Association between sex hormones and innate cytotoxic capacity of circulating and rectal mucosal immune cells.

Due to the previously reported associations between estradiol and innate cytotoxic effector functions, [ 41 – 44 ] we quantified systemic estradiol and testosterone and circulating CD56+CD16+ NK cells as a percentage of CD45+ cells in the four study populations. As expected, serum estradiol concentrations in CGM were significantly lower than that observed in CGW (q=0.0001; Figure 1A ), TGW (q<0.0001), and TGM (q=0.006). Serum concentrations of testosterone in CGM were significantly higher than that observed in CGW and TGW (q<0.0001; Figure 1B ), while values in TGM were significantly higher than CGW (q=0.0002) and TGW (q=0.0012). There were no significant differences in percentages of CD56+CD16+ NK cells between the study populations ( Figure 1C ; q>0.05). Similar to outcomes of previous studies, innate specific lysis capacity of PBMC isolated from CGW was significantly lower than that observed in CGM ( Figure 1D ; q=0.0015). Within this cohort, no significant differences were observed between CGW when stratified by hormonal contraceptive use (p=0.49; median “no contraceptive”=5.6%, “yes contraceptive”=6.1%). Specific lysis of cells isolated from TGW and TGM were not significantly different from the cisgender cohorts, with all values falling between the maximum boundary (CGM, maximum value 32.1%) and minimum boundary (CGW, minimum value 1.52%) exhibited by the cisgender cohorts. While reduced innate cytotoxic capacity in CGW has generally been attributed to estradiol, Spearman correlations identified significant associations between log specific lysis and log estradiol (r=−0.31; q=0.014), testosterone (r=0.29; q=0.015), and the percentage of CD56+CD16+ NK cells (r=0.24; q=0.034) ( Figure 1E – G ). Despite minimal differences in CD56+CD16+ NK percentages between study populations and a modest Spearman correlation with specific lysis, linear regression modeling of these variable revealed that the percentage of CD56+CD16+ NK cells had the most significant influence on specific lysis values (Beta 0.03, p=0.05), followed by estradiol (Beta=−0.27, p=0.12), and testosterone (Beta=0.22, p=0.16). This hierarchy of influence was further supported by the results of a drop-one evaluation model, where Akaike Information Criterion (AIC) of the full model (146.08, R 2 adj =0.142) demonstrated the largest AIC increase/R 2 adj decrease when the percentage of CD56+CD16+ NK cells was removed from the model (AIC=155.7, R 2 adj =0.111), followed by estradiol (AIC=146.6, R 2 adj =0.126) and testosterone (AIC=146.2, R 2 adj =0.130). Assessment of collinearity showed low VIFs for all predictors (log10 testosterone=1.33, log10 estradiol=1.28, CD56+CD16+ NK%=1.09), indicating no evidence of problematic multicollinearity. The predictor correlation matrix showed a moderate inverse correlation between log10 testosterone and log10 estradiol (r=−0.46), while CD56+CD16+ NK percentage showed only modest correlations with log10 testosterone (r=0.35) and log10 estradiol (r=−0.24). Exploratory assays evaluating the innate cytotoxic capacity of cells residing within the rectal mucosal (RM) lamina propria were carried out on a subset of study participants from whom rectal biopsies were available for analyses. Assays revealed that specific lysis values generated with RM cells were significantly higher than values obtained with PBMC from the same study participants (Wilcoxon test; p=0.0003; RM median=13.6%, IQR=7.1–21.2%; PBMC median=5.9%, IQR=4.3–7.1%). While there was a positive association between RM NK specific lysis and PBMC values that did not reach statistical significance (r=0.48; p=0.06) in this smaller subset of participants. There was no association with RM NK specific lysis and circulating estradiol or testosterone concentrations. CD56+CD16+ quantification was not performed with RM cells, as this immune cell subset is generally not present within the rectal mucosa, [ 52 , 53 ] and the phenotype of the precise cells capable of mediating innate cytotoxicity within this tissue compartment are not well described.

