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The Journal of Immunology, 2007, 179, 4473 -4479
Copyright © 2007 by The American Association of Immunologists, Inc.
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Negative Regulation of CD40-Mediated B Cell Responses by E3 Ubiquitin Ligase Casitas-B-Lineage Lymphoma Protein-B1

Guilin Qiao2,*,{ddagger}, Minxiang Lei2,*, Zhenping Li*, Yonglian Sun{dagger},{ddagger}, Andrew Minto*, Yang-Xin Fu{dagger},{ddagger}, Haiyan Ying*,{ddagger}, Richard J. Quigg* and Jian Zhang3,*,{ddagger}

* Sections of Nephrology, Department of Medicine, {dagger} Department of Pathology, {ddagger} Committee on Immunology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It has been documented that CD40 is essential for B cell function. Casitas-B-lineage lymphoma protein-b (Cbl-b), an adapter protein and ubiquitin ligase, has been shown to regulate the activation of T and B cells through their Ag receptors. In this study, we report that CD40-induced B cell proliferation is significantly augmented in mice lacking Cbl-b. Furthermore, Cbl-b–/– mice display enhanced thymus-dependent Ab responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects. Hyper thymus-dependent humoral response in Cbl-b–/– mice is in part due to an intrinsic defect in B cells. Mechanistically, Cbl-b selectively down-modulates CD40-induced activation of NF-{kappa}B and JNK. Cbl-b associates with TNF receptor-associated factor 2 upon CD40 ligation, and inhibits the recruitment of TNF receptor-associated factor 2 to the CD40. Together, our data suggest that Cbl-b attenuates CD40-mediated NF-{kappa}B and JNK activation, thereby suppressing B cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD40,a member of the TNF receptor superfamily, plays central roles in the homeostatic regulation of B cell functions (1). Activated CD4+ T cells express CD40L, and the interaction of CD40 on B cells with the CD40L on activated CD4+ T cells represents the key event in the initiation of B cell proliferation, differentiation, isotype switching, up-regulation of surface molecules contributing to Ag presentation, development of the germinal center (GC),4 and the humoral memory response to thymus-dependent (TD) Ags (1). CD40-CD40L interactions induce association of TNF receptor-associated factor (TRAF)-2, -3, and -6 with CD40, and activate IKKbeta and then NF-{kappa}B (2, 3). CD40 ligation also activates the noncanonical NF-{kappa}B2 processing pathway, that is, IKK{alpha} leads to the phosphorylation and processing of p100 resulting in the formation of an active RelB/p52 heterodimer (4). The other knowing signaling events occurring upon CD40 ligation include activation of MAPKs such as ERK, JNK, and p38 MAPK, and PI3K (5, 6).

Casitas-B-lineage lymphoma protein-b (Cbl-b) belongs to the Cbl family, which consists of a protein tyrosine kinase (PTK)-binding (TKB) domain, linker, and RING finger domains, and extensive proline-rich regions in their COOH-terminal halves. It is now understood that Cbl-b functions as an E3 ubiquitin (Ub) ligase with a RING finger that recruits an Ub-conjugating enzyme (E2) and the TKB domain that recognizes target proteins for Ub conjugation (7, 8). The absence of Cbl-b in mice results in the hyperactivation of autoreactive T and B cells, leading to the development of spontaneous lupus-like disease (9), indicating a critical role of Cbl-b in the regulation of T and B cell activation and, hence, the maintenance of a balance between immunity and tolerance. B cells lacking Cbl-b show augmented proliferation in response to IgM stimulation (9), suggesting that Cbl-b regulates BCR-mediated signaling. In support of this notion, Cbl-b may negatively regulate BCR signaling in mature mouse B cells through the ubiquitination of Syk (10). However, it is largely unknown whether and how Cbl-b modulates CD40-mediated B cell function. In this study, we attempted to investigate the role of Cbl-b in CD40-mediated B cell function. Our data show that Cbl-b negatively regulates CD40-mediated B cell responses in vitro and in vivo. Cbl-b associates with TRAF-2 upon CD40 ligation, thus suppressing the recruitment of TRAF-2 to CD40, which leads to activation of NF-{kappa}B and JNK.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Wild-type (WT), CD40–/–, and Rag-1–/– BALB/c mice were purchased from The Jackson Laboratory. Cbl-b–/– mice were described previously (9), and have been backcrossed to a BALB/c background for 12 generations. Cbl-b–/– mice were crossed with CD40–/–mice, and the offspring were intercrossed to generate Cbl-b–/–CD40–/– mice. All mice were used for experiments at ages of 6 to 10 wk.

