Immunity, Vol. 15, 647–657, October, 2001, Copyright 2001 by Cell Press
TRAF1 Is a Negative Regulator of TNF Signaling:
Enhanced TNF Signaling in TRAF1-Deficient Mice
cleavage product of TRAF1 completely prevents NF-?B
activation induced by TNF, IL-1, or overexpression of
TRAF2 or TRAF6, and therefore may function as a domi-
nant-negative form of TRAF1 (Carpentier and Beyaert,
1999; Irmler et al., 2000; Leo et al., 2000). In contrast,
is prolonged in transfectants overexpressing TRAF1,
whereas overexpression of a TRAF1 mutant in which
the N-terminal part was replaced by green fluorescent
protein interferes with TNF-induced activation of NF-?B
and JNK (Schwenzer et al., 1999). TRAF1, TRAF2, and
the cellular inhibitor-of-apoptosis proteins were identi-
fiedas genetargetsof NF-?B-dependenttranscriptional
genic mice, TRAF1 was shown to play an inhibitory role
in antigen-induced apoptosis of CD8?T lymphocytes
(Speiser et al., 1997).
To gain further insight into TRAF1 function, we have
generated TRAF1 null (TRAF1?/?) mice. Although
TRAF1?/?mice have normal lymphocyte development,
T cells from these mice exhibit increased proliferation
to anti-CD3 stimulation compared with WT T cells. More
importantly, anti-CD3 activated T cells from TRAF1?/?
mice, but not from WT controls, responded to TNF by
proliferation and activation of the NF-?B and AP-1 sig-
naling pathways. Furthermore,skin from TRAF1?/?mice
is hypersensitive to lymphocyte-dependent TNF-induced
skin necrosis. Taken together, these findings indicate
that TRAF1 is a negative regulator of TNF signaling in
Erdyni N. Tsitsikov,1,3Dhafer Laouini,1Ian F. Dunn,1
Tatyana Y. Sannikova,1Laurie Davidson,2
Frederick W. Alt,2and Raif S. Geha1
1Division of Immunology
2Howard Hughes Medical Institute
Children’s Hospital and Department of Pediatrics
Harvard Medical School
Boston, Massachusetts 02115
TNF receptor-associated factor 1 (TRAF1) is a unique
TRAF protein because it lacks a RING finger domain
and is predominantly expressed in activated lympho-
cytes. To elucidate the function of TRAF1, we gener-
ated TRAF1-deficient mice. TRAF1?/?mice are viable
and have normal lymphocyte development. TRAF1?/?
eration to anti-CD3 mAb, which persisted in the pres-
ence of IL-2 or anti-CD28 antibodies. Activated
TRAF1?/?T cells, but not TRAF1?/?T cells, responded
to TNF by proliferation and activation of the NF-?B
and AP-1signaling pathways. This TNFeffect was me-
diatedbyTNFR2 (p75)butnotby TNFR1(p55).Further-
more, skin from TRAF1?/?mice was hypersensitive to
TNF-induced necrosis. These findings suggest that
TRAF1 is a negative regulator of TNF signaling.
TRAF1 and TRAF2 were originally discovered due to
their ability to bind to TNFR2 (Rothe et al., 1994). Six
TRAFs have been described to date (Arch et al., 1998).
Members of the TNF receptor superfamily, including
TNFR2, CD27, CD30, and CD40, may associate with one
or several TRAFs (Arch et al., 1998). TRAF2, TRAF3,
TRAF5, and TRAF6 were shown to be important for the
activation of both NF-?B and AP-1 transcription factors
finger, several zinc fingers, and a C-terminal TRAF do-
main, which is important for interactions with receptors
and other TRAF proteins. TRAF1 is a unique member of
the TRAF family; it contains a single zinc finger and a
TRAF domain. Its expression is restricted to spleen,
lung, and testis, in contrast to the more ubiquitous ex-
pression of other TRAFs (Mosialos et al., 1995; Rothe
et al., 1994). TRAF1 can be recruited to a number of
distinct members of the TNFR superfamily, including
TNFR2, CD27 (Yamamoto et al., 1998), CD30 (Tsitsikov
OX-40 (Kawamata et al., 1998), HVEM/ATAR (Marsters
et al., 1997), TRANCE-R (Wong et al., 1998), XEDAR
(Yan et al., 2000), and also to Epstein-Barr virus latent
infection membrane protein 1 (Mosialos et al., 1995).