This study was approved by Emory University Institutional Review Board, and written informed consent was obtained from all participants. Participants included here represent a subset of individuals enrolled in a larger parent study which focused on evaluating the role of GAHT on the systemic and gut mucosal immune environment in TGW as compared to CGM, as well as additional individuals enrolled for this smaller, gender inclusive sub study (CGW, TGM, and additional TGW and CGM). Participants were healthy individuals recruited from the Atlanta area who tested negative for HIV and bacterial sexually transmitted infections at the time of sampling. Study participants included self-identified CGM (n=30; median age 35 years) and CGW (n=14; median age 32 years) who reported no exogenous hormone use (with the exception of hormonal contraceptive modalities: OCP=5, IUD=2, implant=1), as well as TGW (n=30; median age 38 years) and TGM (n=8; median age 25 years) who reported use of gender affirming hormone therapy for >6 months without a change in dose for >3 months. Specific GAHT formulations, doses, and modalities were diverse and functionally participant-specific. Broadly, TGW regimens included estrogen (pill, patch, injectable)=29, spironolactone=16, cyproterone=2, progesterone=14, the precise modality was unavailable for one participant. While TGM regimens generally only included testosterone (injectable=8), three also reported hormonal contraceptive use (injection=1, IUD=1, implant=1). Serum was collected via SST tubes and cryopreserved for batch analyses. Serum hormone concentrations were quantified via Estradiol (DRG, #75871–318) and Testosterone (DRG, #75871–120) ELISA kits, according to manufacturer instructions. PBMC were isolated via CPT tubes and cryopreserved for batch analyses. PBMC were thawed, washed of freeze media, and allowed to rest overnight in complete media (RPMI+10% FBS+antibiotic/antimycotic) at 37C, 5% CO2. K-562-GFP (ATCC, #CCL-243-GFP) were maintained in IMDM (ATCC, #30–2005) with 10% FBS and 0.5 ug/ml puromycin. Day of assay, PBMC count and viability were assessed and cells were diluted to 1,500,000 viable cells/ml. K-562-GFP count and viability were assessed and cells were diluted to 60,000 viable cells/ml. To a V-bottom 96-well plate, 50 ul of K-562-GFP cells were added to 100 ul of PBMC, or, to 100 ul media for background target cell death controls, in replicates of five. This resulted in 150,000 PBMC to 3,000 K-562-GFP, for a 50:1 Effector:Target ratio. One well of PBMC only with 50 ul media was included as a negative control. Plates were spun 100xg for one minute, then placed in 37C, 5% CO2 incubator for four hours. SYTOX Red (Invitrogen, 501137614) was used to stain dead/dying cells for 20 minutes before flow cytometry acquisition. To determine the innate cytotoxic capacity of cells residing with the rectal mucosa, 8 to 10 fresh rectosigmoid biopsies were collected 3 to 10 cm from the anal verge via rigid sigmoidoscopy from a subset of individuals (CGM=3, CGW=4, TGW=3, TGM=6). Biopsies were processed into a single-cell suspension via gentleMACS program 37C_h_TDK_2 in 3 ml of digestion media (RPMI with Collagenase IV, HEPES, DNAse). Cell count and viability were assessed after filtering, debris removal, and red blood cell lysis. Rectal mucosal cells were then resuspended to a concentration of 600,000 viable cells/400 ul for use in K-562-GFP killing assay, in triplicate, except when cell availability precluded three replicates (P-032, P-058). For each sample, the percentage of SYTOX Red+ GFP+ cells (dead/dying K-562 target cells) were calculated as a percentage of GFP+ cells (generally 1,000 to 2,000 K-562-GFP events collected). In PBMC-only control wells, GFP+ cells (i.e. autofluorescence) was negligible (<10 cells) for all participants. Values from replicate cells were averaged, and normalized to background target cell death to determine sample specific lysis: (Average % Death – Average Background % Death)/(100 – Average Background % Death). Median Average Background K-562-GFP death was 1.7%. Separate from K-562-GFP killing assays, cell staining to quantify innate immune cell subsets was performed, and included LIVE/DEAD (Thermo, # L34966 ), CD45 (Biolegend, #304050), CD3 (Biolegend, #300424), and markers for B cells (CD20, Biolegend #302314; HLA-DR, #307604), monocytes (CD14, Biolegend #301812), and myeloid cells (CD11c, Biolegend #301606; CD1c, #331526; CD123, #306034), and included routine markers for blood Natural Killer cells (CD56, Biolegend #362510; CD16, BD #563172). NK subsets were gated as follows: Lymphocytes, singlets, live, CD45+, CD3−, CD20−, HLA-DR+/−, CD14−, then CD56+CD16+ or CD16−CD56+, and reported as a percentage of CD45+ cells. Multiple comparisons between the four study groups were performed with Kruskal-Wallis test with