NF-κB has been implicated in the development, activation, and function of B and T lymphocytes. We have evaluated the in vivo effects of deletion of IκB-α, a major inhibitor of NF-κB, on lymphocyte development, proliferation, and function. To elucidate the long term role of IκB-α in lymphocytes, fetal liver cells of 14.5-day-old IκB-α−/− or wild-type embryos were transplanted into irradiated recombinase-activating gene-2-deficient mice. Within 4 wk, the IκB-α−/− fetal liver cells reconstitute mature B and T cell populations in the recipients comparable to those produced by wild-type fetal liver cells. However, the proliferative responses of IκB-α−/− B cells are enhanced, whereas those of IκB-α−/− T cells are reduced. The levels of IgG1, IgG2a, IgA, and IgE produced by IκB-α−/− B cells are elevated relative to those produced by IκB-α+/+ or IκB-α+/−. Moreover, the specific immune responses to OVA and the generation of germinal centers are impaired in recipients of IκB-α−/− fetal liver cells. These results indicate that IκB-α plays a vital role in signal transduction pathways regulating lymphocyte proliferation and also in the production of specific Ig isotypes.

The ubiquitous transcription factor family, NF-κB, plays a critical role in a wide variety of cellular functions, including proliferation, differentiation, and programmed cell death. NF-κB complexes are segregated in the cytoplasm by association with an inhibitor IκB. The current model for the activation of NF-κB is that second messenger cascades activate the IκB kinases (IKKs) that phosphorylate IκB-α on specific serines. Hyperphosphorylation of IκB-α leads to inactivation and destruction of IκB by proteasome-mediated proteolysis, which results in the release of NF-κB, translocation into nucleus, and its binding to specific DNA elements (1, 2, 3, 4). The NF-κB family contains five mammalian members (RelA (p65), c-Rel, RelB, p52, and p50) and three Drosophila homologues (dorsal, dif, and relish) (5, 6, 7). These proteins complex together as a variety of homodimers or heterodimers (8) that can coexist simultaneously in a given cell type. The activity of such dimers must be regulated coordinately or independently. IκB, an evolutionarily conserved multigene family, is comprised of nine members, including IκB-α, IκB-β, Bcl-3, p100, p105, IκB-γ, IκB-ε, and two Drosophila homologues (cactus and relish). These proteins share three to seven repeats of a conserved motif known as an ankyrin repeat, which is required for association with the NF-κB complex and inhibition of the DNA binding activity of the NF-κB complex (9). The different IκB forms may differentially regulate distinct NF-κB dimers. IκB-α associates with and blocks the activity of two major transcription activators, c-Rel and RelA. Hence, IκB-α is one of the primary inhibitors of inducible NF-κB binding activity.

Gene disruption has been used to generate mice lacking individual NF-κB members. As predicted, deficiency of Rel/NF-κB members results in defects of various immune functions (reviewed in Ref. 10). However, the variety of heterodimeric combinations that occur within the NF-κB family makes it difficult to interpret the function of NF-κB in the immune system by studying the deletion of a single NF-κB member. In this case, more information might be gained by altering the genes that control NF-κB activity, namely IκBs. It was proposed that gene disruption of IκB-α would result in constitutive NF-κB activity. Indeed, NF-κB DNA binding activity is elevated in several tissues from IκB-α-deficient mice. They display granulopoiesis and severe skin disease and typically die within 9 days after birth, although they develop normally until birth (11, 12). However, lymphoid cells and immune responses in these mice were not characterized.

The neonatal lethality exhibited by these animals precludes the study of immune system development and function in adult mice. Therefore, we have used fetal liver cell adoptive transfer to extend our characterization to include effects of IκB-α deficiency on the development and function of lymphocytes. IκB-α deficiency results in alterations in the B and T cell populations consistent with more differentiated and activated states. Elevated NF-κB activity was observed in several tissues, including hemopoietic cells of IκB-α-deficient mice, and the expression of several cytokines was altered. Proliferation of IκB-α−/− lymphocytes is affected, and alterations in specific isotype Igs are observed. Our results demonstrate for the first time that IκB-α−/−-deficient lymphocytes display defects in proliferation and hypermaturation and a profound alteration in their ability to mount an immune response.

A mouse IκB-α gene, mad3, fragment was cloned from a mouse 129 genomic library into the gt11 phage. An Acc65I fragment from phage clone 4 was subcloned into pBluescript (Stratagene, La Jolla, CA). Plasmid containing both 5′ and 3′ regions οf IκB-α cDNA was used to create the targeting construct. NarI was used to remove a 300-bp region containing 5′ untranslated region and part of exon I region and was replaced with a PGK-Neo cassette to construct the targeting vector MAD3 PGK-Neo. This single selection construct, PGK-Neo, was verified by Southern blot using external and internal probes. To create the double selection construct, an XbaI fragment containing PGK-Neo was subcloned into a PGK-tk plasmid to create a PGK-tk Neo IκB-α construct.

Linearized targeting vector (50 μg), was introduced into TL-1 ES cells (a gift from Trish Labosky, University of Pennsylvania, Philadelphia, PA). Cells were cultured in the presence of G418 for 7 days, and resistant colonies were selected and cultured as described by Hogan et al. (13). Genomic DNA isolated from individual colonies was digested with HindIII and SmaI, screened by Southern blot analysis using a 500-bp KpnI/HindIII fragment as an external probe (probe a) and Neo cassette as internal probe (probe b). Targeted clones were used for microinjection into C57BL/6 blastocysts. Chimeric males with >80% agouti coloring were backcrossed to C57BL/6 mice, and IκB-α+/− mice were verified by Southern blot analysis. IκB-α−/− mice were subsequently generated from crosses of IκB-α+/− siblings. To generate IκB-α−/− bearing a luciferase reporter of NF-κB activity, IκB-α+/− animals were mated with HLL transgenics, then IκB-α+/− HLL+ siblings were mated. HLL transgenic mice contain a reporter gene encoding luciferase under the control of HIV long terminal repeat region, which includes NF-κB DNA binding elements. Animals carrying HLL were identified by PCR or Southern blot analyses as well as luciferase activity from the brain tissue (F. E. Yull et al., manuscript in preparation). Mice were kept in a pathogen-free environment, provided with autoclaved food, water, and cage.

Single-cell populations from thymus, spleen, lymph node, peripheral blood, and bone marrow were isolated and surface stained as previously described (14, 15), with directly fluoresceinated mAbs (PharMingen, San Diego, CA). Stained cells were subjected to analysis by FACS flow cytometer (FACS caliber at Howard Hughes Medical Institute; flow cytometry at Vanderbilt University). The mAbs used were anti-B220 (PharMingen 01128A), anti-IgD (02214D), anti-CD4 (01065A), anti-CD8 (01048A), anti-CD3 (01084D), anti-CD44 (01224D), anti-CD69 (01575A), anti-heat-stable Ag (anti-HSA; 01575A), and anti-IgM (1021-09; Southern Biotechnology Associates, Birmingham, AL). FACS analysis was performed using the CellQuest program (Becton Dickinson, Mountain View, CA).

