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 ␤ 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.

Inflammatory Muscle Diseases

“…42 Analysis of T-cell-receptor molecules expressed by the infiltrating CD8+ T cells reveals clonal expansion of T-cell-receptor chains and conserved sequences in the antigen-binding region, which suggests an antigendriven T-cell response. 43,44 This is further supported by the expression of costimulatory molecules and up-regulation of adhesion molecules, chemokines, and cytokines [45][46][47] (Fig. 2G).…”

Reconstitution of paired T cell receptor α- and β-chains from microdissected single cells of human inflammatory tissues

“…The ␤-chain may choose from 13 different J elements, but the ␣-chain may use 50 (10). Therefore, if rearranged genomic DNA is used as PCR template, ''only'' 13 J␤-specific reverse primers are required (7,8,29), whereas a comparable ␣-chain analysis would require 50 reverse J primers. The advantage of DNA as a more stable template than RNA is therefore outweighed by the disadvantage of the huge number of primers that would be required.…”

“…Only in very rare cases of monoclonal expansions (24)(25)(26)(27) can paired TCR chains be identified from tissue homogenates. Early pioneering studies described the Ig repertoire of B cells in single-cell experiments (28), but unbiased amplification from single-tissue-infiltrating T cells has so far been achieved only for TCR ␤-chains (7,8,29). Both the ␣-and ␤-chain may select from almost 50 different V elements.…”

“…In previous studies we successfully amplified TCR ␤-chains from individual, morphologically characterized, tissue-infiltrating T cells from brains of patients with multiple sclerosis and from muscles of patients with myositis (7)(8)(9). Although these studies provided insight into intralesional TCR ␤-chain repertoires, they did not allow the identification of complete TCR ␣␤-heterodimers expressed by tissue-infiltrating T cells because information on the coexpressed ␣-chains was lacking, a result of the very limited number of available anti-␣-chain antibodies and higher variability of ␣-chain genes (10,11).…”

Intraepithelial CD8-positive T lymphocytes predict survival for patients with serous stage III ovarian carcinomas: relevance of clonal selection of T lymphocytes

“…As CD8 expression is not observed in classical gd T cells, our results suggest that the presence of clonal CD8-positive ab T cells may be primarily involved in a possible anti-tumoural immune response in ovarian carcinomas. Our data encourage further advanced studies on the nature of TIL clonality in ovarian carcinomas, for example, also including complementary determining region (CDR3) spectratyping of (single) microdissected TILs and reconstitution of TCR chains (Hofbauer et al, 2003;Seitz et al, 2006). In view of the potential clonal restriction of CD8-positive T lymphocytes actively counteracting the tumour and its progression, we investigated whether the tumour-cell-specific expression of Her2/neu protein in ovarian carcinomas may represent a candidate target antigen of TILs, as suggested for breast cancer 10 TCRγ9, 11 Figure 2B) and two non-neoplastic ovaries (cases 9 and 10).…”