Báo cáo khoa học: Intrabodies against the EVH1 domain of Wiskott–Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells potx

Intrabodies against the EVH1 domain of Wiskott–Aldrich
syndrome protein inhibit T cell receptor signaling in
transgenic mice T cells
Mitsuru Sato
1
, Ryo Iwaya
1,2
, Kazumasa Ogihara
1,3
, Ryoko Sawahata
1,3
, Hiroshi Kitani
1
, Joe Chiba
2
,
Yoshikazu Kurosawa
4
and Kenji Sekikawa
1,5
1 Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, Ibaraki, Japan
2 Department of Biological Science and Technology, Tokyo University of Science, Chiba, Japan
3 Institute for Antibodies Co., Ltd, National Institute of Agrobiological Sciences, Ibaraki, Japan
4 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan
5 Kitasato University School of Veterinary Medicine and Animal Sciences, Aomori, Japan
Intracellular antibodies (intrabodies) may be useful
tools for not only clinical applications such as viral
neutralization and cancer therapy but also functional
analysis of proteins inside cells. A variety of intrabody
formats have been used. Single-chain variable frag-
ments (scFvs) consist of one heavy chain variable

region (V
H
) linked through a flexible peptide spacer,
usually a repeated motif of 3 · GGGGS, to one light
Keywords
cytosolic protein; functional knockdown;
intrabody; T-cell receptor signaling; Wiskott–
Aldrich syndrome protein (WASP)
Correspondence
K. Sekikawa, Department of Molecular
Biology and Immunology, National Institute
of Agrobiological Sciences, 3-1-5,
Kannondai, Tsukuba, Ibaraki 305-0856,
Japan
Tel ⁄ Fax: +81 29 8386039
E-mail:
(Received 2 August 2005, revised 4 October
2005, accepted 10 October 2005)
doi:10.1111/j.1742-4658.2005.05011.x
Intracellularly expressed antibodies (intrabodies) have been used to inhibit
the function of various kinds of protein inside cells. However, problems
with stability and functional expression of intrabodies in the cytosol remain
unsolved. In this study, we show that single-chain variable fragment (scFv)
intrabodies constructed with a heavy chain variable (V
H
) leader signal
sequence at the N-terminus were translocated from the endoplasmic reti-
culum into the cytosol of T lymphocytes and inhibited the function of
the target molecule, Wiskott–Aldrich syndrome protein (WASP). WASP
resides in the cytosol as a multifunctional adaptor molecule and mediates

actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor
(TCR) signaling pathway. It has been suggested that an EVH1 domain in
the N-terminal region of WASP may participate in IL-2 synthesis. In trans-
genic mice expressing anti-EVH1 scFvs derived from hybridoma cells pro-
ducing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and
binding between anti-EVH1 scFvs and native WASP in T cells were detec-
ted by immunoprecipitation analysis. Furthermore, impairment of the pro-
liferative response and IL-2 production induced by TCR stimulation which
did not affect TCR capping was demonstrated in the scFv transgenic
T cells. We previously described the same T-cell defects in WASP trans-
genic mice overexpressing the EVH1 domain. These results indicate that
the EVH1 intrabodies inhibit only the EVH1 domain function that regu-
lates IL-2 synthesis signaling without affecting the overall domain structure
of WASP. The novel procedure presented here is a valuable tool for in vivo
functional analysis of cytosolic proteins.
Abbreviations
BrdU, 5-bromo-2¢-deoxyuridine ER, endoplasmic reticulum; intrabody, intracellular expressed antibody; EVH1, enabled ⁄ vasodilator-stimulated
phosphoprotein (Ena ⁄ VASP) homology 1; FITC, fluorescein isothiocyanate; GST, glutathione S-transferase; IL, interleukin; scFv, single-chain
variable fragment; TCR, T cell receptor; VH, heavy chain variable; VL, light chain variable; WASP, Wiskott–Aldrich syndrome protein; WIP,
WASP-interacting protein.
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6131
chain variable (V
L
). They are able to fold and retain
the antigen-binding specificity and affinity of the paren-
tal antibody [1,2]. scFvs are expressed more easily than
whole antibodies assembled with heavy and light chains
by disulfide bonds. In general, the antibody fragments
for assembling the scFvs are isolated from either anti-
body phage display libraries [3] or well-characterized

hybridoma producing mAbs. Although screening of
phage libraries allows the selection of antibody frag-
ments directed against a variety of antigens, the
screened antibody fragments often show low or inter-
mediate affinity for the antigen. Therefore, large-scale
libraries and extensive screening are required for the
selection of the antigen-specific antibody fragments. On
the other hand, the antibody fragments isolated from
hybridomas have high affinity and specificity for the
target molecules. However, the cloning of heavy and
light chain variable regions by RT-PCR can be difficult
because of the presence of nonspecific variable region
transcripts produced by myeloma cells that are fused to
the antibody-producing cells.
In functional proteomics, comprehensive protein
analyses have been demonstrated [4]. However, the
development of a new procedure for domain analysis
of protein is necessary. Gene knock-out technologies
that rely on developing a phenotype from null muta-
tion of the gene in embryonic stem cells are powerful
tools for understanding gene function. Recently, RNA
interference (RNAi) which can eliminate specific
mRNA and lead to gene silencing has been developed
[5]. However, these gene knock-out and silencing tech-
niques cannot be used to analyze domain structures
and functions and post-translationally modified protein
functions. Dominant negative gene knock-out proce-
dures succeed in inhibiting the targeted domain func-
tions of proteins, but not in all cases.
Antibodies have been used for various purposes for a

long time. For example, they have been used as reagents
for Western blotting, immunostaining, immunoprecipi-
tation and blocking of protein function. Therefore, if
intrabodies retain their specificity and high-affinity bind-
ing properties, they may be useful tools for inhibition of
protein function inside the cell. In fact, much attention
has been paid to intrabodies for clinical applications.
The functional knockdown of target proteins, such as
HIV gp120, chemokine receptor, growth factor recep-
tors, MHC class I, Ras oncogene, p53 tumor suppres-
sor, and protein kinases has been demonstrated [6–12].
If the target proteins are synthesized and processed in
the endoplasmic reticulum (ER), scFvs are expressed
with the signal peptide at the N-terminus of V
H
and V
L
with the ER retention signal KDEL (Lys-Asp-Glu-Leu)
at the C-terminus. Folded scFvs can bind to the target
proteins on the lumen side and inhibit transport of tar-
get proteins in the process of functional maturation
[6,8]. If the targets are cytosolic proteins, scFvs without
the signal peptide are used for expression in the cytosol.
However, expression levels of scFvs are low in the cyto-
sol, and binding of scFv intrabodies to target molecules
is difficult to detect [13]. A small quantity of intrabodies
in the cytosol may explain the low translational effi-
ciency and low stability of intrabodies in the cytosol.
Wiskott–Aldrich syndrome protein (WASP), the
causal gene product of the X-linked immunodeficiency

(WAS) [14,15], participates in TCR signaling as a cyto-
solic adaptor molecule [16–18]. It is well known that
TCR stimulation activates various signaling cascades
accompanied by recruitment of adaptor molecules,
protein kinases and regulatory molecules into the
membrane-receptor complexes, resulting in the correct
initiation and amplification of the signaling reaction.
WASP is an adaptor molecule containing multiple
domains: for example, a GTPase-binding domain,
which is thought to interact with Cdc42, and a pro-
line-rich region, which interacts with the Src homol-
ogy 3 domain of the adaptor Nck, Grb2 and several
kinases [19–22]. Furthermore, WASP is also associated
with the actin-related protein (Arp2 ⁄ 3) complex
through its C-terminal region. The association of
WASP and the Arp2 ⁄ 3 complex activates the actin
nucleation activity of the Arp2 ⁄ 3 complex [23].
To investigate further the function of the WASP-
EVH1 domain in the TCR signaling pathway, we
developed transgenic (Tg) mice that express
intrabodies that specifically bind to the WASP-EVH1
domain. The cDNA fragments that encode variable
regions of heavy and light chains were isolated from
two established hybridomas producing WASP-EVH1-
specific mAbs. We constructed several scFvs consisting
of V
H
and V
L
regions with ⁄ without the V

