Immune dysregulation in endometriosis: the T cell perspective

Endometriosis (EM) is an estrogen-dependent gynecological disease in which endometrial-like tissue grows outside of the uterus. Impacting around 200 million reproductive-age women (assigned female sex at birth) ( 2 ) and an estimated 17–33% of transgender men ( 3 ), EM commonly presents as chronic inflammation, debilitating pelvic pain, and irregular/heavy menstruation. While exact mechanisms are not entirely known, 30–50% of patients experience infertility, with 25–50% of infertile individuals having EM ( 4 ). Due to high heterogeneity in disease presentation ( 5 ), invasive disease diagnosis, and stigmatization of reproductive disorders, patients often experience a 7–9 year diagnostic delay globally ( 6 , 7 ), with nearly 75% of patients receiving a misdiagnosis during this period ( 8 ). Notably, EM symptomology substantially overlaps with several gynecological disorders, such as adenomyosis and polycystic ovary syndrome (PCOS) ( 9 ), further contributing to diagnostic delay and misdiagnosis of patients. Despite being a leading cause of hysterectomy ( 10 ), current therapeutics are highly focused on EM symptom management and have significant side effects. While surgical excision of lesions may initially relieve symptoms, recurrence of lesions and pain symptoms is high, with recurrence rates ranging from 30–50% and 27–58% of patients undergoing re-operation within 7 years ( 8 , 11 ). Patients often receive hormonal therapy as a first-line treatment to manage symptoms and/or prevent disease recurrence following surgical excision of lesions. However, this is not a long-term treatment option and cannot be used for patients aiming to conceive. The debilitating pain experienced by patients results in a significant patient, social, and economic burden, often interfering with day-to-day activities, career success, workplace productivity, mental health, and overall quality of life. Indeed, EM patients experience significantly higher health care costs compared to controls ( 12 ), with annual overall direct medical cost of EM per patient ranging from $1,459–20,239 USD in 2022 ( 13 ), and the annual economic burden of EM in the USA estimated at $69.4 billion USD in 2009 ( 14 , 15 ). Given the substantial disease burden and lack of viable, effective therapeutic options, there is an urgent, unmet need to develop novel diagnostic and therapeutic targets that preserve patient fertility, reduce disease recurrence, and improve quality of life. Though the etiology and pathogenesis of EM remain largely unknown, immune dysfunction is well-established to play a key role in the establishment and survival of lesions. Namely, altered proportions of T cell subsets, such as T regulatory (Treg) and T helper (Th)17 cells ( 16 ), have been observed systemically (peripheral blood; PB) and locally (eutopic/ectopic tissues, peritoneal fluid; PF) in EM, and are thought to drive associated chronic inflammation and immune dysregulation. Immune dysfunction, particularly changes in Treg and Th subsets, are also speculated to largely contribute to EM-associated infertility ( 17 , 18 ). However, despite their clinical relevance, the contributions of specific T cell subsets to EM-associated infertility, pain, and fibrosis remain poorly characterized, representing an important gap in literature. Immune phenotypes greatly vary in EM dependent on compartment, disease stage, hormonal contraception/therapeutics, and menstrual cycle, underscoring the need to stratify results to improve comparability across studies. As EM is a hormone-dependent disease, hormonal signaling, as well as EM-associated menstrual cycle alterations, can influence T cell function and phenotype. However, hormone-mediated effects were not explored in depth as this was beyond the scope of this review. Here, we provide a comprehensive overview of key alterations in T cell subsets across compartments in EM ( Table 1 ). Alterations in T cell subsets across compartments in EM. DIE, deep infiltrating endometriosis; DN, double negative; ELISA, enzyme-linked immunosorbent assay; EM, endometriosis; FF, follicular fluid; GI, gastrointestinal; GSEA, gene set enrichment analysis; IFN, interferon; IHC, immunohistochemistry; iNKT, invariant natural killer T cells; iTreg, inducible T-regulatory cells; MAIT, mucosal-associated invariant T cells; ME, menstrual effluent; N/A, not applicable/available; nTreg, natural T-regulatory cells; OMA, ovarian endometrioma lesions; PB, peripheral blood; PCR, polymerase chain reaction; PF, peripheral fluid; sFGL2, soluble fibrinogen-like protein 2; SPE, superficial peritoneal endometriosis; TEM, effector memory T cells; TNF, tumor necrosis factor; TSLP, thymic stromal lymphopoietin. † Animal model. ‡ Inferred; requires mechanistic study confirmation. – No change. ↓↑ Increased/decreased.

Th9 cells have emerged to characterize CD4+ T cells that secrete IL-9. Previously IL-9 production was included under the banner of Th2 cell secretions, however, studies have shown that CD4+ IL-9 producing cells are independent in both their secretory and transcriptional profiles from the other T-helper subsets ( 156 , 157 ). Th9 cells can be differentiated through two pathways, either from naïve CD4+ T cells in the presence of IL-4 and TGF-β1, or from Th2 cells in the presence of TGF-β1. For this reason, Th9 cells are thought to be a subset of Th2 cells ( 158 ). Due to the requirement for IL-4 and TGF-β1 in differentiation, these cytokines complimentary surface receptors, IL-4R and TGF-β RII, are commonly used for phenotypic characterization of Th9 cells ( 157 ). As with Th2 cells, IRF4 is an important transcription factor in Th9 development, in addition to PU.1, BATF3 ( 156 ), and PPARγ ( 158 ). Th9 cells function to enhance protective immunity at mucosal surfaces through IL-9 on mast cells, epithelial cells, and smooth muscle cells. Adversely however, Th9 cells have been identified in asthma, inflammatory gut reactions, and autoinflammation, with a particular role in fibrosis due to IL-9 production ( 159 ). In EM, IL-9 has been identified as significantly elevated in the PF ( 37 ) and plasma ( 160 ) of patients compared to controls. However, the primary source of IL-9 remains to be interrogated. Tarumi et al., identified significantly higher presence of Th9 cells (CD4+IL-9+) in the PF of patients with ovarian EM, but did not find a corresponding increase of IL-9 in the PF compared to healthy controls ( 38 ). A critical relationship that requires continued interrogation in EM is that of mast cells and Th9 cells. Our lab has identified mast cells as contributors to EM pathophysiology ( 161 ). In a mouse model of EM, adoptively transferred Th9-like cells induced no alterations to mast cell numbers ( 162 ). However, Th9 cells infiltrated the lesion and promoted reduced proliferation and increased lesion-associated inflammation through upregulation of IL-1α. In pathological contexts such as asthma, IL-9 drives proliferation and recruitment of mast cells to sites of lung inflammation ( 38 ). Thus, whether mast cell and Th9 cell cross talk shapes the EM lesion microenvironment and associated alterations remain to be explored. As aforementioned, EM is characterized by high recurrence rates post excision surgery ( 8 , 11 ). Persistent immunological dysfunction and chronic inflammation may contribute to recurrence despite surgery or medical treatment ( 163 ). Notably, memory T cells, characterized by their long lifespan and enhanced recall responses, may play a key role in driving and maintaining immune dysfunction in EM recurrence. These memory T cells can be either CD4+ or CD8+ and are mainly divided into three subpopulations: central memory T cells (TCM), effector memory T cells (TEM) and tissue resident memory T cells (TRM), depending on their ability to migrate within SLOs and non-lymphoid tissues (NLT) ( 164 , 165 ) through the expression of CCR7 and CD62L ( 166 , 167 ). Like TEM, TRM cells do not express SLO homing markers, but do exhibit prolonged expression of the activation marker CD69, which blocks S1P1-mediated tissue egress ( 168 ), and retention surface proteins to their tissue of residency including CD49a and CD103 ( 169 , 170 ). TCM and TEM cells exhibit functionally distinct responses to TCR stimulation. TEM primarily release effector molecules including IFNγ and perforin, enabling rapid immune response. In contrast, TCM predominantly secrete IL-2, supporting T-cell proliferation and survival and can differentiate into effector T cells and TEM ( 171 , 172 ). At local inflammatory sites, TRM cells play the role of front-line defense, as they effectively detect pathogens and exert typical memory functions including cytotoxic granzyme B and IFNγ secretion ( 173 , 174 ). Memory T cells, including TRM, have been extensively studied in various mucosal and non-mucosal tissues in both healthy and disease contexts ( 175 – 177 ). However, these memory cell types are surprisingly overlooked in the upper female reproductive tract and in the context of EM. Some literature suggests differing levels of CD8+ memory T cell subsets within the eutopic endometrium and PB of EM patients compared to controls, though, data overall appears to be inconsistent ( 137 ). For instance, two GSEA studies depicted CD8+ TEM gene enrichment scores were increased in the eutopic endometrium of EM patients compared to controls ( 51 , 63 ). While, in contrast, another GSEA evaluation demonstrated decreased TEM and TCM scores ( 50 ). Though, this study did not appear to delineate TEM or TCM subsets by CD4/CD8 expression. Another study found elevated levels of peripheral CD8+ TCM but reduced number of terminally differentiated CD8+ TEM cells in EM patients ( 178 ). Ultimately, further studies need to address these controversial