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The Journal of Immunology, 2007, 178: 1426-1432.
Copyright © 2007 by The American Association of Immunologists, Inc.

B7-DC/PD-L2 Cross-Linking Induces NF-{kappa}B-Dependent Protection of Dendritic Cells from Cell Death

Suresh Radhakrishnan, Loc T. Nguyen, Bogoljub Ciric, Virginia P. Van Keulen and Larry R. Pease1

Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cross-linking cell surface molecules with IgM Abs is a specific approach for activating cells in vitro or in vivo. Dendritic cells (DC) activated with a human B7-DC (PD-L2)-specific IgM Ab can induce strong antitumor responses and block inflammatory airway disease in experimental models, yet the Ab-mediated molecular events promoting these responses remain unclear. Analysis of human or mouse DC treated with the B7-DC cross-linking Ab revealed PI3K-dependent phosphorylation of AKT accompanied by mobilization of NF-{kappa}B. Ab-activated DC up-regulated expression of cytokine and chemokine genes in an NF-{kappa}B-dependent manner. Importantly, PI3K->AKT->NF-{kappa}B activation was found to be indispensable for B7-DC cross-linking Ab-mediated protection of DC from cell death caused by cytokine withdrawal. Although other DC activators similarly protect DC from cell death, a synergy between cross-linking B7-DC and ligating RANK was observed. The parallel signaling events induced in human and mouse DC demonstrate that activation of cells using IgM Ab results in a response governed by a common mechanism and support the hypothesis that B7-DC cross-linking using this Ab may provide beneficial therapeutic immune modulation in human patients similar to those seen in animal models.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have recently described a new approach for modulating the activity of dendritic cells (DCs)2 that is distinct from previously defined mechanisms of DC activation. DCs are key targets in schemes to regulate immunity (1). Activation of DC through the TLR family initiates DC maturation and migration to regional lymph nodes, where naive T cells are activated (2, 3). As DC mature, the cell surface expression of costimulatory molecules critical for the activation of naive T cells is up-regulated (4, 5, 6). The activated DC also produce immunomodulating cytokines that influence the polarity of the ensuing immune response, determining the array of effector mechanisms brought to bear at the site of infection (7, 8). Among the transcription factors activated by the TLR gene family, NF-{kappa}B is a key regulator of the expression of molecules that mediate intercellular communication among leukocytes (9, 10). TNF-{alpha} and CD40L can also activate the maturation process of DC, inducing signaling pathways mediated by TNFR or CD40 coupled TNFR-associated factor adaptors (11, 12).

Cross-linking B7-DC (PD-L2) with the human IgM Ab B7-DC cross-linking Ab increased a wide variety of important functions by DC, including enhanced survival, ability to process and present soluble Ag by class I molecules, ability to activate naive T cells, efficiency of seeding draining lymph nodes, and expression of IL-12 (13, 14). DC treated with B7-DC cross-linking Ab did not display traditionally defined maturation phenotypes (14). There was no ensuing up-regulation of the costimulatory markers CD80 or CD86, or a concomitant increase in cell surface expression of class II molecules. Instead, treatment of immature DC with B7-DC cross-linking Ab resulted in increased Ag uptake and even restored the ability of TLR ligand-matured DC to take up Ag (15). Furthermore, combination treatment with a TLR9 ligand and B7-DC cross-linking resulted in a synergistic CTL response against peptide Ag (15). These differences in maturation lead to important biological distinctions following activation by traditional approaches or by cross-linking B7-DC.

B7-DC belongs to a subfamily of B7 costimulatory molecules and serves as a ligand for the receptor PD-1, expressed by activated T cells. B7-DC interaction with PD-1 has been shown to result in either a positive response (16) or a negative response (17). The nature of the responses observed in these experiments could be due either to the different model systems used in the studies or to the ability of B7-DC to interact with more than one receptor that differentially govern T cell responsiveness (18). Although it is theoretically possible that B7-DC cross-linking Ab may stimulate immune responsiveness by blocking a negative signal, adoptive transfer experiments using DC activated in vitro under conditions in which B7-DC cross-linking Ab did not physically block receptor access to B7-DC demonstrated full immunomodulatory capabilities (19).

