Enhanced Toll-like receptor (TLR)
responses of TNFR-associated factor 3
(TRAF3)-deficient B lymphocytes
Ping Xie,*,1Jayakumar Poovassery,* Laura L. Stunz,* Sonja M. Smith,* Mark L. Schultz,†
Lindsey E. Carlin,‡and Gail A. Bishop*,†,‡,§,2
*Department of Microbiology, Graduate Programs in†Molecular and Cellular Biology and‡Immunology, The University of
Iowa, and the§Iowa City Veterans Affairs Medical Center, Iowa City, Iowa, USA
RECEIVED JANUARY 24, 2011; REVISED AUGUST 22, 2011; ACCEPTED AUGUST 24, 2011. DOI: 10.1189/jlb.0111044
The key role of TRAF6 in TLR signaling pathways is well
known. More recent evidence has implicated TRAF3 as
another TRAF family member important to certain TLR
responses of myeloid cells. Previous studies demon-
strate that TRAF3 functions are highly context-depen-
dent, displaying receptor and cell-type specificity. We
thus examined the TLR responses of TRAF3?/?mouse B
lymphocytes to test the hypothesis that TRAF3 plays
distinct roles in such responses, depending on cell
type. TRAF3?/?DC are known to have a defect in type 1
IFN production and here, showed diminished produc-
tion of TNF and IL-10 and unaltered IL-6. In marked con-
trast, TRAF3?/?B cells made elevated amounts of TNF
and IL-6 protein, as well as IL-10 and IP-10 mRNA, in re-
sponse to TLR ligands. Also, in contrast to TRAF3?/?
DC, the type 1 IFN pathway was elevated in TRAF3?/?B
cells. Increased early responses of TRAF3?/?B cells to
TLR signals were independent of cell survival or prolif-
eration but associated with elevated canonical NF-?B
activation. Additionally, TRAF3?/?B cells displayed en-
hanced TLR-mediated expression of AID and Ig isotype
switching. Thus, TRAF3 plays varied and cell type-spe-
cific, biological roles in TLR responses. J. Leukoc. Biol.
90: 1149–1157; 2011.
TLRs are a large family, mediating signals important to devel-
opment of the innate immune response . It has been
known for over one decade that one of the key players in TLR
signaling is a member of the TRAF adaptor protein family,
TRAF6 . For many years after this discovery, TRAF6 was the
only TRAF implicated in TLR signaling pathways, and other
TRAFs were known primarily for their roles as adapters, which
associate directly with cell surface receptors of the TNFR su-
perfamily . However, reports published in 2006 indicate a
role for TRAF3 in regulating a subset of TLR signals delivered
to macrophages and DCs [4, 5], and this role was validated
recently in a rare human disorder featuring a loss-of-function
TRAF3 allele . These signals are primarily implicated in
TLR influence, particularly TLR3, on type 1 IFN production
and antiviral responses.
TLRs play major roles in regulating the function of cells of
the myeloid lineage, so it is not surprising that the majority of
studies of TLR function focuses on these cells [7–9]. However,
B lymphocytes also express most of the known TLRs and are
highly responsive to TLR ligands . TLR signaling to B cells
has also been associated with autoimmune disease [11, 12].
Recently, it was shown that B cells play an important role in
innate-protective responses to bacterial sepsis . Thus, it is
important to understand how TLRs signal and function in the
B cell component of innate and adaptive immune responses.
It has become clear over the years since their discovery that
TRAFs function in a manner strongly influenced by cell type,
stimulating receptor(s), and the presence or absence of addi-
tional TRAF family members and other proteins in the signal-
ing complex . TRAF3 function appears to be particularly
context-dependent. TRAF3, in contrast to TRAF2, -5, and -6,
does not activate a NF-?B reporter gene when overexpressed
in the 293 epithelial cell line [15–18]. In these cells, trans-
fected TRAF3 also inhibits TRAF2 and TRAF5 overexpression-
mediated, noncanonical NF-?B2 activation associated with a
variety of transfected TNFR superfamily members . In B
lymphocytes, endogenous TRAF3 serves as an inhibitor of
TRAF2-dependent CD40 signals [20, 21], as well as BAFF-R
signals [22, 23]. In sharp contrast, TRAF3 is a key positive reg-
ulator of signals delivered to B cells by the EBV-encoded CD40
mimic and latent membrane protein 1  and of signals de-
1. Current address: Department of Cell Biology and Neuroscience, Rutgers
University, Piscataway, NJ 08854, USA.
