Pathways regulating plasmacytoid dendritic cells in Peyers Patches

描述（由申请人提供）：浆细胞样树突状细胞（pDC）是骨髓和淋巴器官中主要的I型干扰素（IFN）产生细胞，被认为在抗病毒免疫中起关键作用。在通过Toll样受体（TLR）激活后，pDC分泌IFN并成熟至抗原呈递状态，在该状态下它们能够引发包括CD 4 + T细胞极化的适应性免疫应答。pDC发育途径通过共同的DC祖细胞（CDP）进行，其产生pDC和常规DC两者。PDC的产生依赖于受体酪氨酸激酶Flt 3和转录因子STAT 3和E2-2的信号传导。在肠道中的派伊尔集合淋巴结（PP）内鉴定了在TLR触发时不能产生I型IFN的pDC子集。该亚群涉及口服耐受性;然而，PP pDC在引发适应性免疫应答中的发育起源和作用在很大程度上是未知的。我们发现PP pDC的积累需要通过I型IFN和STAT 1的细胞自主信号传导，这表明与BM或脾pDC相比，PP pDC具有不同的发育或调节途径。此外，我们发现PP pDC与PP中分泌IL-17的CD 4 + T（Th 17）细胞相关。我们假设PP pDC与位于BM和脾中的pDC来自相同的发育途径（即，通过响应Flt 3-STAT 3信号传导的CDP），然而，肠道微环境内的调节可赋予不同的功能属性，包括诱导Th 17细胞产生的能力。我们将通过研究调节PP内pDC累积的途径来检验这一假设（目的1）。我们将评估纯化的BM祖细胞如CDPs是否在致死性照射的小鼠中或在使用缺乏PP pDC的Ifnar-/-动物的新型移植模型中重建PP pDC。PP pDC产生所需的分子途径将通过移植实验来检查，所述移植实验使用来自稳态或响应于引发BM和脾pDC产生的细胞因子的Stat 1-/-、STAT 3-缺陷、Ifnar-/-或Tcf 4-/-（E2-2-缺陷）小鼠的BM祖细胞（即，Flt3L、IFN-1）。在目的2中，我们将使用纯化的PP、BM和脾pDC和naove CD 4 + T细胞的体外CD 4 + T细胞极化测定来确定PP pDC在引发CD 4 + T细胞应答方面是否在功能上不同于BM或脾pDC。此外，PP内的CD 4 + T细胞群将与目的1中的PP pDC重建相关，以进一步检查PP pDC和Th细胞应答之间的关系，并且PP pDC的活化谱将通过测量候选细胞因子产生、活化和抑制分子表达以及转录谱与BM和脾pDC进行比较。该项目的成功完成将提供PP pDC发育途径的机制观点，因此可能有助于开发能够在与胃肠道炎症相关的疾病中重定向免疫应答的治疗应用。

DESCRIPTION (provided by applicant): Plasmacytoid dendritic cells (pDCs) are principal type I interferon(IFN)-producing cells in bone marrow and lymphoid organs that are thought to play critical roles in anti-viral immunity. Upon activation through Toll-like receptors (TLRs), pDCs secrete IFN and mature to an antigen-presenting state in which they are able to elicit adaptive immune responses including CD4+ T cell polarization. The pDC developmental pathway proceeds through a common DC progenitor (CDP), which generates both pDCs and conventional DCs. PDC production depends on signaling by the receptor tyrosine kinase Flt3 and the transcription factors STAT3 and E2-2. A pDC subset was identified within Peyer's Patches (PP) in the gut that is unable to produce type I IFNs upon TLR triggering. This subset is implicated in oral tolerance; however the developmental origin and role for PP pDCs in eliciting adaptive immune responses are largely unknown. We found that PP pDC accrual required cell autonomous signaling via type I IFNs and STAT1, suggesting a distinct developmental or conditioning pathway compared to BM or splenic pDCs. Furthermore, we found that PP pDCs correlate with IL-17-secreting CD4+ T (Th17) cells in PPs. We hypothesize that PP pDCs arise from the same developmental pathway as pDCs localized in BM and spleen (i.e., via CDPs in response to Flt3-STAT3 signaling), however conditioning within the gut microenvironment may render distinct functional attributes including the ability to induce Th17 cell generation. We will test this hypothesis by investigating the pathways that regulate the accrual of pDCs within PPs (Aim 1). We will assess whether purified BM progenitors such as CDPs reconstitute PP pDCs in lethally irradiated mice or in a novel transplant model using Ifnar-/- animals that lack PP pDCs. Molecular pathways required for PP pDC generation will be examined by transplant experiments with BM progenitors from Stat1-/-, STAT3-deficient, Ifnar-/- or Tcf4-/- (E2-2-deficient) mice in steady state or in response to cytokines that elicit BM and spleen pDC generation (i.e., Flt3L, IFN-1). In Aim 2, we will determine whether PP pDCs are functionally distinct from BM or splenic pDCs in terms of eliciting CD4+ T cell responses, using in vitro CD4+ T cell polarization assays with purified PP, BM and splenic pDCs and naove CD4+ T cells. In addition, CD4+ T cell populations within PPs will be correlated with PP pDC reconstitution in Aim 1 to further examine the relationship between PP pDCs and Th cell responses, and the activation profiles of PP pDCs will be compared to BM and spleen pDCs by measuring candidate cytokine production, activating and inhibitory molecule expression and transcriptional profiles. Successful completion of this project will provide a mechanistic view of the PP pDC developmental pathway, and thus may contribute to development of therapeutic applications that enable redirection of the immune response in diseases associated with gastrointestinal inflammation.