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Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.

Watanabe N, Nisitani S, Ikuta K, Suzuki M, Chiba T, Honjo T - J. Exp. Med. (1999)

Bottom Line: Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains.These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion.By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.

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Tg Id expression in B cells of heterozygous or homozygous H+L5, H+L6 and H3 Tg mice. Cells were isolated from bone marrow (A and B) and spleen (A and D) of mice at 10–14 wk of age and stained with FITC-conjugated antiallotypic antibody for IgMa (Tg) and biotin-conjugated anti-Id (A, B, and D) or biotin-conjugated anti-λ antibody (A) coupled with PE-conjugated SA. The percentages of the cells of each gated region in total viable cells are indicated. Note that results from different mice are shown for A than for B–E. Total numbers of the bone marrow (C) and spleen (E) cells expressing endogenous L chain and Tg H chain in heterozygous or homozygous H+L5 and H+L6 Tg mice. Cells were analyzed from at least three mice in each line. Numbers of indicated cells were calculated by (percentage of indicated cells in viable cells) × (No. of viable cells). Results are expressed as mean ± SD.
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Figure 3: Tg Id expression in B cells of heterozygous or homozygous H+L5, H+L6 and H3 Tg mice. Cells were isolated from bone marrow (A and B) and spleen (A and D) of mice at 10–14 wk of age and stained with FITC-conjugated antiallotypic antibody for IgMa (Tg) and biotin-conjugated anti-Id (A, B, and D) or biotin-conjugated anti-λ antibody (A) coupled with PE-conjugated SA. The percentages of the cells of each gated region in total viable cells are indicated. Note that results from different mice are shown for A than for B–E. Total numbers of the bone marrow (C) and spleen (E) cells expressing endogenous L chain and Tg H chain in heterozygous or homozygous H+L5 and H+L6 Tg mice. Cells were analyzed from at least three mice in each line. Numbers of indicated cells were calculated by (percentage of indicated cells in viable cells) × (No. of viable cells). Results are expressed as mean ± SD.

Mentions: Next, we asked whether homozygosity of Tg loci also reduces endogenous L chain expression. Since the allotypic specificity for the Tg Lκ chain is not known, we estimated the degree of endogenous L chain expression by difference in expression of Tg H chain (IgMa) and Id (the combined epitope of the H and L chains of the 4C8 anti-RBC mAb) that is recognized by the S54 mAb. In the H+L5 line, two populations of IgMa+ cells were observed: one stained with both anti-IgMa and anti-Id mAbs diagonally, and the other stained more weakly with anti-Id mAb than with anti-IgMa mAb (Fig. 3a, Fig. B, and Fig. D, tops). As a control, IgMa+ cells from H3 mice did not contain the population stained with the S54 mAb (Fig. 3B and Fig. D, bottoms). We presume that the cells that stained diagonally with both mAbs express the Tg H and L chains equally, and that the IgMa+Idlow cells express the Tg H chain with both endogenous and Tg L chains. In support of this assumption, IgMa+ bone marrow cells in heterozygous H+L6 mice, which consisted of almost exclusively IgMa+Id+ cells and only few IgMa+Idlow cells, contained <1.2% λ+ cells, which represent a fraction of B cells that express endogenous L chains (Fig. 3 A). In contrast, IgMa+ bone marrow cells in heterozygous H+L5 mice, which consisted of ∼28% IgMa+Id+ and ∼72% IgMa+Idlow cells, contained >5.5% λ+ cells (Fig. 3 A). Furthermore, IgMa+ spleen cells in heterozygous H+L5 mice, which consisted of ∼4% IgMa+Id+ and ∼96% IgMa+Idlow cells, contained >14% λ+ cells (Fig. 3 A). The total number of cells expressing endogenous L chains (IgMa+Idlow) in bone marrow (Fig. 3B and Fig. C) and spleen (Fig. 3D and Fig. E) was less in homozygous than in heterozygous Tg lines, indicating that homozygosity of the Tg loci enhances the inhibition of endogenous L chain expression as compared with heterozygosity.


Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.

