HLA-Releasing Metalloproteinase in Allograft Rejection

我们先前已经描述了金属蛋白酶介导的可溶性MHC I类分子释放途径 并提出了其在移植中的作用。我们发现，可溶性MHC I类分子的释放是由一种免疫调节因子介导的。 解整合素和金属蛋白酶家族成员，ADAM 17。内皮细胞（EC）与 同种异体T细胞上调特异性活化标志物以及ADAM 17的表达和活性。 这种激活是由干扰素γ驱动的，并在可溶性MHC I类蛋白的释放中达到高潮。 的EC。然而，至少一种不同于ADAM 17的其他金属蛋白酶完全能够释放 可溶性MHC I类。它的活性可以通过细胞因子以组织特异性方式调节。筛选 用我们的阻断可溶性MHC I类释放的mAb构建人白血病表达文库， 鉴定了一种功能未知新蛋白质BC 036469。这种广泛表达的蛋白质 可能通过介导特异性酶/底物参与可溶性MHC I类释放的机制 在不同的组织中，其相互作用及其功能可由细胞特异性细胞因子调节。三 为解决这些问题制定了独立的目标。首先，我们将确定尼古丁诱导的 通过使用一组ADAM缺陷型细胞系和通过 DNA微阵列分析。第二，BC 036469蛋白的功能将通过过量表达来确定。 野生型和缺失突变体，并通过破坏内源性蛋白质的表达， siRNA。最后，我们将确定生产酶/底物相互作用所需的网站内， α 3和跨膜结构域的MHC I类，并测试特异性肽的抑制能力， 互动可溶性MHC I类在抗原呈递中的预测作用将使用反式- 在HLA-A2免疫调节的连锁抑制模型中的体内迟发型超敏反应测定- 限制性CD 8低亲合力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.