Antibody deciency associated with an inherited

autosomal dominant mutation in TWEAK

Hong-Ying Wang
a,1
, Chi A. Ma
a,1
, Yongge Zhao
a
, Xiying Fan
a
, Qing Zhou
b
, Pamela Edmonds
a
, Gulbu Uzel
c
,
Joao Bosco Oliveira
d
, Jordan Orange
e
, and Ashish Jain
a,2
a
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases (NIAID),
b
Inammatory Disease Section, National Human Genome Research
Institute,
c
Immunopathogenesis Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, and
d
Department of
Laboratory Medicine, Clinical Center, National Institutes of Health (NIH), Bethesda, MD 20892; and
e
Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104
Edited by Rebecca H. Buckley, Duke University Medical Center, Durham, NC, and approved January 29, 2013 (received for review December 11, 2012)
Mutations in the TNF family of proteins have been associated with
inherited forms of immune deciency. Using an array-based se-
quencing assay, we identied an autosomal-dominant deciency in
TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred
with recurrent infection and impaired antibody responses to protein
and polysaccharide vaccines. This mutation occurs in the sixth exon
of TWEAK and results in the amino acid substitution R145C within
the conserved TNF-homology domain of the full-length protein.
TWEAK mutant protein formed high molecular weight aggregates
under nonreducing conditions, suggesting an increased propensity
for intermolecular interactions. As a result, mutant TWEAK associ-
ated with B-cellactivating factor (BAFF) protein and down-regu-
lated the BAFF-mediated activation of the noncanonical NF-B
pathway through inhibition of p100 processing to p52, resulting
in inhibition of BAFF-dependent B-cell survival and proliferation.
As BAFF mediates T-cellindependent isotype switching and B-cell
survival, our data implicate TWEAK as a disease-susceptibility gene
for a humoral immunodeciency.
apoptosis
|
B cell lymphopenia
|
cysteine mutation
|
heteromers
M
utations in the TNF superfamily (TNFSF) and their cognate
receptors have been identied in humans with inherited
forms of autoimmune or immune deciency disorders (1), for
example, autoimmune lymphoproliferative syndrome [FAS (CD95
or TNFRSF6) mutations], TNF receptor-associated periodic syn-
drome (TNFR1 mutations), hyper IgM syndrome (CD154/CD40
mutations), and EBV sensitivity (CD27 mutations). Common
variable immunodeciency (CVID) represents a group of heter-
ogenous primary antibody deciency disorders characterized by
a marked reduction in serum Ig levels with impaired antibody
responses to common bacteria and viruses that result in re-
current infections (2). Single-gene defects in B-cellactivating
factor receptor BAFF-R (TNFRSF13C) (3) and TACI (trans-
membrane activator and calcium-modulating cyclophilin li-
gand interactor, CD267, TNFRSF13B) (4, 5) are associated
with some cases of CVID.
Both BAFF-R and TACI bind to BAFF (B-cell activating fac-
tor, BLYS, TNFSF13B). BAFF is expressed primarily by mono-
cytes and dendritic cells (6) and may induce T-cellindependent
isotype switching in naive B cells in the absence of CD40 en-
gagement and exogenous cytokines (7). However, BAFF seems
to play a major role in B-cell survival through interaction with
BAFF-R (8) based on studies of mice decient in either BAFF
or BAFF-R, which both display severe defects in peripheral B-
cell maturation and decreased levels of immunoglobulins (9, 10).
BAFF can form active heterotrimeric molecules when coex-
pressed with the TNF ligand APRIL (a proliferation-inducing
ligand, TNFSF13), and circulating BAFF/APRIL heterotrimers
are present in serum samples from patients with autoimmune
disorders (11, 12). These ndings raise the possibility that BAFF
may form heteromeric complexes with other members of the
TNF ligand superfamily and play a role in other autoimmune or
immunodecient disease states.
In a recent array-based resequencing study, we screened 148
candidate genes implicated in B-cell development, class switch
recombination, and NF-B signaling in 35 patients with defective
antibody production (13). In one kindred with impaired humoral
immunity and recurrent infections we identied a heterozygous
mutation in the sixth exon of TWEAK (TNF-like weak inducer of
apoptosis, APO3L, TNFSF12) that leads to a single amino acid
substitution of arginine 145 to cysteine (position 52 in the secreted
form). Similar to BAFF and many other TNFSFs, TWEAK can
function as a type II transmembrane protein or as a secreted
soluble cytokine that is cleaved by furin proteases (14). In general,
TWEAK is widely expressed in many tissues and cell types, in-
cluding monocytes/macrophages, dendritic cells, natural killer
(NK) cells, and T cells, and its expression is increased during in-
ammation (15). Although the precise physiologic function of
TWEAK awaits further delineation, it has been proposed that
TWEAK may promote proliferation in endothelial cells (16) and
modulate innate immunity transition to adaptive Th1 immunity
(17) through its receptor Fn14 (TWEAKR, TNFRSF12A) (18).
We found that mutant R145C TWEAK has impaired signaling
function in the Fn14 pathway and may also affect B-cell de-
velopment through interaction with BAFF.
Results
Case Report. The proband patient P2 developed pneumococcal
meningitis complicated by multiple infarcts resulting in left-sided
hemiparesis at 3 y of age. A review of the family medical history
revealed that both siblings (P1 and P2 in the family pedigree; Fig.
1A) and their father (B1) had a history of frequent ear or sino-
pulmonary infections since infancy. The father had been hospital-
ized several times in infancy with pneumonia and had a prolonged
hospitalization for osteomyelitis, although the frequency and se-
verity of his infections had decreased in adulthood. The father had
chronic thrombocytopenia, and both children had intermittent
neutropenia in the peripheral blood. The three affected family
members had multiple warts. Subsequent immunologic evaluation
in both siblings and the father revealed the absence of antibody
responses to T-celldependent and polysaccharide antigens (Table
1). Serum Ig proles and lymphocyte phenotypes were further
analyzed for the father and the two proband patients (Tables 13).
All three affected members had low levels of IgM and IgA. Low
levels of IgG were revealed in patients P1 and P2, especially P2.
Although the absolute IgG level of the father was considered
normal, both IgG2 and IgG4 subclasses were far below normal
Author contributions: H.-Y.W., C.A.M., and A.J. designed research; H.-Y.W., C.A.M., Y.Z.,
X.F., Q.Z., P.E., G.U., J.B.O., and A.J. performed research; H.-Y.W. and J.O. contributed new
reagents/analytic tools; H.-Y.W., C.A.M., Y.Z., X.F., Q.Z., G.U., J.B.O., J.O., and A.J. analyzed
data; and H.-Y.W. and A.J. wrote the paper.
The authors declare no conict of interest.
This article is a PNAS Direct Submission.
1
H.-Y.W. and C.A.M. contributed equally to this work.
2
To whom correspondence should be addressed. E-mail: ajain@niaid.nih.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1221211110/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1221211110 PNAS Early Edition | 1 of 6
I
M
M
U
N
O
L
O
G
Y
range (Table 1). The father and patient P1 had reduced number of
B cells, and nearly all the B cells (>94%) in the three affected
patients were IgM
+
IgD
+
naive cells (Table 2, column 6). Fur-
thermore, all three affected members had a slight increase in the
percentage of TCR
+
CD4