results and elucidate the pathophysiological roles of memory T cells in EM. Conventional alpha beta (αβ) T cells are the focus of most reports on the role of T cells in EM, while gamma delta (γδ) T cells are often overlooked. γδ T cells possess a different TCR pattern consisting of γ and δ chains and generally lack both CD4 and CD8 expression on their cell surface, distinguishing them from their αβ counterparts ( 179 ). γδ T cells can recognize a wide range of antigens without relying on major histocompatibility complex (MHC)-dependent antigen presentation ( 180 , 181 ), serving as a linking arm between the innate and adaptive immune response. In humans, Vδ1+, Vδ2+, and Vδ3+ are the three main subsets of γδ T cells with Vδ2+ T cells most prevalent in PB and Vδ1+ and Vδ3+ T lymphocytes predominantly located in tissues ( 182 ). Unlike human γδ T cells categorization, their murine counterpart is divided into six distinct subsets (Vγ1, Vγ2, Vγ4, Vγ5, Vγ6, Vγ7) based on their γ-chain locus ( 183 ). Despite the difference in their subpopulation determination, human and murine γδ T cells share common features such as early fetal development, restricted TCR diversity, and antigen recognition by butyrophilins ( 184 , 185 ). γδ T cells are widely dispersed throughout mucosal tissues, including the female reproductive system, where their numbers may vary in response to hormonal shifts ( 182 ). About 5–10% of human uterine T cells and 60–80% of mouse uterine T cells have γδ T cells spread throughout the endometrium ( 186 – 188 ). Estrogen receptors (ERs) are highly expressed by γδ T cells in the uterus, which promote cell expansion and trigger the release of CXCR3 and IL-17A in response to estrogen signalling ( 186 , 189 , 190 ). Thus, the dysregulated immunological milieu that characterizes estrogen-driven diseases, such as EM, may be significantly influenced by γδ T cells. Hudecek et al. confirmed the presence of γδ T cells in both PF and PB of EM patients ( 48 ). Specifically, authors observed a significant decrease in circulating Vδ1+ T cells (PB) in EM as compared to healthy controls, whereas circulating Vδ2+ T cell numbers remained unchanged. Another study found that Vδ1+ T cells in EM patient PB exhibited a heightened activation state and possible signs of exhaustion due to elevated PD-1 levels ( 80 ). In contrast, Vδ2+ T cells in EM patient PB had reduced activation and cytotoxic capabilities. Peripheral Vδ1+ T cells were also positively correlated with EM disease severity and presence of pain symptoms, while peripheral Vδ2+ T cells were negatively correlated with these aspects of EM. Overall, as authors found reduced frequencies of Vδ2+ in EM PB, as well as higher frequencies of PD-1+Vδ1+ and PD-1+Vδ2+ T cells in EM compared to controls, they suggest that γδ T cells may develop an exhausted phenotype after activation due to the prolonged chronic inflammation in EM ( 80 ). While further research is necessary to confirm this, these studies provide evidence of an imbalance of Vδ1+ and Vδ2+ T cells in EM, which may be associated with disease severity and pain symptoms. In the context of endometrial and ovarian cancers, γδ T cells have been shown to infiltrate tumors and contribute to a protective response through their tumor-killing activity. However, this effect can be suppressed by factors, such as EphA2, which inhibit their tumor lysis function ( 48 , 191 ). The specific functions of γδ T lymphocytes in EM are still largely unknown, despite evidence of their varied participation in the uterine endometrium and several gynecological cancers. As γδ T cells are one of the most abundant T cell populations at mucosal surfaces, including the uterus, where they enact unique MHC independent cytotoxic response and recognition of stress ligands, future studies are required to understand their specific contributions to EM, as these cells form a unique bridge between innate and adaptive immunity. More specifically, given their abundance in the uterus and Sampson’s theory of retrograde menstruation ( 143 ), research should aim to assess whether γδ T cells are maintaining similar roles in eutopic and ectopic tissues. Natural killer T (NKT) cells are a unique T cell subset with a highly restricted, invariant TCR that recognizes both self and foreign lipid-based antigens presented by CD1d. Thus, they are commonly referred to as invariant NKT (iNKT) cells, which can be CD4+/-. Human iNKT cells predominantly express CD8α, while mouse iNKT cells generally lack CD8 expression ( 192 , 193 ). CD4+ iNKT cells share similar transcriptional and subpopulation features with conventional CD4+ T cells ( 194 ). Upon activation, iNKT cells secrete a variety of cytokines and chemokines, including Th1 cytokines (IFNγ, TNF), Th2 cytokines (IL-4, IL-10, IL-13), IL-2, IL-3, TGF-β ( 195 – 197 ), and IL-17 specifically from NK1.1 lacking subsets ( 198 ). iNKT cells are critical for successful implantation and maternal-fetal tolerance, by proliferating in the decidua (endometrium) and recognizing CD1d-presented fetal glycolipids ( 199 , 200 ). Indeed, abnormal NKT levels in human and mouse decidua are linked to recurrent miscarriages ( 201 – 203 ). Though, the role of NKT cells is still largely underexplored in EM pathophysiology. Reports demonstrate that the overall number of iNKT cells and double-negative (DN) iNKT cells are decreased in EM patient PB, particularly during the secretory phase, compared to healthy controls. Moreover, patients with severe dysmenorrhea observed an increase in CD4+ iNKT cells expressing IL-17 ( 49 ). These findings align with a previous study by Guo et al. that reported a decrease in NKT cells number in both PB and PF of EM patients compared to healthy individuals. Interestingly, higher levels of IFNγ and IL-4 levels were positively associated with the NKT cell numbers while an inverse correlation was observed with disease stage ( 39 ). Moreover, a transcriptome meta-analysis by Poli-Neto et al. revealed NKT gene signatures are increased in the eutopic endometrium of EM patients, most predominantly stage I–II patients, as compared to healthy controls, suggesting an increased local abundance of NKT cells in early disease ( 64 ). However, potential contributions of NKT cells in EM pathophysiology remain largely unexplored. DN T cells are characterized by expression of TCRαβ but absence of CD4/CD8 co-receptors and NK T cell markers. These cells are thought to originate from either thymus exit or the loss of CD8 expression in the periphery after TCR and cytokine stimulation ( 204 – 207 ), residing in PB, SLOs, and a variety of tissues (tissue-resident DN T cells), including liver, lungs, skin, intestine, and genital tract. Circulating DN T cells can also migrate to infiltrate non-lymphoid tissues (NLTs), contributing to tissue-specific immune responses ( 208 ). Like Th17 cells, DN T cells exhibit functional plasticity, whereby they can act as a major source of IL-17, contributing to chronic inflammation and tissue damage in autoimmune disease ( 209 ), or can produce a marked level of IL-10 and exert regulatory roles as a specific peripheral subset of DN Tregs ( 210 ). This plasticity is demonstrated in cancer, where DN T cells can be tumor-promoting or -suppressing depending on cancer type and the tumor microenvironment, with their presence noted in both PB and solid tumors (lung, liver, glioma, pancreatic cancer) ( 211 – 214 ). In an autoimmune-prone lpr (fas-deficient) murine model, DN T cells exhibited immunosuppressive activity via IL-10 secretion ( 215 ). Therapeutic approaches targeting DN T cells show promise in treating EM-associated comorbidities, including cancer and autoimmune conditions ( 216 , 217 ), suggesting potential for EM-specific treatment. Mucosal-associated invariant T (MAIT) cells are unconventional T cells with a highly conserved TCR repertoire ( 218 ), primarily found in mucosal tissues but also present in PB. MAIT cells are identified by the TCR α-chain (Vα7.2–Jα33 in humans, and Vα19–Jα33 in mice) and restricted TCR β-chain repertoire ( 219 , 220 ), with high expression of C-type lectin CD161 in humans. These cells contribute to both innate and adaptive immunity and are implicated in autoimmune and immune-mediated diseases ( 218 ). Upon IL-18-dependent activation, MAIT cells produce proinflammatory molecules, including IL-17, IFNγ, TNF, and cytotoxic factor granzyme B ( 221 ), with IL-12, IL-15 and type I interferons further enhancing activation ( 222 ). In autoimmune conditions, including MS ( 223 ), IBD ( 224 ), and RA ( 225 , 226 ), circulating MAIT cells are reduced in patient PB, but markedly increased in affected tissues, reflecting their migration to inflamed sites. Decreased circulating MAIT cells may also be attributed to prolonged activation leading to cell death, as MAIT cells expressed increased exhaustion markers (CD38, PD-1) and apoptosis indicators (FAS, intracellular active caspase 3) ( 218 ). MAIT cells migrate and accumulate in inflamed tissues via chemokine receptors (CCR5, CCR6 and CXCR6), integrins (α4β1 integrin, CD49a and CD103), and adhesion molecules (PSGL1 and sLex). Notably, IL-18, the major activator of MAIT cells, is enriched in both the PB and PF of EM patients ( 227 ). A pilot study measured several factors crucial to MAIT cell functioning and observed enriched levels of IL-8, IL-12 and in the PF of EM patients compared to healthy controls. Interestingly, MAIT cells were increased in both PF and PB of EM patients. Specifically, CD4+ and CD8+ MAIT populations expanded, while DN MAIT cells were reduced. CD8+ MAIT cells in EM patients were highly activated by expressing more robust levels of CD38, while lower DN MAIT cell number was attributed to elevated PD-1 expression ( 40 ). However, no study has yet explored the functional role of MAIT cells in EM.

