New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy
© The Author(s) 2016
Received: 29 May 2016
Accepted: 11 August 2016
Published: 24 August 2016
Th17 cells are permissive to HIV-1 infection and their depletion from the gut of infected individuals leads to microbial translocation, a major cause for non-AIDS co-morbidities. Most recent evidence supports the contribution of long-lived Th17 cells to HIV persistence during antiretroviral therapy (ART). However, the identity of long-lived Th17 cells remains unknown.
Here, we performed an in-depth transcriptional and functional characterization of four distinct Th17 subsets and investigated their contribution to HIV reservoir persistence during ART. In addition to the previously characterized CCR6+CCR4+ (Th17) and CCR6+CXCR3+ (Th1Th17) subsets, we reveal the existence of two novel CCR6+ subsets, lacking (double negative, CCR6+DN) or co-expressing CXCR3 and CCR4 (double positive, CCR6+DP). The four subsets shared multiple Th17-polarization markers, a fraction of cells proliferated in response to C. albicans, and exhibited lineage commitment and plasticity when cultured under Th17 and Th1 conditions, respectively. Of note, fractions of CCR6+DN and Th17 demonstrated stable Th17-lineage commitment under Th1-polarization conditions. Among the four subsets, CCR6+DN expressed a unique transcriptional signature indicative of early Th17 development (IL-17F, STAT3), lymph-node homing (CCR7, CD62L), follicular help (CXCR5, BCL6, ASCL2), and self-renewal (LEFI, MYC, TERC). Cross sectional and longitudinal studies demonstrated that CCR6+DN cells were the most predominant CCR6+ subset in the blood before and after ART initiation; high frequencies of these cells were similarly observed in inguinal lymph nodes of individuals receiving long-term ART. Importantly, replication competent HIV was isolated from CCR6+DN of ART-treated individuals.
Together, these results provide new insights into the functional heterogeneity of Th17-polarized CCR6+CD4+ T-cells and support the major contribution of CCR6+DN cells to HIV persistence during ART.
The Th17 cells represent a subset of CD4+ T-cells that act as the first line of defense against pathogens at barrier surfaces . Th17 cells are defined by the production of unique effector cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-26, CCL20) under the transcriptional regulation of multiple lineage-specific transcription factors (IRF4, BATF, STAT3, RORγt) [2, 3]. Their anatomic localization and effector functions, together with their unique developmental plasticity, position Th17 cells at the very core of the immune system [2, 4]. CCR6 is a well-established marker for Th17 cells [5, 6]. CCR6 mediates recruitment into specific tissues including Peyer’s patches (CCR6 ), skin  and brain . Although not all CCR6+ T-cells produce IL-17A upon stimulation ex vivo, the majority of these cells are prone to acquire Th17 effector functions . The co-expression of CCR6 and CCR4 identifies cells producing IL-17A (Th17 profile) specific to Candida albicans and Staphylococcus aureus [5, 11], while CCR6+CXCR3+ cells produce both IL-17A and IFN-γ (Th1Th17 profile) in response to Mycobacterium tuberculosis or upon polyclonal stimulation [ ,  ]. These advances in the identification of surface markers for functionally distinct CD4+ T cell subsets proved to be instrumental for understanding the contribution of Th17 cells to human pathologies including rheumatoid arthritis , multiple sclerosis , cancer , and HIV-infection [15, 16].
The existence of functionally distinct IL-17A-producing CD4+ T-cells cells was originally reported in the context of autoimmunity, with CCR6+CXCR3+Th1Th17 and CCR6+CCR4+Th17 cells being considered pathogenic and non-pathogenic, respectively [3, 17]. This discovery led to the identification of molecular signatures associated with Th17 pathogenicity in mice [18–20] and most recently in humans . In contrast, during HIV-1 infection, we previously demonstrated that both CCR6+CCR4+Th17 and CCR6+CXCR3+Th1Th17 cells are pathogenic since they are permissive to viral infection in vitro, carry integrated HIV-DNA in vivo, and their frequency is significantly reduced in HIV-infected individuals, including those with undetectable plasma viral load under antiretroviral therapy (ART) . Considering the fact that IL-17A plays a critical role in maintaining epithelial barrier integrity at intestinal level [22, 23], the depletion of Th17 and Th1Th17 cells from gut-associated lymphoid tissues (GALT) is considered as a major cause for microbial translocation, chronic immune activation and occurrence of non-AIDS co-morbidities in HIV-infected individuals . Thus, features of Th17 pathogenicity are unique in the context of HIV infection. In addition, long-lived Th17 cells exist and were reported to promote cancer progression . The possibility that long-lived Th17 cells contribute to HIV reservoir persistence under ART, as supported by recent findings by our group (Gosselin et al, unpublished observations) and others , adds to the complexity of Th17 pathogenicity concept and position these cells as a major barrier for HIV eradication.
