Abstract
Clonal expansions of CD8+ T cells have been identified in muscle and blood of polymyositis patients by PCR techniques, including T cell receptor (TCR) complementarity-determining region (CDR)3 length analysis (spectratyping). To examine a possible pathogenic role of these clonally expanded T cells, we combined CDR3 spectratyping with laser microdissection and single-cell PCR of individual myocytotoxic T cells that contact, invade, and destroy a skeletal muscle fiber. First, we screened cDNA from muscle biopsy specimens by CDR3 spectratyping for expanded TCR β chain variable region (BV) sequences. To pinpoint the corresponding T cells in tissue, we stained cryostat sections with appropriate anti-TCR BV mAbs, isolated single BV+ T cells that directly contacted or invaded a muscle fiber by laser-assisted microdissection, and amplified their TCR BV chain sequences from rearranged genomic DNA. In this way, we could relate the oligoclonal peaks identified by CDR3-spectratype screening to morphologically characterized microdissected T cells. In one patient, a large fraction of the microdissected T cells carried a common TCR-BV amino acid CDR3 motif and conservative nucleotide exchanges in the CDR3 region, suggesting an antigen-driven response. In several cases, we tracked these T cell clones for several years in CD8+ (but not CD4+) blood lymphocytes and in two patients also in consecutive muscle biopsy specimens. During immunosuppressive therapy, oligoclonal CDR3-spectratype patterns tended to revert to more polyclonal Gaussian distribution-like patterns. Our findings demonstrate that CDR3 spectratyping and single-cell analysis can be combined to identify and track autoaggressive T cell clones in blood and target tissue. This approach should be applicable to other inflammatory and autoimmune disorders.
The detection and tracking of autoaggressive T cells represents a major challenge in human autoimmune diseases. The only way to identify antigen-specific clones of T cells is via their cell-associated clonally unique antigen-specific T cell receptors (TCRs). In this regard, spectratyping of the TCR complementarity-determining region (CDR)3 has proved to be a sensitive and practical screening technique for studying T cell repertoires (1). However, the mere occurrence of clonal expansions of T cells in blood and/or target tissue does not imply their pathogenic relevance. Clonal T cell expansions, especially of CD8+ T cells, are commonly observed in blood of normal, especially older subjects (2–5), and blood T cells might easily “contaminate” tissue samples. Furthermore, activated T cells of irrelevant specificity may be nonspecifically trapped at sites of inflammation (6, 7). To formally relate clonal expansions to potentially pathogenic T cells, a screening technique like CDR3 spectratyping should be combined with a method that allows the identification and characterization of individual pathogenically relevant T cells in morphologically intact tissue.
In the present study, we addressed this problem in polymyositis (PM), an inflammatory muscle disorder that represents an ideal paradigm of a CD8+ T cell-mediated disease. In PM, CD8+ T lymphocytes surround, invade, and apparently destroy HLA class I-expressing muscle fibers (8–12). Typically, the inflammatory infiltrate contains putative effector CD8+ T cells, which make tight membrane contact with the muscle fiber, orient their cytotoxic granules toward the contact zone (13), and sometimes deeply invade into the HLA class I-positive target fiber (14). Bystander cells, which are not in direct contact with muscle fibers, are located in interstitial spaces. Because extensive searches for viral proteins and genetic material were negative in the past (15–18), it is now assumed that myocytotoxic CD8+ T cells recognize genuine, as yet unknown, autoantigen(s) presented on the myofiber surface. Consistent with an autoimmune T cell-mediated pathogenesis, immunosuppressive treatment with corticosteroids and/or cytotoxic agents is usually at least partially effective.
We used a scaled combination of CDR3 spectratyping and single-cell PCR analysis of muscle-infiltrating cells. First, we screened paired blood and muscle samples from PM patients by CDR3-spectratyping analysis of all TCR β chain variable region (BV) (Vβ) families to identify candidate T cell clonal expansions shared between the two compartments. Next, we stained muscle sections with the corresponding anti-TCR BV mAbs to identify T cells expressing the appropriate TCR BV family member. We selected T cells that showed the typical autoaggressive behavior, i.e., directly contacted or invaded a muscle fiber, for laser microdissection and single-cell PCR. In this way, we could relate the clonal expansions identified by CDR3 spectratyping to individual T cells contacting or invading a muscle fiber.
Methods
Clinical Samples.
