F: median difference: −1.66%, 95% CI: −1.88% to 0.12%; for IL-22: median difference: −1.37%, 95% CI: −2.59% to −0.17%) individuals compared to HDs (Fig. 3c–e). In addition, the Boolean gating strategy analysis showed significantly lower polyfunctional (IL-17A+IL-17F+IL-22+) memory CCR6+ T-cells in STs compared to HD individuals, whereas higher frequencies of cells with no cytokine production (IL-17A−IL-17F−IL-22-) were also found in STs and EC compared to HD individuals (Supplementary Figure S2). Furthermore, both STs and ECs showed significantly lower levels of Th1/Th17 cell cytokines IFN-γ+, IFN-γ+IL-17A+, IFN-γ+IL-17F+, and IFN-γ+IL-22+ memory CCR6+ T-cells than HD individuals (Fig. 3f–i). It is also known that autocrine TGF-β1 contributes to Th17 cell differentiation40,41; however, lower TGF-β1 production by memory CCR6+ T-cells were detected in STs compared to both HDs and EC individuals (Fig. 3j). Overall, consistent with dysregulated proportions of memory CCR6+ sub-populations in ST and EC participants, we observed impaired Th17 cytokine production in these individuals despite their undetectable plasma VL.Fig. 3 Diminished Th17 cell-specific cytokine production by memory CCR6+CD4+T-cells in PWH under successful ART and elite controllers compared to HIV-uninfected controls. a) Schematic representation of in vitro stimulation protocol with PMA/Ionomycin. Cells were stimulated for 4 h with phorbol myristate acetate (PMA) at 100 ng/ml and ionomycin (1 μg/ml), followed by an additional 4 h in the presence of brefeldin A and monensin. b) Gating strategy in flow cytometry to determine IL-17A, IL-17F, IL-22, IFN-γ, and TGF-β1 production by PMA/Ionomycin-stimulated memory CCR6+ CD4+ T-cells. Flow cytometry analysis was used to determine the percentages of expression of IL-17A (c), IL-17F (d), IL-22 (e), IFN-γ (f), IFN-γ+IL-17A+ (g), IFN-γ+IL-17F+ (h), IFN-γ+IL-22+ (i), and TGF-β1 (j) within memory CCR6+ CD4+ T-cells.

mRNA-seq identified transcriptional changes in memory CCR6+ CD4+ T-cells in PWH under successful ART and elite controllers compared to uninfected controls

In order to assess how natural or ART-induced suppression of HIV replication affect Th17 cell biology, mRNA-seq analysis was performed to investigate differences in the transcriptional profile of freshly FACS-sorted memory CCR6+ CD4+ T-cells in STs, ECs, and HDs (Fig. 1a). Tregs (CD25highCD127low) were excluded from our analysis to avoid contamination with CCR6+ Tregs in the sorted population.16

To determine an enrichment in Th17 cell-specific gene module in our FACS-sorted memory CCR6+ population, we first analysed the expression of T-helper-specific and Tregs-specific genes. We found that sorted memory CCR6+ cells were enriched in transcripts encoding Th17 cell-specific molecular signatures (RORC, KLRB1, RORA, IL6R, IL17RA, CCL20) and expressed lower levels of Th1- (IFN-g, t-bet/TBX21, IL12RB2), Th2- (GATA3), and Tregs-specific (FoxP3, ICOS, IL2RA, TIGIT, CTLA-4, IL-10, and TNFRSF18) transcripts; confirming that the FACS-sorted memory CCR6+ T-cells are enriched in Th17 cells (Fig. 1b), as well documented by our group and others previously.4,9 DEGs were identified based on significant p-values and fold change ratios (cut-off 1.3). Memory CCR6+ T-cells were transcriptionally different between study groups, with 1066 DEGs in STs versus HDs, 560 DEGs in ECs versus HDs, and 427 DEGs in ECs versus STs (Fig. 1c). Among DEG in the ST versus HD comparison, 204 were shared with EC versus HD and 102 with EC versus ST (Fig. 1c). Indeed, PCA grouped samples from STs and HDs separately, whereas samples from EC clustered with ST individuals (Fig. 1d), highlighting major differences between STs and ECs versus HDs. Similarly, the volcano plots representation showed higher differences in the comparison of ST versus HD, followed by EC versus ST, and lastly, EC versus HD (Fig. 1e).

Among the top 50 DEG in both ST and EC groups compared to HD there was a predominance of transcripts related to (1) Th17 cell differentiation, stability and functions, including nuclear receptor subfamily 1 group D member 1 (NR1D1), and Semaphorin (Sema)4D; (2) susceptibility to HIV infection, including, interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) and Krueppel-like factor 3 (KLF3); (3) HIV latency and persistence, including, transcription factor AP-4 (TFAP4), embryonic ectoderm development (EED), and tumor suppressor p53-binding protein 1 (TP53BP1); and (4) DNA replication, DNA damage repair, cell cycle and cell proliferation, such as nuclear autoantigenic sperm protein (NASP) (Fig. 1f). Supplementary Table S5 includes others DEGs beyond the top 50 which are also related to (1) Th17 cell differentiation, stability, and functions; (2) susceptibility to HIV infection; (3) HIV latency and persistence; and (4) DNA replication, DNA damage repair, cell cycle and cell proliferation, for which a cut-off FC ≥ 2 was set for upregulated genes and a cut-off ≤2 was set for downregulated genes.

