Clonal tracking of autoaggressive T cells in polymyositis by combining laser microdissection, single-cell PCR, and CDR3-spectratype analysis.
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.
Hofbauer
Monika
M
Institute for Clinical Neuroimmunology, Ludwig Maximilians University, D-81377 Munich, Germany.
Wiesener
Solveigh
S
Babbe
Holger
H
Roers
Axel
A
Wekerle
Hartmut
H
Dornmair
Klaus
K
Hohlfeld
Reinhard
R
Goebels
Norbert
N
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000032:analysis / Q000097:blood; D001483:Base Sequence; D001706:Biopsy; D017931:DNA Primers; D004210:Dissection / Q000379:methods; D005260:Female; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods; D017285:Polymyositis / Q000276:immunology* / Q000473:pathology; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics*; D013601:T-Lymphocytes / Q000276:immunology* / Q000473:pathology
D015703:Antigens, CD; D017931:DNA Primers; D017260:Receptor-CD3 Complex, Antigen, T-Cell