淋巴瘤相关抗原肽刺激所获细胞毒性T细胞克隆的特征

目的研究抗肿瘤细胞毒性T淋巴细胞(CTL)克隆识别靶细胞的肽特异性和人类白细胞抗原(HLA)限制性,以及其赖以识别抗原肽的T细胞受体(TCR)基因表达序列的分子特征。方法利用体外CTL刺激扩增体系,应用免疫球蛋白重链可变区(IgHV)框架区上的B淋巴瘤相关抗原九肽(IgHV1QLVQSGAEV和IgHV3SLYLQMNSL)负荷的抗原递呈细胞(APC),刺激正常HLAA0201供者的外周血单个核细胞(PBMC),每周1次共4次,用流式细胞仪检测体外培养细胞的免疫表型的变化,并用肽/主要组织相容性基因复合体(MHC)四聚体方法检测肽特异性的CTL增殖情况。应用酶联免疫吸附实验(ELISA)检测CTL与不同的靶细胞共同孵育时释放干扰素γ(IFNγ)的能力。应用逆转录多聚酶链反应(RTPCR)和T细胞受体(TCR)基因指纹谱型图方法检测培养前后的淋巴细胞的克隆变化,并用直接测序的方法得到CTL克隆的TCR基因序列。结果B淋巴瘤相关抗原肽4次刺激体外培养的PBMC后CD8+CTL大量增殖,CD4/CD8明显下降(0.10vs1.43,P<0.05)。IgHV1QLVQSGAEV/HLAA0201四聚体和CD8双阳性的肽特异性CTL数量(49.38%)比刺激前(0.04%)明显上升。ELISA检测IFNγ的分泌表明其识别靶细胞是肽特异性和HLA限制性的。TCR基因指纹谱型图显示抗原肽反复刺激获得的CTL的TCR表达集中于个别基因家族,呈克隆性增殖。结论应用IgHV基因框架区来源的B淋巴瘤相关抗原九肽可以使特异性CTL克隆增殖,这些克隆以肽特异性和HLA限制性的方式识别靶细胞。了解这些CTL的作用和TCR识别相关抗原肽的分子结构,将有助于确定体内T细胞和B细胞相互调节的机制。

The characteristics of anti-B-cell malignance cytotoxic T lymphocytic clones induced by lymphoma relative peptides

OBJECTIVE: To analyze if the cytotoxic T-cell (CTLs) are peptide-special and HLA restrict, and what is the sequence characteristic of TCRbeta genes. METHODS: Using an antigen-specific T-cell expansion system in vitro, the peripheral blood mononuclear cells (PBMCs) from healthy HLA-A0201 positive donor were stimulated by PBMCs and T2 cells loading the IgHV1-QLVQSGAEV nonapeptide or IgHV3-QLVQSGAEV, B-lymphoma-related nonapeptides, as antigen presenting cells (APCs) once a week for four weeks so as to obtain peptide-specific CTLs. PBMCs from non-HLA-A * 0201 positive donors were used as controls. The immunophenotypes of the CTLs (CD3, CD4, or CD8) were identified by flow cytometry. The proportions of CD8 and peptide/tetramer double positive cells were assayed by using peptide/ HLA tetramer method, The IFN-gamma-releasing capacity of the CTLs incubated together with different target cells was assayed by ELISA. The changes of lymphocyte clones were analyzed TCR beta genes were identified by RT-PCR and spectral type method and then sequenced. RESULTS: After four times stimulation, the CD4/CD8 ratio of the cultured cell decreased obviously from 1.43 to 0.10 (P < 0.05), showing a proliferation of CD8(+) CTLs. The frequency of CD8 and IgHV1-QLVQSGAEV/ HLA-A * 0201 tetramer double positive CTLs was 49.83%, significantly higher than that before stimulation (0.04%). The IFN-gamma secretion detected by ELISA indicated that these CTLs were capable of recognizing the target cells in a peptide-specific and MHC-restricted way. Spectral type method showed that the TCRbeta repertoires were skewed in only a few TCR families. CONCLUSION: The peptides derived from IgHV succeeds in generation of peptide-specific CTLs in vitro in a clonality manner. These CTLs are capable of recognizing the target cells in a peptide-specific and MHC-restricted way, Understanding the function of these CTLs and the molecular structures of these TCR identification-related antigen peptides help discover the interaction between the B-cell and T-cell clones.