Impaired B Cell Development and Function in the Absence of IκBNS

Maki Touma, Derin B. Keskin, Fumiko Shiroki, Ibuki Saito, Shigeo Koyasu, Ellis L. Reinherz and Linda K. Clayton
J Immunol October 15, 2011, 187 (8) 3942-3952; DOI: https://doi.org/10.4049/jimmunol.1002109

Abstract

IκBNS has been identified as a member of the IκB family of NF-κB inhibitors, which undergoes induction upon TCR signaling. Mice carrying a targeted gene disruption of IκBNS demonstrate dysregulation of cytokines in T cells, macrophages, and dendritic cells. IκBNS mediates both positive and negative gene regulation, depending on individual cell type and/or cytokine. In this study, we demonstrate an additional role for IκBNS in the B cell lineage. B cells from IκBNS knockout (KO) mice were impaired in proliferative responses to LPS and anti-CD40. IgM and IgG3 Igs were drastically reduced in the serum of IκBNS KO mice, although IκBNS KO B cells exhibited a higher level of surface IgM than that found in wild-type mice. Switching to IgG3 was significantly reduced in IκBNS KO B cells. The in vitro induction of plasma cell development demonstrated that progression to Ab-secreting cells was impaired in IκBNS KO B cells. In agreement with this finding, the number of Ab-secreting cells in the spleens of IκBNS KO mice was reduced and production of Ag-specific Igs was lower in IκBNS KO mice after influenza infection as compared with wild-type mice. Additionally, IκBNS KO mice lacked B1 B cells and exhibited a reduction in marginal zone B cells. Thus, IκBNS significantly impacts the development and functions of B cells and plasma cells.

Nuclear factor κB is a transcription factor that was first identified in B cells (1) but is now recognized as a master controller of multiple genes in virtually every cell type. In particular, NF-κB plays a key role in the overall regulation of the immune system and the inflammatory response. NF-κB consists of homodimers or heterodimers formed by five different NF-κB family members (2–5). Transcriptional control via NF-κB is extremely rapid due to the unique mechanism of regulation of NF-κB by inhibitory proteins termed IκBs. The first IκB protein identified and best characterized, IκBα, binds NF-κB proteins and positions them in the cytoplasm and away from the nucleus, thus preventing DNA binding and gene regulation (6, 7). Upon triggering of cell surface receptors that signal through NF-κB, including the TCR, BCR, TNFR1/2, or many other input signals, the IκBα protein is phosphorylated by IκB kinase and then ubiquitinated, leading to degradation of IκBα and release of the NF-κB heterodimer, enabling nuclear translocation followed by gene regulation. In turn, IκBα is itself a target of NF-κB regulation such that degradation of IκBα releases NF-κB, which then acts to induce synthesis of new IκBα proteins that can begin another cycle of sequestering NF-κB and shutting down the transcriptional activity. This mechanism of NF-κB activation has been termed the “classical” activation pathway. A pathway activating relB, termed the “nonclassical” pathway, involves the partial proteolysis of p100 to p52, which translocates to the nucleus with relB (reviewed in Refs. 8, 9). Thus, the extremely rapid response to the >150 stimuli that induce NF-κB activity is a result of the release from IκB inhibition of the preexisting NF-κB proteins, and NF-κB activation occurs without the need for transcription or translation, allowing cells to respond very rapidly to cell surface signals.

Five forms of NF-κB proteins have been identified (2–5), but detailed analysis of their specific individual roles has been complicated due to the overlapping tissue distributions and redundant functions of the various homo- and heterodimeric NF-κB pairs. The p65, c-Rel, and relB NF-κB proteins contain transactivation domains capable of activating gene transcription, whereas p50 and p52 lack transactivation domains. Thus, homodimers of p50 and p52 are thought to inhibit gene transcription by blocking κB-binding sites. Strict regulation of NF-κB activation is necessary for proper immune cell function and avoidance of tumor formation (10), and abnormal levels of NF-κB subunits lead to a variety of cancers (reviewed in Ref. 11), including various B cell leukemias (reviewed in Ref. 12). Targeted gene disruption or transgenic overexpression of NF-κB genes has aided more precise delineation of the roles of each of these subunits. In particular, deletion of each of the NF-κB subunits affects the immune response in some way, underscoring the importance of NF-κB in cells of the immune system (reviewed in Ref. 13). The phenotypes of mice carrying deletions of NF-κB genes include effects on both T and B cells, but in this study our concentration is on B lymphocytes. Deletion of rela is embryonically lethal, and reconstitution of rag-1−/− or SCID mice with embryonic day 13 rela−/− fetal liver cells demonstrates that RelA is required for mitogen-induced lymphocyte proliferation and isotype switching. Various defects in B cell activation result from the deletion of rel, and isotype switching is also affected, perhaps as a result of reduced transcription through the H chain locus. Isotype switching and Ig secretion are normal when assayed in vitro on relb−/− cells, indicating that the humoral defect of impaired IgG responses to T cell-dependent Ags observed in relb−/− mice is most likely a secondary consequence due to the absence of specific dendritic cell populations. Thus, although normal hemopoiesis does not require any of the other NF-κB proteins, the development of specific dendritic cell populations requires relB. In nfkb1−/− mice, Ig secretion, Ig switching, and proliferation are impaired, and quiescent nfkb1−/− B cells turnover more rapidly, suggesting that p50 is required for survival of nonactivated B cells. Mice lacking p52/p100 proteins develop normally but display disruption of splenic and lymph node architecture. However, nfkb2−/− lymphocytes activated in vitro exhibit only mildly impaired proliferative responses coupled with normal Ab or cytokine production. Thus, although the failure of nfkb2−/− mice to mount a normal T cell-dependent Ab response is associated with an inability to form germinal centers (GCs), intrinsic B cell defects are excluded.

With regard to the NF-κB family of proteins no new family members have been identified for some time. In contrast, the family of IκB inhibitors has continued to expand with the identification of IκBNS (14) and IκBζ (15–17). Prior to this, seven NF-κB inhibitor proteins were known: IκBα, IκBβ, IκBε, IκBγ, Bcl-3, p100, and p105 (reviewed in Ref. 2). IκBNS and IκBζ show more sequence homology to each other and to Bcl-3 than to IκBα, and, similar to Bcl-3, these proteins appear to be nuclear rather than cytoplasmic. IκBζ (18), Bcl-3 (2, 19, 20), and IκBNS (21–23) have been shown to regulate promoters of cytokines controlled by NF-κB and to be involved in inflammatory responses. Their mechanisms of action are different from those of IκBα since their expression is transcriptionally rather than posttranslationally regulated and is more restricted in cell expression patterns (14–19, 24, 25), unlike the more ubiquitous NF-κB and other IκB family members.

We previously created a knockout (KO) mouse line of IκBNS and demonstrated that T cells in IκBNS KO mice exhibit defective proliferation and cytokine production (23). For example, IκBNS KO T cells produce less IL-2, a key cytokine in survival and activation of T cells during the immune response (23). Additional cytokines, including IFN-γ, are also affected by IκBNS (23). Others have shown independently that IκBNS suppresses the inflammatory cytokines IL-6 and IL-12 in macrophages and dendritic cells (21, 22). In contrast, IκBζ increases expression of IL-6 and IL-12 as well as other inflammatory cytokines (18). Both IκBNS and IκBζ bind the p50 NF-κB family member (14, 18). Thus, these newly identified members of the IκB family serve to focus the action of the ubiquitous NF-κB transcription factors. Their tissue specificity explains the mechanism by which various cell surface receptors act through the NF-κB pathway to induce different genes in different cell types.

In this study, we describe the effects of the deletion of IκBNS on B cells. Our initial analysis of the IκBNS KO mice showed slightly lower total B cell numbers, but the difference was not significant between wild-type (WT) and IκBNS KO mice (23). However, further detailed analysis has revealed a disturbance in individual B lineage subsets of IκBNS KO mice. Furthermore, we find that IκBNS KO B cells are deficient in proliferation, Ig production, and plasma cell differentiation.

Materials and Methods

Mice

IκBNS KO mice on a C57BL/6 (B6) background were established and maintained as described (23). B6 and B6Ly5.1 mice were purchased from Taconic (Germantown, NY). Mice were maintained and bred under specific pathogen-free conditions in the animal facilities of the Dana-Farber Cancer Institute and Keio University under protocols reviewed and approved by the Institutional Animal Care and Use Committees.

Abs and flow cytometric analysis

The following mAbs were used: FITC-anti–Ly5.1 (A20), FITC-anti–CD19 (1D3), FITC-anti–CD5 (53-7.3), FITC- or PE-Cy7-anti–IgM (R6-60.2), PE-anti–CD45.2 (104), PE-anti–CD21/35 (7G6), PE-anti–CD138 (syndecan-1) (281-2), PE- or allophycocyanin-Cy7-anti–B220 (RA3-6B2), biotin-anti–CD23 (B3B4), PE-Cy7- or allophycocyanin-streptavidin (BD Biosciences). Single-cell suspensions were prepared in a FACS buffer solution (1× PBS containing 2% FCS and 0.05% NaN3). Cells were pretreated for 10 min at 4°C with anti-FcγRII/III (clone 2.4G2; 1 μg/ml) to reduce nonspecific staining. Staining for cell-surface Ag expression was performed at saturating Ab concentrations for 20 min at 4°C. Cells were washed once in FACS buffer solution and incubated with second-step reagent if necessary. A FACScan (BD Biosciences) and CellQuest software were used for analysis of triple-stained samples, and FACSAria and FlowJo software (Tree Star) were used for five-color samples. Dead cells were excluded from the analysis by forward and side scatter gating. Although B6 mice do not have the IgG2a isotype but, rather, the IgG2c isotype, the anti-IgG2a Ab crossreacts with IgG2c and therefore detects IgG2c in our mice.