It is well-known that T cells are crucial regulators of the immune response and play a key role in maintaining immune homeostasis. Imbalance in T cell subsets and their functions are often associated with a variety of health complications such as autoimmune disease, chronic inflammation, and infection. As there are numerous subsets of T cells, each with distinct functions, it is crucial to obtain an understanding of how each of these populations individually and collectively play a role in health and disease. It is also important to note that around 95% of women with EM report at least 1 comorbidity, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), asthma, and rheumatoid arthritis (RA) ( 8 ). As several of these associated comorbidities are driven by T cell dysfunction ( 67 – 70 ), it is highly likely that perturbed T cell functions overlap between EM and associated comorbidities. CD4+ T cells, also known as effector/T helper (Th) cells, are termed due to their “helper function”, as they are responsible for activating other immune cells, including B cells and CD8+ T cells, to orchestrate a broader immune response. Dependent on their environment, naïve CD4+ T cells can be activated and differentiated into various subsets, such as Th1, Th2, Th9, Th17, T follicular helper cells (Tfh), and Tregs, each with distinct functions. CD4+ T cells have been increasingly examined in the context of EM due to their central role in regulating immune responses, including those promoting inflammation, a hallmark feature of EM. Literature depicts an increased percentage of CD4+ T cells in EM patient PB as compared to the CD8+ T cell population, with CD4+ T cells comprising 67% of total T cells ( 71 ). Though, proportions of T cell subsets appear to be distinct to sample type, as within patient PF CD4+ T cells only comprised 37% of total T cells ( 71 ). Within patient tissues, others report increased CD4+ T cell gene signatures within the eutopic endometrium of EM patients as compared to healthy controls ( 50 ), as well as increased infiltration of CD4+ T cells into ectopic lesions compared to controls ( 27 ). While these local and systemic alterations have been reported in literature, a systematic review of CD4+ T cells in EM found no clear correlation between EM and systemic deviations of CD4+ T cells ( 72 ). Th1 cells play a critical role in eliciting a pro-inflammatory type 1 immune response, primarily focused on protecting hosts against intracellular pathogens. Th1 cells produce distinct cytokines, including IL-2, TNFα, and pro-inflammatory IFNγ, which help to promote T cell growth and differentiation as well as activate M1 macrophages and cytotoxic T cells to eliminate foreign cells/debris/pathogens. While Th1 cells do aid in host defense, overreactive Th1 responses can induce various inflammatory and organ-specific autoimmune diseases, such as RA, multiple sclerosis (MS), and type 1 diabetes ( 42 ). It has long been debated whether EM is an autoimmune disease ( 73 ), with some reporting increased Th1 cells in patient PB ( 41 ) and within the PF of murine models of EM ( 20 ). EM also commonly co-occurs with autoimmune disease ( 73 ), however, EM has not yet been classified as an autoimmune disease. While the role of Th1 cells in EM is not entirely understood, Th1 cells are postulated to contribute to disease pathophysiology by promoting chronic inflammation and lesion growth. In a bioinformatic study examining differentially expressed genes in EM patient ectopic tissues as compared to healthy endometrium, Th1 cells, along with memory B-cells, were the top cell types associated with the highest number of hub genes ( 74 ). As hub genes are those that are highly connected to other genes and play influential roles in these immune networks, Th1 cells may then play an essential and regulatory role in EM-associated immune dysfunction. Recent literature suggests that Th1 cells may play a particularly influential role in the early stages of EM ( 75 ), contributing to the initial inflammatory response and lesion establishment. Indeed, a gene set enrichment analysis (GSEA) by Poli-Neto et al. identified increased Th1 cell signatures in the eutopic endometrium of early stage (I–II) EM patients, suggesting a more active Th1-associated immune response in early disease ( 51 ). Though, it should be noted that another study by Wu et al. reported decreased Th1 gene signatures in patient eutopic endometrium compared to controls ( 50 ). Others also report that TNFα, a Th1 cytokine, is increased EM patient serum and PF in early disease stages and these levels significantly decrease as EM disease severity increases ( 21 ). The inflammatory lesion microenvironment is then thought to switch to an anti-inflammatory, Th2 dominant environment in later stages of EM ( 51 , 75 , 76 ), allowing for the survival and growth of lesions. However, mechanistic studies are required to delineate the precise roles of these immune cell subsets in the chronological development of EM lesions. Despite aforementioned reports of increased Th1 cells in EM, some report decreased Th1 cells in ectopic tissue ( 41 ) as well as no differences in Th1 cell numbers in patient PB as compared to controls ( 28 ). Additionally, recent research indicates that Th1 cytokines, IFNγ and IL-2, may serve as independent protective factors for EM ( 77 ). Overall, literature surrounding the role of Th1 cells in EM is not entirely standardized and, at times, is conflicting. While definitive markers identifying/characterizing Th1 cells are largely agreed upon, such as IFNγ, T-bet, and CXCR3, there is a lack of standardization in the usage of these markers to conclusively identify Th1 cells. Th2 cells are a subset of CD4+ T cells that protect against parasitic infection and promote tissue repair through their contribution to the type 2 immune response. Naïve CD4+ T cells differentiate into Th2 cells upon antigenic stimulation in the presence of IL-4 and secrete type 2 cytokines including IL-4, IL-5, and IL-13. IL-4 signaling upregulates the canonical type 2 transcription factor GATA3, which in partnership with STAT5 leads to IL-4 and IL-13 expression ( 78 ). Additional transcription factors are required to regulate Th2 programming and are reviewed extensively elsewhere ( 78 , 79 ). Type 2 cytokines drive both non-hematopoietic (goblet cell hyperplasia, smooth muscle contraction, and fibroblast extracellular matrix production) and hematopoietic cell changes (activation of mast cells, eosinophilia, B cell proliferation and production of immunoglobulin E (IgE), and M2 macrophage polarization). Due to these influences, Th2 cells are associated with disease states that have exacerbated type 2 responses such as asthma/allergic disease and other chronic inflammatory autoimmune diseases including systemic lupus erythematosus (SLE) and IBD. It is however important to note that group two innate lymphoid cells (ILC2s) exhibit a similar secretory cytokine profile as Th2s (IL-5 and IL-13). Thus, delineation is required between these cell types as to their individual and/or combined contribution to disease pathophysiology. Support for EM as a type 2 inflammatory disease arises from increased detection of type 2 cytokines (IL-4 and IL-10) in patient ectopic tissue ( 52 – 54 ), PF ( 24 , 25 ), and PB/serum ( 43 , 77 ), compared to healthy controls. Consistent with this, several studies report increased presence of Th2 cells in EM-associated compartments. Notably, phenotypic characterization of CD4+ cells isolated from PF ( 22 ) and PBMCs ( 80 ) demonstrated significantly elevated Th2 cells in EM compared to controls, with no apparent influence of disease stage on Th2 cell frequency. Further, intracellular cytokine production of type 1 cytokines, IL-2 and IFNγ, was significantly reduced in CD4+ T cells from EM patient PF compared to controls ( 23 ), suggesting a shift to a type 2 response. Contrary to this, Guo et al., found reduced T cell numbers in the PF of stage I and II patients compared to controls, yet no changes in Th2 cell (CD4+CCR4+) frequency ( 19 ). However, CD69+ T cells were significantly increased in patient PF compared to controls, with a significant increase in the frequency of CD69+ Th2 cells. Notably, CD69+ T cells were greatest in the PF compared to PBMCs. As CD69 is known as an activation marker, this may suggest an activated T cell phenotype localized to the lesion microenvironment. When examining eutopic tissues in a GSEA study, others identified a decrease in Th2-associated gene signatures in EM patients relative to healthy controls, suggesting a decreased local abundance of Th2 cells ( 50 , 51 ). This compartment-specific variance highlights a possible disconnect between functional immune phenotypes and transcriptional profiles within the endometrium. Such divergence also highlights