Phenotypic and functional properties of B cells with receptors for blood group A carbohydrate determinants in mice. PerC and Spl cells were prepared from Balb/c mice that were either untreated or immunized with 5 × 108 A-RBCs. The assay was performed after 7 days after the immunization. The cells were stained with FLUOS-BSA–synthetic A determinant (GalNAcα1–3Fucα1–2Gal; A-BSA) or control FLUOS-conjugated BSA and biotinylated anti-mouse IgM mAb along with various PE-conjugated mAbs (CD5, CD19, or CD11b). The biotinylated mAb were visualized using allophycocyanin-streptavidin. To ensure statistical significance, date on 105 PerC cells and 2 × 105 Spl cells were collected for each sample. (A) Representative contour plots obtained by FCM analysis show increased A-BSA–binding B cells in the Spl and PerC. Percentages given are of Spl or PerC cells in each region (average values ± SD). (B) Carbohydrate determinant-binding sIgM+ B cells were selected by gating and analyzed for the expression of various B cell markers. Dotted lines represent negative control staining with isotype-matched Abs. The results were consistent in 4 independent experiments. (C) Anti-A IgM-producing cells were detected by the ELISPOT assay. Spl, BM, PerC, cervical lymph node cells, and mesenteric lymph node cells were prepared from Balb/c mice that were either untreated or immunized with A-RBCs. A representative image of wells with 8 × 105 seeded cells is shown. The results are representative of 5 mice in each group. Anti–A carbohydrate determinant IgM-producing cells are localized mainly in the Spl.
