Tertiary Lymphoid Structures in Endometriosis.

OBJECTIVE: To determine whether tertiary lymphoid structures (TLSs), which reflect organized immune cell aggregates present in non-lymphoid tissues, are consistent features of endometriosis lesions. DESIGN: Detailed histopathological analysis of endometrial and lesion tissue from endometriosis patients and controls was performed. Multiplex immunofluorescence on select samples was then conducted to identify canonical cell populations present within TLSs: CD3+ and CD8+ T-cells, CD79a+ B-cells, CD208+ dendritic cells, CD21+ follicular dendritic cells (fDC), and PNAd+ high endothelial venules (HEVs). PATIENT(S): Histologically confirmed endometriosis patients (N=113; 44.3± 6.0) and control individuals (N=110; 44.6±7.1). INTERVENTION: Not applicable. MAIN OUTCOME MEASURE(S): Detection of TLSs as characterized by the presence of all canonical cell types that constitute TLS and structure morphology. RESULT(S): Of the selected samples (N=18; 6 ectopic/eutopic/control), mature TLSs were identified in 3 ectopic tissue samples present on the ovary and fallopian tube, with immature TLSs (lacking fDC networks and HEVs) present throughout eutopic and control endometrial samples. CONCLUSION: These findings demonstrate the presence of TLSs across various endometriosis phenotypes, prompting further research into their significance within disease pathophysiology and the prognostic implications for patients.

Endocannabinoids and their receptors modulate endometriosis pathogenesis and immune response.

Endometriosis (EM), characterized by the presence of endometrial-like tissue outside the uterus, is the leading cause of chronic pelvic pain and infertility in females of reproductive age. Despite its high prevalence, the molecular mechanisms underlying EM pathogenesis remain poorly understood. The endocannabinoid system (ECS) is known to influence several cardinal features of this complex disease including pain, vascularization, and overall lesion survival, but the exact mechanisms are not known. Utilizing CNR1 knockout (k/o), CNR2 k/o, and wild-type (WT) mouse models of EM, we reveal contributions of ECS and these receptors in disease initiation, progression, and immune modulation. Particularly, we identified EM-specific T cell dysfunction in the CNR2 k/o mouse model of EM. We also demonstrate the impact of decidualization-induced changes on ECS components, and the unique disease-associated transcriptional landscape of ECS components in EM. Imaging mass cytometry (IMC) analysis revealed distinct features of the microenvironment between CNR1, CNR2, and WT genotypes in the presence or absence of decidualization. This study, for the first time, provides an in-depth analysis of the involvement of the ECS in EM pathogenesis and lays the foundation for the development of novel therapeutic interventions to alleviate the burden of this debilitating condition.

Atypical B Cells Promote Cancer Progression and Poor Response to Bacillus Calmette-Guérin in Non-Muscle Invasive Bladder Cancer.

Poor response to Bacillus Calmette-Guérin (BCG) immunotherapy remains a major barrier in the management of patients with non-muscle invasive bladder cancer (NMIBC). Multiple factors are associated with poor outcomes, including biological aging and female sex. More recently, it has emerged that a B-cell-infiltrated pretreatment immune microenvironment of NMIBC tumors can influence the response to intravesically administered BCG. The mechanisms underlying the roles of B cells in NMIBC are poorly understood. Here, we show that B-cell-dominant tertiary lymphoid structures (TLSs), a hallmark feature of the chronic mucosal immune response, are abundant and located close to the epithelial compartment in pretreatment tumors from BCG non-responders. Digital spatial proteomic profiling of whole tumor sections from male and female patients with NMIBC who underwent treatment with intravesical BCG, revealed higher expression of immune exhaustion-associated proteins within the tumor-adjacent TLSs in both responders and non-responders. Chronic local inflammation, induced by the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen, led to TLS formation with recruitment and differentiation of the immunosuppressive atypical B-cell (ABC) subset within the bladder microenvironment, predominantly in aging female mice compared to their male counterparts. Depletion of ABCs simultaneous to BCG treatment delayed cancer progression in female mice. Our findings provide evidence indicating a role for ABCs in BCG response and will inform future development of therapies targeting the B-cell-exhaustion axis.

Comparative analysis of syngeneic mouse models of high-grade serous ovarian cancer.

