Institute for Transfusion Medicine, Hannover Medical School, Germany.
Int Immunol. 2012 Sep;24(9):561-72. doi: 10.1093/intimm/dxs059. Epub 2012 Jun 27.
Multimers of soluble peptide-major histocompatibilty complex (pMHC) molecules are used in both basic and clinical immunology. They allow the specific visualization and isolation of antigen-specific T cells from ex vivo samples. Adoptive transfer of antigen-specific T cells sorted by pMHC multimers is an effective strategy for treatment of patients with malignancies or infectious diseases after transplantation. We developed a new reversible pMHC multimer called 'Histamer' to enable the specific detection and isolation of antiviral T cells from peripheral blood. HLA-A*02:01/CMVpp65 (495-503) Histamer (A02/CMV Histamer) was generated by coupling 6xHis-tagged pMHC molecules onto cobalt-based magnetic beads. The specificity of the Histamer was evaluated by flow cytometry. Sorting of antiviral CD8(+) cytotoxic T lymphocytes (CTLs) was performed by magnetic cell separation, followed by the monomerization of the Histamer after addition of the competitor L-histidine. Sorted T cells were analyzed for phenotype and function. The reversible pMHC Histamer proved to be highly specific and sensitive. CMV-specific T cells of up to 99.6% purity were isolated using the Histamer technology. Rapid and complete disassembly of the T-cell surface-bound A02/CMV Histamer followed by the subsequent dissociation of the pMHC monomers from CD8(+) CTL receptors was achieved using 100 mM L-histidine. The function of CMV-specific T cells enriched by Histamer staining did not differ from CTLs induced by standard T-cell assays. This reversible T-cell staining procedure preserves the functionality of antigen-specific T cells and can be adapted to good manufacturing practice conditions. The pMHC Histamer technology offers full flexibility and fulfills all requirements to generate clinical-grade T lymphocytes.
可溶性肽-主要组织相容性复合物（pMHC）分子的多聚体在基础和临床免疫学中都有应用。它们允许从体外样本中特异性地可视化和分离抗原特异性 T 细胞。通过 pMHC 多聚体分选的抗原特异性 T 细胞的过继转移是治疗移植后恶性肿瘤或传染病患者的有效策略。我们开发了一种新的可逆 pMHC 多聚体，称为“Histamer”，用于从外周血中特异性检测和分离抗病毒 T 细胞。HLA-A*02:01/CMVpp65（495-503）Histamer（A02/CMV Histamer）是通过将 6xHis 标记的 pMHC 分子偶联到钴基磁性珠上而生成的。通过流式细胞术评估了 Histamer 的特异性。通过磁性细胞分离对抗病毒 CD8(+)细胞毒性 T 淋巴细胞（CTL）进行分选，然后在添加竞争物 L-组氨酸后对 Histamer 进行单体化。对分选的 T 细胞进行表型和功能分析。结果表明，可逆的 pMHC Histamer 具有高度的特异性和敏感性。使用 Histamer 技术可分离高达 99.6%纯度的 CMV 特异性 T 细胞。使用 100 mM L-组氨酸可快速且完全地拆卸 T 细胞表面结合的 A02/CMV Histamer，随后从 CD8(+) CTL 受体上解离 pMHC 单体。通过 Histamer 染色富集的 CMV 特异性 T 细胞的功能与通过标准 T 细胞测定诱导的 CTL 没有差异。这种可逆的 T 细胞染色程序保留了抗原特异性 T 细胞的功能，并且可以适应良好生产规范条件。pMHC Histamer 技术提供了充分的灵活性，并满足了生成临床级 T 淋巴细胞的所有要求。