CD8

T cells [i.e., double-negative T

(DNT) cells] and an increase in total T cell numbers, especially
CD8
+
T cells, whereas the level and percentage of CD4
+
T cells
were generally within the normal range (Table 3). The maternal
medical history was unrevealing. The paternal grandmother died
of lymphoma, and the paternal grandfather was not available for
detailed analysis.
Identication of Rare Heterozygous Mutation in TWEAK. By using a
custom resequencing array (13), we identied a single-base het-
erozygous mutation, c.433C>T, in the sixth exon of TWEAK in the
two siblings P1 and P2 (Fig. 1 B and C). This mutation results in
the amino acid substitution from arginine (R) to cysteine (C) at
position 145 in full-length TWEAK protein or position 52 in the
secreted protein. The p.R145C mutation is located in the con-
served TNF-homology domain (THD) of TWEAK and is close to
the only N-glycosylation site (N125), which may be responsible for
trimerization of the TWEAK monomer and receptor binding (Fig.
1 D and E); in addition, PolyPhen prediction (http://genetics.bwh.
harvard.edu/pph/) indicated that the R-to-C change may be del-
eterious (score of 1.992). Other than this mutation, the two sib-
lings had no unusual nucleotide changes in known CVID disease-
susceptibility genes such as BAFF, BAFF-R, or TACI (19), or in
the related genes FAS, FASLG (FAS ligand, TNFSF6), CASP3
(caspase 3, apoptosis-related cysteine peptidase), and CASP8 (cas-
pase 8) (20). The same heterozygous mutation was conrmed in
their father, B1, but was not detected in more than 200 internally
sequenced control samples, or in the latest build of the National
Center for Biotechnology Information Single Nucleotide Poly-
morphism database.
Mutant TWEAK Fails to Induce Cell Death and Activate NF-B and
MAPK Pathways. As a TNF-like weak inducer of apoptosis,
TWEAK is able to induce apoptosis in certain cell lines through
induction of TNF- secretion (21) or through other independent
mechanisms (22). We therefore tested whether the R145C mu-
tation affected the ability of TWEAK to induce cell death in
TWEAK-sensitive tumor cell lines. We rst puried Flag-tagged
soluble WT or mutant TWEAK protein from stable-transfected
HEK293 cells, and an equal amount of each protein was used to
treat HT-29 cells, an adenocarcinoma cell line that is sensitized
to TNF-induced cell death. Only WT TWEAK induced signi-
cant apoptosis in HT-29 cells in the presence of IFN-; mutant
TWEAK showed little apoptotic effect (Fig. 2A, compare col-
umns 2 and 3). The proapoptotic effect of TWEAK was clearly
blocked by preincubation of the protein with recombinant Fn14:
Fc, the TWEAK receptor (Fig. 2A, column 5), but not by non-
specic human IgG (column 4). Moreover, this proapoptotic
effect of the WT TWEAK protein was dose-dependent, whereas
the mutant protein had little effect at any dose tested (Fig. 2B).
The loss of apoptotic function of mutant TWEAK protein may
result from structural changes induced by the R145C mutation.
As this mutation removes a positive charge from the extracellular
domain but leaves a free thiol group in the cysteine residue, an
increase in intermolecular binding to itself or to other proteins is
expected. Indeed, SDS/PAGE under nonreducing conditions
revealed high molecular weight aggregates in lysates of cells
expressing the secreted form of mutant TWEAK protein
(Fig. 2C).
As previously reported, high molecular weight aggregates of
TWEAK appear to be nonfunctional under some conditions
A B1
P1 P2
aa1 249: membrane-bound form
aa94 249: secreted form
D
H O O C 2 H N
249 1 21 42
Cytoplasmic domain
Transmembrane domain
TNF Homology domain (THD)
94
R145C
Extracellular stalk region
B
C/T
A-Fwd/T-Rev
C-Fwd/G-Rev
G-Fwd/C-Rev
T-Fwd/A-Rev
C
C C T C T G G C T A C A A C C
C/T
***.*************::****.****: * *:* * **********
Human
Mouse
Rat
Cattle
Wild boar
Horse
Chimpanzee
Rhesus Macaque
Dog
120 166
QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ--
QDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQ--
QDGAQAGVDGTVSGWEETKINSSSPLRYDRQIGEFTVIRAGLYYLYCQ--
QDGAQAGVDGTVSGWEEAKINSSNPLRYDCQTGQFTVTRAGLYYLYCQ--
QDGAQAGVDGTVSGWEEAKINSSNPLRYDRQSGQFLVTRAGLYYLYCQ--
QDGAQAGVDGTVSGWEEAKINSSNPLRYDRQSGEFIVIRAGLYYLYCQ--
QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ--
QDGAQAGVDGTVSGWEEARINSSSPLRYNRQIGEFIVTRAGLYYLYCQ--
QDGGQAGVDGTVSGWEEAKINSSNPLRYDRQSGEFIVTRAGLYYLYCQ--
TWEAK
E
Fig. 1. Identication of a rare heterozygous mutation in the TWEAK gene in a family diagnosed with CVID. (A) Pedigree of the affected family members.
Highlighted, mutation carrier; square, male; circle, female. (B) Representative image of resequencing array signals showing the heterozygous mutation in the
TWEAK coding region. (C) Conrmation of the heterozygous mutation by Sanger sequencing. (D) Schematic representation of TWEAK structure showing full-length
and secreted forms and location of the mutation site. (E) Sequence conservation of the TWEAK gene among mammals. Highlighted box indicates the mutation site.
Table 1. Serum Ig characterization of three patients carrying the heterozygous mutation in TWEAK
Patient Age at diagnosis, y Sex IgM, mg/dL IgG, mg/dL IgA, mg/dL
Pneumococcal
antibodies, g/mL
Diphtheria
antibodies, IU/mL
Tetanus
antibodies, IU/mL
Normal 34342 6421,730 91499 >0.1 >0.1 >0.6
B1 31 M 61 672* 34

