Author response: The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli. A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function. Editor's evaluation This manuscript reports important findings regarding the highly variable immune environments along the epididymis. Using multiple mouse models (bacterial infection and parabiosis between WT and Ccr2 KO) in conjunction with scRNA-seq analyses, the authors provided solid evidence supporting the notion that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for sperm maturations and elimination of pathogens ascending the urogenital tract. https://doi.org/10.7554/eLife.82193.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Within the male reproductive tract, the epididymis plays an essential role in post-testicular sperm maturation and storage. Immotile spermatozoa released from the seminiferous epithelium of the testis enter the epididymis via the efferent ducts and undergo distinct consecutive biochemical maturation processes required to gain motility and fertilization capacities (Belleannee et al., 2011; Skerget et al., 2015; Björkgren and Sipilä, 2019; Barrachina et al., 2022). The sequential maturation process is orchestrated by the pseudostratified epithelium composed of several different epithelial (principal, basal, narrow/clear) and immune cell types that creates an unique luminal milieu. The barrier function of the epididymal epithelium highly depends on epithelial integrity (Breton et al., 2019). Intraepithelial immune cells, particularly mononuclear phagocytes (MP), are highly abundant within the epididymal epithelium and perform a key role in the preservation of epithelial integrity (Smith et al., 2014). From an immunological perspective, the epididymis performs a functionally complex role by providing an immunotolerant environment for transiting immunogenic spermatozoa, while maintaining the capacity to effectively combat invading pathogens ascending from the urethra and vas deferens. Previous investigations in rodents revealed differences in the immune reactions of opposing ends of the epididymis toward ascending bacterial infection and other local and systemic inflammatory stimuli. In this regard, the proximal regions appear to be almost unresponsive, while the distal regions are prone to intense immune responses resulting in persistent tissue damage (Michel et al., 2016; Silva et al., 2018; Klein et al., 2019; Wang et al., 2019; Klein et al., 2020; Wijayarathna et al., 2020). As the epididymis consists of a single highly convoluted duct that meanders through structurally different regions (initial segment [IS], caput, corpus, cauda), inflammation-associated tissue damage and fibrotic remodeling result in epididymal duct obstruction which has a direct impact on the maturation and passage of sperm and, thereby, fertility. The histopathological observations in rodent models replicate many of the clinical manifestations in epididymitis patients (Pilatz et al., 2015; Fijak et al., 2018). Epididymitis in humans is mostly caused by urogenital tract infections with coliform bacteria (i.e. uropathogenic Escherichia coli [UPEC]) or pathogens linked to sexually transmitted diseases (e.g. Chlamydia trachomatis, Pilatz et al., 2015; Pleuger et al., 2020) and can effectively be treated with antibiotics. However, up to 40% of epididymitis patients exhibit a persistent sub- or infertility (Rusz et al., 2012), most likely due to epididymal duct stenosis/obstruction and concomitant oligo- or azoospermia. The reasons underlying differences in different immune responsiveness, with strong pro-inflammatory immune response largely confined to the cauda, are not well understood. Within the last few decades, initial steps have been made in characterizing the immunological landscape within the epididymis and understanding how the epididymis is prepared for its immunological challenges (Nashan et al., 1989; Flickinger et al., 1997; Serre and Robaire, 1999; Da Silva et al., 2011; Shum et al., 2014; Pierucci-Alves et al., 2018; Voisin et al., 2018; Battistone et al., 2020; Mendelsohn et al., 2020; Wang et al., 2021). The murine epididymis is populated by various myeloid and lymphoid cell populations that are differentially distributed along the epididymal duct. Subsets of the MP system are the most prominent group within the epididymis and form a dense network within and around the epididymal duct, especially within the IS which is the site of spermatozoa entry (Da Silva et al., 2011; Battistone et al., 2020). Generally, the MP system comprises