Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

doi:10.15302/J-FASE-2014015

Front. Agr. Sci. Eng.

2014, Vol. 1

Issue (2) : 158-173 https://doi.org/10.15302/J-FASE-2014015

RESEARCH ARTICLE

Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice

Min ZHANG¹,Xueqian CHENG¹,Dan CHU¹,Jingwen LIANG¹,Yi SUN¹,Li MA¹,Beilei XU¹,Min ZHENG²,Meili WANG²,Liming REN¹,Xiaoxiang HU¹,Qingyong MENG¹,Ran ZHANG¹,Ying GUO¹,Yunping DAI¹,Robert AITKEN³,Ning LI¹,Yaofeng ZHAO^1,^4,^*(

)

1. State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China
2. GeneProtein Biotech Ltd, Beijing 100193, China
3. School of Life Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
4. Key Laboratory of Animal Reproduction and Germplasm Enhancement in Universities of Shandong, College of Animal Science and Technology, Qingdao Agricultural University, Qingdao 266109, China

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Abstract

In this study, we introduced the bovine immunoglobulin μ heavy-chain gene (the orphaned gene on BTA11) into mouse germline cells. Bovine IgM was highly expressed in selected transgenic lines, and it largely inhibited rearrangements of the endogenous immunoglobulin heavy chain (IgH) genes in these lines. The forced expression of bovine IgM resulted in reduced numbers of pro- and pre-B cells but increased the number of immature B cells in the transgenic mice. Bovine IgM-expressing B cells can migrate from the bone marrow to the spleen, but most of the cells are arrested at the T1 transitional B cell stage, leading to a significantly lower number of T2 transitional and mature B cells in the spleen. Although the serum concentrations of endogenous IgM and IgG in the transgenic mice were significantly decreased, the IgA levels were slightly increased compared to the WT mice. The bovine IgM level in the serum was only one-tenth to one-fifth of that of endogenous mouse IgM, suggesting that most of the serum immunoglobulin were contributed by endogenous IgH gene-expressing B cells. These transgenic mice also exhibited a lower frequency of unique complementarity determining region 3 (CDR3) sequences in their VH repertoire and Vκ repertoire but exhibited an increased frequency of unique CDR3 in their Vλ repertoire. Compared to the WT mice, the transgenic mice had a significantly higher percentage of mouse IgM-expressing B cells that expressed λ chains. Finally, we showed that the transgenic mice were deficient in a specific antibody response to antigen stimulation.

Keywords bovine Ig μ heavy-chain transgenic mice B cell development allelic exclusion immune response Ig repertoire

Corresponding Author(s): Yaofeng ZHAO

Online First Date: 22 September 2014 Issue Date: 10 October 2014

Cite this article:

Min ZHANG,Xueqian CHENG,Dan CHU, et al. Depletion of conventional mature B cells and compromised specific antibody response in bovine immunoglobulin μ heavy-chain transgenic mice[J]. Front. Agr. Sci. Eng. , 2014, 1(2): 158-173.

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https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2014015
https://academic.hep.com.cn/fase/EN/Y2014/V1/I2/158

Fig.1 Schematic map of the two modified transgenic BAC vectors. (a) mVH-bBAC vector; (b) mPL-bVH-bBAC vector. Gene elements are indicated by different boxes. P, L and RSS denote the promoter, leader peptide and recombination signal sequence, respectively. Squares with differently hatched fills, abbreviated as H-A, indicate homologous arms. The FRT elements are depicted as triangles. Heavy chain variable (VH), diversity (DH), joining (JH) and constant (CH) region coding segments are shown as black squares. The proportional scales for the two transgenes are also indicated.

Fig.2 Southern blot analysis of the transgenic mice. (a) Analysis of the founders of the mVH-bBAC vector transgenic lines; (b) analysis of the founders of the mPL-bVH-bBAC vector transgenic lines. The hybridized μ and pseudo-δ genes are 3.5 and 5.9 kb in length, respectively; The genomic DNA of a WT mouse is used as a negative control (NC); Line 7 was shown to be transgene-positive by PCR and another Southern blotting experiment.

