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Department of Molecular Neuropathology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and ß-chains of the TCR (1, 2).
Furthermore, the complementarity-determining region 3 (CDR3) of TCR of
in vitro-established 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 Vß8.2 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. 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myosin-reactive carditogenic T cells using cyanogen bromide (CNBr)-treated soluble myosin have been unsuccessful (our unpublished observation).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 ß-chain genes were screened by CDR3 spectratyping and the sequence of their CDR3 region was determined after cloning. Then immunotherapy with Vß-specific mAbs was performed to see whether this treatment suppresses the development of EAC. Consequently, we found that combined immunotherapy with anti-Vß8.2 plus Vß10 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 Vß8.2 and Vß10. 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 (anti-TCR ß) (10), R78 (anti-Vß8.2),
B73 (anti-Vß8.5), G101 (anti-Vß10) (11), and
HIS42 (anti-Vß16). R78, B73, and G101 were kindly provided 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
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 µg 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
ß (R73), Vß8.2 (R78), Vß8.5 (B73), and Vß10 (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 anti-mouse 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.
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 heart-infiltrating T cells were isolated by the proteolytic enzyme treatment and density gradient method as described previously (14). Cells were incubated with one of the Vß-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 FITC-R73 (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 Vß1–20 were the same as those used previously (15). Two types of Cß primers, Cß outer (5'-TGTTTGTCTGCGATCTCTGC-3') and Cß 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 Vß-specific and rhodamine-labeled Cß 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 µl 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 Vß and unlabeled Cß 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 Cß 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 Vß segments and ending before the GXG box in the Jß region as described previously (17).
In vivo administration of mAbs
Protein G-purified R78, G101, or both mAbs at a dose of 100 µg 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 Vß8.2 (5'-CAAAACACATGGAAGCTGCAG-3'), Vß10 (5'-TTATGAGCTATAGGCTCCTAAGCTGTGTGG-3') or Vß12 (5'-AAATGGGCATCCAGACCCTCTGTTGTATGA-3') and Cß 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 µl/g body weight; Sigma) by injecting it into tibialis anterior muscle 1 wk before vaccination. Then 100 µg 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 Cß 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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 TCRß+ T cells and macrophages
infiltrated the outer one-third of the muscle. In severe cases, there
was extensive necrosis of muscle fibers (Fig. 1
A). 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).
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C). The percentages of Vß8.2, Vß8.5, Vß10, and
Vß16 in lymph nodes, PBL, pericardial effusion, and heart were
determined and proportions to total T cells were calculated
(numbers in parentheses). The percentages of Vßs 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 Vß
in the target organ. CDR3-spectratyping analysis of heart-infiltrating T cells
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 Vß8.2
(arrow in Fig. 2A) and Vß10 (arrowhead in Fig. 2A) spectratypes. Expansion of Vß8.2 and Vß10 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, Vß12 was oligoclonally expanded at this
stage (double arrowhead in Fig. 2B). Thus,
CDR3-spectratyping analysis suggests that three spectratypes, Vß8.2,
Vß10, and Vß12, are candidates for EAC-inducing TCR.
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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 Vß8.2, 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 Vß8.2 spectratype became diverse at a later stage (Table I).
Vß12 spectratype showed diverse CDR3 sequences (data not shown). In
sharp contrast, sequencing analysis of Vß10 revealed a very striking
finding. As shown in Table II, all of the clones isolated from Vß10
on day 8 PI possessed the sequence ERTDERLFF (Table II), and 85.7% of
Vß10 on day 14 PI showed this sequence (Table II). These findings
suggest that Vß8.2 and Vß10, rather than Vß12, are more likely
effector TCRs because they have unique sequences in their CDR3
region.
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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-Vß8.2) or G101 (anti-Vß10) or
both, were administered to rats by i.p. injections for 21 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 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).
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). Plasmid DNA encoding
Vß8.2, Vß10, or Vß8.2 plus Vß10 was injected i.m. twice and
challenged for EAC. Vß8.2 DNA vaccination was ineffective (Table IV,
group A). Vß10 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 Vß8.2 and Vß10 DNA significantly reduced the
incidence and histological severity (group C). These findings indicate
that both Vß8.2 and Vß10 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
Vß8.2, Vß10, and Vß12 spectratypes showed oligoclonal expansion
(Fig. 2). To our surprise, virtually all of the clones (100% at the
early stage and 85.7% at the plateau stage of EAC) derived from Vß10
spectratype possessed an identical CDR3 region, i.e., ERTDERLFF (Table II), whereas Vß12 spectratypes were heterogeneous. Vß8.2 showed
oligoclonal expansion only at the early stage (Table I). These findings
strongly suggest that Vß10 and possibly Vß8.2 are carditogenic TCR
Vß 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 single-strand
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 (Vß8.2) is observed in the spinal cord by this approach (15), that of Vß8.2 and Vß10 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-Vß8.2 or anti-Vß10 mAbs or both before and
after the challenge for EAC. As nicely demonstrated in Table III,
combined therapy with anti-Vß8.2 and anti-Vß10 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 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 Vß8.2 or
Vß10 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 cell-mediated 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.
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Acknowledgments |
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Footnotes |
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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:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalitis; EAC, experimental autoimmune carditis; CDR, complementarity-determining region; CNBr, cyanogen bromide; PI, postimmunization.
Received for publication October 4, 1999. Accepted for publication December 9, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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