The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mehl, A. M.
Right arrow Articles by Brennan, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mehl, A. M.
Right arrow Articles by Brennan, P.
The Journal of Immunology, 2001, 167: 6388-6393.
Copyright © 2001 by The American Association of Immunologists

Characterization of a CD40-Dominant Inhibitory Receptor Mutant1

Anja M. Mehl2, Matthew Jones, Martin Rowe and Paul Brennan3

Infection and Immunity, University of Wales College of Medicine, Cardiff, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD40 is an important mediator of immune and inflammatory responses. It is a costimulatory molecule for B cell proliferation and survival. Blockade of CD40 has been shown to induce tolerance and its role in other pathogenic conditions has led to the proposal that CD40 inhibition could be valuable therapeutically. As a first step to this end, we have characterized a CD40-dominant negative receptor. This inhibitory mutant lacks the identified CD40 signaling domains. It inhibits both cotransfected and endogenous CD40 activation of NF-{kappa}B. This mutant is specific, as it does not affect TNF or latent membrane protein 1 signaling. Its potential usefulness is illustrated by its ability to inhibit the CD40 ligand-stimulated increases of HLA and CD54 expression, molecules involved in Ag recognition and lymphocyte recruitment leading to organ rejection. The inhibitory mutant has no TNFR-associated factor 2-binding capabilities and inhibits the recruitment of TNFR-associated factor 2 to the CD40 signaling complex after stimulation. These studies show that the CD40 inhibitory receptor molecule is effective, specific, and useful both for research and potentially as a clinical tool. And furthermore, it is likely that similar dominant inhibitory receptors can be generated for all of the members of the TNFR superfamily.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD40 is a member of the TNFR superfamily of cytokine receptors. It binds CD40 ligand (CD40L4 or CD154) during germinal center formation and provides important signals for B cell activation and survival. It is also involved in dendritic cell-B cell interactions. CD40 is expressed on a range of other cell types where it plays diverse roles including inhibition of cell growth and the induction of proinflammatory cytokines (for review, see Ref. 1).

CD40 signaling pathways and their effects on cellular phenotype have been intensively studied. CD40 consists of an extracellular CD40L-binding domain and an intracellular signaling domain with two distinct functional regions, one of which binds TNFR-associated factor (TRAF) 6 (2) and the other binds TRAF1, TRAF2, and TRAF3 molecules (3, 4, 5). Ligation of CD40 leads to the association of TRAF complexes and activation of various kinase cascades which in turn activate transcription factors, such as NF-{kappa}B (6, 7, 8) and AP-1 (9). Deletion studies of the regions further demonstrated the importance of the signaling molecules and their role in transcriptional activator functions (10, 11). CD40 signaling results in the expression of adhesion molecules (12), cytokines (13), and apoptotic mediators (14), which are associated with pathogenic processes of chronic inflammatory diseases such as autoimmune disease and graft-vs-host disease (1, 15).

Despite the importance of CD40 in numerous pathogenic conditions, few tools to specifically inhibit CD40 function are available. Abs to CD40L have been used to block CD40-CD40L interaction and have been shown to induce tolerance in transplant models. However, administration of Abs as therapy can cause cytotoxic problems (16, 17). This study was initiated to investigate the dominant inhibitory properties of a CD40 molecule in which the signaling domains have been mutated or removed. We have demonstrated the effectiveness, specificity, and functionality of the CD40-dominant inhibitory mutant. Therefore, we believe that the mutant will be useful for characterizing cell-specific effects of CD40 and has also the potential to be used as a therapeutic tool.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Gene transfection

For transient expression, 0.5–1 x 107 cells from a suspension culture were transfected by electroporation using a Bio-Rad Genepulser II electroporator (Bio-Rad, Hercules, CA) at 280 V and 950 µF at room temperature in 500 µl of growth medium. The cells were reseeded in 5 ml of fresh growth medium and were then incubated under normal conditions. Transfection efficiency ranged from 10 to 20% for Eli-BL (EBV-positive Burkitt lymphoma cell line, Ref. 18) and DG75 (EBV-negative Burkitt lymphoma cell line Ref. 19), and up to 40–50% for Jurkat (T cell line, Ref. 20), as assessed by cotransfection with the EGFP-C1 expression vector and flow cytometry analysis. At 24 h after transfection, cells were stimulated with CD40L-expressing fibroblasts (21), which were irradiated (5000 rad) and incubated for another 24 h. As a control for CD40L-specific effect, incubation of transfected DG75 cells with nontransformed fibroblasts (22) had no effect on NF-{kappa}B induction (data not shown).

Assay for NF-{kappa}B activity

The activity of the NF-{kappa}B reporter plasmid was measured 24 h posttransfection. Cells were washed twice in PBS and lysed in 150 µl of lysis buffer containing 100 mM HEPES (pH 8.0), 2 mM magnesium chloride, 5 mM DTT, and 2% Triton X-100. Luciferase activity in 50 µl of clarified lysate was analyzed in a Berthold LB9501 luminometer following injection of 100 µl of 0.5 mM luciferin (USB, Cleveland, OH) dissolved in luciferin assay reagent (30 mM glycylglycin (pH 7.9), 1 mM MgCl2, 0.1 mM EDTA, 30 mM DTT, 0.3 mM CoA, and 0.5 mM ATP). Light release was integrated over 10 s.

