IFNgamma-driven skewing towards Th1 over Th17 differentiation underlies CRS and neutropenia in CAR-T therapy

CAR-T (Chimeric antigen receptor T cell) therapy has led to significant improvements in patient survival. However, a subset of patients experience high-grade toxicities, including cytokine release syndrome (CRS) and immune cell-associated hematological toxicity (ICAHT). We utilized IL-2Ralpha knockout mice to model toxicities with elevated levels of IL-6, IFNgamma, and TNFalpha and increased M1-like macrophages. Onset of CRS was accompanied by a reduction in peripheral blood neutrophils due to disruption of bone marrow neutrophil homeostasis characterized by an increase in apoptotic neutrophils and a decrease in proliferative and mature neutrophils. Both non-tumor-bearing and Emu-ALL tumor-bearing mice recapitulated the co-occurrence of CRS and neutropenia. IFNgamma-blockade alleviated CRS and neutropenia without affecting CAR-T efficacy. Mechanistically, a Th1-Th17 imbalance was observed to drive co-occurrence of CRS and neutropenia in an IFNgamma-dependent manner leading to decreased IL-17A and G-CSF, neutrophil production, and neutrophil survival. In patients, we observed an increase in the IFNgamma-to-IL-17A ratio in the peripheral blood during high-grade CRS and neutropenia. We have uncovered a biological basis for ICAHT and provide support for the use of IFNgamma-blockade to reduce both CRS and neutropenia. We performed single cell sequencing to compare differences in Neutrophils and Macrophages from Bone marrow derived mononuclear cells isolated at week 4 from IL-2Ralpha knockout mice treated with wild type CD1928zeta CAR-T(Group 1, n=2), IFNgamma knockout CAR-T(Group 2, n=2) and wild type CAR-T+Th17(Group 4, n=2).