Limits...
NF-kappaB activity marks cells engaged in receptor editing.

Cadera EJ, Wan F, Amin RH, Nolla H, Lenardo MJ, Schlissel MS - J. Exp. Med. (2009)

Bottom Line: Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens.We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells.Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

ABSTRACT
Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

Show MeSH
Markers of receptor editing are increased in IκBα-expressing cells. (A) The top diagram shows that the κ locus is in its germline configuration with the primary break intermediate shown below. The bottom diagram is the κ locus after a Vκ has rearranged to Jκ1, with the secondary break intermediate shown below. (B) Agarose gel analysis of PCR assays for RS rearrangements to either IRS (RS-IRS) or Vκ (RS-Vκ) in DNA from pre–B cells sorted for β-gal expression. PCR amplification of the APRT locus was used as a template control, and H20 indicates PCR reactions without template. (C) LM-PCR assays to detect primary and secondary double-stranded DNA RSS break intermediates in DNA from sorted β-gal–positive and –negative pre–B cells. An ethidium-stained agarose gel analysis of LM-PCR products is shown. Pooled bone marrow from five to six mice was used for each of two repetitions of this experiment yielding similar results.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2722169&req=5

fig3: Markers of receptor editing are increased in IκBα-expressing cells. (A) The top diagram shows that the κ locus is in its germline configuration with the primary break intermediate shown below. The bottom diagram is the κ locus after a Vκ has rearranged to Jκ1, with the secondary break intermediate shown below. (B) Agarose gel analysis of PCR assays for RS rearrangements to either IRS (RS-IRS) or Vκ (RS-Vκ) in DNA from pre–B cells sorted for β-gal expression. PCR amplification of the APRT locus was used as a template control, and H20 indicates PCR reactions without template. (C) LM-PCR assays to detect primary and secondary double-stranded DNA RSS break intermediates in DNA from sorted β-gal–positive and –negative pre–B cells. An ethidium-stained agarose gel analysis of LM-PCR products is shown. Pooled bone marrow from five to six mice was used for each of two repetitions of this experiment yielding similar results.

Mentions: Considering what process might divide the pre–B cell compartment into nonequivalent subpopulations, we proceeded to test the idea that NF-κB might be specifically activated in cells undergoing receptor editing. To test this idea, we examined β-gal–positive and –negative pre–B cells for various markers of receptor editing. Rearrangements involving the RS element, which lies 25 kb 3′ of Cκ, and either a Vκ gene segment or the IRS sequence in the J-C interval are considered hallmarks of receptor editing (Fig. 3 A; Tiegs et al., 1993; Retter and Nemazee, 1998; Vela et al., 2008). Using a PCR assay, we detected more RS-IRS and RS-Vκ rearrangements in the β-gal+ than in the β-gal− pre–B cell population (Fig. 3 B). Thus, RS rearrangements are enriched in pre–B cells expressing IκBα.


NF-kappaB activity marks cells engaged in receptor editing.

Cadera EJ, Wan F, Amin RH, Nolla H, Lenardo MJ, Schlissel MS - J. Exp. Med. (2009)

Markers of receptor editing are increased in IκBα-expressing cells. (A) The top diagram shows that the κ locus is in its germline configuration with the primary break intermediate shown below. The bottom diagram is the κ locus after a Vκ has rearranged to Jκ1, with the secondary break intermediate shown below. (B) Agarose gel analysis of PCR assays for RS rearrangements to either IRS (RS-IRS) or Vκ (RS-Vκ) in DNA from pre–B cells sorted for β-gal expression. PCR amplification of the APRT locus was used as a template control, and H20 indicates PCR reactions without template. (C) LM-PCR assays to detect primary and secondary double-stranded DNA RSS break intermediates in DNA from sorted β-gal–positive and –negative pre–B cells. An ethidium-stained agarose gel analysis of LM-PCR products is shown. Pooled bone marrow from five to six mice was used for each of two repetitions of this experiment yielding similar results.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2722169&req=5

fig3: Markers of receptor editing are increased in IκBα-expressing cells. (A) The top diagram shows that the κ locus is in its germline configuration with the primary break intermediate shown below. The bottom diagram is the κ locus after a Vκ has rearranged to Jκ1, with the secondary break intermediate shown below. (B) Agarose gel analysis of PCR assays for RS rearrangements to either IRS (RS-IRS) or Vκ (RS-Vκ) in DNA from pre–B cells sorted for β-gal expression. PCR amplification of the APRT locus was used as a template control, and H20 indicates PCR reactions without template. (C) LM-PCR assays to detect primary and secondary double-stranded DNA RSS break intermediates in DNA from sorted β-gal–positive and –negative pre–B cells. An ethidium-stained agarose gel analysis of LM-PCR products is shown. Pooled bone marrow from five to six mice was used for each of two repetitions of this experiment yielding similar results.
Mentions: Considering what process might divide the pre–B cell compartment into nonequivalent subpopulations, we proceeded to test the idea that NF-κB might be specifically activated in cells undergoing receptor editing. To test this idea, we examined β-gal–positive and –negative pre–B cells for various markers of receptor editing. Rearrangements involving the RS element, which lies 25 kb 3′ of Cκ, and either a Vκ gene segment or the IRS sequence in the J-C interval are considered hallmarks of receptor editing (Fig. 3 A; Tiegs et al., 1993; Retter and Nemazee, 1998; Vela et al., 2008). Using a PCR assay, we detected more RS-IRS and RS-Vκ rearrangements in the β-gal+ than in the β-gal− pre–B cell population (Fig. 3 B). Thus, RS rearrangements are enriched in pre–B cells expressing IκBα.

Bottom Line: Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens.We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells.Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

ABSTRACT
Because of the extreme diversity in immunoglobulin genes, tolerance mechanisms are necessary to ensure that B cells do not respond to self-antigens. One such tolerance mechanism is called receptor editing. If the B cell receptor (BCR) on an immature B cell recognizes self-antigen, it is down-regulated from the cell surface, and light chain gene rearrangement continues in an attempt to edit the autoreactive specificity. Analysis of a heterozygous mutant mouse in which the NF-kappaB-dependent IkappaB alpha gene was replaced with a lacZ (beta-gal) reporter complementary DNA (cDNA; IkappaB alpha(+/lacZ)) suggests a potential role for NF-kappaB in receptor editing. Sorted beta-gal(+) pre-B cells showed increased levels of various markers of receptor editing. In IkappaB alpha(+/lacZ) reporter mice expressing either innocuous or self-specific knocked in BCRs, beta-gal was preferentially expressed in pre-B cells from the mice with self-specific BCRs. Retroviral-mediated expression of a cDNA encoding an IkappaB alpha superrepressor in primary bone marrow cultures resulted in diminished germline kappa and rearranged lambda transcripts but similar levels of RAG expression as compared with controls. We found that IRF4 transcripts were up-regulated in beta-gal(+) pre-B cells. Because IRF4 is a target of NF-kappaB and is required for receptor editing, we suggest that NF-kappaB could be acting through IRF4 to regulate receptor editing.

Show MeSH