Analysis of cell surface protein expression

The induction of CD40, CD54, and HLA proteins in transfected cells was assayed by immunofluorescence staining of viable cells by flow cytometry using a BD Biosciences FACSCalibur. Briefly, at 48 h posttransfection the cells were washed and stained with a PE-conjugated mAb to either human CD54 (MCA675PE; Serotec, Oxford, U.K.) or CD40 (MCA1590PE; Serotec) at 4°C for 60 min. For HLA staining, we used W632 supernatant at 4°C for 60 min followed by mouse IgRPE (R0439; DAKO, Glostrup, Denmark) at 4°C for 45 min. The transfected population was marked by the expression of cotransfected EGFP-C1 plasmid and gated for analysis of surface protein staining.

Immunoprecipitations and Western blotting

For each immunoprecipitation, 15 x 106 cells of the Jurkat or DG75 cell line in 0.5 ml of growth medium were electroporated at 280 V and 950 µF with either latent membrane protein 1 (LMP1; control), CD40 wild-type (wt), or CD40delA-expressing plasmid DNA along with 6 µg of TRAF2-expressing plasmid. At 24 h posttransfection, half of the cells were incubated with CD40L-expressing fibroblast as described earlier. At 48 h posttransfection, the cells were washed twice with PBS, then lysed for 45 min on ice in 800 µl of Nonidet P-40 lysis buffer (0.5% Nonidet P-40, 50 mM HEPES buffer, 0.25 M NaCl, and 2 mM EDTA), to which a 5% protease inhibitor mixture (P8340; Sigma-Aldrich, St. Louis, MO) was added before use. The lysates were clarified by centrifugation at 13,000 x g for 5 min, and the soluble fraction was then precleared for 4 h with 20 µl of Sepharose-protein G, to which 1 µg of control Ab had been covalently cross-linked. The precleared lysate was then incubated overnight with 20 µl of Sepharose-protein G, to which 1 µg of CD40 rabbit Ab (sc975; Santa Cruz Biotechnolgy, Santa Cruz, CA) or 1 µg of CD40 mouse Ab (815002; Ancell, Bayport, MN) or a mixture of the LMP1 Abs CS.3 (0.5 µg) and CS.4 (0.5 µg) had been covalently cross-linked. The immunoprecipitates were washed three times and eluted by boiling in 40 µl of SDS gel sample buffer. Half of the eluate was separated by SDS-PAGE and analyzed for TRAF2 expression by Western blotting using a TRAF2 rabbit polyclonal Ab (sc876; Santa Cruz Biotechnology).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The CD40 mutant, CD40delA, is nonfunctional and can act as a dominant negative

This project was initiated to test the hypothesis that a CD40 molecule, which lacked signaling properties, could be used as a dominant inhibitory molecule. The CD40 mutant used in this study, CD40delA, is mutated and deleted at the critical sequences for binding the TRAF signaling molecules (2, 4, 5, 23). This mutant therefore lacks the ability to bind any signaling molecules (10, 24) and was recently used to identify the region involved in CD40-induced apoptosis (11). The features of the CD40delA mutant with the deleted TRAF6-binding region (del 219–239) and the point-mutated second TRAF-binding domain (T254A) (11) are shown in Fig. 1Goa.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 1. Features and functionality of the CD40 protein. a, Schematic of the mutant used in this study. Relevant signaling protein-binding domains are boxed, and the positions of deleted or mutated amino acids are shown. b, Assay for NF-{kappa}B luciferase reporter activity in Jurkat cells transfected with different amounts (0–8 µg) of either CD40 ({blacksquare}) or CD40delA ({square}) along with NF-{kappa}B reporter plasmid. Luciferase was measured after 24 h, and the results are representative of at least three independent experiments. c, Assay for NF-{kappa}B activation in Jurkat cells cotransfected with variable amounts (0–8 µg) of CD40delA mutant along with wt CD40 plasmid (3 µg) and NF-{kappa}B reporter plasmid (3 µg). The luciferase activity was measured after 24 h, and the results, shown relative to wt CD40 activity (set to 100%, 2336.3 ± 209.3), are representative of at least three independent experiments.

 
CD40 induces many different signal-transducing pathways, such as mitogen-activated protein kinase pathways and the NF-{kappa}B pathway, which are important for the regulation of cell survival and apoptosis (25). However, one of the most important signaling cascades involved in CD40 signaling is the activation of the transcription factor NF-{kappa}B. Hence, our first step was to characterize the ability of CD40delA to activate a NF-{kappa}B luciferase reporter in Jurkat cells, a T lymphoblastoid cell line negative for CD40. Fig. 1Gob shows that while expression of wt CD40 efficiently induces NF-{kappa}B activity, CD40delA caused only a very small increase in luciferase activity. This suggested that CD40delA is a good candidate for a dominant inhibitory CD40 molecule. We tested this possibility by transient transfection of Jurkat cells with increasing amounts of CD40delA and a constant dose of CD40-expressing plasmid (Fig. 1Goc). The results show a dose-dependent inhibition of CD40-induced NF-{kappa}B activation by CD40delA with a maximal down-regulation of NF-{kappa}B activation below 10% of CD40 activity (Fig. 1Goc). It should also be noted, that transfection of other plasmids did not nonspecifically inhibit CD40-induced NF-{kappa}B activation (see Fig. 3Go). These results clearly demonstrate that CD40delA is able to effectively inhibit CD40-induced NF-{kappa}B activation and has therefore the potential to act as a dominant negative inhibitor for the CD40 receptor.