multiple subsets that can share similar cell surface markers, yet possess distinct functions related to tissue homeostasis and pathogen-specific immunity. Despite accumulating information about the localization and antigen presentation and antigen-processing properties (Da Silva et al., 2011; Da Silva and Smith, 2015; Battistone et al., 2020; Mendelsohn et al., 2020), the identity of MP subgroups within the epididymis as well as the full extent of their heterogeneity is still not well understood mainly due to the general similarities between macrophage and DC subpopulations. In view of the fundamentally different immunological requirements of the epididymis, maintaining both a stable and immunotolerant microenvironment for sperm maturation in the proximal regions and the ability to mount adequate immune responses toward invading bacteria at the distal end, detailed investigation of the phenotypes, localization, and function of resident immune cells is essential. In this regard, we hypothesized that strategically positioned resident immune cells that function as both ‘scavengers’ and ‘guardians’ create distinct immunological landscapes within epididymal regions. These, in turn, are responsible for the observed differences in the intensity of the immune responses toward infectious or inflammatory stimuli as well as for tissue homeostasis and the maintenance of epithelial function that is essential for regulating the sequential steps of sperm maturation. Therefore, in this study, we aimed to both (i) analyze the differential immune responses to UPEC-elicited epididymitis and (ii) uncover the immune diversity among epididymal regions, by using an unbiased single-cell RNA sequencing (scRNASeq) analysis complemented by flow cytometry and immunofluorescence analysis to localize identified populations in situ. Results Caput and cauda epididymides react fundamentally differently during acute bacterial epididymitis To better understand the different immune responses within the epididymal regions and to expand on our previous studies, an experimental bacterial mouse epididymitis model was used to monitor disease progression up to 10 days post infection (p.i., Figure 1A). Bacteria were found in all epididymal regions (IS, caput, corpus, cauda) and in the testis 1 day p.i. (Figure 1B), but persisted at high numbers only for up to 10 days in the cauda (Figure 1B). Later time points were not examined, as it is known that bacteria are cleared toward day 30 p.i. (Klein et al., 2019). In line with previous reports (Klein et al., 2020), the caput showed no gross morphological alterations (Figure 1C, Figure 1—figure supplement 1A–D), although slight histopathological changes, including mild focal epithelial damage and minor connective tissue deposition within the interstitium, were observed 5 days p.i. (Figure 1C) resulting in an elevated disease score that returned to normal values at 10 days p.i. (Figure 1D). In accordance with previous studies from our group (Klein et al., 2020) severe tissue remodeling was seen in the cauda (Figure 1E, Figure 1—figure supplement 2) characterized by infiltration of immune cells, loss of epithelial integrity, connective tissue deposition in the interstitium, reduction of luminal diameter, and ultimately, epididymal duct destruction resulting in a significantly increased and persistent overall disease score (Figure 1F). Initially, immune cell infiltrates were predominantly located peripherally within the cauda (5 days p.i.) before larger leukocytic conglomerates/granulomas developed within the entire cauda region (Figure 1—figure supplement 2). Sham control mice initially showed histopathological alterations in the cauda that were milder than in infected animals and returned to a level comparable to untreated epididymis toward day 10 p.i. (Figure 1E and F, Figure 1—figure supplement 2). Figure 1 with 3 supplements see all Download asset Open asset Analysis of differential immune responses of caput and cauda epididymides following uropathogenic Escherichia coli (UPEC) infection in C57BL/6J wild type mice. (A) Male C57BL/6J mice (10–12 weeks of age) were intravasally injected with UPEC or saline vehicle alone (sham) after ligation of the vas deferens. For the study organs were harvested and analyzed at the indicated time points. (B) Bacterial loads were assessed by determining colony forming units per mg tissue at the indicated time points within testis and the four main epididymal regions (initial segment [IS], caput, corpus, cauda; n=4 per time point, mean ± SD). (C and