(A) Confocal microscopy of thick lymph node (LN) sections prepared from mice that had received adoptively transferred B cells (previous day), injected with fluorescently labeled gp120, and immunostained as indicated. LN section image (sagittal, tiled) shows gp120, green; CD169, pink; CD21/35, cyan; and B cells, red and blue. Scale bar is 200 μm (top). A zoomed image of the white boxed area is shown. Scale bar is 60 μm (middle). Images of an IFC are shown: gp120, green; CD169, red; SIGN-R1, cyan; CD21/35, blue; adoptively transferred B cells, pink; and CD169, red (bottom, left). SIGN-R1 signal removed (bottom, right). Arrows indicate gp120 positive cells. Scale bars is 50 μm. (B) Intravital two-photon laser scanning microscopy (TP-LSM) images of the inguinal LN from a mouse injected with fluorescent gp120 and the indicated antibodies. The top images over the IFC show gp120, white; CD169, red; F4/80, green; and adoptively transferred B cells, blue, (left panel). SIGN-R1 antibody, red, used instead of CD169 (right panel). Scale bars are 100 μm. The bottom images are from the follicular-medullary junction and show gp120, white; SIGN-R1, red; F4/80, green; and adoptively transferred B cells, blue. Scale bar is 50 μm. (C, D) Flow cytometry of LN cells immunostained and gated as indicated using inguinal LN cells from a mouse injected with fluorescent gp120 1.5 hr previously, or not. LiveGr-1−CD11c− gated population plotted for F4/80 vs CD11b. Gates ‘a’, ‘b’, and ‘c’ as indicated were re-plotted to show SIGN-R1 vs gp120 in right three plots (top 2 rows). (C). LiveGr-1−CD11c+ population is shown plotted for SIGN-R1 vs CD11b. Histogram of indicated three populations (a, b, and c) plotted as gp120 signal (black line) vs % of maximum intensity compare to gp120 negative control (shaded). Numbers are % gp120 positive cell population in gate (D). (E) In vitro binding by LN cells incubated with fluorescent gp120, or not, and in the presence of non-labeled gp120 or non-labeled SIGN-R1 antibody (different epitope) and then analyzed by flow cytometry. LiveGr-1−CD11c−CD11b+ cells were analyzed for gp120 vs SIGN-R1 (not shown) and the CD11b+ cells, gray contour; the SIGN-R1+gp120+ cells, green dots; SIGN-R1−gp120− cells, black dots; and SIGN-R1+gp120− cells, gray dots, were plotted to show CD169 vs SIGN-R1. (F) Interferon-γ intracellular flow cytometry of cells prepared from the inguinal LNs of mice administered gp120 near the tail base, or not, 3 hr prior to collection. LiveGr-1−CD11c−CD11b+ cells were analyzed for SIGN-R1 vs CD169 and separated into three populations (left panels). The levels of intracellular interferon-γ are shown as histograms of maximum intensity in cells from the gp120 non-exposed (gray) and gp120 injected mice (white, outlined by black lines). Unstained control is delineated by a gray line.