CCR5 blockade inhibits menstrual-driven proinflammatory T cell infiltration into the cervicovaginal mucosa of women

This cohort included a total of 30 healthy women of reproductive age who were recruited in Atlanta, Georgia, from 2013 to 2015. Participants were randomly assigned to treatment groups of 10 participants per group. The demographics are shown in Table 1 . Genital infection status was monitored throughout immune profiling. All samples were included in the cellular analyses, with additional comparisons performed by infection status to assess potential contributions to the discoveries. Determined using Nugent score (0 to 10): normal (0 to 3), intermediate (4 to 6), and yes (7 to 10). To investigate immune properties by FRT-localized cells under menstrual cycle regulation in humans that would allow characterizations with low risk of contamination by blood circulating cells, we performed immunophenotyping of the cervicovaginal lumen (CVL) through lavage sampling ( 26 ) throughout the menstrual cycle in participants ( Fig. 1 ). Measurements were stratified according to the date of a participant’s last menses onset (set as day 1) in addition to progesterone kinetics ( Fig. 1, A and B ) ( 27 ). The timeframe comprising the follicular phase, when menstruation has ended and blood progesterone levels are typically low, was identified as days 4 to 12; the transitional time frame generally comprising ovulation was identified as days 13 to 16; and the luteal phase, when progesterone levels peak, was identified as days 17 to 20. On the basis of previous immune observations using the pig-tailed macaque animal model ( 28 ), the late luteal phase was further separated to distinguish the time frame during which progesterone was decreasing (days 21 to 25 in participants) and then at the end of the menstrual cycle or premenstrual phase at days 26+ up until the next menses onset when progesterone returned to low levels. ( A ) A schematic of the menstrual cycle with corresponding progesterone fluctuations and study sampling windows: follicular (F; days 4 to 12), the transitional time frame typically comprising ovulation (T; days 13 to 16), luteal (L; days 17 to 20), late luteal (LL; days 21 to 25), and premenstrual (PM; days 26+). Created in BioRender. Kohlmeier, A. (2026) https://BioRender.com/faqjblt . ( B ) Mean progesterone concentrations across the cycle, stratified by sampling day plotted with the standard error of the mean (SEM). ( C ) CVL cell yields by population and cycle phase, displayed as box-and-whiskers plots with individual participant values. ( D ) Surface expression frequency of indicated molecules on CD4 T cells. For (C) and (D), pairwise comparisons were analyzed using a generalized estimating equation (GEE) models ( n  = 10). ( E ) Mean fold deviation with SEM in total CD4 T cells and CD4 T cells expressing ( F ) CCR5, ( G ) CXCR3, and ( H ) CD38 across the menstrual cycle. The change in indicated immune properties was calculated as the number of cells at a given time point normalized to the average value over the cycle (the mean value of 1.0 is indicated by a dotted line on each graph). Statistical significance was determined using the Wilcoxon signed rank test ( n  = 10 participants). ( I ) Sensitivity analysis stratified by infection status comparing T cell numbers and ( J ) CD4 T cell phenotypes. (C to H) Significance: * P  ≤ 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001. Using this grouping strategy, we compared T cell yields, which showed the highest numbers detected at the late luteal phase ( Fig. 1C and table S1). This increase was statistically significant compared with the transitional and premenstrual phases. Subset analysis revealed distinct temporal patterns for CD4 and CD8 T cells. CD4 T cells showed little detection at the transitional phase but then peaked during the late luteal phase. In contrast, relatively no change was observed with the CD8 T cells, with the exception of a significant increase at the follicular phase compared with the luteal phase. Given the late luteal-specific CD4 T cell increase, a time frame associated with endometrial remodeling and the detection of a type 1 immune response using macaque modeling ( 25 ), we further measured the T H 1-specific inflammatory trafficking chemokine receptors CCR5 and CXCR3 in addition to T cell activation properties ( Fig. 1D and table S2). This approach showed that CD4 T cell expression of both chemokine receptors increased at the late luteal phase along with an increase in the activation-associated markers CD38 and HLA-DR ( 29 , 30 ). Because the CD4 T cell trends over the menstrual cycle suggested that these cells were exhibiting an immune effector response, we constructed kinetic profiles to visualize this trajectory ( Fig. 1, E to H ). Using the average value of CD4 T cell yields (per individual participant) collected from samples over the menstrual cycle, each time point measurement was then used to determine the fold deviation or change ( Fig. 1E ). This strategy showed a consistent trend in the behavior of CD4 T cells, describing that once the follicular phase had ended, cells tended to decrease to the lowest value at the transitional phase. From this point, CD4 T cell populations began to increase at the luteal phase and then peaked at the late luteal phase. By the end of the cycle, mean