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
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1432 B7-DC CROSS-LINKED Ab-INDUCED NF-?B PROTECTS FROM CELL DEATH