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Department of Bioregulatory Medicine and Rheumatology, Graduate School, Tokyo Medical and Dental University, 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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TCR heterodimers dominate at the sites of the muscle injury and that
some of them surround and invade the nonnecrotic muscle fibers
(1). The infiltrating CD8 T cells express activation
markers such as CD45RO and ICAM-1, whereas the invaded myocytes
overexpress HLA class I molecules on their cell surface
(2, 3, 4). In addition, these CD8 T cells express cytotoxic
effector molecules including perforin and granzyme B (5, 6). Furthermore, it has been found that the perforin molecules
in the CD8 T cells are oriented toward the target muscle fibers
(7). Thus, the tissue injury in PM appears to be mediated
by infiltrating cytotoxic CD8 T cells. Using biopsy specimens of the muscles, PCR analysis of TCR transcripts and immunohistochemical analysis with anti-TCRV mAbs were conducted to show that the T cells in the muscles are diverse but, compared with the peripheral lymphocytes, use a restricted set of V genes (8, 9). Subsequent nucleotide sequence analyses of the PCR-amplified TCR transcripts have shown that the infiltrating T cells contained clonally expanded populations. These results suggest that Ag-specific immune responses operate in the muscles affected by PM (10, 11). However, the pathological roles of the individual clones are obscure because clonally expanded T cells in the site of autoimmune inflammation could be either autoaggressive or regulatory (12).
Unavailability of large pieces of the PM-affected muscles for research
studies has hampered identification and isolation of myocytotoxic T
cell clones. Past studies on the T cells in PM have relied on the
analysis of cDNA derived from the muscles en bloc. Inevitably, the two
chains of the TCR heterodimers used by the expanded clones have never
been identified, nor have the effector molecules expressed by these
cells been characterized. The only exception is a variant type of PM,
where CD4-CD8- T cells
with TCR 
heterodimers dominated at the sites of inflammation
(13). Although molecular analysis has revealed nucleic
acid sequences of both chains of the heterodimers, it has not led to
determination of the responsible autoantigen (14). Thus,
autoantigens targeted by the aberrant immune reaction in PM have yet to
be identified.
It is known that CD8 T cells, unlike CD4 T cells, often expand clonally in the peripheral blood. This clonal expansion of CD8 T cells is commonly observed in healthy individuals, and is more frequent in the elderly than in the young (15, 16, 17, 18). Phenotypically, the expanded clones are confined in a CD28-CD57+ subset, which is known to be a group of cytotoxic effector T cells (17, 19, 20, 21, 22, 23, 24). Indeed, the CD8 T cells with this phenotype are common in individuals with previous exposure to viruses, and have responded well to in vitro stimulation with the viral Ags to which the hosts had been exposed (21, 24, 25).
Recently, tetrameric MHC/peptide complexes were developed to quantitate Ag-specific T cells with flow cytometry (26). This technique revealed that peripheral CD8 T cells expanded massively during the acute phase of viral infection and that expansion persisted during the chronic phase (26, 27, 28, 29). The expansion continued even after the viruses were cleared from the hosts (27). Similarly, clonal expansion of CD8 T cells specific to tumor Ags was found in peripheral blood from tumor-carrying patients (30). Thus, when CD8 T lymphocytes are stimulated by Ags, they tend to expand massively in the peripheral blood and to persist as large populations in an Ag-independent manner. In this regard, several reports have shown that memory CD8 T cells could survive or proliferate without specific MHC/Ag complexes that triggered them (31, 32, 33). Accordingly, it has been conceived that the clonal expansion of peripheral CD8 T cells represents footprints of the past and/or persistent viral infection and anti-tumor immunity.
