Abstract 4800: Modulation of Th1/Th17 axis through IFNγ-blockade alleviates Anti-CD19 CAR-T associated toxicities in a pre-clinical immune competent model

Abstract CD19-targeted chimeric antigen receptor T cell (CAR-T) therapy for B cell malignancies has led to tremendous improvement in patient survival, yet a subset of patients experience high-grade cytokine release syndrome (CRS) and prolonged cytopenias leading to poor clinical outcomes. Owing to IL2Rα’s role in moderating inflammation and supporting Treg survival, we used IL2Rα knockout (KO) mice to emulate the inflammatory conditions that drive CAR-T associated CRS and neutropenia (NP). KO mice injected with a B cell leukemia cell line, followed by CAR-T show elevated levels of IL6 (p=0.046), IFNγ (p=0.006), and TNFα (p=0.048) in serum compared to WT, which coincided with increase (p<0.0001) in CRS causing M1 macrophages (Mϕ) in bone marrow (BM). Onset of CRS was accompanied by a reduction in peripheral blood neutrophils (NEUT) (p=0.004) due to a disruption of BM NEUT homeostasis characterized by an increase in apoptotic NEUTs (p=0.0002), and a decrease in proliferative (p=0.0052) and mature (p<0.0001) NEUTs. Due to the established role of IFNγ in monopoiesis (Mϕ production) over granulopoiesis (NEUTs production), we treated KO mice with IFNγ-/-CD19-CART (IFNγKO-CART) that resulted in restoration of CRS-NP symptoms. Interestingly, IFNγ-blockade restored levels of NEUT regulating cytokine IL-17. In a Th0 to Th1/Th17 in vitro differentiation assay, we noted an increase (p=0.008) in Th17 cells and a decrease (p<0.0001) in Th1 cells in the presence of IFNγKO-CART compared to CAR-T. KO mice treated with CAR-T and adoptively transferred Th17 cells displayed a significant recovery of CRS-NP symptoms with improved survival (p=0.002). Conversely, KO mice treated with IFNγ KO-CART and adoptively transferred Th1 cells reversed the benefit of IFNγ blockade on CRS-NP by triggering polarization of M1 Mϕ (p=0.048) that significantly reduced their survival (p<0.0001). Serum analysis of 27 patients diagnosed with hematologic malignancies and treated with standard of care CAR-T products showed that CRS-NP patients had a decline in IL-17A levels post CAR-T therapy (p=0.049), compared to non-CRS-NP patients. Furthermore, CRS-NP patients had higher baseline levels of IL-17A, typical of KO mice, putatively serving as a biomarker for CRS (Area under the curve of receiver operator characteristic curve, ROC-AUC=0.773) and prolonged NP (ROC-AUC=0.821). Our study shows that Th1-Th17 imbalance under inflammatory conditions triggered by CAR-T cells drive CRS-NP in an IFNγ-dependent manner. Finding ways to fine-tune IFNγ release during CAR-T therapy will benefit B-ALL patients by reducing their chances of developing toxicities without compromising their anti-tumor efficacy. To this end, single-cell sequencing analyses are being conducted of BM mononuclear cells of KO mice treated with CAR-T, IFNγKO-CART, CAR-T+Th17, and IFNγKO-CART+Th1 to identify sources of IFNγ production. Citation Format: Payal Goala, Marco Davila, Yongliang Zhang, Nolan Betty, Shannon McSain, Sailer Cooper, Muhammad Junaid Tariq, Showkat Hamid. Modulation of Th1/Th17 axis through IFNγ-blockade alleviates Anti-CD19 CAR-T associated toxicities in a pre-clinical immune competent model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4800.