2. Correspondence: 2193B MERF, The University of Iowa, Iowa City, IA
52242, USA. E-mail: firstname.lastname@example.org
Abbreviations: AID?activation-induced deaminase, B-TRAF3–/–?B cell-spe-
cific TRAF3-deficient mice, BAFF-R?B cell activation factor receptor,
BCM?B cell medium, BMDC?bone marrow-derived DC, DC-TRAF3–/–?
selectively deleted TRAF3 in DC mice, IP-10?IFN-inducible protein 10,
IRF3?IFN regulatory factor 3, LMC?littermate control, MZ?marginal zone,
NIK?NF-?B-inducing kinase, poly(I:C)?polyinosinic:polycytidylic acid,
rm?recombinant mouse, TRAF3?/??TRAF3-deficient
0741-5400/11/0090-1149 © Society for Leukocyte Biology
Volume 90, December 2011
Journal of Leukocyte Biology 1149
livered to 293 cells by a transfected ectodermal dysplasia re-
ceptor . Interestingly, a novel human CD40 polymorphism
that confers a gain-of-function to B cells in vitro  also uses
and requires TRAF3 as an inducer rather than inhibitor of
CD40 signals . TRAF3 is also important to apoptotic sig-
nals mediated by the lymphotoxin-? receptor . TRAF3 ex-
pression in B cells plays a critical restraining role in B cell sur-
vival and homeostasis [23, 29], which may be related to its
ability in this cell type to negatively regulate the noncanonical
NF-?B2 pathway [19, 23, 29, 30]. TRAF3?/?T lymphocytes
also display increased NF-?B2 activation. However, in marked
contrast to TRAF3?/?B cells, mice lacking TRAF3 specifically
in T cells have impaired CD4?and CD8?T cell responses and
display defects in early TCR signaling .
Given the varied roles played by TRAF3 in immune cells, an
important knowledge gap includes the distinct functions of
TRAF3 in TLR signaling to B cells. To address this question,
we examined TLR-mediated functions in B cells from TRAF3-
conditional knockout mice . We determined that most
TLR-mediated signals and functions in B cells, in contrast to
several of those reported previously for myeloid cells, were
markedly enhanced in TRAF3?/?B cells, in a survival-inde-
pendent manner. This elevation was seen in MZ and non-MZ
B cells. Additionally, TLR-mediated Ig isotype switching and
production of AID, a key enzyme involved in combinatorial
switch recombination, were elevated in TRAF3?/?B cells.
These results add new information to understanding the pleio-
tropic functions of this interesting and functionally important
adaptor protein in cells of the immune system.
MATERIALS AND METHODS
TRAF3flox/floxmice  were crossed with CD19?/Cremice  obtained
from The Jackson Laboratory (Bar Harbor, ME, USA) and subsequently
backcrossed onto TRAF3flox/floxmice, as described previously, to produce B
cell-specific B-TRAF3?/?mice . To create a DC-TRAF3?/?mouse,
TRAF3flox/floxmice were crossed with CD11c-Cre mice  provided by Dr.
Richard Hodes (NIH, Bethesda, MD, USA) with permission from Dr. Boris
Rezis (Columbia University, New York, NY, USA), and it was verified that
DCs from these mice are TRAF3?/?. The phenotype of these mice will be
described in detail elsewhere (unpublished results); they breed and de-
velop normally. The mice are on a mixed genetic background between
129/SvJ and C57Bl/6, and Cre-negative littermates, designated LMC, were
used as controls in all experiments.
BMDCs were generated by culturing RBC-depleted BM cells in RPMI-1640
medium supplemented with 10% FCS, penicillin/streptomycin, 10 ?M
2-ME, 1000 U/ml rGM-CSF, and 25 U/ml rIL-4 (R&D Systems, Minneapo-
lis, MN, USA). BMDCs were harvested on Day 10 of culture and were puri-
fied further using anti-CD11c, mAb-conjugated microbeads (Miltenyi Bio-
tec, Auburn, CA, USA). The purified BMDCs were ?90% CD11c?, as as-
sessed by flow cytometric analysis. Resting splenic B cells were purified by
Percoll density gradient centrifugation as described previously , fol-
lowed by negative selection using anti-mouse CD43-coated magnetic beads
and a MACS separator (Miltenyi Biotec), as recommended by the manufac-
turer. The purified B cells were ?95% B220?, as assessed by flow cytomet-
ric analysis of stained cells.
Splenocytes from 2- to 3-month-old LMC or B-TRAF3?/?mice were stained
with the fluorescent antibodies rat anti-B220-allophycocyanin (clone RA3-
6B2, eBioscience, San Diego, CA, USA) and rat anti-CD1d-FITC (clone
1B1, BD PharMingen, Mountain View, CA, USA) at 0.07 ug/106cells. Cells
were sorted into B220?CD1dhiand B220?CD1dlopopulations using a BD
FACSDiva at The University of Iowa Flow Cytometry Facility (Iowa City, IA,
USA). MZ B cells are CD1dhi. Following the sort, MZ and non-MZ B
cells were equilibrated on ice for 1 h, after which, TLR stimuli were added.