Reagents

Anti-mouse IgM F(ab') 2 were purchased from Zymed Laboratories. Abs against Cbl-b (sc-8006), p52 (sc-298), TRAF-2 (sc-876), TRAF-3 (sc-1828), and TRAF-6 (sc-7221) were purchased from Santa Cruz Biotechnology. Phospho-Abs were purchased from Cell Signaling Technology. A murine B cell isolation kit was obtained from Miltenyi Biotec. The fluorescence conjugated and nonfluorescence conjugated mAbs were purchased from BD Biosciences. MG-132 was obtained from Sigma-Aldrich. CD40L (CRC 803B) was purchased from Cell Sciences.

B cell isolation and activation

Splenic B cells from WT, CD40–/–, and Cbl-b–/– mice were isolated (purity >95% as determined by FACS analysis of B220 cell surface expression) using a B cell isolation kit using negative selection (Miltenyi Biotec). Contaminating T cells were <1% as determined by CD3 staining. For in vitro activation, B cells (2 x 107/ml) were stimulated for various time periods indicated by CD40L or anti-CD40 mAbs. The cells were lysed in 1% Nonidet P-40 lysis buffer or radioimmunoprecipitation assay buffer (11, 12) where indicated.

Assays of B cell proliferation and survival

Splenic B cells from WT and Cbl-b–/– mice were cultured for 56 h at 37°C in round-bottom 96-well plates with F(ab')2 anti-IgM with or without anti-CD40 mAb as indicated. The cells were pulsed with 1µCi [3H]thymidine, and harvested 16 h later. The radioactivity was quantitated as previously described (11, 12). For viability assays, B cells were stimulated as above and cell death was measured through FACS analysis after 72 h of culture by double staining with propidium iodide (PI) and FITC-conjugated-annexin V. The percentage of cells in the double-negative quadrant was recorded as the viable fraction.

Immunoprecipitation and Western blotting

The conditions for immunoprecipitation and immunoblotting were described previously (11, 12).

Detection of surface receptors and activation markers by flow cytometry

Splenocyte suspensions were incubated with anti-CD32/CD16 for 30 min at 4°C before staining with the Ab mixture. For the detection of cycling, WT and Cbl-b–/– B cells were stimulated with CD40L for 72 h, and fixed in cold 70% ethanol, treated with RNase, and stained with PI for 30 min at room temperature. Cells were analyzed by flow cytometry.

Induction of TD and thymus-independent (TI) Ab responses

For TD Ag-induced responses, mice were immunized i.p. with 50 µg keyhole limpet hemocyanin (KLH; Calbiochem) in CFA (Sigma-Aldrich). For secondary responses, KLH-primed mice were challenged with 50 µg KLH in IFA i.p. on day 21 after primary immunization. Mice were bled on days 14 and 28 after primary immunization. For TI Ag analysis, mice were injected i.p. with 25 µg TNP-Ficoll (Biosearch Technologies). We bled tails and prepared serum on days 7, 14, and 28 after immunization. Titers of KLH-specific IgG1, IgG2b, and IgM Abs, or TNP-specific IgG1 and IgM Abs were assessed using sandwich ELISA (Southern Biotechnology Associates).

Adoptive transfer

For Rag-1–/– reconstitution experiments, CD4+ T cells were purified from spleens and lymph nodes via negative selection and sorted for CD44lowCD62Lhigh naive cells. Splenic B cells were isolated via negative selection and sorted for CD19+ cells. Five x 106 naive CD4+ T cells and 10 x 106 CD19+ B cells from WT or Cbl-b–/– mice in different combinations were transferred i.v. into Rag-1–/– hosts, which were permitted to equilibrate 30 days after transfer to avoid effects of homeostatic proliferation (13). The mice were immunized with KLH as above and sacrificed at day 14.

