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

弓形虫热休克蛋白70（Toxoplasma gondii-derived heat shock protein 70，T.g.HSP70）是一种速殖子特异性的毒力分子，在宿主死亡前表达增加，具有致病性，可引起宿主防御能力下降。我们克隆了T.g.HSP70，并部分成功地在体内培养了T. G.利用基因枪靶向皮肤的未成熟树突状细胞（DC）构建HSP 70基因疫苗，DC是一种专职的抗原提呈细胞（APC），其成熟是诱导T细胞有效活化的必要条件。因此，本研究利用重组T.g.HSP70，探讨T.g.HSP70对DC体外成熟的影响，获得以下结果。(1)通过用IL-4和GM-CSF培养从骨髓细胞获得未成熟DC。(2)T.g.HSP70诱导DC成熟，这通过MHC I类和II类分子以及共刺激分子如CD 40、CD 80和CD 86的表面表达增加来证明。在功能上，T.g.HSP70刺激的DC中观察到吞噬能力降低和同种异体反应性T细胞刺激能力增加。(3)T.g.HSP70刺激的DC成熟在WT和Toll样受体（TLR）2缺陷型而非TLR 4缺陷型C57 BL/6小鼠中得到证实。此外，来自WT和TLR 2缺陷但不是TLR 4缺陷小鼠的DC在T.g.HSP70刺激后产生IL-12。(4)在TLR 4分子下游，T.g.HSP70刺激的DC成熟在MyD 88缺陷小鼠中得到证实，但在TRIF缺陷小鼠中没有。此外，来自WT、TLR 2缺陷和MyD 88缺陷但TLR 4缺陷和TRIF缺陷小鼠的T.g.HSP70刺激的DC表达IFN-mRNA。这些数据表明，T.g.HSP70通过TLR 4通过MyD 88独立的TRIF缺陷信号转导级联刺激小鼠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.