<0.1

0.04

<0.6

P1

6 F <21

562

<10

<0.03

<0.1

0.02

P2

4 M <21

16

<10

<0.03

<0.1

<0.10

*Low IgG2 (85 mg/dL; normal range, 171632 mg/dL) and IgG4 (0.3 mg/dL; normal range, 2.4121 mg/dL).

Abnormal values.

Blood samples for subjects P1 and P2 were drawn before IVIG replacement therapy.
2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1221211110 Wang et al.
(23), possibly because of changes in protein conformation or loss
of receptor binding activity. As the proapoptotic function of
TWEAK is dependent on its receptor Fn14, we tested whether
the loss of function in this mutant was a result of loss of binding
capability to Fn14. To our surprise, 293T cells transfected with
mutant TWEAK seemed to have slightly increased binding to
FITC-labeled Fn14 compared with cells transfected with WT
TWEAK (Fig. 2D). This indicates that the failure of R145C
TWEAK to induce cell death in TWEAK-sensitive cell line is not
a result of diminished interaction with Fn14.
TWEAK has been proposed to activate cell death pathways
through multiple pathways involving activation of NF-B and
MAP kinases (22). To determine whether mutant TWEAK
affects IB degradation, which leads to NF-B release and ac-
tivation, we treated THP1 cells (a human acute monocytic leu-
kemia cell line) with puried Flag-tagged WT or mutant
TWEAK soluble protein. As expected, WT TWEAK induced
IB- degradation in the presence of cycloheximide (CHX)
whereas the mutant protein failed to induce IB- degradation
(Fig. 2E). Similarly, mutant TWEAK lost the ability to induce
MAPK pathways, as evidenced by minimal induction of JNK
phosphorylation in THP1 cells treated with mutant TWEAK
protein (Fig. 2F). Taken together, these data suggest that mutant
TWEAK is a partially functional ligand that can still bind to its
receptor but has lost the capability to stimulate NF-B activation
and induce cell death.
Mutant TWEAK Associates with BAFF. The free thiol group at the
mutant position C145 may allow TWEAK protein to readily bind
to other TNFSFs with structurally similar THDs through for-
mation of an additional disulde bridge. BAFF, a critical B-cell
activation and survival factor, contains many cysteine residues
and is prone to form homotrimers, oligomers, or heterotrimers
with other proteins such as APRIL (11). In fact, active BAFF/
APRIL heterotrimers were detected in serum samples from
patients with systemic immune-based rheumatic diseases (11). As
the TWEAK receptor Fn14 resembles BCMA (B cell maturation
antigen, TNFRSF17), one of the BAFF receptors, we reasoned
that the THD of TWEAK, which shares structural similarity with
that of BAFF, might interact with BAFF, and thus the TWEAK
mutation might affect this binding. To test this hypothesis, we
cotransfected 293T cells with full-length BAFF together with
either WT or mutant TWEAK. After 48 h, cells were lysed and
immunoprecipitated with anti-BAFF or anti-TWEAK antibodies
and then immunoblotted with antibodies against TWEAK or
BAFF, respectively. Both experiments clearly demonstrated that
mutant TWEAK has stronger binding to BAFF than WT
TWEAK (Fig. 3 A and B). In contrast, mutant TWEAK does not
bind to CD40L (Fig. S1).
To conrm the in vivo association between BAFF and TWEAK,
we differentiated monocytes of the two siblings into dendritic
cells that express TWEAK and BAFF and performed similar
coimmunoprecipitation (IP) experiments. Using a monoclonal
antibody against BAFF, BAFFTWEAK association was ob-
served in activated dendritic cells from patient samples but not in
those from normal controls (Fig. 3C). IP experiments with an-
tibody against TWEAK gave a similar result (Fig. S2). We also
conducted hybrid ELISA tests on serum samples of the two
patients (P1 and P2) and their father (B1). Serum samples from
20 normal individuals served as controls. The ELISA plates were
coated with anti-TWEAK antibody and detected by using a spe-
cic HRP-conjugated anti-BAFF antibody. A low level of BAFF
TWEAK complex was generally detected in normal control serum
samples, whereas serum from the two patients displayed signi-
cantly higher levels of BAFFTWEAK protein complex, with
patient P2 showing the highest levels (Fig. 3D). Interestingly, the
BAFFTWEAK complex level was lower in the father, B1, than in
the two siblings, which may explain the reduced severity of im-
munodecient symptoms in the father. The incomplete pene-
trance of the phenotype in the kindred could relate to differences
in expression of the mutant protein as well as differences in ge-
netic background of the affected individuals.
Mutant TWEAK Inhibits BAFF Signaling Function in B Cells. Associa-