Little is known about the biochemical function of
Generation of TRAF1-Deficient Mice
To elucidate the function of TRAF1 in vivo, we disrupted
the murine traf1 gene by gene targeting. A portion of
the murine traf1 gene and the targeting construct are
shown in Figure 1A. Exons 2 to 5, including the first
coding exon (exon 4), were replaced by the neomycin
resistance gene following homologous recombination
in embryonic stem cells. Genomic DNA from individual
neoresistant ES clones was prepared, digested with
EcoRI, and used for Southern blotting analysis with a
3? probe (Figure 1A). ES clones with targeted disruption
of the novel 10 kb fragment derived from the targeted
allele in addition to the 12 kb fragment derived from the
WT allele (Figure 1B). Of 38 analyzed ES clones, three
were identified to contain a disrupted allele. TRAF1?/?
by PCR and/or Southern blotting of tail DNA, as de-
scribed in Experimental Procedures. Western blotting
analysis with polyclonal antibody against a C-terminal
TRAF1 peptide revealed that CD40-stimulated spleno-
cytes from TRAF1?/?mice have no detectable TRAF1
expression (Figure 1D). TRAF1?/?mice were raised in
a germ-free environment and did not display apparent
differences from WT littermates in growth, weight, or
Figure 1. Generation of TRAF1-Deficient Mice
(A) Partial structure of the murine TRAF1 gene (above) and of the targeting construct (below). Exons are represented by bold segments.
Neomycin resistance (neo) and thymidine kinase (TK) genes are indicated. PCR primers are represented by small arrows.
(B) Southern blot analysis of DNA from ES cell clones. Genomic DNA was digested by EcoRI and probed with the HindIII/EcoRI fragment
shown in (A). The wild-type allele is represented by the 12 kb band, whereas the knockout allele is represented by the 10 kb band.
(C) Western blot analysis of TRAF1 expression in 50 ? 106splenocytes from wild-type (WT) and TRAF1-deficient (KO) mice. Cells were
stimulated with anti-CD40 antibodies overnight, lysed, and TRAF1 expression was evaluated with polyclonal anti-TRAF1 antibodies.
Normal Development of T and B Lymphocytes
TRAF1?/?mice have thymi of normal size and architec-
ture. Detailed FACS analysis revealed no difference in
the numbers of CD4, CD8, TCR??, TCR??, CD2, and
CD3 positive thymocytes between WT and TRAF1?/?
mice (data not shown). Bone marrow from TRAF1?/?
mice has normal numbers of IgM?B cells with normal
expression of CD43 and B220 (data not shown). Taken
together, these results indicate that TRAF1 expression
is not necessary for T or B lymphocyte development.
Spleens from TRAF1?/?mice are of normal size and
have normal T and B cell numbers; normal expression
of the B cell surface markers B220, sIgM, sIgD, CD5,
CD21, CD23, CD40, and HSA; and normal expression
of the T cell surface markers CD4, CD8, TCR??, TCR??,
CD2, and CD3 molecules (data not shown).
mice (2.74 ? 0.37 ? 106in TRAF1?/?mice versus 1.09 ?
0.32 ? 106in WT littermates, p ? 0.05), as well as an
increased T/B cell ratio, but a normal CD4/CD8 ratio
(data not shown).
TRAF2, TRAF3, TRAF5, and TRAF6 knockout (KO)
micehave impairedantibodyresponses toT-dependent
et al., 1999; Xu et al., 1996). To determine the role of
TRAF1 protein in antibody immune responses, we im-
munized TRAF1?/?mice and WT littermates with the
T-dependent antigen ovalbumin. Figure 2 shows that
TRAF1?/?mice have normal IgG1, IgG2a, and IgE anti-
T cell help and intact immunoglobulin isotype switching
in B cells. The antibody responses of TRAF1?/?mice to
the type 1 T-independent antigen TNP-LPS and to the
type 2 T-independent antigen TNP-Ficoll (Figures 2E
and 2F) were also normal. These results suggest that
antigen specific antibody responses are not dependent
Enhanced Proliferation of TRAF1?/?
T Cells to Anti-CD3 mAb
To determine whether TRAF1 is important for T cell pro-
liferation, the in vitro responses of TRAF1?/?T cells to
ligation of the TCR/CD3 complex was compared with
that of T cells from WT littermates. Purified spleen T
cells from TRAF1?/?mice exhibited higher proliferation
to immobilized anti-CD3 mAb than WT T cells (Figure
3A). The increased T cell proliferation in response to
anti-CD3 mAb was not due to impaired activation-
induced cell death because the fraction of annexin V
stainingT cellsfollowing anti-CD3activation wassimilar
in TRAF1?/?and WT mice (Figure 3B).
Interactions between IL-2 and its receptor play an
tion of IL-2 by T cells requires costimulation via CD28
(Sharpe, 1995). IL-2R? (CD25) chain expression follow-
ing anti-CD3 stimulation was comparable in TRAF1?/?
and WT T cells (Figure 3C). Furthermore, intracellular
IL-2 protein content as assessed by FACS was equiva-
Normal B Cell Proliferation and Antibody Responses
in TRAF1-Deficient Mice
Stimulation through the IgM B cell receptor or CD40
induces TRAF1 expression in B cells (Dunn et al., 1999;
Zapata et al., 2000). Furthermore, TRAF1 has been re-
ported to be associated with CD40 (Pullen et al., 1998).
