CAR T cell-driven induction of iNOS in tumor associated macrophages promotes CAR T cell resistance in B cell lymphoma

Abstract Chimeric antigen receptor (CAR) T cell therapies have revolutionized B cell malignancy treatment, but subsets of patients with large B cell lymphoma (LBCL) experience primary resistance or relapse after CAR T cell treatment. To uncover tumor microenvironment (TME)-induced resistance mechanisms, we performed bulk RNA sequencing on pre-treatment tumor biopsies from LBCL patients receiving Axicabtagene ciloleucel (axi-cel). Compared to patients who remained in remission for at least 6 months after treatment (durable responders; n=18), those who relapsed within 6 months (non-durable responders; n=26) exhibited enriched expression of M2 macrophage-associated gene sets and increased frequency of immunoregulatory (M2-like) macrophages (p=0.0017). This was associated with worse progression-free survival (PFS; p=0.0183), with a median PFS of 3.17 months in patients with high M2-like macrophages versus not reached PFS in those with low M2-like macrophages. Using a murine in vitro model, we found that M2-polarized macrophages (generated with IL-4/IL-10; “imMac”) inhibited CD19-targeted CAR T cell cytotoxicity against Eμ-myc B cell lymphoma cells (p=0.0109). Macrophage expression of inducible nitric oxide synthase (iNOS) increased dramatically from 1.0% to 92.7% in coculture with CAR T cells (p<0.0001). This effect required CAR T-derived interferon-gamma (IFN-γ), as iNOS expression on macrophages was reduced to 0.9% when cocultured with Ifng-/- CAR T cells (p< 0.0001). CAR T cell anti-tumor function was restored with genetic deletion of iNOS in imMac, pharmacological inhibition of iNOS, or blockade of IFNγ (all p<0.001). To understand the mechanistic underpinnings of CAR T cell inhibition by iNOS-expressing macrophages, we performed proteomic and metabolomic analyses on CAR T cells cocultured with imMac, unpolarized macrophages, or no macrophages. CAR T cells cultured with imMac upregulated the p53 pathway, mediating apoptosis and cell cycle arrest in CAR T cells, while downregulating the MYC pathway involved in ribosome biogenesis and protein synthesis. Furthermore, when cocultured with imMac, CAR T cell metabolism was compromised by depletion of glycolytic intermediates and rewiring of the TCA cycle. Extracellular flux analysis corroborated the attenuation of glycolytic and oxidative metabolic activities in CAR T cells cocultured with imMac. Proteomic and metabolomic changes were reversed with iNOS inhibition. To understand the role of CART-derived IFNγ and iNOS+ macrophages on CAR T cell efficacy in vivo, Eμ-myc B cell lymphoma tumors were implanted intraperitoneally into Rag1-/- mice, which lack B and T cells, to reduce the effects of secondary sources of IFNγ. Treatment with wildtype CAR T cells increased the frequency of iNOS+ macrophages to 4.1% compared to 0.03% with Ifng-/- CAR T cells (p=0.0100), and iNOS inhibition enhanced overall survival with wildtype CAR T cell treatment from 29 to 38 days (p<0.0411). Notably, elevated levels of iNOS+CD14+ monocytes were observed in leukaphereses of LBCL patients with non-durable responses to CAR T cell therapy (n=32, iNOS+=91.6%) compared to those with durable responses (n=19, iNOS+=85.1%; p=0.0207). These findings support blocking IFN-γ secretion from CAR T cells to mitigate iNOS in tumor-associated macrophages to improve durable responses to CAR T cell therapy in patients with hematologic malignancies.