| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires,
Service de Médecine Interne, and
Laboratoire de Neuropathologie, Hôpital Pitié-Salpêtrière, Paris, France;
Service de Dermatologie, Hôpital Saint-Louis, Paris, France;
¶ Service de Médecine Interne, Hôpital Avicenne, Bobigny, France; and
||
Service de Biostatistiques, Hôpital Tenon, Paris, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A sensitive method to study the T cell repertoire is to determine the
distribution of lengths of the TCR 
-chains (19).
Indeed, the generation of TCR -chain diversity occurs during
differentiation of T cell precursors in the thymus by rearrangement of
germline variable (V), diversity (D) and joining (J) gene segments of
the TCRB (or TCR ) locus (20). As a result of this
somatic recombination process, the length of the third
complementarity-determining region (CDR3) of the TCR -chain can vary
between two TCRs by up to eight amino acids. This CDR3 region is the
part of the TCR that is responsible for most of the interaction with
the peptide Ag presented by a MHC molecule, and thus confers most of a
given T cell’s antigenic specificity (21, 22).
Physiologically, CDR3 lengths follow a gaussian-like distribution that
reflects the great diversity of polyclonal TCR specificities within a
given BV (or V) family (23). By providing a global
representation of the T cell repertoire, this method, generally
referred to as immunoscope or spectratype, is particularly well suited
to detect clonal expansions associated with Ag-driven immune responses
(24). For instance, it has been used to demonstrate
clonally expanded T cells in the blood during acute viral infection
(25). The presence of such expansions results in an
overrepresentation of peaks of given lengths that accumulate above the
gaussian-like background of polyclonal T cells and may ultimately lead
to the appearance of a single peak in a given BV family.
By using the immunoscope approach, we observed severe perturbations of the peripheral T cell repertoire in the blood of PM but not DM patients. These perturbations occurred in the CD8+ subpopulation and reflected a subset of the clonal expansions present in muscle infiltrates of PM patients. These results indicate that the pathogenesis of PM and DM is indeed different and support the view that PM but not DM is an autoimmune CD8+ T cell-mediated disease. They also open perspectives for a noninvasive TCR-based follow-up of PM patients.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Twenty consecutive patients with a diagnosis of PM
(n = 10) or DM (n = 10) (Table I
), and
a group of healthy control subjects (n = 17,
male:female = 7:10, age = 32 ± 9 years) were included
in the study between December 1998 and February 2001. The institutional
ethics committee approved the study and patients gave written informed
consent. None of the patients had inclusion body myositis, cancer, or
other connective tissue disorders. The criteria for diagnosis of PM and
DM were based on studies by Bohan and colleagues (26, 27):
symmetric muscle weakness, increased serum muscle enzyme, myopathic
changes on electromyography, and typical histological findings on
muscle biopsy and/or characteristic dermatological manifestations
(heliotrope rash, periungual erythema, Gotron papules, and
poikiloderma) for DM. The diagnosis of PM or DM was considered definite
in all cases since each patient presented at least four manifestations.
All PM patients and 8 of 10 DM patients had a muscle biopsy. Since the
diagnosis based on the above-mentioned criteria was evident for DM04
and DM06, it was considered nonethical to perform muscle biopsy. Six of
10 PM and 7 of 10 DM patients were included at onset of their disease
before any treatment. Four of 10 PM and 3 of 10 DM patients were
included at relapse. The latter had previously been treated with
prednisone (seven of seven) with or without immunomodulator agents
(methotrexate, three of seven; i.v. Ig, two of seven; azathioprine, one
of seven; or cyclosporine, one of seven). At inclusion, they either had
no treatment (four of seven) or received only low-dose corticosteroids
(prednisone 
6 mg/day for at least 6 mo). Muscle weakness was
evaluated using a modified grading scale of the British Medical
Research Council: values from 0 to 11 were assigned to the strength of
8 muscles (neck flexors, trapezius, deltoid, biceps, psoas, maximus and
medius gluteus, quadriceps), yielding a theoretical maximum score of 88
points (28). Age, disease duration, muscle strength, and
creatine kinase levels were not statistically different between PM and
DM patients.
|
PBMC were separated on a Ficoll-Hypaque gradient (density 1.077 g/ml), washed in RPMI medium, and resuspended in sterile PBS. A minimum of 7 x 106 PBMC was used for RNA extraction. Available samples of liquid nitrogen-frozen muscle biopsies performed for diagnostic purposes in PM patients were used for RNA extraction and further repertoire analysis using BV-BC PCR amplification and BJ-primed run-off reactions.