In this report we dissect the mechanistic basis of cross-linking B7-DC-induced survival of DCs in a cytokine-deprived environment. We document that cross-linking B7-DC results in nuclear translocation of canonical NF-{kappa}B. Moreover, the activation of this NF-{kappa}B pathway is different from some TLR-mediated pathways of NF-{kappa}B activation as it is not dependent on MyD88. Importantly, cross-linking B7-DC on DCs leads to the synthesis of IL-6 and TNF-{alpha}, and cytokines that are dependent on activation of NF-{kappa}B. Finally, the mechanism of NF-{kappa}B activation is mediated through activation of a PI3K->AKT pathway resulting in B7-DC cross-linking Ab-induced DC survival.


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

Mice 6- to 8-wk-old C57BL/6J, B6.129s4-CD80–/–CD86–/– knockout strains of mice were obtained from The Jackson Laboratory and were maintained according to Institutional Animal Care and Use Committee guidelines. Class II–/–, MyD88–/–, and C57BL/10ScN mice (TLR4 mutant) were gifts from Dr. C. S. David, Dr. E. Celis, and Dr. J. L. Platt, respectively, all of the Mayo Clinic (Rochester, MN). B7-DC knockout mice containing GFP were obtained from Dr. D. M. Pardoll (Johns Hopkins University of Medicine, Baltimore, MD).

Reagents

Abs against the C-terminal portion of NF-{kappa}B (sc372) and the N-terminal portion of NF-{kappa}B, I{kappa}B{alpha}, and I{kappa}Bbeta were obtained from Santa Cruz Biotechnology. Phospho-AKT-Ser473 (193H12) rabbit Ab (catalog no. 4058) was obtained from Cell Signaling Technology. The mouse class II-specific IgM Ab 25-9-3 was obtained from BD Biosciences. The Abs against relB and p65 used in supershift assays were gifts from Dr. A. D. Badley (Mayo Clinic). Purified Ab against mouse CD40 (IC10) was purchased from eBioscience. Goat anti-rabbit secondary Abs were obtained from BD Biosciences. Polymyxin B sulfate, DAPI (4',6'-diamidino-2-phenylindole), and LPS were obtained from Sigma-Aldrich. Vitamin D3 (1{alpha},25-dihydroxyvitamin D3) was a gift from Dr. M. Griffin (Mayo Clinic). Receptor activator of the NF-{kappa}B (RANK) ligand was purchased from Chemicon International. TNF-{alpha} was obtained from R&D Systems and was used at 1 µg/ml. Control IgM or serum-derived human IgM 12 (B7-DC cross-link Ab) were purified as described (14) and used at 10 µg/ml. All the inhibitors used in this experiment were purchased from Calbiochem.

Generation of DC

DC from the mouse bone marrow were generated as described (14). Bone marrow cells were plated at the density of 1 x 106/ml in RPMI 1640 containing 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4 (PeproTech). The cells were incubated at 37°C with 5% CO2. After 48 h, the cells were washed and replated in the same medium for another 5 days. Human DC were derived from CD14+ mononuclear cells isolated from peripheral blood using magnetic bead sorting (Miltenyi Biotec). The isolated cells were incubated with IL-4 (800 U/ml; R&D Systems) and GM-CSF (1000 U/ml; Berlex Laboratories) for 7 days (immature cells).

Confocal microscopy

DC were stimulated with 10 µg of Ab for different lengths of time before being fixed and permeabilized using Cytofix/Cytoperm kit (BD Pharmingen). Subsequently, Ab against a C-terminal peptide of NF-{kappa}B was added followed by anti-rabbit FITC. Nuclei were stained with DAPI (Sigma-Aldrich) before being observed with a LSM510 laser scanning confocal microscope (Carl Zeiss) at magnification of x40.

Flow cytometry

Phosphorylated AKT was monitored by flow cytometry. Briefly, permeabilized mouse DC were stained at 4°C with phosphospecific AKT Ab followed by appropriate secondary Ab-labeled with FITC. The cells were analyzed using a FACSCalibur (BD Biosciences). Data collected as log10 fluorescence were analyzed using CellQuest (BD Biosciences).

Immunoblot

For visualizing NF-{kappa}B members, nuclear extracts and cytoplasmic protein fractions were prepared from stimulated DC using the NE-PER nuclear and cytoplasmic extraction kit (Pierce). Protein extracts resolved on a SDS-PAGE gel were transferred to polyvinylidene difluoride membranes. Membranes were incubated with the anti-NF-{kappa}B Ab mixture, and membranes were developed with ECL (Pierce).

