We examine the immunomodulatory impact of a mechanism-of-action drug library on human PBMC responses to both lipopolysaccharide (LPS) and interferon beta (IFN-b). Adapting our computational framework to distinguish the effects of different stimulants and drugs, as well as their interactions, on cellular features, we uncover how 90 different compounds influence LPS & IFN-b responses across several PBMC subsets in parallel through an analysis of over 111,000 single-cell transcriptomes, validating select observations. 

We tested the effects of 90 chemical compounds with a wide-range of known MOAs curated by the Broad Drug Repurposing Hub (https://github.com/jump-cellpainting/JUMP-MOA) on immune responses among PBMCs. To execute these compressed screens, we generated identical pool sets (R = 3 replicates/drug, P = 6 drugs/well) in three 96-well plates with DMSO controls, added fresh PBMCs, incubated overnight, and then stimulated with IFNb or LPS for 4 hours the following day while leaving one plate unstimulated. Following an evaluation of cell viability, each well was hashed using oligo-tagged antibodies and scRNA-seq was performed following 10x Feature Barcoding Technology and Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Surface Protein protocols.