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Anti-DNA B cells in MRL/lpr mice show altered differentiation and editing pattern.

Li Y, Li H, Ni D, Weigert M - J. Exp. Med. (2002)

Bottom Line: This transgene codes for a heavy chain that forms anti-double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vkappa repertoire, but certain L chains, referred to as Vkappa editors, do not sustain dsDNA binding in combination with 3H9H/56R.In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is associated with a Vkappa editor.Anti-dsDNA B cells use noneditor kappas but, in addition, most anti-dsDNA B cells have edited the heavy chain transgene. lambda1 B cells (without the coexpression of a kappa editor) are found and the kappa/lambda1 MZ population is absent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
We have studied the regulation of anti-DNA B cells in transgenic mice with a heavy chain transgene (3H9H/56R). This transgene codes for a heavy chain that forms anti-double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vkappa repertoire, but certain L chains, referred to as Vkappa editors, do not sustain dsDNA binding in combination with 3H9H/56R. In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is associated with a Vkappa editor. A minor population of B cells (30%) bind dsDNA and express the lambda1 light chain (known to sustain 3H9H/56R DNA binding). These 3H9/56R/lambda1 B cells coexpress a kappa editor, and we propose that the down-regulation of the anti-DNA BCR caused by the dual L chain expression may prevent activation of this kappa/lambda population. These kappa/lambda B cells are sequestered in the marginal zone. Here, we studied the influence of autoimmunity on expression and regulation of 3H9H/56R. In 3H9H/56R MRL/lpr mice, the expression of anti-dsDNA is vastly accelerated. Anti-dsDNA B cells use noneditor kappas but, in addition, most anti-dsDNA B cells have edited the heavy chain transgene. lambda1 B cells (without the coexpression of a kappa editor) are found and the kappa/lambda1 MZ population is absent. Our results suggest that improper editing and failure to sequester autoreactive B cells may contribute to the breakdown of tolerance in MRL/lpr mice.

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Heavy chain sequence of B cells that have edited the H chain transgene. (A) The nucleotide and deduced amino acid sequences of the V regions of heavy chains from hybridomas that are transgene-negative (clones 15, 46, 26, and 75) are shown. VH gene usage and point mutations compared with each of their germline sequences from the NCBI database are indicated. (B) CDR3 regions of the heavy chains of hybridomas (clones 46, 26, and 75) that had VH replacement are shown. The N-additions at the newly formed D-junctions, the remaining D and J4 region of the 3H9H/56R transgene and point mutations are indicated. Another heavy chain from clone 15 is generated by endogenous rearrangement on the untargeted allele, indicated by the differences at the JH region (marked by asterisk) between the transgene (a-allotype) and endogenous JH4 (j-allotype). The mutations compared with its germline sequence are also shown.
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fig3: Heavy chain sequence of B cells that have edited the H chain transgene. (A) The nucleotide and deduced amino acid sequences of the V regions of heavy chains from hybridomas that are transgene-negative (clones 15, 46, 26, and 75) are shown. VH gene usage and point mutations compared with each of their germline sequences from the NCBI database are indicated. (B) CDR3 regions of the heavy chains of hybridomas (clones 46, 26, and 75) that had VH replacement are shown. The N-additions at the newly formed D-junctions, the remaining D and J4 region of the 3H9H/56R transgene and point mutations are indicated. Another heavy chain from clone 15 is generated by endogenous rearrangement on the untargeted allele, indicated by the differences at the JH region (marked by asterisk) between the transgene (a-allotype) and endogenous JH4 (j-allotype). The mutations compared with its germline sequence are also shown.

Mentions: In the anti-dsDNA IgG clones with VH replacements (clones 46, 26, and 75), mutations are also found (Fig. 3) . It is interesting to note that the arginine mutation in the FR2 of clone 75 is shared by clone 57 (and other anti-DNAs in the literature; reference 26). This site is superficial and could contribute to DNA binding. In these VH genes, arginines are found in the newly formed D junctions due to either N-addition or point mutation (Fig. 3 B). Another source of an arginine codon is a VH replacement that captures a short sequence from the end of the recipient VH gene (in this case the transgene). In the transgene as well as most VH genes, this sequence includes an arginine codon (Fig. 3 B, clones 46 and 75). These mechanisms by which anti-DNAs are modified or created play a central role in autoantibody formation in the non-tg MRL/lpr. It is surprising that the sd-tg resorts to these mechanisms because the inherited 3H9H/56R (along with most L chains) contributes to high affinity DNA binding. Therefore, it appears the emerging 3H9H/56R B cells must have undergone prior modifications that necessitate de novo development of anti-DNA. These could include VH replacement, somatic mutation (in two instances critical Args in 3H9H/56R have mutated to a neutral amino acid), and L chain editing.


Anti-DNA B cells in MRL/lpr mice show altered differentiation and editing pattern.

