Chersi Alberto, Galati Rossella, Accapezzato Daniele, Francavilla Vittorio, Barnaba Vincenzo, Butler Richard H, Tanigaki Nobuyuki
Department of Biochemistry, Regina Elena Institute for Cancer Research, Rome, Italy.
J Immunol Methods. 2004 Aug;291(1-2):79-91. doi: 10.1016/j.jim.2004.05.001.
Cell-sized microbeads carrying single peptide-loaded HLA class I molecules were prepared for HLA-A2 and HLA-B7 by a simple procedure which transfers single peptide-loaded HLA class I molecules from cultured cells to polystyrene beads using anti-peptide antibodies directed to an intracellular segment of HLA-A alpha chains. The surface density of peptide-loaded HLA class I molecules on beads was comparable to that on the peptide-loaded cells. HLA-A2 beads loaded with an HCV peptide HCV1073 were tested for stimulation activity on an HCV1073-specific CD8+ T cell clone NS3-1. A substantial level of gamma-IFN production was induced. The stimulation was peptide-specific. The efficiency was dependent on the bead concentration and the surface HLA class I density on beads and enhanced significantly by co-coupling of anti-CD28 to peptide-loaded beads. The peptide-loading efficiency on HLA class I molecules and the transfer efficiency of HLA class I molecules to polystyrene beads were reasonably high for HLA-A2 and HLA-B7. Thus, polystyrene beads carrying these single peptide-loaded HLA class I molecules are potentially useful in further analysis of the co-stimulatory or inhibitory factors involved in CD8+ T cell responses and eventually in detection of cytotoxic T cells in PBLs.
通过一种简单的程序制备了携带单个负载肽的HLA I类分子的细胞大小的微珠，该程序使用针对HLA-Aα链细胞内片段的抗肽抗体将单个负载肽的HLA I类分子从培养细胞转移到聚苯乙烯珠上。针对HLA-A2和HLA-B7进行了该制备。珠上负载肽的HLA I类分子的表面密度与负载肽的细胞上的密度相当。对负载丙型肝炎病毒（HCV）肽HCV1073的HLA-A2珠进行了对HCV1073特异性CD8 + T细胞克隆NS3-1的刺激活性测试。诱导了相当水平的γ-干扰素产生。该刺激是肽特异性的。效率取决于珠浓度和珠上的表面HLA I类密度，并通过将抗CD28共偶联到负载肽的珠上而显著提高。对于HLA-A2和HLA-B7，HLA I类分子上的肽负载效率以及HLA I类分子向聚苯乙烯珠的转移效率相当高。因此，携带这些单个负载肽的HLA I类分子的聚苯乙烯珠在进一步分析CD8 + T细胞反应中涉及的共刺激或抑制因子以及最终在检测外周血淋巴细胞中的细胞毒性T细胞方面可能有用。