two-stage step-up Benjamini, Krieger, and Yekutieli correction for multiple comparisons. Significant discoveries were reported when q values <0.05. Correlations were calculated via nonparametric Spearman, with two-stage step-up Benjamini, Krieger, and Yekutieli FDR=5%, q<0.05 considered significant. Analyses were performed with GraphPad Prism 7.0. To assess factors associated with NK cytotoxic activity, linear regression models were constructed with specific lysis as the dependent variable. Because specific lysis, testosterone, and estradiol exhibited right-skewed distributions, all three variables were log10-transformed prior to modeling. The initial full model included log10 testosterone, log10 estradiol, and the frequency of CD56+CD16+ cells among CD45+ leukocytes as candidate predictors. Model fit and statistical significance were evaluated using estimated coefficients and two-sided p-values. To further assess potential multicollinearity among predictors included in the model, variance inflation factors (VIFs) and the predictor Spearman correlation matrix were calculated. To quantify the relative contribution of each predictor, drop-one variable comparisons were performed, in which each predictor was removed from the model individually. Resulting changes in Akaike Information Criterion (AIC) and adjusted R2 were used to evaluate the influence of each variable on model performance, with larger increases in AIC and greater decreases in adjusted R2 interpreted as stronger influence on specific lysis.

In summary, our study concludes that innate cytotoxic capacity is an amalgam of factors, including an individual’s CD56+CD16+ NK percentages and serum sex hormone concentrations.

Here, we enrolled a sex- and gender-inclusive cohort of participants to begin to elucidate relationships between sex hormones, circulating innate cytotoxic cells, and innate cytotoxic effector functions. Defining significant contributors to observed sex-based clinical outcomes in humans requires untangling complex, dynamic interactions between the sex hormonal milieu, sex chromosomes, and gender-based social behaviors, each of which could play a role in immune cell differentiation and maturation in vivo in a manner that cannot be modeled ex vivo . [ 45 , 46 ] Our data reveal that the reported significant reduction of circulating innate specific lysis in CGW, [ 41 – 44 ] also reported here, can be partially attributed to increased systemic concentrations of estradiol, but also decreases in systemic testosterone and the proportion of circulating cells with NK CD56+CD16+ phenotypes. By also including data from TG persons, we were also able to demonstrate, for the first time, that GAHT could also contribute to diverse phenotypes in circulating innate cytotoxic capacity. That is, while specific lysis was significantly higher in CGM, as compared to CGW, there was no significant difference between TGW and CGW, nor was there a difference between CGM and TGM. Indeed, specific lysis values from TG individuals fell between the apparent sex-binary CGW (low), CGM (high) specific lysis spectrum. These data illustrate that consideration of sex as a binary variable (male/female), or strict categorization of individuals in human subjects’ research by sex designation at birth, obfuscates the potential biological influence of gender on immune phenotypes. Beyond categorization by sex and gender, our data suggests that each of these three assayed metrics (systemic estradiol, testosterone, CD56+CD16+ NK proportion) is unique within every individual, which culminates in person-specific patterns of innate cytotoxic potential. Thus, as opposed to simple categorization of study participants based on birth sex designation, each of these quantitative metrics should be considered in design, analyses, and interpretation of outcomes in future basic, translational, and clinical intervention studies which focus on innate cytotoxic effector functions. It is well accepted that systemic concentrations of sex hormones vary between and within cisgender individuals, [ 54 ] and diversity in systemic estradiol concentrations in CGW (the menstrual cycle, endometriosis, pregnancy, and menopause) have each been associated with changes in innate cytotoxic capacity. [ 44 , 46 , 55 – 59 ] This cohort of CGW was heterogeneous, as samples were not collected during a defined stage of menstruation and, diverse modalities of contraception were used by some CGW. Hormonal contraceptives often contain synthetic hormones, such ethinyl estradiol, which are not quantifiable through the assays utilized here and could have differential immunomodulatory effects from endogenous or bioidentical estradiol. [ 60 ] While categorized as “CGW”, each CGW participant in this study represents a unique data point. Similarly, there is not a singular GAHT regimen for TGW