Induction of an Altered CD40 Signaling Complex by an Antagonistic Human Monoclonal Antibody to CD40 Katherine C. Bankert, Kyp L. Oxley, Sonja M. Smith, John P. Graham, Mark de Boer, Marielle Thewissen, Peter J. Simons and Gail A. Bishop J Immunol published online 20 March 2015 http://www.jimmunol.org/content/early/2015/03/20/jimmun ol.1402903

Supplementary Material

http://www.jimmunol.org/content/suppl/2015/03/20/jimmunol.140290 3.DCSupplemental.html

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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This information is current as of March 23, 2015.

Published March 20, 2015, doi:10.4049/jimmunol.1402903 The Journal of Immunology

Induction of an Altered CD40 Signaling Complex by an Antagonistic Human Monoclonal Antibody to CD40 Katherine C. Bankert,*,1 Kyp L. Oxley,*,1,2 Sonja M. Smith,*,3 John P. Graham,†,2 Mark de Boer,‡ Marielle Thewissen,‡ Peter J. Simons,x and Gail A. Bishop*,†,{,‖

C

D40, a member of the TNFR superfamily, is expressed by multiple immune and nonimmune cell types and plays key roles in stimulating effective Ag presentation and humoral immunity (1). CD40 signaling is implicated in the pathogenesis of both autoimmunity (2, 3) and transplant rejection (4, 5), and disruption of CD40-mediated activation has been of considerable therapeutic interest (reviewed in Ref. 5). The first interventions tested used mAbs specific for the CD40L, CD154, to block the receptor–ligand interaction. This approach showed great

*Department of Microbiology, University of Iowa, Iowa City, IA 52242; †Interdisciplinary Graduate Program in Immunology, University of Iowa, Iowa City, IA 52242; ‡ Fast Forward Pharmaceuticals BV, Utrecht, the Netherlands; xBioceros Holding BV, Utrecht, the Netherlands; {Department of Internal Medicine, University of Iowa, Iowa City, IA 52242; and ‖VA Medical Center, Iowa City, IA 52242 1

K.C.B. and K.L.O. contributed equally to this work.

2

Current address: The Jackson Laboratory, Bar Harbor, ME.

3

Current address: Department of Pediatrics, University of Iowa, Iowa City, IA.

Received for publication November 17, 2014. Accepted for publication February 20, 2015. This work was supported in part by funds from the Holden Chair in Cancer Biology (to G.A.B.), PanGenetics BV (Utrecht, the Netherlands), and Fast Forward Pharmaceuticals. G.A.B. is a Senior Research Career Scientist of the Veterans Administration. This material is based upon work supported in part by the Office of Research and Development, Veterans Health Administration, Department of Veterans Affairs. Address correspondence and reprint requests to Dr. Gail A. Bishop, University of Iowa, 2193B MERF, 375 Newton Road, Iowa City, IA 52242. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: BCM, B cell medium; ch5D12, chimeric form of 5D12; cIAP1, cellular inhibitor of apoptosis-1; DC, dendritic cell; hCD40, human CD40; hCD154, human CD154; HOIP, HOIL1-L interacting protein; IKK, IkB kinase; R10, RPMI 1640 medium with 10% FCS, 1% HEPES, 1% L-glutamine, 1% penicillin/ streptomycin, and 1% essential amino acids; TRAF, TNFR-associated factor. Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402903

promise in preclinical mouse models of arthritis (6) and allograft transplant (7). However, the first human clinical trial using antiCD154–blocking mAb was halted as the result of several fatal thromboembolic events (8), which may have been due to the effect of immune complexes upon activated platelets expressing CD154. Although a variety of approaches to blocking CD40–CD154 interactions have since been explored and may prove successful (5), an attractive alternative is direct inhibition of the CD40-mediated activation pathway. The mAb PG102 was derived from the previously described antihuman CD40 (hCD40) mAb 5D12 (9), which inhibits the proliferation of human B cells stimulated with activated T cells or agonistic CD40-specific mAbs (10, 11). Subsequently, a chimeric form of 5D12 (ch5D12) containing human IgG4 constant regions was produced. The ch5D12 mAb prevents development of experimental autoimmune encephalomyelitis in marmosets (12), as well as prolongs the survival of kidney allografts and allows repeated systemic administration of adenoviral gene therapy vectors in rhesus monkeys (13, 14). In an open-label dose-escalation phase I/IIa study of Crohn’s disease patients, ch5D12 was well tolerated and showed promising clinical benefit (15). Several amino acids in both H and L chains of the ch5D12 mAb were subsequently changed to remove potential MHC-binding epitopes and thus reduce possible immunogenicity (M. de Boer, personal communication); the resultant mAb is PG102. Previous studies showed that ch5D12 inhibits CD40-induced production of proinflammatory cytokines (16), and we observed this as well with PG102 in B cell cultures (K.L. Oxley and G.A. Bishop, unpublished observations). The present study was undertaken to test the hypothesis that PG102 inhibits CD40 signaling by inducing a signaling cascade that is distinct from that initiated by binding of CD154 or agonistic anti-CD40 Abs to CD40. Results reveal that engagement of CD40

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Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating autoimmune diseases and transplant rejection. CD154-blocking mAbs used in human clinical trials resulted in unanticipated vascular complications, leading to heightened interest in the therapeutic potential of antagonist mAbs specific for human CD40. Abs that do not require physical competition with CD154 to inhibit CD40 signaling have particular therapeutic promise. In this study, we demonstrate that the antagonist anti-human CD40 mAb PG102 fails to trigger CD40-mediated activation, as well as impairs CD154mediated CD40 activation, via a distinct nonstimulatory CD40 signaling mechanism. PG102 did not induce early CD40-induced signaling events, and it inhibited early kinase and transcription factor activation by CD154 or agonist anti-CD40 mAbs. However, PG102 stimulated normal CD40-mediated TNFR-associated factor (TRAF)2 and TRAF3 degradation. PG102 induced the formation of a CD40 signaling complex that contained decreased amounts of both TRAF2 and TRAF3 and TRAF2-associated signaling proteins. Additionally, PG102-induced CD40 signaling complexes failed to recruit TRAF6 to detergent-insoluble membrane fractions. Fab fragments of PG102, while retaining CD40 binding, did not induce TRAF degradation, nor could they inhibit CD154stimulated B cell signaling, indicating that CD40 aggregation is required for the signaling inhibition induced by PG102. The antagonistic impact of PG102 on CD40 signaling reveals that the manner of CD40 ligation can determine sharply different outcomes for CD40 signaling and suggests that such information can be used to therapeutically manipulate these outcomes. The Journal of Immunology, 2015, 194: 000–000.

