Figure 5
Figure 5. Induction of LMP-1 by activated CD4+ T cells in cocultures with EBV-positive BL cells. (A) Immunoblot analysis of LMP-1 expression in total cell extracts of the Daudi cells after 24 hours of coculture with nonactivated, PHA (5 μg/mL) activated, or SEB (5 μg/mL) activated peripheral blood (PB) CD4+ T cells (mixed in a 1:1 or 1:4 ratio). (B) Double immunofluorescence staining for LMP-1 and CD20 of the Daudi cells after 24 hours of coculture with nonactivated or PHA (5 μg/mL) activated PB CD4+ T cells (mixed in a 1:4 ratio). Nuclei were visualized with Hoechst 33258. The red staining of the white squared cells are shown at a higher magnification in the lower row. Images were generated with a Leitz DM RB microscope (Leica Microsystems) using a 63×/1.32 NA oil immersion objective lens. Images were captured with a Hamamatsu dual-mode cooled charged coupled device camera (C4880) and Hipic 6.4.0 software (Hamamatsu Photonics Deutschland). Pictures were edited for opitmal color contrast with Adobe Photoshop 7 (Adobe Systems). (C) Expression of LMP-1 and EBNA-1 in total cell lysates prepared from the Daudi cells cocultured for 24 hours with PHA (5 μg/mL) activated PB CD4+ T cells (ratio 1:1). In parallel cultures the Daudi cells were separated from the CD4+ T cells through a semipermeable membrane and cultured either in the lower or upper chamber of the Transwell plate. (D) Expression of LMP-1 in total cell lysates prepared from the Daudi cells cocultured for 24 hours with nonactivated, PHA (5 μg/mL) activated, or SEB (5 μg/mL) activated tonsillar total T cells (ratio 1:1). (E) Expression of LMP-1 and β-actin in total cell lysates prepared from the Daudi, Mutu I cl.59, Jijoye M13, or KMH2-EBV cells cocultured for 12 hours with nonactivated or PHA (5 μg/mL) activated CD4+ tonsillar T cells (ratio 1:1). As control the KMH2-EBV cells were also treated with 25 ng/mL IL-4 for 12 hours. (F) Immunoblot analysis of LMP-1 and Pax5 expression in total cell lysates prepared from the Jijoye M13 cells cocultured for 6 or 12 hours with nonactivated or PHA (5 μg/mL) activated CD4+ tonsillar T cells (ratio 1:1).

Induction of LMP-1 by activated CD4+ T cells in cocultures with EBV-positive BL cells. (A) Immunoblot analysis of LMP-1 expression in total cell extracts of the Daudi cells after 24 hours of coculture with nonactivated, PHA (5 μg/mL) activated, or SEB (5 μg/mL) activated peripheral blood (PB) CD4+ T cells (mixed in a 1:1 or 1:4 ratio). (B) Double immunofluorescence staining for LMP-1 and CD20 of the Daudi cells after 24 hours of coculture with nonactivated or PHA (5 μg/mL) activated PB CD4+ T cells (mixed in a 1:4 ratio). Nuclei were visualized with Hoechst 33258. The red staining of the white squared cells are shown at a higher magnification in the lower row. Images were generated with a Leitz DM RB microscope (Leica Microsystems) using a 63×/1.32 NA oil immersion objective lens. Images were captured with a Hamamatsu dual-mode cooled charged coupled device camera (C4880) and Hipic 6.4.0 software (Hamamatsu Photonics Deutschland). Pictures were edited for opitmal color contrast with Adobe Photoshop 7 (Adobe Systems). (C) Expression of LMP-1 and EBNA-1 in total cell lysates prepared from the Daudi cells cocultured for 24 hours with PHA (5 μg/mL) activated PB CD4+ T cells (ratio 1:1). In parallel cultures the Daudi cells were separated from the CD4+ T cells through a semipermeable membrane and cultured either in the lower or upper chamber of the Transwell plate. (D) Expression of LMP-1 in total cell lysates prepared from the Daudi cells cocultured for 24 hours with nonactivated, PHA (5 μg/mL) activated, or SEB (5 μg/mL) activated tonsillar total T cells (ratio 1:1). (E) Expression of LMP-1 and β-actin in total cell lysates prepared from the Daudi, Mutu I cl.59, Jijoye M13, or KMH2-EBV cells cocultured for 12 hours with nonactivated or PHA (5 μg/mL) activated CD4+ tonsillar T cells (ratio 1:1). As control the KMH2-EBV cells were also treated with 25 ng/mL IL-4 for 12 hours. (F) Immunoblot analysis of LMP-1 and Pax5 expression in total cell lysates prepared from the Jijoye M13 cells cocultured for 6 or 12 hours with nonactivated or PHA (5 μg/mL) activated CD4+ tonsillar T cells (ratio 1:1).

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