Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses

Abstract

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

Figure 1

Generation of nfkb2-null mice. (A) The targeting vector pNF-κB2 is shown at the top. The thin line depicts plasmid sequences, the thick line represents intron sequences, and the closed boxes show exons of the nfkb2 gene. The open boxes correspond to the PGK-neo and PGK-tk cassettes. The long arm contains a fragment of the 5′ region, exons 1 to 3, and 33 nucleotides of exon 4 (EcoRI site). The short arm contains the rest of exon 4 to exon 15. A homologous recombination event between the targeting vector and wild-type genomic DNA introduces the PGK-neo cassette into exon 4, disrupting the open reading frame of the gene. (B) Southern Blot analysis of genomic DNA isolated from mouse tails. 10 μg of DNA from mice of different genotypes were digested with SpeI, run in 0.7% agarose gels, blotted according to standard procedures, and hybridized with the 3′ external probe A. The 8.0- and 5.3-kb fragments correspond to the wild-type and targeted alleles respectively. (C) Thymocytes from control (+/+) and nfkb2-null (−/−) mice were labeled with [³⁵S]methionine for 3 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of PMA/PHA (see Materials and Methods). Cell extracts were immunoprecipitated with NF-κB2–specific antiserum and analyzed by SDS-PAGE. (D) EMSA profiles of the κB binding complexes in whole cell extracts of thymus (Th) and spleen (Sp) from control (+/+) and nfkb2-null mice (−/−). Extracts were incubated with preimmune serum (p.i.), or antisera against p50 (αp50), p52 (αp52), or both, before the addition of the κB palindromic oligo.

Figure 2

Histopathologic and immunohistochemical analysis of the spleen of naive control and nfkb2 (−/−) mice. (A and C) Spleen sections stained with hematoxylin and eosin from 8-wk-old control and nfkb2 (−/−) mice, respectively. F, follicles. Perifollicular marginal zones are indicated by arrows. (B and D) Immunohistochemistry of consecutive spleen sections from A and C, respectively, stained with the B cell–specific mAb B220. GC, germinal center. Bar equals 100 μm.

Figure 3

Reduction of the B cell population in the spleen of the nfkb2-deficient mice. (A) Scatter plots of splenocytes stained with B220 versus Thy 1.2; CD4 versus CD8; IgM versus B220; and IgD versus B220 antibodies. Single cell suspensions of splenocytes from 12-wk-old control (+/+) and mutant (−/−) mice were analyzed by flow cytometry as described in Materials and Methods. Representative diagrams from four independent experiments are shown with the percentage of positive cells in relevant quadrants. (B) Diagram showing the decrease in the total B cell population in spleen of the nfkb2 (−/−) mice respective to the control littermates at different time points. (C) Total T cells in the spleen of the nfkb2-null mice at different time points.

Figure 4

Changes in the B cell population in the bone marrow of the nfkb2-deficient mice. Scatter plots of bone marrow cells stained with IgM versus B220 from 6-wk-old (A) or 10-wk-old (B) control (+/+) and mutant (−/−) mice were analyzed by flow cytometry as described in Materials and Methods.

Figure 5

In vitro proliferation of B cells lacking NF-κB2. Purified resting B cells from nfkb2-null (open circles) and control mice (closed circles) were cultured during 72 h at 10⁵ cells/ml in the presence of different concentrations of (A) LPS (0.09–20 μg/ml); (B) anti–IgD-dextran (0.1–10 ng/ml); or (C) anti-CD40 mAb (0.08–5 μg/ml) + IL-4 (3,000 U/ml) + IL-5 (150 U/ml). [³H]thymidine was added for 12 h and cells were then harvested for determination of [³H]thymidine incorporation. Values shown represent the means of triplicate cultures ± SEM.

Figure 6

NF-κB2 (−/−) B cells have normal maturation to Ig secretion and class switching in vitro. Control (+/+) and NF-κB2 (−/−) B cells were stimulated as described in Materials and Methods. Concentrations of secreted immunoglobulins were measured in culture supernatants by ELISA 6 d after initiation of the cuture. The values represent the mean of triplicate cultures.

Figure 7

Decreased immunoglobulin production in nfkb2-null mice. (A) Resting immunoglobulin isotype levels in unimmunized mice. Serum immunoglobulin levels in naive 7-wk-old littermates were determined by isotype-specific ELISA. Closed circles correspond to control (+/+), open circles to heterozygous (+/−), and open squares to mutant (−/−) mice. (B) Immune response to the T cell–dependent antigen NP-KLH. Five littermates of control (+/+, closed circles) and mutant (−/−, open squares) mice were used for this assay. Serum samples were collected 7, 14, and 21 d after immunization as indicated. (C) Immune response to the T cell–independent antigen NP-LPS. Five control (+/+) and five mutant (−/−) mice were used in this assay. Serum samples were collected at 7, 14, and 21 d after immunization. Horizontal lines indicate the mean values.

Figure 8

Impaired formation of GC in nfkb2 (−/−) mice. Control (+/+) and mutant (−/−) mice were injected with SRBCs. Animals were killed 10 d after immunization, and then spleens were collected and processed for frozen tissue sections. A and B correspond to PNA, C and D to anti-IgD, E and F to MOMA-1, and G and H to FDC-M1 staining of spleen sections. A–D have the same magnification (bar in D equals 75 μm). E and F have the same magnification (bar in F equals 100 μm). Magnification in G and H is identical (bar in H equals 50 μm).