Progesterone Levels Associate with a Novel Population of CCR5 +CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential This information is current as of September 28, 2021. Alison Swaims-Kohlmeier, Richard E. Haaland, Lisa B. Haddad, Anandi N. Sheth, Tammy Evans-Strickfaden, L. Davis Lupo, Sarah Cordes, Alfredo J. Aguirre, Kathryn A. Lupoli, Cheng-Yen Chen, Igho Ofotukun, Clyde E. Hart and Jacob E. Kohlmeier Downloaded from J Immunol 2016; 197:368-376; Prepublished online 27 May 2016; doi: 10.4049/jimmunol.1502628 http://www.jimmunol.org/content/197/1/368 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2016/05/27/jimmunol.150262 Material 8.DCSupplemental References This article cites 59 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/197/1/368.full#ref-list-1 by guest on September 28, 2021

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential

Alison Swaims-Kohlmeier,* Richard E. Haaland,* Lisa B. Haddad,† Anandi N. Sheth,‡ Tammy Evans-Strickfaden,* L. Davis Lupo,* Sarah Cordes,† Alfredo J. Aguirre,‡ Kathryn A. Lupoli,x Cheng-Yen Chen,x Igho Ofotukun,‡ Clyde E. Hart,* and Jacob E. Kohlmeier{

The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell Downloaded from populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7hi, consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7hi CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7hi CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7hi CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines http://www.jimmunol.org/ CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7hi memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7hi memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7hi FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7hi CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells. The Journal of Immunology, 2016, 197: 368–376.

uman immunodeficiency virus infection is one of the course is the predominant mode of HIV acquisition in women and gravest health challenges worldwide with an estimated accounts for an estimated 1 million new infections annually (1), by guest on September 28, 2021 H 35 million people currently infected (1). Sexual inter- yet the mechanisms by which HIV penetrates the female genital tract (FGT) mucosa to establish a systemic infection are not fully understood. Potential routes of genital infection are CCR5 *Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30329; †Department of Gynecology and Obstetrics, Emory University dependent and are thought to involve Langerhans cells and/or + School of Medicine, Atlanta, GA 30322; ‡Division of Infectious Diseases, Depart- CCR5 CD4 T cells at the superficial vaginal mucosa (2). T cell x ment of Medicine, Emory University School of Medicine, Atlanta, GA 30322; Di- characterization data show increased CCR5 expression on CD4 vision of STD Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30329; and {Department of Microbiology and Immunology, Emory University T cells in the vagina (3) and cervix (4). Nonhuman primate and School of Medicine, Atlanta, GA 30322 human explant models show CD4 T cells in the FGT are directly ORCIDs: 0000-0001-6508-0038 (A.S.-K.); 0000-0002-6668-6389 (L.B.H.); 0000- and productively infected following virus inoculation (5, 6), and 0002-6305-2727 (A.N.S.); 0000-0002-3875-425X (S.C.); 0000-0001-6766-1522 virus transmission of SIV in both rectal and vaginal mucosal (K.A.L.); 0000-0003-2735-1903 (I.O.). tissues are proportional to the number of CCR5+ CD4+ T cells at Received for publication December 21, 2015. Accepted for publication April 27, 2016. these sites (7). Recently, a study using cervical CD4 T cells showed that HIV preferentially targets populations of CD4 This work was supported by the U.S. Centers for Disease Control and Prevention and Emory University and in part by the National Center for Advancing Transla- T cells with increased expression of the HIV coreceptor CCR5 in tional Sciences of the National Institutes of Health (Grants KLR2TR000455 and combination with the activation marker CD69 and mucosal UL1TR000454). This work was also supported by Grants K23HD078153 (to L.B.H.), K23AI114407 (to A.N.S.), and HL122559 (to J.E.K.) from the National Institutes integrins a4b7 and a4b1 (8). Taken together, these studies dem- of Health and facilitated by the Center for AIDS Research at Emory University onstrate that FGT CD4 T cells express HIV susceptibility markers (Grant P30AI050409). and are direct targets of HIV infection. However, it is unclear The findings and conclusions in this report are those of the authors and do not whether the population of CD4 T cells at the surface of the genital necessarily represent the views of the U.S. Centers for Disease Control and Preven- tion or the Department of Health and Human Services. tract can directly contribute to establishment of a systemic HIV infection. Furthermore, there is currently limited information on Address correspondence and reprint requests to Dr. Jacob E. Kohlmeier, Emory University School of Medicine, 1510 Clifton Road, RRC 3133, Atlanta, GA the CD4 T cell subsets present in the FGT of healthy women, the 30322. E-mail address: [email protected] impact of sex hormones on the composition of the FGT CD4 The online version of this article contains supplemental material. T cell population, and whether distinct FGT CD4 T cell subsets Abbreviations used in this article: cRPMI, complete RPMI; CVL, cervicovaginal may have differential contributions to HIV transmission. lavage; FGT, female genital tract; TCM, central memory T cell; TEM, effector memory The FGT is an immune-restricted site where T cell trafficking T cell; T , resident memory T cell; T , terminally differentiated T cell. RM TD is tightly regulated (9, 10). Immune-restricted tissues primarily Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 contain memory T cells of an effector memory phenotype (TEM; www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502628 The Journal of Immunology 369

