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Editing anti-DNA B cells by Vlambdax.

Li Y, Louzoun Y, Weigert M - J. Exp. Med. (2004)

Bottom Line: However, the lambda locus, either of man or mouse, does not allow V gene replacement.We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of lambda1 in association with an anti-DNA heavy chain often coexpress a kappa chain that prevents DNA binding.Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vlambda family, Vlambdax.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, NJ 08544, USA. yijinli@uchicago.edu

ABSTRACT
Receptor editing is performed by replacement of Vkappa genes that contribute to autoreactivity. In addition, the Ckappa locus can be deleted by Vkappa rearrangement to intronic or 3' of Ckappa RS sequences (also referred to as kappa deletion elements). B cells that delete the Ckappa can then express lambda light chains. However, the lambda locus, either of man or mouse, does not allow V gene replacement. Nor does it appear to be deleted. Therefore, editing of autoreactive lambda B cells may require alternative pathways. We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of lambda1 in association with an anti-DNA heavy chain often coexpress a kappa chain that prevents DNA binding. We speculate that such isotypically included cells may have low anti-DNA receptor densities, a feature that may lead to self-tolerance. Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vlambda family, Vlambdax. The lambdax B cells of the tgs also express CD25 and may represent B cells that have exhausted light chain editing possibilities.

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The CD25+ B cells from VH3H9/56R express the transgene allele (Iga). Spleen cells from BALB/c (Iga), CB17 (Igb), and CB17 mice carrying the 3H9/56R transgene (Iga) were stained with antibodies to B220, CD25, IgDa, and IgDb. The top panel shows the frequency of CD25+ B220+ from the VH3H9/56R CB17. BALB/c or CB17 mice have background levels of these cells. The bottom panel (left to right) shows the IgDa/IgDb staining of the B220+ cells from BALB/c, CB17, VH3H9/56R CB17, and CD25+ B220+ cells of the VH3H9/56R CB17.
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fig5: The CD25+ B cells from VH3H9/56R express the transgene allele (Iga). Spleen cells from BALB/c (Iga), CB17 (Igb), and CB17 mice carrying the 3H9/56R transgene (Iga) were stained with antibodies to B220, CD25, IgDa, and IgDb. The top panel shows the frequency of CD25+ B220+ from the VH3H9/56R CB17. BALB/c or CB17 mice have background levels of these cells. The bottom panel (left to right) shows the IgDa/IgDb staining of the B220+ cells from BALB/c, CB17, VH3H9/56R CB17, and CD25+ B220+ cells of the VH3H9/56R CB17.

Mentions: The CD25+/λx population appears to depend on the presence of the 3H9/56R transgene. As discussed above, λx B cells in non-tgs are CD25−. However, hybridoma panels from this tg show that 17% of the hybridomas have undergone replacement of the transgene VH (17). Therefore, it was important to confirm that the CD25+ B cells coexpress λx and the 3H9/56R H chain. We tested transgene expression by allotype analysis. The Iga tgs were crossed to CB17 mice (Igb) and the B cells from the allotype heterozygous mice were stained with allotype-specific antibodies. Nearly all of the CD25+ B cells express the transgene allotype (Fig. 5). This, however, does not rule out the possibility that the transgene VH is replaced and that the Iga C region is retained (33). To address this, we tested the expression of the 3H9/56R H chain together with Vλx by single cell PCR. 22 of 48 (45.8%) CD25+ cells are positive for the Vλx-Jλ2 PCR, whereas only 4 of 48 (8.3%) CD25− cells sorted from the same mice are positive. 12 of the 22 CD25+/λx cells (54.5%) are positive for the PCR detecting the 3H9/56R H chain. We do not know if this is the actual percentage of transgene+ cells because the efficiency of the assay has not been determined. However, this result shows that at least 50% of the cells in this population retain the transgene.


Editing anti-DNA B cells by Vlambdax.