Diagnostic muscle biopsy specimens were obtained from eight patients with PM (patients PM1–8) and two patients with myositis associated with antibodies against tRNA synthetase (patients Jo1 and Jo2) (Table 1).
Table 1.
Overview of the patients and clinical material investigated in the study
| Patient code | Age, sex | Type sample, mo/yr | Concurrent treatment |
|---|---|---|---|
| PM 1 | 66, m | Muscle* (08/91) | ø |
| PM 2 | 76, m | Muscle* (83) | ø |
| Muscle (10/98) | C + AZA (since 6/98) | ||
| CD8 (11/98) | |||
| CD4 (11/98) | |||
| CD8 (06/99) | C + AZA | ||
| CD4 (06/99) | |||
| PM 3 | 70, m | Muscle (04/92) | ø |
| Muscle (05/99) | C + AZA | ||
| CD8 (05/00) | C + AZA (until 02/00) | ||
| CD4 (05/00) | |||
| PM 4 | 63, m | Muscle (06/00) | ø |
| CD8 (06/00) | |||
| CD8 (02/02) | C | ||
| CD4 (02/02) | |||
| PM 5 | 47, m | Muscle* (07/98) | ø |
| CD8 (07/98) | |||
| CD8 (06/99) | AZA | ||
| PM 6 | 78, m | Muscle (03/99) | Ø |
| CD8 (06/99) | AZA | ||
| CD8 (08/00) | |||
| PM 7 | 42, f | Muscle (07/98) | ø |
| CD8 (07/98) | |||
| CD8 (09/98) | C + AZA | ||
| PM 8 | 85, f | Muscle (11/97) | ø |
| CD8 (01/98) | ø | ||
| CD4 (01/98) | |||
| CD8 (10/98) | ø | ||
| CD4 (10/98) | |||
| Jo 1 | 41, m | Muscle* (06/96) | ø |
| CD8 (06/96) | |||
| CD4 (06/96) | |||
| CD8 (07/96) | C | ||
| CD4 (07/96) | |||
| CD8 (02/98) | Cy | ||
| Jo 2 | 63, f | Muscle (05/97) | ø |
| CD8 (05/97) | |||
| CD8 (03/98) | C | ||
| CD8 (09/98) | AZA |
AZA, azathriopine; C, corticosteroids; CD, cluster of differentiation; Cy, cyclophosphamide; Jo 1, myositis associated with antibodies against tRNA synthetase; PM, polymyositis; ø, no treatment; f, female; m, male.
Biopsy specimens analyzed by single-cell analysis and CDR3 spectratyping; all other samples were investigated by CDR3 spectratyping.
Isolation of RNA and cDNA Synthesis.
Total RNA was extracted from muscle and blood cells by using the TRIzol-LS reagent (GIBCO/BRL). For cDNA synthesis, oligo-(dT) and reverse transcriptase (SuperScript II, GIBCO/BRL) were used according to the recommendations of the suppliers.
CDR3 Spectratyping.
CDR3-spectratype (“immunoscope”) analysis of TCR BV families was performed essentially as described (19–21). For the detection of selected clones (as indicated) on an oligoclonal background, clone-specific β chain joining region (BJ) primers, complementary to part of the J and part of the N-D-N region, were used. The sequence identity of the amplification product was determined by direct sequencing as above. The sequences of the clone-specific BJ primers used were: PM2-BV13.1-BJ1.5A, ATG CTG GGG CTG GCT TAA TCC; and PM2-BV13.1-BJ2.5B, GAA GTA CTG GGT CTT CTT CCC.
Immunohistochemical Staining of Cryostat Sections.
Immunohistochemical staining of 10-μm muscle cryostat sections was performed by using standard PAP or APAAP procedures with the primary antibodies indicated.
Microdissection of Single T Cells in Tissue Sections.
Laser microdissection (22) was used for the isolation of pathogenically relevant T cells from muscle sections (Fig. 1 b and c). The localization of each excised cell was documented with a video camera. As negative controls, myofibers not invaded by lymphocytes were excised and subjected to single-cell PCR (Table 2).
Figure 1.
