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

HLA class I typing of endothelial cells.

<p>ND : Not determined.</p

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

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