BCR signal through
4 is involved in S6 kinase activation and required for B cell maturation including isotype switching and V region somatic hypermutation
Seiji Inui,
Kazuhiko Maeda,
Ding Rong Hua,
Takeshi Yamashita,
Hideyuki Yamamoto1,
Eishichi Miyamoto1,
Shinichi Aizawa2 and
Nobuo Sakaguchi
Departments of Immunology and
1 Pharmacology I, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
2 Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan
Correspondence to:
N. Sakaguchi; E-mail: nobusaka{at}kaiju.medic.kumamoto-u.ac.jp
 |
Abstract
|
---|
4 potentially mediates BCR signals through a rapamycin-sensitive TOR pathway. To investigate a potential role for
4 in B cell activation, the
4 gene was disrupted conditionally in B cells by mating male CD19-Cre mice with female
4-floxed mice. CD19-Cre+/
4flox mice showed loss of
4 protein in B lineage cells and a decreased number of phenotypically normal mature B cells. Compared to normal B cells,
4- B cells showed a decreased proliferation in response to the B cell stimulants (anti-IgM antibody plus IL-4, anti-CD40 mAb and lipopolysaccharide), and a reduced S6 kinase activation and rapamycin sensitivity. While CD19-Cre+/
4flox mice showed impaired antibody responses to both T cell-independent and T cell-dependent (TD) antigens, the TD antigen response was markedly impaired as demonstrated by reduced isotype switching, reduced germinal center formation and reduced V region somatic hypermutation. These results show that
4 plays a pivotal role in antigen-specific signal transduction during B cell activation and differentiation in vivo.
Keywords: class switch, gene knockout mouse, germinal center, S6 kinase, V region somatic hypermutation
 |
Introduction
|
---|
Antigen binding to a specific BCR triggers activation of B cells for further proliferation and differentiation with secondary co-stimulatory signals provided in the environment of peripheral lymphoid organs (13). The direct TB interaction or soluble cytokines secreted by Th2 cells that are activated during the immune response provide the secondary co-stimulatory signals (4,5). The first signal results when antigens interact with the BCR. Immediately following this interaction the cytoplasmic ITAM motifs of the BCR-associated molecules Ig
and Igß undergo tyrosine phosphorylation. This is one of the earliest biochemical events in the BCR-induced signal transduction cascade (69). The ITAM tyrosine phosphorylation of Ig
and Igß is catalyzed by Src-type tyrosine kinase Lyn and non-Src-type tyrosine kinase Syk. This phosphorylation induces the recruitment or subsequent phosphorylation of various downstream second messengers and adaptor molecules. This initial tyrosine phosphorylation reaction in the BCR complex nearby cell surface molecules leads to phospholipase C
activation, Ras/Sos activation resulting in activation of the MAP kinase pathway, phosphatidylinositol-3 kinase (PI-3K) activation, Ca2+ mobilization response, protein kinase C (PKC) activation, serine/threonine kinase activation and presumably the activation of other unknown pathways (1014). These signal transduction pathways are involved in cell proliferation, differentiation and maturation by activation of cell cycle machinery, regulation of DNA replication, induction of various nuclear transcription factors and promotion of protein synthesis of various key molecules involved in the initial cell activation (3,15,16).
Despite the enormous effort put into studies of BCR-mediated signal transduction, it remains unclear precisely how the molecules required for the initial cell activation process are induced in B cells after stimulation with antigens. The initial activation molecules are presumably induced by the stimulation of protein synthesis mechanisms such as those mediated by S6 kinase (S6K) and other associated components (17). In previous work, we identified a phosphoprotein component p52 that co-immunoprecipitates with Ig
. Phosphorylation of p52 is induced by PKC activation with phorbol myristate acetate (18). We determined the structure of p52 by the cloning of cDNA and named the gene
4 (19). It has been suggested that
4 plays a role in signal transduction through the BCR (18,19).
An
4 homologue, Tap42 from Saccharomyces cerevisiae, was found to be associated with the catalytic subunit of protein phosphatase 2A (PP2Ac) and Sit4 in a rapamycin-sensitive manner (20). Tap42, phosphorylated by TOR (21), is essential for survival and is involved in cell proliferation induced by nutrients (20). Rapamycin inhibits the growth of yeast cells by suppressing kinase activities of TOR1 and TOR2. Several studies including ours have shown that
4 is associated with PP2Ac in mammalian cells (22,23). We further showed that the binding of
4 enhances PP2A activity, and rapamycin inhibits the growth of rapamycin-sensitive cells with reduced PP2A activity by dissociating
4 and PP2Ac (22). However, it is still controversial whether signal transduction through
4 is associated with rapamycin sensitivity. It is also controversial whether the binding of
4 with PP2Ac is affected by rapamycin or not (24,25), because the dissociation of
4 and PP2Ac in eukaryotic cells proceeds gradually as compared with cells from lower organisms. This might have raised the ambiguity regarding the role of
4 in mammalian cells.
The rapamycin-sensitive signal transduction pathway involves S6K and 4EBP1 in mammalian cells. The mammalian homologue of TOR, mTOR (also called RAFT1 or FRAP), controls mRNA translation by regulating phosphorylation states of S6K and 4EBP1 (2628). Activation of S6K is involved in mRNA translation required for growth factor-induced proliferation (2931). 4EBP1 inhibits translation of 5' TOP mRNAs by interacting with eIF4E and mTOR-induced phosphorylation causes dissociation of 4EBP1 from eIF4E (32,33). It is necessary to clearly demonstrate a function of
4 in the signal transduction of the BCR signal leading to the initiation of protein synthesis that is required for the further activation processes.
In this study, we employed a conditional gene-targeting strategy to clarify the function of
4 by using
4-floxed mice crossed with CD19-Cre knockin mice to inactivate the
4 gene in a B cell-specific manner. The
4- B cells show impaired growth response with reduced S6K activation, Ig production and isotype switching. The results indicate that
4 plays a role in the antigen-induced signal in B cell activation in vitro and in vivo.
 |
Methods
|
---|
Generation of CD19-Cre+/
4flox mice
A targeting vector was constructed using the mouse
4 gene in which the neomycin resistance (neo) gene flanked by two loxP sites was introduced downstream of exon 2 (Fig. 1
). An additional loxP site was inserted upstream of exon 1. Embryonic stem cells were electroporated with this vector and from >500 colonies growing under G418 selection, a clone was positive in a Southern blot screen for a homologously recombined
4 locus. Chimera mice were generated from this embryonic stem cell clone and germline-transmitted mice were then obtained. To disrupt the
4 gene specifically in B cells, we crossed a female
4-floxed mouse (
4flox/WT) with a male CD19-Cre mouse in which the cre cDNA is inserted into the mouse CD19 gene by the knockin approach (34). All the experiments were done with male mice carrying the CD19-Cre allele and a single X chromosome, which showed either only a floxed allele or a normal allele of
4 gene locus.