important methodological considerations, as markers used to classify Th2 cells differ across publications, highlighting the dynamic nature of these cells yet also potentially leading to discrepancies in reporting. Additionally, many studies report only on total CD4+ T cells without further characterization as to the particular T helper phenotypes. The EM lesion microenvironment is highly complex with intertwined hormonal, epithelial, stromal, and immune cell cross talk. Local environmental factors at mucosal sites, including epithelial cell-derived alarmin cytokines, such as IL-25, IL-33, and thymic stromal lymphopoietin (TSLP), regulate and fine tune the Th2 response. Indirectly, these cytokines drive Th2 cell differentiation through upregulation of the dendritic cell (DC) co-stimulatory molecule, OX40L ( 78 ), and through activation of ILC2s that act on monocytes and macrophages, ultimately regulating Th2 cytokine production and associated downstream functions. These inflammatory cytokines play a large role in Th2-dominant pathological conditions, such as asthma and allergic disease, and have also been found to be elevated in EM. Indeed, our lab has shown that TSLP protein expression is significantly higher in ectopic lesions compared to control endometrium as detected on a tissue microarray of ovarian EM lesions ( 55 ). When explored in a mouse model of EM, TSLP treatment increased the PF CD3+ IRF4/T-bet ratio, suggesting an enrichment of Th2 cells with treatment ( 55 ). Further, we and others have shown that IL-33 is elevated in EM patient plasma/serum, PF, and ectopic lesions compared to corresponding healthy control samples, which correlates with disease severity ( 26 , 56 ). This is poignant as pathogenic memory Th2 cells that highly express the IL-33 receptor, suppressor of tumorigenicity 2 (ST2), have been identified in asthma. These cells are known for their high secretion of type 2 cytokines (predominantly IL-5) and amphiregulin which contribute to fibrosis ( 81 ). Fibrosis is an important pathological feature of EM thought to contribute to chronic pelvic pain, yet its mechanisms of development remain incompletely understood ( 82 ). Thus, mechanistic studies are needed to determine whether Th2 cells are playing a protective or pathogenic role within EM pathophysiology, as there is potential to leverage immunologic therapeutics utilized in other type 2 dominant pathologies. Finally, the co-occurrence of EM with autoimmune and allergic diseases underscores potential overlapping immunological dysfunction that contributes to the comorbid nature of these conditions ( 83 , 84 ). A Th2 cell dominant response supports B cell activation and antibody production yet this relationship in EM remains poorly understood. In EM, a positive association between PB and ectopic tissue IL-4 protein and mRNA expression and B cell presence (CD20+CD5+ and HLA-DR+CD20+) has been identified ( 52 ), yet Th2 cells were not characterized. Further studies are warranted to explore the relationship between Th2 and B cell activation within EM as Th2 cells may provide a critical link between EM, autoimmune, and allergic disease. Th17 cells are a subset of CD4+ T cells regulated by the RORγt transcription factor. Known for their production of pro-inflammatory IL-17 and their highly plastic nature, Th17 cells are key regulators of inflammation and immune homeostasis ( 85 ), allowing them to contribute to the pathogenesis of various inflammatory and autoimmune diseases ( 86 , 87 ). The local cytokine milieu shapes Th17 cell phenotype. Namely, IL-23 drives and maintains a “pathogenic” Th17 cell phenotype, producing pro-inflammatory cytokines, such as IL-17, IL-22, and IL-21 ( 44 , 87 – 89 ). Pathogenic Th17 cells also co-produce IFNγ, which can in-turn promote Th1 differentiation and inflammation ( 90 ). In contrast, “non-pathogenic” Th17 cells differentiate in the absence of IL-23 and produce more anti-inflammatory cytokines, such as IL-10, to regulate/suppress immune responses ( 44 , 91 ). Indeed, non-pathogenic Th17 cells can play a beneficial/protective role, supporting mucosal barrier integrity and aiding in host defense against bacterial/fungal infections ( 91 ). Dependent on their environment, Th17 cells can also acquire characteristics of other Th subsets, such as Th1 (in presence of IL-12), Th2 (in presence of IL-4), and Tfh (in Peyer’s patches) to induce the development of IgA-producing germinal center B cells ( 92 ). Notably, in the presence of TGF-β1, Th17 cells can transdifferentiate into Tregs during an immune response ( 93 ). Tregs can also differentiate back into Th17 cells dependent on their environment ( 94 ). As a balance of Tregs and Th17 cells ensures immune homeostasis, skewing of this balance can result in the development and/or exacerbation of various chronic inflammatory/autoimmune diseases ( 94 – 96 ). This is particularly relevant to diseases affecting mucosal barrier surfaces ( 94 ), such as IBD/IBS, asthma, and EM, as both Th17 and Tregs are commonly found at mucosal surfaces. Ultimately, while it is clear that Th17 cells can act as key modulators of both inflammation and immune tolerance, it is still not completely understood whether Th17 cells completely adopt these various Th lineages, as their transformation is not irreversible, meaning they may still retain characteristics of a Th17 phenotype ( 97 ). The Th17/IL-17 axis has become increasingly researched in the context of EM. Various reports depict increased and/or dysregulated levels of IL-17, Th17 cells, and other key mediators in this axis, within EM patient PF, plasma, menstrual effluent, and/or ectopic/eutopic tissues ( 27 – 32 , 41 , 44 – 46 , 65 , 98 ), as well as within animal models ( 33 , 44 , 47 , 99 ), which is reported to be correlated with disease severity ( 29 ). Our lab has shown that EM lesions produce IL-17 and patient plasma has significantly elevated IL-17 compared to controls ( 45 ). Surgical removal of lesions led to a significant decline in IL-17 levels in plasma, suggesting that EM lesions and associated inflammation may contribute to elevated IL-17 in EM patients. We further depict that IL-17 influences macrophage recruitment and polarization, inducing a M2 phenotype, which aids in lesion development ( 99 ). Our lab also depicts dysregulated gene expression of key mediators in the IL-23/Th17 axis in EM ectopic and eutopic tissues, as well as significantly increased IL-23 in patient plasma, compared to controls ( 44 ). Recombinant IL-23 treatment of cell lines representative of the endometriotic lesion microenvironment also significantly increased cytokines and growth factors known to play a role in lesion establishment and maintenance ( 44 ). Various other reports highlight a dysregulated Treg/Th17 ratio in EM ( 16 ), supporting the theory that Th17 cells may skew immune homeostasis to promote inflammation in EM and exacerbate hallmark symptoms of this disease. Ultimately, while Th17 cells and downstream IL-17 have been moderately investigated in EM pathophysiology, a gap in literature remains as to whether/how upstream IL-23 may drive pathogenic Th17 cells in EM to exacerbate disease. A 2022 study by Jiang et al. reveals Th17 cells to have a significantly different gene expression profile in EM patient PF compared to controls ( 31 ). Interestingly, within EM patients, Th17 cells had unique gene expression signatures dependent on disease stage (stages I–II as compared to stages III–IV). The top 10 significantly up-regulated genes were found to encode metabolic proteins, signaling proteins, and cell surface receptors ( 31 ). Authors suggest that these results indicate that PF Th17 cells are activated in EM and undergo metabolic reprogramming to provide themselves with sufficient energy. Moreover, the receptor for activated c kinase 1 (RACK1) scaffolding protein was particularly enhanced in EM Th17 cells relative to controls. RACK1 plays a critical role in the regulation of T cell activation, proliferation, and migration ( 100 – 102 ). Indeed, Qiu et al. report that loss of RACK1 increases T cell apoptosis ( 103 ). Interestingly, RACK1 expression was significantly increased in Th17 cells in later stages (III–IV) of EM compared to earlier stages (I–II) ( 31 ). Thus, as Th17 cells are associated with EM severity ( 29 ), this stage-dependent upregulation of RACK1 is hypothesized to maintain and/or promote Th17 cell activation and in-turn promote Th17 cell-mediated inflammation in EM, exacerbating disease in a stage-dependent manner ( 31 ). A study by Kang et al. also suggests that IL-17 is able to confer a resistance to natural killer (NK) cell-mediated cytotoxicity via activation of ERK1/2 signaling, promoting endometrial cell survival in EM ( 27 ). Recent research on the relationship and association of gut microbiota dysbiosis and EM has also