Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Am Mühlenberg 1, 14476 Potsdam, Germany; Freie Universität Berlin, Institute of Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Arnimallee 22, 14195 Berlin, Germany.
Max Planck Institute of Colloids and Interfaces, Department of Biomolecular Systems, Am Mühlenberg 1, 14476 Potsdam, Germany; Freie Universität Berlin, Institute of Chemistry and Biochemistry, Department of Biology, Chemistry and Pharmacy, Arnimallee 22, 14195 Berlin, Germany.
J Control Release. 2014 Feb 10;175:36-42. doi: 10.1016/j.jconrel.2013.12.011. Epub 2013 Dec 22.
Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.
髓系 C 型凝集素受体 (CLRs) 在天然免疫中代表了一类模式识别受体超家族，能够识别病原体和自身抗原上的碳水化合物结构。抗原呈递细胞与病原体的初次相互作用决定了随后的免疫反应。因此，鉴定既能增强又能调节免疫反应的 CLR 配体是很有意义的。我们开发了一种基于聚糖阵列的筛选平台，用于鉴定 CLR 的免疫调节性碳水化合物配体。通过将每个 CLR 的胞外部分（包含碳水化合物识别结构域 (CRD) 的部分）与人类 IgG1 分子的 Fc 片段融合，表达了一个全面的 CLR 文库。CLR-Fc 融合蛋白以二聚体形式展示 CRD，糖基化正确，并可通过缀合荧光团的二抗检测。因此，它们是高通量筛选的有价值的工具。我们能够使用聚糖阵列技术鉴定 CLR 的新型碳水化合物结合物。然后，将这些 CLR 结合碳水化合物共价连接到模型抗原卵清蛋白上。在树突状细胞/T 细胞共培养测定中使用卵清蛋白糖基化缀合物来体外刺激转基因 T 细胞。此外，用这些缀合物免疫小鼠，以分析体内 CLR 配体的免疫调节特性。CLR 配体在体外诱导 Th1 细胞因子产生增加，并调节体内体液免疫反应。这里描述的平台允许鉴定 CLR 配体，以及评估每种配体的细胞特异性靶向和免疫调节特性。
