Investigating the effects of IFN-α and intrinsic RIG-I stimulation on viability, CAR expression, expansion, and cytotoxicity of CD19 CAR T cells

Cytotoxic CD8⁺ T cells play a central role in eliminating pathogen-infected and malignant cells through the release of effector cytokines, such as TNF and IFN-γ, and by exerting direct cytotoxic activity. Type I interferons, particularly IFN-α, enhance CD8⁺ T-cell proliferation, survival, and effector differentiation, thereby strengthening antiviral and antitumor immunity. Antiviral sensing pathways, including nucleic acid recognition by the RIG-I helicase, further shape T-cell responses by inducing type I IFN secretion and downstream interferon-stimulated gene expression. Previous studies have shown that IFN-α treatment and RIG-I stimulation improve human CD8⁺ T-cell proliferation and effector activity, suggesting potential utility for engineered T-cell therapies. CAR T-cell therapy, which involves engineering T cells with chimeric antigen receptors (CARs) to recognize tumor-associated antigens, has revolutionized treatment of hematological malignancies. However, challenges remain, including limited proliferation, suboptimal persistence, and functional exhaustion of CAR T cells. Based on existing evidence that IFN-α and RIG-I activation enhance CD8⁺ T-cell responses, we hypothesized that stimulating these pathways during CAR T-cell expansion or prior to cytotoxic assays would enhance CAR T-cell proliferation and cytotoxicity. In this project, we investigated the effects of IFN-α treatment and intrinsic RIG-I activation (via synthetic 3p-dsRNA) on viability, CAR expression, expansion, and cytotoxicity of CD8⁺ and CD3⁺ CD19 CAR T cells. Then, we compared CD8⁺ CAR and CD3⁺ CAR T cells to identify the most effective strategy for CAR T-cell generation. We found that both IFN-α treatment and RIG-I activation significantly enhanced CD3⁺ CAR T-cell cytotoxicity against CD19⁺ target cells following a double stimulation at days 5 and 9; whereas viability, CAR expression, and expansion were not significantly affected. Furthermore, unstimulated CD3⁺ CAR T cells exhibited stronger cytotoxicity than CD8⁺ CAR T cells, likely reflecting the supportive role of CD4⁺ T-cell help. These findings demonstrate that activating intrinsic antiviral pathways through IFN-α exposure or RIG-I stimulation can potentiate CAR T-cell cytotoxic function. Integrating such stimulation strategies into CAR T-cell manufacturing protocols may improve functional potency and support the development of CAR T-cell therapies with enhanced antitumor activity.