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by PG102 induced formation of a dysfunctional CD40 signaling complex characterized by altered TNFR-associated factor (TRAF) recruitment and association, with resultant ineffective induction of kinase and transcription factor activation. Importantly for potential therapeutic applications, although PG102 did not inhibit CD154– CD40 binding, it inhibited and prevented CD154-induced CD40 activation.

Materials and Methods Cells

mAbs and CD154 The agonistic anti-hCD40 mouse IgG1 mAb G28.5 (18) was produced by saturated AmSO4 precipitation of supernatant from a hybridoma obtained from the American Type Culture Collection (Manassas, VA). The antagonistic humanized anti-hCD40 mAb PG102 is a less immunogenic form of the humanized anti-hCD40 mAb ch5D12 (12) and was provided by Fast Forward Pharmaceuticals. The humanized anti-hCD40 control mAb 4D11 was used in Fig. 1 (19). The isotype-control Abs used were mIgG1 (Southern Biotech, Birmingham, AL) and hIgG4k (Sigma-Aldrich, St. Louis, MO) for G28.5 and PG102, respectively. The source of human CD154 was membranes of CD154-expressing Hi5 insect cells produced in the Bishop laboratory, as previously described (20). Hi5 cells grow at 25˚C; they die and form membrane fragments when cultured at 37˚C. The following primary Abs were used for Western blotting and are listed by their species and recognition specificities: rabbit anti–phospho-JNK, rabbit anti–phospho-IkBa, rabbit anti-total IkBa, mouse anti–IkB kinase (IKK)g, rabbit anti–phospho-p38, rabbit anti–phospho-ERK, rabbit antiTRAF6 (all from Cell Signaling Technology, Danvers, MA), rabbit antitotal JNK and rabbit anti-TRAF3 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-actin (Millipore, Billerica, MA), rabbit anti-TRAF2 (Medical and Biological Laboratories, Nagoya, Japan), and mouse anti– cellular inhibitor of apoptosis-1 (cIAP1; R&D Systems, Minneapolis, MN). The rabbit anti–HOIL1-L interacting protein (HOIP) Ab was obtained from Dr. B. Hostager (University of Iowa). The sheep anti-hCD40 Ab (21) was generated in the Bishop laboratory. The secondary Abs used included goat anti-mouse IgG, goat anti-rabbit IgG, goat anti-chicken IgG, donkey antisheep IgG (all from Jackson ImmunoResearch, West Grove, PA), and goat anti-hamster IgG (Santa Cruz Biotechnology).

Simultaneous binding of PG102 and human CD154 to hCD40 ELISA. Flat-bottom 96-well EIA/RIA plates (Corning Life Sciences, Amsterdam, the Netherlands) were coated with human recombinant CD40Fc fusion protein (Bioceros) at 0.25 mg/ml in PBS (pH 7.2). After blocking with 1% w/v BSA, saturating (2 mg/ml) human CD154 (hCD154)–mouse CD8 fusion protein (Ancell, Bayport, MN) was added, followed by saturating (1 mg/ml) PG102 or 4D11 (or vice versa). hCD154 or PG102/4D11 was detected with a combination of biotinylated rat anti-mouse CD8 Ab (Ancell) and HRP-labeled streptavidin (Jackson ImmunoResearch) or with HRP-labeled goat anti-human k L chain Ab (The Jackson Laboratory), respectively. Optical densities were read at 450 nm after development with a ready-to-use tetramethylbenzidine solution, according to the

Signaling assays A total of 1–2 3 106 cells was washed in RPMI 1640 medium, resuspended in 1 ml BCM in 1.5-ml Eppendorf tubes, and rested for 45 min at 37˚C. Cells were stimulated for the time periods indicated in the figure legends with anti-hCD40 or isotype-control mAbs at 10 mg/ml. Alternatively, cells were incubated with Hi5 insect cells, either wild-type or expressing hCD154, as described previously (20). Whole-cell lysates were prepared as described (21).

Immunoprecipitation and Western blotting For immunoprecipitation of CD40 and associated proteins, Dynal protein G magnetic beads (Invitrogen) were coated with anti-hCD40 mAb (10 mg/ 10 ml beads), as described previously (22). Abs were conjugated to beads according to the manufacturer’s protocols. A total of 2 3 107 cells was incubated in 1 ml BCM with mAb-coated beads for 10 min at 37˚C. Beads and cells were then pelleted and lysed, as described previously (22). Beadbound proteins were resuspended in 23 SDS-PAGE loading buffer and boiled for 3–5 min at 95˚C prior to loading samples for PAGE. For Western blotting, 10–15 ml sample was resolved on 10% SDS-PAGE. Proteins were transferred to Immobilon-P membranes (Millipore). Membranes were blocked with 10% dry milk in TBST for 1 h, washed in TBST, incubated with primary Abs overnight, washed in 10% dry milk in TBST, and incubated with secondary Abs for 1–2 h or overnight. Protein bands were visualized using a chemiluminescent detection reagent (Pierce, Rockford, IL). Images of blots were recorded with a low-light imaging system (LAS3000; Fujifilm Medical Systems Stamford, CT). Immunoblots were reprobed with appropriate Abs specific for control proteins, such as actin, to verify equal protein loading in each lane.

TRAF-degradation assay Cells were placed in a 24-well plate at 1 3 106 cells/ml/well. Anti-CD40 mAbs were added to appropriate wells at 10 mg/ml, and cells were incubated at 37˚C for 5 h. Cells were then transferred to 1.5-ml Eppendorf tubes and pelleted (7000 rpm, 1 min, 4˚C). Cells were resuspended in 23 SDS sample dye and sonicated (Branson Sonifier 450, 1.5 output, 90% duty cycle), and samples were boiled at 95˚C for 3 min.

Analysis of detergent-soluble and insoluble membrane signaling complexes Cells (1 3 107/condition) were stimulated with mAbs or insect cell membranes, as described above, or left unstimulated and then pelleted (400 3 g, 1 min). The cell pellets were resuspended in lysis buffer (1% Brij, 150 mM NaCl, 20 mM Tris [pH 7.5], 50 mM b-glycerophosphate, 2 mini complete EDTA-free tablets, and sodium vanadate) and incubated for 30 min on ice. After incubation, cell lysates were centrifuged (30 min, 4˚C, 400 3 g), and supernatants were transferred to a new tube. Lysis buffer + 0.5% SDS and 1% 2-ME were added. Pellets were sonicated (1.5 output, 90% duty cycle), and 23 SDS- PAGE buffer was added to both pellet and supernatant. Both fractions were boiled (3 min, 95˚C).

