Limits...
Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

Derudder E, Cadera EJ, Vahl JC, Wang J, Fox CJ, Zha S, van Loo G, Pasparakis M, Schlissel MS, Schmidt-Supprian M, Rajewsky K - Nat. Immunol. (2009)

Bottom Line: During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing.In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain.This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Immune Disease Institute, Boston, Massachusetts, USA. derudder@idi.harvard.edu

ABSTRACT
By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

Show MeSH

Related in: MedlinePlus

B cell development in the absence of NF-κB signaling. (a) Transitional (B220+CD93+) and mature (B220+CD93−) B cells (top) and T1 (IgM+CD23−), T2 (IgM+CD23+) and T3 (IgMloCD23+) transitional subsets (bottom) in splenocytes of the indicated mice (n=7) were examined by flow cytometry. Numbers indicate percentages of cells within each gate. Gated on lymphocytes (top) or transitional B cells (bottom). (b) BM leukocytes from the indicated mice were stained for B220 and IgM to identify proand pre-B cells (B220loIgM−), immature B cells (B220loIgM+) and recirculating (B220hiIgM+) B cells. Numbers indicate percent cells within each gate. Gated on lymphocytes. (c) Electrophoretic mobility shift assay (EMSA) of NF-κB DNA-binding activity in total extracts of B cell subsets from indicated mice. Pro-B (B220loIgM−c-Kit+), pre-B (B220loIgM−CD25+), immature (B220+IgM+) and mature (B220+CD93−) B cells were purified by FACS from 6−8 mice per experimental group. EMSA has been performed twice. I and II, NF-κB complexes; n.s, non-specific.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2742984&req=5

Figure 1: B cell development in the absence of NF-κB signaling. (a) Transitional (B220+CD93+) and mature (B220+CD93−) B cells (top) and T1 (IgM+CD23−), T2 (IgM+CD23+) and T3 (IgMloCD23+) transitional subsets (bottom) in splenocytes of the indicated mice (n=7) were examined by flow cytometry. Numbers indicate percentages of cells within each gate. Gated on lymphocytes (top) or transitional B cells (bottom). (b) BM leukocytes from the indicated mice were stained for B220 and IgM to identify proand pre-B cells (B220loIgM−), immature B cells (B220loIgM+) and recirculating (B220hiIgM+) B cells. Numbers indicate percent cells within each gate. Gated on lymphocytes. (c) Electrophoretic mobility shift assay (EMSA) of NF-κB DNA-binding activity in total extracts of B cell subsets from indicated mice. Pro-B (B220loIgM−c-Kit+), pre-B (B220loIgM−CD25+), immature (B220+IgM+) and mature (B220+CD93−) B cells were purified by FACS from 6−8 mice per experimental group. EMSA has been performed twice. I and II, NF-κB complexes; n.s, non-specific.

Mentions: In accord with previous work4,5, ablation of NEMO in the B cell lineage largely abolished the generation of mature (B220+CD93−) peripheral B cells (Fig. 1a, Fig. 2). This effect was even more pronounced upon ablation of both canonical and alternative NF-κB signaling, in mb1-cre Ikk1f/fIkk2f/f mice. Indeed, the B220+CD93− cells in these animals were presumably mostly non-B cells, as only a few of them expressed IgM or IgD (Supplementary Fig. 2a, online). Ablation of IKK-mediated NF-κB signaling also affected the generation of transitional (T) 1 and T2 B cells, identified according to the classification of Allman et al.17. Whereas some T2 cells, characterized by CD93 and CD23 expression, could still be identified in mb1-cre Nemof mice (carrying a single (X-linked) Nemof allele in males, and two such alleles in females), such cells were undetectable in mice lacking IKK1 and IKK2 in the B cell lineage (Fig. 1a, Fig. 2). This could have resulted from CD23 regulation by NF-κB18. However, the impaired T2 cell generation in the latter mice was also evident from the bright staining of the entire population of transitional cells for CD93, indicating that only the most immature (T1) subset of transitional B cells was present (Supplementary Fig. 2b, online). T1 B cells on the other hand were only slightly reduced in mb1-cre Nemof and mb1-cre Ikk1f/fIkk2f/f mice.


Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

Derudder E, Cadera EJ, Vahl JC, Wang J, Fox CJ, Zha S, van Loo G, Pasparakis M, Schlissel MS, Schmidt-Supprian M, Rajewsky K - Nat. Immunol. (2009)

B cell development in the absence of NF-κB signaling. (a) Transitional (B220+CD93+) and mature (B220+CD93−) B cells (top) and T1 (IgM+CD23−), T2 (IgM+CD23+) and T3 (IgMloCD23+) transitional subsets (bottom) in splenocytes of the indicated mice (n=7) were examined by flow cytometry. Numbers indicate percentages of cells within each gate. Gated on lymphocytes (top) or transitional B cells (bottom). (b) BM leukocytes from the indicated mice were stained for B220 and IgM to identify proand pre-B cells (B220loIgM−), immature B cells (B220loIgM+) and recirculating (B220hiIgM+) B cells. Numbers indicate percent cells within each gate. Gated on lymphocytes. (c) Electrophoretic mobility shift assay (EMSA) of NF-κB DNA-binding activity in total extracts of B cell subsets from indicated mice. Pro-B (B220loIgM−c-Kit+), pre-B (B220loIgM−CD25+), immature (B220+IgM+) and mature (B220+CD93−) B cells were purified by FACS from 6−8 mice per experimental group. EMSA has been performed twice. I and II, NF-κB complexes; n.s, non-specific.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742984&req=5

Figure 1: B cell development in the absence of NF-κB signaling. (a) Transitional (B220+CD93+) and mature (B220+CD93−) B cells (top) and T1 (IgM+CD23−), T2 (IgM+CD23+) and T3 (IgMloCD23+) transitional subsets (bottom) in splenocytes of the indicated mice (n=7) were examined by flow cytometry. Numbers indicate percentages of cells within each gate. Gated on lymphocytes (top) or transitional B cells (bottom). (b) BM leukocytes from the indicated mice were stained for B220 and IgM to identify proand pre-B cells (B220loIgM−), immature B cells (B220loIgM+) and recirculating (B220hiIgM+) B cells. Numbers indicate percent cells within each gate. Gated on lymphocytes. (c) Electrophoretic mobility shift assay (EMSA) of NF-κB DNA-binding activity in total extracts of B cell subsets from indicated mice. Pro-B (B220loIgM−c-Kit+), pre-B (B220loIgM−CD25+), immature (B220+IgM+) and mature (B220+CD93−) B cells were purified by FACS from 6−8 mice per experimental group. EMSA has been performed twice. I and II, NF-κB complexes; n.s, non-specific.
Mentions: In accord with previous work4,5, ablation of NEMO in the B cell lineage largely abolished the generation of mature (B220+CD93−) peripheral B cells (Fig. 1a, Fig. 2). This effect was even more pronounced upon ablation of both canonical and alternative NF-κB signaling, in mb1-cre Ikk1f/fIkk2f/f mice. Indeed, the B220+CD93− cells in these animals were presumably mostly non-B cells, as only a few of them expressed IgM or IgD (Supplementary Fig. 2a, online). Ablation of IKK-mediated NF-κB signaling also affected the generation of transitional (T) 1 and T2 B cells, identified according to the classification of Allman et al.17. Whereas some T2 cells, characterized by CD93 and CD23 expression, could still be identified in mb1-cre Nemof mice (carrying a single (X-linked) Nemof allele in males, and two such alleles in females), such cells were undetectable in mice lacking IKK1 and IKK2 in the B cell lineage (Fig. 1a, Fig. 2). This could have resulted from CD23 regulation by NF-κB18. However, the impaired T2 cell generation in the latter mice was also evident from the bright staining of the entire population of transitional cells for CD93, indicating that only the most immature (T1) subset of transitional B cells was present (Supplementary Fig. 2b, online). T1 B cells on the other hand were only slightly reduced in mb1-cre Nemof and mb1-cre Ikk1f/fIkk2f/f mice.

Bottom Line: During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing.In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain.This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Immune Disease Institute, Boston, Massachusetts, USA. derudder@idi.harvard.edu

ABSTRACT
By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

Show MeSH
Related in: MedlinePlus