Splenocytes from recipients of fetal liver cells were first depleted of erythrocytes by hypotonic shock, followed by incubation at 37°C in complete medium for 1 h to allow macrophages to attach to the bottom of the plate. After macrophage depletion, the suspended cells were gently transferred to 15-ml tubes and counted. Cells (3 × 107) were incubated with 1.5 ml of anti-Thy1.2, anti-CD4 (G.K 1.5) and anti-CD8 (T1B105) hybridoma culture supernatant on ice for 40 min followed by washing twice with fresh cold medium. Cells were then transferred onto plates coated with mouse anti-rat IgG (115-006-075, Jackson ImmunoResearch, West Grove, PA) to deplete the T cell population at 4°C for 1 h. Spleen cells (3 × 107) were incubated in anti-IgM (BioSource, Camarillo, CA)-bound plates at 4°C for 1 h to deplete B cells. After panning out B and T cells, the purity of T and B cell populations was verified by FACS analysis using anti-CD3, anti-B220, and anti-Mac-1 (PharMingen). Normally, 80–85% purity of B or T cells was obtained.

All primary cells were cultured in complete medium containing RPMI 1640 supplemented with 10% heat-inactivated FCS, 50 μM 2-ME, and 2 mM l-glutamine. Spleen cells were cultured at an initial concentration of 1 × 106 lymphocytes/ml. Purified B and T cells were cultured at concentrations of 5 × 105 cells/ml. For proliferation assays, the purified B or T lymphocytes were dispensed into 96-well microtiter plates at 200 μl/well. B lymphocytes were stimulated with anti-IgM (Jackson ImmunoResearch), anti-CD40 (PharMingen, HM 40-3), or LPS (Sigma, St. Louis, MO) at the indicated concentrations. T lymphocytes were stimulated with Con A (Pharmacia, Piscataway, NJ), anti-CD3 (culture medium from 1452C11 hybridoma), or OVA in the presence of irradiated splenic cells from C57BL/6 at the indicated concentration. After 48 h of stimulation, cells were pulsed with 0.75 mCi of [3H]thymidine (Amersham, Arlington Heights, IL)/well for the next 16 h. Cells were harvested with a cell harvester (Tomtec orange), and uptake of radioactivity was measured with a betaplate recorder (Wallac, Gaithersburg, MD).

Samples representing preimmune sera for analysis of basal levels of Ig isotypes were collected from 6-, 7-, and 8-wk-old recipients of fetal liver cells from IκB-α−/− embryos and control littermates. For analysis of Ig levels, prechallenge serum was collected at 7 wk after fetal liver cell transfer. The mice were then immunized with 100 μg/ml OVA emulsified in CFA (Becton Dickinson) via i.p. injection. Sera were obtained at 7-day intervals after immunization for a period of 3 wk. The immunized mice were boosted with OVA emulsified together with IFA at wk 3. Mice were bled 1 wk after secondary challenge, and sera were collected for Ig isotype analyses. Lymph nodes and part of the spleen were taken for immunohistochemical analyses. T cells were isolated from part of the spleen and used for the proliferative response of OVA stimulation. The level of specific Ig in each sample was determined by ELISA using goat anti-Ig (H+L) polyclonal Ab as the capture agent and goat anti-mouse isotype-specific polyclonal Ab conjugated with HRP (both from Southern Biotechnology Associates, Birmingham, AL) as detecting agents. The levels of Ag-specific Ig isotypes in immune sera and serum IgE were determined by ELISA as described by Singh et al. (16). The concentrations of Ig isotypes from immunized or immune sera were determined using purified myeloma proteins as standards (Sigma).

Spleens were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 6 μm. Sections were stained with hematoxylin and eosin or used for immunohistochemistry. For immunohistochemistry, rehydrated serial sections were treated for Ag retrieval and quenching of endogenous peroxidase activity as previously described (17). The slides were blocked at room temperature with unchallenged mouse serum for 1 h and then labeled with peroxidase-conjugated peanut agglutinin (PNA; EY Laboratories, San Mateo, CA) or anti-B220 for 1 h at room temperature. Peroxidase-conjugated anti-rat Abs were used as secondary Ab for B220 staining. FDC-M1 (anti-FDC, a gift from Dr. M. H. Kosco-Vibois) Ab staining was performed as described by Schubart et al. (18). After washing, diaminobenzidene was added to form precipitate. Peanut agglutinin-positive germinal centers were counted, and pictures were taken at ×100 magnification (FDC-M1 40X).

The IκB-α locus was disrupted by homologous recombination in ES cells using two alternative targeting vectors, PGK-Neo-IκB-α and PGK-tk-Neo-IκB-α, each replacing the promoter and first exon of the IκB-α gene with a PGK-Neo cassette. This strategy is designed to disrupt IκB-α transcription and translation. Consistent with other groups’ findings (11, 12), IκB-α−/− mice were comparable in size and behavior to wild-type or heterozygous mice at birth, and their growth and development were equal to those of their wild-type and heterozygous littermates for the first 3 days. At this point, IκB-α-deficient pups stop gaining weight and by 6 days, their weight is approximately one third that of wild-type pups. The IκB-α −/− pups develop a dry, red, flaky skin; suffer from severe runting; and typically die within 9 days after birth. Six-day-old IκB-α−/− pups have formed internal organs. The spleens of IκB-α null mice appear smaller than those of wild-type or heterozygous littermates. Thymic atrophy is also observed in IκB-α−/− mice, whereas spleen and liver appear anemic. The cause of the neonatal lethality in IκB-α−/− mice remains unknown. In agreement with other groups’ findings, a significant increase in specific protein:DNA complexes was observed in nuclear extracts prepared from the thymocytes and splenocytes of IκB-α−/− compared with those from IκB-α+/+ and IκB-α+/− animals (data not shown). The transcriptional levels of several cytokines, including TNF-α, IL-6, macrophage inflammatory protein-1α, and GM-CSF, were elevated in the IκB-α-deficient skin, whereas the transcriptional levels of IL-2 and IFN-γ remained comparable to those in wild-type animals (data not shown). To examine the effect of IκB-α deficiency on NF-κB transcriptional activity in vivo, heterozygous mice were mated with transgenic mice in which the expression of a luciferase reporter gene is under the control of the HIV-long terminal repeat region, which contains NF-κB-responsive elements. This line of luciferase reporter animals is termed HLL and is a useful reporter of NF-κB activity as a measure of luciferase activity. The phenotype of IκB-α−/− HLL+/− mice is identical with that of IκB-α−/− mice. Luciferase activity, indicative of NF-κB transcriptional activity, was elevated in the organs tested (Fig. 1). These observations confirm that lack of IκB-α results in elevated NF-κB activity. This unregulated NF-κB activity may contribute to the neonatal lethality caused by IκB-α−/− deficiency (12).