D) Modified Masson-Goldner trichrome staining of caput (C) and cauda (D) epididymides showing histological differences between sham- and UPEC-infected mice at day 1, day 5, and day 10 post infection. Scale bar 50 µm. (E and F) Pearson’s correlation plot of infection time point (days post infection) and disease score of caput (E) and cauda (F). The average ± SEM disease score per time point (n=4 per time point) for sham- and UPEC-infected mice is shown. Pearson’s correlation was considered to be statistically significant at p<0.05. (G) Volcano plot of differentially expressed genes (DEG) identified between sham- and UPEC-infected mice within caput and cauda epididymides by RNASeq analysis. Numbers of DEG are indicated below the respective plot. Cut-off criteria: FDR ≤0.05, –1 < logFC > 1. (H) Top 30 DEG by comparing caput and cauda epididymides of sham- and UPEC-infected mice. Cut-off criteria: FDR ≤0.05, –1 < logFC > 1. (I) Gene set enrichment analysis using DEG between caput and cauda epididymides of UPEC-infected mice. Cut-off criteria: FDR < 0.2, Top up/downregulated gene sets based on gene ontology. Whole transcriptome and tissue analysis reveal fundamentally different immune responses in caput and cauda epididymides following infection Initial examination pointed to different gene signatures in caput and cauda epididymides under physiological conditions, but examination under infectious conditions was not performed (Klein et al., 2019). We employed whole transcriptome analysis by RNA sequencing of total caput (including the IS), corpus and cauda 10 days p.i. to investigate the principal changes in the whole transcriptome of the different epididymal regions under pathological conditions in vivo. In line with the minimal histopathological alterations, almost no transcriptional differences were identified between the caput of sham- and UPEC-infected mice (in total 5 differentially expressed genes (DEG), cut-off: FDR ≤0.05, –1 < logFC > 1, Figure 1G, Figure 1—figure supplement 3A). Intriguingly, although the transcriptional profiles of the caput in sham- and UPEC-infected mice were very similar, upregulation of a few infection-related genes such as S100a8, S100a9, and Slfn4 was indicative for the presence of UPEC in the infected caput (Figure 1—figure supplement 3B). In contrast, the cauda of sham- and UPEC-infected mice showed considerable transcriptional differences (in total 5082 DEG, cut-off: FDS ≤0.05, –1 < logFC > 1, Figure 1g, Figure 1—figure supplement 3C). As shown by principal component analysis (PCA), the transcriptional changes in the corpus were intermediate compared with those in caput and cauda epididymides (Figure 1—figure supplement 3A), an observation that was reflected in a comparable magnitude of histopathological alterations (Figure 1—figure supplement 2). To analyze principal differences, we focused on caput and cauda in subsequent studies as these regions displayed greater differences in gene expression levels and histopathology. Compared to the cauda, the caput was highly enriched in transcripts encoding immunomodulatory factors, such as β-defensins, bactericidal permeability-increasing protein, and indoleamine 2,3-dioxygenase 1, with no changes in the high levels observed in sham- and UPEC-infected mice (Figure 1H). In contrast, compared to sham control mice, the cauda of UPEC-infected mice was characterized by an upregulation of numerous transcripts encoding pro-inflammatory mediators, including pro-inflammatory cytokines (e.g. Il-1α, Il-6, Il-17) and chemoattractants (e.g. Ccl2, Ccl3, Ccl4, Cxcl2, Cxcl5) as well as inflammasome-associated transcripts (e.g. Nlrp3, Il1b) (Figure 1H, Figure 1—figure supplement 3D). By grouping transcripts according to their gene ontology and pathway contribution, the cauda of UPEC-infected mice 10 days p.i. displayed upregulation of gene sets associated with fibrotic tissue remodeling and pro-inflammatory immune responses (e.g. positive regulation of NF kappa B – transcription factor activity, collagen fibril organization, and positive regulation of the ERK1 and ERK2 cascade, Figure 1I). Further pathway analyses revealed an upregulation of gene sets associated with B and T cell activation, indicating a transition from the innate to the adaptive immune response at this stage of infection within the cauda (Figure 1—figure supplement 3). The caput epididymidis of sham and infected mice were enriched with gene sets related to sperm maturation (e.g. protein localization in cilium, cellular component assembly involved in morphogenesis, and