Genetic deletion of vasoactive intestinal peptide (VIP) and PI3K signaling pathways impair CAR T cell function Free

Introduction: The clinical efficacy of chimeric antigen receptor (CAR) T cell therapy remains limited in solid tumors, with immunosuppressive and hypoxic tumor microenvironments. Enhancing CAR T cell function and harnessing the endogenous immune response through novel signaling pathways may help overcome this limitation. We previously showed that targeting the PI3K signaling pathway improves CAR T cell efficacy in hematologic cancers, however, its therapeutic potential in solid tumors has yet to be fully explored. Vasoactive intestinal peptide (VIP) is a neuropeptide which exerts its function via interaction with two receptors, VPAC1 and VPAC2. VIP signaling in T cells is known to suppress T cell proliferation and effector function. Therefore, making this pathway an intriguing target for cancer immunotherapy, and pharmacological inhibition of PI3K and VPAC increased the expansion of T_CMin vitro. Thus, dual targeting of PI3K and VIP signaling pathways may be a novel approach to enhance CAR T cell function against solid tumors. CAR T cells generated from VIP-knockout (VIP-KO), and VPAC-knockout (VPAC-KO) mice were expanded using PI3Kδ/γ inhibition,metabolism and in vivo anti-cancer activity were assessed.

Methods: Splenocytes isolated from C57BL/6 wild-type (WT), VIP-KO, and VPAC-KO mice were activated ex vivo using concanavalin A and IL-2, followed by retroviral transduction via spinoculation with a CAR-expressing vector. During expansion, cells were cultured in the presence or absence of the PI3Kδ/γ inhibitor, Duvelisib. Transduction efficiency and T cell phenotypes—including central memory, stem-like, and exhausted subsets—were analyzed by spectral flow cytometry using surface markers. Metabolic function was assessed using a Seahorse extracellular flux analyzer. CAR T cell function was assessed using syngeneic pancreatic ductal adenocarcinoma (PDAC) tumor model.

Result: VIP-KO and VPAC-KO CAR T cells have higher frequencies of CD4⁺ and CD8⁺ naïve and stem-like memory T_scm subsets compared to WT CAR T cells. Treatment with Duvelisib further enhanced these memory subsets in both VIP-KO and VPAC-KO CAR T cells. Duvelsisib also significantly reduced effector memory Tcells and reduced phenotypically-exhausted CAR T cell populations. Seahorse metabolic analysis revealed that VIP-KO and VIPKO-DUV CAR T cells had increased basal respiration and greater ATP production through mitochondrial oxidative phosphorylation compared to WT CAR T cells. This metabolic reprogramming promoted a more quiescent phenotype in VIP-KO CAR T cells, characterized by enhanced mitochondrial function and greater metabolism up-regulation upon antigen stimulation. Despite enhanced metabolic fitness and increased frequencies of memory T cell subsets in VIPKO-DUV and VPACKO-DUV CAR T cells, in vivo studies using a syngeneic PDAC mouse model demonstrated only modest tumor control and no significant improvement in overall survival of any group compared to WT CAR T cells. Co-culture impedance experiments revealed that the addition of macrophages enhanced the function of DUV-CAR T cells, suggesting that endogenous immune cells within the tumor microenvironment may be critical for full therapeutic efficacy.

Conclusion: Dual targeting of PI3Kδ/γ and VIP signaling enhances CAR T cell memory phenotype and mitochondrial metabolic fitness, promoting a less exhausted and more quiescent T cell state. These in vivo results demonstrate that infusion of greater frequencies of memory CAR T cells with greater reliance on oxidative phosphorylation were insufficient to achieve durable tumor control in a solid tumor model, highlighting the critical role of the tumor microenvironment and endogenous immune cells in CAR T cell efficacy. The genetic absence of VIP/VPAC in the context of PI3K inhibitors may have a more profound effect on T cell survial than pharmacological inhibition of VPAC and PI3K. Future studies will include evaluation of CAR T cell infiltration and persistence in solid tumors, as well as profiling of immune cell populations in tumors from responders versus non-responders to better understand the cellular context that supports effective tumor control. These findings demonstrated the need for integrated strategies that consider both intrinsic T cell programming and extrinsic immune context to optimize CAR T cell therapy for solid tumors.