Decreased Th17 cell differentiation, stability, proliferation, and functions in PWH under successful ART and elite controllers compared to uninfected controls

In line with our observations in the top 50 DEG, further GSVA identified differentially expressed C2 pathways previously reported to be associated with promoting Th17 cell differentiation, stability, proliferation, and functions (Fig. 4a). The significantly downregulated pathways related to Th17 cell differentiation, stability, proliferation, and functions included, among others, the TGF-β/smad pathways, Notch, and Semaphorin 4D (Fig. 4a). Moreover, Semaphorin 4D, sphingolipid metabolism, and S1P pathways were also downregulated in ECs compared to HDs, which could also indicate impairment in Th17 cell differentiation, stability, proliferation, and functions in these individuals (Fig. 4a). A detailed description of all pathways can be found in Supplementary Table S6.Fig. 4 The differentiation, stability, proliferation, and functions of memory CCR6+ CD4+ T-cells are dysregulated in successfully ART-Treated HIV+individuals and elite controllers. a) Top-regulated canonical pathways (C2) related to Th17 cell differentiation, stability, proliferation, and functions were identified using gene set enrichment analysis in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 5), successfully ART-Treated HIV+ individuals (ST, n = 6), and HIV elite controllers (EC, n = 3). Relative expression of NR1D1 (b), NOTCH1 (c), Semaphorin 4D (CD100) (d), and DDIT4L (e) RNA relative to the ACTB housekeeping gene in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 9), successfully ART-Treated HIV+ individuals (ST, n = 7), and HIV elite controllers (EC, n = 4). f) Gating strategy in flow cytometry to determine TGFBR2+, CD81+, CD82+, CD37+, and Ki-67+ cells within CCR6+CD4+ T-cells. Percentages determined in flow cytometry of TGFBR2+ (g), CD81+ (h), CD82+ (i), CD37 (j), and Ki-67 (k) within CCR6+ CD4+ T-cells. Following Kruskal–Wallis analysis, the differences among the three study groups were determined by nonparametric Mann–Whitney rank test for unpaired variables. Sample size in flow cytometry analysis: HD, n = 20; ST, n = 23; and EC, n = 18.

As demonstrated in Fig. 4b–d, RT-PCR quantifications confirmed increased expression of NR1D1 (among the top 50 DEG, Fig. 1e) along with decreased expression of NOTCH1 (canonical pathway analysis, Fig. 4a) and Semaphorin 4D (among the top 50 DEG, Fig. 1e; canonical pathways analysis, Fig. 4a) in STs and ECs individuals compared to HDs, which is in line with our observations in the mRNA-seq analysis. In addition, the mTOR inhibitor, DNA-damage-inducible transcript 4-like (DDIT4L), which negatively regulates Th17 cell differentiation, was significantly upregulated in STs compared to HDs (Supplementary Table S5) and was confirmed by RT-PCR (Fig. 4e). Interestingly, frequencies of total memory CCR6+ CD4+ T-cells and IL-17A-producing cells were both negatively correlated with mRNA levels of NR1D1, whereas a positive correlation was found with mRNA levels of Semaphorin 4D, which is in line with decreased memory CCR6+ CD4+ T-cells and IL-17A-producing cells in STs and ECs (Supplementary Table S7).

The expression of TGF-β receptor 2 (TGFBR2), part of the TGF-β1 signalling pathway (canonical pathway analysis, Fig. 4a), which is necessary for Th17 cell differentiation,5 was significantly lower in memory CCR6+ T-cells from STs compared to HD controls (Fig. 4f and g). Furthermore, reduced expression of tetraspanins CD81, CD82, and CD37—known to contribute to TCR-mediated activation and functions and cell proliferation—were observed in HIV-infected individuals compared to uninfected HD controls (Fig. 4f, h–j). Importantly, CD81 also contributes to forming the functional heterodimer between the TGFBR1 and TGFBR2.42 Additionally, memory CCR6+ T-cells from STs and ECs, compared to HD individuals, showed decreased proliferation assessed by their expression of Ki-67 (Fig. 4f and k). Furthermore, the expression of CCR9, a marker of T-cell gut homing, was also diminished in ST individuals versus HDs in our transcriptomic analysis (Supplementary Table S5) and was validated by flow cytometry (Supplementary Figure S3).

Altogether, our results demonstrated that memory CCR6+ T-cells from STs and ECs have impaired signatures of differentiation, proliferation, stability, and functions despite successful ART or natural control of infection.

