volume 10 number 4 april 2009 nature immunology
Genentech, Inc., South San Francisco, California, USA. Correspondence
should be addressed to V.D. (firstname.lastname@example.org) or A.A. (email@example.com).
Published online 19 March 2009; doi:10.1038/ni.1714
Death receptor signal transducers: nodes of
coordination in immune signaling networks
Nicholas S Wilson, Vishva Dixit & Avi Ashkenazi
Death receptors (DRs) are members of the tumor necrosis factor receptor superfamily that possess a cytoplasmic death domain
(DD). DRs regulate important operational and homeostatic aspects of the immune system. They transmit signals through apical
protein complexes, which are nucleated by the DD adaptors FADD and TRADD, to control cellular outcomes that range from
apoptosis to gene activation. FADD and TRADD also nucleate several distal signaling complexes, which mediate cross-talk
between distinct DR signaling pathways. Moreover, together with other DR signal transducers, FADD and TRADD participate
in functional complexes assembled by certain non-DR immune cell receptors, such as pattern-recognition receptors. Thus, DR
signal transducers may provide important nodes of coordination in immune signaling networks.
The tumor necrosis factor (TNF) ligand superfamily acts through cog-
nate TNF receptors (TNFRs) to control diverse biological functions that
include key aspects of immune modulation1. Most TNFRs are type 1
transmembrane proteins, but some are anchored to the plasma mem-
brane by glycophospholipid moieties or secreted as soluble molecules.
Several of these TNFRs, dubbed death receptors, share a relatively con-
served, 80-amino-acid death domain motif in the cytoplasmic tail2. DR
signals induce diverse, context-dependent outcomes in immune cells,
ranging from apoptosis to proliferation to survival to secretion of proin-
Six human DRs and their cognate ligands have been identified, except
for a ligand for DR6, which has not yet been reported (Table 1). Mice
have orthologs of all human DRs and ligands, with the exception of
mouse DR5, which acts as a single ortholog of human DR4 and DR5 and
shares respectively 76% and 79% amino acid identity with the human
Upon binding of their specific ligands, DRs recruit one of two pivotal
DD-containing adaptor proteins: Fas-associated DD (FADD) or TNF
receptor–associated DD (TRADD) (Fig. 1). Other binding modules
in these adaptors serve to recruit apical effector enzymes that trigger
different intracellular signaling cascades. FADD controls cell death by
recruiting the apoptosis-initiating proteases caspases 8 and 10. TRADD
controls non-apoptotic functions by recruiting the DD-containing
kinase receptor-interacting protein-1 (RIP1)4, and the E3 ubiquitin
ligases TNF receptor–associated factor 2 (TRAF2) and cellular inhibi-
tor of apoptosis proteins (cIAPs)5. These components activate phos-
phorylation cascades involving IKK—a kinase that phosphorylates IκB,
the inhibitor of nuclear factor (NF)-κB—and the mitogen-activated
protein kinases (MAPKs) c-Jun N-terminal kinase (JNK) and p38. The
resulting events initiate transcriptional programs that modulate cellular
function and/or fate.
DRs can be divided into two categories based upon the primary
adaptor protein to which they bind. CD95, DR4 and DR5 bind FADD
and show mainly proapoptotic function (Fig. 1a). In contrast, TNFR1
and DR3 bind TRADD and mediate mainly proinflammatory and
immune-stimulatory activity (Fig. 1b)6. DR6 can bind TRADD when
overexpressed7, although its primary physiological adaptor has yet to be
determined. Notably, however, cross-talk may occur between DR signal
transducing components. For example, TNFR1 can trigger apoptosis
through TRADD-dependent or RIP1-dependent recruitment of FADD
and caspase-8 (refs. 8,9), whereas DR4 or DR5 can activate the NF-κB
and MAPK pathways through FADD- and caspase-8-dependent activa-
tion of RIP1 (ref. 10). In addition, DR signal transducers participate in
non-DR signaling complexes downstream of the T cell antigen receptor
(TCR)11–13 and pattern-recognition receptors (PRRs) such as Toll-like
receptors (TLRs)14–18 and retinoic acid–inducible gene I (RIG-I)-like
helicases (RLHs)19,20. Here we discuss the emerging evidence for involve-
ment of DR signal transducers as points of cross-talk between immune
FADD-mediated DR signaling
Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells express
proapoptotic DR ligands such as CD95L or Apo2 ligand, also known as
TNF-related apoptosis-inducing ligand (Apo2L/TRAIL), as part of their
armamentarium against infected or transformed cells. The apoptosis
pathway induced by these DR ligands is called the cell-extrinsic path-
way. Binding of the cognate homotrimeric ligand drives DR clustering
and binding of DR to FADD. FADD recruits caspase-8 and caspase-10,
closely related apical proteases, to form the death-inducing signaling
complex (DISC) (Fig. 1a)2,21. Specific post-translational DR modifica-
tions such as palmitoylation and O-linked glycosylation can facilitate
DISC activation22–24. The DISC mediates autocatalytic processing of
caspases 8 and 10 and releases active enzyme into the cytoplasm. Active
caspases 8 and 10 stimulate the executioner caspases 3, 6 and 7, which in
turn cleave many cellular substrates, culminating in apoptotic cell death.
