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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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Western blot analysis of λx. (A) Verifying the specificity of anti-Vλx antibody. Cell lysates of three different λx hybridomas (lanes 1–3; chosen because they are positive for Vλx-Jλ2 by PCR but negative in both κ and λ ELISA), a λ1 (lane 4), and a κ hybridoma (lane 5) were used for the analysis. (B) Detection of λx protein expression in CD25+ cells. Cell lysates of purified CD25+ and CD25−/B220+ B cells from the tg and B220+ B cells from non-tg BALB/c mice were used for the Western blot. The positive control (lane 1) is the supernatant collected from a λx hybridoma. The lower 23-kD band is most likely a degradation fragment of λx.
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fig4: Western blot analysis of λx. (A) Verifying the specificity of anti-Vλx antibody. Cell lysates of three different λx hybridomas (lanes 1–3; chosen because they are positive for Vλx-Jλ2 by PCR but negative in both κ and λ ELISA), a λ1 (lane 4), and a κ hybridoma (lane 5) were used for the analysis. (B) Detection of λx protein expression in CD25+ cells. Cell lysates of purified CD25+ and CD25−/B220+ B cells from the tg and B220+ B cells from non-tg BALB/c mice were used for the Western blot. The positive control (lane 1) is the supernatant collected from a λx hybridoma. The lower 23-kD band is most likely a degradation fragment of λx.

Mentions: To detect the expression of Vλx at protein level, a polyclonal antibody was generated by immunizing a rabbit with a peptide corresponding to a sequence spanning the FWR2 and CDR2 region of Vλx, which has no homology to Vλ1 or Vλ2. Western blot using cell lysate of λx, λ1, and κ hybridomas demonstrates that the antibody recognizes a single band with the molecular weight of 27 kD from the lysates of λx hybridomas and does not cross talk with λ1 or κ L chains (Fig. 4 A). The observed molecular weight of λx is about the same as that which has been reported by another group using their antibody (32). We then did the Western blot using cell lysates from purified CD25+ B cells, CD25−/B220+ B cells from the tg, and B220+ B cells from non-tg BALB/c mice. The CD25+ cells shows a strong band of λx. The CD25−/B220+ cells sorted from the same mice show a much weaker band and B220+ cells from nontransgenic BALB/c mice show no signal (Fig. 4 B). We were not able to use the antibody to perform flow cytometry as it only recognizes the denatured λx protein.


Editing anti-DNA B cells by Vlambdax.

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

Western blot analysis of λx. (A) Verifying the specificity of anti-Vλx antibody. Cell lysates of three different λx hybridomas (lanes 1–3; chosen because they are positive for Vλx-Jλ2 by PCR but negative in both κ and λ ELISA), a λ1 (lane 4), and a κ hybridoma (lane 5) were used for the analysis. (B) Detection of λx protein expression in CD25+ cells. Cell lysates of purified CD25+ and CD25−/B220+ B cells from the tg and B220+ B cells from non-tg BALB/c mice were used for the Western blot. The positive control (lane 1) is the supernatant collected from a λx hybridoma. The lower 23-kD band is most likely a degradation fragment of λx.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211795&req=5

fig4: Western blot analysis of λx. (A) Verifying the specificity of anti-Vλx antibody. Cell lysates of three different λx hybridomas (lanes 1–3; chosen because they are positive for Vλx-Jλ2 by PCR but negative in both κ and λ ELISA), a λ1 (lane 4), and a κ hybridoma (lane 5) were used for the analysis. (B) Detection of λx protein expression in CD25+ cells. Cell lysates of purified CD25+ and CD25−/B220+ B cells from the tg and B220+ B cells from non-tg BALB/c mice were used for the Western blot. The positive control (lane 1) is the supernatant collected from a λx hybridoma. The lower 23-kD band is most likely a degradation fragment of λx.
Mentions: To detect the expression of Vλx at protein level, a polyclonal antibody was generated by immunizing a rabbit with a peptide corresponding to a sequence spanning the FWR2 and CDR2 region of Vλx, which has no homology to Vλ1 or Vλ2. Western blot using cell lysate of λx, λ1, and κ hybridomas demonstrates that the antibody recognizes a single band with the molecular weight of 27 kD from the lysates of λx hybridomas and does not cross talk with λ1 or κ L chains (Fig. 4 A). The observed molecular weight of λx is about the same as that which has been reported by another group using their antibody (32). We then did the Western blot using cell lysates from purified CD25+ B cells, CD25−/B220+ B cells from the tg, and B220+ B cells from non-tg BALB/c mice. The CD25+ cells shows a strong band of λx. The CD25−/B220+ cells sorted from the same mice show a much weaker band and B220+ cells from nontransgenic BALB/c mice show no signal (Fig. 4 B). We were not able to use the antibody to perform flow cytometry as it only recognizes the denatured λx protein.

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