8 research outputs found
Localization of CD8<sup>+</sup> 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-CD8Ī² (red) and Alexa 488-labeled anti-CD134 (green) antibodies. (<b>AāC</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>DāF</b>): Laser microdissection of T cells stained for CD8Ī² (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>DāF</b>). The scale bars correspond to 20 Āµm.</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(āø, 200-fold magnification in an inverted microscope).</p
Functional validation of the primer sets for the simultaneous amplification of the TCR Ī±- and Ī²-gene repertoires by multiplex RT-PCR. cDNA from PBL was amplified with 24 VĪ± and 9 VĪ² primers in a multiplex RT-PCR.
<p>(<b>A</b>) nine forward VĪ²-primers (Vp1 to Vp9) were used together with the CĪ²-<i>out</i> reverse primer (lanes 1ā9). (<b>B</b>) 24 VĪ±-<i>out</i> forward primers were used together with the reverse CĪ±-<i>out</i> primer (lanes 1ā24) in individual reactions (see ref. (2) for Ī±-primer sequences and correlation of the lanes to VĪ±-families). Each VĪ²- (<b>A</b>) and VĪ±-primer (<b>B</b>) yielded a PCR product of the expected size. M, molecular weight marker. (<b>C</b>) To validate that all TCR VĪ²-gene families were covered, the pre-amplification multiplex RT-PCR product was amplified using 23 VĪ²-primers specific for the functional VĪ²-gene repertoire together with the FAM-labelled CĪ²-<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
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 āUPā sequence at their 5ā²-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
Ī±Ī²-TCR rearrangements, frequency and tissue localization of clonal CD8<sup>+</sup> 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
Ī±Ī²-TCR rearrangements and frequency of clonal CD4<sup>+</sup> or CD8<sup>+</sup> 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; +/ā, TCR rearrangement also/not identified in cDNA derived from biopsy part A, peripheral blood lymphocytes (PBL), tonsil of patient; N, not available.</p
Ī±Ī²-TCR rearrangements, frequency and tissue localization of clonal CD8<sup>+</sup> 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>ā”</b>: T cells were in direct contact with antigen-presenting cells of dendritic or epithelial morphology; N, not analyzed.</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 Ī²-chains (upper panel) and Ī±-chains (lower panel) from particular T cell clones were detected.</p