Effect of αGalCer treatment on activation and numbers of MZBs <i>in vivo</i> and <i>in vitro</i>.

<p><b>A–D</b>. Four-mo-old female CD1d^−/−, WT and Vα14^Tg BALB/c mice were injected i.p. with 4 µg of αGalCer or vehicle. Their spleens were harvested 3–4 d later and analyzed by flow cytometry and immunohistochemistry for MZBs. Results using three mice per group from one representative of at least three independent experiments are shown. <b>A</b>. CD86 expression on gated CD19⁺CD21^hiCD23^−/low (MZBs) and CD19⁺CD21⁺CD23⁺ (FoBs) in αGalCer (thick line) or vehicle (shaded area) injected mice. <b>B</b>. MZBs (CD21^hiCD23^−/low) were analyzed on gated CD19⁺ lymphocytes. MZB frequency is expressed as % of CD19⁺ lymphocytes in representative dotplots (<b>B</b>) and as the mean±SE from three each of vehicle or αGalCer-injected WT, CD1d^−/− and Vα14^Tg mice (<b>C</b>). A significant reduction of MZBs was found in αGalCer-treated WT (*p<0.05) and Vα14^Tg mice (**p<0.01), but not in CD1d^−/− mice. <b>D</b>. Frozen spleen sections from αGalCer or vehicle treated mice were stained for APC-IgM (blue) and FITC-MOMA1 (green). Confocal images show IgM⁺ cells (blue) in the marginal zone between red pulp and MOMA-1 (green) in CD1d^−/− and vehicle-injected Vα14^Tg mice. IgM⁺ cells in the marginal zone are reduced in αGalCer-treated Vα14^Tg mice (as indicated by a blue arrow). MZ, marginal zone; RP, red pulp; WP, white pulp. 20× and 40× magnification. <b>E</b>, <b>F</b>, Spleen cells (2×10⁶ cells per ml) from 3-mo-old BALB/c mice were cultured with or without LPS in the absence or presence of αGalCer (100 ng/ml). Results represent five independent experiments, each time using cells from one mouse per group. <b>E</b>. Expression of CD69 and CD86 are shown on gated MZBs (CD19⁺CD21^hiCD23^−/low) in spleen cells cultured with medium alone (shaded area), αGalCer (blue line), LPS (green line) or LPS+αGalCer (red line) for 24 h. The MFI of CD69 and CD86 are shown. <b>F</b>. Spleen cells from WT and CD1d^−/− BALB/c mice were cultured without or with LPS and/or αGalCer for 72 h. MZB cells are expressed as % of mature B cells (AA4.1⁻ IgM⁺).