In type I cells (for example, thymocytes), the latter events are sufficient to
immune signaling cross-talk
© 2009 Nature America, Inc. All rights reserved.
nature immunology volume 10 number 4 april 2009
trigger apoptosis. In type II cells (for example, B cells), apoptosis requires
further amplification by means of caspase-8-mediated processing of the
Bcl-2–family protein Bid, which engages the cell-intrinsic mitochondrial
pathway (Fig. 1a). Mice encode a single relative of human caspases 8
and 10 (ref. 25).
Signal modulation within the DISC involves the cellular FLICE
inhibitory protein (c-FLIP) (Fig. 1a). Like caspases 8 and 10, c-FLIP
contains two tandem N-terminal death effector domains (DEDs) and a
C-terminal region that resembles the enzyme segment of caspases but
lacks a critical catalytic cysteine residue26,27. c-FLIP has two short splice
variants (c-FLIPS and c-FLIPR) and one long variant (c-FLIPL). Mice
express c-FLIPL and c-FLIPR (ref. 28). Knockdown of c-FLIPS or c-FLIPL
by short interfering RNA sensitizes various transformed cell lines to
DR-mediated apoptosis29. Short c-FLIP variants inhibit DR-mediated
apoptosis by competing with caspase-8 for binding to FADD. When
present in high amounts, c-FLIPL interferes with caspase recruitment to
the DISC30; however, in low quantities, c-FLIPL can form heterodimers
with caspase-8 that support enzymatic activation31,32.
TRADD-mediated DR signaling
Upon recruitment to TNFR1, TRADD provides a scaffold to assemble
‘complex I’ at the plasma membrane, through binding of RIP1, TRAF2
(or TRAF5) and cIAPs 1 and 2 (Fig. 1b). This core complex has the
signaling capacity to activate the NF-κB and the JNK and p38 MAPK
pathways. Recent studies with TRADD-deficient mice verify the role
of TRADD in DR-proximal events; however, they indicate that a strict
dependence on TRADD for RIP1-mediated
NF-κB signaling is cell-type specific14–16.
Association of complex I at TNFR1 trig-
gers polyubiquitination of RIP1, mediated by
cIAPs (refs. 33–35). This process is negatively
regulated by A20-dependent deubiquitinating
activity36 and by caspase 8/10-associated ring
protein 2 (CARP2)-mediated Lys48 polyubiq-
uitination of RIP1 (Fig. 1b)37. Ubiquitinated
RIP1 interacts with transforming growth
factor-β–activated kinase 1 (TAK1) through
TAK1-binding proteins 1 and 2 (TAB1,2), and
consequently, TAK1 activates the IKK complex,
which contains IKKα, IKKβ and IKKγ (also
called NEMO)38. Once phosphorylated by the
IKK complex, IκB—which retains NF-κB in
the cytoplasm—is subjected to Lys48 polyubiq-
uitination and proteasomal degradation, liber-
ating NF-κB to move into the nucleus. NF-κB
induces transcription of many genes, includ-
ing those encoding proinflammatory cytokines
and chemokines as well as antiapoptotic factors
such as cIAPs and c-FLIP. Complex I also acti-
vates JNK and p38 (Fig. 1b)39,40, stimulating
genes that regulate proliferation, differentia-
tion, inflammation or apoptosis.
