Bone marrow Single cell sequencing data of CAR-T treated IL-2Ra -/- mice

Despite breakthrough research and success in CAR-T therapy in heme
malignancies, adverse events caused during CAR-T infusion remain a
challenge. Among the various toxicities, the co-occurrence of cytokine
release syndrome (CRS) along with neutropenia is the leading cause of
severe life-threatening infections, increased hospitalizations, and the
leading cause of non-relapse mortality. To this end, we developed an IL-2R
alpha knockout model (where exons 2 and 3 that bind to IL-2 are replaced
by a neomycin resistance gene) that showed the recapitulation of CRS and
neutropenia seen in the clinic. This novel pre-clinical model helped us
identify IFNg (one of the CRS-associated cytokines) to drive this
co-occurrence. Through adoptive transfer studies, we show that excess IFNg
released during CAR-T expansion triggers the inflammatory cascade that
fuels the etiology of both CRS and neutropenia by triggering Th1/Th17
imbalance. IFNg is a Th1 cytokine that inhibits Th17 differentiation,
which is crucial for neutrophil regulation. To corroborate the impact of
IFNg on CRS-neutropenia, we processed bone marrow samples of tumor-bearing
IL-2Rα -/- mice treated with CAR-T (Group 1) and IFNγ -/- CAR-T (Group 2)
in the presence or absence of Th17 (Group 4) and Th1 cells (Group 5). The
samples were processed for sequencing at week 4 post CAR-T infusion to
study the mechanistic underpinnings of the impact of IFNg blockade on
neutrophils and macrophages (M1 macrophages are known to drive CRS) that
are involved in CRS and neutropenia co-occurrence during CAR-T therapy.