Induction of colitis in the absence of functional Foxp3. A, Weight change following the adoptive transfer of 4 × 10⁵ CD4⁺EGFP⁻Thy1.2⁺CD45RB^high T cells isolated from Foxp3^EGFP (dashed red lines; n = 47) and Foxp3^ΔEGFP (dashed black lines; n = 20) mice into Rag^−/− recipients. Control Rag^−/− littermates (gray lines; n = 11) did not receive transferred cells. Color-matched linear regression lines are plotted for each data set. B, Kaplan-Meier survival curves for the mice in A. C, Representative histological sections from the colons of randomly selected mice in A stained with H&E. D, Scatter plot showing the colitis score for each mouse where histology was obtained. E, Representative flow cytometry analysis of the mesenteric lymph nodes from each group (n = 7, 14, and 17, left to right). Numbers denote means for the adjacent gate. F, The total number of T_reg cells found in the mesenteric lymph nodes of healthy Foxp3^EGFP mice (green) and in mice with colitis with either functional (red) or nonfunctional (black) in situ-derived iT_reg cells. G, Representative data from in vitro suppression assays (n = 3) using sorted in situ-derived iT_reg cells (red), sorted nT_reg cells from healthy mice (green), or a 50:50 mix of each population (blue) to inhibit the anti-TCR-induced proliferation of unfractionated splenocytes. The ratio of T_reg cells to CD4⁺ responder splenocytes is indicated. H, Representative cell surface marker analysis of CD4⁺EGFP⁺ mesenteric lymph node T_reg cells from E above stained as indicated.

Treatment of colitis in the presence of in situ-derived iT_reg cells. A, Weight change following colitis induced by adoptive transfer of 4 × 10⁵ CD4⁺EGFP⁻Thy1.2⁺CD45RB^high T cells isolated from Foxp3^EGFP mice. After losing 5–7% of their body weight, recipients were treated by i.p. injection of 1 × 10⁶ sorted in vitro-derived Thy1.1⁺EGFP⁺ iT_reg cells (red; n = 9) or by 1 × 10⁶ sorted Thy1.1⁺EGFP⁺ nT_reg cells (green; n = 6). Color-matched solid lines represent the polynomial fit based on a random coefficient regression model. B, Kaplan-Meier survival curves for the mice in A. C, Representative histological sections from the colons of the mice in A stained with H&E. D, Scatter plots showing colitis scores for each group. E, Representative flow cytometry analysis of the mesenteric lymph nodes from mice treated with nT_reg cells (left panel) or iT_reg cells (right panel). Numbers denote mean values for the quadrant. F, Bar graphs depicting the total number of T_reg cells found in the mesenteric lymph nodes and spleens of treated mice. The proportion of in situ-derived iT_reg cells (red), nT_reg cells (green), and in vitro-derived iT_reg cells (pink) is indicated.

Treatment of colitis in the absence of in situ-derived iT_reg cells. A, Weight change following colitis induced by adoptive transfer of 4 × 10⁵ CD4⁺EGFP⁻ Thy1.1⁺CD45RB^high T cells isolated from Foxp3^ΔEGFP mice. After 5–7% weight loss, recipients were treated by i.p. injection of 0.25 × 10⁶ Thy1.2⁺EGFP⁺ nT_reg cells (red; n = 6), by 0.5 × 10⁶ Thy1.2⁺EGFP⁺ nT_reg cells (orange; n = 6), by 1 × 10⁶ Thy1.2⁺EGFP⁺ nT_reg cells (green; n = 5), or by a mixture of 0.5 × 10⁶ Thy1.1⁺ iT_reg and 0.5 × 10⁶ Thy1.2⁺nT_reg cells (blue; n = 5). Color-matched solid lines represent the polynomial fit based on a random coefficient regression model. B, Kaplan-Meier survival curves for the mice in A. C, Representative histological sections from the colons of the mice in A stained with H&E. D, Scatter plots depicting the colitis scores for each group. E, Representative flow cytometry analysis of the mesenteric lymph nodes from mice treated with nT_reg cells (left panels) or a mixture of both cell types (right panel). Numbers denote mean values for the quadrant or gate. F, Bar graphs depicting the total number of T_reg cells found in the mesenteric lymph nodes and spleens of treated mice. The proportion of nonfunctional in situ-derived ΔiT_reg cells (black), nT_reg cells (green), and in vitro-derived iT_reg cells (pink) is indicated. ns, Not significant).

Overlap among the gene expression profiles of iT_reg cells and nT_reg cells. A comparison of the expression profiles of iT_reg and nT_reg cells with that of T_conv cells identified commonly (A) and selectively (B) expressed genes. All cells were derived from Foxp3^EGFP mice. The Venn diagrams illustrate the comparison groups and the number of distinct and shared genes. The bar graphs show a few of the genes that are differentially expressed in iT_reg and nT_reg cells as mean fold change values derived from three to five independent experiments, each representing mRNA pooled from 4 to 10 mice and scoring p < 0.05 by Student’s unpaired two-tailed t test. C, Scatter plots of mean gene expression values of iT_reg cells vs nT_reg cells (top panel) or activated nT_reg cells (bottom panel). D, Scatter plots of mean gene expression values of iT_reg cells vs ΔiT_reg cells (top panel) or iT_reg cells vs ΔiT_reg cells cultured for 7 days in 50 IU/ml IL-2 (bottom panel). Gene comparisons that are found significant in C and D are highlighted in red.