Table_3_Rapid Multiplex Genotyping of 20 HLA-A*02:01 Restricted Minor Histocompatibility Antigens.XLSX

A subset of MHC-associated self-peptides presented by the recipient's cells and immunologically foreign to the donor can induce an allogeneic immune response after hematopoietic stem cell transplantation (HSCT). These immunogenic peptides originate from the genomic polymorphisms and are known as minor histocompatibility antigens (MiHA). MiHA mismatches trigger the post-transplant immune response, which could manifest in both the deleterious “graft-vs.-host” disease and the beneficial “graft-vs.-leukemia” effect. Importantly, some MiHAs are considered to be promising targets for posttransplant T-cell immunotherapy of hematopoietic malignancies. This creates a demand for a robust and fast approach to genotyping MiHA-encoding polymorphisms. We report a multiplex real-time PCR method for the genotyping of 20 polymorphisms that are encoding HLA-A*02:01-restricted MiHAs. This method uses allele-specific primers and gene-specific hydrolysis probes. In 1 h it allows for the detection of MiHA mismatches in a donor-recipient pair without the need for electrophoresis, sequencing, or other time-consuming techniques. We validated the method with Sanger and NGS sequencing and demonstrated good performance over a wide range of DNA concentrations. We propose our protocol as a fast and accurate method of identifying mismatched MiHAs. The information on the MiHA mismatches is useful for studying the allogeneic immune response following HSCT and for selecting the targets for post-transplant T-cell therapy.

MHC相关自身肽段，由受者细胞呈现且对供体免疫学上为异物，能够在造血干细胞移植（HSCT）后诱发同种免疫反应。这些具有免疫原性的肽段源于基因组多态性，被称为小分子组织相容性抗原（MiHA）。MiHA的不匹配会引发移植后免疫反应，该反应可能表现为有害的“移植物抗宿主病”或有益的“移植物抗白血病”效应。值得注意的是，某些MiHA被视为移植后T细胞免疫治疗血液恶性肿瘤的潜在靶点。这催生了对一种强大且快速的方法进行MiHA编码多态性基因分型的需求。我们报道了一种针对编码HLA-A*02:01限制性MiHA的20个多态性的多重实时PCR基因分型方法。此方法使用等位特异性引物和基因特异性水解探针。在1小时内，它允许在供体-受者对中检测到MiHA不匹配，而无需电泳、测序或其他耗时技术。我们使用Sanger和NGS测序验证了该方法，并在广泛的DNA浓度范围内展示了良好的性能。我们提议将我们的方案作为识别不匹配MiHA的快速准确方法。关于MiHA不匹配的信息对于研究HSCT后的同种免疫反应以及选择移植后T细胞疗法的靶点具有重要意义。