FIGURE 1.

Disruption of expression of IκB-α leads to constitutive NF-κB activity in multiple tissues. Lack of IκB-α results in the increase in κB transcriptional activity in several tissues. Cells from different organs were collected from 5.5-day-old IκB-α−/−HLL+/− pups and their littermates. Tissues were homogenized and used for luciferase assay. Results are expressed as relative light units (R.L.U.) per protein amount (micrograms). Shown are the mean values of four pups.

FIGURE 1.

Disruption of expression of IκB-α leads to constitutive NF-κB activity in multiple tissues. Lack of IκB-α results in the increase in κB transcriptional activity in several tissues. Cells from different organs were collected from 5.5-day-old IκB-α−/−HLL+/− pups and their littermates. Tissues were homogenized and used for luciferase assay. Results are expressed as relative light units (R.L.U.) per protein amount (micrograms). Shown are the mean values of four pups.

Close modal

To determine whether constitutive NF-κB activation perturbs T and B cell development, several stage-specific or function-specific cell surface markers were selected to examine T and B cell development in IκB-α−/− mice. Consistent with the observed thymic atrophy in IκB-α−/− mice, the total cell number of thymocytes was dramatically decreased in IκB-α deficient mice.

An increased apoptotic rate could result in a reduced total cell number of thymocytes. Therefore, the percentage of apoptotic cells was measured in stimulated wild-type and null T and B cell populations. T cells (2 × 106) were stimulated with either bound anti-CD3 (10 μg/ml) or Con A (2.5 μg/ml). After 48 h, TUNEL assays were performed, and the percentage of apoptotic cells in CD4 and CD8 populations was determined by FACS analysis. No significant differences between null and wild-type cells were detected (data not shown).

Staining of thymic cells with anti-CD4 or anti-CD8 revealed an alteration in the T cell population of IκB-α−/− mice (Fig. 2 A). The population of single-positive (CD4+ or CD8+) cells was increased in IκB-α−/− mice, whereas the population of double-positive (CD4+/CD8+) cells was decreased, relative to those in littermate controls.

FIGURE 2.

Constitutive NF-κB activation perturbs T and B cell development in IκB-α−/− mice. FACS analysis of thymocytes from IκB-α−/− pups was performed. A reduction of total thymocyte numbers and an increase in single-positive thymocyte population in IκB-α−/− pups were found. Thymocytes were collected from 5-day-old pups and stained with fluorescence-conjugated Abs. The stained cells were subsequently subjected to flow cytometric analysis (gated on 15,000 live cells). Shown is one representative experiment of three independent experiments. B, CD8+ thymocytes of IκB-α−/− are skewed toward more mature and activated stages. To determine the differentiation status of thymocytes, cells were stained with fluorescence-conjugated CD4 and CD8 in the presence of CD3, HAS, or CD69 Abs. Shown are histogram analysis for surface expression of CD3, HSA, and CD69 gated on CD8 single-positive cells. Numbers indicate the percentage of the gated area. C, FACS analysis of B cells from spleen of IκB-α−/− pups. To define the B cell population, splenic cells stained with fluorescence-labeled B220 and IgM Abs were subjected to FACS analysis (gated on 12,000 live lymphocytes). D, The level of expression of IgM gated on B220-positive cells. Numbers are the percentage of gated cells. Shown are representative results reproduced by at least two mice from each IκB-α−/− line and its littermates. T, Total cell number from thymus or spleen.

FIGURE 2.

Constitutive NF-κB activation perturbs T and B cell development in IκB-α−/− mice. FACS analysis of thymocytes from IκB-α−/− pups was performed. A reduction of total thymocyte numbers and an increase in single-positive thymocyte population in IκB-α−/− pups were found. Thymocytes were collected from 5-day-old pups and stained with fluorescence-conjugated Abs. The stained cells were subsequently subjected to flow cytometric analysis (gated on 15,000 live cells). Shown is one representative experiment of three independent experiments. B, CD8+ thymocytes of IκB-α−/− are skewed toward more mature and activated stages. To determine the differentiation status of thymocytes, cells were stained with fluorescence-conjugated CD4 and CD8 in the presence of CD3, HAS, or CD69 Abs. Shown are histogram analysis for surface expression of CD3, HSA, and CD69 gated on CD8 single-positive cells. Numbers indicate the percentage of the gated area. C, FACS analysis of B cells from spleen of IκB-α−/− pups. To define the B cell population, splenic cells stained with fluorescence-labeled B220 and IgM Abs were subjected to FACS analysis (gated on 12,000 live lymphocytes). D, The level of expression of IgM gated on B220-positive cells. Numbers are the percentage of gated cells. Shown are representative results reproduced by at least two mice from each IκB-α−/− line and its littermates. T, Total cell number from thymus or spleen.

Close modal

To investigate the differentiation stage of IκB-α null T cells, samples were stained with CD3 and HSA. During the maturation of single-positive cells the TCR level increased, while the level of expression of HSA normally decreased. By comparison to control littermates, FACS analysis showed that CD8 single-positive T cells from IκB-α-deficient thymus expressed higher levels of CD3 but lower levels of HSA than T cells from control littermates (Fig. 2 B, gated on CD8+). However, CD4 single-positive T cells expressed levels of CD3 and HSA in IκB-α−/− pups comparable to those observed in control littermates (data not shown). These results indicate that constitutive NF-κB activation accelerates T cell development toward a single-positive state and, in particular, toward a more mature CD8 single-positive state.

The functional status of IκB-α−/− T cells was determined by staining with anti-CD69 Abs, a surface marker indicating transient activation of T cells. Interestingly, FACS analysis revealed that 59% of IκB-α−/− CD8+ T cells express a high level of CD69 compared with 6% in wild-type T cells. The level of CD69 expression was comparable in CD4 single-positive cells. These results indicate that although there are fewer T cells present, constitutive NF-κB activity produces a higher proportion of mature, activated CD8+ cells (Fig. 2 B, gated on CD8).

To determine whether constitutive NF-κB activity perturbs the differentiation of B cells, spleen cells were isolated from 5-day-old pups and stained with fluorescence-conjugated B220 and IgM Abs. A larger population of B cells expressed high levels of IgM in IκB-α−/− spleen compared with the wild-type spleen (Fig. 2, C and D). However, fewer total splenic B cells were harvested from IκB-α−/− mice, consistent with the reduction in the size of the spleen of IκB-α−/− mice compared with wild-type mice. To investigate whether the reduction in total B cell numbers was due to increased apoptosis, B cells (2 × 106) were stimulated with either anti-IGM (2 μg/ml) or LPS (10 μg/ml). After 48 h TUNEL assays were performed, and the percentage of apoptotic cells in B220 populations was determined by FACS analysis. No significant differences between null and wild-type cells were detected (data not shown).

