EBV infection of ex vivo tongue explants. EBV-infected tongue explants were grown in vitro for 3 days and then sectioned and coimmunostained with EBV-specific human serum (green) and markers for CD14 (A), CD68 (B), CD1a (C), and CD86 (D) (all in red). Nuclei were stained in blue. Yellow in merged panels shows colocalization of EBV proteins with immune cell markers.

Cocultivation of tongue and buccal tissue explants with EBV-infected monocytes. Purified CD14⁺ monocytes were infected with EBV and 3 days later were cocultured with oral explants. At 2, 3, 4, and 7 days after cocultivation, cryosections of explants were coimmunostained with anti-EBV human serum and markers for macrophages (CD68) or LC (CD1a). EBV-positive intraepithelial macrophages and LC were counted, and the average numbers from five sections are presented. n, number of explants. Horizontal lines indicate median values.

Detection of EBV-infected monocytes and epithelial cells in tongue tissue explants. Tongue explant was infected with cell-free EBV and at 3 days postinfection was coimmunostained for EBV proteins and markers for CD14 or keratin 1. Upper panels show costaining with mouse MAb to gp350/220 (green) and rabbit antiserum to keratin 1 (red). Middle panels show costaining with mouse MAb to gp350/220 and rabbit antiserum to CD14 (red). Lower panels show costaining with mouse MAb to BZLF-1 (green) and rabbit antiserum to CD14 (red). White arrowheads and insets in upper panels show EBV-infected WBC in close proximity to EBV-infected keratinocytes. Nuclei were stained in blue.

Spread of EBV between B lymphocytes and monocytes in vivo and in vitro. (A) CD14⁺ monocytes and CD19⁺ B lymphocytes were isolated from PBMC of HIV-positive and -negative individuals. Detection and quantification of EBV DNA were performed using a QC-PCR assay. EBV DNA copy numbers are expressed on the logarithmic scale as numbers of copies per 1 million cells. Each data symbol indicates one sample. (B) Purity of monocytes was examined using antibodies to CD14 and CD20 and isotype control antibodies to CD14. (C) (Top and middle) Freshly isolated B lymphocytes and monocytes from HIV-negative individuals were coimmunostained with antibodies to EBV VCA p18 and CD20 or CD14 markers, respectively. (Bottom) B lymphocytes were infected with EBV at 10 virions/cell for 3 days, and cells were coimmunostained for CD20 and VCA p18. Yellow in the merged panel shows EBV-infected B lymphocytes. (D) EBV-infected B lymphocytes were cocultivated with monocytes from the same donor at a ratio of 1:1 for 3 days. Cocultured cells were triply coimmunostained for CD20, CD14, and VCA p18. The light blue signals (red arrowheads) in the merged section show EBV-infected B lymphocytes, and yellow signals (yellow arrowheads) show EBV-infected monocytes.

Analysis of EBV-infected immune cells in HL lesions. (A) Total proteins of purified EBV virions were analyzed using a Western blot assay with EBV-positive and -negative human sera. MW, molecular weight (in thousands). (B) HL sections were immunostained with EBV-negative human serum. GR, granulosum; SP, spinosum; BL, basal lamina. (C) Normal tongue sections were coimmunostained with EBV-positive human serum (green) and MAbs to CD68 (red). Only merged panels are shown. (D and E) HL sections were costained with EBV-specific immune serum (green) and MAbs to CD68 or CD1a markers (red). (F) HL section costained with MAb to BZLF-1 (green) and rabbit antibodies to CD14 (red). (D to F) Yellow in merged panels shows colocalization of EBV proteins with immune cell markers. (B to F) Cell nuclei were stained in blue. (G) Detection of EBV-infected WBC by electron microscopy in the suprabasal layer of HL tissue (green arrowheads). The inset shows a magnified EBV virion.

Ex vivo cell-free EBV infection of tongue and buccal tissue explants. Tongue and buccal tissue explants were infected with cell-free EBV for 1, 2, 3, and 7 days. Then the explants were fixed, sectioned, and coimmunostained with EBV-specific human serum and antibodies to B lymphocytes (CD20), monocytes (CD14), macrophages (CD68), LC (CD1a), and activated macrophages/LC (CD86). The intraepithelial EBV-positive immune cells were counted within the oral epithelium, and their average numbers from five sections are presented. Each data symbol indicates one sample. n, number of tissue explants. Horizontal lines represent median values.

