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Blood, Vol. 109, Issue 7, 2806-2814, April 1, 2007
Expression and release of soluble HLA-E is an immunoregulatory feature of endothelial cell activation
Blood Coupel et al.
109: 2806
Supplemental materials for: Coupel et al, Vol 109, Issue 7, 2806-2814
Files in this Data Supplement:
- Table S1. HLA-E expression in various normal tissues (PDF, 14.5 KB) -
HLA-E protein expression was investigated in the following normal tissues: kidney, spleen, lymph node, salivary gland, urinary bladder, thyroid, endometrium, skin, liver, and stomach. For each tissue, at least 3 independent samples were immunostained and analyzed.
- Figure S1. Expression and release of HLA-E by PBMCs and cell lines (PDF, 31.3 KB)
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PBMCs from random healthy volunteers were purified by Ficoll/Hypaque density centrifugation. NK cells were purified (> 95% of CD3-CD56+ and/or CD16+) by negative selection using the NK Cell Isolation kit according to the manufacturer’s recommendations (Miltenyi Biotec, Paris, France). U937, Raji, C1R, K562, and Jurkat cell lines were obtained from the American Tissue Culture Collection (Rockville, MD). The NKL cell line was kindly provided by Dr Eric Vivier (Centre d’Immunologie Marseille-Luminy CIML, Marseille-Luminy, France). (A) For flow cytometry analysis of cell-surface HLA-E expression, untreated cells were double-stained with FITC-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD14, or PE-labeled anti-CD19 mAbs and anti–HLA-E (MEM-E/8) mAbs, revealed using PE- or FITC-labeled antimouse secondary Abs. Results are expressed as dotplots after subset selection according to cytometric side-scatter and forward-scatter parameters. Results are representative of 3 independent experiments. (B) Fresh PBMCs and purified NK cell subsets were cultured for 48 hours with Con A, IL-2, and anti-CD28 mAb, or IFN . At the end of treatment, cells and culture supernatants were collected for Western blot and flow cytometry analyses. Membrane-bound HLA-E and sHLA-E were then detected by western blot in lysates (15 µg/sample) and supernatants (10 µL/sample), respectively. Immunoblots were reprobed with anti-GAPDH mAb to compare protein loading within samples. (C) Cell-surface expression of HLA-E on monocytoid (U937), T (Jurkat), B (Raji), and NK (NKL) cell lines analyzed by flow cytometry. HLA-E staining (solid histograms) was compared with labeling obtained using an isotype-matched irrelevant mAb (empty histograms). Detection by Western blotting of sHLA-E in culture supernatants from cell lines treated for 24 hours with or without 150 U/mL rIL-2.
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