Figure 3

Signalling pathways involved in Treg proliferation induced by G-BMDCs. (a) Total CD4⁺ and CD4⁺CD25⁺ T-cells isolated from NOD mice spleen were CellTrace violet-labelled, incubated for 6 hrs in the presence of various kinase inhibitors, and then co-cultured with WT-BMDCs in the absence (total CD4+ T cells) or presence (CD4⁺CD25⁺ T-cells) of exogenous IL-2 (5 U/ml). Cells were harvested on day 5 and analysed by flow cytometry for Treg proliferation. Representative dot plots for Treg proliferation are shown for indicated inhibitors. (b) WT and OX40-KO splenic CD4+ T-cells were isolated, labelled with CellTrace violet and co-cultured with MHC-class II-KO-BMDCs in presence of exogenous IL-2 (1 U/ml). After 5 days, cells were harvested and analysed by flow cytometry for activation of NF-kB pathway. Mean fluorescence intensity (MFI) of the indicated molecules are shown in bar graphs. (c) CD4⁺ T-cells isolated from splenocytes were treated with IL-2 alone or with IL-2 and OX40L for 24 h. Cells were lysed and analysed for NF-kB-p65 phosphorylation using total and phospho-p65 antibodies by western blot. Values show average ± SD, *P < 0.05, **p < 0.005, and ***p < 0.0005.