Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.
Department of Medical Microbiology, Radboud university medical center, Nijmegen, The Netherlands.
Sci Rep. 2017 Mar 7;7:43486. doi: 10.1038/srep43486.
Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.
血蓝蛋白（KLH）被用作各种临床应用和疫苗开发中的免疫原性新抗原。为了对基于 KLH 的干预措施进行高级监测，我们开发了一种基于流式细胞术的体外检测、表型分析和 KLH 特异性 B 细胞分离的方法。作为原理验证，我们分析了 10 名接受 KLH 治疗的黑色素瘤患者在接受抗癌树突状细胞疫苗接种期间的情况。我们的检测方法能够在患者外周血中灵敏且特异性地检测到 KLH 特异性 B 细胞，并且 KLH 特异性 B 细胞的频率与抗 KLH 血清抗体滴度强烈相关。在整个疫苗接种过程中对 B 细胞亚群的分析表明，KLH 特异性 B 细胞从幼稚 B 细胞成熟为类别转换的记忆 B 细胞，证实了对新抗原的典型 B 细胞反应。我们得出结论，流式细胞术检测和 KLH 特异性 B 细胞的深入表型分析具有特异性、敏感性和可扩展性。我们的发现为监测 KLH 特异性免疫反应提供了新的机会，并为新的流式细胞术方案的开发提供了蓝图。
