Figure 2. Defective capping and MIIC targeting of IgβKΔR BCRs. (A and B) WT and IgβKΔR splenocytes were stimulated with FITC-conjugated IgG and IgM (H+L) F(ab)2 antibodies for 30 min in vitro, then fixed, stained with antibodies specific for Lamp-1 and H2-M, and visualized by confocal microscopy. Representative images. Quantitation of WT (red) or IgβKΔR (blue) samples (n = 3; B) based on percentage of cells with >25% overlap between the indicated markers (top) or percent colocalization of total immunofluorescence (Manders’ Coefficient; bottom). Top: *, P = 1.3 × 10−12; **, P = 3.6 × 10−15; ***, P = 0.0061. Bottom: *, P = 4.0 × 10−5; **, P = 10−6. (C and D) Cells were stimulated as in A for 2 min, fixed, and visualized by confocal microscopy. Representative images (n = 3) provided in C with quantitation of percentage of cells displaying capping provided in D; *, P = 1.4 × 10−5. (E) Internalization of BCRs from surface of WT (closed circles) or IgβKΔR (open squares) B splenocytes after stimulation with PE-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies; P = 0.0291 (two-way ANOVA; n = 3). (F and G) WT BM large pre–B cell, small pre–B cell, immature, and mature B cell populations were isolated by flow cytometry. Pre-BCRs were stimulated with biotin-conjugated anti-SL156, and BCRs were stimulated FITC-conjugated IgG and IgM (H+L) F(ab)2 antibodies, respectively, for 30 min at 37°C, and then fixed and counterstained with anti–Lamp-1 antibodies. The pre-BCR was stained with rabbit antibiotin, followed by donkey anti–rabbit Alexa Fluor 488, and all cells were visualized by confocal microscopy. Representative images are provided in F, whereas the fraction of each cell population demonstrating >25% colocalization between the receptor and Lamp-1 are provided in G (n = 3). *, P = 8.10−10 versus large pre–B; **, P = 9.8 × 10−16 versus large pre–B; ***, P = 1.03 × 10−9 versus small pre–B; †, P = 1.4 × 10−15 versus small pre–B. (H) WT (red) or IgβKΔR (blue) B splenocytes were stimulated with FITC-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for the indicated times in vitro, fixed, and stained with EEA1-specific antibodies, and the percentage of cells demonstrating >25% BCR colocalization with EEA1 was plotted as a function of time. *, P = 0.293; **, P = 6.22 × 10−6; ***, P = 0.0004. (I and J) WT or IgβKΔR B splenocytes were stimulated with FITC-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for 30 min in vitro, fixed, and stained with antibodies specific for Lamp-1 and Cathepsin L. Representative images. Quantitative assessment of colocalization of the indicated markers for WT (red) or IgβKΔR (blue) cells (J; n = 3). *, P = 1.16 × 10−16; **, P = 4.92 × 10−12; ***, P = 0.0010. (K and L) Cells were loaded with 1 µM LysoSensor and then stimulated with Texas red–conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for 30 min. (K) Cells were visualized by confocal microscopy. (I) Provided are the percentage of cells demonstrating >25% colocalization. WT (red bar) and IgβKΔR (blue bar); error bars represent mean ± SD (n = 3). Bars, 5 µm.
Figure 2.

Defective capping and MIIC targeting of IgβKΔR BCRs. (A and B) WT and IgβKΔR splenocytes were stimulated with FITC-conjugated IgG and IgM (H+L) F(ab)2 antibodies for 30 min in vitro, then fixed, stained with antibodies specific for Lamp-1 and H2-M, and visualized by confocal microscopy. Representative images. Quantitation of WT (red) or IgβKΔR (blue) samples (n = 3; B) based on percentage of cells with >25% overlap between the indicated markers (top) or percent colocalization of total immunofluorescence (Manders’ Coefficient; bottom). Top: *, P = 1.3 × 10−12; **, P = 3.6 × 10−15; ***, P = 0.0061. Bottom: *, P = 4.0 × 10−5; **, P = 10−6. (C and D) Cells were stimulated as in A for 2 min, fixed, and visualized by confocal microscopy. Representative images (n = 3) provided in C with quantitation of percentage of cells displaying capping provided in D; *, P = 1.4 × 10−5. (E) Internalization of BCRs from surface of WT (closed circles) or IgβKΔR (open squares) B splenocytes after stimulation with PE-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies; P = 0.0291 (two-way ANOVA; n = 3). (F and G) WT BM large pre–B cell, small pre–B cell, immature, and mature B cell populations were isolated by flow cytometry. Pre-BCRs were stimulated with biotin-conjugated anti-SL156, and BCRs were stimulated FITC-conjugated IgG and IgM (H+L) F(ab)2 antibodies, respectively, for 30 min at 37°C, and then fixed and counterstained with anti–Lamp-1 antibodies. The pre-BCR was stained with rabbit antibiotin, followed by donkey anti–rabbit Alexa Fluor 488, and all cells were visualized by confocal microscopy. Representative images are provided in F, whereas the fraction of each cell population demonstrating >25% colocalization between the receptor and Lamp-1 are provided in G (n = 3). *, P = 8.10−10 versus large pre–B; **, P = 9.8 × 10−16 versus large pre–B; ***, P = 1.03 × 10−9 versus small pre–B; †, P = 1.4 × 10−15 versus small pre–B. (H) WT (red) or IgβKΔR (blue) B splenocytes were stimulated with FITC-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for the indicated times in vitro, fixed, and stained with EEA1-specific antibodies, and the percentage of cells demonstrating >25% BCR colocalization with EEA1 was plotted as a function of time. *, P = 0.293; **, P = 6.22 × 10−6; ***, P = 0.0004. (I and J) WT or IgβKΔR B splenocytes were stimulated with FITC-conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for 30 min in vitro, fixed, and stained with antibodies specific for Lamp-1 and Cathepsin L. Representative images. Quantitative assessment of colocalization of the indicated markers for WT (red) or IgβKΔR (blue) cells (J; n = 3). *, P = 1.16 × 10−16; **, P = 4.92 × 10−12; ***, P = 0.0010. (K and L) Cells were loaded with 1 µM LysoSensor and then stimulated with Texas red–conjugated IgG- and IgM (H+L)-specific F(ab)2 antibodies for 30 min. (K) Cells were visualized by confocal microscopy. (I) Provided are the percentage of cells demonstrating >25% colocalization. WT (red bar) and IgβKΔR (blue bar); error bars represent mean ± SD (n = 3). Bars, 5 µm.

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