regulation of cilia beating frequency, Figure 1I), indicative of normal epididymal function. Flow cytometry analysis of immune cell populations in UPEC-infected mice In line with the observed histopathological alterations and the transcriptional profile of the cauda of UPEC-infected mice, disease progression correlated positively with the appearance and degree of immune cell infiltration in this region (Figure 2A, Figure 1—figure supplement 1D). Notably, we observed an increase in the total immune cell population (CD45+) in both the caput and cauda of sham mice with most immune cell infiltrates observed at day 5, which returned to normal levels by day 14 (Figure 2B). This indicated that an immune response was elicited in the absence of pathogens by surgery-associated trauma and ductal pressure due to the ligation of the vas deferens. In the context of infection with UPEC, an increased infiltration of immune cells was observed in the cauda, whereas the caput showed a significant increase of immune cells compared to sham injected mice only at day 14 p.i. (Figure 2B). Immune cell infiltration peaked at 10 days p.i., which correlated with disease score of the infected animals (Figure 2C). Figure 2 with 3 supplements see all Download asset Open asset Analysis of changes in immune cell populations following infection with uropathogenic Escherichia coli (UPEC) in C57BL/6J wild type mice. (A) Pearson’s correlation plot of infection time points (days post infection) and the area of immune cell infiltration within the total cauda area (%) determined by histological evaluation. Mean ± SD of at least two independent experiments with each n=4 are plotted per time point for sham- and UPEC-infected mice. Pearson’s correlation was considered to be statistically significant at p<0.05 (*p<0.05, **p<0.005, ***p<0.001). (B) Percentage of CD45+ cells in single live cells within caput and cauda assessed by flow cytometry at different time points (days) post infection (mean ± SD, n=4, two-way ANOVA with Bonferroni post hoc test, *p<0.05, **p<0.005, ***p<0.001). (C) Pearson’s correlation plot showing disease score and percentage of CD45+ cells in single live cells. Pearson’s correlation was considered to be statistically significant at p<0.05. (D) FltSNE plots of CD45+ populations in naïve, sham- and UPEC-infected mice 10 days after infection. Cells were gated as described in Figure 2—figure supplement 1 and downsampled to equal cell numbers for each segment. Samples from all biological groups (three biological replicates, respectively) were concatenated, FltSNE plots (perplexity: 20, max. iterations 1000, exaggeration factor: 12) were generated and individually gated cell populations were overlaid using FlowJo software and colored according to the legend on the right. (E) Bar diagram showing the ratio of neutrophils (GR-1+SSChi cells) within single live cells in initial segment (IS), caput, corpus, cauda of naïve, sham- and UPEC-infected mice 10 days after infection, 4–6 biological replicates from two independent experiments were grouped, mean ± SD, two-way ANOVA with Bonferroni post hoc test, *p<0.05, **p<0.005, ***p>0.001. (F) Bar diagram showing the ratio of monocytes (GR-1+SSClo cells) within single live cells in IS, caput, corpus, cauda of naïve, sham- and UPEC-infected mice 10 days after infection (4–6 biological replicates from two independent experiments were grouped, mean ± SD, two-way ANOVA with Bonferroni post hoc test, *p<0.05, **p<0.005, ***p>0.001). (G) Stacked bar diagrams showing the ratio of analyzed GR-1- immune cells within single live cells in IS, caput, corpus, cauda of naïve, sham- and UPEC-infected mice 10 days after infection (4–6 biological replicates from two independent experiments were grouped, mean ± SD, two-way ANOVA with Bonferroni post hoc test, *p<0.05, **p<0.005, ***p>0.001). Identified immune cells are colored equally to the FltSNE plots shown in (D). In both panels indicated immune cells were identified according to the gating strategy displayed in Figure 2—figure supplement 1. (H) Bar diagram showing the ratio of CCR2+ cells in the total macrophage population (F4/80+CX3CR1+/-), 4–6 biological replicates from two independent experiments were grouped, mean ± SD, two-way ANOVA with Bonferroni post hoc test, *p<0.05, **p<0.005, ***p>0.001. (I) Confocal microscopy images showing the location of Ly6G+Ly6C+ cells (GR-1+, red) within caput and cauda of UPEC-infected mice 5, 10, and 14 days post infection (nuclei in gray) including bar diagrams showing the semi-quantified summary of all