Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice

Abstract

The pre-B cell receptor consists of immunoglobulin (Ig) mu heavy chains and surrogate light chain, i.e., the VpreB and lambda5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1(-/-) VpreB2(-/-) mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2-containing receptor in this process.

Figure 1

Homologous recombination of VpreB1 and VpreB2. (A) VpreB2 genomic locus before and after homologous integration. The targeting vector consists of 8.4 kb of VpreB2 genomic sequences and the VpreB2 coding sequence replaced by the hygromycin (hygro) resistance gene. Shown is the location of primers used for detection of homologous recombination (nos. 1 and 2), giving rise to a 1.8-kb PCR product. Offspring were later screened by PCR with primer nos. 6 and 7 and nos. 8 and 9, and the expected size of the respective product is indicated. The probe for Southern blotting and expected fragments are indicated. Restriction enzymes: R, EcoRI; H, HindIII; K, KpnI; S, SalI. (B) The arrow indicates the 1.8-kb PCR product (primer nos. 1 and 2) upon homologous recombination of the VpreB2 locus. E14, original nontargeted ES cells; Y2, VpreB1-targeted E14 cells; ES94 and ES163, VpreB2-targeted Y2 cells; ES94 and ES163 mice, mice established from these ES clones. (C) The VpreB1 genomic locus before and after homologous integration. The VpreB1 targeting vector consisted of 5.3 kb of VpreB1 genomic sequences and the neomycin (neo) resistance gene which replaced the VpreB1 coding sequence. Primer nos. 3, 4, and 5 were used to screen for genotype and the expected size of the respective product is shown. The probe used for Southern blotting is indicated and expected fragments. Restriction enzymes: R, EcoRI; X, XbaI. (D) Homologous recombination of the VpreB1 locus using the probe in C. Samples are as indicated in B, except ES65. The asterisk (*) shows the extra band detected in ES163 ES cells and in ES163 +/− and −/− mice.

Figure 2

Lack of VpreB expression in VpreB1 ^{− /}−VpreB2 ^{− /}− pre-B cells. (A) Total RNA from bone marrow cells of the indicated mice. RT-PCR was performed for HGPRT, VpreB1, VpreB2, and λ5. C indicates negative control. (B) In vitro cultured pre-BI cells were stained for B220 in combination with VP245 (anti-VpreB), LM34 (anti-λ5), or c-kit and analyzed by FACS^®. The histograms from the VP245, LM34, and CD117 stains are shown.

Figure 3

Impaired B cell development in VpreB1 ^{− /}− VpreB2 ^{− /}− mice. (A) FACS^® analysis of bone marrow cells from VpreB1⁺/−VpreB2⁺/− (top) and VpreB1 ^{− /}−VpreB2 ^{− /}− (bottom) ES94-derived 10-d-old littermates. Cells were stained for B220 (CD45R) in combination with either CD19, c-kit (CD117), CD25, IgM, or IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (B) FACS^® analysis of spleen cells from the mice in A. Cells were stained for B220 in combination with CD19, IgM, IgD, or Igλ, and also with IgM in combination with IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (C) FACS^® analysis of peritoneal cells from VpreB1⁺/+VpreB2⁺/+ and VpreB1 ^{− /}−VpreB2 ^{− /}− ES94 mice stained for B220 and CD5 aged 12 d and 12 wk, respectively. Cells falling within the lymphocyte gate are displayed. The numbers indicate the percentage of cells within these regions.

Figure 3

Impaired B cell development in VpreB1 ^{− /}− VpreB2 ^{− /}− mice. (A) FACS^® analysis of bone marrow cells from VpreB1⁺/−VpreB2⁺/− (top) and VpreB1 ^{− /}−VpreB2 ^{− /}− (bottom) ES94-derived 10-d-old littermates. Cells were stained for B220 (CD45R) in combination with either CD19, c-kit (CD117), CD25, IgM, or IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (B) FACS^® analysis of spleen cells from the mice in A. Cells were stained for B220 in combination with CD19, IgM, IgD, or Igλ, and also with IgM in combination with IgD. Cells falling within the lymphocyte gate are displayed. Numbers indicate percentage of cells within this quadrant. (C) FACS^® analysis of peritoneal cells from VpreB1⁺/+VpreB2⁺/+ and VpreB1 ^{− /}−VpreB2 ^{− /}− ES94 mice stained for B220 and CD5 aged 12 d and 12 wk, respectively. Cells falling within the lymphocyte gate are displayed. The numbers indicate the percentage of cells within these regions.

Figure 4

T cell–dependent and –independent immune responses. 4 to 5-mo-old mice of the indicated genotypes, were immunized with either NP-Ficoll or alum-precipitates of either oxazolone-ovalbumin (Ox) or ovalbumin (Ov). 2 wk later, the mice were bled and the sera analyzed in ELISA. The data are represented as titers. Preimmune sera were <1. wt, wild-type; het, heterozygous; homo, homozygous.

Figure 4

T cell–dependent and –independent immune responses. 4 to 5-mo-old mice of the indicated genotypes, were immunized with either NP-Ficoll or alum-precipitates of either oxazolone-ovalbumin (Ox) or ovalbumin (Ov). 2 wk later, the mice were bled and the sera analyzed in ELISA. The data are represented as titers. Preimmune sera were <1. wt, wild-type; het, heterozygous; homo, homozygous.

Figure 4

T cell–dependent and –independent immune responses. 4 to 5-mo-old mice of the indicated genotypes, were immunized with either NP-Ficoll or alum-precipitates of either oxazolone-ovalbumin (Ox) or ovalbumin (Ov). 2 wk later, the mice were bled and the sera analyzed in ELISA. The data are represented as titers. Preimmune sera were <1. wt, wild-type; het, heterozygous; homo, homozygous.

Figure 5

Allelic exclusion of Ig μH chain expression on IgM^aIgM^b F1 B cells. FACS^® analysis of bone marrow cells from ES94-derived VpreB1/VpreB2 double-mutant littermates of the indicated genotype at the age of 74 d. All cells from both femurs were stained for B220 in combination with IgD, IgM^a, and IgM^b. Cells falling both within the lymphocyte and the B220⁺IgD⁻ gate were analyzed for expression of IgM^a and IgM^b. The numbers indicate the percentage of cells within these regions. Only 10% of the gated cells are shown for wild-type.