Molecular mechanisms To protect malaria infection by vaccine using antigen / hsp70 fusion protein

点击翻译按钮获取中文摘要

APCs can internalize some types of exogenous antigens for processing and presentation on MHC class I molecules, which is referred to as cross-presentation. It is known that peptides associated with hsp70 can be presented in this manner to induce specific CTLs. To determine the cross-presentation pathway of hsp70 associated peptides, we generated hsc70-OVA_<257-264> (hsc70-OVA) fusion protein, which could induce specific CTL in vivo. CD8^+ T cells (OT-1 cells) were isolated from K_b-restricted OVA_<257-264> specific TCR transgenic mice (OT-1). RMA, RMA-S (TAP-) or bone-marrow derived DC, which were generated from C57BL/6 or TAP KO mice, were used as ARC. To determine whether APC can cross-present hsc70-OVA, we measured IFN-γ production from OT-1 cells in response to hsc70-OVA-pulsed ARC, and the expression of K_b-OVA_<257-264> complex on APC using 25D1.16 mAb. Our results showed that, 1) OT-1 cells produced IFN-γ in response to hsc70-OVA-pulsed TAP-negative 8M-DC, 2) High levels of K_b-OVA_<257-264> complex were detected on hsc70-OVA-pulsed RMA. The levels of K_b-OVA_<257-264> complexes detected on hsc70-OVA-pulsed RMA-S were similar to RMA. 3) The expression level of K_b-OVA_<257-264> complexes on RMA was only partially Inhibited by brefeldin A. 4) The expression of K_b-OVA_<257-264> complexes on RMA was completely inhibited by pretreatment of pCMBS, which is the inhibitor of fluid phase endocytosis (macropinocytosis). However, IFN-γ production from OT-1 T cells were not inhibited when BM-DC were treated with pCMBS. 5) IFN-γ production from OT-1 T cells were reduced when BM-DC were pulsed with hsc70-OVA in the presence of hsc70. Our results suggest that there are two distinct pathways of endocytosis of hsc, macropinocytosis and receptor-mediated endocytosis, and that there existed TAP-independent cross-presentation pathway of hsc associated peptide.