Also shown (Figure 2D), which demonstrates a remarkable overlap
These final results indicate that there's a network of SIGN-R1 good macrophages inside the LN IFC that give a platform for nearby B cells and DCs to obtain gp120 and that a subset of SIGN-R1+ cells in the marginal zone of your spleen are also poised to provide gp120 to Een alternative conformations determined by synchrotron crystallography and NMR experiments splenic marginal zone and trafficking follicular B cells. Overlay of gp120 visualizes DC-SIGN+/CD163+ macrophages inside a human LN section. DOI: 10.7554/eLife.06467.Park et al. eLife 2015;four:e06467. DOI: 10.7554/eLife.6 ofResearch articleImmunologywe immunostained one particular for CD163, a human macrophage marker (Martens et al., 2006), CD11c, and DC-SIGN; and the other for gp120 and DC-SIGN. This allowed the identification of a group of CD163+, DC-SIGN+, and CD11c- cells close to the LN follicle that bound the overlaid gp120 (Figure 2--figure supplement two). These results indicate that in human LN DC-SIGN expressing macrophage close to the follicle may well uptake gp120 similar towards the mouse IFC SIGN-R1+ macrophages.The SIGN-R1+ subcapsular macrophages uptake gp120 before the SIGN-R1+ IFC macrophagesTo determine the kinetics of gp120 uptake by SIGN-R1+ subcapsular macrophages plus the underlying SIGN-R1+ IFC macrophages, we intravitally imaged for 3.five hr following gp120 injection.Also shown (Figure 2D), which demonstrates a remarkable overlap involving the Also shown (Figure 2D), which demonstrates a remarkable overlap among the SIGN-R1 and gp120 signals. These outcomes indicate that there is a network of SIGN-R1 positive macrophages inside the LN IFC that provide a platform for nearby B cells and DCs to acquire gp120 and that a subset of SIGN-R1+ cells within the marginal zone of the spleen are also poised to provide gp120 to splenic marginal zone and trafficking follicular B cells. The capability to especially detect gp120 binding cells applying an overlay assay prompted us to determine regardless of whether we could detect equivalent macrophages in human LN. To recognize the LN follicles and T cell zone we immunostained a LN section for CD19 and CD4 expression. Utilizing two adjacent sectionsPark et al. eLife 2015;four:e06467. DOI: ten.7554/eLife.five ofResearch articleImmunologyFigure two. IFC cell processes bearing gp120 straight get in touch with B cells plus a subset of splenic marginal zone cells also bind gp120. (A) Confocal microscopy of a thick LN section from a mouse previously injected with fluorescent gp120 and immunostained for B220 and CD3. Scale bar is 30 m. Boxed locations in left image were enlarged and shown inside the ideal panels. Scale bars are 10 m. (B) Confocal microscopy of a thick splenic section from a mouse previously injected intravenously with fluorescent gp120 and immunostained together with the indicated markers. Zoomed photos shown below. Scale bars are 100 m, above, and 30 m, under. (C) Confocal microscopy of a thick splenic section overlaid with fluorescent gp120 and immunostained for the indicated markers. Zoomed photos are shown beneath. Scale bars are 100 m, above, and 40 m, below. (D) Tiled confocal microscopy images of a spleen section immunostained with CD169 (red) and SIGN-R1 (cyan) and overlaid with fluorescent gp120 (green). As indicated the pictures show CD169 alone, CD169 and SIGN-R1; CD169 and gp120; and overlay of all 3. Scale bar is 300 m.