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. Author manuscript; available in PMC: 2010 Dec 8.

Published in final edited form as: Nat Immunol. 2008 Nov 9;9(12):1347–1355. doi: 10.1038/ni.1677

(a) Suppression assay with naïve CD4⁺CD62L⁺ T cells from IL-10.GFP reporter mice activated with or without cytokines (IL-4 + TGF-β) and non-treated IL-10.GFP⁺ T cells. Naïve wild-type Foxp3⁻ responder T cells were cultured alone, together with natural-occurring T_regs or with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells); when indicated, anti-IL-9 or anti-IL-10 was added to the culture. Proliferation was assessed in triplicate wells by ³H-thymidine incorporation and is presented as mean ± s.d. Data represent one of three experiments. (b) RAG-1-deficient mice were reconstituted with sorted CD4⁺Foxp3-GFP⁻CD45RB^hiT cell sub populations in PBS. Mice received CD4⁺Foxp3-GFP⁻CD45RB^hiT cell alone or in combination with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells) or CD4⁺Foxp3⁺ naturally occurring T_reg cells. Mice were examined for their weight loss for 9 weeks. Each time point represents an average of the calculated weight of all mice present per group (5–8 mice) of 2 independent experiments. The total colonic score was calculated as the average of the individual scores from the sections of proximal colon, mid-colon, and distal colon²⁸. The mice in Group 1 (CD45RB^hi effector T) had a pathology score of 7.5 ± 3.5; Group 2 (CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells): 8.8±2.3; Group 3, (T4-IL-10.GFP⁺ cells): 5.7±2.6 and Group 4, (CD45RB^hi T_eff cells + T_reg cells) : 3.4±1.5. The difference in the mean score was statistically different between Group 2 and 4 (P = 0.01) and Group 3 and 4 (P = 0.03) (Mann-Whitney one tailed U test). (c) Representative histopathology of colitis and peripheral neuritis in Rag1^−/−mice reconstituted with the following cell types. Group 1, CD45RB^hi effector T cells (mononuclear cell inflammation in the lamina propria, muscularis and adventitial tissues (arrow)). Group 2, CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells (T4-IL-10.GFP⁺ cells); inflammation in the lamina propria and submuscularis (arrow) and blue staining of myelin; higher power of a nerve root (middle) with inflammatory cell infiltration (arrows) and myelin breakdown product in vacuoles (right, arrow). Group 3, T4-IL-10.GFP⁺ cells alone; inflammation in the lamina propria, muscularis mucosa and adventitial tissues (left, arrow); a spinal nerve root shows vacuolation with myelin breakdown products (right, arrows). Group 4, CD45RB^hi T_eff cells + T_reg cells; intact mucosal architecture and the muscularis (left), intact spinal cord white matter and nerve root (middle) and higher power of intact myelin in cauda equina nerve root (right) a–d. Luxol fast blue-H&E, e–h. Bar in Group 1 corresponds to 200 μm, in 100 μm in c and h. Bar in D corresponds to 100 μm in D, F and H.

(a) Suppression assay with naïve CD4⁺CD62L⁺ T cells from IL-10.GFP reporter mice activated with or without cytokines (IL-4 + TGF-β) and non-treated IL-10.GFP⁺ T cells. Naïve wild-type Foxp3⁻ responder T cells were cultured alone, together with natural-occurring T_regs or with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells); when indicated, anti-IL-9 or anti-IL-10 was added to the culture. Proliferation was assessed in triplicate wells by ³H-thymidine incorporation and is presented as mean ± s.d. Data represent one of three experiments. (b) RAG-1-deficient mice were reconstituted with sorted CD4⁺Foxp3-GFP⁻CD45RB^hiT cell sub populations in PBS. Mice received CD4⁺Foxp3-GFP⁻CD45RB^hiT cell alone or in combination with IL-10.GFP⁺ cells stimulated with TGF-β plus IL-4 (T4-IL-10.GFP⁺ cells) or CD4⁺Foxp3⁺ naturally occurring T_reg cells. Mice were examined for their weight loss for 9 weeks. Each time point represents an average of the calculated weight of all mice present per group (5–8 mice) of 2 independent experiments. The total colonic score was calculated as the average of the individual scores from the sections of proximal colon, mid-colon, and distal colon²⁸. The mice in Group 1 (CD45RB^hi effector T) had a pathology score of 7.5 ± 3.5; Group 2 (CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells): 8.8±2.3; Group 3, (T4-IL-10.GFP⁺ cells): 5.7±2.6 and Group 4, (CD45RB^hi T_eff cells + T_reg cells) : 3.4±1.5. The difference in the mean score was statistically different between Group 2 and 4 (P = 0.01) and Group 3 and 4 (P = 0.03) (Mann-Whitney one tailed U test). (c) Representative histopathology of colitis and peripheral neuritis in Rag1^−/−mice reconstituted with the following cell types. Group 1, CD45RB^hi effector T cells (mononuclear cell inflammation in the lamina propria, muscularis and adventitial tissues (arrow)). Group 2, CD45RB^hi effector T cells + (TGF-β + IL-4) IL-10.GFP⁺ cells (T4-IL-10.GFP⁺ cells); inflammation in the lamina propria and submuscularis (arrow) and blue staining of myelin; higher power of a nerve root (middle) with inflammatory cell infiltration (arrows) and myelin breakdown product in vacuoles (right, arrow). Group 3, T4-IL-10.GFP⁺ cells alone; inflammation in the lamina propria, muscularis mucosa and adventitial tissues (left, arrow); a spinal nerve root shows vacuolation with myelin breakdown products (right, arrows). Group 4, CD45RB^hi T_eff cells + T_reg cells; intact mucosal architecture and the muscularis (left), intact spinal cord white matter and nerve root (middle) and higher power of intact myelin in cauda equina nerve root (right) a–d. Luxol fast blue-H&E, e–h. Bar in Group 1 corresponds to 200 μm, in 100 μm in c and h. Bar in D corresponds to 100 μm in D, F and H.