Generation of PG102 Fab fragments Fab fragments from PG102-intact IgGs were generated using the Pierce Fab Preparation Kit (Thermo Fisher Scientific, Boston, MA), according to the manufacturer’s instructions. After this procedure, only PG102 Fab fragments were found by standard SDS-PAGE and Coomassie Blue staining (data not shown). Subsequently, the binding of these PG102 Fabs on hCD40-expressing JY B cells was compared with PG102-intact IgGs using flow cytometry. Results are presented in Supplemental Fig. 1B and confirm effective binding of Fabs to hCD40, indistinguishable from the binding of intact PG102.

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The human B cell line T5-1 was described previously (17). PBMCs from human blood donors were isolated from discarded Leukocyte Reduction System cones obtained from the DeGowin Blood Center (University of Iowa). The Blood Center has University of Iowa Institutional Review Board approval to provide these deidentified samples to investigators. Cells were cultured in RPMI 1640 medium with 10% heat-inactivated FCS (Atlanta Biologicals, Atlanta, GA), 10 mM 2-ME (Invitrogen, Grand Island, NY), 2 mM L-glutamine, penicillin, and streptomycin (B cell medium [BCM]). Human primary monocyte-derived dendritic cells (DCs) were obtained from the above-described human blood samples, as follows. Monocytes were isolated from these PBMCs by culturing for 30 min and washing away nonadherent cells. A cell scraper was used to dislodge monocytes, which were washed and resuspended in R10 (RPMI 1640 medium with 10% FCS, 1% HEPES, 1% L-glutamine, 1% penicillin/ streptomycin, and 1% essential amino acids) at 1 3 106 cells/3ml with GM-CSF (100 ng/ml) and recombinant human IL-4 (10 ng/ml). Cells were cultured in six-well tissue culture plates at 3 ml/well. At days 2 and 5, 900 ml culture medium was removed and replenished with 1 ml fresh R10 containing 300 ng/ml GM-CSF and 30 ng/ml IL-4. To generate mature DCs, immature DCs were harvested on day 5, washed with PBS, seeded (5 3 105 cells/ml/well) in fresh R10 supplemented with GM-CSF (100 ng/ml) and recombinant human IL-4 (10 ng/ml), and stimulated with LPS (1 mg/ml) for 48 h.

manufacturer’s instructions (Invitrogen), using an ELISA reader (BioRad, Hercules, CA). All binding reactions were performed at room temperature in the presence of 1% w/v BSA (Roche) and 0.05% v/v Tween-20 detergent. FACS. After blocking with 50 mg/ml human IgG (Sigma-Aldrich), hCD40expressing JY human B lymphoblastoid cells (American Type Culture Collection) were incubated with saturating (15 mg/ml) hCD154–mouse CD8 fusion protein (Ancell), followed by saturating (10 mg/ml) biotinylated PG102 (or vice versa). Subsequently, hCD154 and PG102 were detected with FITC-labeled rat anti-mouse CD8 Ab (Ancell) and PE-labeled streptavidin (Jackson ImmunoResearch), respectively. Fluorescent signals were measured using a flow cytometer (Becton Dickinson, Mt. View, CA). All binding reactions were performed at 4˚C in the presence of 0.1% v/v BSA (Sigma-Aldrich) and 0.05% w/v sodium azide.

The Journal of Immunology Statistical analysis The statistical significance of the results from multiple experiments was determined by the Student t test. The ranges of p values are indicated in the figure legends.

Results Independent CD40 binding by PG102 and CD154

internalization of the CD40 complex; after 2 h of incubation with PG102 at 37˚C, no detectable differences in CD40 surface levels were observed (Supplemental Fig. 1A). Lack of effective induction of early CD40 signaling by PG102 Engagement of CD40 by agonistic anti-CD40 Abs in B lymphocytes and myeloid cells results in rapid induction of activation of MAPKs, including JNK, ERK, and p38, as well as activation of the canonical NF-kB1 transcriptional pathway (23, 24). Because the 5D12/PG102 mAb inhibits human B cell proliferation and Ig production induced by either activated T cells or agonistic antihCD40 Ab (10, 11), we investigated whether this corresponds to a lack of CD40-mediated early signals induced by PG102. Supplemental Fig. 2A demonstrates that the agonistic anti-hCD40 mAb G28.5, as expected, stimulated robust activation of MAPK and NF-kB1 in human primary DCs. In contrast, PG102 stimulated detectable, but quite modest, MAPK activation and little to no NF-kB1 activation. Initial studies of PG102 examined CD40-mediated B cell activation, so we focused upon this cell type for our subsequent experiments. Fig. 2 shows that PG102 did not induce detectable phosphorylation of either JNK or IkBa in the human B cell line T5-1, which was previously reported as a robust model of CD40 signaling to human B cells (20, 25). Fig. 2A compares the phosphorylated forms of JNK and IkBa in a representative Western

FIGURE 1. Lack of CD154–PG102 binding competition. (A) PG102 and hCD154 binding to plate-bound CD40. Binding assays were performed as described in Materials and Methods, using binding of HRP-labeled secondary reagents as a detection method. Cells were incubated first with PG102 or control mAb, followed by CD154, and CD154 was detected (upper panel). The initial incubations were with CD154, followed by PG102 or control mAbs; PG102 binding was detected (lower panel). Results represent the mean 6 SD of triplicate cultures from a representative of three experiments. (B) PG102 or 4D11 mAb and hCD154 binding to cell-expressed hCD40. hCD40-expressing human B lymphoblastoid cells (JY) were incubated with FITC-labeled hCD154, followed by PE-labeled PG102 or vice versa, as described in Materials and Methods (left panel). Cells were incubated first with PE-4D11 mAb and then FITC-hCD154 (right panel). Flow cytometry of stained cells from a representative of three experiments is shown. BG, background staining.

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Most reagents designed to inhibit CD40 signaling for therapeutic purposes block the binding of CD40 to CD154 (reviewed in Refs. 2, 5). Thus, it was of interest to determine whether the previously reported inhibitory impact of the hCD40-specific mAb PG102 on CD40-mediated immune cell activation results from blocking of the CD40–CD154 interaction. This was addressed using two complementary approaches, described in detail in Materials and Methods. An ELISA approach used plates coated with soluble hCD40, incubated with various combinations of soluble hCD154, PG102, or control Abs. Fig. 1A shows that, regardless of whether hCD154 or PG102 was added first, neither inhibited the binding of the other. The same results were obtained using intact human B cells, analyzed by flow cytometry (Fig. 1B). Fig. 1B also presents, for comparison, results with the 4D11 anti-hCD40 mAb, which blocked hCD154 binding to CD40. Thus, PG102 does not impair CD154 binding to CD40 and vice versa. We also found that PG102 binding to hCD40 on human B cells did not result in rapid

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blot of lysates from B cells stimulated with G28.5 or PG102 antihCD40 mAbs for the indicated minutes. Fig. 2B shows quantification of phospho-JNK relative to total JNK, as well as phosphoIkBa relative to actin (because total IkBa is degraded following activation), providing the mean values obtained from three individual experiments.