Increased HIV susceptibility and viral persistence in PWH under successful ART and elite controllers compared to uninfected controls

Following our observations that Top 50 DEG included genes encoding factors linked to HIV susceptibility (IFIT2, THAP11, and KLF3) and viral persistence (TFAP4, EED, ETS1, and TP53BP1), we used GSVA to identify dysregulated pathways associated with HIV biological terms in GSVA. The analysis of canonical pathways (C2) revealed a downregulation in pathways associated with increased protection against HIV infection, including the estrogen signalling pathway, Wnt, and NRF2ARE in STs and/or ECs versus HD individuals (Fig. 5a). The Wnt pathway was also downregulated in EC compared to HD. A detailed description of all pathways related to susceptibility and permissiveness to HIV infection is provided in Supplementary Table S8. Other dysregulated genes associated with HIV infection are presented in Supplementary Table S5. RT-PCR quantifications confirmed decreased expression of HIV restriction factors and infection inhibitors by memory CCR6+ T-cells in both ST and EC study groups compared to HD controls, including KLF3 (top 50 DEG, Fig. 1e), serine incorporator 3 (SERINC3), and ring finger protein 125 (RNF125), along with increase expression of a positive regulator of HIV infection, MRE11 (Fig. 5b–e). The KLF3, SERINC3, RNF125, and MRE11 transcripts were validated by RT-PCR according to previous reports in the literature about their role in HIV susceptibility. Interestingly, we also confirmed increased expression of HIV restriction factor IFIT2 (top 50 DEG, Fig. 1e) (Fig. 5f), which might indicate the activation of additional antiviral mechanisms in memory CCR6+ T-cells in response to their preferential HIV infection, along with decreased expression of a promoter of HIV infection cyclin-dependent kinase 6 (CDK6) (Fig. 5g) in both ST and EC individuals compared to HD controls.Fig. 5 Gene expression of memory CCR6+CD4+T-cells from successfully ART-Treated HIV+individuals and elite controllers is associated with higher susceptibility to HIV infection. a) Top-regulated canonical pathways (C2) related to HIV susceptibility were identified using gene set enrichment analysis in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 5), successfully ART-Treated HIV+ individuals (ST, n = 6), and HIV elite controllers (EC, n = 3). Relative expression of KLF3 (b), SERINC3 (c), RNF125 (d), MRE11 (e), IFIT2 (f), and CDK6 (g) RNA relative to the ACTB housekeeping gene from HIV-uninfected individuals (HD) (n = 9), successfully ART-Treated HIV+ individuals (ST) (n = 7), and HIV elite controllers (EC) (n = 4). Following Kruskal–Wallis analysis, the differences among the three study groups was determined by nonparametric Mann–Whitney rank test for unpaired variables.

ST versus HD individuals showed downregulation in canonical pathways previously associated with HIV persistence, including calcium and calcineurin, RUNX1, CREB, NFAT, estrogen signaling pathway, nitric oxide and nitric oxide synthase, and p38 (Fig. 6a). A detailed description of all pathways associated with HIV persistence can be found in Supplementary Table S9. RT-PCR quantifications confirmed increased expression of viral latency-promoting factors, including EED (top 50 DEG, Fig. 1e) and TP53BP1 (top 50 DEG, Fig. 1e), along with decreased expression of TFAP4 (top 50 DEG, Fig. 1e) (Fig. 6b–d) in STs and ECs compared to HDs, which are in line with the presence of HIV-1 reservoirs in memory CCR6+ T-cells of ST, and at a lower extend in ECs as demonstrated in Fig. 2h. Levels of total HIV-DNA in Mem CCR6+ T-cells negatively correlated with CD4 T-cell counts, whereas only in ST individuals they positively correlated with the time of ART initiation and mRNA expression levels of SERINC3 (Supplementary Table S10). On the other hand, levels of integrated HIV DNA negatively correlated with mRNA levels of CDK6 and RNF125, while only in STs, negatively correlated with CD4 T-cell counts and mRNA levels of KLF3 (Supplementary Table S10). Altogether, our findings demonstrated that memory CCR6+ T-cells in ST and EC individuals showed a transcriptional signature compatible with elevated HIV susceptibility and viral persistence despite successful ART or natural control of the infection.Fig. 6 Pathways and genes associated with HIV persistence are dysregulated in memory CCR6+CD4+T-cells from successfully ART-Treated HIV+individuals and elite controllers. a) Top-regulated canonical pathways (C2) related to HIV persistence were identified using gene set enrichment analysis in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 5), successfully ART-Treated HIV+ individuals (ST, n = 6), and HIV elite controllers (EC, n = 3). Relative expression of EED (b), TP53BP1 (c), and TFAP4 (d) RNA relative to the ACTB housekeeping gene in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 9), successfully ART-Treated HIV+ individuals (ST, n = 7), and HIV elite controllers (EC, n = 4). Following Kruskal–Wallis analysis, the differences among the three study groups was determined by nonparametric Mann–Whitney rank test for unpaired variables.