Immunohistochemistry

Mice immunized with KLH in CFA were sacrificed on day 28. The spleens were collected and frozen, and 5-µm thick sections were cut. The frozen sections of spleens were immunostained with biotinylated-PNA followed by avidin-biotin complex-immunoalkaline phosphatase for GC and FITC-labeled anti-B220 followed by HRP-conjugated anti-FITC for B cells.

NF-{kappa}B activity assay

A TransAM ELISA was used to determine the nuclear activity of the p65 subunit (Active Motif). In brief, the nuclear extracts of WT and Cbl-b–/– B cells (2 x 106) treated with 2 µg/ml CD40L were prepared as previously described (14). Ten µg of nuclear protein diluted in lysis buffer was loaded in triplicate onto a 96-well plate coated with an oligonucleotide containing an NF-{kappa}B p65 consensus binding site (5'-GGGACTTTCC-3'). The wells were rinsed with washing buffer, and a NF-{kappa}B p65 Ab was added to each well at a dilution of 1/1000. After rinsing, a final incubation with HRP-conjugated secondary Ab resulted in a colorimetric reaction, which was then quantified by spectrophotometry at 450 nm with a reference wavelength of 655 nm.

Statistical analysis

Statistical analysis was done by two-tailed Student’s unpaired t test. Results are shown as the mean ± SD. A difference between experimental groups was considered significant when the p value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cbl-b–/– B cells display hyperproliferation and increased survival in response to CD40 stimulation

It has previously been shown that Cbl-b–/– B cells are hyperproliferative in response to IgM stimulation (9), suggesting that Cbl-b may negatively regulate the threshold for B cell activation. To extend this finding, we measured proliferation of splenic B cells from naive WT and Cbl-b–/– BALB/c mice in response to IgM and CD40 stimulation. Cbl-b–/– B cells proliferated highly in response to stimulation of IgM and CD40 (Fig. 1A, upper panel), suggesting that Cbl-b regulates not only BCR- but also CD40-induced B cell activation. Consistent with this observation, CD40 ligation promoted cell cycle progression more in Cbl-b–/– B cells than WT B cells (Fig. 1A, lower panel). Note that the expression of BCR, CD40, CD69, B7-1, and I-Ek was comparable between WT and Cbl-b–/– B cells (Fig. 1B), suggesting that heightened B cell proliferation induced by BCR or CD40 does not result from enhanced expression of BCR or CD40 receptor, or an activated phenotype.


Figure 1
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  		FIGURE 1. Cbl-b–/– B cells are hyperproliferative and display an increased rate of survival in response to CD40 ligation. A, Purified B cells from WT and Cbl-b–/– mice were stimulated with various concentrations of F(ab')2 anti-IgM or anti-CD40, or constant F(ab')2 anti-IgM with various concentrations of anti-CD40 at 37°C for 72 h, and B cell proliferation was determined by [3H]thymidine incorporation (upper panel). The cell cycle in CD40L-activated WT and Cbl-b–/– B cells was examined by PI staining and flow cytometry. Histograms represent the mean fluorescence intensity of PI incorporation. Numbers represent the percentage of cells in each phase (lower panel). B, WT and Cbl-b–/– B cells were stained with Abs against CD69, I-Ek, B7-1, and CD40, and analyzed by flow cytometry. C, WT and Cbl-b–/– B cells were cultured for 3 days with anti-CD40 (8 µg/ml), and the B cell survival was determined by Annexin-V and PI staining, and was defined as Annexin-V–PI– cells. D, WT and Cbl-b–/– B cells were stimulated with anti-CD40 for 0, 24, 48, and 72 h, and lysed. The cell lysates were blotted with anti-Bcl-xL and anti-actin, respectively. Data shown in this figure are pooled from two experiments.