CCR7lo, CD45RAlo), which recent studies have shown are mostly relation to these emerging studies, we found this T cell population composed of long-lived, noncirculating tissue-resident memory more concentrated during the luteal phase of the menstrual cycle + + hi cells (TRM:CD103CD69 ) that provide tissue-specific protection and upon longitudinal analysis the frequency of FGT CCR7 CD4 from invading pathogens (11, 12). In murine models, resident T cells positively correlated to blood progesterone levels. Our data populations of CD4 T cells in the FGT predominantly comprise a identify an unexpected recirculating memory CD4 T cell population TEM phenotype expressing CD69 but with little to no CD103 ex- at the surface of the FGT and suggest a novel mechanism of HIV pression in comparison with FGT CD8 TRM (13, 14). Interestingly, transmission and establishment of systemic infection through the hi the protective functions of TRM cells at mucosal sites can be me- migration of infected, recirculating FGT CCR7 CD4 T cells into diated by cells embedded in the tissue as well as cells located on the underlying lymphoid tissues. Furthermore, our data show these luminal side of the epithelium (15–18). For example, in the lung, target cells are more accessible to HIV during the luteal phase of the CD8 TRM have differential effector functions based on whether they menstrual cycle. are localized to the airway lumen or the underlying tissue, and both populations contribute to optimal immune protection (19). There- Materials and Methods fore, to obtain a complete picture of the resident T cell population at Specimen collection and processing mucosal sites, it is important to characterize immune cells on both the apical and basal side of the epithelium. Paired blood and cervicovaginal lavage (CVL) specimens were collected hi from women participating in a study of oral maraviroc pharmacokinetics in Although CCR7 T cells are not characteristically found in im- HIV-negative volunteers (Atlanta, GA) at visits without detectable mar- mune restricted tissue, recirculating memory T cells are a recently aviroc. Additional CVL only specimens for the FGT T cell characterizations 2 identified subset of CCR7 expressing CD69 memory CD4 T cells were obtained from a convenience sample of women attending a normal Downloaded from detected in cutaneous tissue under steady-state conditions (20). well-woman visit at Emory Clinic and used in some experiments where only CVL cells were analyzed. Protocols were approved by the Emory University These recirculating CD4 T cells migrate from peripheral tissue into and U.S. Centers for Disease Control and Prevention Institutional Review draining lymph nodes in a CCR7-dependent fashion and are thought Boards and the Grady Research Oversight Committee. All participants to function in immune surveillance. Even though the identification provided informed consent. of recirculating memory CD4 T cells in humans is relatively recent Menstrual cycle follicular (,10 d following start of previous menses) and luteal phase (.18 d following start of previous menses) were determined (21), prior mechanistic studies in murine models have shown that http://www.jimmunol.org/ according to self-report. Progesterone and estradiol were measured from CD4 T cells located in the lumen of mucosal tissues can traverse the blood samples using the estradiol, progesterone Milliplex kit for Luminex epithelium and migrate to lymph nodes (22–24). Importantly, this (EMD Millipore, Billerica, MA), according to manufacturer’s instructions. process required expression of CCR7 because CCR7-deficient CD4 Blood was collected in 8 ml sodium citrate–containing CPT tubes (BD T cells failed to migrate out of the lumen (20). Thus, memory CD4 Biosciences, Franklin Lakes, NJ) and separated into plasma and PBMC by centrifugation. During a speculum examination, CVL specimens were col- T cells located on the apical surface of the mucosal epithelium have lected by directing two series of 10 ml PBS toward the endocervix and the potential to migrate across the epithelium and re-enter the vaginal walls according to the protocol described by the Microbicide Trials lymphatics in a CCR7-dependent manner. Network (www.mtnstopshiv.org/node/773). Briefly, 1/3 PBS directed at To date, investigations of tissue-resident immune cell populations cervical os and endocervix, 1/3 PBS directed at right vaginal wall, and 1/3 at the human FGT mucosal surface are complicated by collection PBS directed at left vaginal wall were then allowed to pool into the posterior by guest on September 28, 2021 fornix and then aspirated into a 15-ml conical containing 0.5 ml human AB procedures that sample small surface areas that may not represent serum (Cellgro, Manassas, VA). The CVL was transported in a 15-ml conical areas most susceptible to HIV infection (25, 26); moreover, such tube containing 0.5 ml human AB serum (Cellgro). CVL was centrifuged sampling has the potential to introduce blood-borne leukocytes into into cell-free supernatant and cellular fractions at 300 3 g for 10 min. CVL resident cell samples (27). Notably, these methods would sample cell fractions were enriched for leukocytes by centrifugation through a 40/ 80% Percoll (GE Healthcare, Pittsburgh, PA) gradient at 900 3 g for 20 min both luminal T cells as well as T cells embedded in the underlying at room temperature without break. Enriched CVL leukocytes were collected tissue, whereas isolation of FGT immune cells by lavage allows for at the gradient interface and passed through a 70-mm cell strainer. Leuko- the specific examination of the FGT T cell population located on cytes were washed with complete RPMI (cRPMI; RPMI containing 10% the apical surface of the epithelium. We developed an atraumatic FBS, penicillin, and streptomycin) and stained for cell surface markers as enhanced lavage-enrichment method to characterize local T cell indicated below. Separation of PBMC and CVL cell fractions was performed within 4 h of sample collection. CVL samples were measured for semen populations at the apical surface of the mucosal epithelium in the using the ABACard p30 Ag Detection Test (Abacus Diagnostics, West Hill, lower FGT for the purpose of better understanding T cell–mediated CA) to identify samples containing cells from a male sexual partner that susceptibility to HIV infection from sexual exposure. Using this might affect immune characterization. method, we determined the majority of FGT memory CD4 T cells Sexually transmitted infection testing (CD69+/2CD45RAlo) in healthy women express the lymphoid homing chemokine receptor CCR7, and approximately half of the DNA was extracted from DrySwab (Lakewood Biochemical) using the CCR7hi CD4 T cells did not express the residency marker CD69. In Qiagen DNA Mini kit to amplify targets from gonorrhea, Chlamydia, hi syphilis, HSV types 1 and 2, pelvic inflammatory disease, Candida, and addition, FGT CCR7 CD4 T cells expressed the HIV susceptibility HIV using real-time duplex PCR and Qiagen Rotor-Gene Q and 6000 real- markers CCR5 and CD38 and display chemotaxis to CCL19 and time PCR instruments. Qiagen Rotor-Gene Q Series software was used to CCL21. Using a CCR5-tropic GFP-HIV reporter, we further show analyze data. that CCR7hi FGT CD4 T cells are targets of infection and separate Chemotaxis assays migration studies of productively infected CCR7-expressing T cells show no altered chemotaxis to CCL19 and CCL21. These data Chemotactic activity to CCL19 and CCL21 was measured using a standard suggest CCR7hi memory CD4 T cells resident in the genital tract migration assay as described previously (30). In brief, CCL19 (600 ng/ml) and CCL21 (600 ng/ml) chemokines (R&D Systems, Minneapolis, MN) could be infected at the earliest stages of HIV transmission and were added to the lower chamber of 24-well Transwell plates (5-mm pore serve as a mechanism for viral entry and dissemination to lymphoid size; Corning, Tewksbury, MA) and incubated 20 min at 37˚C. Cells (1 3 tissues. In addition, the sex hormones estradiol and progesterone 106) were added to the upper chamber and incubated 3 h at 37˚C. Lower that regulate the menstrual cycle also impact the immune environ- chamber contents were analyzed by flow cytometry as indicated using Trucount tubes (BD Biosciences, San Jose, CA) to calculate the absolute ment in the FGT, and some evidence suggest these hormones may number of cells. Chemotactic index was calculated as the number of cells influence HIV sexual transmission in the FGT, although the mech- migrating in response to indicated chemokines divided by the number of anism of this increased susceptibility are unclear (28, 29). In cor- cells migrating in response to medium lacking additional chemokines. 370 HIV TARGET T CELLS IN THE FGT ARE LYMPHOID HOMING

