TD antigen-specific immunoglobulin and long-lived plasma cell production are impaired in TRAF6-ΔB mice.

<p>(a–c) Defective antigen-specific IgG1 and IgG2b production in response to TD antigens for primary response to the TD Ag NP-KLH (“1^st”, 7days after immunization) and also for the secondary response (“2^nd”, 14 days after immunization as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004736#s4" target="_blank">Methods</a>). The level of high-affinity (clear bars) and total (black bars) Abs are compared: (a), antigen-specific IgM levels; (b), IgG1; (c), IgG2b. Inset in (b) shows the primary response plotted on a smaller scale. Data are presented as mean±SD. *; <i>p</i><0.05, compare total and high affinity Igs, **; <i>p</i><0.05, compare control and TRAF6-ΔB mice. (c) Reduced number of long-lived plasma cells producing NP-specific IgG1 in the BM of TRAF6-ΔB mice collected 60 days after immunization (as described in a–c), detected by ELISpot assay.</p

Generation of B cell specific TRAF6 KO mice.

<p>(a) Southern blot analysis to confirm the <i>Cre</i>-mediated deletion of the floxed fragment. (b) Western blot analysis to demonstrate specific deletion of TRAF6 in B cells. Lysates blotted with anti-TRAF6 or anti-actin from B cells and non-B cells are shown.</p

TRAF6 is required for mature B cell homeostasis.

<p>(a) Flow cytometric analysis of BM from control and TRAF6-ΔB mice stained with the indicated antibodies (top-most and middle panels). The percentages of encircled areas are indicated. Pro+Pre, pro-B and pre-B cells; Imm, immature B cells; Rec, recirculating B cells. Results are representative of 5 mice. Lowest panel, absolute cell numbers; *, <i>p</i><0.005. (b) Similar analysis of total spleen cells. Total B cells are shown in the topmost panels; mature (Mat) and immature (Imm) B cells are compared in the middle panels. The lowest panels compare the follicular (FO) and MZ sub-populations within the mature B cell gate shown in the middle panels. Cell numbers of each B cell subset in the spleen are shown in the lowest panels. Results are representative of at least 4 mice; *, <i>p</i><0.05. (c) Splenic microarchitecture visualized by immunohistochemical staining with anti-B220 (red), anti-CD3 (blue) and anti-MOMA1 (green) antibodies. Results are representative of at least 5 mice.</p

Peritoneal CD5⁺ B cell population is absent in TRAF6-ΔB mice.

<p>(a) Total peritoneal exudate cells (PECs) numbers from control and TRAF6-ΔB mice. (control; n = 5, TRAF6-ΔB; n = 5) *: <i>p</i><0.05 (b) Peritoneal exudate cells (PECs) from control and TRAF6-ΔB mice analyzed by flow cytometry. Total CD19⁺ B cells (top panels) are subdivided into B-1 and B-2 subsets (middle panels), and the CD23⁻CD11b⁺ B-1 subset is further divided into B-1a and B-1b (bottom panels). Results are representative of at least 8 mice. (c) The ratios of each B cell subset in the PECs from control, CD40 KO and TRAF6-ΔB mice are shown. Data are presented as mean±SD. (control; n = 12, CD40 KO; n = 4, TRAF6-ΔB; n = 8) *: <i>p</i><0.01 compare to control. (d) Profiles of CD5 expression in B-1 cells from CD40-deficient mice and MyD88/TRIF-doubly deficient mice are analyzed as described in (b).</p

Germinal center formation in response to TD antigen is intact in TRAF6-ΔB mice.

<p>(a) GC formation in the spleen in response to PBS or sheep erythrocyte (SRBC) immunization examined by flow cytometric analysis using fluorescent PNA and antibodies to B220, CD19 and GL7. Profiles of PNA vs. GL7 in B220⁺CD19⁺ B cell populations are shown. Ratios of GL7^hi and PNA⁺ B cells in the encircled areas are indicated. (b) Frequencies of GL7^hi and PNA⁺ B cells are quantified. Data are presented as mean±SD of five samples of one representative experiment out of 2 independent experiments. (c) Spleen sections were examined for GC formation by immunohistochemical staining with fluorescent PNA (green) and antibodies to B220 (red) and CD3 (blue).</p