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PubMed1995

Functional expression of CD40 antigen on human epidermal Langerhans cells.

It is now well established that interactions of CD40 on the B cells, along with its ligand (CD40-L) on the T cells, regulate B cell proliferation and differentiation. However, the functional significance of CD40 expression on cells known for most efficient Ag-presenting function, i.e., dendritic cells, is not so clear. In this study, we demonstrate that CD40 is expressed on human dendritic Langerhans cells (LC) freshly isolated from epidermis. Using CD40-L transfected cells, CD40 triggering was found to enhance LC viability when cultured and to result in phenotypic alterations. Thus, a 2-day CD40 activation induced up-regulation of CD54 and CD86 at the LC surface, while it did not significantly affect the levels of HLA-DR, CD1a, CD58, and CD80 expression. These phenotypic changes correlate with enhanced LC allostimulatory property, as shown by the use of paraformaldehyde-fixed LC. Furthermore, mAbs against CD40, as well as CD40-L, strongly inhibit the primary T cell response to allogeneic LC. Collectively, these data support a role for CD40/CD40-L pair in the development of normal T cell functions.

Binding Sites, Antibody↗
PubMed1998

Stimulation of CD40 in human bladder carcinoma cells inhibits anti-Fas/APO-1 (CD95)-induced apoptosis.

CD40 and the CD95 (Fas/APO-1 antigen) are both members of the tumor necrosis factor receptor family. Whereas CD40 mediates a strong growth stimulatory signal in B cells, engagement of the CD95 receptor leads to growth inhibition and induction of apoptosis. As it has been reported that CD40 activation may rescue B cells from undergoing apoptosis, we were interested to see whether it had a similar effect in other cells expressing the CD40 receptor. We used epithelial tumor cells from the urinary bladder, a cell type that frequently expresses CD40 but for which no clear function of the molecule has been assigned. We found that the ligation of CD95 with the antibody anti-APO-1 induced apoptosis in most of the cell lines tested. Stimulation of CD40 with antibodies or a soluble construct of the CD40 ligand was shown to protect cells from apoptosis, as demonstrated by their ability to suppress the growth inhibition exerted by the anti-APO-1 antibody. Our results show that CD40 stimulation make cells less vulnerable to apoptosis induced via CD95 and suggest that CD40 expression on epithelial tumors may be associated with cell survival.

Animals↗
PubMed1998

CD40 induces resistance to TNF-mediated apoptosis in a fibroblast cell line.

CD40, a member of the TNF receptor family, has been characterized as an important T-B cell interaction molecule. In B cells it co-stimulates isotype switching, proliferation, adhesion and is involved in cell death regulation. In addition to B cells, CD40 expression was found on transformed cells and carcinomas. However, little is known about its functions in these cell types. Recent studies show that CD40 mediates the production of pro-inflammatory cytokines in non-hematopoietic cells, inhibits proliferation or induces cell death. In some cell types the apoptotic program triggered by CD40 is only executed when protein synthesis is blocked, suggesting the existence of constitutively expressed resistance proteins. Here we demonstrate that CD40, similar to the 55-kDa TNF receptor (p55TNFR), has a dual role in the regulation of apoptosis in such cells. In the fibroblast cell line SV80 both CD40 and the p55TNFR trigger apoptosis when protein synthesis is blocked with cycloheximide (CHX). Simultaneous activation of both receptors results in markedly enhanced cell death. However, CD40 activation more than 4 h prior to a challenge with TNF/CHX paradoxically conferred resistance to TNF-induced cell death. Protection correlated with NF-kappaB induction and up-regulation of the anti-apoptotic zinc finger protein A20. Overexpression of A20 in turn rendered SV80 cells resistant to TNF cytotoxicity. In conclusion, our data provide evidence that CD40 may regulate cell death in non-hematopoietic cells in a dual fashion: the decision upon apoptosis or survival of a CD40-activated cell seems to depend on its ability to up-regulate resistance factors.

Animals↗
PubMed1998

LY294002-mediated inhibition of phosphatidylinositol 3-kinase activity triggers growth inhibition and apoptosis in CD40-triggered Ramos-Burkitt lymphoma B cells.