CDR3 TCR BV spectratype analysis and single-cell PCR of myocytotoxic T cells from patient PM2. (a) CDR3 peaks appeared in the BV13.1-BJ1.5, BV13.1-BJ2.5, and BV13.1-BJ2.7 reactions. The top two lines are from two consecutive muscle biopsies, obtained in 1983 and 1998. The third and fourth lines are from CD8+ PBMC, obtained in 1998 and 1999. The bottom line is from CD4+ T cells obtained in 1998. The BV13.1-BJ2.5 and BV13.1-BJ2.7 peaks (Center and Right) occurred in both muscle biopsies. The BV13.1-BJ2.7 peak was clearly detectable in the first (1998) blood sample (sequence confirmed by cycle sequencing Δ) but had essentially disappeared in the 1999 blood sample after continued immunosuppressive therapy. In contrast, the BV13.1-BJ2.5 peak was barely detectable in the blood samples. However, the corresponding CDR3 sequence could be amplified from the 1998 and 1999 CD8 cDNA samples by PCR with clone specific primers (□). The BV13.1-BJ1.5 peak (Left) was undetectable in the 1998 muscle and all blood samples, and the corresponding sequence could not even be amplified with clone-specific primers. Note that none of these clones was detectably expanded in CD4+ T cells, which showed a Gaussian CDR3 pattern (Bottom). Double peaks (asterisks) are caused by a partially degenerated primer. Triangles indicate peaks that were directly sequenced. A square indicates a peak that was confirmed with a clone-specific primer. (b) Example of laser-microbeam dissection. The T cell (arrow), stained with anti-BV13.1 mAb, invades a muscle fiber (Left). The T cell was excised (Middle) and catapulted into a PCR tube (Right). The morphological features are slightly blurred, because frozen sections were mounted on a polyethylene foil and viewed without coverslip for microdissection. (c) Examples of muscle-invading or -contacting T cells from the biopsy specimen (1983) of patient PM2. The T cells were stained with anti-TCR BV13.1 mAb. They contained the same sequences that were identified by CDR3 spectratyping in a. RFI, relative fluorescence intensity.
Table 2.
Overview of the results obtained by single-cell analysis
| Patient code | No. of catapulted cells | No. of obtained TCR sequences (% of catapulted cells) | Amino acid sequences of expanded clones (CDR3− region)† | No. of catapulted cells containing expanded sequence (% of identified sequences) |
|---|---|---|---|---|
| PM 1 | 112 | 22/112 (19.6) | BV13.1-CASSYSPQGWGYT-BJ 1.2 | 8/22 (36.4) |
| BV13.1-CASGVRGGYQPQ-BJ 1.5 | 4/22¶ (18.2) | |||
| PM 2 | 587 | 93/587* (15.8) | BV13.1-CASASASGNT-BJ 1.3‡ | 3/93 (3.2) |
| BV13.1-CASSYSGGLSQPQ-BJ1.5 | 12/93 (12.9) | |||
| BV13.1-CASSYSGGYQPQ-BJ1.5‡ | 2/93 (2.2) | |||
| BV13.1-CASSYRATDT-BJ2.3‡ | 6/93 (6.5) | |||
| BV13.1-CASSWGGKKTQY-BJ 2.5 | 20/93 (21.5) | |||
| BV13.1-CASSYR_§ YEQ-BJ2.7 | 17/93 (18.3)§ | |||
| PM 5 | 57 | 5/57 (8.8) | BV11-CASSGQGVSLGNT-BJ1.3 | 4/5 (80) |
| Jo 1 | 402 | 48/402 (11.9) | BV13.1-CASSRTDRDGYT-BJ1.2‡ | 5/48‖ (10.4) |
| BV13.1-CASSYSGTVGQPQ-BJ1.5 | 6/48 (12.5) | |||
| BV13.1-CASSRTNLGDTGE-BJ2.2‡ | 6/48 (12.5) | |||
| Negative controls | ||||
| Muscle fibers | 20 | 0 | ||
| Reagent controls | 55 | 0 |
Four samples revealed two distinct TCR sequences.
Bold amino acids represent the CDR3 N-D-N region, followed by the first three amino acids of the indicated BJ region.
Sequence identified only by single-cell PCR but not detectably expanded in CDR3 spectratyping.
Variable amino acids occurred in this position (see Table 3).
One cell without muscle contact.
Two sequences identifiable but not fully readable.
PCR Amplification of TCR BV-Gene Rearrangements from Single Cells.
