Analysis of the paired TCR α- and β-chains of single human T cells.
Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV) and multiple sclerosis (MS). In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+) T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+) T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.
Department of Dermatology, Ludwig-Maximilian-University, Munich, Germany.
Song-Min
SM
Kim
Latika
L
Bhonsle
Petra
P
Besgen
Jens
J
Nickel
Anna
A
Backes
Kathrin
K
Held
Sigrid
S
Vollmer
Klaus
K
Dornmair
Joerg C
JC
Prinz
D000938:Antigen-Presenting Cells / Q000276:immunology; D017931:DNA Primers / Q000235:genetics; D005434:Flow Cytometry; D019673:Genes, T-Cell Receptor alpha / Q000235:genetics*; D019674:Genes, T-Cell Receptor beta / Q000235:genetics*; D006801:Humans; D009103:Multiple Sclerosis / Q000276:immunology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D011565:Psoriasis / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012867:Skin / Q000276:immunology; D013601:T-Lymphocytes / Q000378:metabolism*
D017931:DNA Primers