Antibodies and reagents
Phycoerythrin (PE)B220, PECD8, biotinCD3, biotinanti-IgD, biotinCD23, biotinCD19, biotinCD43, biotinCD4, biotinSyndecan1 and FITCanti-IgM antibodies were purchased from Becton Dickinson (Mountain View, CA) for FACS analysis. CD40 antibody was prepared in our laboratory (35). Goat anti-mouse IgM F(ab')2 antibody was purchased from ICN Pharmaceuticals (Costa Mesa, CA). Lipopolysaccharide (LPS) was purchased from Sigma (St Louis, MO). IL-4 was a kind gift from Dr Nakanishi (Hyogo Medical College, Hyogo, Japan). Rapamycin was purchased from Wako Chemicals (Osaka, Japan). Anti-S6K antibody was purchased from Upstate Biotechnology (Lake placid, NY). Anti-mouse
4 antibody was prepared as previously (19). Biotinpeanut agglutinin (PNA) was purchased from Vector (Burlingame, CA) and anti-IgM antibody was a kind gift from Dr Miyake (Saga Medical School, Japan). Biotinylated anti-IgG1 and anti-GL7 antibodies were purchased from Becton Dickinson for immunohistochemical analysis.
RT-PCR analysis
Genomic DNA was purified from the tail of each mouse as described previously (36). Primers used to amplify the deleted
4 allele were primer 1 (5'-CCTTGAATCACTAATTGAGA-3') and primer 2 (5'-CCCATACAGCTCCCTCCCAGCTGTTTATTT-3'). Each PCR cycle consisted of denaturation at 94°C, annealing for 2 min at 54°C and extension for 2 min at 72°C. After 30 cycles of PCR, the products were visualized by means of electrophoresis on 1% agarose gels stained with ethidium bromide. Primers 1 and 2 detect a ~780-bp fragment only on the
4-deleted allele, because the distance between primer 1 and 2 is ~4 kbp on the targeted allele.
FACS analysis
Cell surface markers were analyzed with mAb by flow cytometry. Single-cell suspensions of lymphoid tissues were incubated with a saturating amount of each mAb conjugated either to fluorochrome or to biotin. Two-color analyses were performed with fluorochromes conjugated with FITC and PE or with Red670 and PE. Lymphoid cell populations were analyzed after gating by forward and side scatters using a FACScan using CellQuest software (Becton Dickinson). For cell cycle analysis, treated cells were fixed in 70% methanol, incubated overnight at 20°C and stained with 50 µg of propidium iodide (PI) for 30 min at room temperature before analysis.
In vitro assay of cell proliferation and antibody production
Single-cell suspensions of spleen cells were treated with 0.1 M ammonium chloride solution to lyse red blood cells and were incubated at 37°C for 1 h to let macrophages adhere. Non-adherent cells were recovered and incubated with Dynabeadsanti-mouse Thy1.2 antibody (Dynal, Oslo, Norway) to remove T cells by a magnet. More than 90% of the purified cells were positive for the B220 marker. For the proliferation assay, purified B cells were cultured at 1x105/well in 96-well microtiter plates with or without stimulation in the presence of 200 µl of RPMI 1640 culture medium containing 10% heat-inactivated FCS (Biowhittaker, Walkersville, MD), 2 mM L-glutamine and 5x10-5 M 2-mercaptoethanol. After 2 days, cells were pulsed with 0.5 µCi = 18.5 kBq of [3H]thymidine/well (Amersham Pharmacia, Little Chalfont, UK) for the last 16 h. Cells were harvested onto glass fiber filters and the incorporated radioactivity was measured. For in vitro antibody production, purified B cells were cultured at 1x104 cells/well in 96-well plates with 20 µg/ml of LPS in the presence or absence of 10 U/ml of IL-4. After 5 days, ELISA measured the Ig concentration of culture supernatants in the same way as used for serum Ig (see below).
S6K assay and Western blot analysis
Cells were lysed in the lysis buffer containing 1% Nonidet P-40, 150 mM NaCl, 10 mM TrisHCl (pH 7.8), 1 mM EDTA, 0.05% NaN3, 100 mM NaVO4, 1 mM PMSF and 10 µg/ml aprotinin. The lysates were centrifuged for 5 min at 12,000 g at 4°C to remove nuclei and the supernatants were used for immunoprecipitation. For in vitro kinase assay, the lysates of 1x107 cells were incubated with anti-S6K antibody for 2 h at 4°C. Immune complexes were collected with 30 µl of Protein ASepharose beads (Pharmacia Biotech, Uppsala, Sweden), washed 4 times with the lysis buffer, and then resuspended in the assay buffer containing 100 mM HEPES (pH 7.5), 10 mM magnesium acetate, 0.1 mM [
-32P]ATP (30005000 c.p.m./pmol) and 67 µM Kemptide as a substrate. After 10 min of incubation at 30°C, the radioactive incorporation was measured by spotting onto a phosphocellulose filter. Protein contents of the aliquots were measured after separation by SDSPAGE. Separated proteins were transferred onto nitrocellulose filters by electroblotting. PP2Ac and
4 were detected by the anti-PP2Ac and anti-
4 antibodies at a dilution of 1/1000. The blots were developed using an ECL kit (Amersham Pharmacia) according to the manufacturer's protocol.