tion of mutant TWEAK with BAFF may affect binding of BAFF
to its receptors. However, neither WT nor mutant TWEAK binds
BAFF-R (Fig. S3). BAFF has three receptors: a high-afnity
receptor (BAFF-R), a moderate-afnity receptor (TACI), and
a low-afnity receptor (BCMA). Cotransfection of full-length
BAFF with WT or mutant full-length TWEAK slightly enhanced
binding of BAFF to BAFF-R (Fig. 4A) but not to TACI (Fig. 4B);
however, the enhancement effect was indistinguishable between
WT and mutant TWEAK (Fig. 4A). Because an important
function of BAFF is to provide continuous survival signaling to
antigen-activated B cells through BAFF-R, we reasoned that
mutant TWEAK might affect downstream signaling of BAFF-R.
Activation of BAFF-R induces the noncanonical NF-B pathway
by promoting processing of a precursor protein p100 (NF-B2)
to an active p52 subunit (24). Indeed, membrane fraction prep-
arations from BAFF stably expressing HEK293 cells transfected
with mutant TWEAK showed decreased BAFF-induced pro-
cessing of p100 in naive B cells as evidenced by an increase in
Table 2. B lymphocyte and platelet phenotype report for the three patients
Patient Total CD19
+
B cells CD19
+
B cells, % CD27
+
B cells, %
CD19
+
IgD

CD27
+
cells, %
IgM
+
IgD
+
B cells/total
B cells, % Platelets, 10
3
/L
Normal 47409 3.57.1 0.76.3 0.42.3 6394 161347
B1 74 2.2* 0.9 0.4* 97* 55*
P1 19* 0.9* ND
*
ND
*
95* 226
P2 126 4.6 0.8 0.1* 98* 206
ND, not determined (i.e., too low to be determined accurately).
*Abnormal values.
Table 3. T lymphocyte phenotype report for the three patients
Patient Total CD3
+
T cells CD3
+
T cells, % Total CD4
+
T cells CD4
+
T cells, % Total CD8
+
T cells CD8
+
T cells, %
Total /
DNT cells
/ DNT
cells, %
Normal 6502,108 57.386.4 3621,275 29.057.3 344911 25.250.8 217 <1.4
B1 3,036* 88.8* 1,294 37.8 1,795* 52.5* 33* 1.4*
P1 2,061 92.9* 698 31.5 1,302* 58.7* 31* 1.7*
P2 2,235* 82.5 810 30.0 1,346* 49.7 63* 3.2*
*Abnormal values.
Wang et al. PNAS Early Edition | 3 of 6
I
M
M
U
N
O
L
O
G
Y
p100 levels and a decrease in p52 levels (Fig. 4C, lane 4). In
contrast, BAFF-expressing stable cell lines transfected with WT
TWEAK showed enhanced p100 processing with increased p52
levels compared with BAFF-expressing stable transfectants alone
(Fig. 4C, lane 3).
To further test whether down-regulation of BAFF-R signaling
by mutant TWEAK is associated with a decreased proliferation
response in activated B cells, we performed an in vitro B-cell
proliferation assay using [
3
H]thymidine incorporation. WT
TWEAK, mutant TWEAK, or empty vector were transfected
into BAFF-expressing stable HEK293 cell lines. After 36 h, cells
were irradiated and cocultured with puried human B cells that
were stimulated with anti-IgM F(ab)2 fragment. B cells cultured
with WT TWEAK transfectants showed a slightly higher pro-
liferation response than B cells cultured with control transfectants,
whereas B cells cultured with mutant TWEAK transfectants
showed a decreased proliferation response (Fig. 4D). BAFF-me-
diated B-cell survival is primarily mediated by BAFF-R (25, 26).
To test whether mutant TWEAK down-regulates BAFF-de-
pendent B-cell survival, human B cells were treated with
recombinant BAFF with or without puried soluble TWEAK
WT or mutant, or with puried BAFF/mutant TWEAK hetero-
mers (Fig. S4). As expected, addition of WT or mutant soluble
TWEAK did not affect BAFF-induced B-cell survival, whereas
addition of BAFF/mutant TWEAK heteromers decreased cell
viability to the levels seen in medium alone at all time points
tested. Finally, to analyze whether mutant TWEAK affects the
BAFF-induced B-cell isotype switching response, human B cells
were treated with recombinant BAFF with or without puried WT
or mutant soluble TWEAK, or with puried BAFF/mutant
TWEAK heteromers. BAFF alone promoted secretion of IgG
and IgA, whereas addition of BAFF/mutant TWEAK hetero-
mers decreased BAFF-mediated IgG and IgA secretion in cul-
tured B cells (Fig. S5).
Taken together, these data indicate that mutant TWEAK may
dominantly inhibit B-cell survival and proliferation in addition to
Ig class switching through association with BAFF and down-
regulation of the noncanonical BAFF-induced NF-B pathway.
Discussion
The combination of candidate-gene sequencing and protein func-
tional analysis used in this study provides an effective strategy for
identifying genes responsible for rare monogenic immunode-
ciency disorders (27). Here, we describe an autosomal dominant
TWEAK mutation that is associated with impaired antibody
responses, reduced IgM and IgA levels, and an increased number
of DNT cells (i.e., TCR
+
CD4

