CTL诱导及杀伤实验.doc

CTL诱导及杀伤实验.doc

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一、抗原肽诱导CTL制备

1.PBMCswereseparatedfromthewholebloodofHLA-A2+healthycontrols.PBMCs（2x106/ml）wereculturedwitheachoftheHLAA*0201refoldingpeptidesataconcentrationof10uMinRPMI1640mediumcontaining10%FCSand20U/mlrecombinanthumanIL-2（rhIL-2）in24-wellcultureplate.Halfofthemediumwaschangedatday4withsupplementationofrhIL-2at20U/ml.Atday7,cellswereharvestedandtestedforthepresenceofpeptide-specificCD8+TcellsbyanIFN-γreleaseELISPOTassay.

---------------ZhouM,XuD,LiX,LiH,ShanM,TangJ,WangM,WangFS,ZhuX,

TaoHetal:

Screeningandidentificationofsevereacuterespiratory

syndrome-associatedcoronavirus-specificCTLepitopes.JImmunol2006,

177（4）:

2138-2145.

2.PBMCswereseparatedfromheparinizedvenousbloodbyFicoll-HypaquedensitygradientcentrifugationfromHLAA*0201normaldonorsandHCCpatients.DCsweregeneratedfromperipheralbloodmonocytesasdescribedbyRomani.Briefly,PBMCswereseededintosix-wellcultureplatescontaining3mlRPMI-1640and10%FCSat5–10x106/well.Plateswereincubatedina37oCincubatorfor2h,thenthenon-adherentcellswereremovedandtheadherentcellswereculturedat37oCinRPMI-1640supplementedwith10%FCS,1000U/mlhumanrecombinantGM-CSFand500U/mlhumanrecombinantIL-4.AllTcellstimulationwaswithday7DCs.Asdescribedbelow,DCswerematuredwithlipopolysaccharide（LPS）onday6andHCA587antigenwasaddedforeffectiveprocessingatthistime.

Day6DCswereresuspendedinRPMI-1640with1000U/mlGM-CSF,500U/mlIL-4and10ng/mlLPSandincubatedat37oC.After24hcultivationtheDCswerecollected,washedandpulsedwith10mg/mlHCA587peptidefor3hat37oC.TheDCswerewashedtwiceforthefollowingstimulation.

CD8+TcellswereisolatedbypositiveselectionwithCD8-Dynalimmunomagneticbeadsaccordingtothemanufacturer’sinstructions.Afterwashing,CD8+Tcellswereco-culturedwithHCA587peptide-loadedDCsin2mlRPMI-1640mediumsupplementedwith10%ABserum,recombinanthumanIL-2（10ng/ml）andIL-6（500U/ml）.Sevendayslater,theculturedTcellswererestimulatedwithfreshlypreparedpeptide-pulsedDCsandculturedforanother7days.Afterfourconsecutiveroundsofstimulation,culturesweretestedforthepresenceofHCA587-specificCTLs.

-----------LiB,WangY,ChenJ,WuH,ChenW:

IdentificationofanewHLA-A*0201-restrictedCD8+Tcellepitopefromhepatocellularcarcinoma-associatedantigenHCA587.ClinExpImmunol2005,140

（2）:

310-319.

3.GenerationofCTLsinPBMCsfromhealthydonors：

Dendriticcells（DCs）havetheuniquecapacityofactivatingnaiveTcellsandinitiatingprimaryT-cellresponse.Antigen-specificT-cellresponsesfromperipheralbloodmononuclearcells（PBMCs）canbeelicitedwithantigenicpeptide-pulsedautologousDCs.WeobtainedPBMCsfromthebuffycoatofheparinizedwholebloodsamplesofhealthydonorsbydensitygradientcentrifugationontheHistopaque1077.Thesecellswereresuspendedinserum-freeRPMI1640andallowedtoadheretosixwellplatesatafinalconcentrationof1x107cells/3ml/well.After2hofincubationat37°C,non-adherentcellsweregentlyremovedwithwarmmediumbygentlypipetting.Thenon-adherentcells（effectorlymphocytes）werecryopreservedinFCSsupplementedby10%DMSO.TheresultantadherentcellscontainingDCswereculturedinmediumsupplementedwith800U/mlGMCSFand1,000U/mlIL-4in37°C/5%CO2.Every2days,one-halfofthemediumwasreplacedbyfreshmediumcontainingadoubleconcentrationofGM-CSFandIL-4asindicatedabove.Onday5,10ng/mlofrecombinanthumantumornecrosisfactora（TNF-a）wasaddedtothemediumtoinducephenotypicandfunctionalmaturationofDCs.After48h,DCswerepulsedwith20ug/mlpeptideinthepresenceof3ug/mlb2-microglobulinat37°Cfor3handirradiatedat30Gybeforeuse.Thethawed2x106ofnon-adherenteffectorlymphocyteswerecoculturedwith2x105peptidepulsedirradiatedautologousDCsina24-wellplateinthepresenceof10ng/mlrecombinanthumaninterleukin-7（IL-7）.After7days,lymphocyteswererestimulatedwithpeptide-pulsedautologousPBMCsinmediumcontaining10ng/mlIL-7and20U/mlIL-2.About20U/mlofIL-2wasadded24hlateratregularintervals,2daysaftereachrestimulation.Lymphocyteswererestimulatedeachweekinthesamemanner.Ontheseventhday,afterthethreeroundsofrestimulation,cellswereharvestedandtestedbyELISPOTassay.

--------------AnalteredpeptideligandfornavecytotoxicTlymphocyteepitopeofTRP-2（180–188）enhancedimmunogenicity.

CancerImmunolImmunother（2007）56:

319–329第三军医大吴玉章

4.GenerationofCTLsinhealthydonors

DendriticcellsarecharacterizedbytheuniquecapacitytoactivatenaiveTcellsandinitiateprimaryT-cellresponse.Antigen-specificT-cellresponsesfromperi