Transgenic mice expressing dominant-negative bright exhibit defects in B1 B cells

Abstract

The transcription factor Bright up-regulates Ig H chain production from select V region promoters and requires Bright dimerization, Bruton's tyrosine kinase (Btk), and the Btk substrate, TFII-I, for this activity. Defects in Btk cause X-linked immunodeficiency disease in mice and humans. Btk-deficient mice exhibit decreased serum IgM production, B cell developmental blocks, absence of peritoneal B1 cells, and subnormal immune responses against Ags, including phosphorylcholine, which confer protection against Streptococcus pneumoniae. Transgenic mice expressing dominant-negative Bright share similarities with Btk-deficient mice, including decreased serum IgM, poor anti-phosphorylcholine responses, and slightly reduced numbers of mature B cells. Although dominant-negative Bright mice developed B1 B cells, these were functionally deficient in Ig secretion. These data suggest a mechanistic explanation for the abnormal responses to phosphorylcholine observed in Btk-deficient mice, and indicate that Bright functions in a subset of Btk-dependent pathways in vivo, particularly those responses dominated by B1 B cells.

Figure 1. Generation of DN Bright transgenic mice

A. A schematic diagram depicts the DNA construct used for DN Bright expression from the B cell-specific CD19 promoter with the associated C-terminal his-myc tag, SV40 polyA, and LoxP sequences. Amino acids mutated to alanine (underlined and in bold) in the Bright DNA binding domain are shown. B. DNA from five potential founder mice (denoted X, B, Y, Z and H) was PCR amplified for the Bright transgene. The lower band was an unrelated product confirming the sample DNA integrity. C. A representative experiment of transgene expression from DN Bright positive lines by RT-PCR of splenic RNA with and without reverse transcriptase (RT) is shown for transgenic line D (TG-D) and a negative control (CON). The smudge in the RT⁻ lane was not consistent and was due to probe background.

Figure 2. Serum IgM is reduced in DN Bright TG mice

Sera obtained from non-immunized 8–12 week old male control (CON) and DN Bright homozygous transgenic (TG) mice were analyzed by ELISA for total IgM (A), IgG_2a (B), IgG_2b (C), IgG₃ (D) and IgA (E). Points indicate values obtained for individual mice. Data are representative of 6 experiments using two to three transgenic lines. Means and standard error bars are indicated. Statistically significant differences are indicated by asterisks (p value = *0.0132, **0.019) as determined by the unpaired two-tailed Student’s t test.

Figure 3. Peripheral lymphocytes develop in DN Bright transgenic mice

A. Representative flow cytometric analyses of whole spleen from a transgenic (TG-D) and control (CON) mouse indicate relative percentages of CD19⁺ B and CD3⁺ T cells. B. Spleen sections from non-immunized transgenic and control mice were stained with antibodies to IgM (red) and MOMA-1 (green) to identify IgM⁺ B cells and metallophyllic macrophages. Data are representative of three mice per group. C. Representative flow cytometric analyses of thymic tissue from transgenic and control mice indicate percentages of CD4 and CD8 single and double positive T cells.

Figure 4. DN Bright protein expression is highest in immature B cell subpopulations

A. DN Bright protein was not detected in immunoblots (IB) of whole thymus (Thy), heart (Hrt), lung (Lg), kidney (Kd), liver (Lv), or brain (Br) cells from transgenic TG-D mice and was barely detectable in spleen (Spl). Lane 1 contains 10 μg of wild type transgenic (WT-TG) Bright splenic protein demonstrating both transgenic (T) and endogenous (E) Bright. Relative actin levels are indicated below. Data are representative of two experiments from two transgenic lines. B. Representative flow cytometric analyses of bone marrow from DN Bright transgenic (TG-D) and control (CON) mice indicate relative percentages of pro-B (Pro), pre-B (Pre), immature (Imm) and recirculating (Rec) B cells. C. TG-D, CON and WT-TG bone marrow B cell subpopulations were isolated as in (B) and assessed for Bright proteins (left panel). WT-TG spleen (+) was used in lane 1. An independent experiment (right panels) shows corresponding actin levels for Imm and Rec B cells. Data are representative of three experiments. D. Representative flow cytometric analyses of transitional (T1, T2 and T3), marginal zone (MZ) and follicular (FOL) splenic B cells are shown for transgenic and control mice. E. Immunoblots of sorted splenic T1, T2, MZ and FOL B cells from DN Bright (TG-D and TG-B), control (CON) and WT Bright (WT-TG) transgenic lines are shown. Data are representative of 4 experiments. F. DN Bright and actin transcripts were amplified by semi-quantitative RT-PCR from T1 and FOL B cell RNA. Actin levels are quantified below.

Figure 5. DN Bright functions in peritoneal B1 cells

A. Representative flow cytometric analyses of peritoneal cells from transgenic (TG-D) and control (CON) mice show similar proportions of B1a, B1b and B2 B cells. B. Western blot of wild type transgenic spleen (WT Spl) and peritoneal cavity cells from CON and DN Bright (TG-D) mice show transgenic (T) and endogenous (E) Bright protein. C. Peritoneal B cell extracts from two control mice (C1 and C2) and two DN Bright transgenic mice (DN1 and DN2) were assessed for Bright binding activity by EMSA with and without anti-Bright antibody. The Bright complex is indicated with an arrow. Data are representative of 5–7 control and transgenic mice assessed. D. The extracts used in (C) were compared for relative protein levels using an EMSA for octamer protein binding. Oct2 levels, as shown by the arrow, were quantified as indicated by the numbers below each lane.

Figure 6. DN Bright expression inhibits B1 cell function

A. Peritoneal B cells from CON and TG-D mice (left panel) or WT-Br mice (right panel) were cultured with 20 μg/ml LPS for 72 hours and secreted IgM was measured by ELISA. Symbols represent individual cultures. Data shown are representative of 2–3 experiments performed. Means and standard error bars are indicated. p=0.0183, Student’s T test for TG-D compared to CON. B. ELISPOT analyses of LPS stimulated peritoneal B cells from CON, TG-D and WT-Br mice show numbers of IgM⁺ cells obtained per input cell. Standard error bars and means are shown. Data are representative of 3 experiments. C. Peritoneal B cell mRNAs from control (CON), DN and WT transgenic mice were stimulated with LPS as in (A) and assessed by PCR for relative levels of μ membrane (mem) versus secretory (sec) IgM. Ratios were quantified and are listed below. Actin levels for each sample are shown and quantified. Data are representative of samples from 4 to 7 mice each.

Figure 7. DN Bright expression causes deficient anti-PC responses and increased susceptibility to S. pneumoniae

Sera from five control (CON) and four transgenic (TG-D) preimmune and post-immunized 8–12 week old male mice were analyzed by ELISA for PC-specific IgM (A). The last positive dilution from triplicate samples was the defined endpoint. At day 7, p= 0.0268 by two-tailed Student’s t test. At day 25, p=0.0483 by one-tailed Student’s t test. B. Mice immunized with NP-KLH were analyzed similarly for anti-NP-specific antibodies. Standard error bars are indicated. C. RNA from CON and TG-D DN Bright follicular B cells was amplified for S107 V1 IgM and actin by RT-PCR. D. CON and TG-D mice were infected with S. pneumoniae and monitored for survival. Symbols represent the hours survived by one mouse. All samples fall within two standard deviations from the mean. Means and standard error bars are indicated. p= 0.019, Mann-Whitney analysis and 0.0079 by Student’s T test.