Cross-talk between DR signaling adaptors
In addition to complex I, ligation of TNFR1
can lead to TRADD-mediated formation of
two cytoplasmic complexes: IIA and IIB (Fig.
2a)8,9,40. After receptor internalization, confor-
mational changes in RIP1 and TRAF2 facili-
tate their dissociation from TRADD, allowing
TRADD to recruit caspase-8 through FADD
and to assemble complex IIA (refs. 9,41). FADD also recruits c-FLIP,
which in this capacity determines whether effective caspase-8 activa-
tion occurs within complex IIA and apoptosis ensues. c-FLIP quantities
are tightly regulated by interplay between the signaling pathways that
emanate from complex I. Survival signals such as NF-κB and phos-
phatidylinositol-3-OH kinase activation increase c-FLIP expression9,42,
whereas stress signals can cause JNK-mediated phosphorylation of the
E3 ubiquitin ligase ITCH, which drives ubiquitination and proteasomal
degradation of c-FLIPL (refs. 43,44).
In accordance with evidence that RIP1 can regulate both proapop-
totic and antiapoptotic cascades45,46, recent work shows that RIP1 can
associate with FADD to activate caspase-8 and trigger apoptosis down-
stream of TNFR1 through complex IIB (Fig. 2a)9,47. This latter pathway
is negatively regulated by cIAPs and is amplified by the IAP antagonist
Smac or by Smac mimetics that facilitate cIAP autoubiquitination and
proteasomal degradation5,48. When associated with TRADD in complex
I, RIP1 is modified by Lys63-linked polyubiquitin; however, in complex
IIB, RIP1 is not ubiquitinated. Moreover, the RIP1 deubiquitinating
enzyme cylindromatosis gene product (CYLD) is required for caspase-8
activation by complex IIB (ref. 9), suggesting that deubiquitination sup-
ports RIP1 oligomerization and binding to FADD. TRADD and TRAF2
are not detected in complex IIB (ref. 9), and TRADD knockdown aug-
ments formation of complex IIB, implicating RIP1 as an alternative
apical adaptor for TNFR1 (Fig. 1b)9,15,49,50.
The specific contribution of complex IIA or IIB to proapoptotic
TNFR1 signaling in immune cells remains to be discerned. Macrophages
a FADD: CD95, DR4, DR5
b TRADD: TNFR1, DR3, (DR6?)
Type II Type I
Figure 1 Primary FADD- and TRADD-dependent DR signaling complexes. FADD and TRADD nucleate
distinct apical signaling complexes to coordinate proapoptotic and non-apoptotic pathways downstream
of DRs. (a) Receptor-bound FADD forms a death-inducing signaling complex (DISC) with caspase-8 (as
well as with caspase-10 in human cells). The amount of caspase-8 activity generated in type I cells is
sufficient to activate effector caspases and trigger apoptosis. In contrast, apoptotic commitment in
type II cells requires further signal augmentation through mitochondria-based amplification mediated
by caspase-8-dependent cleavage of the Bid protein to its active form, t-Bid. (b) TRADD signaling is
best characterized downstream of TNFR1. TRADD assembles an apical complex that contains RIP1,
TRAF2 or TRAF5, and cIAPs 1 and 2, to activate diverse gene expression programs through the NF-κB
and AP-1 transcription factors.
© 2009 Nature America, Inc. All rights reserved.
volume 10 number 4 april 2009 nature immunology
and dendritic cells (DCs) constitutively express c-FLIP and cIAPs, which
negatively regulate complex IIA and IIB51–53. Therefore, TNFR1 signal-
ing through complex I is likely to dominate in these cell types (Fig. 1b).
Genetic ablation of cIAP2 renders macrophages sensitive to lipopoly-
saccharide (LPS)-induced apoptosis, which may be caused by autocrine
TNF signaling52. It is conceivable that conditions of cellular stress could
lead to JNK-mediated ITCH activation, which would downregulate
c-FLIP and further facilitate TNFR1-dependent stimulation of complex
IIA (Fig. 2a). In thymocytes, TNFR1 seems important for determina-
tion of proliferative versus apoptotic fate, depending on developmental
stage54; however, the involvement of complex IIA or IIB in this process
has yet to be defined.