We therefore examined the proliferative response of B
2A shows that B cells from TRAF1?/?mice have normal
proliferation to anti-IgM or anti-CD40 antibodies. Fur-
thermore, electrophoretic mobility shift assay (EMSA)
analysis revealed normal activation of the transcrip-
tion factors NF-?B and AP-1 following CD40 ligation in
TRAF1?/?B cells (data not shown).
TRAF1 Is a Negative Regulator of TNF Signaling
Figure 2. B Cell Proliferation and Antibody
Responses in TRAF1-Deficient Mice
TRAF1?/?(striped bars) B lymphocytes to
anti-IgM and anti-CD40 stimulation. For pro-
liferation, purified spleen B cells were cul-
tured and activated by F (ab)?2fragments of
polyclonal anti-IgM antibodies or anti-CD40
mAb HM40-3 for 72 hr at 1 ? 105/well. [3H]-
thymidine was added (1 ?Ci /well) during the
last 6 hr of culture. Values represent mean ?
(B–D) Response to the T-dependent antigen
ovalbumin. Wild-type (open circles) and
TRAF1?/?(closed circles) littermate mice
were intravenously immunized with 20 ?g
ovalbumin/alum at day 0 and boosted with
the same dose at day 21. Ovalbumin-specific
IgG1 (B), IgG2a (C), and IgE (D) were mea-
sured at day 28. Isotype-specific mAbs were
absorbed to 96-well plates. Sera were diluted
(1/1000 for IgG1, 1/50 for IgG2a, and 1/25 for
IgE,) and added to the wells. Next, biotinyl-
ated-ovalbumin was added to the wells and
revealed with the strepavidin-peroxidase
TNP-LPS. Mice were immunized intraperito-
neally with 10 ?g TNP-LPS in PBS at day 0.
(F) Response to the type 2 T-independent an-
tigen TNP-Ficoll. Mice were immunized intra-
peritoneally with 10 ?g TNP-Ficoll in PBS at
day 0. Sera were diluted 1/1000 and levels of
antigen-specific antibody responses of the
indicated isotypes were analyzed by TNP-
specific ELISA. Mean values ? SEM obtained
for at least four mice per group are shown.
higher proliferation of TRAF1?/?T cells persisted when
T cells were stimulated with submitogenic concentra-
tions of anti-CD3 mAb and increasing concentrations of
anti-CD28 mAb or of recombinant IL-2 (Figures 3E and
3F). These results suggest that pathways other than
the IL-2 pathway may be responsible for the enhanced
proliferation of TRAF1?/?T cells to TCR ligation.
and 4C). Also, as expected, small compensatory
changes could be detected in the V?6?CD4?and
V?6?CD8?subsets,which are not engagedby SEB (Fig-
ures 4B and 4D). Following injection of SEB, TRAF1?/?
mice displayed changes in the V?8?and V?6?subsets
identical to those of WT mice.
We also assessed in vivo proliferation of T cells by
V?8-bearing proliferating T cells showed detectable dif-
ferences between WT and TRAF1?/?mice (data not
shown). Apoptosis in the CD4?and CD8?populations
was estimated by surface staining with annexin V-FITC.
No detectable differences in activation dependent cell
death in response to SEB injection were observed be-
tween WT and TRAF1?/?T cell populations (data not
Superantigen-Induced Clonal Expansion and Deletion
Is Normal in TRAF1-Deficient Mice
The enhanced proliferation of T cells from TRAF1?/?
mice in response to TCR ligation in vitro prompted us
to evaluate the role of TRAF1 in T cell proliferation and
apoptosis in vivo in a superantigen-induced clonal
expansion/deletion model (Kawabe and Ochi, 1991;
MacDonald et al., 1991; Wahl et al., 1993). In mice, the
bacterial superantigen staphylococcal enterotoxin B
(SEB) is recognized by T cells bearing V?8.1 or V?8.2
T cell receptors. SEB injection causes first an early (day
transient proliferative expansion of V?8?T cells, which
peaks on day 2. Finally, there is deletion of the V?8?
T cell subset by apoptosis (days 5–30). We injected WT
and TRAF1?/?mice with 20 ?g of SEB and analyzed
changes of V?8?CD4 and CD8 T cell populations in
the lymph nodes. As expected, all three phases of
the response were observed in both V?8?CD4?and
V?8?CD8?subsets of T cells in WT mice (Figures 4A
TNF-Mediated Signaling Is Enhanced
in TRAF1?/?T Lymphocytes
A possible pathway that may underlie the hyperrespon-
siveness of TRAF1?/?T cells to CD3 stimulation may
ciate with TRAF1 and to be expressed on T cells. These
include CD27 (Hintzen et al., 1995), CD30 (Horie and
Watanabe, 1998), 4-1BB (Hurtado et al., 1995), OX-40
(Gramaglia et al., 1998), HVEM/ATAR (Tamada et al.,
2000), AITR (Kwon et al., 1999), and TNFR2 (Cope et al.,
1995; Zheng et al., 1995). Addition of TNF had no effect
Figure 3. Response of TRAF1-Deficient T Lymphocytes in Response to Anti-CD3 Stimulation
(A) Proliferation of T cells from wild-type (open circles) and TRAF1?/?(closed circles) littermate mice in response to anti-CD3 mAb. Purified
spleen T cells were cultured for 72 hr at 1 ? 105/well in plates coated with different concentrations of immobilized anti-CD3 mAb 145-2C11.