numbers had slightly decreased, although no significant change was detected at the premenstrual phase. A similar evaluation of activation-associated properties over time revealed consistent tendencies, described by an initial decrease in CCR5, CXCR3, and CD38-expressing CD4 T cells (HLA-DR T cell frequencies were too low to be detected by this approach) at the transitional phase, which was followed by an increase and peak in populations at the late luteal phase ( Fig. 1, F to H ). Last, by the premenstrual phase, the levels of CCR5 and CXCR3 CD4 T cells had decreased. To better understand potential contributions to these observations, we compared T cell yields and properties by infection status but found no significant differences ( Fig. 1, I and J ). Furthermore, although we did not detect significant changes in epithelial cell numbers, fluctuations over the cycle were noted. Thus, as the predominant cell population in CVL, we tested whether epithelial cell abundance might have interfered with the T cell measurements by performing linear correlation analyses (fig. S1); however, this approach did not detect any relationship between the epithelial cell and T cell yields, further indicating that these T cell activation–associated patterns were instead the result of menstrual cycle regulation. The identification of a CD4 T cell immune response in the lower FRT across the menstrual cycle suggested that local immune signaling interactions might be influencing this response. To investigate tissue-localized cues associated with this response, we measured cervicovaginal secretions from uninfected samples for a panel of 10 chemokines. By comparing concentration levels according to menstrual cycle phases ( Table 2 ), we found significant changes that showed an association with chemokine fluctuations, with an overall peak occurring during the late luteal phase ( Fig. 2A ). To better understand this change, we measured individual variations, which identified that interleukin-8 (IL-8), macrophage inflammatory protein 1β (MIP1β), and MIP1α increased at the late luteal phase ( Fig. 2B ). During the premenstrual phase, MIP1α levels remained elevated, whereas MIP1β decreased. The percent of samples within the upper quartile ranges from each chemokine is indicated. Statistical significance was determined using the Fisher’s exact test. ( A ) Heatmap showing mean chemokine concentrations in CVL samples across the menstrual cycle. ( B ) Heatmap depicting fold changes in individual chemokines across cycle phases. Statistical significance was assessed using a one-sample proportion test. ( C ) Heatmap correlation matrix of chemokines; significance was determined using a two-tailed Pearson correlation coefficient test. ( D ) XY linear regression plots with trends showing associations between indicated chemokines and IL-8 concentrations. Mip1β and Mip1α are shown with shaded confidence intervals. Statistical significance was determined using a two-tailed Spearman test. (B to E) Significance: * P  ≤ 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001. NS, not significant. Because IL-8, in the context of the menstrual cycle, functions to facilitate endometrial tissue remodeling in preparation for menstruation ( 31 , 32 ), we further investigated linear relationships by creating a correlation matrix ( Fig. 2C ). This approach showed IL-8 changes positively associated with both MIP1α and MIP1β. This was further confirmed by individual correlations, which exhibited positive trends only with MIP1α/β levels ( Fig. 2D and table S3). Overall, these data identified increased CCR5 signaling during CD4 T cell recruitment at the late luteal phase. To elucidate how the menstrual cycle was driving CD4 T cell recruitment into the FRT, we characterized longitudinal changes in tissue-localized FRT T cell populations using a murine menstruation model with intravital vascular labeling to distinguish tissue-localized cells over the course of decidualization, endometrial remodeling, and menstruation ( Fig. 3, A and B ) ( 33 ). This analysis revealed increased CD4 T cell numbers at day 8 of pseudopregnancy, in both the apical lumen and underlying cervicovaginal tissues ( Fig. 3, C and D ). This time point coincided with the detection of progesterone withdrawal, consistent with our observations in women. CVL analysis showed that luminal CD4 T cells were significantly increased at day 8 compared with all other time points, including control mice treated with medroxyprogesterone acetate (MPA) to suppress estrous cycling. CD4 T cells within cervicovaginal tissues were also increased during peak progesterone at day 6, when endometrial remodeling is initiated ( 33 ), which further increased and peaked at day 8. Following endometrial remodeling and menstruation, CD4 T cell populations were decreased and showed no significant change from cell measurements taken at the start of pseudopregnancy. ( A ) Top: a schematic of the C57Bl/6 pseudopregnancy model aligned with human endometrial changes. Bottom: Progesterone levels are shown over time, with withdrawal at day 8 ( n  = 6). ( B ) Representative flow cytometry gating strategy for identifying CD4 T cells from spleen (top) and CVL (bottom). ( C ) CD4 T cell yields from CVL and ( D ) cervicovaginal tissues. ( E ) Fluorescent microscopy images of vaginal tissues at day 8 taken at ×20 