Immunizations and ELISA
The
4- and control littermate mice were prebled and immunized by i.p. injection with 25 µg of 2,4,6-trinitrophenol (TNP)-conjugated Ficoll (Biosearch Technologies, Novato, CA) in PBS or 50 µg of TNP-keyhole limpet hemocyanin (KLH) (Biosearch Technologies) with complete Freund's adjuvant (Difco, Detroit, MI). At 14 days after immunization, mice were bled, the sera collected, and assayed by TNP-specific and isotype-specific ELISA to determine the levels of the antigen-specific immune response. Briefly, ELISA plates were coated with 5 µg of TNP-BSA (Biosearch Technologies), and blocked with 1% of BSA and 0.05% of Tween 20. Sera were serially diluted in triplicate and detected with horseradish peroxidase-conjugated rabbit anti-mouse isotype-specific antisera (Southern Biotechnology Associates, Birmingham, AL). The significance of the difference was calculated by Student's t-test and P < 0.05 was considered statistically significant.
Immunohistochemistry
Mice were immunized with nitrophenyl-conjugated chicken
-globulin (NP-CGG) (20 µg) with complete Freund's adjuvant as described above. Spleen tissue was frozen in Tissuetek OCT compound (Miles, Elkhart, IN), and tissue sections (6-µm thick) were prepared and fixed in cold acetone for 5 min. After washing with PBS, the presence of PNA which stains germinal center (GC) B cells was revealed by biotinPNA or biotinIgG1 followed by streptavidinhorseradish peroxidase. Anti-IgM antibody (a kind gift from Dr Miyake, Saga, Japan) or anti-GL7 antibody was counterstained with alkaline phosphatase-conjugated goat anti-rat IgG antibody.
Somatic hypermutation analysis
RNA samples were purified from the spleen of mice immunized with NP-CGG and the cDNAs were synthesized by RT-PCR using random primers according to the manufacturer's protocol (PE Biosystems, Warrington, UK). The VH186.2 transcripts of IgM class were amplified by PCR using the primers VH186.2F (5'-TTCTTGGCAGCAACAGCTACA-3') and CmR (5'-GAAGACATTTGGGAAGGACTGACT-3') (37). Amplified products were cloned into pCRII vector (Invitrogen, Carlsbad, CA), and sequence reactions were performed by a dye terminator cycle sequencing premix kit (Amersham Pharmacia) and analyzed on an Applied Biosystems automatic 373A DNA sequencer (Applied Biosystems, Foster City, CA). Thirty clones each from wild-type and CD19-Cre+/
4flox mice were sequenced. With the manufacture's data on Taq polymerase, the error rate is estimated <1/1000 in the process of PCR for the V gene sequencing.
 |
Results
|
---|
Generation of B cell-specific
4 knockout mice
To clarify the functional involvement of
4 in B cell activation, we deleted the
4 gene specifically in B cells by conditional gene targeting using Cre/lox P-mediated recombination (38,39) (Fig. 1A
). Southern blot analysis confirmed the homologous recombination and showed that a male mouse bearing the targeted
4 locus lost the wild-type (WT) locus (Fig. 1B
). This indicates that the mouse
4 gene is on the X chromosome as in human (40). To disrupt the
4 gene specifically in B cells, we crossed a female
4-floxed mouse (
4flox/WT) with a male CD19-Cre mouse in which the cre cDNA was inserted into the mouse CD19 gene by a knockin approach (34). Cre appears exclusively in B lineage cells in the CD19-Cre mouse. In the male CD19-Cre+/
4flox mouse, therefore, Cre-mediated deletion of the
4 gene was expected to occur only in B cells. PCR, using the primers indicated in Fig. 1
(A), showed that Cre-mediated deletion of the
4 gene had occurred specifically in B lineage cells (Fig. 1C
). The DNA bands seen in Fig. 1
(C) are of the size predicted when exons 1 and 2 were deleted. To circumvent the effect of the CD19 heterozygous knockout phenotype, all the following experiments compared male CD19-Cre+/
4flox mice and littermate CD19-Cre+/
4WT mice. The CD19-Cre+/
4flox mice developed normally with normal architecture of organs and tissues of the brain, liver, heart, kidney and the thymus (data not shown). Cell numbers of the bone marrow (BM) and the spleens of CD19-Cre+/
4flox mice were comparable to wild-type littermates. Splenic B cells showed no
4 mRNA (Fig. 1D
) or
4 protein (Fig. 1E
), while non-B cells expressed both of them (data not shown), indicating that
4 is specifically targeted under the regulation of the CD19 promoter.
Maturation of B cells in mutant mice
Flow cytometry using various B lineage differentiation markers characterizes the maturation of B cells in wild-type (littermate) and CD19-Cre+/
4flox mice. Spleen cells of CD19-Cre+/
4flox mice revealed a decrease in the number of B220+ peripheral B cells, but do contain a population with the normal maturation phenotype (Fig. 2A
). This decrease is marked in the IgM+IgDhi mature B cell population. Among the mature B cells, CD23+IgM+ follicular B cells are reduced (from 37.0 to 10.6%) and the number of CD40+ cells is also reduced (from 32.1 to 12.6%) in the spleen of CD19-Cre+/
4flox mice. While the number of CD19+ cells is also reduced, the expression level of CD19 on B cells is not affected in CD19-Cre+/
4flox mice compared to wild-type mice both in the spleen (Fig. 2A
) and BM (Fig. 2B
), indicating no down-regulation of CD19 expression occurred. In the BM, pro-B cells defined by B220-CD43+ are present at a normal number in CD19-Cre+/
4flox mice; however, the number of pre-B cells defined by B220+CD43- was reduced (from 51.6 to 34.4%) (Fig. 2B
). In CD19-Cre+/
4flox mice there was a slight decrease in B220+IgM- pre-B cells, and marked decreases in B220+IgM+ B cells (from 21.7 to 7.7%) and in the mature recirculating IgM+IgD+ B cells (from 12.0 to 2.5%). The total number or the ratio of CD4+ and CD8+ T cell populations in the thymus showed no apparent change (Fig. 2C
). The surface analysis did not detect any obvious abnormality in the maturation of B cells in the BM and the spleen of mutant mice.
