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

HLA class I typing of LCL.

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

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

Gene network composed of immune-related genes potentially targeted by miR-142-5p

<p>A) This gene network was built with IPA software and down-regulated potential targets are highlighted in grey. Corresponding gene expression based on microarrays data, in relative fluorescent units (RFU), are displayed for XCL1 (B), STK17 (C), CD69 (D), MCL1 (E) and PDE4D (F) (CAMR = 12 and STA = 12).</p

miR-142-5p as a blood biomarker of CAMR

<p>A) TLDA and individual qPCR measurements performed in same PBMC samples (10 STA and 9 CAMR) are displayed for miR-142-5p. B) miR-142-5p over-expression in CAMR compared to STA only was validated on additional PBMC samples (30 STA and 18 CAMR; p = 0.0058). The expression of miR-142-5p was measured in the blood of patients with different clinical statuses (9AR, 10RF and 8HV). Non parametric Dunn's ad hoc test was used to compare all groups to STA. C) ROC curve analysis of qPCR data for these validation samples (30 STA and 18 CAMR) revealed an area under the curve (AUC) of 0.74 (CI_95% = [0.59 to 0.89]; p = 0.0056). D) miR-142-5p expression was rapidly decreased in the PBMC of 3 HV after PHA (2 µg/mL) and Il-2 (150 U/mL) stimulation. Mean ±SEM miR-142-5p expression relative to miR-374b is represented.</p

Unsupervised clustering (A) and PCA (B) of STA and CAMR groups with the 10 selected miRNAs.

<p>The heatmap represents normalized and color-coded relative expression values (2^−ΔΔCq) in which red values indicate over-expression and green values indicate under-expression. The PCA graph represents the first (x axis) and second (y axis) components of the PCA analysis.</p