Nuclear extraction and EMSA

Extraction of nuclei and EMSA were conducted as previously described (20). Briefly, DCs were stimulated with control Ab or B7-DC cross-linking Ab for 30 min before lysis. Nuclei were isolated by high-speed centrifugation and lysed. The 5 µg of nuclear extract was incubated with [{gamma}-32P]ATP-labeled double-stranded probe for 30 min. When Abs were used for supershift analysis, lysates were preincubated with Ab before incubating with the labeled probe. All samples were resolved on 6% acrylamide gels under nondenaturing conditions and visualized by autoradiography.

Inhibition experiments

DCs were preincubated with inhibitors at the indicated concentrations for 30 min before addition of B7-DC cross-linking Ab or isotype control Ab. Subsequently, the cells were lysed and subjected to analysis by immunobloting. For NF-{kappa}B inhibition studies using the inhibitor NEMO-binding domain (NBD) peptide (sequence of NBD), cells were treated with 10 µM inhibitory peptide for 2 h before stimulation with Ab for 15 min. Cells were harvested and analyzed for nuclear localization of NF-{kappa}B by confocal microscopy. Alternatively, for studies involving NBD inhibition of B7-DC cross-linking Ab induced gene expression, mouse DC were treated with 10 µg/ml B7-DC cross-linking Ab after preincubation with NBD inhibitor for 30 min. Cells were harvested 90 min later and RNA was prepared using TRIzol reagent (Invitrogen Life Technologies). Radioactive probes were generated and hybridized to GEArray Q Series Mouse Inflammatory Cytokines & Receptors Gene Arrays (SuperArray Bioscience) according to the manufacturer’s instructions. Gene arrays were imaged using a STORM 840 imager (Molecular Dynamics), and results were normalized to the control gene cyclophilin A. For inhibition of PI3K, AKT, and NF-{kappa}B, DC were pretreated with the inhibitors LY294002, AKT inhibitor IV, or NBD in a dose-dependent fashion before being stimulated with B7-DC cross-linking Ab. For inducing cell death,

Cell viability assay

DCs were tested for their viability in a cytokine-deprived environment as previously described (13). In brief, day 5 DCs from mice or human were plated into 96-well plates at 2 x 104 cells/well. Cells were cultured with the indicated concentration of B7-DC cross-linking Ab, isotype control Ab, or RANK ligand to a final concentration of 10 µg/ml in RPMI 1640 without GM-CSF and IL-4. In experiments using vitamin D3 to induce cell death (21), DC were matured with 200 ng/ml LPS together with 10 nM vitamin D3. After 1 h of culture, Alamar Blue (BioSource International) was added to a final concentration of 10% (v/v). Readings were taken at 24 h of culture using a Spectra Max M2 Multi Detection Reader (Molecular Devices). The fluorescence plate reader was set to an excitation wavelength of 520 nm and an emission reading of 590 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
B7-DC cross-linking results in activation of AKT

We previously observed increased viability of DC in a cytokine-deprived environment if the cells were activated with B7-DC cross-linking Ab (13). The kinase AKT/PKB has been shown to function in the regulation of DC survival (22). The importance of AKT/PKB in DC survival following treatment with B7-DC cross-linking Ab was indicated by its rapid phosphorylation in Ab-treated cells (Fig. 1, A and B). As a specificity control, class II molecules expressed on the surface of DC were ligated with a commercially available IgM Ab, failing to induce phosphorylation of AKT (Fig. 1A). Ligation of TLR4, but not stimulation with a second isotype control IgM Ab, also led to the phosphorylation of AKT (data not shown).


Figure 1
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  		FIGURE 1. A prosurvival protein, AKT is activated upon cross-linking B7-DC. A, Mouse DC were stimulated with control IgM Ab specific for MHC class II molecules or B7-DC cross-linking Ab for the indicated times. Cells were lysed, immunoprecipitated for AKT, and probed for phosphorylated AKT using Ab Ser473 (193H12). The presence of AKT is shown in lanes in which phospho-AKT was not originally detected by reprobing the blot with AKT-specific Ab. B, Mouse DC stimulated with control Ab (filled histogram) or B7-DC cross-linking Ab (open histogram) was analyzed for phosphorylated forms of AKT by intracellular staining using Ab Ser473 followed by flow cytometry. C, Whole cell lysates from mouse DC stimulated with control Ab, B7-DC cross-linking Ab, or LPS for the indicated time points were subjected to immunoblot analysis. The membrane was probed for I{kappa}B{alpha} using Ab sc-847 (top lane) as described in Materials and Methods and for I{kappa}Bbeta using Ab sc-9130 (bottom lane).