CD8

T cells). The TWEAK p.

R145C mutation changes a charged arginine residue to a cysteine
at a position close to the receptor binding sites in the THD.
15
20
25
30
C
e
l
l

s
u
r
v
i
v
a
l

i
n
d
e
x
(
L
U

x

1
0
-
4
)
Purified TWEAK protein
level in the medium
35
40
B
BSA
Twt
Tmu
None 3 ng/ml 6 ng/ml 9 ng/ml
4
8
12
16
0
0.25 0.5 1 1.5 2
[Fn14:Fc-FITC] (g/ml)
F
I
T
C

M
F
I
0
5
15
C
e
l
l

s
u
r
v
i
v
a
l

i
n
d
e
x
(
L
U

x

1
0
-
4
)
EV
Twt
Tmu
IgG
Fn14:Fc
25
35
A
*
*
D
Time(h)
actin
P-JNK IKB-
actin
Twt
Time(h) 1 2 3 0 0 1 2 3 0.5 1 2 0 0 0.5 1 2
Tmu Twt Tmu
E F
0.5 0.5
*
*
High-
molecular
weight
aggregates
TWEAK (soluble)
EV Twt TmuTwt Tmu Transfection:
Reducing Condition
C
15
25
45
72
95
MW
170
EV
Twt
Tmu
EV
Twt
Tmu
Fig. 2. Mutant TWEAK fails to induce cell death and activate NF-B pathways.
(A) Mutant TWEAK fails to induce cell death in HT-29 cells. Flag-tag
recombinant WT and mutant TWEAK soluble proteins were puried by M2
anti-Flag agarose gel and quantied by ELISA. HT-29 cells were cultured in the
presence of WT TWEAK, mutant soluble TWEAK, or control BSA (3 ng/mL)
together with IFN- (10 ng/mL). In some experiments, the WT soluble TWEAK
protein was preincubated with human IgG or recombinant Fn14:Fc for 2 h
before addition to HT-29 cells. Cell viability was determined in triplicate after 3
d. EV, empty vector; Tmu, TWEAK mutant; Twt, TWEAK WT (*P < 0.05 vs. BSA
control). (B) Puried mutant TWEAK protein showed minimal capability to
induce cell death in HT-29 cells. Equal amounts of WT TWEAK, mutant TWEAK,
or control BSA were added in triplicate to HT-29 cell cultures, and cell viability
was determined after 3 d (*P < 0.05 vs. BSA control). (C) Mutant TWEAK forms
high molecular weight aggregates. Lysates of Escherichia coli transformed
with WT or mutant soluble TWEAK were subjected to SDS/PAGE and Western
blotting under reducing and nonreducing conditions. (D) The R145C mutation
does not reduce binding of TWEAK to its receptor Fn14. 293T cells were
transfected with RFP fusion constructs expressing full-length WT TWEAK or
mutant TWEAK. Cells transfected with RFP empty vector served as controls.
Transfection efciency of each construct was monitored by RFP expression.
Surface expression of TWEAK was measured by FACS analysis using ve dif-
ferent concentrations of FITC-conjugated Fn14:Fc and gating on RFP
+
cells.
MFI, mean uorescence intensity. Data in the plots were based on four in-
dependent experiments. (E) Mutant TWEAK protein was unable to induce NF-
B activation in THP1 cells. THP1 cells were treated with 6 ng/mL puried WT
or mutant TWEAK soluble protein at 37 C for the indicated time periods. To
observe the effect on IB degradation, 50 g/mL cycloheximide (CHX) was
added to the cell culture. At the indicated times, cells were lysed and subjected
to Western blotting with the indicated antibodies. (F) Western blot analysis of
phospho-JNK expression was performed as in A but without CHX treatment.
IB:
anti-BAFF
IB:
anti-TWEAK
IP: anti-BAFF
Lysate
IB:
anti-TWEAK
IB:
anti-BAFF
BAFF
Twt
Tmu
A
IB:
anti-TWEAK
IB:
anti-BAFF
IP: anti-TWEAK
IB:
anti-BAFF
IB:
anti-TWEAK
BAFF
Twt
Tmu
B
Lysate
Normal
*
P1
*
P2 B1
D
0
5
100
150
T
W
E
A
K
-
b
o
u
n
d

