Young TILs with favorable cell subset composition can be expanded directly from fresh tumor digests using aAPCs. (a) Fresh ovarian cancer digests (PRE-EXP) are comprised by a heterogenous mix of EpCAM+ tumor cells and CD45+ leukocytes, containing CD14+ monocytes and CD3+ T cells; aAPC expanded digests (POST-EXP) contain only CD45+ CD3+ T cells. Lower dot plots are CD45+ gated. (b) 106 total tumor digest cells were stimulated with 106 aAPC loaded with anti-CD3 and anti-CD28 agonist antibody in CM containing 100 IU/mL IL-2, or cultured in 600 IU/mL IL-2 alone. Mean viable cell counts ± SEM are shown (n = 7). (c) Fold expansion of CD3+ TILs. Calculated viable absolute T cell numbers are shown (Total T cell number times % viable CD3+). (d) Ratio of CD4 + TILs to CD8+ TILs pre- and post-expansion with either aAPC or IL-2 alone; (e) percentage of CD3+ TILs expressing CD27; (f) or CD28; (g) percentage of CD4+ CD3+ TILs expressing FOXP3. Values in (d-g) represent the mean expression of the indicated molecule by 4 independently expanded TILs. (h) TILs expanded directly from enzyme-digested tumor specimens using KT64/BBL aAPC demonstrated autologous tumor reactivity. 105 aAPC-expanded TILs or 105 TILs outgrown in 600 IU/mL of IL-2 were co-cultured overnight with 105 autologous tumor cells or not stimulated (none). Anti-CD3/28 bead stimulation was applied as positive control. Mean concentration of IFN-g (pg/mL ± SEM) detected in supernatants from paired aAPC- and IL-2-expanded TIL cultures from 4 independent ovarian cancer specimens with anti-tumor reactivity is shown.