Ovarian cancers exhibit high rates of recurrence and poor treatment response. Preclinical models that recapitulate human disease are critical to develop new therapeutic approaches. Syngeneic mouse models allow for the generation of tumours comprising the full repertoire of non-malignant cell types but have expanded in number, varying in the cell type of origin, method for transformation, and ultimately, the properties of the tumours they produce. Here we have performed a comparative analysis of high-grade serous ovarian cancer models based on transcriptomic profiling of 22 cell line models, and intrabursal and intraperitoneal tumours from 12. Among cell lines, we identify distinct signalling activity, such as elevated inflammatory signalling in STOSE and OVE16 models, and MAPK/ERK signalling in ID8 and OVE4 models; metabolic differences, such as reduced glycolysis-associated expression in several engineered ID8 subclones; and relevant functional properties, including differences in EMT activation, PD-L1 and MHC class I expression, and predicted chemosensitivity. Among tumour samples, we observe increased variability and stromal content among intrabursal tumours. Finally, we predict differences in the microenvironment of ID8 models engineered with clinically relevant mutations. We anticipate that this work will serve as a valuable resource, providing new insight to help select models for specific experimental objectives.

A Step Closer to Predicting Progression After Bacillus Calmette-Guérin Immunotherapy in High-risk Non-muscle-invasive Bladder Cancer.

Treatment-naïve tumors from patients who then experience recurrence or progression after bacillus Calmette-Guérin (BCG) therapy exhibit elevated expression of genes associated with basal differentiation and immune suppression. Three tumor molecular subtypes have been associates with distinct clinical outcomes and will allow early identification of patients unlikely to respond to BCG immunotherapy.

Adaptive Immunity in Genitourinary Cancers.

CONTEXT: While urothelial and renal cell cancers have exhibited modest responses to novel immune checkpoint inhibitors targeting the programmed death ligand 1 and its receptor, response rates in patients with prostate cancer have remained poor. The factors underlying suboptimal outcomes observed in patients treated with novel immunotherapies are still to be resolved. OBJECTIVE: To review the literature and describe the key adaptive immune physiological events associated with cancer progression and therapeutic response in genitourinary (GU) cancers. EVIDENCE ACQUISITION: We performed a nonsystematic, collaborative narrative review to highlight recent advancements leading to the current state of knowledge on the critical mediators of antitumor adaptive immunity to GU cancers. Further, we discuss the findings on the pre- and post-treatment immunological events that either are unique to each of the three cancer types or exhibit overlapping clinical associations. EVIDENCE SYNTHESIS: Aging-associated immune function decline is a major factor underlying poor outcomes observed in patients treated with both conventional and novel immunotherapies. Other cancer immunobiological aspects associated with suboptimal responses in GU cancers include the overall tumor mutational burden, mutations in specific tumor suppressor/DNA damage repair genes (KDM6A, PTEN, STAG2, TP53, ATM, and BRCA2), and abundance of multiple functional states of adaptive immune cells and their spatiotemporal localization within the tumor immune microenvironment. Understanding these mechanisms may potentially lead to the development of prognostic and predictive biomarkers such as immune cell infiltration profiles and tertiary lymphoid structures (TLSs) that associate with variable clinical outcomes depending on the nature of the novel immunotherapeutic approach. Implementation of newer immune-monitoring technologies and improved preclinical modeling systems will augment our understanding of the host and tumor intrinsic factors contributing to the variability of responses to immunotherapies. CONCLUSIONS: Despite the tremendous progress made in the understanding of dynamic and static adaptive immune elements within the tumor immune landscape, several knowledge gaps remain. A comprehensive knowledge thus gained will lead to precision immunotherapy, improved drug sequencing, and a therapeutic response. PATIENT SUMMARY: We performed a collaborative review by a diverse group of experts in the field to examine our understanding of the events and crosstalk between cancer cells and the patient's immune system that are associated with responses to novel immunotherapies. An evolving understanding of tumor-intrinsic and host-related immune alterations, both before and after therapy, will aid in the discovery of promising markers of responses to immunotherapy as well as the development of unique therapeutic approaches for the management of genitourinary cancers.