Proinflammatory versus proapoptotic CD95 signaling
The association between genetic alterations in various DR signaling
components and a range of immunological disorders indicates that DRs
are critical for immune modulation. Mutations in the genes encoding
CD95 or CD95L underlie human autoimmune lymphoproliferative syn-
drome (ALPS)55,56, a disease characterized by a gradual accumulation
of atypical lymphocytes and autoantibody production. A similar syn-
drome occurs in mice with either of two non-allelic, autosomal recessive
mutations in the genes encoding CD95 (Fas) or CD95L (Fasl)57. CD95
enforces self-tolerance by eliminating chronically stimulated T cells. In
contrast, acute T cell contraction depends primarily on Bim-mediated
activation of the intrinsic apoptosis pathway58. CD95 also provides an
important checkpoint in the elimination of autoreactive B cells and in
DC homeostasis, although other proapoptotic pathways not involving
CD95 contribute to these functions as well59–61.
Besides proapoptotic signaling, CD95 serves to coordinate vari-
ous proinflammatory responses in immune cells27,62. These include
chemokine and cytokine production by monocytes, macrophages and
inflammatory DCs (DCs derived from monocytes or bone marrow pre-
cursors with GM-CSF)63. Activation of the NF-κB and MAPK path-
ways is a salient feature of non-apoptotic CD95 signaling in immune
cells, and recruitment of c-FLIP isoforms into the CD95 DISC probably
favors these proinflammatory outcomes (Fig. 2b)27,64. Many immune
cell types, including DCs, macrophages and B and T cells, constitu-
tively express c-FLIP, and this expression is dynamically modulated by
cytokine-receptor or antigen-receptor stimulation27. When present at
high concentrations, c-FLIPL incorporates into the CD95 DISC to pre-
vent apoptosis and may promote proliferation and cytokine production
by recruiting RIP1 and TRAF2 (refs. 26,65,66). T cells can upregulate
the c-FLIPS isoform by means of the transcription factor NFAT, thereby
becoming more resistant to CD95-induced apoptosis67,68.
The outcome of CD95 ligation can be determined by coordinated
signaling. For example, interleukin-7 (IL-7) primes peripheral T cells
for CD95-mediated apoptosis by inducing the accumulation of CD95
at the cell surface69. Coordinated signaling with TCR stimulation modi-
fies this response, causing T cells to proliferate and produce more IL-2
upon combined stimulation by IL-7 and CD95L (ref. 70). ‘Reverse
signaling’ by membrane-associated CD95L provides another level of
modulation, promoting T cell proliferation in response to CD95 binding
in conjunction with TCR stimulation71. This latter phenomenon may
account for the impairment of T cell proliferation in a CD95-deficient
background72, although the molecular mechanism of ‘reverse signaling’
by CD95L remains obscure.
Other determinants of proapoptotic versus proinflammatory CD95
signaling include the form of ligand encountered (the potently agonistic
plasma membrane–associated or the weakly agonistic soluble CD95L)
and compartmentalization of CD95 in plasma membrane lipid rafts,
as well as the receptor’s route of internalization and endosomal traf-
ficking41. Cell surface CD95L is expressed by immune cells, epithelial
cells, transformed cells and paracortical high endothelial venules of the
lymph node73–75. Although T cell hybridomas rapidly produce surface
CD95L after TCR stimulation, CD95L expression by primary T cells
requires prolonged co-stimulation through the TCR and CD28 (refs.
76,77). Moreover, primary T cells activated by antigen-presenting cells
displaying MHC–peptide complexes do not produce CD95L (ref. 77).
Thus, T cell hybridomas may not faithfully reflect the expression of
CD95L by primary T cells.
