Zhu Bo, Chen Zheng-tang, Cheng Xiao-ming, Wu Yu-zhang
Cancer Center of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China.
Zhonghua Zhong Liu Za Zhi. 2004 Dec;26(12):709-12.
To explore the in vitro immune response to taxol resistance associated antigen 3 (TRAG-3)-derived cytotoxic T lymphocyte (CTL) epitope-pulsed dendritic cell (DC).
The HLA-A2.1 restricted CTL epitope of TRAG-3 was previously identified to be a nonameric peptide sequence from 58 to 66 amino acid residues (TRAG-3(58-66)). The peptide was synthesized according to Fmos procedure, purified and molecular weight determined. Peripheral blood mononuclear cells (PBMC) of normal HLA-A2.1(+) individuals were used to isolate DC and DCs' phenotype identified by flow cytometry. Induction of CTL was achieved by in vitro stimulation of HLA-A2.1(+) PBMC with peptide-pulsed DC. CTL activity against HLA-A2.1(+)/TRAG-3(+) melanoma cell line LB373-MEL was assessed by (51)Cr release assay and IFN-gamma released by ELISA.
The synthetic nonameric peptide was above 90% pure and the determined values of molecular weight were conformed to their theoretical values. The phenotype of the isolate DCs was characteristic for mature ones. PBMC stimulated in vitro by TRAG-3-derived epitope-pulsed DCs for three times could specifically kill the target cells and secreted high concentration of IFN-gamma.
TRAG-3-derived epitope-pulsed DC can elicit specific anti-tumor immune response in vitro. Clinical trials using it as tumor vaccine may be feasible as specific immunotherapy for patients with TRAG-3 expressing cancers.
探讨对紫杉醇耐药相关抗原3（TRAG-3）衍生的细胞毒性T淋巴细胞（CTL）表位脉冲树突状细胞（DC）的体外免疫反应。
TRAG-3的HLA-A2.1限制性CTL表位先前已被鉴定为一个由58至66个氨基酸残基组成的九聚体肽序列（TRAG-3(58-66)）。该肽按照Fmos程序合成、纯化并测定分子量。使用正常HLA-A2.1(+)个体的外周血单个核细胞（PBMC）分离DC，并通过流式细胞术鉴定DC的表型。通过用肽脉冲DC体外刺激HLA-A2.1(+) PBMC来诱导CTL。通过(51)Cr释放试验评估针对HLA-A2.1(+)/TRAG-3(+)黑色素瘤细胞系LB373-MEL的CTL活性，并通过ELISA测定释放的IFN-γ。
合成的九聚体肽纯度高于90%，测定的分子量值与理论值相符。分离的DC的表型具有成熟DC的特征。用TRAG-3衍生的表位脉冲DC体外刺激PBMC三次可特异性杀伤靶细胞并分泌高浓度的IFN-γ。
TRAG-3衍生的表位脉冲DC可在体外引发特异性抗肿瘤免疫反应。将其用作肿瘤疫苗的临床试验作为针对表达TRAG-3的癌症患者的特异性免疫疗法可能是可行的。