magnification, showing epithelial cells (cadherin: white), blood vessels (CD31: red), T cells (TCR β chain: green), and cell nuclei [4′,6-diamidino-2-phenylindole (DAPI): blue]. Arrows indicate T cells at the apical surface. Scale bars, 100 μm. ( F ) Left: uterine vascularization during decidualization in wild-type (WT) and CCR5KO mice. Right: quantification of vascularization at days 6 and 8. ( G ) Total CD4 T cells (top) and leukocytes (bottom) from CVL (left graph), cervicovaginal tissue (middle graph), and uterine horns (right graph) at days 6 and 8, shown with SEM. ( H ) Left: bone marrow chimera mouse strategy to assess CCR5-dependent effects following progesterone withdrawal. Right: representative uterine vascularization plots (top: induced to undergo a decidual reaction, n  = 10) or MPA control mice (bottom, n  = 5), 4 days postinduction. ( I ) Representative contour plots demonstrating CD4 T cell chimerism. ( J ) Ratios of CCR5KO to WT CD4 T cells and Ly6G + leukocytes in the lower and upper FRT (box and whisker plots). Statistical analysis used multiple comparisons with false discovery rate (FDR) correction (C to G) or Wilcoxon signed rank tests (J). Significance: * P  ≤ 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001. (A and H) Created in BioRender. Kohlmeier, A. (2026) https://BioRender.com/yxx4oa8 . To better visualize the process of CD4 T cell recruitment into the lower FRT during endometrial remodeling, we used immunofluorescence microscopy at day 8 of pseudopregnancy ( Fig. 3E ). This approach identified T cells positioned within the stromal layer of vaginal tissues, forming clusters in proximity to both blood vessels and the basal layer of the epithelium. T cells were also found progressing through the vaginal epithelium into the apical lumen, often radiating from those T cell clusters in the underlying tissues. Next, we examined the contribution of CCR5 to this response by inducing menstruation in CCR5-deficient (CCR5KO) mice. During endometrial remodeling, CCR5KO mice exhibited reduced CD4 T cell numbers in the lower FRT (cervicovaginal tissues) compared with wild-type controls ( Fig. 3, F and G ). Luminal CD4 T cells were consistently reduced throughout endometrial remodeling, whereas reductions within cervicovaginal tissues were observed only at day 8 of pseudopregnancy. No significant differences in CD4 T cell numbers were detected in uterine tissues at either time point. To test whether this reduction reflected a broader defect in leukocyte recruitment, we quantified total FRT leukocyte populations and observed a corresponding decrease in total leukocytes specifically within the lower FRT of CCR5KO mice at day 8. To determine the specific influence of CCR5 on the FRT CD4 T cells, we generated mixed bone marrow chimeras and induced progesterone withdrawal to stimulate immune breakdown of the endometrium ( Fig. 2H ). The ratio of tissue-localized CCR5KO to wild-type cells (normalized to spleen ratios) revealed reduced luminal and cervicovaginal CCR5KO CD4 T cells from chimeric mice undergoing a decidual immune reaction ( Fig. 3I and fig. S2). This decrease was not observed in uterine tissues or any FRT tissue site in control MPA mice (CVL from MPA mice did not yield detectable CD4 T cells). In contrast, CCR5 deficiency did not impair neutrophil recruitment, which is mediated by IL-8–dependent chemotaxis during endometrial remodeling (fig. S3) ( 31 , 32 , 34 ). Together, these data demonstrate that the murine menstruation model recapitulates cervicovaginal CD4 T cell dynamics observed across the menstrual cycle and identify that the endometrial remodeling process regulates this response in a CCR5-dependent manner. To assess the effects of maraviroc on FRT CD4 T cells, participants received oral maraviroc at either 300 mg twice daily (2× MVC) or 300 mg once daily (1× MVC) for 7 days, starting in the follicular phase ( Fig. 4A ). Longitudinal analysis of CCR5 expression on peripheral blood CD4 T cells showed that both dosing regimens increased CCR5 frequency, which remained elevated through the transitional and luteal phases ( Fig. 4B ). Although maraviroc has been previously reported to increase cell surface CCR5 stability and inhibit receptor internalization ( 35 ), the luteal phase is also associated with naturally higher CCR5 expression on circulating CD4 T cells ( 25 , 28 ). Cross-sectional comparisons by the treatment group confirmed that CCR5 levels remained largely similar across cycle phases, except for a transient increase at the transitional phase in the 2× MVC group ( Fig. 4C ). ( A ) Schematic diagram depicting the time points of blood and CVL collection for immune profiling over the menstrual cycle in drug treatment groups. The time frame of maraviroc (MVC) dosing is shown by red shading. Created in BioRender. Kohlmeier, A. (2026) https://BioRender.com/faqjblt . ( B ) Symbol line graphs with SEM bars comparing the mean cell surface CCR5 expression on PBMC CD4 T cells from untreated participants (top graph) and participants in the 2× MVC (middle graph) or 1× MVC (bottom graph) group. A vertical red dotted line indicates the drug dosing time frame; samples were collected before drug dosing at these time points. The mean value at the follicular phase (before drug initiation) is shown as a black horizontal dotted line. Statistical significance was determined