1. Volume 132, Issue 7, Pages 2346-2358 (June 2007)
CD4+NKG2D+ T Cells in Crohn’s Disease Mediate Inflammatory and Cytotoxic Responses Through MICA Interactions 
Matthieu Allez, Vannary Tieng, Atsushi Nakazawa, Xavier Treton, Vincent Pacault, Nicolas Dulphy, Sophie Caillat–Zucman, Pascale Paul, Jean–Marc Gornet, Corinne Douay, Sophie Ravet, Ryad Tamouza, Dominique Charron, Marc Lémann, Lloyd Mayer, Antoine Toubert 
Gastroenterology 
Volume 132, Issue 7, Pages (June 2007)
DOI: /j.gastro
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2. Figure 1
Increased expression of MICA on IECs in CD. (A) MICA specific expression (MFI with anti-MICA mAb [AMO1] vs MFI IgG1 isotype control) on IECs from patients with CD (n = 13) and patients with UC (n = 8) compared with controls (n = 10) matched for the intestinal area studied (ileum/right colon or left colon). ¤P ≤ .005; §P < .05. (B) IECs were isolated from the surgical specimen (ileocecal resection) of a patient with CD. Isolation was performed from macroscopically inflamed as well as noninflamed areas. IECs were stained with anti-MICA (AMO-1), anti-MHC II (L243), anti-MHC I (W6/32), and isotype controls and analyzed by flow cytometry. Analysis was gated on IECs (ESA-positive cells). This figure is representative of data obtained in 7 patients with CD. (C) Paraffin-embedded tissue was stained with anti-MICA SR99 mAb or isotype control. Anti-MICA staining in patients with CD is shown: (C) noninflamed and (D and E) inflamed areas in the ileum and (F) noninflamed and (G) inflamed areas in the left colon. Anti-MICA staining of (H) noninflamed and (I) inflamed areas in the left colon of a patient with UC is shown. This figure is representative of staining performed in 6 patients with IBD. (Original magnification: C and E, 40×; D, F, G, H, and I, 100×.)
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
CD4+NKG2D+ T cells in CD. PBLs and LPLs were isolated from patients with IBD and controls. Analysis was gated on CD3+CD4+ T cells. (A) Representative data from a patient with CD and a control are shown. (B) There was a significant increased expression of NKG2D on CD4+ T cells isolated from the PB and the mucosa (LPL) in patients with CD (n = 21 and n = 12, respectively) compared with patients with UC (n = 10 and n = 8) and controls (n = 18 and n = 8). P < .005.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
Phenotype and cytokine profile of PB and lamina propria CD4+NKG2D+ T cells in patients with CD. (A) The phenotype of CD4+NKG2D+ T cells in patients with CD was compared with the phenotype of CD4+NKG2D− T cells. Analysis was gated on CD3+CD4+NKG2D+ and NKG2D− T cells. (B) Intracellular staining for perforin was performed in CD4+NKG2D+ and CD4+NKG2D− PBLs and LPLs of patients with CD. (C and D) Intracellular staining for cytokines was performed on CD4+NKG2D+ and CD4+NKG2D− PBLs and LPLs stimulated with PMA/ionomycin. These data are representative of results obtained in 8 patients with CD.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
CD4+NKG2D+ T-cell clones produce IFN-γ through MICA interaction. (A) CD4+NKG2D+ T-cell clones obtained from patients with CD were cultured alone, with C1R and C1R-MICA transfected lines for 24 hours. IFN-γ concentration was measured in the supernatant. C1R-MICA transfected lines were also preincubated with anti-MICA (AMO1), anti-MHC II (L243), or isotype control (IgG1) mAb at 10 μg/mL for 30 minutes. CD4+NKG2D+ T-cell clones were preincubated with anti-NKG2D (1D11) or isotype control (IgG1) mAb at 10 μg/mL for 30 minutes. The data represent means of 4 independent experiments. The IFN-γ concentrations are expressed as percentage of the maximal production obtained from the cultures of CD4+NKG2D+ T-cell clones stimulated by C1R-MICA. The mean value of IFN-γ concentrations in the 4 independent experiments was 1866 ± 1356 pg/mL. (B) CD4+NKG2D+ T-cell clones were cultured with soluble MICA (at 0, 2 or 10 pg/mL) coated on plates, in the presence of anti-NKG2D blocking mAb (1D11) or isotype control for 24 hours. IFN-γ concentration was measured in the supernatant. These data are representative of 3 independent experiments. P ≤ .005; §P < .05.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