Expanded TCR BV candidates were first identified by CDR3 spectratyping. This allowed us to identify cells expressing the corresponding TCR BV family by immunohistochemistry. Positively stained (TCR BV+) cells were isolated by micromanipulation (see above). Subsequently, the first round of PCR could be limited to the corresponding BV-specific primer, plus a BJ-primer mix.
Rearranged TCR BV chains were amplified from DNA of single T cells essentially as described (19, 23), with minor modifications. Positive products were sequenced as described above. Negative controls containing myofibers or reagents alone documented the absence of contaminating material from tissue sections and reagents (Table 2).
Results
Combination of CDR3 Spectratyping and Single-Cell PCR Analysis for Identification of Potentially Autoaggressive T Cells in Muscle Tissue.
To validate the combination of TCR CDR3 spectratyping and single-cell PCR analysis, we first studied a case of PM that we had previously investigated by using a combination of immunocytochemistry, PCR analysis of whole tissue, and subsequent cloning of the PCR products (24) (patient PM1 in the previous and present study). In this patient, a few clones of BV13.1+ cells predominated in the population of autoaggressive (myocytotoxic) T cells (24). Using CDR3 spectratyping, we confirmed the same TCR sequences that we had previously identified with inverse and family PCR (data not shown). Next we applied single-cell microdissection to pinpoint the TCR BV13.1+ autoaggressive T cell clone(s) in muscle tissue. To this end, we stained muscle sections from patient PM1 with anti-BV13.1 mAb and identified individual BV13.1+ T cells that directly contacted or invaded a muscle fiber. A total of 112 T cells were individually picked from muscle sections of patient PM1, and TCR BV chain rearrangements of single molecules of genomic DNA were amplified by single-cell PCR. Twenty-two cells yielded TCR sequences. Eight of the 22 cells contained the previously described (24) BV13.1-YSPQGW-BJ1.2 sequence and four cells, the previously described BV13.1-GVRGGY-BJ1.5 sequence (Table 2). The other 10 cells expressed BV13.1 sequences with different CDR3 regions that had not been previously identified (six cells), or the CDR3 regions could not be read by direct sequencing (four cells). Thus, CDR3-spectratyping screening of whole tissue correctly predicted the dominant sequences that we subsequently pinpointed in individual tissue-infiltrating T cells. This allowed us to unequivocally identify two pathogenic T cell clones, each with a characteristic TCR BV CDR3 sequence.
CDR3 Spectratyping and Single-Cell PCR Analysis of Muscle-Infiltrating T Cells in Additional Patients: Evidence for Clonal Persistence and Antigen-Driven Expansion.
We analyzed muscle biopsy specimens from three additional patients (PM2, PM5, and Jo1; Table 1). From patient PM2, two muscle biopsies were available, one obtained in 1983 and a second in 1998 on the occasion of a relapse after tapering of immunosuppressive medication (Table 1). Comparison of the CDR3 spectratypes of the first (1983) and second (1998) specimens revealed persisting clonal peaks in several BV families (BV3, -9, -13.1, and -21; Table 4). In the first biopsy, within the BV13.1 family, peaks appeared in the BV13.1-BJ1.5, BV13.1-BJ2.5, and BV13.1-BJ2.7 spectragrams at the BJ level (Fig. 1a). The second (1998) specimen yielded identical peaks (and CDR3 sequences) in the BV13.1-BJ2.5 and BV13.1-BJ2.7 spectragrams and a different peak in the BV13.1-BJ1.5 spectragram (Fig. 1a). The findings demonstrate that clonal expansions may persist (or reinvade) for >15 years in the target tissue. Clonal persistence was confirmed in a second patient, PM3. In this patient, repeat biopsy specimens, 7 years apart, were available (Table 1). Also in this patient, CDR3 spectratyping revealed the persistence of several clonal expansions in different BV families (BV3, -5.2, -7, -13.1, -16, and -22) in muscle for many years (Table 4).
Table 4.
