Figure 2.

NKp44 negative sorting removed aberrant NK activity of CD8αβ iPSC-CTLs and enriched early memory phenotype

(A−C) NK activity (A) and NKp44 expression (B) of iPSC-CTLs re-differentiated from independent iPSC clones and healthy donor-derived primary CTLs. ⁵¹Cr release assay against peptide-unpulsed K562-HLA-A24 cells at E/T = 10 (A) and flow cytometry (B) was performed after one PHA-PBMC-feeder expansion. (C) The relationship between the NK activity (A) and NKp44⁺ ratio (B) of different iPSC-CTLs (n = 8). r is Pearson’s correlation coefficient. Representative of two independent experiments using TKT3V1-7 (#1, #2)-, H254SeV3 (#3, #4, #6)-, and H2531SeV3 (#5, #7, #8)-derived iPSC-CTLs. (D) Antigen-dependent and -independent cytotoxic activities of sorted NKp44⁺ and NKp44^– iPSC-CTLs. NK-strong iPSC-CTLs (H2531SeV3) were expanded once with the PHA-PBMC feeder for 2 weeks, sorted for NKp44 expression, and underwent the ⁵¹Cr release assay against peptide-pulsed or -unpulsed K562-HLA-A24 cells. (E−H) RNA sequencing (E and F) and cytokine production (G and H) of iPSC-CTLs and the indicated subsets of healthy donor-derived primary CTLs. (E) The gene expressions were analyzed by RNA sequencing, and the correlation analysis was performed based on the 776 T cell-associated genes indicated in Table S1 (gene list from Muranski et al.²⁶). (F) Messenger RNA expression levels (reads Per Kilobase of exon per Million mapped reads [RPKM]) of TCF7, LEF1, TAF4B, EOMES, TBX21, PRDM1, and GZMA in the indicated populations (iPSC-CTLs, n = 2; primary CTL subsets, n = 3 each; mean ± SEM). TKT3V1-7- and H254SeV3-derived iPSC-CTLs were used in (E) and (F). (G and H) Intracellular IL-2 and IFN-γ were analyzed with flow cytometry after 4 h of stimulation with 50 ng/mL PMA and 1 μg/mL ionomycin. Shown in (H) is the mean percent of the indicated IL-2- and/or IFN-γ-producing cells in three independent experiments using TKT3V1-7- and H254SeV3-derived iPSC-CTLs. (Data from one experiment of TKT3V1-7-derived iPSC-CTLs are shown in G. Data from other experiments are shown in Figure S12.)