or TGM. TG persons work with their physicians to choose, and change, GAHT modalities which best suit their unique needs and goals. [ 31 ] This was reflected in participant-specific GAHT regimens in our TG cohorts, where we observed a broad range of systemic sex hormones. For example, approximately half of our cohort of TGW exceeded the standard clinical goal of 200 pg/ml estradiol, while approximately a quarter of TGW were not yet effectively suppressing endogenous testosterone production. Similarly, our cohort of TGM had systemic testosterone concentrations comparable to the cohort of CGM, however endogenous estradiol was still detectable. The outcomes reported here represent diversity in GAHT hormonal outcomes in TG persons, and the relationship between of these individualized therapeutic hormone concentrations and specific lysis activity. In summary, considering the diversity in sex hormones in CG and TG persons, incorporation of these values in our mathematical modeling approaches facilitated our ability to rank the contributions of each of these assayed metrics in systemic specific lysis outcomes, further illustrating the relative importance of systemic estradiol, testosterone, and CD56+CD16+ NK proportion in innate specific lysis phenotypes. We are also among the first to explore innate cytotoxic capacity in the context of immune cells residing within the rectal mucosal lamina propria. The assays performed here with K-562 cells are well established within the field. However, use of this protocol with tissue-resident immune cells is limited. [ 61 – 63 ] The exploratory assays performed with samples from a subset of study participants here demonstrated that cells residing within the rectal lamina propria are able to potently target cells that lack MHC Class I for cell death via innate pathways. This outcome must be explored within a larger cohort in greater detail, as it could have profound influence on modern strategies to enhance innate cytotoxic capacity to treat conditions where innate responses, or, downregulation of MHC Class I, is key, such as colon cancer, or targeting virally infected cells within the lamina propria tissue compartment. [ 64 – 66 ] A substantial limitation of the data presented here, is that innate cytotoxic cells such as NK cells differ in both phenotype and function depending on the tissue of residence. [ 53 ] The precise phenotype of the cells mediating this effector function are not well described and was not defined here, though the CD56+CD16+ NK mediating cell death in the PBMC assays described here are generally not present within the rectal mucosa. [ 52 ] We also note that there was an insignificant but positive trend between PBMC and rectal mucosal cytotoxic capacity. This suggests evaluation of specific lysis of circulating cells is not a suitable proxy for estimating the innate cytotoxic potential of rectal mucosal immune environment, however limited power within these exploratory assays tempers this conclusion. Likewise, we also did not identify a significant correlation between rectal mucosal specific lysis and circulating estradiol or testosterone concentrations, while these associations were observed in PBMC. It is possible that sex hormone concentrations within these tissue compartments is distinct from systemic concentrations. Or, the innate cells mediating assayed cytotoxicity might be distinct from those in PBMC, thus are differentially affected by sex hormones. Thus, while the rectal mucosal assays performed here were exploratory, larger future studies which seek to expand upon our observations should seek to identify and characterize the cells mediating this observed cytolysis and the role of sex hormones in their function. This will allow advancements towards personalized therapeutic interventions specific for the immune cells that reside within the rectal mucosa and our mechanistic understanding of innate cytotoxicity within this critical immune and mucosal barrier. There are a number of limitations within this small study, which should be considered in future studies. This study is cross-sectional, and, GAHT is unique to each individual. Outcomes observed here could be influenced by an individual’s pre-GAHT baseline, however that data was not captured within this study. Future studies which evaluate systemic hormones and innate cytotoxic capacity longitudinally, in the same individuals before and at multiple time-points after GAHT, will be capable of incorporating these data into analyses. [ 19 , 67 ] And, while the data reported here is from a heterogeneous cohort of individuals, precisely defined and controlled hormonal regimens with murine and non-human primate models could provide additional immunological insight and causal support for the role of GAHT in functional immunological outcomes, in support of the correlative