Increased DNA damage, methylation, and repair in memory CCR6+ CD4+ T-cells in PWH under successful ART and elite controllers compared to uninfected controls

The canonical pathways analysis indicated an upregulation in STs versus HDs in pathways linked to increased DNA damage and modifications, as well as those related to DNA damage repair mechanisms (Fig. 7a, Supplementary Table S11). RT-PCR quantifications confirmed increased expression of two transcripts implicated in DNA damage repair, protein replication protein A2 (RPA2) (Fig. 7b) and 8-oxoguanine DNA glycosylase (OGG1) (Fig. 7c) in STs and ECs compared to HDs, which might be a cellular response to elevated DNA damage in memory CCR6+ cells in these individuals. Similarly, increased RNA expression of histone-binding protein NASP was also found in ST and EC individuals compared to HDs (Fig. 7d). The increased NASP expression is in line with increased expression of genes related to nucleosome/chromatin structure since NASP binds to histones and contribute to their intracellular transport (Supplementary Table S5). Furthermore, higher expression levels of DNA methylases DNA-methyltransferase 1 (DNMT1) (Fig. 7e) and DNMT3A (Fig. 7f) in memory CCR6+ Th17 cells from ST individuals versus HD controls were also found in our RT-PCR validation, which might contribute to increased DNA methylation and gene silencing in these cells. Finally, using flow cytometry, we observed increased levels of phosphorylated H2AX and cleaved PARP in ST individuals compared to HDs (for PARP: median difference: 3.34%, 95% CI: 0.020% to 6.43%; for H2AX: median difference: 1.20%, 95% CI: 0.040% to 4.59%) and ECs (for PARP: median difference: −1.27%, 95% CI: −2.41% to 0.46%; for H2AX: median difference: −0.56%, 95% CI: −1.28% to −0.040%), indicating increased double-strand DNA breaks and general DNA damage in these individuals (Fig. 7g–j). Importantly, we did not observe a relationship between the duration of the infection and levels of expression of PARP and H2AX. However, a negative correlation was observed with the duration of ART, while a positive correlation was found with the time of ART initiation (years post-infection), suggesting that early ART initiation and its longer duration might reduce DNA damage in Th17 cells (Supplementary Table S12). Overall, our findings showed that memory CCR6+ T-cells in STs and ECs showed a transcriptional signature related to elevated DNA damage, methylation, and impairment in the mechanisms of DNA repair.Fig. 7 Dysregulation of markers associated with DNA damage, DNA modifications, and DNA repair in memory CCR6+CD4+T-cells from successfully ART-Treated HIV+individuals and elite controllers. a) Top-regulated canonical pathways (C2) related to DNA damage, DNA modifications, and DNA repair mechanisms were identified using gene set enrichment analysis in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 5), successfully ART-Treated HIV+ individuals (ST, n = 6), and HIV elite controllers (EC, n = 3). Relative expression of RPA2 (b), OGG1 (c), NASP (d), DNMT1 (e), and DNMT3a (f) RNA relative to the ACTB housekeeping gene in FACS-sorted memory CCR6+CD4+ T-cells from HIV− healthy donors (HD, n = 9), successfully ART-Treated HIV+ individuals (ST, n = 7), and HIV elite controllers (EC, n = 4). g) Gating strategy in flow cytometry to determine PARP/H2AX expression within memory CCR6+ cells. Percentages determined in flow cytometry of PARP+ (h), H2AX+ (i), and H2AX+PARP+ (j) cells within memory CCR6+ cells. After Kruskal–Wallis analysis, the differences among the three study groups were determined by nonparametric Mann–Whitney rank test for unpaired variables. Sample size in flow cytometry analysis: HD, n = 20; ST, n = 23; and EC, n = 18.

Increased immune activation, exhaustion, and senescence in PWH under successful ART compared to uninfected controls

In our GSVA we observed various C2 pathways dysregulated in STs and/or ECs compared to HDs. These dysregulated pathways include 1) Calcium/calcineurin, which are involved in T-cell activation and immune response regulation and the dysregulation of these pathways can contribute to immune exhaustion; 2) NFAT, which is crucial for T-cell activation and differentiation, and dysregulation of this pathway might contribute to immune exhaustion (Fig. 4a, Supplementary Table S6); 3) CREB, that can initiate the transcription of genes that promote cell survival, such as anti-apoptotic genes, growth factors, and proteins involved in stress responses further impacting immune exhaustion (Fig. 4a, Supplementary Table S6); 4) NRF2-ARE, which is involved in oxidative stress responses and can modulate immune cell function and exhaustion (Fig. 4a, Supplementary Table S6); 5) histone modifications and epigenetic regulations, which directly impact immune exhaustion (Fig. 7a, Supplementary Table S11); 6) G2/M checkpoints since cell cycle checkpoints are crucial for regulating cell proliferation and can indirectly affect immune cell function and exhaustion (Fig. 7a, Supplementary Table S11); and 7) DNA damage and repair mechanisms, which also directly influence immune exhaustion (Fig. 7a, Supplementary Table S11). Moreover, we also observed various dysregulated pathways in STs and/or ECs compared to HDs, including 1) the Reactome DNA damage and telomere stress-induced senescence and 2) Reactome senescence-associated secretory phenotype (SASP), which are both well-known pathways related to immuno-senescence (Fig. 7a, Supplementary Table S11). These pathways specifically involve senescence-associated processes, including the induction of senescence in response to DNA damage and SASP, which involves the secretion of various inflammatory mediators by senescent cells.