Antigen-induced proliferation is mediated through a rapamycin-sensitive and the
4-unique signal transduction pathways of B cells
Because our previous study showed the involvement of
4 in antigen receptor-mediated signaling, we tested the effects of BCR cross-linkage on proliferation of
4- B cells in vitro (19). Purified spleen B cells of CD19-Cre+/
4flox mice showed severe impairment of B cell proliferation induced by anti-IgM antibody plus IL-4 in comparison to wild-type (6000 versus 35,000 c.p.m. of [3H]thymidine incorporation) (Fig. 3A
). Proliferative responses to LPS and anti-CD40 antibody showed an impairment in
4- B cells, indicating that
4 plays an important role in the proliferation of B cells induced by various mitogenic stimuli. Next, we assessed the effect of rapamycin on proliferation of normal or
4- B cells. The in vitro proliferation assay by [3H]thymidine incorporation demonstrated the relative impairment of purified B cells stimulated with anti-IgM antibody in the presence or absence of various concentrations of rapamycin for 48 h (Fig. 3B
). The proliferative response of
4- B cells was comparable to that of wild-type B cells that had been exposed to rapamycin (1 nM), supporting the involvement of
4 in the rapamycin-sensitive signal transduction pathway. As was previously shown (41), rapamycin inhibited the growth of normal B cells induced by anti-IgM antibody, LPS or anti-CD40 antibody in a dose-dependent manner and the sensitivity of
4- B cells to rapamycin showed a reduction in every stimulatory signal (Fig. 3C
). The
4- B cells showed less sensitivity to rapamycin treatment, but were still inhibitable with a high dose of rapamycin. This suggests that, although the major rapamycin-sensitive signaling pathway involves the TOR pathway,
4 also contributes to this pathway. The above results suggested that anti-IgM antibody induces signals for B cell proliferation through
4-associated and non-associated rapamycin-sensitive pathways, and showed that
4 is also important in the proliferation of B cells induced by other stimulatory signals.
Since rapamycin affects cell cycle progression of lymphocytes at the G1/S stage (42), the next approach studied the cell cycle transition after stimulation with anti-IgM antibody plus IL-4 (Fig. 4A
). The
4- B cells stayed mostly at the G1/S stage even after 48 h stimulation (6.0%) as compared with wild-type B cells, many of which are stimulated to enter the cell cycle (29.6%). This shows that
4- B cells have an impaired G1/S transition that is consistent with the impaired proliferative response (measured by [3H]thymidine incorporation). Recent studies suggested the interaction of the TOR-mediated pathway with activation of S6K (31). To examine downstream signals, we examined S6K activity in
4- B cells. A previous study reported that the stimulation with mitogens induced S6K activity in normal B cells as early as 2 h after stimulation (17). Our observation, however, showed that the response is more marked at 48 h after stimulation in vitro. Purified B cells were stimulated with anti-IgM antibody and the cellular S6K activity was measured by in vitro kinase assay after immunoprecipitation of S6K (Fig. 4B
). BCR cross-linkage of the
4- B cells failed to induce S6K activity, in marked contrast to the response in
4+ B cells, although both cell types showed similar levels of basal S6K activity in the absence of stimulation. These results proved the correctness of our previous suggestion that
4 is potentially involved in antigens-induced signal transduction for B cell activation in association with a rapamycin-sensitive pathway (19).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 4. (A) Cell cycle analysis of splenic B cells. Purified B cells were cultured with medium alone or anti-IgM antibody plus IL-4 for 48 h. DNA was stained with 50 µg/ml of PI and the DNA content was measured by FACS analysis. The percentages of S/G2/M cells are indicated. (B) BCR-induced S6K activity was measured. Purified B cells were cultured in the presence of 10 µg/ml of anti-IgM antibody for 2 or 48 h. Cells were then lysed with lysis buffer and immunoprecipitated by anti-S6K antibody. S6K activity was measured using Kemptide as a substrate. The S6K activity of unstimulated B cells of each type was defined as control.
|
|
Humoral immune response of CD19-Cre+/
4flox mice to antigen stimulation
Under non-immunized conditions at 8 weeks after birth, the CD19-Cre+/
4flox mice bear comparable levels of serum IgM but slightly reduced levels of serum IgG compared to age-matched control littermates (Fig. 5A
). Next, we studied the antigen-specific responses of CD19-Cre+/
4flox mice in comparison with control littermate (wild-type) mice. CD19-Cre+/
4flox mice showed reduced IgM, IgG1 and IgG3 responses to T cell-independent (TI)-2 antigens, TNP-Ficoll, at day 14 (Fig. 5B
), but the responses were not severely impaired in IgG2a and IgG2b antibodies. Before the immunization, the titers of anti-TNP antibody were lower than the measurable threshold in both CD19-Cre+/
4flox and wild-type mice (data not shown). Interestingly, immunization with T cell-dependent (TD) antigen, TNP-KLH, induced much lower TNP-specific responses of IgM, IgG1, IgG2a, IgG2b and IgG3 at day 14 than in wild-type mice (Fig. 5C
). These results suggested that
4 is more important for TD differentiation of antigen-driven B cells than in the maturation of TI B cell stimulation. The number of peritoneal CD5+ B cells was decreased in CD19-Cre+/
4flox mice, which accords with the fact that CD5+ B cells are major producers of IgM, IgG1 and IgG3 classes, and are responsible for antibody production against TI-2 antigens in vivo (data not shown).
To investigate the differentiation and maturation of B cells after antigen immunization, we studied spleen cells of immunized mice by flow cytometric analysis (Fig. 5D
). The size of the splenic B cell population was smaller in CD19-Cre+/
4flox mice after immunization. Expression of CD23, a characteristic marker of follicular B cells, was poorly inducible in CD19-Cre+/
4flox mice and the differentiation to Syndecan-1+ cells was severely impaired in CD19-Cre+/
4flox mice (Fig. 5D
), which accords well with the observation of a low antibody response to TD antigens (Fig. 5C
). Next, we investigated whether
4- B cells also showed impairment of differentiation into antibody-secreting cells by stimulation in vitro. The
4- B cells mounted much lower antibody production in vitro by stimulation with LPS or LPS plus IL-4 compared to wild-type B cells (Fig. 5E
). The impairment was marked in IgM, IgG1 and IgG3 responses. These results clearly demonstrated that
4 is involved in the signal transduction in B cells leading to antibody production in vitro.
To clearly demonstrate the impairment of B cell differentiation, we examined the follicular region of the spleen by immunohistochemical methods after immunization with the TD antigen, NP-CGG. CD19-Cre+/
4flox mice showed an impairment of GC formation in contrast to wild-type mice because there was no accumulation of PNAhi+ cells, which are characteristic of GC B cells at day 19 after immunization (Fig. 6
). Slight expression of PNA staining was detected in the follicle-like region of CD19-Cre+/
4flox mice. These areas mostly contained IgM+ but almost no IgG1+ cells in contrast to wild-type mice. Absence of GL7 expression, another differentiation marker for mature GC B cells (43), further supports that mature GC formation is impaired in CD19-Cre+/
4flox mice. These results demonstrated that
4 is quite important for the development of GC with functionally mature B cells undergoing differentiation with IgG isotype switching and expression of the GL7 marker.