 
It has been shown that CD40–/– and CD40L–/– mice showed defective T cell-dependent B cell activation, proliferation, and survival (1). To test the role of Cbl-b in CD40-mediated B cell survival, resting mature B cells were purified from spleen and cultured with or without anti-CD40 Ab for 3 days. The B cell survival was determined by Annexin-V and PI staining, and was defined as Annexin-V–PI– cells. As shown in Fig. 1C, the absence of Cbl-b led to a significant enhancement of B cell survival induced by CD40 ligation. This result suggests that increased B cell survival may contribute to the aberrant CD40-mediated B cell proliferation in Cbl-b–/– mice. To better understand the capacity of CD40 ligation to elicit significant survival in Cbl-b–/– B cells, we compared the ability of anti-CD40 treatment to regulate the induction of Bcl-xL, a prosurvival protein which has been implicated in CD40-mediated B cell survival (15, 16) in WT and Cbl-b–/– B cells. Cbl-b deficiency greatly enhanced CD40-induced Bcl-xL expression in B cells (Fig. 1D).

Cbl-b exerts its regulatory role in TD- and TI-humoral immune responses

To investigate the role of Cbl-b in CD40-mediated B cell function in vivo, we measured TD humoral responses in WT, Cbl-b–/–, CD40–/–, and Cbl-b–/–CD40–/–mice. To this end, the mice were challenged with a TD Ag, KLH, and specific Ab responses, and GC formation were monitored. Consistent with previous reports (17, 18), CD40 function was dispensable for IgM production in response to KLH immunization but was required for Ig class switching for the production of IgG1 and IgG2b as CD40–/– mice produced very low levels of KLH-specific IgG1 and IgG2b Abs (Fig. 2A). In contrast, primary KLH immunization elicited a strong TD immune response resulting in increased production of anti-KLH IgG1 and IgG2b Abs in Cbl-b–/– mice. We note that IgG2a production was undetectable in all groups of mice immunized with KLH (data not shown). Overall secondary responses to KLH were comparable between WT and Cbl-b–/– mice (Fig. 2A). To determine whether Cbl-b indeed exerts its regulatory role in humoral immune responses through the CD40-dependent pathway, we compared Ab responses to KLH immunization in Cbl-b–/–CD40–/– mice with WT, CD40–/–, and Cbl-b–/– mice. Introduction of the CD40 deficiency into Cbl-b–/– mice significantly abrogated anti-KLH IgG1 and IgG2b production by these mice (Fig. 2A). Consistent with this, heightened formation of GCs in Cbl-b–/– mice upon KLH challenge was diminished by introduction of the CD40 deficiency (Fig. 2B). These findings suggest that Cbl-b modulates the humoral immune responses through its interaction with the CD40-dependent pathway.


Figure 2
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  		FIGURE 2. Cbl-b down-regulates TD- and TI-humoral responses. A, WT, CD40–/–, Cbl-b–/–, and Cbl-b–/–CD40–/– mice (n = 5) were immunized i.p. with KLH and challenged on day 21. All mice were sacrificed on day 28. Serum was collected from peripheral blood on days 0, 14, and 28 after initial immunization. Circulating Abs were measured by an isotype-specific, Ag-specific ELISA. B, The spleens were removed and frozen sections of spleens were immunostained with biotinylated-PNA followed by ABC-AP for GC (blue) and FITC-labeled anti-B220 followed by HRP-conjugated anti-FITC for B cells (brown). C, WT and Cbl-b–/– mice (n = 5) were injected i.p. with TNP-Ficoll. All mice were sacrificed on day 28. Serum was collected from peripheral blood on days 0, 7, 14, and 28 after immunization. Circulating Ag-specific Abs were measured by ELISA. Original magnification, x40. The data are representative of three independent experiments. D, Either WT or Cbl-b–/– CD4+ T cells were adoptively transferred in conjunction with either Cbl-b–/– or WT B cells into Rag-1–/– mice (n = 5), which were permitted to equilibrate 30 days after transfer to avoid effects of homeostatic proliferation. The mice were immunized with KLH, and sacrificed on day 14. Serum KLH-specific Ab (IgG1) was measured by ELISA. The results are shown as the mean ± SD (*, p < 0.05; **, p < 0.01 for indicated comparison. ns, no significant difference), and the splenocytes were stained with PE-Cy5-anti-CD4 and FITC-anti-B220. One of three independent experiments is shown.

