Allorecognition of human endothelial cell cultures by CD8 T cell lines enriched in herpesvirus-specific T cells.

<p>T cell lines from D01 and D03, sorted with pp65_495–503/A*0201 multimers, were tested against HAEC #4373, #3315, and #3376, while T cell line from D15, sorted with BZLF1_54–64/B*3501 multimers, was tested against HAEC #3643 (Cw*0602+) and #1415 (Cw*0602-) for TNF-α production (<b>A</b>) and cytotoxicity (Effector-Target ratio 15∶1) (<b>B</b>). TNF-α production was measured as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012120#pone-0012120-g002" target="_blank">Figure 2</a> legend. Only the endothelial cell lines expressing the relevant allogeneic HLA allele induced cytotoxicity and TNF production by the CD8 T cell lines tested. The data are representative of 3 different experiments.</p

HLA class I typing of LCL.

<p>ND : Not determined. LCL and the HLA triggering allorecognition are indicated in bold.</p

HLA class I typing of endothelial cells.

<p>ND : Not determined.</p

Enrichment in pp65_495–503/A*0201- or BZLF1_54–64/B*3501-specific T cells after sorting of CD8 T cells with pMHC magnetic multimers.

<p>(<b>A</b>) CD8 T cells, derived from the PBL from D01 and D03 were sorted with 245V mutated pp65_495–503/A*0201 multimers, then expanded in culture, and stained with PE-conjugated pp65_495–503/A*0201 tetramers and FITC-conjugated anti-CD3. (<b>B</b>) CD8 T cells derived from the PBL from D15 were sorted with 245V mutated BZLF1_54–64/B*3501 multimers, and then expanded in culture. Unsorted and sorted T cells were stained with PE-conjugated BZLF1/B35 tetramers and FITC-conjugated anti-CD3. The percentage of positive cells is indicated in the upper right quadrant.</p

Screening of CD8 T cell lines enriched in HCMV- or EBV-specific T cells for cross-reactivity to allogeneic MHC molecules.

a<p>CD8 T cell lines were screened on COS-7 cells transfected with individual HLA-encoding cDNA and TNF-α production was measured after a 6h-coculture. All T cell lines were PBL-derived. ND : not determined.</p

Screening of HCMV- or EBV-specific CD8 T cell lines for cross-recognition of allogeneic MHC molecules.

<p>CD8 T cell lines, sorted with recombinant pMHC multimeric complexes specific to HCMV (pp65_495–503/A*0201) or EBV lytic epitopes (BMLF1_259–267/A*0201 or BZLF1_54–64/B*3501), were tested: (A) for cytotoxicity toward a panel of 30 HLA-typed LCL, in a 4-h chromium release assay (Effector-Target ratio 15∶1). HLA-specific killing of LCL was observed for 3 of the 11 pp65/A2-specific T cell lines tested: T cell line from D01 killed A*3101⁺ LCL, T cell line from D03 killed A*3201⁺ LCL, and T cell line from D08 killed A*3001⁺ LCL. The BZLF1/B*3501-sorted T cell line from D15 killed LCL sharing Cw*0602 expression. Only T cell lines that showed alloresponse are displayed. LCL triggering an alloresponse are shown as well as some of the tested LCL which do not elicit a cytotoxic response. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. Data are presented as the mean percentage lysis and are representative of 3 different experiments. (<b>B</b>) for TNF-α production toward COS-7 cells transfected with plasmids encoding class I HLA alleles. T cells were added 2 days after the transfection, and the TNF-α content of the supernatant, expressed in pg/mL, was estimated 6 h later by testing the toxicity of the supernatants for TNF-α sensitive WEHI-164 clone 13 cells. TNF-α production was observed for the pp65/A*0201-sorted T cell line from D03, that recognized selectively COS cells expressing HLA-A*3201, and for the BZLF1/B*3501 T cell line from D15 that recognized selectively COS cells expressing HLA-Cw*0602. Plasmids encoding class I HLA alleles that elicits a response are shown as well as some of the plasmid encoding class I HLA alleles that do not induce TNF-α secretion. T cell lines that did not produce TNF-α are not represented. Cognate peptide-HLA complexes (pp65_495–503/A*0201 or BZLF1_54–64/B*3501) were used as positive control. One out three independent experiments is shown.</p

Analysis of the TCR Vb repertoire of pp65_495–502/A*0201- or BZLF1/B*3501-sorted T cell lines, using TCR Vβ-specific mAb.