been growing, with numerous reports of gut dysbiosis in EM ( 104 ). It is known that shifts in the composition of gastrointestinal and/or urogenital microbiota can regulate differentiation of Th17 and Treg cells to promote either a pathological/pro-inflammatory (Th17) or protective/tolerant (Treg) response ( 47 ). Using a baboon model of EM, Le et al. reveal that surgical induction of EM alters mucosal microbiota in both gastrointestinal and urogenital tracts ( 47 ). In fact, each stage of EM disease progression was associated with a unique signature of T cell subset dominance and microbial diversity. Namely, authors found immune tolerant Tregs to be associated with microbial diversity during early and late stages of EM progression, while inflammatory Th17 cells were associated with microbial diversity in mid-stages of disease. Though, it should be noted that no sham control was used to confirm results were not due to surgical interventions rather than EM itself. A 2024 study by Li et al. also highlights the correlation between gut microbiota dysbiosis and IL-17 levels in EM, shedding light on how alterations in gut microbiota in EM patients has profound changes in both the intestinal and peritoneal environments ( 33 ). Briefly, authors suggest that EM-associated gut dysbiosis compromises intestinal barrier integrity, promotes EM progression, and alters intestinal Th17/Treg balance, subsequently increasing IL-17A levels in patient PF. Due to the influence of gut dysbiosis on Treg/Th17 cell commitment, this can in-turn influence patient susceptibility to IBD ( 94 ), which is associated with EM ( 84 ). Though, it is unclear whether alterations in immune and microbial phenotypes are a consequence of EM, or if these factors may play a causative role. Collectively, IL-17-producing Th17 cells may promote endometrial cell survival and are reprogrammed under EM-conditions to further promote inflammation and exacerbate disease. T follicular helper (Tfh) cells are a subset of CD4+ T cells essential for germinal center (GC) B cell maturation and antibody responses ( 105 ). Outside of the GC, Tfh cells can fuel cytotoxic CD8+ T cell responses ( 106 ) and contribute to tertiary lymphoid structures (TLSs) in chronic inflammation ( 106 ), such as in EM. Tfh cell differentiation requires T cell receptor (TCR) and inducible T-cell co-stimulator (ICOS) activation, often provided by DCs, in the presence of IL-6, which upregulates Bcl6 and CXCR5 expression, enabling Tfh cell trafficking to inflammatory structures, including secondary lymphoid organs (SLOs) and ectopic TLSs, through the receptor ligand CXCL13 ( 105 ). Once in TLSs, B cells replace DCs as the primary antigen presenting cell and Tfh cells maintain B cells via IL-21 and further produce CXCL13 to recruit additional T and B cells ( 106 ). Tfh cells are often characterized as CD4+CXCR5+Bcl6hi ( 107 ). Though, Tfh cells exhibit plasticity, with subsets resembling Th1, Th2, and Th17 cells (Tfh1, Tfh2, Tfh17) ( 105 ), which is of interest due to aforementioned roles of these Th cells in EM pathophysiology. Tfh cells also play a key role in various autoimmune diseases including SLE and Sjögren’s syndrome, in which there is extensive autoantibody production ( 105 ). It is suggested that their ability to support B cell antibody production may contribute to autoantibody development ( 108 ). As EM is associated with autoantibodies ( 109 ), Tfh cells may play a similar role in EM pathophysiology. Tfh cells also play a crucial role in the formation and function of TLSs, which are organized immune cell aggregates known to form in cases of chronic inflammation and/or persistent antigenic exposure ( 110 ). This is of interest as our group recently discovered the presence of TLSs across varying phenotypes of EM ( 110 ). While T cell markers (CD3 and CD8) were used, Tfh cells were not specifically highlighted by the multiplex immunofluorescence panel used to classify TLSs within our study ( 110 ). Thus, continued characterization of EM-associated TLSs is critical to determine the contribution of diverse immune phenotypes to these dynamic structures. While the role of Tfh cells is understudied in EM, literature depicts significant differences in local proportions of Tfh cells in EM tissues compared to controls ( 111 ). Indeed, Wu et al. revealed an enrichment of Tfh cell-associated gene signatures in eutopic tissues of patients compared to healthy controls, suggesting an increased abundance of local Tfh cells ( 50 ). Notably, a 2022 study found Tfh cells were significantly reduced in EM lesions compared to matched eutopic tissues of patients ( 57 ). Moreover, Tfh cells were correlated with aquaporin 1 (AQP1) and ZW10 binding protein (ZQINT), two proposed diagnostic markers of EM, and showed positive correlations with Tregs and activated NK cells, but negative correlations with M2 macrophages. Others report significantly increased Tfh cells in EM compared to healthy controls ( 80 , 112 ). Though, these authors observed Tfh cell proportions in PB ( 80 ), underscoring the need to better define Tfh cell presence within lesion tissue. Tregs contribute to immune homeostasis by suppressing exaggerated immune responses and promoting immunological self-tolerance. Consequently, reduced Treg cell function is implicated in autoimmune disease ( 113 ). Tregs carry out their effector functions via direct cell-cell interaction, cytotoxic action, and release of suppressive cytokines, including IL-10, TGF-β1, and IL-35 ( 114 ). These cytokines can act to inhibit effector T cell activation, while granzyme and perforin produced by Tregs can act to kill effector T cells ( 115 ). IL-10 can also function to inhibit DC maturation, further contributing to immune tolerance ( 116 ). Moreover, the immunosuppressive actions of Tregs are primarily regulated by FoxP3, a transcription factor crucial for Treg cell differentiation and function ( 117 ). Efficient Treg function requires the expression of FOXP3 , in addition to other signature Treg genes, such as IL2RA (CD25) and CTLA4 ( 118 ). Three distinct subpopulations of Tregs exist in humans: resting suppressive Tregs (CD45RA+FoxP3 low ), high-activated suppressive Tregs (CD45RA-FoxP3 high ), and cytokine-secreting, non-suppressive Tregs (CD45RA-FoxP3 low ) ( 118 ). In mice and humans, Tregs may also be categorized by origin/developmental context into natural Tregs, developed in the thymus, and induced Tregs, differentiated from naïve CD4+ T cells in the periphery/outside the thymus ( 119 ). Tregs may contribute to EM pathophysiology in part through the prevention of adequate immune surveillance and lesion clearance by other immune cell types, allowing for lesion establishment and growth. Alterations in Treg homeostasis, both systemically and locally within ectopic and eutopic endometrium, have been noted in EM ( 116 ). However, differing methodology and characterization of Treg cells has resulted in variable results. A single-cell ligand-receptor analysis revealed upregulation of CTLA4 , which was interacting with CD86 , in ectopic lesions of stage II–IV EM patients, indicative of highly activated immunosuppressive Tregs ( 58 ). Thus, Tregs may facilitate immune evasion in ectopic tissues, promoting local lesion persistence. This is further supported by findings of Treg density in patient PF being correlated with EM stage and severity ( 120 ). In a flow cytometric analysis of PB and PF from EM patients, an increase in Tregs (CD25 high FoxP3+) was noted in PF compared to controls, while a decrease in Tregs was noted in PB ( 34 ), suggesting altered Treg compartmentalization in EM. Khan et al. similarly reported increased Tregs in patient PF, though, no significant differences were found in PB ( 30 ). Through IL-10-mediated inhibition of DC maturation ( 58 ), Tregs may also contribute to the tolerogenic, immature DC phenotype observed in peritoneal lesions. Thus, Tregs may support immune evasion observed in EM, facilitating lesion establishment and preventing effective clearance. Treg secretion of soluble fibrinogen-like protein 2 (sFGL2) appears to promote an anti-inflammatory M2 phenotype, which supports tissue repair/remodeling ( 121 ). Notably, sFGL2 levels are increased in EM patient PF, which may locally promote this alternatively activated M2 phenotype within the lesion microenvironment ( 121 ). This suggests a positive feedback loop between Tregs and macrophages that perpetuates EM progression. As M2 macrophages play a crucial role in EM-associated fibrogenesis, Tregs may also indirectly contribute to fibrosis ( 122 ), a key feature of EM linked to pain and infertility ( 82 ). However, contradictory findings exist. Tanaka et al. reported a significant reduction in activated Tregs (CD45RA-FoxP3 high ) in both ectopic and eutopic EM tissues compared to controls ( 35 ), which was not observed in the PF or PB. Though, as only ovarian endometrioma tissues were utilized, this finding may be specific to this type of EM. This study also used a mouse model of EM, whereby Tregs were selectively depleted in FoxP3 DTR (diphtheria toxin receptor) C57BL/6 mice ( 35 ), resulting in increased systemic (PB) and local (PF, lesions) levels of inflammatory cytokines and angiogenic factors, as well as increased lesion number and weight compared to controls. As the lack of Tregs facilitated EM progression, this further supports aforementioned theories of an initial pro-inflammatory, Th1/Th17-dominant microenvironment which transitions into a later anti-inflammatory, Th2/Treg-dominant microenvironment to allow for EM lesion growth/maintenance. Tregs also play a crucial role in regulating angiogenesis, another key hallmark feature of EM. Indeed, their angiogenic role is well-known during embryo implantation and pregnancy, though, whether they play an anti- or pro-angiogenic role is highly tissue and context specific. In an ovarian cancer study, Tregs were recruited to tumor sites via CCL28, where they contributed excess VEGFA and promoted tumor angiogenesis ( 123 ). Notably, Treg depletion in mice using an anti-CD25 antibody resulted in a significant reduction in tumor microvascular density within intraperitoneal tumors ( 123 ). Moreover, in another study of abortion-prone mice, adoptive transfer of Tregs improved spiral artery remodeling and placental development, influencing vascularization ( 124 ). Thus, Tregs may function in a similar fashion in EM, promoting angiogenesis and lesion establishment. This is supported by in vitro evidence in which co-culture of primary human endometrial stromal cells (ESCs) and monocytes increased CCL17 and CCL22 production, promoting Treg recruitment, enhanced immunosuppressive function, and increased TGF-β1 secretion, which may collectively support angiogenesis in endometrial cells ( 125 ). However, this study had limitations, such as using eutopic ESCs rather than ectopic cells. Ultimately, further research is required to elucidate the exact role of Tregs in EM-associated angiogenesis. Our lab and others have demonstrated increased IL-33 in EM, which may drive disease progression by perpetuating inflammation, angiogenesis, and lesion proliferation ( 26 , 56 , 126 ). IL-33 also influences Treg cell fate and function, which may then impact EM. Indeed, in a mouse study on skin transplantation, IL-33-stimulated Tregs displayed potent anti-inflammatory activity ( 127 ). Further, in the presence of TGF-β1, Tregs can be differentiated from naïve CD4+ T cells through the IL-33/ST2 pathway, via regulation of FoxP3 expression ( 128 ). IL-33 binding to ST2 both promotes Treg fate and increases ST2 expression, creating a positive feedback loop for Treg expansion ( 127 ). Others have also shown that IL-33 promotes functional stability of Tregs in vivo , allowing for enhanced immune evasion ( 129 ). Ultimately, one may draw parallels from these findings, suggestive that a similar mechanism may exist in EM whereby IL-33 may mediate Treg stability, allowing for evasion of EM lesion clearance by the immune system. CD8+ T cells play a key role in immune surveillance and produce pro-inflammatory cytokines, including TNFα and IFNγ, to facilitate immune cell recruitment and sustain chronic inflammation, respectively ( 130 , 131 ). Cytotoxic CD8+ T cells are a critical component of the adaptive immune response, targeting and eliminating infected, malignant, and foreign cells. In healthy states, cytotoxic T cells control viral infections and provide tumor surveilance ( 132 ). However, their activity is context dependent. While vital for clearing infections, they can also promote inflammation and tissue/joint damage, as observed in autoimmune diseases, such as RA ( 133 , 134 ). Notably, CD8+ T cells exhibit functional plasticity ( 62 , 135 ), with research identifying an IL-17-producing CD8+ subset known as Tc17 cells, which are increased in EM patients, potentially driven by high IL-23 levels ( 44 , 80 , 136 ). This plasticity allows them to modulate immune responses, contributing to both protective immunity and immunopathology. The involvement of CD8+ T cells in the initial establishment of EM lesions remains under investigation. Literature depicts an increased proportion of CD8+ T cells in EM lesions when compared to matched eutopic endometrium from EM patients and/or healthy eutopic endometrium ( 59 ). Overexpression of CD8+ T cells in peritoneal EM lesions, as compared to both eutopic endometrium and healthy control peritoneum, has been established ( 60 , 61 , 137 ). However, in a study aiming to investigate cellular heterogeneity in EM, the proportion of CD8+ T cells was reduced in EM lesions, with the cytotoxicity of ectopic T cells being depressed ( 62 ). Interestingly, CD8 inhibition in an EM mouse model resulted in visually larger lesions, while mice supplemented with CD8+ T cells showed reduced weight in EM lesions ( 62 ). Evidence suggests that these cells play a significant role in the maintenance and progression of established lesions ( 62 ). Indeed, a 2023 systematic review highlighted alterations in the function and number of CD8+ T cells within EM, finding consistent indications of CD8+ T cell enrichment within EM lesions, indicating their likely contribution to lesion persistence ( 137 ). Though, no consistent, significant alterations were identified in CD8+ T cell populations within PB, PF, or eutopic endometrium samples from EM patients/controls ( 137 ). In the context of EM, disease progression is marked by impaired immune surveillance and ineffective clearance of ectopic endometrial cells within the peritoneal cavity, ultimately promoting lesion establishment. Chronic antigenic stimulation, as observed in persistent infections and tumors, drives the development of CD8+ T cell exhaustion ( 135 , 138 ). Although persistent exposure to non-self-antigens is the most studied cause of T cell exhaustion, exhaustion can also arise in response to self-antigen presentation, particularly in the context of chronic inflammation ( 139 ). Under steady-state conditions, classical tolerance mechanisms such as deletion and anergy maintain self-reactive T cells in check ( 140 ). However, when self-antigens are presented in an inflammatory environment, exhaustion may be observed as an additional regulatory mechanism ( 139 , 141 ). In EM, persistent self-antigen exposure, coupled with inflammation and immune activation, may promote CD8+ T cell exhaustion as a means of limiting tissue damage. While this response can help suppress excessive inflammation, it may also impair the cytotoxic capacity of CD8+ T cells, reducing their ability to clear ectopic endometrial debris and developing lesions ( 135 , 138 ). Exhausted CD8+ T cells exhibit impaired cytotoxicity due to dysregulated perforin and granzyme expression and sustained expression of inhibitory receptors, notably PD-1, which enforce their dysfunctional state ( 135 , 138 ). These cells undergo a progressive loss of effector functions, beginning with compromised/lost production of IL-2, followed by TNF−α and IFN−γ, ultimately resulting in terminally exhausted cells with limited proliferative capacity and reduced persistence ( 142 ). A study by Schmitz et al. demonstrated that CD8+ T cells isolated from menstrual effluent of EM patients showed significantly lower perforin expression compared to controls, indicating a reduction in cytotoxic potential ( 66 ). The reduced number of perforin-positive CD8+ T cells may be explained by incomplete CD8+ T cell differentiation, potentially caused by inhibitory cytokine signaling (e.g. IL-10 and TGF-β) or cytotoxic activation leading to granule exocytosis ( 66 ). Given that the most widely accepted theory for the etiology of EM involves retrograde menstruation ( 143 ), it is possible that the lack of perforin-positive CD8+ T cells in the menstrual effluent allows for lesion establishment. Similarly, Iwasaki et al. found a reduction in cytotoxic T cells in both the PB and PF of EM patients, further supporting the notion of compromised immune function in this condition ( 36 ). In addition to their cytotoxic functions, CD8+ T cells secrete key cytokines, including IFNγ, TNFα, and lymphotoxin-α (LT-α), which play crucial roles in modulating host defense responses ( 135 ). IFNγ has been observed to be significantly upregulated in the PF of EM patients relative to fluid from healthy controls ( 24 ). A study conducted by Wu et al. found that in response to IFNγ stimulation, intercellular adhesion molecule-1 (ICAM-1) expression was elevated in cultured cells isolated from both eutopic endometrium and peritoneal EM lesions ( 144 ). ICAM-1 is a prominent cell adhesion molecule displaying widespread distribution