Dendritic cell subsets in autoimmune diabetes

利用FMS样酪氨酸激酶3配体(Flt3L)刺激的骨髓细胞培养，我们发现IL-2，一种在小鼠和人类中都存在的自身免疫性糖尿病易感基因，抑制DC的发展。IL-2可能在MDP阶段起作用，因为这些是表达IL-2ra的前体，而MDP在含有Flt3L和IL-2的培养中积累。我们现在发现，当IL2加入Flt3L BMDC培养时，单核细胞和巨噬细胞的前体增加，表明IL2可以将DC的发育转变为单核细胞系。我们检测了在IL-2存在下发展的DC的基因表达变化，发现编码bim的基因bcl211随着IL-2的上调而上调，CD135(Flt3)的表达随着IL-2的下调而下调。这两种变化在蛋白质水平上都得到了证实。此外，我们还发现，在IL-2存在的情况下发育的DC在Flt3L刺激下表现出较低的磷酸化STAT3，这可能解释了DC发育受阻的原因。 我们还有兴趣确定NOD小鼠的DC与非自身免疫菌株相比有何不同，以及不同糖尿病发病状态和疾病部位的NOD小鼠的DC有何不同。我们比较了8-10周龄NOD和B6、脾和淋巴结树突状细胞对TLRL的反应，TLRL为我们提供了慢性自身免疫小鼠DCs在显性疾病开始之前的基线和刺激活性的信息。读数包括基因表达分析、流式细胞仪和酶联免疫吸附试验。我们已经确定了NOD DC在细胞因子和共刺激反应方面的差异。我们还发现，在CpG刺激后，NOD DC中诱导的干扰素反应基因较少，并正在对此进行跟踪，以确定哪些途径导致这种改变的反应，以及对DC-T细胞相互作用和糖尿病发病机制的影响。

Using cultures of bone marrow cells stimulated with FMS-like tyrosine kinase 3 ligand (Flt3L), we have shown that IL-2, an autoimmune diabetes susceptibility gene in both mouse and human, inhibits DC development. IL-2 is likely acting at the MDP stage because those are the precursors that express the IL-2Ra, and MDPs accumulate in cultures with Flt3L and IL-2. We now find that when IL2 is added to Flt3L BMDC cultures, the precursor of monocytes and macrophages increases, suggesting that IL2 can shift development from the DC to monocyte lineage. We measured gene expression changes in DCs that develop in the presence of IL-2 and found the bim-encoding gene bcl2l11 up with IL-2 and CD135 (flt3) down with IL-2. Both of these changes were confirmed at the protein level. Furthermore, we showed that DCs that develop in the presence of IL-2 display lower phospho-STAT3 in response to Flt3L stimulation, which may explain the block in DC development. We are also interested in determining how DCs differ in NOD mice compared to non-autoimmune strains, and how DCs are different in NOD mice at different diabetes pathogenesis states and disease sites. We have compared responses of 8-10 week old NOD and B6 spleen and lymph node DCs to TLRL which gives us information about both baseline and stimulated activity of DCs in mice with chronic autoimmunity but before overt disease initiates. Readouts include gene expression analysis, flow cytometry and elisa. We have identified differences in the cytokine and costimulatory responses of NOD DCs. We also find that IFN-responsive genes are induced less in NOD DCs after CpG stimulation, and are following up on this to determine what pathways are responsible for this altered response, as well as what the consequences are for DC-T cell interactions and diabetes pathogenesis.