dendrtitic cell subsets in autoimmune diabetes

DEC-205是由不同的CD 8+树突状细胞亚群表达的内吞受体。作为甘露糖受体家族的一员，DEC-205内吞抗原并允许它们呈递在MHC I类和II类上。通过嵌合抗体构建体将蛋白质靶向DEC-205导致免疫正常小鼠中抗原特异性CD 4+和CD 8 + T细胞的克隆缺失或无反应性。最近的工作（Mukhopadhaya A，et al. PNAS 2008）已经显示β细胞特异性CD 8 + T细胞可以从NOD小鼠（其在大约12-15周龄发展自身免疫性糖尿病）中耗尽。我们现在正在评估DEC-205靶向自身免疫NOD小鼠中耗尽或无反应性CD 4 + T细胞的能力。 向NOD小鼠注射识别未知β细胞抗原的BDC2.5 T细胞受体（TCR）转基因CD 4 + T细胞，然后用连接至BDC模拟位肽的抗DEC-205处理。在抗DEC-205处理后三天，BDC T细胞在脾、外周淋巴结和胰腺淋巴结中增殖，与正常小鼠中的结果相似。 然而，在用抗DEC-205治疗10天后，当正常小鼠中的T细胞被删除时，抗原特异性细胞保留在自身免疫小鼠中并且不是无反应性的（它们保留产生干扰素的能力）。鉴于耐受性可以通过在正常小鼠中注射toll样受体（TLR）配体来打破，我们假设内源性TLR配体可能是改变自身免疫小鼠中树突状细胞表型的原因。因此，我们测试了MyD 88缺陷小鼠是否会删除T细胞。在用抗DEC-205处理后10天，在NOD和NOD.MyD88-/-小鼠中保持相似水平的抗原特异性T细胞，尽管仅在NOD小鼠中观察到来自小鼠的CpG处理的增殖增加。因此，内源性Myd 88依赖性TLR配体不抑制NOD小鼠中DEC介导的CD 4耐受性诱导。我们目前正在研究第二种识别胰岛素的β细胞特异性TCR转基因系，以及通过利用与抗DEC-205连接的低亲和力BDC模拟位来观察细胞信号强度。 另一种类型的DC，浆细胞样DC（pDC），由于其表达TLR 7/TLR 9和其在刺激时分泌大量1型IFN的能力，传统上已被鉴定为DC的不同子集，其作为先天免疫细胞参与病毒免疫。 然而，最近，pDC也显示出向CD 4 T细胞呈递抗原，并且在诱导免疫耐受中至关重要。 我们现在通过1）研究NOD小鼠中糖尿病进展的不同阶段的pDC的数量和2）耗尽已被诱导发展成糖尿病的NOD.scid小鼠中的pDC来解决pDC在自身免疫性糖尿病发病机制中的作用。

DEC-205 is an endocytic receptor expressed by a distinct CD8α+ dendritic cell subpopulation. A member of the mannose receptor family, DEC-205 endocytoses antigens and allows them to be presented on MHC class I and II. Targeting of proteins to DEC-205 through chimeric antibody constructs causes clonal deletion or anergy of antigen-specific CD4+ and CD8+ T cells in immunologically normal mice. Recent work (Mukhopadhaya A, et al. PNAS 2008) has shown that beta cell-specific CD8+ T cells can be depleted from NOD mice (which develop autoimmune diabetes around 12-15 weeks of age). We are now assessing the ability of DEC-205 targeting to deplete or anergize CD4+ T cells in autoimmune NOD mice. NOD mice were injected with BDC2.5 T cell receptor (TCR) transgenic CD4+ T cells that recognize an unknown beta cell antigen, followed by treatment with anti-DEC-205 attached to a BDC mimeotope peptide. Three days after anti-DEC-205 treatment, BDC T cells had proliferated in the spleen, peripheral lymph nodes and pancreatic lymph nodes, similar to results in normal mice. However, ten days after treatment with anti-DEC-205, when T cells are deleted in normal mice, antigen-specific cells remained in the autoimmune mice and were not anergic (they retained capacity to produce interferon-γ). Given that tolerance can be broken by injection of toll-like receptor (TLR) ligands in normal mice, we hypothesized that endogenous TLR ligands could be responsible for changing the phenotype of dendritic cells in the autoimmune mice. We therefore tested if MyD88-deficient mice would delete the T cells. Ten days after treatment with anti-DEC-205, similar levels of antigen-specific T cells remained in NOD and NOD.MyD88-/- mice, although increased proliferation from CpG treatment of mice was only observed in NOD mice. Therefore, endogenous Myd88-dependent TLR ligands, are not inhibiting DEC-mediated induction of CD4 tolerance in NOD mice. We are currently examining a second beta cell-specific TCR transgenic line that recognizes insulin, as well as looking at cell signal strength through utilization of a lower-affinity BDC mimeotope attached to anti-DEC-205. Another type of DC, Plasmacytoid DCs (pDC) have traditionally been identified as a distinct subset of DCs involved as an innate immune cell in viral immunity due to their expression of TLR7/TLR9 and their ability to secrete large amount of type 1 IFN upon stimulation. However, more recently, pDCs have also been shown to present antigen to CD4 T cells and to be vital in the induction of immune tolerance. We are now addressing the role of pDC in the pathogenesis of autoimmune diabetes by 1) studying the number of pDCs at different stages of diabetes progression in the NOD mice and 2) depleting pDCs in NOD.scid mice that have been induced to develop diabetes.