In this study, we used a systems biology approach an revealed phenotypic, functional and transcriptional features of two previously uncharacterized human CD4+ T-cell subsets expressing the Th17 marker CCR6 and lacking or co-expressing the homing receptors CCR4 and CXCR3: CCR4−CXCR3− (double negative; CCR6+DN) and CCR4+CXCR3+ (double positive; CCR6+DP). Our results provide new insights into the diversity of Th17 subsets during homeostasis and HIV-1 infection, thus adding a novel piece of complexity to the recent understanding of Th17 functional heterogeneity and clonotype sharing in humans . We reveal that CCR6+DN are distinguished from Th17, Th1Th17 and CCR6+DP by their expression of markers of early Th17 development, lymph node trafficking, follicular help and self-renewal. We also demonstrate that CCR6+DN represent the most predominant Th17 subset in the blood and lymph nodes of HIV-infected ART-treated individuals and carry replication-competent integrated HIV-DNA. These findings support the newly emerged concept that HIV takes advantage of the long-lived properties of specific Th17 subsets [25, 28, 29] to ensure its persistence during ART. Thus, permissiveness to HIV-DNA integration compatible with survival represents a new previously unrecognized feature of pathogenic Th17 cells during HIV infection
Two novel subsets of memory CCR6+ T-cells exhibit Th17-features
For subsequent functional studies, sufficient numbers of highly pure CCR6+ and CCR6− cells with differential CCR4 and CXCR3 expression were sorted by flow cytometry from large numbers of PBMCs (109) obtained by leukapheresis (Additional file 1: S1 Figure) from HIV-uninfected individuals. To determine the polarization profile of CCR6+DN and CCR6+DP, the expression of lineage-specific cytokines was measured in FACS-sorted cells exposed to CD3/CD28 triggering. Compared to Th17 and Th1Th17, CCR6+DN and CCR6+DP produced high and low levels of IL-17A, respectively. In contrast to Th17, CCR6+DN and CCR6+DP produced IFN-γ but at significantly lower levels compared to Th1Th17; as expected, the Th2 cytokine IL-5 was produced by CCR6− but not CCR6+ cells (Fig. 1e).
To further investigate the Th17 polarization profile of CCR6+DN and CCR6+DP, their transcriptome was compared to that of Th17 and CCR6−DN using the Illumina technology. The list of 1020 transcripts differentially expressed in CCR6+DN versus CCR6−DN (adjusted p value >0.05; FC cut-off 1.3), included well-established markers for Th17 (IL-17F, KLRB1/CD161, CCL20, KLF2, CTSH, IL-22, CCR6, RORA, IL-23R, RORC, PTPN13, IL-26, S100A4, ARNTL, IL-6R) and Th1 lineages (CCL3L3, CCL3, CCL3L1, CCL5, CD300a), potentially new Th17 markers (CD96, LGMN, ANTXR2, CXCR6, MAP3K4, TNFSF25, LY9, S1PR1), together with negative regulators of Th17 polarization (IL-9, IFITM3, OSM, IRF8, MX1, NFIL3, PTK2, IL4R, SOCS1) (Fig. 1f). Clustering analysis of these transcripts grouped all CCR6+ subsets together, indicative of their common Th17 polarization. These results identify CCR6+DN and CCR6+DP as two novel Th17 subsets, with CCR6+DN producing high levels of IL-17A and being as preponderant as the previously described Th17 and Th1Th17 in the peripheral blood of healthy humans .
Th17-polarized CCR6+DN and CCR6+DP subsets exhibit unique transcriptional profiles
To extract functional meaning from these transcriptional dissimilarities, Ingenuity Pathway Analysis (IPA), Gene Set Variation Analysis (GSVA), and Gene Ontology (GO) tools were used to identify differentially expressed pathways and biological functions. IPA identified the most significant differences for the CCR6+DN versus CCR6+DP and Th17 versus CCR6+DP contrasts, and revealed similarities between CCR6+DN and Th17 (Fig. 2b). Among top modulated pathways, PPAR/RXR (previously associated with the negative regulation of Th17 polarization ), cyclin and cell cycle regulation, and apoptosis pathways were under represented in CCR6+DN versus CCR6+DP. In contrast, IL-6, phospholipase C (both involved in Th17 differentiation  CXCR4, thrombin, p38/ERK/MAPK (involved in cell activation/proliferation ) and Tec Kinase (involved in TCR signaling ) were over expressed in CCR6+DN versus CCR6+DP and in CCR6+DN versus Th17. Other pathways specifically up-regulated in CCR6+DN versus CCR6+DP and Th17 versus CCR6+DP included IL-2, Jak/STAT, PKC, sphingosine-1-phosphate (involved in cell migration ), NANOG (implicated in the maintenance of stem cells ), IL-3 (involved in lymphopoiesis ), PI3 K (involved in Th17 differentiation , April (involved in Th17 polarization , and NFAT (involved in IL-17A production ) (Fig. 2b).
GSVA identified canonical pathways (C2) and biological processes (C5) differentially expressed in CCR6+DN versus CCR6+DP and Th17 versus CCR6+DP (Additional file 3: S2A-B Figure). Among the top 50 differentially-expressed canonical pathways, the IL-23 pathway, which is key for Th17 polarization , was found to be enriched in CCR6+DN (Fig. 2c). Among the top 50 differentially-expressed biological processes, the expression of pathways such as lymphocyte differentiation, protease inhibitor and response to steroid hormone receptor were over represented in CCR6+DN (Fig. 2d).