As was reviewed earlier, PM seems to be mediated by cytotoxic CD8 T cells. The target striated muscles occur throughout the body. This made us assume that the autoaggressive CD8 T cells damaging the muscles might be expanded clonally in the peripheral blood from the patients. In the present report, we show that the clonal expansion of CD8 T cells was indeed more frequent in patients with PM than in age-matched controls. The CD8 T cell expansion in the PM patients persisted over time like that of virus Ag-specific CD8 T cells. Analysis of the TCR transcripts in the muscle biopsy specimens showed that some of the expanded clones were present in the inflamed muscles. These results show that organ-specific autoimmunity directly biases repertoire of T cells in systemic circulation, and argue that the expanded cells are candidates of autoaggressive T cells damaging the muscles. Using a flow cytometer, peripheral lymphocytes were readily isolated at a single-cell level. Subsequent sensitive RT-PCR techniques enabled us to show that these cells indeed express cytotoxic effector molecules. Thus, our findings should provide a direct clue to studying T cells involved in the immunopathology of PM and probably other CD8 T cell-mediated organ-specific autoimmune diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were isolated from seven normal donors (N1-N7) and eight
patients diagnosed to suffer from PM (P1-P8) (34). Age of
the patients ranged from 35 to 64 years old (mean 46), and the controls
were age-matched (mean 48). No patients had major complications except
for patient P2, who had bronchitis obliterans organizing pneumonia. The
muscle tissues were obtained from patients P1 and P2 during diagnostic
biopsy. All procedures in the present study were approved by the ethics
committee of the Tokyo Medical and Dental University. PBMC were also
collected from five children who had been already shown to have no
detectable clonal expansion in their peripheral T cell pool
(35). PBMC were stained with CD4 (SFCI12T4D11; Beckman
Coulter, Hialeah, FL) and CD8 (SFCI21Thy2D3; Beckman Coulter) Abs, and
300,000–800,000 CD4- or CD8-positive cells were collected by an EPICS
ELITE cell sorter (Beckman Coulter). To collect individual CD8 T cells
carrying TCR V2 or V17, the cells were stained with the CD8 Ab
together with anti-V2 (MPB2D5; Beckman Coulter) or V17
(E17.5F3.15.3; Beckman Coulter) Ab, and sorted by the autoclone
function of the same cell sorter.
Polymerase chain reaction
Total RNA were prepared from the sorted single positive cells and serial cryosections of the muscle specimens, and were converted to cDNA. Individual cells, which were sorted into microwells, were lysed in 8 µl of lysis buffer together with 50 JY cells (a gift from Dr. Miyuki Azuma, Tokyo Medical and Dental University) (36). First-strand cDNA were directly synthesized in the same wells (36).
The cDNA of the CD4 and CD8 cells were subjected to 20 cycles of
primary anchored PCR to amplify TCRB (37). The products
were reamplified with nested primers to determine TCRBV gene repertoire
(37). For TCR CDR3 length spectratyping (CLS)
(3), the anchored PCR products were amplified using a
panel of sense oligonucleotide primers specific to different BV subset
genes, and a fluoresceinated BC-specific antisense primer (Cb)
(37). The amplification reaction consisted of 25 cycles of
1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, with final
extension at 72°C for 7 min.
TCR clonotype-specific PCR for T cells in the muscle tissues and sorted
peripheral lymphocytes was performed with one of the sense primers
specific to TCRB CDR3 sequences of the clonally expanded T cells
(P1/1s: GCC AGC AGC GTA GAG GAC C, P1/2s: TCT GCA GTG CGC CCC CCG,
P1/6S1s: CAG CAG CTT GGC GGG GGG, P1/6S6s: GTG CTA GCA GCC CAG ACC C,
P1/16s: TGC CAG CAG CCA GCT GTA TG, P1/17s: GCC AGT AGT ACT AGC GGG AG,
P1/24s: GCC ACC AGC AGA GAT CGG G, P2/6S1As: TGT GTC CCC ATG GAG GCT
AG, P2/6S1Bs: GCC AGC AGC TCC CTC CAG, P2/6S5s: CCA GCA GAC GGG GCT TCG
A, P2/12S1s: CAT CAG CAA AAT CGG ACA GGG, P2/15s: CAC CAG TGA TTA TGG
GGG AAG, P2/16s: GCC AGC AGC CAA GGG TTC G, P2/18s: GTG CCA GCT CGT CGA
CCT TA, and P2/23s: GCA GCC TTC TAG CGG GGA AA) and a TCRBC-specific
antisense primer (Cd: CCT ATC CTG GGT CCA CTC GT). The reaction
consisted of 40 cycles of 30 s at 94°C, and 90 s at 50°C
or 59°C, followed by final extension at 72°C for 5 min.
