STAT3对免疫缺陷鼠过继转移CD8^+T细胞的调节作用

Study the regulatory role of STAT3 on CD8+T cells using adoptive transfer model in immunodeficient mice

目的·探讨转录因子STAT3对CD8^+T细胞在免疫缺陷Rag1^(-/-)小鼠中的增殖及活化的内在调控作用。方法·流式分选CD45.1小鼠和CD45.2-CD8(ΔStat3)小鼠的Na?ve CD8^+T细胞,等数量混合并用羟基荧光素二醋酸盐琥珀酰亚胺酯(carboxyfluorescein diacetate succinimidyl ester,CFSE)标记后尾静脉过继转移到Rag1^(-/-)小鼠。细胞转移10 d后,分离脾脏和淋巴结并收集细胞,用流式细胞术检测脾脏、淋巴结中的CD8^+T细胞的比率、增殖情况;用佛波酯(phorbol myristate acetate,PMA)和离子霉素(ionomycin,Ion)刺激后检测细胞因子和杀伤性分子的表达情况;运用t检验分析Stat3敲除后对CD8^+T细胞活化和效应分子表达的影响。结果·在脾脏、腹股沟和肠系膜淋巴结中,野生型(wild type,WT) CD8^+T细胞的比例显著高于ΔStat3 CD8^+T细胞(76.2%vs23.4%,82.1%vs 17.4%,64.5%vs 32.3%,均P=0.008);腹股沟淋巴结中WT CD8^+T细胞活化的CD44^+T细胞比例和细胞数量显著高于ΔStat3 CD8^+T细胞(均P=0.008)。脾脏、腹股沟淋巴结和肠系膜淋巴结中,WT CD8^+T细胞的TNF-α、IFN-γ、Gra B、CD107a的表达水平显著高于ΔStat3 CD8^+T细胞(均P<0.05)。结论·Rag1^(-/-)小鼠过继转移Na?ve CD8^+T细胞是研究信号通路/转录因子调控CD8^+T细胞功能的理想体内实验模型,STAT3可内在调控CD8^+T细胞的活化、增殖和效应功能。

Objective·To investigate the intrinsically regulatory role of transcription factor STAT3 on the activation and proliferation of CD8+T cells in immunodeficient Rag1-/-mice.Methods·The CD8+CD44-CD62L+Naïve T cells,from CD45.1 wild type(WT)mice and CD45.2-CD8(ΔStat3)mice,were sorted by flow cytometry cell sorter,equally mixed and labelled with Carboxyl Fluorescein diacetate Succinimidyl Ester(CFSE),and then transferred(i.v.)to the Rag1-/-mice.Ten days after CD8+T cells transfer,the proportion and proliferation of CD8+T cells in the spleen,mesenteric lymph nodes(mLNs)and popliteal lymph nodes(pLNs)were determined by flow cytometry.TNF-α,IFN-γ,FasL and granzyme B(GraB)produced by the CD8+T cells were measured with flow cytometry after PMA and ionomycin stimulation.The statistical significance of activation and effectors expressed between WT andΔStat3 CD8+T cell was analyzed by t test.Results·The percentages of WT CD8+T cells in spleen,mLNs and pLNs were significantly higher than that inΔStat3 CD8+T cells(76.2%vs 23.4%,82.1%vs 17.4%,64.5%vs 32.3%,with all P=0.008).The percentage and cell number of activated CD44+CD8+T cells in WT CD8+T cells were much higher than those inΔStat3 CD8+T cells in pLNs(all P=0.008).In spleen,pLNs and mLNs,the levels of TNF-α,IFN-γ,GraB and CD107a expressed in WT CD8+T cells were higher than those inΔStat3 CD8+T cells(all P<0.05).Conclusion·Rag1-/-mice can work as an ideal model to evaluate the survival,proliferation,activation and function of CD8+T cells in vivo.STAT3 intrinsically regulates the proliferation,activation and function of CD8+T cells in vivo.