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

apc可内化某些外源抗原，在MHC I类分子上加工和呈递，称为交叉呈递。众所周知，与hsp70相关的肽可以以这种方式呈现以诱导特异性ctl。为了确定hsp70相关肽的交叉呈递途径，我们制备了hsc70-OVA_<257-264> (hsc70-OVA)融合蛋白，该融合蛋白可以在体内诱导特异性CTL。从k_b限制性OVA_<257-264>特异性TCR转基因小鼠（OT-1）中分离到CD8^+ T细胞（OT-1细胞）。以C57BL/6或TAP- KO小鼠产生的RMA、RMA- s （TAP-）或骨髓来源DC作为ARC。为了确定APC是否可以交叉呈递hsc70-OVA，我们测量了响应hsc70-OVA脉冲ARC的OT-1细胞产生的IFN-γ，并使用25D1.16 mAb检测了K_b-OVA_<257-264>复合物在APC上的表达。我们的研究结果表明，1)OT-1细胞在hsc70- ova脉冲的tap -阴性8M-DC下产生IFN-γ， 2)在hsc70- ova脉冲的RMA上检测到高水平的K_b-OVA_<257-264>复合物。hsc70- ova脉冲RMA- s检测到的K_b-OVA_<257 ~ 264>复合物水平与RMA相似。3) brefeldin a仅部分抑制RMA上K_b-OVA_<257-264>复合物的表达水平。4)pCMBS预处理可完全抑制RMA上K_b-OVA_<257-264>复合物的表达，pCMBS是液相内吞噬（巨胞饮）抑制剂。然而，pCMBS处理BM-DC时，OT-1 T细胞的IFN-γ产生并未受到抑制。5)在hsc70存在的情况下，用hsc70- ova刺激BM-DC后，OT-1 T细胞的IFN-γ产生减少。我们的研究结果表明，hsc的内吞作用有两种不同的途径，即巨噬细胞作用和受体介导的内吞作用，并且存在与tap无关的hsc相关肽交叉呈递途径。

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.