Mature GC formation is associated with affinity maturation of antibodies against TD antigens (44,45). We therefore investigated whether
4-mediated signal transduction leads to the generation of somatic hypermutation of the V region. After immunization with NP-CGG in CD19-Cre+/
4flox mice, RT-PCR study of cDNAs showed the nucleotide sequences of the expressed Ig determined at VH186.2, which has been shown to be the germline VH gene utilized in the NP-CGG response in C57BL/6 mice (46,47). RNA samples were obtained from the half of the spleens of wild-type and mutant mice after antigen immunization. Ongoing humoral immune responses were confirmed by ELISA of the serum samples and by an immunohisotochemical study carried out with the rest of the spleen (data not shown). Somatic mutation of the V region was observed at a lower frequency in CD19-Cre+/
4flox mice than in wild-type mice. More meaningfully, higher frequencies of CDR1 and CDR2 mutations were not observed in CD19-Cre+/
4flox mice in comparison to the rest of the V region. Wild-type mice bear frequent somatic hypermutations in the CDR1 and CDR2 regions, including the common hypermutation at amino acid position 33 (CDR1), 57 and 66 (CDR2) (4648) after antigen immunization for 14 days (data not shown) and 28 days (Fig. 7
). The CD19-Cre+/
4flox mice showed much lower mutation rates in CDR1 and CDR2 regions than in the other V region residues. This result indicates that the
4-mediated BCR signal is required for elaboration of somatic hypermutation through activation, proliferation and differentiation of B cells in GC.
 |
Discussion
|
---|
In yeast, rapamycin treatment or inhibition of the TOR pathway results in a severe decrease in translation initiation and an arrest in the early G1 phase of the cell cycle (33,42). The
4 homologue yeast Tap42 is essential for nutrient-induced growth signals through a rapamycin-sensitive TOR pathway via association with the catalytic subunits of type 2A or type 2A-related protein phosphatases (20). Loss of Tap42 causes a defect of cell proliferation as observed in the rapamycin treatment (20). For mammalian lymphocytes, however, rapamycin does not inhibit the cell proliferation completely even at the highest dose of 100 nM (22). Rapamycin only partially inhibited the proliferation of mouse B cells and human lymphoid cell lines such as Jurkat and Ramos (22,41). This might suggest that the TOR pathway is not completely necessary for the growth of mammalian lymphoid cells.
In this study, CD19-Cre+/
4flox mutant mice were used to demonstrate that
4 is involved in the signal transduction of a rapamycin-sensitive pathway as predicted by previous results (22). However, the inhibitory effect was more drastic in the
4- spleen B cells than that observed with rapamycin treatment in vitro. The in vitro proliferation responses of
4- B cells are sensitive to rapamycin treatment, but these cells are also functionally impaired in the differentiation of B cells to antibody-secreting cells upon antigen stimulation.
In mammalian cells, TOR controls the translation initiation stimulated by growth factors through the activation of S6K and the inhibition of the elF4E inhibitor 4E-BP1, leading to protein synthesis of a small family of molecules of primary ribosomal proteins and components of the translation apparatus. The mutant mice clearly demonstrated that mammalian
4 plays a role in the activation of S6K after antigen stimulation of B cells. Here, it becomes evident that antigen simulation involves a signal transduction pathway from BCR to S6K activation via
4/PP2Ac association. The molecular mechanism of S6K regulation via
4 remains to be elucidated in future analysis. How does the activation of S6K through the TOR pathway occur in BCR signaling? One of the main signaling pathways in the other nutrient-stimulating system of insulin/insulin receptor involves TOR-mediated PI-3K activation leading to the activation PDK1, PKB (Akt) and S6K. Recently, it was reported that PP2A associates and controls the S6K activity (49). The association of
4 with PP2Ac potentially results in the regulation of the phosphatase activity, which might suggest a direct regulation of the S6K activity by altering the phosphorylation state of S6K itself or the associated components. Alternatively,
4/PP2Ac might regulate the kinase activity of the upstream signal transduction molecule that activates S6K and the other regulatory components.
The results suggested that
4 is important for the clonal expansion of antigen-stimulated B cells in the immune response. The impairment of B cell proliferation in vitro by treatment with other B cell stimulants indicated that
4-mediated signal transduction is not specific for the BCR signal only, but is also important for various signal transduction pathways in B cells. The
4- mutation results in a severe defect in the proliferation of B cells in vivo. Other gene-targeted mice also showed a similar reduction of proliferation in response to LPS and CD40, especially for SLP-65, which was thought to be a BCR signal-specific adaptor molecule (50).
The
4- B cells exist and are maintained in the spleen, where they produce similar levels of serum Ig of several isotypes in comparison to the wild-type mice under non-immunized conditions. CD19-Cre+/
4flox mice bear comparable levels of serum IgM, IgG2a and IgG2b, but decreased levels of IgG1 and IgG3. In agreement with this observation, peritoneal CD5+ B cells are also reduced in CD19-Cre+/
4flox mice (data not shown). CD5+ B cells are the major producers of IgG1 and IgG3, and are often compromised in knockout mice of BCR signal-transducer molecules. Immunization with TI-2 antigens, e.g. TNP-Ficoll, induced a slightly decreased but reasonable level of antigen-specific antibody responses, notably with the higher levels in IgG2a and IgG2b, and with lower levels in IgM, IgG1 and IgG3, suggesting that the isotype switching nonetheless occurs in the
4- B cells. Gene-targeting experiments of several other BCR signal-transducer molecules often result in reduced responses to TI-2 antigens. Responses to TD antigens showed no reduction in most cases in comparison with wild-type littermates (5156). CD19-Cre+/
4flox mutant mice, however, showed marked abnormalities in response to TD antigens of TNP-KLH, NP-CGG and sheep red blood cells in vivo by both single and boosting immunization until 28 days (some representative results are shown in the figures). In the experiments with all of the above antigens, the humoral immune responses to TD antigen showed impairment, especially in the isotype switch to IgG. This marked difference in the responses against TI-2 antigens and TD antigens suggests that
4 plays a role in B cell differentiation with the co-stimulation provided by the T cell-dependent stimulation.