TCR clonotype-specific PCR for the individual cells was performed in
two steps. Sense primers specific to BV subset genes used by the
expanded T cell clones and BC-specific antisense primer (Ce: ACT GGT
CGA CTG TGG GAG ATC TCT GCT TC) were used in primary PCR, which
consisted of 30 cycles of 45 s at 94°C, 45 s at 53°C, and
60 s at 72°C, followed by final extension at 72°C for 5 min.
One-fiftieth of the products was amplified with the corresponding
clonotype-specific primers described above and a nested BC-specific
antisense primer (Cc) (37). The reaction consisted of
30 cycles of 45 s at 94°C, 45 s at 59°C, and 60 s at
72°C, followed by final extension at 72°C for 5 min.
Perforin mRNA were PCR-amplified first with a perforin-specific sense primer (Pfos: TGG ACG TGA CTC CTA AGC CC) and an antisense primer (Pfoas: GCC GTG GAT GCC TAT GTT GAC). The reaction consisted of 45 cycles of 45 s at 94°C, 45 s at 59°C, and 60 s at 72°C, followed by final extension at 72°C for 5 min. One part per 5000 of the products was subjected to the nested PCR using a sense primer (Pfis: TGC ATG TGT CTG TGG CCG GC) and an antisense primer (Pfias: CAG TCA GGC AGT CCT CCA CC). The reaction consisted of 45 cycles of 45 s at 94°C, 45 s at 61°C, and 60 s at 72°C, followed by final extension at 72°C for 5 min.
TCR CLS
The fluoresceinated PCR products representing TCRB cDNA of the individual TCRBV subsets were fractionated on denaturing 7% polyacrylamide gel in a Hitachi SQ-5500 sequencer (Hitachi Electronics Engineering, Tokyo, Japan). The data were analyzed with the associated software to display histograms. Relative percentage of the TCRB transcripts of a given length to total TCRB transcripts in the BV subsets was calculated by dividing the fluorescence intensity of the corresponding peaks by the sum of the intensity of all peaks.
Standard profiles of TCRB CLS
Although TCR CLS profiles of T cell populations without clonal biases display roughly Gaussian-like distribution, we realized that the typical profiles of the individual TCRBV subsets had subtle differences (J. Nishio, M. Suzuki, N. Miyasaka, and H. Kohsaka, manuscript in preparation). This urged us to establish a standard profile for each BV subset by analyzing peripheral CD4 T cells from five children. Our previous studies showed that they had no detectable signs of clonal T cell expansion (35). The average distribution profiles derived from these donors displayed symmetrical patterns and were used as standard profiles.
It has been shown that expansion of a specific clone makes a peak of
the corresponding CDR3 length stand out of the standard distribution.
Arbitrarily, we set up criteria to identify such biased profiles; we
considered TCRBV subsets to contain clonal expansion when the CLS
profiles implied that a specific clone occupies >50% of the subsets.
This is when the frequency of a dominant peak (d%) is >50% plus half
of a standard peak of the corresponding CDR3 length (s%): d% > 50%
+ s/2%. This formula was derived from the fact that quantity of the
dominant peak should be Q x s/100 + 
Q, and also (Q +
Q) x d/100, where Q and Q stand for total original
population and increase owing to the expansion of a specific clone,
respectively. When d and s fulfill the formula, the relative frequency
of the expanded clone in the BV subset should be greater than
50%.
Deduction of clonal size
When TCRBV subsets are considered to contain clonal expansion,
the frequencies of the expanded clones in the total CD8 T cells were
estimated by multiplying the frequency of the TCRBV gene usage by
Q/(Q + Q), which is equal to (d - s)/(100 -
s)%.
In quantifying frequencies of TCRBV family gene usage by the PCR-ELISA, a few oligonucleotide probes were mixed to detect BV5, 6B, 12, 13, and 21 family genes (37). The probes for the BV5 or BV21 family genes hybridize to the same framework site of the corresponding TCRBV. However, the three BV6B oligonucleotides hybridize to different sites, yielding PCR products of different sizes. The same is true for the two BV12 oligonucleotides and the six BV13 oligonucleotides. To use these oligonucleotides for TCR CLS, we subdivided the three families according to the hybridization sites (BV6S2/4, BV6S3, and BV6S6 subsets; BV12S1 and BV12S2 subsets; BV13S1/4/6 and BV13S2/3/5 subsets). When estimating the frequencies of these subsets, the BV family gene usage measured with PCR-ELISA was divided in proportion to the fluorescence intensity of the PCR products generated with the corresponding primers.
