Impairment of Mature B Cell Maintenance upon Combined Deletion of the Alternative NF-κB Transcription Factors RELB and NF-κB2 in B Cells

Abstract

BAFF is critical for the survival and maturation of mature B cells. BAFF, via BAFFR, activates multiple signaling pathways in B cells, including the alternative NF-κB pathway. The transcription factors RELB and NF-κB2 (p100/p52) are the downstream mediators of the alternative pathway; however, the B cell-intrinsic functions of these NF-κB subunits have not been studied in vivo using conditional alleles, either individually or in combination. We in this study report that B cell-specific deletion of relb led to only a slight decrease in the fraction of mature splenic B cells, whereas deletion of nfkb2 caused a marked reduction. This phenotype was further exacerbated upon combined deletion of relb and nfkb2 and most dramatically affected the maintenance of marginal zone B cells. BAFF stimulation, in contrast to CD40 activation, was unable to rescue relb/nfkb2-deleted B cells in vitro. RNA-sequencing analysis of BAFF-stimulated nfkb2-deleted versus normal B cells suggests that the alternative NF-κB pathway, in addition to its critical role in BAFF-mediated cell survival, may control the expression of genes involved in the positioning of B cells within the lymphoid microenvironment and in the establishment of T cell-B cell interactions. Thus, by ablating the downstream transcription factors of the alternative NF-κB pathway specifically in B cells, we identify in this study a critical role for the combined activity of the RELB and NF-κB2 subunits in B cell homeostasis that cannot be compensated for by the canonical NF-κB pathway under physiological conditions.

Figure 1. Generation of mice with conditional deletion of relb or nfkb2 in B-cells and simultaneous expression of eGFP

(A and D) Targeting strategy showing the status of relb and nfkb2 before (top) and after (bottom) Cre-mediated recombination. Numbers indicate the respective exons. (B and E) Western blot analysis of RELB and p52 protein levels in purified splenic B cells of the indicated genotypes, and (C) of flow-sorted eGFP⁺ B-cells from relb^fl/flCD19-Cre mice and the corresponding eGFP⁺ rel^fl/+CD19-Cre and eGFP⁻ CD19-Cre control mice.

Figure 2. Reduced fractions of splenic B-cells in the absence of alternative NF-κB subunits

(A–C, top) The fractions of splenic B-cells from relb^fl/flCD19-Cre, nfkb2^fl/flCD19-Cre or relb^fl/flnfkb2^fl/flCD19-Cre mice and the corresponding heterozygous and CD19-Cre control mice were determined by flow cytometry (for absolute B-cell numbers, see Supplementary Fig. 2). Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by one-way ANOVA (***, P<0.001). (A–C, bottom) Flow cytometry of eGFP expression in splenic B-cells of the indicated genotypes. The number above the gate indicates the percentage of eGFP⁺ B-cells among B220⁺ B cells of relb^fl/flnfkb2^fl/flCD19-Cre mice.

Figure 3. relb^fl/flnfkb2^fl/flCD19-Cre mice display fewer B-cell follicles within the white pulp, and exhibit more heterogeneity in the size of B-cell follicles, compared to control mice

Spleen sections from mice of the indicated genotypes were analyzed via immunohistochemistry for the expression of CD3 and IgM. One representative mouse out of 3 per group is shown. 40x magnification (left) and 100x magnification (right).

Figure 4. Relb/nfkb2-deleted B-cells show a developmental block at the T1 stage

IgM and CD23 expression of AA4.1⁺ splenic B cells from mice of the indicated genotypes were analyzed by flow cytometry. Numbers besides gates indicate the percentage of T1 (AA4.1⁺IgM^hiCD23⁻), T2 (AA4.1⁺IgM^hiCD23⁺) and T3 (AA4.1⁺IgM^lowCD23⁺) B-cells (left). Summary of the frequencies of T1–T3 B-cells (right). Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by one-way ANOVA (**, P<0.01; ***, P<0.001).

Figure 5. Counter selection against relb/nfkb2-deleted follicular and MZ B-cells in relb^fl/flnfkb2^fl/flCD19-Cre mice

(A) CD21 and CD23 expression of splenic B cells from mice of the indicated genotypes were analyzed by flow cytometry. Numbers besides gates indicate the percentage of follicular (CD23⁺CD21^int) and MZ (CD21^hiCD23⁻) B-cells (top). Summary of the frequencies of follicular and MZ B-cells (bottom). Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by one-way ANOVA (*, P<0.05; **, P<0.01; ***, P<0.001). (B) The fractions of eGFP⁺ cells among splenic follicular (CD23⁺CD21^int) and MZ (CD21^hiCD23⁻) B-cells in relb^fl/flnfkb2^fl/flCD19-Cre mice were determined by flow cytometry. Numbers above gates indicate the percentage of eGFP⁺ B-cells among the indicated B-cell subsets (top). Summary of the frequency of eGFP⁺ cells among the corresponding B-cell subsets (bottom). (C) Spleen sections from mice of the indicated genotypes were analyzed for the expression of eGFP and IgM by immunohistochemistry. FO, follicular area; MZ, marginal zone area. One representative mouse out of 3 per group is shown. 400x. Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (***, P<0.001).

Figure 6. Differential expression of BAFF-responsive genes controlled by the alternative NF-κB pathway

(A) Flow cytometric analysis of BAFF or CD40-stimulated B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice at d3 for the expression of ICOSL. Staining of eGFP⁺ and eGFP⁻ B-cells from relb^fl/flnfkb2^fl/flCD19-Cre mice and B-cells from CD19-Cre mice (top). Summary of the corresponding median fluorescence intensities (MFI) (bottom). eGFP⁺ and eGFP⁻ identifies relb/nfkb2-deleted and non-deleted B-cells from relb^fl/flnfkb2^fl/flCD19-Cre mice, respectively. Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by one-way ANOVA (*, P<0.05; **, P<0.01; ***, P<0.001). (B) Flow cytometric analysis of BAFF or CD40-stimulated purified B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice at d3 for DNA content by propidium iodide (PI) staining (top) and summary of the corresponding percentage Sub-G1 (bottom). (B) Flow cytometric analysis of BAFF or CD40-stimulated purified B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice at d3 for apoptotic/dead cells by annexin V/7AAD staining (top) and summary of the corresponding percentage of annexin V/7AAD cells (bottom). (D) Flow cytometric analysis of ex vivo B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice for BAFFR expression (right) and summary of the corresponding MFIs (right). eGFP⁺ identifies relb/nfkb2-deleted B-cells from relb^fl/flnfkb2^fl/flCD19-Cre mice. (E) Flow cytometric analysis of ex vivo B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice for β₂ expression (left) and summary of the corresponding MFIs (right). eGFP⁺ identifies relb/nfkb2-deleted B-cells from relb^fl/flnfkb2^fl/flCD19-Cre mice. (F) Flow cytometric analysis of ex vivo B-cells from relb^fl/flnfkb2^fl/flCD19-Cre and CD19-Cre mice for CD21 expression (left) and summary of the corresponding MFIs (right). eGFP⁺ identifies relb/nfkb2-deleted B-cells from relb^fl/flnfkb2^fl/flCD19-Cre mice. (B–F) Each symbol represents a mouse. Data are shown as mean ± standard deviation. Statistical significance was determined by Student’s t test (*, P<0.05; **, P<0.01; ***, P<0.001).