Brucella epitope recognition by CD8+T cells

描述(申请人提供)：CD8+T细胞很重要，因为它们清除了细胞内的感染；然而，颠覆这些免疫细胞对长期感染有影响。我们将研究布氏杆菌，一种兼性细胞内细菌，如何在动物体内长期存在于免疫反应中。我们假设低数量和无效的CD8+T细胞允许持续感染。利用生物发光布鲁氏菌，我们发现BALB/c小鼠感染布氏杆菌可导致持续12个月的慢性感染。对这些小鼠的CD8+T细胞记忆池的评估显示，未能维持CD8+T细胞记忆表型(LFA1hi、KLRG1lo和CD127hi)或多功能细胞因子的表达(IL-2、干扰素？和肿瘤坏死因子？)。这些发现表明CD8+T细胞表现出一种“耗尽”的表型，这表明布鲁氏菌避开了这一已知的清除细胞内病原体的效应机制。此外，布鲁氏菌蛋白TcpB在体内可抑制CD8+T细胞对表达布鲁氏菌多肽的靶细胞的杀伤作用。我们的长期目标是通过研究以下目标来了解细菌在免疫反应存在的情况下如何保持慢性状态：目的1：确定BALB/c小鼠在急性和慢性感染期间的CD8+T细胞反应。我们将通过CD8+T效应分子和记忆标记物、转录因子表达、细胞因子产生和体内杀伤来比较小鼠在急性和慢性感染期间CD8+T细胞反应的幅度和有效性。影响：我们将确定CD8+T细胞在急性和慢性感染之间的表型差异，并假设“耗尽”表型和无效的CD8+T细胞与慢性布鲁氏菌病有关。目的：检测布鲁氏菌诱导的记忆CD8+T细胞的保护作用。我们将检测过继转移的CD8+T细胞免受急性和慢性感染的能力，以保护幼小动物免受首次感染。影响：我们预计来自急性感染小鼠的CD8+T细胞将比来自慢性感染小鼠的细胞更好地保护幼年小鼠，支持慢性感染小鼠失去功能的CD8+T细胞。目的：检测布鲁氏菌TcpB对细胞毒CD8+T细胞的抑制作用。我们将确定TcpB蛋白对感染细胞的细胞毒性CD8+T细胞杀伤的能力，以及在感染期间这种抑制的体内动力学。影响：我们预计TcpB蛋白通过抑制CD8+T细胞杀伤来调节适应性免疫反应，从而允许布鲁氏菌感染细胞的长期存活。我们的研究是阐明布鲁氏菌感染如何塑造CD8+T细胞效应器和记忆反应的关键的第一步。这项工作将填补在了解CD8+T细胞在布鲁氏菌病中的作用方面的一个严重空白，这些细胞可能参与TIS疾病的解决。 与公共卫生相关：布鲁氏菌病是一种慢性致衰性细菌性疾病，是一种在世界范围内流行的人畜共患病。CD8+T细胞可能在控制细胞内感染中起关键作用，但对这些CD8+T细胞在感染过程中的效应和记忆反应知之甚少。我们将研究效应和记忆CD8+T细胞在急性和慢性布鲁氏菌感染中的反应，我们假设这些研究将确定功能CD8+T细胞的下降是布鲁氏菌病从早期感染过渡到晚期感染的过程。我们的发现将为增强功能性CD8+T细胞的发育和持久性提供未来的机会，从而改善细菌清除，并设计能够增强CD8+T记忆细胞的疫苗。

DESCRIPTION (provided by applicant): CD8+T cells are significant because they clear intracellular infections; however, subverting these immune cells has implications to long-term infections. We will study how Brucella melitensis, a facultative intracellular bacterium, chronically persists in animals in the presence of an immune response. We hypothesize that low numbers and ineffectual CD8+T cells permit continuing infection. We have found BALB/c mice infected with B. melitensis results in chronic infection lasting >12 months using bioluminescent Brucella. Evaluation of the CD8+T cell memory pool from these mice reveals a failure to maintain the CD8+T cell memory phenotype (LFA1hi, KLRG1lo, and CD127hi), or polyfunctional cytokine expression (IL-2, IFN-? and TNF-?). These findings indicate CD8+T cells express an "exhausted" phenotype suggesting that Brucella evades this known effector mechanism for removing intracellular pathogens. Further, a Brucella protein, TcpB, can inhibit CD8+T cell killing of Brucella peptide expressing target cells in vivo. Our long-term goal is to understand how the bacteria remain in a chronic state in the presence of an immune response, by investigating the following Aims: Aim 1: To determine the CD8+T cell response in BALB/c mice during acute and chronic infection. We will compare the magnitude and effectiveness of CD8+Tcell responses in mice during acute and chronic infection using CD8+Teffector and memory markers, transcription factor expression, cytokine production, and in vivo killing. Impact: We will identify CD8+T cell phenotypic differences between acute and chronic infection and hypothesize an "exhausted" phenotype and ineffectual CD8+Tcells contribute to chronic brucellosis. Aim 2: To determine the protective capacity of Brucella-induced memory CD8+T cells. We will examine the capacity of adoptively transferred CD8+T cells from acute versus chronic infections to protect naive animals from a first infection. Impact: We expect CD8+Tcells from acute infected mice will protect naive mice better than cells from chronically infected mice supporting a loss of functional CD8+Tcells with chronic infection. Aim 3: To determine the inhibitory ability of Brucella TcpB on cytotoxic CD8+T cells. We will determine the ability of the TcpB protein to inhibit cytotoxic CD8+T cell killing of infected cells and the kinetics of this inhibition in vivo during infection. Impact: We expect that TcpB protein modulates the adaptive immune response by inhibiting CD8+T cell killing permitting long-term survival of Brucella-infected cells. Our studies represent a critical first step in elucidating how Brucella infection shapes CD8+T cell effector and memory responses. This work will fill a serious void in understanding the role of CD8+T cells in brucellosis that likely participate in the resolution of tis disease. PUBLIC HEALTH RELEVANCE: Brucellosis is a chronic debilitating bacterial disease and a prevalent zoonosis worldwide. CD8+ T cells likely play a critical role in controlling the intracellular infection, but little is known regarding effector and memory responses of these CD8+T cells during infection. We will study how effector and memory CD8+T cells respond in acute versus chronic Brucella infection, and we hypothesize that these studies will identify a decline in functional CD8+T cells as brucellosis transitions from early to late infection. Our findings will provide future opportunities to enhance development and persistence of functional CD8+T cells leading to improved bacterial clearance and designing vaccines that can potentate CD8+T memory cells.