Figure 4

Cytotoxic and tumor-recognizing capacity of TIL expanded with IL-15, IL-21 or no cytokine producing aAPC. Cytotoxic capacity of the IL-2, IL-15 and IL-21 aAPC-expanded TIL was determined by flow cytometry-based re-directed cytotoxicity assay. To this end, CFSE- and anti-CD3-pulsed no cytokine aAPC cells were cultured in a 1:2 ratio in triplicate for 20 hours with CD8⁺ T cells derived from IL-2, IL-15 or IL-21 aAPC-expanded total TIL, after which percentage of killing was determined by 7-AAD staining. A) Data is presented as percentage of 7-AAD⁺ target cells for three patients. B) MHC-I restricted autologous tumor recognition of expanded TIL from one donor was determined by IFN-γ secretion assay. To this end, autologous/CD45-depleted tumor cells were cultured in a 1:2 ratio in triplicate for 20 hours with CD8⁺ T cells derived from IL-2, IL-15 or IL-21 aAPC-expanded total TIL in the presence of either irrelevant IgG2a antibody (left) or anti-MHC class I blocking antibody (w6/32; right), after which supernatants were harvested and tested by IFN-γ ELISA. Differences were compared using repeated measures ANOVA’s with a Tukey post test, and considered significant when p < 0.05, as indicated with an asterisk (* p < 0.05, ** p < 0.01), *** p < 0.001).