可溶性CD83促进耐受性DCs和T_(reg)细胞增殖和免疫耐受的分子机制

The mechanisms of soluble CD83 in promoting proliferation ability of tolerant DCs and T_(reg) cells and in sustaining immune tolerance status

目的 探索可溶性CD83(sCD83)发挥免疫抑制活性的分子机制。方法 分离6~8周龄C57B/6小鼠骨髓祖细胞培养骨髓衍生树突细胞(DCs)和脾脏内CD4^(+)T细胞。分别向细胞培养物中加入10μl PBS处理(Control组)或10μl 10μg/ml的可溶性CD83处理(sCD83组)。流式细胞术测定DCs中的非成熟DCs(imDCs)和CD4^(+)T细胞中的CD4^(+)CD25^(+)FOXP3^(+)T细胞,即调节性T(T_(reg))细胞的细胞计数所占百分比。Western blot测定imDCs胞内吲哚胺2,3-双加氧酶(IDO)的表达水平及T_(reg)细胞内磷酸酶及张力蛋白同源物(PTEN)、磷酸化蛋白激酶B(p-Akt)、蛋白激酶B(Akt)、细胞核和细胞质核因子-κB亚基RelB和程序性细胞死亡蛋白-1(PD-1)的表达水平。ELISA测定DCs培养上清液中犬尿酸的水平以及imDCs和CD4^(+)T细胞共培养系统中白细胞介素(IL)-10、转化生长因子-β(TGF-β)和IL-12的水平。结果 与Control组相比,sCD83组表达表面标志物CD40、CD83、CD86、Ⅱ类主要组织相容性复合物(MHCⅡ)的DCs细胞计数所占百分比降低(P<0.05);DCs培养上清液中犬尿酸的水平上升(P<0.05)。与Control组相比,sCD83组Treg细胞计数所占百分比升高(P<0.05);胞内PTEN和细胞质RelB的表达增强(P<0.05),而p-Akt、细胞核RelB和PD-1的表达降低(P<0.05);Akt的表达水平差异无统计学意义。在imDCs与CD4^(+)T细胞共培养物上清液中,与Control组相比,sCD83组IL-10和TGF-β的水平升高(P<0.05),IL-12的水平降低(P<0.05)。结论 sCD83能够抑制CD4^(+)T细胞核内RelB的水平调节相关细胞因子的分泌,影响DCs和T_(reg)细胞的细胞间通讯,促进耐受性imDCs和T_(reg)细胞的增殖。

Objective To explore the molecular mechanisms of immunosuppressive activity of soluble CD83(sCD83).Methods Bone marrow-derived dendritic cells(DCs)and CD4^(+)T cells in the spleen were isolated from bone marrow progenitor cells of 6~8-week-old C57B/6 mice.DCs and CD4*T cells were treated with 10μl PBS(Control group)or 10μl 10μg/ml soluble CD83(sCD83 group),respectively.Immature DCs(imDCs)in DCs and CD4^(+)CD25^(+)FOXP3^(+)T cells(T_(reg) cells)in CD4^(+)T cells were determined by flow cytometry.Western blot was used to determine the expression levels of indoleamine 2,3-dioxygenase(IDO)in imDCs and the expression levels of phosphatase and tensin homolog(PTEN),phosphorylated-Akt serine/threonine kinase 1(p-Akt),Akt,nucleus NF-kB subunit(RelB),cytoplasm RelB and programmed cell death 1(PD-1)in T_(reg) cells.ELISA was used to determine the level of kynurenine in DCs culture supernatant,and the levels of interleukin-10(IL-10),transforming growth factor-β(TGF-β)and IL-12 in imDCs and CD4^(+)T cells co-culture system.Results Compared with Control group,the percentage of DCs cell counts expressing the surface markers of CD40,CD83,CD86 and major histocompatibility complex class-II(MHC II)in sCD83 group decreased(P<0.05),with the level of kynurenine in DCs culture supernatant increased(P<0.05).Compared with Control group,the percentage of T_(reg) cell counts in sCD83 group increased(P<0.05),with the expression levels of PTEN and cytoplasm RelB enhanced(P<0.05),while the expression levels of p-Akt,nucleus RelB and PD-1 decreased(P<0.05).There was no significant difference in the expression level of Akt between the two groups.In the imDCs and CD4^(+)T cells co-cultured supernatants,compared with Control group,the levels of IL-10 and TGF-βincreased,and the level of IL-12 decreased in sCD83 group(P<0.05).Conclusion sCD83 can inhibit CD4^(+)T cells nucleus RelB levels and regulate cytokines secretion so as to have an effects on cellular crosstalk of imDCs and T_(reg) cells and promote imDCs and T_(reg) cells proliferation.