Analysis of the paired TCR α- and β-chains of single human T cells

Abstract

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.

Figure 1. Strategy to identify paired TCR α- and β-chains from single T cells.

Step 1: one step multiplex pre-amplification RT-PCR composed of step 1A, reverse transcription using a Cα-out and Cβ-out primer and step 1B, amplification of the α and β TCR rearrangements by a pool of 9 oligonucleotide Vβ primers (Vp1– Vp9) for the Vβ repertoire and a pool of 24 oligonucleotide Vα primers for the Vα repertoire. Subsequently, the PCR products of the Vβ (step 2) and Vα (step 3) gene rearrangements are handled separately. Step 2: Introduction of a universal primer (UP) site at the 5′-site of each Vβ-rearrangement using primers Vp1-UP to Vp9-UP by a run off reaction (step 2A), followed by amplification of the Vβ gene rearrangement by seminested PCR with the UP primer and a Cβ-in primer (step 2B) and sequencing (step 2C). Step 3: Amplification (step 3A) and sequencing (step 3B) of the Vα gene rearrangement by nested PCR from the pre-amplification PCR product using five different nested Vα-in primer pools and a Cα-in primer.

Figure 2. Functional validation of the primer sets for the simultaneous amplification of the TCR α- and β-gene repertoires by multiplex RT-PCR. cDNA from PBL was amplified with 24 Vα and 9 Vβ primers in a multiplex RT-PCR.

(A) nine forward Vβ-primers (Vp1 to Vp9) were used together with the Cβ-out reverse primer (lanes 1–9). (B) 24 Vα-out forward primers were used together with the reverse Cα-out primer (lanes 1–24) in individual reactions (see ref. (2) for α-primer sequences and correlation of the lanes to Vα-families). Each Vβ- (A) and Vα-primer (B) yielded a PCR product of the expected size. M, molecular weight marker. (C) To validate that all TCR Vβ-gene families were covered, the pre-amplification multiplex RT-PCR product was amplified using 23 Vβ-primers specific for the functional Vβ-gene repertoire together with the FAM-labelled Cβ-in reverse primer in individual reactions as described . The PCR-products were analyzed by spectratyping their fragment-lengths on a genetic sequencer.

Figure 3. Different antigen presenting cells may present the same antigens to different T cells.

Two lesional psoriatic T cells (*) labelled with CD8 beads are seen in direct contact with dendritic-like cells with antigen presenting cells of dendritic phenotype(▸, 200-fold magnification in an inverted microscope).

Figure 4. Localization of CD8⁺ T cells infiltrating the MS brain and their isolation by laser microdissection.

Cryosections of a frozen biopsy sample from the MS patient were stained with Cy3-labeled anti-CD8β (red) and Alexa 488-labeled anti-CD134 (green) antibodies. (A–C): Visualization of activated T cells (white arrows) that are double positive (C) for CD8 (A) and CD134 (B). (D–F): Laser microdissection of T cells stained for CD8β (red; D) and CD134 (green; E). Activated single cells are dissected at the indicated yellow circles and catapulted out of the tissue directly into the cap of a PCR tube for subsequent TCR analysis. We show the corresponding bright-light image of the tissue after (F) laser microdissection. The numbers in the yellow field refer to apparatus parameters (D–F). The scale bars correspond to 20 µm.