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

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

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-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

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