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 3. Specificity of the CD40delA and LMP1dn mutants. Jurkat cells were transfected with empty vector, LMP1dn (3 µg), or CD40delA plasmid (3 µg) along with NF-{kappa}B reporter plasmid (3 µg) and either wt LMP1 (3 µg) or wt CD40 (3 µg) constructs. The cells were incubated overnight to allow protein expression and, in the case of the TNF-{alpha} stimulation (left panel), the cells were treated for 6 h with TNF-{alpha} (10 ng/ml) before luciferase activity was measured. The data, shown in relation to stimulated control transfectants (set to 100%, representing a mean value of 1856 ± 324 luciferase units for TNF-{alpha} (+ column in left panel), 23531 ± 782 U for LMP1 (+ column in middle panel), and 4128 ± 444 U for CD40 (+ column in right panel), are representative of at least three independent experiments.

 
The CD40delA mutant is an effective inhibitor of endogenous CD40 signaling

CD40 signaling is important for B cell activation. The effect of CD40delA on endogenous CD40 was investigated in B cells that express CD40. There are many B cell lines described which respond to CD40L-induced stimulation in terms of NF-{kappa}B activation and induction of cell surface molecules (26). We chose the DG75 and Eli-BL B cell lines, both of which can be transfected and effectively stimulated using CD40L-expressing fibroblasts. First of all, we analyzed the expression levels of CD40 on cells transfected with control plasmid and CD40delA-expressing plasmid (Fig. 2Go, a and b). For this purpose, DG75 cells were transfected with different amounts of CD40delA-expressing plasmid along with a fixed amount of green fluorescence protein (GFP)-expressing plasmid. At 48 h posttransfection, the cells were stained for CD40 and the positive transfected population was analyzed by flow cytometry. Transfection of 5 µg of CD40delA results in a three times higher mean fluorescence intensity compared with basal CD40 levels in those cells, and transfection with high amounts of the CD40delA (such as 20 µg) shows a 6-fold higher CD40 mean fluorescence intensity than control transfected cells (Fig. 2Gob).



View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 2. Inhibition of endogenous CD40 signaling by CD40delA. DG75, or Eli-BL cells were transfected with increasing amounts of CD40delA plasmid along with a constant amount (1 µg) of GFP-expressing plasmid. After 8 h posttransfection, half of the cells were incubated with irradiated (5000 rad) CD40L-expressing fibroblasts until they were assayed. The controls only transfected with empty vector and GFP, lacking the CD40delA plasmid, were always treated in the same way. a, CD40 expression was determined in nonstimulated DG75 cells 48 h posttransfection by fluorescence staining and flow cytometry (FACS). The histogram shows the number of CD40-positive cells either transfected with control vector (dark gray) or with 5 µg of CD40delA (light gray) relative to basal levels (black) of the GFP-transfected cell population. b, CD40 expression levels (mean fluorescence intensity, MFI) were determined in nonstimulated DG75 cells transfected with a range of 0–20 µg of CD40delA plasmid. The results shown are representative of at least three independent experiments. c and d, NF-{kappa}B luciferase activity was measured in CD40L-stimulated ({blacksquare}) and nonstimulated ({square}) responder cell lines. DG75 (c) and Eli-BL (d) cells were transfected with NF-{kappa}B reporter plasmid (3 µg) and with a range of 0–20 µg or 0–8 µg of CD40delA plasmid, respectively. The plasmid concentration was kept constant by adding empty vector to the final concentration of 20 or 8 µg. Unstimulated cells and cells exposed to control fibroblasts showed no NF-{kappa}B induction. The results shown are representative of at least three independent experiments.

 
After determining the expression levels of transfected and endogenous CD40, we analyzed the NF-{kappa}B activation by CD40L-induced CD40 stimulation in the transfected cells expressing CD40delA. For this purpose, DG75 and Eli-BL cells were transfected with a range of concentrations of CD40delA-expressing plasmid (0–20 µg) along with NF-{kappa}B luciferase reporter plasmid, and the luciferase activity in the CD40L-stimulated cells was measured (Fig. 2Go, c and d). The results show that DG75 cells have a much higher basal NF-{kappa}B level than Eli-BL cells and, more importantly, that CD40delA is able to reduce the luciferase activity in both stimulated cell lines down to basal NF-{kappa}B activity levels. This demonstrates that CD40delA effectively inhibits endogenous CD40-induced NF-{kappa}B activation in a dose-dependent manner down to the basal NF-{kappa}B activity levels in CD40 responder cell lines.

