Fig 2: Sentinel macrophages mediate capture of incoming viruses at the injection site during subcutaneous challenge.(A) Images of footpad cryosections from B6 mice 15 min after intrafootpad (i.f.p.) administration of murine leukemia virus (MLV) Gag-GFP virus particles (green). Macrophages and dendritic cells (DCs) were identified using antibodies to CD169 (blue) and CD11c (red), respectively. Green arrows denote MLV-laden CD169+ macrophages, and white arrows denote contacts between CD169+ macrophages and DCs. (B) Electron tomography of footpads from B6 mice prepared 15 min post-challenge with WT FrMLV. MLV virions can be seen associated with plasma membranes (PM) invaginations of macrophages (mF) as well as via a presumed CD169 tether in the magnified inset. (C) Electron tomography showing synaptic, MLV-containing contacts between macrophages and DCs in footpads of B6 mice at 15 min post-challenge (i.f.p.) WT FrMLV. Green arrowheads indicate macrophage-associated MLV particles, and blue arrowheads indicate DC-associated MLV particles. Scale bars as indicated.

Fig 3: Gut-infiltrating murine leukemia virus (MLV) is captured by Peyer’s patch (PP)-resident CD169+ macrophages.(A) Images of cryosections from ligated gut tissue containing a PP from B6 mice that was challenged for 1 hr with MLV Gag-GFP (green) corresponding to 1–4 × 105 IU based on comparative western blot analyses with antibodies to Gag with equivalent amounts of WT FrMLV. Tissue sections were stained with phalloidin (red) to visualize actin in the PP tissue structure. Dotted lines demarcate the epithelium from intestinal lumen. Free, adhered, and infiltrating MLV Gag-GFP particles are as indicated with arrows. (B) Images of cryosections from PP for an experiment as in (A). Macrophages identified using antibodies to CD169 (magenta) were predominantly located at the serosal side of PP. MLV Gag-GFP (green) viruses within marked insets of PP were identified and modeled into spheres (modeled virions), which were then merged to depict close association with CD169+ macrophages. Scale bars as indicated.

Fig 4: CD8+ CTL Response Is Compromised in CD169-/- Mice(A) Comparison of percent CD8+ T cell population in the spleen of B6 and CD169-/- mice (n = 4) after i.p. administration of CD8a T cell depleting antibodies.(B) FVC-infected cells in the spleen of B6 and CD169-/- mice (n = 4, 8 dpi, 2,500 SFFU s.c.) with and without CD8 T cell depletion for an experiment as in (A).(C) Experimental design for estimating in vivo CTL activity using a 1:1 ratio of FrMLV Gag peptide pulsed dsRed+ and non-pulsed GFP+ splenocytes in FVC-infected (r.o., 2,500 SFFU) mice.(D) Representative FACS plots showing comparative killing of Gag peptide pulsed dsRed+ splenocytes in uninfected (n = 5) and infected B6.Fv2s/s (n = 10) and B6.Fv2s/sCD169-/- (n = 5) mice for an experiment as in (C).(E) The graph in the left panel shows the ratio of non-pulsed to pulsed peptide cells in uninfected and infected mice for the experiment shown in (D) in pLN and spleen. The right panel shows specific CTL killing activity of peptide-pulsed cells after normalization to uninfected mice.(F) Specific CTL activity determined using in vitro assay at indicated effector-to-target ratios using purified CD8+ T cells from spleens of uninfected or infected B6 or CD169-/- mice (7 dpi, 2,500 SFFU r.o.). 1:1 ratio of peptide pulsed dsRed+ and non-pulsed GFP+ splenocytes were used as targets and CTL activity monitored as in (D) after culturing cells for 48 hr.(G–K) 2 × 106 splenocytes from FVC-infected B6.Fv2s/s (n = 5) and B6.Fv2s/sCD169-/- (n = 5) (8 dpi, 2,500 SFFU s.c.) or uninfected mice were cultured in vitro with 6 µM Gag peptide for 15–18 hr. The plots show a comparison of cells that stained positive for indicated markers in the CD8+ T cell population.(L) Experimental design to test the in vivo efficacy of adoptively transferred primed CD8+ T cells from B6 or CD169-/- mice to target FVC-infected cells.(M) FVC-infected cells in the spleen of CD169-/- mice for an experiment depicted in (L) (n = 4, 7 dpi, 2,500 SFFU r.o.). CD169-/- mice that did not receive exogenous CD8+ T cells were used as control.p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line, error bars denote SD. See also Figures S4–S6.

Fig 5: CD169 Function Is Required at Both pLN and Spleen for Limiting Retrovirus Dissemination(A) The upper panel depicts a scheme showing administration of isotype control or CD169 blocking antibodies via s.c. (5 µg) or r.o. (20 µg) route to elicit site-specific blocking at pLN (n = 7–9) or spleen (n = 5), respectively, in BALB/cJ mice. The graph in the lower panel shows percentages of CD169-positive cells in pLN or spleen, 1 hr after CD169 blockade via indicated routes.(B) Scheme showing administration of CD169 blocking antibodies via mentioned routes as in (A) followed by s.c. inoculation of BALB/cJ mice (n = 6; 2,500 SFFU) with FVC after 1 hr. The splenocytes were harvested 3 dpi and co-cultured with DFJ8 cells to determine the levels of infection by FACS analyses of Glycogag+ cells.(C) Image of spleens from BALB/cJ mice that were uninfected or infected with FVC (500 SFFU) 15 dpi via mentioned routes.p values derived from non-parametric Mann-Whitney test; mean values denoted by horizontal line.