Signaling and Selection in Germinal Center B Cells

生发中心(GC)反应是产生长寿体液免疫的核心，也是 有效的疫苗接种。GC B细胞(GCBC)广泛重塑其转录因子(TF)网络和基因 表情。GCBC经历V区体细胞超突变，产生循环过程的底物 正选择和负选择：存活的GCBC每天分3-4次。其中最关键的是 GC生物学中的问题是，信号如何导致更高亲和力的B细胞的积极选择，无论是通过输入 细胞分裂的新周期和/或从细胞死亡中解救出来。GC中的选择是如何平衡死亡和 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 可能控制这些事件的网络因素。一条重要的线索来自我们最近的研究表明 早期的GC反应倾向于产生MBC，而后期的反应主要产生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通路如何相互作用来决定GCBC的命运 (选择和MBC/PC分化，并阐明GCBC及其后代的前体-产物关系。 在我们第一轮资金支持的工作中，我们一直专注于银离子依赖信号-无论是 直接，通过BCR，或间接通过T细胞的招募，通过抗原提呈帮助-控制 GC中正选择过程中的GCBC。我们发现BCR和CD40信号显著地 在GCBC中重新编程，而不是幼稚的B细胞(NBC)，来自两个受体的信号基本上 但选择性地减弱了。与NBC不同的是，GCBC需要BCR和CD40来触发正面选择 通过c-myc的表达和p-S6的产生而读出的信号。这些观察结果提出了一些 悬而未决的问题。首先，GCBC对IL-21信号的解释是否也不同，因为我们发现IL-21和 CD40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 积极选择和确定GCBC如何区分，如果是，如何区分？从机械上讲，因为我们知道 信号协同作用，我们想要了解BCR，CD40和细胞因子信号是如何 互动，或“相声”。阐明信号重新编程和串扰的机制将揭示信号如何 GCBC在分子水平上被整合，以确定这些细胞的适当反应。我们将测试一个 假设模型假设在反应早期，银是丰富的，BCR信号占主导地位，这 有利于MBC的产生和GC的维持，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，我们将在目标1中研究IL-21R信号是如何在GCBC中重新连接的；在目标1中 2、BCR/CD40和IL-21/CD40信号在基因表达、表观遗传学和 Tf网络重构；最后在目标3中，选择性BCR和T细胞信号如何决定患者的命运结局 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.