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. 2018 May 15;9:984. doi: 10.3389/fmicb.2018.00984

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Copyright © 2018 Nanbo, Ohashi, Yoshiyama and Ohba.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Direct cell-contact facilitates the expression of TGF-β receptors on the surface of B cells and their susceptibility to TGF-β. (A,B) Effect of cell contact on expression of TGF-β receptors in B cells. Akata⁻ EBV-eGFP cells were cocultured with AGS cells directly (A) or through a trans-well (B) for 2 or 5 days. B cells were harvested and the expression of TβRI (white) and TβRII(gray) was analyzed by means of flow cytometry. The data were normalized to their expression in Akata⁻ EBV-eGFP without cocultivation. The experiment was performed three times independently and the average values and their SD are shown for each condition. ^*P < 0.05; ^**P < 0.01 vs. respective control; N.S., not significant (Student's t-test). (C) Effect of cell contact on TGF-β-mediated induction of lytic cycle in Akata cells. Akata⁻ GFP-EBV cells were cocultured with (gray) or without (white) AGS cells for 2 days. B cells were harvested and treated with a recombinant TGF-β (100 pg/ml) for 48 h. The percentage of gp350-positive cells was analyzed by immunofluorescence staining. The experiment was performed three times independently and the average values and their SD are shown for each condition. ^**P < 0.01 vs. respective control (Student's t-test). (D) The amount of TGF-β released from cocultured epithelial cells. AGS (gray) or NU-GC-3 (black) cells were cultured with or without Akata⁻ EBV-eGFP cells for 5 days and the supernatants of cultured cells were harvested. The amount of TGF-β in the supernatants was analyzed by ELISA. The experiment was performed three times independently and the average values and their SD are shown for each condition. N.S.; not significant (Student's t-test). (E) Downregulation of TGF-β by siRNA. Total RNA was isolated from AGS cells at 72 h-post transfection of control siRNA or siRNA against TGF-β. Downregulation of expression for TGF-β was analyzed by ELISA. The experiment was performed three times independently and the average and its SD are shown in each condition. ^*P < 0.05 vs. respective control (Student's t-test). (F) Effect of downregulation of TGF-β in AGS cells on cell-to-cell contact-mediated EBV transmission. Akata⁻ EBV-eGFP cells were cocultured with AGS cells, in which TGF-β was downregulated for 5 days. The percentages of eGFP-positive, infected epithelial cells were analyzed by means of flow cytometry. The experiment was performed three times independently and the average and its SD are shown in each condition. ^**P < 0.01 vs. respective control (Student's t-test).