H
leader
sequence at the N-terminal and with ⁄ without the
C
L
(j) region behind the V
L
region. None of the con-
structs contained the KDEL sequence at the C-termi-
nus. We compared the quantity of scFv intrabodies
and assessed their binding activity to the WASP-EVH1
domain in the scFv gene-transfected T cells. Finally,
we succeeded in expressing the functional scFv intra-
bodies in the cytosol and precisely knocking down the
targeted protein domain in scFv transgenic mice.
Results
Construction of anti-WASP-EVH1 scFvs
To assess the binding activity to native WASP in
T cells, mAb clones (17, 18 and 21) were confirmed
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.
6132 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
by immunoprecipitaion. Clones 18 and 21 were able
to bind to the native form of WASP expressed in
T cells, but clone 17 was not able to immunoprecipi-
tate native WASP (Fig. 1A). On the basis of this
result, clones 18 and 21 were selected for construc-
tion of scFv intrabodies. For the design of primers
for PCR amplification of cDNA that encodes sub-
type-specific V
H

and V
L
regions, mAbs were checked
by an isotyping test. Clones 18 and 21 were classi-
fied as IgG3 ⁄ j and IgG2b ⁄ j, respectively. The
appropriate cDNA fragments of the V
H
and V
L
regions were then generated by RT-PCR. A compar-
ison of the V
H
and V
L
amino-acid sequences of
clones 18 and 21 is shown in Fig. 1B. All of the V
H
regions and the complementarity-determining region
3 of the V
L
regions differed strongly between the
two clones.
Generation of scFv from hybridomas was achieved
by well-established molecular engineering methods.
The four-step PCR using appropriate primers allowed
amplification and assembly of the V
H
and V
L
regions

(Fig. 2A). To investigate the stability of scFvs, we
designed several scFv constructs with and without the
N-terminal leader signal sequence of the V
H
region
and with and without the C
L
(j) region following the
V
L
region, which are described as HL, SHL, HL-CL
and SHL-CL in Fig. 2B.
Expression of scFv intrabodies and binding
to WASP
In all scFv gene-transfected T cells, expression of scFv
intrabodies was detected by Western blot analysis.
However, scFvs containing the V
H
signal peptide
sequence and C
L
region (SHL or SHL-CL) were highly
expressed in T cells (Fig. 3A). These results strongly
suggest that the addition of the V
H
signal peptide
sequence and C
L
(j) region to scFvs increases the sta-
bility of the scFv intrabodies in T cells.

An in vitro binding assay was performed using gluta-
thione S-transferase (GST) pull-down to detect the
binding activity of anti-WASP scFvs. Constructs con-
taining the V
H
signal peptide sequence, 18SHL ⁄
SHL-CL and 21SHL ⁄ SHL-CL, were able to bind to
GST-WASP15 (see Experimental procedures for defini-
tion of WASP15), whereas no binding activity of scFvs
that did not contain the signal peptide sequence
of the V
H
region was detected (Fig. 3B). Although
the expression levels of 18SHL ⁄ SHL-CL and
21SHL ⁄ SHL-CL were almost the same in the scFv
gene-transfected T cells, 21SHL ⁄ SHL-CL bound more
strongly to GST-WASP15 than 18SHL ⁄ SHL-CL
(Fig. 3A,B). Furthermore, to examine the interaction
in vivo between scFv intrabodies and the target mole-
cule, WASP, scFv gene-transfected T cells were lysed
Fig. 1. Selection of WASP EVH1 mAbs for
assembling scFvs and aligned amino-acid
sequences of the V
H
and V
L
regions. (A)
Immunoprecipitation of T cell lysates with
WASP EVH1 mAbs produced by established
hybridomas. T cell lysates were immunopre-

cipitated with 5 lgÆmL
)1
control mouse IgG
(lane 1), clone 17 (lane 2), clone 18 (lane 3),
clone 21 (lane 4) or commercially available
WASP mAb (lane 5) and analyzed by West-
ern blotting with WASP polyclonal antibody.
Control T cell lysates were loaded in lane 6.
The 30-kDa bands (arrowhead) indicated
secondary antibody cross-reactive nonspe-
cific proteins. (B) Comparison of deduced
amino-acid sequences of the V
H
and V
L
frag-
ments derived from WASP EVH1 mAbs 18
and 21. Shared amino acids are indicated by
bars. Leader signal sequences and three
complementarity-determining regions are
shown in gray boxes. Four framework
regions (FR) are marked above the
sequence.
M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6133
and immunoprecipitated with WASP mAb. A strong
interaction between WASP and 21SHL ⁄ SHL-CL scFvs
was detected by Western blot analysis with Myc tag
antibody, whereas 18SHL ⁄ SHL-CL scFvs and other
scFvs were not able to associate with native WASP

(Fig. 3C). The binding specificity for the WASP EVH1
domain was demonstrated by in vivo interaction
between T7-tagged WASP15 and 21SHL ⁄ SHL-CL
scFvs (Fig. 3D). These results suggest that 21SHL and
21SHL-CL are stably expressed as intrabodies with
domain-specific binding capabilities, and are able to
associate with native WASP in T cells.
To detect cleavage of the V
H
signal peptide sequence
from the N-terminal scFv-V
H
region, the N-terminal
amino-acid sequence of scFv 21SHL-CL expressed in
T cells was determined. Unfortunately, we could not
detect the N-terminal sequence by the well-established
Edman method because the N-terminal amino-acid
residue was blocked. Moreover, we could not detect a
signal
V
H
V
L
(G
4
S)
3
V
H
V

L
(G
4
S)
3
signal
V
H
V
L
(G
4
S)
3
V
H
V
L
(G
4
S)
3
Myc
Myc
C
L
C
L
Myc
Myc

SHL
HL
SHL-CL
HL-CL
signal V
H
V
L
(G
4
S)
3
1, 4 3, 6 2, 5 7, 9 8, 10
11, 12
13, 14
A
B
Fig. 2. Constructions of anti-WASP EVH1 scFvs. (A) Cloning of vari-
able region of immunoglobulin heavy and light chains from hybri-
doma cells producing WASP EVH1 mAb. The arrows represent
the following primers used to amplify the antibody fragments: pri-
mer 1, 5¢-CACCC
AAGCTTGCCACCATGGGCAGACTTACTTCTTCATTC-3¢;
primer 2, 5¢-CAGAACCACCACCCCCTGAGGAGACGGTGACTGAGG
ATCC-3¢; primer 3, 5¢-CACCC
AAGCTTGCCACCATGCAGGTTACTCT
GAAAGAGTC-3¢; primer 4, 5¢-CACCC
AAGCTTGCCACCATGAAATG
CAGCTGGGTTATCTTC-3¢; primer 5, 5¢-CAGAACCACCACCCCCTG
AGGAGACGGTGACTGAGGTTCC-3¢; primer 6, 5¢-CACCCAAGCTT

GCCACCATGGAGGTTCAGCTGCAGCAGTCTG-3¢; primer 7, 5¢-GGT
GGAGGAGGTTCTGATGTTTTGATGACCCAAACTCCAC-3¢; primer 8,
5¢-CGAAT
GCGGCCGCCCGTTTGATTTCCAGCTTGGTGC-3¢; primer 9,
5¢-GGTGGAGGAGGTTCTGATGTTGTTCTGACCCAAACTCCACTC-3¢;
primer 10, 5¢-CGAAT
GCGGCCGCCCGTTTCAGCTCCAGCTTGGTCC-3¢;
primer 11, 5¢-TCAAAACATCAGAACCTCCTCCACCGGATCCTCCAC
CTCCAGAACCACCACCCCC-3¢; primer 12, 5¢-GAACAACATCAGAA
CCTCCTCACCGGATCCTCCACCTCCAGAACCACCACCCCC-3¢; pri-
mer 13, 5¢-CGTCTCCTCAGGGGGTGGTGGTTCTGGAGGTGGAG
GATCCGGTGGAGGAGGTTCT-3¢; primer 14, 5¢-CGTCTCCTCA
GGGGGTGGTGGTTCTGGAGGTGGAGGATCCGGTGGAGGAGG
TTCT-3¢. In all primers, underlined sequences indicate restriction site
of HindIII and NotI, and bold letters indicate full or part of the (Gly
4
-
Ser)
3
linker sequence. (B) Schematic representation of the four scFv
formats (SHL, HL, SHL-CL, and HL-CL). Shown are the leader signal
sequence, V
H
region, polypeptide linker (G
4
S)
3
,V
L
region, light chain

constant [C
L
(j)] region and Myc tag sequence.
G W G W G W G W G W G W G W G W G W
49.9
32.3
49.9
49.9
32.3
32.3
(kD)
(kD)
(kD)
WASP
A
B
C
G: GST
W: GST-WASP15
87
49.9
T7-WASP15
D
21SHL-CL-Myc
21SHL-Myc
49.9
32.3
(kD)
28.8
22

vector
18HL
18SHL
18HL
-
CL
18SHL
-
CL
21HL
21SHL
21HL
-
CL
21SHL
-
CL
vector
18HL
18SHL
18HL
-
CL
18SHL
-
CL
21HL
21SHL
21HL
-

