A closer look at T cells across the female reproductive tract

From the Prlic and Lund labs, Vaccine and Infectious Disease Division

Barrier tissues, such as oral mucosa, lung, and female reproductive tract (FRT), have a unique role in providing the first line of defense against pathogens. Therefore, mucosal tissues are home to distinct immune environments that are specialized to quickly respond to viral or bacterial exposure. A subset of memory T cells that reside in non-lymphoid peripheral tissues, called tissue resident memory T cells (TRM), are important for pathogen control at mucosal barriers.  These TRM cells rapidly respond during re-exposure to pathogens and are able to control infections. Although both CD4+ and CD8+ Trm are known to be necessary for barrier tissue protection, CD8+ Trm have been studied more extensively than CD4+ Trm, and the full extent of CD4+ Trm phenotype and function remains unexplored. Additionally, the CD4+ Trm compartment in the FRT—the site of many sexually transmitted infections—is further unclear. Drs. Amanda Woodward-Davis (Prlic Lab) and Sarah Vick (Lund Lab), along with colleagues from the Vaccine and Infectious Disease Division and UW/Fred Hutch Cancer Consortium, defined the CD4+ Trm compartment across different tissues within the human FRT in a recent Mucosal Immunology publication.

Although FRT generally describes the female genital barrier tract, the tissues within the FRT are quite distinct: the upper FRT (endocervix [EndoCx]) and lower FRT (ectocervix [EctoCx] and vaginal tract [VT]) each have specific physiologic functions and are comprised of differing mucosal surfaces. Therefore, the authors were first interested in characterizing the distribution of CD4+ Trm across FRT tissues. Using matched FRT tissue samples from healthy women undergoing elective hysterectomy or reconstructive surgery, the authors performed flow cytometry analysis on CD4+ T cells within the VT, EndoCx, and EctoCx, as well as donor-matched blood controls. CD69 and CD103 are T cell surface markers that are widely used to identify Trm. CD4+ T cells in all three FRT tissues –but not blood—expressed CD69 and CD103, suggesting that these cells are resident in the tissue and are not simply circulating T cells passing through. Interestingly, a larger fraction of VT compartment CD4+ T cells expressed CD69 and CD103, suggesting that the first point of pathogen entry requires elevated CD4+ Trm numbers.

Overview of the tissues and CD4+ T cells subsets within the female reproductive tract.
Overview of the tissues and CD4+ T cells subsets within the female reproductive tract. Figure provided by Sarah Vick.

Next, the team assessed the expression of other known Trm or barrier tissue T cell markers. Expression of PD-1, a context-dependent surface marker known to be expressed by Trm, was largely restricted to FRT CD4+ T cells and not blood, and PD-1 was increased specifically in the CD69+ CD103+ fraction, especially in VT and EctoCx, further suggesting that the CD4+ T cells within the FRT are likely true Trm. Additionally, CCR5, a chemokine receptor known to be stably maintained on Trm, was expressed by at least half of all CD4+ T cells within the three FRT tissues, especially on CD69+ CD103+ cells. Together, these findings further support that CD4+ Trm are important in barrier immunity, especially in the lower FRT.

Although the phenotypes of CD4+ T cells were largely conserved across FRT tissues, the authors’ earlier finding that a higher frequency of vaginal CD4+ expressed Trm markers prompted them to hypothesize that CD4+ T cells may have unique functions in the vagina. To interrogate, they sorted CD4+ T cells from blood, EndoCx, EctoCx, and VT into CD69+ CD103-, CD69- CD103+, CD103- CD69-, and CD69+ CD103+ fractions. They then assessed the transcriptional profiles through bulk RNA-sequencing of each population. Within the VT, the two fractions defined by CD69+ were largely similar, but had large numbers of genes expressed at significantly higher levels compared to CD69- fractions. One interesting finding was that CD69+ CD103+ cells highly expressed RORC—the transcription factor that defines a subset of T cells called Th17—along with other genes associated with IL-17 cytokine. IL-17, which is secreted by Th17 CD4+ T cells, is important for both anti-bacterial immunity as well as preserving barrier mucosa by promoting tight junctions. Compared to a previously published Th17 gene signature, CD69+ CD103+ cells had an enriched Th17 signature over CD69+ CD103- cells, suggesting that vaginal Trm are skewed towards a Th17 phenotype. To confirm if the transcript-based Th17 phenotype was maintained at the protein level, the authors next stimulated VT CD4+ T cells in ex vivo assays and measured IL-17 production. As predicted, VT T cells produced more IL-17 cytokine than blood CD4+ T cells, especially in the CD69+ CD103+ Trm subset, suggesting that IL-17-secreting CD4+ Trm may play a unique role in FRT immunity.

Finally, to compare the transcriptional profile of VT CD4+ T cells to those in the EctoCx and EndoCx, they compared the Th17 gene set across the cervical tissues and found that the Th17 phenotype was conserved across all FRT tissues. Likewise, the general transcriptional profile was shared across the CD4+ T cells from all three FRT tissues, suggesting that although general CD4+ Trm transcriptional profiles are shared across the tissues of the FRT, tissue localization drives changes in CD4+ T cell subset distribution, with a bias for more tissue resident, Th17-skewed cells in the vagina. “The finding that proportions are different, but phenotypic subsets have a shared function despite location adds important context for interventions against microbiome disruption, STDs and malignancies in the FRT,” explained Dr. Woodward-Davis. Going forward, more work is needed to assess how sex hormones such as progesterone, which is known to have anti-inflammatory effects on immune cells, are distributed across the FRT and how these signals may influence FRT tissue microenvironments.

Woodward Davis AS, Vick SC, Pattacini L, Voillet V, Hughes SM, Lentz GM, Kirby AC, Fialkow MF, Gottardo R, Hladik F, Lund JM, Prlic M. The human memory T cell compartment changes across tissues of the female reproductive tract. Mucosal Immunology. 2021. https://doi.org/10.1038/s41385-021-00406-6

UW/Fred Hutch Cancer Consortium members Florian Hladik, Raphael Gottardo, and Martin Prlic contributed to this work.

This work was supported by the National Institutes of Health and the Doug and Maggie Walker Fellowship.