 
The enhanced TD humoral responses in Cbl-b–/– mice may be the result of greater help from T cells rather than an intrinsic defect in B cells. However, the hyperproliferation of Cbl-b–/– B cells in response to BCR or CD40 suggests an intrinsic defect in these B cells. To further address whether B cells lacking Cbl-b indeed have an intrinsic defect, we then monitored the Ab responses in Cbl-b–/– mice to TNP-Ficoll, a TI Ag. Upon in vivo challenge with TNP-Ficoll, Cbl-b–/– mice produced much higher titers of total TNP-specific IgG1 Ab, but showed no change in TNP-specific IgM production (Fig. 2C). However, TNP-specific IgG2a and IgG2b production was undetectable in all mice. The hyper TI Ag-specific responses observed in Cbl-b–/– mice suggest that Cbl-b also plays a negative regulatory role in TI-humoral immune responses, and loss of Cbl-b leads to an intrinsic defect in B cells.

To further clarify T versus B cell contributions to the aberrant humoral responses in Cbl-b–/– mice, either WT or Cbl-b–/– naive CD4+ T cells were adoptively transferred in conjunction with either Cbl-b–/– or WT B cells into Rag-1–/– hosts, which were permitted to equilibrate 30 days after transfer to avoid effects of homeostatic proliferation (13). Although Rag-1–/– mice reconstituted with Cbl-b–/– CD4+ T cells together with Cbl-b–/– B cells displayed a robust increase in the Ab production after KLH immunization, transfer of WT CD4+ T cells in conjunction with Cbl-b–/– B cells or Cbl-b–/– CD4+ T cells together with WT B cells into Rag-1–/– mice also produced higher Ab titers than those reconstituted with WT CD4+ and B cells (Fig. 2D). No statistical significance in Ab production was observed in Rag-1–/– mice reconstituted with WT CD4+ T cells and Cbl-b–/– B cells compared with those receiving Cbl-b–/– CD4+ T cells together with WT B cells. These data strongly support that the aberrant TD B cell responses are in part due to an intrinsic B cell defect in mice deficient for Cbl-b.

Cbl-b selectively modulates CD40-mediated activation of NF-{kappa}B and JNK but not ERK, p38 MAPK, and Akt during B cell activation

CD40 ligation in B cells is known to activate multiple signaling pathways including NF-{kappa}B, MAPKs, and PI3K (5, 6). We then determined which signaling pathways are perturbed in the absence of Cbl-b. Loss of Cbl-b resulted in the hyperphosphorylation of CD40-induced I{kappa}B{alpha} and JNK, but phosphorylation of ERK, p38 MAPK, and Akt was comparable between WT and Cbl-b–/– B cells (Fig. 3A). To further confirm CD40-induced NF-{kappa}B activation is regulated by Cbl-b, the NF-{kappa}B p65 binding activity was assayed using an ELISA kit. NF-{kappa}B p65 binding activity was significantly enhanced in B cells lacking Cbl-b upon CD40 ligation (Fig. 3B). Because CD40 triggers both canonical and noncanonical NF-{kappa}B pathway (16), we then detected whether Cbl-b also affected noncanonical NF-{kappa}B pathway. Cbl-b–/– B cells showed equal p100 processing to p52 in response to CD40 ligation (Fig. 3C). These data implicate a negative regulatory role of Cbl-b in canonical but not noncanonical NF-{kappa}B activation pathway. Taken together, our data indicate that Cbl-b negatively regulates CD40-induced activation of NF-{kappa}B and JNK in B cells.


Figure 3
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  		FIGURE 3. Cbl-b selectively inhibits activation of NF-{kappa}B and JNK in B cells. A, WT and Cbl-b–/– B cells were stimulated for 5 and 15 min with CD40L or anti-CD40 and lysed. The cell lysates were blotted with phospho-Abs against ERK, JNK, p38 MAPK, Akt, and I{kappa}B{alpha}, respectively. The membrane was stripped and reprobed with anti-actin. The phosphorylation of I{kappa}B{alpha} upon CD40L or anti-CD40 stimulation was semiquantitated by densitometric scanning and normalized to the corresponding beta-actin bands. Data are presented as fold change as compared with WT unstimulated cells (**, p < 0.01, Student’s t test). B, WT and Cbl-b–/– B cells were stimulated with CD40L for 30 min. The nuclear extracts were prepared, and the nuclear activity of the p65 subunit was determined by a TransAM ELISA kit. C, WT and Cbl-b–/– B cells were stimulated with anti-CD40 for 2, 4, 8, and 16 h, and lysed. The cell lysates were blotted with anti-p52 Ab. Similar results were obtained from two independent experiments.

