Systematic Single Cell Pathway Analysis (SCPA) reveals novel pathways engaged during early T cell activation

Pathway analysis is a key analytical stage in the interpretation of omics data, providing a powerful method for detecting alterations in cellular processes. We recently developed a sensitive and distribution-free statistical framework for multisample distribution testing, which we implement here in the open-source R package Single Cell Pathway Analysis (SCPA). We demonstrate the effectiveness of SCPA over commonly used methods, generate a scRNA-seq T cell dataset, and characterize pathway activity over early cellular activation. This reveals regulatory pathways in T cells, including an intrinsic type I interferon system regulating T cell survival and a reliance on arachidonic acid metabolism throughout T cell activation. A systems-level characterization of pathway activity in T cells across multiple tissues also identifies alpha defensin expression as a hallmark of bone marrow derived T cells. Overall, this work provides a widely applicable tool for single-cell pathway analysis and highlights unexpected regulatory mechanisms of T cells. Overall design: CD4+ and CD8+ T cells were isolated from the peripheral blood of healthy donors via negative selection using the EasySep kits (Stemcell). These populations were then further sorted into naïve and memory populations by FACS, generating four populations in total: CD45RA+CD4+ cells (naive CD4), CD45RA+CD8+ cells (naive CD8), CD45RO+CD4+ (memory CD4), and CD45RO+CD8+ (memory CD8). Each cell population was then either left unstimulated, or stimulated for 12 or 24 hours with anti-CD3 and anti-CD28 before scRNA-sequencing. Splenic CD4+ T cells from WT and Ifnar1-/- mice were stimulated with anti-CD3 and anti-CD28 (2ug/ml) for 24 hours then harvested for bulk RNA-sequencing