B cell proliferation assay

Splenic B cells from IκBNS KO or B6 WT mice were purified by negative selection using anti-CD43–conjugated microbeads (Miltenyi Biotec). The CD43− cells were suspended in PBS containing 0.5% BSA, stained with 1 μM CFSE at 37°C for 10 min, washed three times in complete medium, and cultured in the presence of 0.1, 1, or 10 μg/ml LPS (Sigma-Aldrich), 0.1, 1, or 5 μg/ml anti-CD40 (BD Biosciences), or 10 μg/ml anti-IgM (μ-chain) (Jackson ImmunoResearch Laboratories). The cells were harvested on different days as indicated, and CFSE intensity was monitored by flow cytometry. For determination of B cell proliferation by [3H]thymidine incorporation, assays were carried out as previously described (23) and 37 kBq/well [3H]thymidine was added for the last 18 h of culture, and the incorporated radioactivity was measured by a scintillation counter (PerkinElmer MicroBeta2 LumiJET).

Immunizations

To measure the immune response to T-independent and T-dependent Ags, 8- to 12-wk-old mice were inoculated with an i.p. injection of 100 μg 2,4,6-trinitrophenol (TNP)-Ficoll or 75 μg TNP-keyhole limpet hemocyanin (KLH) (Bioresearch Technologies) mixed with Imject Alum (Pierce) as an adjuvant. Serum was collected every 7 d after immunization and analyzed for Ag-specific Abs.

For analysis of GC formation, five WT and five IκBNS KO mice received an i.p. injection of 5 × 105 SRBCs (Innovative Research). Mice were sacrificed 5 or 10 d later and spleens examined for peanut agglutinin (PNA) reactivity.

Immunohistochemical analysis

Spleens were removed, frozen in Tissue-Tec OCT compound (Sakura Finetek), and subjected to immunofluorescent staining. Cryosections (5 mm thick) were mounted onto slides, air-dried for 1 h, and fixed with ice-cold acetone for 10 min. Sections were stained with tetramethylrhodamine isothiocyanate-anti–mouse IgM (SouthernBiotech) and FITC-MOMA-1 (AbD Serotec). The slides were mounted with low fluorescent glycerol (Invitrogen) and observed with a fluorescent microscope (Olympus).

Surface and intracellular Ig staining

Cells were stained for surface Ig with biotinylated Abs and streptavidin-allophycocyanin. Purified anti-mouse Ig Abs were then used to block surface Ig. The membrane-labeled cells were treated with fixation and permeabilization reagents (BD Pharmingen) and then stained with FITC-labeled anti-Ig diluted in permeabilization medium (0.05% saponin, 2% FBS, PBS). The cells were washed and resuspended in PBS containing 2% FBS and immediately analyzed on a flow cytometer.

Influenza virus inoculation

Mice received an i.p. injection of 104 multiplicity of infection (MOI) influenza A virus (PR8). Fifteen days after the inoculation, serum was collected to measure the virus-specific Ab levels, and mice were intranasally infected with 104 MOI PR8. Survival was monitored for 20 d after the infection.

Serum Ig titration

Ig isotype levels of serum were measured by ELISA with 2.5 μg/ml anti-IgGAM–coated immunoplates and HRP-conjugated mouse Ig isotype-specific Abs (SouthernBiotech). For TNP-specific Abs, the plates were precoated with 10 μg/ml TNP-BSA. The TMB substrate set (BD Pharmingen) was used, and absorbance at 450 nm was measured. For detection of influenza-specific Ab, plates coated overnight with 100 μl 106 MOI/ml UV-inactivated influenza virus in PBS were used.

Adoptive transfer of bone marrow cells

Bone marrow cells were prepared from the tibias and femurs of 2- to 3-mo-old WT or IκBNS KO mice. T and B cells were eliminated from total bone marrow cells by CD4-, CD8-, and B220-negative separation using magnetic beads. Bone marrow cells (2 × 106) were i.v. injected into irradiated (900 rad) Ly5-congenic mice (B6Ly5.1). Six weeks after the injection, recipient mice were sacrificed, and spleen, peripheral blood, and peritoneal cavity cells were FACS analyzed for donor-derived Ly5.2+ cells. For competitive reconstruction assays of B cell subsets, one million bone marrow cells prepared from B6Ly5.1 and IκBNS KO mice were mixed and transferred together into 900 rad irradiated B6 Ly5.1 mice.

Reconstitution of B and T cells in Rag-2–deficient mice

B cells were purified from splenocytes of WT and IκBNS KO mice by positive selection of B220-expressing cells using anti-B220–conjugated MACS magnetic beads (Miltenyi Biotec). T cells were purified from lymph nodes of WT mice by depleting B cells with anti-B220–conjugated magnetic beads. FACS analysis with B220, CD4, and CD8 staining showed that B cell preparations contained >98% B220+ cells, whereas T cell preparations contained >95% CD4+ and CD8+ cells. The purified B cells from WT and IκBNS KO mice were mixed with T cells from WT mice (1 × 107 each) and injected i.v. into rag-2−/− mice. The reconstituted mice were challenged with 75 μg alum-precipitated TNP-KLH given i.p. 24 h after the transfer. Serum was collected every 7 d after immunization, and the spleen was harvested on day 14 postimmunization.

ELISPOT assay for Ab-secreting cells

Nitrocellulose membranes were coated with anti-IgGAM Ab at 2.5 μg/ml in PBS for 2 h and then blocked with 5% BSA in PBS for 4 h, both at room temperature. Single-cell suspensions were prepared from spleens, RBCs were depleted, and the cells were incubated with the membranes in 10% FBS, 50 μM 2-ME, and RPMI 1640 at 37°C for 18 h. Membranes were washed in PBS and incubated with HRP-conjugated goat anti-mouse IgG or IgM for 1 h at room temperature followed by detection using an ECL kit (GE Healthcare/Amersham Biosciences). The results are expressed as the mean of Ig-secreting cells per 106 spleen cells for three to five experiments.

Semiquantitative RT-PCR analysis

Semiquantitative RT-PCR analysis was performed to compare the relative mRNA levels of μM, μS, γ3, and γ1 in stimulated B cells from WT and IκBNS KO mice. Purified splenic B cells from B6 or IκBNS KO mice were incubated with LPS (5 μg/ml) alone or in combination with IL-4 (10 ng/ml) for 3–5 d and RNA was isolated using TRIzol reagent (Invitrogen). One microgram total RNA was used for cDNA synthesis (Roche). Semiquantitative RT-PCR was performed using β-actin or hypoxanthine phosphoribosyltransferase (HPRT) as an internal control. Primers for μM and μS (26) and for germline and postswitch γ3 and γ1 (27) were as previously described.

Real-time PCR

RNA isolated using TRIzol from purified WT or IκBNS KO B cells was used for both semiquantitative and quantitative real-time PCR analysis. Real-time PCR was performed using the Applied Biosystems TaqMan Universal PCR Master Mix. TaqMan Gene Expression premade probes were used for Blimp-1, Xbp1, Irf4, and β-actin. Probes for IκBNS were custom ordered and sequences are available upon request. The expression level of β-actin was used to normalize the template input. When the relative gene expression levels in WT and IκBNS KO B cells were compared, the mean value of WT B cells was set at 1. Assays were performed in triplicate.

Statistical analysis

The results are shown as the mean ± SD of values obtained from three to five separate experiments. For proliferation, results of a single representative experiment are provided. ELISA data were analyzed by the Student t test to assess the significance of the differences between IκBNS KO and WT groups.

Results

Altered B cell compartments in the absence of IκBNS

We had previously noted a modest reduction in the number of B cells in the spleen and lymph nodes of the IκBNS KO mice (23). This was confirmed after backcrossing the IκBNS KO mice onto a B6 background for 11 generations. Analyses of cells isolated from the lymph node and spleen of B6 and IκBNS KO mice using an Ab against the B cell marker B220 further demonstrated that the percentage of B cells is lower in the IκBNS KO spleen and lymph node compared with WT mice. This reduction was even more dramatic in the blood of these animals (Fig. 1A). Furthermore, within the IκBNS KO B220+ population, the distribution of IgM was skewed compared with WT mice. The geometric mean fluorescence intensity of IgM was 216 on IκBNS KO peripheral blood B220+ cells and 37 on WT B cells (Fig. 1A, Supplemental Fig. 1). These observations led us to perform a more detailed analysis of IκBNS KO B cells as follows.

FIGURE 1.
FIGURE 1.

