AUTOIMMUNE DIABETES: EARLY EVENTS IN ISLETS OF LANGERHANS

 描述（由申请人提供）：该提议基于最近发表的研究，该研究表明，树突状细胞的一个小亚群，即CD 11 c + CD 103 + DC，即Batf 3转录因子控制下的树突状细胞，是NOD小鼠糖尿病发展绝对需要的。这些DC在胰岛和胰腺淋巴结中都有发现，并充当糖尿病起始的守门人。我们的目标集中于发现CD 103 +DC在胰岛中定位的过程。我们结合联合收割机检查趋化因子，抗体，和现场成像与新的初步发现，指出NOR小鼠有一个缺陷，在胰岛CD 103 + DC，但不是在pLN。这些重点领域可能指向可以转化为人类T1 D的策略。目的1探讨CD 103 + DC进入胰岛的机制。将在体外和体内检查趋化因子在CD 103 + DC募集中的作用。将在体内测试趋化因子阻断。体外迁移试验将确定具有将NOD衍生的CD 103 + DC募集到胰岛的潜力的趋化因子。RT-PCR将用于确定NOD小鼠胰岛中在不同时间存在哪些趋化因子。目的2将产生并检测几种用于在NOD小鼠中耗尽或追踪CD 103 + DC的试剂。我们刚刚使用Crispr/Cas技术产生了Xcr 1-/-小鼠，这应该带来关于这种趋化因子受体在CD 103 + DC的胰岛定位中的作用的明确信息。我们将遵循与刚刚发表的Batf 3-/-小鼠相同的方法。在第二子目标中，将产生NOD.Xcr1-mOrange报告小鼠，并使用实时成像方法追踪胰腺中CD 103 + DC的运动。我们想检测胰腺中CD 103 +DC的运输，并确定它是否可能是迁移到pLN的细胞。最后，将测试抗Xcl 1单克隆抗体对CD 103 + DC迁移和预防糖尿病的影响。目的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CD 103 + DC，但在LN中有它们。通过比较Batf 3-/-小鼠与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CD 103 + 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.