Cancer cell genotype associated tumor immune microenvironment exhibits differential response to therapeutic STING pathway activation in high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy characterized by resistance to chemotherapy and high rates of recurrence. HGSC tumors display a high prevalence of tumor suppressor gene loss. Given the type 1 interferon regulatory function of BRCA1 and PTENgenes and their associated contrasting T-cell infiltrated and non-infiltrated tumor immune microenvironment (TIME) states, respectively, in this study we investigated the potential of stimulator of interferon genes (STING) pathway activation in improving overall survival via enhancing chemotherapy response, specifically in tumors with PTEN deficiency. METHODS: Expression of PTEN protein was evaluated in tissue microarrays generated using pretreatment tumors collected from a cohort of 110 patients with HGSC. Multiplex immunofluorescence staining was performed to determine spatial profiles and density of selected lymphoid and myeloid cells. In vivo studies using the syngeneic murine HGSC cell lines, ID8-Trp53 -/-; Pten -/- and ID8-Trp53 -/-; Brca1 -/-, were conducted to characterize the TIME and response to carboplatin chemotherapy in combination with exogenous STING activation therapy. RESULTS: Patient tumors with absence of PTEN protein exhibited a significantly decreased disease specific survival and intraepithelial CD68+ macrophage infiltration as compared with intact PTEN expression. In vivo studies demonstrated that Pten-deficient ovarian cancer cells establish an immunosuppressed TIME characterized by increased proportions of M2-like macrophages, GR1+MDSCs in the ascites, and reduced effector CD8+ cytotoxic T-cell function compared with Brca1-deficient cells; further, tumors from mice injected with Pten-deficient ID8 cells exhibited an aggressive behavior due to suppressive macrophage dominance in the malignant ascites. In combination with chemotherapy, exogenous STING activation resulted in longer overall survival in mice injected with Pten-deficient ID8 cells, reprogrammed intraperitoneal M2-like macrophages derived from Pten-deficient ascites to M1-like phenotype and rescued CD8+ cytotoxic T-cell activation. CONCLUSIONS: This study reveals the importance of considering the influence of cancer cell intrinsic genetic alterations on the TIME for therapeutic selection. We establish the rationale for the optimal incorporation of interferon activating therapies as a novel combination strategy in PTEN-deficient HGSC.

Role of the endocannabinoid system in the pathophysiology of endometriosis and therapeutic implications.

Endometriosis patients experience debilitating chronic pain, and the first-line treatment is ineffective at managing symptoms. Although surgical removal of the lesions provides temporary relief, more than 50% of the patients experience disease recurrence. Despite being a leading cause of hysterectomy, endometriosis lacks satisfactory treatments and a cure. Another challenge is the poor understanding of disease pathophysiology which adds to the delays in diagnosis and overall compromised quality of life. Endometriosis patients are in dire need of an effective therapeutic strategy that is both economical and effective in managing symptoms, while fertility is unaffected. Endocannabinoids and phytocannabinoids possess anti-inflammatory, anti-nociceptive, and anti-proliferative properties that may prove beneficial for endometriosis management, given that inflammation, vascularization, and pain are hallmark features of endometriosis. Endocannabinoids are a complex network of molecules that play a central role in physiological processes including homeostasis and tissue repair, but endocannabinoids have also been associated in the pathophysiology of several chronic inflammatory diseases including endometriosis and cancers. The lack of satisfactory treatment options combined with the recent legalization of recreational cannabinoids in some parts of the world has led to a rise in self-management strategies including the use of cannabinoids for endometriosis-related pain and other symptoms. In this review, we provide a comprehensive overview of endocannabinoids with a focus on their potential roles in the pathophysiology of endometriosis. We further provide evidence-driven perspectives on the current state of knowledge on endometriosis-associated pain, inflammation, and therapeutic avenues exploiting the endocannabinoid system for its management.

Estrogen mediates inflammatory role of mast cells in endometriosis pathophysiology.

Endometriosis is an estrogen dependent, chronic inflammatory disease characterized by the growth of endometrial lining outside of the uterus. Mast cells have emerged as key players in regulating not only allergic responses but also other mechanisms such as angiogenesis, fibrosis, and pain. The influence of estrogen on mast cell function has also been recognized as a potential factor driving disease pathophysiology in number of allergic and chronic inflammatory conditions. However, precise information is lacking on the cross talk between endocrine and immune factors within the endometriotic lesions and whether that contributes to the involvement of mast cells with disease pathophysiology. In this study, we observed a significant increase in mast cell numbers within endometriotic lesions compared to matched eutopic endometrium from the same patients. Compared to eutopic endometrium, endometriotic lesions had significantly higher levels of stem cell factor (SCF), a potent growth factor critical for mast cell expansion, differentiation, and survival for tissue resident mast cells. Targeted mRNA Q-PCR array revealed that the endometriotic lesions harbour microenvironment (upregulation of CPA3, VCAM1, CCL2, CMA1, CCR1, and KITLG) that is conducive to mast cells recruitment and subsequent differentiation. To examine cross-talk of mast cells within the endometriotic lesion microenvironment, endometriotic epithelial cells (12Z) and endometrial stromal cells (hESC) incubated with mast cell-conditioned media showed significantly increased production of pro-inflammatory and chemokinetic cytokines. To further understand the impact of estrogen on mast cells in endometriosis, we induced endometriosis in C57BL/6 mice. Mature mast cells were significantly higher in peritoneal fluid of estrogen-treated mice compared to untreated mice within the sham operated groups. Mouse endometriotic lesion tissue revealed several genes (qRT-PCR) relevant in mast cell biology significantly upregulated in the estrogen treated, endometriosis-induced group compared to control endometrium. The endometriotic lesions from estrogen treated mice also had significantly higher density of Alcian blue stained mast cells compared to untreated lesions or control endometrium. Collectively, these findings suggest that endometriotic lesions provide a microenvironment necessary for recruitment and differentiation of mast cells. In turn, mast cells potentially release pro-inflammatory mediators that contribute to chronic pelvic pain and endometriosis disease progression.