Migration of EBV-infected submucosal monocytes into oral epithelium. (A) Tongue tissue sections at 24 h post-cocultivation with EBV-infected monocytes were coimmunostained with EBV-specific serum (green) and MAb to CD68 (red). Artificial white lines were added to the basement membrane to show translocation of EBV-infected submucosal cells to the epithelium. Yellow indicates colocalization of EBV proteins with CD68⁺ cells (arrowhead). (B) The same tongue tissue as in panel A was coimmunostained with MAb to EBV gp350/220 (green), rat antibody to CD1a (blue), and rabbit antibody to ZO-1 (red). White in the merged panel indicates colocalization of gp350/220, CD68, and ZO-1. (C) EBV-infected monocytes and B lymphocytes were cocultivated with tongue tissue for 2 days, and a section of this tissue was coimmunostained with EBV-specific human serum (green) and mouse MAb to CD20 (red) (left panels) and with mouse MAb to EBV BZLF-1 (green) and rabbit antiserum to CD14 (red) (right panels). Yellow in the merged panels shows colocalization of EBV proteins with CD20 and CD14. (A and C) Only merged panels are shown. Nuclei were stained in blue. ME, mucosal epithelium; GR, granulosum; SP, spinosum; BL, basal lamina; LP, lamina propria.

EBV infection of oral keratinocytes by transmigrated EBV-infected monocytes. (A) Tongue explants were cocultivated with EBV-infected CD14⁺ monocytes for 4, 7, and 10 days, and cryosections of these explants were immunostained with EBV-positive human serum (green). Uninfected tongue tissue was immunostained with the same EBV-positive human serum and served as a control. White lines show basement membranes, and cell nuclei were stained in blue. d p.i., days postinfection. (B) A tongue explant that was cocultivated with EBV-infected CD14⁺ monocytes for 7 days was analyzed by FISH assay using EBV BMRF-2 probe (red). An uninfected tongue explant and HL tissue served as negative and positive controls, respectively. Cell nuclei were stained in blue, and only merged panels are shown. White lines indicate basement membranes. LP, lamina propria; ME, mucosal epithelium. (C) Tongue tissue was cocultivated with EBV-infected CD14⁺ cells for 7 days; sectioned; and immunostained for keratin 1 (blue), EBV-positive human serum (green), and CD1a or CD68 (red). Images were obtained from the spinosum layer. Yellow and light blue in the merged section indicate colocalization of EBV signals with CD1a or CD68 and keratin 1, respectively. (D) The tongue biopsy samples were cut in two pieces, one of which was treated with antibodies to MCP-1 and the other of which was untreated. Freshly isolated monocytes were infected with EBV, and at 3 days postinfection cells were divided between two tubes. Cells in one tube were treated with CCR2 antibodies, and those in the other tube were not. Then antibody-treated monocytes were cocultivated with antibody-treated explants (experimental explants) for 7 days. As a control, untreated monocytes were cocultured with untreated explants. Experimental and control explants were coimmunostained for CD68 and EBV VCA p18. The numbers of EBV-infected intraepithelial macrophages (top) and oral keratinocytes (bottom) were counted and are presented as number of infected cells per section. #1, #2, and #3, independently obtained tongue tissue biopsy samples from three individuals. a, cocultivation of untreated explants with untreated monocytes; b, cocultivation of treated explants with treated monocytes. Data are presented as the means ± standard errors.

Model of EBV infection in stratified oral mucosal epithelium. In immunocompromised individuals, particularly those with HIV-mediated immunodeficiency, EBV replication may reactivate in latently infected B lymphocytes, and infectious progeny virions from B lymphocytes may infect monocytes in peripheral blood or in the lamina propria of oral mucosal epithelium. Subsequently these monocytes will differentiate into EBV-infected macrophages and LC. EBV-infected macrophages/LC may migrate into mucosal epithelium and transmit virus to terminally differentiated epithelial cells in the spinosum layer. The epithelial cells of the spinosum layer are highly susceptible to lytic EBV infection and therefore may produce a large number of infectious progeny virions. Productive EBV infection in the spinosum layer may lead to spread of virus to upper granulosum layers of oral epithelium and cause development of the HL. Mφ, macrophage; GR, stratum granulosum; SP, stratum spinosum; BL, stratum basale; TJ, tight junction.