GO identified differentially-expressed genes in CCR6+DN versus CCR6+DP (p < 0.05, cut-off 1.3-fold) involved in cell migration, chemotaxis, cell differentiation, and transcription. Transcripts associated with migration/chemotaxis into/outside lymph nodes, including CCR7, CXCR5, the CXCR5 ligand CXCL13, SELL, SIRP1, JAM3 and AIF1 were enriched in CCR6+DN, whereas adhesion molecules/chemokine receptors mediating homing into peripheral tissues such as integrin β7 (gut), CXCR3 (inflammatory sites), CCR2 and CCR4 (skin), and integrin β1 (cervix) were enriched in both Th17 and/or CCR6+DP (Additional file 3: S2C-D Figure). Well-established markers of Th17 (IL-8, IL-23R, SOSC3, TIM3, CD26/DPP4) and follicular helper T cells (Tfh) (CXCR5, ICOS) were enriched in CCR6+DN (Additional file 3: S2E Figure). Also, transcription factors related to the control of Th17 (STAT3, RORA) and Tfh (BLC6, ASCL2 [44, 45]) polarization/function were enriched in CCR6+DN (Additional file 3: S2E-F Figure). In addition, other transcripts enriched in CCR6+DN versus CCR6+DP include LEF1 and MYC, key markers of human stem-cells and long-lived Th17 cells , together with the anti-senescence marker TERC  (Additional file 2: Table S1). RT-PCR quantifications confirmed superior expression of STAT3, BCL6, LEF1 and TERC in CCR6+DN versus Th17 and CCR6+DP (Fig. 2e). These transcriptional analyses suggest that CCR6+DN, Th17 and CCR6+DP represent distinct stages of Th17 differentiation with specific migration potential, immunological functions, and transcriptional regulation. Among these subsets, CCR6+DN exhibit markers of early Th17 commitment, lymph-node tropism, follicular help, and self-renewal.
CCR6+DN are a major source of IL-17F, IL-8 and IL-21
CCR6+DN proliferate and produce IL-17A in response to C. albicans
CCR6+DN and CCR6+DP exhibit Th17-commitment and lineage plasticity
CCR6+DN acquire CCR4/CXCR3 expression in response to Th17- or Th1-polarizing signals
Th17-polarized CCR6+DN and CCR6+DP are present in the blood of HIV-infected individuals
To determine whether CCR6+DN and CCR6+DP exist in the blood of HIV-infected individuals and are stably Th17-polarized, the four memory CCR6+ T-cell subsets as well as Th1 cells were identified and then sorted by flow cytometry (as in Additional file 1: S1 Figure) from three chronically infected aviremic ART-treated individuals (CI on ART) (Additional file 2: Table S2). The intracellular expression of IL-17A and IFN-γ was assessed upon long-term culture in vitro. Briefly, cells were stimulated via CD3/CD28 for 4 days and then cultured in the presence of IL-2 (Th0 polarizing conditions) for additional 9 days (Additional file 7: S6A Figure). Similar to HIV-uninfected donors, Th17 and CCR6+DN subsets from CI on ART individuals included a major fraction of IL-17A+IFN-γ− cells, while Th1Th17, CCR6+DN, and CCR6+DP exhibited a similar frequency of IL-17A+IFN-γ+ cells (Additional file 7: S6B-C Figure). Of note, similar to Th1, Th1Th17 and CCR6+DP included a major fraction of IL-17A−IFN-γ+ cells (Additional file 7: S6B-C Figure). In conclusion, the two newly identified Th17-polarized subsets, CCR6+DN and CCR6+DP, exist in the peripheral blood of HIV-infected individuals and exhibit a Th17- and Th1Th17-polarization profile.
CCR6+DN predominate in the blood of HIV-infected individuals during ART
Circulating Th17 and Th1Th17 are depleted upon HIV-infection and their frequency is not restored with viral-suppressive ART [15, 16]. Thus, we investigated alterations in the frequencies of CCR6+DN and CCR6+DP, relative to Th17 and Th17Th17, during HIV infection. In contrast to uninfected controls (Fig. 1b), the frequency of CCR6+DN in CI on ART individuals (Additional file 2: Table S2) was superior to that of Th17 and Th1Th17, while CCR6+DP were the least preponderant subset (Fig. 6a). As in uninfected controls (Fig. 1d), the four CCR6+ subsets from CI on ART individuals were enriched in cells with a CM phenotype (Fig. 6b). Similar to Th17 and Th1Th17 , the frequency of memory CCR6+DP was significantly reduced in CI on ART versus uninfected individuals (Fig. 6c). In contrast, no significant differences were observed in the frequency of CCR6+DN in CI on ART versus uninfected individuals. Similar observations were made when absolute cell counts were compared (Fig. 6d), indicative that CCR6+DN are preserved during HIV infection.