 
Cbl-b associates with TRAF-2 upon CD40 ligation and regulates the recruitment of TRAF-2 to CD40 in B cells

The recruitment of TRAFs to the CD40 cytoplasmic domain is essential for CD40-mediated activation of multiple signaling pathways including NF-{kappa}B and JNK/p38 MAPK, thus regulating B cell responses (19, 20). The enhanced recruitment of TRAF-2 to CD40 in TRAF-3–/– B cells upon CD40 ligation suggests that TRAF-3 may exert its suppressive effects on CD40 signaling by competing with TRAF-2 for binding to CD40 (20). To elucidate the molecular mechanism of Cbl-b’s action, we investigated whether Cbl-b can associate with TRAF-2, -3, or -6 upon CD40 ligation. To this end, WT B cells were stimulated with CD40L for 5, 15, and 30 min at 37°C and lysed. The cell lysates were immunoprecipitated with anti-TRAF-2, anti-TRAF-3, and anti-TRAF-6, and blotted with anti-Cbl-b. Interestingly, we found that CD40 ligation induced association of Cbl-b with TRAF-2, which became evident at 15–30 min after stimulation. No association between Cbl-b and TRAF-3 or TRAF-6 was observed in WT B cells before and after CD40 ligation (Fig. 4A). These data raised a possibility that Cbl-b may selectively regulate the recruitment of TRAF-2 to CD40. Indeed, the absence of Cbl-b indeed resulted in enhanced and sustained association of TRAF-2 with the CD40, whereas very little TRAF-3 was found to bind to CD40 in Cbl-b–/– B cells (Fig. 4B). We failed to detect TRAF-6 in CD40 immunoprecipitates (data not shown). Note that CD40 was found in both TRAF-2 and TRAF-3 immunoprecipitates upon CD40 ligation, suggesting that at least two distinct complexes, namely CD40-TRAF-2-Cbl-b and CD40-TRAF-3, are formed.


Figure 4
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  		FIGURE 4. Cbl-b regulates the recruitment of TRAF-2 to the CD40. A, WT B cells were stimulated with CD40L for 5, 15, and 30 min at 37°C, and lysed in 0.5% Nonidet P-40 lysis buffer. The cell lysates were immunoprecipitated with anti-TRAF-2, anti-TRAF-3, and anti-TRAF-6, respectively, and blotted with anti-Cbl-b. The membranes were stripped and reprobed with Abs against TRAF-2, TRAF-3, and TRAF-6, respectively. The B cell lysates from WT and Cbl-b–/– mice were used as positive and negative controls. The membranes were stripped and reprobed with Abs against TRAF-2, TRAF-3, and TRAF-6, respectively. B, WT and Cbl-b–/– B cells were stimulated with CD40L as above. The cell lysates were immunoprecipitated with biotinylated anti-CD40, blotted with anti-TRAF-2, anti-TRAF-3, and anti-Cbl-b, respectively. The membrane was stripped and reprobed with streptavidin-HRP. C, WT B cells were stimulated with CD40L for 5 and 15 min and lysed. The cell lysates were immunoprecipitated with anti-TRAF-3, blotted with biotinylated anti-CD40, and visualized by streptavidin-HRP. One representative experiment from four independent experiments is shown.

 
Cbl-b promotes CD40-induced TRAF-2 ubiquitination and degradation in primary B cells

Both TRAF-2 and Cbl-b are RING finger E3 Ub ligases, and TRAF-2 undergoes self-ubiquitination (21). We then hypothesized that Cbl-b may target TRAF-2 for ubiquitination and subsequent degradation. To visualize endogenous TRAF-2 ubiquitination, we treated WT and Cbl-b–/– B cells with MG-132, a proteasome inhibitor, to block proteasome-mediated degradation, allowing ubiquitinated proteins to accumulate in the cytoplasm. Using this approach, we clearly showed that CD40-induced TRAF-2 ubiquitination was defective in B cells lacking Cbl-b (Fig. 5A). We should note that TRAF-3 also underwent ubiquitination upon CD40 ligation (Fig. 5A). Consistent with these observations, TRAF-2 and TRAF-3, but not TRAF-6 underwent degradation upon CD40 ligation in a time-dependent manner in WT B cells, and this degradation was significantly inhibited in B cells lacking Cbl-b (Fig. 5B).


