DC vaccine for T.gondii infection by T.gondi-derived virulent molecule and Th2-suppressive siRNA

弓形虫来源的热休克蛋白70(T.G.HSP70)是一种速殖子特异的毒力分子，在具有致病性的宿主死亡前表达增加，从而导致宿主防御能力的恶化。我们克隆了T.g.HSP70，并利用该基因成功地在体内研制了针对朗格汉斯细胞(皮肤的未成熟树突状细胞)的T.g.HSP70基因疫苗。DC是一种专业的抗原提呈细胞，DC的成熟是诱导有效的T细胞激活所必需的。因此，本研究利用重组T.g.HSP70研究了T.g.HSP70对DC体外成熟的影响，获得了以下结果。(1)骨髓细胞经IL-4和GM-CSF培养获得未成熟DC。(2)T.g.HSP70诱导DC成熟，表现为MHC I、II类分子及CD40、CD80、CD86等共刺激分子表面表达增加。在功能上，T.G.HSP70刺激的DC吞噬能力降低，同种异体反应性T细胞刺激能力增强。(3)在WT和Toll样受体(TLR)2缺陷的C57BL/6小鼠中，存在T.g.HSP70刺激的DC成熟，但未发现TLR4缺陷的C57BL/6小鼠。此外，来自WT和TLR2缺陷而非TLR4缺陷小鼠的DC在T.G.HSP70刺激后产生IL-12。(4)在TLR4分子下游，在MyD88缺陷小鼠中存在T.g.HSP70刺激的DC成熟，而在TRIF缺陷小鼠中未发现。此外，T.g.HSP70刺激的来自WT、TLR2缺陷和MyD88缺陷但既没有TLR4缺陷也没有TRIF缺陷的小鼠的DC表达干扰素-mRNA。这些数据表明，T.g.HSP70通过MyD88非依赖的TRIF缺陷信号转导通路，通过TLR4刺激小鼠DC成熟。

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) is a tachyzoite-specific virulent molecule which expression increases just before the death of hosts possessing the pathogenicities to cause deterioration of the host defense. We cloned T.g.HSP70 and partially succeeded to develop in vivo T. g. HSP70 gene vaccine targeting the Langerhans cells (immature dendritic cells (DCs) of skin) by using the gene-gun.DC is a professional antigen presenting cell (APC) and DC maturation is essential to induce efficient T cell activation. Therefore, in this research, we investigated the role of T.g.HSP70 on DC maturation in vitro by using recombinant T.g.HSP70, and following results were obtained. (1) immature DC were obtained from bone-marrow cells by culturing with IL-4 and GM-CSF. (2) T.g.HSP70 induced DC maturation as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. (3) The T.g.HSP70-stimulated DC maturation was demonstrated in WT and Toll-like receptor (TLR) 2-deficient but not TLR4-deficient C57BL/6 mice. Furthermore, DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70-stimulation. (4) Concerning the downstream of TLR4 molecule, the T.g.HSP70-stimulated DC maturation was demonstrated in MyD88-deficient but not TRIF-deficient mice. Furthermore, T.g.HSP70-stimulated DCs from WT, TLR2-deficient and MyD88-deficient but neither TLR4-deficient nor TRIF-deficient mice expressed IFN-mRNA. These data indicated that T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent TRIF-deficient signal transduction cascade.