To further explore the preservation of CCR6+DN during HIV infection, we performed longitudinal studies in HIV-infected individuals enrolled in the Montreal HIV Primary Infection (HPI) cohort. The dynamics of the four CCR6+ subsets were studied in relationship with plasma viral load at different time-points post-infection, before and after ART initiation (Fig. 6e, f). Of note, in all five HIV+ individuals, CCR6+DN were the most predominant CCR6+ subset before and after ART initiation. ART initiation during the first year of infection efficiently decreased plasma viral load to undetectable levels in all individuals (Fig. 6e) and increased CD4 counts (Additional file 8: Table S3). We observed a tendency for increased Th17, Th1Th17 and CCR6+DN counts following treatment (Fig. 6e), indicative of the positive effects of ART in increasing Th17 levels during early chronic infection. The counts of CCR6+DP were increased in 4/5 individuals following treatment but remained stable in one individual. Interestingly, the counts of CCR6+DN were superior at all time-points in 4/5 and 5/5 individuals, respectively, when compared to Th17 and CCR6+DP. In contrast, CCR6+DP counts were the lowest in all patients before/after ART initiation. These results demonstrate that CCR6+DP but not CCR6+DN are depleted in CI on ART individuals.
CCR6+DN are enriched in the lymph nodes of HIV-infected individuals on ART
CCR6+DN are permissive to viral infection in vitro and in HIV-infected individuals
CCR6+DN carry replication-competent integrated HIV-DNA in CI on ART individuals
The concept of Th17 pathogenicity is well defined in the context of autoimmunity [18, 19, 21]. During HIV-1 infection, Th17 cells may be considered pathogenic because they are permissive to HIV-1 and subsequently depleted [15, 16]. Alternatively, long-lived Th17 cells [25, 28, 29] may carry integrated HIV-DNA and contribute to viral reservoir persistence under ART (Gosselin et al, unpublished observations) . In this manuscript we used a systems biology approach to characterize the heterogeneity of memory subsets expressing the Th17 marker CCR6 at homeostasis and during HIV-1 infection. We reveal the existence of two new subsets of Th17-polarized CCR6+ T-cells, CCR6+DN (CCR4−CXCR3−) and CCR6+DP (CCR4+CXCR3+), that share functional characteristics with the previously characterized Th17 and Th1Th17 [5, 15, 16] in terms of lineage-specific cytokines, antigenic specificity, lineage specification versus plasticity and HIV permissiveness. Genome-wide transcriptional profiling revealed a unique molecular signature in CCR6+DN, suggesting they represent an early stage of Th17 differentiation. In contrast to the other Th17-polarized subsets, the CCR6+DN was the most predominant in the blood and the lymph nodes of HIV-infected subjects with undetectable plasma viral load under ART. Finally, we demonstrated that CCR6+DN carry replication competent HIV reservoirs in ART-treated individuals. Our results support the concept that long-lived Th17 cells, i.e., CCR6+DN, contribute to HIV persistence during ART.
Similar to Th17 cells, CCR6+DN were a major source of IL-17A, while CCR6+DP and Th1Th17 cells produced low IL-17A levels. Nevertheless, CCR6+DN and CCR6+DP expressed both typical Th17 transcripts (i.e., IL-26, CCL20, IL-22, IL-23R, RORC, IL-17F, RORA, KLRB1/CD161, IL6R ) and potentially new Th17 markers (CXCR6, PTPN13, MAP3K4, S100A4, CD96, Ly9, KLF2, LGMN, TNFRSF25, ANTXR2, and CTSH). PTPN13, a phosphatase associated with Fas , was previously identified as Th17-specific [21, 55] and may contribute to the regulation of cell activation/apoptosis. ARNTL, a component of the circadian clock machinery, was proved to regulate RORγt expression and Th17 development in mice . Finally, the expression of ANTXR2 is consistent with recent findings that Th17 cells govern adaptive immune responses against Bacillus anthracis .
Despite several transcriptional similarities, CCR6+DN, CCR6+DP and Th17 expressed distinct molecular signatures linked to specific pathways and biological processes. CCR6+DN were enriched in molecules associated with lymph node chemotaxis (CCR7, CXCR5, CXCL13, SELL, SIRP1, JAM3, AIF1). In contrast, Th17 and CCR6+DP expressed molecules related to homing into peripheral tissues including the gut (integrin β7 or CXCR3), skin (CCR2, CCR4) and cervix (integrin β1). Several signaling pathways involved in Th17 differentiation, including IL-6, phospholipase C, April and NFAT, were upregulated in CCR6+DN versus Th17 and/or CCR6+DP. CCR6+DN expressed higher levels of STAT3 mRNA and were enriched in other Th17-associated markers including IL-17F, RORA , IL-23R , SOSC3 , TIM3  and CD26/DPP4 . STAT3 is one of the first transcription factors to be up-regulated within the first hours of Th17 polarization . Cell fate mapping experiments demonstrated IL-17F is expressed during early stages of Th17 differentiation . Further, CCR6+DN were found to express a stem-cell-like molecular signature including RORA, STAT3, LEF1, MYC and TERC [25, 46, 63, 64] as well as the NANOG signaling pathway, important for the maintenance of stem cells . LEF1 and MYC characterize long-lived Th17 cells with self-renewal properties . TERC is linked to telomerase activity, which is up-regulated in stem cells, cancer cells and proliferating lymphocytes . In contrast, CCR6+DP  together with Th17 expressed high levels of LMNA, a senescence marker . These findings feed the idea that CCR6+DN versus Th17 and CCR6+DP represent a less advanced stage of Th17 differentiation with superior survival potential.
