Characterization of a Human Cervical CD4⁺ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

There were >1.8 million new HIV infections in sub-Saharan Africa in 2009, almost three quarters of the global total, and most of these infections were in women (1). Despite the scale of the epidemic, the probability of HIV transmission during a single act of unprotected receptive penile-vaginal sex with an HIV⁺ man is <0.2% (2). The fact that our natural mucosal immune defenses are >99% protective is very fortunate from the perspective of epidemic spread, but this very low per-act transmission probability makes it difficult to define the mucosal immune basis of virus acquisition or altered host susceptibility (3).

HIV transmission within stable, serodiscordant couples is associated with a higher HIV RNA virus level in both the blood and the genital tract of the infected partner (4, 5). In an analogous fashion, HIV susceptibility could also be enhanced by factors that increase the number of potential HIV target cells in the genital mucosa of the uninfected partner, which is one of several proposed mechanisms to explain the increase in HIV transmission associated with infection by herpes simplex type 2 (6). These observations suggest that two thresholds must be overcome for HIV transmission to occur. First, there may be a threshold level of infectious virus needed in the genital secretions of the infected partner; second, there may be a threshold number and/or density of susceptible target cells required in the genital mucosa of the uninfected partner (7). Nonhuman primate models support this concept, documenting initial foci of infected mucosal CD4⁺ T cells that expand prior to systemic viremia, and pilot studies demonstrated that a microbicide blocking mucosal CD4⁺ T cell recruitment prevented SIV infection (8). A better definition of the initial mucosal cells present in the female genital tract that are targeted by HIV during human sexual transmission might assist in the design of better biomedical prevention strategies.

In addition to functional heterogeneity, not all CD4⁺ T cells are equally susceptible to HIV infection (9). The near-uniform transmission during sex of HIV strains that use the CCR5 coreceptor (R5 tropic strains) implies that HIV preferentially infects mucosal target cells that express CCR5 (10). It was also demonstrated that HIV replicates preferentially within activated T cells (11). The integrin α4β7, induced on T cells after priming by mucosally derived dendritic cells, mediates T cell trafficking to gut mucosal sites via a cascade of receptor interactions on gut endothelial cells that includes the addressin MadCAM (12). α4β7 was shown to bind HIV gp120 directly and correlates with enhanced virus infection and cell–cell spread (13, 14). In addition, α4β7⁺ CD4⁺ T cells were reported to be preferentially infected during acute SIV infection in nonhuman primates (15).

The CD4⁺ Th17 subset is defined by the production of IL-17A and plays a key role in antimicrobial defense at mucosal sites, as well as in some autoimmune diseases (16). Overactivity of Th17 cells is associated with the gastrointestinal pathology observed in Crohn’s disease (17), whereas Th17 deficiency underpins chronic mucocutaneous candidiasis (18, 19). As might be expected from their inflammatory nature, HIV replicates preferentially within Th17 cells (20). Not surprisingly, these cells are depleted during HIV infection (21), particularly in the gut mucosa (22), but are maintained in HIV⁺ individuals with an undetectable viral load (23). However, the presence and correlates of Th17 cells within the genital tract have not been examined and neither has their potential role as an HIV target cell at this mucosal site.

In this article, we demonstrate that a subset of activated cervical CD4⁺ T cells express α4β7, CCR5, IL-17A, and IFN-γ. We further show that these cells preferentially bind HIV-1 gp120 in vitro and are almost entirely depleted from the cervix in vivo during HIV infection. The presence of multiple susceptibility markers on a subset of genital tract CD4⁺ T cells suggests that these cells may play a key role as HIV targets during sexual transmission.

Informed written consent was obtained prior to participation, and Institutional Review Boards at Kenyatta National Hospital (Nairobi, Kenya) and Universities of Manitoba and Toronto (Canada) approved the study. The described studies were conducted according to the principles expressed in the Declaration of Helsinki.

Participants were recruited for the study from existing female sex worker (FSW) cohorts located in Nairobi, Kenya. Each participant was tested for HIV-1 serology, peripheral CD4 counts, bacterial vaginosis (BV) by Gram stain, Chlamydia trachomatis and Neisseria gonorrhea by PCR (Roche), and Trichomonas vaginalis by InPouch. Demographic and behavioral data were also collected. HIV⁺ participants were said to be in the early stage of infection if sampled <6 mo from the suspected infection date (midpoint between last HIV⁻ and first HIV⁺ visit).

