Download TCR After Rapid Identification of Pathogenic Vaccines Autoimmune

Successful TCR-Based Immunotherapy for
Autoimmune Myocarditis with DNA Vaccines
After Rapid Identification of Pathogenic TCR
This information is current as
of August 12, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2000; 164:2248-2254; ;
doi: 10.4049/jimmunol.164.4.2248
http://www.jimmunol.org/content/164/4/2248
This article cites 19 articles, 11 of which you can access for free at:
http://www.jimmunol.org/content/164/4/2248.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
References
Yoh Matsumoto, Youngheun Jee and Mayumi Sugisaki
Successful TCR-Based Immunotherapy for Autoimmune
Myocarditis with DNA Vaccines After Rapid Identification of
Pathogenic TCR1
Yoh Matsumoto, Youngheun Jee, and Mayumi Sugisaki
E
xperimental organ-specific autoimmune diseases can be
induced in animals by immunization with organ-specific
autoantigens and serves as a model for human autoimmune diseases. Recent analysis mainly using experimental autoimmune encephalomyelitis (EAE)3 indicates that autoimmune disease-inducing T cells bear CD4 molecules and use a limited
number of ␣- and ␤-chains of the TCR (1, 2). Furthermore, the
complementarity-determining region 3 (CDR3) of TCR of in vitroestablished encephalitogenic T cell clones is rather short, and some
amino acid residues are conservatively preserved (3, 4). We have
recently demonstrated by CDR3 spectratyping that only V␤8.2
spectratype shows oligoclonal expansion in the spinal cord
throughout the course of EAE induced in Lewis rats, whereas irrelevant TCRs become more diverse at later stages of the disease
(5, 6). Importantly, the CDR3 sequence of the majority of clones
derived from EAE-specific spectratype is the same as that of encephalitogenic T cell clones. These findings imply that although
the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus,
CDR3 spectratyping is a powerful tool for the screening of autoimmune disease-inducing T cells whose pathomechanism is poorly
known.
Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan
Received for publication October 4, 1999. Accepted for publication December
9, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported in part by grants-in-aid (10357005, 09480216, and
09670682) from the Ministry of Education, Japan. Y.J. was supported by the research
subsidy of Japan Foundation for Neuroscience and Mental Health.
2
Address correspondence and reprint requests to Dr. Yoh Matsumoto, Department of
Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Musashidai 2-6 Fuchu, Tokyo 183-8526, Japan. E-mail address: [email protected]
3
Abbreviations used in this paper: EAE, experimental autoimmune encephalitis;
EAC, experimental autoimmune carditis; CDR, complementarity-determining region;
CNBr, cyanogen bromide; PI, postimmunization.
Copyright © 2000 by The American Association of Immunologists
In some types of organ-specific autoimmune diseases such as
experimental autoimmune myocarditis (EAC), it is difficult to establish autoantigen-reactive disease-inducing T cell lines and
clones. Like EAE, EAC is inducible in Lewis rats by immunization
with cardiac myosin (7, 8) or adoptive transfer of sensitized T cells
activated in vitro with Con A (9). Therefore, EAC is judged to be
a T cell-mediated autoimmune disease. However, so far, attempts
to establish cardiac myosin-reactive carditogenic T cells using cyanogen bromide (CNBr)-treated soluble myosin have been unsuccessful (our unpublished observation). These findings suggest that
carditogenic epitope(s) resides in the cleavage site of CNBr. In
such a case, it is impossible to identify disease-inducing TCR by
determining the TCR phenotype of in vitro-established carditogenic T cell clones.
In the present study, we have extended our strategy to identify
EAC-inducing TCRs on the basis of the findings obtained with the
EAE system by direct analysis of heart-infiltrating T cells. For this
purpose, candidate TCR ␤-chain genes were screened by CDR3
spectratyping and the sequence of their CDR3 region was determined after cloning. Then immunotherapy with V␤-specific mAbs
was performed to see whether this treatment suppresses the development of EAC. Consequently, we found that combined immunotherapy with anti-V␤8.2 plus V␤10 mAbs, but not with either
alone, significantly reduced the histological severity of EAC and
completely suppressed the inflammation in some animals. More
important, essentially the same results were obtained using DNA
vaccines encoding V␤8.2 and V␤10. Collectively, the determination of candidate TCR genes by CDR3 spectratyping and subsequent immunotherapy serves not only to elucidate the pathomechanism, but also to provide a systematic therapeutic strategy for T
cell-mediated autoimmune diseases.