CL
21SHL
-
CL
vector
18HL
18SHL
18HL
-
CL
18SHL
-
CL
21HL
21SHL
21HL
-
CL
21SHL
-
CL
T7
-
WASP15
T7
-
WASP15
+ 21HL
T7
-

WASP15
+ 21SHL
T7
-
WASP15
+ 21HL
-
CL
T7
-
WASP15
+ 21SHL
-
CL
Fig. 3. Expression of anti-WASP scFvs and detection of their bind-
ing activity to WASP in T cells. (A) Western blot analysis of protein
extracts of anti-WASP scFv DNA-transfected T cells. The immuno-
blot was probed with Myc tag mAb. (B) In vitro binding assay using
GST pull-down. All anti-WASP scFv DNA-transfected T cells were
lysed and incubated with GST (G) or GST-WASP15 (W) fusion pro-
tein noncovalently bound to glutathione–Sepharose beads. Bound
proteins were analyzed by Western blotting with Myc tag mAb. (C)
In vivo association between scFvs and WASP. All scFv DNA-trans-
fected cell lysates were immunoprecipitated with WASP mAb and
analyzed by Western blotting with Myc tag mAb (top panel) or
WASP mAb (bottom panel). (D) EVH1 domain-specific binding of
scFv T7-WASP15 and scFv DNA cotransfected cell lysates were
immunoprecipitated with biotinylated T7 tag mAb. Immunocom-
plexes were recovered by on streptavidin–agarose and analyzed by
Western blotting with Myc tag mAb (top panel) or T7 tag mAb (bot-

tom panel). Arrowheads indicate secondary antibody cross-reactive
nonspecific proteins.
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.
6134 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
V
H
signal peptide sequence by MS analysis of 21SHL-
CL digested with lysyl endopeptidase (data not
shown). These results suggest that the V
H
signal pep-
tide sequence was cleaved from the N-terminal V
H
region. The culture supernatant of 21SHL-CL scFv-
expressed T cells was examined but the scFv could not
be detected (data not shown). These results suggest
that, even if scFvs are expressed with a signal
sequence, they do not enter the secretory pathway.
Generation of anti-WASP scFv transgenic mice
scFv 21SHL and 21SHL-CL vector DNAs were chosen
as the transgenes for development of transgenic mice
with the functional knockdown WASP-EVH1 domain.
High expression of 21SHL and 21SHL-CL was
detected in T and B cells from the spleens of the
21SHL ⁄ 21SHL-CL scFv transgenic mice (Fig. 4A).
Eight 21SHL transgenic founders and 10 21SHL-CL
transgenic founders carrying the scFv intrabody
expression vectors were obtained. In four of eight
21SHL lines and five of 10 21SHL-CL lines, the same
levels of expression of 21SHL and 21SHL-CL were

detected (data not shown). Furthermore, T and B cells
from the spleens of both scFv transgenic mice were
solubilized with 1% digitonin buffer and immuno-
precipitated with WASP mAb and Myc tag mAb to
examine the in vivo interaction between scFvs and
endogenous WASP. Binding of intracellular scFvs and
WASP was detected in both T and B cells from scFv
transgenic spleens by immunoprecipitation (Fig. 4B–D).
Impaired antigen receptor-induced proliferation
in anti-WASP scFv transgenic T cells, but not
B cells
To assess the effects of the anti-WASP scFvs 21SHL
and 21SHL-CL on T-cell function, the proliferative
response to stimulation with CD3e antibody (2c11)
was examined. Compared with the wild-type, T cells
from 21SHL transgenic mice and 21SHL-CL trans-
genic mice were impaired in their proliferative response
to CD3e antibody stimulation to the same extent as in
WASP15 transgenic T cells [24] (Fig. 5A). These find-
ings indicate that the function of the WASP N-ter-
minal EVH1 domain is blocked by scFv 21SHL and
21SHL-CL intrabodies in the T cells. In contrast with
T cells, proliferative responses to antigen receptor sti-
mulation with anti-IgM Ab F(ab¢)
2
or CD40 antibody
were normal in the scFv transgenic B cells (Fig. 5B).
Therefore, the EVH1 domain of WASP is not func-
tional, at least in the Ag receptor-induced proliferative
response of B cells.

T cells from the other three 21SHL transgenic lines
and the other four 21SHL-CL transgenic lines were
also impaired in their proliferative response to stimula-
tion with CD3e antibody (data not shown), confirming
that there were no problems in the integration site of
the transgene.
Lymphoid development in anti-WASP scFv
transgenic mice
T-cell development in the spleen can be followed by
examining the expression patterns of the CD4 and
CD8 surface antigens. The population of mature
single-positive thymocytes (either CD4
+
CD8
–
or
CD4
–
CD8
+
) was almost the same in wild-type, 21SHL
transgenic, and 21SHL-CL transgenic mice (Fig. 5C).
Likewise the expression pattern of CD3 was nearly the
same. Furthermore, the percentages of splenic T and B
lineage cell populations were normal (Fig. 5C). In
addition, T lineage cell populations in the thymus and
B lineage cell populations in the bone marrow were
almost the same for wild-type, 21SHL transgenic and
21SHL-CL transgenic mice (Fig. 5D,E). These results
T cell B cell

21SHL-CL-Myc
21SHL-Myc
21SHL-CL-Myc
21SHL-Myc
WASP
WASP
49.9
32.3
49.9
32.3
(kD)
21SHL
21SHL
-
CL
21SHL
21SHL
-
CL
WB: anti-Myc tag
IP: anti-WASP
WB: anti-Myc tag
IP: anti-Myc tag
WB: anti-WASP
WB: anti-WASP
A
B
C
D
Fig. 4. Expression of anti-WASP scFvs and in vivo interaction

between scFvs and WASP in scFv transgenic mice T and B cells.
(A) Western blot analysis of protein extracts of T and B cells from
the spleens of the 21SHL and 21SHL-CL scFv transgenic mice. The
immunoblot was probed with Myc tag mAb. (B, C) In vivo associ-
ation between scFvs and WASP. The scFv 21SHL and 21SHL-CL
transgenic T and B cell lysates were immunoprecipitated with
WASP mAb and Myc tag mAb and analyzed by Western blotting
with Myc tag mAb and WASP mAb. Arrowheads indicated secon-
dary antibody cross-reactive nonspecific proteins. (D) Both scFv
transgenic mice T and B cell lysates were analyzed by Western
blotting with WASP antibody.
M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6135
indicate that anti-WASP scFvs do not have a marked
effect on lymphocyte development.
Impaired interleukin (IL)-2 production induced
by TCR stimulation, but not antigen receptor
capping
To assess whether the 21SHL and 21SHL-CL scFvs
affect IL-2 production induced by TCR stimulation,
purified T cells from spleens of wild-type, WASP15
transgenic, 21SHL transgenic and 21SHL-CL trans-
genic mice were stimulated with immobilized CD3e
antibody and IL-2 in the culture supernatant and deter-
mined by ELISA. T cells expressing 21SHL and
21SHL-CL scFvs were impaired in IL-2 production
induced by TCR stimulation, whereas the defect in IL-2
production of scFv transgenic T cells was slight com-
pared with the WASP15 transgenic T cells (Fig. 6A).
In addtion, purified T cells were incubated in vitro

with fluorescein isothiocyanate (FITC)-conjugated
CD3e antibody at either 37 °Cor4°C (stimulation or
nonstimulation) to assess whether the 21SHL and
21SHL-CL scFvs affect TCR-induced capping. The
rate of antigen-receptor capping of T cells was the
same in all the mice (Fig. 6B). These results indicate
that the anti-WASP scFvs 21SHL and 21SHL-CL inhi-
bit the signaling cascade of IL-2 production via TCR
stimulation without affecting the regulation of the
cytoskeleton, including antigen-receptor capping. These
findings strongly indicate that IL-2 synthesis is medi-
ated directly by the WASP EVH1 domain and not by
secondary events resulting from WASP-mediated actin
cytoskeletal rearrangements induced by TCR signaling.
Subcellular localization of anti-WASP scFvs
To examine the subcellular localization of anti-WASP
scFvs 21SHL and 21SHL-CL in T cells, cell extracts
of their scFv-transgenic T cells were fractionated into
the subcellular compartments, cytosolic proteins and
CD4
CD8
CD3
B220
CD4
CD8
I
g
M
B220
10.7

22.1
36.3
31.5
3.6
5.5
7.1
0.4
11.7
24.5
37.9
33.4
4.1
7.0
9.7
0.1
13.4
25.8
35.4
34.5
3.7
8.0
9.1
0.1
7.3 9.5 7.6
85.9 83.6 82.3
3.6 4.7 4.7
1.5 1.5 1.5
wild 21SHL Tg 21SHL-CL Tg
A
B

C
D
E
Fig. 5. Antigen receptor-induced proliferation in anti-WASP scFv
transgenic T and B cells, and lymphoid development in anti-WASP
scFv transgenic mice. (A) T-cell proliferation. Splenic T cells from
anti-WASP scFv 21SHL transgenic, 21SHL-CL transgenic, WASP15
transgenic and wild-type mice were cultured in medium alone or in
the presence of CD3e antibody. (B) B-cell proliferation. Splenic
B cells from anti-WASP scFv 21SHL transgenic, 21SHL-CL trans-
genic, WASP15 transgenic and wild-type mice were cultured in
medium alone or in the presence of IgM antibody F(ab¢)
2
or CD40
antibody. Each stimulation was performed in the presence of exo-
genous IL-4. In each experiment, cells were cultured for 48 h, then
10 l
M BrdU was added to the T and B-cell cultures. The cells were
reincubated for an additional 16 h, and BrdU incorporation was
quantified by ELISA. Values represent means ± SE of triplicate
cultures and are representative of three independent experi-
ments. Statistical significance is indicated by *(P<0.05) and
**(P<0.005). (C)–(E) FACS analyses of lymphocytes from wild-
type, anti-WASP scFv 21SHL transgenic and 21SHL-CL transgenic
mice. Two-color flow cytometric analyses were performed on
spleen (C), thymus (D) and bone marrow (E). Percentages of repre-
sentative lymphoid populations are noted. The results shown are
representative of at least three male mice for each analysis at the
age of 8 weeks.
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.

