Tremante Elisa, Lo Monaco Elisa, Ingegnere Tiziano, Sampaoli Camilla, Fraioli Rocco, Giacomini Patrizio
Laboratory of Immunology, Regina Elena National Cancer Institute, Rome, Italy.
Eur J Immunol. 2015 Aug;45(8):2356-64. doi: 10.1002/eji.201545446. Epub 2015 Jun 5.
Since HLA-E heavy chains accumulate free of their light β2 -microglobulin (β2 m) subunit, raising mAbs to folded HLA-E heterodimers has been difficult, and mAb characterization has been controversial. Herein, mAb W6/32 and 5 HLA-E-restricted mAbs (MEM-E/02, MEM-E/07, MEM-E/08, DT9, and 3D12) were tested on denatured, acid-treated, and natively folded (both β2 m-associated and β2 m-free) HLA-E molecules. Four distinct conformations were detected, including unusual, partially folded (and yet β2 m-free) heavy chains reactive with mAb DT9. In contrast with previous studies, epitope mapping and substitution scan on thousands of overlapping peptides printed on microchips revealed that mAbs MEM-E/02, MEM-E/07, and MEM-E/08 bind three distinct α1 and α2 domain epitopes. All three epitopes are linear since they span just 4-6 residues and are "hidden" in folded HLA-E heterodimers. They contain at least one HLA-E-specific residue that cannot be replaced by single substitutions with polymorphic HLA-A, HLA-B, HLA-C, HLA-F, and HLA-G residues. Finally, also the MEM-E/02 and 3D12 epitopes are spatially distinct. In summary, HLA-E-specific residues are dominantly immunogenic, but only when heavy chains are locally unfolded. Consequently, the available mAbs fail to selectively bind conformed HLA-E heterodimers, and HLA-E expression may have been inaccurately assessed in some previous oncology, reproductive immunology, virology, and transplantation studies.
由于HLA - E重链在没有轻链β2 -微球蛋白（β2m）亚基的情况下积累，因此制备针对折叠的HLA - E异二聚体的单克隆抗体一直很困难，并且单克隆抗体的表征也存在争议。在此，对单克隆抗体W6/32和5种HLA - E限制性单克隆抗体（MEM - E/02、MEM - E/07、MEM - E/08、DT9和3D12）在变性、酸处理和天然折叠（β2m相关和β2m游离）的HLA - E分子上进行了测试。检测到四种不同的构象，包括与单克隆抗体DT9反应的异常、部分折叠（且β2m游离）的重链。与先前的研究不同，对印在微芯片上的数千个重叠肽进行的表位作图和取代扫描显示，单克隆抗体MEM - E/02、MEM - E/07和MEM - E/08结合三个不同的α1和α2结构域表位。所有这三个表位都是线性的，因为它们仅跨越4 - 6个残基，并且在折叠的HLA - E异二聚体中是“隐藏”的。它们包含至少一个HLA - E特异性残基，该残基不能被多态性HLA - A、HLA - B、HLA - C、HLA - F和HLA - G残基的单取代所替代。最后，MEM - E/02和3D12表位在空间上也是不同的。总之，HLA - E特异性残基具有主要的免疫原性，但仅当重链局部未折叠时才具有。因此，现有的单克隆抗体不能选择性地结合构象化的HLA - E异二聚体，并且在一些先前的肿瘤学、生殖免疫学、病毒学和移植研究中，HLA - E的表达可能被不准确地评估。