CD40delA is specific for CD40-induced signaling pathways

CD40 shares intracellular signal-transducing molecules and pathways with other members of the TNFR superfamily. Our strategy, using a dominant negative CD40 receptor molecule, should allow inhibition of CD40/CD40L signaling without interfering with other receptors using the same signal transduction pathways. To test this hypothesis, we examined the effect of CD40delA upon NF-{kappa}B activation by two related mechanisms, the TNFR/TNF-{alpha} and the LMP1 of the EBV. Both molecules have been shown to induce NF-{kappa}B transcriptional activation after stimulation (6, 27) and are able to bind several cytosolic signaling factors, including the TRAFs (28, 29, 30). Given the similarity among CD40, TNF-{alpha}, and LMP1 signaling, we tested the specificity of the CD40delA mutant in these related systems using a NF-{kappa}B luciferase reporter assay and comparing CD40-induced NF-{kappa}B activation with TNF-{alpha} and LMP1. With this system, we also analyzed the effects of a dominant negative form of the LMP1 (LMP1dn) (31), which has lost its signaling molecule binding function by point-mutating amino acids at positions 204, 206, 208, and 384 in the two so-called C-terminal activator regions. The experiments were conducted as transient transfection reporter assays, analyzing NF-{kappa}B luciferase activity in Jurkat cells transfected with CD40delA or LMP1dn-expressing plasmid, which were either stimulated with TNF-{alpha} or cotransfected with wt LMP1 or CD40, respectively (Fig. 3Go). The results shown here clearly demonstrate that CD40delA does not interfere with the ability of either TNF-{alpha} or LMP1 to activate the NF-{kappa}B signaling pathway in the transfected cells. Furthermore, the LMP1-dominant negative mutant, which was designed using a similar strategy to CD40delA, does not inhibit TNF-{alpha} or CD40-induced NF-{kappa}B. Thus, we conclude that the CD40delA and LMP1dn mutants are specific inhibitors and do not interfere with the signaling capabilities of the other receptors. This implies a range of applications for specific dominant negative receptor molecules and should lead to the development of other TNFR-based dominant negative mutants.

CD40delA inhibits CD40-induced phenotypical changes

NF-{kappa}B is a major effector molecule involved in CD40 signaling and is responsible for many cell surface changes due to CD40 ligation, such as expression of CD54 and HLA, which are involved in immune responses (1, 32, 33). HLA, for example, is one of the most important molecules involved in immune responses directed against alloantigens on grafts; thus, inhibition of CD40-mediated up-regulation by CD40delA could have great potential. In the case of CD54 it is known that, in acute rejection, CD40 stimulation and CD54 expression are important mediators of lymphocyte recruitment into rejecting allografts (34). Therefore, the effects of CD40delA on the CD40-induced stimulation of CD54 and HLA were analyzed in the high responder cell line DG75 (26). These cells were transfected with different amounts of CD40delA-expressing plasmid along with GFP-expressing plasmid. After CD40 stimulation, the expression levels of the cell surface proteins CD54 and HLA in the positive transfected population were analyzed by flow cytometry (Fig. 4Go). In the case of both markers, the basal levels of expression are not altered in the cells transfected with different amounts of the CD40delA mutant ( in Fig. 4Gob). Stimulation with CD40L resulted in an up-regulation of CD54 and HLA expression as shown. Most importantly, however, this activation is effectively down-regulated in cells expressing the CD40delA mutant. This illustrates the fact that the CD40delA mutant is able to inhibit CD40-induced phenotypical changes.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 4. Analysis of the biological function of CD40delA. DG75 cells were transfected with increasing amounts of CD40delA plasmid along with GFP-expressing plasmid (1 µg). At 24 h posttransfection, half of the cells were incubated with irradiated (5000 rad) CD40L-expressing fibroblasts for another 24 h before analyzing surface marker expression by fluorescence staining and flow cytometry (FACS). The controls lacking the CD40delA plasmid were treated in the same way. a, CD54 mean fluorescence intensity (MFI) of GFP-positive cells in nonstimulated (No Ligand) and stimulated (+ CD40 Ligand) cultures. The results shown are representative of at least three independent experiments. b, HLA mean fluorescence intensity (MFI) of GFP-positive cells in the nonstimulated (- Ligand) and stimulated (+ Ligand) cultures. The results shown are representative of at least three independent experiments.