Materials and Methods
Rats and reagents
Lewis rats were obtained from Seiwa (Fukuoka, Japan). All of the rats were
used at the age of 8 –12 wk. The mAbs used in this study were R73 (antiTCR ␣␤) (10), R78 (anti-V␤8.2), B73 (anti-V␤8.5), G101 (anti-V␤10)
(11), and HIS42 (anti-V␤16). R78, B73, and G101 were kindly provided
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing
TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditisassociated TCRs, V␤8.2 and V␤10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis
and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both V␤8.2 and
V␤10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and
completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does
not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary
therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune diseaseassociated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose
pathomechanism is poorly understood. The Journal of Immunology, 2000, 164: 2248 –2254.
The Journal of Immunology
by Dr. T. Hünig (Würzburg, Germany). R73 and HIS42 were obtained
from Serotec (Oxford, U.K.).
Cardiac myosin preparation
Cardiac myosin was partially purified according to the method of Perry
(12) with a few modifications. Human heart kept at ⫺80°C was thawed,
minced, and weighed. A total of 300 ml of chilled 0.3 M KCl-0.15 M
sodium phosphate buffer (pH 6.5) was added to 100 g of minced heart
tissue and kept on ice for 20 min. This homogenate was centrifuged at 5000
rpm for 20 min at 4°C, and the supernatant was collected by filtration
through Toyo No. 2 filter paper (Toyo Roshi, Tokyo, Japan). The filtrate
was then diluted with 15 volumes of chilled Mili-Q-filtered (Millipore
Japan, Tokyo, Japan) purified water to aggregate myosin. Aggregated myosin was collected by centrifugation at 5000 rpm, dissolved in 0.5 M KCl,
and stored at ⫺20°C with the same volume of glycerin.
EAC induction and histological evaluation
Flow cytometric analysis
Under ether anesthesia, blood was aspirated via cardiac puncture and the
heart and the popliteal lymph node were removed. Then PBL and heartinfiltrating T cells were isolated by the proteolytic enzyme treatment and
density gradient method as described previously (14). Cells were incubated
with one of the V␤-specific mAbs followed by PE-conjugated anti-mouse
IgG (Biomeda, Foster City, CA). To saturate free binding sites of the secondary Ab, cells were incubated with normal mouse serum. Then FITCR73 (Serotec) was applied in the second step. Ten thousand cells were
analyzed in each sample by FACScan (Becton Dickinson, Mountain View,
CA) flow cytometry. In preliminary studies, it was shown that the profile
of staining using irrelevant mAbs plus the secondary Ab or the secondary
Ab alone was essentially the same as that of unstained controls. Therefore,
Ab controls were omitted in subsequent analysis.
cDNA synthesis and PCR amplification
RNA was extracted from isolated heart-infiltrating T cells using RNazol B
(Biotecx Laboratories, Houston, TX). cDNA was then synthesized by reverse transcription using a SuperScript Preamplification System (Life
Technologies, Gaithersburg, MD) and amplified in a thermal cycler (Perkin-Elmer/Cetus, Norwalk, CT) using primer pairs for TCR. Primers for
V␤1–20 were the same as those used previously (15). Two types of C␤
primers, C␤ outer (5⬘-TGTTTGTCTGCGATCTCTGC-3⬘) and C␤ inner
(5⬘-TCTGCTTCTGATGGCTCA-3⬘), were used in this study. They were
labeled with Cy5 or rhodamine or remained unlabeled.
CDR3 spectratyping and sequencing of
spectratype-derived DNA
CDR3 spectratyping was performed as described previously (16) with a
few modifications. cDNA was amplified with V␤-specific and rhodaminelabeled C␤ outer primers, and undiluted or diluted PCR products were
added to an equal volume of formamide/dye loading buffer and heated at
94°C for 2 min. A total of 2 ␮l of the samples was applied to a 6%
acrylamide-sequencing gel. Gels were run at 30 W for 3 h and 30 min at
50°C. Then the fluorescence-labeled DNA profile on the gel was directly
recorded using a FMBIO fluorescence image analyzer (Hitachi, Yokohama, Japan).
cDNA extracted from spectratypes of interest on the acrylamide gel was
reamplified with V␤ and unlabeled C␤ inner primers. Then PCR products
were ligated into pT-Adv vector and cloned using the AdvanTAge PCR
Cloning kit (Clontech Laboratories, Palo Alto, CA) according to the manufacturer’s instructions. The plasmid DNA was then sequenced using a
Cy5-labeled C␤ inner primer and Thermo Sequenase Fluorescent-labeled
Primer Cycle sequencing kit on an ALFexpress DNA sequencer (Pharmacia Biotech, Tokyo, Japan). CDR3 length is defined as the region starting from
the amino acid residue after the CASS sequence of most V␤ segments and
ending before the GXG box in the J␤ region as described previously (17).
