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Blood, Vol. 112, Issue 4, 1259-1268, August 15, 2008
PILAR is a novel modulator of human T-cell expansion
Blood Huarte et al.
112: 1259
Supplemental materials for: Huarte et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 68.5 KB)
- Figure S1. PILAR (JPG, 0.98 MB)
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(A) Incubation of 106 peripheral CD8+ T cells/ml with phytohemagglutinin (10 µg/ml; left) resulted in a transient upregulation of PILAR. (B) We confirmed that the rabbit polyclonal anti-PILAR antibody (left, thicked), but not pre-immune serum (left, dotted) specifically recognized PILAR expressed by stably transfected K562 cells. Endotoxin levels were below the limit of detection of the Cambrex Limulus amebocyte lysate 514 QCL-1000 kit (0.15 EU/ml). Identical results were obtained with a mouse anti-PILAR monoclonal antibody (right). (C) Comparable proportions of naïve (Day 0) peripheral blood PILAR+CD3+ T cells stimulated for up to 5 days with aAPCs coated with agonistic CD3/CD28 Abs (CD3/CD28; 100 ng/ml each) or 10 µg/ml of phytohemagglutinin (PHA) were detected using a rabbit polyclonal or a mouse monoclonal Ab. Basal expression on a small proportion of naïve CD4 peripheral blood T cells is also shown (Day0). These data are representative of 3 independent experiments. (D) Peripheral CD3+ T cells stimulated for 3 days with aAPCs coated with agonistic anti-CD3/CD28 Abs (100 ng/ml) upregulated intracellular PILAR protein. This staining was performed with a PILAR-specific monoclonal Ab, plus a rat anti-mouse IgG, after fixation plus permeabilization using the Cytofix/Cytoperm kit (BD Biosciences). Second Ab, rat anti-mouse IgG in the absence of primary Ab. These data are representative of 2 independent experiments. (E) Magnetically purified CD3+ T cells were stimulated for 9 hours with CD3/CD28-coated beads, then incubated with 5 µg/ml actinomycin D (Sigma) and harvested for the extraction of RNA and cDNA production at 45 and 60 min. PILAR mRNA levels, normalized by GAPDH expression, were determined by Real-Time Quantitative PCR. (F) Unstimulated peripheral CD3+ T cells exhibit negligible levels of CD161. (G) Unstimulated T cells were nucleofected using the Human T Cell Nucleofector Kit and a pcDNA3.1 plasmid encoding the PILAR open-reading frame (PILAR plasmid), or with the empty vector (control plasmid), following the manufacturer’s protocol. Forty-eight hours post nucleofection the cells were stained for PILAR (using a polyclonal Ab) and analyzed by flow cytometry. A control plasmid encoding GFP was used as a positive control (GFP plasmid). (H) IFN-γ ELISPOT analysis of PBMCs stimulated for 7 days with autologous monocyte-derived dendritic cells (10:1 ratio) pulsed with the CEF peptide pool (Mabtech, 2 µg/mL of each peptide), in the presence of 20 µg/ml of anti-PILAR or an irrelevant Ab. These results are representative of 3 independent experiments. (I) Blockade of PILAR on naïve T cells for 3 days results in the transcriptional upregulation of IL-10, but not IFN-γ or TGF-β (left). The increase of IL-10 in cell culture supernatants was also confirmed at the protein level (right). This experiment was repeated twice with identical results. (J) Tumor CD8 T cells infiltrating 7 unselected human ovarian carcinoma samples exhibit a predominant effector memory phenotype (gate on CD45+CD3+CD8+ cells). Single cell suspensions were prepared by mechanical dissociation of fresh specimens.
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