Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
Sci Rep. 2022 May 9;12(1):7608. doi: 10.1038/s41598-022-11774-9.
Anti-interferon gamma autoantibodies (anti-IFN-γ autoAbs) neutralize the IFN-γ-mediated functions, contributing to immunodeficiency. A particular autoAb in patient serum had been previously demonstrated to recognize the same determinant on IFN-γ as the neutralizing anti-IFN-γ monoclonal antibody clone B27 (B27 mAb). This study explored the epitope recognized by B27 mAb. The specific peptide sequence recognized by B27 mAb, TDFLRMMLQEER, was retrieved from a phage display random peptide library. Sequence alignment and homology modeling demonstrated that the queried phage peptide sequence and structure were similar to amino acids at position 27-40 (TLFLGILKNWKEES) of the human IFN-γ. This determinant resides in the contact surface of IFN-γ and interferon gamma receptor 1. To elucidate the crucial amino acids, mutations were introduced by substituting T27 and T27F29L30 with alanine or deleting the amino acid residues T27-L33. The binding of B27 mAb to IFN-γ T27A using western blotting was lesser than that to wild-type. The interaction with triple mutant and T27-L33 deletion mutant using western blotting and sandwich ELISA was abolished. The finding demonstrated that T27, F29, and L30 are critical residues in the B27 antigenic determinant. Identification of the functional domain of IFN-γ decrypted the relevance of neutralizing autoAb in adult-onset immunodeficiency.
抗干扰素 γ 自身抗体（抗 IFN-γ 自身 Abs）中和 IFN-γ 介导的功能，导致免疫缺陷。先前已经证明患者血清中的一种特殊自身抗体识别与中和抗 IFN-γ 单克隆抗体克隆 B27（B27 mAb）相同的 IFN-γ 决定簇。本研究探索了 B27 mAb 识别的表位。从噬菌体展示随机肽文库中检索到 B27 mAb 识别的特定肽序列 TDFLRMMLQEER。序列比对和同源建模表明，所查询的噬菌体肽序列和结构与 IFN-γ 位置 27-40（TLFLGILKNWKEES）的氨基酸相似。该决定簇位于 IFN-γ 和干扰素 γ 受体 1 的接触表面。为了阐明关键氨基酸，通过用丙氨酸取代 T27 和 T27F29L30 或删除氨基酸残基 T27-L33 引入突变。使用 Western blot 法，B27 mAb 与 IFN-γ T27A 的结合比与野生型的结合少。使用 Western blot 和夹心 ELISA 法，与三突变体和 T27-L33 缺失突变体的相互作用被废除。该发现表明 T27、F29 和 L30 是 B27 抗原决定簇的关键残基。IFN-γ 功能域的鉴定揭示了成人免疫缺陷中中和自身抗体的相关性。