6136 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
membrane ⁄ membrane organelles. In general, the scFv
intrabodies (V
H
–linker–V
L
format) with heavy chain
signal peptide sequences cross the rough ER mem-
brane and enter the secretory pathway through the
trans-Golgi network. However, equivalent amounts of
scFv 21SHL were detected in both the cytosol and
membrane fractions, and most of the scFv 21SHL-CL
was detected in the cytosol fraction in anti-WASP scFv
transgenic T cells (Fig. 7A). To confirm the presence
of cross-contamination in both fractions of scFv
21SHL-CL transgenic T cells, each fraction was exam-
ined by Western blotting with WASP antibody and
Ribophorin I antibody specifically expressed in the
cytosolic and membrane fractions, respectively. These
results show that neither fraction was cross-contamin-
ated (Fig. 7A).
When scFv intrabodies were expressed in NIH-3T3
fibroblastic cells, the scFvs were localized in the subcel-
lular compartments. NIH-3T3 cells were transfected
with scFv 21SHL-CL (with leader signal sequence) or
21HL-CL (without leader signal sequence) genes and
then their subcellular fractions were subjected to West-
ern blotting with Myc tag antibody. The majority of
the intrabodies expressed without signal sequence were
detected in the cytosol, whereas most of the intrabodies

expressed with the signal sequence were detected in the
membrane fraction (Fig. 7B). These results indicate
that the post-translational processing of ER-coupled
protein synthesis must be different among cell types
such as lymphocytes and fibroblasts.
On immunostaining, colocalization of 21SHL-CL
scFv and endogenous WASP was observed in the cyto-
sol of the scFv DNA transfected T cells (Fig. 7C).
Again these results indicate that scFv intrabodies
expressed with the V
H
signal peptide sequence are
localized in the cytosol of T cells. Taken together, the
results strongly suggest that scFv intrabodies synthes-
ized in the ER are released from the ER membrane
into the cytosol by retro-translocation in lymphocytes
including T cells [25].
In general, when proteins synthesized in the ER are
misfolded or incompletely assembled into oligomeric
forms, they are transferred from the lumen of the ER
into the cytosol, so-called retro-translocation. In the
cytosol, the retro-translocated proteins are polyubiqui-
tinated and degraded by proteasomal proteolysis
[26–29]. Our results suggest that the WASP scFv intra-
bodies expressed with the V
H
signal sequence are
translocated across the ER membrane into the cytosol
without degradation. The cell lysates or immunopre-
wild WASP-15 21SHL 21SHL-CL

unstimulate
stimulate
20µm
20µm20µm
20µm
20µm
20µm
20µm
20µm
B
A
Fig. 6. IL-2 production was impaired, but
not antigen receptor capping induced by
TCR stimulation. (A) Splenic T cells from
anti-WASP scFv 21SHL transgenic, 21SHL-
CL transgenic, WASP15 transgenic and
wild-type mice were cultured in medium
alone or in the presence of anti-CD3e Ab.
Each cell culture supernatant was collected
at 24 h. IL-2 in the supernatant was quanti-
fied by ELISA. Values are mean ± SE from
triplicate cultures and are representative of
three independent experiments. Statistical
significance is indicated by *(P<0.005) and
**(P<0.001). (B) Splenic T cells from anti-
WASP scFv 21SHL transgenic, 21SHL-CL
transgenic, WASP15 transgenic and wild-
type mice were incubated with FITC-conju-
gated CD3e antibody at either 4 °Cor37°C
for 30 min. The treated cells were placed on

polyethylenimine coated eight-well tissue
culture glass slides, fixed, analyzed and
photographed at · 100 using confocal micro-
scopy. The rate of capping of unstimulated
and stimulated T cells was determined by
counting the number of caps in  200 cells ⁄
experiment. The wild-type and transgenic
mice used for these experiments were
8 weeks old.
M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6137
cipitates with Myc tag antibody were immunoblotted
with ubiquitin antibody to determine if polyubiquitina-
tion of the anti-WASP scFv 21SHL and 21SHL-CL
occurs in the T cells. 21SHL and 21SHL-CL were not
polyubiquitinated in the scFv-transgenic T cells. How-
ever, the polyubiquitination of nonspecific proteins
was observed in the scFv-transgenic T cell lysate
(Fig. 7D). These results indicate that the scFv genes
with signal sequence are translated in the ER, and,
after cleavage of the signal peptide sequence, are trans-
located from the ER into the cytosol without poly-
ubiquitination and degradation.
Discussion
In this study, we show that the scFv intrabodies con-
structed with a leader signal sequence at the N-termi-
nus inhibited the domain function of a cytosolic
protein, and preserved the strong binding activity for
target molecules under the reducing conditions of the
cytosol in scFv-transgenic lymphocytes.

This study also demonstrates that the successful
expression of intrabodies in the cytosol is related to
translational efficiency, post-translational processing,
and modification of scFvs. We constructed several
scFvs with or without the N-terminal leader signal
sequence of the V
H
region and with or without the
C
L
(j) region following the V
L
region (Fig. 2B). Fusion
of scFvs with the C
L
(j) region has already been shown
to increase intracellular stability and target protein
inactivation in some cases, but not all [30,31]. scFvs
containing the V
H
signal sequence and C
L
(j) region
(SHL or SHL-CL formats) were highly expressed in
T cells compared with scFvs not containing the V
H
signal sequence (HL or HL-CL formats) (Fig. 3A).
Binding activity of 18SHL ⁄ SHL-CL and 21SHL ⁄
SHL-CL scFvs was detected, but not in the HL ⁄
HL-CL formats, by in vitro binding assay using GST

pull-down (Fig. 3B). These results strongly suggest that
scFvs with the native V
H
signal sequence and C
L
(j)
region increase the binding capabilities of scFv intra-
bodies in T cells.
In this study, we established two hybridoma cell
lines (clones 18 and 21) producing WASP EVH1 mAbs
which were able to equivalently immunoprecipitate
with native WASP in T cells. Then we isolated cDNA
fragments for assembling anti-WASP scFvs from them.
Although the expression levels of 18SHL ⁄ SHL-CL and
21SHL ⁄ SHL-CL were almost the same in the scFv
gene-transfected T cells, 21SHL ⁄ SHL-CL bound more
strongly to GST-WASP15 than 18SHL ⁄ SHL-CL
(Fig. 3A,B). Furthermore, a strong interaction between
native WASP and 21SHL ⁄ SHL-CL scFvs was detected
1 2 3 4 1 2 3 4
anti-Myc tag anti-Ub
21SHL
21SHL-CL
WASP
Ribophorin I
C: Cytosolic
M: Membrane/
Organelle
21HL-CL 21SHL-CL
anti-WASP scFv-Tg T cell

anti-WASP scFv transfected NIH-3T3
anti-Myc tag anti-WASP merged image
(kD)
199
133
87
40.1
31.6
40.1
32.3
49.9
40.1
87
50.7
87
50.7
(kD)
CM
CMC M
49.9
40.1
(kD)
A
B
C
D
Fig. 7. Subcellular localization of anti-WASP scFvs. Cell extracts of
(A) anti-WASP scFvs transgenic T cells and (B) anti-WASP scFv
DNA-transfected NIH-3T3 cells were fractionated into the subcellu-
lar compartments, cytosolic proteins and membrane ⁄ membrane

