Enumeration, Phenotyping, and Clinical Associations of Tissue‐Resident T Cells in the Ecto‐ and Endocervix of Women Attending a Colposcopy Clinic

ABSTRACT Problem Tissue‐resident memory (T RM ) cells represent important immune sentinels that mount rapid recall responses to pathogens and cancers. However, there are limited data in humans on genital tract T RM collected by clinically feasible sampling methods, limiting a full understanding of their role in immunity and clinical disease. Method of Study We used flow cytometry and single cell RNA sequencing (scRNAseq) to characterize T cells isolated from ectocervical biopsies and endocervical cytobrushes collected from women attending a colposcopy clinic in Winnipeg, Canada. Results The ectocervix generally contained a higher frequency and abundance of immune cells and T‐cells compared to the endocervix. CD4+ and CD8+ T RM were more approximately 5‐times more frequent and abundant in the ecto‐ compared to endocervix, even after accounting for higher T‐cell recovery from the ectocervix. Phenotypically, CD4+ T RM showed higher Th17‐ and comparable regulatory‐associated marker expression compared to non‐T RM in both the ecto‐ and endocervix. Cervical dysplasia and ectropion were both associated with several immune cell differences in the ecto‐ and endocervix including lower CD4+ T RM. Single‐cell RNAseq analyses confirmed broad CD69 and core T RM ‐related gene expression and captured several heterogeneous CD4+ and CD8+ T RM subsets with diverse gene expression and pathways associated with host immunity, homeostasis, and nonimmune cell interactions. Conclusions Our data suggest that T RM are more abundant in ecto‐ versus endocervical samples, which may reflect differences in commonly used sampling methods. Location and heterogeneous expression profiles underscore the need to better understand their role in microbial interactions, inflammation, and genital infection susceptibility in women.