NKG2D-mediated cytotoxicity of CD4+NKG2D+ T-cell clones. (A) Cytotoxic activity of CD4+NKG2D+ T-cell clones on MICA-expressing targets. 51Chromium-release assays of CD4+NKG2D+ T-cell clones and C1R-MICA transfectants or control C1R as targets at a 50:1 effector/target ratio in triplicate wells were performed in the presence of anti-MICA (AMO1), anti-NKG2D (1D11), or isotype control (IgG1) at 10 μg/mL. Chromium release was measured after 4 hours of incubation and indicated as percentages of specific cytotoxicity. Data representative of 5 independent experiments are shown. (B and C) Redirected cytotoxicity of Fcγ-R P815 cell lines by CD4+NKG2D+ T-cell clones (50:1 E/T ratio). Cell lines were incubated in the presence of IgG1 (5 μg/mL), anti-CD3 (0.05 μg/mL), and/or anti-NKG2D (5 μg/mL) mAb. Clones were tested after stimulation with IL-2 for (B) 3 days and (C) 8 days. Assays were performed in triplicate. Results are expressed as mean ± SD from 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Decrease of CD4+NKG2D+ T cells in the PB of patients with CD after resection of intestinal inflammatory lesions. Flow cytometric analysis of PB CD4+NKG2D+ T cells was performed in 5 patients with CD before ileocolonic resection, 3 days and 6 months after surgery. Analysis was gated on CD3+CD4+ T cells, and results are given as percentage of CD4+ T cells expressing NKG2D.
Gastroenterology  , DOI: ( /j.gastro )
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8. Figure 7
Expansion of CD4+NKG2D+ T cells in culture with IECs from patients with CD. Freshly isolated IECs obtained from patients with IBD and controls were cultured for 7 days with allogenic PB T cells from healthy donors stained with CFSE. A representative experiment using IECs from a patient with CD is shown. Analysis was gated on CD3+ T cells. The percentage of CD4+ and CD4− proliferating (CFSElo) and nonproliferating (CFSEhi) is shown. The percentage of NKG2D+ cells in each quadrant is indicated.
Gastroenterology  , DOI: ( /j.gastro )
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9. Figure 8
Effect of cytokines on CD4+NKG2D+ T-cell expansion and activation. (A) CFSE-labeled PB T cells from normal individuals were cultured with normal serum (NS), different cytokines (IL-2 at 30 U/mL, IL-7 at 10 ng/mL, IL-12 at 5 μg/mL, IL-15 at 15 ng/mL), or phytohemagglutinin (PHA) (1 μg/mL) for 5 days. The analysis was gated on CD3+ T cells. The percentage of CD4+ proliferating (CFSElo) T cells expressing NKG2D is shown in all cases. This figure is representative of 4 independent experiments. (B) CD4+NKG2D+ LPLs isolated from the mucosa of patients with CD highly express IL-15Rα. LPLs were isolated from the mucosa of patients with CD and analyzed by flow cytometry. The analysis is gated on CD3+CD4+ T cells. The expression of IL-15Rα, as well as staining with isotype controls (thin gray line), is shown in CD4+NKG2D+ (thick black line) and CD4+NKG2D− (thin black line) LPLs, respectively. These data are representative of staining performed in 3 patients with CD. (C) The effect of IL-15 was tested on CD4+NKG2D+ and CD4+NKG2D− T-cell clones obtained from patients with CD. Analysis was performed after 3 days of culture with (thick black line) or without (thin black line) IL-15 (15 ng/mL). Staining with isotype controls (thin gray line) is shown. These data are representative of experiments performed on 2 CD4+NKG2D− clones and 4 CD4+NKG2D+ clones. (D) Increased DAP10 and NKG2D expression in CD4+NKG2D+ T-cell clones cultured with IL-15. CD4+NKG2D+ T-cell clones were cultured with IL-15 (15 ng/mL). Staining for surface NKG2D and intracellular DAP10 was performed at days 0, 3, and 8. These data are representative of 3 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
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