IL-10-deficient T_reg cells prevent colitis. (a) Body weight of Rag1^−/− mice given sorted Il10^−/− or wild-type (WT) CD4⁺CD25⁺ T_reg cells, together with CD4⁺CD45RB^hi T cells, or of Rag1^−/− mice given CD4⁺CD45RB^hi T cells alone (control; None), presented relative to initial body weight. Data are pooled from two independent experiments with six mice each (error bars, s.d.). (b) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. NS, not significant. Data are pooled from at least two independent experiments.

Rag1^−/− host IL-10 is required for T_reg cell function. (a) Body weight of Rag1^−/− or Il10^−/−Rag1^−/− hosts given CD4⁺CD45RB^hi T cells plus sorted Foxp3^gfp T_reg cells, presented relative to initial body weight. Data are pooled from two independent experiments with ten mice each (error bars, s.d.). (b) Proximal colon of Rag1^−/− and Il10^−/−Rag1^−/− mice at 6 weeks after the donor cell transfer described in a; sections are stained with hematoxylin and eosin. Original magnification, ×100; scale bars, 100 µm. Data are representative of one of three independent experiments. (c) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s t-test). Data are pooled from three independent experiments with a total of nine mice.

Foxp3 is downregulated in Il10^−/−Rag1^−/− recipients. (a) Composite ratios of CD45.1⁺ to CD45.2⁺ TCRβ⁺CD4⁺ cells in the spleen (Spl), PLNs, MLNs and LPL of Rag1^−/− or Il10^−/−Rag1^−/− recipient mice at 6 weeks after injection of 4 × 10⁵ CD4⁺CD45RB^hi T cells derived from C57BL/6 (CD45.1⁺) mice, plus 1 × 10⁵ Foxp3^gfp (CD45.2⁺) T_reg cells. (b) Foxp3 expression in the cells in a, gated on TCRβ⁺CD4⁺ CD45.2⁺ cells. Bracketed lines indicate the Foxp3⁻ population. max, maximum. (c) Foxp3⁻ cells in the gated TCRβ⁺CD4⁺ CD45.2⁺ populations in b. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s t-test). Data are pooled from three independent experiments with a total of nine mice (a (mean and s.d.) and c) or are representative of one of three independent experiments (b).

Loss of function by T_reg cells from Il10^−/−Rag1^−/− recipients. (a,b) Flow cytometry of intracellular IFN-γ in CD4⁺CD45RB^hi T cells derived from C57BL/6 (CD45.1⁺) mice transferred with Foxp3^gfp (CD45.2⁺) T_reg cells into Rag1^−/− or Il10^−/−Rag1^−/− hosts; plots are gated on the TCRβ⁺CD4⁺ CD45.2⁻ progeny of donor CD4⁺CD45RB^hi T cells (a) and the TCRβ⁺CD4⁺ CD45.2⁺ progeny from donor Foxp3^{gfp +} T_reg cells (b), isolated from spleen (Spl), MLNs and LPL in mice at 6 weeks after donor cell injection and then stimulated with PMA and ionomycin. Numbers in plots indicate percent IFN-γ-producing cells. (c) Suppressive function in vitro of sorted TCRβ⁺CD4⁺ CD45.2⁺ cells from MLNs of Rag1^−/− or Il10^−/−Rag1^−/− recipients of CD45.1⁺ CD4⁺CD45RB^hi and CD45.2⁺ CD4⁺CD25⁺CD45RB^lo T_reg cell populations, cultured for 4 d together with CFSE-labeled CD45.1⁺ naive T cells; after stimulation of cultures, CFSE dilution was assessed by flow cytometry. Data are representative of one of three (a,b) or two (c) independent experiments.

Il10rb^−/− T_reg cells fail to prevent colitis. (a) Body weight of Rag1^−/− recipients of C57BL/6 (CD45.1⁺) CD4⁺CD45RB^hi T cells transferred together with wild-type or Il10rb^−/− (CD45.2⁺) T_reg cells, presented relative to initial body weight. Data are pooled from two independent experiments with a total of ten mice (error bars, s.d.). (b) Proximal colon of recipient mice at 6 weeks after injection of cells as described in a; sections are stained with hematoxylin and eosin. Original magnification, ×100; scale bars, 100 µm. Data are representative of one of three independent experiments. (c) Histology scores of sections of the large intestine at 6 weeks after the cell transfer described in a. Each symbol represents an individual mouse; small horizontal lines indicate the mean. *P < 0.001 (two-tailed Student’s t-test). Data are pooled from three independent experiments with a total of nine mice. (d) Foxp3 expression by cells isolated from the spleen, PLNs, MLNs and LPL of the recipient mice in a, with gating on TCRβ⁺CD4⁺CD45.2⁺ cells. Bracketed lines indicate the Foxp3⁻ population. Data are representative of one of three independent experiments with a total of nine mice. (e) Foxp3⁻ cells in the TCRβ⁺CD4⁺ CD45.2⁺ T lymphocyte populations described in d. Each symbol represents an individual mouse; small horizontal bars indicate the mean. *P < 0.001 (two-tailed Student’s t-test). Data are pooled from three independent experiments with a total of nine mice.

