A flow cytometry-based assay to assess minute frequencies of CD8+ T cells by their cytolytic function

A flow cytometry-based assay to assess minute frequencies of CD8+ T cells by their cytolytic function
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  Research paper A  fl ow cytometry-based assay to assess minute frequencies of CD8 + T cellsby their cytolytic function  Jonas Stanke a , Corinna Hoffmann a , Ulrike Erben b , Helmut von Keyserling a , Stefan Stevanovic c ,Guenter Cichon a , Achim Schneider a , Andreas M. Kaufmann a, ⁎ a Gynecology, Gynecologic Tumor Immunology, Campus Benjamin Franklin and Mitte, Charité-Universitätsmedizin, Berlin, Germany b Gastroenterology, Infectiology and Rheumatology, Campus Benjamin Franklin, Charité-Universitätsmedizin, Berlin, Germany c Department of Immunology, Institute for Cell Biology, University of Tübingen, Tübingen, Germany a r t i c l e i n f o a b s t r a c t  Article history: Received 21 January 2010Received in revised form 20 May 2010Accepted 7 June 2010Available online 15 June 2010 Limited sample size and low sensitivity of currently used functional assays challenge directanalysis of cytotoxic CD8 + T lymphocyte activity to quantify antigen-speci fi c immunity afterinfection or vaccination. Our  fl ow cytometry-based assay reproducibly detects at least threeepitope-speci fi cCD8 + Tlymphocytesbytheircytolyticfunction.Asexempli fi edforviralepitopesrestrictedtothehumanleukocyte antigen(HLA)-A2,the HLA-A2 + humansomaticcellhybridT2provided an about 10-fold more sensitive readout as compared to autologous B-lymphoblastoidcells or the human erythroleukemia cell line K562 transfected to express HLA-A2 when used astargetcells.WenamedourassayVITAL-FRassay,referringtoHermansetal.(2004)andindicatingthemodi fi cationofusingFarRed(FR)dyeinsteadofCMTMR.UnderoptimalconditionstheVITAL-FR assay proved 30 times more sensitive than the  51 chromium-release assay to assess epitope-speci fi c target cell lysis. The high overall sensitivity of the VITAL-FR assay basically depended onthe negligible spectral overlap of the emission of a stable Far Red  fl uorescent reporter with thegreentracerfortarget celllabelling.It alsopro fi tedfromlongco-incubationofeffectorandtargetcellsofupto72 h,from prior in-vitro cultureincreasingthe frequencyofepitope-speci fi cCD8 + Tcells and from generic, easily accessible standardized target cells that were used with only 10 3 speci fi cand10 3 controltargetcellsperindividualexperimentalreaction.Ourfunctionalapproachwiththe VITAL-FR assay thereforeideally suits for monitoring CD8 + T cell-mediated cytotoxicityin e.g. vaccination studies with known MHC-restricted immunogenic peptides in scienti fi c anddiagnostic applications.© 2010 Elsevier B.V. All rights reserved. Keywords: Cytotoxicity assayImmuno monitoringKill assayChromium releaseFar RedCFSE In-vitro  incubation 1. Introduction Identi fi cation and characterisation of disease-associatedspeci fi c CD8 + cytotoxic T lymphocytes (CTL) and antigenicpeptide epitopes involved are central aims in T cell immunol-ogy. Once induced, these lymphocytes represent the mostimportant quickly recallable defence against viral infections.Quantifying these cells and assessing their functionality is of paramount importance for speci fi c immune therapies or formonitoringtheimmunestatus.Frequenciesofepitope-speci fi cCTLvaryfrompercentagestopartspermillionthereforeassaystodetectCTLfunctionhavetobehighlysensitive(Katheretal.,2003). The classical  51 chromium-release assay directly mea-surestheextentofeffectorcell-mediatedlysisthatcorrelatestothe release of target cell-bound