免疫球蛋白重链框架区的抗原九肽诱导特异性细胞毒T细胞增殖

目的 探索免疫球蛋白重链可变区(IgHV)是否存在T细胞识别的表位。方法 PCR扩增108例急性淋巴细胞白血病(ALL)的7个IgHV家族的重排基因,PCR产物直接测序并翻译成氨基酸。利用互联网生物信息资源分析B-ALL IgHV基因的重排类型和基因变异,利用SYFPEITHI和BIMAS软件预测IgHV基因编码蛋白质的T细胞表位。合成代表性九肽IgHV1家族的QLVQSGAEV,进行12结合分析、合成九肽诱导的T细胞扩增、扩增细胞的HLA-A*0201／QLVQSGAEV四聚体检测等实验,确定合成九肽的免疫原性。结果 66％的B-ALL检测到IgHV基因重排克隆。在40份测序得到的B-ALL IgHV序列中,选出26份进行HLA-A*0201分子结合九肽的预测,发现7个IgHV家族共有12条抗原九肽。除1条位于第3互补决定区(CDR3)外,10条(83％)位于框架区(FR),并与相应胚系VH家族的代表序列相同;为同一IgHV家族的白血病细胞所共有。合成的IgHV1抗原九肽可将12细胞表面HLA-A*0201分子的表达提高1．63倍。HLA-A*0201正常供者的外周血淋巴细胞接受该九肽负荷的自身PBMC的1轮刺激,CD8^ 四聚体^ 细胞达1．64％,继续接受该九肽负荷的12细胞的2轮刺激,CD8^ 四聚体^ 细胞增至82．57％。结论 B淋巴细胞的免疫球蛋白重链框架区有CTL识别的IgHv家族特异的抗原九肽,正常人外周血T细胞库存在这些抗原九肽特异的CTL,这些抗原九肽可诱导特异性CTL的增殖。

Proliferation of specific cytotoxic T lymphocytes induced by immunoglobulin heavy chain framework region-derived antigenic nonapeptides

OBJECTIVE: To identify that immunoglobulin heavy chain framework regions-derived peptides function as cytotoxic T lymphocytes epitopes. METHODS: Seven IgHV gene families were respectively amplified by PCR and directly sequenced for 108 acute lymphoblastic leukemia cases. Sequences available were translated into amino acid sequences. Bioinformatics were applied for analyzing recombination patterns and gene mutations of the IgHV genes. Softwares SYFPEITHI and BIMAS were used for predicting the T cell epitopes in the immunoglobulin heavy chain variable regions. To determine whether the predicted peptides have immunogenicity, a IgHV1 family nonapeptide QLVQSGAEV was synthesized as a representation and T2 binding assay of this peptide was performed, inducing proliferation of T cells from normal HLA-A * 0201 peripheral blood lymphocytes (PBLs) with QLVQSGAEV-loaded antigen presenting cells, and detecting the proliferating T cells by HLA-A * 0201/QLVQSGAEV tetramers. RESULTS: Complete IgHV gene rearrangements were identified in 66% cases. Among 40 B-ALL IgHV sequences available, 26 were predicted for antigenic nonapeptides that are likely to bind to HLA-A * 0201 molecule. Twelve peptides were acquired. Except one peptide derived from CDR3, 10 (83%) peptides were located in the immunoglobulin heavy chain framework regions. Moreover B-ALL belonging to the same IgHV family shared 1 - 2 peptides. Synthesized peptide QLVQSGAEV up-regulated HLA-A * 0201 expression 1.63 times on T2 cell surface. PBLs from a normal HLA-A * 0201 donor were stimulated with QLVQSGAEV-loaded autologous PBMCs and T2, the CD8(+) tetramer(+) cells in gated lymphocyte population increased from 1.64% after the first stimulation to 82.57% after the third stimulation. CONCLUSION: Immunoglobulin heavy chain framework region genes encode IgHV family-specific peptides recognized by CTLs. Specific CTLs remain in human peripheral T cell repertoire. Immunoglobulin heavy chain framework-derived peptides function as T cell epitopes to induce the proliferation of specific CTLs.