Cells of the Epstein-Barr virus genome-negative Ramos-Burkitt lymphoma (Ramos-BL) B cell line can be rescued from antigen receptor (AgR)-triggered growth inhibition and apoptosis by signals transduced through their surface CD40. This study investigates whether phosphatidylinositol 3-kinase (PI3-kinase), which has been reported to be intimately involved in the regulation of normal and neoplastic cell growth, plays a role in CD40-promoted Ramos-BL B cell survival and uses the selective and reversible PI3-kinase inhibitor, LY294002 (LY). LY-mediated inhibition of PI3-kinase activity triggers growth inhibition and leads to the processing of caspase-3, caspase-3-like activity, cleavage of the death substrate poly(ADP-ribose) polymerase (PARP), and apoptosis from the G1 phase of cell cycle. These data indicate that constitutive PI3-kinase activity is critical for Ramos-BL B cell progression through the cell cycle such that if this PI3-kinase-dependent pathway(s) is inhibited, the cells default to apoptosis. Signals transduced through CD40 abrogate LY-triggered caspase-3-like activity and PARP cleavage but fail to inhibit LY-triggered growth inhibition, processing of caspase-3, and apoptosis. Likewise, in the presence of LY, signals transduced through CD40 abrogate AgR-triggered caspase-3-like activity and PARP cleavage but fail to inhibit AgR-triggered growth inhibition, caspase-3 processing, and apoptosis. The LY-mediated induction of growth inhibition and apoptosis occurs in the presence of the CD40-induced anti-apoptotic protein Bcl-XL. Taken together these data indicate that the CD40 of Ramos BL B cells is linked to PI3-kinase-independent and -dependent routes of survival: CD40-mediated inhibition of AgR-triggered caspase-3-like activity, PARP cleavage, and CD40-triggered Bcl-XL expression are PI3-kinase-independent, whereas PI3-kinase is critical for CD40-mediated rescue of this cellular population from AgR-triggered growth inhibition, caspase-3 processing, and apoptosis.

Apoptosis↗
PubMedDate not supplied

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors.

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, IC10) can actually promote the in vitro generation of CTL activity by CD8+ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220+ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220+ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220+ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8+ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220+ cells.

Animals↗
PubMed1996

Induction of costimulatory molecules B7-1 and B7-2 in murine B cells. the CBA/N mouse reveals a role for Bruton's tyrosine kinase in CD40-mediated B7 induction.

The binding of CD40 ligand on activated T cells to CD40 on resting B cells induces the expression of costimulatory molecules B7-1 (CD80) and B7-2 (CD86). The induction of B7 molecules by CD40 ligand-CD40 interaction represents a critical step in rendering B cells competent for antigen presentation. The CBA/N mouse has a defect in CD40 signalling which has been attributed to a mutation in Bruton's tyrosine kinase. We have compared the ability of murine CD40 ligand to induce B7-1 and B7-2 expression on B cells isolated from CBA/N and wild-type CBA/J mice. We find that the CBA/N defect partially impairs both B7-1 and B7-2 induction via CD40. Subsequent experiments investigated the roles of different second messenger systems in B7-1 and B7-2 induction in normal B cells. In M12 B lymphomas either CD40 cross-linking or cAMP treatment can induce B7 molecules. Here we report that treatment with dibutyryl-cAMP also induces B7 molecules in normal B cells provided that they have been preactivated by CD40 cross-linking. We also find that PMA and ionomycin treatment of B cells induces B7-2 but not B7-1 expression. Our data therefore show roles for BTK, cAMP and PMA/ionomycin in B7 induction, as well as providing further evidence for differential regulation of B7-1 and B7-2 induction in B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1994

Regulation of immunoglobulin production by nerve growth factor: comparison with anti-CD40.

Nerve growth factor (NGF) is a well-known neurotrophic factor acting on both the peripheral and the central nervous systems. In addition, it has been shown to play a role in the function of the immune system through specific receptors. The low-affinity NGF receptor (NGFR) is present on human B lymphocytes and has been shown to have structural homology with another specific B cell surface molecule, CD40. NGF and anti-CD40 have been shown to modulate B-cell proliferation and Immunoglobulin (Ig) secretion. However, there have been no studies directly comparing the properties of these putative B-cell growth factors, particularly similarities in receptor expression or their role in B cell function. In this study, we examined the expression of NGFR and CD40 in a number of B-lymphoblastoid cell lines, representative of various stages of differentiation. We found that NGFR and CD40 are expressed on all B-cell lines to various degrees with the exception of plasma cells. Using two Ig secreting cell lines, both NGF and anti-CD40 decreased Ig secretion in a dose-dependent manner. The interaction of NGF and anti-CD40 with interleukin-4 (IL-4) was surprisingly different. Whereas IL-4 reversed the inhibitory effect of NGF on Ig secretion, it did not reverse that of anti-CD40. In addition, differences were observed at the level of receptor expression; IL-4 decreased the expression of NGFR, but increased that of CD40. These data indicate that although NGFR and CD40 are expressed in a co-ordinate fashion on B cells and exert similar effects on Ig secretion, differences in interaction with other growth factors may be important in their activities at different stages of B-cell differentiation.