organelles. The fractionated cell extracts were analyzed by Western
blotting with Myc tag, WASP or Ribophorin I antibodies. (C)
Co-localization of anti-WASP scFv and endogenous WASP in the
cytosol of T cells. Anti-WASP scFv 21SHL-CL DNA electroporated
T cells were fixed and incubated with Myc tag antibody or WASP
mAb. After being washed, the cells were stained with FITC-conju-
gated anti-rabbit IgG or Alexa Fluor 546-conjugated anti-mouse IgG.
The treated cells were analyzed and photographed at · 100 using
immunofluorescence microscopy. (D) Anti-WASP scFvs were not
polyubiquitinated in the scFv transgenic mice T cells. Immunopre-
cipitates with Myc tag antibody (lanes 1 and 2) and cell lysates
(lanes 3 and 4) from anti-WASP scFv 21SHL transgenic (lanes 1
and 3) or 21SHL-CL transgenic (lanes 2 and 4) mice T cells were
analyzed by Western blotting with Myc tag or ubiquitin antibody.
The smear bands (arrow) indicate polyubiquitination of nonspecific
proteins in the T cells. The arrowhead indicates secondary antibody
cross-reactive nonspecific proteins.
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.
6138 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
by immunoprecipitaion analysis, whereas 18SHL ⁄
SHL-CL scFvs and other scFvs were not able to asso-
ciate with the native WASP in vivo (Fig. 3C). Also, the
EVH1 domain-specific binding of 21SHL ⁄ SHL-CL
was demonstrated (Fig. 3D). These results indicate that
the differences in in vivo binding activity between
18SHL ⁄ SHL-CL and 21SHL ⁄ SHL-CL may be due to
folding. This structural property is necessary for anti-
gen binding, when antibody fragments are converted
into the scFv format and expressed in the reducing
environment of the cytosol.

The primary mechanism for protein degradation of
misfolded proteins is the ubiquitin–proteasome system
which governs the quality control of proteins. Mis-
folded proteins synthesized by cotranslational or post-
translational events in the ER are retro-translocated
from the ER into the cytosol and rapidly degraded
after polyubiquitination [26–29]. Immunoglobulin
heavy and light chains are cotranslated and assembled
with disulfide bonds in the ER lumen. An ER resi-
dent chaperone, Bip, binds to the constant region of
immunoglobulin and stabilizes and maintains the
integrity of the immunoglobulin form [32,33]. Bip
contains the ER retention signal sequence KDEL
(Lys-Asp-Glu-Leu) at the C-terminus and elaborates
the tertiary structure of immunoglobulin during trans-
location from the ER to the secretory pathway. In
general, it was thought that the scFv intrabodies con-
structed with leader signal sequences cross the rough
ER membrane and enter the secretory pathway
through the trans-Golgi network. However, in this
study the majority of scFvs were detected in the cyto-
solic fraction (Fig. 7A), and not in the T-cell culture
supernatant (data not shown). These results indicate
that the scFvs constructed with the signal sequence
can be transferred from the ER to the cytosol by
retro-translocation without polyubiquitination and
proteasome degradation. We propose two possible
interpretations of these results. One is that ubiquitina-
tion is not coupled with retro-translocation as has
been shown for cholera toxin release from the ER

into the cytosol accompanied by rapid folding [34].
The other is that the scFv modifications of the N-ter-
minal residues occur after cleavage of the signal
sequence in the ER. In MyoD, which is a tissue-spe-
cific transcriptional activator that acts as a master
switch for muscle development, modification of the
N-terminal residue protects it from ubiquitination and
protein degradation irrespective of the presence of
internal lysine residues [35]. In T cells, scFvs con-
structed with the V
H
signal sequence seem to be
modified at the N-terminal residue after cleavage of
the signal peptide sequence in the ER. However, we
have not yet confirmed the N-terminal amino-acid
sequence of scFvs by the Edman method.
Interestingly, when NIH-3T3 cells were transfected
with the 21SHL-CL scFv (containing the V
H
signal
sequence) or the 21HL-CL scFv (not containing the
V
H
signal sequence), most of the 21SHL-CL scFv was
detected in the membrane fraction, whereas the 21HL-
CL scFv accumulated in the cytosol (Fig. 7B). These
results indicate that the mechanisms of retro-transloca-
tion differ among different cell types. Although we do
not know the mechanisms leading to retro-transloca-
tion without proteasome degradation, the scFv intra-

bodies constructed with signal sequences may be
designed for practical use in functional knockdown of
cytosolic proteins in T cells.
T cells from WASP-deficient mice showed a marked
reduction in antigen receptor capping accompanied by
actin polymerization and IL-2 production induced by
TCR stimulation. It has been hypothesized that defects
in IL-2 production in WASP-deficient T cells may be a
secondary phenomenon resulting from defects in actin
remodeling and immune synapse formation induced by
TCR stimulation [17,18,36,37]. However, we previously
demonstrated that T cells from WASP15 transgenic
mice that overexpress WASP-EVH1 domain were
impaired with respect to proliferation and IL-2 pro-
duction induced by TCR stimulation, but antigen
receptor capping and actin polymerization were nor-
mal [24]. This suggest the direct involvement of the
EVH1 domain in the IL-2 synthesis pathway. In the
present study, purified anti-WASP scFv 21SHL and
21SHL-CL transgenic T cells were impaired with
respect to proliferation and IL-2 production induced
by CD3e antibody stimulation (Figs 5A and 6A). In
terms of cytoskeletal rearrangement, normal antigen
receptor capping induced by CD3e antibody stimula-
tion was observed similar to the wild-type and
WASP15 transgenic mice T cells (Fig. 6B). These
results indicate that the role of WASP in regulating
IL-2 production is independent of its role in immune
synapse formation. The following experimental data
support this hypothesis. WASP-deficient T cells form

conjugates with antigen-specific B cells normally and
can form immune synapses accompanied by polariza-
tion of cytoskeleton-regulating proteins, but defects in
IL-2 production are observed [38]. Furthermore, analy-
sis of a series of WASP-deletion mutants shows that
the WASP homology-1 (WH1) ⁄ EVH1 domain is res-
ponsible for NF-AT transcriptional activation [39].
These findings indicate that the functions of WASP
may be more complex than previously believed.
The inability of WASP-deficient, WASP15 trans-
genic, and anti-WASP scFv 21SHL ⁄ SHL-CL transgenic
M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6139
T cells to proliferate in response to TCR stimulation is
similar to the defects observed in T cells from Vav-
deficient mice [40,41]. It has been previously shown
that Vav is a potent regulator of the IL-2 promoter, in
particular NF-AT ⁄ AP-1-mediated gene transcription
[42]. Furthermore, the WASP-interacting protein
(WIP) and WASP interaction is important for Vav-
mediated activation of NF-AT ⁄ AP-1 gene transcrip-
tion induced by TCR stimulation [43]. WIP-deficient
T cells were impaired in proliferation and immune syn-
apse formation induced by TCR stimulation [44]. It is
possible that the overexpressed WASP15 and anti-
WASP 21SHL ⁄ SHL-CL scFvs inhibit WIP and endog-
enous WASP interactions, because the WIP-binding
site in endogenous WASP is included in WASP15 and
may overlap the target region of our anti-WASP
scFvs. The molecules that interact with the EVH1

domain which overlaps our scFv intrabody-binding
epitope need to be identified.
We examined the effects of anti-WASP 21 SHL ⁄
SHL-CL scFvs on lymphocyte development and B-cell
function. The anti-WASP scFvs did not have a marked
effect on lymphocyte development (Fig. 5C–E). Fur-
thermore, B cells from anti-WASP 21 SHL ⁄ SHL-CL
scFv transgenic mice proliferated normally in response
to stimulation by IgM and CD40 antibodies (Fig. 5B).
B cells from WASP-deficient and WASP15 transgenic
mice also proliferated normally after IgM antibody
stimulation [17,18,24]. We have not yet clarified the
significance of WASP expression in B lymphocytes.
Finally, we demonstrate here that scFv intrabodies
bind to the EVH1 domain of WASP and inhibit IL-2
synthesis in T cells. Therefore, scFv intrabodies should
be valuable tools for identifying novel protein func-
tions, and transgenic mice that express scFv intrabod-
ies may be useful in functional knockdown models.
Experimental procedures
Construction of GST fusion protein and mAb
preparation
A cDNA fragment for mouse WASP exon 1–5 (amino acids
1–171) which includes the EVH1 domain (designated
WASP15) was generated by PCR (sense primer, 5¢-CGA
ATGCGGCCGCAATGAATAGTGGCCCTG-3¢; reverse
primer, 5¢-CGAATGCGGCCGCTCACTCCTCATTGATT
GG-3¢) [24], digested with NotI, and subcloned into the
pGEX-4T-2 expression vector (Amersham Biosciences, Pis-
cataway, NJ, USA). The GST-WASP15 fusion protein was

