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Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Mandik-Nayak L, Bui A, Noorchashm H, Eaton A, Erikson J - J. Exp. Med. (1997)

Bottom Line: Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire.We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate.These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

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Phenotypic analysis of VH3H9/λ B cells. Spleen (A–C) and bone marrow (D) B cells were stained with anti-B220-biotin/streptavidin-Red670, anti-λ-PE or -FITC, and either anti-HSA, CD19, CD21/35, CD22, CD23, CD44, CD62L-FITC, or anti–class II–PE. (A) Dot plots showing  B220 versus λ staining in the spleen. (B) Developmental markers and (C) activation markers on the total splenic B cell population (gating on B220+ cells)  in tg(−) mice (thin lines in B and C) and B220+λ+ B cells in tg(−) mice (bold line, top), B220+λ+ B cells in VH3H9 mice (bold line, middle), or B220+κ+  B cells in VH3H9 tg mice (bold line, bottom). The underlayed histograms (thin lines) were scaled down to allow for the comparison to the λ+ B cells  (which are present at ∼0.1 the frequency of total B cells) in the upper and middle panels. (D) Histograms showing developmental and activation markers  on the λ+ B cells in the spleen (thin line) and bone marrow (bold line) in tg(−) (top panels) and VH3H9 tg mice (bottom panels). These are representative  plots from n = 4 mice of each genotype.
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Figure 2: Phenotypic analysis of VH3H9/λ B cells. Spleen (A–C) and bone marrow (D) B cells were stained with anti-B220-biotin/streptavidin-Red670, anti-λ-PE or -FITC, and either anti-HSA, CD19, CD21/35, CD22, CD23, CD44, CD62L-FITC, or anti–class II–PE. (A) Dot plots showing B220 versus λ staining in the spleen. (B) Developmental markers and (C) activation markers on the total splenic B cell population (gating on B220+ cells) in tg(−) mice (thin lines in B and C) and B220+λ+ B cells in tg(−) mice (bold line, top), B220+λ+ B cells in VH3H9 mice (bold line, middle), or B220+κ+ B cells in VH3H9 tg mice (bold line, bottom). The underlayed histograms (thin lines) were scaled down to allow for the comparison to the λ+ B cells (which are present at ∼0.1 the frequency of total B cells) in the upper and middle panels. (D) Histograms showing developmental and activation markers on the λ+ B cells in the spleen (thin line) and bone marrow (bold line) in tg(−) (top panels) and VH3H9 tg mice (bottom panels). These are representative plots from n = 4 mice of each genotype.

Mentions: Using flow cytometry, we assessed the developmental and activation status of anti-dsDNA B cells in the spleen. The panel of developmental markers, shown in Fig. 2, includes CD19, CD21/35, CD22, CD23, HSA, and B220. We compared the expression levels of these markers on λ+ B cells from VH3H9 tg mice with those on the tg(−) λ+ B cells, as well as the total B cell population, from tg(−) mice (Fig. 2). B220 (CD45R) increases with maturity and, in conjunction with HSA, has been used to define the immature to mature stages of B cell development (25). HSA is expressed at high levels on immature (newly emerging) B cells and at a lower level on mature B cells in the spleen (26). As is shown in Fig. 2 B, the VH3H9/λ B cells express a slightly reduced level of B220 and a level of HSA intermediate between the HSAhigh and HSAlow cells in the tg(−) spleen. CD22 is expressed at a low level on immature B cells and increases with maturity, whereas CD21/35 and CD23 become surface positive at the mature B cell stage (38–41). The VH3H9/λ B cells have a dramatically reduced level of CD21/35, as well as lower levels of CD22 and CD23 on their surface (Fig. 2 B). Because CD21/35 (complement receptors 1 and 2) and CD22 play a role in modulating the response through the Ig receptor (42–45), the low expression levels of these coreceptors on VH3H9/λ B cells may alter the signaling threshold of these cells when stimulated through membrane Ig. CD19 is a B cell–specific marker that is expressed on all B cells starting at the pro–B cell stage (46). As is shown in Fig. 2 B, CD19 expression on the surface of λ+ B cells in VH3H9 tg mice is higher than on B cells from tg(−) mice. CD40 was also examined and has a similar expression level to tg(−) B cells (data not shown). In contrast to VH3H9 tg mice, the λ+ B cells in tg(−) mice have equivalent levels of all surface markers tested (Fig. 2), suggesting that there is nothing inherently different about B cells with λ L chains. Taken together, these data suggest that the VH3H9/λ B cells are phenotypically immature.


Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle.

