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Figure 3 | Reciprocal expression of Foxp3 and IL-17 in T cells during differentiation. FACS-sorted naive CD4*CD62L"'GFP~ T cells from 2D2 X Foxp3—GFP knockin mice were stimulated with soluble anti-CD3, antigen-presenting cells and the indicated cytokines. a, Foxp3 (GFP) and cytokine (IL-17 or IFN-y) intracellular expression in CD4* T cells after 5 days. b, Expression of IFN-y, Foxp3 and IL-17 mRNA relative to control actin (in triplicate wells +s.d.). ¢, Proliferation in triplicate wells (+s.d.) of CD4*CD25° cells and Tror-g+it-6 cells (T cells differentiated in the presence of TGF-8 and IL-6) in the presence of angen presenting cells, soluble anti-CD3 and different concentrations of CD4*CD25* T cells or Trep. g cells (T cells generated in the presence of TGF-8) measured by P H]thymidine incorporation.

Figure 3 | Reciprocal expression of Foxp3 and IL-17 in T cells during differentiation. FACS-sorted naive CD4*CD62L"'GFP~ T cells from 2D2 X Foxp3—GFP knockin mice were stimulated with soluble anti-CD3, antigen-presenting cells and the indicated cytokines. a, Foxp3 (GFP) and cytokine (IL-17 or IFN-y) intracellular expression in CD4* T cells after 5 days. b, Expression of IFN-y, Foxp3 and IL-17 mRNA relative to control actin (in triplicate wells +s.d.). ¢, Proliferation in triplicate wells (+s.d.) of CD4*CD25° cells and Tror-g+it-6 cells (T cells differentiated in the presence of TGF-8 and IL-6) in the presence of angen presenting cells, soluble anti-CD3 and different concentrations of CD4*CD25* T cells or Trep. g cells (T cells generated in the presence of TGF-8) measured by P H]thymidine incorporation.

Related Figures (4)