[3H]-thymidine was added (1 ?Ci /well) during the last 6 hr of culture. Values represent mean ? SE.
(B–D) FACS analysis of wild-type (WT) and TRAF1?/?(KO) T cells in response to immobilized anti-CD3 mAb (1 ?g/ml). After 24 hr of activation,
T cells were stained for the binding of Annexin V (B), the expression of CD25 (IL-2R?) (C), and synthesis of intracellular IL-2 (D). The dotted
line indicates cells stained with isotype control antibodies, and the solid line indicates cells stained with anti-CD25 or anti-IL-2 antibodies.
Each experiment shown is representative of experiments performed on at least four pairs of mice.
(E and F) T cells were also examined for proliferation to immobilized anti-CD3 mAb (coating concentration of 0.1 ?g/ml) in the presence of
immobilized anti-CD28 37.51 mAb at the indicated coating concentrations (E) or increasing concentrations of recombinant IL-2 (F).
on the proliferation of WT or TRAF1?/?freshly isolated
T cells in response to anti-CD3 mAb (data not shown).
This was not surprising, because resting T cells express
low or no detectable amounts of either TNFR2 or TRAF1
(Dunn et al., 1999; Scheurich et al., 1987). In contrast,
WT and TRAF1?/?T cells that were preactivated with
immobilized anti-CD3 mAb for 3 days expressed similar
in agreement with a previous report describing TNFR2
et al., 1995). Figure 5B shows that addition of TNF had
little effect on the proliferation of activated WT T cells
but significantly enhanced the proliferation of activated
TRAF1?/?T cells. To determine which of the two known
TNFRs mediates the proliferative response of activated
TRAF1?/?T cells to TNF, we examined the capacity of
TNFR1 and TNFR2 specific mAbs to block the effect of
TNF. Figure 5C shows that anti-TNFR2 mAb TR75-54,
but not anti-TNFR1 mAb 55R170, blocked proliferation
of activated TRAF1?/?T cells to TNF. These results sug-
gest that in WT T cells, TRAF1 negatively regulates pro-
Enhanced TNF-Dependent Activation
TNFR2 engagement leads to the activation of the NF-
?B and AP-1 transcription factors, which play important
roles in TNF-mediated cell activation (Aggarwal et al.,
1999). We examined the I?B kinase (IKK) activity in anti-
CD3 activated T cells by phosphorylation of a GST-
I?B(1-66) fusion protein after TNF stimulation. Figure
6A shows that GST-I?B(1-66) was phosphorylated by
lysates from TNF-treated TRAF1?/?T cells (lanes 5–8)
but not by lysates of TNF-treated WT T cells (lanes 2–4).
This phosphorylation was specific to the I?B serine resi-
ylated in TRAF1?/?T cells.
NF-?B binding activity was measured by EMSA in
nuclear extracts prepared from anti-CD3 preactivated
WT and TRAF1?/?T cells treated with TNF. Figure 6B
shows that TNF caused enhanced activation of NF-?B
in TRAF1?/?T cells (lane 5). In contrast, TNF caused no
detectable increase of NF-?B activation in WT T cells
TRAF1 Is a Negative Regulator of TNF Signaling
Figure 4. Clonal Deletion and Expansion of Peripheral V?8-Bearing
T Cells in SEB-Injected TRAF1-Deficient Mice
On day 0, wild-type (closed circles) and TRAF1?/?(open circles)
mice received a single intraperitoneal injection of SEB (20 ?g). On
days 0, 1, 2, 5, and 8, percentage of lymph node CD4V?8?(A),
CD4V?6?(B), CD8V?8?(C), or CD8V?6?(D) cells was determined
by FACS analysis. Each point represents the mean ? SE for six
mice from two separate experiments.
indicating that there is no difference in the intrinsic in-
duction of NF-?B in TRAF1?/?T cells. Figure 6C shows
that TNF induced the NF-?B-dependent transcription of
I?B and A20 mRNAs in anti-CD3 activated TRAF1?/?but
not in WT T cells.
TNF induces phosphorylation and activation of the
stress-activated protein kinase/JNK (SAPK/JNK), which
is critical for activation of the transcription factor AP-1.
Figure 6D shows that TNF induced the JNK phosphory-
lation in activated TRAF1?/?cells but not in WT cells.
We also examined AP-1 binding activity in these cells.
Figure 6E shows that TNF caused enhanced activation
of AP-1 in TRAF1?/?T cells (lane 5) but not in WT T cells
(lane 2), although both types of cells activated AP-1
comparably in response to PMA. Taken together, these
findings suggest that TRAF1 inhibits TNFR2-mediated
activation of NF-?B and AP-1in T cells.
