Institut D'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, 08036, Spain.
Julia McFarlane Diabetes Research Centre (JMDRC) and Department of Microbiology, Immunology and Infectious Diseases, Snyder Institute for Chronic Diseases and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Alberta, T2N 4N1, Canada.
Nat Commun. 2019 Oct 29;10(1):4917. doi: 10.1038/s41467-019-12902-2.
Assembly of soluble peptide-major histocompatibility complex class II (pMHCII) monomers into multimeric structures enables the detection of antigen-specific CD4 T cells in biological samples and, in some configurations, their reprogramming in vivo. Unfortunately, current MHCII-αβ chain heterodimerization strategies are typically associated with low production yields and require the use of foreign affinity tags for purification, precluding therapeutic applications in humans. Here, we show that fusion of peptide-tethered or empty MHCII-αβ chains to the IgG1-Fc mutated to form knob-into-hole structures results in the assembly of highly stable pMHCII monomers. This design enables the expression and rapid purification of challenging pMHCII types at high yields without the need for leucine zippers and purification affinity tags. Importantly, this design increases the antigen-receptor signaling potency of multimerized derivatives useful for therapeutic applications and facilitates the detection and amplification of low-avidity T cell specificities in biological samples using flow cytometry.
可溶性肽-主要组织相容性复合体 II 类（pMHCII）单体组装成多聚体结构，可在生物样本中检测抗原特异性 CD4 T 细胞，并且在某些构型下，可在体内对其进行重编程。不幸的是，当前 MHCII-αβ 链异源二聚化策略通常与低产量相关联，并且需要使用外来亲和标签进行纯化，从而排除了在人类中的治疗应用。在这里，我们表明，肽连接的或空的 MHCII-αβ 链与 IgG1-Fc 的融合突变形成了旋钮进入孔结构，导致高度稳定的 pMHCII 单体的组装。该设计能够以高产率表达和快速纯化具有挑战性的 pMHCII 类型，而无需亮氨酸拉链和纯化亲和标签。重要的是，该设计增加了用于治疗应用的多聚化衍生物的抗原受体信号转导效力，并通过流式细胞术促进了生物样本中低亲和力 T 细胞特异性的检测和扩增。