using one-sample t tests. ( C ) Bar graphs with the SD comparing PBMC CD4 T cell CCR5 expression frequency from the participant groups over the course of the menstrual cycle. ( D ) Pseudo-color dot plots from a representative PBMC sample depicting the flow cytometry gating strategy for cell surface CCR5 and receptor occupancy measurement from PBMC CD4 T cells following ex vivo coculture in the presence or absence of MIP1β. ( E ) A bar graph with SEM comparing PBMC CD4 T cell receptor (TCR) occupancy across each treatment group at each menstrual cycle phase. ( F ) A box and whiskers graph comparing the percent of CCR5 occupancy measured from PBMC CD4 T cells longitudinally per group. (C, E, and F) Statistical significance was determined using a two-way ANOVA with FDR testing: * P  ≤ 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001. To evaluate drug binding into the active site of the CCR5 receptor, we performed receptor occupancy (CCR5 blocking) assays using peripheral blood mononuclear cell (PBMC) CD4 T cells incubated ex vivo with and without the CCR5 ligand, MIP1β ( Fig. 4D ) ( 36 ). Compared with untreated controls, CCR5 blocking was increased in both treatment groups at the transitional and luteal phases ( Fig. 4E ). At the late luteal phase, a significant mean increase was detected in the 1× MVC group, and by the end of the cycle, no significant increases were discernible when compared with the untreated group. To control for person-to-person cell heterogeneities in these measurements, we compared receptor occupancy longitudinally in each group ( Fig. 4F ). These data showed that both drug treatment arms exhibited increased CCR5 blocking throughout the remainder of the menstrual cycle, although, on average, this began to decrease at the premenstrual phase ( Fig. 4E ). Our murine model showed that endometrial remodeling drives CD4 T cell trafficking into the lower FRT mucosa in a CCR5-dependent manner ( Fig. 3 ). We therefore hypothesized that sustained CCR5 antagonism with maraviroc would inhibit this response in women. To test this, we compared FRT T cell properties from participants in the drug treatment arms with those from untreated participants (referenced to Fig. 1 ). Comparison of overall CVL T cell and CD4 T cell yields across the cycle showed no statistically significant differences, although mean counts trended lower in both drug treatment groups during the late luteal phase. ( Fig. 5, A and B ). We next examined CD4 T cell characteristics, focusing on T H 1-associated chemokine receptors and activation markers (CCR5, CXCR3, CD38, and HLA-DR) at each cycle phase ( Fig. 5, C to F ). Compared with untreated participants, both maraviroc groups showed consistent decreases in these markers during the late luteal phase. Additional decreases were also observed for CXCR3 and CD38 during the luteal phase and, for CD38, during the transitional phase; in the 2× MVC group, CXCR3 was increased during the transitional phase. Longitudinal analysis within the drug treatment arms revealed no changes in T cell yields or proinflammatory marker expression over time, in contrast to the untreated group ( Fig. 1, C and D ). This analysis showed that, following maraviroc administration, proinflammatory phenotypes tended to exhibit stable expression frequencies throughout the menstrual cycle, in contrast to increases observed at the late luteal phase, due to CD4 T cell recruitment. ( A ) A box and whiskers graph overlaid with individual symbols ( n values) comparing CVL T cell and ( B ) CD4 T cell yields from the control arm (black), 2× MVC arm (red), and 1× MVC arm (blue) in each treatment group, stratified according to the menstrual cycle phase at sample collection. Open circles represent values of zero. ( C ) A bar graph with SEM comparing the cell surface frequency of CVL CCR5, ( D ) CXCR3, ( E ) CD38, or ( F ) HLA-DR expressed on CD4 T cells by treatment group and cycle phase. ( G ) Symbol line graphs comparing the mean fold deviation and SEM in the number of CD4 T cells and CD4 T cells expressing ( H ) CCR5, ( I ) CXCR3, or ( J ) CD38 from CVL over one menstrual cycle by treatment group: 2× MVC group (red lines) and 1× MVC group (blue lines) with the untreated group [black lines previously shown in Fig. 1 (E to H) ]. [(G) to (F)] Figures are overlaid with the mean data points in Fig. 1 (E to H) . [(A) to (F)] Statistical significance was determined using a two-way analysis of variance (ANOVA) with FDR testing and [(G) to (J)] Wilcoxon signed rank tests: * P  ≤ 0.05, ** P  < 0.01, *** P  < 0.001, and **** P  < 0.0001. To test whether maraviroc influenced CD4 T cell trafficking into the lower FRT mucosa, we created a kinetic profile of CVL CD4 T cells over the course of the menstrual cycle ( Fig. 5G ) and compared these changes to those in the untreated arm [referenced to Fig. 1 (E to H) ]. This approach showed that at the late luteal phase, both the 1× MVC and 2× MVC groups exhibited a significant decrease. Corresponding to CD4 T cell yields, CCR5 and CXCR3 all exhibited reductions specifically at the late luteal phase compared to the control arm, while CD38 was decreased only in the 1× MVC group ( Fig. 5, H to J ). Together, these data show that oral maraviroc administration in women inhibited menstrual cycle–driven recruitment of bystander-activated T H 1 cells into the tissue barrier.