 
Phosphorylation of AKT is known to lead to NF-{kappa}B activation in multiple model systems featuring increased cell survival (23, 24). Moreover, NF-{kappa}B has been shown to be an important transcription factor in preventing cell death (25). To determine whether NF-{kappa}B activation in B7-DC cross-linked DC follows a defined regulatory pathway, levels of I{kappa}B{alpha} and I{kappa}Bbeta were monitored following treatment of DC with B7-DC cross-linking Ab. The observed drop in levels of I{kappa}B{alpha} at 30 min and both I{kappa}B{alpha} and I{kappa}Bbeta at 60 min indicates that known regulators of NF-{kappa}B activation are affected by cross-linking B7-DC (Fig. 1C).

B7-DC cross-linking results in nuclear localization of NF-{kappa}B

First, to directly visualize NF-{kappa}B activation following B7-DC cross-linking of mouse DC, we assayed for nuclear translocation of NF-{kappa}B by monitoring for p65 protein. Nuclear extracts from B7-DC cross-linking Ab-treated DC were found to contain increased levels of p65-RelA complexes compared with levels found in nuclei from sham-treated DC. The kinetics of NF-{kappa}B mobilization following B7-DC cross-linking Ab treatment indicate that this response is intermediate, with elevated p65/RelA levels peaking at 15 min after cross-linking (Fig. 2A). RelA/p65 levels in cytoplasmic fractions were not significantly different between the treatment groups (data not shown). Nuclear localization of NF-{kappa}B was further verified in mobility shift assays using p65 Ab (Fig. 2B). Again addition of an IgM Ab that binds to class II molecules on the DC failed to activate NF-{kappa}B (Fig. 2B), demonstrating the specificity of this response.


Figure 2
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  		FIGURE 2. Activation of NF-{kappa}B results in p65 nuclear translocation. Bone marrow-derived DC were stimulated with 10 µg/ml B7-DC cross-linking Ab, control Ab, or TNF-{alpha} for the indicated times. A, Nuclear extracts of cells activated with either an irrelevant IgM Ab or B7-DC cross-linking Ab were analyzed by using the Western blot technique using a mix of Abs specific for N-terminal (sc-109) and C-terminal (sc-372) p65 peptides. B, Nuclear extracts of cells activated with a control MHC class II-specific IgM Ab or B7-DC cross-linking Ab were analyzed by EMSA using a p65-specific Ab. C, Human DC, after treatment with control IgM Ab (left) or B7-DC cross-linking Ab (right) for 15 min, were probed for "free" NF-{kappa}B as described in Materials and Methods. Subsequently, cells were stained with goat anti-rabbit FITC (green). Nuclei were visualized by staining with DAPI (blue). Images were obtained by confocal microscopy using a x40 apochromat lens magnification. Merged images (bottom panel) are shown of each cluster. D, Mouse bone marrow-derived DC were treated, stained, and images the same as described in C. E, GFP-positive B7-DC–/– DC were treated, stained, and imaged as in C. Goat anti-rabbit Texas Red was used as secondary Ab. F, NF-{kappa}B activation was inhibited by pretreatment of mouse DC for 30 min with a peptide inhibitor of I{kappa}B kinase-{gamma}, NBD peptide (50 µM), before stimulation with B7-DC cross-linking Ab. Cells were stained for the active free form of NF-{kappa}B and analyzed as in C. G, Mouse DC were stimulated with B7-DC cross-linking Ab only (Figure 2) or were stimulated after pretreatment with the NF-{kappa}B inhibitor NBD ({blacksquare}) as described in F. Total mRNA was extracted and was used to prepare radiolabeled cDNA for subsequent analysis of dot blot gene arrays. H and I, Human DC were stimulated with control Ab or B7-DC cross-linking Ab for 12 h. The supernatants were analyzed for IL-6 and TNF-{alpha} by ELISA.

 
Second, to see whether activation of NF-{kappa}B in mouse DC is paralleled in human DC, day-7 mouse or human DC were stimulated with control Ab or B7-DC cross-linking Ab at several time points and were evaluated by confocal microscopy for the presence of activated NF-{kappa}B using an Ab (sc-372) that recognizes human and mouse p65 molecules not bound by I{kappa}B. As visualized within the defined three-dimensional structure of p65 complexes with I{kappa}B, the peptide recognized by Ab sc-372 appears buried in the interior of the complex (26). Upon release of p65 from the inhibitor, this region becomes exposed. Activation of the p65 complexes was not seen upon treatment with control Ab (Fig. 2, C and D, left panels). Treatment of both human and mouse DC with B7-DC cross-linking Ab resulted in NF-{kappa}B activation that peaks at 15 min (Fig. 2, C and D, right panels) and is dependent on B7-DC because DCs from B7-DC knockout mice failed to activate NF-{kappa}B in response to cross-linking B7-DC. B7-DC-deficient cells were capable of activating NF-{kappa}B upon ligation of CD40 or activation with LPS (Fig. 2E). These findings document the ability of the B7-DC cross-linking Ab to activate NF-{kappa}B in both human and mouse DC.

To determine the consequences of NF-{kappa}B activation in mouse DC following cross-linking of B7-DC, DC were stimulated with B7-DC cross-linked Ab, and at various time points total RNA was extracted and tested using dot blot arrays containing 96 cytokine and receptor genes associated with inflammatory responses. These experiments revealed an increase in message for several chemokines and cytokines, notably IL-6, IL-1{alpha}, MIP1{alpha}, MIP1beta, TNF-{alpha}, TARC/CCL17, and Scya6/CCL6 (Fig. 2F). Similarly, cross-linking B7-DC on human DC resulted in secretion of IL-6 and TNF-{alpha} (Fig. 2, H and I). Competitive inhibition of the binding of I{kappa}K{gamma} to I{kappa}K{alpha}-I{kappa}Kbeta complex using the NBD peptide (27, 28) blocked NF-{kappa}B activation for up to 2 h (Fig. 2F). The presence of the inhibitor did not alter global tyrosine phosphorylation following FcR{gamma} cross-linking (data not shown). More importantly, treatment of the DC with peptide inhibitor 120 min before activation with B7-DC cross-linking Ab reduced Ab-induced mRNA accumulation for IL-1{alpha} and IL-6 by 50% and TNF-{alpha}, MIP1{alpha}, MIP1beta by >70% (Fig. 2G). Notably, Scya6/CCL6 was not inhibited. These results establish that activation of the NF-{kappa}B family of transcription factors by cross-linking B7-DC leads to a rapid increase in mRNA encoding several key chemokines and cytokines in Ab-treated DC.

NF-{kappa}B activation by B7-DC cross-linking is MyD88-independent

NF-{kappa}B can be activated by several different signaling pathways (29). MyD88 is an adaptor protein that is associated with various receptors including IL-1R, IL-18R, and TLR family members linking these receptors to downstream mediators of activation of NF-{kappa}B (30). Cross-linking of MyD88-deficient DC with B7-DC cross-linked Ab resulted in NF-{kappa}B activation to levels comparable to those seen using wild-type cells, whereas LPS treatment was an ineffective stimulus in MyD88-deficient cells (Fig. 3A). To further analyze the difference in activation pathways involving cross-linking B7-DC vs TLR ligation, DCs from TLR4 mutant mice were used. Again, cross-linking B7-DC still resulted in activation of NF-{kappa}B in both wild-type and TLR4 mutant DCs, whereas the TLR4 ligand, LPS, was able to activate NF-{kappa}B only in wild-type DCs (Fig. 3B). Potential contamination with LPS in the B7-DC cross-linking Ab preparation was ruled out because cross-linking B7-DC in DCs in presence of polymyxin B was still able to activate NF-{kappa}B, whereas preparations of LPS-treated with polymyxin B could not (data not shown).


Figure 3
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  		FIGURE 3. Activation of NF-{kappa}B by B7-DC cross-linking Ab is independent of adaptor protein MyD88 and TLR4. A, Mouse DC from wild-type mice or mice lacking the adaptor protein MyD88 was stimulated with control Ab, B7-DC cross-linking Ab, or LPS for 15 min. Cells were probed for free NF-{kappa}B as described in Materials and Methods. Subsequently as in Fig. 2C, cells were stained with goat anti-rabbit FITC (green). Nuclei were visualized by staining with DAPI (blue). Cells were analyzed for activation of NF-{kappa}B by confocal microscopy using a x40 apochromat lens magnification. B, Mouse DC from wild-type mice (left) or TLR4 mutant mice (right) were stimulated with control Ab (upper), B7-DC cross-linking Ab (middle), or LPS (lower) for 15 min and were stained with C-terminal-specific Ab sc-372 followed by anti-rabbit FITC (x-axis). The cells were double stained with CD11c PE (y-axis) and analyzed by flow cytometry.

 
Activation of NF-{kappa}B by cross-linking B7-DC is dependent on PI3K->AKT activation

Next, we addressed the mechanism of activation of NF-{kappa}B by cross-linking B7-DC. A common pathway that can lead to NF-{kappa}B activation is through activation of PI3K, an inducer of AKT-mediated NF-{kappa}B activation (31, 32). Because AKT is phosphorylated following B7-DC cross-linking Ab treatment (Fig. 1A), the possibility exists that signals generated by cross-linking B7-DC are mediated by PI3K regulated pathways. Consistent with this hypothesis, pharmacological inhibition of PI3K activity using LY294002 before treatment with B7-DC cross-linking Ab blocked activation of AKT in a dose-dependent manner (Fig. 4).


Figure 4
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  		FIGURE 4. Activation of AKT is dependent on PI3K. Bone marrow-derived mouse DCs were stimulated with control Ab or B7-DC cross-linking Ab and were permeablized for analysis of phospho-AKT, using the phospho-AKT-specific Ab Ser473 (193H12), followed by rabbit Ab (no. 4058). Cells were analyzed by flow cytometry. A, Control Ab treatment at 0 min (filled histogram) vs 30 min (open histogram). B, Same treatment as in A except in presence of 10 µM concentration of LY294002 preincubated for 30 min. C–G, Stimulation with control Ab (filled histogram) vs B7-DC cross-linking Ab (open histogram) in presence of 10, 5, 2.5, 1 µM LY294002, respectively. H, Dose response inhibition by LY294002 as measured by mean fluorescent intensity (MFI) of phospho-AKT.

 
To examine the relationships of PI3K and AKT to the activation of NF-{kappa}B, we used pharmacological inhibitors of this signaling cascade. As shown in Fig. 5, control Ab did not activate NF-{kappa}B (Fig. 5A), whereas B7-DC cross-linking Ab induced robust NF-{kappa}B activation (Fig. 5B). Inhibition of PI3K by LY294002 resulted in prevention of B7-DC cross-linking Ab-induced activation of NF-{kappa}B in a dose-dependent fashion (Fig. 5, C and D). Furthermore, prevention of AKT activation with the pharmacological AKT inhibitor IV prevented NF-{kappa}B activation following B7-DC cross-linking (Fig. 5, E and F). Finally, I{kappa}B kinase-{gamma} inhibition by NBD peptide also resulted in inhibition of NF-{kappa}B activation (Fig. 5, G and H). Taken together, these experiments demonstrate PI3K-mediated activation of AKT plays a crucial role in activation of NF-{kappa}B in an I{kappa}B kinase-{gamma}-dependent manner following cross-linking of B7-DC.


Figure 5
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  		FIGURE 5. Activation of NF-{kappa}B is dependent on AKT -> PI3K activation and is important for DC survival. Bone marrow-derived DC were stimulated with control Ab or B7-DC cross-linking Ab in absence or presence of inhibitors of PI3K, AKT, or NF-{kappa}B. Cells were assayed for activation of NF-{kappa}B by staining with goat anti-rabbit FITC (green). Nuclei were visualized by staining with DAPI (blue). Images were obtained by confocal microscopy using a x40 apochromat lens magnification as in Fig. 2C. NF-{kappa}B activation upon control Ab treatment is shown in A, whereas B shows NF-{kappa}B activation upon B7-DC cross-linking Ab treatment. C and D are NF-{kappa}B activation upon cross-linking B7-DC in cells that were pretreated with AKT inhibitor at 10 (C) or 1 µM (D) concentration, PI3K inhibitor at 10 (E) and 1 µM (F) concentration, and NF-{kappa}B inhibitor at 50 (G) and 10 µM (H) concentration. I, Bone marrow-derived DCs were assessed for protection against cell death after cytokine withdrawal upon cross-linking B7-DC in the presence or absence of PI3K, AKT, or NF-{kappa}B inhibitors as indicated. J, Same as in I except human DC were used. K, Same as in I except mouse DC were matured with LPS and were assayed for survival upon vitamin D3-mediated cell death in presence of different doses of B7-DC cross-linking Ab. L, Depicts synergistic effects mediated by RANK ligand ligation and cross-linking B7-DC upon withdrawal of cytokines.

 
PI3K->AKT->NF-{kappa}B pathway mediates survival upon cytokine withdrawal in DCs activated by cross-linking B7-DC

PI3K-mediated activation of AKT resulting in activation of NF-{kappa}B has been shown to induce survival signals in multiple cell types (31, 32). Because we found activation of NF-{kappa}B is dependent on PI3K->AKT, we tested the importance of this signaling pathway in protecting DC against cytokine withdrawal. Thus, day-5 bone marrow-derived DC were deprived of the cytokines GM-CSF plus IL-4 and were monitored for cell survival by culturing in the presence of control Ab, B7-DC cross-linking Ab, or B7-DC cross-linking Ab plus DC pretreated 30 min with 10 or 1 µM concentration of the PI3K inhibitor, LY294002, AKT inhibitor IV, and the NF-{kappa}B inhibitor NBD at 10 µM or 1 µM for 8 h in presence of the indicator dye, Alamar Blue, a commonly used indicator of cell viability (33). Data from this set of experiments revealed B7-DC cross-linking Ab mediated protection of DC upon cytokine withdrawal and that this response is dependent on PI3K, the activity of AKT, and NF-{kappa}B, thereby underscoring the importance of this signaling pathway for DC survival induced by provided by cross-linking B7-DC (Fig. 5I). In parallel experiments using human DC, the pharmacological inhibitors blocked the ability of B7-DC cross-linking Ab to protect the cells from cell death in an identical manner as seen using mouse DC (Fig. 5J). Apart from cytokine withdrawal, signaling through MHC class II (34), retinoids (35), and vitamin D3 (21) can induce cell death of DCs. As shown in Fig. 5K, addition of 1 µg/ml B7-DC cross-linking Ab was capable of rescuing DC from vitamin D3-induced cell death.

A number of DC activators, including CD40 (36) and RANK agonists (37), are known to protect DC from cell death. We evaluated whether CD40-specific Ab or RANK ligand will enhance the survival of cells in combination with B7-DC cross-linking. Although we found no evidence of interactions between B7-DC cross-linking Ab and the CD40-activating Ab IC10 (data not shown), a synergistic effect between suboptimally cross-linked B7-DC and ligated RANK in the protection of DC from cell death was consistently observed (Fig. 5L), while the class II specific IgM Ab 25-9-3 did not protect the DC from cell death.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Exposing cells to a depleted environment devoid of growth promoting cytokines can result in alterations of cellular metabolism leading to cell death (38). Several studies demonstrate the role of PI3K for protection of a variety of cell types from cell death (38, 39). Moreover, PI3K-mediated activation of AKT is also known to provide antiapoptotic signals (40, 41). AKT functions by activating NF-{kappa}B, a transcription factor that induces prosurvival factors (42, 43, 44). Although the precise pathway by which AKT activates NF-{kappa}B is not known in DC, a recent report demonstrates a role of COT (Tpl-2), a serine threonine kinase belonging to MAPK kinase kinase family of T cells (45). Moreover, dominant negative COT abolishes AKT mediated activation of NF-{kappa}B. Together, these studies define a PI3K->AKT->NF-{kappa}B signaling pathway that regulates apoptosis.

In this report we demonstrate that the B7-DC cross-linking Ab-mediated protection against DC death is dependent on activation of NF-{kappa}B, involving a PI3K->AKT->NF-{kappa}B signaling pathway. NF-{kappa}B in this system is dependent on PI3K, as the PI3K inhibitor Ly294002 blocked both AKT and NF-{kappa}B activation. Inhibition of AKT with AKT inhibitor IV also blocked NF-{kappa}B activation, indicating that these three signaling intermediates are organized in a canonical pathway. Importantly, blockade of each of these signaling intermediates prevented B7-DC cross-linking Ab-mediated protection against cell death induced by the stress of cytokine deprivation.

Other signals that activate DC also function through NF-{kappa}B. For example, TLR ligation can lead to activation of NF-{kappa}B. However, our studies indicate that B7-DC cross-linking Ab and TLR agonists use different strategies to achieve this common goal. Most TLRs signal through the adaptor MyD88, whereas B7-DC cross-linking Ab induces signals that are MyD88-independent. In addition, we found an absence of NF-{kappa}B activation 30 min after TLR4 ligation in MyD88-deficient DC, whereas B7-DC cross-linking Ab strongly activates NF-{kappa}B in this time frame. This finding is consistent with the reported differences in the kinetics of activation of NF-{kappa}B observed in MyD88-deficient DC by EMSA, where there was a delay of 10 min in MyD88-deficient macrophages in comparison to NF-{kappa}B in wild-type macrophages (46).

An important question addressed by our study is whether B7-DC cross-linking Ab can activate human and mouse DC in an equivalent manner. We had previously reported that B7-DC cross-linking Ab binds both human and mouse DC (14). In this study we show that B7-DC cross-linking Ab activates NF-{kappa}B in DC from both species. In the mouse system, we have found that NF-{kappa}B activation leads to up-regulation of cytokine messenger RNAs, including the proinflammatory cytokines IL-6 and TNF-{alpha}. We also found that activation of human DC by cross-linking B7-DC resulted in increased production of IL-6 and TNF-{alpha}, demonstrating the functional significance of the parallel pathways induced in DC from both species by this treatment.

An unanswered question is whether cross-linking of B7-DC is a normal cellular process that leads to NF-{kappa}B activation. To date, the described phenotypes of B7-DC knockout mice are not severely deviated from normal (47). The number of DC in the spleens of knockout mice is approximately normal, suggesting there is not a significant defect in cell longevity. As we show, NF-{kappa}B can be readily activated in B7-DC-deficient cells through cell surface receptors such as CD40. Whether or not activation of DC through B7-DC cross-linking is the usual way DC receive survival or activation signals, our findings that activation of DC through this mechanism delivers strong immunomodulatory signals provides compelling reasons to understand this process.

Our current view is that Ab-induced cross-linking of B7-DC is a stimulus that has distinct physiological consequences that might be exploited in the treatment of a variety of diseases including the treatment of cancer and allergic asthma. In this regard we have found that systemic treatment of mice with disseminated B16 melanoma with B7-DC cross-linking Ab protects the animals from the development of lung nodules characteristic of metastasis and leads to their long-term survival (48). Similarly, treatment of mice with 6-day established B16 melanoma grafts completely protects the animals from tumor growth and rapidly induces tumor-specific CTL to melanoma, lymphoma, and breast cancer cell lines (our unpublished observations). Our finding that systemic treatment of Th2-sensitized mice protects them from developing inflammatory airway disease upon repeated intranasal stimulation with Ag indicates that the Ab can modulate recall responses (49). Because these results can be mimicked by adoptive transfer of DC activated by B7-DC cross-linking Ab ex vivo (19), and we find evidence of DC activation in situ using Ag uptake assays (15), we surmise that systemically administered Ab is activating native DC in vivo. The observations in this report demonstrate that the human IgM Ab B7-DC cross-linking Ab activates the NF-{kappa}B pathways in human DC in a manner that closely mirror the activated pathway in the mouse. Although the relationship between the activation of NF-{kappa}B and the complex phenotypes induced in vivo remains to be established, this finding provides a basis for our hypothesis that B7-DC cross-linking Ab will be a potent modulator of human immune responses as well.


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


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

1 Address correspondence and reprint requests to Dr. Larry R. Pease, Professor and Chair, Department of Immunology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905. E-mail address: pease.larry{at}mayo.edu Back

2 Abbreviations used in this paper: DC, dendritic cell; RANK, receptor activator of the NF-{kappa}B; NBD, NEMO-binding domain; DAPI, 4',6'-diamidino-2-phenylindale. Back

Received for publication July 19, 2006. Accepted for publication October 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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