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. 2021 Jan 18;10:e59350. doi: 10.7554/eLife.59350

Figure 11. HCMV, HSV-1, and HSV-2 also limit cell surface expression of ICOSL on antigen-presenting cells (APCs).

Figure 11.

(A) Primary monocyte-derived macrophages (upper panels) and PMA-treated THP-1 cells (bottom panels) were mock-infected or infected for 72 hr with HCMV-GFP at an moi of 10 and analyzed by flow cytometry for cell-surface expression of human ICOSL or CD70 using specific mAbs against each of these receptors. Gray histograms represent the expression of mock-infected cells, green histograms represent the expression on HCMV-infected (GFP+) cells, and blue histograms represent the expression on uninfected (GFP-) cells from the same culture. (B) PMA-treated THP-1 cells were mock-infected (time 0) or infected with HCMV-GFP as in A and analyzed by flow cytometry for surface expression of ICOSL at the different time points after infection indicated. (C) Same as in A, except that an moi of 20 was used, and THP-1 cells were also exposed for 72 hr to the same amount of HCMV-GFP UV-inactivated (red histogram). (D) Equal amounts of lysates from PMA-treated THP-1 cells mock-infected (lane 1) or infected for 72 hr at an moi of 20 with HCMV-GFP (lanes 2 and 3), and when indicated, treated with 250 μM leupeptin and 20 nM of bafilomycin A1 (lane 3), were lysed and analyzed by western blot with antibodies against ICOSL and actin, followed by anti-rabbit IgG-HRP (ICOSL) or anti-mouse IgG-HRP (actin). (E) PMA-treated THP-1 cells were mock-infected or infected with HSV-1-GFP, HSV-1-GFP UV-inactivated, HSV-2-GFP or HSV-2-GFP UV-inactivated at an moi of 100 (HSV-1) or 200 (HSV-2). Twenty-four hr later, the expression of human ICOSL and CD70 were analyzed as in A. Green histograms represent the expression of ICOSL on infected cells, red histograms represent the expression on UV-inactivated HSV infected cells, and gray histograms represent the expression on mock-infected cells. The isotype for the ICOSL antibody was used as a negative control (dotted lines). A representative experiment out of three performed is shown. (F) PMA-treated THP-1 cells were mock-infected (lane 1) or infected with HSV-1-GFP (lane 2) and HSV-2-GFP (lane 3) as indicated in E. Cell lysates were prepared, and subjected to western blot analysis using anti-human ICOSL or anti-actin mAbs as in D. The molecular weight of the band corresponding to ICOSL is indicated in D and E in kilodaltons on the right margin.