Reconstitution of paired T cell receptor alpha- and beta-chains from microdissected single cells of human inflammatory tissues.

Abstract:

: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) beta-chains in muscle sections of patients with inflammatory muscle diseases. By immunohistochemistry, we identified those Vbeta-positive T cells that fulfilled the morphological criteria of myocytotoxicity and isolated them by laser microdissection. Next, we identified coexpressed pairs of TCR alpha- and beta-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-alpha-chain antibodies and the great heterogeneity of the alpha-chain genes. From muscle tissue of a patient with polymyositis, we isolated 64 T cells that expressed an expanded Vbeta1 chain. In 23 of these cells, we identified the corresponding alpha-chain. Twenty of these 23 alpha-chains were identical, suggesting antigen-driven selection. After functional reconstitution of the alphabeta-pairs, their antigen-recognition properties could be studied. Our results open avenues for combined analysis of the full TCR alpha- and beta-chain repertoire in human inflammatory tissues.

authors

Seitz S,Schneider CK,Malotka J,Nong X,Engel AG,Wekerle H,Hohlfeld R,Dornmair K

doi

10.1073/pnas.0604247103

subject

Has Abstract

pub_date

2006-08-08 00:00:00

pages

12057-62

issue

32

eissn

0027-8424

issn

1091-6490

pii

0604247103

journal_volume

103

pub_type

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