Figure 2.

IL-27 is required for the maintenance of Treg population. (A-C) 4×10⁵ CD45.1⁺ WT CD4⁺CD45RB^high cells were co-injected to Rag1^−/− mice with 1×10⁵ CD45.2 CD4⁺CD25^high cells sorted from WT (filled symbols) or IL-27Rα^−/− (open) spleens. (A) Relative body weight compared to the original body weight at each time point, averaged from a total of seven recipients. Data shown are combined from two independent experiments. (B) Representative H&E staining of the large intestine of recipients that received CD4⁺CD45RB^high cells and WT (top) or IL-27Rα^−/− (bottom) Treg at 4:1 ratio. (C) Histological scores of large intestine. Each dot represents the average of 4 specimens obtained from a single recipient. Horizontal lines indicate the mean. * P < 0.001 (two-tailed Student’s t-test). (D-E) T lymphocytes were transferred as in (A). Four weeks post transfer, cells isolated from MLN and large intestine were stained for Foxp3. A representative dot plot showing Foxp3 and CD45 staining of CD4⁺TCRβ⁺ donor cells isolated from the recipients that received a 4:1 ratio of CD45.1⁺ CD4⁺CD45RB^high T cells and either WT (left) or IL-27Rα^−/− (right) CD45.2⁺ Treg. Numbers indicate the percentage of the cells in each quadrant. (E) Compilation of the percentages of CD45.1⁻ cells in CD4⁺TCRβ⁺ total donor cells isolated from MLN and large intestine of the recipients that received WT (filled circles) or IL-27Rα^−/− (open) Treg at the indicated ratio with CD4⁺CD45RB^high naïve T cells. Small horizontal lines indicate the mean. Differences between WT and IL-27Rα^−/− Treg were significant. * P < 0.001 (two-tailed Student’s t-test). (F-G) Analysis at three days after transfer to Rag1^−/− recipients of 8×10⁵ CD45.1⁺ CD4⁺CD45RB^high cells with 2×10⁵ WT or IL-27Rα^−/− Treg (CD45.1⁻). (F) Top panels represent CD4 and TCRβ staining of total spleen cells at 3 days post transfer. Numbers in the plots indicate the percentage of CD4⁺TCRβ⁺ cells gated as shown. CD4⁺TCRβ⁺ splenic (middle panels) or MLN (bottom panels) cells gated as above were further analyzed for Foxp3 expression by CD45.1⁺ or CD45.1⁻ cells. Percentages are indicated in each quadrant. (G) Total number of spleen cells (top) or CD4⁺ T cells (bottom) isolated from the recipients of either WT Treg (black bar) or IL-27Rα^−/− Treg (blank bar) with WT CD45.1⁺ naïve CD4⁺ T cells, or naïve CD4⁺ T cells only (gray bar). Each bar indicates the average of 4 recipients in each condition (error bar; s.d.). *P < 0.001 (two-tailed Student’s t-test).