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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.

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Impaired generation of immature NEMO-deficient Igλ+ B cells in the absence of rearrangements at Igk loci. (a) BM cells from iEκT homozygous mice (iEκT/T)— which cannot undergo Igk rearrangements— on wild-type (n=7) or NEMO-deficient (n=9) genetic backgrounds were stained with anti-B220 and anti-IgM to identify immature (B220loIgM+) and recirculating (B220hiIgM+) B cells. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on lymphocytes. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (b) Immature B cells (B220loIgM+CD93+CD23−) in the BM of mb1-cre Nemof iEκT/T (n=6) and Nemof iEκT/T (n=5) mice over-expressing Bcl2 were examined by flow cytometry. (The combination of fluorochromes in this analysis allowed inclusion of CD93 and CD23 as additional markers to separate immature B cells from an interfering B220+IgMhi B cell population present in Bcl2 Tg mice) Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on CD93+CD23− cells. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (c) BrdU incorporation in Igκ+ and Igλ1+ immature B cells (B220loIgM+). Mice were injected intra-peritoneally with BrdU and analyzed at the indicated time points thereafter. The percentage of BrdU+Igκ+ and BrdU+Igλ1+ immature B cells in iEκT/T and littermate control mice (129/Sv) was determined by flow cytometry.
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Figure 6: Impaired generation of immature NEMO-deficient Igλ+ B cells in the absence of rearrangements at Igk loci. (a) BM cells from iEκT homozygous mice (iEκT/T)— which cannot undergo Igk rearrangements— on wild-type (n=7) or NEMO-deficient (n=9) genetic backgrounds were stained with anti-B220 and anti-IgM to identify immature (B220loIgM+) and recirculating (B220hiIgM+) B cells. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on lymphocytes. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (b) Immature B cells (B220loIgM+CD93+CD23−) in the BM of mb1-cre Nemof iEκT/T (n=6) and Nemof iEκT/T (n=5) mice over-expressing Bcl2 were examined by flow cytometry. (The combination of fluorochromes in this analysis allowed inclusion of CD93 and CD23 as additional markers to separate immature B cells from an interfering B220+IgMhi B cell population present in Bcl2 Tg mice) Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on CD93+CD23− cells. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (c) BrdU incorporation in Igκ+ and Igλ1+ immature B cells (B220loIgM+). Mice were injected intra-peritoneally with BrdU and analyzed at the indicated time points thereafter. The percentage of BrdU+Igκ+ and BrdU+Igλ1+ immature B cells in iEκT/T and littermate control mice (129/Sv) was determined by flow cytometry.

Mentions: While our results have established that gene rearrangements and receptor editing at Igk proceeded efficiently in B cell progenitors lacking NEMO or IKK1 and IKK2, our findings do not exclude some positive impact of NF-κB signals on these processes; this possibility would be consistent with the slightly enlarged pre-B cell compartment in the mutant mice. It therefore appeared possible that the impaired generation of Igλ+ B cells in the absence of NF-κB signals might result from an extended time window in which the mutant pre-B cells attempt to rearrange their Igk loci. To address this possibility, we investigated the impact of NEMO ablation on B cells of iEκT homozygous (iEκT/T) mice, in which the Igk locus is developmentally “frozen”, so that neither Vκ-Jκ rearrangements nor RS recombination takes place and the mice produce exclusively Igλ+ B cells14. Similar to what we had observed in the κ-macroself model, the development of B cells was impaired in mb1-cre Nemof iEκT/T mice, but was fully rescued by a Bcl2 transgene (Fig. 6a,b and Supplementary Fig. 9, online).


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)

Impaired generation of immature NEMO-deficient Igλ+ B cells in the absence of rearrangements at Igk loci. (a) BM cells from iEκT homozygous mice (iEκT/T)— which cannot undergo Igk rearrangements— on wild-type (n=7) or NEMO-deficient (n=9) genetic backgrounds were stained with anti-B220 and anti-IgM to identify immature (B220loIgM+) and recirculating (B220hiIgM+) B cells. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on lymphocytes. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (b) Immature B cells (B220loIgM+CD93+CD23−) in the BM of mb1-cre Nemof iEκT/T (n=6) and Nemof iEκT/T (n=5) mice over-expressing Bcl2 were examined by flow cytometry. (The combination of fluorochromes in this analysis allowed inclusion of CD93 and CD23 as additional markers to separate immature B cells from an interfering B220+IgMhi B cell population present in Bcl2 Tg mice) Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on CD93+CD23− cells. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (c) BrdU incorporation in Igκ+ and Igλ1+ immature B cells (B220loIgM+). Mice were injected intra-peritoneally with BrdU and analyzed at the indicated time points thereafter. The percentage of BrdU+Igκ+ and BrdU+Igλ1+ immature B cells in iEκT/T and littermate control mice (129/Sv) was determined by flow cytometry.
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Related In: Results  -  Collection

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Figure 6: Impaired generation of immature NEMO-deficient Igλ+ B cells in the absence of rearrangements at Igk loci. (a) BM cells from iEκT homozygous mice (iEκT/T)— which cannot undergo Igk rearrangements— on wild-type (n=7) or NEMO-deficient (n=9) genetic backgrounds were stained with anti-B220 and anti-IgM to identify immature (B220loIgM+) and recirculating (B220hiIgM+) B cells. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on lymphocytes. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (b) Immature B cells (B220loIgM+CD93+CD23−) in the BM of mb1-cre Nemof iEκT/T (n=6) and Nemof iEκT/T (n=5) mice over-expressing Bcl2 were examined by flow cytometry. (The combination of fluorochromes in this analysis allowed inclusion of CD93 and CD23 as additional markers to separate immature B cells from an interfering B220+IgMhi B cell population present in Bcl2 Tg mice) Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on CD93+CD23− cells. Right, immature B cell numbers in indicated mice. Each symbol represents one mouse and black bars depict mean. (c) BrdU incorporation in Igκ+ and Igλ1+ immature B cells (B220loIgM+). Mice were injected intra-peritoneally with BrdU and analyzed at the indicated time points thereafter. The percentage of BrdU+Igκ+ and BrdU+Igλ1+ immature B cells in iEκT/T and littermate control mice (129/Sv) was determined by flow cytometry.
Mentions: While our results have established that gene rearrangements and receptor editing at Igk proceeded efficiently in B cell progenitors lacking NEMO or IKK1 and IKK2, our findings do not exclude some positive impact of NF-κB signals on these processes; this possibility would be consistent with the slightly enlarged pre-B cell compartment in the mutant mice. It therefore appeared possible that the impaired generation of Igλ+ B cells in the absence of NF-κB signals might result from an extended time window in which the mutant pre-B cells attempt to rearrange their Igk loci. To address this possibility, we investigated the impact of NEMO ablation on B cells of iEκT homozygous (iEκT/T) mice, in which the Igk locus is developmentally “frozen”, so that neither Vκ-Jκ rearrangements nor RS recombination takes place and the mice produce exclusively Igλ+ B cells14. Similar to what we had observed in the κ-macroself model, the development of B cells was impaired in mb1-cre Nemof iEκT/T mice, but was fully rescued by a Bcl2 transgene (Fig. 6a,b and Supplementary Fig. 9, online).

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