Overview of expanded and persisting TCR BV sequences in muscle and blood of all investigated patients
| Patient code | Amino acid sequences of expanded clones (CDR3 region) | Muscle, mo/yr | Blood CD8 subset | |||
|---|---|---|---|---|---|---|
| 1983 | 10/98 | 11/98 | 06/99 | |||
| PM 2 | BV3-CASSPPDRGAFF-BJ1.1 | (X) | (X) | X | X | |
| BV9-CASSQDSGRAQHF-BJ1.5 | X | X | PC | PC | ||
| BV13.1-CASSYSGGLSQPQ-BJ1.5 | X | — | PC | PC | ||
| BV13.1-CASSWGGKKTQYF-BJ2.5 | X | X | X° | X° | ||
| BV13.1-CASSYRSSYE-BJ2.7 | (X) | (X) | (X) | PC | ||
| BV21-CASSPSDGSFRGDTQ-BJ2.3 | X | — | X | PC | ||
| 04/92 | 05/99 | 05/00 | ||||
| PM 3 | BV3-CASTFGSSGA-BJ2.6 | X | X | PC | ||
| BV5.2-CASSAEGTGHEQY-BJ2.7 | X | X | PC | |||
| BV13.1-CASSSSMGRGDTQ-BJ2.3 | X | X | PC | |||
| BV16-CASSAFREKL-BJ1.4 | X | X | PC | |||
| BV16-CASSTGQYTGE-BJ2.2 | X | X | PC | |||
| BV22-CASSLMTPSGANV-BJ2.6 | X | X | PC | |||
| 06/00 | 06/00 | 02/02 | ||||
| PM 4 | BV6-CASSLGQSNQP-BJ1.5 | X | X | X | ||
| BV7-CASSQDLRGPNYG-BJ1.2 | X | X | (X) | |||
| BV14-CASSLGQNNEQ-BJ2.1 | X | (X) | (X) | |||
| BV16-CASSQGVRGYG-BJ1.2 | X | X | PC | |||
| BV22-CASSDWSISQET-BJ2.5 | X | X | X | |||
| BV23-CASSLGGRWDQPQ-BJ1.5 | X | X | X | |||
| BV24-CATSSAGAKETQ-BJ2.5 | X | X | PC | |||
| 07/98 | 07/98 | 06/99 | ||||
| PM 5 | BV5.2-CASSQAVEQF-BJ2.1 | X | X | X | ||
| BV11-CASSGQGVSLSGNT-BJ1.3 | X | X | PC | |||
| BV11-CASSTPGQNTGE-BJ2.2 | X | X | X | |||
| BV13.2-CASSHSYE-BJ2.7 | X | (X) | PC | |||
| 03/99 | 06/99 | 08/00 | ||||
| PM 6 | BV2-CSARDFRTGTNQP-BJ1.5 | X | X | X | ||
| BV12-CAIGTGDSNQP-BJ1.5 | (X) | X | X | |||
| BV16-CASSHEAQGFYGY-BJ1.2 | X | X | X | |||
| BV18-CASSRPTRGSGANV-BJ2.6 | X | X | PC | |||
| BV23-CASSLGQVHQPQ-BJ1.5 | X | X | X | |||
| BV23-CASSPQTGRNEQ-BJ2.1 | X | X | X | |||
| 07/98 | 07/98 | 09/98 | ||||
| PM 7 | BV14-CASSLWGQET-BJ2.5 | X | X | X | ||
| 11/97 | 01/98 | 10/98 | ||||
| PM 8 | BV1-CASSGLSGANV-BJ2.6 | X | X | X | ||
| BV7-CASSQDMGGGQPQ-BJ1.5 | (X) | X | X | |||
| BV17-CASSTGQIGNQP-BJ1.5 | X | (X) | (X) | |||
| 06/96 | 06/96 | 07/96 | 02/98 | |||
| Jo 1 | BV4-CSVEGTGRLGTQY-BJ2.3 | X | (X) | (X) | PC | |
| BV7-CASSQDPTGVNTE-BJ1.1 | X | X | X | PC | ||
| BV13.1-CASSYSGTVGQPQ-BJ1.5 | X | X | (X) | PC | ||
| BV16-CASSQENRPRDWSRVIEQF-2.1 | X | X | (X) | PC | ||
| BV22-CANRWDYNE-BJ2.1 | X | (X) | PC | PC | ||
| 05/97 | 05/97 | 03/98 | 09/98 | |||
| Jo 2 | BV3-CASSSIQGSNEQ-BJ2.1 | X | X | X | X | |
| BV7-CASSQEEGTGNT-BJ1.3 | X | X | ND | X | ||
| BV13.2-CASSYSIRGMNTE-BJ1.1 | X | X | ND | X | ||
ND, not done; PC, polyclonal (demonstrated by CDR3 spectratyping and/or direct sequencing); X, completely readable sequence; (X), incompletely readable sequence, but clonal identity clearly discernible; °, with clone-specific primer; —, peak represents different sequence.
