A Newly Developed TGF-Β-Responsive CAR T Cell for Enhanced Proliferation and Cytokine Secretion.

Purpose: Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising cancer treatment. Nevertheless, the tumor microenvironment (TME) of solid tumors provides substantial challenges to CAR T cell efficacy. Tumor growth factor-beta (TGF-β), a potent immunosuppressive cytokine in the TME, impedes T cell activation, proliferation, and cytotoxicity, diminishing the anti-tumor potency of CAR T cells. This study investigates whether TGF-βRII CAR T cells can overcome these barriers and remain functional in TGF-β-rich environments. Methods: We developed a novel TGF-βRII CAR T cell (TGF-βRII-CD28CD3z) and a dominant-negative TGF-β receptor (dnTβRII) T cell utilizing Jurkat cells. Transduction efficiency and surface expression were confirmed using flow cytometry. T cell activation and proliferation were assessed by CD69 and Ki-67 expression, respectively. IL-2 and IFN-γ secretion were quantified using ELISA kits. Results: Flow cytometry confirmed the successful cell surface expression of the designed receptors: 62% and 24% for TGF-βRII CAR and dnTβRII, respectively. TGF-βRII CAR T cells were markedly activated in a dose-dependent manner, with optimal responses at 10 ng/mL TGF-β. The Ki-67 expression of CAR T cells, used as a proliferation marker, increased 1.21-fold (from 79.5% to 96%) upon exposure to 10 ng/mL TGF-β. At 5 ng/mL TGF-β, the cells' proliferation was maintained at a 1.04-fold increase. Cytokine analysis revealed a 1.9-fold increase in IL-2 (130±4 pg/mL) and a 2.7-fold increase in IFN-γ (146±21.9 pg/mL) secretion at 10 ng/mL TGF-β. Additionally, at 5 ng/mL TGF-β, IL-2 secretion increased 1.6-fold (110±10.7 pg/mL), and IFN-γ secretion increased 1.7-fold (94.3±10.2 pg/mL). In contrast, dnTβRII T cells also produced IL-2 (95 pg/mL±22, 2.7-fold increase) but failed to sustain proliferation or IFN-γ production at 10 ng/mL TGF-β. Conclusion: Our findings demonstrate that the TGF-βRII CAR T cells not only resist TGF-β-mediated suppression but also promote activation, proliferation, and cytokine release in the presence of TGF-β. This underscores their therapeutic potential as an innovative approach to overcome TGF-β-driven immunosuppression and improve the CAR T cell therapy efficacy in solid tumors.