Figure 5
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  		FIGURE 5. Cbl-b promotes CD40-induced TRAF-2 degradation in primary B cells. A, WT and Cbl-b–/– B cells were pretreated with MG-132 for 30 min at 37°C, stimulated with CD40L for 15 and 30 min, and lysed in radioimmunoprecipitation assay buffer. The cell lysates were immunoprecipitated with anti-TRAF-2 or anti-TRAF-3, and blotted with anti-Ub and anti-TRAF-2 or anti-TRAF-3. B, WT and Cbl-b–/– B cells were stimulated with CD40L for 0.25, 0.5, 1, 2, and 4 h, and lysed. The cell lysates were blotted with anti-TRAF-2, anti-TRAF-3, and anti-TRAF-6. The membrane was stripped and reprobed with anti-actin. One of three independent experiments is shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we analyzed the role of Cbl-b in CD40-mediated B cell responses and signaling. Our results show that CD40-induced B cell proliferation in vitro and TD humoral responses are significantly increased in Cbl-b–/– mice (Fig. 1A and 2, A and B). HyperTD humoral response in Cbl-b–/– mice is in part due to an intrinsic defect in B cells (Fig. 2D). Introduction of CD40 mutation abrogates hyperTD humoral immune responses in Cbl-b–/– mice (Fig. 2, A and B), supporting the notion that Cbl-b functions as a major negative mediator in CD40 signaling pathway in B cells. Our data indicate that Cbl-b selectively affects CD40-mediated NF-{kappa}B and JNK activation possibly via inhibiting TRAF-2 degradation, thus regulating the recruitment of TRAF-2 to the CD40 (Figs. 4 and 5).

We show that Cbl-b–/– mice display heightened KLH-specific IgG production and GC formation in response to TD Ag (Fig. 2, A and B). These data suggest that Cbl-b is indeed involved in regulation of TD humoral responses. However, it was not defined whether the hyperTD Ab responses in Cbl-b–/– mice are B cell intrinsic, or are secondary to hyperactivity of T cells. The evidence that Cbl-b–/– B cells are hyperproliferative in response to CD40 ligation in vitro and that Cbl-b–/– mice display hyperhumoral responses induced by TI Ag challenge suggests an intrinsic defect in Cbl-b–/– B cells. The direct evidence confirming the intrinsic B cell defect in Cbl-b–/– mice comes from the Rag-1–/– mice reconstitution experiments. Rag-1–/– mice reconstituted with WT T cells plus Cbl-b–/– B cells produced more IgG1 than those receiving WT B cells together with WT T cells. These data clearly indicate that there is a B cell intrinsic defect in Cbl-b–/– mice. However, the hyperTD Ab responses in Cbl-b–/– mice cannot be ascribed to B cells only because Rag-1–/– mice receiving Cbl-b–/– T cells and WT B cells produced the modest levels of IgG1. This notion is further supported by the evidence that loss of Cbl-b in both T and B cells acts synergistically to increase IgG1 production. Therefore, the hyperTD Ab production in Cbl-b–/– mice results from intrinsic defects in both T and B cells which lead to enhanced interaction between CD40 in B cells and CD40L in activated T cells.

It was reported that loss of Cbl-b rescues impaired T cell proliferation and IL-2 production in CD28–/– mice (9, 22). We then tested whether this is the case with genetic inactivation of Cbl-b in CD40–/– mice. CD40 mutation completely abrogates heightened TD responses in Cbl-b–/– mice (Fig. 2, A and B), indicating that Cbl-b exerts its negative regulatory role in TD responses via a CD40-dependent manner, and Cbl-b is one of the important negative regulators in CD40-signaling pathway in B cells. Therefore, our data suggest that manipulating the levels of Cbl-b expression in B cells may have potential therapeutic use in Ab-mediated autoimmune disease such as myasthenia gravis. Indeed, blocking CD40-CD40L interaction completely suppresses the development of experimental autoimmune myasthenia gravis (23, 24).