Blood was collected by venipuncture using heparin as an anticoagulant. Cervical sampling included the collection of samples in the following order: urine; cervicovaginal lavage using 2 ml sterile PBS; two Dacron swabs for assessment of cervicovaginal viral shedding, collected into viral-transport medium; a vaginal swab for BV; and cervical cytobrush and cell scraper for collection of cervical immune cells. The present study focused on cytobrush samples to define cell populations. Cytobrushes were inserted into the cervical os and rotated 360°, followed by a gentle scraping of the ectocervix and a mini-lavage to collect cells. Cytobrushes were transferred to a conical vial containing PBS and transported on ice to the laboratory for processing.

Cervical immune cells were obtained from the cytobrush and filtered through a 100-μm filter (Becton Dickinson [BD]). Tubes were vortexed first several times to remove as many cells as possible from each cytobrush. After filtering, cells were washed in warm RPMI 1640 10% FBS 1% antibiotic/antimycotic and used in the assays described below.

We used the following flow cytometry Abs for staining PBMC and cervical cells in a number of experiments: Act-1–PE (α4β7, National Institutes of Health AIDS Reagent program), CD3-Alexa Fluor 700, CD4-ECD (Beckman Coulter), CCR9-Alexa Fluor 647, CD103-FITC, CCR5–PE–Cy5, allophycocyanin-Cy7, CXCR4–PE–Cy5, CD69–PE–Cy7, IL-17A–FITC, IL-22–allophycocyanin, IFN-γ–PE–Cy7, and TNF-α–Alexa Fluor 700. All Abs were from BD, unless otherwise indicated.

Washed cervical cells were stained for surface markers, including pretitrated amounts of Vivid (Invitrogen) to discriminate dead from live cells, for 20 min at room temperature. Samples were acquired on a BD LSRII Flow Cytometer configured for detection of 10 colors. All events were collected for cervical samples.

Cells were resuspended in IMDM supplemented with 10% Serum replacement factor (Invitrogen) 1% antibiotic/antimycotic and then stimulated with PMA (1 ng/ml) and ionomycin (1 μg/ml) for 6 h. After the first hour, protein secretion was inhibited through addition of pretitrated amounts of BD GolgiStop and GolgiPlug. Surface staining was carried out as above, followed by washing, permeabilization using BD Fix/Perm, and staining with a mixture of Abs to intracellular markers for 15 min at room temperature. Cells were then washed and fixed in 1% paraformaldehyde and acquired as described above. Results are reported following background subtraction.

Samples were processed and assayed via quantitative real-time PCR (qPCR) as previously described (24). RNA was extracted from cell pellets, and genomic DNA was eliminated using the Qiagen RNeasy Plus Kit (Qiagen). cDNA was created by adding 10 ng RNA to the RT master mix and was reverse transcribed using the Superscript III Kit (Invitrogen). Genomic DNA standards were isolated from purified placental tissue and used as a universal standard over a 7-log dilution range to calculate relative gene-expression levels (25). Single intraexon gene-specific primers were generated using Primer Express Software (Perkin Elmer Applied Biosystems) or OligoPerfect (Invitrogen). Specific primers were designed to target IL-17A (forward, 5′-CATGAACTCTGTCCCCATCC-3′; reverse, 5′-CCCACGGACACCAGTATCTT-3′) and IL-22 (forward, 5′-TGCATTTGACCAGAGCAAAG-3′; reverse, 5′-AGTTTGGCTTCCCATCTTCC-3′) mRNA induction. SYBR green fluorescent dye was used to detect amplification under previously published amplification conditions (24). All reactions were run in triplicate. Quantitative PCR values crossing threshold were obtained during the exponential amplification phase using SDS 2.3 Software (Applied Biosystems). The housekeeping genes GAPDH and β-actin were used to assess target gene expression in the cervix and blood, respectively (24). Gene quantities were calculated from standard curves in arbitrary units and normalized by dividing the amplified gene target by the housekeeping gene for each sample and reported as the normalized gene induction ratio. All kits were used per the manufacturer’s instructions.