We studied muscle tissue (obtained in 1983) of patient PM2 with the single-cell approach to morphologically identify the VB13.1 expansions observed by CDR3 spectratyping. T cells that contacted or invaded muscle fibers and stained positive with the anti-BV13.1 mAb were isolated by laser-assisted microdissection, catapulted into PCR tubes, and subjected to single-cell PCR for amplification of the corresponding TCR gene rearrangement from genomic DNA. Ninety-three of 587 isolated T cells yielded TCR sequences (Fig. 1c; Table 2). A few clones of T cells dominated in this population of endomysial, autoaggressive T cells: 20 cells contained a BV13.1-WGGKK-BJ2.5 rearrangement and 12 cells, a BV13.1-YSGGLS-BJ1.5 rearrangement (Fig. 1c; Table 2). Three additional BV13.1 rearrangements were obtained from two, six, and three cells, respectively (Table 2).
Notably, in 57 of 60 TCR sequences the CASS consensus was followed by a large aromatic amino acid (W, Y) and then often by a hydrophilic amino acid (S, R, G) (Table 2). Furthermore, 17 cells contained a closely related motif with fixed and variable amino acids in the CDR3 region (BV13.1-YR_-BJ2.7; Table 2). The corresponding nucleotide sequences of all these rearrangements are shown in Table 3. Single-nucleotide differences in the CDR3 region did not alter the amino acid sequences. The oligoclonal nature of the BV13.1+ infiltrate and the pattern of conserved CDR3 “motifs” and conservative nucleotide exchanges suggest that the BV13.1 TCRs recognize a common antigen.
Table 3.
TCR-CDR3− motifs in BV13.1-BJ2.7 T cells from patient PM 2 (see Table 2)
 |
CDR3 N-D-N nucleotides and amino acids are given in boldface.
One cell with identifiable sequence despite minor PCR artifact.
Differences in nucleotide sequences are highlighted in big letters.
See Table 2.
We applied the combination of CDR3-spectratyping and single-cell analysis to muscle of two additional patients, PM5 and Jo1. In PM5, CDR3 spectratyping revealed a conspicuous peak in the BV11-BJ1.3 reaction. Of 57 BV11+ cells, five cells yielded a sequence; four of these cells contained a BV11-GQGVSL-BJ1.3 sequence (Table 2). We also analyzed muscle of a patient with myositis associated with antibodies against tRNA synthetase (Jo1). CDR3 spectratyping revealed a clonal peak in the BV13.1-BJ1.5 reaction. Of 402 BV13.1+ cells contacting or invading a muscle fiber, 48 cells yielded a sequence. Six cells contained the BV13.1-YSGTVG-BJ1.5 rearrangement previously identified by CDR3 spectratyping. Additional rearrangements were found in five cells (BV13.1-RTDRD-BJ1.2) and six cells (BV13.1-RTNLGD-BJ2.2), respectively (Table 2).
Clonal Tracking of Potentially Autoaggressive CD8+ T Cells in Blood: Repertoire Dynamics and Response to Immunosuppressive Therapy.
If possible, we compared the CDR3 spectragrams of muscle specimens with those of blood samples from the same patients to track the potentially autoaggressive T cells in the peripheral circulation. Because the myocytotoxic T cells in PM belong to the CD8+ population (14, 25), we isolated CD8+ T cells from PBMC by magnetic bead sorting and analyzed cDNA from these cells by CDR3 spectratyping. In all three patients studied by single-cell PCR analysis of tissue-infiltrating T cells (PM2, PM5, and Jo1; see above section; Table 1), we identified corresponding clonal expansions in CD8+ PBMC.
In patient PM2, a BV13.1-BJ1.5 rearrangement appeared in the initial muscle biopsy in 1983 and was later lost (Fig. 1a). In contrast, a BV13.1-BJ2.7 rearrangement appeared in both muscle biopsy specimens and was also conspicuous in CD8+ T cells obtained in 1998 (sequence confirmed by cycle sequencing, Δ in Fig. 1a). Further, an expanded BV13.1-BJ2.5 clone was detected in both muscle biopsies. Although not detectably expanded by CDR3 spectratyping, this clone could be PCR amplified from CD8+ T cells in 1998 and 1999 using clone specific primers (□ in Fig. 1a). CD4+ T cells showed a Gaussian distribution of CDR3 lengths, without evidence for clonal expansions (Fig. 1a). The findings indicate that the distribution of individual clones of T cells is dynamic. Similar fluctuations occurred with additional clonal expansions observed in muscle in the BV3, -9, -13.1, and -21 families (Table 4). The changes between 1998 and 1999 occurred during a period of intensified immunosuppressive therapy, with increased doses of corticosteroids and azathioprine because of muscle weakness (see Table 1).
