tyrosine kinases (Kurosaki and Hikida, 2009). However, BCR ligation in purified splenic BMKO B cells resulted in a normal tyrosine phosphorylation pattern (Fig. 3 D). Thus, Mule is unlikely to be involved in BCR-mediated proximal signaling. Consistent with this finding, Ca2+ flux in BMKO B cells treated with anti-IgM was also normal (Fig. 3 E). Further investigation is required to dissect how Mule is in- volved in BCR-induced proliferation and activation. Mule deficiency severely reduces serum immunoglobulins and humoral immune responses The involvement of Mule in B cell proliferation and activation prompted us to investigate serum Ig production and humoral JEM Vol. 209, No. 1 A r t i c l e Figure 4. Impaired humoral responses in BMKO mice. (A) Serum from control and BMKO mice (n = 7–10) was ana- lyzed by ELISA to determine levels of the indicated Igs. Hori- zontal bars are the geometric mean. (B) Littermate control and BMKO mice (n = 6) were i.p. immunized with TNP-Ficoll and serum was analyzed as for A on the indicated days after im- munization to detect anti-TNP IgM (left) and anti-TNP IgG3 (right). Results are expressed as a serum dilution factor. Hori- zontal bars are the geometric mean. (C) Littermate control and BMKO mice (n = 6) were i.p. immunized with NP(15)-CGG. Left, serum was analyzed as in B to detect NP-specific IgG1. Right, representative flow cytometric analysis of GC B cells (PNA- hiCD38lo) among gated B220+ cells from control and BMKO mice before immunization (No-imm) and at 14 d after immu- nization (NP-CGG). Numbers indicate the percentage of GC B cells. Data shown are from a single experiment involving four to five mice per genotype. Horizontal bars are the geometric mean. (D) Littermate control and BMKO mice (n = 5–6) were i.v. injected with 105 pfu VSV. Serum was analyzed on day 0 (before infection) and on days 2, 4, 6, 10, and 12 after VSV infection to detect the total anti-VSV IgM plus IgG titer (left) or the VSV-specific IgG titer (right). Results are the combined mean ± SD (n = 5–6; *, P < 0.05; **, P < 0.005). Ab titration data are from a single experiment involving 6–10 mice per geno- type (A and B), or from two independent experiments involving two to three mice per genotype analyzed together (D). immune responses in our BMKO mice. Apart from IgG2b, serum levels of IgM, IgG1, IgA, IgG2a, and IgG3 were reduced by 2.5-, 2-, 4.5-, 32-, and 37-fold, respectively, in nonimmunized BMKO mice com- pared with controls (Fig. 4 A). However, plasma cell numbers in the BM of mutants and controls were similar (unpublished data), indicating that Mule is involved in Ig production rather than plasma cell differentiation or maintenance. To determine whether Mule-deficient B cells could mount normal humoral responses, we im- munized control and BMKO mice with either the T-independent (Ti) antigen TNP-Ficoll or the T-dependent (Td) antigen alum-precipitated 4-hydroxy-3-nitrophenylacetyl–chicken -globulin (NP(15)-CGG). When serum was analyzed at 2–3 wk after immunization, Ti-immunized BMKO mice failed to mount a TNP-specific IgM or IgG3 response, whereas Ti-immunized control mice showed normal IgM and IgG3 responses (Fig. 4 B). In contrast, although Td-immunized BMKO B cells produced significantly less NP-specific IgG1 compared with controls at 7 d after immunization, this anti- body (Ab) titer was normal at 14 and 21 d after immunization (Fig. 4 C, left). Consistent with this finding, the frequency of GC B cells in BMKO spleens was comparable to controls at day 14 after Td immunization as determined by flow cytometry (Fig. 4 C, right). To examine the role of Mule in humoral responses in a more physiological setting, we infected BMKO and control mice with vesicular stomatitis virus (VSV).WT mice infected with VSV mount strong humoral responses characterized by 177