Figure 2
Figure 2. Human IgM and IgA PCs express a functional BCR. (A) Increase in phospho-SykY525-526 staining in BM-PCs (black bars), memory B cells (white bars), and LP-PCs (gray bar) following cross-linking with 2.5 μg/mL anti–light chain F(abʹ)2 antibody fragments. Mean of rMFI + SEM of duplicates. PC gates as in Figure 1. One experiment of 3. (B) ERK1/2 phosphorylation of BM-PCs (black circles) and memory B cells (white triangles) 15 minutes after stimulation with increasing doses of anti–light chain antibodies; 1 experiment of 3. (C) Levels of ERK1/2 phosphorylation in LP-PCs cultured for 15 minutes in the presence or absence of anti-IgA F(abʹ)2 antibodies. Mean of rMFI + SEM of duplicates; 1 representative experiment of 2. (D) Levels of ERK1/2 phosphorylation of κ (black bars) or λ (white bars) IgA BM-PCs after 15 minutes in the presence of 2.5 μg/mL anti-κ or anti-λ light chain F(abʹ)2 antibodies. Mean + SEM of duplicate; 1 experiment of 3. (E) Internalization of IgM and IgA on BM-PCs and memory B cells cultured with 2.5 μg/mL anti-IgM or IgA heavy chain F(abʹ)2 fragments. Shown is the surface staining of light chains after 20 minutes at 4°C (black bars) or 37°C (white bars). Mean of fold expression to control (concomitant staining of heavy and light chains at 4°C) + SEM of duplicates; 1 experiment of 3. (F) Confocal images of IgA BM-PCs and memory B cells incubated for 20 minutes with anti-IgA-Cy5 (green) and counterstained with CD45 Alexa Fluor 700 (red) as a membrane marker and 4,6 diamidino-2-phenylindole (blue). Images are representative of 2 independent experiments. Quantification of the ratio of internal vs membrane IgA (right panel). (G) Expression of phospho-AKT (Ser 473) on IgA and IgM BM-PCs cultured for 20 minutes with 2.5 μg/mL anti–light chain F(abʹ)2 antibodies. Mean of rMFI + SEM of duplicates; 1 representative experiment of 3. (H) Expression of CD44 on IgG, IgA, and IgM BM-PCs cultured for 12 hours with increasing doses of F(abʹ)2 anti–light chain antibodies. Mean of rMFI + SEM of duplicates; 1 experiment of 3. (I) Survival of IgA and IgM BM-PCs cultured for 4 days in the presence of increasing doses of anti–light chain F(abʹ)2 antibodies. Mean of % input cells + SEM of duplicates; 1 representative experiment of 3. (J) Cell tracer dilution of IgA BM-PCs after 4 days in culture with (lower histogram) or without (upper histogram) 0.5 μg/mL of anti–light chain F(abʹ)2 antibodies (left panel). One experiment out of 2. Survival of κ (black bars) or λ (white bars) IgA BM-PCs upon being cultured for 4 days in the presence of 10 μg/mL anti-κ or anti-λ light chain F(abʹ)2 antibodies. Mean + SEM of duplicate; 1 experiment of 3 (right panel). *P < .05; **P = .01 in Student t test.

Human IgM and IgA PCs express a functional BCR. (A) Increase in phospho-SykY525-526 staining in BM-PCs (black bars), memory B cells (white bars), and LP-PCs (gray bar) following cross-linking with 2.5 μg/mL anti–light chain F(abʹ)2 antibody fragments. Mean of rMFI + SEM of duplicates. PC gates as in Figure 1. One experiment of 3. (B) ERK1/2 phosphorylation of BM-PCs (black circles) and memory B cells (white triangles) 15 minutes after stimulation with increasing doses of anti–light chain antibodies; 1 experiment of 3. (C) Levels of ERK1/2 phosphorylation in LP-PCs cultured for 15 minutes in the presence or absence of anti-IgA F(abʹ)2 antibodies. Mean of rMFI + SEM of duplicates; 1 representative experiment of 2. (D) Levels of ERK1/2 phosphorylation of κ (black bars) or λ (white bars) IgA BM-PCs after 15 minutes in the presence of 2.5 μg/mL anti-κ or anti-λ light chain F(abʹ)2 antibodies. Mean + SEM of duplicate; 1 experiment of 3. (E) Internalization of IgM and IgA on BM-PCs and memory B cells cultured with 2.5 μg/mL anti-IgM or IgA heavy chain F(abʹ)2 fragments. Shown is the surface staining of light chains after 20 minutes at 4°C (black bars) or 37°C (white bars). Mean of fold expression to control (concomitant staining of heavy and light chains at 4°C) + SEM of duplicates; 1 experiment of 3. (F) Confocal images of IgA BM-PCs and memory B cells incubated for 20 minutes with anti-IgA-Cy5 (green) and counterstained with CD45 Alexa Fluor 700 (red) as a membrane marker and 4,6 diamidino-2-phenylindole (blue). Images are representative of 2 independent experiments. Quantification of the ratio of internal vs membrane IgA (right panel). (G) Expression of phospho-AKT (Ser 473) on IgA and IgM BM-PCs cultured for 20 minutes with 2.5 μg/mL anti–light chain F(abʹ)2 antibodies. Mean of rMFI + SEM of duplicates; 1 representative experiment of 3. (H) Expression of CD44 on IgG, IgA, and IgM BM-PCs cultured for 12 hours with increasing doses of F(abʹ)2 anti–light chain antibodies. Mean of rMFI + SEM of duplicates; 1 experiment of 3. (I) Survival of IgA and IgM BM-PCs cultured for 4 days in the presence of increasing doses of anti–light chain F(abʹ)2 antibodies. Mean of % input cells + SEM of duplicates; 1 representative experiment of 3. (J) Cell tracer dilution of IgA BM-PCs after 4 days in culture with (lower histogram) or without (upper histogram) 0.5 μg/mL of anti–light chain F(abʹ)2 antibodies (left panel). One experiment out of 2. Survival of κ (black bars) or λ (white bars) IgA BM-PCs upon being cultured for 4 days in the presence of 10 μg/mL anti-κ or anti-λ light chain F(abʹ)2 antibodies. Mean + SEM of duplicate; 1 experiment of 3 (right panel). *P < .05; **P = .01 in Student t test.

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