Cell culture supernatants were saved following the same stimulation as above for mRNA quantification. The Searchlight chemiluminescent multiplex-ELISA assay (Aushon Biosystems, Billerica, MA) was used to quantify IL-17A and IL-22 cytokines and was run per the manufacturer’s instructions.

Cervical cells were collected and isolated as described and resuspended in buffer either containing divalent cations (Mn⁺⁺Ca⁺⁺) or EDTA. Following blocking with mouse and human IgG, unlabeled SK3 mAb was added to all wells to block CD4 binding. The CD4-blocked wells contained divalent buffers, as this is shown to facilitate integrin binding. EDTA was used as an integrin⁻ control (i.e., α4β7 block). Two to five micrograms of biotinylated gp120 Z205F (26) was incubated with the cells for 25 min, followed by washing and incubation with pretitrated amounts of neutravidin-PE and surface Abs for 20 min at room temperature. Cells were washed again, fixed using 1% paraformaldehyde, and acquired on the LSRII. Results are presented as gated on CD3⁺ CD8⁻ cells (CD4 is blocked).

Coexpression of markers was assessed using paired Student t tests. Differences in levels of expression between groups was determined by the Mann–Whitney U test. Correlations were determined using Spearman rank correlation. All statistical tests were run on SPSS v. 17.0 for Mac. Flow cytometry data were analyzed in FlowJo v. 8.6.6. and Microsoft Excel prior to statistical testing.

Convenience samples were obtained from HIV⁻ and HIV⁺ FSW participants recruited through two research clinics in Nairobi, Kenya between July and December 2009. Twenty HIV⁻ FSWs were enrolled for characterization of surface marker expression. An additional 56 women were studied for cytokine responses following stimulation (demographics below). Given the limited cell yield obtained from a cervical cytobrush (27), it was necessary to perform the ex vivo and stimulation studies on different groups of participants. For the surface-expression study (n = 20), the median age, duration of sex work, and clients/wk for these participants was 30.8 y, 5.2 y, and 12, respectively. All screened negative for C. trachomatis, N. gonorrhea, T. vaginalis, and syphilis; 63% were BV-negative on Gram’s stain. For the cytokine studies, the median age for HIV⁻ (n = 41) participants was 30.3 y, with 2.9 y in sex work and 8.2 clients/wk. Sexually transmitted infections were rare (3/41, 7%), and included one case of N. gonorrhea, one case of syphilis, and one case of T. vaginalis. Most participants (66%) were BV-negative.

We performed surface phenotyping of CD4⁺ T cells from the blood and cervix, examining ex vivo expression of a number of surface receptors after gating on live CD3⁺ CD4⁺ T cells derived from cervical cytobrush and blood samples. These included the mucosal homing markers CCR9, α4β7, and CD103; the early immune activation marker CD69; and the HIV coreceptors CCR5 and CXCR4 (Fig. 1A). There was a wide range of α4β7 expression on cervical CD4⁺ T cells (median 26.0, IQR 15.5–39.6%). The majority of α4β7 expression (median 91.8% of α4β7⁺ T cells) was intermediate in intensity (Fig. 1B), as has been described in ex vivo studies of intestinal tissue (28), with the remainder being α4β7^hi. Expression of the integrin CD103 (αEβ7) was less common (median 9.6%), corroborating a previous report by our group (27), but was significantly higher in the cervix than in peripheral blood (0.6%, p < 0.0001). Moderate levels of the gut homing marker CCR9 expression were expressed (median, 17.9%; interquartile range [IQR], 13.1–33.3%, compared with 5.3% in blood). Although CCR9 and α4β7 have both been described as gut-homing markers, their expression levels did not correlate (p = 0.46), and coexpression analysis demonstrated that these molecules were often expressed on different cells (data not shown). Relatively high levels of CXCR4, CCR5, and CD69 were observed on cervical CD4⁺ T cells (medians of 38.7, 81.5, and 53.6%, respectively), all of which were at higher levels in the cervix compared with blood (all p < 0.01; Fig. 1A). CCR5 was particularly overexpressed in the cervix compared with the blood (>10-fold higher); understanding further the phenotypic characteristics of these CD4⁺ T cells has obvious implications for HIV transmission, given that CCR5-tropic strains are responsible for establishing HIV infection during male–female vaginal transmission.