Sample staining and flow cytometry TCM and TEM subsets (Fig. 1E, 1F) (12, 35). Notably, although previously characterized mucosal sites contain a predominant TEM All cells were stained for viability using the Zombie Fixable Viability Kit hi (BioLegend), incubated with anti-CD16/32 Fc-block (BioXCell), and stained population, the primary population of FGT T cells was CCR7 with the indicated Abs: CD19 allophycocyanin, CD3 allophycocyanin-H7 or CD4 memory cells (p # 0.0001), consistent with a TCM phenotype BV450, CD4 AF700, CD8 V500, CCR7 PE-CF594, CD69 BV785, CCR5 (CD45RAloCCR7hi). FGT CD8 T cells, in contrast, expressed a PE, CD103 FITC (BD Biosciences), CD38 PE-Cy7, CD11c BV711, CD14 hi lo predominant TEM phenotype (CD45RA CCR7 )(p # 0.0001). BV650, and CD45RA BV605 (BioLegend). Stained samples were run on hi lo an LSRII flow cytometer, and data were acquired using FACSDiva software TTD (CD45RA CCR7 ) cells were reduced within the CD4 (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, (p = 0.279) and CD8 (p = 0.0313) T cell populations (Fig. 1F). OR). These results show that human mucosal T cell populations in the lower FGT are primarily memory CD4 T cells expressing CCR7, a HIV infection of T cells chemokine receptor important for T cell trafficking to the lym- 5 PBMC from healthy donors were infected overnight with 2 3 10 tissue phatics from peripheral tissue sites (20). culture-infective dose50 CCR5-tropic subtype C HIV (NG201530; AIDS Research and Reference Reagent Program, Division of AIDS, National In- FGT CCR7hi CD4 T cells populations express increased CD69 stitute of Allergy and Infectious Diseases, National Institutes of Health, expression yet display chemotaxis to CCL19 and CCL21 Bethesda, MD), and p24 levels were measured as described previously (31). PBMC collected from healthy blood donors were incubated in cRPMI-10 The expression of CCR7 on human CD4 T cells in the lower FGT is with 20 U/ml IL-2 (Roche), then activated with Cell Stimulation Cocktail distinct from characterization studies using human hysterectomy (eBioscience, San Diego, CA) overnight. Cells were washed in cRPMI, rested for 4 d, and incubated in cRPMI with 20 U/ml IL-2. Cells (2 3 106) tissue (36) as well as previous studies in mice (14). To better