Altered B cell profiles in IκBNS KO mice. A, IκBNS KO mice exhibit a reduction in the percentage of B220+ B cells in the periphery. Cells prepared from the lymph nodes, spleens, or blood of 9-mo-old B6 or IκBNS KO mice were examined for B220 and IgM surface expression by FACS. Numbers in boxes indicate the percentage of B220+ cells, and the mean fluorescence intensity of IgM-FITC on B220+ cells is given for each sample. B, Abnormal generation of MZ B cells in IκBNS KO mice. B220+ splenic B cells from 4-wk-old or 10-mo-old WT and IκBNS KO mice were examined for CD21 and CD23 by flow cytometry. Young IκBNS KO mice show a reduced percentage of CD21highCD23low MZ B cells. C, IκBNS KO mice lack B1 B cells in the peritoneal cavity. Peritoneal cavity cells isolated from WT or IκBNS KO mice were analyzed for B220+CD5+ B cells. Gates show B2 (CD5−B220high), B1a (CD5+B220int/low), and B1b (CD5−B220low) cells. B1a and B1b cell populations are absent from the IκBNS KO mouse peritoneal cavity. Three age-matched animals were examined in each set of experiments.

B cells consist of a number of different subpopulations, including terminally differentiated plasma cells, GC B cells, and marginal zone (MZ) B cells. In the periphery, surface IgMhigh B cells contain immature or “transitional” B cells and MZ B cells. Therefore, we further analyzed the developmental status of B cells in the absence of IκBNS. As shown in Supplemental Fig. 2, the IκBNS KO has a lower percentage of sIgDhighsIgMlow cells, which contain the most mature B cells, but B cells from the IκBNS KO spleen have slightly more CD93 (AA4.1)-positive immature B cells (7.0% in IκBNS WT, 8.2% in IκBNS KO at 2–3 months of age; n = 3). Examination of MZ B cells (B220+CD21highCD23low) revealed a deficit of this population in the spleens of 4-wk-old IκBNS KO mice (Fig. 1B). However, as 10-mo-old IκBNS KO mice have MZ B cells, the development of this population seems delayed rather than blocked in the absence of IκBNS. Histochemical analysis of frozen sections from the spleens of WT and IκBNS KO mice was performed to further examine MZ B cells. In both WT and IκBNS KO mice, the MZ could be observed as a layer outside of the B cell-rich follicle separated by a layer of MOMA-1+ MZ metallophilic macrophages. The MZ area of IκBNS KO spleens was reduced compared to WT in young mice (Supplemental Fig. 3). Additionally, IκBNS KO mice lacked B1 B cells, a B220low population found mainly in the peritoneal and pleural cavities (28) (Fig. 1C). Neither the B1a (CD5+) or B1b (CD5−) cells were detected in the absence of IκBNS.

When bone marrow cells from IκBNS KO mice were adoptively transferred to irradiated Ly5.1+ congenic WT hosts, the donor-derived Ly5.2+ cells recapitulated the MZ B cell deficiency observed in the IκBNS KO mice (Fig. 2A), demonstrating that this developmental defect is inherent to the IκBNS KO cells. Additionally, we noted reduced generation of GC (Fas+PNA+) B cells when donor bone marrow cells were derived from IκBNS KO mice (Fig. 2A). These results are confirmed in competitive reconstitution assays. When donor cells consisted of Ly5.1+ WT plus Ly5.2+ IκBNS KO bone marrow cells, the resulting B220+CD21highCD23low MZ cells and Fas+PNA+ GC cells were Ly5.1+ and thus derived from WT but not IκBNS KO bone marrow (Fig. 2B). Furthermore, analysis of peritoneal cavity cells after adoptive transfer of WT or IκBNS KO bone marrow showed that CD5+ and CD5−B220low cells were not repopulated when the donor cells were from IκBNS KO mice (Fig. 2A).

FIGURE 2.
FIGURE 2.

A developmental defect of MZ, GC, and B1 B cells is an intrinsic property of IκBNS KO B cells. A, T- and B-depleted bone marrow cells (2 × 106) from WT or IκBNS KO mice (Ly5.2) were adoptively transferred to lethally irradiated (900 rad) B6Ly5.1 hosts, and Ly5.2+ donor-derived cells were examined at 6 wk posttransfer. IκBNS KO-derived bone marrow produced reduced CD21highCD23low MZ and PNAhighFas+ GC B cells after gating on B220+ cells. Additionally, KO-derived bone marrow failed to repopulate B1a (CD5+B220int/low) and B1b (CD5−B220low) cells. Two independent experiments were performed and similar results obtained. B, Competitive reconstitution of B cell populations in bone marrow chimera mice. T- and B-depleted bone marrow cells (2 × 106) from WT (Ly5.1) or IκBNS KO mice (Ly5.2) were adoptively transferred to lethally irradiated (900 rad) B6Ly5.1 hosts and donor-derived B220+ cells were examined as described in A. In the upper panel, spleen cells from one bone marrow chimera show Ly5.1 WT- and Ly5.2 KO-derived populations, although fewer KO cells are present. WT Ly5.1+ bone marrow-derived cells repopulated both MZ (CD21highCD23low) and GC (PNA+Fas+) subsets, whereas IκBNS KO Ly5.2+ bone marrow-derived cells did not. Two recipient animals of each type were analyzed.

IκBNS KO B cells are altered in proliferation

The expression of IκBNS is induced by TCR-mediated signals in T cells (23) and TLR-mediated signals in macrophages and dendritic cells (21, 22). We therefore examined the ability of various stimuli to induce IκBNS expression in B cells. Purified splenic B cells were exposed to LPS, anti-CD40, and anti-IgM. Using real-time PCR we demonstrated that IκBNS RNA expression is induced upon stimulation with each of these mitogens (Fig. 3A). Splenic B cells stimulated with LPS, anti-CD40, and anti-IgM increased expression of IκBNS within 0.5–6 h, and RNA expression peaked at ∼2 h. The increased RNA levels led to increased IκBNS protein levels (Fig. 3B).

FIGURE 3.
FIGURE 3.

Induction of IκBNS and proliferation of IκBNS KO and WT B cells in response to LPS, anti-IgM, and anti-CD40. A, RNA was isolated from WT B cells 0, 0.5, 2, and 6 h after stimulation by the indicated mitogens and examined for IκBNS expression by real-time PCR. B, Splenic B cells (CD43−) were treated for 2 h with the indicated reagent or left untreated (None). Nuclear lysates (50 μg) were immunoprecipitated using anti-IκBNS antisera. After transfer to polyvinylidene difluoride membranes, IκBNS was identified using an anti-IκBNS mAb for Western blotting. Arrows indicate two isoforms. HC, H chain of the immunoprecipitating IgG. C, Splenic B cells (CD43−) were incubated for 48 h in the presence of media only (None) or the indicated reagents (0.1, 1, or 10 μg/ml LPS; 0.1, 1, or 5 μg/ml anti-CD40; 10 μg/ml anti-IgM). Proliferation was measured by [3H]thymidine incorporation. *p < 0.01. D, BrdU cell cycle analysis. Cells were pulsed with BrdU, then stained with anti-BrdU Ab and 7-aminoactinomycin D. Percentages of BrdU+ S-phase cells are indicated. Two independent experiments were performed and similar results obtained.

Given that we observed a defect in proliferation of the IκBNS KO T cells (23) and that known B cell mitogens induce IκBNS expression, we next analyzed the proliferation of IκBNS KO B cells. As seen in Fig. 3C, proliferation measured by incorporation of [3H]thymidine in response to LPS and anti-CD40 is reduced in IκBNS KO B cells whereas the response to anti-IgM is slightly higher. This experiment was performed five times and the response of the IκBNS KO B cells to IgM was consistently equivalent or higher. The alterations in proliferation in the IκBNS KO B cells were further confirmed using flow cytometery and CFSE labeling (data not shown). The lower proliferative response for LPS and anti-CD40 in IκBNS KO B cells results from reduced S-phase progression in the absence of IκBNS (Fig. 3D). Signaling induced by LPS, anti-CD40, and anti-IgM involves the NF-κB pathway (reviewed in Ref. 4), consistent with the fact that disruption of IκBNS impacts the proliferative response in B cells. Because all three agents induce expression of IκBNS, the fact that the proliferation induced by anti-IgM is not reduced in the IκBNS KO seems to be a contradiction. However, as described above and as shown in Fig. 1, IκBNS KO B cells exhibited an altered distribution of IgM compared with WT B cells. The increased number of IgM molecules on a cell may mask a decreased proliferative response to the crosslinking of IgM in the IκBNS KO B cells.

Defective Ig production in IκBNS KO mice

Because a primary function of B cells is to produce Ab, we determined the level of Igs in sera from IκBNS KO and WT mice (Fig. 4A). Levels of IgM and IgG3 were dramatically lower in the IκBNS KO mice and levels of IgG2c appeared slightly reduced. IgG1 and IgG2b Ig levels were equal to those in WT mice, however. Although the surface levels of IgM appeared higher on the IκBNS KO B cells, less IgM was secreted in vivo. To examine the possibility that the regulation of membrane versus secretory IgM production was defective in IκBNS KO B cells, we performed flow cytometric analysis of surface versus intracellular IgM in B cells purified from the spleens of WT and IκBNS KO mice. Whereas cytoplasmic IgM was virtually undetectable in stimulated and unstimulated IκBNS KO B cells, the surface IgM levels were much higher compared with WT (Supplemental Fig. 4), suggesting a defect in IgM secretion. Both the lack of B1 B cells and the delayed differentiation of MZ B cells as described above, two major cellular sources of serum IgM (28, 29), as well as a decreased level of the secreted form of IgM mRNA (Fig. 4B) likely account for the low serum IgM in IκBNS KO mice.

FIGURE 4.
FIGURE 4.