B
A
F
F

(
p
g
/
m
l
)
200
C1 C2 P1 P2
IB: TWEAK
IB: BAFF
IP: BAFF
Input
BAFF
C
1 3
1 5
Fig. 3. Mutant TWEAK associates with BAFF. (A and B) In vitro association of
mutant TWEAK with BAFF. Full-length BAFF and WT (Twt) or mutant TWEAK
(Tmu) constructs inpIRES vector werecotransfectedinto293Tcells. After 48h, cells
were lysed and immunoprecipitated with anti-BAFF (A) or anti-TWEAK (B) anti-
bodies, andtheresultingprecipitates weresubjectedtoWesternblot analysis with
the opposing antibodies. Numbers under the bands indicate relative intensity of
BAFF:TWEAKcomplex against BAFF-IP or TWEAK-IP input. (C) Associationof BAFF
and TWEAK in dendritic cells frompatients and normal controls. Monocytes from
patients and control subjects were separated and cultured in the presence of GM-
CSF and IL-4 for 6 d. The resulting dendritic cells were activated with lipopoly-
saccharide andsubjectedtoIP andimmunoblottingwiththeindicatedantibodies.
(D) Detection of BAFFTWEAK heteromers in serum samples of patients. Serum
samples from affected family members and normal control subjects were added
to an ELISA plate coated with anti-TWEAK monoclonal antibodies and detected
by HRP-conjugated monoclonal antibody against BAFF. Results from the two
siblings (P1 andP2) and their father (B1) are shown (*P <0.05 vs. normal controls).
4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1221211110 Wang et al.
Although this mutation does not affect binding of TWEAK to its
receptor, it appears to impair its ability to induce apoptosis in
TWEAK-sensitive cell lines by decreasing activation of NF-B
and MAPK pathways. The demonstration that mutant TWEAK
associates with BAFF indicates that the mutant protein might
also dominantly inhibit B-cell function by forming noneffective
ligand trimers or oligomers, thereby blocking effective receptor
binding and downstream signaling.
Of particular interest among the observations in these patients
is the increased number of DNT cells and presence of cutaneous
papillomatosis. Previous reports suggest that TWEAK works
with other proapoptotic TNFSF ligands such as FASLG, TRAIL
(TNF-related apoptosis inducing ligand, TNFSF10), and TNF-
to facilitate cytotoxicity in many cell types, including activated
monocytes (28), dendritic cells (29), NK cells (30), and T cells
(31). Autoimmune lymphoproliferative syndrome caused by im-
paired FAS-mediated cell death is characterized by an accumu-
lation of DNT cells and autoimmunity (32). It seems that the loss
of apoptotic function of TWEAK protein is correlated to the
increase in peripheral DNT cells and CD8
+
T cells in patients
carrying the mutant R145C allele; however, the exact link and
underlying apoptotic mechanism awaits further study. Because
the patients have papillomatosis, we were intrigued by the fact
that TWEAK protein can be up-regulated by IFN- or phorbol
myristate acetate in cultured human peripheral NK cells (17, 28).
Although further investigation is warranted, TWEAK expression
by NK cells and its subsequent engagement of Fn14 on the
surface of epithelial cells may be important for controlling local
immune responses to papilloma virus.
Mutations in BAFF-R in humans have been associated with
reduced antibody production. The ndings in our patients of
absent antibody responses to T-celldependent and poly-
saccharide antigens, as well as reduced B cell numbers, have also
been reported in BAFF-Rdecient patients (3). However, in
contrast to the patients presented here, BAFF-Rdecient
patients have normal or even elevated IgA serum concentrations.
Differences in genetic background among patient groups may
inuence the development of some of these phenotypes. It is
possible that R145C TWEAK/BAFF heterotrimers or oligomers
also bind to TACI in a dominant-negative manner and limit the
receptors association with another TNF ligand, APRIL. Con-
sistent with this hypothesis are the observations of reduced se-
rum IgA levels in TACI-decient humans and mice decient in
APRIL (4, 5, 33). Thus, the phenotype that results from the
R145C TWEAK mutation may not only be inuenced by its
interaction with BAFF, but also by association of the mutant
heteromeric complex with multiple TNF family receptors.
TWEAK is located on human chromosome 17p13.1 approxi-
mately 878 bp upstream of APRIL, and these two genes are
tightly linked in human and mouse with similar intronexon
organization. APRIL is also an important regulator of T-cell
independent class-switch pathways in B cells. An intergene
splicing transcript TWE-PRIL, which combines the rst six exons
of TWEAK and the last ve exons of APRIL, was identied in
activated T cells and monocytes (34). TWE-PRIL is a membrane-
anchored protein that possesses the intracellular transmembrane
stalk region of TWEAK but the receptor-binding domain of
APRIL, thus signaling through the APRIL binding domain may
be linked to downstream pathways shared with the TWEAK
intracellular domain. As the R145 residue is also present in
TWE-PRIL, the possibility that the R145C mutation may in-
terfere with the signaling pathways of APRIL cannot be ruled
out. However, we were not able to detect a band corresponding
to the TWE-PRIL protein in activated monocytes or T cells
using monoclonal antibodies against APRIL or TWEAK in
normal subjects. Furthermore, we prepared single-cell suspen-
sions from lymph nodes of normal subjects and did not detect
TWE-PRIL expression on the surface of T cells and monocytes.
These results call into question whether TWE-PRIL exists as a
functional protein in human leukocytes.
These ndings lead us to consider the physiologic function of
naturally occurring heteromeric TNF ligands. Further study of these
complexes could lead to the rational design of immunosuppressive
agents that target specic aspects of immunity. In addition, further
analysis of cysteine mutations in other TNF family members and
their contribution to the formation of heteromeric complexes
might provide a mechanism for some of the phenotypic variability
associated with immune deciency disorders.
Materials and Methods
Patients and Protocols. All patients and healthy controls were studied with
informed consent at the NIH clinical center under clinical protocol (00-I-006),
which was approved by the NIAID institutional review board. A detailed of
summary of research methods (7, 13, 23, 35), reagents, and statistical analysis
is available in the SI Materials and Methods.
ACKNOWLEDGMENTS. We thank the patients families for their participa-
tion in this study; Ron Hornung and Margaret Brown for immunologic eval-
uations; Mary Derry for editoral assistance; and Ivona Aksentijevich, Linda
Burkly, and Warren Strober for helpful discussions. This research was sup-
ported by the Intramural Research Program of the NIH/NIAID.
0
1
2
[
3
H
]

