Volume 141, Issue 1, Pages 217-226.e2 (July 2011) Activation of the Receptor NKG2D Leads to Production of Th17 Cytokines in CD4+ T Cells of Patients With Crohn's Disease  Benjamin Pariente, Iulia Mocan, Matthieu Camus, Charles–Antoine Dutertre, Julien Ettersperger, Pierre Cattan, Jean–Marc Gornet, Nicolas Dulphy, Dominique Charron, Marc Lémann, Antoine Toubert, Matthieu Allez  Gastroenterology  Volume 141, Issue 1, Pages 217-226.e2 (July 2011) DOI: 10.1053/j.gastro.2011.03.061 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 CD4+NKG2D+ T cells are a major source of IL-17 in CD. (A–D) PBLs (n = 42) and LPLs (n = 16) were isolated from patients with active CD and stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3+ CD4+, CD4+NKG2D+, and CD4+NKG2D− T-cell populations. Scattergrams show percentages of overall IL-17–positive cells among (A) LP and PB CD4+ T cells (n = 13), (B) LP CD4+NKG2D+ and CD4+NKG2D− T cells (n = 16), and (C) PB CD4+NKG2D+ and CD4+NKG2D− T cells (n = 42). Median values are indicated by a bar. *P < .05, **P < .005. (D and E) These figures represent flow cytometric analysis of IL-17 intracellular staining in (D) LP and (E) PB CD4+NKG2D+ (right dot plot) and CD4+NKG2D− (left dot plot) T cells in 2 patients. Plots are representative of (D) 16 and (E) 42 independent patients with CD. Isotype control antibody staining was used for gate settings. (F) LP mononuclear cells from patients with active CD (n = 4) were fluorescence-activated cell sorted for CD4−, CD4+NKG2D+, and CD4−NKG2D− T cells, and gene expression was profiled. RORC (white bars) and IL-17A (black bars) gene expression was assessed by Q-RT-PCR and normalized to CD3e RNA. *P < .05. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 CD4+NKG2D+ T cells exhibit Th17 and NK markers. Flow cytometry analysis of CCR6, IL-23R, CD161, and NKG2D expression was performed on (A and B) LPLs (n = 12) and (C–E) PBLs (n = 26) isolated from patients with active CD. Scattergrams indicate percentages of (A) CCR6 and (B) IL-23R expression on CD3+ CD4+NKG2D+, CD4+NKG2D−, and CD8+NKG2D+ LP T-cell populations. (C) This figure represents flow cytometric analysis of IL-23R staining in PB CD4+NKG2D+ and CD4+NKG2D− T cells in one patient. The plot is representative of n = 26 independent CD patients. Isotype control antibody staining was used for gate settings. (D) Graphics indicate proportions of PB CD4+ T cells expressing CD161 in patients with CD (n = 26) as compared with patients with UC (n = 12) and HCs (n = 10). (E) Scattergrams indicate percentages of CD161-positive cells among CD3+ CD4+NKG2D+, CD4+NKG2D−, and CD8+NKG2D+ PB T-cell populations. Median values are indicated by a bar. *P < .05, **P < .005. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 NKG2D expression identifies IL-17–producing cells. PBLs were stimulated with PMA/ionomycin before IL-17 intracellular staining. Analysis of IL-17 production was gated on CD4+ T cells. (A) This figure is representative of analysis performed in 10 patients with active CD. NKG2D and CD161 expression in IL-17–negative (left dot plot) and IL-17–positive (right dot plot) CD4+ T cells in one patient is shown. (B and C) Expressions of CD161 (gray bars) and NKG2D (black bars) on (B) CD4+IL-17− and (C) CD4+IL-17+ lymphocytes from 10 patients with CD are plotted. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 NKG2D costimulation significantly enhances IL-17 secretion triggered by the TCR in CD4+ T cells. (A–D) PBLs isolated from 7 patients with CD were cultured in the presence of P815 cell lines transfected or not with MICA coated with an immunoglobulin G1 antibody (isotype) or with an anti-CD3 agonist antibody at 1 μg/mL concentration and/or with an anti-CD28 antibody at 5 μg/mL concentration. Analysis of (A) IL-17, (B) IFN-γ, and (C) TNF-α intracellular staining was gated on CD3+ CD4+ T cells. (D) Analysis of IL-17 intracellular staining was gated on CD3+ CD4+NKG2D+ T cells. Histograms represent percentage of CD4+ T cells expressing IL-17 under different stimulations. *P < .05, **P < .005. (E) In this experiment, PBLs of one patient were cultured in the presence of P815 cell lines transfected or not with ULBP2, coated with an IgG1 antibody (isotype) or with an anti-CD3 agonist antibody. IL-17 production versus IFN-γ and TNF-α production and CD107a expression were analyzed in gated CD3+CD4+ T cells. The dot plots are representative of 7 independent experiments. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 IL-23 drives IL-17 secretion by CD4+NKG2D+ T cells. LP mononuclear cells isolated from 5 patients with CD were cultured with anti-CD2/anti-CD3/anti-CD28 activation beads and with the addition of IL15, IL-12, IL-1β, and/or IL-23 as indicated. After 3 days of culture, the cells were stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3+ CD4+NKG2D+ and CD4+NKG2D− T-cell populations. Individual symbols represent individual patients with CD to visualize trends per patient. Horizontal bars represent mean values. *P < .05. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 IL-17/IL-22– and IL-17/IFN-γ–producing cells are mainly found among CD4+NKG2D+ T cells. LP lymphocytes were stimulated with PMA/ionomycin before IL-17, IL-22, and IFN-γ intracellular staining. Analysis of IL-17/IL-22 and IL-17/IFN-γ production was assessed in CD3+ CD4+NKG2D+, CD4+NKG2D−, and CD8+NKG2D+ LP T cells. This figure is representative of analysis performed in 8 patients with active CD. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 IL-17 secretion by ex vivo PB CD4+ T cells from patients with CD, patients with UC, and HCs. PBLs were isolated from patients with CD (n = 42), patients with UC (n = 9), and HCs (n = 10) and stimulated with PMA/ionomycin. Intracellular staining for IL-17 was analyzed by flow cytometry on gated CD3+ CD4+ T cells. (A) Scattergrams show the percentages of overall IL-17–positive cells among PB CD4+ T cells. Median values are indicated by a bar. *P < .05, **P < .005. (B) This figure represents flow cytometric analysis of IL-17 intracellular staining in PB CD4+ T cells in one patient with CD (left dot plot), one patient with UC (middle dot plot), and one HC (right dot plot). Gates have been set regarding isotype control antibody staining. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 NKG2D expression by PB CD4+ T cells from patients with CD, patients with UC, and HCs. PB lymphocytes were isolated from patients with CD (n = 28), patients with UC (n = 12), and HCs (n = 10). NKG2D staining was analyzed by flow cytometry on gated CD3+ CD4+ T cells. Scattergrams show the percentages of overall NKG2D-positive cells among PB CD4+ T cells. Median values are indicated by a bar. *P < .05, **P < .005. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 3 IL-17 production under NKG2D stimulation through its different ligands expressed on the P815 cell line is different from patient to patient. PBLs isolated from patients with CD were cultured in the presence of P815 cell lines transfected or not with MICA, MICB, ULBP1, ULBP2, or ULBP3, coated with an immunoglobulin G1 antibody (isotype) or with an anti-CD3 agonist antibody at 1 μg/mL concentration. Analysis of IL-17 intracellular staining was gated on CD3+ CD4+ T cells. Histograms represent percentage of CD4+ T cells expressing IL-17 under stimulation through each ligand in 3 different patients. In each patient, IL-17 production was maximal under stimulation of both NKG2D and TCR when NKG2D was triggered by (A) MICA, (B) MICB, or (C) ULBP2 interaction. Gastroenterology 2011 141, 217-226.e2DOI: (10.1053/j.gastro.2011.03.061) Copyright © 2011 AGA Institute Terms and Conditions