IκBNS KO mice manifest B cell defects in serum Ig levels and IgG3-switch recombination. A, Basal Ig isotype levels in the sera from naive IκBNS KO (−) mice and age-matched WT/HET mice (+) were measured by ELISA. Ig levels <0.1 μg/ml were plotted on the base line. The serum IgA levels of IκBNS KO mice were similar to those of B6 mice (data not shown). p < 0.05 for IgG2c; p < 0.01 for IgM and IgG3. All mice are on a B6 background: one mouse was WT, four mice were IκBNS heterozygous, and four mice were IκBNS KO. B, Splenic B cells (CD43−) from WT and IκBNS KO mice were stimulated in vitro with LPS (5 μg/ml) alone or LPS plus cytokines (10 ng/ml) as indicated for 4 d. Semiquantitative RT-PCR analysis of μM, μS, and HPRT mRNA was performed. C, Germline and postswitch transcripts for IgG1 and IgG3 were assessed in cDNA prepared from cells cultured in the presence and absence of LPS alone or in combination with with IL-4, respectively. Lower panels, AID and HPRT expression before and after stimulation with LPS or LPS plus IL-4 were examined. AID expression confirms that switch recombination processes have initiated and HPRT expression confirms equivalent loading of RNA in each sample. For B and C, two independent experiments were performed and similar results were obtained.

The production of classes of Igs other than IgM requires switching at the DNA level, and we therefore investigated switching using an in vitro assay. Spleen B cells were exposed to LPS or LPS plus IL-4 to induce production of IgG3 or IgG1, respectively (Fig. 4C). Measurement of both germline and postswitch transcription in B cells was carried out by PCR using primers designed to produce distinct DNA products arising from either the germline or recombined DNA configurations. Treatment of both WT and IκBNS KO B cells with LPS or LPS plus IL-4 resulted in increased transcription of activation-induced cytidine deaminase (AID) to an equal extent relative to the HPRT control (Fig. 4C, lower panel). AID is required for the process of IgH class-switch recombination (CSR) (27), and the equal induction of this enzyme in both WT and IκBNS KO B cells implies that the process of class switching has been successfully initiated, at least with respect to this enzyme. Transcription from the germline configuration was approximately equal for the WT and IκBNS KO B cells, and the level of transcription from the germline configuration was increased by the addition of LPS plus IL-4 for IgG1 and by addition of LPS alone for IgG3 in both WT and IκBNS KO cells (Fig. 4C). The level of transcription from the postswitch configuration of IgG1 appeared to be reduced in the IκBNS KO cells, whereas transcription from the postswitch IgG3 configuration was undetectable. The defect in class switching was confirmed when anti-CD40 was used to stimulate IgG3 and IgG1 class switching in the presence and absence of IL-4, respectively (Supplemental Fig. 5). Thus, the absence of IκBNS dramatically impaired class switching to the IgG3 H chain gene as reflected in the lack of detectable IgG3 in the IκBNS KO mouse serum.

Given these deficiencies, are the IκBNS KO mice capable of mounting a humoral immune response? We performed ELISA assays to measure the TNP-specific IgM and IgG produced by IκBNS KO and WT mice before and after i.p. immunization with TNP-Ficoll, a type II T-independent Ag. At 1 and 2 wk postimmunization, IκBNS KO mice produced no detectable TNP-specific IgM or IgG3, whereas such Abs were detected in WT mice at both time points (Fig. 5A). Using TNP-KLH as a T-dependent Ag, we next measured the TNP-specific IgM and IgG1 produced by IκBNS KO and WT mice 1 and 2 wk after immunization. As shown in Fig. 5B, the IκBNS KO mice produced little, if any, TNP-specific IgM. Production of Ag-specific IgG1 appeared to be delayed by a week, although the final levels of IgG1 were equivalent to those of WT mice. Because we previously showed that the absence of IκBNS also alters T cell functions, we performed adoptive transfer assays into B6.Rag-2−/− mice using purified B cells from WT or IκBNS KO spleen mixed with WT T cells. Upon immunization with TNP-KLH, the production of IgM by IκBNS KO B cells remained decreased, showing that the decreased production of IgM was a B cell-intrinsic defect (Supplemental Fig. 6). However, in the presence of WT T cells, production of IgG1 from IκBNS KO B cells was equivalent to that of WT B cells, indicating that the defective IκBNS T cells altered the production of IgG1 by the IκBNS KO B cells.

FIGURE 5.
FIGURE 5.

Defective production of Ag-specific Abs in IκBNS KO mice. Eight- to 12-wk-old WT and IκBNS KO mice were injected i.p. with (A) 100 μg TNP-Ficoll (T-independent Ag) or (B) 75 μg TNP-KLH (T-dependent Ag). Sera were collected at 7 and 14 d postimmunization. TNP-specific IgM and IgG3 or TNP-specific IgM and IgG1 in the sera were determined by ELISA. One representative data set from two independent experiments is shown. Similar results were obtained in both experiments. C, Twelve age-matched (8- to 12-wk-old) male WT and IκBNS KO mice were immunized i.p. with influenza virus PR8 at an MOI of 1 × 104. Fifteen days after immunization, mice were intranasally infected with 1 × 104 PR8 virus and survival was followed. Day 1 on the survival graph is the day of infection. D, Virus-specific IgG2c Abs were determined by ELISA in WT and IκBNS KO mice before immunization (Prebleed) and 15 d postimmunization (PI). Filled circles represent WT samples; open circles represent IκBNS KO samples. The asterisk in C and D denotes one WT mouse that succumbed to viral infection and did not produce IgG2c in response to PR8. At 15 d postimmunization, p < 0.01 for WT versus KO. E, Spleen sections were prepared from WT and IκBNS KO mice 5 d after i.p. injection of SRBCs. Sections were stained with PNA and with H&E. Original magnification ×20. Arrows indicate PNA+ areas in active GCs in WT mice. IκBNS KO mice did not develop active GCs. Five WT and five KO mice were examined with similar results.

The humoral response to infection in the absence of IκBNS was assayed using a murine-adapted influenza A virus, PR8. WT and IκBNS KO mice were immunized i.p. and then infected intranasally ∼2 wk later with influenza virus. The survival curve in Fig. 5C shows that IκBNS KO mice were more severely affected by influenza A, with 50% succumbing to infection. The asterisk in Fig. 5C indicates the single death of one WT mouse after receiving a sublethal viral challenge. Ag-specific Ab production in response to influenza immunization was then determined (Fig. 5D). Whereas IκBNS KO mice were deficient in production of influenza-specific Abs of the IgM, IgG1, and IgG2b classes (data not shown), the most significant deficiency was in IgG2c Ig, as shown in Fig. 5D. This is noteworthy as this class of Igs affords a prominent protective response against influenza A in the murine system (30, 31).

To examine the B cell response histologically, we assayed GC formation using immunization with SRBCs in WT and IκBNS KO mice (Fig. 5E). Five days after immunization, spleens were removed and sections prepared by staining with H&E followed by PNA staining to visualize GCs where core-1 glycans are O-linked without sialic acid adducts and, hence, are PNA-reactive. Although GCs were clearly evident in the spleens of WT mice, no GC formation was detected in the spleens of the five IκBNS KO mice examined. Given the fact that the TNP-KLH IgG1 response appeared delayed, this experiment was repeated and GC formation examined at 10 d after immunization. Again, no GC formation was observed in the IκBNS KO mice whereas areas of PNA reactivity were clear in the WT mice (data not shown).

IκBNS KO B cells exhibit reduced plasma cell differentiation

One path of B cell differentiation culminates in the production of plasma cells, which are terminally differentiated, nondividing Ab-secreting cells (ASCs). Determination of the ex vivo number of ASCs from the spleens of WT and IκBNS KO mice showed that in the IκBNS KO spleen there were fewer B cells secreting all of the types of the Igs assayed, but the most dramatic differences were in IgM-, IgG3-, and IgG2b-producing splenic cells. Fig. 6 shows results of an individual ELISPOT assay and statistics compiled from three to five experiments in spleen (Fig. 6B) and bone marrow (Fig. 6C). Note that IgM and IgG3 ASCs were reduced in both organs.

FIGURE 6.
FIGURE 6.

Splenic ASCs are reduced in IκBNS KO mice. A, Ig-secreting cells from WT and IκBNS KO mice were compared using an ELISPOT assay on total splenocytes after RBC lysis. The numbers of ASCs per 106 spleen cells (B) and bone marrow cells (C) are shown graphically. Results are a compilation of three to five independent assays. Statistical analysis was performed using the Student t test. *p < 0.01.

The finding that there are fewer ASCs in the spleens of IκBNS KO mice led us to examine the process of plasma cell differentiation in IκBNS KO B cells. B cells from spleens were cultured for 5 d in the presence of LPS to induce plasma cell differentiation. Proliferation was followed using CFSE staining, whereas plasma cell differentiation was measured by CD138 (syndecan-1) expression. As shown in Fig. 7A, LPS-stimulated plasma cell differentiation was decreased in IκBNS KO B cells. Addition to the culture of IL-4, IFN-γ, or TGF-β in combination with LPS resulted in production of IgG1+, IgG2c+, or IgG2b+ cells, respectively, in the culture as determined by FACS of cell surface IgG (Supplemental Table I). Cells incubated with LPS alone expressed surface IgG3 (Supplemental Table I). The percentage of IgG3+ or IgG2c+ cells was lower in the IκBNS KO cultures than in the WT cultures, but the percentages of IgG1+ or IgG2b+ cells were approximately equivalent (Supplemental Table I). However, the percentage of CD138+ cells was 4- to 6-fold lower in the IκBNS KO cultures under every culture condition (Fig. 7B), indicating that IκBNS KO B cells were defective in plasma cell differentiation.