Nucleotide sequence determination of TCRB transcripts
TCRB transcripts of BV subsets that displayed the biased
profiles were PCR-amplified with a BV-specific sense primer and the
Cc primer. The products were subcloned to generate BV
subset-specific TCRB cDNA plasmid libraries. Ten to 20 plasmid clones
of the TCRB CDR3 length of the dominant clonotypes were selected and
subjected to sequence determination by the Hitachi SQ-5500
sequencer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral CD4 and CD8 T cells from eight patients with active PM and seven age-matched normal donors were examined for clonal expansion with CLS of the TCRB transcripts. The TCR CLS technique has been applied, in many reports, to the analysis of T cell clonality (38). We sorted CD4 and CD8 T cell populations from the peripheral blood of patients and normal donors, and extracted their RNA, which were converted to cDNA. The cDNA were PCR-amplified with a fluoresceinated antisense TCRBC-specific primer and a panel of sense TCRBV-specific primers that were used previously for PCR-ELISA to analyze TCRBV repertoire (37). The products were fractionated on denaturing polyacrylamide gel. The gel was then analyzed by an automatic gel reader and associated software to generate TCR CLS profiles of individual BV subsets.
The results revealed that the spectratyping profiles of most BV subsets
in CD4 T cells displayed Gaussian-like distribution. In contrast, the
spectratyping profiles of the CD8 T cells often contained prominent
peaks that bias Gaussian-like distribution. This is consistent with the
previous observation that clonal expansion of CD8 T cells, but not that
of CD4 T cells, is common in the peripheral blood. To assess the bias
of each profile, we defined standard profiles for individual BV subsets
by analyzing peripheral CD4 T lymphocytes from children who had no
detectable clonal expansion in their peripheral blood
(35). Based on these standard profiles, we set up criteria
for "biased" profiles indicating that the TCRBV transcripts of an
expanded T cell clone account for more than half of total TCR
transcripts in the corresponding BV subset. The criteria were used to
enumerate BV subsets that should contain clonally expanded T cells.
Representative TCR CLS profiles of peripheral CD4 and CD8 T cells from
a normal donor (N1) and those from a patient (P1) are shown in Fig. 1
. The CD8 T cells from the patients had
more biased profiles than those from the normal donors. Fig. 2 summarizes the number of BV subsets
that showed the biased profiles. No more than one subset of CD4 T cells
from the normal donors fulfilled the criteria (the mean number ±
SD of the subsets: 0.1 ± 0.4). A similar number of the BV subsets
in CD4 T cells from the patients had the biased profiles (0.7 ±
0.5). The difference was not statistically significant (Fig. 2A). In contrast, many profiles of CD8 T cells were biased.
The numbers of the biased subsets in CD8 T cells from the normal donors
and from the patients were 2.0 ± 1.4 and 6.4 ± 2.1
(mean ± SD), respectively. The difference was statistically
significant (p < 0.001, Fig. 2B).
Thus, clonal expansions of CD8 T cells were more frequent in peripheral
blood from the patients than from the normal donors.
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. No particular BV subsets contained
clonally expanded populations.
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The cDNA of CD8 T cells from patients P1, P2, P3, and P4 were used
to generate BV-specific TCRB cDNA plasmid libraries. Sequence analyses
of 10–20 clones of the same TCRB CDR3 length as the peaks revealed
that 50–100% had an identical nucleotide sequence (Table II). An exception was the BV6S1 subset of
P2, where two different TCR sequences of the same CDR3 length dominated
with the ratio of 58:42%. Because no other clones appeared in the
subsets, both should have expanded. These results demonstrated that the
BV subsets with the biased profiles based on our criteria actually
contained expanded T cell clones. They also showed the TCRB junctional
sequences derived even from the same patients were diverse, and that no
conserved CDR3 motifs were shared by the clones (Table II).