TLR agonists used were: TLR3 ? poly(I:C), 5 ug/ml for B cells; 30 ?g/ml for
DCs (Invivogen, San Diego, CA, USA); TLR2/4 ? LPS 0111:B4, 20 ug/ml for
B cells or 1 ug/ml for BMDCs (Sigma Chemical, St. Louis, MO, USA);
TLR7 ? R848, 100 ng/ml (Alexis Biochemicals, San Diego, CA, USA);
TLR9 ? 100 nM phosphorothioate oligonucleotide CpG 1826 (for DCs) or
CpG 2084 (for B cells; Integrated DNA Technologies, Coralville, IA,
Antibodies specific for phospho-I?B? and phospho-IRF3 (Ser396) were
from Cell Signaling Technologies (Danvers, MA, USA). Antibodies to
TRAF3 and YY1 were from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). Antibody to actin was from Chemicon International (Temecula, CA,
USA). Antibody to AID and anti-mouse TNF-?, IL-6, IL-10, and IL-12p40
ELISA antibody pairs were from eBioscience. The agonistic anti-CD40 mAb,
used as a control in Ig isotype-switching experiments, was HM 40-3 (eBio-
science). Alkaline phosphatase-conjugated polyclonal goat antibodies spe-
cific for mouse IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, and IgE were from
Southern Biotechnology Associates (Birmingham, AL, USA).
Cytokine ELISA assays
Purified, resting splenic B cells, unseparated or sorted as above, were cultured
in 96-well plates at 1 ? 106cells/ml in 0.2 ml vol RPMI-1640 culture medium,
supplemented with 5% FCS, 10 ?M 2-ME, 10 mM HEPES (pH 7.55), 1 mM
sodium pyruvate, 2 mM L-glutamine, and 0.1 mM nonessential amino acids
(BCM). BMDCs were cultured at 5 ? 105cells/ml in RPMI 1640 supple-
mented with 10% FCS, penicillin/streptomycin, and 2-ME. As designated in
figures, some cultures included TLR agonists, agonistic anti-mouse CD40 mAb
(HM40.3, eBioscience), and/or rmIL-4 (100 ng/ml; PeproTech, Rocky Hill,
NJ, USA). Quantitative mouse IL-6, IL-12, IL-10, and TNF-? ELISAs were per-
formed on 24-h DC culture supernatants [72h for poly (I:C)] as described pre-
viously . B cell cytokine production was assessed similarly, except for
TNF-? production. To measure TNF-?, B cells were resuspended in BCM with
10% FCS (1?106cells/ml) following the wash, rested for 1 h, and then placed
in an anti-TNF-?-coated 96-well flat-bottom plate with the appropriate antibod-
ies. This "in-plate" assay is necessary, as B cells express TNFR2 and rapidly bind
the TNF-?, which they secrete. Thus, TNF ELISAs were performed on super-
natants of cells cultured for 4 h as described previously .
For detection of phospho-IRF3 and AID in LMC or TRAF3?/?B cells, puri-
fied, resting splenic B cells (0.5?106cells/ml) were cultured in 24-well plates,
with or without 20 ?g/ml poly(I:C) or 100 nM CpG 2084 and 100 ng/ml
rIL-4. Nuclear extracts were prepared from cells for detection of phospho-IRF3
as described . Whole cell lysates were prepared for AID experiments. Cells
were resuspended in 2? SDS lysis buffer and sonicated, and phospho- and
total I?B? were detected as described . Actin blotting was used as a load-
ing control, and total protein amounts were determined for equal lane load-
ing using the BCA assay from Pierce (Rockford, IL, USA).
Taqman assay of IL-10 and IP-10 mRNA expression
Splenic B cells were purified using negative selection with CD43 beads, as
described above, and then stimulated with TLR ligands, as detailed above
Journal of Leukocyte Biology
Volume 90, December 2011
for 0, 2, or 4 h. Total cellular RNA was extracted using TRIzol reagent (In-
vitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol.
cDNA was prepared from RNA using the high-capacity cDNA RT kit (Ap-
plied Biosystems, Foster City, CA, USA). Quantitative real-time PCR was
performed using the TaqMan gene assay kit (Applied Biosystems). TaqMan
primers and probes (FAM-labeled) specific for individual mouse cytokines
were used in the PCR reaction to detect IL-10 or IP-10 mRNA. Reactions
were performed on a 7500 Fast Real-Time PCR system (Applied Biosys-
tems). Each reaction also included primers and the probe (2?-chloro-7?-
phenyl-1,4-dichloro-6-carboxyfluorescein-labeled) specific for mouse ?-actin
mRNA, which served as endogenous control. Relative mRNA expression
levels of cytokines were analyzed using sequencing detection software (Ap-
plied Biosystems) and the ?? Ct, comparative Ct threshold method follow-
ing the manufacturer’s procedures. For each biological sample, duplicate
PCR reactions were performed.
