DC_VIPs induce T cell anergy in alloreactive cytotoxic CD8 T cells. (A) Human CD8 T cells (5 × 10⁶) obtained from the PBMCs primed with allogeneic fibroblasts (donor 1) were cultured with or without syngeneic DC_controls or DC_VIPs (5 × 10⁵) obtained from the same donor pulsed without (unpulsed) or with the necrotic fibroblasts used in the priming culture (pulsed 1) or unrelated donor-derived necrotic fibroblasts (pulsed 2). After 3 days of coculture, CD8⁺ cells (5 × 10⁵) were recovered, rested, and assayed for cytotoxicity against the allogeneic fibroblasts used in the priming culture (donor 1) at different effector-target (E/T) cell ratios. The established allogeneic fibroblast-specific CD8 T cells showed cytotoxicity only against allogeneic fibroblasts used in their generation (donor 1), indicating that their cytotoxicity was antigen specific, because none of the CD8⁺ cells showed cytotoxicity against allogeneic fibroblasts from an unrelated donor (donor 2). Each result is the mean ± SD of 3 experiments performed in duplicate. (B) Human allogeneic fibroblast-specific CD8 T cells (5 × 10⁶) were cocultured without (none) or with unpulsed DC_controls or DC_VIPs (5 × 10³, 5 × 10⁴, or 5 × 10⁵ cells) derived from the indicated donors (allogeneic 1 and syngeneic 2) for 3 days. These CD8 T cells were then rescued and subsequently assayed (5 × 10⁵ cells) for cytotoxicity against the allogeneic fibroblasts (10⁴ cells) used in the priming culture (donor 1). The value of spontaneous release cpm was less than 10% of the total release cpm. Each result is the mean ± SD of 4 experiments performed in duplicate.