CD27-γδT细胞主要分泌IL-17，在疾病早期发挥关键作用。如何调控其功能和发育尚不清楚。我们预实验显示骨桥蛋白（OPN）基因敲除小鼠胸腺和脾CD27-（IL-17+）γδT的比例和数量较WT小鼠增多。OPN有sOPN和OPN-i两种，功能不同。哪种OPN发挥上述调控作用有待研究。现拟用Cre-Loxp系统构建的转基因小鼠OPN-i-KI（只表达OPN-i）、OPN-KO(两种OPN均不表达)：检测外周淋巴器官CD27-IL-17+γδT的比例和数量，确定发挥关键作用的OPN类型；进而研究该种OPN对脾CD27-和CD27+γδT产生IL-17和IFN-γ，表达RORγt和T-bet及体内外杀伤功能的调控及机制；检测不同OPN对CD27-和CD27+γδT在胸腺发育的调控及机制。本项目将为阐明OPN调控CD27-γδT的功能及机制，寻求新的靶向IL-17+γδT细胞的药物提供实验基础。

γδ T cells are found to be a very important source of IL-17 which plays a key role in early phase of some diseases. How to regulate γδ T cells to produce IL-17 remains unclear. According to CD27 expression, γδ T cells are grouped into two populations: CD27- and CD27+ γδ T cells, which produce IL-17 and IFN-γ respectively. Our preliminary results showed that: the higher percentage and numbers of CD27-γδT cells in thymus and spleen from adult osteopontin (OPN) gene knockout mice compared with WT mice were observed; moreover, splenic CD27- γδT cells produced higher frequency of IL-17. OPN has two isoforms, one is sOPN(secreted OPN) and the other is OPN-i (intracellular OPN). These two isoforms have different function. Using Cre-Loxp system to produce transgenic mice OPN-i-KI (only expression of OPN-i) and OPN-KO(express neither OPN isoform), this project aims to further define the potential contribution of OPN isoforms to regulate the function and development of CD27- (IL-17+) and CD27+ (IFN-γ+) γδ T cells. Aim 1. Investigate which OPN isoform plays a key role in regulation of CD27- and CD27+γδT cells: the percentage, counts, phenotype, IL-17 and IFN-γ production of CD27- and CD27+γδT cells from peripheral lymphoid organs of adult OPN-i-KI, OPN-KO and WT mice using FACS. Aim 2. To investigate the effects and mechanisms of OPN isoforms on adult CD27- and CD27+ γδT cells. If the above results show that sOPN plays the key role: splenocytes from OPN-i-KI/WT mice cultured with sOPN, IL-17 and IFN-γ production, activation, proliferation and apoptosis, and killing function, and OPN receptors expression are determined. Furthermore, the above are determined when adding OPN receptor or sOPN neutralizing Abs. If the above results show that OPN-i plays the key role: RORγt and T-bet expression, IL-17 and IFN-γ production, activation, proliferation, apoptosis of CD27- and CD27+γδT cells, and in vitro cytotoxic function of γδT cells from adult OPN-i-KI, OPN-KO and WT mice. Moreover, transfer OPN-i-KI, OPN-KO and WT γδT cells into nude mice respectively to observe the in vivo cytotoxic function of γδT cells using the model of lung metastasis of melanoma. Aim 3. To observe the potential regulatory effects of OPN isoforms on the development of CD27- and CD27+γδT cells in the thymus. The percentage and number of thymus CD4-CD8- double negative cell subsets in embryonic stage, the frequency, counts, phenotype, IL-17 and IFN-γ production of thymus γδ27,25+ progenitor cells, CD27- and CD27+γδT cells of OPN-i-KI, OPN-KO and WT mice in different development stages were determined. Evaluate the proliferation and apoptosis of above subsets cells, and Blk、RelA、RelB、Hes1 mRNA level of thymus γδ T cells by real-time PCR to explore the mechanism of OPN. The above experiments will characterize the effects of osteopontin on the function and development of IL-17-producing CD27-γδT cells, and might help to find a new way to target IL-17+γδT cells in the treatment of some diseases.