HLA-Releasing Metalloproteinase in Allograft Rejection

描述(申请人提供)：我们之前已经描述了金属蛋白酶介导的可溶性MHC-I类释放的途径，并提出了它在移植中的作用。我们发现可溶性MHC-I类分子的释放是由去整合素和金属蛋白水解酶家族成员ADAM17介导的。内皮细胞(EC)与同种异体T细胞共培养后，可上调ADAM 17的表达和活性。这种激活是由干扰素-γ驱动的，最终由EC释放可溶性的MHC-I类蛋白。然而，至少有一种与ADAM17不同的金属蛋白酶完全能够释放可溶性MHC-I类分子，其活性可能受细胞因子以组织特异性方式调节。用我们的单抗筛选人白血病表达文库，阻断可溶性MHC-I类分子的释放，从而鉴定出一个功能未知的新蛋白BC036469。这种普遍表达的蛋白可能通过介导特定的酶/底物相互作用参与了可溶性MHC-I类分子的释放，其功能可能受到不同组织中细胞特异性细胞因子的调节。我们设计了三个独立的目标来解决这些问题。首先，我们将通过使用一组ADAM缺失的细胞系和DNA微阵列分析来鉴定能够处理MHC I类的细胞因子诱导的金属蛋白酶。其次，BC036469蛋白的功能将通过过度表达野生型和缺失突变体，以及通过用特定的siRNA干扰内源蛋白的表达来确定。最后，我们将确定在MHC I类分子的α3和跨膜区内产生酶/底物相互作用所需的位点，并测试特定多肽抑制相互作用的能力。在由控制移植耐受的HLA-A2限制性CD8低亲和力T调节细胞控制的免疫调节的连锁抑制模型中，将使用体内迟发型超敏反应试验来检验可溶性MHC I类在抗原提呈中的预测作用。

DESCRIPTION (provided by applicant): 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.