FIGURE 7.
FIGURE 7.

In vitro induction of CD138+ plasma cells from WT and IκBNS KO B cells. A, Splenic CD43− B cells from IκBNS KO and WT mice were CFSE labeled and then cultured for 3 d in the presence of 5 μg/ml LPS. Cells were examined for proliferation and CD138 expression by flow cytometry. B, Cells were cultured as described for A. On day 3, 10 ng/ml IL-4, IFN-γ, or TGF-β was added for 2 additional days to stimulate isotype switching to IgG1, IgG2a, or IgG2c, respectively. Cultures were then examined for CD138 and IgG expression. The graph indicates the percentage of CD138+ cells under each condition in the WT (filled bar) and IκBNS KO (open bar) cultures. C, Enriched CD43− splenic B cells (1 × 105 cells/200 μl/well) were stimulated with LPS (5 μg/ml) for the indicated periods, and total RNA was isolated for quantitative real-time PCR analysis. All data were normalized to β-actin expression. Fold differences relative to day 0 WT levels are shown. Open circles represent KO samples; closed circles represent WT samples. Two independent experiments were performed in A–C and similar results were obtained.

The differentiation of plasma cells involves a well-studied transcriptional program (reviewed in Refs. 32, 33), including Prdm-1 (Blimp-1), Xbp1, and Irf4. Given the role of IκBNS in gene regulation, we used real-time PCR to examine the levels of expression of these transcription factors during LPS-stimulated culture of B cells from WT and IκBNS KO mice. Splenic B cells from WT and IκBNS KO mice were stimulated with LPS for 24, 48, and 72 h. RNAs prepared at these time points were examined for levels of Blimp-1, Xbp1, and Irf4. As shown in Fig. 7C, levels of Prdm-1/Blimp-1 and Xbp1 increased during 72 h in the WT B cells, but in the absence of IκBNS the increase in levels of these RNAs was greatly diminished. By 72 h, the levels of Prdm-1/Blimp-1, Xbp1 and Irf4 were 4- to 5-fold higher in WT than in IκBNS KO B cells. Although IκBNS KO B cells showed an increase, albeit much reduced, in Prdm-1/Blimp-1 and Xbp1 levels over time, there was no change in Irf4.

These results suggest that IκBNS plays a role in the induction of the transcriptional program leading to a terminally differentiated plasma cell. In the absence of IκBNS, the requisite increases in the plasma cell transcriptome, namely Prdm-1/Blimp-1, Xbp1, and Irf4, did not occur, thereby resulting in decreased numbers of ASCs (Fig. 7).

Discussion

Our findings that deletion of IκBNS, a member of the IκB family of NF-κB inhibitors, affects the development and function of B cells is consistent with the important role of NF-κB in this cell lineage. As stated above, NF-κB was first identified in B cells (1) and plays a major role in cells involved in inflammation and the immune response. B cells are primarily divided into B1 cells, follicular B cells, and MZ B cells (reviewed in Ref. 34). The origins of each cell type, as well as the transitions that may occur between these cell types, have long been an area of intense analysis. Follicular and MZ B cells are the most abundant mature B cells, with most peripheral B cells being of the follicular type. Follicular B cells are mobile, circulate within the periphery, and are found in the follicles of the splenic white pulp. They participate in both T cell-dependent and T cell-independent immune responses (35). MZ B cells, in contrast, remain at the marginal sinus of the spleen (29, 36), although they migrate to the follicles and back, transporting immune complexes containing Ag to the follicles (37, 38). MZ cells participate in T cell-independent responses and respond rapidly to blood-borne pathogens, a property shared with B1 B cells (29, 39). B1 B cells comprise most of the B cells in the peritoneal and pleural cavities in mice (40, 41) and produce IgM and IgG3 serum Abs (29, 42). The B1 subset is divided into B1a and B1b cells based on the levels of CD5 on these two cell populations. B1a cells, which express CD5, comprise the earliest B cells arising in the neonate to become a self-replenishing population (43, 44) that is the main source of “natural” IgM Abs (44). B1b cells arise from the neonate but are derived from adult bone marrow as well (45, 46). In addition to these developmental differences, B1a and B1b cells differ in function, with B1a contributing to innate immunity and B1b to adaptive immunity (47).

As is true for T cells (23), the absence of IκBNS affects B cell proliferation in response to multiple stimuli, including LPS, anti-CD40, and anti-IgM (Fig. 3). The diminution in LPS-triggered activation in the IκBNS KO is most obvious in [3H]thymidine incorporation, whereas that of anti-CD40 is more pronounced in the BrdU experiments. Consistent with this result, each of these stimuli induced IκBNS at both the protein and mRNA level (Fig. 3). LPS also induced IκBNS in gut and peritoneal macrophages (21, 22), presumably acting through the TLR4 (48) in a MyD88-dependent manner (22). However, IκBNS KO B cells sustained a high level of surface IgM expression compared with WT B cells and, thus, even if the signal via each IgM molecule is weaker than in WT B cells, the abundance of IgM on the surface may compensate for the lack of IκBNS. These opposing effects may account for preservation of the anti-IgM signaling in IκBNS KO B cells. Activation via CD40 induces NF-κB, and the reduced proliferation in the absence of IκBNS is consistent with this. Furthermore, the reduced response to anti-CD40 correlates with the reduction (or delay) in the response of IκBNS KO B cells to T-dependent Ags as shown in Fig. 5.

Production of Ig is also altered in IκBNS KO mice. Measurement of steady-state serum levels (Fig. 4) show that IgM and IgG3 were drastically reduced, IgG2c was somewhat reduced, and IgG1 and IgG2b were approximately equal compared with levels found in WT mice. As B1 B cells are the source of serum levels of IgM and IgG3 (reviewed in Ref. 49), the absence of these Igs can be partially explained by the lack of B1 B cells in these mice. Additionally, B cells from the IκBNS KO mice produced less secreted IgM as evident by the lack of serum IgM and as assayed by PCR for the secreted versus membrane form of IgM mRNA (Fig. 4B). Given that the reduction in the response appears more profound than the reduction in MZ B cell numbers, perhaps this implies functional defects in residual cells.

CSR to IgG3 at the DNA level was not detected in IκBNS KO B cells (Fig. 4) using an in vitro system that mimics CSR (reviewed in Ref. 50). In these experiments, LPS was used with or without additional cytokines to direct CSR to particular H chain isotypes. However, a low level of IgG3 protein expression was observed (Fig. 7). Presumably, the LPS/cytokine milieu directs germline transcription to distinct isotypes (reviewed in Ref. 51), resulting in modulation of CSR. LPS in combination with IL-4 induces CSR to IgG1 in both WT and IκBNS KO B cells, and induction of AID is equivalent in both cell types, indicating that at least this component of the CSR machinery is operational in IκBNS KO B cells. Clearly a specific effect of deletion of IκBNS is an alteration in IgG3 CSR. This does not appear to be due to a reduction in transcription through the IgG3 locus, as germline transcription before and after LPS addition appears to be equivalent for both WT and IκBNS KO mice (Fig. 4). There is precedence for transcription factors regulating CSR. For example, T-bet acts either by controlling germline transcripts or by mediating IgH locus access to other transcription factors and recombination-inducing machinery (52) and affects T-independent IgG2a production (53). Ikaros was shown to repress transcription of IgG2a and IgG2b, thus increasing transcription from and recombination to other H chain genes (54). Other proteins affecting CSR specificity include TGF-β, which induces Smad and Runx that promote CSR to IgG2b and IgA, and IL-4, which induces the transcriptional activator Stat6 whose activity promotes CSR to IgG1 and IgE (reviewed in Ref. 55). Perhaps more pertinent to our finding of the role of IκBNS in CSR, NF-κB proteins and, in particular, NF-κB p50 have been shown to contribute to isotype specificity in CSR (56–60). In addition to proteins that contribute to the isotype specificity of CSR, the switch region sequences that lie upstream of each CH gene also function to mediate Ab class switching (Ref. 61 and references therein), and the roles of the various proteins and switch regions are an area of active investigation. The finding that IκBNS contributes to isotype switching extends further the list of involved proteins.

Along with the observed serum Ig and class-switch phenotypes in the IκBNS KO mice, we found a reduction in ASCs assayed ex vivo from the spleens of IκBNS KO mice (Fig. 6). The pattern of reduction of ASCs corroborates the observed differences in serum Ig values in the IκBNS KO mice. Furthermore, the IκBNS KO mice failed to produce any Ag-specific IgM or IgG3 Abs in response to TNP-Ficoll. Given that T-independent responses are predominantly mediated by MZ B cells (29, 39, 62), the lack of response to TNP-Ficoll correlates with the reduction in MZ B cells in the IκBNS KO mouse. Furthermore, the lack of Ag-specific IgM may result in part from the reduction in MZ B cells but also from the inability of IκBNS KO B cells to produce secreted IgM (Fig. 4). This is observed as well in the response to TNP-KLH, a T-dependent Ag, as there is a delayed production of Ag-specific IgG1 but no production of IgM (Fig. 5), although this may also be affected by intrinsic alterations in the IκBNS KO T cells (Supplemental Fig. 6). When WT T cells were mixed with either WT or IκBNS KO B cells and these mixtures were used to reconstitute rag-2−/− mice, the IgG1 response was delayed and the response in IκBNS KO mice reached levels similar to that in WT mice, albeit the kinetics of the response were a bit slower in the reconstitution experiment (Supplemental Fig. 6). The phenotype of the reduced secretion of IgM is quite clearly represented in both Fig. 5 and Supplemental Fig. 6, however, and is thus an intrinsic B cell defect. Additionally, flow cytometric analysis of spleen cells in the recipient mice in Supplemental Fig. 6 showed 12.5 ± 1.1% CD138+B220low plasma cells in recipients of WT B and T cells and 2.8 ± 0.4% CD138+B220low plasma cells in recipients of IκBNS KO B and T cells (data not shown).

