The female cervicovaginal tract (CVT) is the site of entry for many viral infections, especially sexually transmitted infections (STIs) such as HIV or herpes simplex virus (HSV). Although the CVT functions as a barrier tissue and requires prompt anti-pathogen responses, it must also facilitate immune tolerance to resident microbiota as well as fetal and partner antigens. Given these opposing roles, the local immune environment within the CVT must balance dichotomous immune responses depending on circumstances in order to eliminate pathogens while preventing collateral tissue damage. Within mucosal barrier non-lymphoid compartments, tissue resident memory T cells (Trm) are a subset of non-circulating T cells that have been shown to be important for protection from and control of viral pathogens. Contrarily, an influx of inflammatory immune cells in response to one infection can increase the risk of another infection as the infiltrates may cause epithelial barrier damage or provide additional targets for viral infection. However, it is unknown which subset of immune cells is responsible for chronic inflammation in response to infection in the CVT. To investigate the inflammatory role of CVT Trm in HIV infection, Dr. Laura Pattacini (formerly of the Lund lab, Vaccine and Infectious Disease Division) conducted a study to define CD8+ Trm in the CVT, and recently published this work in Mucosal Immunology.
This research “started with the idea of characterizing memory T cells that express the residency markers [activation marker] CD69 and [integrin] CD103 in the cervico-vaginal tract.” Pattacini said, as Trm typically express increased amounts of these molecules that promote retention in the tissue. The authors used flow cytometry to phenotype T cells within blood, vaginal, endocervical, and ectocervical samples from healthy donors. As expected, blood and tissue T cells expressed distinct cell surface marker profiles. They found tissue-derived T cells express double-negative (DN), double positive (DP) and single-positive (SP) combinations of CD69 and CD103 in every compartment of the CVT.
To further compare these CVT CD8+ subsets to each other as well as to circulating CD8+ T cells, they performed bulk RNA-sequencing on each of the three CD8+ subsets (CD69 SP, CD69 CD103 DN, CD69 CD103 DP) in both blood and tissue. These results corroborated the flow cytometry data, demonstrating that these populations define transcriptionally distinct CD8+ subsets in the CVT, but that all CVT subsets were also transcriptionally distinct from blood SP and DN subsets. The CVT contained a minor but significant fraction of DN CD8+ T cells, prompting the authors to ask if these cells were simply leftover from T cells that were traveling in blood through CVT vasculature or instead a distinct, undescribed population. They used their RNA sequencing data to make gene expression comparisons between CVT double negative populations and either blood or tissue CD8+ T cells and found that double negative CVT CD8+ differed from blood CD8+ T cells, suggesting that they were not derived from blood contamination and were instead a distinct tissue population. Pathways analysis software identified an enrichment of pro-inflammatory as well as cell migration and localization pathways. Specifically, the DN population had increased expression of molecules that reduce cell migration out of the tissue, and using a chemotaxis assay, confirmed that tissue DN CD8+ T cells had a reduced potential to migrate out of the tissue. These results demonstrated that a transcriptionally and phenotypically distinct DN CD8+ population exists in the CVT and potentially explained how this subset could remain in the CVT despite lacking classical tissue-retention molecules.
After discovering that this unexpected DN CD8+ population had reduced migration capacity, the authors questioned which signals would initially bring these cells to the CVT. They measured soluble chemokines and cytokines from cervicovaginal fluid samples from healthy women and found a correlation between the abundance of DN CD8+ T cells and chemokine CCL3, suggesting a migration signal that may recruit this population to the tissue. Together, their analysis of DN CD8+ in the CVT revealed that a distinct subset with increased inflammatory potential and decreased tissue egress ability are maintained in healthy CVT, which they termed inflammatory mucosal T cells (Tim).
Having identified Tim as an unexpected and potentially inflammatory subset of CD8+ T cells in the CVT, they then asked if Tim were increased in women with chronic mucosal infections. They compared the phenotype of CD8+ T cells between women with and without HIV, some of whom from both groups were also positive for HSV or bacterial vaginosis (BV). They found that Tim were increased in women who were HIV-positive, and that Tim frequency was further stratified by BV but not HSV status. In total, their results suggest that a population of CD8+ T cells lacking classical Trm markers reside in the CVT, “expands during chronic infection and might have a role in perpetuating inflammation and creating a favorable environment for further infections and neoplastic transformation.” Pattacini explained. Although their work suggests that Tim possess a reduced capacity for tissue egress, further experiments are needed to functionally demonstrate this putative reduction in migration. It also remains unclear if Tim exist in other non-lymphoid barrier tissues and how these cells may function in anti-pathogen responses. Importantly, CVT Tim require further investigation to define their role along the axis of detrimental local inflammation and beneficial pathogen control.
This work was supported by the National Institutes of Health.
UW/Fred Hutch Cancer Consortium members Martin Prlic and Olliver Hyrien contributed to this work.
Pattacini L, Davis AW, Czartoski J, Mair F, Oresness S, Hughes SM, Hyrien O, Kentz GM, Kirby AC, Fialkow MF, Hladik F, Prlic M, Lund JM. 2019. A pro-inflammatory CD8+ T-cell subset patrols the cervicovaginal tract. Mucosal Immunology. 12:1118-1129; https://doi.org/10.1038/s41385-019-0186-9.