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To estimate the frequencies of the expanded clones in the total
CD8 T lymphocyte pool, frequencies of the BV gene usage
were multiplied by calculated frequencies of the particular clones in
the BV subsets. The BV gene frequencies were quantitated by the
PCR-ELISA technique (37). The deduced
frequencies of the clones at the initial analyses ranged
from 0.05 to 19%. To study further whether the expanded CD8 T cell
clones persist for a long term, we examined the expanded clones from
patient P3 6 and 12 mo after the initial examination. Also, the
expanded clones from patient P5 6 mo later were examined. The myositis
had been ameliorated by corticosteroid administration at the time of
these follow-up analyses. We found that all clones that had been
expanded at the initial analysis still occupied considerably large
parts of the peripheral CD8 lymphocyte pool although they tended to
shrink during the long interval (Fig. 3).
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To investigate pathological relevance of the expanded T cell
clones, the inflamed muscles from patients P1 and P2 were examined for
presence of the expanded clones. First, serial cryostat sections were
prepared from biopsy specimens and stained with H&E. The tissues from
the two patients displayed marked infiltration of mononuclear cells,
but contained no large vessels or extravascular erythrocytes suggesting
hemorrhage (data not shown). Unstained sections were used for isolation
of RNA, which were converted to cDNA. Based on the TCRB CDR3 nucleic
acid sequences of the expanded clones, sense oligonucleotide primers
specific to TCR CDR3 sequences of clones 1–7 from P1 and clones 1–8
from P2 were synthesized. One of these primers together with an
antisense BC-specific primers (Cd) were used for TCRB
clonotype-specific PCR to detect the expanded clones. The same PCR was
performed to amplify the TCRB clonotypes in peripheral CD8 T cells from
patient P1 and in peripheral lymphocytes from patient P2. Quantities of
the template cDNA were standardized with respect to the amount of the
products generated by PCR amplification of the TCRBC gene.
After the PCR amplification, the products were fractionated with
agarose gel electrophoresis (Fig. 4).
Fifteen clonotype-specific PCR of peripheral lymphocyte cDNA generated
14 distinct bands of expected sizes. The amplification for clone 8 in
the peripheral lymphocytes from patient P2 did not yield visible
products. However, when the same PCR was performed as nested PCR after
preamplifying the cDNA with BV23- and BC-specific primers, it generated
significant amounts of the appropriate products (data not shown). The
requirement of such a sensitive PCR technique to detect this clone is
consistent with the fact that it was the least prevalent clone
(
0.05% in the total CD8 pool).
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Perforin expression by the expanded clones
It has been reported that virus-specific CD8 T cells expanded
clonally in the peripheral blood are functionally competent cytotoxic
cells, expressing perforin (24, 39). To investigate
whether the clonally expanded T cells in the PM patients also express
the effector molecules, clones 2 and 6 from P1 were examined for mRNA
expression using single-cell RT-PCR. From peripheral lymphocytes of the
patient, individual TCRV2- or V17-positive CD8 T cells were
sorted at a single-cell level. Each cell was lysed and used to
synthesize cDNA. The expanded clones were identified by TCRBV-specific
PCR followed by clonotype-specific PCR (Fig. 5A). PCR analysis of the same
cells revealed that both express perforin messages (Fig. 5B). Thus, it is probable that the expanded cells in the
patients, regardless of the localization in the muscle, were
differentiated CTL.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Unlike previous studies, our approach has given us a direct clue to isolating the myocytotoxic T clones from the peripheral blood. The results, derived from isolation of individual candidate T clones from the peripheral blood and subsequent analyses of gene expression, implied that they are functional cytotoxic cells. Using the nested PCR technique, we further succeeded in determining expressed TCRA gene (data not shown). Partly because of limited availability of the large muscle pieces, it has been difficult to study pathogenic T cells in PM. However, our methods facilitate examining function and phenotype of candidate pathogenic T cells involved in PM and probably in other CD8 T cell-mediated autoimmune diseases.
Clonal CD8 T cell expansion in normal donors was conceivably due to past viral infections or other immune insults. Because patients should have similar levels of clonal expansion irrelevant to the disease, the increase in the expansions should be attributable to the cytotoxic autoimmunity against the muscles. In accordance with this notion, we found that approximately half of the expanded T cell clones in the periphery were present in the inflamed muscles. Also, the mRNA analysis revealed that clonally expanded T cells, regardless of their presence in the inflamed muscles, expressed cytotoxic effector molecules. This is consistent with the previous notion that donors exposed to virus often carry clonally expanded virus-specific CTL in the peripheral blood (21, 24).