To validate how altered transcriptomic signature and in vitro functions of Th17-polarised memory CCR6+ T-cells in PWH might be linked to their immune exhaustion and senescence, we assessed the expression of cellular markers of activation (CD38/HLA-DR) and senescence/exhaustion (CTLA-4, PD-1, CD57) in all study participants. Increased immune activation was observed in memory CCR6+ T-cells from ST individuals compared to HD controls, represented by their higher frequencies of CD38+, HLA-DR+, and CD38+HLA-DR+ cells (Fig. 8a–d). The expression of immune checkpoints PD-1 and CTLA-4 by memory CCR6+ T-cells was higher in STs and ECs compared to HD individuals (Fig. 8a, e-g). Additionally, increased levels of immune senescence (PD-1+CD57+ phenotype) were also observed in memory CCR6+ T-cells from STs (median difference: 0.29%, 95% CI: 0.050% to 0.48%) and ECs (median difference: 0.44%, 95% CI: 0.020% to 0.60%) compared to HD individuals (Fig. 8a and h), which can be associated with the increased DNA damage found in these individuals. Finally, increased levels of exhausted memory CCR6+ T-cells (CD28−CD57+) were detected in ECs (median difference: 0.046%, 95% CI: 0.016% to 0.18%) compared to HD individuals (Fig. 8a and i).Fig. 8 Higher expression of cellular markers of immune activation, exhaustion, and senescence in memory CCR6+CD4+T-cells from successfully ART-Treated HIV+individuals and elite controllers. a) Gating strategy in flow cytometry to determine CD38/HLA-DR, PD-1/CTLA-4, PD-1/CD57, and CD28/CD57 expression within memory CCR6+ cells. Percentages determined in flow cytometry of CD38+ (b), HLA-DR+ (c), CD38+HLA-DR+ (d), PD-1+ (e), CTLA-4+ (f), PD-1+CTLA-4+ (g), PD-1+CD57+ (h), and CD28-CD57+ (i) cells within memory CCR6+ cells. After Kruskal–Wallis analysis, the differences among the three study groups was determined by nonparametric Mann–Whitney rank test for unpaired variables. Sample size in flow cytometry analysis: HD, n = 20; ST, n = 23; and EC, n = 18.

Discussion

In this study, using combined approaches including mRNA-seq analysis on freshly FACS-sorted memory CCR6+ CD4 T-cells from leukapheresis, in addition to ex vivo gene validation by RT-PCR or flow cytometry, as well as in vitro functional assays, we demonstrated significant alterations in Th17-polarised memory CCR6+ T-cell frequencies, biology and function in PWH under suppressive ART and elite controllers.

Even though the frequencies of total memory CCR6+ T-cells CD4 T-cells seem to be similar in HD and ST individuals, the frequencies of memory CCR6+ subpopulations, such as Th17 cells, remain lower in ST individuals, which is in line with previous reports on the failure of ART in restoring their frequencies.4,21,24 In ECs, total memory CCR6+ frequencies were significantly lower compared to HDs and correlated with the duration of infection, as we previously reported in the same study cohort.16 Importantly, CCR6+ CD4 T-cells, in contrast to CCR6-, exhibit Th17 features and functions,43 and CCR6 expression is linked to Th17 lineage commitment.10,44 Furthermore, we found decreased expression of Th17-associated markers CD26 and CD161 in ST individuals.9 Consistent with lower frequencies of Th17 polarised frequencies despite ART or HIV natural control in ECs, we also observed that memory CCR6+ T-cells from PWH produce less Th17-specific cytokines in vitro, including IL-17A, IL-17F, and IL-22, in addition to their decreased IFN-γ production, indicating the impairment in their immunological functions compared to uninfected individuals, in line with previous reports.22,25 Moreover, increases in CCR6+ (CCR4−CXCR3-) DN cells, which are one of the major IL-17A/IL-17F producers,9 along with an overall decrease in IL-17A/IL-17F production in STs and ECs might indicate that CCR6+ DN cells are somehow dysfunctional in these individuals. The latter could be associated with the high permissiveness of DN cells to HIV infection and viral persistence in this subpopulation, which was previously shown by our group. In addition, compared to IL-17A, the expression of IL-17F is a preferable marker to identify the early CD4 T-cell commitment toward Th17 lineage.45 Thus, decreased IL-17F+ memory CCR6+ T-cells in STs and ECs compared to HDs might also indicate a reduction in early committed Th17 cells and reduced stemness properties in PWH compared to HDs.