IκBNS KO B cells also exhibited a reduced response to influenza virus. WT mice immunized by i.p. injection of influenza successfully withstood influenza infection and produced influenza-specific IgG2c Abs, the major isotype found in mice surviving influenza infections (30, 31). IκBNS KO mice, in contrast, produced less influenza-specific IgG2c and were more susceptible to infection after immunization. The lack of formation of GCs in IκBNS KO mice upon immunization is additional evidence of a functional defect in IκBNS KO B cells and may explain the reduction in the Ag-specific immune response, as the GC is where B cell affinity maturation and class switching occur, resulting in Ag-specific ASCs (reviewed in Ref. 63). Impairment of GC formation is demonstrated by the lack of PNA staining after immunization with a T-dependent Ag, SRBCs (64). The presence of a low level of Ag-specific IgG2c Abs can be explained by the fact that some Ab responses can be generated in the absence of GC formation (65). Note, however, that mice deficient for Bcl-3, an IκB family member to which IκBNS has a strong homology, also fail to develop a GC response and fail to produce influenza-specific Abs, although, in contrast to the IκBNS KO mice, they do produce influenza-specific IgM Abs (66). Additionally, the serum levels of all Ig types were similar between WT and Bcl-3 KO mice.

The GC response is dependent on T cell help provided to B cells through CD40 (reviewed in Ref. 63). We have shown that proliferation of IκBNS KO B cells through CD40 is reduced and the dampening of CD40 signaling in the absence of IκBNS may affect the ability of IκBNS KO B cells to participate in a GC response. Furthermore, B cells express TLRs, which participate in the innate and adaptive immune response (reviewed in Ref. 67). IκBNS plays a role in the downstream signaling of those TLRs that work through MyD88-dependent mechanisms (22). Thus, the absence of IκBNS could affect both innate and adaptive immunity by altering TLR activation. In particular, B1 and MZ B cells respond to TLR ligands in T-independent responses, resulting in production of low-affinity IgM (reviewed in Ref. 67).

Young IκBNS KO mice exhibited a reduced MZ B cell population, but this population reached WT levels after the mice were >6 mo old (Fig. 1 and data not shown). MZ B cell development is blocked in mice carrying a targeted gene deletion of the NF-κB p50 protein (56, 68, 69). We and others have shown that IκBNS interacts with p50 (14, 21), suggesting that this complex and genes regulated by it play a role in MZ B cell development. In addition to NF-κB p50, alterations in the expression of NF-κB p65 (RelA), c-Rel (68) and RelB (70) also affect MZ B cell development.

The absence of IκBNS also affects the development of plasma cells. Plasma cells are a final differentiation state of B cells and are basically an Ig-secreting factory. These cells emerge from a GC reaction and express Ag-specific high-affinity Ig, having undergone somatic hypermutation within the GC. Plasma cells are the source of persistent Ag-specific Ab titers (71) and, thus, the lack of development of plasma cells may contribute to the fact that the IκBNS KO mice have reduced anti-influenza specific Abs. The differentiation of plasma cells has a defined transcriptional program, including Prdm-1/Blimp-1, Xbp1, and Irf4 (reviewed in Refs. 32, 33). Our analysis of these factors in WT and IκBNS KO B cells during in vitro differentiation shows that in the absence of IκBNS, these transcription factors were decreased and in vitro plasma cell differentiation was impaired (Fig. 7). The frequency of ASCs found in the spleen of the IκBNS KO mice was also reduced (Fig. 6). The development and survival of plasma cells in special niches in the bone marrow is only partially understood, and the reduction in expression of Prdm-1/Blimp-1, Xbp1, and Irf4 in the absence of IκBNS suggests that IκBNS may control a point upstream of the plasma cell transcriptional program.

B1 B cells are missing from IκBNS KO mice. The origin of the B1 B cell lineage is controversial (42, 72), and recent findings suggest that signal strength drives B cell fate (73) or that a specific progenitor exists for B1 B cells (46). Altered expression of molecules that affect signal strength through the BCR affect the B1 cell population in mice. For example, construction of transgenic mice using the LMP2A transgene in place of Igh demonstrated that mice with a high copy number of the transgene, which mimics strong BCR signaling, have higher numbers of B1 cells, whereas mice carrying fewer LMP2A transgenes, consistent with a lower intensity BCR signal, have lower numbers of B1 B cells (73). Similarly, more B1 cells are also found in mice that lack negative regulators of BCR signaling (42) or express higher levels of BCR (72, 74). This suggests that IκBNS may play a role in strengthening the BCR signal and that in the absence of IκBNS a reduced BCR signal results in the lack of B1 B cells. A similar role for IκBNS was suggested in T cells (23). IκBNS was cloned as a gene that was induced in thymocytes after N15TCRtg rag-2−/− mice were injected with VSV8, a negatively selecting peptide for this TCR (14, 75). Positively selecting peptides did not induce expression of IκBNS (75). As negative selection is associated with a strong TCR signal and positive selection is associated with a weaker TCR signal, the expression of IκBNS appeared to correlate with a strong TCR signal. Furthermore, production of IL-2 by T cells is thought to require a strong TCR signal, and in the absence of IκBNS, IL-2 production is reduced (23), again associating IκBNS with receptor signal strength.

The phenotype of p50 KO mice has some commonalities with the phenotype of IκBNS KO mice, which is to be expected, as IκBNS has been demonstrated to interact with p50 (14, 21). Specifically (as reviewed in Ref. 13), p50 KO mice display normal development but are abnormal in immune function. As in the IκBNS KO, p50 KO B cells proliferate poorly to LPS and CD40 and display abnormal H chain switching due to lack of transcription through that region. In fact, p50 and p65 were found to bind the IgG3 switch region, and p50 KO mice are specifically impaired in switching to IgG3 (59, 60, 76) and have a reduced MZ B cell population (68).

In summary, characterization of B cells in our IκBNS KO mouse uncovers a pleiotropic phenotype, which is consistent with other factors that affect NF-κB activity. B cell functions, including Ig production, plasma cell development, and isotype switching, are impaired and particular B cell populations such as B1 cells and MZ B cells are reduced in comparison with WT mice. Although not shown, the development of pro-B and pre-B cells in young adult bone marrow was not affected by IκBNS deletion, arguing that the function of this regulator is targeted to the mantle zone and follicular B cell compartments, Ig switching processes, and plasma cell development. The phenotype overlaps that of p50 KO mice, correlating with the fact that IκBNS and p50 interact. That the IκBNS KO phenotype also overlaps in part that of the Bcl-3 KO is noteworthy because IκBNS and Bcl-3 are homologous members of the IκB family of NF-κB inhibitors. These results reveal a significant role for IκBNS in development of the B cell arm of the immune system and the humoral response in addition to the already defined role of IκBNS in T cells, macrophages, and dendritic cells.

Disclosures

S.K. is a consultant for Medical and Biological Laboratories, Co., Ltd. The remaining authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Klaus Rajewsky for helpful discussions and suggestions during the course of this work and Drs. Garnet Kelsoe, Emmanuel Derudder, and Klaus Rajewsky for constructive criticism of the manuscript.

Footnotes

  • This work was supported by National Institutes of Health Grants AI19807 (to E.L.R.) and AI51779 (to L.K.C.) and by KAKENHI Grant 22790457, the Uehara Memorial Foundation, and the Hayashi Memorial Foundation for Female Natural Scientists (to M.T.). F.S. is a postdoctoral fellow of the Global Center of Excellence Program supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AID
    activation-induced cytidine deaminase
    ASC
    Ab-secreting cell
    B6
    C57BL/6
    CSR
    class-switch recombination
    GC
    germinal center
    HPRT
    hypoxanthine phosphoribosyltransferase
    KLH
    keyhole limpet hemocyanin
    KO
    knockout
    MOI
    multiplicity of infection
    MZ
    marginal zone
    PNA
    peanut agglutinin
    TNP
    2,4,6-trinitrophenol
    WT
    wild-type.

  • Received June 25, 2010.
  • Accepted August 4, 2011.