Diverse immune functions of DR5
Mounting evidence indicates that mouse DR5 (the ortholog of human
DR4 and DR5) is important in innate immune surveillance. NK cells
participate in the elimination of virally infected, transformed or dam-
aged cells78,79. Of the several developmentally and functionally distinct
subsets of NK cells80,81, immature NK cells have poorer cytolytic activ-
ity than their phenotypically mature counterparts. In addition, only
immature NK cells show surface expression of the DR5 ligand Apo2L
(also called TRAIL)/TRAIL. Such cells were originally identified in the
adult liver82, although existence of a similar population in the bone
marrow has been reported as well81. Antibody blockade or genetic dele-
tion of Apo2L increases liver metastasis in several mouse tumor models,
implicating this proapoptotic ligand in NK cell–mediated antitumor
immune surveillance83–85. Similarly, DR5-deficient mice crossed with
strains that harbor tumorigenic genetic lesions show an increased inci-
dence of tumor metastases in the lung and liver86. Type I interferons,
TLR ligands and viruses promote expression of Apo2L on NK cells and
other innate immune-cell populations87–89, suggesting that this ligand
contributes to initial clearance of virus-infected cells. Consistent with
this notion, inhibition of Apo2L in virus-infected mice is associated with
higher viral titers and a diminished survival rate90,91.
In addition to mediating immune surveillance, mouse DR5 signaling
in immune cells seems to restrict the production of proinflammatory
cytokines in response to pathogen challenge86,92. DCs and macrophages
from DR5 knockout mice show delayed de novo IκB synthesis after TLR
stimulation, suggesting that DR5 signaling serves to temporally limit
TLR-induced NF-κB activation92. How mouse DR5 promotes IκB
transcription remains to be defined. Human monocytes express DR5,
as do mouse inflammatory DCs87,88,93. Lung NK cells are reported to
eliminate adoptively transferred inflammatory DCs by a mechanism
involving Apo2L, implicating mouse DR5 in NK cell–mediated control
of DC function93; however, the physiological importance of this phe-
nomenon remains to be examined. Some NK and T cell populations
upregulate mouse DR5 in response to stimulation, although c-FLIP
expression renders these cells refractory to the proapoptotic activity of
the cognate ligand94.
Soluble recombinant human Apo2L (corresponding to the extracel-
lular portion of the transmembrane ligand) and agonistic antibodies
that stimulate DR4 or DR5 trigger apoptosis in various cancer cell types
Table 1 Death receptors and their ligands and apical adaptors
HUGO Pivotal adaptor
TNFR1TNFRSF1ATNF, TNFB TRADD
DR3 TNFRSF25TL1A TNFSF15TRADD
DR4 (TRAILR1) TNFRSF10A Apo2L/TRAIL TNFSF10 FADD
DR5 (TRAILR2) TNFRSF10B Apo2L/TRAIL TNFSF10 FADD
DR6 TNFRSF21? TRADD?
Reviewed in refs. 2,6.
aAssigned by the HUGO gene nomenclature committee (http://www.genenames.org).
© 2009 Nature America, Inc. All rights reserved.
nature immunology volume 10 number 4 april 2009
but not in most normal cells; investigation of these agents for cancer
therapy in clinical trials is underway95. In addition to the primary DISC,
a secondary complex induced by Apo2L, involving the core DISC com-
ponents FADD and caspase-8 as well as TRAF2 and RIP1, occurs in cer-
tain transformed cell lines10 (Fig. 2b). This FADD-dependent complex
II may further augment caspase-8 activation and cell death49,96; alterna-
tively, it may activate NF-κB, JNK and p38, thereby inducing production
of cytokines and chemokines such as CXCL8 and CCL2 (ref. 10). These
secreted factors may serve to recruit monocytes that facilitate clearance
of the apoptotic tumor cells and may promote presentation of tumor-
associated antigens in regional lymph nodes97. Indeed, DR5-mediated
apoptosis induces robust antitumor adaptive immune responses in a
variety of mouse models98.
Although some work implicates mouse DR5 in negative selection of
thymocytes99, this role remains controversial100,101. Other data implicate
mouse DR5 signaling in the elimination of ‘helpless’ CD8+ T cells (that
is, cells that have not received requisite signals from CD4+ T cells)102–105.
However, the importance of DR5 in this process has been debated and
may depend on the antigenic challenge and the protocol used to ablate
CD4+ T cell help106. CD4+CD25+ regulatory T cells seem to mediate
some of their immunosuppressive effects by expressing surface Apo2L,
which stimulates mouse DR5 on CD4+ T cells107.
