Immunological, genetic and therapeutic studies have implicated T lymphocytes in the pathogenesis of Spondyloarthritis (SpA). GM-CSF is emerging as a cytokine that marks out pathogenic T lymphocytes. Inhibition of this cytokine pathway in the SKG mouse model of SpA leads to improvement of joint scores.Methods
Matched blood (PBMC) and synovial fluid (SFMC) from patients with SpA was studied ex-vivo using a 40 colour surface and intracellular CyTOF panel. CyTOF findings were validated by flow cytometry in a cohort of patients with SpA, rheumatoid arthritis (RA) controls and healthy donors. CyTOF data was analysed by multi-dimensional viSNE mapping in order to understand the heterogeneity of the synovial fluid inflammatory response. Novel triple cytokine capture was used to purify GM-CSF producing lymphocytes to high purity and RNA sequencing used to interrogate their transcriptional profile.Results
CyTOF revealed ex-vivo GM-CSF production from multiple lymphoid cell lineages, with CD4 and CD8 cells the main producers in PBMC and SFMC. GM-CSF production overlaps with multiple other cytokines including IL-17A, IFN- and TNF-. 43% (SEM 3.98) of all GM-CSF producing cells in SpA PBMC were CD4 cells. The percentage of CD4 cells producing GM-CSF was significantly increased in SpA PBMC ex-vivo compared to healthy controls (mean 7.73% vs 4.96% n=38, p<0.005). The mean percentage of GM-CSF-positive CD4 cells from ex-vivo SFMCs was 32.2%, and was significantly higher compared to matched PBMC (n=5, p=0.005). RNA sequencing of capture-sorted GM-CSF-positive human CD4 cells showed a unique transcriptional profile and validated key genes previously shown to drive pathogenicity in mouse models of type-17 immune-mediated disease.Conclusions
The synovial fluid of patients with SpA shows an expanded GM-CSF signature. This corresponds with an expansion of GM-CSF producing T lymphocytes in the peripheral blood of patients with SpA compared to controls. Human GM-CSF-positive CD4 cells have a unique ‘pathogenic’ transcriptional profile.