Inhibition of CD154-mediated B cell activation by PG102 PG102 did not bind to or block binding of CD154 (Fig. 1). Thus, we wished to test the hypothesis that binding of PG102 induces an altered CD40 signaling complex that is refractory to agonistic stimuli. Therefore, we treated human B cells with membrane-bound CD154 alone, together with PG102, or subsequent to PG102 addition.

FIGURE 3. PG102-mediated inhibition of CD154 signaling. (A) T5-1 human B cells were stimulated with hCD154 alone, CD154+PG102, or PG102 alone for 30 min, followed by CD154 for the indicated minutes. Phosphorylation of JNK and IkBa was assessed as in Fig. 2. A representative of three experiments is shown. (B) Quantification, as in Materials and Methods, for three replicates of the experiment shown in (A), expressed as fold increase over values at the zero time point. Data are mean 6 SD. (C) T5-1 human B cells were stimulated with hCD154 alone, CD154+PG102, PG102 for 30 min + CD154, or CD154 for 30 min + PG102 for the indicated number of minutes. Phosphorylation of JNK and IkBa was assessed as in (A); line graphs represent fold increase over values at the zero time point. **p , 0.01. Iso, cells treated for 30 min with isotype-control Ab for PG102.

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FIGURE 2. Failure of PG102 to effectively activate early CD40-mediated signaling events. (A) Cells of the human B cell line T5-1 were incubated with the hCD40-specific mAbs G28.5 or PG102 for the indicated minutes (Min), as described in Materials and Methods. Cell lysates were subjected to SDS-PAGE, and Western blotting was performed for total or p-JNK, p-IkBa, and actin (as a loading control; total IkBa decreases with activation so cannot serve this function). A representative of three experiments is shown. (B) Relative quantification, as in Materials and Methods, of CD40-induced phosphorylation of JNK and IkBa, normalized to total JNK or actin, respectively. Data are mean 6 SD of three experiments. **p , 0.01. Iso, cells treated for 30 min with isotype-control Abs for PG102 or G28.5.

The Journal of Immunology

CD40-mediated TRAF degradation induced by PG102 CD40 signals to B cells rapidly induce recruitment of TRAF2 and TRAF3 to the CD40 cytoplasmic domain, followed by TRAF2dependent K48-linked polyubiquitination and proteasome-mediated degradation of both of these TRAFs (21, 26–28). Because PG102 could not induce CD40-mediated activating signals, we examined whether the mAb induced this regulatory event. Data presented in Fig. 4 indicate that PG102 induced CD40-mediated degradation of both TRAF2 and TRAF3 in human B cells, to an extent that was not detectably different from degradation induced by the agonistic mAb G28.5. This was also true in primary human DCs (Supplemental Fig. 2B). Consistent with these findings, relative levels of ubiquitination of CD40-associated TRAF2 induced by PG102 versus G28.5 in human B cells were indistinguishable (K.C. Bankert and G.A. Bishop, unpublished observations). CD40 signaling complex formation induced by PG102 Data shown in Fig. 4 indicate that PG102 showed selective induction of CD40-mediated signaling events, with normal degradation of TRAF2 and TRAF3. We next examined recruitment of signaling proteins known to play major roles in CD40-mediated signals to B cells (24, 29–31). Following stimulation with either G28.5 or PG102 mAbs, CD40 was immunoprecipitated from human B cells, and associated proteins were normalized to CD40 to determine their relative amounts. Although amounts of TRAF6 associated with the signaling complex were not statistically different, CD40 from PG102-stimulated B cells had significantly less associated TRAF2 and TRAF3 (Fig. 5). Representative Western blots in Fig. 5A show that this was not due to the decreased ability of PG102 to bind or precipitate hCD40. In addition, the important TRAF2-associated signaling proteins cIAP1, IKKg, and HOIP showed significantly reduced amounts in the PG102induced CD40 signaling complex. Although T5-1 cells are a well-validated model for CD40 signaling to human B cells, this is a transformed cell line, and CD40 is also biologically important in a variety of immune cells, particularly myeloid cells. Thus, we examined CD40 signaling complex formation in primary human PBMCs. Fig. 5C presents a representative Western blot demonstrating that the marked decrease in TRAF2 and with CD40 in PG102- versus G28.5-stimulated cells is also seen in primary human immune cells. The decrease in TRAF3 was more modest and did not reach statistical significance, as it did in the T5-1 cells (Fig. 5B). However, the trend of

FIGURE 4. CD40-mediated TRAF degradation. (A) T5-1 human B cells were incubated for 5 h with mAbs G28.5 or PG102 or BCM alone. Cell lysates were prepared and separated by SDS-PAGE, followed by Western blotting for TRAF2, TRAF3, and actin, as described in Materials and Methods. A representative of three experiments is shown. Arrows indicate size markers (kDa). (B) Quantification of TRAF band intensities, as in Materials and Methods, normalized to actin. Data are mean 6 SD of three experiments. *p , 0.05, **p , 0.01. ns, not significant.

reduced TRAF2/3 binding was consistent, and Fig. 5C indicates that this is seen for PBMCs from multiple healthy human blood donors. Another feature of CD40-induced signaling complex formation is the localization of the complex to cholesterol-rich plasma membrane fractions (“rafts”), separated by their relative detergent insolubility in cell lysates (21, 32). Stimulation with either CD154 or PG102 induced movement of TRAF2 and TRAF3 to these membrane rafts; however, because their overall recruitment to CD40 was decreased (Fig. 5), the amounts in the rafts were also lower (data not shown). Fig. 5 shows that there was not a significant decrease in total TRAF6–CD40 binding induced by PG102 versus CD154. However, although CD40 translocates to the detergent-insoluble fraction following PG102 stimulation, PG102, when used alone to stimulate B cells, was unable to induce TRAF6 localization to this fraction (Fig. 6). Notably, PG102 treatment for 30 min or 2 h prior to the addition of hCD154 also inhibited hCD154-induced TRAF6 translocation (Fig. 6). Thus, the CD40 signaling complex induced by PG102 displayed altered associations of membrane CD40 with TRAF2, TRAF3, and TRAF6. Requirement for CD40 cross-linking to induce PG102-mediated signaling The results described above show that PG102-mediated CD40 engagement induced normal degradation of TRAF2 and TRAF3 but inhibited CD154-mediated CD40 signaling, without interfering with CD154 binding to CD40. Taken together, these data support the hypothesis that PG102-mediated inhibition of CD40 signaling is not passive, but instead induces formation of an abnormal signaling complex that inhibits CD154-induced CD40 signaling. Normal CD40 signaling requires cross-linking of membrane CD40. To determine whether aggregation of CD40 by PG102 is required for PG102-induced effects, we prepared Fab fragments of PG102, which bound normally to CD40 (Supplemental Fig. 1B). Fig. 7 demonstrates that, similar to intact mAb, PG102 Fabs did not induce CD40-mediated JNK or NF-kB1 activation, but the Fabs were ineffective in impairing agonist-induced activation of these events. Additionally, in contrast to intact PG102, Fab fragments did not induce TRAF degradation in human B cells. Thus, PG102 must induce CD40 aggregation to inhibit CD40 signaling by alteration of the signaling pathway.