FADD, caspase-8 and c-FLIP
Unlike mice lacking CD95 function, those with T or B cell–specific
ablation of FADD do not develop excessive lymphoproliferation;
instead, these mice manifest defective lymphocyte homeostasis and
proliferation18,108,109. A similar phenotype is shown by mice with T cell–
specific expression of a FADD mutant transgene that lacks the DED but
retains the DD region110,111 and by T cells lacking caspase-8 (ref. 112) or
c-FLIP113,114. Mice and humans expressing mutant forms of caspase-8
also show defects in lymphocyte proliferative responses12,13. Taken
together, these studies are consistent with the
hypothesis that FADD, caspase-8 and c-FLIP
control not only proapoptotic DR functions but
also DR-independent proliferative responses
of lymphocytes. c-FLIPL contains an activa-
tion loop in its caspase-like domain that binds
and opens the enzymatic pocket of caspase-8
when the two molecules heterodimerize31,32.
Thereby, c-FLIPL can directly support the
processing of caspase-8 into its p43 frag-
ment. Precisely how c-FLIP variants modulate
proapoptotic versus non-apoptotic caspase-8
activity remains unclear27. One supposition is
that heterodimeric complexes comprising the
p43 fragments of caspase-8 and c-FLIPL can
recruit RIP1 and TRAF2 to activate prosurvival
NF-κB signaling (Fig. 2b)66,115,116. Consistent
with this model, c-FLIP expression modulates
T-cell proliferative responses114,117,118. c-FLIPL
and c-FLIPS can be cleaved by the unprocessed
form of caspase-8 to release the p22-FLIP pro-
cessing intermediate, which is found in acti-
vated primary B and T cells and inflammatory
DCs119. The p22-FLIP fragment shows dual
function: it can inhibit apoptosis by incorpo-
rating into the DISC and forming heterodim-
ers with caspase-8; alternatively, it can promote
NF-κB activation by interacting and interfering
with IκB (Fig. 2b).
The involvement of FADD in supporting lymphocyte prolifera-
tion seems to be linked to its phosphorylation state. FADD contains a
C-terminal phosphorylation site (Ser191 in mouse or Ser194 in human
FADD), which is dispensable for proapoptotic function but impor-
tant for modulation of T cell proliferation120,121. Reconstitution of
FADD-deficient mice with a phosphorylation-deficient S191A mutant
of FADD rescues the lethality associated with FADD gene ablation. In
contrast, reconstitution with an S191D mutant, which mimics consti-
tutive FADD phosphorylation, leads to death within 4 weeks of birth
or to runting. Whereas T cells harboring wild-type FADD show FADD
phosphorylation within hours of TCR activation and coincidentally
with entry into the cell cycle, T cells expressing the S191D mutant
show defective cell cycle progression upon TCR stimulation120,121. The
modulation of T cell proliferation by FADD correlates with sustained
activation of ribosomal protein S6 kinase (S6K)122. Consistent with
this observation, T cells expressing a DED-deleted FADD mutant or
lacking caspase-8 show cell cycle defects in association with dimin-
ished S6K phosphorylation122. It has been proposed that caspase-8
facilitates T cell expansion and survival by activating NF-κB12,13,123,
but this remains controversial124. A recent study demonstrates that
caspase-8 deficiency does not affect TCR-mediated NF-κB activation,
suggesting instead that caspase-8 inhibits a particular type of RIP1-
dependent necrotic T cell death, termed necroptosis124 (Fig. 3a). Upon
TCR stimulation, protein kinase C-θ (PKCθ) is recruited to the immu-
nological synapse125. Consequently, a specialized signaling complex
(dubbed CBM) is formed, comprising three proteins: caspase recruit-
ment domain–containing membrane-associated guanylate kinase
protein-1 (CARMA1), Bcl10 and the ‘paracaspase’ mucosa-associated
lymphoma translocation gene 1 (MALT1), which possesses a DD and
a caspase-like region126. Within the CBM complex, Bcl10 and MALT1
undergo Lys63 polyubiquitination, which facilitates NF-κB activation
by means of the IKK complex126,127. Alternatively, TAK1 may mediate
Figure 2 Secondary FADD-, TRADD- and RIP1-dependent signaling complexes. FADD, TRADD and
RIP1 can form distal signaling complexes that coordinate non-apoptotic and proapoptotic pathways.
