Department of Endocrinology，The First Affiliated Hospital of Nanjing Medical University
The National Natural Science Foundation of China (General Program, Key Program, Major Research Plan)
目的：探究负载Hybrid Insulin Peptides（HIPs）的耐受性树突状细胞（Tolerogenic dendritic cells，tolDCs）对致糖尿病性BDC2.5 T细胞的免疫调节作用。方法：采用细胞因子诱导生成NOD（Non-obese Diabetic）小鼠骨髓细胞来源的未成熟树突状细胞（Immature DCs，iDCs），培养过程中额外加入维生素D3可生成tolDCs，脂多糖（Lipopolysaccharide，LPS）刺激24h后可以分别获得LPS-iDC、LPS-tolDCs，收集其上清液检测细胞因子IL-12p70及TGF-β，通过形态学及流式细胞术鉴定上述DCs的表型。将致糖尿病性T细胞即BDC2.5 CD4+ T细胞与负载HIPs的四组DCs共孵育3天后，检测T细胞增殖、活化以及Tregs（Regulatory T cells）的产生情况。结果：表型鉴定结果显示tolDCs呈现低表达共刺激分子CD80、CD86分子、高表达共抑制分子PD-L1的耐受性表型，在脂多糖的刺激下仍保持稳定，且与LPS-iDCs相比，LPS-tolDCs 的IL-12p70分泌减少、TGF-β分泌增加。负载HIPs的tolDCs及LPS-tolDCs均可以抑制BDC2.5 T细胞的增殖和活化，诱导抗原特异性Tregs的产生。结论：负载HIPs的tolDCs可以通过其稳定的耐受表型及功能抑制致糖尿病性BDC2.5 T细胞的增殖活化，诱导抗原特异性Tregs的产生。
Objective: To investigate the immunoregulatory effect of tolerogenic dendritic cells (tolDCs) loaded with hybrid insulin peptides (HIPs) on diabetogenic BDC2.5 T cells. METHODS: Bone marrow derived imature DCs（iDCs）of Non-obese Diabetic（NOD）mice were induced with cytokines，and additional vitamin D3 was added to generate tolDCs. After 24h stimulation with lipopolysaccharide (LPS), The supernatant of LPS-iDCs and LPS-tolDCs was collected to detect cytokines IL-12p70 and TGF-β, and the phenotype of the above DCs was identified by morphology and flow cytometry. Diabetogenic T cells, namely BDC2.5 CD4+ T cells were incubated with HIPs-loaded DCs for 3 days, and the proliferation, activation and Tregs production were detected. RESULTS: Phenotypic identification results showed that tolDCs had low expression of costimulatory molecules CD80、CD86 and high expression of coinhibitory molecule PD-L1.The phenotype of tolDCs remained stable under the stimulation of LPS, and the secretion of IL-12p70 was decreased while the secretion of TGF-β was increased compared with LPS-iDCs. Both tolDCs and LPS-tolDCs loaded with HIPs could inhibit the proliferation and activation of BDC2.5 T cells and induce the production of antigen-specific regulatory T cells（Tregs）. Conclusion: HIPs loaded tolerogenic dendritic cells can inhibit the proliferation and activation of diabetogenic BDC2.5 T cells and promote the production of Tregs through their stable tolerance phenotype and function.