 
CD40delA prevents TRAF2 recruitment to wt CD40

One possible mechanism for the observed dominant negative effect of the CD40delA molecule could be its inability to bind signaling molecules important for CD40 function. The TNF adapter molecule, TRAF2, is one of the major components of the CD40 signaling cascade and because the CD40delA construct is deleted and mutated for the TRAF binding sites it should be unable to bind TRAF2. To investigate this possibility, coimmunoprecipitation experiments were conducted to analyze the binding of TRAF2 to CD40 and CD40delA and to determine whether CD40delA could inhibit the recruitment of TRAF2 to wt CD40. For this purpose, Jurkat and DG75 cells were transfected with LMP1, CD40 wt, CD40delA, or both CD40wt- and CD40delA-expressing plasmids in the presence of TRAF2 (Fig. 5Go). The cells were left for 24 h to allow expression before they were stimulated with CD40L for another 24 h. The cells were then lysed and the lysates were precleared with Sepharose-protein G. LMP1 was immunoprecipitated with the Abs CS.3 and CS.4 as a positive control for a protein that binds TRAF2. CD40 was immunoprecipitated with either a specific polyclonal rabbit CD40 Ab (Fig. 5Goa) or with a specific monoclonal mouse CD40 Ab (Fig. 5Gob). The immunoprecipitates were resolved by SDS-PAGE and the presence of TRAF2 was detected by Western blotting. The results from a representative experiment in Jurkat cells are shown in Fig. 5Goa (top panel). It shows that TRAF2 can be coimmunoprecipitated with both LMP1 and CD40. However, CD40delA, which lacks the TRAF binding site, did not coimmunoprecipitate TRAF2. In the cells cotransfected with wt CD40 and CD40delA, coprecipitated TRAF2 could not be detected. Similar results were obtained in DG75 cells (Fig. 5Gob). However, TRAF2 binding was only observed after stimulation of the cells with CD40L. These results support the hypothesis that CD40delA inhibits the binding of TRAF2 to CD40 via a mechanism whereby CD40delA binds to wt CD40, preventing TRAF2 binding; thus inhibiting CD40-induced signal transducing pathways. These data provide a mechanism for inhibition of signaling by CD40delA, which result in the inhibition of phenotypical changes.



View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 5. Analysis of TRAF2 recruitment of CD40delA. To analyze the TRAF2 binding abilities, Jurkat (a) and DG75 (b) cells were transfected with empty vector, LMP1 (positive control), CD40 (wt), CD40delA (delA), or CD40 along with CD40delA (wt + delA) together with TRAF2-expressing plasmid (6 µg). At 24 h posttransfection, the cells were incubated with irradiated (5000 rad) CD40L-expressing fibroblasts for another 24 h. The cells were lysed and samples for input lysates were taken. The precleared extracts were incubated with cross-linked protein G-Sepharose using either LMP1 (positive control) or rabbit polyclonal CD40 (a) or mouse monoclonal CD40 (b) Abs. After 12 h of incubation, the beads were washed and the proteins were eluted by boiling in gel sample buffer. The proteins were separated using SDS-PAGE and the presence of TRAF2 was investigated by Western blotting probing with rabbit antiserum to TRAF2.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study describes the characterization of a dominant inhibitory CD40 molecule, CD40delA. This CD40 mutant is effective and specific for CD40 signaling, inhibiting the activation of the transcription factor, NF-{kappa}B, and the induction of gene expression changes. Our choice of CD40delA as a dominant inhibitory molecule was inspired by our previous experience with the EBV-encoded protein LMP1, which functions like a constitutive member of the TNFR superfamily. We have generated a dominant inhibitory form of LMP1 (31) by mutating the TRAF binding sites in the molecule. The coexpression of dominant inhibitory LMP1 prevents the binding of TRAF2 to wt LMP1. CD40delA lacks the ability to bind any signaling molecules (10, 24). Our experiments showed that CD40delA could prevent coimmunoprecipitation of TRAF2 with wt CD40, suggesting that dominant inhibitory CD40 and LMP1 share a similar mechanism. TRAF2 binding requires a conserved binding motif (35) and receptor oligomerization (36, 37). Crystallographic analysis shows that the trimeric receptor complex recognizes a TRAF trimer (38). Our proposed mechanism is that incorporation of CD40delA into the CD40 receptor complex prevents the binding of TRAFs and thus the downstream signals. The observation that CD40delA can also inhibit endogenous CD40 signaling is a substantial advance and demonstrates the effectiveness of this approach.

This study combined with our work on LMP1 has led to a hypothesis that all members of the TNFR superfamily can be inhibited by such an approach. A study of lymphoproliferative syndromes identified dominant interfering Fas mutations that are similar to the ones that we have generated for LMP1 and CD40 (39). Furthermore, a receptor-based mutant has also been generated for TNF (40), although this was a receptor truncation, which may have less efficiency than receptors with smaller mutations. The generation of receptor-based mutants for LMP1, CD40, Fas, and TNF implies that all members of the TNFR superfamily may be exploited in this way.

One of the major benefits of a receptor-based dominant inhibitor approach is specificity. We have shown that the introduction of CD40delA does not affect either TNF signaling through its endogenous receptor or cotransfected LMP1 signaling while effectively inhibiting coexpressed CD40. Similar specificity was observed for the dominant negative LMP1. This approach has the potential for more selective inhibition than that provided by the use of dominant inhibitory forms of shared signaling molecules, such as TRAFs, TNFR-associated death domain, or I{kappa}B, the inhibitor of NF-{kappa}B. Although they are useful, these mutants lack selectivity since these signaling molecules are shared by many different cell stimuli.

NF-{kappa}B is one of the most important signaling pathways stimulated by CD40. CD40delA effectively inhibits NF-{kappa}B activation. We chose not to test each individual CD40-stimulated pathway but instead to focus on gene expression changes. CD40 increases the expression of MHC class I and ICAM-1 (CD54), which are known to be important for immune responses (1, 32, 33). CD40delA inhibits the induction of both of these proteins. ICAM-1 is particularly interesting, as maximal induction of ICAM-1 by LMP1 has been shown to involve, as yet, uncharacterized signaling pathways (41). Although we have not investigated the pathways that CD40 utilizes for the regulation of ICAM-1, the parallels between LMP1 and CD40 signaling suggests that all their pathways may be shared. Thus, the inhibition of ICAM-1 and HLA induction by CD40delA suggests that pathways beyond NF-{kappa}B activation are effectively inhibited.

