Signaling and Selection in Germinal Center B Cells

生发中心（GC）应答在产生长寿命体液免疫中是中心的，并且是免疫应答的基础。 有效的疫苗。GC B细胞（GCBC）广泛重塑其转录因子（TF）网络和基因表达。 表情GCBC经历V区体细胞超突变，产生用于细胞周期过程的底物， 阳性和阴性选择，其中存活的GCBC每天分裂3-4次。其中最关键的 GC生物学中的一个问题是信号如何导致更高亲和力的B细胞的阳性选择，无论是通过进入一个 新的细胞分裂周期和/或从细胞死亡中拯救。GC中的选择如何与死亡平衡， 分化为两种可能的长寿命运之一：记忆B细胞（MBC）和长寿浆细胞（LLPC）。 最近的研究已经确定了某些假定的表面标记以及相反和相互作用的转录 这些网络可以控制这些事件。一个重要的线索来自我们最近的研究， 早期GC反应倾向于产生MBC，而晚期阶段主要产生LLPC。在本提案中，我们将 使用新的方法来询问不同的信号和下游途径如何相互作用，以确定GCBC的命运 （选择和MBC/PC分化，并阐明GCBC和后代的受体-产物关系。 在我们第一轮资金支持的工作中，我们一直专注于银依赖信号-无论是 直接地，通过BCR，或间接地，通过募集T细胞帮助通过抗原呈递控制的命运， GC中阳性选择期间的GCBC。我们发现BCR和CD 40信号转导显著地 与幼稚B细胞（NBC）相比，GCBC中的重编程，来自两种受体的信号传导基本上都是 但选择性地减弱。与NBC相反，GCBC需要BCR和CD 40两者来引发阳性选择 信号，如通过c-MYC的表达和p-S6的产生所读出的。这些意见提出了一些 悬而未决的问题。首先，IL-21信号是否也被GCBC不同地解释，因为我们发现IL-21和 CD 40刺激仅协同诱导GCBC中的c-MYC？第二，细胞因子信号是否有助于 正选择和确定GCBC如何区分，如果是，如何区分？从机制上讲，既然我们知道 我们希望了解BCR、CD 40和细胞因子信号是如何协同作用的， 相互作用或“串扰”。阐明信号重编程和串扰的机制将揭示信号如何 GCBC在分子水平上进行整合，以确定这些细胞的适当反应。我们将测试 假设模型假定在反应早期，Ag是丰富的，BCR信号占主导地位， 有利于MBC生成和GC维持，而在后期IL-21信号占优势，这有利于血浆 细胞生成为了测试这个模型，我们将在Aim 1中研究IL-21 R信号如何在GCBC中重新连接;在Aim 1中， 2、BCR/CD 40和IL-21/CD 40信号在基因表达、表观遗传学和免疫学方面的机制如何不同。 TF网络重塑;最后在目标3中，选择性BCR和T细胞信号如何决定TF网络的命运结果。 体外和体内。这些实验处于B细胞生物学的概念和技术前沿。

The germinal center (GC) response is central in generating long-lived humoral immunity and the basis for effective vaccination. GC B cells (GCBC) extensively remodel their transcription factor (TF) networks and gene expression. GCBC undergo V region somatic hypermutation that generates the substrate for a cyclic process of positive and negative selection in which surviving GCBC divide 3-4 times per day. Among the most pivotal questions in GC biology is how signals lead to positive selection of higher affinity B cells, either by entering a new cycle of cell division and/or rescue from cell death. How is selection in GC balanced against death and differentiation into one of two possible long-lived fates: memory B cell (MBC) and long-lived plasma cell (LLPC). Recent studies have identified certain putative surface markers as well as opposing and interacting transcription factor networks that may control these events. One important clue comes from our recent studies showing that the early GC reaction tends to spawn MBC while the late phase largely generates LLPC. In this proposal we will use novel approaches to ask how different signals and downstream pathways interact to determine GCBC fate (selection and MBC/PC differentiation and to elucidate precursor-product relationships of GCBC and progeny. In work supported by our first cycle of funding, we have been focusing on how Ag-dependent signals—either directly, via BCR, or indirectly, via recruitment of T cell help through antigen presentation—control the fate of GCBC during positive selection in the GC. We found that BCR and CD40 signaling are dramatically reprogrammed in GCBC compared to naïve B cells (NBC), with signaling from both receptors being substantially yet selectively attenuated. In contrast to NBC, GCBC require both BCR and CD40 to ignite a positive selection signal, as read out by expression of c-MYC and generation of p-S6. These observations raise a number of outstanding questions. First, are IL-21 signals also interpreted differently by GCBC, as we found that IL21 and CD40 stimulation only synergistically induce c-MYC in GCBC? Second, are cytokine signals contributing to positive selection and determining how GCBC differentiate and if so, how? Mechanistically, since we know that signals act in concert and synergistically, we would like to understand how BCR, CD40 and cytokine signals interact, or “crosstalk”. Elucidating mechanisms of signal reprogramming and crosstalk will reveal how signals to GCBC are integrated at the molecular level to determine appropriate responses of those cells. We will test a hypothetical model that posits that early in the reaction, Ag is abundant and BCR signals predominate, which favors MBC generation and GC maintenance, while at later stages IL-21 signals prevail, which favors plasma cell generation. To test this model we will investigate, in Aim 1, how IL-21R signals are rewired in GCBC; in Aim 2, how, mechanistically, BCR/CD40 and IL-21/CD40 signals differ in terms of gene expression, epigenetics and TF network remodeling; and finally in Aim 3, how selective BCR and T cell signals determine fate outcome in vitro and in vivo. These experiments are at the conceptual and technical forefront of B cell biology.