References

  1. ↵
    1. Sen R.,
    2. D. Baltimore
    . 1986. Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46: 705–716.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Ghosh S.,
    2. M. J. May,
    3. E. B. Kopp
    . 1998. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16: 225–260.
    OpenUrlCrossRefPubMed
    1. Karin M.,
    2. Y. Ben-Neriah
    . 2000. Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu. Rev. Immunol. 18: 621–663.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Hayden M. S.,
    2. S. Ghosh
    . 2004. Signaling to NF-κB. Genes Dev. 18: 2195–2224.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Sun S.-C.,
    2. S. C. Ley
    . 2008. New insights into NF-κB regulation and function. Trends Immunol. 29: 469–478.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Baeuerle P. A.,
    2. D. Baltimore
    . 1988. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor. Cell 53: 211–217.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Baeuerle P. A.,
    2. D. Baltimore
    . 1988. IκB: a specific inhibitor of the NF-κB transcription factor. Science 242: 540–546.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Pomerantz J. L.,
    2. D. Baltimore
    . 2002. Two pathways to NF-κB. Mol. Cell 10: 693–695.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Beinke S.,
    2. S. C. Ley
    . 2004. Functions of NF-κB1 and NF-κB2 in immune cell biology. Biochem. J. 382: 393–409.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Shishodia S.,
    2. B. B. Aggarwal
    . 2004. Nuclear factor-κB: a friend or a foe in cancer? Biochem. Pharmacol. 68: 1071–1080.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Bassères D. S.,
    2. A. S. Baldwin
    . 2006. Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression. Oncogene 25: 6817–6830.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Goetz C. A.,
    2. A. S. Baldwin
    . 2008. NF-κB pathways in the immune system: control of the germinal center reaction. Immunol. Res. 41: 233–247.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Gerondakis S.,
    2. M. Grossmann,
    3. Y. Nakamura,
    4. T. Pohl,
    5. R. Grumont
    . 1999. Genetic approaches in mice to understand Rel/NF-κB and IκB function: transgenics and knockouts. Oncogene 18: 6888–6895.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Fiorini E.,
    2. I. Schmitz,
    3. W. E. Marissen,
    4. S. L. Osborn,
    5. M. Touma,
    6. T. Sasada,
    7. P. A. Reche,
    8. E. V. Tibaldi,
    9. R. E. Hussey,
    10. A. M. Kruisbeek,
    11. et al
    . 2002. Peptide-induced negative selection of thymocytes activates transcription of an NF-κB inhibitor. Mol. Cell 9: 637–648.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kitamura H.,
    2. K. Kanehira,
    3. K. Okita,
    4. M. Morimatsu,
    5. M. Saito
    . 2000. MAIL, a novel nuclear IκB protein that potentiates LPS-induced IL-6 production. FEBS Lett. 485: 53–56.
    OpenUrlCrossRefPubMed
    1. Haruta H.,
    2. A. Kato,
    3. K. Todokoro
    . 2001. Isolation of a novel interleukin-1-inducible nuclear protein bearing ankyrin-repeat motifs. J. Biol. Chem. 276: 12485–12488.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Yamazaki S.,
    2. T. Muta,
    3. K. Takeshige
    . 2001. A novel IκB protein, IκB-ζ, induced by proinflammatory stimuli, negatively regulates nuclear factor-κB in the nuclei. J. Biol. Chem. 276: 27657–27662.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Yamamoto M.,
    2. S. Yamazaki,
    3. S. Uematsu,
    4. S. Sato,
    5. H. Hemmi,
    6. K. Hoshino,
    7. T. Kaisho,
    8. H. Kuwata,
    9. O. Takeuchi,
    10. K. Takeshige,
    11. et al
    . 2004. Regulation of Toll/IL-1-receptor-mediated gene expression by the inducible nuclear protein IκBζ. Nature 430: 218–222.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Kuwata H.,
    2. Y. Watanabe,
    3. H. Miyoshi,
    4. M. Yamamoto,
    5. T. Kaisho,
    6. K. Takeda,
    7. S. Akira
    . 2003. IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-α production in macrophages. Blood 102: 4123–4129.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Wessells J.,
    2. M. Baer,
    3. H. A. Young,
    4. E. Claudio,
    5. K. Brown,
    6. U. Siebenlist,
    7. P. F. Johnson
    . 2004. BCL-3 and NF-κB p50 attenuate lipopolysaccharide-induced inflammatory responses in macrophages. J. Biol. Chem. 279: 49995–50003.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Hirotani T.,
    2. P. Y. Lee,
    3. H. Kuwata,
    4. M. Yamamoto,
    5. M. Matsumoto,
    6. I. Kawase,
    7. S. Akira,
    8. K. Takeda
    . 2005. The nuclear IκB protein IκBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria. J. Immunol. 174: 3650–3657.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Kuwata H.,
    2. M. Matsumoto,
    3. K. Atarashi,
    4. H. Morishita,
    5. T. Hirotani,
    6. R. Koga,
    7. K. Takeda
    . 2006. IκBNS inhibits induction of a subset of Toll-like receptor-dependent genes and limits inflammation. Immunity 24: 41–51.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Touma M.,
    2. V. Antonini,
    3. M. Kumar,
    4. S. L. Osborn,
    5. A. M. Bobenchik,
    6. D. B. Keskin,
    7. J. E. Connolly,
    8. M. J. Grusby,
    9. E. L. Reinherz,
    10. L. K. Clayton
    . 2007. Functional role for IκBNS in T cell cytokine regulation as revealed by targeted gene disruption. J. Immunol. 179: 1681–1692.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Ohno H.,
    2. G. Takimoto,
    3. T. W. McKeithan
    . 1990. The candidate proto-oncogene bcl-3 is related to genes implicated in cell lineage determination and cell cycle control. Cell 60: 991–997.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Bhatia K.,
    2. K. Huppi,
    3. T. McKeithan,
    4. D. Siwarski,
    5. J. F. Mushinski,
    6. I. Magrath
    . 1991. Mouse bcl-3: cDNA structure, mapping and stage-dependent expression in B lymphocytes. Oncogene 6: 1569–1573.
    OpenUrlPubMed
  26. ↵
    1. Ren H.,
    2. A. Schmalstieg,
    3. D. Yuan,
    4. R. B. Gaynor
    . 2002. I-κB kinase β is critical for B cell proliferation and antibody response. J. Immunol. 168: 577–587.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Muramatsu M.,
    2. K. Kinoshita,
    3. S. Fagarasan,
    4. S. Yamada,
    5. Y. Shinkai,
    6. T. Honjo
    . 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102: 553–563.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Montecino-Rodriguez E.,
    2. K. Dorshkind
    . 2006. New perspectives in B-1 B cell development and function. Trends Immunol. 27: 428–433.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Martin F.,
    2. J. F. Kearney
    . 2002. Marginal-zone B cells. Nat. Rev. Immunol. 2: 323–335.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Coutelier J. P.,
    2. J. T. van der Logt,
    3. F. W. Heessen,
    4. G. Warnier,
    5. J. Van Snick
    . 1987. IgG2a restriction of murine antibodies elicited by viral infections. J. Exp. Med. 165: 64–69.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Coutelier J. P.,
    2. J. T. van der Logt,
    3. F. W. Heessen,
    4. A. Vink,
    5. J. van Snick
    . 1988. Virally induced modulation of murine IgG antibody subclasses. J. Exp. Med. 168: 2373–2378.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Shapiro-Shelef M.,
    2. K. Calame
    . 2005. Regulation of plasma-cell development. Nat. Rev. Immunol. 5: 230–242.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Martins G.,
    2. K. Calame
    . 2008. Regulation and functions of Blimp-1 in T and B lymphocytes. Annu. Rev. Immunol. 26: 133–169.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Allman D.,
    2. S. Pillai
    . 2008. Peripheral B cell subsets. Curr. Opin. Immunol. 20: 149–157.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Rajewsky K.
    1996. Clonal selection and learning in the antibody system. Nature 381: 751–758.
    OpenUrlCrossRefPubMed
  36. ↵
    1. MacLennan E. C. M.,
    2. D. Gray,
    3. D. S. Kumararatne,
    4. H. Bazin
    . 1982. The lymphocytes of splenic marginal zones: a distinct B-cell lineage. Immunol. Today 3: 305–397.
    OpenUrlCrossRef
  37. ↵
    1. Ferguson A. R.,
    2. M. E. Youd,
    3. R. B. Corley
    . 2004. Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells. Int. Immunol. 16: 1411–1422.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Cinamon G.,
    2. M. A. Zachariah,
    3. O. M. Lam,
    4. F. W. J. Foss Jr..,
    5. J. G. Cyster
    . 2008. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat. Immunol. 9: 54–62.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Pillai S.,
    2. A. Cariappa,
    3. S. T. Moran
    . 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161–196.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Hayakawa K.,
    2. R. R. Hardy,
    3. L. A. Herzenberg,
    4. L. A. Herzenberg
