Figure 1.

DP-to-CD8SP maturation was optimized to guide iPSC-CTLs from innate to adaptive CD8αβ⁺CCR7⁺CD45RA⁺ cells

(A) Flow cytometric profile of CD8αβ and CD8αα iPSC-CTLs after modified maturation culture as described in Materials and methods. (B) Frequency of CD8αβ⁺CD5⁺ iPSC-CTLs after DP-to-CD8SP transition with or without RetroNectin coating in the presence of 1 μg/mL CD3ε antibody and 10 ng/ml IL-7 but not IL-21 (n = 3; mean ± SEM; ∗p < 0.05). (C and D) The effect of cytokine supplements on the maturation and subsequent expansion efficacy (C) and surface phenotype (D). (C) The CD8SP cell yield against the starting DP cell number was calculated as the maturation efficacy. The same CD8SP cells were subsequently expanded side by side in a CD3/CD28-based PBMC feeder-free condition, and the expansion efficacy was calculated at day 14 (top, n = 6; bottom, n = 10; mean ± SEM; one-way ANOVA comparing mean log10 of all groups with Tukey’s multiple comparisons test; ∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.0001). (D) The expression of innate-, adaptive-, and naive-associated markers in healthy donor-derived primary naive CTLs and iPSC-CTLs matured in the presence of the indicated cytokines at the DP-to-CD8SP transition step. (E) Schematic depiction of the modified maturation and expansion culture. (F) Flow cytometric profile of pre- and post-DP-to-CD8SP maturation iPSC-CTLs. Data are representative of three (A, B, D, and F) or more than six (C) independent experiments using TKT3V1-7-, H254SeV3-, and H2531SeV3-derived iPSC-CTLs.