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Results presented in Fig. 3 indicate that either simultaneous or pretreatment of B cells with PG102 abrogated CD154-mediated JNK and IkBa phosphorylation. Fig. 3A (representative Western blot) and Fig. 3B (quantification of results from three experiments) show that either PG102 pretreatment or simultaneous addition of PG102 and hCD154 to B cells resulted in marked inhibition of early signaling events. Fig. 3C further demonstrates that this inhibition lasted throughout the course of JNK or NF-kB activation and could be mediated by PG102, even when this mAb was added to cells subsequent to an hCD154 stimulus. Similar findings were obtained in myeloid cells (M. Thewissen and M. de Boer, unpublished observations). These data suggest several potential mechanisms, which are not mutually exclusive. One explanation is that a distinct, negative signal delivered via PG102 to the CD40 signaling complex counteracted and prevented normal agonist-mediated activating signals. Another possibility is that PG102 induced formation of a defective CD40 signaling complex, which competed with the functional signaling complex induced by CD154. We next examined the nature of CD40 signals induced by PG102.

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Discussion Initial discovery of the CD40 receptor preceded that of its ligand. Although agonistic mAbs to CD40 initially suggested its role as an immune cell activator (18, 33), the discovery of its key physiological roles in immune responses followed identification of the ligand for CD40 (initially called gp39 or CD40L, now designated CD154) (34–36). This, in turn, led to elucidation of the critical role for defective CD40 signaling in the human immunodeficiency disease X-linked hyper-IgM syndrome (37–40). Soon after the appreciation of CD40’s important functions in host immune defenses, studies implicated an important contribution of CD40mediated activation in pathogenic processes, including transplant rejection, autoimmune diseases, and B cell malignancies (reviewed in Refs. 2–4, 41–43). Thus, CD40, and its activation by CD154 became attractive targets for these clinical problems. The earliest and most frequent approach to inhibiting CD40mediated signaling in vivo is to interfere with CD40–CD154 interactions. Blocking Abs to CD154 interfere with the development of collagen-induced arthritis, a mouse model of human rheumatoid arthritis (6), as well mouse models of human systemic lupus erythematosus (44, 45). These findings were followed by many studies in various laboratory models, showing promise for blocking CD40– CD154 signaling in the alleviation of autoimmune and chronic inflammatory diseases (reviewed in Ref. 5). Additionally, blocking Abs

to CD154 showed efficacy in the suppression of transplant rejection and graft-versus-host disease in both mouse and preclinical primate models (4, 7, 46–48). However, initial human clinical trials of the anti-hCD154 mAb hu5c8/BG9588/ruplizumab in systemic lupus erythematosus patients, based upon these promising preclinical results, resulted in unexpected and serious thromboembolic complications in some patients. This problem also emerged in trials of other anti-CD154 mAbs (reviewed in Ref. 5). The cause of these complications is not entirely clear, but it may be related to the expression of CD154 on human platelets, together with FcRs that can bind the therapeutic mAbs. Thus, alternative approaches to inhibit CD40 signaling are needed. This could include development of CD154-blocking Abs or Ab fragments that cannot bind FcgRs but retain high affinity for CD154 and show normal, unaccelerated serum clearance. However, if direct ligation of platelet CD154, not just FcR binding, is responsible for certain complications, this drawback will remain. Peptidomimetics can also provide a way to block CD40–CD154 interactions (49). However, peptide-binding affinities can frequently be modest, requiring large and potentially impractical quantities to effectively compete with CD154 for CD40 binding. An approach that circumvents the limitations of both of the prior strategies is the use of nonagonistic CD40 mAbs. A number of such reagents act by competing directly with CD154 for CD40 binding

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FIGURE 5. CD40 signaling complex composition. (A) T5-1 human B cells were stimulated with magnetic beads coated with mAbs G28.5 or PG102; these beads were also used to immunoprecipitate the CD40 signaling complex, as described in Materials and Methods. CD40-associated proteins were subjected to SDS-PAGE, and Western blotting was performed for the indicated proteins. A representative of five experiments is shown. (B) Relative quantities of CD40-associating proteins in signaling complexes induced by G28.5 versus PG102. Amounts are normalized to the amount of hCD40 immunoprecipitated. The number of experiments averaged for each panel is represented by the “n” value. The p values were calculated as described in Materials and Methods. (C) CD40 signaling complex induced by G28.5 or PG102 in primary human PBMCs. PBMCs from normal human blood donors were stimulated, and hCD40 immunoprecipitates were analyzed as in (A). The “n” value refers to the number of human blood donors tested.

The Journal of Immunology

(reviewed in Ref. 5); thus, they must be delivered at sufficiently high concentration to outcompete CD154 binding. However, the nonagonistic mAb to hCD40 described in this report, PG102, does not require or act by blocking CD154 binding. Importantly, binding of hCD40 by PG102, although it does not remove hCD40 from the cell surface, renders B cells refractory to subsequent stimulation with CD154. This raises the attractive possibility that smaller amounts of PG102 can be therapeutically effective in inhibiting CD40-mediated immune activation in vivo, while also avoiding any complications arising from ligation of CD154. There is precedent for the concept that ligation of different epitopes on the CD40 molecule can induce distinct signaling cascades (50, 51), and even agonistic anti-CD40 Abs are not equivalent to CD154 stimulation in all regards (52). The results demonstrate that strong binding to CD40, as is the case with PG102, does not necessarily predict agonistic activity. The striking inability of PG102mediated CD40 engagement to trigger early signaling pathways in immune cells is consistent with its reported inhibition of CD40mediated B cell effector functions (10, 11). This also correlates with PG102-mediated inhibition of in vivo CD40-dependent promotion of inflammation and transplant rejection in animal models (12–14) and early clinical human studies (15). In some regards, this PG102-induced altered CD40 signaling resembles the “alterna-

