Unmasking Endometriosis: T-Cell Receptor Signatures as Roadmaps to Immunotherapy

INTRODUCTION: Endometriosis involves pathologic transplantation of endometrial-like tissue from the internal lining of the uterus to a location outside of the uterus. Presently, treatment regimens center around symptom management and sequelae, such as pelvic pain and infertility, respectively. Current treatment, including analgesics, hormonal suppression, and surgical excision, primarily target symptom relief and recurrent prevention but rarely provide durable resolution. Increasing evidence suggests that immune dysregulation plays a central role in disease pathogenesis. While prior studies have identified generalized immune alterations, there remains a critical gap in understanding antigen-specific T-cell receptor (TCR) repertoires in endometriosis. Mapping these antigen-specific TCRs could provide unique insights into disease mechanisms and serve as the foundation for novel, targeted immunotherapeutic strategies. OBJECTIVE: The purpose of this pilot study is to establish a fast, efficient, and economical workflow for cloning, sequencing, synthesizing, and testing antigen-specific TCR. Additionally, we aim to generate targeted TCR sequences from endometriotic tissues and peripheral blood in efforts to determine dominant TCR biomarkers that may serve in the development of immunotherapies. METHODS: English-speaking, non-pregnant women between 18 and 45 years old who presented to a single, tertiary-care center were broken down into the following two groups: 1) patients with ovarian endometriomas or deep pelvic endometriosis and 2) controls. Pelvic peritoneal tissue and blood samples were collected from individual women with ovarian endometriomas or deep pelvic endometriosis undergoing laparoscopic or robotic treatment of endometriosis and from control patients without endometriosis undergoing laparoscopic or robotic hysterectomy. Women who were on immunosuppressants were excluded from this study. Endometriosis tissues and non-disease (control) tissue had total RNA extracted, which was used for targeted TCR cDNA synthesis and amplification utilizing the Lightning TCR protocol, with each sample being run in triplicate. The resulting amplicons were barcoded and sequenced. The specimen reads were individually aligned to germline V, D, and J gene sequences from the IMGT database for identification. Error corrected sequences were then used to determine the complementarity-determining region 3 (CDR3) sequence for the TCRs. Over a 24-hour sequencing run, ∼798K reads were generated, of which ∼180K of the highest quality barcoded reads were used for TCR analysis. RESULTS: There were ∼4x–13x more TCR reads from endometriosis tissues (4857, 1990, and 7837 average reads per sample) as compared to the control tissue (584 average reads per sample). Endometriosis tissues also showed ∼5x increase in the number of unique V beta – J beta gene recombination’s relative to the control tissue (155 unique V-J recombination on average in diseased tissue as compared to 29 unique recombination in control tissue). Furthermore, one deep endometriotic sample shows an enriched, unique sequence at ∼13% of total TCR beta reads, suggesting a clonal antigen-specific T cell response in the diseased tissue. CONCLUSIONS: Endometriosis tissues may have higher T cell infiltration due to observed increases in T cell diversity and TCR RNA. Lightning TCR is not only feasible for rapid sequencing of tumor-infiltrating lymphocytes in tissues, but also at helping identify unique biomarkers in endometrial tissues for potential immunotherapies to be developed.