In vivo administration of mAbs
Protein G-purified R78, G101, or both mAbs at a dose of 100 ␮g was
injected i.p. once a day for 21 consecutive days from day ⫺7 to ⫹14
postimmunization except on the day of challenge.
DNA vaccination
DNA vaccine therapy was performed as reported previously with modifications (18). Total RNA was extracted from normal rat PBL and reverse
transcribed into cDNA. This cDNA was then amplified using Amplitaq
Gold (Perkin-Elmer/Cetus) with one of primers specific for V␤8.2 (5⬘CAAAACACATGGAAGCTGCAG-3⬘), V␤10 (5⬘-TTATGAGCTATAG
GCTCCTAAGCTGTGTGG-3⬘) or V␤12 (5⬘-AAATGGGCATCCAGA
CCCTCTGTTGTATGA-3⬘) and C␤ inner primer. All of the forward
primers were designed to include an ATG in-frame. PCR products were
cloned into pTargeT plasmid (Promega, Madison, WI) according to the
manufacturer’s instructions. Colonies grown in competent cells were
picked and recombinant plasmid DNA was isolated using Mini prep (Promega). By restriction enzyme digestion with PstI, colonies with an insert
with right direction and length were screened, and the nucleotide sequence
of each clone was determined to confirm that inserts had the right sequence
with ATG in-frame.
Large-scale preparation of plasmid DNA was done using Mega prep
(Promega). For DNA vaccination, animals were pretreated with 0.75%
bupivacaine (1 ␮l/g body weight; Sigma) by injecting it into tibialis anterior muscle 1 wk before vaccination. Then 100 ␮g of DNA was injected
into the same site according to the indicated protocol. Two weeks after the
last vaccination, rats were challenged with human cardiac myosin emulsified in CFA. To verify the expression of RNA and protein, the muscle
tissue was removed from the injected site after extensive perfusion with
PBS, and RNA and protein extracts were prepared. Using a forward primer
specific for the plasmid sequence upstream to the insert and C␤ inner
primer, we identified that transcripts corresponding to the plasmid sequence plus insert existed in the muscle. We also verified the presence of
the TCR protein by Western blot analysis (data not shown).
Results
Histological and flow cytometric analysis of EAC
Lewis rats were immunized with human cardiac myosin in CFA
along with an i.p. injection of pertussis toxin. At various time
points, the heart was removed under ether anesthesia and processed for histological examination. At the early stage of EAC,
mononuclear cells which mainly consisted of TCR␣␤⫹ T cells and
macrophages infiltrated the outer one-third of the muscle. In severe
cases, there was extensive necrosis of muscle fibers (Fig. 1A).
Multinucleated giant cells were occasionally seen in the lesion
(arrows in Fig. 1A) as reported previously (8). Using hematoxylin
and eosin-stained sections, the histological severity of the disease
during the course was scored (Fig. 1B). Inflammatory lesions appeared at around day 7 postimmunization (PI), increased in severity gradually, and reached a maximal level on day 12 PI. The
severity of inflammation remained unchanged during the examination period until day 20 PI (Fig. 1B).
We next examined the TCR V␤ phenotype of infiltrating T cells
by flow cytometry with currently available anti-V␤ mAbs (Fig.
1C ). The percentages of V␤8.2, V␤8.5, V␤10, and V␤16 in lymph
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
EAC was induced in Lewis rats as described previously (7) with modifications. Each rat was immunized in the hind footpads on both sides with an
emulsion containing 1.5 mg of cardiac myosin in CFA (Mycobacterium
tuberculosis H37Ra, 5 mg/ml) along with an i.p. injection of 2 ␮g pertussis
toxin (Sigma, St. Louis, MO). Immunized rats were weighed and observed
daily.
