Clonal composition and T cell receptor (TCR) repertoire of CD4+ and CD8+ T cells infiltrating actively demyelinating multiple sclerosis (MS) lesions were determined with unprecedented resolution at the level of single cells. Individual CD4+ or CD8+ T cells were isolated from frozen sections of lesional tissue by micromanipulation and subjected to single target amplification of TCR-β gene rearrangements. This strategy allows the assignment of a TCR variable region (V region) sequence to the particular T cell from which it was amplified. Sequence analysis revealed that in both cases investigated, the majority of CD8+ T cells belonged to few clones. One of these clones accounted for 35% of CD8+ T cells in case 1. V region sequence comparison revealed signs of selection for common peptide specificities for some of the CD8+ T cells in case 1. In both cases, the CD4+ T cell population was more heterogeneous. Most CD4+ and CD8+ clones were represented in perivascular infiltrates as well as among parenchymal T cells. In case 2, two of the CD8+ clones identified in brain tissue were also detected in peripheral blood. Investigation of the antigenic specificities of expanded clones may help to elucidate their functional properties.
We surveyed the T cell receptor repertoire in three separate compartments (brain, cerebrospinal fluid, and blood) of two multiple sclerosis patients who initially had diagnostic brain biopsies to clarify their unusual clinical presentation but were subsequently confirmed to have typical multiple sclerosis. One of the brain biopsy specimens had been previously investigated by microdissection and single-cell PCR to determine the clonal composition of brain-infiltrating T cells at the single-cell level. Using complementarity-determining region 3 spectratyping, we identified several identical, expanded CD8 ؉ (but not CD4 ؉ ) T cell clones in all three compartments. Some of the expanded CD8 ؉ T cells also occurred in sorted CD38 ؉ blood cells, suggesting that they were activated. Strikingly, some of the brain-infiltrating CD8 ؉ T cell clones persisted for >5 years in the cerebrospinal fluid and͞or blood and may thus contribute to the progression of the disease.
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  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.
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