Antigen specificity as measured by anti-IFNγ ELISpot, TNFα and IFNγ intracellular cytokine staining, and cytolytic function of the TAA-T products. (A) Target antigen specificity of the TAA-T product (n = 25) as determined by IFNγ production, measured by ELISpot. Target antigens were WT1, PRAME, survivin, and TAA (WT1, PRAME, and survivin pepmixes combined). The bottom dotted line denotes the median for negative control (actin = 12 SFU), the top dotted line denotes the median for positive control (SEB = 732 SFU). Mean antigen responses were statistically significantly different from actin for WT1 (P = .0469), PRAME (P = .0001), and TAA (P < .0001) but not for survivin (P = .7028). (B) TNFα and IFNγ intracellular cytokine staining (ICS) demonstrates antigen specificity for WT1 and PRAME shown for products (P6, P9). Antigen specificity measured by TNFα and IFNγ ICS of CD8⁺ T cells (C) and CD4⁺ T cells (D) of the TAA-T-cell product in evaluable samples (n = 7). SEB is used as the positive control and actin as negative control. (E) In vitro cytolytic activity of the HLA A*02⁺ TAA-T product against an HLA A*02⁺ AML cell line (THP-1) as compared with a donor lymphocyte infusion (donor PBMCs) product. (F) Superior cytolytic activity against THP-1 Violet⁺ CD33⁺ cells of the TAA-T product as compared with donor lymphocyte infusion (PBMC) is reproducible in the majority of A*02⁺ donor TAA-T products evaluated (as shown for P2, P6, P12). SEB, staphylococcal enterotoxin B.