The results from the FACS analysis suggest that although fewer B cells are present in IκB-α−/− mice, these B cells are more differentiated than their wild-type counterparts. Together, our results lead us to propose that hyperactivation of NF-κB is sufficient for accelerating differentiation of T cells and activating T cells (as assessed by certain cell surface markers). B cell maturation is affected by the removal of IκB-α, which suggests that NF-κB may be involved in distinct mechanisms of differentiation in T and B cells.

Although the lack of both p50 and p65 resulted in the blockade of B and T cell development, normal lymphocyte development occurred when p50/65-deficient fetal liver cells were adoptively transferred together with wild-type bone marrow cells (19). This observation suggests that some effects of NF-κB activity on B and T cell development are extrinsic. To extend our investigation of the development of lymphocytes from IκB-α−/− pups into adult mice and to examine whether the effects are intrinsic or extrinsic, adoptive transfers of fetal liver cells were performed. The same number (5 × 106) of fetal liver cells isolated from 14.5-day-old embryos from a heterozygous mating were transferred into lethally irradiated 12-wk-old recombinase-activating gene-2-deficient mice. Reconstitution of B and T cells by IκB-α−/− fetal liver cells was confirmed by FACS analysis, using selected markers 5 wk after transfer. No severe phenotype was observed in the recipients, and the thymus and spleen appeared to develop normally upon gross morphologic and histologic examination. Mice sacrificed at 6, 7, and 8 wk were used to investigate the development of B and T cells collected from bone marrow, thymus, spleen, and peripheral blood. Total numbers of reconstituted B and T cells in the spleen were comparable between recipients of wild-type and IκB-α−/− cells (Fig. 3,A). Single-positive T cell populations in thymus, spleen, or peripheral blood were normal in IκB-α−/− recipients (Fig. 3B). Populations of mature B cells were normal in bone marrow, spleen, and peripheral blood (Fig. 3 C). Several surface markers, including HSA, CD3, CD69, and CD44, indicative of differentiation and activation status were examined, and no abnormalities were observed (data not shown). To investigate the population of granulocytes, bone marrow cells and peripheral cells were stained with Mac-1. The results reveal slightly elevated granulocytosis (data not shown). Our results suggest that the observed defects in lymphocyte development in IκB-α−/− newborns are not cell intrinsic. We hypothesize that factors extrinsic to the IκB-α−/− lymphocytes and myeloid cells (i.e., cytokines, contacting accessory cells, etc.) contribute to the more pronounced phenotypes in the homozygous animals.

FIGURE 3.

Normal B and T cell development in IκB-α−/− fetal liver cell recipients. A, Total T or B cell numbers in spleen from recipients. Equal amounts of fetal liver cells (5 × 106) from 14.5-day-old embryos were injected into lethally irradiated 12-wk-old recombinase-activating gene-2-deficient mice. Recipients were sacrificed at 6, 7, and 8 wk after transfer. B and C, Cells from the indicated organs were isolated, stained with fluorescence-conjugated Abs, and subjected to FACS analysis. The total numbers of thymocytes from IκB-α−/− and wild-type animals are 7 × 107 and 9 × 107, respectively. FACS analysis was performed by gating on 15,000 live lymphocytes. Shown are representatives of five mice from two independent transfers.

FIGURE 3.

Normal B and T cell development in IκB-α−/− fetal liver cell recipients. A, Total T or B cell numbers in spleen from recipients. Equal amounts of fetal liver cells (5 × 106) from 14.5-day-old embryos were injected into lethally irradiated 12-wk-old recombinase-activating gene-2-deficient mice. Recipients were sacrificed at 6, 7, and 8 wk after transfer. B and C, Cells from the indicated organs were isolated, stained with fluorescence-conjugated Abs, and subjected to FACS analysis. The total numbers of thymocytes from IκB-α−/− and wild-type animals are 7 × 107 and 9 × 107, respectively. FACS analysis was performed by gating on 15,000 live lymphocytes. Shown are representatives of five mice from two independent transfers.

Close modal

To investigate the role of IκB-α in the regulation of lymphocytes, we performed ex vivo proliferation assays using spleen cells from recipients of IκB-α−/− or wild-type fetal liver cells. Splenic B and T cells were purified, then equal numbers of enriched B or T cells were treated with specific activators to stimulate proliferation (14, 20, 21). The loss of IκB-α alters the proliferative response of B and T cells compared with that of wild-type cells. Interestingly, IκB-α deficiency affects the proliferative response of B cells differently than it affects T cells. In response to the T cell stimuli (anti-CD3 or Con A), IκB-α−/− T cells exhibit a 50% decreased proliferative response compared with controls (Fig. 4 A). Medium from these proliferation assays was used for ELISA to investigate potential differences in levels of cytokines critical for T cell proliferation. Levels of IL-2, IFN-γ, and IL-4 were measured, and no significant differences were detected between medium collected from wild-type or null cells when stimulated with anti-CD3 or Con A (data not shown).

FIGURE 4.

Proliferative responses of IκB-α−/− B and T cells are perturbed. A, Proliferation of IκB-α−/− T cells is impaired in response to T cell-specific stimuli compared with control littermates. T cells isolated from recipients of fetal liver cells of embryos derived from IκB-α+/− mating were purified as described in Materials andMethods and then stimulated with anti-CD3 or Con A in the presence of irradiated spleen cells at the indicated dilutions or concentrations. The anti-CD3 used here is provided by conditioned medium from hybridoma (1615-2C11). B, Proliferation of IκB-α−/− splenic B cells in response to B cell stimuli. Purified B cells (105) were stimulated with anti-IgM, anti-CD40, or LPS at the indicated concentrations. The error bars are generated from five independent experiments. Each experiment was performed in triplicate.

FIGURE 4.

Proliferative responses of IκB-α−/− B and T cells are perturbed. A, Proliferation of IκB-α−/− T cells is impaired in response to T cell-specific stimuli compared with control littermates. T cells isolated from recipients of fetal liver cells of embryos derived from IκB-α+/− mating were purified as described in Materials andMethods and then stimulated with anti-CD3 or Con A in the presence of irradiated spleen cells at the indicated dilutions or concentrations. The anti-CD3 used here is provided by conditioned medium from hybridoma (1615-2C11). B, Proliferation of IκB-α−/− splenic B cells in response to B cell stimuli. Purified B cells (105) were stimulated with anti-IgM, anti-CD40, or LPS at the indicated concentrations. The error bars are generated from five independent experiments. Each experiment was performed in triplicate.

Close modal

In sharp contrast to results from T cells, in response to B cell stimuli (anti-CD40, anti-IgM, LPS), IκB-α−/− B cells displayed a 2- to 4-fold increase in proliferation (Fig. 4 B). T and B cell numbers were comparable between recipients of IκB-α−/− and wild-type fetal liver cells.