    . 1985. Progenitors for Ly-1 B cells are distinct from progenitors for other B cells. J. Exp. Med. 161: 1554–1568.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Lalor P. A.,
    2. A. M. Stall,
    3. S. Adams,
    4. L. A. Herzenberg
    . 1989. Permanent alteration of the murine Ly-1 B repertoire due to selective depletion of Ly-1 B cells in neonatal animals. Eur. J. Immunol. 19: 501–506.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Berland R.,
    2. H. H. Wortis
    . 2002. Origins and functions of B-1 cells with notes on the role of CD5. Annu. Rev. Immunol. 20: 253–300.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Kantor A. B.,
    2. L. A. Herzenberg
    . 1993. Origin of murine B cell lineages. Annu. Rev. Immunol. 11: 501–538.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Baumgarth N.,
    2. J. W. Tung,
    3. L. A. Herzenberg
    . 2005. Inherent specificities in natural antibodies: a key to immune defense against pathogen invasion. Springer Semin. Immunopathol. 26: 347–362.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Hardy R. R.,
    2. K. Hayakawa
    . 1991. A developmental switch in B lymphopoiesis. Proc. Natl. Acad. Sci. USA 88: 11550–11554.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Montecino-Rodriguez E.,
    2. H. Leathers,
    3. K. Dorshkind
    . 2006. Identification of a B-1 B cell-specified progenitor. Nat. Immunol. 7: 293–301.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Haas K. M.,
    2. J. C. Poe,
    3. D. A. Steeber,
    4. R. F. Tedder
    . 2005. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. Immunity 23: 7–18.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Hoshino K.,
    2. O. Takeuchi,
    3. T. Kawai,
    4. H. Sanjo,
    5. T. Ogawa,
    6. Y. Takeda,
    7. K. Takeda,
    8. S. Akira
    . 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162: 3749–3752.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Manz R. A.,
    2. A. E. Hauser,
    3. F. Hiepe,
    4. A. Radbruch
    . 2005. Maintenance of serum antibody levels. Annu. Rev. Immunol. 23: 367–386.
    OpenUrlCrossRefPubMed
  50. ↵
    1. Chaudhuri J.,
    2. U. Basu,
    3. A. Zarrin,
    4. C. Yan,
    5. S. Franco,
    6. T. Perlot,
    7. B. Vuong,
    8. J. Wang,
    9. R. T. Phan,
    10. A. Datta,
    11. et al
    . 2007. Evolution of the immunoglobulin heavy chain class switch recombination mechanism. Adv. Immunol. 94: 157–214.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Manis J. P.,
    2. M. Tian,
    3. F. W. Alt
    . 2002. Mechanism and control of class-switch recombination. Trends Immunol. 23: 31–39.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Peng S. L.,
    2. S. J. Szabo,
    3. L. H. Glimcher
    . 2002. T-bet regulates IgG class switching and pathogenic autoantibody production. Proc. Natl. Acad. Sci. USA 99: 5545–5550.
    OpenUrlAbstract/FREE Full Text
  53. ↵
    1. Gerth A. J.,
    2. L. Lin,
    3. S. L. Peng
    . 2003. T-bet regulates T-independent IgG2a class switching. Int. Immunol. 15: 937–944.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    1. Sellars M.,
    2. B. Reina-San-Martin,
    3. P. Kastner,
    4. S. Chan
    . 2009. Ikaros controls isotype selection during immunoglobulin class switch recombination. J. Exp. Med. 206: 1073–1087.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    1. Stavnezer J.,
    2. J. E. J. Guikema,
    3. C. E. Schrader
    . 2008. Mechanism and regulation of class switch recombination. Annu. Rev. Immunol. 26: 261–292.
    OpenUrlCrossRefPubMed
  56. ↵
    1. Snapper C. M.,
    2. P. Zelazowski,
    3. F. R. Rosas,
    4. M. R. Kehry,
    5. M. Tian,
    6. D. Baltimore,
    7. W. C. Sha
    . 1996. B cells from p50/NF-κB knockout mice have selective defects in proliferation, differentiation, germ-line CH transcription, and Ig class switching. J. Immunol. 156: 183–191.
    OpenUrlAbstract
    1. Zelazowski P.,
    2. D. Carrasco,
    3. F. R. Rosas,
    4. M. A. Moorman,
    5. R. Bravo,
    6. C. M. Snapper
    . 1997. B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching. J. Immunol. 159: 3133–3139.
    OpenUrlAbstract
    1. Wuerffel R. A.,
    2. L. Ma,
    3. A. L. Kenter
    . 2001. NF-κB p50-dependent in vivo footprints at Ig Sγ3 DNA are correlated with μ→γ3 switch recombination. J. Immunol. 166: 4552–4559.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    1. Kenter A. L.,
    2. R. Wuerffel,
    3. C. Dominguez,
    4. A. Shanmugam,
    5. H. Zhang
    . 2004. Mapping of a functional recombination motif that defines isotype specificity for μ→γ3 switch recombination implicates NF-κB p50 as the isotype-specific switching factor. J. Exp. Med. 199: 617–627.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    1. Wang L.,
    2. R. Wuerffel,
    3. A. L. Kenter
    . 2006. NF-κB binds to the immunoglobulin Sγ3 region in vivo during class switch recombination. Eur. J. Immunol. 36: 3315–3323.
    OpenUrlCrossRefPubMed
  61. ↵
    1. Zarrin A. A.,
    2. P. H. Goff,
    3. K. Senger,
    4. F. W. Alt
    . 2008. Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching. J. Exp. Med. 205: 1567–1572.
    OpenUrlAbstract/FREE Full Text
  62. ↵
    1. Martin F.,
    2. A. M. Oliver,
    3. J. F. Kearney
    . 2001. Marginal zone and B1 B cells unite in the early response against T-independent blood-borne particulate antigens. Immunity 14: 617–629.
    OpenUrlCrossRefPubMed
  63. ↵
    1. Allen C. D. C.,
    2. T. Okada,
    3. J. G. Cyster
    . 2007. Germinal-center organization and cellular dynamics. Immunity 27: 190–202.
    OpenUrlCrossRefPubMed
  64. ↵
    1. MacLennan I. C. M.
    1994. Germinal centers. Annu. Rev. Immunol. 12: 117–139.
    OpenUrlCrossRefPubMed
  65. ↵
    1. Matsumoto M.,
    2. S. F. Lo,
    3. C. J. Carruthers,
    4. J. Min,
    5. S. Mariathasan,
    6. G. Huang,
    7. D. R. Plas,
    8. S. M. Martin,
    9. R. S. Geha,
    10. M. H. Nahm,
    11. D. D. Chaplin
    . 1996. Affinity maturation without germinal centres in lymphotoxin-α-deficient mice. Nature 382: 462–466.
    OpenUrlCrossRefPubMed
  66. ↵
    1. Franzoso G.,
    2. L. Carlson,
    3. T. Scharton-Kersten,
    4. E. W. Shores,
    5. S. Epstein,
    6. A. Grinberg,
    7. T. Tran,
    8. E. Shacter,
    9. A. Leonardi,
    10. M. Anver,
    11. et al
    . 1997. Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions. Immunity 6: 479–490.
    OpenUrlCrossRefPubMed
  67. ↵
    1. Iwasaki A.,
    2. R. Medzhitov
    . 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987–995.
    OpenUrlCrossRefPubMed
  68. ↵
    1. Cariappa A.,
    2. H.-C. Liou,
    3. B. H. Horwitz,
    4. S. Pillai
    . 2000. Nuclear factor κB is required for the development of marginal zone B lymphocytes. J. Exp. Med. 192: 1175–1182.
    OpenUrlAbstract/FREE Full Text
  69. ↵
    1. Ferguson A. R.,
    2. R. B. Corley
    . 2005. Accumulation of marginal zone B cells and accelerated loss of follicular dendritic cells in NF-κB p50-deficient mice. BMC Immunol. 6: 8.
    OpenUrlCrossRefPubMed
  70. ↵
    1. Weih D. S.,
    2. Z. B. Yilmaz,
    3. F. Weih
    . 2001. Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines. J. Immunol. 167: 1909–1919.
    OpenUrlAbstract/FREE Full Text
  71. ↵
    1. Radbruch A.,
    2. G. Muehlinghaus,
    3. E. O. Luger,
    4. A. Inamine,
    5. K. G. C. Smith,
    6. T. Dörner,
    7. F. Hiepe
    . 2006. Competence and competition: the challenge of becoming a long-lived plasma cell. Nat. Rev. Immunol. 6: 741–750.
    OpenUrlCrossRefPubMed
  72. ↵
    1. Lam K.-P.,
    2. K. Rajewsky
    . 1999. B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development. J. Exp. Med. 190: 471–477.
    OpenUrlAbstract/FREE Full Text
  73. ↵
    1. Casola S.,
    2. K. L. Otipoby,
    3. M. Alimzhanov,
    4. S. Humme,
    5. N. Uyttersprot,
    6. J. L. Kutok,
    7. M. C. Carroll,
    8. K. Rajewsky
    . 2004. B cell receptor signal strength determines B cell fate. Nat. Immunol. 5: 317–327.
    OpenUrlCrossRefPubMed
  74. ↵
    1. Watanabe N.,
    2. S. Nisitani,
    3. K. Ikuta,
    4. M. Suzuki,
    5. T. Chiba,
    6. T. Honjo
    . 1999. Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment. J. Exp. Med. 190: 461–469.
    OpenUrlAbstract/FREE Full Text
  75. ↵
    1. Clayton L. K.,
    2. Y. Ghendler,
    3. E. Mizoguchi,
    4. R. J. Patch,
    5. T. D. Ocain,
    6. K. Orth,
    7. A. K. Bhan,
    8. V. M. Dixit,
    9. E. L. Reinherz
    . 1997. T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection. EMBO J. 16: 2282–2293.
    OpenUrlAbstract
  76. ↵
    1. Horwitz B. H.,
    2. P. Zelazowski,
    3. Y. Shen,
    4. K. M. Wolcott,
    5. M. L. Scott,
    6. D. Baltimore,
    7. C. M. Snapper
    . 1999. The p65 subunit of NF-κB is redundant with p50 during B cell proliferative responses, and is required for germline CH transcription and class switching to IgG3. J. Immunol. 162: 1941–1946.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section