A Computational Approach to Identification of Candidate Biomarkers in High-Dimensional Molecular Data.

Complex high-dimensional datasets that are challenging to analyze are frequently produced through '-omics' profiling. Typically, these datasets contain more genomic features than samples, limiting the use of multivariable statistical and machine learning-based approaches to analysis. Therefore, effective alternative approaches are urgently needed to identify features-of-interest in '-omics' data. In this study, we present the molecular feature selection tool, a novel, ensemble-based, feature selection application for identifying candidate biomarkers in '-omics' data. As proof-of-principle, we applied the molecular feature selection tool to identify a small set of immune-related genes as potential biomarkers of three prostate adenocarcinoma subtypes. Furthermore, we tested the selected genes in a model to classify the three subtypes and compared the results to models built using all genes and all differentially expressed genes. Genes identified with the molecular feature selection tool performed better than the other models in this study in all comparison metrics: accuracy, precision, recall, and F1-score using a significantly smaller set of genes. In addition, we developed a simple graphical user interface for the molecular feature selection tool, which is available for free download. This user-friendly interface is a valuable tool for the identification of potential biomarkers in gene expression datasets and is an asset for biomarker discovery studies.

T helper 17 axis and endometrial macrophage disruption in menstrual effluent provides potential insights into the pathogenesis of endometriosis.

OBJECTIVE: To identify immune cells, cytokines, and immune cell transcriptome in the menstrual effluent (ME) of women with endometriosis compared with that of healthy donors. DESIGN: Live immune cells were isolated from human ME samples and were analyzed by flow cytometry to identify various immune cell populations. Selected cytokines from the same patients were evaluated using multiplex cytokine analyses. The transcriptome of the immune cell population was subsequently profiled using NanoString nCounter's PanCancer Immune panel. SETTING: Academic institution. PATIENT(S): Surgically confirmed endometriosis patients (n = 14) and healthy fertile donors (n = 19). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): In-depth immune cell profiling of ME obtained from women with endometriosis compared with that of healthy donors. RESULT(S): ME analysis revealed that the number of T helper 17 (TH17) cells was significantly lower in patients with endometriosis compared with that of healthy donors; the number of macrophages was also lower (P=.06) in the former. Multiplex cytokine analysis revealed significantly lower transforming growth factor α in the ME "serum" of patients with endometriosis. Transcriptomic analysis of CD45+ cells revealed 47 differentially expressed genes, mainly associated with the TH17 axis (IL10, IL23A, and IL6), as well as genes associated with macrophage signaling/activation (CD74, CD83, CXCL16, and CCL3). CONCLUSION(S): We demonstrate for the first time that the levels of TH17 axis, macrophages, and transforming growth factor α were altered in the ME of women with endometriosis compared with that of healthy donors. These findings shed light on the potential immune pathways that could partly explain the pathogenesis and progression of endometriosis. Future large-scale studies on ME samples are warranted to exploit the use of these markers to study the pathogenesis of endometriosis.

IL-33-ILC2 axis in the female reproductive tract.