Various cytokines are responsible for selective induction of Ig isotypes both in vivo and in vitro. IL-4 and IL-5 are associated with isotype switch to IgG1, while IFN-
induces isotype switch to IgG2a (57,58).
4 might directly participate in regulation of signal transduction for isotype switch that is induced by cytokines from Th cells or by the cognate TB cell interaction. The fact that the proliferative response to anti-IgM antibody plus IL-4 was impaired in
4- B cells supports the idea that IL-4 receptor signaling necessary for IgG1 class switching is blocked in CD19-Cre+/
4flox mice. It would be interesting to study whether the phosphatase activity of
4-associated PP2Ac might directly or indirectly influence the IL-4 receptor signaling which is also rapamycin sensitive. STAT3 is regulated by mTOR and the activation of STAT3 is rapamycin sensitive (59), but little is known regarding the regulation of mTOR on the other STAT molecules.
Antigen binding to the BCR of mature B cells initiates activation processes necessary for maturation into antibody-producing cells. Following immunization with a TD antigen, an oligoclonal population of B cells is activated within the border of the T cell area adjacent to B cell follicles and gathers to form GC. GC will serve for the expansion of antigen-specific B cells, the affinity maturation by somatic hypermutation and the selection of high-affinity B cells by apoptosis (44,45,60). The fact that GC B cells express IgM during primary immune response and IgG during secondary response suggested that GC are also the place where the isotype switch reaction occurs. Recent studies, however, demonstrated that the GC formation is not necessarily required for the immune response to TD antigens. Some B cells may undergo isotype switching without affinity maturation (61,62) and there are some IgM+ memory cells (63). Isotype switching and affinity maturation can appear even in GC- mice (64). Therefore, somatic mutation and isotype switching are two independent processes that do not depend exclusively on the formation of GC (65).
The PNA+ GC were no clearer in CD19-Cre+/
4flox mice than those observed in wild-type spleen. However, the PNA staining showed that the area with IgM+ B cells existed in the follicle-like region of mutant mice but was less compact in comparison to the wild-type GC. These results might suggest that the
4- B cells gather in the GC-like architecture upon immunization with TD antigen but are incapable of responding to the stimuli provided in the GC to differentiate into mature B cells with isotype switching. A similar phenomenon is observed in AID knockout mice, which also displayed a larger GC formation without isotype switching of Ig and reduced somatic hypermutation of the V region upon TD antigen stimulation (37). As an interesting difference from the AID knockout mice, the CD19-Cre+/
4flox mice do not demonstrate the complete absence of V region somatic hypermutation. The CD19-Cre+/
4flox mice showed a marked loss of higher mutation rates in the regions of CDR1 and CDR2 related to affinity maturation. The results suggest that
4-mediated signal transduction is an important pathway not only for clonal expansion but also for affinity maturation of antibodies against TD antigens. Taking these observations into consideration, the following model of B cell activation is proposed. B cell activation after TD antigen immunization induces the formation of GC in the follicular area with the accumulation of PNA+ antigen-driven B cells. This first stage is followed by a secondary maturation stage, with the expression of mature surface markers such as CD23 and GL7, during which isotype switching and V region somatic hypermutation would occur. At the second stage,
4 might play a critical role in B cell maturation.
The results clearly demonstrated that the rapamycinsensitive
4 pathway is pivotal for proliferation and differentiation of B cells undergoing isotype switching and somatic hypermutation of V region genes in the response to TD antigens. This mutant mouse provides a useful model for the study of the signals that stimulate the
4 pathway, which is mostly provided by the GC architecture acting upon antigen-driven B cells undergoing differentiation into plasma cells in order to produce high-affinity IgG antibodies.
 |
Acknowledgments
|
---|
We thank Dr R. C. Rickert (Department of Biology and the Cancer Center, University of California, San Diego, CA) for providing us the CD19-Cre knockin mouse. We thank Dr T. Kaisho for helpful discussion and Dr H. Tanaka for helping the preparation of cryosections of the spleen. We also thank Dr S. Bauer for the critical reading of our manuscript. The work was supported by grants from The Ministry of Education, Culture, Sports, Science and Technology of Japan.
 |
Abbreviations
|
---|
BM bone marrow |
CGG chicken globulin |
GC germinal center |
KLH keyhole limpet hemocyanin |
LPS lipopolysaccharide |
neo neomycin resistant gene |
NP nitrophenyl |
PE phycoerythrin |
PI propidium iodide |
PI-3K phosphatidylinositol-3 kinase |
PKC protein kinase C |
PNA peanut agglutinin |
PP2Ac catalytic subunit of protein phosphatase 2A |
S6K ribosomal S6 protein kinase |
TD T cell dependent |
TI T cell independent |
TNP 2,4,6-trinitrophenyl |
 |
Notes
|
---|
Transmitting editor: T. Watanabe
Received 6 September 2001,
accepted 23 October 2001.
 |
References
|
---|
-
Liu, Y.-J. and Banchereau, J. 1997. Regulation of B-cell commitment to plasma cells or to memory B cells. Semin. Immunol. 9:235.[Medline]
-
Dal Porto, J. M., Haberman, A. M., Shlomchik, M. J. and Kelsoe, G. 1998. Antigen drives very low affinity B cells to become plasmacytes and enter germinal centers. J. Immunol. 161:5373.[Abstract/Free Full Text]
-
Kurosaki, T. 1999. Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 15:555.
