Figure 5.

Modified CD8αβ iPSC-CTLs exert superior anti-viral and anti-tumor activity with superior proliferative capacity than parental T cell clone

Cytotoxicity and cytokine production against K562/A24 cells, cytotoxicity (A−C) against HIV-1-infected human CD4 T cell lines (D), in vitro proliferation (E), long-term coculture killing (F), in vivo persistence (G), and in vivo killing assay (H and I) of iPSC-CTLs and parental T cell clones. All T cells were thawed and expanded for 2 weeks once in feeder-free condition side by side before the functional assays. (A−C) ⁵¹Cr release assay (A) and cytometric bead array (CBA) cytokine assay for IL-2 and IFN-γ production (B) were performed after coculture for 5 h with K562-A24 cells pulsed with the indicated dose of peptide at an E/T = 1 (B), 9 (A, left), or as indicated (A, right). Shown in (C) is a summary of independent experiments when iPSC-CTLs or T cell clones were cocultured with 1 μM (IL-2 and cytotoxicity) or 10 nM (IFN-γ) peptide-pulsed K562-A24 cells (cytotoxicity, n = 5; IL-2 and IFN-γ, n = 4 per group; mean ± SEM; ∗p < 0.05; NS, not significant). (D) Cytotoxicity of T cells against HIV-1-infected human CD4 T cell lines based on the number of surviving target cells after co-culture with T cells at the indicated E/T ratio. p24 (ACH-2) or inducible-GFP (GXR) was used to calculate the number of HIV-1-infected cells. The relative percentages of p24-positive or GFP-positive cells in the target cells to that of the control, which was normalized to 100%. (E) Carboxyfluorescein succinimidyl ester (CFSE) dilution of iPSC-CTLs and the parental T cell clone during 1-week culture with 100 pg/mL or 10 ng/mL IL-15 without TCR stimulation. (F) Fold expansion of T cells and K562-A24-N138Rluc in 2-week coculture, calculated using flow cytometry. (G) IVIS images of NOD-SCID IL2Rγc^null (NSG) mice engrafted with 2 × 10⁶ luciferase-transduced iPSC-CTLs or the parental T cell clone intraperitoneally. 100 ng/head recombinant hIL-15 was infused weekly. Representative of two independent experiments using H254SeV3-derived iPSC-CTLs (5 mice per group). (H and I) IVIS images (H) and Kaplan-Meier survival curves (I) of NSG mice with ∼2 cm³ large tumors after subcutaneous inoculation with 2 × 10⁵ cognate-Nef-peptide expressing K562-A24-N138Rluc followed by the injection of PBS, iPSC-CTLs, or the parental clone 4 days and 7 days later (2 × 10⁶ T cells at each time point). Mice were euthanized when the tumor volume exceeded 2 cm³ on either side. At day 4 immediately before the T cell infusion, top and bottom photos are identical but processed with different color scales. Combined results of two independent experiments using H254SeV3-derived iPSC-CTLs are shown (12 mice per group [5 and 7 mice for each experiment]; ∗p < 0.05 by the log-rank [Mantel-Cox] test; tumor volume = [length × width²]/2⁴¹).