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Reconstitution of paired T cell receptor [alpha]- and ß-chains from microdissected single cells of human inflammatory tissues
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 targe...
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| Published in: | Proceedings of the National Academy of Sciences - PNAS 2006-08, Vol.103 (32), p.12057 |
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| container_issue | 32 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
| container_volume | 103 |
| creator | Seitz, Sabine Schneider, Christian K Malotka, Joachim Xiao Nong |
| description | 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) ß-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vß-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 ß-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 Vß1 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 αß-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR α- and ß-chain repertoire in human inflammatory tissues. [PUBLICATION ABSTRACT] |
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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) ß-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vß-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 ß-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 Vß1 chain. 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| ispartof | Proceedings of the National Academy of Sciences - PNAS, 2006-08, Vol.103 (32), p.12057 |
| issn | 0027-8424 1091-6490 |
| language | eng |
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| source | PubMed Central（OA）; JSTOR Archival Journals and Primary Sources Collection |
| subjects | Disease Muscular system Pathogens T cell receptors Tissues |
| title | Reconstitution of paired T cell receptor [alpha]- and ß-chains from microdissected single cells of human inflammatory tissues |
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