in the human endometrium, regulating leukocyte trafficking into the tissue and linking adhesive interactions with epithelial and endothelial function ( 145 ). Various studies have shown a significant increase in ICAM-1 in the serum, PF, and lesions of EM patients, relative to controls ( 144 , 146 , 147 ). Together, these may indicate that an increase in IFNγ may promote the adhesive and invasive activity of ectopic lesions, leading to EM progression ( 144 , 146 ). Elevated TNFα levels are commonly observed in both acute and chronic inflammatory conditions, including EM and numerous autoimmune diseases. Notably, Liu et al. found that increased localized expression of TNFα correlates with the presence of active (vascular, inflammatory, proliferative ( 148 )) EM lesions ( 149 ). Similarly, LT-α, a member of the TNF superfamily, is implicated in the pathogenesis of various chronic inflammatory diseases. In advanced stages of EM, serum LT-α levels are significantly elevated compared to healthy controls, underscoring its potential role in disease progression ( 150 ). Cross-sectional and case-controlled studies report a significant association between EM and increased incidence of RA ( 84 , 151 – 153 ). Patients with active RA exhibit increased unstimulated CD8+ T cells expressing TNFα and granzyme B ( 154 ), as well as increased serum TNFα relative to healthy controls ( 155 ). As TNFα is also elevated in EM serum, this suggests a potential shared inflammatory mechanism, where CD8+ T cell-derived TNFα may recruit pro-inflammatory cells that amplify cytokine-drive inflammation, exacerbating EM. CD8+ T cell dysregulation may also contribute to EM-associated infertility ( 137 ), as impaired cytotoxic function can foster a pro-inflammatory environment, adversely affecting reproductive processes ( 137 ). While the exact role of CD8+ T cells remains unclear, there are correlations between this cell type’s associated cytokines and infertility ( 50 ). This aspect is extensively covered elsewhere ( 137 ).

Given the central role of T cells in EM disease pathophysiology and emerging evidence successfully targeting T cells in other autoimmune and chronic inflammatory conditions, there is a growing interest in exploring how T cells could be harnessed for therapeutic purposes in EM. A recent study by Zhang et al. identified ectopic endometrial T cells as the primary source of active IL-16 ( 98 ), a key initiator of EM-associated inflammation. Indeed, IL-16 knockout mice exhibited reduced inflammation and lesion growth ( 98 ). As iron overload is observed in EM ( 238 ), authors recapitulated these conditions in vitro and found this drives the caspase-3-GSDME-mediated pyroptosis axis in ectopic endometrial T cells, initiating IL-16 activation and release. Targeting this pathway, authors developed Z30702029, a GSDME-NTD inhibitor, depicting that Z30702029 alleviated EM lesion development and associated pain in vivo ( 98 ). However, this has yet to be examined in human patients. Another study by Park et al. examined the therapeutic potential of S-Aallyl-L-cysteine (SAC) in EM, a large component of aged garlic extracts, using a mouse model of EM and found that SAC treatment significantly reduced lesion growth ( 239 ). Moreover, treatment was shown to alter splenic CD4+ and CD8+ T cells and inflammatory cytokine production within samples from spleen and EM lesions ( 239 ). While PD-1 and PD-L1 proteins have been extensively studied in cancer due to their regulation of immune tolerance within the tumor microenvironment ( 240 ), the role of the PD-1/PD-L1 pathway in EM remains unclear. Sobstyl et al. recently discovered that expression of PD-1 and PD-L1 is significantly increased on circulating T and B lymphocytes within EM patient PB as compared to controls ( 241 ). This was also reflected when analyzing serum concentrations of soluble (s)PD-1 and sPD-L1 forms in EM patients (correlated with stage) as compared to controls. Another study by Abramiuk et al. found significantly increased levels of CTLA-4+ T cells in advanced cases of EM compared to patients with less advanced disease ( 242 ). This is of interest as CTLA-4, a membrane receptor that facilitates lymphocyte anergy, may play a role in the dysregulated immune response within the endometriotic microenvironment. Thus, further research is required to comprehensively investigate the role of PD-1/PDL-1 and CTLA-4 in EM and their potential for therapeutic targeting. Another study by Tian et al. aimed to examine the impact of T cell Ig and mucin domain-containing molecule-3 (TIM-3), an immunomodulatory molecule found on the surface of Th1 cells which mediates Th1 cell apoptosis, in EM ( 243 ). Using both in vitro and in vivo studies, authors depict that TIM-3 promotes EM lesion proliferation via the brain-derived neurotrophic factor (BDNF)-mediated PI3K/AKT axis, providing another possible therapeutic target of interest in EM. Tregs are integral in maintaining immune tolerance and modulating the immune response, and thus, targeting Treg activity offers a potential avenue of interest in EM. Indeed, a study by Li et al. found significantly increased Tregs, along with overactivation of estrogen-ERα, within EM patient PF ( 244 ). Estrogen-ERα was also found to promote Treg expansion and cytokine production, namely IL-10 and TGF-β1 cytokines, which promote the survival and growth of EM lesions ( 120 ). Authors further showed that treatment with SCM-198 (a synthetic derivative of leonurine) significantly reduced number and expansion of peritoneal Tregs, decreased levels of immunosuppressive cytokines (IL-10 and TGF-β1), and attenuated estrogen secretion by lesions ( 244 ). Moreover, using a Treg-depleted mouse model of EM, adoptive transfer of Tregs significantly reduced number, volume, and weight of EM lesions ( 245 ), as well as reduced mRNA expression of Th1-, Th2-, and Th17-related cytokines, such as IFNγ, IL-4, and IL-17, and pro-inflammatory IL-6 within EM lesions of Treg-depleted mice. As aforementioned, almost all patients with EM report at least 1 comorbid disorder ( 8 ), of which, a large majority appear to be driven by T cell dysfunction. A study by Shigesi et al. suggests an association between EM and autoimmune diseases, including systemic lupus erythematosus, Sjögren’s syndrome, RA, autoimmune thyroid disorder, celiac disease, MS, and IBD ( 84 ). Interestingly, the IL-23/Th17/IL-17 axis plays a role in each of these EM-associated autoimmune diseases ( 246 – 250 ), suggesting a possible overlapping/shared pathogenic pathway. This is of high interest for therapeutics, particularly as IL-23- and IL-17-targetted therapeutics (several monoclonal antibodies) have already undergone clinical trials and are commonly used clinically to treat cases of psoriasis, ankylosing spondylitis, arthritis, and Crohn’s disease ( 251 ). Moreover, due to the known role of IL-17 in EM-associated infertility ( 252 ), this pathway is of high interest for therapeutic targeting. Ultimately, it is speculated that particular autoimmune diseases may be largely facilitated by a dysregulated IL-23/Th17/IL-17 axis, and as this now also being depicted in EM, blocking IL-23 and/or IL-17 systemically/locally in EM patients may suppress lesion development/progression as well as associated infertility. EM and asthma are also often comorbid and have a positive association ( 253 ). While this association is not completely understood, this may be due to an exaggerated type 2 immune response/mast cell activation ( 161 ). Indeed, it is postulated that the exacerbated type 2 response (Th2-dominant) in EM downregulates the pro-inflammatory type 1 response (Th1-dominant) and promotes aberrant wound-healing leading to lesion maintenance and progression. Thus, interruptions in these pathways could be beneficial to lesion clearance. As previously highlighted, epithelial cell-derived cytokines (IL-33, IL-25, TSLP) act as instigators of the type 2 immune response and both these cytokines and downstream cytokine products (IL-4, IL-5, IL-13) are elevated within EM. This provides an opportunity for therapeutic targeting of upstream and/or downstream components to dampen the type 2 response, and in asthma, such biologic therapeutics are already in clinical trials or approved for patient use ( 254 – 256 ). Given the overlapping immunological pathways between these chronic inflammatory conditions, existing therapeutics could be leveraged for EM patients. For instance, retrospective analyses of EM patients with asthma, or other associated comorbidities, could be conducted to determine whether these existing therapeutics also impact EM symptomology or disease progression. Such findings could inform a personalized medicine approach, enabling targeted therapies for EM patients based on their specific immunological and comorbidity profiles.