EAC lesions were evaluated using hematoxylin and eosin-stained sections according to the following criteria: grade 1, focal inflammatory lesions mainly located in the outer layer of the cardiac muscle; grade 2,
diffuse inflammation involving the outer layer of the muscle; grade 3, grade
2 plus focal transmural inflammation; and grade 4, diffuse inflammation
with partial necrosis.
A single immunoperoxidase staining was performed using mAbs
against TCR ␣␤ (R73), V␤8.2 (R78), V␤8.5 (B73), and V␤10 (G101) (11)
as described previously (13). Briefly, frozen sections of the heart were air
dried and fixed in ether for 10 min. After incubation with normal horse
serum, sections were allowed to react with mAb, biotinylated horse antimouse IgG (Vector Laboratories, Burlingame, CA), and HRP-labeled
Vectstain Elite ABC kit (Vector Laboratories). HRP binding sites were
detected in 0.005% diaminobenzidine and 0.01% hydrogen peroxide.
2249
DNA VACCINE THERAPY FOR AUTOIMMUNE MYOCARDITIS
2250
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
The Journal of Immunology
2251
nodes, PBL, pericardial effusion, and heart were determined and
proportions to total T cells were calculated (numbers in parentheses). The percentages of V␤s in lymph nodes and PBL were essentially the same as those in normal controls which were reported
previously (15). Moreover, there was no preferential infiltration of
T cells bearing a particular type of V␤ in the target organ.
CDR3-spectratyping analysis of heart-infiltrating T cells
Nucleotide sequence of the CDR3 region of oligoclonally
expanded spectratypes
Based on findings obtained by CDR3 spectratyping, bands representing candidate spectratypes were cut out from the gel and extracted cDNA was reamplified by nested PCR. PCR products were
then cloned and the nucleotide sequences of the clones were determined. The results are listed in Tables 1 and 2. With regard to
V␤8.2, DSSYEQYF, which is a predominant sequence in EAE,
was also recognized in 50% of clones on day 8 PI (Table I). However, CDR3 sequences of the clones from V␤8.2 spectratype became diverse at a later stage (Table I). V␤12 spectratype showed
diverse CDR3 sequences (data not shown). In sharp contrast, sequencing analysis of V␤10 revealed a very striking finding. As
shown in Table II, all of the clones isolated from V␤10 on day 8
PI possessed the sequence ERTDERLFF (Table II), and 85.7% of
V␤10 on day 14 PI showed this sequence (Table II). These findings suggest that V␤8.2 and V␤10, rather than V␤12, are more
likely effector TCRs because they have unique sequences in their
CDR3 region.
Immunotherapy of autoimmune carditis with mAbs and DNA
vaccines
We finally tested whether TCR-based immunotherapy with mAbs
and DNA vaccines is effective for suppression of EAC. Purified
mAbs against candidate TCRs, R78 (anti-V␤8.2) or G101 (antiV␤10) or both, were administered to rats by i.p. injections for 21
FIGURE 2. CDR3 spectratyping of heart-infiltrating T cells on days 8
(A) and 14 (B). On day 8, oligoclonal expansion of spectratypes with a
short CDR3 was noted in V␤8.2 and V␤10 (indicated by arrow and arrowhead in A, respectively). However, on day 14, there was additional
expansion in V␤10 and V␤12 (open arrow and double arrowhead in B,
respectively).
consecutive days starting from day ⫺7, i.e., 7 days before the
immunization. On day 0, rats were immunized with human cardiac
myosin, and heart pathology was examined on day 14 PI. The
results are summarized in Table III. R78 treatment alone did not
alter the severity of EAC (Table III, group A). G101 suppressed
FIGURE 1. Histological and flow cytometric analysis of inflammatory lesions in the heart during EAC. Upon immunization with cardiac myosin, rats
developed severe inflammatory lesions mainly comprising mononuclear cells with focal necrosis. In severe cases, there was extensive necrosis of muscle
fibers (A). Residual muscle fibers and multinucleated giant cells are indicated by arrowheads and arrows, respectively. Histological grading of the lesions
at various time points revealed that inflammatory lesions developed around day 6 PI, reached a plateau phase on day 12 PI, and persisted throughout the
examination period (B). Standard errors are within 10% of the mean values at each time point. Flow cytometric analysis of inflammatory cells isolated from
the heart during EAC revealed that no predominant V␤ usage was detected using currently available anti-V␤-specific mAbs (anti-V␤8.2, V␤8.5, V␤10,
and V␤16) (C). Numbers in parentheses indicate percentages of particular V␤-positive cells in the total TCR ␣␤-positive cells.