-
Parker, D. C. 1993. T cell-dependent B cell activation. Annu. Rev. Immunol. 11:331.[ISI][Medline]
-
Clark, E. A. and Ledbetter, J. A. 1994. How B and T cells talk to each other. Nature 367:425.[ISI][Medline]
-
Venkitaraman, A. R., Williams, G. T., Dariavach, P. and Neuberger, M. S. 1991. The B-cell antigen receptor of the five immunoglobulin classes. Nature 352:777.[ISI][Medline]
-
Sakaguchi, N., Matsuo, T., Nomura, J., Kuwahara, K., Igarashi, H. and Inui, S. 1993. Immunoglobulin receptor-associated molecules. Adv. Immunol. 54:337.[ISI][Medline]
-
Reth, M. and Wienands, J. 1997. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15:453.[ISI][Medline]
-
Tamir, I. and Cambier, J. C. 1998. Antigen receptor signaling: integration of protein tyrosine kinase functions. Oncogene 17:1353.[ISI][Medline]
-
Casillas, A., Hanekom, C, Williams, K., Katz, R. and Nel, A. E. 1991. Stimulation of B-cells via the membrane immunoglobulin receptor or with phorbol myristate 13-acetate induces tyrosine phosphorylation and activation of a 42-kDa microtubule-associated protein-2 kinase. J. Biol. Chem. 266:19088.[Abstract/Free Full Text]
-
Clark, M. R., Campbell, K. A. S., Kazlauskas, A., Hertz, J. M., Potter, T. A., Pleiman, C. and Cambier, J. C. 1992. The B cell antigen receptor complex: association of Ig-
and Ig-ß with distinct cytoplasmic effectors. Science 258:123.[ISI][Medline]
-
Gold, M. R., Chan, V. W.-F., Turck, C. W. and DeFranco, A. L. 1992. Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes. J. Immunol. 148:2012.[Abstract/Free Full Text]
-
Lin, J. and Justment, L. B. 1992. The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases. J. Immunol. 149:1548.[Abstract/Free Full Text]
-
Saxton, T. M., van Oostveen, I., Bowtell, D., Aebersold, R. and Gold, M. R. 1994. B cell antigen receptor cross-linking induces phosphorylation of the p21ras oncoprotein activators SHC and mSOS as well as assembly of complexes containing SHC, GRB-2, mSOS1, and a 145-kDa tyrosine-phosphorylated protein. J. Immunol. 153:623.[Abstract/Free Full Text]
-
DeFranco, A. L. 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9:296.[ISI][Medline]
-
Campbell, K. S. 1999. Signal transduction from the B cell antigen-receptor. Curr. Opin. Immunol. 11:256.[ISI][Medline]
-
Li, H.-L., Davis, W. and Pure, E. 1999. Suboptimal cross-linking of antigen receptor induces Syk-dependent activation of p70S6 kinase through protein kinase C and phosphoinositol 3-kinase. J. Biol. Chem. 274:9812.[Abstract/Free Full Text]
-
Kuwahara, K., Matsuo, T., Nomura, J., Igarashi, H., Kimoto, M., Inui, S. and Sakaguchi, N. 1994. Identification of a 52-kDa molecule (p52) coprecipitated with the Ig receptor-related MB-1 protein that is inducibly phosphorylated by the stimulation with phorbol myristate acetate. J. Immunol. 152:2742.[Abstract/Free Full Text]
-
Inui, S., Kuwahara, K., Mizutani, J., Maeda, K., Kawai, T., Nakayasu, H. and Sakaguchi, N. 1995. Molecular cloning of a cDNA clone encoding a phosphoprotein component related to the Ig receptor-mediated signal transduction. J. Immunol. 154:2714.[Abstract/Free Full Text]
-
Di Como, C. J. and Arndt, K. T. 1996. Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev. 10:1904.[Abstract]
-
Jian, Y. and Broach, J. R. 1999. Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO 18:2782.[Abstract/Free Full Text]
-
Inui, S., Sanjo, H., Maeda, K., Yamamoto, H., Miyamoto, E. and Sakaguchi, N. 1998. Ig receptor binding protein 1(
4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood 92:539.[Abstract/Free Full Text]
-
Murata, K., Wu, J. and Brautigan, D. L. 1997. B cell receptor-associated protein
4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc. Natl Acad. Sci. USA 94:10624.[Abstract/Free Full Text]
-
Chen, J., Peterson, R. T. and Schreiber, S. T. 1998.
4 associates with protein phosphatase 2A, 4, and 6. Biochem. Biophys. Res. Commun. 247:827.[ISI][Medline]
-
Nanahoshi, M., Nishiuma, T., Tsujishita, Y., Hara, K., Inui, S., Sakaguchi, N. and Yonezawa, K. 1998. Regulation of protein phosphatase 2A catalytic activity by alpha4 protein and its yeast homolog Tap42. Biochem. Biophys. Res. Commun. 251:520.[ISI][Medline]
-
Brown, E. J., Alberts, M. W., Shin, T. B., Ichikawa, K., Lane, W. S. and Schreiber, S. L. 1994. A mammalian protein targeted by G1-arresting rapamycinreceptor complex. Nature 369:756.[ISI][Medline]
-
Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. and Snyder, S. H. 1994. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78:35.[ISI][Medline]
-
Sabers, C. J., Martin, M. M., Brunn, G. J., Williams, J. M., Dumont, F. J., Wiederrecht, G. and Abraham, R. T. 1995. Isolation of a protein target of the FKBP12rapamycin complex in mammalian cells. J. Biol. Chem. 270:815.[Abstract/Free Full Text]
-
Pullen, N. and Thomas, G. 1997. The modular phosphorylation and activation of p70s6k. FEBS Lett. 410:78.[ISI][Medline]
-
Burnett, P. E., Barrow, R., Cohen, N. A., Snyder, S. H. and Sabatini, D. M. 1998. RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc. Natl Acad. Sci. USA 95:1432.[Abstract/Free Full Text]
-
Dufner, A. and Thomas, G. 1999. Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res. 253:100.[ISI][Medline]
-
Brown, E. J. and Schreiber, S. L. 1996. A signaling pathway to translational control. Cell 86:517.[ISI][Medline]
-
Schmelzle, T. and Hall, M. N. 2000. TOR, a central controller of cell growth. Cell 103:253.[ISI][Medline]
-
Rickert, R. C., Roes, J. and Rajewsky, K. 1997. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 25:1317.[Abstract/Free Full Text]
-
Nomura, J., Inui, S., Yamasaki, T., Kataoka, S., Maeda, K., Nakanishi, K. and Sakaguchi, N. 1995. Anti-CD40 monoclonal antibody induces the proliferation of murine B cells as a B-cell mitogen through a distinct pathway from receptors for antigens or lipopolysaccharide. Immunol. Lett. 45:195.[ISI][Medline]
