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 (IgM^loCD23⁺) 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 (B220^loIgM⁻), immature B cells (B220^loIgM⁺) and recirculating (B220^hiIgM⁺) 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 (B220^loIgM⁻c-Kit⁺), pre-B (B220^loIgM⁻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.

Impaired generation of Igλ⁺ B cells in the absence of IKK-mediated NF-κB activation. (a) BM immature B cells (B220^loIgM⁺) from mb1-cre Nemo^f (n=7), mb1-cre Ikk1^f/fIkk2^f/f (n=7) and littermate control (n=9) mice were stained for Igκ and Igλ₁ expression. Left, representative contour plots. Numbers indicate percent cells in each gate. Gated on immature B cells. Right, proportion of Igλ₁⁺ immature B cells. Each symbol represents one mouse, and black bars depict arithmetic mean. (b) Pro- and pre-B (IgM⁻B220^lo), immature (B220^loIgM⁺) and recirculating (B220^hiIgM⁺) B cells in the BM of mb1-cre Nemo^f (n=3) and littermate control (n=3) mice expressing the κ-macroself transgene were determined by flow cytometry. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on lymphocytes. Right, percent total BM IgM⁻B220^lo and immature B cells (B220^loIgM⁺) from indicated mice. Each symbol represents one mouse, and black bars depict mean.

Over-expression of Bcl2 in the B cell lineage rescues Igλ⁺ B cell generation in absence of IKK-mediated NF-κB activation. (a) Igκ⁺ and Igλ₁⁺ BM immature B (B220^loIgM^lo) cells from mb1-cre Nemo^f (n=7) mb1-cre Ikk1^f/fIkk2^f/f (n=5) and littermate controls (n=8) expressing a Bcl2 transgene (Bcl2 Tg) were examined by flow cytometry. Immature B cells were defined as B220^loIgM^lo cells to exclude from the analysis an interfering B220⁺IgM^hi B cell population present in Bcl2 Tg mice. Top, representative contour plots. Numbers indicate proportions of cells in each gate. Gated on immature B cells. Bottom, percent Igλ₁⁺ immature B cells in indicated mice. Each symbol represents one mouse and black bars indicate mean. (b) Pim2 mRNA expression in B220^loCD93⁺IgM⁻CD25⁺ pre-B cells sorted from the indicated mice was determined by quantitative fluorescence real-time PCR. mRNA abundance was normalized to γ-actin mRNA. Mean and s.d. was calculated for 3 mice per genotype. (c) BM immature B cells (B220^loIgM⁺) from Pim1^+/−Pim2^−/− (n=4) and Pim1^+/−Pim2^+/− (n=4) mice were stained for Igκ and Igλ₁ expression. Left, representative contour plots. Numbers indicate percent cells within each gate. Gated on immature B cells. Right, proportions of Igλ₁⁺ immature B cells in indicated mice. Each symbol represents one mouse and black bars depict mean.

B cell numbers are reduced from the T2 stage of B cell development onwards in the absence of NF-κB signaling. Absolute numbers of BM pro-B (B220^loIgM⁻c-Kit⁺), pre-B (B220^loIgM⁻CD25⁺), and immature B (B220^loIgM⁺) cells, as well as splenic T1 (B220⁺CD93⁺IgM⁺CD23⁻), T2 (B220⁺CD93⁺IgM⁺CD23⁺) and mature B (B220⁺CD93⁻) cells from littermate control (n=7), mb1-cre Nemo^f (n=7) and mb1-cre Ikk1^f/fIkk2^f/f (n=7) mice. One control mouse was excluded from the analysis because splenic B cell numbers were aberrantly higher than in the other control mice.

Receptor editing at the Igk locus is not affected in the absence of IKK-mediated NF-κB activation. (a) LM-PCR analysis of primary and secondary breaks at the Igk locus of pre-B cells purified from mb1-cre Nemo^f, mb1-cre Ikk1^f/fIkk2^f/f and littermate control mice. The sensitivity of the assay was tested by diluting wild-type pre B cell DNA with wild-type mature B cell DNA that should not contain DNA breaks at the Igk locus. Genomic DNA quantity was determined by optical density and equal loading of template DNA was confirmed by APRT amplification. The cartoon details the strategy used to analyze primary and secondary breaks at the Igk locus. (b) PCR amplification of endogenous V_κ-RS and IRS-RS rearrangements in pre-B cells (B220^loIgM⁻CD25⁺) sorted from the indicated mice. The sensitivity of the assay was tested by diluting wild-type BM IgM⁺ B cell DNA with thymus DNA that should not contain RS rearrangement. PCR amplification of APRT served as a genomic DNA template control. The cartoon depicts rearrangements between the RS and either a V_κ segment or the IRS sequences. (a) and (b) were repeated, using two different sets of samples. (c) Rag1 and Rag2 mRNA expression in pre-B cells (B220^loCD93⁺IgM⁻CD25⁺) from the indicated mice and from the liver of Rag2^−/− mice was determined by quantitative fluorescence real-time PCR. mRNA abundance was normalized to γ-actin mRNA expression. Mean and s.d. was calculated from mRNA isolated from 3 mice per genotype except for the liver control. N.D., not detected.

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 (B220^loIgM⁺) and recirculating (B220^hiIgM⁺) 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 (B220^loIgM⁺CD93⁺CD23⁻) in the BM of mb1-cre Nemo^f iEκT/T (n=6) and Nemo^f 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⁺IgM^hi 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 (B220^loIgM⁺). 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.