Mandik-Nayak L, Bui A, Noorchashm H, Eaton A, Erikson J - J. Exp. Med. (1997)

Phenotypic analysis of VH3H9/λ B cells. Spleen (A–C) and bone marrow (D) B cells were stained with anti-B220-biotin/streptavidin-Red670, anti-λ-PE or -FITC, and either anti-HSA, CD19, CD21/35, CD22, CD23, CD44, CD62L-FITC, or anti–class II–PE. (A) Dot plots showing  B220 versus λ staining in the spleen. (B) Developmental markers and (C) activation markers on the total splenic B cell population (gating on B220+ cells)  in tg(−) mice (thin lines in B and C) and B220+λ+ B cells in tg(−) mice (bold line, top), B220+λ+ B cells in VH3H9 mice (bold line, middle), or B220+κ+  B cells in VH3H9 tg mice (bold line, bottom). The underlayed histograms (thin lines) were scaled down to allow for the comparison to the λ+ B cells  (which are present at ∼0.1 the frequency of total B cells) in the upper and middle panels. (D) Histograms showing developmental and activation markers  on the λ+ B cells in the spleen (thin line) and bone marrow (bold line) in tg(−) (top panels) and VH3H9 tg mice (bottom panels). These are representative  plots from n = 4 mice of each genotype.
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: Phenotypic analysis of VH3H9/λ B cells. Spleen (A–C) and bone marrow (D) B cells were stained with anti-B220-biotin/streptavidin-Red670, anti-λ-PE or -FITC, and either anti-HSA, CD19, CD21/35, CD22, CD23, CD44, CD62L-FITC, or anti–class II–PE. (A) Dot plots showing B220 versus λ staining in the spleen. (B) Developmental markers and (C) activation markers on the total splenic B cell population (gating on B220+ cells) in tg(−) mice (thin lines in B and C) and B220+λ+ B cells in tg(−) mice (bold line, top), B220+λ+ B cells in VH3H9 mice (bold line, middle), or B220+κ+ B cells in VH3H9 tg mice (bold line, bottom). The underlayed histograms (thin lines) were scaled down to allow for the comparison to the λ+ B cells (which are present at ∼0.1 the frequency of total B cells) in the upper and middle panels. (D) Histograms showing developmental and activation markers on the λ+ B cells in the spleen (thin line) and bone marrow (bold line) in tg(−) (top panels) and VH3H9 tg mice (bottom panels). These are representative plots from n = 4 mice of each genotype.
Mentions: Using flow cytometry, we assessed the developmental and activation status of anti-dsDNA B cells in the spleen. The panel of developmental markers, shown in Fig. 2, includes CD19, CD21/35, CD22, CD23, HSA, and B220. We compared the expression levels of these markers on λ+ B cells from VH3H9 tg mice with those on the tg(−) λ+ B cells, as well as the total B cell population, from tg(−) mice (Fig. 2). B220 (CD45R) increases with maturity and, in conjunction with HSA, has been used to define the immature to mature stages of B cell development (25). HSA is expressed at high levels on immature (newly emerging) B cells and at a lower level on mature B cells in the spleen (26). As is shown in Fig. 2 B, the VH3H9/λ B cells express a slightly reduced level of B220 and a level of HSA intermediate between the HSAhigh and HSAlow cells in the tg(−) spleen. CD22 is expressed at a low level on immature B cells and increases with maturity, whereas CD21/35 and CD23 become surface positive at the mature B cell stage (38–41). The VH3H9/λ B cells have a dramatically reduced level of CD21/35, as well as lower levels of CD22 and CD23 on their surface (Fig. 2 B). Because CD21/35 (complement receptors 1 and 2) and CD22 play a role in modulating the response through the Ig receptor (42–45), the low expression levels of these coreceptors on VH3H9/λ B cells may alter the signaling threshold of these cells when stimulated through membrane Ig. CD19 is a B cell–specific marker that is expressed on all B cells starting at the pro–B cell stage (46). As is shown in Fig. 2 B, CD19 expression on the surface of λ+ B cells in VH3H9 tg mice is higher than on B cells from tg(−) mice. CD40 was also examined and has a similar expression level to tg(−) B cells (data not shown). In contrast to VH3H9 tg mice, the λ+ B cells in tg(−) mice have equivalent levels of all surface markers tested (Fig. 2), suggesting that there is nothing inherently different about B cells with λ L chains. Taken together, these data suggest that the VH3H9/λ B cells are phenotypically immature.

Bottom Line: Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire.We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate.These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

View Article: PubMed Central - PubMed

Affiliation: The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Systemic lupus erythematosus (SLE) and the MRL-lpr/lpr murine model for SLE are characterized by the presence of serum anti-double-stranded (ds)DNA antibodies (Abs), whereas nonautoimmune individuals have negligible levels of these Abs. To increase the frequency of anti-DNA B cells and identify the mechanisms involved in their regulation in nonautoimmune mice, we have used Ig transgenes (tgs). In the present study, we used the VH3H9 heavy (H) chain tg which expresses an H chain that was repeatedly isolated from anti-dsDNA Abs from MRL-lpr/lpr mice. Because the VH3H9 H chain can pair with endogenous L chains to generate anti-single-stranded DNA, anti-dsDNA, and non-DNA B cells, this allowed us to study the regulation of anti-dsDNA B cells in the context of a diverse B cell repertoire. We have identified anti-dsDNA B cells that are located at the T-B interface in the splenic follicle where they have an increased in vivo turnover rate. These anti-dsDNA B cells exhibit a unique surface phenotype suggesting developmental arrest due to antigen exposure.

Show MeSH
Related in: MedlinePlus