FIGURE 7. CD40 cross-linking requirement for PG102 signaling. (A) CD40-mediated degradation of TRAF2 and TRAF3 was assessed in T5-1 human B cells, as in Fig. 4, comparing intact mAbs with equimolar amounts of PG102 Fab fragments, prepared as described in Materials and Methods. Relative quantification of amounts of TRAFs in cell lysates was determined from three experiments, as in Fig. 4. (B) PG102 inhibition of CD154-mediated JNK and IkBa phosphorylation was measured in T51 human B cells, as in Fig. 3, comparing intact PG102 with Fab fragments, as in (A). Values represent the mean 6 SD of three experiments, quantified as in Fig. 3. *p , 0.05. ns, not significant.

tive” signaling pathways that bacterial superantigens are reported to trigger when they bind TCR/MHC complexes (reviewed in Ref. 53). To gain further insight into how possible allosteric effects of PG102 binding may alter formation of the CD40 signaling complex, we examined two key features of this process. CD40 ligation results in immediate redistribution of CD40 into cholesterol-rich detergent-insoluble fractions of the plasma membrane (21, 32), to which it rapidly recruits TRAF2 and TRAF3, followed by a somewhat slower recruitment of TRAF6 (21, 54). The TRAF2/3 recruitment facilitates TRAF2-dependent K48-mediated polyubiquitination of these TRAFs, leading to their subsequent proteasomal degradation (26–28). This degradation limits and prevents prolonged CD40 signaling (31, 55, 56). Thus, we examined each of these events in response to PG102 compared with agonistic stimuli. The results revealed that PG102 induced levels of TRAF2 and TRAF3 degradation that were indistinguishable from those induced by an agonistic CD40 stimulus in both human B cells and DCs (Fig. 4, Supplemental Fig. 2). However, the amounts of TRAF2 and TRAF3 recruited to total cellular CD40 were decreased significantly in both a human B cell line and freshly isolated PBMCs from normal donors. This resulted in a concomitant decrease in PG102-induced CD40 binding of TRAF2-associated signaling proteins, including cIAP1, IKKg, and the ubiquitination regulator HOIP. The latter findings are complex and intriguing. Reduced HOIP and IKKg recruitment is consistent with the inability of PG102 to stimulate CD40-mediated NF-kB activation. However, although cIAP1 was reported to participate in CD40-mediated ubiquitination and subsequent degradation of TRAF2 and TRAF3, we found no detectable impairment of this regulatory event following PG102 ligation. Thus, although PG102 effectively stimulated events involved in normal restraint of CD40 signaling, this mAb did not stimulate activation events. This suggests that the reduced recruitment of TRAF2, cIAP1, and HOIP was nonetheless sufficient to mediate TRAF2 and TRAF3 degradation downstream of PG102 ligation and/or that additional unidentified CD40-binding molecules can also mediate these events. Recruitment of CD40 and associated TRAF molecules to detergent-insoluble membrane fractions was also altered when PG102 was used to engage hCD40

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FIGURE 6. Altered TRAF6 recruitment to the detergent-insoluble fraction of the plasma membrane by PG102. T5-1 human B cells were incubated, as in Materials and Methods, with control or hCD154-expressing insect cell (Hi5) membranes or PG102, as indicated, for 30 min. Cell lysates were separated into detergent-soluble (Sol) and insoluble (In) fractions prior to Western blotting, as in Fig. 5. Cell fractions were blotted for TRAF6 and hCD40. A representative of three total experiments is shown.

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ALTERED CD40 SIGNALING COMPLEX INDUCED BY AN ANTAGONIST mAb

Acknowledgments We thank Dr. Bruce Hostager for valuable discussion and critical review of the manuscript.

Disclosures M.d.B. and M.T. are employees of Fast Forward Pharmaceuticals, which owns the PG102 mAb. G.A.B. has consultant arrangements with, and has received payments for travel expenses from, Fast Forward Pharmaceuticals. The other authors have no financial conflicts of interest.

References 1. Van Kooten, C., and J. Banchereau. 1996. CD40-CD40 ligand: a multifunctional receptor-ligand pair. Adv. Immunol. 61: 1–77. 2. Toubi, E., and Y. Shoenfeld. 2004. The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity 37: 457–464. 3. Peters, A. L., L. L. Stunz, and G. A. Bishop. 2009. CD40 and autoimmunity: the dark side of a great activator. Semin. Immunol. 21: 293–300. 4. Larsen, C. P., and T. C. Pearson. 1997. The CD40 pathway in allograft rejection, acceptance, and tolerance. Curr. Opin. Immunol. 9: 641–647. 5. Law, C.-L., and I. S. Grewal. 2009. Therapeutic interventions targeting CD40L (CD154) and CD40: The opportunities and challenges. In Therapeutic Targets of the TNF Superfamily. I. S. Grewal, ed. Landes Bioscience, Austin, TX, p. 8–36. 6. Durie, F. H., R. A. Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993. Prevention of collagen-induced arthritis with an antibody to gp39, the ligand for CD40. Science 261: 1328–1330.