Foxp3 is lost ‘preferentially’ by Il10rb^−/− T_reg cells in mice with colitis. (a) Ratio of CD45.1⁺ to CD45.2⁺ CD90.1⁻CD4⁺TCRβ⁺ cells isolated from spleen, PLNs, MLNs and LPL of Rag1^−/− recipients at 6 weeks after injection of 8 × 10⁵ C57BL/6 (CD90.1⁺) CD4⁺CD45RB^hi T cells, transferred with 2 × 10⁴ wild-type (CD45.1⁺) T_reg cells and 2 × 10⁴ Il10rb^−/− (CD45.2⁺) T_reg cells. Data are pooled from two independent experiments with a total of four mice (mean and s.d.). (b) Flow cytometry of Foxp3 expression by cells isolated from a Rag1^−/− recipient mouse as described in a, with gating on CD90.1⁻CD4⁺TCRβ⁺CD45.2⁻ cells (wild-type T_reg cells) or CD90.1⁻CD4⁺TCRβ⁺CD45.2⁺ cells (Il10rb^−/− T_reg cells). Bracketed lines indicate the Foxp3⁻ population. Data are representative of one of two independent experiments. (c) Foxp3⁻ cells in the CD90.1⁻CD4⁺TCRβ⁺CD45.2⁻ (wild-type T_reg) and CD90.1⁻CD4⁺TCRβ⁺CD45.2⁺ (Il10rb^−/− T_reg) populations isolated from Rag1^−/− mice as described in a. Each symbol represents an individual mouse; small horizontal bars indicate the mean. Data are pooled from two independent experiments with a total of four mice. (d) Foxp3⁻ cells in CD90.1⁻CD4⁺TCRβ⁺CD45.2⁻ (wild-type T_reg) or CD90.1⁻CD4⁺TCRβ⁺CD45.2⁺ (Il10rb^−/− T_reg) populations isolated from Rag1^−/− recipients of 4 × 10⁵ C57BL/6 (CD90.1⁺) CD4⁺CD45RB^hi T cells, transferred together with 1 × 10⁵ wild-type (CD45.1⁺) and 1 × 10⁵ Il10rb^−/− (CD45.2⁺) T_reg cells. Each symbol represents an individual mouse; small horizontal bars indicate the mean. * P < 0.01; ** P < 0.001 (two-tailed Student’s t-test). Data are pooled from two independent experiments with a total of four mice.

Kinetics of IL-10 expression by T_reg cells and host cells. (a) GFP⁺ cells in IL-10 reporter mice. Numbers adjacent to outlined areas (top row) indicate percent GFP⁺ cells among gated naive splenocytes (TCRβ⁺CD4⁺CD45RB^hi) and T_reg splenocytes (TCRβ⁺CD4⁺CD45RB^loCD25⁺) from Il10^gfp mice; numbers in top right quadrants (bottom row) indicate percent CD45⁺GFP⁺ cells in tissues from Il10^gfpRag1^−/− mice. MFI, mean fluorescence intensity. (b) Flow cytometry of GFP⁺ cells in tissues 7 d after transfer of a mixture of CD45.1⁺ CD4⁺CD45RB^hi T cells and CD45.2⁺ Il10^gfp T_reg cells (ratio, 4:1). Top row, T_reg cells gated as CD45.2⁺ TCRβ⁺CD4⁺ cells; bottom row, gated TCRβ⁻CD4⁻ nonlymphoid cells. Numbers adjacent to outlined areas and in top right quadrants indicate percent GFP⁺ cells. (c) Real-time PCR analysis of Il10 mRNA in sorted T_reg cells (left), CD11b⁺ cells (middle) and CD11c⁺ dendritic cells (right) from various sites (keys) before transfer (0) or at 1, 2 and 6 weeks after transfer as in b. Data are from representative one of two independent experiments with six mice (a,b) or are pooled from two independent experiments with six mice (c; mean and s.d.).

IL-10-producing CD11b⁺ myeloid cells prevent the downregulation of Foxp3. Flow cytometry of Foxp3 expression 3 weeks after the injection of 5 × 10⁶ Rag1^−/− or Il10^−/−Rag1^−/− intestinal CD11c⁺CD11b⁺F4/80⁺ cells (a) or CD11c⁺CD11bF4/80⁻ cells (b) into Il10^−/−Rag1^−/− recipients, transferred intravenously on days 0 and 7 (where ‘day 0’ is the day of T cell transfer) with 4 × 10⁵ CD4⁺ CD45RB^hi (CD45.1⁺) cells in the presence of 1 × 10⁵ (CD45.2⁺) T_reg cells from Foxp3^gfp mice. Plots are gated on TCRβ⁺CD4⁺CD45.2⁺ splenocytes. Data are representative of one of two independent experiments with a total of three mice.