The recruitment of TRAFs to the CD40 cytoplasmic domain is essential for CD40-mediated B cell responses. CD40 transduces signals via these adaptor proteins to mediate the activation of multiple signaling pathways including NF-{kappa}B and JNK/p38 MAPK, which in turn phosphorylate and activate downstream transcription factors (19, 20). Although a TRAF-2-deficent B cell line has been shown to retain CD40-mediated activation of the canonical NF-{kappa}B pathway (25), TRAF-2 is essential for CD40-mediated activation of this pathway in primary B cells (26, 27). These studies indicate that the signaling milieu in B cell lines differs significantly from that of primary B cells. TRAF-3 has been shown to play a negative regulatory role in CD40 signaling. TRAF-3–/– B cells display enhanced CD40-mediated JNK activation. Furthermore, the recruitment of TRAF-2 to CD40 is increased in TRAF-3–/– B cells upon CD40 ligation, suggesting that TRAF-3 may exert its suppressive effects on CD40 signaling by competing with TRAF-2 for binding to CD40 (20). The evidence that Cbl-b associates with TRAF-2 but not with TRAF-3 and TRAF-6, suggests that Cbl-b may regulate the recruitment of TRAF-2 to CD40. Indeed, the absence of Cbl-b led to increased recruitment of TRAF2 but not TRAF-3 to the CD40 (Fig. 4B).

The association of Cbl-b with TRAF-2 suggests that Cbl-b may target TRAF-2 for ubiquitination. Indeed, treating primary B cells with MG-132 which allows ubiquitinated proteins to accumulate in the cytoplasm, we clearly show that CD40-induced TRAF2 ubiquitination is impaired in B cells lacking Cbl-b (Fig. 5A). Although the structural requirement for Cbl-b-TRAF-2 association has not been characterized, based upon the structural analysis of both Cbl-b and TRAF-2, we predict that Cbl-b may interact with TRAF-2 via its TKB domain or ubiquitin-associated domain with tyrosine residues or Ub chains of TRAF-2. Currently, the experiments designed to investigate how Cbl-b associates with TRAF-2 and regulates TRAF-2 degradation are underway.

We also showed that TRAF-3 degradation is impaired in Cbl-b–/– B cells upon CD40 ligation in a manner similar to TRAF-2. However, we could not detect the physical association of Cbl-b with TRAF-3 before and after CD40 ligation. It has been shown that CD40-induced TRAF-3 degradation is dependent upon TRAF-2 (26, 28). Given that Cbl-b only transiently associates with TRAF-2 and CD40 upon CD40 ligation, one might speculate that TRAF-2 that dissociates from CD40 and Cbl-b may then target TRAF-3 for ubiquitination. Our data also suggest that TRAF-2 may have higher affinity to CD40 than TRAF-3.

In conclusion, our data strongly indicate that B cells lacking Cbl-b display an intrinsic defect in CD40 signaling pathway. Cbl-b exerts its negative regulatory function on TD humoral responses via a CD40-dependent manner. Mechanistically, Cbl-b associates with TRAF-2, and regulates the recruitment of TRAF-2 to CD40 which leads to activation of NF-{kappa}B and JNK.


   Acknowledgments
 
We thank Dr. Josef M. Penninger for providing the Cbl-b-deficient mice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The project described was supported by Grants K02 AR049047 and R01AR049775 (to J.Z.) from the National Institutes of Health, and Grants-in-aid (0355509Z and 0650181Z to J.Z.) from the American Heart Association. Back

2 G.Q. and M.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jian Zhang, Section of Nephrology, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: jzhang{at}medicine.bsd.uchicago.edu Back

4 Abbreviations used in this paper: GC, germinal center; TD, thymus dependent; Cbl-b, Casitas-B-lineage lymphoma protein-b; PTK, protein tyrosine kinase; TKB, PTK binding; Ub, ubiquitin; TRAF-2, TNFR-associated factor 2; WT, wild type; PI, propidium iodide; TI, thymus independent; KLH, keyhole limpet hemocyanin. Back

Received for publication May 15, 2007. Accepted for publication July 30, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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