produced in BL21 Escherichia coli cells and purified on a
glutathione–Sepharose 4B affinity chromatography column
(Amersham Biosciences) according to the manufacturer’s
instructions. mAbs were prepared from mice immunized with
GST-WASP15 fusion protein by the conventional procedure.
Cloning and construction of WASP-EVH1 scFv
intrabodies
We identified subtype mAbs (18, 21) using a mouse mAb
isotyping kit IsoStrip (Roche Diagnostics, Mannheim, Ger-
many). We performed a four-step PCR to generate appro-
priate cDNA fragments that encoded the V
H
and V
L
region. Total RNA from hybridoma cells was reverse-tran-
scribed using the SMART
TM
RACE cDNA Amplification
Kit (Clontech, Palo Alto, CA, USA). The cDNA fragments
for the V
H
and V
L
regions containing the leader signal
sequence and CH1 or C
L
constant region sequences were
generated by PCR using subtype-specific primers (heavy
chain, clone 18, IgG3: sense primer 5¢-CTAATACGACTC
ACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3¢

and reverse primer 5¢-GTACTGGGCTTGGGTATTCT
AGGCTC-3¢; clone 21, IgG2b: sense primer 5¢-AAGCAG
TGGTATCAACGCAGAGTACGCG-3¢ and reverse pri-
mer 5¢-GGACAGGGGTTGATTGTTGAAATGGG-3¢;
light chain, clone 18 and 21, j: sense primer 5¢-CTAATAC
GACTCACTATA GGGCAA GC AGTGGT ATCAA CGCA
GAGT-3¢ and reverse primer 5¢-CCTGTTGAAGCTCTTG
ACAATGGGTG-3¢). The second PCR products for V
H
region were classified into two forms containing the native
V
H
leader signal sequence at the 5¢ end of the V
H
fragment
(SV
H
form) or no V
H
leader signal sequence (V
H
form).
The second PCR amplification was performed with the fol-
lowing primers: 18SV
H
, sense primer 1 and reverse primer
2; 18V
H
, sense primer 3 and reverse primer 2; 21SV
H

, sense
primer 4 and reverse primer 5; 21V
H
, sense primer 6 and
reverse primer 5; 18V
L
, sense primer 7 and reverse primer
8; 21V
L
, sense primer 9 and reverse primer 10. Primer
sequences are shown in the legend to Fig. 2. The third PCR
products were amplified using the following primers:
18SV
H
–linker, sense primer 1 and reverse primer 11; 18V
H
–
linker, sense primer 3 and reverse primer 11; 21SV
H
–linker,
sense primer 4 and reverse primer 12; 21V
H
–linker, sense
primer 6 and reverse primer 12; linker)18V
L
, sense primer
13 and reverse primer 8; linker)21V
L
, sense primer 14 and
reverse primer 10. The third PCR products were mixed in

the following combinations: 18SV
H
–linker and linker)
18V
L
, 18V
H
–linker and linker)18V
L
, 21SV
H
–linker and
linker)21V
L
, 21V
H
–linker and linker)21V
L
and single-
chain antibodies, scFvs, designated 18SHL, 18HL, 21SHL,
and 21HL assembled by the fourth PCR amplification
using the following primers: 18SHL, sense primer 1 and
reverse primer 8; 18HL, sense primer 3 and reverse primer
8; 21SHL, sense primer 4 and reverse primer 10; 21HL,
sense primer 6 and reverse primer 10. The fourth PCR
products were digested with HindIII–NotI and cloned into
the pCAGGS-MCS expression vector [45,46]. The Myc tag
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.
6140 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
(EQKLISEEDL) was generated as follows: coding linker

containing a NotI site at the 5¢ end of the linker (5¢-GGC
CGCAGGTTCGGAGCAGAAGCTGATCAGCGAGGAG
GACCTGTAG-3¢) and noncoding linker containing an
EcoRI site at the 5¢ end of the linker (5¢-AATTCTACAGG
TCCTCCTCGCTGATCAGCTTCTGCTCCGAACCTGC-3¢)
were annealed and inserted into the NotI ⁄ EcoRI site of all
pCAG ⁄ anti-WASP EVH1 scFvs. All anti-WASP scFvs
were fused with the Myc tag at the C-terminus. Moreover,
to generate a cDNA fragment for the C
L
(j) region, total
RNA from hybridoma producing clone 18 mAb was
reverse-transcribed and a two-step PCR amplification
performed using the following primers: first PCR, sense pri-
mer 5 ¢-GAGGCACCAAGCTGGAAATCAAACGG-3¢ and
reverse primer 5¢-TGGTGGTGGCGTCTCAGGACCT
TTG-3¢; second (nested) PCR, sense primer 5¢-CGAATGC
GGCCGCAGCTGATGCTGCACCAACTGTATCC-3¢ and
reverse primer 5¢ -CGAATGCGGCCGCACACTCATTCC
TGTTGAAGCTCTTGAC-3¢. The PCR product for the
C
L
(j) region was digested with NotI and cloned into the
NotI site between the scFv and Myc tag sequences. These
constructs were designated pCAG ⁄ 18SHL-CL, 18HL-CL,
21SHL-CL and 21HL-CL, respectively. All scFv constructs
were confirmed by DNA sequencing analysis. A DNA con-
struct, pCAG ⁄ T7-WASP15 [24], which contained T7-tagged
WASP exon 1–5, was used for evaluation of EVH1
domain-specific binding.

Cells and electroporation ⁄ transfection
The T-cell hybridoma DO-11.10 [47] was maintained in
RPMI 1640 medium supplemented with 10% fetal calf
serum, 100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomycin,
4mml-glutamine, 50 lm 2-mercaptoethanol (2-ME) and
10 mm Hepes (all obtained from Gibco, Carlsbad, CA,
USA). DO-11.10 cells, adjusted to a concentration of 5 · 10
6
cells ⁄ 400 lL culture medium with 1.25% dimethyl sulfoxide
per cuvette, were electroporated using a Gene Pulser (Bio-
Rad, Hercules, CA, USA) with 40 lg plasmid DNA at 290V
and 960lF. NIH-3T3 cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal calf
serum, 50 UÆmL
)1
penicillin, 50 lgÆmL
)1
streptomycin and
4mml-glutamine. NIH-3T3 cells adjusted to a concentration
of 1 · 10
6
cells ⁄ 10 cm dish were transfected with 10 lg plas-
mid DNA using FuGENE 6 Transfection Reagent (Roche
Diagnostics).
Immunoprecipitation and Western blot analysis
The electroporated T cells and anti-WASP scFv transgenic T

and B cells were lysed with digitonin buffer (10 m m trietha-
nolamine, 10 mm iodoacetoamide, 1% digitonin, 0.15 m
NaCl, 1 mm EDTA, and Complete
TM
protease inhibitor
cocktail; Roche Diagnostics), incubated with 5 lgÆmL
)1
WASP mAb (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), Myc tag mAb (MBL, Nagoya, Japan) or biotinylated
T7 tag mAb (Novagen, Madison, WI, USA) and immuno-
precipitated by the addition of 40 lL protein G–Sepharose
or streptavidin–agarose (Upstate, Charlottesville, VA, USA).
The cell lysates and immunoprecipitates were separated by
SDS ⁄ PAGE (12.5% gel) and transferred to a polyvinylidene
difluoride membrane (Bio-Rad). Blots were probed with Myc
tag mAb, WASP mAb, T7 tag mAb, Ribophorin I mAb and
ubiquitin mAb (Santa Cruz Biotechnology), followed by
horseradish peroxidase-conjugated anti-mouse or anti-rabbit
IgG (Dako, Glostrup, Denmark). Immunoreactive proteins
were detected by ECL (Amersham Biosciences).
Assay of GST fusion protein binding
After 48 h of electroporation, cell lysates (1 · 10
7
cells)
were prepared by lysis with 10 mm Tris/HCl, pH 7.8, 1%
NP-40, 0.15 m NaCl, 1 m m EDTA and Complete
TM
pro-
tease inhibitor cocktail (TNE) buffer, cleared by centrifuga-
tion, and treated with excess glutathione–Sepharose beads