A deeper understanding of how the human menstrual cycle shapes immune function may provide critical insights into infection risk, the etiology of associated inflammatory disorders, and identify promising biomedical intervention strategies. Here, we conducted a longitudinal evaluation of T cells at the mucosal barrier site of exposure to genitourinary pathogens in cycling women of reproductive age, with and without the CCR5 antagonist maraviroc. Through cellular profiling and statistical modeling, we identified two key trends in these cell populations across the menstrual cycle. First, the transitional time frame, typically spanning ovulation, was associated with an overall reduction in T cells and their expression of immune activation properties. Then, following the fertility window at time points corresponding to the late luteal phase, there was an infiltration by immune-activated T H 1 cells. These trends were observed regardless of infection status, and although high bacterial vaginosis (BV) detection precluded direct assessment of microbial dysbiosis, our murine menstruation model, together with prior macaque studies, indicates that this CD4 T cell recruitment is driven by endometrial remodeling ( 25 , 28 ). Thus, although the vaginal microbiome and changes in microbial diversity might also influence tissue-resident T cells ( 37 , 38 ), our findings demonstrate that the immune response identified in this study is primarily governed by the menstrual cycle. Using a murine model of menstruation, we found that this immune response was more compartmentalized to the lower FRT and CCR5 dependent. Although few studies have examined in vivo bystander T cell activation in mucosal tissues in the absence of infection or antigen presentation, exposure to inflammatory cytokines can drive T cell responses independent of T cell receptor (TCR) engagement, including CCR5 up-regulation ( 39 – 42 ). Consistent with this, we and others have shown that the luteal phase and progesterone withdrawal are associated with increased systemic cytokines capable of inducing bystander T H 1 activation ( 25 , 43 – 46 ). To further define the local drivers of this response, we assessed immune signaling within the vaginal environment and found CCR5 chemokine levels closely associated with progesterone withdrawal and CD4 T cell recruitment, suggesting a key role for the vaginal mucosa in regulating this process. Bystander T cell activation can contribute to immune defense against pathogens and cancers while also implicated in the development of autoimmune inflammatory diseases ( 45 , 47 , 48 ). Yet, whether this response plays a biological role during the menstrual cycle is unknown. Some evidence suggests that immune-mediated vaginal remodeling is critical for fertility ( 49 – 52 ), and we previously found that these CD4 T cells are enriched for gene pathways involved in both hormonal regulation and T cell effector responses in macaques ( 28 ). Thus, we believe that future studies focused on uncovering the roles of these cells during fertility and in immune defense against genital infections may help advance our understanding of menstrual cycle biology. In the context of infection risk, previous studies from our group and others have identified CCR5 signaling as an immune correlate of increased HIV infection risk at mucosal sites and in circulation ( 25 , 53 , 54 ). Our findings in this study suggest that one potential mechanism behind this observation is the increased trafficking opportunities by CCR5 + CD4 T cells (HIV target cells) into tissue sites of sexual exposure. While long-acting injectable preexposure prophylaxis (PrEP) drugs are showing great promise for preventing HIV infection in women ( 55 , 56 ), long-acting PrEP does not modulate intrinsic immune vulnerabilities, such as the menstrual cycle–driven target cell influx identified in this study. Moreover, these immunologic risk windows may still influence breakthrough susceptibility, particularly in the presence of a local infection or inflammation. Notably, CCR5 blockade is emerging as a potential therapeutic option for many inflammatory disorders, including various cancers, inflammatory bowel disease, COVID-19, and atherosclerosis ( 57 ). As our findings demonstrate, CCR5 signaling is critical for these FRT T H 1 bystander responses during the menstrual cycle. Targeting this pathway with CCR5 antagonists such as maraviroc may provide an additional layer of protection when breakthrough risk is elevated in women. Limitations of our findings include the small number of participants in a focused prospective cohort, particularly during infection, which was insufficiently powered to draw conclusions about the immune response to these pathogens. Although key demographics obtained did not influence our findings, the absence of a broader range of potential confounders, including variations in menstrual cycle kinetics, may have augmented the trends observed, and we clarify