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
To identify oligoclonal expansion of TCRs with a particular CDR3
size, CDR3-spectratyping analysis was performed using heart-infiltrating T cells isolated at different times during EAC. In previous
studies, we have shown that EAE-specific spectratype has several
characteristics (5). First, clonal expansion of EAE-specific spectratype is observed throughout the course of the disease, whereas
expansion of irreverent spectratypes is detectable only over a short
period. Second, EAE-specific spectratype has a short CDR3. Finally, a predominant CDR3 sequence is found in EAE-specific
spectratype throughout the disease course. On the basis of these
criteria, we searched for EAC-specific spectratypes and representative results are shown in Fig. 2. At the early stage of EAC (day
8 PI), oligoclonal expansion was noted in V␤8.2 (arrow in Fig. 2A)
and V␤10 (arrowhead in Fig. 2A) spectratypes. Expansion of
V␤8.2 and V␤10 was also detectable at a later stage (day 14 PI)
(arrow and arrowhead in Fig. 2B) when the histological severity
was maximal (Fig. 1B). In addition, V␤12 was oligoclonally expanded at this stage (double arrowhead in Fig. 2B). Thus, CDR3spectratyping analysis suggests that three spectratypes, V␤8.2,
V␤10, and V␤12, are candidates for EAC-inducing TCR.
2252
DNA VACCINE THERAPY FOR AUTOIMMUNE MYOCARDITIS
Table I. Amino acid and nucleotide sequences of the CDR3 region of TCR clones extracted from V␤8.2 spectratype showing oligoclonal expansion
in EACa
V
2190H: EAC day 8, V␤8.2
CASS
CASG
CASS
2196H: EAC day 14, V␤8.2
CASS
CASS
CASS
CASS
CASG
Frequency
D
gac
G
ggt
N
aat
S
agc
S
tct
S
tct
S
tcc
G
gga
G
gga
Y
tat
N
aat
N
aat
E
gag
V
gtg
V
gtg
Q
cag
L
ctc
L
ctc
Y
tat
Y
tat
Y
tat
F
ttc
F
ttt
F
ttt
N
aat
D
gac
A
ggc
S
tct
L
ctt
D
gac
S
tct
S
tcg
S
agc
S
tct
S
tct
S
agc
G
gga
G
ggg
S
tcc
G
gga
G
gga
S
tcc
N
aat
G
ggt
Y
tat
N
aat
N
aat
Y
tat
V
gtg
E
gag
E
gag
V
gtg
V
gtg
E
gat
L
ctc
Q
cag
Q
cag
L
ctc
L
ctc
Q
cag
Y
tat
Y
tat
Y
tat
Y
tat
Y
tat
Y
tat
F
ttt
F
ttc
F
ttc
F
ttt
F
ttt
F
ttc
GPG
3/6 (50.0%)
GEG
2/6 (33.3%)
GEG
1/6 (16.7%)
GEG
2/7 (28.6%)
GPG
1/7 (14.3%)
GPG
1/7 (14.3%)
GEG
1/7 (14.3%)
GEG
1/7 (14.3%)
GAG
1/7 (14.3%)
a
CDR3 spectratyping was performed using PCR products amplified with TCR V␤1-20-specific primers. cDNA was extracted from bands showing oligoclonal expansion and
reamplified by nested PCR, cloned, and sequenced.
inflammation in the heart to some extent but the difference between
test and control groups (groups B and D) was statistically insignificant. Combination therapy with R78 and G101 significantly
reduced the severity of EAC and completely suppressed inflammation in two of four rats examined (Table III, group C).
We also examined the effects of DNA vaccination on the development of EAC (Table IV). Plasmid DNA encoding V␤8.2,
V␤10, or V␤8.2 plus V␤10 was injected i.m. twice and challenged
for EAC. V␤8.2 DNA vaccination was ineffective (Table IV,
group A). V␤10 DNA reduced the histological severity slightly
compared with that of the control (group D, empty vector) but the
difference was not significantly different. In contrast, combination
therapy using V␤8.2 and V␤10 DNA significantly reduced the
incidence and histological severity (group C). These findings indicate that both V␤8.2 and V␤10 screened by CDR3-spectratyping
analysis are pathogenic TCRs and that TCR-based immunotherapy, especially using DNA vaccines, is effective for the treatment
of T cell-mediated autoimmune diseases.
