Fig 2: CD169 Limits Dissemination of FVC into Erythroblast Niche of the Splenic Red Pulp(A and B) Merged immunostaining images of splenic tissue sections from B6 and CD169-/- mice 5 dpi after s.c. administration (2 × 106 IU) of Ypet expressing FVC (green). B cells, erythroblasts, and metallophilic macrophages were identified using antibodies to surface markers B220 (blue), CD71 (red), and CD169 (pink), respectively. The B cell follicular area (white pulp) and extrafollicular erythroblast rich areas (red pulp) are demarcated by dashed white lines. Magnified images of merged and individual channels of insets are shown on the right.(C) Merged immunostaining images of splenic tissue sections from B6 and CD169-/- mice 5 dpi after r.o. administration of FVC (2,500 SFFU). Metallophilic macrophages lining the white pulp and FVC-infected cells were identified using antibodies to surface marker CD169 (red) and viral protein Glycogag (green).(D) Electron tomography of a spleen section from CD169-/- mice for an experiment as in (A). The image shows a cluster of clonally expanded FVC-infected erythroblasts (labeled E). Insets show details from serial tomographic reconstructions, demonstrating nascent viruses (red arrowheads) budding from the surfaces or invaginations of infected erythroblasts.See also Figures S2 and S3 and Video S1. Scale bars as indicated.

Fig 3: CD169 Limits Retrovirus Dissemination from pLN to Spleen and Is Required for Efficient FrMLV Infection(A) Scheme indicating a possible path of virus dissemination from popliteal lymph node (pLN) to blood and spleen after subcutaneous (s.c.) footpad administration of luciferase expressing FrMLV.(B–D) The indicated organs and plasma were harvested 1 hr after virus administration as in (A). The graphs show viral loads measured as relative luciferase units at indicated locations after performing highly sensitive virus load assay in which plasma (n = 5), pLN (n = 7), and splenocyte (n = 5) cell suspensions were incubated with DFJ8 cells for 36–48 hr before measuring luciferase activity.(E and F) FrMLV-infected cells 5 dpi (s.c., 4 × 105 IU) at pLN (n = 10) and spleen (n = 5) in B6 and CD169-/- mice.(G) A model depicting FrMLV dissemination and subsequent levels of infection 5 dpi from pLN to blood and the spleen following subcutaneous challenge in B6 and CD169-/- mice to show the infection-promoting role of CD169.p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line.

Fig 4: CD169 Reduces FVC Spread to Erythroblasts in the Red PulpFluorescence-activated cell sorting (FACS) plots showing the gating strategy and graphs depicting the numbers of FVC-infected cells, erythroblasts, B cells, and ratios of infected erythroblasts and B cells in splenocytes of B6 and CD169-/- mice (n = 4) 5 days after s.c. (A–E) or r.o. (F–J) administration (2,500 SFFU). Erythroblasts (CD71+ Ter119+ CD19-), B cells (CD19+), and FVC-infected cells (Glycogag+) were identified using the indicated fluorophore conjugates. p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line.

Fig 5: CD169 Plays a Crucial Role in Activating cDC1s to Elicit Effective CD8+ T Cell Response during FVC Infection(A and B) CD80+-activated dendritic cells in CD11chi (A) and CD11c+CD8a+ (cDC1) (B) populations from splenocytes of FVC-infected B6.Fv2s/s (n = 5) and B6.Fv2s/sCD169-/- (n = 5) (8 dpi, 2,500 SFFU s.c.).(C) FVC-infected cells in the spleens of B6 (with and without CD8 T cell depletion), Batf3-/-, and CD169-/- mice (n = 4, 7 dpi, 2,500 SFFU r.o.).p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line; error bars denote SD. Scale bars as indicated. See also Figure S7 and Video S2.