CD40 induction of HLA and ICAM-1 plays a role in graft rejection (34). Blockade of CD40 has been shown to induce tolerance (42, 43, 44). The inhibition of these important molecules by the CD40delA mutant suggests that it may be as effective as Ab blockade. However, it is important to verify the use of CD40delA in an animal model. Experiments to investigate the in vivo applicability of CD40delA to inhibit immune responses will be conducted in a rodent model using a viral-based gene delivery used previously (45). The effectiveness of the dominant interfering Fas in a transgenic model provides some in vivo evidence for this approach (46). The validation of the CD40delA mutant in vivo is the next step toward the exploitation of this molecule in the clinic.

In conclusion, the functionality, specificity, and efficiency of CD40delA shows that this mutant could serve as a dominant negative tool to inhibit CD40 signaling involved in pathogenic conditions. The potential of the CD40delA mutant for therapy is particularly interesting in light of the questions about the application of anti-CD40L Ab treatment to humans and the lack of synthetic inhibitors for the CD40-CD40L interaction (1, 43). Thus, dominant negative receptor mutants may be useful tools for additional therapeutic strategies.


   Acknowledgments
 
We are grateful to Dr. A. Eliopoulos for providing the CD40 mutant.


   Footnotes
 
1 This work was supported by grants from the Leukemia Research Fund, London (9533 and 9842), the Wellcome Trust, and the Leverhulme Trust. Back

2 Current address: Cellular Immunology of Autoimmune Reactions, Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Strasse 10, 13125 Berlin, Germany. Back

3 Address correspondence and reprint requests to Dr. Paul Brennan, Department of Medicine, University of Wales College of Medicine, Tenovus Building, Cardiff, CF14 4XX, U.K. E-mail address: brennanp{at}cardiff.ac.uk Back

4 Abbreviations used in this paper: CD40L, CD40 ligand; GFP, green fluorescence protein; LMP1, latent membrane protein 1; LMP1dn, dominant negative form of LMP1; wt, wild type; TRAF, TNFR-associated factor. Back