Extensive additional followup studies in other patients are summarized in Table 4. In many cases, clonal expansions submerged in more polyclonal patterns during intense immunosuppressive therapy (see Tables 1 and 4). By contrast, in patient PM8, who was not treated with immunosuppressive agents, all peaks persisted (Table 4).
Discussion
Autoreactive CD8+ T cells have recently emerged as important mediators of tissue injury and potential targets for therapeutic intervention in murine models of diabetes and in experimental autoimmune encephalomyelitis and notably also in human autoimmune diseases like multiple sclerosis and PM (reviewed in refs. 26 and 27). Human PM is probably one of the best paradigms to study the immunopathology mediated by CD8+ T cells (8–12), because there is a conspicuous endomysial inflammatory exudate of CD8+ T cells that surround and focally invade muscle fibers (25, 28). Immunoelectron microscopy revealed that CD8+ T cells and macrophages traverse the basal lamina, firmly attach to and invade muscle fibers, and sometimes replace entire segments of a muscle fiber (14). All of the attacked muscle fibers express increased amounts of HLA-class I antigens but no detectable MHC class II (29, 30). By contrast, normal muscle fibers do not express detectable amounts of MHC class I or II antigens (29, 30).
The autoaggressive CD8+ T cells seem to attack muscle fibers via a perforin-dependent mechanism. Perforin was localized in inflammatory T cells by immunohistochemistry (13, 31), and in situ hybridization (32) and confocal laser microscopy demonstrated that the contacting autoinvasive T cells orient their perforin-containing granules toward the target muscle fiber. Functional studies revealed that CD8+ T cells expanded from muscle showed low but significant cytotoxicity against autologous cultured myotubes (33). All these features strongly suggest an MHC class I-restricted, cytotoxic T lymphocyte-mediated response against antigen(s) expressed on muscle fibers.
Previous studies of the TCR repertoire demonstrated clonally expanded T cell populations in muscle of patients with PM (24, 34–38). Most recently, CDR3 spectratyping has been applied to blood and muscle samples from patients with myositis (37, 38). These studies revealed remarkable expansions of CD8+ T cells in PM but not dermatomyositis patients (38). However, these previous repertoire studies were unable to relate the presence of expanded T cells to their pathogenic function. For example, clonally expanded CD8+ T cells are commonly observed in the blood of seemingly healthy subjects, especially in older age (2–5). These T cells may persist over years and have been related to persisting infections (4). CDR3 spectratype or conventional PCR screening of muscle would detect such irrelevant clonal expansions, because muscle biopsy tissue is unavoidably contaminated with blood cells. Furthermore, irrelevant clonal expansions may accumulate in inflamed target tissue. For example, expanded Herpes virus-specific T cell clones were detected in the synovial fluid of patients with arthritis of immune and nonimmune etiology, suggesting that these T cells may nonspecifically accumulate at inflammatory sites (6, 7).
For these reasons, it is important to relate the clonal peaks identified by CDR3 spectratyping to individual autoaggressive T cells contacting or invading a muscle fiber. Our strategy was first to identify candidate expansions by CDR3 spectratype screening. Next, we stained the tissue-infiltrating cells with appropriate anti-BV mAbs and applied laser-assisted single-cell microdissection in conjunction with single-cell PCR (39). The single-cell PCR protocol used in our study (19, 23) allows the amplification of TCR BV chain rearrangements from single molecules of rearranged genomic DNA, which is more stable than RNA (23). A drawback of this approach is that the initial spectratype screening may miss expanded clones among muscle-infiltrating T cells. This is exemplified by the analysis of patient Jo1 (Table 2), where the microdissection analysis for T cells using BV13.1 revealed three clones of nearly equal size, only one of which was originally identified in the spectratype analysis.