IL-33 is a member of the IL-1 family and was first identified as an alarmin that acts at mucosal barrier sites. However, IL-33 is now understood to be a pleiotropic cytokine that acts on a variety of immune and non-immune cell types to promote type 2 T helper cell (TH2) inflammation as well as to regulate and suppress homeostatic processes. Of particular interest are group 2 innate lymphoid cells (ILC2s) which are activated by IL-33 and promote many IL-33-specific effects. Considerable investigation has surrounded the integral role of IL-33 and ILC2s in driving inflammation in asthma, allergy, atopic dermatitis, fibrotic diseases, microbial interactions, and more. However, IL-33 and ILC2s have also emerged as key components of a healthy pregnancy and fertility; when dysregulated, they can drastically drive female reproductive pathologies. We first summarize the presence of both IL-33 and ILC2s in the female reproductive tract (FRT) and in healthy pregnancy. We then provide insights into how IL-33 and ILC2s drive female reproductive pathologies and how this axis could be a potential therapeutic target in reproductive disorders including preterm birth, pre-eclampsia, recurrent spontaneous abortion, and endometriosis.

Sex differences in the aging murine urinary bladder and influence on the tumor immune microenvironment of a carcinogen-induced model of bladder cancer.

Sex and age associated differences in the tumor immune microenvironment of non-muscle invasive bladder (NMIBC) cancer and associated clinical outcomes are emerging indicators of treatment outcomes. The incidence of urothelial carcinoma of the bladder is four times higher in males than females; however, females tend to present with a more aggressive disease, a poorer response to immunotherapy and suffer worse clinical outcomes. Recent findings have demonstrated sex differences in the tumor immune microenvironment of non-muscle invasive and muscle invasive bladder cancer and associated clinical outcomes. However, a significant gap in knowledge remains with respect to the current pre-clinical modeling approaches to more precisely recapitulate these differences towards improved therapeutic design. Given the similarities in mucosal immune physiology between humans and mice, we evaluated the sex and age-related immune alterations in healthy murine bladders. Bulk-RNA sequencing and multiplex immunofluorescence-based spatial immune profiling of healthy murine bladders from male and female mice of age groups spanning young to old showed a highly altered immune landscape that exhibited sex and age associated differences, particularly in the context of B cell mediated responses. Spatial profiling of healthy bladders, using markers specific to macrophages, T cells, B cells, activated dendritic cells, high endothelial venules, myeloid cells and the PD-L1 immune checkpoint showed sex and age associated differences. Bladders from healthy older female mice also showed a higher presence of tertiary lymphoid structures (TLSs) compared to both young female and male equivalents. Spatial immune profiling of N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen exposed male and female bladders from young and old mice revealed a similar frequency of TLS formation, sex differences in the bladder immune microenvironment and, age associated differences in latency of tumor induction. These findings support the incorporation of sex and age as factors in pre-clinical modeling of bladder cancer and will potentially advance the field of immunotherapeutic drug development to improve clinical outcomes.

The STING pathway: Therapeutic vulnerabilities in ovarian cancer.

Ovarian cancer is the leading cause of mortality due to gynecologic malignancy. The majority of women diagnosed with the most common subtype, high-grade serous ovarian carcinoma (HGSC), develop resistance to conventional therapies despite initial response to treatment. HGSC tumors displaying DNA damage repair (DDR) gene deficiency and high chromosomal instability mainly associate with higher cytotoxic immune cell infiltration and expression of genes associated with these immune pathways. Despite the high level of immune infiltration observed, the majority of patients with HGSC have not benefited from immunomodulatory treatments as the mechanistic basis of this infiltration is unclear. This lack of response can be primarily attributed to heterogeneity at the levels of both cancer cell genetic alterations and the tumour immune microenvironment. Strategies to enhance anti-tumour immunity have been investigated in ovarian cancer, of which interferon activating therapies present as an attractive option. Of the several type I interferon (IFN-1) stimulating therapies, exogenously activating the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is emerging as a promising avenue. Herein, we highlight our current understanding of how constitutive and induced cGAS-STING pathway activation influences the ovarian tumour microenvironment. We further elaborate on the links between the genomic alterations prevalent in ovarian tumours and how the resultant immune phenotypes can make them more susceptible to exogenous STING pathway activation and potentiate immune-mediated killing of cancer cells. The therapeutic potential of cGAS-STING pathway activation in ovarian cancer and factors implicating treatment outcomes are discussed, providing a rationale for future combinatorial treatment approaches on the backbone of chemotherapy.

Intravesical immunotherapy with a GM-CSF armed oncolytic vesicular stomatitis virus improves outcome in bladder cancer.

