Fig. 2

Combinatorial antigen requirement for T cell activity in vitro. a Modified T cells or wild T cells were incubated at indicated with various tumor cells at an effector/target rations of 8:1, 4:1, 2:1, 1:1, 1:2, 1:4, or 1:8. After a 24-h incubation, target cell lysis was measured by LDH release in the supernatant. The optimal effector/target ratio in this research was determined to be 2:1. In addition, dCAR-T cells could specifically lyse AsPC-1 cells yet do not eliminate HT29 cells, U87 cells, and PANC-1 cells (n = 3, error bars denote standard deviation). b Activation of dCAR-engineered CD4⁺ T cells required cognate target cells. The primary CD4⁺ T cells were modified with dCARs by lentivirus transfection, and cell activation assays were performed with an “AND logic gate” strategy, including cytokines release, marker expression, and T cell proliferation. c Released cytokines in each sample were quantified by enzyme-linked immunosorbent assay, including IL-2, IFNγ, TNFα, IL-4, IL-13, and IL-15. All cytokines were significantly produced when dCAR-T cells were exposed to AsPC-1 cells yet not when exposed to non-cognate tumor cells (HT29 cells, U87 cells, or PANC-1 cells). For conventional CAR-T (CEA-CAR or MSLN-CAR) cell treatment, similar cytokines were obtained (n = 3, error bars denote standard deviation). d Monitoring T cell activation by CD25 and CD69 expression. T cell activation marker, CD25 or CD69, was significantly expressed on dCAR-T cells or conventional CAR-T cells compared with that of other single-receptor modified T cells in the presence of AsPC-1 cells (n = 3). e Combinatorial antigen-dependent T cell proliferation. Data showed that dCAR-T cells have a high proliferation activity in the presence of cognate tumor cells expressing CEA and MSLN, which was similar to that of conventional CAR-T cells against target cells. Interestingly, Cζ-CAR-modified T cells showed a lower proliferation capacity, indicating that the CD3ζ signaling pathway is not sufficient to trigger T cell activation (n = 3). f dCAR-engineered CD8⁺ T cells yield specific target cell killing in vitro. g Cytotoxicity mediated by dCAR-CD8⁺ T cells in a 24-h experiment. After an overnight incubation, significant cytotoxicity was observed in dCAR-T cells co-cultured with AsPC-1 cells, approximately 85% of target cell apoptosis (n = 3, error bars denote standard deviation)