自体EBV-LCL及同种异体单核细胞在ELISPOT试验中的抗原递呈作用研究

Investigation of the antigen presentation by autologous EBV-LCL and allograft monocytes according to ELISPOT

目的探索自体EB病毒(EBV)转化的B淋巴母细胞系(EBV-LCL)细胞及同种异体单核细胞在ELISPOT试验中的抗原递呈功能。方法采用Ficoll分离PBMC,采用CD14磁珠分选CD14单核细胞;采用序列特异性引物PCR扩增HLA等位基因特异性片段,进行HLA分型;复苏冻存的表位特异性的T细胞克隆细胞及相应的自体EBVLCL细胞,采用酶联免疫斑点法(enzyme-linked immunospot assay,ELISPOT)检测自体EBV-LCL细胞及具有表位相关HLA分子的同种异体单核细胞的抗原递呈功能。结果表位特异性的T细胞克隆细胞能够识别由自体EBV-LCL细胞及具有表位相关HLA分子的同种异体单核细胞递呈的抗原肽并分泌IFN-γ。EBV-LCL细胞与单核细胞的抗原递呈能力无明显区别。表位特异性T细胞克隆细胞冻存复苏后其IFN-γ产生细胞的比率小于10%。结论自体EBVLCL细胞及具有表位相关HLA分子的同种异体单核细胞在ELISPOT试验中能发挥抗原递呈功能。

Objective To investigate the antigen presentation by autologous EBV-LCL and allograft monocytes accord- ing to ELISPOT. Methods PBMCs were separated and purified using Ficoll gradient centrifugation and CD14＋ cells were separated using a CD14 microbead kit. HLA typing of EBV-LCL and monocytes was performed with PCR using se- quence-specific primers. Frozen epitope-specific T clone cells and autologous EBV-LCL cells were recovered. Antigen presentation by EBV-LCL and monocytes with epitope-matched HLA molecules was detected with ELISPOT. Results The peptides presented by autologous EBV-LCL and allograft monocytes were recognized by epitope-specific T clone cells. No differences in the antigen-presenting ability of autologous EBV-LCL and allograft monocytes were noted. IFN y producing cells accounted for less than 10% of the total T clone cells that were recovered. Conclusion Autologous EBV LCL and allograft monocytes with epitope-matched HLA molecules have the ability to present antigens according to ELISPOT to detect epitope-specific T cells.