Functional comparison of CD28 and 2B4 intracellular signaling domain of FLT3 CAR in primary cord blood NK cells. (A) Scheme of the FLT3 CAR constructs containing CD28 or 2B4 as the intracellular costimulatory domain. tCD19 is coexpressed to serve as a marker. (B) Functional assessment of the FLT3 CAR NK cells against FLT3⁺ MOLM-13 AML cells using a 4-hour Cr-51 release assay under the indicated effector-to-target (E:T) ratio. NK cells only expressing tCD19 serve as a control group (n = 3 donors). (C) Time-lapse luciferase imaging of AML mouse model established by an infusion of MOLM-13 AML cells expressing luciferase (MOLM-13_luc, 2 × 10⁵ cells per mouse) with a single dose of indicated treatments, administered 2 days after tumor implantation (5 × 10⁶ cells per dose). (D) Quantification of the ventral bioluminescence images (n = 3 mice). Data are shown as mean ± SEM. ∗P < .05;∗∗P < .01. scFv, single-chain variable fragment.