FIGURE 7.

TGFβ-mediated suppressive function of CD4⁺CD25⁺LAP⁺ cells in vitro and in vivo. A, Effect of SiRNA knockdown of TGFβ1 on the in vitro suppressive function of CD4⁺CD25⁺LAP⁺ and CD4⁺CD25⁺LAP⁻ cells. Sorted CD4⁺CD25⁺LAP⁺ and CD4⁺CD25⁺LAP⁻ cells were transfected with 250 ng of SiRNA specific for TGFβ1. Negative controls consist of mock-transfected cells and cells transfected with control SiRNA. The knockdown extent of TGFβ1 siRNA was determined by real-time PCR and was 48 ± 15% and 34 ± 2% with reference to control siRNA transfected cells for CD4⁺CD25⁺LAP⁻ and CD4⁺CD25⁺LAP⁺ subsets, respectively. TGFβ1 expression was normalized to the expression of the housekeeping gene β-actin. Twenty-four hours after transfection, transfected cells were cultured at 1:1 ratio with responder CD4⁺CD25⁻LAP⁻ cells, and in vitro proliferation assays were performed as described in Materials and Methods. The results shown represent the mean ± SD of triplicate wells and are representative of two independent experiments. B, Effect of neutralization of TGFβ on the regulatory function of CD4⁺CD25⁺LAP⁺ cells in vivo. SJL mice were adoptively transferred with 1 × 10⁵ sorted CD4⁺CD25⁺LAP⁺ cells 2 days before EAE induction. Mice then received five i.p. injections of 50 μg of neutralizing TGFβ-specific or control Abs every other day starting 1 day before EAE induction. Values are the mean daily score for each group (five mice per group). Data are representative of at least two independent experiments.