outcomes observed here. [ 39 , 68 , 69 ] Furthermore, while associations between sex hormones and innate cytotoxic capacity described here and elsewhere is relatively consistent, it is notable that the precise molecular and immunological mechanisms contributing to phenotypic differences in innate cytotoxicity have yet to be defined. Animal model systems allow for more invasive, longitudinal sampling, which could provide a pathway to precisely define these mechanisms in a manner that has not been possible with ex vivo assays or human subjects research. Finally, transcriptomic analyses were not performed on immune cells before (with cells at homeostasis) or after K-562-GFP stimulation. There are potentially additional factors, including genes on sex chromosomes, that could play a role in study outcomes, which should be considered in future studies. A recent publication noted that increased expression of X-linked KDM6A (aka UTX) is associated with increased specific lysis with splenic cells isolated from mice, in a variety of hormonal conditions. [ 70 ] Thus, while a substantial body of literature supports the role of estradiol in NK number and function, additional sex-based factors are potentially also contributing to diversity in NK function between individuals. Thus, inclusion of TG persons in future transcriptomic analyses, specifically those that focus on immune cells that reside within the rectal mucosal lamina propria, are a top research priority to further elucidate the intersection of sex hormones and sex chromosomes on immune cells within this critical tissue compartment.

While sex hormones are primarily known for their influence on reproductive systems, it is now well established that sex hormones also contribute to the sexually dimorphic immunological outcomes observed between cisgender women (CGW) and men (CGM). [ 1 – 15 ] Estrogens, progesterone, and testosterone each exert distinct immunomodulatory effects [ 16 – 21 ] over the entire “lifespan” of innate and adaptive immune cells. [ 22 – 30 ] While this growing body of literature is defining the role of sex hormones in immunity in cisgender persons, it could also be hypothesized that gender affirming hormone therapy (GAHT), which suppresses endogenous sex hormones with the goal of maintaining affirmed gender sex hormones within normal physiologic ranges, [ 31 ] is likely also an important contributor to unique immunological outcomes among transgender persons. A majority of transgender women (TGW) utilize GAHT, [ 32 ] which generally includes the use of supplemental estrogen and anti-androgen, [ 33 , 34 ] and can also incorporate supplemental progesterone. [ 35 ] Among transgender men (TGM), GAHT regimens primarily incorporate testosterone, which also acts to suppress endogenous estrogen. [ 36 ] Recent studies seeking to define the immunomodulatory effects of GAHT have reported significant observations, [ 37 – 40 ] critical for design and implementation of personalized, sex- and gender-appropriate biomedical interventions for cisgender and transgender persons. However, studies evaluating immune and clinical outcomes in transgender populations remain scarce. Previous studies have described an apparent role of estradiol in decreased Natural Killer (NK) cell number and cytotoxic effector functions in CGW. [ 41 – 44 ] Interestingly, standard ex vivo strategies, such as culturing of purified immune cells with supraphysiologic concentrations of sex hormones, had no significant effect on innate cytotoxicity. [ 45 , 46 ] This is in contrast to the significant differences reported after sustained hormone replacement therapy in menopausal women, or, oral contraceptive pills (OCP) use in CGW, suggesting that the apparent sex-bias in functional cytotoxicity cannot be solely attributed to a direct effect of estradiol or testosterone on innate cells, but appears to be a cumulative effect of the presence of sex hormones during immune cell differentiation and maturation in vivo . [ 41 , 42 , 46 ] Remarkably, though it is well accepted that sex hormones can influence innate cytotoxicity, and, the increased research focus on NK due to their innate capacity to target and eliminate diverse cells of interest with the goal of therapeutically altering NK-mediated cytotoxicity, [ 47 – 51 ] innate cytotoxic capacity of individuals on GAHT has not been described. To begin to address this gap in our knowledge, here, we expanded upon previous studies to include evaluation of innate cytotoxic capacity of circulating immune cells isolated from both cisgender and transgender people to evaluate the relative roles of systemic estradiol, testosterone, and NK populations in circulating innate cytotoxic capacity, as well as exploratory assays evaluating tissue-specific (rectal mucosal) cytotoxic capacity in a subset of study participants.