Figure 5. TRAF1-Deficient T Cells Proliferate in Response to TNF
(A) Expression of TNF receptors on activated wild-type (WT) and
TRAF1?/?(KO) T cells. Purified spleen T cells were prestimulated
with anti-CD3 mAb (1 ?g/ml) for 72 hr and stained with biotinylated
isotype control (dotted line), anti-TNFR1 (dashed line), and anti-
TNFR2 (solid line) mAbs. Binding of biotinylated antibodies was
revealed by Streptavidin-PE.
(B) Proliferation of activated wild-type (open circles) and TRAF1?/?
(closed circles) T cells in response to stimulation with TNF. Live
activated T cells as described in (A) were cultured at 2 ? 104/well
for an additional 72 hr with indicated concentrations of recombinant
TNF and pulsed with [3H]-thymidine (1 ?Ci /well) for the last 6 hr of
(C) Effect of anti-TNF receptors antibodies on proliferation of acti-
vated wild-type (open bars) and TRAF1?/?(closed bars) T cells. T
cells were activated and stimulated with 100 ng/ml of TNF. Blocking
anti-TNFR1 (55R-170) and anti-TNFR2 (TR75-54) mAbs (2 ?g/ml)
were added separately or together to T cells 30 min before the
addition of TNF (50 ng/ml). Data represent the mean values within
an experiment with error bars representing the SE of the mean.
Every experiment was repeated with at least four pairs of mice.
TNF-Induced Skin Necrosis Is Exaggerated
in TRAF1-Deficient Mice
We sought an in vivo model for enhanced respon-
(Amar et al., 1995; Erickson et al., 1994; Sheehan et al.,
1995). Injection of 3 ?g of TNF for 5 days caused skin
necrosis to a similar degree in TRAF1?/?and WT mice
(Figure 7A) but failed to induce detectable skin necrosis
in RAG-2?/?mice, suggesting that TNF skin necrosis is
dependent on lymphocytes. Injection of a suboptimal
amount (1.5 ?g) of TNF produced barely visible hemor-
rhages in the skin of WT mice. In contrast, the same
dose caused macroscopic ulceration and skin necrosis
in TRAF1?/?mice. There were no visible skin changes
with injection of 0.4 ?g of TNF in either type of mouse
(data not shown).
Histologic examination revealed no differences be-
tween uninjected skin from normal and TRAF1?/?mice
(Figure 7B). Biopsies from skin of WT and TRAF1?/?
mice injected with 3.0 ?g of TNF revealed an almost
complete loss of the epidermis and extensive cellular
lization and disintegration. Identical changes were ob-
served in skin of TRAF1?/?mice injected with 1.5 ?g of
the expression of TRAF1 being restricted mostly to
lymphoid cells. TRAF5-deficient mice, like TRAF1-defi-
cient mice, survive normally (Nakano et al., 1999). In
contrast, TRAF2- andTRAF3-deficient mice exhibit lym-
phopenia and die prematurely (Xu et al., 1996; Yeh et
al., 1997), and TRAF4-deficient mice exhibit tracheal
display severe osteopetrosis, become runted, and die
at the age of 17–19 days (Lomaga et al., 1999; Naito et
Although the T and B phenotype of lymphoid organs
from TRAF1 mice (data not shown) and the function
of TRAF1?/?B cells (Figure 2) appeared to be normal,
TRAF1?/?T cells displayed exaggerated proliferation in
response to stimulation by anti-CD3 (Figure 3). This was
not accompanied by increased apoptosis, enhanced
IL-2R? (CD25) expression, or increased IL-2 production,
and it persisted in the presence of costimulation with
anti-CD28 antibodies and upon addition of IL-2. These
results suggest that TRAF1 normally inhibits TCR/CD3-
mediated activation by interfering with signaling path-
ways different from CD28 or IL-2. Candidate pathways
may include those initiated by the TRAF1-associated
TNFR family members, including TNFR2, CD30, OX40,
some of them was shown to enhance T cell proliferation
to anti-CD3 stimulation (Akiba et al., 1998; Gramaglia et
al., 2000; Hintzen et al., 1995; Hurtado et al., 1997). More
importantly, T cells from OX40?/?and CD27?/?mice
proliferate poorly in response to anti-CD3 stimulation
(Hendriks et al., 2000; Kopf et al., 1999; Pippig et al.,
1999). Therefore, loss of TRAF1 may amplify the costim-
ulatory signal delivered by these molecules, resulting in
experiments will test this hypothesis.
TNF caused marked proliferation of preactivated T
cells from TRAF1?/?mice (Figure 5). In contrast, it
caused no detectable proliferation of preactivated WT
T cells. In agreement with the selective expression of
TNFR2, but not TNFR1, on activated T cells, the re-
gated by antagonistic antibodies to TNFR2 but not by
inhibits activation signals delivered via TNFR2. The role
of TNF in the enhanced proliferation of TRAF1?/?T cells
to anti-CD3 will be examined by studying mice double
deficient for TRAF1 and TNF.
