IL-6 is a potent pro-inflammatory signaling molecule in our immune system, with important roles in diseases such as rheumatoid arthritis and cytokine storm. Under less extreme conditions, IL-6 is a key regulator of the balance between T cell subtypes in the immune system, promoting CD4 T cell differentiation to Th17 cells, and inhibiting Treg differentiation. These, and other, diverse effects of IL-6 signaling can be attributed to the ways that IL-6 signals propagate through downstream signaling pathways, including the JAK/STAT pathway, to alter T cell function.
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To better understand IL-6/STAT3 pathway in human T cells, we isolated T cells from PBMCs, placed them in a 96-well plate, then treated them with a panel of 88 JAK/STAT pathway inibitors for 1 hour. After drug treatment, we stimulated the T cels with IL-6. We then profiled the response to IL-6 using two assays: a phospo-STAT flow cytometry assay, and 10x Genomics Flex v2 scRNA-seq. For scRNA-seq, each sample in the 96-well plate was labeled with a distinct Barcode oligo, and cells from all 96 wells were then pooled and processed simultaneously on 4 wells of a GEM-X chip. This resulted in a dataset of ~4.2 million single cells with a panel of ~1900 genes, and paired flow cytometry data which showcases our high-throughput, high-plexity screening capabilities as well as the capabilities of the new 10x Genomics Flex v2 GEM-X chemistry.
Visualize phospho-flow results, differential expression, and gene set enrichment for comparisons between treatments and unstimulated controls.
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