(Amersham Biosciences). The precleared cell lysates were
incubated at 4 °C overnight with glutathione–Sepharose
beads bound to 50 lg GST fusion proteins. Beads were
washed with TNE buffer, lysed with SDS sample buffer,
and immunoblotted with Myc tag mAb.
Generation of transgenic mice
The transgene was excised from the plasmid vector with
SalI ⁄ NheI restriction enzyme, purified by agarose gel elec-
trophoresis and a QIAquick Gel Extraction kit (Qiagen,
Hilden, Germany), adjusted to a final concentration of
3 lgÆmL
)1
, and microinjected into the fertilized egg pronu-
clei of C57BL ⁄ 6J inbred strain mice. The injected eggs were
then transferred into the oviducts of pseudopregnant female
ICR mice.
Antigen receptor stimulation
T and B cells were purified from WASP15 transgenic,
anti-WASP scFv transgenic mice spleens or age-matched
wild-type control mice by magnetic cell sorting using auto-
MACSTM (Miltenyi Biotec, Bergisch Gladbach, Germany)
according to the manufacturer’s instructions. T and B cells
were isolated by negative or positive selection using micro-
beads conjugated to mouse CD45R (B220) antibodies (Milt-
enyi Biotech). The cell purity of both the resulting
populations exceeded 90%, as confirmed by FACS analysis.
For the T-cell proliferation assay, CD3e antibodies (145–
2C11; BD PharMingen, San Diego, CA, USA) were adhered
to 96-well tissue culture plates by incubating 20 lgÆmL
)1

in
NaCl ⁄ P
i
, pH 8.0, at 4 °C for 6 h, after which the plates were
washed with NaCl ⁄ P
i
, pH 7.2. Purified T cells were added to
the antibody-coated wells (5 · 10
4
cells ⁄ well) and cultured at
M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6141
37 °C in RPMI 1640 medium containing 10% fetal calf
serum. For the B-cell proliferation assay, B cells were cul-
tured in 96-well tissue culture plates (5 · 10
4
cells ⁄ well) in
culture medium alone or in the presence of mouse IgM anti-
body F(ab¢)
2
(10 lgÆmL
)1
; Jackson ImmunoReseach Labor-
atories, West Grove, PA, USA) and CD40 antibody
(10 lgÆmL
)1
; BD Pharmingen). Each stimulation was
performed in the presence of exogenous IL-4 (2 ngÆmL
)1
;

PeproTech, London, UK). After 48 h of incubation, 10 lm
5-bromo-2¢-deoxyuridine (BrdU) was added to the T and B
cell cultures. The cells were reincubated for an additional
16 h, and then BrdU incorporation during DNA synthesis in
proliferating cells was quantified by Cell Proliferation ELISA
(Roche Diagnostics) as described by the manufacturer. For
evaluation of cytokine production, purified T cells from the
spleens of wild-type, WASP15 transgenic or anti-WASP scFv
transgenic mice were cultured on CD3e antibody-coated
48-well tissue culture plates. The cell culture supernatant was
collected at 24 h. IL-2 in the supernatant was quantified by
ELISA using OptEIA set for mouse cytokine (BD Pharmin-
gen) according to the manufacturer’s instructions.
FACS analysis
Single-cell suspensions of lymphoid cells were prepared and
stained with antibodies following standard procedures.
Antibodies to CD3, CD4, CD8, IgM or B220 (polyethylene
or FITC conjugated; Immunotech, Marselle, France) were
used to stain the cells.
T-cell capping
Purified T cells from the spleens of wild-type, WASP15
transgenic or anti-WASP scFv transgenic mice were incuba-
ted in RPMI 1640 culture medium containing 5 lgÆmL
)1
FITC-conjugated CD3e antibody (145–2C11; BD Pharmin-
gen) at either 37 °Cor4°C for 30 min. The treated cells
(5 · 10
4
cells) were placed on polyethylenimine-coated
eight-well tissue culture glass slides (BD Falcon, Bedford,

MA, USA) that were preincubated with 0.01% polyethylen-
imine (Sigma-Aldrich, St Louis, MO, USA) at room tem-
perature for 1 h and dried at 4 °C overnight. They were
then fixed in 3.5% paraformaldehyde (Sigma-Aldrich).
After being washed with NaCl ⁄ P
i
, cells were sealed with
coverslips and immediately analyzed and photographed
at · 100 by confocal microscopy (FV300; Olympus, Tokyo,
Japan). The rate of capping of unstimulated and stimulated
T cells was determined by counting the number of caps in
 200 cells ⁄ experiment.
Subcellular localization of scFv intrabodies
Cell extracts of anti-WASP scFv transgenic T cells and
scFv DNA-transfected NIH-3T3 cells were fractionated
into the subcellular compartments, cytosolic proteins and
membrane ⁄ membrane organelles, by differential solubilities
using a ProteoExtract
TM
Subcellular Proteome Extraction
Kit (Calbiochem, San Diego, CA, USA) according to the
manufacturer’s instructions. Subcellular localization of scFv
intrabodies was analyzed by Western blotting using the
fractionated extracts.
Immunostaining
After 48 h of scFv DNA electroporation, cells were
placed on polyethylenimine-coated eight-well tissue culture
glass slides (5 · 10
4
cells ⁄ well), fixed with 95% ethanol ⁄

acetic acid (99 : 1, v ⁄ v) at 4 °C for 15 min, and blocked
with NaCl ⁄ P
i
containing 1% BSA and 5% normal goat
serum (Sigma-Aldrich) for 15 min. Then cells were incu-
bated with Myc tag mAb or WASP mAb and stained
with FITC-conjugated anti-rabbit IgG (Biosource, Cama-
rillo, CA, USA) and Alexa Fluor 546-conjugated anti-
mouse IgG (Molecular Probes, Eugene, OR, USA) after
being washed with NaCl ⁄ P
i
⁄ 0.05% Tween 20. The cells
were photographed at · 100 by immunofluorescence
microscopy (DM RBE; Leica Microsystems, Wetzlar,
Germany).
Statistical analysis
Statistical significance was assessed using Student’s t test.
The differences were considered significant when P values
were less than 0.05.
Acknowledgements
We thank Dr H. Ohba for helpful discussions. This
work was supported by a Coordination Fund from the
Ministry of Education, Culture, Sports, Science and
Technology, Japan (to K.S. and Y.K.). Dr R. Iwaya
died in September 2004. This paper is dedicated to his
memory.
References
1 Bird RE, Hardman KD, Jacobson JW, Johnson S,
Kaufman BM, Lee SM, Lee T, Pope SH, Riordan GS
& Whitlow M (1988) Single-chain antigen-binding

proteins. Science 242, 423–426.
2 Huston S, Levinson D, Mudgett-Hunter M, Tai MS,
Novotny J, Margolies MN, Ridge RJ, Bruccoleri RE,
Haber E, Crea R & Oppermann H (1988) Protein engi-
neering of antibody binding sites: recovery of specific
activity in an anti-digoxin single-chain Fv analogue pro-
duced in Escherichia coli. Proc Natl Acad Sci USA 85,
5879–5883.
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.
6142 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
3 Barbas CF III, Kang AS, Lerner RA & Benkovic SJ
(1991) Assembly of combinatorial antibody libraries on
phage surfaces: the gene III site. Proc Natl Acad Sci
USA 88, 7978–7982.
4 Zhu H, Biligin M & Snyder M (2003) Proteomics. Annu
Rev Biochem 72, 783–812.
5 Tuschl T (2002) Expanding small RNA interference.
Nat Biotechnol 20, 446–448.
6 Marasco WA, Haseltine WA & Chen SY (1993) Design,
intracellular expression, and activity of a human anti-
human immunodeficiency virus type 1 gp120 single-
chain antibody. Proc Natl Acad Sci USA 90, 7889–7893.
7 Steinberger P, Andris-Widhopf J, Buhler B, Torbett BE
& Barbas CF III (2000) Functional deletion of the
CCR5 receptor by intracellular immunization produces
cells that are refractory to CCR5-dependent HIV-1
infection and cell fusion. Proc Natl Acad Sci USA 97,
805–810.
8 Richardson JH, Sodroski JG, Waldmann TA & Mar-
asco WA (1995) Phenotypic knockout of the high-

affinity human interleukin 2 receptor by intracellular
single-chain antibodies against the alpha subunit of the
receptor. Proc Natl Acad Sci USA 92, 3137–3141.
9 Mhashilkar AM, Doebis C, Seifert M, Busch A, Zani
C, Soo Hoo J, Nagy M, Ritter T, Volk HD & Marasco
WA (2002) Intrabody-mediated phenotypic knockout of
major histocompatibility complex class I expression in
human and monkey cell lines and in primary human
keratinocytes. Gene Ther 9, 307–319.
10 Tanaka T & Rabbitts TH (2003) Intrabodies based on
intracellular capture frameworks that bind the RAS
protein with high affinity and impair oncogenic trans-
formation. EMBO J 22, 1025–1035.
11 Caron de Fromentel C, Gruel N, Venot C, Debussche
L, Conseiller E, Dureuil C, Teillaud JL, Tocque B &
Bracco L (1999) Restoration of transcriptional activity
of p53 mutants in human tumour cells by intracellular
expression of anti-p53 single chain Fv fragments. Onco-
gene 18, 551–557.
12 Alvarez RD, Barnes MN, Gomez-Navarro J, Wang M,
Strong TV, Arafat W, Arani RB, Johnson MR, Roberts
BL, Siegal GP, et al. (2000) A cancer gene therapy
approach utilizing an anti-erbB-2 single-chain antibody-
encoding adenovirus (AD 21): a phase I trial. Clin
Cancer Res 6, 3081–3087.
13 Worn A & Pluckthun A (2001) Stability engineering of
antibody single-chain Fv fragments. J Mol Biol 305,
989–1010.
14 Ochs HD, Slichter SJ, Harker LA, Von Behrens WE,
Clark RA & Wedgwood RJ (1980) The Wiskott–Aldrich