To determine coexpression patterns of various susceptibility markers on the same cervical CD4⁺ T cells, we assessed the expression of various markers in conjunction with α4β7 staining. Gating on CD3⁺ CD4⁺ T lymphocytes, we found high levels of double-positive populations for most of the markers assessed (Fig. 2A). Compared with α4β7⁻ cells, expression of CCR5 and CD69 was increased on α4β7⁺ CD4⁺ cervical T cells (Fig. 2B). The median CCR5 expression on α4β7⁺ CD4⁺ cervical T cells was 81% (IQR, 61–93%), compared with 62% (IQR, 46–81%) on α4β7⁻ CD4⁺ T cells (p < 0.0001). Furthermore, the mean fluorescence intensity of α4β7 on CD4⁺ T cells correlated with the levels of CCR5 expression on those cells (r = +0.565, p = 0.009, Fig 2C). Similarly, the median CD69 expression on α4β7⁺ CD4⁺ T cells was 53.4% (IQR, 33–79%), compared with 46.2% (IQR, 25–62%) on α4β7⁻ CD4⁺ T cells (p < 0.0001). Because each of these markers defines a T cell subset that is preferentially infected by HIV in vitro (29), their coenrichment on the surface of the same cervical CD4⁺ cells suggests that this coexpression may represent an important mucosal target cell during sexual HIV transmission. Conversely, CXCR4 was not preferentially expressed on α4β7⁺ CD4+ T cells compared with α4β7⁻ CD4⁺ T cells (median, 44.3 versus 37.6%, p = 0.34, Fig. 2B).

Although Th17 and Th22 cells are gaining prominence for their roles in mucosal immunology of the gut and skin (30, 31), these cell subsets have not been described in the female genital tract. We went on to characterize the frequency and phenotypes of Th17 cells in blood and cervix of HIV⁻ FSWs (n = 41). Representative gating for Th17 responses in the blood and cervix is shown in Fig. 3A and for IL-22, IFN-γ, and TNF-α is shown in Fig. 3B. Following PMA/ionomycin stimulation, IL-17⁺ CD4⁺ T cells coproduced a range of additional cytokines, and we compared coexpression patterns between the blood and cervix (Fig. 3C). Coexpression of IL-17 and IL-22 in the cervix was fairly modest (median, 22.2% of IL-17⁺ cells also expressed IL-22), and cervical Th17 cells tended to produce more IL-22 than did those in the blood (22.2 versus 12.0%; p = 0.07). We also found modest frequencies of the IFN-γ⁺ IL-17⁺ subset (Th1 Th17) in the female genital tract, and coexpression of IFN-γ⁺ was three times higher in the cervix than the blood (median, 29.5 versus 8.9%, p < 0.0001). TNF-α was highly expressed in cervical Th17 cells (median 60.2%, Fig. 3D), albeit with significant heterogeneity between participants, but the proportion of Th17 cells coexpressing TNF-α was substantially higher in the blood than the cervix (median 100 versus 60.2%, p < 0.0001). These data demonstrated substantial functional differences in Th17 populations derived from the blood and genital mucosa.

Next, we tested the hypothesis that Th17 cells from the cervix would preferentially express α4β7 and CCR5, characteristics that would be expected to enhance their susceptibility to HIV infection. CCR5 expression was substantially increased on Th17 cells from the cervix compared with blood samples from the same participants (Fig. 4A, 4B). The median expression of CCR5 on cervical IL-17⁺ T cells was 68.7% compared with 10.8% in the blood (p < 0.0001, Fig. 4B). This was not merely a reflection of higher overall expression of CCR5 in the cervix; indeed, when we compared the IL-17A⁺ subset with other cervical CD4⁺ T cells, there was an increased expression of CCR5 (p = 0.026, Fig. 4C). We then extended this analysis to IL-17⁺ CD4⁺ T cells expressing α4β7. Consistent with our data on total α4β7⁺ CD3⁺ CD4⁺ T cells (Fig. 2), the majority of cervical IL-17⁺ CD4⁺ T cells coexpressed both α4β7 and CCR5 (67.2–75.2%, Fig. 4D).

