Figure 5.

Silencing of one gene (Bcl2) in CD8⁺ T cells from tolerized mice does not effect expression of other selected genes known to be involved (or not) in suppressive capacity of CD8⁺ Treg in our system. CD8⁺ T cells were isolated from pCons-treated mice 1 week after pCons tolerization using antibody-specific microbeads from AUTOMACS. Cells were pooled from 3–4 mice in each group. The cells were transfected with siRNA of Foxp3, CCR7, IFI202b, IFNar1, bcl2 (50–100 nm) or with siRNA controls (GAPDH or scrambled siRNA, 50–100 nm). The negative control scrambled sequence has random sequences with no homology to mouse, rat or human genomes. Incubation with siPORT amine served as a control for the transfection reagent. Then the transfected CD8+ T cells (1 × 105) were cultured for 3 days and RNA was isolated. Real-time PCR for Bcl2 was performed with isolated RNA (100 ng). tolerized CD8=tCD8. Columns: a; tCD8; b; tCD8⁺ Bcl2 siRNA, c; tCD8⁺ Foxp3 siRNA, d; tCD8⁺ p53 siRNA, e; tCD8⁺ IFNar1 siRNA, f; tCD8⁺ ifi202b siRNA, g; tCD8⁺ CCR7 siRNA, h; tCD8⁺ GAPDH siRNA, I; tCD8⁺ scrambled siRNA, j; tCD8⁺ si PORT amine.