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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 determine whether there are epitopes on the framework region (FR) of immunoglobulin heavy chain variable region (IgHV) on the surface of B lymphocyte tumors that can stimulate cytotoxic T lymphocytes (CTLs) Peptides can stimulate family-specific immune responses and explore the possibility of familial immunotherapy based on IgHV genotyping of B lymphocyte tumors. Methods The bioinformatics system was used to predict the epitopes of immunoglobulin sequences on the surface of 40 cases of B lymphocytic tumors. Antigen nonapeptides of different gene families were artificially synthesized, and the predicted antigen peptides and HLA progenitors were detected by T2 cell binding assay Together. The proliferation of CTL cells was induced by CTL stimulation in vitro. The number of specific CTLs was detected by peptide / HLA tetramer. The cytotoxic activity of CTL was tested by LDH release assay and the family specificity was verified. RESULTS: Twelve high-affinity antigen nonapeptides were found in the seven IgHV gene families of B-cell lymphoma, of which 10 (83%) were located in the FR, peripheral blood mononuclear cells from normal donors of HLA-A * 0201 PBMNC) loaded with the nonapeptide as antigen-presenting cells to deactivate its own PBMNC. After 4 rounds of stimulation, double positive cells of CD8 and peptide / HLA tetramer increased from 0.38% to 49.38%. LDH release assay demonstrated that the cytotoxicity against HLA-A * 0201 (+) and IgHV1 (+) target cells was significantly inhibited, and CTL stimulated by IgHV1 peptide did not recognize target cells loaded with IgHV3 peptide. Conclusion The IgHV gene FR antigen nonapeptide can stimulate the production of HLA-restricted and peptide-specific CTLs. At the same time, such cytotoxicity has familial specificity, and it is expected that this target will be used for family-specific immunotherapy of B lymphocytic tumors .