TCR CDR3 size analysis
Experiments were performed as described elsewhere (19, 29, 30). Analysis was performed in 14 different BV families that cover >70% of the T cell repertoire of healthy subjects (31). Briefly, cellular RNA was reverse transcribed into cDNA using oligo(dT) and Moloney murine leukemia virus reverse transcriptase (RT) (Life Technologies, Rockville, MD). After phenol/chloroform extraction, a quantity of cDNA corresponding to 300 ng of total RNA was amplified by PCR in a 50-µl reaction using one primer for each of the 14 BV studied and a common BC primer (Genset, Paris, France). The final concentration was 0.5 µM for each primer, 0.2 mM dNTP (except for 0.4 mM dUTP), 2 mM MgCl2 in PCR buffer (Boehringer Mannheim, Mannheim, Germany) in the presence of 2.5 U of Taq polymerase (Boehringer Mannheim). The amplification was performed on a thermal cycler (Thermo Hybaid, Ashford, U.K.) with an initial denaturation step at 94°C for 5 min, then 40 of the following cycles: 94°C for 1 min, 60°C for 1 min, 72°C for 2 min, and a final step at 72°C for 10 min. Amplification was verified by agarose gel electrophoresis. Each BV-BC PCR product was subjected to 10 run-off cycles primed with a nested fluorophore-labeled BC or a BJ primer in the presence of 1 U of Taq polymerase. Each run-off product was denatured and loaded on a gel for fluorescence analysis using an Applied Biosystems 377 sequencer (PerkinElmer, Norwalk, CT). Raw data were analyzed with Immunoscope 3.01b software (Loginserm, Paris, France) (19).
The method for quantifying T cell repertoire perturbations was adapted
from Ref. 32 . Briefly, CDR3 length profiles are translated
into a p distribution
pk(i) for each peak
i (i = 1–8) of a given
BVk as a function of the area under the curve
(expressed in relative fluorescence intensity, RFI) with normalization
so that 
i
pk(i) = 100%. The extent
of perturbation Dk(i) for each
peak i is computed by the distance
Dk(i) between
pk(i) values from the sample
and
prefk(i)
values from a reference distribution derived from repertoire analysis
of eight cord blood samples. The perturbation
Dk for a given BVk
is then calculated as
i|Dk(i)|/2,
so that Dk = 0 or 100% for a p
distribution equal to or completely nonoverlapping with the reference,
respectively. The mean of individual BVk
perturbations yields the average perturbation D =
k
Dk/n for all n
BVk families (n = 14). Quality
assurance of data processing was provided by parallel data entry by two
independent investigators followed by separate calculations using two
independently programmed Excel spreadsheets (Microsoft, Redman,
WA).
FACS purification of T cell subsets
PBMC were incubated with FITC-labeled anti-CD8 and PE-labeled anti-CD4 mAbs (Coulter Immunotech, Marseille, France), washed, filtered through a nylon Falcon 2350 cell strainer (BD Biosciences, Mountain View, CA) and sorted using a FACStarPlus (BD Biosciences). Sorted CD4+ and CD8+ cells were recovered in RPMI medium supplemented with 50% FCS. Cells were then washed in PBS and placed in RNAble for RNA extraction and further T cell repertoire analysis. The purity of sorted cell populations was always >98%.
Statistical analyses
Statistical analyses were performed using Statview software (SAS Institute, Cary, NC). The Mann-Whitney U test was used to compare data. The confidence interval for specificity and sensitivity was computed with correction for continuity (33, 34).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Using the immunoscope method, the distribution of TCR CDR3
lengths is visualized as a series of peaks separated by a distance of
three nucleotides corresponding to in-frame transcripts. A
physiologically diverse repertoire yields a gaussian-like profile,
whereas the presence of T cell clonal expansions manifests as a
distribution skewed by the accumulation of larger peaks. We studied the
blood T cell repertoire of 10 PM and 10 DM patients. Most of them (17
of 20) were not receiving any treatment at the time of inclusion while
a minority (3 of 20) were receiving only very low-dose prednisone
(Table I
).
