Figure 3.

Effect of membrane proximity of the targeted CD33 epitope on TNF-α expression in CD8⁺ and CD4⁺ CAR T cells

(A) CD8⁺ or (B) CD4⁺ primary human T cells expressing either an My96-based CAR construct or an A33-based CAR construct were incubated with ML-1 or K562 cells with CRISPR-Cas9-mediated deletion of the endogenous CD33 locus (C33^KO) or sublines engineered to overexpress CD33^FL or CD33^ΔE3-4 at various E:T cell ratios as indicated. After 12 h, the percentage of CAR T cells positive for TNF-α was assessed flow cytometrically by intracellular cytokine staining. Shown are mean ± SEM from technical triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns (not significant) by two-way ANOVA with post hoc Tukey correction.