Supplementary Material for: Soluble NKG2D Ligands Are Potential Biomarkers and Sentinels of Immune-Mediated Bone Marrow Injury in Bone Marrow Failure Syndromes

Immune-mediated processes are considered important in the pathogenesis of bone marrow failure syndromes (BFS). We previously reported that natural killer group 2D (NKG2D) ligands were expressed on pathological blood cells of patients with BFS and that NKG2D immunity may be involved in bone marrow failure. In addition to membranous NKG2D ligands on the cell surface, soluble NKG2D ligands can exist in plasma. We therefore examined the relationship between soluble NKG2D ligands and blood cell counts in 86 patients with BFS, including aplastic anemia, myelodysplastic syndrome with single lineage dysplasia, and paroxysmal nocturnal hemoglobinuria. Approximately half of the BFS patients were positive for soluble NKG2D ligands in the plasma by enzyme-linked immunosorbent assay, and soluble NKG2D ligand-positive BFS patients exhibited severe cytopenia regardless of membranous NKG2D ligand expression. In vitro<i></i>analyses demonstrated that soluble ULBP1, an NKG2D ligand, down-regulated NKG2D receptors on CD2-positive cells in peripheral blood. Moreover, soluble ULBP1 attenuated the cytotoxic effects of peripheral blood mononuclear cells on K562, which express membranous ULBP1. Our results suggest that soluble NKG2D ligands can be easy-to-measure biomarkers for the prediction of activity of immune-meditated bone marrow injury in BFS and that soluble NKG2D ligands suppress redundant immune-mediated bone marrow injury.免疫介导过程被认为是骨髓衰竭综合征（bone marrow failure syndromes, BFS）发病机制中的关键环节。我们此前的研究报道显示，骨髓衰竭综合征患者的病理性血细胞可表达自然杀伤细胞2族成员D（natural killer group 2D, NKG2D）配体，且NKG2D免疫通路可能参与骨髓衰竭的发生发展。除细胞表面表达的膜结合型NKG2D配体外，血浆中还可存在可溶性NKG2D配体。为此，我们纳入86例骨髓衰竭综合征患者开展研究，分析可溶性NKG2D配体与血细胞计数的相关性，所纳入患者涵盖再生障碍性贫血、单系发育异常型骨髓增生异常综合征以及阵发性睡眠性血红蛋白尿症。经酶联免疫吸附试验检测，约半数骨髓衰竭综合征患者的血浆可溶性NKG2D配体呈阳性；且无论膜结合型NKG2D配体的表达情况如何，可溶性NKG2D配体阳性的患者均表现为重度血细胞减少。体外实验结果显示，作为NKG2D配体的可溶性UL16结合蛋白1（ULBP1）可下调外周血CD2阳性细胞表面的NKG2D受体。此外，可溶性UL16结合蛋白1可减弱外周血单个核细胞对表达膜结合型UL16结合蛋白1的K562细胞的细胞毒作用。本研究结果表明，可溶性NKG2D配体可作为易于检测的生物标志物，用于预测骨髓衰竭综合征中免疫介导的骨髓损伤活性；同时可溶性NKG2D配体可抑制过度的免疫介导性骨髓损伤。