5 Downloaded from were incubated overnight with 23 10 tissue culture-infective dose50 of characterize FGT CD4 T cells enriched from the mucosal surface, CCR5-tropic subtype C HIV (NG201530; AIDS Research and Reference we measured expression of the tissue retention markers CD69 Reagent Program, Division of AIDS, National Institute of Allergy and In- and CD103 in comparison with CCR7 expression (Fig. 2A–C). fectious Diseases, National Institutes of Health). Infected cells were washed CD69 interferes with sphingosine-1-phosphate receptor function with cRPMI and incubated with cRPMI containing 20 U/ml IL-2 for 4 d. Cells were assayed for chemotactic index using the migration assay de- and CD103 binds E-cadherin to facilitate adherence to the epithe- hi scribed above. Cells were stained with the following Abs: CD19 allophy- lium (37, 38). CCR7 CD4 T cells expressed a greater range in lo cocyanin, CD3 allophycocyanin-H7, CD4 AF700, CD8 V500, (BD CD69 frequency (10–93.8) compared with CCR7 CD4 T cells http://www.jimmunol.org/ biosciences), CD45RA BV605 (BioLegend). For intracellular staining, cells (30–93.3) though the average percentage of CD69+ cells was were permeabilized with Cytofix/Cytoperm kit (BD Biosciences) prior to hi lo staining with p24 PE (KC57-RD1; Beckman Coulter, Indianapolis, IN). similar between CCR7 and CCR7 CD4 T cell subsets (mean For infection of CVL from healthy women, 2–10 3 106 enriched CVL 54.2 and 60.2, respectively) and no significant differences in CD69 cells were infected for 1 h with an R5-tropic GFP-HIV (a gift from expression were detected (Fig. 2B). CD103 expression was detected T. Mempel, Harvard Medical School, Boston, MA) (32), washed, and with a greater frequency on FGT CCR7lo CD4 T cells compared cultured for 48 h in complete medium supplemented with 20 U/ml IL-2. with CCR7hi CD4 T cells (mean 22.8 and 2.3) (p =0.0005) CVL cells were stained as described above with the indicated Abs. Cells were assessed for GFP expression on an LSRII flow cytometer (BD Bio- (Fig. 2C). These data show that CD4 T cells in the FGT comprise hi +/2 sciences) and data analyzed as described above. CCR7 CD69 populations consistent with both CD4 TRM (CD69+) and recirculating memory CD4 T cells (CD692). by guest on September 28, 2021 Statistics The expression of CD69 on T cells at peripheral sites relates to Statistical analysis was performed with Prism 5 software (Graphpad). Unless long-term tissue retention (37), yet the detection of CCR7 on FGT otherwise noted, an unpaired two-tailed Student t test was used to determine CD4 T cell populations indicates these cells may be able to exit significance: *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. the tissue via the lymph in response to CCR7 chemokines. To determine whether CCR7 expression on FGT CD4 T cell pop- Results ulations would enable migratory function and trafficking into af- The lower FGT mucosal surface is an immune restricted site hi ferent lymphatics, we investigated chemotaxis to CCR7-binding with a majority CCR7 CD4 memory T cell population chemokines CCL19 and CCL21 by Transwell assay (39). In all To investigate how T cells at the FGT mucosal surface may influence FGT samples tested (n = 8), CD4 T cells exhibited migration to HIVacquisition, we initiated a study of premenopausal healthy women CCR7-binding chemokines (p = 0.0259) (Fig. 2D). Notably, al- to perform atraumatic broad surface area sampling of the lower FGT. though the chemotactic patterns of FGT CD4 T cells displayed Participants were enrolled and screened for the purpose of collecting variation among participant samples, there was a significant in- genital lavage and matched blood samples. Using standard CVL crease the frequency of migrated cells comparing the media collection procedures, we optimized an enhanced lavage and en- control (average 1.9 6 2.7 SD) with CCL19+CCL21 (average richment technique to increase leukocyte yields while minimizing 11.6 6 11.9 SD) (Fig. 2E). These data show that FGT CCR7hi tissue trauma. To determine whether lavage samples provided char- CD4 T cells are composed of both CD69+ and CD692 memory acterizations representative of an immune restricted environment, we subsets and these populations can migrate to CCR7 chemokines. implemented three criteria to confirm method validity: 1) a low Thus, the combination of migratory ability displayed by FGT proportion of cells from blood circulation (,3% CD19+ B cells de- CD4 T cell populations in addition to phenotypic analysis support tected among lymphocytes) (Fig. 1A) (33); 2) the absence of naive that a subset of these cells are consistent with recirculating T cells (Fig. 1C, 1F); and 3) an increased frequency of the mucosal memory CD4 T cells localized to the FGT. residence marker CD103 on T cells compared with matched pe- hi ripheral blood samples (CD4, p = 0.0181; CD8, p # 0.0001) FGT CCR7 CD4 T cells express HIV susceptibility markers (Fig. 1D) (34). A description of the CVL samples used in the char- Because of their localization at the surface of the genital tract, it acterizations in Figs. 1–3 is provided in Supplemental Table I. was possible that these CCR7hi memory CD4 T cells would also be Initial characterizations found the predominant T cell population early targets of HIV infection from sexual transmission. To in- at the FGT mucosal surface was memory CD4 cells (CD45RAlo) vestigate evidence for HIV susceptibility in CCR7hi CD4 T cell (CD4, p = 0.0002; CD45RAlo, p # 0.0001) (Fig. 1B, 1C). We populations in the FGT, we analyzed FGT CD4 T cells for ex- further measured the frequency of CD45RA and CCR7 to dis- pression of the HIV coreceptor CCR5 and HIV susceptibility tinguish naive and terminally differentiated cells (TTD) as well as marker CD38 (40) based on CCR7 expression. As shown The Journal of Immunology 371