Ig isotype production
To detect TLR-driven isotype-switched Ig production, B cells were cultured
in the presence of TLR agonists or as a positive control, agonistic anti-
mouse CD40 mAb ? rmIL-4 (100 ng/ml; PeproTech) for 5 days. Culture
supernatants were assayed by ELISA for individual Ig isotypes using isotype-
specific reagents from Southern Biotechnology Associates, according to the
manufacturer’s protocols as described previously .
Quantitative data were analyzed by Student’s t test. P values are indicated in
figures above bar graphs by asterisks: *P ? 0.05, **P ? 0.01, ***P ? 0.001.
Effect of TRAF3 deficiency on TLR-mediated
proinflammatory cytokine production by DCs versus
As deletion of TRAF3 from all cells of a mouse is neonatally le-
thal , previous studies reconstituted WT mice with
TRAF3?/?BM. BM-derived macrophages from the recipients
produce elevated IL-12, thought to result from reduced IL-10, in
response to ligands for TLR4 and TLR9 . In the present study,
BMDCs from DC-TRAF3?/?mice also produced elevated IL-12
and decreased IL-10 compared with DCs from their LMC coun-
terparts in response to ligands for TLR4, -7, and -9 (Fig. 1). To
directly compare TRAF3?/?DCs with TRAF3–/–B cells, we ex-
amined two proinflammatory cytokines measurably produced as
secreted protein by both cell types in culture upon TLR stimula-
tion, TNF-? and IL-6. Fig. 1 shows that TRAF3 deficiency resulted
in partial but reproducible decreases in TNF-? production by
BMDCs in response to TLR ligands. TRAF3?/?DCs showed no
significant change in IL-6 production compared with DCs from
LMC mice. In contrast, TRAF3?/?B cells produced markedly
elevated amounts of TNF-? and IL-6 in response to TLR stimula-
tion, compared with LMC B cells (Fig. 2, upper panels). Produc-
tion of both cytokines was assessed at early poststimulation time-
points, when there were no detectable differences in cell viability
or number between TRAF3?/?and LMC B cells (data not
shown). Neither TRAF3?/?nor LMC B cells produced reliably
detectable IL-12 in response to the tested TLR ligands (not
shown). Interestingly, TRAF3?/?B cells showed an early en-
hanced production of IL-10 mRNA in response to signals from
several TLRs, but this enhancement disappeared or decreased
markedly by 4-h poststimulation (Fig. 2, lower panels), and IL-10
protein in B cell cultures was undetectable until 72 h poststimula-
tion, at a time when TRAF3?/?B cells also display a survival ad-
vantage . At this late time post-TLR stimulation, TRAF3?/?B
cells did not show enhanced IL-10 production (data not shown).
Thus, the effect on IL-10 is early and transient. However, it can
be concluded that overall, TRAF3 deficiency has markedly differ-
ent effects upon cytokine production by B cells versus DCs.
Enhanced cytokine production by MZ and non-MZ B
cells in the absence of TRAF3
B-TRAF3?/?mice have increased total B cells, as well as an
increased percentage of transitional and MZ B cells . To
address the possibility that the increased TLR responses seen
Figure 1. Effect of TRAF3 deficiency on
TLR-mediated cytokine production by
DCs. BMDCs were isolated and cultured
with the indicated stimuli and cytokine
production measured, as described in
Materials and Methods. Filled bars are
data from BMDCs of DC-TRAF3?/?mice,
and open bars are data from LMC mice.
Data represent the mean values ? sd of
three experiments. Statistical analysis was
conducted using Student’s t test. *P ?
0.05; **P ? 0.01; ***P ? 0.001.
Xie et al.
TRAF3–/–B cells have enhanced TLR responses
Volume 90, December 2011
Journal of Leukocyte Biology 1151
in Fig. 2 were a result of an enhanced responsiveness selec-
tively of the MZ B cell subset, we separated MZ and non-MZ B
cells as described in Materials and Methods and cultured them
with various TLR ligands as in Fig. 2. Data presented in Fig. 3
demonstrate that MZ B cells of LMC and B-TRAF3?/?mice
produced more IL-6 than non-MZ B cells in response to all
TLR ligands. However, there were statistically significant in-
creases in TLR responses of both subsets of B cells from
B-TRAF3?/?mice; their enhanced responses were not con-
fined to the MZ subset. A similar trend was seen in TNF-? pro-
duction, but TNF production by sorted LMC B cells was too
low to quantify reliably (not shown). We also measured pro-
tein expression of TLR3 and TLR9 (we could not find reliable
antibodies for detecting protein expression of mouse TLR4 or
-7) in B cell subsets of LMC and B-TRAF3?/?mice; no differ-
ences were seen (not shown).