The transcription factors NF-?B and AP-1 are acti-
vated by TNF and play an important role in TNF-medi-
ated cell activation. Activation of the NF-?B pathway in
response to TNF was enhanced in TRAF1?/?T cells as
evidenced by increased IKK activity, enhanced NF-?B
nuclear translocation, and increased expression of
NF-?B-regulated genes (Figures 6A–6C). Moreover,
TRAF1?/?T cells had enhanced SAPK/JNK phosphory-
lation and enhanced nuclear AP-1 binding activity in
is not important in regulating NF-?B and AP-1 activation
One possible mechanism of TRAF1 inhibition of TNF
signaling is that TRAF1 competes with TRAF2 for bind-
ing to TNFR2. To date, attempts to immunoprecipitate
Figure 6. Activated TRAF1-Deficient T Cells Are Hyperresponsive
Signaling by activated wild-type (WT) and TRAF1?/?(KO) T cells
was stimulated with TNF (400 ng/ml) for indicated time points.
(A) Phosphorylation of GST-I?B in vitro. Whole-cell lysates were
prepared, and then in vitro kinase assays were performed with GST-
I?B (1-66) and GST-I?B(AA) fusion proteins.
(B) EMSA with NF-?B oligonucleotide probe and nuclear extracts
from T cells, which were left unstimulated or stimulated with TNF
or PMA for 30 min.
(C) Northern blotting analyses of I?B?, A20, and L32 mRNA ex-
(D) Phosphorylation of SAPK/JNK. After stimulation, T cells were
lysed, and phophorylation of SAPK/JNK was determined with anti-
was ascertained with anti-phospho-SAPK antibody. Two p46 and
p54 isoforms of SAPK/JNK are indicated.
T cells, which were left unstimulated or stimulated with TNF or PMA
for 30 min.
TNF. In contrast, skin of WT mice injected with 1.5 ?g
andmarkedly lesshemorrhageand tissuedisintegration
in the dermis and hypodermis but displayed intense
infiltration by lymphocytes, neutrophils, and macro-
phages. Taken together, these results indicate that
TRAF1?/?mice have increased sensitivity to TNF-
induced skin necrosis and suggest that TRAF1 normally
inhibits the cytotoxic effects of TNF on skin.
In this study we demonstrate that T cells from TRAF1?/?
mice exhibit enhanced proliferation to anti-CD3 mAb
to TNF. Furthermore, skin from TRAF1?/?mice is hyper-
sensitive to TNF-induced skin necrosis. These findings
suggest that TRAF1 is a negative regulator of TNF ac-
TRAF1?/?mice are born normal and do not develop
any visible problems with age, which is consistent with
TRAF1 Is a Negative Regulator of TNF Signaling
Figure 7. TNF-Induced Skin Necrosis Is Ex-
aggerated in TRAF1-Deficient Mice
(A) Skin necrosis in WT, RAG-2?/?, and
of TNF 3 ?g/day and1.5 ?g/day for 5 consec-
(B) Histological examination of the skin from
uninjected and TNF injected (as in [A]) WT
and TRAF1?/?mice, fixed and stained with
hematoxylin and eosin.
TRAF2 with TNFR2 from activated primary T cells have
not been successful. Another possibility is that TRAF1
forms an inactive heterodimer by binding to TRAF2. A
third possibility is that TRAF1 may regulate molecules
other than TRAF2 that are needed for efficient TRAF2
signaling, and a fourth possibility is that TRAF1 may
recruit othermolecules thatnegatively regulateTNF sig-
naling, such as A20 (Lee et al., 2000; Song et al., 1996).
Further work is needed to understand the precise bio-
chemical basis of the inhibition of TNFR2 signaling by
TRAF1 and of the role of TRAF1 in the regulation of
signaling by other TNFR family members.
OX40 and 4-1BB have been reported to be important
in the peripheral T cell expansion (Maxwell et al., 2000;
Takahashi etal., 1999). However, examinationof periph-
eral T cell clonal expansion and deletion following TCR
signaling after injection of the superantigen SEB re-
vealed no differences between TRAF1?/?and WT mice
(Figure 4). Because both Fas/CD95 and TNF have been
implicated in clonal deletion of mature T cells following
T cell receptor engagement (Miethke et al., 1996; Mixter
et al., 1994; Papiernik et al., 1995; Singer and Abbas,
1994; Sytwu et al., 1996), it will be of interest to examine
superantigen-induced apoptosis of mature T cells in
TRAF1?/?mice bredonthe lprbackground todetermine
if TRAF1 plays a role in TNF-mediated cell death.
We used a model of TNF-induced skin necrosis to
C-terminal peptide of the murine TRAF1 (Santa Cruz) overnight at
room temperature, then with goat anti-rabbit polyclonal antibodies
conjugated with peroxidase (Pierce) for 4 hr at room temperature.
