Volume 126, Issue 5, Pages 1347-1357 (May 2004) The expression and function of costimulatory molecules B7H and B7-H1 on colonic epithelial cells  Atsushi Nakazawa, Iris Dotan, Jens Brimnes, Matthieu Allez, Ling Shao, Fumihiko Tsushima, Miyuki Azuma, Lloyd Mayer  Gastroenterology  Volume 126, Issue 5, Pages 1347-1357 (May 2004) DOI: 10.1053/j.gastro.2004.02.004

Fig 1 B7h, B7-H1, and B7-DC mRNA expression in the colonic epithelial cell line HT-29. RNA was isolated, reverse transcribed, and used for PCR analysis. This figure represents the analysis of reverse-transcription PCR-amplified transcripts of B7h, B7-H1, B7-DC, and β-actin from unstimulated, IFN-γ-(100 U/mL), or TNF-α-(10 ng/mL) stimulated (7 hours) HT-29 cells. The predicted product sizes for B7h, B7-H1, B7-DC, and β-actin are 402, 399, 422, and 661 base pairs, respectively. This figure is representative of 4 experiments. Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)

Fig 2 Flow cytometric analysis of B7h and B7-H1 expression on the colonic epithelial cell line HT-29. HT-29 were grown to subconfluence and either left alone in culture medium or stimulated with IFN-γ (100 U/mL) or TNF-α (10 ng/mL) for 48 hours. Cells were stained with biotinylated (A) anti-B7h mAb, (B) anti-B7-H1 mAb, or an isotype-matched control mAb followed by streptavidin/phycoerythrin. This is representative of 5 experiments. Data are shown by histograms. Thin line, unstimulated; bold line, IFN-γ stimulated; dashed line, TNF-α stimulated. Dotted lines reflect the staining with an isotype-matched antibody as a negative control. Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)

Fig 3 B7h, B7-H1, and B7-DC mRNA expression in freshly isolated colonic epithelial cells. This figure represents the analysis of reverse-transcription PCR-amplified transcripts of B7h, B7-H1, B7-DC, and β-actin from cells derived from the colonic mucosa of healthy controls (n = 6; 3 representative shown) and patients with IBD (n = 10; 5 representative shown). PCR product from the reaction without template mRNA as a negative control. The predicted product sizes for B7h, B7-H1, B7-DC, and β-actin are 402, 399, 422, and 661 base pairs, respectively. Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)

Fig 4 Flow cytometric analysis of B7h and B7-H1 expression on freshly isolated colonic epithelial cells from patients with ulcerative colitis, patients with Crohn’s disease, or healthy controls. Cells were stained with biotinylated anti-B7h and anti-B7-H1 mAbs followed by streptavidin/phycoerythrin. The purity of the epithelial cell preparations was confirmed by flow cytometric analysis after gating on fluorescein isothiocyanate-conjugated anti-epithelial cell specific antigen positive cells. Data are shown as histograms (bold lines). Solid lines indicated background staining with an isotype-matched control mAb. Representative data from each of 5 patients with (A) ulcerative colitis, patients with Crohn’s disease, and (B) healthy controls are shown. Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)

Fig 5 IECs from normal or IBD mucosa (0.5 × 106 cells/mL) were cocultured at a ratio of 1:2 with allogeneic normal peripheral blood T cells (1 × 106 cells/mL). Before the coculture, T cells were incubated with CFSE. T-cell proliferation was assessed 5–10 days after incubation by flow cytometric analysis of CFSE-high (nonproliferating) versus CFSE-low (proliferating) CD3+CD8+ versus CD3+CD8− T cells. (A) T cells incubated alone do not proliferate spontaneously. CD8+ versus CD8− T-cell proliferation induced by (A) normal and (B) ulcerative colitis IECs are shown. At the start of the culture, isotype-matched control, anti-B7h and B7-H1 mAbs, or B7h-Ig and B7-H1-Ig fusion proteins were added respectively at a final concentration of 5–10 μg/mL. A representative example of 5 independent experiments is shown. The numbers in the quadrants show the percentage of cells (CD8+, CD8−, CFSEhigh, CFSElow) in each subpopulation. B7h-Ig blocks CD8+ T-cell proliferation (and to a lesser extent CD4+ T-cell proliferation) by normal IECs and both CD4+ and CD8+ T-cell proliferation in IBD IEC:T-cell cocultures. (C) T-cell proliferation induced by non-T cells is shown in conventional MLR cultures. Analysis was gated on CD3+ cells. A representative example of 5 independent experiments is shown. B7h-Ig blocked CD4+ but not CD8+ T-cell proliferation in these cocultures. Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)

Fig 6 IECs from normal or IBD mucosa (1 × 106 cells/mL) were cocultured at a ratio of 1:1 with allogeneic peripheral blood T cells (1 × 106 cells/mL). The IFN-γ concentration in coculture supernatants was determined by ELISA after 120 hours. T cells incubated alone did not secrete IFN-γ. (B) IBD IEC:T-cell cocultures showed significantly higher IFN-γ secretion than (A) normal IEC:T-cell cocultures. At the start of the culture, isotype-matched control, anti-B7h and B7-H1 mAbs, or B7h-Ig and B7-H1-Ig were added respectively at a final concentration of 5–10 μg/mL. Values are the mean ± SD from 6 experiments using different donors. Both anti-B7h mAb and B7h-Ig inhibited IFN-γ production in the 2 types of cocultures. (C) IFN-γ secretion in MLR cultures was determined by ELISA after 72 hours of culture. Values are the mean ± SD from 6 independent experiments using different donors. Similar to IEC:T-cell cocultures, anti-B7h and B7h-Ig blocked IFN-γ production. There was no evidence of toxicity of these agents. ∗Statistically significant difference (P < 0.05). ∗∗Statistically significant difference (P < 0.01). Gastroenterology 2004 126, 1347-1357DOI: (10.1053/j.gastro.2004.02.004)