syndrome: studies of lymphocytes, granulocytes, and
platelets. Blood 55, 243–252.
15 Remold-O’Donnell E, Rosen FS & Kenney DM (1996)
Defects in Wiskott–Aldrich syndrome blood cells. Blood
87, 2621–2631.
16 Molina IJ, Sancho J, Terhorst C, Rosen FS & Remold-
O’Donnell E (1993) T cells of patients with the
Wiskott–Aldrich syndrome have a restricted defect in
proliferative responses. J Immunol 151, 4383–4390.
17 Snapper SB, Rosen FS, Mizoguchi E, Cohen P, Khan
W, Liu CH, Hagemann TL, Kwan SP, Ferrini R,
Davidson L, et al. (1998) Wiskott–Aldrich syndrome
protein · deficient mice reveal a role for WASP in T
but not B cell activation. Immunity 9, 81–91.
18 Zhang J, Shehabeldin A, da Cruz LA, Butler J, Somani
AK, McGavin M, Kozieradzki I, dos Santos AO, Nagy
A, Grinstein S, et al. (1999) Antigen receptor-induced
activation and cytoskeletal rearrangement are impaired
in Wiskott–Aldrich syndrome protein · deficient
lymphocytes. J Exp Med 190, 1329–1342.
19 Symons M, Derry JM, Karlak B, Jiang S, Lemahieu V,
Mccormick F, Francke U & Abo A (1996) Wiskott–
Aldrich syndrome protein, a novel effector for the
GTPase CDC42Hs, is implicated in actin polymeriza-
tion. Cell 84, 723–734.
20 Rivero-Lezcano OM, Marcilla A, Sameshima JH &
Robbins KC (1995) Wiskott–Aldrich syndrome protein
physically associates with Nck through Src homology 3
domains. Mol Cell Biol 15, 5725–5731.
21 She HY, Rockow S, Tang J, Nishimura R, Skolnik EY,

Chen M, Margolis B & Li W (1997) Wiskott–Aldrich
syndrome protein is associated with the adapter protein
Grb2 and the epidermal growth factor receptor in living
cells. Mol Biol Cell 8, 1709–1721.
22 Banin S, Truong O, Katz DR, Waterfield MD, Brickell
PM & Gout I (1996) Wiskott–Aldrich syndrome protein
(WASp) is a binding partner for c-Src family protein-
tyrosine kinases. Curr Biol 6, 981–988.
23 Blanchoin L, Amann KJ, Higgs HN, Marchand JB,
Kaiser DA & Pollard TD (2000) Direct observation of
dendritic actin filament networks nucleated by Arp2 ⁄ 3
complex and WASP ⁄ Scar proteins. Nature 404, 1007–
1011.
24 Sato M, Tsuji NM, Gotoh H, Yamashita K, Hashimoto
K, Tadotsu N, Yamanaka H, Sekikawa K & Hashi-
moto Y (2001) Overexpression of the Wiskott–Aldrich
syndrome protein N-terminal domain in transgenic mice
inhibits T cell proliferative responses via TCR signaling
without affecting cytoskeletal rearrangements. J Immu-
nol 167, 4701–4709.
25 Tsai B, YeY & Rapoport TA (2002) Retro-translocation
of proteins from the endoplasmic reticulum into the
cytosol. Nat Rev Mol Cell Biol 3, 246–255.
26 Hershko A, Ciechanover A & Varshavsky A (2000) The
ubiquitin system. Nat Med 6, 1037–1081.
27 Weissman AM (2001) Themes and variations on ubiqui-
tylation. Nat Rev Mol Cell Biol 2, 169–178.
28 Coux O, Tanaka K & Goldberg AL (1996) Structure
and function of the 20S and 26S proteasomes. Annu Rev
Biochem 65, 801–847.

M. Sato et al. Impaired TCR signaling in anti-WASP scFv Tg mice
FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS 6143
29 Ellgaard L & Helenius A (2003) Quality control in the
endoplasmic reticulum. Nat Rev Mol Cell Biol 4, 181–
191.
30 Mhashilkar AM, Bagley J, Chen SY, Szilvay AM, Hel-
land DG & Marasco WA (1995) Inhibition of HIV-1
Tat-mediated LTR transactivation and HIV-1 infection
by anti-Tat single chain intrabodies. EMBO J 14, 1542–
1551.
31 Zhu Q, Zeng C, Huhalov A, Yao J, Turi TG, Danley
D, Hynes T, Cong Y, DiMattia D, Kennedy S, et al.
(1999) Extended half-life and elevated steady-state level
of a single-chain Fv intrabody are critical for specific
intracellular retargeting of its antigen, caspase-7.
J Immunol Methods 231, 207–222.
32 Munro S & Pelham HR (1986) An Hsp70-like protein
in the ER: identity with the 78 kd glucose-regulated
protein and immunoglobulin heavy chain binding pro-
tein. Cell 46, 291–300.
33 Flynn GC, Pohl J, Flocco MT & Rothman JE (1991)
Peptide-binding specificity of the molecular chaperone
BiP. Nature 353, 726–730.
34 Lencer WI & Tsai B (2003) The intracellular voyage of
cholera toxin: going retro. Trends Biochem Sci 28, 639–
645.
35 Breitschopf K, Bengal E, Ziv T, Admon A & Ciechan-
over A (1998) A novel site for ubiqitination: the
N-terminal residue, and not internal lysines of MyoD, is
essential for conjugation and degradation of the protein.

EMBO J 17, 5964–5973.
36 Dustin ML & Cooper JA (2000) The immunological
synapse and the actin cytoskeleton: molecular hardware
for T cell signaling. Nat Immunol 1, 23–29.
37 Krawczyk C & Penninger JM (2001) Molecular controls
of antigen receptor clustering and autoimmunity. Trends
Cell Biol 11, 212–220.
38 Cannon JL & Burkhardt JK (2004) Differential roles
for Wiskott–Aldrich syndrome protein in immune syn-
apse formation and IL-2 production. J Immunol 173,
1658–1662.
39 Silvin C, Belisle B & Abo A (2001) A role for Wiskott–
Aldrich syndrome protein in T-cell receptor-mediated
transcriptional activation independent of actin poly-
merization. J Biol Chem 276, 21450–21457.
40 Fischer KD, Kong YY, Nishina H, Tedford K, Mar-
engere LE, Kozieradzki I, Sasaki T, Starr M, Chan G,
Gardener S, et al. (1998) Vav is a regulator of cytoskele-
tal reorganization mediated by the T-cell receptor. Curr
Biol 8, 554–562.
41 Holsinger LJ, Graef IA, Swat W, Chi T, Bautista DM,
Davidson L, Lewis RS, Alt FW & Crabtree GR (1998)
Defects in actin-cap formation in Vav-deficient mice
implicate an actin requirement for lymphocyte signal
transduction. Curr Biol 8, 563–572.
42 Wu J, Katzav S & Weiss A (1995) A functional T-cell
receptor signaling pathway is required for p95vav activ-
ity. Mol Cell Biol 15, 4337–4346.
43 Savoy DN, Billadeau DD & Leibson PJ (2000) WIP, a
binding partner for Wiskott–Aldrich syndrome protein,

cooperates with Vav in the regulation of T cell activa-
tion. J Immunol 164, 2866–2870.
44 Anton IM, de la Fu nte MA, Sims TN, Freeman S,
Ramesh N, Hartwig JH, Dustin ML & Geha RS (2002)
WIP deficiency reveals a differential role for WIP and
the actin cytoskeleton in T and B cell activation. Immu-
nity 16, 193–204.
45 Niwa H, Yamamura K & Miyazaki J (1991) Efficient
selection for high-expression transfectants with a novel
eukaryotic vector. Gene 108, 193–199.
46 Takemoto Y, Furuta M, Sato M, Kubo M & Hashimoto
Y (1999) Isolation and characterization of a novel HS1
SH3 domain binding protein, HS1BP3. Int Immunol 11,
1957–1964.
47 Shimonkevitz R, Kappler J, Marrack P & Grey H
(1983) Antigen recognition by H-2-restricted T cells. I.
Cell-free antigen processing. J Exp Med 158, 303–316.
6144 FEBS Journal 272 (2005) 6131–6144 ª 2005 The Authors Journal Compilation ª 2005 FEBS
Impaired TCR signaling in anti-WASP scFv Tg mice M. Sato et al.