Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis

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 beta 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.

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.