Effect of B cell TRAF3 deficiency on the
TLR-mediated type 1 IFN pathway
The predominant effect of TRAF3 deficiency in BM-derived
macrophages and DCs from mice adoptively transferred with
TRAF3?/?BM is a greatly reduced, TLR3-induced type 1 IFN
response [4, 5], which we also observed in DCs from our DC-
TRAF3?/?mouse (unpublished results). This specific defect is
also implicated in the impaired response to Herpes simplex
virus of a patient with TRAF3 deficiency . We thus exam-
ined B cells from conditional TRAF3?/?mice for their type 1
IFN pathway response to poly(I:C), a TLR3 ligand. Levels of
type 1 IFN protein in B cell culture supernatants were too low
to be reliably quantifiable, but we investigated an upstream
regulator and a downstream target of the type 1 IFN signaling
pathway. Phosphorylation of IRF3, a transcription factor that
regulates type I IFN production, proved a useful measure of B
cell TLR3 responses. Fig 4A shows that TRAF3?/?B cells ex-
hibited elevated phosphorylation of IRF3 within 1 h of
poly(I:C) treatment, and this amount was more than double
1 1 1
3 3 3
Figure 4. Effect of TRAF3 deficiency on activation of the TLR-medi-
ated type 1 IFN pathway in B cells. B cells from LMC or B-TRAF3?/?
mice were cultured with the TLR ligands, indicated as described in
Materials and Methods, for the indicated times. For all bar graphs,
filled bars represent results from B-TRAF3?/?B cells and open bars
data from LMC B cells. (A) Western blots of cell nuclear lysates were
performed as described in Materials and Methods. Blotting for the
resident nuclear protein YY1 served as a lane-loading control. Data are
representative of three similar experiments. Chemiluminescence of
phospho-IRF3 (P-IRF3) bands was quantitated and normalized to the
intensity of the YY1 band signals in the right bar graph. Graphs depict
the results of three independent experiments with duplicate samples
in each experiment (mean?sd). (B) mRNA for IP-10 was quantitated
as described in Materials and Methods. Graphs depict the results of
three independent experiments with duplicate samples in each experi-
ment (mean?sd). Statistical analysis was conducted using Student’s t
test. *P ? 0.05; **P ? 0.01; ***P ? 0.001.
BCM R848 CpG
BCM Poly(I:C)R848 CpG
Figure 2. Effect of TRAF3 deficiency on TLR-mediated cytokine pro-
duction by B cells. Resting B cells were isolated and cultured ? the
indicated stimuli and cytokine production measured, as described in
Materials and Methods. Measurement of mRNA production for IL-10
was as described in Materials and Methods. Filled bars are data from B
cells of B-TRAF3?/?mice, and open bars are data from LMC mice.
Data represent the mean values ? sd of three experiments. Statistical
analysis was conducted using Student’s t test. *P ? 0.05; **P ? 0.01;
***P ? 0.001.
CPoly(I:C) LPSR848 CpG
Figure 3. Effect of TRAF3 deficiency on TLR responses of B cell sub-
sets. Splenic B cells were isolated from LMC and B-TRAF3?/?mice
and then separated by flow cytometric sorting into non-MZ (Cd1dlo)
and MZ (CD1dhi) populations, as described in Materials and Methods.
Sorted cells were cultured with the indicated stimuli and IL-6 mea-
sured as in Fig. 2. Data represent the mean values ? sd of three ex-
periments. Statistical analysis was conducted using Student’s t test.
*P ? 0.05; **P ? 0.01; ***P ? 0.001.
Journal of Leukocyte Biology
Volume 90, December 2011
the phospho-IRF3 produced by LMC B cells after 3 h of TLR3
stimulation. We also measured B cell production of mRNA for
the type 1 IFN-regulated chemokine IP-10 in response to all
four of the TLR ligands. Fig. 4B shows that TRAF3?/?B cells
gave elevated responses at 2 h and 4 h poststimulation (except
for LPS, where 2-h measurements were not detectable), com-
pared with responses of LMC B cells. Thus, B cells again
showed a divergence from DCs in the role played by TRAF3 in
the type 1 IFN pathway in response to TLR stimulation.