All PM patients exhibited severely perturbed CDR3 distributions (Fig. 1). One or more discrete peaks
accumulated within different BV families of each PM patient. These T
cell expansions appeared to be oligoclonal and, for a given patient,
occurred in several BV families. From one patient to another, these
expansions did not concern the same BV and no recurrence of a
particular peak was found. In marked contrast, all patients with DM
presented a gaussian-like distribution in the vast majority of their BV
families (Fig. 2). Nevertheless, in a
minority of DM patients, some expansions were occasionally observed in
a few BV families.
|
|
To perform statistical analyses, we quantified the extent of
perturbation of the T cell repertoire. For this, we first established
reference values for the CDR3 length p distribution of each
BV family in eight cord blood samples. Cord blood was chosen because it
provides a representation of the theoretical unperturbed repertoire of
naive preimmune T cells (35). The extent of perturbation,
Dk, for each of the
BVk families of a given sample, is calculated as
a function of the difference between the p distribution of
this sample and the reference p distribution (Fig. 3a). Furthermore, the average
perturbation D can be calculated as the mean of the
Dk values of the 14
BVk families studied. This approach provides an
objective and quantitative determination of repertoire perturbations
with D values varying from 0 (absence of perturbation) to
100% (theoretical maximum perturbation).
|
b). Furthermore,
there was no overlap in the distribution of D values from PM
and DM patients (range, 11.4–20.9% for PM and 4.4–10.7% for DM).
Thus, a threshold Dthres = 11% could
be defined which allowed to distinguish PM and DM with an estimated
sensitivity and specificity of 100% (confidence interval,
66–100%).
At the individual BV level, statistically significant perturbations of
the repertoire of PM patients as compared with DM patients were
observed in 12 of 14 BV families (Fig. 3c). In BV2 and BV15
families, although the difference did not reach statistical
significance, there was a trend toward a higher level of
perturbation in PM as compared with DM patients. Together, the
results of qualitative and quantitative repertoire analyses
strongly suggest that the physiopathological process underlying PM and
DM is different, with a central role for clonally expanded T cells in
PM but not in DM.
Repertoire analysis of blood CD4+ and CD8+ T cell subsets
The simplest explanation for the results observed in PM patients
is that cytotoxic T cells responsible for muscle cell injury
recirculate from muscle to blood where they can be detected by the
immunoscope method. This implies that the observed repertoire
perturbations should be primarily due to clonal expansion of
CD8+ T cells, since this subset is largely
predominant in muscle lesions (8, 16, 17). To test this
hypothesis, we studied the repertoire of sorted
CD4+ and CD8+ T cells in
three patients for whom sufficient numbers of PBMC were available
(PM-05, PM-06, and PM-09). This analysis revealed that the
perturbations observed in unsorted PBMC were due to clonal expansions
of CD8+ but not CD4+ T
cells (Fig. 4a). In the
CD8+ subset, the average perturbation
D reached the high value of 37.3 ± 1.3%, which was
significantly greater than that in unsorted T cells (Fig. 4b). In contrast, in the CD4+
subset, D values were significantly lower than in unsorted T
cells and, notably, remained inferior to
Dthres = 11%. Thus, blood T cell
repertoire perturbations in PM patients are due to the clonal expansion
of CD8+ T cells.
|
The presence of clonally expanded CD8+ T cells in the blood of PM patients suggests that they belong to the same population of autoaggressive T cells that cause muscle injury. We therefore aimed to determine whether the blood T cell expansions were due to recirculation of muscle-infiltrating T cells. For this, we analyzed the repertoire of muscle infiltrates in four PM patients for whom a frozen biopsy was available and compared it to their blood repertoire. Since two different clones using the same BV gene segment can fortuitously share the same CDR3 length, which would therefore yield undistinguishable peaks, analyses were performed at the BV-BJ rather than the BV-BC resolution so as to better ascertain clonal identity.
