Peptide-specific suppression of cocultured T cells induced by anergic T cells.

Tyrosinase-specific CD8⁺ T-cell lines or HA-specific CD4⁺ T cells of the HA1.7 clone were cultured and restimulated as described in “Materials and methods.” For anergy induction, these cells were cocultured with IL-10–treated DCs of HLA-A.2 (tyrosinase) or HLA-DR1⁺ (HA1.7) donors. Subsequently, (5 × 10⁴) anergic tyrosinase-specific CD8⁺ T cells (ATs) (panel A) or CD4⁺ T cells of the clone HA1.7 (ATs) (panel B) were cocultured with (5 × 10⁴) syngeneic T cells (CTs) of the same specificity and restimulated with syngeneic mature DCs (1 × 10⁴) (pulsed with the specific antigen) (hatched bars). A coculture of control activated (tyrosinase- or HA-specific) T cells (1 × 10⁵) (CTs) with mature DCs served as controls (black bars). To test the peptide specificity, control CD8⁺MART-1 or TT CD4⁺ T cells (5 × 10⁴) were cocultured with anergic syngeneic tyrosinase CD8⁺ or anergic syngeneic HA-specific CD4⁺ T cells (5 × 10⁴) and stimulated with syngeneic mature DCs (1 × 10⁴). As controls, anergic (ATs) or activated (Tyr or MART CTs) (5 × 10⁴) T cells were stimulated with syngeneic (5 × 10³) DCs in an MLR (dotted bars). T-cell proliferation was measured after 3 days of incubation and an additional 16-hour pulse with ³H-Tdr (37 kBq/well) by means of a liquid scintillation counter. Similiar results were measured in 5 independent experiments.