In all cases that we studied with the combined approach, including a myositis patient with anti-tRNA synthetase autoantibodies, we could relate spectratype-defined clonal expansions to morphologically identifiable T cells that directly contacted or invaded a muscle fiber. Some T cells persisted for many years in affected muscle, as revealed by CDR3-spectratype analysis of repeat muscle biopsy specimens obtained after 15 years in patient PM2, and after 7 years in patient PM3. The single-cell data provide strong evidence for the pathogenic relevance of the persisting expanded T cells. Remarkably, at least some of our microdissected clonally expanded T cells seem to react to a common antigen. As shown in Tables 2 and 3, 17 VB13.1-positive cells individually picked from muscle of patient PM2 contained a closely related VB13.1-YR_-BJ2.7 motif in the CDR3 region. Interestingly, there were several conservative single nucleotide differences in or around the CDR3 region, which did not alter the amino acid sequences (Table 3). This observation suggests that these cells, all of which directly contacted or invaded a muscle fiber, represent different T cell clones selected for common antigen specificities. Consistent with our results, the persistence of clonally expanded T cells over time has also been demonstrated in repeated muscle biopsies of patients with sporadic inclusion body myositis (40). The nature of the stimulating antigen(s) is unknown, but heat-shock proteins or viral antigens would be possible candidates (8, 10, 11).
We applied CDR3-spectratype analysis for the tracking of the putative effector T cells in the peripheral blood of the same patients. In most patients investigated, CDR3 spectratyping revealed the presence of one or more expanded CD8+ T cell clones with identical CDR3 length and sequence in corresponding cDNAs from muscle biopsy tissue and CD8+ T cells isolated from PBMC. In contrast, spectratype analysis of CD4+ T cells yielded polyclonal patterns, consistent with the existing evidence for a cytotoxic T lymphocyte-mediated immunopathology in PM (8–12).
Longitudinal analyses allowed us to investigate the effect of immunosuppressive treatment on the blood T cell repertoire of individual patients. PBMC were reanalyzed by CDR3 spectratyping after immunosuppressive therapy of various lengths and intensities. In most patients, at least some of the clonal peaks that were present in the initial CDR3 spectragrams (obtained before immunosuppressive therapy was started) disappeared during immunosuppressive therapy. In contrast, in patient PM8, who did not receive immunosuppressive treatment, all expanded CD8+ T cell clones persisted in the blood. It is tempting to speculate that the loss of expanded CDR3 peaks during therapy relates to its immunosuppressive effect, but we cannot exclude spontaneous fluctuations. Further studies are needed to assess whether the persistence or disappearance of myocytotoxic T cell clones in the blood correlates with the clinical response to immunosuppressive therapy.
To our knowledge, this is the first study that relates the population dynamics of the TCR repertoire to the pathogenic role of individual autoaggressive effector cells. A recent cooperative study by Rajewsky's and our group applied the same single-cell PCR protocol to analyze CD8+ and CD4+ T cell infiltrates in brain of two patients with multiple sclerosis (19). In one of the MS patients, two clones of CD8+ T cells could be detected in blood by CDR3 spectratyping. However, in contrast to our present study, the peripheral T cell repertoire could not be systematically explored, nor was it possible to track individual clones of autoaggressive T cells during therapy. In contrast to PM, it is not possible in MS to morphologically distinguish between bystander cells and pathogenic effectors.
The combination of CDR3 spectratyping with laser-assisted microdissection and single-cell PCR allows the identification and convenient tracking of pathogenically relevant T cell clones, making them attractive potential targets for specific, TCR-targeted immunotherapy. This approach should help to further characterize pathogenic CD8+ cell populations in PM and in other inflammatory and autoimmune diseases (41–43).
Acknowledgments
We thank Prof. Klaus Rajewsky for support and comments, Prof. Dieter Pongratz for supplying clinical material, and Ms. Ingrid Eiglmeier for expert technical assistance. The technical advice of Dr. K. Schütze of PALM, Bernried, Germany, is greatly appreciated. Part of this study was performed in fulfillment of the Ph.D. thesis of M.H. and the M.D. thesis of S.W. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 571, Projects A1, A3, and SFB 243).
Abbreviations
- TCR
T cell receptor
- CDR
complementarity-determining region
- PM
polymyositis
- PBMC
peripheral blood mononuclear cells
- BV
β chain variable region
- BJ
β chain joining region
Footnotes
This paper was submitted directly (Track II) to the PNAS office.
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