immunostained tissues (by counting Ly6G+Ly6C+ cells within caput and cauda of sham- and UPEC-infected mice, n=4, for each biological replicate three representative areas were counted, mean ± SD). Scale bar 50 µm. As the RNASeq data indicated a transition from innate to adaptive immune responses 10 days after infection, which also correlated with the peak of immune cell infiltration in several segments, we aimed to further characterize immune cell populations within all epididymal regions (IS, caput, corpus, cauda) of naive, sham- and UPEC-infected mice. We designed a flow cytometry panel that allowed us to simultaneously identify different populations of innate (neutrophils, monocytes, macrophages, dendritic cells, NK cells) and adaptive immune cells (B and T cells, Figure 2—figure supplement 1). CX3CR1+ macrophages represented the most dominant immune cell population in the IS and caput, whereas immune cell composition was more diverse in the corpus and cauda (Figure 2D). While neutrophils were absent in samples from naive mice, infiltrates of Gr-1+SSChi neutrophils (Figure 2E) and GR-1+SSClo monocytes (Figure 2F) were most pronounced in the corpus and cauda upon UPEC infection (Figure 2D–F. Figure 2—figure supplement 2). Furthermore, both corpus and cauda showed a significant increase in adaptive immune cell populations (B and T lymphocytes), which were present after sham injection and UPEC infection (Figure 2G) and correlated with the observed enrichment of gene sets associated with B and T cell activation at 10 days p.i. (Figure 1—figure supplement 3E). Compared to naive mice the ratio of CX3CR1+ macrophages significantly decreased in sham- and UPEC-infected mice, particularly within the proximal regions (IS and caput, Figure 2G). In contrast to the proximal regions, the decrease of CX3CR1+ macrophages was accompanied by an increased ratio of CX3CR1- macrophages within the distal regions (corpus, cauda), indicating a shift in the macrophage pool (Figure 2G). Notably, we observed a significantly increased ratio of CCR2+ cells within the total macrophages (CX3CR1+ and CX3CR1-) in the corpus and cauda of UPEC-infected mice (Figure 2H), which indicates a potential contribution of monocytes to the macrophage pool within distal but not proximal regions upon UPEC infection. Overall, the corpus and cauda developed a highly inflammatory immune environment in which subgroups of innate antigen-presenting myeloid (macrophages and cDC) and effector lymphoid cells co-existed. In line with histological observations, also sham-infected mice developed an inflammatory response with similar, yet milder changes in the immune cell composition (Figure 2D–H). As seen by immunofluorescence analysis, numbers of Ly6G+ and Ly6C+ cells (including neutrophils and monocytes) were progressively increasing within the interstitium of the cauda, but not the caput, and also could be identified in the epididymal epithelium (Figure 2I) at time points when epithelial integrity was disturbed. Simultaneous exposure to an inflammatory stimulus in vitro results in differential immune responsiveness of the epididymal regions To examine whether the observed differential immune responses within epididymal regions were merely a consequence of microbial ascension and thus the longer exposure of the cauda to the pathogens, we have utilized an ex vivo organ culture model that allows simultaneous challenge with an inflammatory stimulus. Cytokine production profiles of the different epididymal regions (IS, caput, corpus, and cauda) were analyzed separately after stimulation with ultrapure lipopolysaccharide (LPS). While the IS and caput were still mostly unreactive, both corpus and cauda showed a significant upregulation of IL-1α, IL-1β, TNFα, MCP-1 (CCL2), IL-6, and IL-10 (Figure 2—figure supplement 3A). Intriguingly, IS and caput showed a higher intracellular bacteria load compared to corpus and cauda after ex vivo co-culture of organ pieces with UPEC, indicative for a higher and faster bacterial uptake and clearance potential (Figure 2—figure supplement 3B). Overall, these data suggest that the fundamentally different immunological responses observed in vivo within different regions of the epididymis are an inherent feature of the region, and thus independent of the administration route of the inflammatory stimulus. Single-cell transcriptomic analysis of immune cells in the epididymis demonstrates regional heterogeneity in steady state The above described observations indicated the possibility of differential immunological landscapes in