Type I Interferon Impairs the Function of CAR-T Cells By Disrupting Immune Synapse Assembly
Authors: Huang Meijuan , Zhao Lei , Cheng Yuhang , Huang Liang , Zhou Jianfeng , Li Dengju , Wang Jue , Cao Yang 
Published: December 2024

Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated promise in treating haematological malignancies, but resistance and limited durable responses are common, potentially due to T-cell intrinsic fitness. To better understand these differences in efficacy, we conducted STRT-seq sequencing on pre-infusion CAR-T products from B-ALL and DLBCL patients receiving CAR19/22 T-cell cocktail therapy. Patients were classified as responders and non-responders based on clinical response, and subsequent analysis revealed differences in T-cell transcriptional profiles between the two groups. Notably, non-responders exhibited an enriched type I IFN signaling pathway. A previous study has shown that upregulated type I IFN signaling in baseline CAR-T cell products is associated with CAR-T cell dysfunction and failure in the clinic (Gregory M. Chen, et al. Cancer Discovery, 2021). Therefore, we hypothesized that type I IFN may be a key regulator of CAR-T cell dysfunction.

To evaluate the impact of type I IFN on CAR-T cells, we conducted in vitro experiments to assess the phenotypic and functional changes in CD19 CAR-T cells after exposure to recombinant human IFN-β. Following 48 hours of IFN-β treatment, CAR-T cells co-cultured with Nalm6 cells showed significantly reduced cytotoxicity while secreting increased effector cytokines such as IL-2, IFN-γ, Granzyme B and TNF-α compared to controls. However, pretreated CAR-T cell showed no deficiencies in proliferation or degranulation. Subsequently, we examined the activity of pretreated or control CD19 CAR-T cells in a xenograft mouse model with NALM6-Luc cells. In vivo, IFN-β pretreated CAR-T cell displayed significantly weaker anti-tumour effects than the control group, resulting in a survival disadvantage for the mice. These findings suggested a potential role for type I IFN in potentiating CAR T cell dysfunction. To gain insight into the molecular mechanisms involved, bulk RNA-seq analyses were performed on unstimulated and antigen-stimulated CAR-T cells with or without IFN-β exposure. The analysis revealed increased expression of genes associated with response to type I interferon and negative regulation of T cell mediated immunity in both cell states, indicating ongoing type I IFN signaling. Particularly in antigen-stimulated CAR-T cells, IFN-β treatment suppressed multiple pathways related to cell-cell adhesion, regulation of Rho protein signaling, and modification of postsynaptic actin cytoskeleton. Confocal imaging further revealed that IFN-β treatment impaired the binding of CAR-T cells to tumor cells, inhibited immune synapse (IS) assembly and function, as shown by significantly impaired F-actin polymerization, and defective polarization of centrosomes and lytic granule to the IS. Mechanistically, type I IFN targets T-cell Rho-GTPase activation signaling-key regulators of actin dynamics at the T-cell synapse. IFN-β treatment resulted in a decrease in the level of activated RhoA and Rac1 in CAR-T cell stimulated with CD19 protein. Conversely, these deficiencies were reversed by CAR-T cells in which IFNAR1 had been knocked out.

Furthermore, we investigated the potential of utilizing the CRISPR/Cas9 strategy to delete IFNAR1 in order to enhance CAR-T cell potency. Our results indicate that human IFNAR1-edited CAR-T cells exhibited significantly resistant to immunosuppressive effects of type I IFN in vitro and in vivo, without any detrimental impact on the phenotype or function of CAR-T cells. Additionally, preclinical animal models showed that IFNAR1-edited CAR-T cells were well tolerated and safe.

In conclusion, our study reveals a novel role of type I IFN in modulating CAR-T cell dysfunction by disrupting the stabilization of IS formation. We propose the use of IFNAR1-edited CAR-T cells as a potential approach to enhance CAR-T cell function.