In addition to IL-17F, CCR6+DN are distinguished from the other CCR6+ subsets by their relatively high production of IL-8 and IL-21. IL-8 is critical for neutrophil recruitment at inflammatory sites , indicative of superior effector functions for CCR6+DN. IL-21 is the hallmark cytokine for follicular helper T cells (Tfh) . IL-21 up-regulates expression of the Tfh-specific transcription factor BCL6 and the chemokine receptor CXCR5 . Consistently, CCR6+DN express the highest levels of BCL6 and CXCR5 mRNA, and are also enriched in ASCL2 mRNA, another Tfh-specific transcription factor . Thus, CCR6+DN cells share Tfh features. This is in line with a proposed differentiation model in which Tfh development occurs subsequent to Th1, Th2 or Th17 polarization . Similar to Th17 but in contrast to Th1Th17 , CCR6+DN and CCR6+DP produce low/undetectable levels of IL-2, a cytokine known to promote cell survival . IL-21 acts as a survival factor for Th17 cells . IL-21 supplementation leads to the restoration of IL-17A-producing cells in SIV-infected macaques . Therefore, IL-21 produced by CCR6+DN may act in autocrine manner to support superior survival of these cells and compensate for their limited IL-2 expression.
Th17 cells mediate immunity against pathogens localized at barrier surfaces including C. albicans [3, 5]. Here, we reveal that CCR6+DN, similar to Th17, proliferate and produce IL-17A in response to C.albicans but not CMV. We also observed proliferation of Th1Th17 and Th1 in response to C. albicans and CMV. These results are consistent with recent studies from Becattini et al. in which Th17, Th1Th17 and Th1 share C.albicans clonotypes, while expressing distinct effector cytokines . Clonotype sharing between Th17 subsets is consistent with previous reports on the acquisition of Th1 features by Th17 cells under inflammatory conditions . Whether CCR6+DN and Th17 share C.albicans clonotypes remains to be investigated. The antigenic specificity of CCR6+DP was not tested in our study. However, Becattini et al. identified a population similar to CCR6+DP that shared with Th1Th17 M. tuberculosis clonotypes , thus suggesting a potential differentiation relationship between CCR6+DP and Th1Th17.
In contrast to CCR6−, CCR6+ T-cells are prone to express Th17 functions . Consistently, the frequency of cells expressing IL-17A, IL-17F and IL-22 was dramatically increased in all CCR6+ subsets upon long-term exposure to Th17-polarizing signals. Notably, CCR6+DN and Th17 were distinguished from CCR6+DP and Th1Th17 by their superior IL-17A/IL-17F and reduced IFN-γ expression. Th17 cells are known to be extremely plastic and can dedifferentiate into other lineages . As such, all Th17 subsets diminished expression of Th17 cytokines and enhanced IFN-γ expression in response to Th1-polarizing signals. This is consistent with other studies in mice and humans demonstrating suppression of Th17 effector cytokines upon IL-12 exposure [6, 69]. Our results reveal that CCR6+DN and CCR6+DP, together with Th17 and Th1Th17, are four distinct Th17-commited subsets that exhibit lineage plasticity. Noteworthy, a fraction of CCR6+DN and Th17 preserved production of Th17 cytokines under Th1 conditions, providing evidence that stably-committed Th17 cells exist in humans, as previously predicted . Our results also confirm the stability of the Th1 transcriptional program .
The chemokine receptors CCR6, CCR4, and CXCR3 mediate T-cell recruitment into gut-associated lymphoid tissues [7, 70], skin , and various other inflammatory sites . We observed that the expression of these receptors represents stable signatures for a fraction of CM Th17-subsets upon exposure to Th17/Th1-polarization signals. In contrast to CCR4 and CXCR3, the expression of CCR6 was particularly stable under Th17-polarizing conditions, consistent with the identification of stable epigenetic mechanisms involved in the regulation of CCR6 expression . CCR6 expression was mainly decreased upon exposure to Th1-polarizing cytokines, consistent with the reported ability of IL-12 to down-regulate CCR6 . The expression of CCR4 and CXCR3 was stable on >70 % Th17 and >80 % Th1Th17, respectively, regardless of the polarization conditions. Interestingly, CCR6+DN cultured under Th17- or Th1-conditions acquired a “classical” Th17-phenotype (CCR6+CCR4+), while CXCR3, a marker for Th1Th17 and Th1-cells [5, 15], was similarly acquired by CCR6+DN and Th17 under Th17-/Th1-conditions. It is reported that Th1Th17-cells derive from Th17-cells that acquired Th1-features under inflammatory conditions [75–77]. Our results demonstrate that CCR6+DN acquire Th17 and/or Th1Th17 phenotypic features. In contrast to CCR6+DN, a significant fraction of CCR6+DP lost CCR4 and/or CXCR3, regardless of polarization conditions, and acquired a Th1Th17 but not Th17 phenotype. All these findings reveal flexibility in the expression of CCR6, CCR4 and/or CXCR3 by these Th17-subsets, flexibility that may be relevant for their differential recruitment into various tissues. This flexibility suggests a developmental relationship between these four Th17-subsets via molecular mechanisms that remain to be further investigated.
