Figure 2.

STAb-CD1a T cells display enhanced tumor cell killing by recruiting bystander T cells. (A, B) Schematic representation of the direct contact (A) and the non-contacting Transwell (B) co-culture systems used to study the ability of secreted CD1a-STAb to induce bystander T cell cytotoxicity. (C) Decreasing numbers of activated effector T (AT) cells (NT, CD1a-CAR or CD1a-STAb) were co-cultured with 5×10⁴ MOLT4^Luc target cells and increasing numbers of NT T cells from the same donor (bystander T cells), resulting in the indicated AT:T ratios but maintaining a constant 2:1 effector (AT+bystander):Target ratio. (D) 5×10⁴ MOLT4^Luc cells and 1×10⁵ bystander T cells were plated in the bottom well and decreasing numbers (from 1×10⁵ to 1 x 10¹) of activated T (AT) cells (NT, CD1a-CAR or CD1a-STAb) in the upper well. After 48 hours, the percentage of specific cytotoxicity was calculated by adding D-luciferin to detect bioluminescence, and IFNγ secretion was determined by ELISA (C, D). (E) MOLT4 cells were co-cultured with NT, CD1a-CAR or CD1a-STAb T cells at the indicated E:T ratios, and the expression of CD3 and CD1a was analyzed by flow cytometry after 4 and 11 days to assess potential leukemia escape. Data represent mean±SEM of at least three independent experiments by triplicates. Significance was calculated by a two-way ANOVA test corrected with a Tukey’s multiple comparisons test (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001). ANOVA, analysis of variance; E:T, effector:target; NT, non-transduced; CAR, chimeric antigen receptor; STAb, secreting T cell-redirecting antibodies.