We assumed that the development and maintenance of myocytotoxic CD8 T cells should be analogous to those of virus- and tumor-specific CD8 T cells. Self-Ags, like viral Ags, are presented to CD8 T cells by bone marrow-derived APC at the lymph nodes, but not by nonprofessional APC expressing the antigenic peptides (40). Together with promiscuity of the Ag specificity required for survival and proliferation of the CD8 T lymphocytes, this may account for systemic expansion of the CD8 T cells specific to local Ags.
Clonal expansions in the CD4 T cell populations from the patients and the normal controls were far smaller than those in the CD8 T cells. This agrees with previous observations that clonal expansion of peripheral CD4 T cells is infrequent even in the elderly (15, 18). However, the results do not necessarily stand against the roles of CD4 T cells in the pathology of PM. Animal experiments have shown that CD4 T cells are essential for priming of naive autoreactive CD8 T cells, although effector CD8 T cell clones can often transfer the autoimmune tissue destruction adoptively (41, 42). Clonal expansion of peripheral CD4 T cells in the acute phase of viral infection was revealed by a sensitive MHC/peptide tetramer technique. However, the amplitude of the expansion was smaller than that of CD8 T cell expansion (43). Thus, it is most likely that the clonal perturbation of CD4 T cells did occur in the patients, but was under the detection threshold of our technique.
We analyzed peripheral CD4 and CD8 T lymphocytes from patients with Sjögren’s syndrome in the same manner. This disease is mediated by autoimmune destruction of the lacrimal and salivary glands, where CD4 T cells infiltrate dominantly. The results showed that clonal expansions in both populations were comparable to those in age-matched controls (data not shown). Additionally, peripheral lymphocytes from a few patients with dermatomyositis were also analyzed. Although it has been reported that CD4 T cells, in addition to CD8 T cells, contribute to tissue damage in DM, increase in clonal expansions was significant only in the CD8 T cell population (data not shown). These results altogether suggested that CD4 T cell involvement in the tissue-specific autoimmune diseases does not significantly bias clonal diversity of the peripheral CD4 T cells.
It has been reported that clonal expansion of peripheral CD8 T cells from patients with rheumatoid arthritis (RA) is frequent (18, 44, 45, 46). This is intriguing because CD4 T cells appear to be primarily involved in the pathology of RA. This view is based on the facts that the CD4 T cells infiltrate dominantly in the rheumatoid synovial tissues and that the most dominant genetic factor of the disease is the HLA class II gene, the products of which interact directly with CD4 T cells. Unlike in PM, the expanded CD8 T cell clones in RA do not appear to exert cytotoxic effects on the target organs, i.e., the synovial tissues. Alternatively, the CD8 T cell expansion in RA patients may reflect impaired homeostasis of peripheral T cell diversity and/or curious involvement of CD8 T cells in rheumatoid synovial inflammation (47, 48).
The nature of clonal expansion of CD8 T cells in the periphery has been delineated mainly by investigation of viral Ag-specific CD8 T cells. These studies used MHC/peptide tetramers to quantitate Ag-specific T cells, whereas we used a new method that combined the TCR CLS and PCR-ELISA. The size of the clonally expanded CD8 T cells in PM patients was comparable to that of the virus-specific T cell clones (27, 28, 49). Our method is useful when Ags recognized by clones of interest are unknown.
Previous studies of the peripheral lymphocytes from PM patients with a
PCR assay showed that they were clonally diverse (9, 10, 11).
This is consistent with our observation that TCR transcripts of all BV
subsets were successfully amplified by PCR. Subsequent TCR CLS was
required to reveal clonal expansions. This technique turned out to be
powerful to detect clonal expansions, especially when used with our
criteria. However, it has potential drawbacks. First, it cannot detect
two or more clonal expansions of different TCR length within one BV
subset because they individually cannot occupy >50%. Second, two
clones of the same TCR length will be counted as one. An example of
this was in clones 1 and 2 from patient P2 (Table II
). These events
seem rare and should be equally common among patients and controls.