Of particular importance, we demonstrate that the four CCR6+ T-cell subsets exist in the peripheral blood of HIV-infected individuals and exhibit a Th17-polarization profile similar to subsets from uninfected individuals. The CCR6+DN were the most preponderant among Th17 subsets in the peripheral blood of ART-treated individuals as their frequency/counts were preserved at levels similar to uninfected individuals. Furthermore, in a longitudinal follow-up in five HIV-infected individuals, CCR6+DN proved to be the most predominant CCR6+ subset before/after ART initiation. This raised the possibility that CCR6+DN are resistant to infection.
In contrast to the above prediction, we demonstrated that all four Th17 subsets, including CCR6+DN, were permissive to HIV infection in vitro and in vivo. CCR6+DN and CCR6+DP expressed the HIV coreceptors CCR5 and CXCR4 at levels similar to Th17 and Th1Th17, respectively. Accordingly, all Th17 subsets were permissive to HIV in vitro. Furthermore, CCR6+DN, Th17 and Th1Th17 harbored proviral HIV-DNA in RI individuals, indicative of their HIV permissiveness in vivo. Of particular interest, all four CCR6+ subsets were found to carry relatively high levels of integrated HIV-DNA in virologically suppressed ART-treated individuals. We further demonstrated that viral transcription and production, reflected by the detection of HIV-RNA in cell culture supernatants, occurred in all four Th17 subsets, including CCR6+DN cells. Finally, we demonstrate active viral replication in CCR6+DN cells cultured for 13 days under Th0 polarizing conditions in vitro. Of note, HIV-p24+ CCR6+DN cells from CI on ART individuals co-expressed IL-17A and were highly poly-functional, and a fraction of them were able to proliferate in response to repetitive IL-2 stimulation or TCR triggering in vitro. These results support the contribution of CCR6+DN cells to HIV reservoir persistence under ART. This is consistent with a model in which integrative infection is compatible with survival  and CCR6 triggering via CCL20 promotes HIV latency  and suggests that permissiveness to HIV integration compatible with survival represents a new previously unrecognized feature of pathogenic Th17 cells during HIV infection. Future functional validations on the host cell machinery components used by HIV to insure its persistence in specific Th17 subsets such as CCR6+DN cells are important for the design of new targeted “shock and kill” strategies. Alternatively, other strategies may involve the interference with the CCL20-CCR6 interaction using small molecule inhibitors that may limit CCL20-mediated establishment of HIV latency in CCR6+ T-cells, consistent with the model proposed by Lewin et al. . The fact that CCR6+DN exhibit CM features and are located in the lymph nodes emphasized the complexity/challenge of HIV eradication.
Our results that CCR6+DN express Tfh markers are in line with a recent study demonstrating the restoration of CCR6+ T cells expressing a Tfh signature (CCR7highCXCR5highPD-1high) under ART . CD4+ T cells with CM features constitute the main reservoir of latent HIV . Indeed, we demonstrate that CCR6+DN are enriched in cells with CM phenotype and they are the most predominant CCR6+ T-cell subset in the inguinal lymph nodes of three ART-treated individuals. Future studies are required to determine whether CCR6+DN from lymph nodes exhibit Tfh features and carry replication-competent HIV reservoirs.
Study individuals and biological samples
Human individuals were recruited at the Montreal Chest Institute, McGill University Health Centre, and the Universite de Montreal Hospital Centre (CHUM), Montreal, Quebec. Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis and cryopreserved until use . Cytomegalovirus (CMV) infection was determined upon detection of CMV-specific Abs using chemiluminescent microparticle immunoassay (CMIA) . In parallel, matched blood and inguinal lymph node samples were collected from three HIV-infected individuals from the SCOPE study (UCSF School of Medicine, CA, USA) who were on stable ART with undetectable viremia (<40 HIV RNA copies/ml) for at least 8 years. After careful removal of the surrounding fatty tissue, lymph nodes were mechanically disrupted using frosted glass slides. The single cell suspension obtained was filtered and benzonase digested (25 U/mL) before to be used for flow cytometry analysis.
Antibodies and polychromatic flow cytometry analysis
Detailed description available in Online supplemental material.
Magnetic (MACS) and fluorescence activated cell sorting (FACS)
Total CD4+ T-cells were sorted from PBMCs by negative selection using magnetic beads (Miltenyi Biotec) , and stained with CD45RA, CCR4, CXCR3 and CCR6 Abs, and a cocktail of FITC–conjugated CD8, CD19, and CD56 Abs. Memory CD4+ T-cells (CD45RA−) with differential expression of CCR6, CCR4 and/or CXCR3 were sorted by FACS (BDAria II; BD Biosciences): CXCR3−CCR4+ (CCR4+Th17 [5, 15]), CXCR3+CCR4− (CXCR3+Th1Th17 [5, 15]), CXCR3−CCR4− (CCR6+DN), and CXCR3−CCR4− (CCR6+DP). Total CCR6− or CXCR3+CCR4− (Th1 [5, 15]) subsets were sorted in parallel. Sorting gates were set on FITC- to exclude CD8+ T-cells, CD19+ B-cells, and CD56+ NK-cells. For specific experiments, central (CM, CD45RA−CCR7+) and effector memory (EM, CD45RA−CCR7−) subsets with differential expression of CCR6, CCR4 and/or CXCR3 were sorted. Sorting gates were set based on FMO controls . Quality control analysis post-sort indicated an average purity >95 % (Additional file 1: S1 Figure).