<p>The percentage of pp65/A*0201- or BZLF1/B*3501-specific T cells stained by the various anti-TCR Vβ mAb is mentioned according to the IMGT nomenclature (Beckman Coulter anti-TCR Vβ name is indicated in bracket). All T cell lines were derived from PBL, either from healthy donors or from RA patients.</p>a<p>Percentage determined by TCR sequencing (ref. 28). T cell lines exhibiting alloreactivity are marked in bold.</p

Characterization of CMV/HLA-I-derived peptides recognized by HLA-E-restricted CD8 T cells.

<p>A/TNF production in response to stimulation with.221 cells pulsed with synthetic peptides.221 cells were incubated for 1 h with range concentrations of the indicated peptides before addition of MART.22 T cells. After 6 h, T cells were fixed, permeabilized and stained for intracellular TNF-α. Results are expressed as percentage of TNF-producing T cells. B/Peptide-MHC tetramer staining of HLA-E-restricted CD8 T cells. MART.22 T cells were incubated for 1 h with biotyniled HLA-E monomers refolded with the indicated peptides and tetramerized with PE-coupled streptavidin. Peptide-HLA-E tetramers staining was assessed by flow cytometry and RFI are indicated.</p

Functional characterization of HLA-E-restricted CD8 T cells.

<p>A/Induction of strong and rapid Ca²⁺ responses within activated HLA-E-restricted CD8 T cells. B-EBV 721.221 cells transfected (.221-E) or not (.221) with HLA-E and the leader sequence of HLA-B*08, were incubated with MART.22 T cells loaded with Fura-2 (1∶1 ratio). T cell intracellular Ca²⁺ levels were monitored by videomicroscopy for the indicated acquisition time. Graphs represent the kinetics of intracellular Ca2⁺ levels (340/380 nm ratio). Values correspond to the mean of emission measured among all T cells present in the field (approximatively 20 cells per experiment). Results are representative of two independent experiments. B/Degranulation of HLA-E-restricted CD8 T cells upon stimulation.221-E cells (thick line) or.221 cells (thin line) were incubated for 4 h with MART.22 T cells in the presence of anti-CD107a antibody. Results are expressed as pourcentages of surface CD107a positives T cells upon stimulation with.221-E cells. C/Cytotoxic activity of HLA-E restricted CD8 T cells. 10³⁵¹Cr-labeled.221-E cells (squares) or.221 cells (circles) were co-cultured for 4 h with MART.22 T cells at various E/T ratios. Cytotoxic activity was assessed through measure of Chromium release in the supernatants. Percentages of specific lysis are indicated. Means and standard deviations of triplicate wells are shown for one out of three comparable experiments. D/Cytokine production analysis of HLA-E restricted CD8 T cells. MART.22 T cells were fixed, permeabilized and stained for intracellular cytokines following 6 h of incubation with.221-E cells (thick line) or.221 cells (thin line). Data are expressed as mean % of intracellular cytokine secreting cells upon stimulation with.221-E cells.</p

Leader sequence peptides derived from HCMV-UL40/HLA-I molecules and recognition by HLA-E-restricted T cell clone.

<p>Autologous HLA class I alleles of the transplant recipient are indicated in bold.</p>a<p>MART.22 HLA-E-restricted T cell clone activity in response to.221 cells pulsed with different peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050951#pone-0050951-g002" target="_blank">Figure 2</a>).</p>b<p>These peptides are identical to peptides contained in the UL40 ORF from various CMV strains.</p>c<p>These pepides have previously been described for their ability to trigger HLA-E restricted CD8 T cell responses.</p