(a) TNFR1-dependent recruitment of TRADD leads to distal formation of complex IIA, which involves
FADD recruitment to TRADD and subsequent activation of caspase-8. An alternative complex, IIB,
proceeds independently of TRADD through a RIP1–FADD scaffold to activate caspase-8. Complex IIA is
regulated by c-FLIP, and complex IIB is regulated by cIAP-dependent ubiquitination and CYLD-mediated
deubiquitination of RIP1. The e3 ubiquitin ligase ITCH, which is modulated by JNK signaling downstream
of TNFR1-based complex I, negatively regulates c-FLIPL quantities, thereby facilitating activation of
caspase-8 by complex IIA. Smac or Smac mimetics facilitate cIAP autoubiquitination and proteasomal
degradation by allosteric activation of the cIAP RING finger ubiquitin ligase domain, thereby promoting
activation of complex IIB. (b) FADD-dependent complex II activates NF-κB and MAPK pathways,
resulting in chemokine and cytokine secretion that may occur concurrently with proapoptotic signaling.
Heterodimers formed between caspase-8 and c-FLIP may distinguish non-apoptotic from proapoptotic
signaling outcomes. p22 and p43 indicate processed fragments of c-FLIP or caspase-8 (C8).
a TRADD- and RIP-dependent complex IIA and IIB
b FADD-dependent complex II
© 2009 Nature America, Inc. All rights reserved.
volume 10 number 4 april 2009 nature immunology
CBM-independent IKK phosphorylation downstream of the TCR128.
The involvement of caspase-8 in TCR-mediated proliferation and
survival may be related to its ability to bind MALT1 within the CBM
complex26,129. This function may involve c-FLIPL, with p43-FLIP–
p43-caspase-8 heterodimers providing a scaffold for NF-κB activation
through RIP1 (refs. 115,129). CD28-mediated activation of ITCH may
further fine-tune the amount of c-FLIPL available to bind caspase-8
within the CBM complex130.
DR adaptors in PRR signaling
The TLR adaptor TIR-domain–containing adaptor inducing interferon-β
(TRIF) can activate NF-κB by binding to RIP1 through a C-terminal RIP
homology interaction motif (RHIM)131,132. TLR3 functions through
TRIF, whereas TLR4 signals through both TRIF and myeloid differen-
tiation primary response gene 88 (MyD88)133 (Fig. 3b). In addition to
defective TNF responses, mouse embryonic fibroblasts (MEFs) from
TRADD-deficient mice show impaired NF-κB and MAPK signaling
in response to TLR3 activation, indicating a requirement for TRADD
downstream of the TLR3–TRIF complex14–16. In contrast, TRADD-
deficient MEFs show only a modest impairment in their cytokine
response to TLR4 activation, owing to TRIF-independent signaling
through MyD88. The TLR–TRIF complex also recruits RIP1, which then
undergoes ubiquitination and activation. Unlike the phenotype in MEFs,
TRADD deficiency in macrophages or DCs does not significantly impair
TRIF-dependent activation of NF-κB and MAPK14–16. This discrepancy
may be explained by the fact that TRIF can use two distinct mechanisms
to activate NF-κB. One involves TRADD and RIP1, whereas the other
involves direct binding to TRAF6 (Fig. 3b)134. Preferential use of TRAF6
in macrophages and DCs could explain why
TRADD is dispensable downstream of TRIF
in these cell types135.
TLR–TRIF signals can activate caspase-8 to
induce both non-apoptotic136 and proapop-
totic137 functions. TRIF-mediated activation of
caspase-8 requires an intact RHIM, suggesting
that RIP1 is required for this function; FADD is
involved in this axis as well137, suggesting that
a RIP1–FADD complex akin to complex IIB is
involved (Fig. 2a). Through this type of com-
plex, caspase-8 may supplant caspase-1 to pro-
cess pro-IL-1β in cells such as macrophages136.
Alternatively, the proapoptotic function of
caspase-8 may help control viral infection by
eliminating infected cells, thus preventing the
dissemination of progeny virions137. Recent
work reveals further that TNF-induced pro-
tein 8-like 2 (TIPE2), a negative regulator of
innate and adaptive immunity, binds caspase-8
through a putative DED138. Notably, TIPE2 is
not found within the DISC, nor is it required
for DISC recruitment of FADD and caspase-8
(ref. 138); indeed, TIPE2-deficient mice
develop fatal inflammatory disease and are
hypersensitive to LPS-induced shock, suggest-
ing that TIPE2 negatively regulates proinflam-
matory caspase-8 signals, including NF-κB and
Another TRADD-associated innate immune
signaling complex comprises the RLHs RIG-I
and MDA-5, which recognize intracellular RNA
through their C-terminal helicase domain.