Given that several susceptibility markers cluster on the same subset of cervical CD4⁺ T cells, one hypothesis is that HIV is able to target these cells preferentially over other CD4⁺ T cells from this site. We tested this by assessing ex vivo binding of biotinylated gp120 cells. We focused on CCR5 to represent the subset of optimal target cells, because this marker coincided with the others and was expressed at high levels, aiding detection despite low cell recovery from the cervix; low cell numbers precluded using markers, such as cytokines, which require cell permeabilization. α4β7–gp120 binding was implied by inhibition experiments in buffer that precludes integrin activation. Using an Env strain that had been isolated very soon after (and therefore relevant to) HIV transmission, we combined surface marker staining with gp120-binding ex vivo, after blocking CD4 with a mAb (Fig. 5). In two HIV⁻ participants, 78 and 83% of CD3⁺ CD8⁻ T cells that bound gp120 also expressed CCR5 compared with 53 and 62% of CD3⁺ CD8⁻ T cells that were gp120⁻. Increased CCR5 expression was particularly evident in the gp120^hi cells, which also held true in experiments in which both CD4 and α4β7 were blocked (Fig. 5A, right panels). In support of this, we observed a strong correlation between the mean fluorescence intensities of gp120 binding and CCR5 staining in cervical T cells from HIV⁻ participants in conditions where CD4–gp120 binding was blocked (n = 6, r = +0.89, p = 0.033, Fig. 5B). Finally, to determine the extent to which α4β7 binding might account for the preferential binding, we inhibited α4β7 and measured the decrease in gp120⁺ staining. In participant 1, this blockade led to a 43.3% decrease in gp120 binding; in participant 2, a 32.8% decrease in gp120 binding was observed (Fig. 5C). These data showed that HIV gp120 preferentially bound to CCR5⁺ cervical CD8⁻ T cells that coexpressed multiple susceptibility markers (above) and that this preference was partly due to α4β7 binding.

Given these in vitro data, we next hypothesized that a subset of CD4⁺ T cells expressing multiple susceptibility markers would be depleted from the cervix of HIV⁺ individuals. Although there are conflicting data regarding the depletion of CD4⁺ Th17 cells from the blood during HIV infection (32), this cell subset is consistently depleted from the gut mucosa of HIV⁺ participants and SIV⁺ primates (22, 33–35). To determine Th17 frequency at another mucosal site, we examined the impact of HIV infection status on the frequency and magnitude of the Th17 subset in the cervix and blood. Nearly all HIV⁻ subjects (23/24) had detectable responses in the blood, at a median frequency of 1.24% of CD3⁺ CD4⁺ T cells (Fig. 6A). Responses were detected less often in the cervix (16/41, 39%), but at a higher median frequency of 7.02% of CD3⁺ CD4⁺ T cells (in those with a positive assay, Fig. 6C). This lower frequency of detection may be associated with higher background, unstimulated cytokine levels in the cervix compared with the blood (median 6.2 versus 0.2% CD4⁺ IL-17⁺ T cells, p < 0.0001). Despite the differences in magnitude, there was a strong correlation between blood and genital tract IL-17 responses (r = +0.536, p = 0.001, data not shown). Similar data were observed for IL-22; although responses could be detected in the blood of 14/18 HIV⁻ participants, only 14/41 cervical samples were IL-22⁺ after background subtraction (Fig. 6D). Similar to IL-17, the magnitude of IL-22 responses was higher in the cervix than in the blood (4.04 versus 0.61%, Fig. 6B, 6D).

To determine whether these CD4⁺ T cell subsets were depleted from the cervix during HIV infection, we tested HIV⁺ FSWs (n = 16) from the same FSW cohorts for IL-17⁺ CD4⁺ T cells in cervical and blood samples, as above. Participants were HIV⁺ chronic antiretroviral therapy (ART) naive (n = 11), HIV⁺ on ART (n = 3), and HIV⁺ early infection (n = 2). IL-17 responses were common in the blood, detected in 12/12 HIV-infected participants at a median magnitude of 0.99% of CD3⁺ CD4⁺ T cells; this was comparable to the Th17 frequencies observed in HIV⁻ participants (median 1.24%, p = 0.9). In contrast, only 1/16 (6%) of HIV⁺ subjects had a detectable IL-17 response in the cervix (mean compared with HIV⁻, p = 0.034, Fig. 6C), and this response was low in magnitude (0.97% of CD4⁺ T cells). Similarly, although CD4⁺ T cell IL-22 responses were common in blood CD4⁺ T cells from HIV⁺ participants (9/12), only 1/16 participants (6%) had detectable IL-22 responses in the cervix (p = 0.099, Fig. 6D). Different HIV⁺ participants made cervical IL-17 and IL-22 responses (ART naive and ART experienced, respectively).

