Figure 3. Effect of peroxynitrite donor on specific CD8⁺ T-cell activity.

(a) OT-1 T cells were pretreated with SIN-1 (0 to10 mM) for 30 min and then 5×10⁴ T cells were cultured for 4 days with DCs at a 10:1 ratio. CP – DCs were loaded with 10 μg/ml control (RAHYNIVTF) peptide, SP – DCs were loaded with 10 μg/ml specific (SIINFEKL) peptide. PHA – OT-1 T cells and DCs were cultured for 3 days in the presence of 1 μg/ml PHA. ³[H]-thymidine (1 μCi/well) was added 18 h prior to cell harvest and radioactivity was measured in triplicates in liquid scintillation counter. Results presented as Mean ± SD.

(b) OT-1 T cells were cultured for 48 h with DCs, peptide and PHA as described in Fig. 3a. The number of IFN-γ producing cells was evaluated in quadruplicates in ELISPOT assay. Results presented as Mean ± SD.

(c) OT-1 T cells were pretreated with 1 mM SIN-1 for 30 min. The binding of control and specific pMHC dimers to CD8⁺ T cells was evaluated using flow cytometry as described in Fig. 1. T – T cells stimulated with specific peptide-loaded DCs; T+Sin-1 – T cells pre-treated with SIN-1.

(d) Cells were treated as described in Fig. 3c and then labeled with anti-CD8 or anti-TCRαβ antibodies. The levels of expression (mean fluorescence intensity, MFI) within the population of CD8⁺ T cells were evaluated by flow cytometry. The results of three experiments are shown.