Li Y, Li H, Ni D, Weigert M - J. Exp. Med. (2002)

Heavy chain sequence of B cells that have edited the H chain transgene. (A) The nucleotide and deduced amino acid sequences of the V regions of heavy chains from hybridomas that are transgene-negative (clones 15, 46, 26, and 75) are shown. VH gene usage and point mutations compared with each of their germline sequences from the NCBI database are indicated. (B) CDR3 regions of the heavy chains of hybridomas (clones 46, 26, and 75) that had VH replacement are shown. The N-additions at the newly formed D-junctions, the remaining D and J4 region of the 3H9H/56R transgene and point mutations are indicated. Another heavy chain from clone 15 is generated by endogenous rearrangement on the untargeted allele, indicated by the differences at the JH region (marked by asterisk) between the transgene (a-allotype) and endogenous JH4 (j-allotype). The mutations compared with its germline sequence are also shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196070&req=5

fig3: Heavy chain sequence of B cells that have edited the H chain transgene. (A) The nucleotide and deduced amino acid sequences of the V regions of heavy chains from hybridomas that are transgene-negative (clones 15, 46, 26, and 75) are shown. VH gene usage and point mutations compared with each of their germline sequences from the NCBI database are indicated. (B) CDR3 regions of the heavy chains of hybridomas (clones 46, 26, and 75) that had VH replacement are shown. The N-additions at the newly formed D-junctions, the remaining D and J4 region of the 3H9H/56R transgene and point mutations are indicated. Another heavy chain from clone 15 is generated by endogenous rearrangement on the untargeted allele, indicated by the differences at the JH region (marked by asterisk) between the transgene (a-allotype) and endogenous JH4 (j-allotype). The mutations compared with its germline sequence are also shown.
Mentions: In the anti-dsDNA IgG clones with VH replacements (clones 46, 26, and 75), mutations are also found (Fig. 3) . It is interesting to note that the arginine mutation in the FR2 of clone 75 is shared by clone 57 (and other anti-DNAs in the literature; reference 26). This site is superficial and could contribute to DNA binding. In these VH genes, arginines are found in the newly formed D junctions due to either N-addition or point mutation (Fig. 3 B). Another source of an arginine codon is a VH replacement that captures a short sequence from the end of the recipient VH gene (in this case the transgene). In the transgene as well as most VH genes, this sequence includes an arginine codon (Fig. 3 B, clones 46 and 75). These mechanisms by which anti-DNAs are modified or created play a central role in autoantibody formation in the non-tg MRL/lpr. It is surprising that the sd-tg resorts to these mechanisms because the inherited 3H9H/56R (along with most L chains) contributes to high affinity DNA binding. Therefore, it appears the emerging 3H9H/56R B cells must have undergone prior modifications that necessitate de novo development of anti-DNA. These could include VH replacement, somatic mutation (in two instances critical Args in 3H9H/56R have mutated to a neutral amino acid), and L chain editing.

Bottom Line: This transgene codes for a heavy chain that forms anti-double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vkappa repertoire, but certain L chains, referred to as Vkappa editors, do not sustain dsDNA binding in combination with 3H9H/56R.In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is associated with a Vkappa editor.Anti-dsDNA B cells use noneditor kappas but, in addition, most anti-dsDNA B cells have edited the heavy chain transgene. lambda1 B cells (without the coexpression of a kappa editor) are found and the kappa/lambda1 MZ population is absent.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.

ABSTRACT
We have studied the regulation of anti-DNA B cells in transgenic mice with a heavy chain transgene (3H9H/56R). This transgene codes for a heavy chain that forms anti-double-stranded DNA (dsDNA) antibody when paired with most members of the endogenous Vkappa repertoire, but certain L chains, referred to as Vkappa editors, do not sustain dsDNA binding in combination with 3H9H/56R. In the nonautoimmune 3H9H/56R BALB/c, most B cells generated do not bind DNA because the transgene itself is edited or is associated with a Vkappa editor. A minor population of B cells (30%) bind dsDNA and express the lambda1 light chain (known to sustain 3H9H/56R DNA binding). These 3H9/56R/lambda1 B cells coexpress a kappa editor, and we propose that the down-regulation of the anti-DNA BCR caused by the dual L chain expression may prevent activation of this kappa/lambda population. These kappa/lambda B cells are sequestered in the marginal zone. Here, we studied the influence of autoimmunity on expression and regulation of 3H9H/56R. In 3H9H/56R MRL/lpr mice, the expression of anti-dsDNA is vastly accelerated. Anti-dsDNA B cells use noneditor kappas but, in addition, most anti-dsDNA B cells have edited the heavy chain transgene. lambda1 B cells (without the coexpression of a kappa editor) are found and the kappa/lambda1 MZ population is absent. Our results suggest that improper editing and failure to sequester autoreactive B cells may contribute to the breakdown of tolerance in MRL/lpr mice.

Show MeSH
Related in: MedlinePlus