-

T
h
y
m
i
d
i
n
e
i
n
c
o
r
p
o
r
a
t
i
o
n

(
c
p
m

x
1
0
-
3
)
3
E
V
+
2
9
3
E
V
+
2
9
3
-
B
A
F
F
T
w
t
+
2
9
3
-
B
A
F
F
T
m
u
+
2
9
3
-
B
A
F
F
p100
p52
-actin
C B B

+

T
w
t
B

+

T
m
u C D
3.9 1.3 1.2 2.7
A
0
20
40
60
80
100
EV
BAFF + Tmu
BAFF
BAFF + Twt
BAFF-R binding (APC)
C
e
l
l

n
u
m
b
e
r
0
20
40
60
80
100
TACI binding (APC)
B
Fig. 4. Mutant TWEAK affects BAFF downstream signaling. (A) Association
of TWEAK with BAFF slightly increases binding of BAFF to BAFF-R. 293T cell
lines were cotransfected with full-length DsRed2BAFF fusion vector to-
gether with empty vector (EV) or WT or mutant TWEAK in pEGFP-C1 vector.
Surface expression of BAFF was measured by FACS analysis using recombi-
nant Fc-BAFF-R and APC-conjugated secondary IgG. (B) TWEAK does not
affect binding of BAFF to TACI. Experiments were conducted as in A except
that surface expression of BAFF was measured by FACS analysis using
recombinant TACI:Fc. (C) Membrane fraction of HEK293 stable BAFF-
expressing cell line cotransfected with mutant TWEAK showed decreased
p100 processing in human B cells. Membrane fractions were prepared from
HEK293 BAFF stable cells transfected with WT or mutant TWEAK or empty
vectors and used to stimulate puried human B cells. After 42 h, B cells were
lysed and subjected to Western blotting using anti-p52 antibodies. B, BAFF;
Tmu, TWEAK mutant; Twt, TWEAK WT. Numbers under the bands indicate
relative intensity of p100 and p52 bands. (D) Mutant TWEAK down-regulates
the BAFF-induced B-cell proliferation response. B cells were puried from
PBMCs and cocultured in the presence of anti-human IgM F(ab)2 fragment
and irradiated HEK293 cells that stably expressed full-length BAFF and were
transfected with WT or mutant TWEAK construct or empty vector. B-cell
proliferation response was measured as described in SI Materials and Methods
(*P < 0.05 vs. 293-BAFF plus EV control).
Wang et al. PNAS Early Edition | 5 of 6
I
M
M
U
N
O
L
O
G
Y
1. Lobito AA, Gabriel TL, Medema JP, Kimberley FC (2011) Disease causing mutations in
the TNF and TNFR superfamilies: Focus on molecular mechanisms driving disease.
Trends Mol Med 17(9):494505.
2. Wood P (2009) Primary antibody deciency syndromes. Ann Clin Biochem 46(pt 2):
99108.
3. Warnatz K, et al. (2009) B-cell activating factor receptor deciency is associated with
an adult-onset antibody deciency syndrome in humans. Proc Natl Acad Sci USA
106(33):1394513950.
4. Castigli E, et al. (2005) TACI is mutant in common variable immunodeciency and IgA
deciency. Nat Genet 37(8):829834.
5. Salzer U, et al. (2005) Mutations in TNFRSF13B encoding TACI are associated with
common variable immunodeciency in humans. Nat Genet 37(8):820828.
6. Craxton A, Magaletti D, Ryan EJ, Clark EA (2003) Macrophage- and dendritic cell
dependent regulation of human B-cell proliferation requires the TNF family ligand
BAFF. Blood 101(11):44644471.
7. Litinskiy MB, et al. (2002) DCs induce CD40-independent immunoglobulin class
switching through BLyS and APRIL. Nat Immunol 3(9):822829.
8. Waldschmidt TJ, Noelle RJ (2001) Immunology. Long live the mature B cella bafing
mystery resolved. Science 293(5537):20122013.
9. Schiemann B, et al. (2001) An essential role for BAFF in the normal development of B
cells through a BCMA-independent pathway. Science 293(5537):21422114.
10. Yan M, et al. (2001) Identication of a novel receptor for B lymphocyte stimulator
that is mutated in a mouse strain with severe B cell deciency. Curr Biol 11(19):
15471552.
11. Roschke V, et al. (2002) BLyS and APRIL form biologically active heterotrimers that
are expressed in patients with systemic immune-based rheumatic diseases. J Immunol
169(8):43144321.
12. Dalakas MC (2008) B cells as therapeutic targets in autoimmune neurological disorders.
Nat Clin Pract 4(10):557567.
13. Wang HY, et al. (2010) A custom 148 gene-based resequencing chip and the SNP ex-