The mRNA-seq analysis provides evidence that memory CCR6+ T-cells from PWH are characterized by impaired differentiation, stability, proliferation, and functional features, as demonstrated by decreased mRNA expression of Semaphorin 4D and NOTCH1, along with increased mRNA expression of NR1D1 and DDITL4 in STs and ECs versus HDs. Indeed, Semaphorin 4D is known to promote the proliferation of CD4 T-cells and their differentiation into Th17 cells by upregulating their master transcription factor RORγt, resulting in increased production of IL-17 and IL-22.46, 47, 48 NOTCH1 activation has also been observed in in vitro-polarised Th17 cells in both mice and humans, and blockage of Notch signalling markedly reduces RORγt/RORC2 expression and the production of cytokines associated with Th17-polarised cells.49, 50, 51 The transcriptional repressor NR1D1/REV-ERBa is expressed in Th17 cells and competes with RORγt/RORC2 for their shared DNA consensus sequence. NR1D1 negatively regulates Th17 cell development by inhibiting RORγt/RORC2-dependent genes, decreasing Th17 cell differentiation and IL-17 production.52, 53, 54 On the other hand, the crucial role of the mTOR pathway in Th17 cell differentiation is well documented.5 Accordingly, we found increased expression of the mTOR inhibitor DDIT4L in both ST and EC individuals, which activates the tuberous sclerosis complex 1/2 complex to suppress the mTOR pathway, ultimately leading to the inhibition of IL-17 expression.55,56 Of note, the increase in DDIT4L could also be a feedback mechanism associated with increased mTOR expression and/or activation during HIV infection.5

As mentioned previously, the combination of cytokines IL-1β, IL-6, IL-21 and IL-23, and TGF-β1 is crucial for Th17 cell differentiation.6 In this sense, the decreased production of TGF-β1 following in vitro stimulation with PMA/Ionomycin, in addition to the lower expression of TGFBR2 in ST individuals, can directly impact the maintenance and differentiation of memory CCR6+ T-cells. The autocrine production of TGF-β1 is likely needed for Th17 cell differentiation, and TGFBR2 will sense TGF-β1 in the membrane and subsequently trigger the smad2/smad3 signalling pathway leading to RORC2 expression and maintenance of the Th17 cell phenotype.40,57,58 Importantly, the observed decrease in tetraspanin CD81 expression could also contribute to impairments in the TGF-β1 signalling pathway by impeding the formation of the functional heterodimer between the TGFBR1 and TGFBR2.42 Moreover, we cannot exclude that some of the TGF-β1-producing memory CCR6+ T-cells are also FoxP3+ since FoxP3 expression was not included in the flow cytometry analysis of our PMA/Ionomycin stimulation experiment. Besides, CD81 and CD82 interact with CD3 and CD4 in the cell surface, controlling TCR relocalization, antigen recognition, and TCR-mediated activation and downstream signalling, whereas CD37 is also needed for T cell proliferation.42,59,60

In terms of lower cell proliferation, differentiation, and cell cycle progression, we also found decreased Ki-67 expression in memory CCR6+ T-cells from STs and ECs compared to HD, along with increased RNA levels of NASP and decreased expression of CDK6. In fact, NASP is required for DNA replication, normal cell cycle progression, and cell proliferation.61,62 Moreover, CDK6 activates mTORC1 and E2F transcription factors, which directly impact cell growth, proliferation, and cell cycle progression.63,64 These results indicated the confluence of several factors at the time, acting over different targets, and resulting in decreased memory CCR6+ T-cell proliferation as indicated by Ki-67 expression. Along with lower Ki-67 expression as indicative of proliferation, we also observed an increase expression of cellular markers of immune exhaustion and senescence in memory CCR6+ T-cells from STs versus HDs, which is in line with the observed increases in RNA levels of EED, which is related to HIV latency and persistence, and a previous report on activated T-cells.65 The increased DNA damage and repair impairment are also related to increased immune senescence and exhaustion, which is in line with the higher levels of memory CCR6+ expressing PD-1/CD57 in STs and ECs compared to HDs and lower cytokine production by these cells upon PMA/Ionomycin stimulation.66,67 Accordingly, decreased levels of Semaphorin 4D have also been correlated with higher immune senescence and dysfunctional immune response in CD8 T-cells during HIV infection,68 which could also occur in memory CCR6+ T-cells. We also found decreased CCR9 expression in memory CCR6+ T-cells from STs compared to HDs, indicating their diminished migration towards the gut, which can diminish effective mucosal immune reconstitution despite ART. The elevated expression of immune checkpoints PD-1 and CTLA-4 in both STs and ECs can contribute to immune dysfunction and decrease the proliferation of memory CCR6+ cells. Indeed, increased PD-1 and CTLA-4 expression could act as a negative regulator of memory CCR6+ T-cells’ differentiation and cytokine production, which is in line with our in vitro results and the reduced Th17 cytokine secretion capacity in PWH.69, 70, 71