Discussion
In the present study, we analyzed EAC to verify EAC-inducing
TCRs and to establish TCR-based immunotherapy. EAC is inducible in Lewis rats by immunization with human cardiac myosin as
reported previously (7, 8). Since injection of activated T cells that
had been taken from immunized animals and then stimulated in
vitro with Con A induced severe carditis in naive rats (9), EAC is
judged to be a T cell-mediated autoimmune disease. However,
attempts so far have been unsuccessful in establishing cardiac myosin-reactive carditogenic T cells using CNBr-treated soluble myosin (our unpublished observation). These findings imply that
carditogenic epitope(s) resides in the cleavage site of CNBr. As
such, it would be difficult to identify disease-inducing TCRs by
establishing disease-inducing T cell clones and subsequently determining their TCRs. Alternatively, we tried to identify EACspecific TCRs using heart-infiltrating T cells by CDR3 spectratyping and subsequent sequencing of the CDR3 region of
oligoclonally expanded spectratypes. As reported previously (5), it
was revealed in EAE that TCRs of T cells in the spinal cord show
oligoclonal expansion of V␤8.2 spectratype throughout the course
of the disease and that the majority of clones (⬃70% regardless of
the stage examined) possess an identical sequence which is the
same as that of the encephalitogenic T cell clone. On the basis of
these criteria, we screened the spectratype pattern of TCRs isolated
from heart-infiltrating T cells and found that V␤8.2, V␤10, and
V␤12 spectratypes showed oligoclonal expansion (Fig. 2). To our
surprise, virtually all of the clones (100% at the early stage and
Table II. Amino acid and nucleotide sequences of the CDR3 region of TCR clones extracted from V␤10 spectratype showing oligoclonal expansion
in EAC
V
2190H: EAC day 8, V␤10
CASS
2196H: EAC day 14, V␤10
CASS
CASS
(N)D(N)
Frequency
E
gag
R
agg
T
acg
D
gat
E
gaa
R
aga
L
ttg
F
ttt
F
ttc
GHG
9/9 (100%)
E
gag
V
gtc
R
agg
G
gga
T
acg
P
ccg
D
gat
R
cgg
E
gaa
D
gac
R
aga
T
acc
L
ttg
L
ttg
F
ttt
F
ttc
F
ttc
F
ttc
GHG
6/7 (85.7%)
GAG
1/7 (14.3%)
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
CASS
(N)D(N)
The Journal of Immunology
2253
Table III. Immunotherapy of EAC with mAbsa
mAb
Specificity
Incidenceb
Heart weight (g)
Histological Grade
R78
G101
R78 ⫹ G101
Saline
Anti-V␤8.2
Anti-V␤10
3/3
4/4
2/4
3/3
1.3 ⫾ 0.2
1.2 ⫾ 0.3
0.8 ⫾ 0.1c
1.4 ⫾ 0.3
3.7 ⫾ 0.2
2.6 ⫾ 0.9
0.8 ⫾ 0.8d
3.2 ⫾ 0.2
Group
A
B
C
D
a
Purified mAbs at a dose of 100 ␮g or saline were administered i.p. once a day for 21 consecutive days starting from day ⫺7 of immunization. On
day 0, rats were immunized with human cardiac myosin in CFA in bilateral footpads along with an i.p. injection of pertussis toxin.
b
No. of rats with cardiac inflammation/no. of rats examined.
c
Significantly different from groups A ( p ⫽ 0.006), B ( p ⫽ 0.045), and D ( p ⫽ 0.012) by Student’s t test.
d
Significantly different from groups A ( p ⫽ 0.002), B ( p ⫽ 0.024), and D ( p ⫽ 0.004) by Student’s t test.
Table IV. Immunotherapy of EAC with DNA vaccinesa
Group
A
B
C
D
DNA
V␤8.2
V␤10
V␤8.2 ⫹ V␤10
Empty vector
Incidence Heart weight (g) Histological Grade
4/4
3/4
2/4
4/4
1.1 ⫾ 0.2
1.1 ⫾ 0.2
0.9 ⫾ 0.3
1.3 ⫾ 0.3
3.0 ⫾ 0.7
2.3 ⫾ 1.3
0.9 ⫾ 0.9b
3.0 ⫾ 1.1
a
Lewis rats were pretreated by injection of 0.75% bupivacaine (1 ␮l/g body
weight) in the tibialis anterior muscle and 100 ␮g in 50 ␮l of DNA was injected twice
at 2-wk intervals and challenged for EAC. On day 14 PI, hearts were removed and
processed for hematoxylin and eosin staining. Histological severity was evaluated
according to the criteria described in Materials and Methods.
