Figure 5

Treg isolated from irradiated mice have normal immunosuppressive function. C57BL/6 mice received 0 Gy or 15 Gy whole thorax irradiation. At different time points, immune cells were isolated from lung tissue and stained for flow cytometric analysis. (A) Detection of CTLA-4 on CD4+ FoxP3+ Treg in the lung. (B) Expression of CD103 on CD4+ FoxP3+ Treg in the lung. Timelines are shown as mean values ± SEM of percentages calculated on total lung cells. Cells of 4-6 mice per group were analyzed (* p ≤ 0.05; ** p ≤ 0.01; two-way ANOVA followed by post-hoc Bonferroni test). (C) Gating strategy for FACS-sorting of Treg from cervical lymph nodes and spleen. CD4+ CD25hi cells are 92% FoxP3+. Shown are dotplots from one representative experiment. (D) To determine the suppressive capability of regulatory T cells in vitro CD4+ CD25hi T cells (Treg) from cervical lymph nodes and spleens of 0 Gy or 15 Gy whole thorax irradiated mice were isolated at 21 days post-irradiation by FACS sorting. Treg were co-cultured at a ratio of 1:1 with CFSE-labeled CD4+ responder T cells and with antigen-presenting cells in the presence of αCD3. Proliferation of responder T cells was measured by loss of the fluorescent dye CFSE and inhibition was calculated accordingly. Data from three individual hosts are shown with mean values ± SEM.