应用免疫球蛋白重链可变区上框架区来源的抗原九肽获得的细胞毒T细胞功能分析  

Function analysis of the family-specific CTL induced by peptides derived from IgHV gene framework region of B-cell malignance

作　　者：郭晓玲[1] 朱平[1] 刘英[2] 刘静[1] 朱霞[3] 

机构地区：[1]北京大学第一医院血液科,100034 [2]解放军总医院儿科 [3]郑州大学医学院第三附属医院儿科

出　　处：《中华血液学杂志》2005年第8期453-457,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(39970131;30470739);北京市自然科学基金资助项目(7032028)

摘　　要：目的明确在B淋巴细胞肿瘤表面的免疫球蛋白重链可变区(IgHV)的框架区(FR)上是否存在可以激发细胞毒T淋巴细胞(CTL)的抗原表位,这些来源于FR的抗原肽是否能够激发家族特异性的免疫反应,探讨按照B淋巴细胞肿瘤的IgHV基因分型进行家族性的免疫治疗的可能性。方法利用生物信息学系统预测了40例B淋巴细胞肿瘤表面的免疫球蛋白序列的抗原表位,人工合成不同基因家族的抗原九肽,利用T2细胞结合实验检测预测的抗原肽和HLA分子的亲合力。利用体外CTL刺激扩增体系,诱导特异性的CTL细胞增殖,用肽/HLA四聚体检测特异性CTL的数目,应用LDH释放实验检测CTL的细胞毒活性并验证其家族特异性。结果在B淋巴细胞肿瘤的7个IgHV基因家族中找到了12个高亲和力的抗原九肽,其中10个(83%)位于FR,以HLA-A*0201正常供者的外周血单个核细胞(PBMNC)负荷该九肽作为抗原呈递细胞去刺激自身PBMNC,经过4轮刺激,CD8和肽/HLA四聚体双阳性细胞从0.38%上升到49.38%。LDH释放实验证明对HLA-A*0201(+)、IgHV1(+)靶细胞有明显的杀伤作用,并且被IgHV1肽刺激出的CTL不能识别负荷IgHV3肽的靶细胞。结论IgHV基因FR抗原九肽可以刺激产生具有HLA限制性的并且肽特异性的CTL,同时这样的杀伤作用具有家族特异性,以此为靶位有望对B淋巴细胞肿瘤进行家族特异性的免疫治疗。Objective To identify immune epitopes existed in the framework region (FR) of the IgHV protein of B-cell malignance, and explore the use of these FR-derived peptides to induce the family specific immune response in vitro, in order to explore the possibility of a new IgHV gene family-specific immunotherapy for B-cell malignance. Methods Bioinformatics was used for predicting T cell epitopes in IgHV protein. Peptides of interest were synthesized in vitro. T2 cell binding assay was performed to determine the binding ability of the peptides to HLA-A*0201 molecules. Peptide/HLA tetramer staining was used to detect the number of peptide-specific cytotoxic T lymphocytes (CTLs). Cytotoxicity assay was used to determine killing activity. Results Twelve peptides that were common to seven IgHV gene subfamilies were identified, and 10 (83%) of them were located in the FR of IgHV protein. The synthesized peptides up-regulated HLA-A*0201 molecules fluorescence intensity on cell surfaces of T2. By using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMNC) from a healthy HLA-A0201 donor were stimulated weekly by autologous PBMNC loaded with the peptide as antigen presenting cells (APC), and the peptide-specific CTLs were demonstrated to be generated successfully in the healthy donors. The frequency of CD8 and peptide/HLA tetramer double positive cells in the gated lymphocyte population was 0.38% before stimulation and increased to 49.38 % after four times stimulation. Cytotoxicity assay indicated that these CTLs were capable of killing the HLA-A*0201,IgHV1(+)lymphoma cells. Furthermore, the generated CTL could not kill the target cell loaded with the IgHV3 peptide, indicating that the cytotoxicity is family-specific. Conclusion Peptides derived from the IgHV protein FR can successfully induce the generation of peptide-specific CTLs in vitro. These CTLs are capable of killing the lymphoma cell belonged to the same subfamily in a peptide-specific and MHC-restricted way. These finding

关 键 词：免疫球蛋白重链可变区 框架区 抗原九肽 细胞毒T细胞 淋巴瘤细胞 

分 类 号：R733[医药卫生—肿瘤]