AUTOIMMUNE DIABETES: EARLY EVENTS IN ISLETS OF LANGERHANS

 描述(申请人提供)：这项建议是基于最近发表的一项研究，该研究表明，受BATF3转录因子控制的一小部分树突状细胞CD11c+CD103+DC对于NOD小鼠的糖尿病发展是绝对必要的。这些树突状细胞存在于胰岛和胰腺淋巴结中，在糖尿病发病过程中起着守门人的作用。我们的目标是发现CD103+DC定位于胰岛的过程。我们结合对趋化因子、抗体和活体成像的检查，结合新的初步发现，表明NOR小鼠的胰岛CD103+DC存在缺陷，但PLN没有缺陷。这些重点领域可能指向可能被翻译成人类T1D的策略。目的1探讨CD103+DC进入胰岛的机制。趋化因子在CD103+DC募集中的作用将在体外和体内进行检测。趋化因子阻断将在体内进行测试。体外迁移试验将确定具有向胰岛募集NOD来源的CD103+DC的潜力的趋化因子。RT-PCR将用于确定NOD小鼠不同时间胰岛中存在哪些趋化因子。AIM 2将产生并检测几种试剂，这些试剂旨在耗尽或示踪NOD小鼠中的CD103+DC。我们刚刚利用Crispr/Cas技术获得了一只Xcr1-/-小鼠，这应该会带来关于这种趋化因子受体在CD103+DC的胰岛定位中所起作用的明确信息。我们将使用相同的方法来研究刚刚发表的BATF3/-小鼠。在第二个子目标中，将产生一只NOD.Xcr1-Morange报告小鼠，并使用实时成像方法跟踪CD103+DC在胰腺中的运动。我们想要检测胰腺中CD103+DC的流量，并确定它是否可能是向PLN迁移的细胞。最后，将检测抗Xcl1单抗对CD103+DC迁移和预防糖尿病的影响。AIM 3利用了我们最近的发现，NOR小鼠与NOD共享88%的基因组，缺乏胰岛内CD103+DC，但在LNS中有CD103+DC。通过比较BATF3-/-小鼠和NORs，我们期望了解控制细胞早期进入胰岛的机制。我们研究了该菌株和含有NOR衍生等位基因的亚株，以解决CD103+DC进入胰岛背后的遗传问题。

 DESCRIPTION (provided by applicant): This proposal is based on a recently published study showing that a minor subset of dendritic cells, the CD11c+CD103+ DC, those under the control of the Batf3 transcription factor, are absolutely required for diabetes development in NOD mice. These DCs are found both in islets and pancreatic lymph nodes and act as gate-keepers for diabetes initiation. Our aims center on discovering the processes whereby CD103+DC localize in islets. We combine examination of chemokines, antibodies, and live imaging with new preliminary findings that point to NOR mice having a defect in islets CD103+ DC, but not in pLNs. These areas of emphasis may point to strategies that may be translated to human T1D. Aim 1 investigates the mechanism of CD103+ DC entry into islets. The role of chemokines in CD103+ DC recruitment will be examined both in vitro and in vivo. Chemokine blockade will be tested in vivo. In vitro migration assays will define chemokines with the potential to recruit NOD-derived CD103+ DC to islets. RT- PCR will be used to determine which chemokines exist in islets of NOD mice at different times. Aim 2 will generate and examine several reagents intended to deplete or trace CD103+ DC in the NOD mouse. We have just generated an Xcr1-/- mice using Crispr/Cas technology which should bring definitive information on the role of this chemokine receptor in the islet localization of the CD103+ DCs. We will follow the same approaches used to examine the Batf3-/- mice just published. In a second subaim, a NOD.Xcr1-mOrange reporter mouse will be generated and used to trace the movement of CD103+ DC in the pancreas using live imaging approaches. We want to examine the traffic of CD103+DC in the pancreas and determine whether it may be the cell that transmigrates to the pLN. Finally an anti-Xcl1 monoclonal antibodies will be tested for its effects on CD103+ DC migration and protection from diabetes. Aim 3 takes advantage of our recent finding that NOR mice, which share 88% of their genome with NOD, lack the intra islet CD103+ DC but have them in LNs. By comparing the Batf3-/- mice with the NORs we expect to understand the mechanisms controlling early entrance of cells into islets. We examine this strain, and substrains containing NOR-derived alleles, to address the genetic behind the CD103+ DC entry into islets.