Th17 cells are well-known to be preferentially infected by HIV and are viral reservoirs during ART.13,14,72 In line with these previous observations, we detected total and integrated HIV DNA in FACS-sorted memory CCR6+ T-cells from all ST and EC participants, with increased trends toward STs, as indicators of HIV viral persistence in these cells despite successful ART and natural control of HIV infection. The latter findings coincided with increased RNA levels of MRE11, needed for the early stages of HIV replication and LTR circle formation73, 74, 75 and RPA2, which contributes to enhanced HIV reverse transcription and infectivity.76 Importantly, we observed decreased expression of RNA encoding for host restriction factors and inhibitors of HIV infection such as SERINC3, which is counteracted by Nef to avoid the inhibition of HIV replication77; RNF125, which inhibits viral transcription likely by recruiting host factors controlling viral transcription to the ubiquitin-proteasome pathway78; and KLF3, a protein that promotes epigenetic modifications at the HIV LTR, leading to the suppression of proviral transcription.79 In addition, we showed a decreased expression of tetraspanins CD81, CD82, and CD37 in STs and ECs individuals compared to HDs, which could negatively contribute to HIV endocytosis and infection and virological synapse formation.80,81 Furthermore, we detected increased RNA levels of EED and TP53BP1 along with decreased TFAP4 expression, all factors that promote and/or contribute to viral latency. EED contributes to HIV latency by repressing gene activity via histone deacetylation while inhibiting HIV assembly and release.82, 83, 84 TP53BP1, which is also part of the DNA damage response machinery, is needed for HIV DNA integration into the host genome,85 whereas TFAP4, whose levels decreased during HIV infection,86 contributes to transcriptional latency of the HIV-1 provirus.87 Importantly, p53 expression is also known to be related to decreased HIV infection,88 indicating the simultaneous occurrence of various mechanisms that either promote or inhibit HIV infection, further contributing to viral persistence. The latest could be in line with the observed increased IFIT2 expression, a known inhibitor of HIV infection in macrophages,89 and decreased expression of CDK6, which is implicated in positive regulation of HIV infection.90 Altogether, these observations indicate that while memory CCR6+ T-cells showed features associated with their increased susceptibility to HIV infection, at the same time, additional antiviral mechanisms are activated in response to their preferential HIV infection. Other factors can also contribute to viral latency, including the increased expression of histone proteins and higher expression of NASP, as observed in our study, which contributes to chromatin assembly and further impacts DNA replication, normal cell cycle progression, and cell proliferation.61,91,92 The latest can contribute to a more compacted chromatin that is less accessible for transcription factors, favouring viral latency and persistence. In line with these results, higher RNA levels of DNA methyltransferases DNMT1 and DNMT3a can promote DNA methylation and further gene silencing, contributing to viral persistence.93,94

Our transcriptomic analysis revealed the upregulation in various pathways involved in DNA damage and modifications, as well as impairment in DNA repair mechanisms in PWH versus HDs. Particularly, we observed an upregulation in RNA levels of RPA2, OGG1, DNMT1, and DNMT3a as validated by RT-PCR, and higher expression of pS139-H2AX and cleaved PARP as validated by flow cytometry. RPA2, a component of the Replication Protein A complex, is crucial in DNA metabolism, encompassing essential functions such as DNA replication, repair, recombination, and orchestrating the cellular response to DNA damage.95, 96, 97 OGG1 serves as the principal enzyme necessary during base-excision repair for removing 8-hydroxy-2-deoxyguanine (8-OH-dG), a highly mutagenic by-product that arises from exposure to reactive oxygen species (ROS) and if it remains unrepaired during DNA replication, it can lead to GC to TA transversions.98,99 The histone H2AX is phosphorylated in S139 upon DNA double-stranded breaks or DNA damage. The phosphorylated H2AX plays a vital role in recruiting and localizing DNA repair proteins and cell cycle checkpoint factors to sites of DNA damage, which facilitates DNA repair and the maintenance of genomic stability.100,101 Furthermore, PARP's catalytic activity increases in non-apoptotic cells in response to DNA damage, and it is believed to play a crucial role in the normal cellular response to DNA damage. During apoptosis, PARP undergoes cleavage, in which the amino-terminal DNA-binding domain is separated from the C-terminal catalytic domain, leading to the loss of its normal function, which is considered a marker of apoptosis.102,103 However, we did not find differences in anti-apoptotic Bcl-2 expression among the three study groups (data not shown). Therefore, increased levels of cleaved PARP and phosphorylated H2AX in STs and ECs confirm higher levels of DNA damage in HIV-infected individuals regardless of ART or natural HIV control. Moreover, we did not observe correlations between PARP/H2AX expression and duration of the infection in ST individuals, while earlier timing of ART initiation and longer ART duration were associated with their decreased expression, which indicates that earlier and longer ART might improve DNA damage in memory CCR6+ T-cells. On the other hand, DNA methyltransferases DNMT1 and DNMT3a were also upregulated in STs and ECs, indicating increased DNA methylation in memory CCR6+ T-cells from these individuals. In this sense, increased DNA methylation and epigenetic modifications are well-described for CD4 T-cells during HIV infection.16,104

