受体相互作用蛋白激酶-3参与甲型流感病毒H1N1感染C57BL/6小鼠诱导特异性CD8^+T细胞应答

Receptor-interacting protein 3 plays a role in the induction of influenza viral antigen specific CD8^+T cell responses in C57BL/6 mice upon H1N1 influenza virus infection

目的研究受体相互作用蛋白激酶-3(receptor-interacting protein 3, RIP3)在C57BL/6小鼠感染甲型流感病毒H1N1 PR8后,在特异性CD8+T细胞免疫应答中的作用。方法用剂量为0.7×10^3半数组织细胞感染剂量(50% tissue culture infective dose, TCID 50 )的甲型流感H1N1 PR8病毒株,通过滴鼻方式分别感染RIP3敲除(RIP3 -/-)和野生型(WT)的C57BL/6小鼠。在感染后第8天(day post infection, d.p.i)分离小鼠脾细胞,使用流式细胞术(fluorescence-activated cell sorting, FACS)检测流感抗原特异性CD8+T细胞数量及功能:MHC I表位肽四聚体法(tetramer staining)染色流感特异性CD8+T细胞,细胞内细胞因子染色法(intracellular cytokine staining, ICS)检测CD8 +T细胞产生IFN-γ、TNF-α、IL-2等效应性细胞因子水平和与CD8+T细胞杀伤功能有关的颗粒酶(granzyme B)的表达情况。结果 0.7×10^3 TCID 50 流感病毒感染后,WT小鼠中流感抗原特异性CD8 +T细胞比例为RIP3 -/-小鼠的2.71倍;WT小鼠中CD8+T细胞分泌细胞因子IFN-γ、TNF-α、IL-2的能力均显著高于RIP3 -/-小鼠( P<0.01);且WT小鼠CD8+T表达granzyme B水平显著高于RIP3 -/-小鼠( P<0.01)。结论本研究在甲型流感病毒感染的小鼠体内发现敲除RIP3分子可导致流感感染诱导的特异性CD8+T细胞数量减少、功能降低,表明RIP3是参与流感抗原特异性CD8 +T细胞的诱生及功能发挥的关键分子之一。

Objective To investigate whether RIP3 (receptor- interacting protein 3) is involved in the induction of influenza antigen specific CD8+T cell responses in C57BL/6 mice upon H1N1 influenza virus infection. Methods RIP3 knockout (RIP3 -/-) and wild type (WT) C57BL/6 mice were infected by influenza virus H1N1 PR8 with the dose of 0.7×10 3 TCID 50 (50% tissue culture infective dose). Splenocytes were isolated from the infected mice which were sacrificed at 8 days post infection (d.p.i),and fluorescence-activated cell sorting (FACS) were used for the comparison of quantities and functions of influenza viral antigen specific CD8+T cells between the RIP3 -/- group and WT group. Surface staining of influenza viral MHC I tetramer was performed to quantify the antigen specific CD8+T cells. Intracellular cytokine staining (ICS) was carried out to dissect the production levels of effector cytokines,such as IFN-γ,TNF-α and IL-2. Granzyme B,which is related to the cytotoxic effect of the antigen specific CD8+T cells,was also assayed by ICS. Results On average,the percentages of influenza viral antigen specific CD8+T cells in the WT group of mice were about 2.71 fold comparing with that in the RIP3 -/- group of mice. Furthermore,the production levels of IFN-γ,TNF-α and IL-2 by the antigen specific CD8+T cells in the WT group of mice were significantly higher than those levels in the counterpart group of RIP3 -/- mice ( P<0.01);and in regards to granzyme B,the same trend was showed when comparing the expression levels between those two group mice,i.e.,WT group was higher than the RIP3 -/- group ( P<0.01). Conclusions The data of the present study reported that the deficiency of RIP3 molecule in C57BL/6 mice will decrease the quantity and functionality of the influenza viral antigen specific CD8+T cells at 8 d.p.i. upon influenza virus H1N1 PR8 infection, which indicates that RIP3 is indispensable for the induction of influenza viral antigen specific CD8+T cells at effector stage.