AKNA, germinal center reaction and immunity.

描述（由申请方提供）：体液免疫应答由次级淋巴器官富含T细胞区域中的B细胞激活引发，导致生发中心（GC）前体募集到次级滤泡中，在那里它们分化为记忆细胞。B细胞活化需要同源受体/配体相互作用，其中，T细胞-CD 40配体（CD 154）与B细胞-CD 40的连接为细胞增殖、存活和诱导B细胞记忆提供了必要的刺激。本研究的重点是AT-钩蛋白AKNA是由生发中心B淋巴细胞表达，结合到CD 40和CD 154启动子的A/T丰富的调节元件，并调节其转录。与此观点一致，小鼠AKNA（moAKNA）也是由B和T淋巴细胞表达的核蛋白，表现出AT-钩DNA结合基序，上调CD 154并诱导CD 40的表达。总的来说，这些研究结果表明，AKNA在GC内的Ag依赖性反应的调节中发挥作用。具体目标是：1.在体外评估AKNA在基因转录中的作用。使用小鼠模型，我们将确定moAKNA在CD 40、CD 154和其他基因靶点表达中的作用。我们将研究moAKNA介导的基因调控机制，并评估其DNA结合要求和特异性。2.为了检查树突状细胞（DC）是否表达moAKNA以及其是否在DC成熟和功能中起任何作用，将在体内Fit 3配体（FITL）依赖性DC动员和不同阶段特异性DC亚群的分选之后分析moAKNA表达。我们还将检查AKNA表达是否随着抗原呈递细胞（APC）表型的获得以及CD 40和CD154.3的表达而达到峰值。在体内评估AKNA功能的意义。 将产生AKNA缺陷小鼠以阐明其在免疫应答中的功能。我们将检查免疫后二次免疫反应的发展，检查共刺激分子的表达，生殖中心的形成和抗体的产生。淋巴结构、细胞增殖、表型、CD 40和CD 154表达的组织学分析以及血清IG的测定将评估是否存在功能性成熟B淋巴细胞库。将检测DC成熟及其以Ag和CD 40/CD 154依赖性方式刺激T细胞的能力。将测试胸腺内T细胞分化、选择及其归巢至次级卵泡并提供同源帮助的能力。将在体内和体外检查AKNA缺陷型细胞的免疫功能，以确定其响应Ag刺激、增殖、产生抗体和/或细胞因子、响应存活/死亡信号传导和发展免疫记忆的能力。

DESCRIPTION (provided by applicant): Humoral immune responses are initiated by the activation of B cells in the T cell-rich areas of secondary lymphoid organs that lead to the recruitment of germinal center (GC) precursors into secondary follicles, where they differentiate into memory cells. B cell activation requires cognate receptor/ligand interactions, among which, the ligation of B cell-CD40 by the T celI-CD40 ligand (CD154) provides the essential stimuli for cell proliferation, survival, and induction of B cell memory. The present study focuses on the AT-hook protein AKNA that is expressed by germinal center B lymphocytes, binds to A/T-rich regulatory elements of CD40 and CD154 promoters and regulates their transcription. In keeping with this notion, mouse AKNA (moAKNA) is also a nuclear protein that is expressed by B and T lymphocytes, exhibits the AT-hook DNA-binding motifs, upregulates CD154 and induces the expression of CD40. Collectively these findings suggest that AKNA plays a role in the regulation of Ag-dependent responses within GCs. The specific aims are:1. To assess in vitro the role of AKNA in gene transcription. Using the murine model, we shall determine moAKNA's role in the expression of CD40, CD154 and other gene targets. We shall study the mechanisms of moAKNA-mediated gene regulation and assess its DNA-binding requirements and specificity. 2. To examine whether moAKNA is expressed by dendritic cells (DC) and whether it plays any role in DC maturation and function, moAKNA expression will be analyzed following in vivo Fit3 ligand (FItL)-dependent DC mobilization, and sorting of distinct stage-specific DC subsets. We shall also examine whether AKNA expression peaks with the acquisition of the antigen presenting cell(APC) phenotype and the expression of CD40 and CD154.3. To assess in vivo the significance of AKNA's function. AKNA-deficient mice will be generated to elucidate its function in the immune response. We shall examine the development of secondary immune responses after immunization, examine the expression of costimulatory molecules, formation of germinal centers and antibody production. The histological analysis of lymphoid architecture, cell proliferation, phenotype, CD40 and CD154 expression, and determination of serum Ig will assess the presence or absence of functional mature B lymphocyte repertoires. DC maturation and their ability to stimulate T cells in an Ag andCD40/CD154-dependent manner will be examined. Intrathymic T cell differentiation, selection, and their capacity to home to the secondary follicles and provide cognate help will be tested. Immune functions of AKNA-deficient cells will be examined in vivo and in vitro for their capacity to respond to Ag stimuli, proliferate, produce antibodies and/or cytokines, respond to survival/death signaling, and develop immunological memory.