To confirm the observation that Th17 cells were preferentially depleted from the cervix during HIV infection, we recruited 44 HIV⁺ sex workers and 24 HIV⁻ nonsex worker controls and measured the expression of IL-17A and IL-22 mRNA by qPCR in cell pellets and protein expression in culture supernatants using multiplex ELISA in the cervix and blood after Staphylococcus enterotoxin B stimulation. Cervical analysis was only performed on samples with cell counts >2 × 10⁵. Compared with HIV⁻ participants, HIV⁺ participants had lower expression of both blood and cervical IL-17A mRNA (blood, p = 0.029; cervical mononuclear cells [CMC], p = 0.035), as well as protein (blood, p = 0.001; CMC, p = 0.032, Fig. 7). Similarly, there was lower expression of IL-22 mRNA (blood, p < 0.0001; CMC, p = 0.006) and protein (blood, p = 0 < 0.0001; CMC, p = 0.001, Fig. 7) in HIV⁺ participants.

A better understanding of the early events of HIV-1 sexual transmission, as well as the identification of the cells targeted at this stage, remains an important area of HIV research. It was demonstrated in nonhuman primate models that mucosal CD4⁺ T cells are key initial targets (36). Given that HIV is not transmitted during most sexual exposures, more work is needed to characterize the CD4⁺ T cell subsets present in the female genital tract of high-risk populations, as well as how these relate to HIV susceptibility. We described a subset of cervical CD4⁺ T cells that simultaneously expresses several cellular markers of HIV susceptibility, including CCR5, α4β7, IL-17, and IFN-γ. One hypothesis that accounts for the heterogeneity in the relative risk for HIV transmission during a given exposure is that a certain threshold of target cells is required to overcome innate defenses and establish HIV infection. Because the rate at which HIV infects and replicates in CD4⁺ T cells is heavily dependent on the type and activation state of the target cell in question (37), this threshold may differ depending on the concentration of optimal HIV targets, not only the number of possible target cells. Our data demonstrated that multiple HIV susceptibility factors are expressed in tandem on the same T cells, raising the hypothesis that this population may play an important role in establishing HIV infection. Further work is required to prove this hypothesis; such work could provide a possible new avenue for HIV microbicide research. The relevance of these cells as a highly HIV-susceptible subpopulation is supported by their near-complete depletion from the cervix of HIV⁺ individuals, as well as their preferential binding to gp120 in vitro. One important extension of these studies, although technically difficult for a number of reasons, is demonstration of preferential infection of these cells in vitro. At the moment, we are basing our assessment of susceptibility on a large body of previous research on cell susceptibility to HIV and measuring these factors ex vivo in a mucosal surface highly important for male-to-female transmission of HIV.

To our knowledge, this is the first study to describe CD4⁺ T cells in the human genital tract that produce IL-17 and IL-22. Previous work showed that HIV coreceptors, such as CCR5 and α4β7, are also expressed on Th17 cells in nonhuman primates (38), and our study confirmed this in humans. We showed that these cells are common in the cervix, where they display important phenotypic differences compared with the analogous populations in the peripheral blood. In particular, the dramatically increased expression of CCR5, in parallel with a relative expansion of the IFN-γ⁺ IL-17⁺ subset, provides a plausible explanation as to why this population would be rapidly and consistently depleted in HIV infection, because these cellular characteristics were found to associate with preferential HIV infectivity (39). However, in contrast to some previous reports (32), we found a depletion of IL-17⁺ or IL-22⁺ subsets in the blood of some, but not all, HIV⁺ individuals. Although the reason for this difference is unclear, it may relate to the relatively early stage of HIV infection of our participants; of note, our group showed that Th17 depletion occurs very early in the gut mucosa but only at more advanced HIV clinical stages in the blood (22). Another possibility is differences in assay conditions and cohorts studied between this and previous studies.