radioactivity (Brunner et al.,1968). Necessity to work with radioactive material andprevious time-consuming expansion of epitope-speci fi c CTL populationstolargenumberslimitstheCRAforroutineclinical  Journal of Immunological Methods 360 (2010) 56 – 65  Abbreviations:  CFSE, carboxy fl uorescein succinimidyl ester; CMV, cytomeg-alovirus; CRA, chromium release assay; CTL, cytotoxic CD8 + T cell; ELISpot,enzyme-linked immunosorbent spot; EBV, Epstein – Barr virus; HIV, humanimmunode fi ciencyvirus; HLA,humanleukocyteantigen; HPV,humanpapillomavirus; IMP, in fl uenza matrix protein; IL, interleukin; MHC, major histocompat-ibility complex; PBMC, peripheral blood mononuclear cells; pp65, phosphopro-tein 65. ⁎  Corresponding author. Gynecologic Tumor Immunology, Campus Ben- jamin Franklin, Charité-Universitätsmedizin Berlin, Hindenburgdamm 30,12200 Berlin, Germany. Tel.: +49 30 8445 2756; fax: +49 30 8445 2937. E-mail address: (A.M. Kaufmann).0022-1759/$  –  see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2010.06.005 Contents lists available at ScienceDirect  Journal of Immunological Methods  journal homepage:  use especially for epitopes with low immunogenicity (Michelet al., 2002). A non-radioactive variant assessing releasedlactate dehydrogenase from lysed cells by conversion of achromogenic substrate requires even more effector CTL forproper control (Korzeniewski et al., 1983). Interferon- γ expression is regarded a surrogate marker for CTL function(Ghanekar et al., 2001). Enzyme-linked immunosorbent spot(ELISpot) assay determines granzyme or cytokine secretion of individualcellsbutdoesnotdiscriminatefortheproducingcelltype in mixed cell populations (Shafer-Weaver et al., 2003;Schoenborn and Wilson, 2007; Zaritskaya et al., 2009).Intracellularcytokinestainingfor fl owcytometryalsomonitorssurrogateparametersinsteadofdirectCTLfunction,butallowsevaluationofadditionalspeci fi cmarkerse.g.thedegranulationmarker CD107a to further de fi ne the CTL properties andfrequencies (Betts et al., 2003).Flow cytometry-based assay systems were developed inan effort to overcome the limitations of the other experi-mental methods to assess CTL frequency and function(Ritchie et al., 2000; Hermans et al., 2004; Jedema et al.,2004; Abate et al., 2005; Stambas et al., 2007). As for theVITAL assay speci fi c target cells labelled with  fl uorescentchloromethyl derivatives are exogenously loaded with de- fi ned immunogenic synthetic peptides to be presented in thecontext of major histocompatibility complex (MHC) mole-cules. Control target cells are labelled with an orange-red fl uorescentdyeandapeptideknowntobenon-immunogenicagainst the same MHC background. In co-cultures with CTL and peptide-loaded target cells, lysis can be determined bythe ratio of the remaining viable control and target cells,which are quanti fi ed by  fl ow cytometry after a certainincubation time of 4 – 24 h (Kienzle et al., 2002; Hermans etal., 2004). The VITAL assay uses the chloromethyl-benzoyla-minotetramethylrhodaminederivativeCMTMRforoneofthetarget cell populations (Hermans et al., 2004). The mildlythiol-reactive dye reacts with intracellular components andestablishes  fl uorescent cells.We here aimed to provide an even more sensitive and fl exible  fl ow cytometry-based  in-vitro  assay for clinicallyrelevant speci fi c CTL function. In a model with strong viralantigenic peptides and well described associations with thehuman leukocyte antigen (HLA)-A2 we established an assaywe called VITAL-FR assay as an extension to the assayaccording to Hermans et al. (2004). We studied the effect of prior short term HLA/peptide-speci