A central role for induced regulatory T cells in tolerance induction in experimental colitis

Abstract

In addition to thymus-derived or natural T regulatory (nT(reg)) cells, a second subset of induced T regulatory (iT(reg)) cells arises de novo from conventional CD4(+) T cells in the periphery. The function of iT(reg) cells in tolerance was examined in a CD45RB(high)CD4(+) T cell transfer model of colitis. In situ-generated iT(reg) cells were similar to nT(reg) cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nT(reg) cells resolved the colitis, but only when iT(reg) cells were also present. Although iT(reg) cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nT(reg) "genetic signature," indicative of developmental and possibly mechanistic differences. These results identified a functional role for iT(reg) cells in vivo and demonstrated that both iT(reg) and nT(reg) cells can act in concert to maintain tolerance.

FIGURE 1

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.

FIGURE 2

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.

FIGURE 3

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).

FIGURE 4

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.