b
Significantly different from groups A ( p ⫽ 0.01) and D ( p ⫽ 0.03) by unpaired
Student’s t test.
treatment with mAbs for 21 consecutive days. More important,
potential side effects of xenoantibody administration can be
avoided by this method. Treatment experiments using mAbs and
DNA vaccines clearly indicate that both T cells bearing either
V␤8.2 or V␤10 are EAC-inducing T cells. Since depletion or suppression of one of two types of effector cells does not improve the
severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cellmediated autoimmune diseases.
In the present study, we have shown that rapid identification of
pathogenic TCRs by CDR3 spectratyping and CDR3 sequencing
gives useful information for designing TCR-based immunotherapy
without the culture procedures. The strategy employed in the
present study provides insights into the pathomechanism of, but
also provides a systematic therapeutic strategy for, human autoimmune diseases.
Acknowledgments
We thank Dr. Hünig (Würzberg) for kindly providing us with mAbs. We
also thank Y. Kawazoe, K. Kohyama, and K. Nomura for technical
assistance.
References
1. Burns, J., A. Rosenzweig, B. Zweiman, A. Moskovitz, and R. Lisak. 1984. Recovery of myelin basic protein reactive T cells from spinal cords of Lewis rats
with autoimmune encephalomyelitis. J. Immunol. 132:2690.
2. Acha-Orbea, H., D. J. Mitchell, L. Timmermann, D. C. Wraith, G. S. Tausch,
M. K. Waldor, S. S. Zamvil, H. O. McDevitt, and L. Steinman. 1988. Limited
heterogeneity of T cell receptors from lymphocytes mediating autoimmune encephalomyelitis allows specific immune intervention. Cell 54:263.
3. Gold, D. P., H. Offner, D. Sun, S. Wiley, A. A. Vandenbark, and D. B. Wilson.
1991. Analysis of T cell receptor ␤ chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.
J. Exp. Med. 174:1467.
4. Zhang, X., and E. Heber-Katz. 1992. T cell receptor sequences from encephalitogenic T cells in adult Lewis rats suggest an early ontogenic origin. J. Immunol.
148:746.
5. Kim, G., N. Tanuma, T. Kojima, K. Kohyama, Y. Suzuki, Y. Kawazoe, and
Y. Matsumoto. 1998. CDR3 size spectratyping and sequencing of spectratypederived T cell receptor of spinal cord T cells in autoimmune encephalomyelitis.
J. Immunol. 160:509.
6. Kim, G., K. Kohyama, N. Tanuma, H. Arimoto, and Y. Matsumoto. 1998. Persistent expression of autoimmune encephalomyelitis (EAE)-specific V␤8.2 TCR
spectratype in the central nervous system of rats with chronic relapsing EAE.
J. Immunol. 161:6993.
7. Kodama, M., Y. Matsumoto, M. Fujiwara, F. Masani, T. Izumi, and A. Shibata.
1990. A novel experimental model of giant cell myocarditis induced in rats by
immunization with cardiac myosin fraction. Clin. Immunol. Immunopathol. 57:
250.
8. Kodama, M., Y. Matsumoto, M. Fujiwara, S. Zhang, H. Hanawa, E. Itoh,
T. Tsuda, T. Izumi, and A. Shibata. 1991. Characteristics of giant cells and
factors related to the formation of giant cells in myocarditis. Circ. Res. 69:1042.
9. Kodama, M., Y. Matsumoto, and M. Fujiwara. 1992. In vivo lymphocyte-mediated myocardial injuries demonstrated by adoptive transfer of experimental autoimmune myocarditis. Circulation 85:1918.
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017
85.7% at the plateau stage of EAC) derived from V␤10 spectratype
possessed an identical CDR3 region, i.e., ERTDERLFF (Table II),
whereas V␤12 spectratypes were heterogeneous. V␤8.2 showed
oligoclonal expansion only at the early stage (Table I). These findings strongly suggest that V␤10 and possibly V␤8.2 are carditogenic TCR V␤ phenotypes. Since identification of EAC-associated
TCRs by single-strand conformation polymorphism analysis was
unsuccessful (19), CDR3 spectratyping is more suitable for the
screening of autoimmune disease-associated TCR than singlestrand conformation polymorphism.
