Model.

<p>Wild type plasma cells express higher levels of Blimp1, IRF4 and XBP1, while Bcl6 is repressed. This allows for antibody secretion. In the case of ADAM10^Δ/ΔIgG1-cre^+/− mice, Bcl6 levels are higher than seen in wild type. Moreover, Blimp1, IRF4 and XBP1 expression are decreased, leading to impaired antibody secretion.</p

Fyn KO B cells have impaired STAT3 and STAT5 activation upon IL-4 stimulation.

<p>WT and Fyn KO naive B cells were isolated from mice and stimulated with IL-4 (30 ng/ml) at 37°C for indicated times. Cells were lysed and phosphorylated forms of STAT3 (pY705 and pS727), STAT5 (pY694) and STAT6 (pY641) were assessed by western blot. Non-phosphorylated proteins were used as loading controls. Representative image of 3 independent experiments.</p

Fyn deficient mice have impaired humoral responses.

<p>Serum (a) IgM, (b) IgG1, (c) IgG2c, (d) IgG2b and (d) IgE were measured by ELISA from 8–12 week old naïve mice. Bars represent the mean ± SE of 10 mice per group (***p<0.001). Data represent results obtained in at least two independent experiments.</p

Fyn deficient mice have impaired germinal center formation and reduced T_FH numbers.

<p>Mice were immunized with NP-KLH emulsified in alum and 14 days post-immunization GC formation and T_FH frequency were assessed by flow cytometry. (A) Representative dot plot for GC B cells (gated on B220⁺ cells). (B) Quantification of GC B cells. (C) Representative dot plot for T_FH cells (gated on CD4⁺ cells) (D) Quantification of T_FH cells. Bars represent the mean ± SE of 6 mice per group. Data represent results obtained in at least two independent experiments. (**p<0.01, ***p<0.001).</p

Fyn KO mice have impaired antibody titers upon T-dependent immunization.

<p>WT and Fyn KO mice (n  = 5–9) were immunized i.p. with 10 ìg of NP-KLH emulsified in 4 mg of Alum. NP-specific antibody titers were assessed by ELISA: (a) IgM, (b) IgG2c, (c) IgG1 and (d) IgG2b after 7, 14, 21 and 28 days following immunization. Data shown are mean ± SE of 2 independent experiments. *p<0.05; **p<0.001; ***p<0.0001 based on Student’s t test on Fyn WT and Fyn KO. RU, relative units.</p

ADAM10^Δ/ΔIgG1-cre^+/− mice show impaired recall antibody responses but normal memory B cell development.

<p>(A) ADAM10^Δ/ΔIgG1-cre^+/− (▪) and controls (□) were immunized with NP-KLH emulsified in alum. At the indicated times serum samples were collected and NP-specific antibodies were measured by capture ELISA. Mice were challenged with NP-KLH in alum 6 weeks following primary immunization. Bars represent the mean ± SE of 5–9 mice per group (*p<0.05, **p<0.01). Data represent results obtained in at least two independent experiments. (B–C) ADAM10^Δ/ΔIgG1-cre^+/− (▪) and controls (□) were immunized with NP-KLH emulsified in alum. Fourteen days following immunization, spleens were harvested and memory B cell numbers were analyzed. Memory B cells were defined as B220⁺IgM^lo/−IgG1⁺CD38⁺<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042694#pone.0042694-Aiba1" target="_blank">[44]</a>.(B) Representative dot plot (gated on B220⁺IgM^lo/− cells). (C) Frequency of memory B cells of total B220⁺IgM^lo/− cells, representative of 5 mice from at least two independent studies. ns: non significant.</p

Fyn deficient B cells have reduced antibody levels despite normal proliferation following <i>in vitro</i> stimulation.

<p>B cells were isolated and cultured with IL-4 and anti-CD40L. Eight days following stimulation, supernatant were harvest and analyzed for (a) IgG1 and (b) IgE by ELISA. Three days following challenge, tritiated thymidine was added to cells. Cells were incubated for 24 hours and then harvested. Thymidine incorporation was then measured (c). Experiment has been performed three times with similar results. (*p<0.05, **p<0.01, ***p<0.001).</p

Plasma Cells from ADAM10^Δ/ΔIgG1-cre^+/− mice have altered gene expression.

<p>ADAM10^Δ/ΔIgG1-cre^+/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic PCs were isolated via magnetic bead isolation. mRNA was isolated and (A) <i>Xbp1</i> (B) <i>Prdm1</i>, (C) <i>Irf4</i> and (D) <i>Bcl6</i> message levels were determined by qPCR. (E) The ratio of <i>Prdm1</i> to <i>Bcl6</i> was calculated. Bars represent the mean ± SE of 3 independent studies; cells from 3 mice from each genotype pooled in each study. (*p<0.05, **p<0.01, ***p<0.001).</p

Fyn-KO mice have reduced plasma cell percentages.

<p>Fyn-KO and WT mice were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, spleens were harvested and PC numbers were analyzed. (A) Representative FACS staining. (B) Quantified results from spleen. Bars represent the mean ± SE of 6 mice per group. Data represent results obtained in at least two independent experiments. (**p<0.01).</p

Plasma cells isolated from ADAM10^Δ/ΔIgG1-cre^+/− mice express Bcl6.

<p>ADAM10^Δ/ΔIgG1-cre^+/− and controls were immunized with NP-KLH emulsified in alum. Twenty-one days following immunization, splenic plasma cells were analyzed by Bcl6 protein expression via flow cytometry. (A) Plasma cells were defined as CD138⁺B220^lo/−. After gating on these cells, they were analyzed for (B) non-specific (isotype) or (C) Bcl6-specific staining. (D) Frequency of Bcl6⁺ cells of plasma cells. Bars represent the mean ± SE of 4–5 mice. (**p<0.01).</p