the epididymal regions. To gain a comprehensive understanding, we employed scRNASeq of extravascular CD45+ cells. For this purpose, C57BL/6J wild type mice were intravenously injected with an APC/Cyanine7-conjugated anti-CD45.2 antibody (Figure 3A) prior to killing and organ collection. This allowed a later discrimination of tissue-resident immune cells that were labeled with a PerCP-Cyanine 5.5-conjugated CD45.1 antibody from intravascular CD45.2+ cells (Figure 3—figure supplement 1). In total 12,966 cells were separately isolated from the four main epididymal regions (IS, caput, corpus, cauda). The data were subsequently combined into a single dataset to investigate their regional distribution (Figure 3A, Figure 3—figure supplement 2). Unsupervised clustering and uniform manifold approximation and projection (UMAP) identified 13 different clusters (Figure 3B) with distinct gene expression profiles (Figure 3C). The identity of each cluster was annotated manually based on key marker gene expression (Figure 3D and E, Figure 3—figure supplement 3). Among the clusters, the majority of identified immune cells comprised several myeloid cell populations comprised several myeloid cell populations and included subsets of macrophages (clusters 1, 2, and 7), monocytes (clusters 8 and 10), and dendritic cells (clusters 2, 5, and 13). Macrophages were broadly identified by the co-expression of multiple key marker genes such as C1qa, Fcgr1 (encoding CD64), Adgre1 (encoding F4/80), and Cd68, with alternating levels of markers such as Cx3cr1, Ccr2, H2-Aa (encoding an MHC-II component). Monocytes were broadly characterized by the expression of Ly6c2, Ccr2, and Ace. Dendritic cells (DC, Flt3+ high expression levels of MHC-II transcripts) were segregated into three clusters that were identified as conventional DC 1 (Clec9a+Irf8+), conventional DC 2 (Cd209a+), as well as a small population of migratory DC (Ccr7+, Figure 3B–E). Apart from myeloid cells, all epididymal regions were populated by lymphocytes, including T cells (Cd3e+, clusters 4 and 9), NK cells (Nkg7+Eomes+ , cluster 6), and B cells (Cd79a+, cluster 11, Figure 3D and E). T cells were further discriminated into αβ and γδ T cells based on their alternating expression of Trbc and Trdc, respectively. Figure 3 with 3 supplements see all Download asset Open asset Single-cell RNA sequencing (scRNASeq) of different epididymal regions reveals immune cell heterogeneity within the murine epididymis under physiological conditions. (A) Schematic overview of the experimental procedure for isolating extravascular CD45+ cells from different epididymal regions. (B) Uniform manifold approximation and projection (UMAP) plot of 12,966 FACS-sorted CD45+ cells isolated from the four epididymal regions, showing immune cell populations identified by unsupervised clustering. (C) Heatmap of the Top45 marker by stringent selection of markers (only present in one cluster, 585 in total) showing expression differences among clusters. (D) Dot plot corresponding to the UMAP plot showing the expression of selected subset-specific genes – dot size resembles the percentage of cells within the cluster expressing the respective gene and dot color reflects the average expression within the cluster. (E) UMAP plots showing the expression of selected key markers for the indicated immune cell population (APC – antigen-presenting cells, mdC – monocyte-derived cells, DC – dendritic cells). (F) UMAP plots and pie charts showing regional distribution of identified clusters. We next defined the cluster distribution across epididymal regions (Figure 3F, Figure 3—figure supplement 3). Transcriptomic data of identified immune cell populations and their ratios within the CD45+ population in different epididymal regions were subsequently confirmed at the protein level by flow cytometry (Figure 4, Figure 4—figure supplement 1, gating). The vast majority of resident immune cells in the epididymis were found in the IS (approximately 10–15% CD45+ cells among the single live cells vs. 1–5% CD45+ cells in single live cells in caput to cauda; Figure 4A). Overall, we noted similarities in the composition of resident immune cell populations in the IS and caput that were clearly distinct from that in the more distal corpus and cauda. In this regard, IS and caput were predominantly populated by macrophage subsets (approximately 78% and 66% in CD45+ cells, respectively; Figure 4B) with other leukocytes accounting for <5% for each population (Figure 4B–H). In contrast, the corpus and cauda contained a more heter