This comparative analysis revealed that most of the clonal expansions
observed in the blood of PM patients corresponded to peaks also found
in their muscle infiltrates. For example, this was the case for the
BV3-BJ2.2 peak in PM-01, the BV1-BJ2.7 peak in PM-04, the BV15-BJ2.5
peak in PM-06, and the BV15-BJ2.1, BV15-BJ2.3, and BV15-BJ2.7 peaks in
PM-08 (Fig. 5). Nevertheless, some peaks
present in muscle infiltrates could not be detected in the blood. For
instance, this was the case for the left BV3-BJ1.5 peak in PM-01 muscle
or the right BV1-BJ2.5 peak in PM-04 muscle which were not found in the
blood (Fig. 5). Finally, in some instances a peak present in blood was
not found in muscle, such as the right BV15-BJ2.3 peak in PM-06, but
this situation was rare and it is presumable that such peaks correspond
to clonal expansions unrelated to the PM disease. For PM-04 and PM-06,
the blood sample was obtained at the time of diagnosis. It should be
noted that the blood T cell repertoire was determined at relapse in
PM-08 and was compared with that of a previous muscle biopsy performed
at the time of diagnosis, i.e., 33 mo earlier. Strikingly, the peaks
detected in the blood at relapse were identical to those detected
earlier in muscle. Similarly, several clonal expansions persisted over
a 7-mo period in PM-01 (Fig. 5). Together, these results indicate that
the repertoire perturbations observed in the blood of PM patients
reflect part of the clonal expansions present in the muscle infiltrates
and suggest a long-term persistence of repertoire anomalies in PM.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In contrast to normal muscle tissue, myocytes of PM patients express
HLA class I molecules at their surface (37, 38, 39), allowing
the presentation of muscular autoantigens to cytotoxic
CD8+ T cells. Furthermore, it was recently
reported that forced expression of MHC class I molecules in skeletal
muscle of young mice induces a PM-like disease accompanied by
mononuclear cell muscle infiltrates (40). In the present
study, multiple CD8+ T cell expansions were found
in PM patients, suggesting that these cells may target several
autoantigens, even at an early stage of the disease. Previous studies
had already observed the presence of in situ clonally expanded T cells
in muscle of PM patients. Notably, a histochemical analysis using mAbs
revealed the overrepresentation of V
2 and V3 TCRs within the
endomysium (13). Also, studies based on PCR analyses other
than immunoscope have reported different T cell repertoire biases:
overusage of V1, V5, V1, and V15 (12), V1
and V6 (14), or V33, V5, and V13 TCRs
(15). The T cell expansions identified in the present
study did not preferentially occur in selected BV families but rather
differed from one patient to another, in accordance with these previous
reports. MHC polymorphism may partly account for the diversity of this
T cell response since patients were not selected for HLA groups.
We found that most blood T cell expansions were also detected in the muscle infiltrates. In contrast, some muscle-infiltrating T cells were not detected in the blood, suggesting that only a subset of these cells actually recirculates outside the target tissue. The physiopathological significance of this finding remains to be established. It might be hypothesized that the expansions found in the blood mainly reflect the T cells causing muscle injury. Nevertheless, it cannot be ruled out that some truly aggressive T cell clones do not recirculate because they find high-affinity ligands in muscle. We are aware that our data do not formally prove that the expanded T cells clones found in muscle and blood of PM patients are actually muscular autoantigen-specific cytotoxic T cells. Conclusions on the functional importance of expanded clones to the disease process will benefit from further isolation of these T cells and testing them in functional assays. Nevertheless, along with previous morphological observations (1, 41, 42) and the demonstration that T cell lines from PM patients can be cytotoxic against autologous myotubes (43), our results strongly argue for a muscle-specific T cell-dependent autoimmune mechanism in PM. In support of this, we found that the blood T cell clones which had expanded in vivo responded to IL-2 in cell culture (data not shown), suggesting that they might be effector cells that had been primed in vivo, presumably upon recognition of muscular autoantigens.
When we analyzed the blood BV usage by FACS, a technique that provides
quantitative data on the T cell repertoire through only 
20
parameters, we were unable to discriminate PM patients from DM patients
or healthy controls (data not shown). This indicates that FACS analysis
underestimates the extent of repertoire perturbations as compared with
immunoscope, in agreement with a recent report performed in
HIV-infected patients (44). Using sequencing of
PCR-amplified products, Bender et al. (15) demonstrated
the presence of multiple T cell clonal expansions in muscle
infiltrates. Among these expansions, three TCR sequences were searched
for in the blood but could not be found by the authors. Thus, when
compared with other methods, the immunoscope approach, which analyzes
TCR diversity through 100-2000 parameters (24), allows a
global estimation of the T cell repertoire and a highly sensitive
detection of overrepresented T cell populations.