TLR-induced canonical NF-?B1 activation in
Although, as discussed above, TRAF3?/?B cells have constitu-
tive activation of the noncanonical NF-?B2 pathway, B cells
from B-TRAF3?/?mice show no alteration in CD40-mediated
activation of the NF-?B1 pathway . This canonical NF-?B
pathway plays an important role in rapid poststimulus produc-
tion of proinflammatory cytokines and is also activated by TLR
signal cascades . Additionally, TRAF3 can associate with
components of the TLR-mediated NF-?B1 activation pathway
in transformed cell lines [4, 5]. We thus examined activation
of the canonical NF-?B1 pathway in TRAF3?/?and LMC B
cells stimulated via TLR4 (a membrane TLR) or TLR9 (an
intracellular TLR). Fig. 5 demonstrates that I?B? phosphoryla-
tion in response to TLR4 (upper panels) or TLR9 (lower pan-
els) was elevated in TRAF3?/?B cells evident as early as 2
min after stimulation. The NF-?B1 response to LPS was also
sustained in TRAF3?/?B cells compared with LMC. Thus, in
TLR responses, in contrast to CD40 signals, TRAF3?/?defi-
ciency enhanced NF-?B1 activation.
TLR-induced Ig isotype switching in TRAF3?/?B
A TLR-induced function unique to B cells is Ig production. Ig
isotype switching can be induced by adaptive and innate im-
mune signals to B cells [41–44]. We thus examined the effect
of TRAF3 deficiency on TLR-mediated production of various
isotypes of Ig by LMC and TRAF3?/?B cells.
Consistent with our previous findings with sera from unim-
munized B-TRAF3?/?mice , total IgM production was
elevated modestly, whereas little IgG1, a predominantly T cell-
dependent Ig isotype, was produced in response to TLR li-
gands (Fig. 6). CD40 ? IL-4 stimulation served as a positive
control; TRAF3 has been shown to act as an inhibitor of
CD40-mediated IgM production . As in mouse sera, B cell
in vitro production of other IgG isotypes and IgA in response
to TLR ligands was elevated in the absence of TRAF3. Re-
sponses to LPS were less-predictable and -consistent than those
to other TLR ligands, possibly because LPS can use multiple
signaling pathways [9, 45]. However, the general trend that
emerged was enhanced production of T-independent Ig iso-
types in response to TLR family ligands.
TRAF3 deficiency does not cause increased proliferation of
B cells but does result in the enhanced ability to survive in the
absence of activating stimuli . We did not detect increased
numbers of B cells in cultures measured in Fig. 6 (not shown).
However, to determine if TLR stimulation of TRAF3?/?B
cells increases activity of the isotype-switch process itself, we
examined production of AID, a key factor in combinatorial
switch recombination of Ig genes . Fig. 7 demonstrates
that LMC and TRAF3?/?B cells have enhanced AID produc-
tion following TLR9 ? IL-4 stimulation, but this AID up-regu-
lation was considerably more marked in cells deficient in
TRAF3 in relation to production of actin, a "housekeeping"
The present study showed that signaling by TLR3, -4, -7/8, and
-9 in B lymphocytes is elevated in the absence of TRAF3. This
indicates that TRAF3 normally constrains signaling by these
receptors in B cells, in interesting contrast to its effects on
TLR signaling to myeloid cells. TLRs are one of the most im-
portant groups of PRRs, and our findings suggest that TRAF3
can play essential roles in modulating innate and humoral im-
munity in response to pathogen infections that trigger TLRs.
Furthermore, the hyper-responsiveness of TRAF3?/?B cells to
TLRs may contribute to the autoimmune manifestations ob-
served in B-TRAF3?/?mice as they age . We showed pre-
viously that TRAF3?/?B cells, including those producing au-
toantibodies, exhibit prolonged survival, independent of BAFF.
Our present study suggests that autoreactive TRAF3?/?B cells
are not only increased in number in the host as a result of this
enhanced survival but are also functionally enhanced as a re-
sult of amplified responses to signaling by TLRs. This can also
contribute to autoimmune manifestations .
Our prior studies revealed that B-TRAF3?/?mice have an
elevated number and percentage of MZ B cells . Published
evidence has indicated that MZ B cells play unique roles in
T-independent antibody responses and can interact in distinct
ways with DC and T cells (reviewed in ref. ). Previous re-
ports show that MZ B cells can produce elevated IL-10 in re-
sponse to prolonged TLR stimulation [48, 49], and our data
expand on this by showing that MZ B cells produce enhanced
IL-6 in response to a much-shorter stimulation with ligands for
TLR3, -4, -7, and -9. However, this was the case for MZ B cells,
whether or not they lack TRAF3, and non-MZ B cells from
B-TRAF3?/?mice also produce elevated cytokines in response
pIκ κBα α
102 302 30
pIκ κBα α
5 1515 10
102 302 30
Figure 5. TLR-induced canonical NF-?B1 activation in TRAF3?/?