Emerging evidence suggests that mucosal Th17 depletion plays an important role in HIV pathogenesis (40). These cells are prominent in mucosal surfaces, including the gut, where substantial CD4⁺ T cell depletion has been observed very early in HIV infection (41–43). In nonhuman primates, depletion of Th17 cells following SIV infection was associated with loss of Salmonella typhimurium control, leading to systemic leakage of bacterial components that may enhance immune activation and disease progression (33). Furthermore, the balance of Th1 and Th17 cells in the gut was able to predict the development of disease in SIV⁺ monkeys (34), and Th17 cells were depleted from the gut mucosa in pathogenic, but not in nonpathogenic, SIV infection models (35). In the current study, we used three independent experimental approaches to show that T cells producing IL-17 and IL-22 are also dramatically reduced in the cervix of HIV⁺ women. The clinical impact of this mucosal depletion is not clear, but because Th17 cells are critical mediators of protection against mucosal fungal and bacterial infection (19, 44, 45), this suggests that the lack of Th17 and Th22 cells in the cervix may contribute to the increased frequency of candidiasis and other genital tract infections that is seen in HIV⁺ women (46).

Although we demonstrated that Th17 and Th22 are preferentially depleted from the female genital tract during HIV infection, because these subsets were defined by the production of IL-17 and IL-22, respectively, the possibility remains that these cells were present in the cervix but functionally impaired. Although this is doubtful given the potency of PMA/ionomycin as a cell stimulant, further studies will need to examine this possibility, either by using phenotypic markers of the Th17 subset, such as CCR6 and/or CD161, and/or by directly demonstrating enhanced HIV susceptibility in vitro.

In addition to showing that cervical Th17 cells are physically depleted ex vivo during HIV infection, we demonstrated that HIV-1 gp120, isolated from a patient with acute infection, preferentially bound to cervical CD4⁺ T cells expressing CCR5. On a per-cell basis, gp120 bound to cervical T cells with the highest level of CCR5 expression. Moreover, approximately half of this binding could be ablated in conditions that block binding to the integrin α4β7. Blood CCR6-expressing Th17 cells were demonstrated to be preferentially infected by HIV, independent of α4β7 (47), and our results suggested that one important reason for this may be the expression of multiple other HIV susceptibility markers by these cells.

The gut-homing integrin α4β7 binds to HIV Env directly and correlates with enhanced susceptibility of CD4⁺ T cells to HIV infection (13, 14). In this article, we demonstrated intermediate α4β7 expression on the same cells that express several other markers important for cellular HIV susceptibility. Because MAdCAM-1 and VCAM-1, the ligands for α4β7, display variable expression in the genital tract (48), it is unknown whether α4β7 expression on cervical T cells represents active homing or whether these are cells that are homing to this site through other means. In a mouse model of Chlamydia infection, MAdCAM-1, VCAM-1, and ICAM-1 are temporarily induced in genital tracts of infected, but not uninfected, mice, suggesting active recruitment of CD4 cells to the genital tract via α4β7 (49). Given the potential role of α4β7 in enhancing HIV spread, and its coexpression with other susceptibility markers in our study, blockade of this molecule might represent a possible prophylactic modality against HIV. Blockage of α4β7 has shown promise in the treatment of Crohn’s disease, presumably by blocking recruitment of inflammatory T cells to the gut (50). In a nonhuman primate SIV model, α4β7 blockade was shown to delay the entry of SIV into gut tissues and reduce the SIV set point (51).

Our study is focused on FSWs; therefore, it will be important to look at non-FSW populations because of the possibility that the relatively high frequency of activated target cells observed might relate to increased rates of genital infections or trauma associated with sex work. Also, future work will need to determine the potential role that these T cell subsets may play in mucosal immune defense against genital pathogens, particularly because most sexually transmitted infections have demonstrated effects on increasing HIV transmission risk (52–54).

In conclusion, we defined a subset of CD4⁺ T cells from the cervix of FSWs that appears to be highly susceptible to HIV, with a coalescence of expression of multiple HIV-enhancing factors, such as CCR5, α4β7, CD69, and IFN-γ, within the Th17 cervical subset. Understanding the clinical associations of this highly HIV susceptible T cell subset, as well as the role that it plays in both HIV acquisition and in host defense against genital bacterial/fungal infections, may lead to novel strategies for HIV prevention.

We thank the staff at the Kenya AIDS Control Project who contributed to patient care, data management, administration, and laboratory diagnostics. We also thank the study participants for participation in this study.

The authors have no financial conflicts of interest.