CD8⁺T-APCs activate anti-tumor CD8 T cells of the same antigen specificity.

(A-C) Cytokine production by effector CTLs in response to activation by T-APCs. (A) DiIC₁₈-labeled CD8⁺T-APCs (see materials and methods) were incubated for 6 hours with surface-biotinylated effector CTLs. Cytokine-producing effector CTLs were defined based on intracellular IFN-γ or TNF-α staining of CD8⁺streptavidin⁺ lymphocytes. Numbers in upper right quadrants indicate the percentage of IFN-γ ⁺ (upper panel) or TNF-α ⁺ (lower panel) effector CTLs. Labels indicate cells used as targets for CTLs: CTLs co-cultured with 624mel melanoma cells are designated T-APC; CTLs co-cultured with irrelevant M171 melanoma cells are designated non T-APC. (B) Confocal images of cytokine-producing effector CTLs. Calcein AM labeled CD8⁺T-APCs (green, upper panel) or non T-APCs (green, lower panel) were co-cultured for 6 hours with streptavidin-allophycocyanin-stained effector CTLs (red). Intracellular TNF-α production (blue) by effector CTLs is shown. Scale bars are 10 μm (upper panel) and 20 μm (lower panel). (C) Time period that CD8⁺T-APCs activate effector CTLs. Effector CTLs were co-cultured with CD8⁺T-APCs either immediately or 6, 24 and 48 hours after CD8⁺T-APC purification. Data are mean ± SE (n = 3 replicates/group) percentage of IFN-γ ⁺ effector CTLs, gated on CD8⁺ T cells. (D) CD8⁺T-APCs trigger degranulation of effector CTLs. CD8⁺T-APCs were generated as described above (1A) and co-cultured with effector CTLs. Cytolytic activity of T cells was measured by detection of surface CD107A on CD8⁺streptavidin⁺ effector CTLs. Number in upper right quadrants indicates the percentage of CD107A⁺ streptavidin⁺ effector CTLs. Data are representative of at least three independent experiments.