Bulk culture of Helicobacter pylori-specific CD4+T cells in vitro for identification of Th epitopes
10.3760/cma.j.issn.0254-5101.2014.08.012
- VernacularTitle: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
- Author:
Xiaoyang LI
;
Xueqing GUO
;
Ningyi LI
;
Li CHEN
;
Chao WU
- Publication Type:Journal Article
- Keywords:
Helicobacter pylori;
CD4+T cell;
Epitope
- From:Chinese Journal of Microbiology and Immunology
2014;(8):630-634
- CountryChina
- Language:Chinese
-
Abstract:
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 .