Li Y, Louzoun Y, Weigert M - J. Exp. Med. (2004)

The CD25+ B cells from VH3H9/56R express the transgene allele (Iga). Spleen cells from BALB/c (Iga), CB17 (Igb), and CB17 mice carrying the 3H9/56R transgene (Iga) were stained with antibodies to B220, CD25, IgDa, and IgDb. The top panel shows the frequency of CD25+ B220+ from the VH3H9/56R CB17. BALB/c or CB17 mice have background levels of these cells. The bottom panel (left to right) shows the IgDa/IgDb staining of the B220+ cells from BALB/c, CB17, VH3H9/56R CB17, and CD25+ B220+ cells of the VH3H9/56R CB17.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211795&req=5

fig5: The CD25+ B cells from VH3H9/56R express the transgene allele (Iga). Spleen cells from BALB/c (Iga), CB17 (Igb), and CB17 mice carrying the 3H9/56R transgene (Iga) were stained with antibodies to B220, CD25, IgDa, and IgDb. The top panel shows the frequency of CD25+ B220+ from the VH3H9/56R CB17. BALB/c or CB17 mice have background levels of these cells. The bottom panel (left to right) shows the IgDa/IgDb staining of the B220+ cells from BALB/c, CB17, VH3H9/56R CB17, and CD25+ B220+ cells of the VH3H9/56R CB17.
Mentions: The CD25+/λx population appears to depend on the presence of the 3H9/56R transgene. As discussed above, λx B cells in non-tgs are CD25−. However, hybridoma panels from this tg show that 17% of the hybridomas have undergone replacement of the transgene VH (17). Therefore, it was important to confirm that the CD25+ B cells coexpress λx and the 3H9/56R H chain. We tested transgene expression by allotype analysis. The Iga tgs were crossed to CB17 mice (Igb) and the B cells from the allotype heterozygous mice were stained with allotype-specific antibodies. Nearly all of the CD25+ B cells express the transgene allotype (Fig. 5). This, however, does not rule out the possibility that the transgene VH is replaced and that the Iga C region is retained (33). To address this, we tested the expression of the 3H9/56R H chain together with Vλx by single cell PCR. 22 of 48 (45.8%) CD25+ cells are positive for the Vλx-Jλ2 PCR, whereas only 4 of 48 (8.3%) CD25− cells sorted from the same mice are positive. 12 of the 22 CD25+/λx cells (54.5%) are positive for the PCR detecting the 3H9/56R H chain. We do not know if this is the actual percentage of transgene+ cells because the efficiency of the assay has not been determined. However, this result shows that at least 50% of the cells in this population retain the transgene.

Bottom Line: However, the lambda locus, either of man or mouse, does not allow V gene replacement.We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of lambda1 in association with an anti-DNA heavy chain often coexpress a kappa chain that prevents DNA binding.Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vlambda family, Vlambdax.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, NJ 08544, USA. yijinli@uchicago.edu

ABSTRACT
Receptor editing is performed by replacement of Vkappa genes that contribute to autoreactivity. In addition, the Ckappa locus can be deleted by Vkappa rearrangement to intronic or 3' of Ckappa RS sequences (also referred to as kappa deletion elements). B cells that delete the Ckappa can then express lambda light chains. However, the lambda locus, either of man or mouse, does not allow V gene replacement. Nor does it appear to be deleted. Therefore, editing of autoreactive lambda B cells may require alternative pathways. We have found that in anti-DNA heavy chain transgenic mice (tgs) VH3H9/56R, B cells that express anti-DNA receptors comprised of lambda1 in association with an anti-DNA heavy chain often coexpress a kappa chain that prevents DNA binding. We speculate that such isotypically included cells may have low anti-DNA receptor densities, a feature that may lead to self-tolerance. Here we describe a mechanism of preventing DNA binding by expression of a rarely used member of the Vlambda family, Vlambdax. The lambdax B cells of the tgs also express CD25 and may represent B cells that have exhausted light chain editing possibilities.

Show MeSH
Related in: MedlinePlus