FIGURE 1. (A) Representative stain il- lustrating the gating strategy for FGT T cell characterizations. (B) CD4 and CD8 frequency of CD3 population from blood and FGT samples. (C) CD45RA frequency of CD4 T cell populations from blood and FGT samples. (D) CD103 expression of CD4 and CD8 T cell populations from blood and FGT samples. (E) Representa- Downloaded from tive stain of CCR7 and CD45RA T cell populations on either CD4 T cells (top panels) or CD8 T cells (lower panels) from blood (left panels) or the FGT (right pan- els). (F) CD45RA and CCR7 population frequency in CD4 and CD8 T cells from blood and FGT. Labeled, naive T cells http://www.jimmunol.org/ hi hi lo (TNA) CD45RA CCR7 ,TCM CD45RA hi lo lo CCR7 ,TEM CD45RA CCR7 , and TTD CD45RAhiCCR7lo.*p , 0.05, ***p , 0.001, ****p , 0.0001. by guest on September 28, 2021

(Fig. 3A–C), by comparing CCR5 and CD38 expression on FGT with increased HIV susceptibility markers including CCR5 in and blood CD4 T cells, both CCR7hi and CCR7lo FGT CD4 T cell comparison with similar populations from blood, but whether HIV populations exhibited increased expression. Similarly, CCR5 and preferentially targets FGT CD4 T cells based on CCR7 expression CD38 coexpression was increased on CCR7hi (p = 0.0017) and is unknown. To test HIV infection of FGT CD4 T cell populations CCR7lo (p = 0.002) FGT CD4 T cells compared with blood based on CCR7 expression, we infected CVL cells from eight suggesting that both FGT CD4 T cell subsets, including FGT individual subjects with HIV-GFP and measured for infection in CCR7hi CD4 T cells, would be susceptible targets for HIV in- CCR7hi and CCR7lo CD4 T cells following 48 h of cell culture fection. To distinguish whether FGT CCR7hi CD4 T cells may (Fig. 4A, 4B). The gating strategy used for the identification of exhibit different patterns of HIV susceptibility markers based on a infected cells is shown in Supplemental Fig. 1. We detected GFP resident or recirculating T cell phenotype, we also measured signal with a greater frequency in CCR7hi (mean frequency of CCR5 and CD38 expression on both the CD69+ and CD692 infected CD3+CD82 T cells: 56.1) compared with CCR7lo FGT subsets (Fig. 3D). Despite the differences in CD69 expression, CD4 T cells populations (mean frequency of infected CD3+CD82 these subsets of CCR7hi CD4 T cells express similar levels of T cells: 43.57), although this difference was not significant (Fig. CCR5 and CD38. Thus, based on the surface expression of HIV 4B). These data indicate that both CCR7hi and CCR7lo FGT CD4 susceptibility markers, both CD69+ and CD692CCR7hi CD4 T cells are equally susceptible to HIV infection. T cells in the FGT are likely permissive to HIV infection. CCR7 expression on infected T cells support virus trafficking to FGT CCR7hi memory CD4 T cells are susceptible to HIV lymphoid homing chemokines infection HIV-infected TCM can directly disseminate virus within lymph CD4 TEM may be selectively infected by HIV because of increased nodes upon s.c. transfer in humanized mice (32). However, HIV expression of CCR5 in comparison with CD4 TCM cells (35, 41). viral proteins have been shown to interfere with lymph homing FGT CD4 T cells comprise both CCR7hi and CCR7lo CD4 T cells signaling pathways in infected cells (42). FGT memory CD4 T cells 372 HIV TARGET T CELLS IN THE FGT ARE LYMPHOID HOMING Downloaded from http://www.jimmunol.org/