Antibodies, Monoclonal↗
PubMed2005

TRAF-dependent association of protein kinase Tpl2/COT1 (MAP3K8) with CD40.

Signaling by TNF-family receptor CD40 involves TRAF-family adaptor proteins, leading to activation of protein kinases that induce NFkappaB-family transcription factors. We report here that mitogen activated protein kinase kinase kinase-8 (MAP3K8), Tpl2/COT1, is recruited to the CD40 complex via a mechanism dependent on TRAF-binding sites in CD40. Tpl2/COT1 was shown to participate in CD40 signaling based on the ability of a catalytically inactive mutant to suppress CD40-mediated IkappaB kinase activation and induction of NFkappaB-responsive promoters, without affecting signaling by TNF. Tpl2 (-/-) fibroblasts were also deficient in CD40 but not TNF signaling, further supporting a unique role for Tpl2 in CD40 signaling. Experiments using dominant-negative Tpl2 suggest this kinase functions distal to TRAFs but proximal to the TAK1/TAB1 signaling complex, within the IKK/NFkappaB activation pathway. These results indicate a distinction between TNF Receptor family members CD40 and TNFR1 in their utilization of MAP3Ks, and demonstrate TRAF-dependence of Tpl2 association with the CD40 receptor complex.

CD40 Antigens↗
PubMed2006

Internalization of CD40 regulates its signal transduction in vascular endothelial cells.

The CD40 ligand (CD40L)-CD40 dyad can ignite proinflammatory and procoagulatory activities of the vascular endothelium in the pathogenesis and progression of atherosclerosis. Besides being expressed on the activated CD4(+) T cell surface (mCD40L), the majority of circulating CD40L reservoir (sCD40L) in plasma is released from stimulated platelets. It remains debatable which form of CD40L triggers endothelial inflammation. Here, we demonstrate that the agonistic antibody of CD40 (G28.5), which mimics the action of sCD40L, induces rapid endocytosis of CD40 independent of TRAF2/3/6 binding while CD40L expressed on the surface of HEK293A cells captures CD40 at the cell conjunction. Forced internalization of CD40 by constitutively active mutant of Rab5 preemptively activates NF-kappaB pathway, suggesting that CD40 was able to form an intracellular signal complex in the early endosomes. Internalized CD40 exhibits different patterns of TRAF2/3/6 recruitment and Akt phosphorylation from the membrane anchored CD40 complex. Finally, mCD40L but not sCD40L induces the upregulation of proinflammatory cytokines and cell adhesion factors in the primary human vascular endothelial cells in vitro, although both forms of CD40L activate NF-kappaB pathway. These results therefore may help understand the molecular mechanism of CD40L signaling that contributes to the pathophysiology of atherosclerosis.

Animals↗
PubMed2005

CD40 mediated human cholangiocyte apoptosis requires JAK2 dependent activation of STAT3 in addition to activation of JNK1/2 and ERK1/2.

CD40 is critically involved in Fas-mediated cholangiocyte apoptosis during liver inflammation, but the underlying signalling events are poorly understood. Our recent work implicated AP-1 in CD40-induced cholangiocyte apoptosis, but suggested involvement of other signalling pathways. Because STAT3 has been implicated in liver regeneration we investigated this signalling pathway during CD40 mediated cholangiocyte apoptosis. Western immunoblotting, electrophoretic mobility gel shift assays, In situ DNA end labelling and caspase-3 activity were used to investigate intracellular signalling and apoptosis in primary human cholangiocytes following CD40 activation. CD40-activation induced caspase-3 dependent cholangiocyte apoptosis and 3-fold increases in JNK/ERK phosphorylation (concomitant with increased AP-1 binding activity) and 4-fold increases in pSTAT3, which were sustained for up to 24 h. Protein levels of c-Jun, c-Fos and pSTAT3 confirmed the upregulation. Phosphorylation of p38 remained unchanged suggesting that this MAP kinase was not involved in CD40 mediated apoptosis. Increased JAK2 phosphorylation accompanied increased STAT3 phosphorylation after CD40 ligation. Cholangiocytes were also shown to express JAK1 and 3 which was phosphorylated following control stimulation with TNFalpha or IL2 respectively but not after CD40 ligation. JNK, ERK and JAK2 inhibitors partially abrogated apoptosis and when used in combination reduced it to basal levels. In conclusion, induction of CD40-mediated cholangiocyte apoptosis requires JAK2-mediated phosphorylation of STAT3 as well as sustained JNK1/2, ERK1/2 activation. This study demonstrates that STAT3 can function as a proapoptotic factor in primary human liver epithelial cells.

