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

弓形虫慢性感染可重新激活并导致危及生命的弓形体脑炎 艾滋病患者和其他免疫功能受损的个人。这种慢性感染持续存在的基础是 组织囊肿。因此，在慢性感染者成为弓形虫之前，消除他们的包囊 严重的免疫抑制将能够预防这种严重的疾病。然而，目前还没有 对囊肿病有效的药物。我们最近用小鼠模型进行的研究发现CD8+免疫T细胞 细胞具有强大的活性，可以直接穿透到囊内，并通过穿孔素诱导它们被清除- 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) H-2LD分子呈递的寄生虫蛋白(GRA)6(GRA6Nt)是激活抗-HBs的关键抗原。 囊壁CD8+T细胞。我们的研究还发现，Iba1+小胶质细胞和Ly6C+巨噬细胞积聚到 在T细胞启动的免疫过程中，这些吞噬细胞杀死了体内的弓形虫。 包囊，至少部分是通过吞噬酶体酸化。基于这些观察，建议的研究是 旨在获得继续开发激活CD8+的疫苗所需的信息 T细胞能够靶向弓形虫包囊并根除人类体内这种寄生虫的慢性感染。为 为此，我们需要确定GRA6Nt是否通过人MHC激活抗囊变CD8+T细胞 I类分子，因为MHC I类分子呈递抗原来激活CD8+T细胞。目标1将 通过使用三个表达三个主要MHC类别之一的转基因小鼠品系来解决这一问题 我的超级基序覆盖了90%的人类人口。一旦抗囊T细胞被激活，这些T细胞就需要 被招募到含有包囊的细胞，不仅通过识别弓形虫抗原激活，而且还通过 在含囊细胞上表达适当的共刺激分子。我们最近的研究表明 CXCL10趋化因子和可诱导共刺激分子配体(ICOSL)参与这些过程。因此，目标是 2将确定CXCL10和ICOSL在抗囊CD8+T细胞募集和激活中的作用， 分别进行了分析。另一个关键但尚未解决的问题是，受感染的人的大脑中持续存在少量的包囊。 小鼠即使在抗囊变CD8+T细胞存在下。我们最近的研究显示，这些顽固性的囊肿 与对照组相比，部分GRA和棒状病毒(ROP)蛋白的mRNA表达水平显著提高 在没有T细胞的情况下存在的整个囊肿群。因为Gras和Rop是从 寄生虫进入感染细胞，上调分泌这些选定的GRAS和ROP可能起到至关重要的作用 在调控囊性细胞功能以防止CD8+T细胞攻击中的作用。因此，目标3 重点是确定这些GRAs和Rop是否在逃避T细胞包囊的过程中起关键作用 进攻。我们还将探讨这种逃避过程背后的分子机制。信息来自 这些研究将为开发根除慢性弓形虫感染的新方法奠定基础。

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.