Figure 1| Inhibition of T,.. development by different cytokines. a—c, FACS- sorted CD4+GEP” (Foxp3_ ) cells from Foxp3—GEP knockin mice were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days (a, b) or anti- CD3 plus antigen-presenting cells for 48 h in the presence of different cytokines (c). a, Percentage of GFP-expressing cells induced by various cytokines in the presence of TGF-6 (the percentage of GFP* cells induced in the presence of TGF-8 was kept at 100%). b, GFP expression in CD4* T cells. c, Amount of IL-17 produced in triplicate wells +s.d. MOG)-specific T-cell receptor (TCR) transgenic mouse (hereafter called 2D2)"*. In vitro activation of unsorted splenic T cells from the transgenic mice in the presence MOG35-—55 peptide and recombinant IL-23 resulted in the generation of 6.5% of IL-17-producing CD4* T cells (Fig. 2a). Because the IL-23 receptor is expressed only on activated/memory T cells’*’*, we wanted to investigate further whether IL-23 could guide differentiation of naive T cells into T};17 cells. To address this question, naive 2D2 transgenic T cells (CD4*CD62L")
Figure 1| Inhibition of T,.. development by different cytokines. a—c, FACS- sorted CD4+GEP” (Foxp3_ ) cells from Foxp3—GEP knockin mice were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days (a, b) or anti- CD3 plus antigen-presenting cells for 48 h in the presence of different cytokines (c). a, Percentage of GFP-expressing cells induced by various cytokines in the presence of TGF-6 (the percentage of GFP* cells induced in the presence of TGF-8 was kept at 100%). b, GFP expression in CD4* T cells. c, Amount of IL-17 produced in triplicate wells +s.d. MOG)-specific T-cell receptor (TCR) transgenic mouse (hereafter called 2D2)"*. In vitro activation of unsorted splenic T cells from the transgenic mice in the presence MOG35-—55 peptide and recombinant IL-23 resulted in the generation of 6.5% of IL-17-producing CD4* T cells (Fig. 2a). Because the IL-23 receptor is expressed only on activated/memory T cells’*’*, we wanted to investigate further whether IL-23 could guide differentiation of naive T cells into T};17 cells. To address this question, naive 2D2 transgenic T cells (CD4*CD62L")
Figure 2 | Cytokines required for the generation of T,,17 cells. a, Splenocytes from 2D2 TCR transgenic mice were stimulated with MOG35-55 peptide in neutral conditions or with IL-23. b, c, FACS-sorted naive CD4*CD62L"" T cells from 2D2 TCR transgenic mice were stimulatec with soluble anti-CD3, C57BL/6 irradiated spleen cells and the indicated cytokines plus neutralizing antibodies. Five days after the activation, cells were re-stimulated with PMA/ionomycin and subjected to intracellular cytokine staining. The contour plots represent IL-17 expression in CD4* cells.
Figure 2 | Cytokines required for the generation of T,,17 cells. a, Splenocytes from 2D2 TCR transgenic mice were stimulated with MOG35-55 peptide in neutral conditions or with IL-23. b, c, FACS-sorted naive CD4*CD62L"" T cells from 2D2 TCR transgenic mice were stimulatec with soluble anti-CD3, C57BL/6 irradiated spleen cells and the indicated cytokines plus neutralizing antibodies. Five days after the activation, cells were re-stimulated with PMA/ionomycin and subjected to intracellular cytokine staining. The contour plots represent IL-17 expression in CD4* cells.
Table 1 | EAE in 2D2 and 2D2 x TgTGF-8 mice Mice were immunized with MOG35-55 peptide emulsified in complete Freund's adjuvant and monitored for the development of EAE. Statistical analysis was performed by comparing thre Zroups using one-way analysis of variance (ANOVA) followed by a Newman-Keuls multiple comparison as a post-hoc test. ‘P<0.05. +P < 0.05. tP < 0,001. $P < 0.01.
Table 1 | EAE in 2D2 and 2D2 x TgTGF-8 mice Mice were immunized with MOG35-55 peptide emulsified in complete Freund's adjuvant and monitored for the development of EAE. Statistical analysis was performed by comparing thre Zroups using one-way analysis of variance (ANOVA) followed by a Newman-Keuls multiple comparison as a post-hoc test. ‘P<0.05. +P < 0.05. tP < 0,001. $P < 0.01.
Figure 4 | Enhanced IL-17 production by T cells and CNS autoimmunity in TGF-8 transgenic mice. a, b, Splenocytes from naive C57BL/6 wild-type (WT) mice (a, left panel), wild-type and TgTGF-8 mice immunized with MOG35-55 peptide (a, right panel), and 2D2 and 2D2 X TgTGF-8 mice immunized with MOG35-—55 peptide (b), were stimulated with lipopolysaccharide (for IL-6) or MOG35-55 peptide (for other cytokines). Data represent mean cytokine produced in each group (nm = 3) + s.d. c, Wild-type C57BL/6 (n = 13), 2D2 (n = 11) and 2D2 X TgTGF-B (n = 13) mice were immunized with MOG35-—55 in CFA and monitored for clinical signs of EAE (top panel). Linear regression analysis of the individual disease curves reveals a highly significant difference (P < 0.0001) between each of the EAE groups (bottom panel). d, Frequency of IL-17- and Foxp3-expressing CD4* T cells isolated from the CNS of 2D2 x TgTGF-6 or 2D2 mice at the peak of disease.
Figure 4 | Enhanced IL-17 production by T cells and CNS autoimmunity in TGF-8 transgenic mice. a, b, Splenocytes from naive C57BL/6 wild-type (WT) mice (a, left panel), wild-type and TgTGF-8 mice immunized with MOG35-55 peptide (a, right panel), and 2D2 and 2D2 X TgTGF-8 mice immunized with MOG35-—55 peptide (b), were stimulated with lipopolysaccharide (for IL-6) or MOG35-55 peptide (for other cytokines). Data represent mean cytokine produced in each group (nm = 3) + s.d. c, Wild-type C57BL/6 (n = 13), 2D2 (n = 11) and 2D2 X TgTGF-B (n = 13) mice were immunized with MOG35-—55 in CFA and monitored for clinical signs of EAE (top panel). Linear regression analysis of the individual disease curves reveals a highly significant difference (P < 0.0001) between each of the EAE groups (bottom panel). d, Frequency of IL-17- and Foxp3-expressing CD4* T cells isolated from the CNS of 2D2 x TgTGF-6 or 2D2 mice at the peak of disease.
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