B cells. B cells were stimulated for the times shown with the indicated
TLR ligands. Preparation of whole cell lysates and Western blotting for
phospho-I?B? and actin were as described in Materials and Methods.
Results are representative of three similar experiments for each TLR
Xie et al.
TRAF3–/–B cells have enhanced TLR responses
Volume 90, December 2011
Journal of Leukocyte Biology 1153
to TLR stimulation, so the effects of TRAF3 deficiency do not
only affect, nor are solely attributable to, the MZ subset.
Results shown here reveal that the roles played by TRAF3 in
response to TLR signaling to B lymphocytes are overlapping
yet distinct from those that TRAF3 plays in TLR responses of
DCs. It has been reported recently that in myeloid cells, the
self-catalyzed, K63-linked polyubiquitination of TRAF3 is re-
quired for IRF3 activation induced by TLR-Toll/IL-1R domain-
containing adaptor-inducing IFN-? signaling . Interest-
ingly, we found that in B cells, IRF3 activation is not abolished
but instead enhanced in TRAF3?/?B cells. These results rein-
force the concept that TRAF signaling adapters have highly
context-dependent functions. In addition to receptor or cell
type-specific varied roles of TRAF3 discussed in the Introduc-
tion, we now show that in response to the same receptors in
different cell types, TRAF3 can exert different and even con-
This is the first demonstration, to our knowledge, that
TRAF3 can play distinct roles in signaling by the same
receptors in different cell types of the immune system. How-
ever, there is precedent for such distinction in function in the
actions of TRAF2 and -6. CD40-mediated IL-6 production by
DCs has been reported to depend on TRAF2 , whereas
IL-6 induction in B cells by CD40 requires CD40-TRAF6 associ-
ation but not TRAF2 or -3 [36, 52]. Like B cells, macrophages
primarily rely on TRAF6 for IL-6 production, but in contrast
to B cells, macrophages show little if any use of TRAF2 in
CD40-mediated canonical NF-?B1 activation [53, 54]. As
TRAF2 serves as the principal regulator of CD40-induced
TRAF2 and -3 degradation in B cells [55, 56], this may explain
why CD40 signaling in macrophages does not induce TRAF2
degradation . Finally, in nonimmune cells, TRAF6 appears
to be necessary for TRAF2-mediated CD40 induction of kinase
and NF-?B activation .
Another member of the TNFR superfamily, TNFR2/CD120b,
also shows distinct patterns of TRAF use in various cell types. Co-
operation between CD40 and CD120b in the induction of B cell
Ig production requires TRAF2 , whereas in activation of anti-
microbial activity in macrophages, cooperation between these two
receptors is TRAF6-dependent . A contrasting negative regu-
latory role in CD120b signaling is played by TRAF2 in the trans-
formed epithelial cell line, 293T .
Precisely how TRAFs exert different influences in distinct
situations is likely dependent on a number of interacting fac-
tors, including the strength and nature of receptor associa-
tions, the presence of other TRAFs and additional molecules
in the signaling complex, and which additional receptors and
intracellular proteins are expressed by the cells under the con-
ditions examined. Our previous work  showed that TRAF3
deletion in B cells results in enhanced survival compared with
IgG2a ( ng/ml)
Figure 6. TLR-induced Ig isotype switching in TRAF3?/?
B cells. B cells were cultured with the indicated stimuli
and individual Ig isotypes measured by ELISA, as de-
scribed in Materials and Methods. Data represent the
mean values ? sd of triplicate cultures in an experiment
representative of three similar experiments. Statistical
analysis was conducted using Student’s t test. *P ? 0.05;
**P ? 0.01; ***P ? 0.001.
Journal of Leukocyte Biology
Volume 90, December 2011
normal mouse B cells, which die rapidly in culture in the ab-
sence of stimulation. The noncanonical NF-?B2 pathway,
which promotes B cell survival , is constitutively activated
in TRAF3?/?B cells, although this activation can be en-
hanced further by CD40 signals . However, this same con-
stitutive NF-?B2 activation is seen TRAF3?/?T cells but with-
out increased cell survival . It was thus important to con-
sider differences in cell number and viability as the
explanation for enhanced B cell responses to TLR signals re-
ported here. For many of the parameters measured, we were
able to examine cells at early times poststimulation (Figs. 1–5),
before any detectable survival advantage is displayed by
TRAF3?/?B cells. In experiments examining isotype switch-
ing, a process requiring more time (Figs. 6 and 7), we found
that TLR stimulation enhanced the survival of LMC B cells as
well, so there were no significant differences between B cell
number or viability in the experiments shown. The only excep-
tion to this was in the unstimulated B cell cultures, and in
these samples, we show that no nonspecific elevations in any
of the parameters measured were seen in TRAF3?/?B cells.