We interpret these disparate results between the cell types as being due to intrinsic differences in the ways in which T and B cells use NF-κB to establish proliferation and activation.

To examine further the role of IκB-α in B cell function, the levels of serum Ig isotypes in recipients of fetal liver cells were mea- sured. The results indicate that IκB-α−/− B cells were capable of Ig secretion and class switching of Ig isotypes. The basal serum levels of IgM, IgG2b, IgG3 (Fig. 5,A), and Igκ (data not shown) in the recipients of IκB-α−/− fetal liver cells were comparable to those in wild-type cells. However, there was a 10-fold increase in the levels of IgG2a, IgA, and IgE and a 3-fold increase in IgG1 in the unchallenged mice (Fig. 5 A). Levels of Igλ were also elevated (data not shown). These data suggest that NF-κB is involved in Ig production and in switching of Ig isotypes, activities that are critical for B cell function.

FIGURE 5.

Basal and specific Ab production. A, Basal Ig isotype levels. Each symbol represents the results obtained from one animal by ELISA. B, Immune response to the T cell-dependent Ag OVA. Recipients of IκB-α−/− or control animal fetal liver cells were immunized with OVA and boosted 3 wk after immunization. Sera were collected at 1-wk intervals for a 3-wk period and 1 wk after secondary challenge, and analyzed for OVA-specific IgM, IgG1, IgG2a, and IgG2b levels. Three animals were immunized for each group. Shown are the mean of three animals from one representative of two independent experiments. C, The proliferative response of challenged null T cells to OVA is comparable to that of control cells. T cells were purified from OVA-challenged recipients and stimulated with the indicated concentration of OVA. Shown are the mean of three animals from one representative of two independent experiments. D, Immune response to the T cell-independent Ag DNP-Ficoll. Recipients of IκB-α−/− or control animal fetal liver cells were immunized with DNP-Ficoll. Sera were collected at 1-wk intervals for a 3-wk period.

FIGURE 5.

Basal and specific Ab production. A, Basal Ig isotype levels. Each symbol represents the results obtained from one animal by ELISA. B, Immune response to the T cell-dependent Ag OVA. Recipients of IκB-α−/− or control animal fetal liver cells were immunized with OVA and boosted 3 wk after immunization. Sera were collected at 1-wk intervals for a 3-wk period and 1 wk after secondary challenge, and analyzed for OVA-specific IgM, IgG1, IgG2a, and IgG2b levels. Three animals were immunized for each group. Shown are the mean of three animals from one representative of two independent experiments. C, The proliferative response of challenged null T cells to OVA is comparable to that of control cells. T cells were purified from OVA-challenged recipients and stimulated with the indicated concentration of OVA. Shown are the mean of three animals from one representative of two independent experiments. D, Immune response to the T cell-independent Ag DNP-Ficoll. Recipients of IκB-α−/− or control animal fetal liver cells were immunized with DNP-Ficoll. Sera were collected at 1-wk intervals for a 3-wk period.

Close modal

To test the effects of IκB-α−/− deficiency on Ag-specific immune responses, we challenged recipients of IκB-α−/− or wild-type fetal liver cells with the T cell-dependent Ag, OVA (emulsified in CFA), or the T cell-independent Ag, DNP-Ficoll. The Ags were administered 7 wk after fetal liver cell transfers, then readministered 3 wk later for OVA. Sera collected at 1-wk intervals were used for measuring the specific Ig isotypes by ELISA (Fig. 5, B and C). The OVA-specific anti-IgM levels produced by IκB-α−/− B cells are comparable to those produced by IκB-α+/− or IκB-α+/+ B cells. This result indicates that OVA-specific B cells are present in the IκB-α−/− fetal liver cell recipients. However, the levels of IgG1, IgG2a, and IgG2b specific for OVA in IκB-α−/− recipients were significantly reduced even after the secondary challenge, whereas the levels of these Abs produced by IκB-α+/− or IκB-α+/+ B cells were significantly elevated (Fig. 5,B). To explore the function of T cells in response to challenge, T cells were purified from OVA-challenged mice and stimulated with OVA ex vivo. The proliferative response of IκB-α−/− T cells was comparable to that of IκB-α+/+ T cells (Fig. 5,C). This result suggests that the response of IκB-α−/− T cells to OVA challenge is normal. Therefore, the observed alteration in B cell production of IgG1, IgG2a, and IgG2b in response to T cell-dependent OVA is not due to a defect in T cell priming. To test the B cell function, recipients were challenged with a T cell-independent Ag, DNP-Ficoll. The production of IgG3, specific for DNP, is lower by IκB-α−/− B cells than by IκB-α+/+ or IκB-α+/− B cells (Fig. 5 D). This result indicates that the impaired production of Ig may partly be due to defects in IκB-α−/− B cell function. Our results suggest that the deficiency of IκB-α has a more profound impact on B cell differentiation than on T cell differentiation.

Germinal centers are prominent histological structures found within the secondary lymphoid organs that contain activated B cells undergoing proliferation, differentiation, and programmed cell death (22). Activated germinal center B cells undergoing maturation, hypermutation, and class switching express binding sites for PNA. Since Ag-specific Ab responses are lower in IκB-α−/− fetal liver cell recipients, we investigated the formation of germinal centers in the spleens of recipients of IκB-α−/− fetal liver cells by histological analysis and by histochemical staining for PNA. Although FACS analysis shows that the population of splenic B and T cells remains comparable between the recipients of IκB-α−/− and wild-type fetal liver cells after challenge (Fig. 6,A), results from PNA staining show lack of germinal center formation in IκB-α−/− recipients (Fig. 6,B). The follicular dendritic cell (FDC) clusters, which are normally associated with germinal centers, were comparable between mutant and wild-type control animals (Fig. 6 B, FDC-M1). This suggests that although mature IκB-α−/− B cells are present, IκB-α deficiency interferes with germinal center formation. These results are qualitatively similar to findings for other related knockout animals in which the lack of Bcl-3 or CD40 has been demonstrated to result in the disruption of germinal centers (15, 23). Our results indicate that IκB-α deficiency results in the impairment of B cell differentiation to PNA-positive germinal center B cells.

FIGURE 6.

Impaired formation of germinal centers in the spleen of IκB-α−/− fetal liver cell recipients. The recipients of IκB-α−/− or IκB-α+/+ fetal liver cells were challenged by i.p. injection of 100 μg of OVA at wk 7 post-transfer. A, FACS analysis of splenic cells from challenged mice indicates that the populations of T and B cells are comparable between IκB-α−/− and IκB-α+/+ mice. B, Sections of spleen were stained with hematoxylin-eosin or peroxidase-conjugated PNA, B220, or FDC-M1. The black arrow indicates the white pulp. The white arrows indicate the presence or absence of germinal centers.