FIGURE 3. (A) Representative stain of CCR5 and CD38 expression on memory CD4 T cells from blood (left panel) or FGT (right panel). (B) by guest on September 28, 2021 Representative stain of CCR5 and CD38 on FGT CD4 T cells gated by CCR7 expression. (C) CD4 memory T cells gated by CCR7 expression and measured for expression of CCR5, CD38. (D) FGT CCR7hi CD4 T cells gated by CD69 expression and measured for expression of CCR5 and CD38. **p , 0.01, ****p , 0.0001.

HIV-infected CD4 T cells migrated to CCL19 and CCL21 (p = 0.0025) (Fig. 4C) and did not exhibit impaired chemotaxis (Supplemental Fig. 2). These results support the concept that ex- pression of CCR7 on FGT memory CD4 T cells enables HIV to use these cells for direct trafficking and dissemination of virus into secondary lymphoid tissue. FIGURE 2. (A) Representative stain of CCR7 and CD45RA expression Frequencies of FGT CCR7hi memory CD4 T cells associate on memory CD4 T cells from the FGT (left panel). Cells were gated by with the luteal phase of the menstrual cycle and correlate with CCR7 expression and measured for the expression on CD69 and CD103 blood progesterone levels (right panel). (B and C) FGT CD4 T cells were gated by CCR7 expression and measured for CD69 (B) or CD103 (C) expression. (D) CD4 T cell Previous studies using nonhuman primate models and evidence chemotactic function to CCL19 and CCL21 (gray bar, n = 8). (E) FGT from humans suggest the luteal phase of the menstrual cycle is CD4 T cell migration to CCL19/21 shown by change in the percentage of associated with increased HIV susceptibility (28, 43, 44). To ex- CD4 T cells that migrated in complete medium compared with CD4 T cells plore whether the phase of the menstrual cycle may associate with that migrated to CCL19/21 chemokines (n = 7). (D and E) Dotted line changes in the FGT memory CD4 T cell population, we compared , , represents chemotactic index of 1. *p 0.05, ***p 0.001. lavage samples collected at the follicular phase (4-10 d following start of menses) or luteal phase of the menstrual cycle (18– coexpress CCR7 and markers of HIV susceptibility, but whether 26 d following start of menses) for any differences in T cell these cells could support virus trafficking into lymph nodes via characteristics. By analyzing T cell populations based on self- CCR7-mediated chemotaxis is unknown. To determine whether reported phase of the menstrual cycle, we found the frequency CCR7hi CD4 T cells exhibited trafficking to lymph homing che- of FGT CCR7hi memory CD4 T cells increased at time points mokines postinfection, we infected blood-derived CD4 T cells with corresponding to the luteal phase (p # 0.0001) (Fig. 5A), although a clinically derived R5-tropic subtype C HIV isolate and measured the average frequency of CCR7hi memory CD4 T cell populations migration of infected TCM to CCL19 and CCL21. We observed that in blood remained similar between time points. The Journal of Immunology 373

FIGURE 4. (A and B) Unstimulated CVL cells infected with R5-tropic GFP-HIV ex vivo (2–10 3 106 enriched CVL cells) were measured for GFP signal following 48 h of cell culture. (A) GFP frequency measured from gated FGT CD3+CD82 T cells (frequency range: 9.83–29.4, mean: 17.3). (B) GFP-positive FGT T cells were measured for CCR7 expression. GFP-positive CCR7hiCD3+CD82 T cells (mean frequency: 56, range: 6–89%) and CCR7loCD3+CD82 T cells (mean frequency: 43.57, range: 16–85) are compared with the CCR7 frequency of GFP-negative FGT CD3+CD82 T cell populations. (C)PBMC infected with CCR5 tropic subtype C HIV and then tested for CCL19/21 chemotaxis. HIV-infected T cells (red circles) measured by intracellular p24 ex- pression in comparison with uninfected T cells (gray circles). Absolute number of p24-positive T cells migrated to CCL19/21 or complete medium.

