Fig. 2: Migration of EBV-infected B cells is driven by CCL4 and CCR1.

a Transwell assay with CCL4 as an attractor. 5 × 10E4 infected B cells were seeded in the top chamber. Medium containing CCL4 (10 ng/ml) was placed in the bottom chamber. The number of infected cells that have reached the bottom chamber after 1 h of stimulation is given, relative to the number of spontaneously migrating cells (2.5 × 10E3). Medium devoid of chemokines served as a negative control. The bar graph gives the mean with standard deviation (n = 6 independent B cell samples). b The bar graph shows the mean percentage and standard deviation of motile CCL4^null or CCR1^null EBV-infected cells as observed by time-lapse microscopy (n = 5 independent B cell samples). The impact of exogeneous CCL4 on the mobility of CCL4^null cells is also shown. c EBV-infected B cells were seeded in a collagen matrix at low concentration (3 × 10E4 cells/ml) in the presence or absence of CCL4. Cells were subjected to live cell imaging for 15 min to generate 2D tracks with or without alignment (n = 50 one out of 5 individual experiments). The same cells seeded at high concentration (3 × 10E5 cells/ml) served as a positive control. d We determined the percentage and the velocity of mobile cells analyzed in (c). Results are given as bar graphs with mean and standard deviation (n = 50 one out of 5 individual experiments). e–g EBV latent genes and CCL4-CCR1 expression. e CCR1 surface expression on Burkitt cell line MUTU I and III clones was determined by FACS (left panel) and CCL4 concentration in supernatants from these cells was determined by ELISA (right panel) (n = 3 independent transfections). The results are summarized in bar graphs (mean with standard deviation). f B cells stimulated with CD40L + IL-4 were transfected to express LMP1 or EBNA2. The bar graphs show CCR1 surface expression and CCL4 release in transfected cells and in empty vector controls. Results were normalized for the percentage of transfected cells. Bar graphs show mean and standard error of the mean (n = 3 transfected independent B cell samples). g Differential CCR1 expression at the surface of B cells infected with a LMP1^null virus or wild type controls (left panel) and CCL4 release in the supernatants of these cells (right panel) (n = 3 independent B cell samples). The results are summarized in bar graphs (mean with standard deviation). h EBV-infected B cells were subjected to scanning electron microscopy. i EBV-infected B cells were prefixed in PFA and stained with antibodies specific to CDC42 and phosphoPKC Zeta (green), together with a membrane dye (red) and nuclear dye DAPI (blue). Representative pictures are shown (n = 30, one representative example from 3 experiments with independent B cell samples). Statistical significance was determined using two-sided paired t tests in (a), (e), and (g) and one-way analysis of variance in (b), (d), and (f). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Source data are provided as a Source Data file.