Fyn kinase is required for optimal humoral responses

Abstract

The generation of antigen-specific antibodies and the development of immunological memory require collaboration between B and T cells. T cell-secreted IL-4 is important for B cell survival, isotype switch to IgG1 and IgE, affinity maturation, and the development of germinal centers (GC). Fyn, a member of the Src family tyrosine kinase, is widely expressed in many cell types, including lymphocytes. This kinase is known to interact with both the B cell and T cell receptor (BCR and TCR, respectively). While Fyn deletion does not impair the development of immature T cells and B cells, TCR signaling is altered in mature T cells. The current study demonstrates that Fyn deficient (KO) B cells have impaired IL-4 signaling. Fyn KO mice displayed low basal levels of IgG1, IgE and IgG2c, and delayed antigen-specific IgG1 and IgG2b production, with a dramatic decrease in antigen-specific IgG2c following immunization with a T-dependent antigen. Defects in antibody production correlated with significantly reduced numbers of GC B cells, follicular T helper cells (TFH), and splenic plasma cells (PC). Taken together, our data demonstrate that Fyn kinase is required for optimal humoral responses.

Figure 1. Fyn deficient mice have impaired humoral responses.

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.

Figure 2. Fyn KO mice have impaired antibody titers upon T-dependent immunization.

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.

Figure 3. Fyn-KO mice have reduced plasma cell percentages.

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).

Figure 4. Fyn deficient mice have impaired germinal center formation and reduced T_FH numbers.

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).

Figure 5. Fyn deficient B cells have reduced antibody levels despite normal proliferation following in vitro stimulation.

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).

Figure 6. Fyn KO B cells have impaired STAT3 and STAT5 activation upon IL-4 stimulation.

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.