多肽类结核杆菌抗原激活的γδ t细胞表达抗原提呈细胞表型和功能

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1、多肽类结核杆菌抗原激活的γδT细胞表达抗原提呈细胞表型和功能【摘要】目的:观察用多肽类结核杆菌耐热抗原(MtbHAg)激活的人外周血γδT细胞是否具有抗原提呈细胞的表型和抗原提呈作用。方法:外周血单个核细胞(PBMC)经贴壁法获取单核细胞,加GMCSF,IL4和LPS培养诱导为成熟DC。PBMC用MtbHAg优势扩增γδT细胞后用流式分选纯化γδT(Vδ2+)细胞。PBMC经尼龙毛柱法获得纯化T细胞,用CFSE标记后单独加Mtb分泌性抗原(MtbSAg),或加单核细胞和MtbSAg共同培养,或与经MtbSAg预处理的成熟DC或活化γδT细胞共同培养。第11天时用流式细胞

2、术检测CD4+T细胞的增殖。MtbHAg活化的γδT细胞与FITC标记的MtbSAg孵育不同时间后用流式细胞术和激光共聚焦显微镜检测摄入情况。结果:MtbHAg活化的γδT细胞CD80,CD86和MHCⅡ类分子的表达显著增加。MtbSAg预处理活化的γδT细胞诱导初始T细胞增殖数和CD4+T细胞增殖指数的活性与成熟DC相似。MtbHAg活化的γδT细胞能摄入FITC标记的MtbSAg。结论:用多肽类MtbHAg激活的γδT细胞具有抗原提呈细胞的表型,并具有摄入和提呈可溶性抗原给初始T淋巴细胞的能力。【关键词】结核分枝杆菌;抗原提呈;T细胞亚群;流式细胞术;激光共聚焦显

3、微镜13［Abstract］AIM:ToexplorewhetherthehumanperipheralγδTcellsstimulatedbypolypeptideheatresistantantigenfromMycobacteriumtuberculosis(MtbHAg)expressphenotypeandhavethefuctionofantigenpresentation.METHODS:Themonocytesisolatedbyadhesionmethodfromperipheralbloodmononuclearcells(PBMCs)wereculturedw

4、ithGMCSF,IL4andinducedintothematuredendriticcellsbyaddingLPS.PBMCswerestimulatedwithMtbHAgandIL2toexpandpredominantallyγδTcellsthatwerefurtherpurifiedbyflowsorting.ThepureTcellswereisolatedfromPBMCsusingadhesionrevomalandnyloncolumnmethod,labeledwithCFSE,andculturedinalonewithMtbsecretoryan

5、tigen(MtbSAg),inmonocyteswithMtbSAg,orwithMtbSAgprepulseddendriticcellsorMtbHAgactivatedγδTcellsfor11days.TheproliferationoftheCD4+Tcellsweremeasuredbyflowcytometry.TheMtbHAgactivatedγδTcellswereincubatedwithFITClabelledMtbSAgfordifferenttime,andtheingestionofMtbSAgbyγδTcellswasmeasured

6、byflowcytometryandobservedwithlaserconfocalmicroscopye.RESULTS:TheexpressionsofMHCIIandcostimulatorymoleculesCD80andCD86onγδTcellsthatactivatedbyMtbHAgmarketlyincrasedincomparisonwiththatontherestingγδTcells.Theexpandedcellcountsandproliferationindex13ofthepurenaveTcellsthatinducedbyMtbSAg

7、pretreatedactivatedγδTcellsweresimilartothatinducedbymatureDC.Byusingflowcytometerandlaserconfocalmicroscope,FITClabellaedMtbSAgwasingestedbytheactivatedγδTcells.CONCLUSION:γδTcellsstimulatedbypolypeptideMtbHAgexpressthephenotyp