-
Inui, S., Sakata, A., Maeda, K., Tashiro, F., Miyazaki, J. and Sakaguchi, N. 1999. Introduction of human CD40 gene with immunoglobulin enhancer and promoter creates mice with a population of human CD40+ early B lineage cells in the bone marrow. Transgenics 2:347.[ISI]
-
Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and Honjo, T. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102:553.[ISI][Medline]
-
Gu, H., Zou, Y.-R. and Rajewsky, K. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73:1155.[ISI][Medline]
-
Rajewsky, K., Gu, H., Kuehn, R., Betz, U. A. K., Mueller, W. J., Roes, W. and Schwenk, F. 1996. Perspective series: molecular medicine in genetically engineered animals. J. Clin. Invest. 98:600.[Free Full Text]
-
Onda, M., Inui, S., Maeda, K., Suzuki, M., Takahashi, E. and Sakaguchi, N. 1997. Expression and chromosomal localization of the human
4/IGBP1 gene, the structure of which is closely related to the yeast TAP2 protein of the rapamycin-sensitive signal transduction pathway. Genomics 46:373.[ISI][Medline]
-
Sakata, A., Kuwahara, K., Ohmura, T., Inui, S. and Sakaguchi, N. 1999. Involvement of a rapamycin-sensitive pathway in CD40-mediated activation of murine B cells in vitro. Immunol. Lett. 68:301.[ISI][Medline]
-
Abraham, R. T. and Wiederrecht, G. J. 1996. Immunopharmacology of rapamycin. Annu. Rev. Immunol. 14:483.[ISI][Medline]
-
Han, S., Zheng, B., Schatz, D. G., Spanopoulou, E. and Kelsoe, G. 1996. Neoteny in lymphocytes:Rag1 and rag2 expression in germinal center B cells. Science 274:2094.[Abstract/Free Full Text]
-
MacLennan, I. C. M. 1994. Germinal centers. Annu. Rev. Immunol. 12:117.[ISI][Medline]
-
Kelsoe, G. 1996. Life and death in germinal centers (redux). Immunity 4:107.[ISI][Medline]
-
Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. and Cumano, A. 1988. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO 7:1995.[Abstract]
-
Jacob, J., Przylepa, J., Miller, C. and Kelsoe, G. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178:1293.[Abstract]
-
Tao, W., Hardardottir, F. and Bothwell, L. M. 1993. Extensive somatic mutation in the Ig heavy chain V genes in a late primary anti-hapten immune response. Mol. Immunol. 30:593.[ISI][Medline]
-
Peterson, R., Desai, B. N., Hardwick, J. S. and Schreiber, S. 1999. Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycin-associated protein. Proc. Natl Acad. Sci. USA 96:4438.[Abstract/Free Full Text]
-
Jumaa, H., Wollscheid, B., Mitterer, M., Wienands, J., Reth, M. and Nielsen, P. J. 1999. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity 11:547.[ISI][Medline]
-
Kahn, W. N., Alt, F. W., Gerstein, R. M., Malynn, B. A., Larsson, I., Rathbun, G., Davidson, L., Mueller, S., Kantor, A. B., Rosen, F. S. and Sideras, P. 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3:283.[ISI][Medline]
-
Kerner, J. D., Appleby, M. W., Mohr, R. N., Chien, S., Rawlings, D. J., Maliszewski, C. R., Witte, O. N. and Perlmutter, P. M. 1995. Impaired expansion of mouse B cell progenitors lacking Btk. Immunity 3:301.[ISI][Medline]
-
Suzuki, H., Terauchi, Y., Fujiwara, M., Aizawa, S., Yazaki, Y., Kadowaki, T. and Koyasu, S. 1999. Xid-like immunodeficiency in mice with disruption of the p85
subunit of phosphoinositide 3-kinase. Science 283:390.[Abstract/Free Full Text]
-
Fruman, D. A., Snapper, S. B., Yballe, C. M., Davidson, L., Yu, J. Y., Alt, F. W. and Cantley, L. C. 1999. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85
. Science 283:393.[Abstract/Free Full Text]
-
Wang, D., Feng, J., Wen, R., Marine, J.-C., Sangster, M. Y. Parganas, E., Hoffmeyer, A., Jackson, C. W., Cleveland, J. L., Murray, P. J. and Ihle, J. N. 2000. Phospholipase C
2 is essential in the functions of B cell and several Fc receptors. Immunity 13:25.[ISI][Medline]
-
Hashimoto, A., Takeda, K., Inaba, M., Sekimata, M., Kaisho, T., Ikehara, S., Homma, Y., Akira, S. and Kurosaki, T. 2000. Cutting edge: essential role of phospholipase C-
2 in B cell development and function. J. Immunol. 165:1738.[Abstract/Free Full Text]
-
Snapper, C. M. and Paul, W. E. 1987. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944.[ISI][Medline]
-
Purkerson, J. M. and Isakson, P. C. 1992. Interleukin 5 (IL-5) provides a signal that is required in addition to IL-4 for isotype switching to immunoglobulin (Ig) G1 and IgE. J. Exp. Med. 175:973.[Abstract]
-
Yokogami, K., Wakisaka, S., Avruch, J. and Reeves, S. A. 2000. Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by rapamycin target mTOR. Curr. Biol. 10:47.[ISI][Medline]
-
Liu, Y.-L. and Arpin, C. 1997. Germinal center development. Immunol. Rev. 156:111.[ISI][Medline]
-
Cumano, A. and Rajewsky, K. 1985. Structure of primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in normal and idiotypically suppressed C57BL/6 mice. Eur. J. Immunol. 15:512.[ISI][Medline]
-
Jacob, J. and Kelsoe, G. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl II. A common clonal origin for periarteriolar lymphoid sheath-associated foci and germinal centers. J. Exp. Med. 176:679.[Abstract]
-
White, H. and Gray, D. 2000. Analysis of immunoglobulin (Ig) isotype diversity and IgM/D memory in response to phenyl-oxazolone. J. Exp. Med. 191:2209.[Abstract/Free Full Text]
-
Kato, J., Motoyama, N., Taniuchi, I., Takeshita, H., Yoyoda, M., Masuda, K. and Watanabe, T. 1998. Affinity maturation in lyn kinase-deficient mice with defective germinal center formation. J. Immunol. 160:4788.[Abstract/Free Full Text]
-
Liu, Y. J., Malisan, F., Bouteiller, O., Guret, C., Banchereau, J., Mills, F. C., Max, E. E. and Martinez-Valdez, H. 1996. Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation. Immunity 4:241.[ISI][Medline]