Bound conjugates were detected by ECL Super-Signal-Dura kit
in vivo. We found that RAG-2?/?mice, which lack T
and B cells (Shinkai et al., 1992), are resistant to TNF-
mediated skin necrosis, suggesting that lymphocytes
play an important role in this in vivo effect of TNF.
TRAF1?/?mice were found more susceptible to TNF-
induced skin necrosis than WT mice (Figure 7). This
suggests that TRAF1 normally protects skin from lym-
phocyte-mediated TNF-induced necrosis. It is tempting
to speculate that the hypersensitivity of T cells to TNF
may underlie the increased skin sensitivity to TNF in
TRAF1?/?mice. However, because TRAF1, in addition
to T cells, is expressed in the skin (Zapata et al., 2000),
we cannot at present rule out increased sensitivity of
TRAF1?/?skin cells to cytotoxic damage.
The results of the present study suggest that TRAF1
is a negative regulator of TNF signaling through TNFR2.
Induction of TRAF1 may trigger a feedback regulatory
loop that downregulates signals delivered by TNF. This
is very important, given the critical role TNF plays in
infection, immunity, and cancer.
Proliferation of B and T Cells
Single-cell suspensions from spleen, bone marrow, thymus, and
lymph nodes were isolated on a density gradient of Lympholyte-M
(Accurate). Bcells were prepared bydepletion of T cellsfrom single-
spleen-cell suspension. Splenocytes were incubated with mAbs to
CD4, CD8, and Thy1.1 (1 ?g/ml each), washed twice with PBS, and
incubated with magnetic Dynabeads M-450 conjugated with sheep
anti-rat IgG (Dynal). Purified B cells were cultured at 1 ? 105/well for
72 hr and activated by F(ab)?2fragments of goat anti-IgM polyclonal
antibodies (Rockland) and/or anti-CD40 mAb HM40-3 (PharMingen)
at the indicated concentrations. T cells were prepared by depletion
of B cells from single-spleen-cell suspension. Splenocytes were
incubated with rat mAbs to CD19, CD21, and IgM (1 ?g/ml each)
for 30 min, washed twice with PBS, and incubated with magnetic
Dynabeads M-450 conjugated with sheep anti-rat IgG. Purified T
cells (?95% CD3? cells) were activated by anti-CD3 mAb 145-2C11
(BD Pharmingen) by culturing at 1 ? 105/well for 72 hr. Anti-CD3
mAb in PBS was absorbed at the indicated concentrations onto
plastic wells for 24 hr before addition of T cells. For costimulation,
anti-CD28 mAb 37.51 (BD Pharmingen) was absorbed on plastic
together with anti-CD3 mAb (0.1 ?g/ml). Recombinant murine TNF
and IL-2 (R&D) were used at the indicated concentrations. Prolifera-
tion was assessed by the incorporation of [3H]-thymidine added
(1 ?Ci/well) during the last 6 hr of culture. For proliferation with TNF,
anti-CD3-activated T cells were collected after 3 days incubation,
and live cells were isolated on a density gradient of Lympholyte-M
and activated with indicated concentrations of TNF at 2 ? 104cells/
well for 72 hr. Anti-TNFR1 mAb 55R170 and anti-TNFR2 mAb TR75-
54 were purchased from BD Pharmingen.
Generation of TRAF1-Deficient Mice
Recently, we cloned and characterized the murine TRAF1 gene iso-
lated from a Lambda FIXII library (Stratagene) (Dunn et al., 1999).
The targeting construct was assembled using the pPNT vector,
kindly provided by Dr. Richard Mulligan (Children’s Hospital, Boston
MA). The 6.5 kb XbaI/XbaI fragment and the 4.5 kb HindIII/HindIII
fragment of the TRAF1 gene were used as a 5? arm and a 3? arm,
respectively (Figure 1A). In the final construct, the direction of neo
The construct (20 ?g) was linearized by digestion at the unique NotI
site in pPNT and used to transfect 2 ? 107embryonic stem cells
(J1) obtained from Dr. R. Jaenisch (MIT, Cambridge, MA). ES cells
were selected by growing them in medium containing 0.4 mg/ml
G418 and 10 ?g/ml gancyclovir. ES clones were identified by South-
ern blotting for the presence of homologous recombination. Geno-
mic DNA was isolated, digested with EcoRI, and resolved in 1%
A in Figure 1A). The targeted ES clone was injected into 3.5-day-
old C57BL/6 blastocysts, which were then transferred into Swiss
foster mothers. Resulting chimeric males were crossed with C57BL/
6 females. Tail DNAs of agouti offspring were analyzed by Southern
blotting. TRAF1 heterozygous (?/?) mice from the F1 generation
were used to obtain TRAF1 homozygous (?/?) mice by brother-
sister mating. To identify homozygous TRAF1 KO mice, F2 offspring
from these crosses were genotyped by Southern blotting and PCR
of tail DNA. PCR analysis was performed with two different primer
sets. The MTWT1 (GAGGCTCAGACATATTGAAGA) and MTWT2
(ACCAAATTGAAACTCGTTTTGATC) set was used to amplify a 1.4
kb fragment from the WT allele. The MTEOG (GCCCAATGCGAGCA
GAAG) and NeoS (CGACCACCAAGC GAAACAT) set was used to
amplify a 1.2 kb fragment from the KO TRAF1 allele. PCR was
performed with Taq polymerase (Roche) in 1? PCR buffer with 4%
DMSO, 0.4 mM dNTPs, and 0.5 ?M of each primer. After the hot
start (2 min at 94?C), samples were amplified for 35 cycles: 30 s at
94?C, 30 s at 53.5?C, and 2 min at 72?C.
