HLA-Releasing Metalloproteinase in Allograft Rejection

我们之前已经描述了金属蛋白酶介导的可溶性 MHC I 类释放途径 并提出了其在移植中的作用。我们发现可溶性 MHC I 类的释放是由 解整合素和金属蛋白酶家族成员 ADAM17。内皮细胞 (EC) 与 同种异体 T 细胞上调特定激活标记以及 ADAM 17 的表达和活性。 这种激活由干扰素 γ 驱动，最终导致可溶性 MHC I 类蛋白的释放 由欧共体。然而，至少另一种不同于 ADAM17 的金属蛋白酶完全能够释放 可溶性 MHC I 类。其活性可能受细胞因子以组织特异性方式调节。筛选 我们的单克隆抗体可阻断可溶性 MHC I 类的释放，从而构建人类白血病表达文库 鉴定出功能尚不清楚的新型蛋白质 BC036469。这种普遍表达的蛋白质 可能通过介导特定酶/底物参与可溶性 MHC I 类释放机制 相互作用及其功能可能受到不同组织中细胞特异性细胞因子的调节。三 独立目标旨在解决这些问题。首先，我们将识别细胞因子诱导的 能够通过使用一组 ADAM 缺陷细胞系和通过 DNA 微阵列分析。二、BC036469蛋白的功能将通过过表达来确定 野生型和缺失突变体，并通过用特定的方法破坏内源蛋白的表达 siRNA。最后，我们将确定有效酶/底物相互作用所需的位点 α 3 和 MHC I 类跨膜结构域，并测试特定肽抑制的能力 互动。可溶性 MHC I 类在抗原呈递中的预测作用将使用反式进行测试 HLA-A2-免疫调节连锁抑制模型体内迟发型超敏反应测定 限制性 CD8 低亲和力 T 调节细胞控制移植耐受。

We have previously described the metalloproteinase-mediated pathway of soluble MHC class I release and proposed its role in transplantation. We found that the release of soluble MHC class I is mediated by a disintegrin and metalloprotease family member, ADAM17. Endothelial cells (EC) co-cultured with allogeneic T cells up-regulate specific activation markers and both the expression and activity of ADAM 17. This activation is driven by interferon-gamma and culminates in the release of soluble MHC class I proteins by EC. However, at least one other metalloproteinase distinct from ADAM17 is fully capable of releasing soluble MHC class I. Its activity may be regulated by cytokines in a tissue-specific manner. Screening of a human leukemia expression library with our mAb that blocks the release of soluble MHC class I led to identification of a novel protein BC036469 with yet unknown function. This ubiquitously expressed protein may participate in the mechanism of soluble MHC class I release by mediating specific enzyme/substrate interactions and its function may be regulated by cell-specific cytokines in different tissues. Three independent aims are designed to address these questions. First, we will identify cytokine-inducible metalloproteinases capable of processing MHC class I by using a panel of ADAM-deficient cell lines and by DNA microarray analysis. Second, the function of BC036469 protein will be determined by overexpressing wild-type and deletion mutants, and by disrupting expression of the endogenous protein with specific siRNA. Finally, we will determine the sites required for productive enzyme/substrate interactions within alpha 3 and transmembrane domains of MHC class I and test the ability of specific peptides to inhibit the interaction. The predicted role of soluble MHC class I in antigen presentation will be tested using the trans- vivo delayed-type hypersensitivity assay in the linked suppression model of immune regulation by HLA-A2- restricted CD8 low avidity T regulator cells controlling transplantation tolerance.