FIGURE 6.

Impaired formation of germinal centers in the spleen of IκB-α−/− fetal liver cell recipients. The recipients of IκB-α−/− or IκB-α+/+ fetal liver cells were challenged by i.p. injection of 100 μg of OVA at wk 7 post-transfer. A, FACS analysis of splenic cells from challenged mice indicates that the populations of T and B cells are comparable between IκB-α−/− and IκB-α+/+ mice. B, Sections of spleen were stained with hematoxylin-eosin or peroxidase-conjugated PNA, B220, or FDC-M1. The black arrow indicates the white pulp. The white arrows indicate the presence or absence of germinal centers.

Close modal

Lack of individual members of the NF-κB family has varied effects on regulation of the immune response (reviewed in Ref. 10). A deficiency of an individual family member has minor effects on the development of lymphocytes, which may be due to a significant level of functional redundancy within the family. To overcome this, transgenic mice expressing a transdominant negative inhibitor of NF-κB, IκB-αΔN, in T or B cell lineage were generated (5, 24, 25) (F. E. Yull et al., manuscript in preparation). These studies showed the important role of NF-κB in B and T cell development. Our results suggest that lack of IκB-α has an extrinsic rather than an intrinsic effect on development. Our data show that the population of single-positive cells in the thymus of IκB-α−/− pups is elevated relative to that in control littermates. However, this elevation may be due to the reduced total cell number in IκB-α−/− thymus specifically in CD4+CD8+ double-positive cells. In addition, the surface markers on the CD8 single-positive cells as well as on B cells indicate that the majority of these cells are at an advanced stage of maturation (Fig. 3). We cannot rule out the possibility that the increased level of CD69 on IκB-α−/− T cells represents committed double-positive T cells; however, the changes in measured levels of CD3 and HSA suggest that this effect is due to the transient activation of T cells in IκB-α−/− pups. While the findings from our IκB-α−/− pups also reveal perturbation of lymphocyte populations, the IκB-α−/− fetal liver transfers successfully reconstituted normal lymphocyte populations. Considering the data from fetal liver recipients, we hypothesize that the effects of IκB-α deficiency on lymphocyte development are due to extracellular factors, such as cytokines or cell:cell communications, which are absent in IκB-α-deficient animals. Other studies in which there is failure of lymphopoiesis after adoptive transfer of p50/p65 double-deficient fetal liver into irradiated SCID mice also support this hypothesis (19). These p50/p65 double-deficient fetal liver cells can develop into normal lymphocytes when cotransferred with wild-type bone marrow cells. This suggests that NF-κB mediates the development or survival of early lymphocyte precursors through regulation of extracellular factors.

Lymphocytes lacking either of the two major inducible NF-κB transcription activators of the NF-κB family, c-Rel and RelA, have B and T cell proliferative defects in response to certain stimuli (20, 21). From these previous data the assumption would be that IκB-α−/−-deficient lymphocytes would have a hyperproliferative response to B and T cell mitogens. Our data show that IκB-α−/− B cells indeed have hyperproliferative responses to the B cell-specific stimuli. Intriguingly, T cells exhibit impaired proliferative responses. These observations are consistent with studies of p105-deficient lymphocytes (26). Data accumulating from studies using the transdominant inhibitor, mutant IκB-α, have shown that blocking NF-κB can enhance the number of activated T or B cells undergoing apoptosis in response to stimulation (27). However, we have not detected any difference in the number of activated IκB-α−/− T cells or B cells undergoing apoptosis (data not shown). This finding is consistent with the studies on RelA-deficient lymphocytes, which indicate that the actual numbers of RelA−/− apoptotic cells are not significantly different from those of RelA+/− cells(21). Although we did not detect an effect of IκB-α−/− deficiency on apoptosis, we cannot rule out the possibility that NF-κB activity can prevent apoptosis in activated T and B cells, and that this was not detected due to functional redundancy within the NF-κB/IκB family. An alternative explanation for the proliferative response of IκB-α−/− lymphocytes is the involvement of NF-κB in cell cycle control, a hypothesis that is supported by a growing body of evidence (28). Thus, we speculate that disruption of the IκB-α gene may lead to persistent nuclear NF-κB activity, which results in unregulated entry into the cell cycle in a tissue-dependent manner.

Our data show that the basal serum levels of IgG1, IgG2a, IgA, and IgE produced by IκB-α−/− B cells were significantly elevated, whereas the levels of IgM and κ light chain were comparable to those produced by wild-type lymphocytes. These results are consistent with the measured serum Ig levels in c-Rel-, p50-, and RelA-deficient mice. The production of IgG1 and IgG2a in c-Rel−/− mice; IgG1, IgG2a, and IgE in p50−/− mice; and IgG1 and IgA in the irradiated recipient of p65−/− fetal liver cells is impaired. In addition, we have observed that serum levels of λ light chain produced by IκB-α−/− B cells is elevated (data not shown). This finding is consistent with studies showing that blocking NF-κB activity leads to reduced expression of λ light chain (29).

It is surprising that the levels of Ag-specific Ig produced by IκB-α−/− B cells are significantly reduced. A lack of PNA-positive germinal centers correlates with the poor production of specific Abs in the IκB-α−/− recipients. We hypothesize a lack of hypersomatic mutation and specific Ig clonal expansion as the reason why Ag-specific Igs are significantly reduced. It is unlikely that this result is due to the priming defects of T cells, since the proliferative response of IκB-α−/− T cells from OVA-challenged mice is comparable to that of wild-type T cells (Fig. 6 D). However, defects in the interactions between B cells and T cells cannot be ruled out a contributing as factor, since Ig production by B cells is affected in response to OVA, a T cell-dependent Ag. The observation of impaired production of Ig by IκB-α−/− B cells is very similar to the findings in Bcl-3-deficient mice. The proto-oncogene bcl-3 is another member of the IκB family that may regulate different genes or different physiological processes than IκB-α (30, 31). In contrast with usual inhibitory functions of the other known members of the IκB family, it has been postulated that Bcl-3 facilitates trans-activation (32, 33, 34). However, the available data suggest that it probably retains some biological functions similar to those of the other family members in the proliferation or differentiation of B cells (35).

