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3407 Type I Interferon Impairs the Function of CAR-T Cells By Disrupting Immune Synapse Assembly

Program: Oral and Poster Abstracts
Session: 702. CAR-T Cell Therapies: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, ALL, Translational Research, Lymphomas, B Cell lymphoma, Diseases, Lymphoid Malignancies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Meijuan Huang, MD1*, Lei Zhao, MD, PhD2*, Yuhang Cheng2*, Liang Huang2, Jianfeng Zhou, MD, PhD2*, Dengju Li, PhD2, Jue Wang2* and Yang Cao3*

1Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China, Wuhan, China
2Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
3Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, San Diego, CA

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.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH