B7-DC/PD-L2 Cross-Linking Induces NF-?B-Dependent
Protection of Dendritic Cells from Cell Death
Suresh Radhakrishnan, Loc T. Nguyen, Bogoljub Ciric, Virginia P. Van Keulen,
and Larry R. Pease1
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-?B. Ab-activated DC up-regulated expression of cytokine and chemokine genes in an NF-?B-dependent
manner. Importantly, PI3K3AKT3NF-?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. The Journal of Immunology, 2007, 178: 1426–1432.
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 (4–6). The
activated DC also produce immunomodulating cytokines that influ-
ence the polarity of the ensuing immune response, determining the
8). Among the transcription factors activated by the TLR gene family,
NF-?B is a key regulator of the expression of molecules that mediate
intercellular communication among leukocytes (9, 10). TNF-? and
CD40L can also activate the maturation process of DC, inducing sig-
naling pathways mediated by TNFR or CD40 coupled TNFR-asso-
ciated 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
e have recently described a new approach for modulat-
ing the activity of dendritic cells (DCs)2that is distinct
from previously defined mechanisms of DC activation.
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). Fur-
thermore, combination treatment with a TLR9 ligand and B7-DC
cross-linking resulted in a synergistic CTL response against pep-
tide Ag (15). These differences in maturation lead to important
biological distinctions following activation by traditional ap-
proaches 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
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
ically block receptor access to B7-DC demonstrated full immuno-
modulatory capabilities (19).
In this report we dissect the mechanistic basis of cross-linking
B7-DC-induced survival of DCs in a cytokine-deprived environ-
ment. We document that cross-linking B7-DC results in nuclear
translocation of canonical NF-?B. Moreover, the activation of this
NF-?B pathway is different from some TLR-mediated pathways of
NF-?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-?, and cytokines that are dependent on activation of NF-?B.
Finally, the mechanism of NF-?B activation is mediated through
activation of a PI3K3AKT pathway resulting in B7-DC cross-
linking Ab-induced DC survival.
Materials and Methods
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
Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN 55905
Received for publication July 19, 2006. Accepted for publication October 27, 2006.
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.
1Address 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: email@example.com
2Abbreviations used in this paper: DC, dendritic cell; RANK, receptor activator of
the NF-?B; NBD, NEMO-binding domain; DAPI, 4?,6?-diamidino-2-phenylindale.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
guidelines. Class II?/?, MyD88?/?, and C57BL/10ScN mice (TLR4 mu-
tant) 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 Hop-
kins University of Medicine, Baltimore, MD).
Abs against the C-terminal portion of NF-?B (sc372) and the N-terminal
portion of NF-?B, I?B?, and I?B? were obtained from Santa Cruz Bio-
technology. 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?,25-dihy-
droxyvitamin D3) was a gift from Dr. M. Griffin (Mayo Clinic). Receptor
activator of the NF-?B (RANK) ligand was purchased from Chemicon
International. TNF-? was obtained from R&D Systems and was used at 1
?g/ml. Control IgM or serum-derived human IgM 12 (B7-DC cross-link
Ab) were purified as described (14) and used at 10 ?g/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 ? 106/ml in RPMI 1640 con-
taining 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).
DC were stimulated with 10 ?g of Ab for different lengths of time before
being fixed and permeabilized using Cytofix/Cytoperm kit (BD Pharmin-
gen). Subsequently, Ab against a C-terminal peptide of NF-?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 ?40.
Phosphorylated AKT was monitored by flow cytometry. Briefly, perme-
abilized 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).
For visualizing NF-?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. Mem-
branes were incubated with the anti-NF-?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 centrifu-
gation and lysed. The 5 ?g of nuclear extract was incubated with
[?-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
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 immuno-
bloting. For NF-?B inhibition studies using the inhibitor NEMO-binding
domain (NBD) peptide (sequence of NBD), cells were treated with 10 ?M
inhibitory peptide for 2 h before stimulation with Ab for 15 min. Cells were
harvested and analyzed for nuclear localization of NF-?B by confocal mi-
croscopy. Alternatively, for studies involving NBD inhibition of B7-DC
cross-linking Ab induced gene expression, mouse DC were treated with 10
?g/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 Cy-
tokines & 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-?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 ? 104cells/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 ?g/ml in RPMI 1640 with-
out GM-CSF and IL-4. In experiments using vitamin D3to 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.
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 phosphor-
ylated 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 ana-
lyzed for phosphorylated forms of AKT by intracellular staining using Ab
Ser473followed by flow cytometry. C, Whole cell lysates from mouse DC
stimulated with control Ab, B7-DC cross-linking Ab, or LPS for the indi-
cated time points were subjected to immunoblot analysis. The membrane
was probed for I?B? using Ab sc-847 (top lane) as described in Materials
and Methods and for I?B? using Ab sc-9130 (bottom lane).
A prosurvival protein, AKT is activated upon cross-linking
1427 The Journal of Immunology
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).
Phosphorylation of AKT is known to lead to NF-?B activation
in multiple model systems featuring increased cell survival (23,
24). Moreover, NF-?B has been shown to be an important tran-
scription factor in preventing cell death (25). To determine
whether NF-?B activation in B7-DC cross-linked DC follows a
defined regulatory pathway, levels of I?B? and I?B? were mon-
itored following treatment of DC with B7-DC cross-linking Ab.
The observed drop in levels of I?B? at 30 min and both I?B? and
I?B? at 60 min indicates that known regulators of NF-?B activa-
tion are affected by cross-linking B7-DC (Fig. 1C).
B7-DC cross-linking results in nuclear localization of NF-?B
First, to directly visualize NF-?B activation following B7-DC
cross-linking of mouse DC, we assayed for nuclear translocation of
NF-?B by monitoring for p65 protein. Nuclear extracts from
B7-DC cross-linking Ab-treated DC were found to contain in-
creased levels of p65-RelA complexes compared with levels found
in nuclei from sham-treated DC. The kinetics of NF-?B mobili-
zation following B7-DC cross-linking Ab treatment indicate that
Ab, control Ab, or TNF-? 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-?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 ?40 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-?B activation was inhibited by pretreatment of mouse DC for 30 min with a peptide inhibitor of
I?B kinase-?, NBD peptide (50 ?M), before stimulation with B7-DC cross-linking Ab. Cells were stained for the active free form of NF-?B and analyzed
as in C. G, Mouse DC were stimulated with B7-DC cross-linking Ab only (o) or were stimulated after pretreatment with the NF-?B inhibitor NBD (f)
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-? by ELISA.
Activation of NF-?B results in p65 nuclear translocation. Bone marrow-derived DC were stimulated with 10 ?g/ml B7-DC cross-linking
1428B7-DC CROSS-LINKED Ab-INDUCED NF-?B PROTECTS FROM CELL DEATH
this response is intermediate, with elevated p65/RelA levels peak-
ing 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-?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-?B (Fig. 2B), demonstrating the
specificity of this response.
Second, to see whether activation of NF-?B in mouse DC is
paralleled in human DC, day-7 mouse or human DC were stimu-
lated 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-?B using an Ab (sc-372) that recognizes human
and mouse p65 molecules not bound by I?B. As visualized within
the defined three-dimensional structure of p65 complexes with
I?B, the peptide recognized by Ab sc-372 appears buried in the
interior of the complex (26). Upon release of p65 from the inhib-
itor, this region becomes exposed. Activation of the p65 com-
plexes 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-?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-?B
in response to cross-linking B7-DC. B7-DC-deficient cells were
capable of activating NF-?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-?B in both human and
To determine the consequences of NF-?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 chemo-
kines and cytokines, notably IL-6, IL-1?, MIP1?, MIP1?, TNF-?,
TARC/CCL17, and Scya6/CCL6 (Fig. 2F). Similarly, cross-link-
ing B7-DC on human DC resulted in secretion of IL-6 and TNF-?
(Fig. 2, H and I). Competitive inhibition of the binding of I?K? to
I?K?-I?K? complex using the NBD peptide (27, 28) blocked
NF-?B activation for up to 2 h (Fig. 2F). The presence of the
inhibitor did not alter global tyrosine phosphorylation following
FcR? 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 accumula-
tion for IL-1? and IL-6 by 50% and TNF-?, MIP1?, MIP1? by
?70% (Fig. 2G). Notably, Scya6/CCL6 was not inhibited. These
results establish that activation of the NF-?B family of transcrip-
tion factors by cross-linking B7-DC leads to a rapid increase in
mRNA encoding several key chemokines and cytokines in
NF-?B activation by B7-DC cross-linking is MyD88-independent
NF-?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 link-
ing these receptors to downstream mediators of activation of
NF-?B (30). Cross-linking of MyD88-deficient DC with B7-DC
cross-linked Ab resulted in NF-?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-link-
ing B7-DC vs TLR ligation, DCs from TLR4 mutant mice were
used. Again, cross-linking B7-DC still resulted in activation of
NF-?B in both wild-type and TLR4 mutant DCs, whereas the
TLR4 ligand, LPS, was able to activate NF-?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 acti-
vate NF-?B, whereas preparations of LPS-treated with polymyxin
B could not (data not shown).
Activation of NF-?B by cross-linking B7-DC is dependent on
Next, we addressed the mechanism of activation of NF-?B by
cross-linking B7-DC. A common pathway that can lead to NF-?B
activation is through activation of PI3K, an inducer of AKT-me-
diated NF-?B activation (31, 32). Because AKT is phosphorylated
following B7-DC cross-linking Ab treatment (Fig. 1A), the possi-
bility exists that signals generated by cross-linking B7-DC are me-
diated by PI3K regulated pathways. Consistent with this hypoth-
esis, 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).
To examine the relationships of PI3K and AKT to the activation
of NF-?B, we used pharmacological inhibitors of this signaling
cascade. As shown in Fig. 5, control Ab did not activate NF-?B
(Fig. 5A), whereas B7-DC cross-linking Ab induced robust NF-?B
activation (Fig. 5B). Inhibition of PI3K by LY294002 resulted in
prevention of B7-DC cross-linking Ab-induced activation of
NF-?B in a dose-dependent fashion (Fig. 5, C and D). Further-
more, prevention of AKT activation with the pharmacological
AKT inhibitor IV prevented NF-?B activation following B7-DC
cross-linking (Fig. 5, E and F). Finally, I?B kinase-? inhibition by
NBD peptide also resulted in inhibition of NF-?B activation (Fig.
pendent 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-?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-?B by confocal microscopy using a ?40 apochromat lens magni-
fication. 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-?B by B7-DC cross-linking Ab is inde-
1429 The Journal of Immunology
5, G and H). Taken together, these experiments demonstrate PI3K-
mediated activation of AKT plays a crucial role in activation of
NF-?B in an I?B kinase-?-dependent manner following cross-
linking of B7-DC.
PI3K3AKT3NF-?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-?B
has been shown to induce survival signals in multiple cell types
(31, 32). Because we found activation of NF-?B is dependent on
PI3K3AKT, 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
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 ?M 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 ?M
LY294002, respectively. H, Dose response inhibition by LY294002 as
measured by mean fluorescent intensity (MFI) of phospho-AKT.
Activation of AKT is dependent on PI3K. Bone marrow-
vation and is important for DC survival. Bone marrow-derived DC were
stimulated with control Ab or B7-DC cross-linking Ab in absence or pres-
ence of inhibitors of PI3K, AKT, or NF-?B. Cells were assayed for acti-
vation of NF-?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 ?40 apochromat lens magnification as in Fig.
2C. NF-?B activation upon control Ab treatment is shown in A, whereas B
shows NF-?B activation upon B7-DC cross-linking Ab treatment. C and D
are NF-?B activation upon cross-linking B7-DC in cells that were pre-
treated with AKT inhibitor at 10 (C) or 1 ?M (D) concentration, PI3K
inhibitor at 10 (E) and 1 ?M (F) concentration, and NF-?B inhibitor at 50
(G) and 10 ?M (H) concentration. I, Bone marrow-derived DCs were as-
sessed for protection against cell death after cytokine withdrawal upon
cross-linking B7-DC in the presence or absence of PI3K, AKT, or NF-?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
Activation of NF-?B is dependent on AKT 3 PI3K acti-
1430B7-DC CROSS-LINKED Ab-INDUCED NF-?B PROTECTS FROM CELL DEATH
cross-linking Ab plus DC pretreated 30 min with 10 or 1 ?M
concentration of the PI3K inhibitor, LY294002, AKT inhibitor IV,
and the NF-?B inhibitor NBD at 10 ?M or 1 ?M 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-?B, thereby underscoring the impor-
tance of this signaling pathway for DC survival induced by pro-
vided by cross-linking B7-DC (Fig. 5I). In parallel experiments
using human DC, the pharmacological inhibitors blocked the abil-
ity 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 ?g/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 eval-
uated whether CD40-specific Ab or RANK ligand will enhance the
survival of cells in combination with B7-DC cross-linking. Al-
though 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.
Exposing cells to a depleted environment devoid of growth pro-
moting 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 activat-
ing NF-?B, a transcription factor that induces prosurvival factors
(42–44). Although the precise pathway by which AKT activates
NF-?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-?B. Together, these
studies define a PI3K3AKT3NF-?B signaling pathway that reg-
In this report we demonstrate that the B7-DC cross-linking Ab-
mediated protection against DC death is dependent on activation of
NF-?B, involving a PI3K3AKT3NF-?B signaling pathway.
NF-?B in this system is dependent on PI3K, as the PI3K inhibitor
Ly294002 blocked both AKT and NF-?B activation. Inhibition of
AKT with AKT inhibitor IV also blocked NF-?B activation, in-
dicating that these three signaling intermediates are organized in a
canonical pathway. Importantly, blockade of each of these signal-
ing intermediates prevented B7-DC cross-linking Ab-mediated
protection against cell death induced by the stress of cytokine
Other signals that activate DC also function through NF-?B. For
example, TLR ligation can lead to activation of NF-?B. However,
our studies indicate that B7-DC cross-linking Ab and TLR ago-
nists 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 addi-
tion, we found an absence of NF-?B activation 30 min after TLR4
ligation in MyD88-deficient DC, whereas B7-DC cross-linking Ab
strongly activates NF-?B in this time frame. This finding is con-
sistent with the reported differences in the kinetics of activation of
NF-?B observed in MyD88-deficient DC by EMSA, where there
was a delay of 10 min in MyD88-deficient macrophages in com-
parison to NF-?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-?B in DC from
both species. In the mouse system, we have found that NF-?B
activation leads to up-regulation of cytokine messenger RNAs, in-
cluding the proinflammatory cytokines IL-6 and TNF-?. We also
found that activation of human DC by cross-linking B7-DC re-
sulted in increased production of IL-6 and TNF-?, 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-?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-?B can be
readily activated in B7-DC-deficient cells through cell surface re-
ceptors such as CD40. Whether or not activation of DC through
B7-DC cross-linking is the usual way DC receive survival or ac-
tivation 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 mel-
anoma 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 indi-
cates 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-?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-?B
and the complex phenotypes induced in vivo remains to be estab-
lished, 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.
The authors have no financial conflict of interest.
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1432 B7-DC CROSS-LINKED Ab-INDUCED NF-?B PROTECTS FROM CELL DEATH