Genome-wide transcription profiling
Matched memory CD4+ T-cell subsets were isolated by FACS from different HIV-uninfected donors stimulated with immobilized CD3 and soluble CD28 (1 µg/ml) for 4 days. Total RNA was isolated using RNeasy columns kit (Qiagen) according to the manufacturer’s protocol. RNA quantity was determined by Pearl nanophotometer (Implen, Germany) (106 cells yielded 1–5 µg RNA). Genome-wide analysis of gene expression was performed on total RNA by Génome Québec Innovation Centre (Montreal, Qc, Canada). Briefly, the quality of total RNA was tested using the Agilent 2100 Bioanalyzer chip. High quality RNA was reverse transcribed and hybridized on the Illumina HumanHT-12 v4 Expression BeadChip providing coverage for more then 47,000 transcripts and known splice variants across the human transcriptome. The expression of differentially expressed genes was identified as previously described . The entire microarray dataset and technical information requested by Minimum Information About a Microarray Experiment (MIAME) are available at the Gene Expression Omnibus (GEO) database under accession number GSE66972. Differentially expressed genes (cut-off 1.3-fold; p < 0.05) were classified through Gene Ontology using the NetAffx web-based application (Affymetrix), while differentially expressed pathways were identified using Ingenuity Pathway Analysis (IPA) and Gene Set Variation Analysis (GSVA). Corresponding heat maps for biological function categories were generated using programming language R .
One step SYBR Green real-time RT-PCR was carried out in a LightCycler 480 II (Roche) using Qiagen reagents according to manufacturer’s recommendations, as previously described [55, 85]. Briefly, STAT3, BCL-6, Lef1, and Terc primers were purchased from Qiagen (QuantiTect Primer Assay). The expression of each gene was normalized relative to the internal control 28S rRNA levels (forward 5′-CGAGATTCCTGTCCCCACTA-3′; reverse 5′-GGGGCCACCTCCTTATTCTA-3′ (IDT ). Samples without template or without reverse transcriptase were used as negative controls. Each RT-PCR reaction was performed in triplicata.
Cytokine screening and ELISA quantification
Cell culture supernatants were screened for the expression of 34 cytokines using the Human Th1/Th2/Th17 Antibody Array C series (RayBiotec, Norcross, GA), as previously described . Cytokine levels were quantified by ELISA using commercial kits for IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, TNF-α (Ebioscience) as well as CXCL8/IL-8 and CCL20 (R&D Systems).
Intracellular cytokine staining
Intracellular expression of cytokines was measured by FACS  using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above). Analysis was performed using Diva and FlowJo. Polyfunctional profiles were analyzed using the SPICE (version 5.3; provided by Mario Roederer, VRC/NIAID/NIH) .
Antigen presentation assay
Cell proliferation was measured using the carboxy fluoroscein succinimidyl ester (CFSE) dilution assay , following co-culture with antigen-loaded autologous monocyte-derived dendritic cells (MDDC). Briefly, monocytes isolated from PBMCs by negative selection using magnetic beads (Miltenyi Biotec)  were differentiated into MDDC in the presence of GM-CSF and IL-4 (20 ng/ml; R&D Systems). MDDCs were loaded with SEB (25 ng/ml; Toxin Technologies), CMV-pp65 peptide pool (1 μg/ml; Miltenyi) or Candida albicans hyphae LAM-1  (25 µl protein lysate ) for one hour at 37 °C and cocultured with FACS-sorted CFSE-loaded T-cell subsets. MDDC:T-cell cocultures (1:4 ratio) were maintained for 5 days at 37 °C. MDDC:T-cell co-cultures were stained with CD3 (T-cell marker) and CD1c (DC marker). Proliferating T-cells were identified as cells with a CFSElowCD3+CD1c− phenotype. In parallel, MDDC:T-cell co-cultures were stimulated with PMA and Ionomycin in the presence of brefeldin A and the expression of cytokines in proliferating T-cell subsets was quantified by FACS upon intracellular staining with specific anti-cytokine Abs (See table above). Intracellular expression of cytokines was quantified by FACS .
Th17 versus Th1 polarization
Cells were stimulated via CD3/CD28 and cultured under Th17-polarizing conditions (IL-1β (10 ng/ml), IL-6 (50 ng/ml), and IL-23 (40 ng/ml), and anti-IL-4 (1 μg/ml) and anti-IFN-γ Abs (10 μg/ml) (R&D Systems)) or Th1-polarizing conditions (IL-12 (5 ng/ml) (R&D Systems) and anti-IL-4 Abs (1 μg/ml)) for 4 days. Cells were washed and plated in media containing the Th17- or Th1-polarization cocktails together with recombinant IL-2 (5 ng/ml) for 10 additional days. Polarizing cytokines were replenished every 2–3 days and cells were split to optimal density (<2 × 106 cells/ml).
