Figure 6

PKCθ-KO Treg proliferation, but not OX40−/−, phenotype can be rescued when cultured with WT Teffs. (a–d) Splenic Tregs (CD4⁺CD25⁺) isolated from WT, OX40-KO, and PKCθ-KO mice were labelled with CellTrace violet and then each Treg subtype (WT, OX40-KO & PKCθ-KO) was remixed with the Teffs (CD4⁺CD25⁻) from their own counterpart and the other two cell subtypes as indicated. Subsequently, these T-cell combinations were co-cultured with WT-BMDCs or II-KO-BMDCs+ IL-2 (1 U/ml) for 4 days then harvested and analysed by flow cytometry. (a) Representative dot plots (left) and summarizing bar graphs (right) show the percentage of proliferating Tregs when WT Tregs were remixed with either WT Teffs, OX40-KO Teffs or PKC-θ-KO Teffs then co-cultured with WT-BMDCs. (b) Representative dot plots (left) and summarizing bar graphs (right) show the percentage of proliferating Tregs when WT Tregs were remixed with either WT Teffs, OX40-KO Teffs or PKC-θ-KO Teffs then co-cultured with II-KO-BMDCs. (c) Representative dot plots (top) and summarizing bar graphs (bottom) show the percentage of proliferating Tregs when OX40-KO or PKC-θ-KO Tregs remixed with WT Teffs then co-cultured with WT-BMDCs. (d) Representative dot plots (top) and summarizing bar graphs (bottom) show the percentage of proliferating Tregs when OX40-KO or PKC-θ-KO Tregs remixed with WT Teffs then co-cultured with II-KO-BMDCs. E&F) WT, OX40-KO, and PKCθ-KO mice were treated with soluble OX40L three times (100 µg/mouse/week). Another group of mice received PBS (untreated control). One week after the last treatment, mice were euthanized and spleen and thymus were collected for analysis by flow cytometry. (e) Representative dot plots show the percentage of Tregs from total CD4⁺ splenocyte T-cells (left) and summarizing bar graphs (right). (f) Representative dot plots show the percentage of Tregs from total CD4⁺ thymocyte T-cells (left) and summarizing bar graphs (right). Values showing average ± SD, *P < 0.05, **p < 0.005, and ***p < 0.0005.