To more directly examine the association of FGT CCR7hi memory Our study is the first, to our knowledge, to identify a novel Downloaded from CD4 T cells with the menstrual cycle, we followed a subset population of CCR7hi memory CD4 T cells consistent with both of healthy participants longitudinally, collecting blood and lavage CD4 TRM and recirculating memory CD4 T cell phenotypes in samples at indicated time points throughout the cycle and mea- the FGT mucosa of healthy women. Delineating resident and sured estradiol and progesterone from blood plasma to accurately recirculating FGT memory CD4 T cells based on the residency define menstrual cycle phase (Fig. 5B) (45). The frequency of FGT marker CD69, we showed that both of these subsets are enriched hi CCR7 memory CD4 T cells was highest at time points with in- for the expression of the HIV susceptibility markers CCR5 and http://www.jimmunol.org/ creased plasma progesterone levels, corresponding to the luteal CD38 and thus are likely targets of HIV infection. It should be phase of the menstrual cycle (p = 0.0017) (Fig. 5C). As proges- noted, however, that although CD69 can contribute to tissue terone levels declined, the frequency of FGT CCR7hi memory CD4 residency, it is not a definitive marker for tissue-resident T cells, T cells decreased into ranges detected during the follicular phase and it is possible that the dynamic regulation of CD69 expression (p = 0.0059). By directly comparing CCR7 expression on FGT in the tissue lessens its use as a marker to distinguish resident memory CD4 T cells, a significant positive correlation was ob- and recirculating memory CD4 T cells (37, 54). Nevertheless, we served with progesterone concentrations (p = 0.0024) (Fig. 5D). No demonstrated that CCR7hi memory CD4 T cells isolated from correlations were detected upon comparing FGT CCR7hi memory CVL were susceptible to HIV infection and that CCR7hi memory CD4 T cell frequencies and estradiol concentrations (Fig. 5E). CD4 T cells retained their ability to migrate to CCR7 ligands by guest on September 28, 2021 Notably, no change in the HIV susceptibility markers CCR5 and after HIV infection. Several immune cell types, including Lang- CD38 was detected on FGT CD4 T cells based on phase of the erhans cells and myeloid cells, have been implicated in trans- menstrual cycle (Supplemental Fig. 3A, 3B). porting HIV across the mucosal epithelium (5, 55–57). Our data Increased CCR7 frequency in correlation to plasma progesterone suggest that, in addition to these mechanisms, heterosexual HIV levels was not related to a change in total CD4 T cells detected transmission may also occur by infection of recirculating CCR7hi (Supplemental Fig. 3C). memory CD4 T cells at the luminal surface of the FGT that traffic across the epithelium to lymphoid tissues where a systemic in- Discussion fection can be established. Understanding how HIV can penetrate mucosal surfaces and es- How these FGT CCR7hi CD4 T cells may function in immune tablish a systemic infection is needed to advance promising bio- protection remains to be understood, as do the signals that direct medical interventions. Estimated HIV sexual transmission rates their migration into this site. One interesting finding from our data through the cervical–vaginal mucosa of women are relatively low is the difference between FGT CD4 and CD8 T cells in CCR7 and compared with other exposure routes (46) and are often a result of a CD103 expression. Significantly fewer FGT memory CD8 T cells single founder variant that may be specific to CD4 T cells (47, 48). expressed CCR7 compared with CD4 T cells, whereas the fre- Mucosal barriers involve a wide variety of immune mediators to quency of FGT memory CD8 T cells that expressed CD103 was adequately defend against infection, including TRM populations that significantly greater. Taken together, these data suggest that FGT provide a superior and tissue-specific immune response to invading CD8 memory T cells may be more likely to remain resident in the pathogens (49–53); hence, their maintenance at these sites is vital. tissue, whereas the FGT memory CD4 population may be com- Previous work involving adoptive transfers studies in mice found posed of both tissue resident and recirculating cells. The changing that the expression of CCR7 on CD4 T cells was sufficient to enable frequency of FGT CD4 T cells over the course of the menstrual them to migrate from mucosal barriers into afferent lymphatics (23, cycle suggests that as recirculating FGT CCR7hi CD4 T cells 24), demonstrating that not all memory T cells present at mucosal migrate from the tissue to lymphoid sites; their numbers must sites permanently reside in the tissue. Furthermore, the subsequent be maintained by an influx of new cells into the FGT. One pos- identification of recirculating memory CD4 T cells in cutaneous sibility is that CCR7 mediates both the influx and efflux of tissues in humans demonstrated an even greater complexity to the recirculating FGT CCR7hi CD4 T cells because constitutive ex- maintenance and homeostasis of tissue-resident memory CD4 pression of the CCR7 chemokines CCL19 and CCL21 in human T cells at peripheral sites (21). Taken together, these data suggest endometrium has been reported (58). However, we think it un- that the memory CD4 T cell pool resident in mucosal tissues at likely that CCR7-mediated signals draw recirculating cells into any one time is a dynamic population composed of both stationary the FGT because we and others do not observe any naive CD4 and migratory cells that maintain local immune surveillance. T cells in the FGT, which would also migrate to CCR7 chemo- 374 HIV TARGET T CELLS IN THE FGT ARE LYMPHOID HOMING