In addition to results in Bcl-3−/− mice, the role of NF-κB/IκB in the formation of germinal centers was also shown in p52-deficient mice (36), p52/p50 double-deficient mice (37), and transgenic mice expressing IκB-αΔN in B cell lineages (25). It is interesting that NF-κB activity seems to be capable of opposing roles (agonist or antagonist) in the generation of germinal centers. The molecular mechanism of generation of germinal centers remains largely unknown. Impaired germinal center formation results in poor production of specific Igs. Thus, it is reasonable to speculate that differentiation of B cells and formation of germinal centers may share a common etiology. Studies on the signal transduction pathways have revealed that several molecules, including TNF-α, lymphotoxin-α, CD40, and CD40 ligand, are important for the generation of germinal centers (38, 39). Not surprisingly, the NF-κB/IκB family is involved in the signal transduction pathways of these molecules (27). The role of CD40 signaling in B cell terminal differentiation remains controversial. Animals deficient in either CD40 or its ligands are unable to form germinal centers or make class-switched Abs after immunization with T cell-dependent Ags (23, 40). Several in vitro experiments indicate that CD40 signaling promotes B cell differentiation (41, 42, 43). However, other experiments have indirectly suggested that cross-linking of CD40 may actively inhibit B cells from differentiating into Ab-secreting cells (23, 44, 45). Taking CD40 as an example, we speculate that NF-κB activity may be necessary for B cell differentiation to a certain stage, but after this stage unregulated NF-κB activity may prevent the terminal differentiation of B cells. Although our histochemical analysis of spleen from IκB-α−/− fetal liver recipients did not show a defect in the formation of the network of FDCs, our current data do not rule out the possibility that NF-κB/IκB activity may play an important role in the function of FDCs, and loss of IκB-α−/− may alter FDCs in such a manner as to interfere with germinal center formation. Our current model provides a good system in which to explore the effects of unregulated NF-κB activity on the formation of germinal centers and Ag-specific B cell differentiation.

We thank Annapurna Venkatarishnam from the laboratory of Dr. Timothy Blackwell at Vanderbilt University for providing labeled NF-κB probes for EMSA. We also thank Trish Labosky and Bridgid Hogan for providing ES cells, and David Martin for technical advice. We are grateful to Dr. M. H. Kosco-Vibois for generously providing anti-FDC-M1 Ab, and to Drs. Singh and Rodey for the training in the technique of fetal liver cell transfer. Special thanks to members of the Kerr laboratory and to Drs. Earl Ruley, Luc Van Kaer, Terry Dermody, Barney Graham, Roland Stein, Gene Oltz, and Mark Boothby at Vanderbilt University for helpful comments on the manuscript. Finally, we acknowledge the Vanderbilt Transgenic Mouse/ES Cell Shared Resource for generating the animals used in this study.

1

This work was supported by National Institutes of Health Grant R01GM51249 and a Center grant from the National Cancer Institute (CA68485). The Vanderbilt Transgenic Mouse/ES Cell Shared Resource is supported by National Cancer Institute Grant P30CA68485 and the National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health Grant 5P60DK20593.

3

Abbreviations used in this paper: ES, embryonic stem; PNA, peanut agglutinin; HSA, heat-stable Ag; FDC, follicular dendritic cell.