In marked contrast to PM patients, the level of repertoire perturbations in DM patients remained undistinguishable from that of healthy controls. Rarely, some skewed distributions were observed in a minority of BV families, but this most likely reflects the occurrence of clonal expansions that physiologically accumulate with age (45, 46, 47, 48). Thus, the present results strongly argue for essential differences in the pathogenesis of these two inflammatory myopathies, with a central role for CD8+ T cell-mediated autoimmunity in PM but not DM. It can be argued that anti-synthetase autoantibodies may be present in PM patients while some CD8+ T cells can be found in the inflammatory infiltrates of DM patients. Nevertheless, the frequency of PM patients with anti-synthetase autoantibodies is low (49, 50) and the presence of T cell muscle infiltrates in DM patients does not lead to detectable repertoire anomalies in the blood. Furthermore, since quantitative repertoire analysis allowed us to discriminate PM and DM with high sensitivity and specificity, this approach may potentially be helpful in some cases for the differential diagnosis between PM and DM. So far, the immunoscope method is still relatively labor intensive, but it is likely that the development of more automated systems such as capillary sequencers will simplify repertoire analysis and allow a more routine use in the near future.
Relapse is frequent in the course of PM, notably during the period of corticosteroid dose de-escalation. Prediction of PM recurrence remains challenging because follow-up is based mainly on clinical examination (repeated muscle weakness scales) and creatine kinase levels, all of which worsen concomitantly with relapse. Furthermore, these parameters reflect the consequences rather than the cause of muscle cell injury. The ability to trace autoaggressive T cells in the blood opens perspectives for monitoring repertoire anomalies under immunomodulatory treatments and the possibility of correlating their evolution with clinical status. If it is found that a disappearance or decrease of T cell perturbations correlates with recovery and a reappearance or increase with relapse, immunoscope analysis would provide a helpful tool for follow-up and prediction of recurrence in PM patients. In this respect, the cases of patients PM-01 and PM-08 already contribute some preliminary evidence that the T cell perturbation at relapse may be similar to that found earlier at diagnosis. Finally, the present approach may also facilitate further identification of the still unknown T cell autoantigen(s) in PM.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Olivier Boyer, Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l’hôpital, F-75013 Paris, France. E-mail address: olivier.boyer{at}chups.jussieu.fr
3 Abreviations used in this paper: PM, polymyositis; DM, dermatomyositis; CDR3, third complementarity-determining region; RFI, relative fluorescence intensity.
Received for publication May 17, 2001. Accepted for publication July 19, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, interleukin-1, and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis. Arthritis Rheum. 43:336.[Medline]
/ 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]
chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA 90:4319. T cell receptor locus. Science 272:1755.[Abstract]
T cell receptors. Science 288:1135.
- T-cell repertoire: characteristics of a polyclonal and naive but completely formed repertoire. Blood 91:340. T cell repertoire due to different clonal expansions in the CD4+ and CD8+ subsets. Int. Immunol. 10:1281.This article has been cited by other articles:
|
|
 |
|
  S. Calbo, H. Delagreverie, C. Arnoult, F.-J. Authier, F. Tron, and O. Boyer Functional Tolerance of CD8+ T Cells Induced by Muscle-Specific Antigen Expression J. Immunol., July 1, 2008; 181(1): 408 - 417. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Salajegheh, G. Rakocevic, R. Raju, A. Shatunov, L. G. Goldfarb, and M. C. Dalakas T cell receptor profiling in muscle and blood lymphocytes in sporadic inclusion body myositis Neurology, October 23, 2007; 69(17): 1672 - 1679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Dimitri, O. Benveniste, O. Dubourg, T. Maisonobe, B. Eymard, Z. Amoura, L. Jean, K. Tiev, J.-C. Piette, D. Klatzmann, et al. Shared blood and muscle CD8+ T-cell expansions in inclusion body myositis Brain, April 1, 2006; 129(4): 986 - 995. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Nicolls, L. Taraseviciene-Stewart, P. R. Rai, D. B. Badesch, and N. F. Voelkel Autoimmunity and pulmonary hypertension: a perspective Eur. Respir. J., December 1, 2005; 26(6): 1110 - 1118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Aleksza, A Szegedi, P Antal-Szalmas, B Irinyi, L Gergely, A Ponyi, J Hunyadi, S Sipka, M Zeher, G Szegedi, et al. Altered cytokine expression of peripheral blood lymphocytes in polymyositis and dermatomyositis Ann Rheum Dis, October 1, 2005; 64(10): 1485 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and the DM
(
) and PM (•) patients. Numbers are mean ± SEM for each
group. The difference in average perturbations D between
DM and PM patients is statistically significant (Mann-Whitney
U test), whereas the difference between the control and
the DM groups is not. This analysis revealed a threshold
Dthres = 11% discriminating the
D value distribution of PM patients from that of DM
patients (dotted line). c, Comparison of T cell
repertoire perturbations for each BV. Individual perturbations
Dk for each BVk are
compared between DM (