Watanabe N, Nisitani S, Ikuta K, Suzuki M, Chiba T, Honjo T - J. Exp. Med. (1999)

Tg Id expression in B cells of heterozygous or homozygous H+L5, H+L6 and H3 Tg mice. Cells were isolated from bone marrow (A and B) and spleen (A and D) of mice at 10–14 wk of age and stained with FITC-conjugated antiallotypic antibody for IgMa (Tg) and biotin-conjugated anti-Id (A, B, and D) or biotin-conjugated anti-λ antibody (A) coupled with PE-conjugated SA. The percentages of the cells of each gated region in total viable cells are indicated. Note that results from different mice are shown for A than for B–E. Total numbers of the bone marrow (C) and spleen (E) cells expressing endogenous L chain and Tg H chain in heterozygous or homozygous H+L5 and H+L6 Tg mice. Cells were analyzed from at least three mice in each line. Numbers of indicated cells were calculated by (percentage of indicated cells in viable cells) × (No. of viable cells). Results are expressed as mean ± SD.
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Figure 3: Tg Id expression in B cells of heterozygous or homozygous H+L5, H+L6 and H3 Tg mice. Cells were isolated from bone marrow (A and B) and spleen (A and D) of mice at 10–14 wk of age and stained with FITC-conjugated antiallotypic antibody for IgMa (Tg) and biotin-conjugated anti-Id (A, B, and D) or biotin-conjugated anti-λ antibody (A) coupled with PE-conjugated SA. The percentages of the cells of each gated region in total viable cells are indicated. Note that results from different mice are shown for A than for B–E. Total numbers of the bone marrow (C) and spleen (E) cells expressing endogenous L chain and Tg H chain in heterozygous or homozygous H+L5 and H+L6 Tg mice. Cells were analyzed from at least three mice in each line. Numbers of indicated cells were calculated by (percentage of indicated cells in viable cells) × (No. of viable cells). Results are expressed as mean ± SD.
Mentions: Next, we asked whether homozygosity of Tg loci also reduces endogenous L chain expression. Since the allotypic specificity for the Tg Lκ chain is not known, we estimated the degree of endogenous L chain expression by difference in expression of Tg H chain (IgMa) and Id (the combined epitope of the H and L chains of the 4C8 anti-RBC mAb) that is recognized by the S54 mAb. In the H+L5 line, two populations of IgMa+ cells were observed: one stained with both anti-IgMa and anti-Id mAbs diagonally, and the other stained more weakly with anti-Id mAb than with anti-IgMa mAb (Fig. 3a, Fig. B, and Fig. D, tops). As a control, IgMa+ cells from H3 mice did not contain the population stained with the S54 mAb (Fig. 3B and Fig. D, bottoms). We presume that the cells that stained diagonally with both mAbs express the Tg H and L chains equally, and that the IgMa+Idlow cells express the Tg H chain with both endogenous and Tg L chains. In support of this assumption, IgMa+ bone marrow cells in heterozygous H+L6 mice, which consisted of almost exclusively IgMa+Id+ cells and only few IgMa+Idlow cells, contained <1.2% λ+ cells, which represent a fraction of B cells that express endogenous L chains (Fig. 3 A). In contrast, IgMa+ bone marrow cells in heterozygous H+L5 mice, which consisted of ∼28% IgMa+Id+ and ∼72% IgMa+Idlow cells, contained >5.5% λ+ cells (Fig. 3 A). Furthermore, IgMa+ spleen cells in heterozygous H+L5 mice, which consisted of ∼4% IgMa+Id+ and ∼96% IgMa+Idlow cells, contained >14% λ+ cells (Fig. 3 A). The total number of cells expressing endogenous L chains (IgMa+Idlow) in bone marrow (Fig. 3B and Fig. C) and spleen (Fig. 3D and Fig. E) was less in homozygous than in heterozygous Tg lines, indicating that homozygosity of the Tg loci enhances the inhibition of endogenous L chain expression as compared with heterozygosity.

Bottom Line: Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains.These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion.By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Chemistry, Faculty of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.

Show MeSH
Related in: MedlinePlus