1
Chen, Z., J. Hagler, V. J. Palombella, F. Melandri, D. Scherer, D. Ballard, T. Maniatis.
1995
. Signal-induced site-specific phosphorylation targets IκBα to the ubiquitin-proteasome pathway.
Genes Dev.
9
:
1586
2
Rodriguez, M. S., I. Michalopoulos, F. Arenzana-Seisdedos, R. T. Hay.
1995
. Inducible degradation of IκBα in vitro and in vivo requires the acidic C-terminal domain of the protein.
Mol. Cell. Biol.
15
:
2413
3
Scherer, D. C., J. A. Brockman, Z. Chen, T. Maniatis, D. W. Ballard.
1995
. Signal-induced degradation of IκBα requires site-specific ubiquitination.
Proc. Natl. Acad. Sci. USA
92
:
11259
4
DiDonato, J. A., F. Mercurio, M. Karin.
1995
. Phosphorylation of IκBα precedes but is not sufficient for its dissociation from NF-κB.
Mol. Cell. Biol.
15
:
1302
5
Steward, R..
1987
. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel.
Science
238
:
692
6
Ip, Y. T., M. Reach, Y. Engstrom, L. Kadalayil, H. Cai, S. Gonzalez-Crespo, K. Tatei, M. Levine.
1993
. Dif, a dorsal-related gene that mediates an immune response in Drosophila.
Cell
75
:
753
7
Dushay, M. S., B. Asling, D. Hultmark.
1996
. Origins of immunity: relish, a compound Rel-like gene in the antibacterial defense of Drosophila.
Proc. Natl. Acad. Sci. USA
93
:
10343
8
Ghosh, S., M. J. May, E. B. Kopp.
1998
. NF-κB and Rel proteins: evolutionarily conserved mediators of immune responses.
Annu. Rev. Immunol.
16
:
225
9
Verma, I. M., J. K. Stevenson, E. M. Schwarz, D. Van Antwerp, S. Miyamoto.
1995
. Rel/NF-κB/IκB family: intimate tales of association and dissociation.
Genes Dev.
9
:
2723
10
Sha, S., S. W. C..
1998
. Regulation of immune responses by NF-κB/Rel transcription factor: [published erratum appears in J. Exp. Med. 1998 Feb 16;187(4):661].
J. Exp. Med.
187
:
143
11
Klement, J. F., N. R. Rice, B. D. Car, S. J. Abbondanzo, G. D. Powers, P. H. Bhatt, C. H. Chen, C. A. Rosen, C. L. Stewart.
1996
. IκBα deficiency results in a sustained NF-κB response and severe widespread dermatitis in mice.
Mol. Cell. Biol.
16
:
2341
12
Beg, A. A., W. C. Sha, R. T. Bronson, D. Baltimore.
1995
. Constitutive NF-κB activation, enhanced granulopoiesis, and neonatal lethality in IκBα-deficient mice.
Genes Dev.
9
:
2736
13
Hogan, B, F. Costantini, E. Lacey.
1986
.
Manipulating the Mouse Embryo: A Laboratory Manual
Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
14
Sha, W. C., H. C. Liou, E. I. Tuomanen, D. Baltimore.
1995
. Targeted disruption of the p50 subunit of NF-κB leads to multifocal defects in immune responses.
Cell
80
:
321
15
Schwarz, E. M., P. Krimpenfort, A. Berns, I. M. Verma.
1997
. Immunological defects in mice with a targeted disruption in Bcl-3.
Genes Dev.
11
:
187
16
Singh, N., S. Hong, D. C. Scherer, I. Serizawa, N. Burdin, M. Kronenberg, Y. Koezuka, L. Van Kaer.
1999
. Cutting edge: activation of NK T cells by CD1d and α-galactosylceramide directs conventional T cells to the acquisition of a Th2 phenotype.
J. Immunol.
163
:
2373
17
Coico, R. F., B. S. Bhogal, G. J. Thorbecke.
1983
. Relationship of germinal centers in lymphoid tissue to immunologic memory. VI. Transfer of B cell memory with lymph node cells fractionated according to their receptors for peanut agglutinin.
J. Immunol.
131
:
2254
18
Schubart, D. B., A. Rolink, M. H. Kosco-Vilbois, F. Botteri, P. Matthias.
1996
. B-cell-specific coactivator OBF-1/OCA-B/Bob1 required for immune response and germinal centre formation.
Nature
383
:
538
19
Horwitz, B. H., M. L. Scott, S. R. Cherry, R. T. Bronson, D. Baltimore.
1997
. Failure of lymphopoiesis after adoptive transfer of NF-κB-deficient fetal liver cells.
Immunity
6
:
765
20
Kontgen, F., R. J. Grumont, A. Strasser, D. Metcalf, R. Li, D. Tarlinton, S. Gerondakis.
1995
. Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression.
Genes Dev.
9
:
1965
21
Doi, T. S., T. Takahashi, O. Taguchi, T. Azuma, Y. Obata.
1997
. NF-κB RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses.
J. Exp. Med.
185
:
953
22
Tarlinton, D..
1997
. Germinal centers: a second childhood for lymphocytes.
Curr. Biol.
7
:
R155
23
Kawabe, T., T. Naka, K. Yoshida, T. Tanaka, H. Fujiwara, S. Suematsu, N. Yoshida, T. Kishimoto, H. Kikutani.
1994
. The immune responses in CD40-deficient mice: impaired immunoglobulin class switching and germinal center formation.
Immunity
1
:
167
24
Boothby, M. R., A. L. Mora, D. C. Scherer, J. A. Brockman, D. W. Ballard.
1997
. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-κB.
J. Exp. Med.
185
:
1897
25
Bendall, H. H., M. L. Sikes, D. W. Ballard, E. M. Oltz.
1999
. An intact NF-κB signaling pathway is required for maintenance of mature B cell subsets.
Mol. Immunol.
36
:
187
26
Ishikawa, H., E. Claudio, D. Dambach, C. Raventos-Suarez, C. Ryan, R. Bravo.
1998
. Chronic inflammation and susceptibility to bacterial infections in mice lacking the polypeptide (p)105 precursor (NF-κB1) but expressing p50.
J. Exp. Med.
187
:
985
27
Van Antwerp, D. J., S. J. Martin, I. M. Verma, D. R. Green.
1998
. Inhibition of TNF-induced apoptosis by NF-κB.
Trends Cell Biol.
8
:
107
28
Grumont, R. J., I. J. Rourke, L. A. O’Reilly, A. Strasser, K. Miyake, W. Sha, S. Gerondakis.
1998
. B lymphocytes differentially use the Rel and nuclear factor κB1 (NF-κB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells.
J. Exp. Med.
187
:
663
29
Scherer, D. C., J. A. Brockman, H. H. Bendall, G. M. Zhang, D. W. Ballard, E. M. Oltz.
1996
. Corepression of RelA and c-rel inhibits immunoglobulin κ gene transcription and rearrangement in precursor B lymphocytes.
Immunity
5
:
563
30
Wulczyn, F. G., M. Naumann, C. Scheidereit.
1992
. Candidate proto-oncogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-κB.
Nature
358
:
597
31
Kerr, L. D., C. S. Duckett, P. Wamsley, Q. Zhang, P. Chiao, G. Nabel, T. W. McKeithan, P. A. Baeuerle, I. M. Verma.
1992
. The proto-oncogene bcl-3 encodes an IκB protein.
Genes Dev.
6
:
2352
32
Franzoso, G., V. Bours, S. Park, M. Tomita-Yamaguchi, K. Kelly, U. Siebenlist.
1992
. The candidate oncoprotein Bcl-3 is an antagonist of p50/NF-κB-mediated inhibition.
Nature
359
:
339
33
Bours, V., G. Franzoso, V. Azarenko, S. Park, T. Kanno, K. Brown, U. Siebenlist.
1993
. The oncoprotein Bcl-3 directly transactivates through κB motifs via association with DNA-binding p50B homodimers.
Cell
72
:
729
34
Fujita, T., G. P. Nolan, H. C. Liou, M. L. Scott, D. Baltimore.
1993
. The candidate proto-oncogene bcl-3 encodes a transcriptional coactivator that activates through NF-κB p50 homodimers.
Genes Dev.
7
:
1354
35
Ong, S. T., M. L. Hackbarth, L. C. Degenstein, D. A. Baunoch, J. Anastasi, T. W. McKeithan.
1998
. Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice.
Oncogene
16
:
2333
36
Caamano, J. H., C. A. Rizzo, S. K. Durham, D. S. Barton, C. Raventos-Suarez, C. M. Snapper, R. Bravo.
1998
. Nuclear factor (NF)-κB2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.
J. Exp. Med.
187
:
185
37
Franzoso, G., L. Carlson, L. Xing, L. Poljak, E. W. Shores, K. D. Brown, A. Leonardi, T. Tran, B. F. Boyce, U. Siebenlist.
1997
. Requirement for NF-[κ]B in osteoclast and B-cell development.
Genes Dev.
11
:
3482
38
Pasparakis, M., L. Alexopoulou, V. Episkopou, G. Kollias.
1996
. Immune and inflammatory responses in TNF-α-deficient mice: a critical requirement for TNF α in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response.
J. Exp. Med.
184
:
1397
39
Gonzalez, M., F. Mackay, J. L. Browning, M. H. Kosco-Vilbois, R. J. Noelle.
1998
. The sequential role of lymphotoxin and B cells in the development of splenic follicles.
J. Exp. Med.
187
:
997
40
Castigli, E., F. W. Alt, L. Davidson, A. Bottaro, E. Mizoguchi, A. K. Bhan, R. S. Geha.
1994
. CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.
Proc. Natl. Acad. Sci. USA
91
:
12135
41
Spriggs, M. K., R. J. Armitage, L. Strockbine, K. N. Clifford, B. M. Macduff, T. A. Sato, C. R. Maliszewski, W. C. Fanslow.
1992
. Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.
J. Exp. Med.
176
:
1543
42
Grabstein, K. H., C. R. Maliszewski, K. Shanebeck, T. A. Sato, M. K. Spriggs, W. C. Fanslow, R. J. Armitage.
1993
. The regulation of T cell-dependent antibody formation in vitro by CD40 ligand and IL-2.
J. Immunol.
150
:
3141
43
Maliszewski, C. R., K. Grabstein, W. C. Fanslow, R. Armitage, M. K. Spriggs, T. A. Sato.
1993
. Recombinant CD40 ligand stimulation of murine B cell growth and differentiation: cooperative effects of cytokines.
Eur. J. Immunol.
23
:
1044
44
Silvy, A., C. Lagresle, C. Bella, T. Defrance.
1996
. The differentiation of human memory B cells into specific antibody-secreting cells is CD40 independent.
Eur. J. Immunol.
26
:
517
45
Randall, T. D., A. W. Heath, L. Santos-Argumedo, M. C. Howard, I. L. Weissman, F. E. Lund.
1998
. Arrest of B lymphocyte terminal differentiation by CD40 signaling: mechanism for lack of antibody-secreting cells in germinal centers.
Immunity
8
:
733