7. Larsen, C. P., E. T. Elwood, D. Z. Alexander, S. C. Ritchie, R. Hendrix, C. Tucker-Burden, H. R. Cho, A. Aruffo, D. Hollenbaugh, P. S. Linsley, et al. 1996. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature 381: 434–438. 8. Kawai, T., D. Andrews, R. B. Colvin, D. H. Sachs, and A. B. Cosimi. 2000. Thromboembolic complications after treatment with mAb against CD40L. Nat. Med. 6: 114–120. 9. de Boer, M., L. Conroy, H. Y. Min, and J. Kwekkeboom. 1992. Generation of mAbs to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins. J. Immunol. Methods 152: 15–23. 10. Kwekkeboom, J., M. De Boer, J. M. Tager, and C. De Groot. 1993. CD40 plays an essential role in the activation of human B cells by murine EL4B5 cells. Immunology 79: 439–444. 11. Kwekkeboom, J., D. de Rijk, A. Kasran, S. Barcy, C. de Groot, and M. de Boer. 1994. Helper effector function of human T cells stimulated by anti-CD3 mAb can be enhanced by co-stimulatory signals and is partially dependent on CD40CD40 ligand interaction. Eur. J. Immunol. 24: 508–517. 12. Boon, L., H. P. M. Brok, J. Bauer, A. Ortiz-Buijsse, M. M. Schellekens, S. Ramdien-Murli, E. Blezer, M. van Meurs, J. L. Ceuppens, M. de Boer, et al. 2001. Prevention of EAE in the common marmoset (Callithrix jacchus) using a chimeric antagonist mAb against human CD40 is associated with altered B cell responses. J. Immunol. 167: 2942–2949. 13. Haanstra, K. G., J. Ringers, E. A. Sick, S. Ramdien-Murli, E.-M. Kuhn, L. Boon, and M. Jonker. 2003. Prevention of kidney allograft rejection using anti-CD40 and anti-CD86 in primates. Transplantation 75: 637–643. 14. Haegel-Kronenberger, H., K. Haanstra, C. Ziller-Remy, A. P. Ortiz Buijsse, J. Vermeiren, F. Stoeckel, S. W. Van Gool, J. L. Ceuppens, M. Mehtali, M. De Boer, et al. 2004. Inhibition of costimulation allows for repeated systemic administration of adenoviral vector in rhesus monkeys. Gene Ther. 11: 241–252. 15. Kasran, A., L. Boon, C. H. Wortel, R. A. Hogezand, S. Schreiber, E. Goldin, M. Boer, K. Geboes, P. Rutgeerts, and J. L. Ceuppens. 2005. Safety and tolerability of antagonist anti-human CD40 Mab ch5D12 in patients with moderate to severe Crohn’s disease. Aliment. Pharmacol. Ther. 22: 111–122. 16. Boon, L., J. D. Laman, A. Ortiz-Buijsse, M. T. den Hartog, S. Hoffenberg, P. Liu, F. Shiau, and M. de Boer. 2002. Preclinical assessment of anti-CD40 Mab 5D12 in cynomolgus monkeys. Toxicology 174: 53–65. 17. Krangel, M. S., H. T. Orr, and J. L. Strominger. 1979. Assembly and maturation of HLA-A and HLA-B antigens in vivo. Cell 18: 979–991. 18. Clark, E. A., and J. A. Ledbetter. 1986. Activation of human B cells mediated through two distinct cell surface differentiation antigens, Bp35 and Bp50. Proc. Natl. Acad. Sci. USA 83: 4494–4498. 19. Imai, A., T. Suzuki, A. Sugitani, T. Itoh, S. Ueki, T. Aoyagi, K. Yamashita, M. Taniguchi, N. Takahashi, T. Miura, et al. 2007. A novel fully human antiCD40 mAb, 4D11, for kidney transplantation in cynomolgus monkeys. Transplant. 84: 1020–1028. 20. Peters, A. L., R. M. Plenge, R. R. Graham, D. M. Altshuler, K. L. Moser, P. M. Gaffney, and G. A. Bishop. 2008. A novel polymorphism of the human CD40 receptor with enhanced function. Blood 112: 1863–1871. 21. Hostager, B. S., I. M. Catlett, and G. A. Bishop. 2000. Recruitment of CD40, TRAF2 and TRAF3 to membrane microdomains during CD40 signaling. J. Biol. Chem. 275: 15392–15398. 22. Rowland, S. R., M. L. Tremblay, J. M. Ellison, L. L. Stunz, G. A. Bishop, and B. S. Hostager. 2007. A novel mechanism for TRAF6-dependent CD40 signaling. J. Immunol. 179: 4645–4653. 23. Kehry, M. R. 1996. CD40-mediated signaling in B cells. Balancing cell survival, growth, and death. J. Immunol. 156: 2345–2348. 24. Bishop, G. A., and B. S. Hostager. 2001. Molecular mechanisms of CD40 signaling. Arch. Immunol. Ther. Exp. (Warsz.) 49: 129–137. 25. Peters, A. L., and G. A. Bishop. 2010. Differential TRAF3 utilization by a variant human CD40 receptor with enhanced signaling. J. Immunol. 185: 6555–6562. 26. Brown, K. D., B. S. Hostager, and G. A. Bishop. 2001. Differential signaling and tumor necrosis factor receptor-associated factor (TRAF) degradation mediated by CD40 and the Epstein-Barr virus oncoprotein latent membrane protein 1 (LMP1). J. Exp. Med. 193: 943–954. 27. Brown, K. D., B. S. Hostager, and G. A. Bishop. 2002. Regulation of TRAF2 signaling by self-induced degradation. J. Biol. Chem. 277: 19433–19438. 28. Moore, C. R., and G. A. Bishop. 2005. Differential regulation of CD40-mediated TRAF degradation in B lymphocytes. J. Immunol. 175: 3780–3789. 29. Bishop, G. A., C. R. Moore, P. Xie, L. L. Stunz, and Z. J. Kraus. 2007. TRAF proteins in CD40 signaling. Adv. Exp. Med. Biol. 597: 131–151. 30. Hostager, B. S. 2007. Roles of TRAF6 in CD40 signaling. Immunol. Res. 39: 105–114. 31. Graham, J. P., K. M. Arcipowski, and G. A. Bishop. 2010. Differential B lymphocyte regulation by CD40 and its viral mimic, LMP1. Immunol. Rev. 237: 226–248. 32. Vidalain, P. O., O. Azocar, C. Servet-Delprat, C. Rabourdin-Combe, D. Gerlier, and S. Manie´. 2000. CD40 signaling in human dendritic cells is initiated within membrane rafts. EMBO J. 19: 3304–3313. 33. Bishop, G. A. 2012. The power of mAbs as agents of discovery: CD40 revealed as a B lymphocyte costimulator. J. Immunol. 188: 4127–4129. 34. Hollenbaugh, D., L. S. Grosmaire, C. D. Kullas, N. J. Chalupny, S. BraeschAndersen, R. J. Noelle, I. Stamenkovic, J. A. Ledbetter, and A. Aruffo. 1992. The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell costimulatory activity. EMBO J. 11: 4313–4321. 35. Lane, P., A. Traunecker, S. Hubele, S. Inui, A. Lanzavecchia, and D. Gray. 1992. Activated human T cells express a ligand for the human B cell-associated antigen

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compared with CD154. Membrane subdomain redistribution of all of these molecules was reduced in PG102-stimulated B cells, despite robust binding of PG102 to CD40, as demonstrated by CD40 staining and immunoprecipitation by PG102. This suggests that membrane recruitment to lipid-rich subdomains is of particular importance to initiate CD40-mediated activation signals. It is again possible that this involves facilitation of access of CD40 to signaling proteins localized in these subdomains that have not yet been identified. Although it was shown previously that CD40mediated TRAF6 recruitment to detergent-insoluble membrane fractions of fibroblast cell lines impacts TRAF2-dependent CD40 signals to these cells (57), in B cells, CD40 molecules that cannot bind TRAF6 still induce TRAF2-dependent signals (30), suggesting cell-type specificity in some aspects of CD40 signaling. PG102-induced TRAF2 degradation was not greater than that induced by agonistic CD40 signals (Fig. 4), also indicating that reduced PG102-mediated TRAF6 recruitment to lipid rafts was not due to increased TRAF2 degradation. The present study identified key characteristics of the mechanism by which PG102 interacts with CD40 and its signaling complex. PG102 did not exert its inhibitory functions by competing with CD154; instead, it induced a CD154-refractory state of early signaling pathways in B cells, features that enhance its potential to inhibit CD40 signaling effectively without requiring high concentrations and without the risk for inducing complications related to binding to CD154. The altered signaling complex formation and distribution induced by PG102 can provide clues and predictive value as biomarkers for further development of effective therapeutic reagents to block CD40 signaling. There are numerous clinical conditions for which inhibiting CD40 signaling has considerable potential benefit, as discussed above. In addition, agonist binding to CD40 inhibits the survival and growth of malignant cells of various types (reviewed in Ref. 43). Recently, evidence was presented for an important role for T cell CD40 as an inhibitor of pathogenic T cell function in visceral adipose tissue in diet-induced obesity (58). The ability of PG102 to induce negative regulatory events downstream of CD40, such as TRAF degradation, may allow this reagent to trigger clinically desirable inhibitory pathways without the danger of unintended immune activation. This will be an exciting possibility for future investigation.

