Fig. 3

Switch molecule-controlled activation of CD4⁺ T cells engineered with sdCAR. a We engineered primary human CD4⁺ T cells derived from fresh human peripheral blood with sdCARs by electroporation and evaluated the cells by activation assays with an “AND logic gate” strategy. b, c We quantified production of IL-2 and IFNγ by enzyme-linked immunosorbent assay. The cytokines were produced only when sdCAR-T cells were exposed to cognate tumor cells (MSLN⁺ K562) in the presence of FHBM but not when exposed to non-cognate tumor cells (K562 or CEA⁺ K562). For MSLN⁺ HT29 cells, high levels of cytokines were found only in MζBB CAR-T cells. (n = 3, error bars denote standard deviation.) d Monitoring T cell activation by CD69 expression. CD69 was expressed on sdCAR-T cells in the presence of both MSLN⁺ K562 cells and a switch molecule (FHBM) and also was expressed on MζBB CAR-T cells. For MSLN⁺ HT29 cells, only MζBB CAR-T cells had significant CD69 expression. (n = 3, error bars denote standard deviation.) e–h FHBM and cognate tumor cell-dependent T cell proliferation. As sdCAR-T cells express a fluorescence reporter protein (BFP), we quantified the number of activated cells by flow cytometry after 3, 4, or 5 days of incubation. For cognate tumor cells (K562 or CEA⁺ K562), sdCAR-T cells and MSLN-specific CAR-T cells had no significant proliferation. sdCAR-T cell proliferation was regulated by FHBM in the presence of MSLN⁺ K562 cells. A similar degree of T cell proliferation was found in MζBB CAR-T cells. For HT29 tumor cells, only MSLN-specific CAR-T cells had strong proliferation. (n = 3, error bars denote standard deviation)