plorer software: New tools to study antibody deciency. Hum Mutat 31(9):10801088.
14. Brown SA, Ghosh A, Winkles JA (2010) Full-length, membrane-anchored TWEAK can
function as a juxtacrine signaling molecule and activate the NF-kappaB pathway.
J Biol Chem 285(23):1743217441.
15. Chicheportiche Y, et al. (1997) TWEAK, a new secreted ligand in the tumor necrosis
factor family that weakly induces apoptosis. J Biol Chem 272(51):3240132410.
16. Lynch CN, et al. (1999) TWEAK induces angiogenesis and proliferation of endothelial
cells. J Biol Chem 274(13):84558459.
17. Maecker H, et al. (2005) TWEAK attenuates the transition from innate to adaptive
immunity. Cell 123(5):931944.
18. Winkles JA (2008) The TWEAK-Fn14 cytokine-receptor axis: Discovery, biology and
therapeutic targeting. Nat Rev Drug Discov 7(5):411425.
19. Castigli E, Geha RS (2006) Molecular basis of common variable immunodeciency.
J Allergy Clin Immunol 117(4):740746.
20. Worth A, Thrasher AJ, Gaspar HB (2006) Autoimmune lymphoproliferative syndrome:
Molecular basis of disease and clinical phenotype. Br J Haematol 133(2):124140.
21. Ikner A, Ashkenazi A (2011) TWEAK induces apoptosis through a death-signaling
complex comprising receptor-interacting protein 1 (RIP1), Fas-associated death do-
main (FADD), and caspase-8. J Biol Chem 286(24):2154621554.
22. Nakayama M, et al. (2002) Multiple pathways of TWEAK-induced cell death. J Immunol
168(2):734743.
23. Roos C, et al. (2010) Soluble and transmembrane TNF-like weak inducer of apoptosis
differentially activate the classical and noncanonical NF-kappa B pathway. J Immunol
185(3):15931605.
24. Morrison MD, Reiley W, Zhang M, Sun SC (2005) An atypical tumor necrosis factor
(TNF) receptor-associated factor-binding motif of B cell-activating factor belonging to
the TNF family (BAFF) receptor mediates induction of the noncanonical NF-kappaB
signaling pathway. J Biol Chem 280(11):1001810024.
25. Batten M, et al. (2000) BAFF mediates survival of peripheral immature B lymphocytes.
J Exp Med 192(10):14531466.
26. Patke A, Mecklenbruker I, Erdjument-Bromage H, Tempst P, Tarakhovsky A (2006)
BAFF controls B cell metabolic tness through a PKC beta- and Akt-dependent
mechanism. J Exp Med 203(11):25512562.
27. Wang HY, Jain A (2011) Novel sequencing-based strategies for high-throughput
discovery of genetic mutations underlying inherited antibody deciency disorders.
Curr Allergy Asthma Rep 11(5):352360.
28. Nakayama M, Kayagaki N, Yamaguchi N, Okumura K, Yagita H (2000) Involvement of
TWEAK in interferon gamma-stimulated monocyte cytotoxicity. J Exp Med 192(9):
13731380.
29. Lichtner M, et al. (2004) HIV type 1-infected dendritic cells induce apoptotic death
in infected and uninfected primary CD4 T lymphocytes. AIDS Res Hum Retroviruses
20(2):175182.
30. Petitbarat M, et al. (2011) TWEAK appears as a modulator of endometrial IL-18
related cytotoxic activity of uterine natural killers. PLoS ONE 6(1):e14497.
31. Kaplan MJ, Ray D, Mo RR, Yung RL, Richardson BC (2000) TRAIL (Apo2 ligand) and
TWEAK (Apo3 ligand) mediate CD4+ T cell killing of antigen-presenting macro-
phages. J Immunol 164(6):28972904.
32. Fleisher TA (2008) The autoimmune lymphoproliferative syndrome: An experiment of
nature involving lymphocyte apoptosis. Immunol Res 40(1):8792.
33. Castigli E, et al. (2004) Impaired IgA class switching in APRIL-decient mice. Proc Natl
Acad Sci USA 101(11):39033908.
34. Pradet-Balade B, et al. (2002) An endogenous hybrid mRNA encodes TWE-PRIL,
a functional cell surface TWEAK-APRIL fusion protein. EMBO J 21(21):57115720.
35. Temmerman ST, et al. (2006) Impaired dendritic-cell function in ectodermal dysplasia
with immune deciency is linked to defective NEMO ubiquitination. Blood 108(7):
23242331.
6 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1221211110 Wang et al.