that our stratification approach used in this study was not intended to identify the specific time point of ovulation in these participants. Furthermore, although FRT lavage sampling is now, to our knowledge, the only human sampling method that can provide longitudinal phenotypic characterizations of FRT localized immune cells, which are unlikely to be obstructed by blood contamination, the low T cell yields inevitably limited our interpretations and insights. In addition, because our study was focused on a single menstrual cycle in participants, we did not address potential impacts of long-term drug use, including safety and tolerability concerns and potential immunomodulatory effects in combination with exogenous hormone therapies, which will be important to determine in future studies for the purpose of general prevention use ( 58 , 59 ). Together, we conclude that the human menstrual cycle regulates a periodic CD4 T cell response in the FRT, identifying a shifting target of infection risk by genital pathogens and as a key regulator of inflammation, a potential focus of associated immune dysregulations that influence the development of menstrual-associated diseases. Thus, these findings underscore the need for future studies involving larger and broader participant groups to better understand how the menstrual cycle can be targeted to improve uterine health.

Although periodic menstruation requires immune-mediated tissue remodeling of the female reproductive tract (FRT), its impact on mucosal immunity and susceptibility to infection or associated inflammatory diseases remain poorly understood. While most women and adolescent girls of reproductive age menstruate, the majority will experience at least one menstrual-associated inflammatory disorder during their lifetime, which may be caused or aggravated by an infection ( 1 – 7 ). Despite clear associations between the menstrual cycle and disease risk, the underlying immune mechanisms remain poorly defined, underscoring a critical need to better understand the dynamic relationship between the immune system and the human female reproductive cycle. CD4 T cells, including memory T cells located within immune-restricted tissue sites, such as the FRT, are central regulators of inflammation and vital for protection against infections ( 8 – 12 ). For many urogenital infections, CD4 T helper cell type 1 (T H 1)–specific responses are critical for limiting pathogen load and reducing disease severity ( 8 , 11 , 13 , 14 ). However, while providing a more rapid defense at tissue barriers due to their localization at the sites of exposure ( 8 , 11 ), FRT CD4 T cells can also promote dysbiosis, including Candida albicans infections, and are susceptible targets of HIV infection from sexual contact ( 15 – 20 ). Moreover, CD4 T cell effector responses in the FRT can exacerbate tissue damage associated with diseases and conditions indirectly related to pathogens, including endometriosis, miscarriage, and preeclampsia ( 21 – 23 ). This dichotomy in tissue-localized CD4 T cell contributions to FRT health exposes some of the complexities in developing biomedical strategies to prevent and treat associated chronic conditions and indicates the need for greater insight into the mechanisms regulating FRT T cell functions. To investigate mucosal T cell surveillance behavior during the human menstrual cycle, we performed longitudinal profiling of T cells and immune signaling at the lower FRT of healthy cycling women. This approach identified a T H 1-specific response occurring at the late luteal phase of the menstrual cycle. Using a murine model of menstruation, we further uncovered that the process of endometrial breakdown in preparation for menses also drives proinflammatory CD4 T cell infiltration into the cervicovaginal tissues. This response is CCR5 dependent, as CCR5 knockout (CCR5KO) mice showed a compartmental reduction in FRT CD4 T cell recruitment during this time frame. Furthermore, using bone marrow chimera mice, we found that, in direct competition, CCR5KO mice, compared with wild-type CD4 T cells, were impaired in their ability to traffic into the FRT during this time frame. Notably, in women, oral administration of a CCR5 antagonist drug led to sustained receptor blockade throughout the menstrual cycle that attenuated this inflammatory response in the FRT. Our findings demonstrate that the human menstrual cycle regulates a mucosal immune response in vaginal tissues through oscillations in CCR5 signaling, and we further show that pharmacological targeting of these interactions is achievable in women and may strengthen immune barrier homeostasis and reduce aberrant inflammation in the FRT ( 24 , 25 ). Together, we believe that this study highlights the importance of investigations into menstrual cycle regulation of immunity in the reproductive tract for identifying and understanding how to prevent and treat chronic infections and diseases that affect women.