Our study has some limitations that merit to be acknowledged. We are aware that the relatively low number of ECs in our transcriptomic analysis and RT-PCR validation can affect the potency of the statistical test and results. However, since, as per current Canadian HIV clinical guidelines, all previously known ECs are now receiving ART, we were unable to obtain rare and precious freshly collected leukapheresis specimens for our mRNA-seq analysis right before ART initiation from this limited number of individuals. Importantly, the gene expression validation by RT-PCR and flow cytometry analyses using a higher number of previously bio-banked frozen PBMCs from additional EC individuals confirmed that our findings are consistent with our mRNA-seq data, as well as prior literature knowledge and demonstrate a strong biological rationale. Furthermore, other confounding such as sexual preference/orientation, age, duration of infection, and timing of ART initiation may influence our findings. In this sense, various reports indicate alterations in Th17 cell frequencies with age,105,106 whereas sexual orientation/preference has also been associated with gut microbiome dysbiosis and subsequent alterations in Th17 cell proliferation and functions.107 In addition, the ST individuals included in our study cohort used for flow cytometry analysis presented a lower duration of infection and duration of ART compared to their counterparts included for transcriptomics, gene validation by RT-PCR, and viral reservoir assessment, which might impact some of the analysed markers. The dysregulation on several canonical pathways guided us for further analysis related to Th17 cell differentiation, functions, HIV susceptibility, and viral persistence. However, most of these pathways were not validated in our hands by RT-PCR or flow cytometry, even though they have been characterized in previous studies. Furthermore, we also acknowledge that we did not perform the FACS-sorting of Th17 subsets and viral reservoirs quantification has been performed only in CCR6+ memory cells and no comparison with their CCR6- memory cells counterpart to better demonstrate viral persistence enrichment in non-Tregs memory CCR6+ T-cells. Finally, the in vitro evaluation of the role of specific pathways and genes related to HIV susceptibility in memory CCR6+ T-cells should be assessed in further studies.

In summary, our results indicate that chronically HIV-infected individuals with natural (EC) or ART-controlled (ST) HIV-1 replication displayed an altered transcriptomic and phenotypical signature associated with a compromised memory CCR6+ Th17-polarised differentiation and functions, as well as transcriptional changes reflecting increased HIV permissiveness and viral persistence. These findings contribute to a better understanding of Th17 cell biology and immune functions during chronic HIV infection which is essential for the development of future therapeutic strategies to restore Th17 T-cell immunity and/or HIV cure. Targeting specific pathways identified in our study may potentially improve Th17 cell biology and immune function and reduce HIV reservoir size. This might involve targeted therapies to promote Th17 cell proliferation and differentiation by manipulation of pathways and transcription factors identified in our study and reduction of immunosenescence and exhaustion, as well as the use of immune checkpoint inhibitors.

Contributors

Study design: AY, PA, MAJ. Study conceptualization and supervision: MAJ. Funding acquisition: MAJ. Performance of the experiments and data generation: AY, TS, RSMB, YA, AP and NC. Bioinformatic analysis: JPG. Discussion of results and data interpretation throughout the study: AY, JPG, PA, and MAJ. Access to specimens and clinical data JPR, CT, and CTC. Manuscript writing and formatting: AY and MAJ. All authors contributed to the refinement of the study and reviewed, revised, and approved the final version of the manuscript. AY, JPG, and MAJ have verified the underlying data.

Data sharing statement

The entire RNA-Sequencing data set and the technical information requested by Minimum Information About a Microarray Experiment (MIAME) are available at the Gene Expression Omnibus database under accession GSE252771. Data from this manuscript is available from the corresponding author upon reasonable request.

Declaration of interests

We have no competing interest to declare.

Appendix A Supplementary data

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Table S5

Supplementary Table S6

Supplementary Table S7

Supplementary Table S8

Supplementary Table S9

Supplementary Table S10

Supplementary Table S11

Supplementary Table S12

Acknowledgements

First, the authors express their heartfelt gratitude to all the participants in this study for generously contributing their time and unwavering commitment. We would like to acknowledge M. Mario Legault for his crucial administrative support, Ms. Angie Massicotte for her invaluable assistance and coordination, Ms. Josée Girard for the leukapheresis procedures, and Ms. Stephanie Matte from the Canadian HIV-1 Slow Progressor cohort. The authors would like to thank Dr. Madeleine Durand for her valuable advice on statistical analysis. This study was funded by the 10.13039/501100000024 Canadian Institutes of Health Research (CIHR, operating grant MOP 142294 to MAJ, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064 to CTC, JPR, NC, PA, and MAJ) and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S) to MAJ. AY was supported by an FRQ-S doctoral scholarship. RSMB is supported by a CIHR postdoctoral fellowship. JPR holds the McGill University Louis Lowenstein Chair in Hematology and Oncology. CT colds the Pfizer Chair in Clinical and Translational Research on HIV. CTC holds an FRQ-S Junior 2 Clinician-researcher career award. NC holds an FRQ-S senior researcher career award. MAJ holds the tier 2 CIHR Canada Research Chair in Immuno-Virology. The funding institutions played no role in the design, collection, analysis, and interpretation of data.

Appendix A Supplementary data related to this article can be found at https://doi.org/10.1016/j.ebiom.2024.105274.
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