Fig 2: Detection of HCV viral proteins in B cells of HCV-infected patients. Representative images at × 600 magnification were captured by confocal immunofluorescence microscopy in human normal B cells and HCV-infected B cells. Magnified insets with representative single cells were shown on the left corner of each image for further clarification. DAPI, 4',6-diamidino-2-phenylindole. (a) NS3 (red) and CD19 (green) staining. Merge: merged image of CD19, NS3 and DAPI. (b) HCV core (red) and CD19 (green) staining. Merge: merged image of CD19, core and DAPI. (c) HCV core (red) and CD20 (green) staining. Merge: merged image of CD20, core and DAPI. (d) HCV core (red) and B220 (green) staining. Merge: merged image of B220, core and DAPI.

Fig 3: Reduction of FRC progenitors in the aged lymph nodes. Skin-draining (sdLN) and mesenteric (mLN) lymph nodes from young and aged WT mice were depleted of CD45+ cells before analysis by flow cytometry. (A). Gating of CD45-CD35-B220-ICAM-1+ stroma into fibroblastic reticular cells (FRCs), lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and double negative cells (DNs) in the sdLN and mLN of young and aged mice. (B). Frequency of FRCs, LECs, BECs, and DNs within young and aged sdLN and mLN stroma. (C). Number per lymph node of FRCs, LECs, BECs, and DNs within young and aged sdLN and mLN stroma. (D). Gating of MAdCAM-1- FRCs into VCAM-1lo, VCAM-1int, and VCAM-1hi subsets in the sdLN and mLN of young and aged mice. (E). Frequency of VCAM-1lo, VCAM-1int, and VCAM-1hi FRC subsets within young and aged sdLN and mLN stroma. Relative fluorescence intensity (RFI) of surface markers (F). LTBR and (G). SCA-1 on VCAM-1lo, VCAM-1int, and VCAM-1hi FRC subsets within young and aged sdLN and mLN stroma. RFI normalized to expression on young VCAM-1hi FRCs. Data is representative of lymph nodes retrieved from 6 young and 6 aged mice. Analyzed by two-way ANOVA with adjustment for multiple comparisons, p-values: ns, not significant; * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Fig 4: Lymphotoxin signals are essential for iBALT organogenesis and Ag85B-specific immune responses in the lung. Mice were intra-nasally administered Ag85B-rHPIV2 four times at 2-week intervals with or without LTßR-Fc administration. Lung samples were analyzed 2 weeks after the final immunization with Ag85B-rHPIV2. (A) Lung cells were prepared and examined by flow cytometry. The percentage of GL7+ cells in the B220+ B-cell population is shown. Data are combined from four independent experiments, and each point represents data from an individual mouse. (B) Lung tissues were prepared and examined by means of immunohistochemistry. Eight separate slides were prepared from each lung sample for the counting of iBALT structures. Data are combined from four independent experiments, and each point represents data from an individual mouse. In both plots, center values indicate the median. Statistical significance was evaluated by using one-way ANOVA; ****P < 0.0001; N.S., not significant. (C) Lung cells were prepared and then examined by means of IFN? ELIspot assay. Pearson’s correlation analysis was conducted between the numbers of IFN? spots and iBALT structures. (D) BALF and serum were prepared and examined by means of an ELISA for the production of Ag85B-specific IgA and IgG antibodies. Data are combined from four independent experiments and expressed as mean ± SEM (n = 10–16 per group). Statistical significance was evaluated by using Student’s t-test; *P < 0.05; N.S., not significant.

Fig 5: Characterization of surrounding cells in dome-like structures. (A–C, E, G) Immunofluorescence images of dome-like structures at 2 weeks after tamoxifen injection. Scale bars, 20 µm. (A) The multilayered epithelium overlying basally extruded RasV12-trasformed cells. The dotted areas are shown at higher magnification in the lower panels. Dashed lines indicate the epithelium outside (1) or inside (2) the dome-like structure. Arrowheads indicate stromal cells within the dome-like structure. (B) Disrupted tight junctions in the overlying epithelium. Arrowheads or arrows indicate regions where tight junctions are absent or elongated, respectively. (C, D) Increased cell proliferation in the overlying epithelium within dome-like structures. (D) Quantification of the Ki-67-positive epithelial cells. Data are mean ± SEM n = 222 (not-extruded YFP), 191 (not-extruded RasV12), and 222 (RasV12 in dome-like structures) cells from four CK19-YFP or five CK19-RasV12 mice. n = 1124 (surrounding not-extruded YFP), 1270 (surrounding not-extruded RasV12), and 1708 (surrounding RasV12 in dome-like structures) cells from four CK19-YFP or six CK19-RasV12 mice. *p < 0.05, **p < 0.01, one-way ANOVA with Tukey's test. (E, F) Accumulation of activated fibroblasts in dome-like structures. (E) The dotted area is shown at higher magnification in the lower panels. Arrowheads indicate a-SMA-positive activated fibroblasts within the dome-like structure, whereas arrows indicate a-SMA-positive smooth muscle underneath the epithelium. Note that smooth muscle has much higher a-SMA expression compared with activated fibroblasts. (F) Quantification of a-SMA+ fibroblasts around YFP or RasV12 cells. Data are mean ± SEM n = 89 (not extruded in CK19-YFP), 77 (not extruded in CK19-RasV12), and 107 (dome-like structure in CK19-RasV12) areas from four mice. *p < 0.05, one-way anova with Tukey's test. (G, H) Accumulation of immune cells in dome-like structures. (G) Arrowheads indicate CD45-positive immune cells within the dome-like structure. (H) Quantification of CD45+ cells around YFP or RasV12 cells. Data are mean ± SEM n = 89 (not extruded in CK19-YFP), 39 (not extruded in CK19-RasV12), and 99 (dome-like structure in CK19-RasV12) areas from three mice. *p < 0.05, ****p < 0.001, one-way anova with Tukey's test.