Electrophoretic Mobility Shift Assay
Spleen cells at 106/ml were either left unstimulated or were stimu-
T cells were left unstimulated, stimulated with TNF (100 ng/ml), or
stimulated with PMA (20 ng/ml) for 30 min. Cells (3 ? 106) were
washed twice with ice-cold PBS, resuspended in ice-cold 10 mM
Hepes buffer (pH 7.9) containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
DTT, and EDTA-free CPI, and incubated for 10 min on ice. Nuclei
were pelleted for 2 min at 5000 rpm at 4?C and resuspended in ice-
cold 20 mM Hepes buffer (pH 7.9) containing 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and CPI cocktail.
Oligonucleotides used in these experiments are as follows: NF-?B
sequence of human imunodefficiency virus (HIV) 1 Long terminal
Repeat TCGCTGGGGACTTTCCAGGGA (Nabel and Baltimore,
1987); consensus AP-1 sequence, CGCTTGATGAGTCAGCCG (Pro-
mega). For each reaction, 2 ? 104cpm (?0.1 ng) of radiolabeled
oligonucleotide probe was incubated with 2 ?g of nuclear extract
in 20 ?l of binding buffer (10 mM Tris-HCl [pH 7.5], 50 mM NaCl,
5% glycerol, 50 ng/ml poly [dI-dC], 0.1% NP-40, 1 mM DTT, and
CPI). Samples were run on 5% PAGE in 1? TBE.
Flow Cytometry Analysis
FACS analysis was performed on thymi from 3-week-old mice and
on lymph nodes and spleens cells from 6- to 12-week-old mice.
Lymphoid organs were teased by glass slides, and live cells were
isolated on a density gradient of Lympholyte-M and stained with
appropriate antibodies in 2% rat serum PBS containing Fc-block,
fixed in 2% formaldehyde, and analyzed on a FACSCalibur cytom-
eter (BD Pharmingen). FITC- and PE-labeled antibodies were pur-
chased from Pharmingen. Anti-CD8 and anti-B220 (RA3-6B2) anti-
bodies labeled with QuantumRed were purchased from Sigma. For
to BD Pharmingen recommendations.
Splenocytes (50? 106)from WTand TRAF1?/?mice werestimulated
overnight with anti-CD40 antibody HM40-3 purchased from BD Phar-
mingen. Cells were washed twice in ice-cold PBS, then lysed in
buffer containig 10 mM Hepes, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
DTT, and 1? Complete Protease Inhibitor (CPI) cocktail (Roche).
The cell equivalent of 5 ? 106splenocytes was resolved on 10%
(Gelman), and incubated with blocking solution (0.2% gelatin, 2%
BSA, 0.1% Tween 20 in PBS) for 4 hr at room temperature. Mem-
branes were incubated with rabbit polyclonal antibody S-19 against
To determine the antibody response to the T-dependent antigen
ovalbumin, 10- to 12-week-old mice were immunized intraperitone-
TRAF1 Is a Negative Regulator of TNF Signaling
ally with 20 ?g of ovalbumin precipitated with alum and boosted
the same way at day 21 and bled at days 0, 7, 14, 21, 28, and
35. The magnitude of the anti-ovalbumin antibody response was
detected by ELISA as previously described (Spergel et al., 1998).
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PBS were absorbed to 96-well plates (NuncMaxisorb) for 16 hr at
4?C. For IgG1, IgG2a, and IgE isotype determination, sera were
diluted 1/1000, 1/50, and 1/25, respectively, and added to the wells
for 1 hr at 37?C. Biotinylated-ovalbumin was incubated for 4 hr at
37?C. Bound ovalbumin was revealed with the strepavidin-peroxi-
dase conjugate (BD Pharmingen) according to the manufacturer’s
recommendations. For T-independent antigens, mice were immu-
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10 ?g TNP-Ficoll (a gift of Dr. F.D. Finkelman, Bethesda, MD) at day
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WethankDr. VishvaDixitforthe murineA20cDNA,Dr. SylvieMemet
for the GST-I?B fusion proteins and the murine I ?B cDNA, and Dr.
Atul Bhan for help in reading the skin pathology. This research was
supported by NIH grants AI-42031 and AI-35714.
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