KT64/BBL aAPCs support the expansion of TILs in a CD28-independent manner. (a) TILs cultures established for 3-4 weeks in 600 IU/ml IL-2 were expanded using aAPCs loaded with anti-CD3 and anti-CD28 mAbs at various aAPC to T cell ratios in the continued presence of IL-2 (100 IU/mL). In this representative experiment (one of three), a 62-fold expansion of TILs was achieved 9 days after a single stimulation with aAPCs at the 2:1 aAPC to T cell ratio. A 3-fold expansion occurred after continued culture in IL-2. TILs stimulated with aAPCs underwent greater expansion at all aAPC to TIL ratios compared to continued growth in IL-2 or growth in medium alone. (b) KT64/BBL aAPC-based TIL expansion is CD28 costimulation-independent but augmented by provision of IL-2 support. Established TIL cultures were expanded for 9 days using aAPC loaded with anti-CD3 antibody in the presence or absence of clone 9.3 anti-CD28 antibody, in the presence or absence of IL-2 supplement. (c) CD28 costimulation augments the aAPC-based expansion of peripheral blood T cells, but not autologous TILs. CD3/28 beads do not support TIL expansion (3:1 bead to T cell ratio). Day 9 cell counts are shown. (d) TILs stimulated with KT64/BBL aAPCs with or without anti-CD28 antibody do not secrete IL-2 after overnight culture, but peripheral blood lymphocytes do. IL-2 secretion by PBL is increased by provision of CD28 costimulation and supported by CD3/28 bead stimulation. Mean IL-2 (pg/mL) concentration ± SEM from three independent TIL cultures is shown.