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36.

37.

38.

39.

40.

42. 43. 44.

45.

46.

47.

48.

49.

50. 51.

52.

53. 54.

55. 56.

57.

58.

a critical role during allograft rejection. Suppression of allograft rejection by blockade of the CD40-gp39 pathway. Transplantation 61: 4–9. Shepherd, D. M., and N. I. Kerkvliet. 1999. Disruption of CD154:CD40 blocks generation of allograft immunity without affecting APC activation. J. Immunol. 163: 2470–2477. Van Gool, S. W., J. Vermeiren, K. Rafiq, K. Lorr, M. de Boer, and J. L. Ceuppens. 1999. Blocking CD40 - CD154 and CD80/CD86 - CD28 interactions during primary allogeneic stimulation results in T cell anergy and high IL-10 production. Eur. J. Immunol. 29: 2367–2375. Allen, S. D., S. V. Rawale, C. C. Whitacre, and P. T. P. Kaumaya. 2005. Therapeutic peptidomimetic strategies for autoimmune diseases: costimulation blockade. J. Pept. Res. 65: 591–604. Heath, A. W., W. W. Wu, and M. C. Howard. 1994. Monoclonal antibodies to murine CD40 define two distinct functional epitopes. Eur. J. Immunol. 24: 1828–1834. Bjo¨rck, P., and S. Paulie. 1996. CD40 antibodies defining distinct epitopes display qualitative differences in their induction of B-cell differentiation. Immunology 87: 291–295. Baccam, M., and G. A. Bishop. 1999. Membrane-bound CD154, but not CD40specific antibody, mediates NF-kappaB-independent IL-6 production in B cells. Eur. J. Immunol. 29: 3855–3866. Krakauer, T. 2012. PI3K/Akt/mTOR, a pathway less recognized for staphylococcal superantigen-induced toxicity. Toxins (Basel) 4: 1343–1366. Bishop, G. A., B. S. Hostager, and K. D. Brown. 2002. Mechanisms of TNF receptor-associated factor (TRAF) regulation in B lymphocytes. J. Leukoc. Biol. 72: 19–23. Graham, J. P. 2010. CD40-induced TRAF degradation in immune regulation. Doctoral dissertation, The University of Iowa, Iowa City, IA. Graham, J. P., C. R. Moore, and G. A. Bishop. 2009. Roles of the TRAF2/3 binding site in differential B cell signaling by CD40 and its viral oncogenic mimic, LMP1. J. Immunol. 183: 2966–2973. Davies, C. C., T. W. Mak, L. S. Young, and A. G. Eliopoulos. 2005. TRAF6 is required for TRAF2-dependent CD40 signal transduction in nonhemopoietic cells. Mol. Cell. Biol. 25: 9806–9819. Yi, Z., L. L. Stunz, and G. A. Bishop. 2014. CD40-mediated maintenance of immune homeostasis in the adipose tissue microenvironment. Diabetes 63: 2751–2760.

Downloaded from http://www.jimmunol.org/ at University of California San Francisco on March 23, 2015

41.

CD40 which participates in T cell-dependent activation of B lymphocytes. Eur. J. Immunol. 22: 2573–2578. Noelle, R. J., M. Roy, D. M. Shepherd, I. Stamenkovic, J. A. Ledbetter, and A. Aruffo. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89: 6550–6554. Callard, R. E., R. J. Armitage, W. C. Fanslow, and M. K. Spriggs. 1993. CD40 ligand and its role in X-linked hyper-IgM syndrome. Immunol. Today 14: 559– 564. DiSanto, J. P., J. Y. Bonnefoy, J. F. Gauchat, A. Fischer, and G. de Saint Basile. 1993. CD40 ligand mutations in x-linked immunodeficiency with hyper-IgM. Nature 361: 541–543. Fuleihan, R., N. Ramesh, R. Loh, H. Jabara, R. S. Rosen, T. Chatila, S. M. Fu, I. Stamenkovic, and R. S. Geha. 1993. Defective expression of the CD40 ligand in X chromosome-linked immunoglobulin deficiency with normal or elevated IgM. Proc. Natl. Acad. Sci. USA 90: 2170–2173. Kortha¨uer, U., D. Graf, H. W. Mages, F. Brie`re, M. Padayachee, S. Malcolm, A. G. Ugazio, L. D. Notarangelo, R. J. Levinsky, and R. A. Kroczek. 1993. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature 361: 539–541. Stout, R. D., and J. Suttles. 1996. The many roles of CD40 in cell-mediated inflammatory responses. Immunol. Today 17: 487–492. Bishop, G. A. 2009. The many faces of CD40: multiple roles in normal immunity and disease. Semin. Immunol. 21: 255–256. Loskog, A. S. I., and A. G. Eliopoulos. 2009. The Janus faces of CD40 in cancer. Semin. Immunol. 21: 301–307. Mohan, C., Y. Shi, J. D. Laman, and S. K. Datta. 1995. Interaction between CD40 and its ligand gp39 in the development of murine lupus nephritis. J. Immunol. 154: 1470–1480. Early, G., W. Zhao, and C. Burns. 1996. Anti-CD40 ligand antibody treatment prevents the development of lupus-like nephritis in a subset of New Zealand black x New Zealand white mice. Response correlates with the absence of an anti-antibody response. J. Immunol. 157: 3159–3164. Larsen, C. P., D. Z. Alexander, D. Hollenbaugh, E. T. Elwood, S. C. Ritchie, A. Aruffo, R. Hendrix, and T. C. Pearson. 1996. CD40-gp39 interactions play

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Induction of an altered CD40 signaling complex by an antagonistic human monoclonal antibody to CD40.

Blocking the interaction of CD40 with its ligand CD154 is a desirable goal of therapies for preventing and/or ameliorating autoimmune diseases and tra...
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