We describe a strategy to "revive" putatively pathogenic T cells from frozen specimens of human inflammatory target organs. To distinguish pathogenic from irrelevant bystander T cells, we focused on cells that were (i) clonally expanded and (ii) in direct morphological contact with a target cell. Using CDR3 spectratyping, we identified clonally expanded T cell receptor (TCR) β-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vβ-positive T cells that fulfilled the morphological criteria of myocytotoxicity and isolated them by laser microdissection. Next, we identified coexpressed pairs of TCR α- and β-chains by a multiplex PCR protocol, which allows the concomitant amplification of both chains from single cells. This concomitant amplification had not been achieved previously in histological sections, mainly because of the paucity of available anti-α-chain antibodies and the great heterogeneity of the α-chain genes. From muscle tissue of a patient with polymyositis, we isolated 64 T cells that expressed an expanded Vβ1 chain. In 23 of these cells, we identified the corresponding α-chain. Twenty of these 23 α-chains were identical, suggesting antigen-driven selection. After functional reconstitution of the αβ-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR α- and β-chain repertoire in human inflammatory tissues.
|Original language||English (US)|
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - Aug 8 2006|
- Single-cell PCR
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