Cooperation of CD8+ T cells and phagocytes to eliminate Toxoplasma cysts

弓形虫慢性感染可使人重新激活并引起危及生命的弓形虫脑炎， 艾滋病患者和其他免疫功能低下者。这种慢性感染持续存在的基础是 组织囊肿因此，消除T.慢性感染者的弓形虫囊肿， 严重的免疫抑制将能够预防这种严重的疾病。然而，目前没有 对囊肿有效的药物。我们最近使用小鼠模型的研究发现，CD 8+免疫T细胞 细胞具有直接渗透到胞囊中并通过穿孔素诱导其消除的有效活性， 中介机制。我们还鉴定了致密颗粒的N-末端区域（氨基酸41-152）， 由H-2Ld分子呈递的寄生虫的蛋白（GRA）6（GRA 6 Nt）是激活抗 囊肿CD 8 + T细胞。我们的研究还发现，Iba 1+小胶质细胞和Ly 6C+巨噬细胞积累到 囊肿，这些吞噬细胞杀死T。弓形虫缓殖子 囊肿，至少部分，通过吞噬溶酶体酸化。根据这些观察，拟议的研究是 旨在获得开发激活CD 8+的疫苗所需的信息 能够靶向T.弓形虫囊肿和根除人类的这种寄生虫慢性感染。为 为此，我们需要确定GRA 6 Nt是否通过人MHC激活抗囊肿CD 8 + T细胞 I类分子，因为MHC I类分子呈递抗原以激活CD 8 + T细胞。目标1将 通过使用三种表达三种主要MHC类型之一的转基因小鼠品系来解决这一点 覆盖了90%的人类。一旦抗囊肿T细胞被启动，这些T细胞需要 被募集到含有囊的细胞中，不仅通过识别T。弓形虫抗原， 合适的共刺激分子在含囊细胞上表达。我们最近的研究表明， CXCL 10趋化因子和诱导型共刺激配体（ICOSL）参与这些过程。因此，目标 2将确定CXCL 10和ICOSL在募集和激活抗囊肿CD 8 + T细胞中的作用， 分别另一个关键但尚未解决的问题是，少量的囊肿持续存在于受感染者的大脑中。 小鼠甚至在抗囊肿CD 8 + T细胞的存在下。我们最近的研究表明这些持续存在的囊肿 表达显著更高水平的选定GRA和棒状体（ROP）蛋白的mRNA， 在没有T细胞的情况下存在的整个囊肿群体。由于GRA和ROP是由 当寄生虫进入受感染的细胞时，这些选定的GRA和ROP的分泌上调可能起关键作用。 在操纵含囊细胞的功能以防止CD 8 + T细胞的攻击中起作用。因此，目标3 重点是确定这些GRA和ROP是否在从T细胞逃避囊肿中发挥关键作用 攻击我们还将讨论这种逃避过程的分子机制。的信息 这些研究将为开发一种新的方法来根除慢性T。弓形虫感染

Chronic infection with Toxoplasma gondii can reactivate and cause life-threatening toxoplasmic encephalitis in AIDS patients and other immunocompromised individuals. The basis of persistence of this chronic infection is the tissue cyst. Thus, eliminating T. gondii cysts from chronically infected individuals before they become severely immunosuppressed will be able to prevent this serious disease. However, there are currently no drugs effective against the cysts. Our recent studies using a murine model uncovered that CD8+ immune T cells have a potent activity to directly penetrate into the cysts and induce their elimination through perforin- mediated mechanisms. We also identified that the N-terminal region (amino acids 41-152) of dense granule protein (GRA) 6 (GRA6Nt) of the parasite presented by the H-2Ld molecule is a key antigen to activate the anti- cyst CD8+ T cells. Our studies also identified that Iba1+ microglia and Ly6C+ macrophages accumulate to the cysts during the T cell-initiated immune process, and that these phagocytes kill T. gondii bradyzoites within the cysts, at least in part, by phagolysosome acidification. Based on these observations, the proposed studies are designed to obtain the information required for proceeding towards development of a vaccine to activate CD8+ T cells capable of targeting T. gondii cysts and eradicate chronic infection with this parasite in humans. For this purpose, we need to determine whether GRA6Nt activates anti-cyst CD8+ T cells through human MHC class I molecules, since the MHC class I molecules present antigens to activate CD8+ T cells. Aim 1 will address this point by employing three transgenic mouse strains expressing either of the three major MHC class I supermotifs that cover 90% of human population. Once the anti-cyst T cells are primed, these T cells need to be recruited to cyst-containing cells and activated not only by recognizing T. gondii antigens but also by appropriate costimulatory molecules expressed on the cyst-containing cells. Our recent studies suggested an involvement of CXCL10 chemokine and inducible constimulator ligand (ICOSL) in these processes. Thus, Aim 2 will determine the roles of CXCL10 and ICOSL in recruitment and activation of anti-cyst CD8+ T cells, respectively. Another critical but unsolved issue is that small numbers of cysts persist in the brains of infected mice even in the presence of anti-cyst CD8+ T cells. Our recent study revealed that these persisting cysts express significantly greater levels of mRNA for selected GRA and rhoptry (ROP) proteins when compared to the overall cyst population that exists in the absence of the T cells. Since GRAs and ROPs are secreted from the parasite into infected cells, upregulated secretion of these selected GRAs and ROPs could play crucial roles in manipulating the functions of cyst-containing cells to prevent the attack by CD8+ T cells. Thus, Aim 3 focuses to determine whether these GRAs and ROPs play key roles in the evasion of the cysts from the T cell attack. We will also address the molecular mechanisms underlying this evasion process. The information from these studies will establish the basis for developing a novel method to eradicate chronic T. gondii infection.