kines. More recently, it has been shown that CCR5-mediated signals are necessary for sustaining resident memory CD4 T cells in the FGT (13). Although it is unclear whether CCR5 directs the migration of recirculating CCR7hi CD4 T cells to the FGT under homeostatic conditions, it is an intriguing possibility considering that this potential mechanism may increase the number of permissive HIV targets at the surface of the FGT. We are currently investing the role of CCR5 and other migratory signals in directing the recruitment of recirculating CCR7hi memory CD4 T cells to the FGT. In correlation to emerging studies that suggest a role of sex hormones in sexual transmission of HIV (28), we found an in- creased frequency of CCR7hi cells among the FGT CD4 T cell population during the luteal phase of the menstrual cycle that positively correlated with blood progesterone levels. The exact roles by which progesterone alters HIV susceptibility are still debated and include a thinning of the epithelial layers of the vaginal wall, changes in the vaginal microbiota, and increased

expression of CCR5 on CD4 T cells (59, 60). If future studies Downloaded from demonstrate that CCR5-mediated signals are required for the migration of recirculating memory CD4 T cell into the FGT, in- creased CCR5 expression because of higher progesterone levels during the luteal phase of the menstrual cycle may explain the increased frequency of CCR7hi CD4 T cells we observed. Alter-

natively, the increased frequency of these cells on the surface of http://www.jimmunol.org/ the FGT may result from the thinning of the vaginal epithelium and more direct access to the luminal surface. Regardless of the mechanisms involved, the observed increase in recirculating HIV target cells on the surface of the FGT associated with high blood progesterone levels should be considered when evaluating mech- anisms of potential increased risk during the use of progesterone- based hormonal contraceptives. In summary, our detection of a previously unrecognized pop- ulation of recirculating CCR7hi memory CD4 T cells at the surface by guest on September 28, 2021 of the lower FGT that express HIV susceptibility markers and are permissive to infection suggests a mechanism of systemic virus dissemination that could begin with HIV infection of CCR7hi CD4 T cells in the lumen of the FGT. Taken together, these data em- phasize the need to further define resident immune cell pop- ulations at the site of pathogen exposure to better inform the design of more effective biomedical intervention and vaccination strategies.

Acknowledgments We thank the study participants and colleagues who contributed to study implementation. We also thank the Atlanta Clinical and Translational Re- search Institute Clinical Research Network (Grady site), the KL2-Mentored Clinical and Translational Research Program, the Grady Infectious Diseases Program, and the Emory Center for AIDS Research Clinical Core. We also FIGURE 5. (A) Frequency of CCR7hi CD4 T cells from FGT compared thank Michelle Owen, Kelly Curtis, and Krystin Ambrose in the Division with matched blood measured according to self-reported menstrual cycle of HIV/AIDS Prevention, Centers for Disease Control and Prevention for phase. (B) Average blood plasma levels of progesterone and estradiol assistance with hormone assays. shown at indicated time points throughout menstrual cycle. Day of cycle represent range estimated from self-reported start of menses. (C) Fre- Disclosures hi quency of CCR7 CD4 T cells from the FGT measured at indicated time The authors have no financial conflicts of interest. points progesterone and estradiol were measured. (D) Progesterone levels compared with FGT CCR7hi CD4 T cell frequency. Statistics calculated by Spearman correlation (Pearson correlation also performed and show References significance; r = 0.4079 and p = 0.0032). (E) Estradiol levels compared 1. UNAIDS. 2013. AIDS by the Numbers. United Nations, Geneva. with FGT CCR7hi CD4 T cell frequency. Statistics calculated by Spearman 2. Xu, H., X. Wang, and R. S. Veazey. 2013. Mucosal immunology of HIV in- fection. Immunol. Rev. 254: 10–33. correlation (Pearson correlation also performed and show no significance; + , , 3. Veazey, R. S., P. A. Marx, and A. A. Lackner. 2003. Vaginal CD4 T cells ex- r = 0.3006 and p = 0.2111). **p 0.01, ****p 0.0001. press high levels of CCR5 and are rapidly depleted in simian immunodeficiency virus infection. J. Infect. Dis. 187: 769–776. 4. McKinnon, L. R., B. Nyanga, D. Chege, P. Izulla, M. Kimani, S. Huibner, L. Gelmon, K. E. Block, C. Cicala, A. O. Anzala, et al. 2011. Characterization of The Journal of Immunology 375

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