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

Immunogenicity of Biotherapy Used in Psoriasis:The Science Behind the Scenes

A potential limitation in the use of biologic drugs used to treat psoriasis is the development of anti-drug antibodies (ADAs). Many factors contribute to this unwanted immune response, from the product itself, to its mode of administration, the underlying disease, and patient characteristics. ADAs may decrease the efficacy of biologic drugs by neutralizing them or modifying their clearance and may account for hypersensitivity reactions. This article reviews the scientific basis of immunogenicity and the mechanisms by which it affects clinical outcomes. It also considers testing for immunogenicity and how biologic therapy of psoriasis may be tailored on the basis of immunogenicity

Biologic therapies for psoriasis. A systematic review

Alefacept, efalizumab, etanercept, and infliximab are currently approved for the treatment of adults with moderate to severe plaque psoriasis, and phase 3 trials for adalimumab are ongoing. The high level of evidence from large randomized, double-blind, placebo-controlled clinical studies for each of these biologics allows high-grade recommendations and helps define uncertainties, one of which is longterm safety. For tumor necrosis factor-a blocking agents, safety profiles are available from clinical experience in other indications. In general, biologics are safe and effective in the treatment of psoriasis, with potential to address unmet medical needs. Their distinct profiles allow dermatologists to match the biologic agent to individual characteristics of patients who are candidates for systemic therapy or phototherapy. In this evidence-based review of the literature, we assess the effects on psoriasis of induction therapy with 5 biologics and provide preliminary treatment guidelines

Selection of conserved TCR VDJ rearrangements in chronic psoriatic plaques indicates a common antigen in psoriasis vulgaris

Psoriasis vulgaris is a common HLA-associated inflammatory skin disease. Although its etiology is still unknown, it is thought to involve T cell-mediated inflammatory mechanisms. In examining the lesional psoriatic TCR beta chain (TCRB) usage in a pair of identical twins concordant for psoriasis, we observed repetitive TCR VDJ rearrangements which indicated antigen-specific oligoclonal T cell expansion. Several of these TCRB rearrangements were identical or highly homologous in the amino acid composition of the complementarity determining region 3 (CDR3), suggesting that T cells with these TCR might be important for disease manifestation. This conclusion was strengthened by TCR analysis of other psoriasis patients. Several repetitive lesional TCRB rearrangements were found that were similar to the conserved CDR3 seen in the twins. Since TCR antigen specificity is largely determined by the beta chain CDR3, selection of T cells with conserved TCRB CDR3 motifs could indicate the presence of a common antigen as a major target of the lesional psoriatic immune response

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

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

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 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