HIV infection in vitro
FACS-sorted memory CCR6+ T-cell subsets were activated for 4 days via CD3/CD28 (1 μg/ml) and then exposed to HIV-1 ADA8 (50 ng HIV-p24 per 106 cells) for 3 h at 37 °C. Extensive washing was performed to remove unbound HIV. Cells (106 cells per ml) were cultured in media containing FBS (10 %) and IL-2 (5 ng/ml; R&D Systems). Cell culture supernatants were harvested at day 3 post-infection and HIV replication was measured by HIV-p24 ELISA.
Real-time PCR quantification of integrated HIV-DNA
The quantification of integrated HIV-DNA was performed as we previously described [15, 82]. Briefly, cells were digested in a proteinase K buffer (Invitrogen), and 105 cells/15 µl lysate were used per amplification. Integrated HIV-DNA was amplified first (12 cycles) using two outward-facing Alu primers and one HIV LTR primer tagged with a lambda sequence; the CD3 gene was amplified in the same reaction. The HIV and CD3 amplicons were then amplified in separate reactions (Light Cycler 480, Roche Diagnostics). The HIV-DNA was amplified using a lambda-specific primer and an inner LTR primer in the presence of two fluorescent probes specific for HIV LTR. The CD3 DNA was amplified using inner primers and two fluorescent probes specific for CD3. Amplification reactions were carried out with Light Cycler 480 Probe Master Mix (Roche) and Taq Polymerase (Invitrogen). The ACH2 cells carrying one copy of integrated HIV-DNA per cell (NIAIDS reagent program) were used as standard curve.
HIV reactivation assay
Highly pure matched memory CD4+ T-cell subsets were isolated by FACS from different CI on ART individuals and stimulated with immobilized CD3 and soluble CD28 Abs (1 µg/ml) for 4 days. Cells were washed and plated in media containing recombinant IL-2 (5 ng/ml) for 9 additional days. IL-2 was replenished and cells were split to optimal density (<2 × 106 cells/ml) every 3 days. At day 13, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 6 h. Intracellular staining was performed with cytokine-specific Abs and HIV-p24. Cell culture supernatants were collected at day 4, 7, 11, and 13 post-TCR triggering and used for the quantification of HIV-RNA and/or HIV-p24 levels.
Real-time RT-PCR quantification for HIV-RNA
Viral RNA was isolated from cell-culture supernatants using the QIAmp viral RNA mini kit (Qiagen) according to the manufacturer’s protocol. Collected RNA was treated with DNase, reverse transcribe and then amplified for 20 cycles using primers specific for HIV-LTR (tagged with a lambda sequence) and HIV-GAG. The HIV-DNA was further amplified using a lambda-specific primer and an inner HIV-LTR primer as well as two fluorescent probes specific for HIV-LTR, using a protocol adapted from .
Statistical analyses were performed using the GraphPad Prism 5. Details are included in Figure legends.
VSW performed research, analyzed data, and wrote the manuscript. JPG performed microarray data analysis and generated figures. AG and PM performed experiments. HS contributed to research design and manuscript writing. RF performed experiments. MAJ, SV, SGD, NC, and JPR ensured access to clinical samples/information and/or provided experimental protocols. PA designed research, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.
The authors thank Laurence Lejeune and Dr. Dominique Gauchat for expert technical support in FACS analysis and cell sorting (the CHUM-Research Centre FACS Core Facility), Anne Vassal and Mario Legault (HIV/AIDS and Infectious Diseases Network of the Fonds de Recherche Québec-Santé (FRQ-S)) for help with ethical approvals and informed consents, Dr. Louis de Repentigny and Mathieu Goupil for Candida albicans LAM-1 strain preparation, Dr. Dana Gabuzda (Dana-Farber Cancer Institute, Boston, MA, USA) for the HIV-ADA8 molecular clone, Dr. Mohamed Salah and Tomas Fabre for technical assistance with SPICE, and Dr. Shoukry and Dr. El-Far for critical reading of the manuscript. We thank Josée Girouard and Angie Massicotte (McGill University Health Centre) for their involvement in patient recruitment and leukapheresis collection, and Amélie Pagliuzza and Marion Pardons for assistance with lymph node sample treatment. We finally acknowledge the major contribution to this work of all human donors via their gift of leukapheresis.
The authors declare that they have no competing interests.
The entire microarray dataset and technical information requested by Minimum Information About a Microarray Experiment (MIAME) are available at the Gene Expression Omnibus (GEO) database under accession number GSE66972.
Ethics approval and consent to participate
Written informed consents and Institutional Review Board approvals were obtained from all HIV-infected and uninfected individuals included in this study, in compliance with the Declaration of Helsinki principles.
This work was supported by grants to PA from the CIHR (MOP-82849; MOP-114957). VSW received a CIHR Ph.D. Fellowships. JPR holds a Louis-Lowenstein Chair in Hematology and Oncology, McGill University. Human cohorts were supported by the FRQ-S/AIDS and Infectious Diseases Network.
Online supplemental material
Supplemental information includes Supplemental Table 1 legend, Supplemental Tables 2–3, Supplemental Fig. 1–9 legends, Supplemental Materials and methods, and Supplemental references.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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