Received for publication June 1, 2001. Accepted for publication September 26, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schonbeck, U., P. Libby. 2001. The CD40/CD154 receptor/ligand dyad. Cell. Mol. Life Sci. 58:4.[Medline]
  2. Ishida, T., S. Mizushima, S. Azuma, N. Kobayashi, T. Tojo, K. Suzuki, S. Aizawa, T. Watanabe, G. Mosialos, E. Kieff, et al 1996. Identification of TRAF6, a novel tumor necrosis factor receptor- associated factor protein that mediates signaling from an amino- terminal domain of the CD40 cytoplasmic region. J. Biol. Chem. 271:28745.[Abstract/Free Full Text]
  3. Cheng, G., A. M. Cleary, Z. S. Ye, D. I. Hong, S. Lederman, D. Baltimore. 1995. Involvement of CRAF1, a relative of TRAF, in CD40 signaling. Science 267:1494.[Abstract/Free Full Text]
  4. Rothe, M., S. C. Wong, W. J. Henzel, D. V. Goeddel. 1994. A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor. Cell 78:681.[Medline]
  5. Hu, H. M., K. O’Rourke, M. S. Boguski, V. M. Dixit. 1994. A novel RING finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem. 269:30069.[Abstract/Free Full Text]
  6. Rothe, M., V. Sarma, V. M. Dixit, D. V. Goeddel. 1995. TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. Science 269:1424.[Abstract/Free Full Text]
  7. Lalmanach-Girard, A. C., T. C. Chiles, D. C. Parker, T. L. Rothstein. 1993. T cell-dependent induction of NF-kappa B in B cells. J. Exp. Med. 177:1215.[Abstract/Free Full Text]
  8. Francis, D. A., J. G. Karras, X. Y. Ke, R. Sen, T. L. Rothstein. 1995. Induction of the transcription factors NF-{kappa}B, AP-1 and NF-AT during B cell stimulation through the CD40 receptor. Int. Immunol. 7:151.[Abstract/Free Full Text]
  9. Sakata, N., H. R. Patel, N. Terada, A. Aruffo, G. L. Johnson, E. W. Gelfand. 1995. Selective activation of c-Jun kinase mitogen-activated protein kinase by CD40 on human B cells. J. Biol. Chem. 270:30823.[Abstract/Free Full Text]
  10. Inui, S., T. Kaisho, H. Kikutani, I. Stamenkovic, B. Seed, E. A. Clark, T. Kishimoto. 1990. Identification of the intracytoplasmic region essential for signal transduction through a B cell activation molecule, CD40. Eur. J. Immunol. 20:1747.[Medline]
  11. Eliopoulos, A. G., C. Davies, P. G. Knox, N. J. Gallagher, S. C. Afford, D. H. Adams, L. S. Young. 2000. CD40 induces apoptosis in carcinoma cells through activation of cytotoxic ligands of the tumor necrosis factor superfamily. Mol. Cell. Biol. 20:5503.[Abstract/Free Full Text]
  12. Yellin, M. J., S. Winikoff, S. M. Fortune, D. Baum, M. K. Crow, S. Lederman, L. Chess. 1995. Ligation of CD40 on fibroblasts induces CD54 (ICAM-1) and CD106 (VCAM- 1) up-regulation and IL-6 production and proliferation. J. Leukocyte Biol. 58:209.[Abstract]
  13. Eliopoulos, A. G., M. Stack, C. W. Dawson, K. M. Kaye, L. Hodgkin, S. Sihota, M. Rowe, L. S. Young. 1997. Epstein-Barr virus-encoded LMP1 and CD40 mediate IL-6 production in epithelial cells via an NF-{kappa}B pathway involving TNF receptor- associated factors. Oncogene 14:2899.[Medline]
  14. Smith, C. A., T. Farrah, R. G. Goodwin. 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 76:959.[Medline]
  15. Reul, R. M., J. C. Fang, M. D. Denton, C. Geehan, C. Long, R. N. Mitchell, P. Ganz, D. M. Briscoe. 1997. CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection. Transplantation 64:1765.[Medline]
  16. Wekerle, T., J. Kurtz, H. Ito, J. V. Ronquillo, V. Dong, G. Zhao, J. Shaffer, M. H. Sayegh, M. Sykes. 2000. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat. Med. 6:464.[Medline]
  17. Li, Y., X. C. Li, X. X. Zheng, A. D. Wells, L. A. Turka, T. B. Strom. 1999. Blocking both signal 1 and signal 2 of T-cell activation prevents apoptosis of alloreactive T cells and induction of peripheral allograft tolerance. Nat. Med. 5:1298.[Medline]
  18. Rowe, M., A. L. Lear, D. Croom-Carter, A. H. Davies, A. B. Rickinson. 1992. Three pathways of Epstein-Barr virus gene activation from EBNA1- positive latency in B lymphocytes. J. Virol. 66:122.[Abstract/Free Full Text]
  19. Ben-Bassat, H., N. Goldblum, S. Mitrani, T. Goldblum, J. M. Yoffey, M. M. Cohen, Z. Bentwich, B. Ramot, E. Klein, G. Klein. 1977. Establishment in continuous culture of a new type of lymphocyte from a "Burkitt like" malignant lymphoma (line D.G.-75). Int. J. Cancer 19:27.[Medline]
  20. Brattsand, G., D. A. Cantrell, S. Ward, F. Ivars, M. Gullberg. 1990. Signal transduction through the T cell receptor-CD3 complex: evidence for heterogeneity in receptor coupling. J. Immunol. 144:3651.[Abstract]
  21. Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau. 1994. Activation of human dendritic cells through CD40 cross-linking. J. Exp. Med. 180:1263.[Abstract/Free Full Text]
  22. Arias, M. A., J. E. Rey Nores, N. Vita, F. Stelter, L. K. Borysiewicz, P. Ferrara, M. O. Labeta. 2000. Cutting edge: human B cell function is regulated by interaction with soluble CD14: opposite effects on IgG1 and IgE production. J. Immunol. 164:3480.[Abstract/Free Full Text]
  23. Sarma, V., Z. Lin, L. Clark, B. M. Rust, M. Tewari, R. J. Noelle, V. M. Dixit. 1995. Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis. J. Biol. Chem. 270:12343.[Abstract/Free Full Text]
  24. Tsukamoto, N., N. Kobayashi, S. Azuma, T. Yamamoto, J. Inoue. 1999. Two differently regulated nuclear factor {kappa}B activation pathways triggered by the cytoplasmic tail of CD40. Proc. Natl. Acad. Sci. USA 96:1234.[Abstract/Free Full Text]
  25. Kehry, M. R.. 1996. CD40-mediated signaling in B cells. Balancing cell survival, growth, and death. J. Immunol. 156:2345.[Abstract]
