Profiling the peripheral immune response to ex vivo TNF stimulation in untreated juvenile idiopathic arthritis using single cell RNA sequencing

Kathleen J. Imbach, Nicole J. Treadway, Vaishali Prahalad, Astrid Kosters, Dalia Arafat, Meixue Duan, Talia Gergely, Lori A. Ponder, Shanmuganathan Chandrakasan, Eliver E. B. Ghosn, Sampath Prahalad, Greg Gibson

Juvenile Idiopathic Arthritis (JIA) is an autoimmune disease with a heterogenous clinical presentation and unpredictable response to available therapies. This personalized transcriptomics study sought proof-of-concept for single-cell RNA sequencing to characterize patient-specific immune profiles. Whole blood samples from six untreated children, newly diagnosed with JIA, and two healthy controls were cultured for 24 h with or without ex vivo TNF stimulation and subjected to scRNAseq to examine cellular populations and transcript expression in PBMCs. A novel analytical pipeline, scPool, was developed wherein cells are first pooled into pseudocells prior to expression analysis, facilitating variance partitioning of the effects of TNF stimulus, JIA disease status, and individual donor. Seventeen robust immune cell-types were identified, the abundance of which was significantly affected by TNF stimulus, which resulted in notable elevation of memory CD8 + T-cells and NK56 cells, but down-regulation of naïve B-cell proportions. Memory CD8 + and CD4 + T-cells were also both reduced in the JIA cases relative to two controls. Significant differential expression responses to TNF stimulus were also characterized, with monocytes showing more transcriptional shifts than T-lymphocyte subsets, while the B-cell response was more limited. We also show that donor variability exceeds the small degree of possible intrinsic differentiation between JIA and control profiles. An incidental finding of interest was association of HLA-DQA2 and HLA-DRB5 expression with JIA status. These results support the development of personalized immune-profiling combined with ex-vivo immune stimulation for evaluation of patient-specific modes of immune cell activity in autoimmune rheumatic disease.