PI3?? Kinase and IFN-? pathways, identified in an unbiased kinase drug screen, are key mediators of cytokine release syndrome
Chimeric Antigen Receptor T (CAR-T) cell therapy can induce cytokine release syndrome (CRS), which ranges clinically from mild to life-threatening, and is universally characterized by elevation of multiple cytokines, including IL-6. To clarify mechanisms underlying CRS and identify potential new therapeutic agents, we screened a protein kinase inhibitor library using an in vitro  model system where CART19 cells were co-cultured with CD19+ tumor target cells and cytokine-primed myeloid cells (pMC), and secreted IL-6 levels serving as the CRS readout. We identified duvelisib, a dual PI3Kdg inhibitor, which blocked pMC IL-6 secretion in in vitro and in vivo  without negative impact on CART19 cell function. Immunophenotyping showed increased activation markers on CART19 cells CD137+(4-1BB); CD279+ (PD-1) and pMC cells (CD83+CD86+) when cultured with tumor target cells compared to cultures without target cells; these activation phenotypes were reduced in the presence of duvelisib. Single-cell RNA sequencing analyses identified four transcriptional clusters of pMCs. Among these, the activated pMC1 subset was the primary source of IL-6 expression, which was effectively reduced by duvelisib and by ruxolitinib, a JAK1/2 kinase inhibitor known to block CRS. Transcriptionally, IFNG expression in CART19 cells was significantly downregulated in the presence of duvelisib and ruxolitinib, and cell-cell communication between CART19 cells and pMCs suggests different mechanisms of action for the two inhibitors. Finally, pMC IL-6 production was significantly reduced by CRISPR/Cas9 deletion of IFNG in CART19 cells, the IFN-g receptor in pMC, or treatment with duvelisib which blocks both IFN-g production by CART19 and IFN-g receptor signaling. Our studies suggest that targeting PI3dg and IFN-g signaling pathways reduce CRS without altering CAR-T cell function. Overall design: 1x106 CART19, RamosCG, and primed myeloid cells (pMC), were co-cultured for 24hrs with DMSO control or 1000nM duvelisib or 1000nM ruxolitinib. ~50,000 cells of each experimental groups were subjected to scRNA sequencing (3' V3.1 – Dual Index Kit) from 10XGenomics at the Genome Technology Access Center / McDonnell Genome Institute (GTAC@MGI).