Figure 4: Overexpression of LEF1 improves NKT expansion and tumor control and reduces exhaustion following serial tumor challenge.

A–E, NKTs transduced with GFP.FFLuc or GFP.LEF1 were repeatedly challenged with CD1d⁺ J32 leukemia cells at a 1:1 ratio every three days. (A) NKT cell number was determined using counting beads and flow cytometry following each challenge cycle. FSC-SSC dot plots show approximate live cell gate representing NKTs at early (2^nd) and late (7^th) co-culture cycles. Results from a representative donor (n = 4 donors, two independent experiments) and NKT fold-change at each cycle are shown. (B) GFP expression was monitored over time using flow cytometry as a proxy for transduced cells. Mean GFP⁺ NKT percentage ± SEM is shown at each cycle (n = 4 donors). P = 0.07 for AUC analysis for cycles 6 to 8. C, D, Following cycle 5, NKTs were rested for six days following antigenic stimulation and (C) TIM-3 and (D) CD62L expression were assessed by flow cytometry. Representative results and mean TIM-3 MFI or CD62L percentage (n = 4 donors) are shown. ***P < 0.001, ns: not significant, one-way ANOVA with Sidak’s post-test.