These RNA sensors bind to TRADD by means of homotypic caspase
activation and recruitment domain (CARD) interactions with the
mitochondria-associated protein CARD adaptor inducing interferon-β
(CARDIF) (Fig. 3c)19,139. CARDIF recruits TRAF3 (refs. 140,141) and
TRAF6 (ref. 135). TRAF3 binds the adaptor protein TANK, which acti-
vates interferon regulatory factor (IRF)-3 and IRF7; TRAF6 activates
NF-κB and MAPK. TRADD can also recruit RIP1 to induce further
NF-κB activation by means of FADD and caspase-8 (refs. 19,20). This
pathway might compensate for deficiency in TRAF6-dependent induc-
tion of type I interferons135. Given that c-FLIPL can heterodimerize with
caspase-8 to form a signaling platform, it is reasonable to propose that
c-FLIPL also may contribute to NF-κB activation by this latter mecha-
nism. Furthermore, in the context of low amounts of c-FLIPL, TRADD-
and FADD-dependent caspase-8 activation may provide a proapoptotic
mechanism in response to viral infection. Taken together, these findings
implicate FADD and TRADD and some of their associated signal trans-
ducers in coordinating TLR and RLH signals that help conduct the host
immune response to infection.
Researchers have made substantial progress in delineating the molecular
pathways that connect DRs with their downstream effectors. The core
DR signaling networks are broadly classified by engagement of the clas-
sic proapoptotic adaptor FADD or the non-apoptotic adaptor TRADD.
This distinction served as a general paradigm for understanding the
function of DRs in immune system homeostasis. However, genetic and
biochemical approaches have unraveled considerably greater func-
tional diversity of FADD and TRADD and their associated intracellular
Figure 3 Non-DR signaling complexes involving DR signal transducers. (a) At the immunological
synapse, TCR and CD28 stimulation leads to PKCθ activation, which promotes engagement of the CBM
complex. Ubiquitinated CBM components can activate the IKK complex directly, or indirectly through
TAK1-mediated phosphorylation. The paracaspase MALT1 is reported to bind caspase-8, which may
recruit c-FLIP to activate the IKK complex. Alternatively, CBM-mediated activation of caspase-8 may
inhibit a RIP1-dependent necrotic death called necroptosis. (b) TRIF engagement by TLR3 or TLR4
leads to RIP1 recruitment. TRIF can activate the NF-κB and MAPK pathways through two distinct
mechanisms that involve either RIP1 and TRADD, or TRAF6. The predominance of each pathway
depends on cell type; DCs and macrophages seem to favor the TRAF6 axis. TLR4 also activates parallel
signals through MyD88. (c) The RIG-I and MDA-5 RLHs recognize intracellular RNA, and they interact
with CARDIF, which recruits TRAF3 and TRAF6. TRAF3 binds TANK, which phosphorylates IRF3 and
IRF7. An alternative signaling cascade leading to NF-κB activation proceeds by CARDIF-mediated
recruitment of TRADD, which can activate caspase-8 through RIP1 and FADD. c-FLIP binding to
caspase-8 may facilitate activation of NF-κB. In the absence of c-FLIP, stimulation of caspase-8 may
lead to apoptotic death of cells infected by RNA viruses.
© 2009 Nature America, Inc. All rights reserved.
nature immunology volume 10 number 4 april 2009
signaling components. The structural modularity of DR signal trans-
ducers allows these proteins to participate not only in diverse DR sig-
naling cascades but also in non-DR pathways, such as those emanating
from the TCR or several PRRs. DRs modulate various aspects of the
immune system, including lymphocyte homeostasis, innate and adaptive
immunity and immune surveillance. This interplay may facilitate the
coordination between immune system networks. The challenge now is to
decipher how these interconnections help to orchestrate a more effective
response to infection or malignancy while preventing autoimmunity.
It will doubtlessly be fruitful to apply some of the recent advances in
systems biology research to this fascinating question.
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