1. Volume 142, Issue 7, Pages 1526-1535.e6 (June 2012)
Inhibition of Interleukin-17 Promotes Differentiation of CD25– Cells Into Stable T Regulatory Cells in Patients With Autoimmune Hepatitis 
Maria Serena Longhi, Rodrigo Liberal, Beth Holder, Simon C. Robson, Yun Ma, Giorgina Mieli–Vergani, Diego Vergani 
Gastroenterology 
Volume 142, Issue 7, Pages e6 (June 2012)
DOI: /j.gastro
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2. Figure 1
Frequency of IL-17–producing lymphocytes. Frequency of IL-17–producing lymphocytes was determined by intracellular cytokine staining. (A and B) Mean (+SEM) frequency of (A) IL-17–producing CD4+CD25− and (B) CD4+CD25high cells isolated from PBMCs (left) and from expanded CD4+CD25− cells (right) in patients with AIH (black bars) and HS (gray bars). (C) Dot plots showing the frequency of IL-17–producing CD25− or CD25high cells purified from freshly isolated PBMCs or from expanded CD25− cells from a representative patient with AIH and from a representative HS.
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3. Figure 2
RORC gene expression. RORC gene expression was evaluated by real-time polymerase chain reaction. The panels show mean (+SEM) RORC messenger RNA units of (A) CD4+CD25− and (B) CD4+CD25high cells isolated from PBMCs (left) or from expanded CD4+CD25− cells (right) in patients with AIH (black bars) and HS (gray bars).
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4. Figure 3
Inhibition of CD25− cell proliferation by Tregs generated under different neutralizing antibody/cytokine combinations. The columns represent the percentage inhibition of CD25− target cell proliferation after addition of Tregs generated in the presence of different neutralizing antibody/cytokine combinations. Percentages of inhibition are indicated at the top of each column. The percentage of inhibition obtained in the presence of Tregs treated with anti–IL-6, anti–IL-1β neutralizing antibodies, and rTGF-β was higher than that obtained in the presence of Tregs generated under different antibody/cytokine combinations (P < .05 for all). ngTreg/CD25− cell ratio is 1/8.
Gastroenterology  , e6DOI: ( /j.gastro )
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5. Figure 4
Effect of anti–IL-6, anti–IL-1β neutralizing antibodies, and rTGF-β on phenotype and suppressive function of CD127 depleted ngTregs obtained from CD4+CD25− cells. (A) Mean (+SEM) CD25, FOXP3, and CTLA-4 MFI and (B) mean (+SEM) frequency of IFN-γ–, IL-17–, and IL-10–producing ngTregs before and after CD127 depletion in patients with AIH (left) and HS (right). Black and dark gray bars refer to ngTregs obtained from CD4+CD25− cells in the absence and light gray bars in the presence of the inhibitory cocktail. (C) Mean (+SEM) counts per minute of CD25− cells from patients with AIH (left) and HS (right) cultured either alone or with untreated ngTregs; untreated ngTregs after CD127 depletion; ngTregs obtained from CD25− cells treated with anti–IL-6, anti–IL-1β, and rTGF-β; and ngTregs obtained from CD25− cells treated with anti–IL-6, anti–IL-1β, and rTGF-β after CD127 depletion. ngTreg/CD25− cell ratio is 1/8.
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6. Figure 5
Inhibition of CD25− cell proliferation following addition of ngTregs treated with separate or combined anti–IL-17 maneuvers. Mean (+SEM) counts per minute of CD25− target cells cultured alone; with untreated ngTregs; with ngTregs after neutralizing antibody/cytokine treatment, siRNA treatment, Th17 depletion; or after treatment with the 3 maneuvers combined in patients with AIH (left) and in HS (right). ngTreg/CD25− cell ratio is 1/8.
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7. Supplementary Figure 1
Production of newly generated Tregs. To newly generate CD4+CD25high T cells (ngTregs), immunomagnetic beads were used to separate CD4+CD25− cells from naturally occurring CD4+CD25high T cells within PBMCs. Purified CD4+CD25− cells were then exposed to anti-CD3/anti-CD28 T-cell expander/high-dose IL-2 and cultured for 4 weeks. At the end of culture, newly generated CD4+CD25high cells were immunomagnetically isolated and tested directly or after exposure to one of the following 4 treatments: (1) Th17 depletion (at the end of the second week of culture), (2) treatment with anti–IL-6/anti–IL-1β/rTGF-β (added at the same time of T-cell expander and IL-2), (3) transfection with RORC siRNA (at the end of the second and third week of culture), and (4) a combination of strategies 1–3. Purified ngTregs were then tested for their phenotype, cytokine production, and suppressor function.
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8. Supplementary Figure 2
Phenotype of Th17 cells. Dot plots showing the frequency of CD4 cells positive for (A) CCR6, (B) IL-23R, and (C) IFN-γ within IL-17–producing CD25− cells isolated from one representative patient with AIH (left) and one representative HS (right). Cells in A and B are gated on IL-17+ lymphocytes; cells in C are gated on CD4 lymphocytes.
Gastroenterology  , e6DOI: ( /j.gastro )
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9. Supplementary Figure 3
Phenotype of Th17-depleted ngTregs. Results obtained in a representative patient with AIH and a representative HS are shown. (A and B) Frequency of (A) IL-17–producing and (B) RORC+ cells within Th17-depleted ngTregs (purified from 2-week CD4+CD25− cells stimulated with anti-CD3/anti-CD28 T-cell expander at 1 bead per cell and rIL-2 at 300 U/mL) during a 3-week culture period in the absence or presence of proinflammatory challenge with IL-6 and IL-1β, applied simultaneously. (C) MFI of FOXP3 in Th17-depleted ngTregs during the same culture period in the absence or presence of the challenge.
Gastroenterology  , e6DOI: ( /j.gastro )
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10. Supplementary Figure 4
Transwell experiments. ngTregs from 2 HS and 3 patients with AIH were added to autologous CD4+CD25− cells seeded in the lower chamber of a 24-well plate. ngTregs were added either directly or separated by a Transwell semipermeable membrane. CD25− cell proliferation was tested by thymidine incorporation. The plots show the proliferation of CD25− cells in (A) patients with AIH and (B) HS. Columns in each plot represent the mean (+SEM) counts per minute of CD25− cells cultured on their own and after addition of ngTregs in the presence or absence of Transwell membrane.
Gastroenterology  , e6DOI: ( /j.gastro )
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