Th17_TGF-β1 cells present a regulatory phenotype.

<p>(A) IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and then analyzed by real-time PCR for mRNA expression of several transcription factors and cytokines (n = 3). (B) IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and then reactivated for 4 hrs with PMA plus ionomycin to assess cytokine production by CBA or (C) in the presence of PMA, ionomycin and brefeldin A to analyze GM-CSF production by FACS (n = 5). (D) Percentage of GM-CSF+ cells within IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells (n = 5). Data are presented as mean ± S.E.M. *p<0.05, **p<0.01 and ***p<0.001 determined by t-test (A) or Mann-Whitney test (B and D).</p

Th17_TGF-β1 but not Th17_IL-23 cells hydrolyze ATP to adenosine in a CD39-and CD73-dependent manner and survive in the presence of high doses of ATP.

<p>IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and cultured for 1 hr with 10 μM ATP in the presence of 50 μM ARL67156 or 50 μM APCP. Supernatants were then analyzed by HPLC to assess (A and B) ATP and (C and D) AMP hydrolysis (n = 5). (E) Representative FACS analysis of Th17 cell survival (Annexin V-/PI-) in the presence of graded doses of ATP. (F) Percentage of Th17 cell survival in the presence of ATP (n = 3). (G) IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were sorted and then analyzed by real-time PCR to assess mRNA encoding P2X7 receptor (n = 4). Data are presented as mean ± S.E.M. *p<0.05 and **p<0.01 determined by Mann-Whitney test (B and D), two-way analysis of variance (F) or t-test (G).</p

ATP hydrolysis by CD39 on Th17_TGF-β1 cells promotes their conversion into IL-10-producing cells.

<p>(A) IL-10 production by IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells restimulated for 3 days with anti-CD3 and anti-CD28 antibodies in the presence and absence of Tr1 polarizing cytokines (TGF-β1, IL-21 and IL-27), 50 μM ATP and 250 μM ARL67156. IL-10 production was analyzed by CBA (n = 4). (B) Th17_IL-23 cells from wild-type and P2X7R knockout mice were restimulated for 3 days with anti-CD3 and anti-CD28 antibodies and IL-10 production was analyzed by CBA (n = 3). Data are presented as mean ± S.E.M. *p<0.05; **p<0.01 determined by repeated measures analysis of variance.</p

Th17_TGF-β1 cells are less colitogenic than Th17_IL-23 cells and produce IL-10 and IFN-γ <i>in vivo</i>.

<p>1.3x10⁶ IL-17-GFP+ Th17_TGF-β1 and Th17_IL-23 cells were transferred to Rag1^-/- mice. (A) The weight of mice was measured over the course of 6 weeks after adoptive transfer of Th17 cells (n = 5–8 mice per group). (B) Colon length was measured 6 weeks following transfer of Th17 cells (n = 5). (C) Clinical score was calculated based on weight loss and colon length 6 weeks after adoptive transfer of Th17 cells (n = 5). (D) Colonic histopathology. H&E and alcian blue staining, original magnification 20X. Scale bar 100 μm (E) To determine intestinal cytokine production, intestinal tissues of Th17 recipient mice were cultured for 24 hs at 37°C and 5% CO₂ and production of several cytokines was analyzed by CBA (n = 6). (F and G) Representative FACS analysis of IL-17, IL-10 and IFN-γ production by Th17_TGF-β1 and Th17_IL-23 cells 6 weeks after adoptive transfer to Rag1^-/- mice. Data are presented as mean ± S.E.M. *p<0,05, **p<0,01 and ***p<0,001 comparing Th17_TGF-β1 and Th17_IL-23; ᵜᵜp<0,01 comparing PBS and Th17_TGF-β1; ^¤¤¤p<0,001 comparing PBS and Th17_IL-23 determined by two-way analysis of variance (A). *p<0,05 determined by Kruskal-Wallis test (B,C and E).</p