A significant proportion of non-muscle invasive bladder cancer cases will progress to muscle invasive disease. Transurethral resection followed by Bacillus Calmette Guerin immunotherapy can reduce this risk, while cystectomy prior to muscle invasion provides the best option for survival. Currently, there are no effective treatments for Bacillus Calmette Guerin refractory disease. A novel oncolytic vesicular stomatitis virus containing the human GM-CSF transgene (VSVd51-hGM-CSF) was rescued and tested as a potential bladder-sparing therapy for aggressive bladder cancer. The existing variant expressing mouse GM-CSF was also used. Measurement of gene expression and protein level alterations of canonical immunogenic cell death associated events on mouse and human bladder cancer cell lines and spheroids showed enhanced release of danger signals and immunogenic factors following infection with VSVd51-m/hGM-CSF. Intravesical instillation of VSVd51-mGM-CSF into MB49 bladder cancer bearing C57Bl/6 mice demonstrated enhanced activation of peripheral and bladder infiltrating effector immune cells, along with improved survival and reduced tumor volume. Importantly, virus-mediated anti-tumor immunity was recapitulated in bladder cancer patient-derived organoids. These results suggest that VSVd51-hGM-CSF is a promising viro/immunotherapy that could benefit bladder cancer patients.

Sex and Gender Differences in Cancers of the Genitourinary Tract: The Need to Consider Immune Divergence.

Delineating the processes of immune response underlying sexual dimorphism in cancers of the genitourinary tract will improve outcomes from immunotherapy treatment.

IL-33 activates group 2 innate lymphoid cell expansion and modulates endometriosis.

Chronic inflammation and localized alterations in immune cell function are suspected to contribute to the progression of endometriosis and its associated symptoms. In particular, the alarmin IL-33 is elevated in the plasma, peritoneal fluid, and endometriotic lesions from patients with endometriosis; however, the exact role of IL-33 in the pathophysiology of endometriosis is not well understood. In this study, we demonstrate, in both humans and a murine model, that IL-33 contributes to the expansion of group 2 innate lymphoid cells (ILC2s), and this IL-33-induced ILC2 expansion modulates the endometriosis lesion microenvironment. Importantly, we show that IL-33 drives hallmarks of severe endometriosis, including elevated inflammation, lesion proliferation, and fibrosis, and that this IL-33-induced aggravation is mediated by ILC2s. Finally, we demonstrate the functionality of IL-33 neutralization as a promising and potentially novel therapeutic avenue for treating the debilitating symptoms of endometriosis.

Sexual Dimorphism in Outcomes of Non-muscle-invasive Bladder Cancer: A Role of CD163+ Macrophages, B cells, and PD-L1 Immune Checkpoint.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) is over three times as common in men as it is in women; however, female patients do not respond as well to immunotherapeutic treatments and experience worse clinical outcomes than their male counterparts. Based on the established sexual dimorphism in mucosal immune responses, we hypothesized that the tumor immune microenvironment of bladder cancer differs between the sexes, and this may contribute to discrepancies in clinical outcomes. OBJECTIVE: To determine biological sex-associated differences in the expression of immune regulatory genes and spatial organization of immune cells in tumors from NMIBC patients. DESIGN SETTING AND PARTICIPANTS: Immune regulatory gene expression levels in tumors from male (n = 357) and female (n = 103) patients were measured using whole transcriptome profiles of tumors from the UROMOL cohort. Multiplexe immunofluorescence was performed to evaluate the density and spatial distribution of immune cells and immune checkpoints in tumors from an independent cohort of patients with NMIBC (n = 259 males and n = 73 females). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Transcriptome sequencing data were analyzed using DESeq2 in R v4.0.1, followed by application of the Kruskal-Wallis test to determine gene expression differences between tumors from males and females. Immunofluorescence data analyses were conducted using R version 3.5.3. Survival analysis was performed using survminer packages. RESULTS AND LIMITATIONS: High-grade tumors from female patients exhibited significantly increased expression of B-cell recruitment (CXCL13) and function (CD40)-associated genes and the immune checkpoint genes CTLA4, PDCD1, LAG3, and ICOS. Tumors from female patients showed significantly higher infiltration of PD-L1+ cells and CD163+ M2-like macrophages than tumors from male patients. Increased abundance of CD163+ macrophages and CD79a+ B cells were associated with decreased recurrence-free survival. CONCLUSIONS: These novel findings highlight the necessity of considering sexual dimorphism in the design of future immunotherapy trials in NMIBC. PATIENT SUMMARY: In this study, we measured the abundance of various immune cell types between tumors from male and female patients with non-muscle-invasive bladder cancer. We demonstrate that tumors from female patients have a significantly higher abundance of immunosuppressive macrophages that express CD163. Higher abundance of tumor-associated CD163-expressing macrophages and B cells is associated with shorter recurrence-free survival in both male and female patients.