T cell biology is highly complex and involves a tightly regulated and multifaceted network of cell-cell interactions, signaling cascades, and developmental checkpoints that underpin immune function and homeostasis. T cells not only differentiate into a broad spectrum of specialized subsets including cytotoxic T cells, helper T cells (Th1, Th2, Th17, Tfh), and Tregs, but also exhibit immense diversity in their TCRs, enabling the recognition of a vast array of antigens. While most T cells require MHC-restricted antigen presentation, certain unconventional subsets such as γδ T cells and MAIT cells can function independent of classical MHC pathways. Moreover, memory T cells, including those specific to self-antigens, may persist and re-engage upon repeated exposures, adding another layer of complexity to immune surveillance and tolerance. These features of T cell biology become particularly relevant, and problematic in the context of EM. Ectopic EM lesions are autologous in origin, and yet they escape immune clearance, suggesting systemic and localized immune dysfunction. The lesion microenvironment is comprised of not only immune cells, but also epithelial-stromal-endothelial interactions tightly regulated under endocrine influence. In this ever-changing dynamic microenvironment, immune evasion is facilitated by both passive and active mechanisms. T cells likely become anergic, tolerogenic, or skewed toward anti-inflammatory phenotypes such as Th2. The Th2-dominant response, often associated with tissue remodeling and fibrosis, could be shaped by alarmins such as IL-33, which are secreted by epithelial and stromal cells in response to cellular stress or damage. Indeed, we and others have shown that IL-33 promote Th2 differentiation and facilitate the recruitment of ILC2s ( 56 , 257 ), further amplifying the anti-inflammatory and tissue-supportive milieu. Adding to this complexity is the fact that EM represents a unique form of sterile inflammation. Unlike pathogen induced immune responses, the chronic inflammation observed in EM arises in response to self-tissue likely deposited at ectopic sites via retrograde menstruation. Cells of the innate and adaptive immune system are recruited in response but remain functionally impaired, leading to the persistence of cellular debris. Chemokine gradients play a pivotal role in shaping immune cell infiltration. Specific subsets of chemokines attract corresponding immune cells, including effector T cells and regulatory populations. However, the presence of decoy receptors may modulate these gradients, acting as buffers that regulate the recruitment and localization of immune cells within the lesion microenvironment. This dynamic regulation of chemotaxis likely contributes to the establishment of an immune milieu that supports lesion persistence rather than clearance. Despite significant advances in characterizing individual immune cell populations involved in EM, a mechanistic understanding of how these cells interact, communicate, and influence lesion biology remains incomplete. The advent of high-resolution techniques such as single-cell RNA sequencing, spatial transcriptomics, and advanced imaging modalities now offers unprecedented insight into the cellular composition, functional states, and intercellular communication within endometriotic lesions. These approaches will provide critical insights into the roles of different T cell subsets, including their activation status, effector functions, exhaustion phenotypes, and interactions with other immune and stromal cells. Ultimately, bridging these knowledge gaps is essential not only for elucidating the immunopathogenesis of EM but also for identifying targeted immunotherapies. This requires a systems-level understanding of immune-endocrine-resident cell interactions. Through such integrative, mechanistic studies, we should be able to develop precise, personalized interventions that address both the immunological and hormonal drivers of this highly heterogenous and complex disease affecting more than 200 million women globally ( 2 ).

T cell trafficking to the EM lesion microenvironment is an active area of research that is still not fully understood, though, several key findings have emerged. A study by Kim et al. illustrated that PF from EM patients significantly increased the release of CXCL10 by CD4+ T cells isolated from donor PBMCs, as compared to control PF ( 228 ). As CXCL10, also known as IP-10, is well recognized for its role in recruiting activated T cells to sites of inflammation/tissue damage ( 229 , 230 ), CD4+ T cells within the EM lesion microenvironment may be major drivers of CXCL10 production, and in-turn, play a key role in promoting T cell recruitment to the endometriotic lesion. Another study by Manabe et al. found that both CXCL16 and its receptor, CXCR6, were expressed by endometriotic epithelial and stromal cells, but not normal ovarian stroma ( 231 ). As CXCL16 facilitates T cell migration in other contexts, such as psoriasis ( 232 ) and RA ( 233 ), via its receptor CXCR6, this chemokine may play a similar role in EM. Authors also found that CXCL16 induced IL-8 (CXCL8) production in ESCs in vitro ( 231 ). IL-8 is well-known to be involved in EM-related inflammation and fibrosis ( 234 ), and thus, CXCL16 may further exacerbate the lesion milieu via IL-8. Moreover, others demonstrate that CXCL16/CXCR6 mediates the recruitment of pathogenic Th17 cells and IL-17A+ γδ T cells into atherosclerotic lesions ( 235 ). Thus, CXCL16/CXCR6 may also facilitate T cell migration in EM, however, further research is required to examine these chemokines specifically in this disease context. Th17 cells express CCR6 in EM tissues ( 236 ) and PF ( 31 ). Thus, CCL20 (CCR6 ligand), which is expressed in endometriotic epithelial and stromal cells, can cause the selective migration of Th17 cells in EM via CCR6 ( 236 ). The frequency of CCR6+ Th17 cells are also significantly increased in advanced EM compared to mild EM ( 31 ), suggesting increased susceptibility to CCL20-mediated migration to the lesion microenvironment in more advance cases of disease, whereby Th17 cells may further exacerbate key hallmark features of EM. CCL20 is also known to stimulate the migration of Treg cells in EM ( 237 ). Indeed, when CCL20 is neutralized, this chemotactic activity is inhibited ( 237 ). Moreover, as detailed earlier, a study by Wang et al. depicts that ESCs co-cultured with monocytes produce high levels of CCL17 and CCL22 and that these chemokines cause the recruitment of Treg cells ( 125 ). Thus, while various factors may be at play, these chemokines may be considered as therapeutic targets to regulate Th17 (CCL20) and Treg cell (CCL17/20/22) migration to the EM lesion microenvironment.