In response to TLR stimulation, TRAF3?/?B cells showed a
marked increase in TLR-induced canonical NF-?B1 activation
(Fig. 5), as well as cytokine production (Fig. 2). TRAF3 B cells
also displayed enhanced activation of the type 1 IFN pathway
(Fig. 4), whereas TRAF3?/?myeloid cells show a very differ-
ent phenotype [4–6]. It has been hypothesized that TRAF3
plays a key role in NF-?B activation in canonical and nonca-
nonical pathways via its regulation of the NIK [30, 63–65].
However, TRAF3?/?B cells do not display greater constitutive
or CD40-mediated canonical NF-?B1 activation than do con-
trol B cells [23, 24]. Thus, the TLR specificity of the effect on
NF-?B1 activation shown here is unlikely to be explained solely
by TRAF3 effects on NIK. As we cannot detect measurable
amounts of NIK protein in unstimulated or stimulated B cells
used in this study, we cannot yet definitively address its poten-
tial involvement. It is interesting to note that in an earlier
study, DCs from NF-?B p100-deficient mice, whether or not
they lack TRAF3, show the same defect in IFN-? production as
TRAF3?/?p100?/?mice , suggesting that elevated nuclear
p52 may not explain the differential type 1 IFN response to
Another possible mechanism of the specific effects of
TRAF3 deficiency on B cell TLR responses reported here is
potential interaction between TRAF3 and -6. CD40-mediated
canonical NF-?B1 activation , IL-6 production , and Ig
production [52, 67] are all dependent on TRAF6, and these
functions are interconnected—IL-6 production requires NF-?B
activation and can, in turn, promote Ig production and isotype
switching [68, 69]. In B cells, the cell type-specific homeostatic
function of TRAF3 may extend to cytoplasmic TRAF6, as het-
erotypic TRAF interactions are known to occur [38, 70, 71],
including TRAF3–6 interactions . CD40 signals induce
TRAF3 degradation  and cooperate with TLR signals in B
cells [73, 74], and it is possible that TRAF3 deficiency releases
more TRAF6 to serve TLR signaling pathways. In DCs, in
which TRAF3 does not play a negative regulatory role in cell
survival, interactions between TRAF3 and other signaling pro-
teins appear to have distinct downstream consequences.
Future studies will seek additional details of the mecha-
nism(s) by which TRAF3 plays distinct roles in different cell
types. The work presented here highlights the importance of
studying TRAF functions in specific, normal cell types, particu-
larly when contemplating TRAFs as therapeutic targets in ma-
lignancy and autoimmune disease, as responses to even the
CpG+IL -4, 84 hr
LMC T3-/- LMCLMCT3-/-T3-/- T3-/-
Figure 7. TLR-induced AID production in
TRAF3?/?B cells. B cells were cultured
with the indicated stimuli. (A) Measure-
ment of Ig isotypes was as in Fig. 5. (B) B
cells were cultured for 84 h prior to cell
lysis, SDS-PAGE of lysates, and Western
blotting for AID, as described in Materi-
als and Methods. Actin was used as a
lane-loading control. (C) Chemilumines-
cence of AID bands was quantitated and
normalized to the intensity of the actin
band signals. Data represent mean val-
ues ? sd from three experiments. Statisti-
cal significance of the difference between
stimulated and unstimulated TRAF3?/?
B cells by Student’s t test was a P value of
0.026. (D) AID levels were tested at two
additional time-points: 72 and 90 h of
stimulation. Experiments presented are
each representative of three similar ex-
periments. *P ? 0.05; **P ? 0.01.
Xie et al.
TRAF3–/–B cells have enhanced TLR responses
Volume 90, December 2011
Journal of Leukocyte Biology 1155
same receptors cannot be extrapolated from one cell type to
another. Our present findings also highlight the complex and
pleiotropic biology of this fascinating family of signaling pro-
P.X., J.P., L.L.S., and G.A.B. designed experiments; P.X., J.P.,
L.L.S., S.M.S., M.L.S., and L.E.C. performed experiments; P.X.,
J.P., L.L.S., S.M.S., M.L.S., L.E.C., and G.A.B. analyzed and dis-
cussed data and prepared figures; and P.X., J.P., L.L.S., S.M.S.,
and G.A.B. wrote the manuscript.
This study was supported by NIH R01 AI28847 and VA Merit
Review 383 to G.A.B. and an American Heart Association Na-
tional Scientist Development award to P.X. This work was sup-
ported in part with resources and the use of facilities at the
Iowa City Veterans Affairs Medical Center.
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TRAF3–/–B cells have enhanced TLR responses
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Journal of Leukocyte Biology 1157