Adenine↗
PubMed2005

Human T cell leukemia virus type I Tax activates CD40 gene expression via the NF-kappa B pathway.

The human T cell leukemia virus type I (HTLV-I) is an oncogenic retrovirus that is etiologically linked to the genesis of adult T cell leukemia (ATL) as well as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Emerging evidence suggests that the pathogenicity of HTLV-I involves deregulated activation of immune cells, especially T lymphocytes, although the underlying mechanism remains unclear. In this study, we demonstrate that HTLV-I Tax induces the aberrant expression of CD40, a member of the tumor necrosis factor receptor (TNFR) family that plays an important role in lymphocyte activation and differentiation. In a panel of HTLV-I-transformed T cell lines analyzed, CD40 expression was highly elevated compared to HTLV-I-negative T cells. Using Tax mutants and a genetically manipulated T cell system, we demonstrated that Tax-induced CD40 expression required the NF-kappaB signaling pathway. In addition, ligation of CD40 on T cells with recombinant CD40L elicited NF-kappaB activation, suggesting that the CD40 pathway is intact and may participate in a positive regulatory loop in T cells. CD40 ligation strongly synergized with Tax to activate NF-kappaB, suggesting that CD40 signals may costimulate Tax-mediated NF-kappaB activation, particularly when Tax is expressed at low levels. Collectively, these results indicate that CD40 is a novel Tax-regulated gene, and the regulation of CD40 by Tax may play a role in cellular activation and HTLV-I-induced disease pathogenesis.

CD40 Antigens↗
PubMed1997

Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-kappaB pathway involving TNF receptor-associated factors.

Expression of the Epstein-Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kappaB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187-231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kappaB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an effect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kappaB-dependent. Mutational analysis identified domains in the C-terminus of LMP1 which are important for NF-kappaB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kappaB activation and IL6 secretion. The importance of TRAF interactions in mediating these effects was confirmed using dominant negative TRAF2 and TRAF3 mutants which also identified differences in the signalling events mediated by the two NF-kappaB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kappaB activity, also blocked NF-kappaB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

Amino Acid Sequence↗
PubMed2003

Evidence for modulation of human epidermal differentiation and remodelling by CD40.

BACKGROUND: It is widely accepted that CD40 plays a critical role in the regulation of immune response. However, the significance of CD40 expression on normal human keratinocytes is only partially known. OBJECTIVES: To perform a morphological re-examination of the role of CD40 on the differentiation of human keratinocytes and remodelling of the epidermis. METHODS: Keratinocytes were grown on fibroblasts transfected with the CD40 ligand (CD40L) to investigate the formation of epidermal sheets in culture under the influence of the CD40L. Control experiments were carried out using the same cells but transfected with CD32. Further, three specific anti-CD40 monoclonal antibodies were used as soluble agonists to analyse the effect of CD40 ligation on keratinocyte differentiation. RESULTS: Epidermal sheets developing from keratinocytes cocultured with fibroblasts transfected with CD40L but not with CD32 showed an up to 50% reduction in thickness compared with control sheets. This change depended mostly on cellular flattening and a decrease in the number of cell layers, and was coincident with a transient decrease in cell surface CD40 immunoreactivity. On the other hand, normal epidermis, and freshly isolated and cultured keratinocytes revealed a predominant CD40+/Ki-67- phenotype that was demonstrated by double immunocytochemistry. Consistent with these observations, keratinocytes primed with interferon-gamma responded to the three soluble agonists, but not to control IgG1, producing immunoreactive (pro)filaggrin and displaying morphological changes in shape and size equivalent to those seen in differentiated cells. CONCLUSIONS: As a whole, our findings provide evidence that CD40+ keratinocytes represent a poorly differentiated population, not actively engaged in the cell cycle, which under specific stimulation is committed towards terminal differentiation.

Antibodies, Monoclonal↗
PubMed1997

Effect of B-cell receptor engagement on CD40-stimulated B cells.

