幽门螺杆菌抗原特异性 CD4＋T 细胞体外扩增及其在 Th 表位筛选中的初步应用

Bulk culture of Helicobacter pylori-specific CD4＋T cells in vitro for identification of Th epitopes

目的：建立幽门螺杆菌（ Helicobacter pylori，H．pylori）抗原特异性CD4＋T细胞体外扩增方法，并在Th表位筛选中初步应用。方法从H．pylori感染阳性患者中分离外周血单个核细胞（PBMC），以重组黏附素抗原（HpaA）进行体外刺激，分别摸索不同血清培养基、抗原刺激浓度、培养时间等体外扩增条件，并通过细胞内因子染色和流式细胞技术检测HpaA特异性CD4＋T细胞产生IFN-γ应答水平，从而建立H．pylori感染者抗原特异性CD4＋T淋巴细胞的体外扩增方法。同时，结合合成重叠肽技术，对抗原HpaA中可能的Th表位进行初步筛查。结果在含人AB血清的1640培养基中培养的抗原特异性CD4＋T细胞的扩增效率优于含胎牛血清的1640培养基； HpaA抗原初刺激浓度为0．2μmol/L时，抗原特异性CD4＋T细胞的比例最高；在抗原刺激浓度0．2μmol/L条件下，细胞培养9 d时出现特异性CD4＋T细胞应答，且在第15天时达到高峰值；以不同肽段对特异性CD4＋T细胞的IFN-γ应答水平检测分析中显示：针对本研究H．pylori感染个体，HpaA抗原中可能存在一个优势应答的Th细胞表位（HpaA220-237）。结论在人AB血清的1640培养基，0．2μmol/L为抗原刺激浓度，连续15 d扩增等培养条件下，在体外成功扩增出H．pylori抗原特异性CD4＋T淋巴细胞，可用于H．pylori抗原中Th表位的筛选和鉴定，为表位疫苗研究奠定实验基础。

Objective To screen an optimum method for in vitro culture of Helicobacter pylori-spe-cific CD4＋T cells and apply it to immunodominant Th epitopes screening .Methods PBMCs were isolated from subjects positive for Helicobacter pylori infection and were stimulated with HpaA recombinant protein . Various induction conditions including serum containing mediums , concentrations of antigen and time were screened to obtain an optimum method for in vitro culture of Helicobacter pylori-specific CD4＋T cells.The cells were harvested and stimulated using HpaA synthesized overlapping peptide pool .The percentage of an-tigen-specific CD4＋T cells was evaluated by intercellular cytokine staining of interferon-γand the results were compared under different conditions .The possible immunodominant Th epitopes were screened by using synthetic overlapping peptides .Results Antigen-specific CD4＋T cells were well cultured in RPMI 1640 culture medium containing human AB serum in comparison with those cultured in fetal bovine serum based medium.The highest percentage of antigen-specific CD4＋T cells was achieved when stimulated with HpaA recombinant protein at the concentration of 0.2 μmol/L.CD4＋T cells in response to the stimulation of 0.2μmol/L of HpaA recombinant protein was observed on the ninth day after culture and its peak was reached on the fifteenth day .A possible immunodominant Th epitope （ HpaA220-237 ） was screened in subjects with He-licobacter pylori-infection by using synthetic overlapping peptides .Conclusion Helicobacter pylori-specific CD4＋T cells were successfully cultured in vitro by using RPMI 1640 culture medium containing human AB serum and stimulated with 0.2 μmol/L of HpaA recombinant protein for fifteen consecutive days .This cul-ture method could be applied to immunodominant Th epitopes screening and provide evidences for further in -vestigation on the development of Helicobacter pylori epitope-based vaccine .