  26. Henriquez, N. V., E. Floettmann, M. Salmon, M. Rowe, A. B. Rickinson. 1999. Differential responses to CD40 ligation among Burkitt lymphoma lines that are uniformly responsive to Epstein-Barr virus latent membrane protein 1. J. Immunol. 162:3298.[Abstract/Free Full Text]
  27. Huen, D. S., S. A. Henderson, D. Croom-Carter, M. Rowe. 1995. The Epstein-Barr virus latent membrane protein-1 (LMP1) mediates activation of NF-{kappa}B and cell surface phenotype via two effector regions in its carboxy-terminal cytoplasmic domain. Oncogene 10:549.[Medline]
  28. Floettmann, J. E., A. G. Eliopoulos, M. Jones, L. S. Young, M. Rowe. 1998. Epstein-Barr virus latent membrane protein-1 (LMP1) signalling is distinct from CD40 and involves physical cooperation of its two C-terminus functional regions. Oncogene 17:2383.[Medline]
  29. Eliopoulos, A. G., A. B. Rickinson. 1998. Epstein-Barr virus: LMP1 masquerades as an active receptor. Curr. Biol. 8:R196.[Medline]
  30. Izumi, K. M., E. D. Kieff. 1997. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-{kappa}B. Proc. Natl. Acad. Sci. USA 94:12592.[Abstract/Free Full Text]
  31. Brennan, P., J. E. Floettmann, A. Mehl, M. Jones, M. Rowe. 2001. Mechanism of action of a novel latent membrane protein-1 dominant negative. J. Biol. Chem. 276:1195.[Abstract/Free Full Text]
  32. Kansas, G. S., T. F. Tedder. 1991. Transmembrane signals generated through MHC class II, CD19, CD20, CD39, and CD40 antigens induce LFA-1-dependent and independent adhesion in human B cells through a tyrosine kinase-dependent pathway. J. Immunol. 147:4094.[Abstract]
  33. Lee, H. H., P. W. Dempsey, T. P. Parks, X. Zhu, D. Baltimore, G. Cheng. 1999. Specificities of CD40 signaling: involvement of TRAF2 in CD40-induced NF-{kappa}B activation and intercellular adhesion molecule-1 up-regulation. Proc. Natl. Acad. Sci. USA 96:1421.[Abstract/Free Full Text]
  34. Kamoun, M.. 2001. Cellular and molecular parameters in human renal allograft rejection. Clin. Biochem. 34:29.[Medline]
  35. Ye, H., Y. C. Park, M. Kreishman, E. Kieff, H. Wu. 1999. The structural basis for the recognition of diverse receptor sequences by TRAF2. Mol. Cell 4:321.[Medline]
  36. VanArsdale, T. L., S. L. VanArsdale, W. R. Force, B. N. Walter, G. Mosialos, E. Kieff, J. C. Reed, C. F. Ware. 1997. Lymphotoxin-{beta} receptor signaling complex: role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor {kappa}B. Proc. Natl. Acad. Sci. USA 94:2460.[Abstract/Free Full Text]
  37. Park, Y. C., V. Burkitt, A. R. Villa, L. Tong, H. Wu. 1999. Structural basis for self-association and receptor recognition of human TRAF2. Nature 398:533.[Medline]
  38. McWhirter, S. M., S. S. Pullen, J. M. Holton, J. J. Crute, M. R. Kehry, T. Alber. 1999. Crystallographic analysis of CD40 recognition and signaling by human TRAF2. Proc. Natl. Acad. Sci. USA 96:8408.[Abstract/Free Full Text]
  39. Fisher, G. H., F. J. Rosenberg, S. E. Straus, J. K. Dale, L. A. Middleton, A. Y. Lin, W. Strober, M. J. Lenardo, J. M. Puck. 1995. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935.[Medline]
  40. Tartaglia, L. A., D. V. Goeddel. 1992. Tumor necrosis factor receptor signaling: a dominant negative mutation suppresses the activation of the 55-kDa tumor necrosis factor receptor. J. Biol. Chem. 267:4304.[Abstract/Free Full Text]
  41. Mehl, A. M., J. E. Floettmann, M. Jones, P. Brennan, M. Rowe. 2001. Characterization of intercellular adhesion molecule-1 regulation by Epstein-Barr virus-encoded latent membrane protein-1 identifies pathways that cooperate with nuclear factor {kappa}B to activate transcription. J. Biol. Chem. 276:984.[Abstract/Free Full Text]
  42. Larsen, C. P., T. C. Pearson. 1997. The CD40 pathway in allograft rejection, acceptance, and tolerance. Curr. Opin. Immunol. 9:641.[Medline]
  43. Rothstein, D. M., M. F. Livak, K. Kishimoto, C. Ariyan, H. Y. Qian, S. Fecteau, M. Sho, S. Deng, X. X. Zheng, M. H. Sayegh, G. P. Basadonna. 2001. Targeting signal 1 through CD45RB synergizes with CD40 ligand blockade and promotes long-term engraftment and tolerance in stringent transplant models. J. Immunol. 166:322.[Abstract/Free Full Text]
  44. Rossi, G., J. Sarkar, D. Scandella. 2001. Long-term induction of immune tolerance after blockade of CD40-CD40L interaction in a mouse model of hemophilia A. Blood 97:2750.[Abstract/Free Full Text]
  45. McGrath, Y., G. W. Wilkinson, O. B. Spiller, B. P. Morgan. 1999. Development of adenovirus vectors encoding rat complement regulators for use in therapy in rodent models of inflammatory diseases. J. Immunol. 163:6834.[Abstract/Free Full Text]
  46. Choi, Y., V. R. Ramnath, A. S. Eaton, A. Chen, K. L. Simon-Stoos, D. E. Kleiner, J. Erikson, J. M. Puck. 1999. Expression in transgenic mice of dominant interfering Fas mutations: a model for human autoimmune lymphoproliferative syndrome. Clin. Immunol. 93:34.[Medline]



This article has been cited by other articles:


Home page
 Mol Cancer ResHome page
A. L. Willis, N. L. Tran, J. M. Chatigny, N. Charlton, H. Vu, S. A.N. Brown, M. A. Black, W. S. McDonough, S. P. Fortin, J. R. Niska, et al.
The Fibroblast Growth Factor-Inducible 14 Receptor Is Highly Expressed in HER2-Positive Breast Tumors and Regulates Breast Cancer Cell Invasive Capacity
Mol. Cancer Res., May 1, 2008; 6(5): 725 - 734.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Mehl, A. M.
Right arrow Articles by Brennan, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mehl, A. M.
Right arrow Articles by Brennan, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS