Phenotypic, functional and molecular characterisation of human in vitro-generated IL-17A+ CD8+ T-cells

IL-17A+ CD8+ T-cells, often referred to as Tc17 cells, have been identified at sites of inflammation in several immune-mediated inflammatory diseases including psoriasis and spondyloarthritis. Whilst much of our understanding of IL-17A+ CD8+ T-cells has been discerned from murine studies, human IL-17A+ CD8+ T-cells remain less-well characterised. We optimised an in vitro polarisation protocol to expand human IL-17A+ CD8+ T-cells from PBMC or bulk CD8+ T-cell populations for phenotypic and functional assessment. We show that T-cell activation in the presence of IL-1β and IL-23 significantly increased the frequencies of IL-17A+ CD8+ T-cells, which was not further enhanced by the addition of IL-6, IL-2 or anti-IFNγ mAb. In vitro-generated IL-17A+ CD8+ T-cells from healthy donors displayed a distinct type-17 profile compared with IL-17A- CD8+ T-cells, as defined by transcriptional signature (IL17A, IL17F, RORC, RORA, MAF, IL23R, CCR6, CXCR6); high surface expression of CCR6 and CD161; and polyfunctional production of IL-17A, IL-17F, IL-22, IFNγ, TNFα and GM-CSF. A significant proportion of in vitro-induced IL-17A+ CD8+ T-cells expressed TCRVα7.2 and bound MR1 tetramers, indicative of a MAIT CD8+ T-cell population. Using an IL-17A secretion assay, we demonstrate that the in vitro-generated IL-17A+ CD8+ T-cells were biologically functional and induced pro-inflammatory IL-6 and IL-8 production by synovial fibroblasts from patients with psoriatic arthritis. Collectively, we report an in vitro culture system to expand IL-17A+ CD8+ T-cells and further characterise their phenotype, transcriptional regulation and functional relevance to human health and disease. ### Competing Interest Statement The authors have declared no competing interest.