Human paired blood and CVL specimens were obtained from women participating in a prospective study assessing the pharmacokinetics of the drug maraviroc at Emory Center for AIDS Research Clinical Core located within the Grady Ponce de Leon Center in Atlanta, Georgia (clinical trial ID: NCT01749566 ). This study included women in the control arm of the study (who did not receive drugs) and women who were administered two different doses of maraviroc (two drug treatment arms): (i) 300 mg of maraviroc twice daily for 7 days and (ii) 300 mg of maraviroc once daily for 7 days. Participants were initially screened for study eligibility, including HIV-negative status (OraQuick swab) and no recent STIs [self-report and later tested by reverse transcription polymerase chain reaction (PCR) described below]. Upon enrollment, participants were scheduled for study visits over the menstrual cycle after determining the date of their last menstrual period onset. Additional eligibility for participation required that women were aged at least 18 years, had an intact uterus and cervix, reported regular menses (22- to 35-day cycles for at least three cycles), were not pregnant or breastfeeding, and were not experiencing symptomatic vaginal infection or genital ulcer disease at screening. Participants did not have a history of loop electrosurgical excision procedure, conization, or cryosurgery in the previous year. Protocols were approved by the Emory University and CDC Institutional Review Boards and the Grady Research Oversight Committee (approval number: IRB00059752). All participants provided informed consent. DNA was extracted from DrySwab (Lakewood Biochemical Company) using the QIAGEN DNA Mini kit and used to amplify targets from N. gonorrhoeae , C. trachomatis , herpes simplex virus types 1 and 2, M. genitalium , and T. vaginalis , using real-time duplex PCR and QIAGEN Rotor-Gene Q and 6000 real-time PCR instruments as previously described ( 60 ). QIAGEN Rotor-Gene Q Series software was used to analyze data. CVL samples were inspected for C. albicans infections via wet mount. BV was determined by Nugent scoring from Gram-stained vaginal smears as previously described ( 61 ). A Nugent score of 7 to 10 was considered BV positive, a Nugent score of 4 to 6 was considered intermediate BV, and a Nugent score of 0 to 3 was considered BV negative. CVL specimens were collected and enriched for leukocytes by density centrifugation as previously described ( 60 ). Briefly, 10 ml of phosphate-buffered saline (PBS) was directed toward the cervix and vaginal walls. PBS was allowed to pool into the posterior fornix and then aspirated into a 15-ml conical. Epithelial cells were counted using a hemocytometer before density centrifugation, and total yields were estimated from the CVL volume. Total CVL leukocytes were counted following density centrifugation, with T cell yields estimated using CD3 frequency measurements generated by flow cytometry. CVL samples with the detection of naïve T cells (>5% of CD45RA + CCR7 + from total T cells) and B cells (>2% of CD19 + from lymphocyte-gated cells) were considered contaminated with blood cells and excluded from the analysis (this was most likely to occur at the premenstrual phase). Blood was collected into CPT tubes and processed by density centrifugation according to the manufacturer’s instructions. Blood was collected at each time point of CVL collection and before MVC administration in those treatment groups. Progesterone levels from blood plasma samples were quantified by immunoassay using a Milliplex kit for Luminex (EMD Millipore) according to the manufacturer’s instructions. Menses onset was designated as the start of a participant’s menstrual cycle phase (day 1), and sample collection was scheduled once menses stopped. Comparisons based on the menstrual cycle were also informed by progesterone kinetics. Human and murine cell suspensions were stained for viability using a Zombie Fixable Viability Kit (BioLegend) and then incubated with α-CD16/32 Fc block (BioXcell). Stained samples were run on an LSRII or Cytek Aurora flow cytometer, and data were acquired using FACS DIVA software (BD Biosciences) and analyzed using FlowJo software (TreeStar Inc.). The fluorochrome-conjugated antibodies are listed in the Supplementary Materials (method S1). To measure and profile T cells, the following gating strategy was applied to participant-matched PBMC and leukocyte-enriched CVL cells as compared with PBMC controls acquired from a donor leukopak: Live, singlet, CD45 + , lymphocyte-gated cells were discriminated by CD3 expression (which identifies T cells), followed by CD4 and CD8 expression. CVL