Fig.3 Flow cytometry analysis of B cell populations in the bone marrow, spleen and PBL of transgenic (T_high) and WT mice. (a) Measurements of three cell subsets in bone marrow. The percentages of pro-B (B220⁺CD43⁺IgM^-), pre-B (B220⁺CD43^-IgM^-) and immature B (B220⁺CD43^-IgM⁺) cells are indicated in each gate; (b) measurements of the three cell subsets were taken in the spleen; (c) measurements of the three cell subsets were taken the PBL. The percentages of T1 (B220⁺IgD^-IgM⁺), T2 (B220⁺IgD⁺IgM⁺) and mature (B220⁺IgD⁺IgM^-) B cells are indicated in each gate. Each set contains data from transgenic (T_high) and WT animals, respectively. A total of 20000 events were collected for each sample. Plots are representative of measurements in at least five individual mice; (d) statistical analysis of bone marrow B cell subset percentages in subfigure a with black, gray and white bars representing transgenic mice with anti-bovine IgM antibody, transgenic mice with anti-mouse IgM antibody and WT mice with anti-mouse IgM antibody, respectively; (e) statistical analysis of B cell subset percentages in the spleen based on subfigure b. Significance values were determined by a two-tailed Student t test (*: P<0.05, **: P<0.01, ***: P<0.001).

Fig.4 Flow cytometry analysis of splenic lymphocytes from transgenic (T_high) and WT mice. (a) Cells were stained for B220, IgM (bIgM/mIgM) and CD21, and the proportion of the cell population corresponding to T1 (B220⁺CD21^-IgM⁺), T2 (B220⁺CD21⁺IgM⁺) and mature (B220⁺CD21⁺IgM^-) B cells is indicated for each gate. The data are representative of four independent samples; (b) statistical analysis of T1, T2 and mature B cells in transgenic and WT mice by two-tailed Student t tests (**: P<0.01, ***: P<0.001); (c) FACS analysis of cells stained for B220, bIgM and mIgM. The numbers in each plot correspond to the cell percentages contained within each gate. The data presented are representative of measurements in at least five individual mice.

Fig.5 ELISA measurement of serum Ig concentrations. Serum Ig concentrations were measured both before and after OVA immunization. (a) Concentrations of total endogenous IgA, IgG and IgM; (b) concentrations of bovine IgM, anti-OVA IgM and anti-OVA IgG in the three groups. Each point represents the antibody concentration in the serum of an individual mouse. The units of Ig content are indicated in each figure. The horizontal bars represent the mean, and the error bars represent the standard error of the mean expression level. P values were analyzed using two-tailed Student t tests (*: P<0.05, **: P<0.01).

Fig.6 Flow cytometric analysis of plasma cells and B cells in the bone marrow, spleen and PBL. (a) Gates indicate the plasma cell subset (CD138⁺B220^-) and the B cell subset (CD138^-B220⁺). The proportion of cells within each gate is indicated in the figure. The plots are representative of measurements in at least five individual mice; (b) statistical analysis of percentages of B cells and plasma cells in various tissues based on the data from subfigure a. P values were analyzed using two-tailed Student t tests (*: P<0.05, **: P<0.01, ***: P<0.001).

Fig.7 Expression analysis of light chains in transgenic (T_high) and WT mice. (a) Analysis of mouse κ or λ light chains in sera by western blotting. Four mice from each group were evaluated; (b) FACS analysis of mouse κ and λ light chains on splenic B cells; (c) FACS profiles of splenic lymphocytes stained for B220, two types of IgM and κ or λ. The percentage of each subset is indicated in the appropriate gate. The data presented are representative of at least four individual mice; (d) statistical analysis of κ:λ ratios based on the data from subfigure b. The ratios of κ to λ on splenic B cells in transgenic and WT mice are indicated by bars; (e) statistical analysis of κ:λ ratios based on the data from subfigure c. The ratios of κ to λ on bovine IgM-expressing B cells and mouse IgM-expressing B cells in transgenic or WT mice are indicated by bars (***: P<0.001).

Tab.1 Analysis of CDR3 length and number of N and P nucleotides in transgenic and WT mice

Tab.2 Analysis of CDR3 length and number of N and P nucleotides in IgM positive cells of transgenic and WT mice.

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