Thus, this technique may have made us underestimate the number of the
expanded clones marginally, but should have warranted fair
comparison.
The clonal CD8 T cell expansion in the PM patients was stable over time. Six months after the active phase of the disease, most of the clones shrank slightly, possibly reflecting amelioration of the disease and/or the effects of corticosteroid administration. The persistent expansion is also reminiscent of clonal expansion elicited by viral infection (27, 28, 50). Disease activity of the patients was controlled well at the time of the second examination. It is not clear what kept the expanded clones from damaging the muscles. Probably, because of the treatment, the clones reduced their effector functions and/or the muscle tissues down-modulated proinflammatory molecule expression (51). It should be noted that activation of autoreactive CD8 cytotoxic T cell clones does not necessarily lead to autoaggressive tissue destruction (52). Also, the persistence of the autoaggressive clones may account for recurrence of the disease, which occasionally occurs during tapering of the treatment. As was shown in this follow-up analysis, outcome of the clonal size after 6 mo did not necessarily predict that after 12 mo. The clones found and those not found in the muscles did not show different trends in longitudinal studies (data not shown). Thus, the kinetics in size of the expanded clones did not offer critical information on their function.
The comparative RT-PCR analysis of the peripheral lymphocytes and the muscle biopsy samples from the same patients revealed that some clones were present in the inflamed muscle tissues. This held true whether the analyzed peripheral lymphocytes were sorted or unsorted for CD8 T cells. Although the analysis was, at most, semiquantitative, the results argue that CD8 T cells with specific TCRB clonotypes preferentially infiltrate in the muscles. It is somewhat expected that these T cell clones had diverse TCRB chain structures. We and others have found that the TCR structure of T cells reactive to the identical MHC/peptide combination could be diverse (53, 54). Also, more than one epitope could be involved within a single patient.
We believe that functional analysis of the clonally expanded cells is a key to identifying pathogenic CD8 T cells in PM. Isolation of the candidate myocytotoxic cells from the peripheral blood of PM patients and establishment of immortalized clones for precise functional analysis are now under way.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hitoshi Kohsaka, Department of Bioregulatory Medicine and Rheumatology, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. E-mail address: kohsaka.rheu{at}tmd.ac.jp
3 Abbreviations used in this paper: PM, polymyositis; CLS, CDR3 length spectratyping; RA, rheumatoid arthritis.
Received for publication March 12, 2001. Accepted for publication July 30, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ rearrangements may indicate antigen-driven selection. J. Clin. Invest. 91:2880.
variable and joining gene expression by muscle-infiltrating lymphocytes in the idiopathic inflammatory myopathies. J. Immunol. 152:2569.[Abstract]
/ receptor. N. Engl. J. Med. 324:877.[Abstract]
/ T cell receptor. J. Exp. Med. 176:1785.12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients. J. Exp. Med. 177:1623.3 TCR chains in the CD8+ T cell population of rheumatoid arthritis patients. J. Immunol. 156:852.[Abstract]
spectratyping in RA: evidence of clonal expansions in peripheral blood lymphocytes. Ann. Rheum. Dis. 57:319., interleukin-1, and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis. Arthritis Rheum. 43:336.[Medline]
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 K. Dornmair, N. Goebels, H.-U. Weltzien, H. Wekerle, and R. Hohlfeld T-Cell-Mediated Autoimmunity: Novel Techniques to Characterize Autoreactive T-Cell Receptors Am. J. Pathol., October 1, 2003; 163(4): 1215 - 1226. [Abstract] [Full Text] [PDF]
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M. F.G. van der Meulen, I. M. Bronner, J. E. Hoogendijk, H. Burger, W. J. van Venrooij, A. E. Voskuyl, H. J. Dinant, W. H.J.P. Linssen, J. H.J. Wokke, and M. de Visser Polymyositis: An overdiagnosed entity Neurology, August 12, 2003; 61(3): 316 - 321. [Abstract] [Full Text] [PDF]
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M. Hofbauer, S. Wiesener, H. Babbe, A. Roers, H. Wekerle, K. Dornmair, R. Hohlfeld, and N. Goebels Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis PNAS, April 1, 2003; 100(7): 4090 - 4095. [Abstract] [Full Text] [PDF]
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