Figure 4.

A highly efficient feeder-free expansion culture was developed with the supplementation of IL-21, IL-12, IL-18, and TL1A

(A−D) Fold expansion of iPSC-CTLs after 2 weeks of PBMC-feeder-free expansion supplemented with the indicated cytokines. Independent lots of iPSC-CTLs were once expanded in PHA-PBMC-feeder condition, then stimulated with immobilized anti-CD3 antibody in the presence of the indicated cytokines, and enumerated 2 weeks later to calculate the fold expansion (A, n = 8; C, n = 15; mean ± SEM; one-way ANOVA comparing mean log10 of all groups with Tukey’s multiple comparisons test (A and C); ∗p < 0.05, ∗∗p < 0.005, ∗∗∗∗p < 0.0005). (B and D) Fold expansion in sequential feeder-free stimulation with (+) or without (−) IL-12/IL-18 in addition to IL-7/IL-15/IL-21 (B) or with or without TL1A in addition to IL-7/IL-15/IL-21/IL-12/IL-18 (D). (E) Growth curve of purified or unpurified CTLs in healthy donor-derived PBMC in feeder-free expansion culture. Cells were stimulated first by CD3/CD28 microbeads and biweekly afterward by 1 μg/mL immobilized anti-CD3 antibody in the presence of IL-2 alone or IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A. Plots show 4 (left) and 2 (right) independent experiments. (F) Growth curve of iPSC-CTLs, the parental T cell clone, and healthy donor-derived primary naive CTLs in feeder-free expansion culture supplemented with IL-7, IL-15, IL-21, IL-12, IL-18, and TL1A. iPSC-CTLs and primary naive CTLs experienced one-time PHA-PBMC-feeder expansion followed by repeated feeder-free expansions. The T cell clone experienced two feeder-free expansions after four repeated PBMC-feeder expansions (not shown). (G) The expression of exhaustion- and senescence-associated markers in eight repeated feeder-free expanded iPSC-CTLs. (F and G) Data are representative of two independent experiments using TKT3V1-7- and H254SeV3-derived iPSC-CTLs, whereas data of (A)−(D) are representative of more than eight independent experiments using TKT3V1-7-, H254SeV3-, and H2531SeV3-derived iPSC-CTLs.