Activation of human B cells in vitro either by cross-linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin-10 (IL-10) and interleukin-2 (IL-2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B-cell receptor (BCR) and CD40 [with anti-CD40 monoclonal antibody (mAb)] on B-cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti-CD40 and IL-10/IL-2 or IL-4 in the presence of CD32-transfected L cells. Simultaneous cross-linking of the BCR was achieved by mAb held on CD32-L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL-10/IL-2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti-CD40 and IL-10/IL-2, or compared with B cells stimulated with SA or anti-BCR mAb and IL-10/IL-2. Dual BCR and CD40 ligation with anti-immunoglobulin mAb (anti-kappa + anti-lambda light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40-induced proliferation was increased in the presence of IL-10/IL-2, although not with IL-4. The combination anti-kappa and anti-lambda with anti-CD40 showed a long-term suppression of IgG and IgM production (at least 14 days), while anti-kappa or anti-lambda alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B-cell antigen receptor cross-linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.

Antigens, Bacterial↗
PubMed2003

Pre-assembly of the extracellular domains of CD40 is not necessary for rescue of mouse B cells from anti-immunoglobulin M-induced apoptosis.

CD40 is a tumour necrosis factor receptor (TNFR) family member of central importance for the adaptive immune system. To elucidate the functional role of the different extracellular domains of CD40, we have created a set of truncated CD40 molecules where domains, or parts of domains, have been removed. These CD40 proteins, which contain a peptide tag in the N-terminal end, have been expressed in a murine B-cell line, WEHI 231. It was found that ligation of these engineered CD40 proteins via the peptide tag, was sufficient to rescue as well as to promote proliferation of apoptotic WEHI 231 cells, even when all the extracellular domains of CD40 were absent. Our results suggest that pre association of CD40 in the cell membrane plays no critical role for the CD40 signalling pathway. Furthermore, our data imply that conformational changes initiated in the extracellular domains of CD40 are not essential for signal transduction.

Animals↗
PubMed1999

Characterization of CD40-dependent immunoglobulin class switching.

Previous studies have shown that signalling via CD40 together with cytokines stimulates immunoglobulin (Ig) class switching in B cells. This process includes induction of germline (GL) transcripts and switch recombination. Using an agonistic rat anti-mouse CD40 monoclonal antibody (MoAb), we investigated the role of CD40 signalling in these molecular events. We found that stimulation of murine B cells induced high steady-state levels of germline gamma1, gamma2b and low levels of epsilon transcripts. No detectable gamma2a or alphaRNA were found and the level of gamma3 transcripts was high both in stimulated and unstimulated cells. Although cells treated with anti-CD40 MoAb had high levels of GL gamma1 and gamma3 transcripts, we failed to detect switching to IgG1 or IgG3. However, anti-CD40 MoAb-stimulated cells increased expression of IgG2b. Interestingly, anti-CD40 plus interleukin (IL)-5 induced switching to IgG1. Previous work has demonstrated that CD40 signalling, but not lipopolysaccharide (LPS), induces the gamma1 promoter and that NF-kappaB motifs are important. We show here that both LPS and anti-CD40 activated NF-kappaB proteins binding to the gamma1 promoter. The bound NF-kappaB complexes were different with regard to total concentration and subunit composition. In the light of our data, the mechanism of CD40-mediated Ig class switching is discussed.

Animals↗
PubMed1994

CD40-deficient mice generated by recombination-activating gene-2-deficient blastocyst complementation.

To study the role of B-cell antigen CD40 in immune responses, mouse embryonic stem (ES) cells in which both copies of the gene encoding CD40 had been disrupted by homologous recombination were injected in RAG-2 (recombination-activating gene-2)-deficient blastocysts to generate chimeras in which all mature lymphocytes are derived from the CD40-deficient ES cells. T- and B-cell number and phenotype were normal in the CD40-/- chimeras. However, B cells failed to proliferate and undergo isotype switching in vitro in response to soluble CD40 ligand (sCD40L) with interleukin 4 (IL-4) but responded normally to lipopolysaccharide (LPS) with IL-4. CD40-/- chimeras completely failed to mount an antigen-specific antibody response or to develop germinal centers following immunization with the T cell-dependent (TD) antigen keyhole limpet hemocyanin. In contrast, CD40-/- mutant mice responded normally to the T cell-independent (TI) antigens, 2,4,6-trinitrophenyl (TNP)-LPS and TNP-Ficoll. The most noticeable alteration in the serum immunoglobulin levels of young (6-8 weeks old) CD40-/- animals was absence of IgE and severe decrease of IgG1 and IgG2a. These results confirm the essential role of CD40- CD40L interactions in the antibody response to TD antigens and in isotype switching.

Animals↗
PubMed1996

TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling.

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned. TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B. Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.

Amino Acid Sequence↗