Evidence that tissue resident human enthesis γδ T-cells can produce IL-17A independently of IL-23R transcript expression

Objectives: Murine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of Î 3Î T-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated Î 3Î T-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis. Methods: Human spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal Î 3Î T-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from Î 3Î T-cell subsets and analysed by qPCR. Entheseal Î 3Î T-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR. Results: Entheseal Î 3Î T-cells were confirmed immunohistochemically with VÎ 1 and VÎ 2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal VÎ 2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the VÎ 1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both VÎ 1 and VÎ 2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the VÎ 1 subset following IL-23 stimulation. Conclusion: Spinal entheseal VÎ 1 and VÎ 2 subsets are tissue resident cells with inducible IL-17A production with evidence that the VÎ 1 subset does so independently of IL-23R expression.