S1P1 receptor expression varies on B cells in the LN. (A) Recently arrived B cells have low S1P1 receptor expression. CD45.2 B cells transferred into CD45.1 mice. LN sections from inguinal LNs of recipient mice were analyzed at 2, 4, and 18 hours after transfer by immunostaining for CD45.2 (green), S1P1 receptor (red), and confocal microscopy. For each condition a composite image is shown to the left along with zoomed (2.5-fold) composite (above) and S1P1 receptor (below) images to the right. S1P1 receptor expression is indicated by an arrow. Similar results were obtained from 2 separate adoptive transfers using 3 recipient mice in each experiment. The strong S1P1 receptor signal arises from the HEV endothelial cells (HEVs). (B) Variation in S1P1 receptor expression at different sites in the LN. Confocal images of LN sections immunostained for B220 (blue) versus S1P1 receptor (red) are shown to the right and S1P1 receptor alone to the left. Scale bars are 50 μm. Eight small images (4× zoom) are shown below from the indicated sites in the LN. Both an S1P1 receptor and a B220/S1P1 receptor image are shown for each location as indicated. (C) Quantification of S1P1 receptor expression. Levels of S1P1 receptor on B cells at various sites in the inguinal LN were quantified by transferring the images to Imaris for analysis of fluorescent intensity. The results are representative of 1 of 4 experiments preformed. Each symbol represents the S1P1 receptor fluorescence of an individual B cell from the indicated sites. Shown are the mean and the standard deviation. The pairwise comparison of adjacent columns was performed using Mann-Whitney test. (D) Flow cytometry of S1P1 receptor expression on blood, spleen, and lymph node B cells. Fixed and permeablized blood, spleen, and lymph node B cells were immunostained for S1P1 expression and analyzed by flow cytometry. Gray line is isotype control. (E) Immunocytochemistry for S1P1 receptor expression on blood and LN B cells. Fixed and permeablized blood cells (first and second panels) were immunostained for B220 (green) and S1P1 receptor (magenta). B220 alone and B220 plus S1P1 are shown. Fixed and permeablized LN cells (third, fourth, and fifth panels) were immunostained for CD4 (red), B220 (green), and S1P1 receptor (magenta). Individual panels are shown. Fixed and permeablized LN cells (sixth and seventh panels) were immunostained for B220 (green) and a control antibody (magenta). Individual panels are shown. All scale bars are 7 μm unless specified otherwise.