Flow cytometric analysis of TCRs of T cells isolated from the
peripheral blood and target organ is not always useful for the
screening of autoimmune disease-associated T cells. Although the
predominance of T cells bearing encephalitogenic TCR (V␤8.2) is
observed in the spinal cord by this approach (15), that of V␤8.2
and V␤10 in the heart was not detectable in the present, as well as
the previous (19), study. This discrepancy may be attributable to
the difference in histopathology between EAC and EAE. Compared with EAE, T cells in the heart lesion are much fewer and the
predominant population of inflammatory cells is macrophages.
Therefore, it may be difficult to obtain a sufficient number of T
cells from the lesion to show the predominance of a particular type
of TCR.
Finally, we performed treatment experiments with mAbs and
DNA vaccines based on the data obtained by CDR3 spectratyping.
Rats were treated with either anti-V␤8.2 or anti-V␤10 mAbs or
both before and after the challenge for EAC. As nicely demonstrated in Table III, combined therapy with anti-V␤8.2 and antiV␤10 mAbs, but not with either alone, significantly suppressed
autoimmune inflammation in the heart. Furthermore, essentially
the same results were obtained using DNA vaccines (Table IV).
Compared with mAb administration, DNA vaccination is an effective and easy therapeutic approach for the treatment of autoimmune diseases because remarkable suppressive effect was obtained
by vaccination twice. This effect was almost the same as that after
2254
DNA VACCINE THERAPY FOR AUTOIMMUNE MYOCARDITIS
10. Hünig, T., H.-J. Wallny, J. K. Hartley, A. Lawetzky, and G. Tiefenthaler. 1989.
A monoclonal antibody to a constant determinant of the rat T cell antigen receptor
that induces T cell activation: differential reactivity with subsets of immature and
mature T lymphocytes. J. Exp. Med. 169:73.
11. Torres-Nagel, N. E., D. P. Gold, and T. Hünig. 1993. Identification of rat TcrbV8.2, 8.5, and 10 gene products by monoclonal antibodies. Immunogenetics 37:
305.
12. Perry, S. V. 1955. Myosin adenosintriphosphatase. Methods Enzymol. 2:582.
13. Ohmori, K., Y. Hong, M. Fujiwara, and Y. Matsumoto. 1992. In situ demonstration of proliferating cells in the rat central nervous system during experimental
autoimmune encephalomyelitis: evidence suggesting that most infiltrating T cells
do not proliferate in the target organ. Lab. Invest. 66:54.
14. Tanuma, N., T. Kojima, T. Shin, Y. Aikawa, T. Kohji, Y. Ishihara, and
Y. Matsumoto. 1997. Competitive PCR quantification of pro- and antiinflammatory cytokine mRNA in the central nervous system during autoimmune
encephalomyelitis. J. Neuroimmunol. 73:197.
15. Tsuchida, M., Y. Matsumoto, H. Hirahara, H. Hanawa, K. Tomiyama, and
T. Abo. 1993. Preferential distribution of V␤8.2-positive cells in the central nervous system of rats with myelin basic protein-induced autoimmune encephalomyelitis. Eur. J. Immunol. 23:2399.
16. Gorski, J., M. Yassai, X. Zhu, B. Kissella, C. Keever, and N. Flomenberg. 1994.
Circulating T cell repertoire complexity in normal individuals and bone marrow
recipients analyzed by CDR3 size spectratyping. J. Immunol. 152:5109.
17. Rock, E. P., P. R. Sibbald, M. M. Davis, and Y.-H. Chien. 1994. CDR3 length in
antigen-specific immune receptors. J. Exp. Med. 179:323.
18. Waisman, A., P. Ruiz, D. L. Hirschberg, A. Gelman, J. R. Oksenberg, S. Brocke,
F. Mor, I. R. Cohen, and L. Steinman. 1996. Suppressive vaccination with DNA
encoding a variable region gene of the T-cell receptor prevents autoimmune
encephalomyelitis and activates Th2 immunity. Nat. Med. 2:899.
19. Hanawa, H., T. Inomata, H. Sekikawa, T. Abo, M. Kodama, T. Izumi, and
A. Shibata. 1996. Analysis of heart-infiltrating T-cell clonotypes in experimental
autoimmune myocarditis. Circ. Res. 78:118.
Downloaded from http://www.jimmunol.org/ by guest on August 12, 2017