supernatants from participants testing STI negative throughout the study were measured and analyzed for cytokines/chemokines through the Emory Multiplexed Immunoassay Core using the Meso Scale Discovery platform using the Human chemokine panel one kit in one batch run. CVL samples with detectable blood were excluded from the analysis. The percent of CD4 T cell CCR5 receptor occupancy by maraviroc was determined by MIP-1β internalization as previously described ( 36 ). Briefly, 1 × 10 6 PMBCs were incubated with and without 20 ng of MIP-1β (recombinant human CCL4/MIP-1β isoform LAG-1; R&D Systems) for 30 min at 37°C before cell surface measurement of CCR5 using flow cytometry. C57BL/6 (B6), B6.129P2- Ccr5 tm1Kuz /J (CCR5KO), B6.SJL- Ptprc a Pepc b /BoyJ (CD45.1), B6.PL- Thy1 a /CyJ (CD90.1) (the Jackson Laboratory), and Swiss Webster outbred mice (Taconic Biosciences) were housed under specific Animal Biosafety Level 2 conditions at Emory University. All experiments were performed in accordance with the Emory University Institutional Animal Care and Use Committee guidelines (approval number: PROTO202200018). Six- to 12-week-old female C57BL/6 were mated with Swiss Webster vasectomized males to induce pseudopregnancy and then intrauterine injected with sesame seed oil (Sigma-Aldrich) using mNSET devices (Paratechs) as previously described ( 62 ). As a control for reproductive cycling, a subset (not undergoing pseudopregnancy) received a subcutaneous injection of 3 mg of MPA (Prasco) 2 weeks before necropsies. Intravital vascular staining was performed before euthanasia and tissue harvest as previously described ( 63 ). Briefly, 1.5 μg of fluorophore-conjugated α-CD45 in 200 μl PBS was tail vein injected and allowed to circulate for 15 min. Mice were euthanized using Avertin (2,2,2-tribromoethanol; Sigma-Aldrich) and exsanguinated before tissue collection. Fifty microliters of PBS was deposited and retracted into the vaginal vault at equal repetitions lasting about 30 s. FRT tissues were digested using collagenase type II (62.5 U/ml) and deoxyribonuclease I (0.083 U/ml) (STEMCELL Technologies) and processed into single cell suspensions. Blood was collected by tail vein lancet into 1.3-ml EDTA blood tubes (Thermo Fisher Scientific) and then centrifuged for plasma collection. Progesterone levels were quantified by immunoassay in one batch. Assay services were provided by the Biomarkers Core Laboratory at the Yerkes National Primate Research Center. Tissues were flash-frozen in Tissue-Tek Optimal Cutting Temperature Compound (Sakura) and stored at −80°C. Tissue blocks were sectioned onto microslides at 6- to 8-μm thickness using a cryostat. For immunofluorescent staining, slides were fixed in 75:25 acetone/ethanol and then blocked with donkey and rabbit serum. The following antibodies were used: α-E-cadherin (Abcam clone: DECMA-1) paired with α-rat immunoglobulin G Alexa Fluor 488 (Abcam), α-TCR β chain Alexa Fluor 594 (BioLegend clone: H57-957), and α-CD31 Alexa Fluor 647 (Abcam clone: MEC 7.46). Mounting medium with 4′,6-diamidino-2-phenylindole (Abcam) was added before microscopy. Imaging was performed on a Zeiss Axio Observer Z1 with an Axiocam 506 monochromatic camera. Image processing was performed using Zen 2 software. Chimeras were generated by euthanizing donor mice and harvesting femur and tibia bones for bone marrow cell isolation, which was then intravenously injected into irradiated recipient mice. Recipients received two doses of gamma irradiation (550 rad) 6 hours apart using a Small Animal Radiation Therapy (SmART+) system. Mice were kept in sterile housing and underwent antibiotic treatment with neomycin for 6 weeks by administering 1 g into 300 ml of sterile drinking water. Chimerism was confirmed after 8 to 10 weeks via flow cytometry from blood samples. Animals were rested for at least 12 weeks before treatment. To induce a decidual reaction in chimeras, animals were administered 2 mg of MPA and, after 1 week, received intrauterine sesame oil 4 days before necropsy. In the event of comparisons with repeated time point collections (used only in the study’s control arm due to an overlapping phase), cellular immune marker comparisons by cycle phase were fit using generalized estimating equations (GEEs) with an exchangeable working correlation structure as previously described ( 25 , 64 ). A two-way analysis of variance (ANOVA) was performed with matching for longitudinal comparisons between participant groups. Other statistical testing approaches are indicated in the respective figure legends. GraphPad Prism software version 10.2.2 was used for statistical modeling in non-GEE approaches.