Fig 2: Protein expression of CX3CL1 and CX3CR1 evaluated in hepatoblastomas and hepatoblastoma lung metastasis by immunohistochemistry assay. (A–D) Show CX3CL1 data, and (A1–D1), CX3CR1. (A) TCH361, CX3CL1 strong positivity of infiltrated lymphocytes (indicated by arrow 1) in necrotic regions of the tumor, and (A1), CX3CR1 strong positivity of infiltrated lymphocytes (indicated by arrow 2) in necrotic regions of the tumor; (B,B1) TCH327, positivity in tumor cells (indicated by arrows 3 and 5) and infiltrated lymphocytes negative (indicated by arrows 4 and 6) for both proteins. (C) TCH321, positivity in the osteoblast component and strong positivity in the fetal type (indicated by arrow 7); infiltrated lymphocytes are negative (indicated by arrow 8); (C1) positivity in tumor cells and lymphocytes negative; (D,D1) TCH360, lung metastasis showing positivity in tumor cells (indicated by arrows 9 and 11), and no expression in infiltrated lymphocytes (indicated by arrows 10 and 12), for both proteins.

Fig 3: Role of the CX3CR1-CX3CL1 axis on intercellular interactions. Human microglia were adhered with JEV (Nakayama isolate used at an MOI of 10 TCID50/cell) at 37 °C for 2 hours. Cells were then intensively washed with cold PBS and left in culture at 37 °C for 48 hours. Cells were intensively washed with PBS and co-cultured with BHK-21 cells. In (a), co-cultures were performed in the presence of 1:100 Ctrl serum for 6 hours. In (b), co-cultures were performed in the presence of 1:100 Ctrl serum and in the absence or the presence of CX3CR1 antagonist for 6 hours. Cells were stained for the indicated markers and analysed using confocal microscopy. (a,b) Micrographs showing fluorescence signals on bright field overlay focusing on intercellular interactions. Zoom of white square in left panel is presented. Scale bars are of 20 μm in left panels and of 8 μm in zoom panels. Micrographs are representative out of 3 independent experiments.

Fig 4: Role of the CX3CR1-CX3CL1 axis in cell-to-cell virus transmission and recovery. (a) Human microglia were treated with Mock and JEV (Nakayama isolate used at an MOI of 10 TCID50/cell) at 37 °C for 6 days. Representative density plots of cell surface expression of CX3CR1. Frequency of CX3CR1+ cells is indicated. (b) BHK-21 were maintained in culture at 37 °C. Representative density plots of steady state surface expression of CX3CL1 by BHK-21 cells. Frequency of CX3CL1+ cells is indicated. Representative plots are out of 3 independent experiments. (c–e) Human microglia were pre-treated with Mock and JEV (Nakayama isolate used at an MOI of 10 TCID50/cell) at 37 °C for 6 days. Cells were then washed with cold PBS and subsequently co-cultured with BHK-21 cells for 2 additional days in the presence of 1:100 Ctrl serum and indicated concentrations of CX3CR1 antagonist. In (c,d), cells were analysed by flow cytometry for dsRNA and JEV expression in BHK-21 cells and microglia identified as in Fig. 4a. (c) Histogram bars representing frequencies of BHK-21 cells and microglia expressing dsRNA. The bar represents the mean value; the error bars the standard deviation. (d) Box plots representing frequencies of BHK-21 cells and microglia expressing JEV E. The symbols represent replicates where each colour is an individual donor. The black line represents the median value, the red line the mean value and the error bars the standard deviation. (e) Scatter dot plots representing virus titres in supernatants. The symbols represent replicates where each colour is an individual donor and the black line represents the mean value. Data are of 2 or 4 independent experiments with each condition performed in triplicate or duplicate or triplicate cultures. Asterisks on top of a condition show significant differences compared to mock; asterisks on black line show significant differences between the indicated conditions. Statistics are calculated with (c,d) the t-test or (e) the Mann-Whitney test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Fig 5: Protein expression of CX3CL1 and CX3CR1 evaluated in hepatoblastoma samples by immunohistochemistry assay. (A–C) Show CX3CR1 data, and (A1–C1), CX3CL1 from the same tumor samples. (A) HB17, example of negative labeling for CX3CR1 (A) and CX3CL1 (A1). (B) HB32T, positive for nuclear and cytoplasmic CX3CR1 (B) and CX3CL1 (B1). (C) HB33T, positive for cytoplasmic CX3CR1 labeling (C) and positive for nuclear and cytoplasmic CX3CL1 (C1).