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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.

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Expression of NF-κB target genes in IκBαΔ-transduced pre–B cells. (A) cDNA from sorted β-gal–positive and –negative pre–B cells was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT transcripts to control for any differences in cDNA template. β-gal–negative cell result was arbitrarily set to 1, with error bars indicating range of triplicate assays. (B) cDNA from CD19+IgD− cells from B1-8 α-HEL-κ and B1-8low α-HEL-κ mice was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT, with error bars indicating range of triplicate assays. These results are representative of two independent repetitions of this experiment using pools of sorted bone marrow cells from five to six mice.
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fig7: Expression of NF-κB target genes in IκBαΔ-transduced pre–B cells. (A) cDNA from sorted β-gal–positive and –negative pre–B cells was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT transcripts to control for any differences in cDNA template. β-gal–negative cell result was arbitrarily set to 1, with error bars indicating range of triplicate assays. (B) cDNA from CD19+IgD− cells from B1-8 α-HEL-κ and B1-8low α-HEL-κ mice was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT, with error bars indicating range of triplicate assays. These results are representative of two independent repetitions of this experiment using pools of sorted bone marrow cells from five to six mice.

Mentions: IRF4 is a target of NF-κB that has been previously shown to be important in both pre–B cell development and receptor editing. (Lu et al., 2003; Muljo and Schlissel, 2003; Ma et al., 2006; Saito et al., 2007; Johnson et al., 2008; Pathak et al., 2008). We used real-time RT-PCR to quantify IRF4 transcripts in pro–/pre–B cells sorted based on β-gal activity. The signal was normalized to hypoxanthine-guanine phosphoribosyl transferase (HPRT) to account for differences in template quality. We observed a fourfold increase in IRF4 transcripts in β-gal+ pre–B cells (Fig. 7 A). A twofold increase was observed in IRF4 expression in B1-8low α-HEL-κ CD19+IgD− cells compared with B1-8 α-HEL-κ CD19+IgD− cells (Fig. 7 B). IRF4 has binding sites in the 3′κ enhancer as well as in both λ enhancers (Pongubala et al., 1992; Eisenbeis et al., 1995). This increase in IRF4 in the β-gal–positive population implies that NF-κB could be acting through IRF4 to regulate receptor editing.


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)

Expression of NF-κB target genes in IκBαΔ-transduced pre–B cells. (A) cDNA from sorted β-gal–positive and –negative pre–B cells was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT transcripts to control for any differences in cDNA template. β-gal–negative cell result was arbitrarily set to 1, with error bars indicating range of triplicate assays. (B) cDNA from CD19+IgD− cells from B1-8 α-HEL-κ and B1-8low α-HEL-κ mice was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT, with error bars indicating range of triplicate assays. These results are representative of two independent repetitions of this experiment using pools of sorted bone marrow cells from five to six mice.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig7: Expression of NF-κB target genes in IκBαΔ-transduced pre–B cells. (A) cDNA from sorted β-gal–positive and –negative pre–B cells was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT transcripts to control for any differences in cDNA template. β-gal–negative cell result was arbitrarily set to 1, with error bars indicating range of triplicate assays. (B) cDNA from CD19+IgD− cells from B1-8 α-HEL-κ and B1-8low α-HEL-κ mice was analyzed by real time RT-PCR for IRF4 transcripts and the data were normalized to HPRT, with error bars indicating range of triplicate assays. These results are representative of two independent repetitions of this experiment using pools of sorted bone marrow cells from five to six mice.
Mentions: IRF4 is a target of NF-κB that has been previously shown to be important in both pre–B cell development and receptor editing. (Lu et al., 2003; Muljo and Schlissel, 2003; Ma et al., 2006; Saito et al., 2007; Johnson et al., 2008; Pathak et al., 2008). We used real-time RT-PCR to quantify IRF4 transcripts in pro–/pre–B cells sorted based on β-gal activity. The signal was normalized to hypoxanthine-guanine phosphoribosyl transferase (HPRT) to account for differences in template quality. We observed a fourfold increase in IRF4 transcripts in β-gal+ pre–B cells (Fig. 7 A). A twofold increase was observed in IRF4 expression in B1-8low α-HEL-κ CD19+IgD− cells compared with B1-8 α-HEL-κ CD19+IgD− cells (Fig. 7 B). IRF4 has binding sites in the 3′κ enhancer as well as in both λ enhancers (Pongubala et al., 1992; Eisenbeis et al., 1995). This increase in IRF4 in the β-gal–positive population implies that NF-κB could be acting through IRF4 to regulate receptor editing.

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