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

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

<p>Two lesional psoriatic T cells (<b>*</b>) 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).</p

Distribution of T cells isolated by laser microdissection from morphologically distinct regions of the MS lesion.

<p>We list the numbers of T cells and tissue blocks where TCR β-chains (upper panel) and α-chains (lower panel) from particular T cell clones were detected.</p

αβ-TCR rearrangements and frequency of clonal CD4⁺ or CD8⁺ T cells isolated from lesional full thickness skin biopsies of PV patients #1 to #3.

*<p>deduced amino acid sequence, one letter amino acid code; +/−, TCR rearrangement also/not identified in cDNA derived from biopsy part A, peripheral blood lymphocytes (PBL), tonsil of patient; N, not available.</p

αβ-TCR rearrangements, frequency and tissue localization of clonal CD8⁺ T cells isolated from lesional dermis and epidermis of PV patient #4.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037338#pone-0037338-t002" target="_blank">Table 2</a> for details. <b>‡</b>: T cells were in direct contact with antigen-presenting cells of dendritic or epithelial morphology; N, not analyzed.</p

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

<p>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. (<b>A–C</b>): Visualization of activated T cells (white arrows) that are double positive (<b>C</b>) for CD8 (<b>A</b>) and CD134 (<b>B</b>). (<b>D–F</b>): Laser microdissection of T cells stained for CD8β (red; <b>D</b>) and CD134 (green; <b>E</b>). 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 (<b>F</b>) laser microdissection. The numbers in the yellow field refer to apparatus parameters (<b>D–F</b>). The scale bars correspond to 20 µm.</p

Sequence, localisation and specificity of oligonucleotide primers.

*<p>Arden nomenclature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037338#pone.0037338-Arden1" target="_blank">[27]</a>; degenerate primers contain bold letters to indicate nucleotide exchanges. All primers Vp1 to Vp9 were also synthesised with a “UP” sequence at their 5′-end (termed Vp1-UP to Vp9-UP). We only show Vp2-UP and Vp9-UP because nucleotides exchanges (underlined) were introduced there to avoid primer interactions.</p

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.

<p>(<b>A</b>) nine forward Vβ-primers (Vp1 to Vp9) were used together with the Cβ-<i>out</i> reverse primer (lanes 1–9). (<b>B</b>) 24 Vα-<i>out</i> forward primers were used together with the reverse Cα-<i>out</i> primer (lanes 1–24) in individual reactions (see ref. (2) for α-primer sequences and correlation of the lanes to Vα-families). Each Vβ- (<b>A</b>) and Vα-primer (<b>B</b>) yielded a PCR product of the expected size. M, molecular weight marker. (<b>C</b>) 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-<i>in</i> reverse primer in individual reactions as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037338#pone.0037338-Besgen1" target="_blank">[14]</a>. The PCR-products were analyzed by spectratyping their fragment-lengths on a genetic sequencer.</p

αβ-TCR rearrangements, frequency and tissue localization of clonal CD8⁺ T cells isolated from lesional dermis and epidermis of PV patient #5.

<p>See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037338#pone-0037338-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037338#pone-0037338-t003" target="_blank">3</a> for details.</p