fi c CTL activation, alter-native  fl uorescent cell dyes with optimized emission wave-length for discrimination by  fl ow cytometry and with lowtoxicity,as well as generictarget cell types with de fi ned HLA-class I variants on the speci fi city and sensitivity for detectionof epitope-speci fi c CTL activity. Under optimal conditions, bythe VITAL-FR assay we were able to signi fi cantly detectcytotoxicity caused by approximately three speci fi c CTL. 2. Materials and methods  2.1. Cell lines and primary cells The human erythroleukemia cell line K562 (DSMZ ACC 10)and the monkey lymphocyte cell line B95-8 (DSMZ ACC 100)releasing high titers of Epstein – Barr virus (EBV) were obtainedfrom the Deutsche Sammlung für Mikroorganismen undZellkulturen (Braunschweig, Germany). The HLA-A2 + T2human – human somatic cell hybrid (ATCC CRL-1992) wasfrom the American Type Culture Collection (Bethesda, MD,USA). Peripheral blood mononuclear cells (PBMC) were pre-pared from buffy coats of HLA-A2 + healthy donors (DeutschesRotes Kreuz, Berlin, Germany) by Ficoll density gradientcentrifugation (  ρ =1.078 g/ml; GE Healthcare, Freiburg,Germany). Autologous B-lymphoblastoid cell lines (B-LCL)were established as described earlier (Pelloquin et al., 1986).In brief, 2×10 6 freshly prepared PBMC were cultured in 1mlstandard culture medium consisting of RPMI1640 and 10% fetalbovine serumsupplementedwith1 μ  g/mlofcyclosporinA,and1 ml culture supernatant of B95-8 cells. Weekly, 1 ml culturesupernatant was replaced by 1ml fresh standard medium withcyclosporin A. Proliferating cells were stained with a CD19-speci fi c monoclonal antibody (clone HIB19; BD Biosciences,Heidelberg, Germany) and assessed by  fl ow cytometry (FACS-CaliburwiththeCellQuestProsoftware;BDBiosciences).When98% of the cells were CD19 + , B-LCL were used as autologoustarget cell populations. Cell morphology was monitored byphasecontrastmicroscopy(Zeiss,Oberkochen,Germany).Ifnotnoted otherwise, cell culture reagents were from Invitrogen(Karlsruhe, Germany). Chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany) or Merck (Darmstadt,Germany) and were of the highest purity available.  2.2. Generation of K562 cells stably expressing HLA-A2 Recombinant constitutive expression of HLA-A2 by K562cells allows for presentation of HLA-A2-restricted peptides(Britten et al., 2002). In brief, complete cDNA of the heavychain of the human HLA-A*0201 allele (NCBI accessionAY365426) was a kind gift of Dr. Wolfgang Herr (UniversityMedical Center, Mainz, Germany). The sequence was clonedinto the pVITRO2-mcs plasmid (Invivogen, San Diego, CA,USA) under the control of the ferritin promoter. K562 cellstransfectedbyelectroporationwiththeplasmidtoectopicallyexpress HLA-A*0201 were incubated with 200 μ  g/ml Hygro-mycin to select for stable transfectants. A HLA-A2-speci fi cmonoclonal antibody (clone BB7.2; Parham and Brodsky,1981) coupled to pan mouse anti-human immunoglobulin Gparamagnetic beads (BD Biosciences) was used to enrich bymagnet-activated cell sorting for HLA-A*0201 expression.About 95% of the cells of this population were HLA-A2 + andthe cell line now called K562-A2 was routinely maintained instandard culture medium, and was used as target cells forpeptide presentation to CTL.  2.3. Generation of epitope-speci  fi c T cell lines CD8 + T cells speci fi c for immunodominant epitopes of thecytomegalovirus (CMV)-derived phosphoprotein 65 (NCBIaccession ACN52454.1) CMV pp65 495 – 503  (NLVPMVATV), thepolyprotein from the human immunode fi ciency virus (HIV;NCBI accession NC001802) HIV pol 510 – 518  (ILKEPVHGV), orfrom the matrix protein-M1 of the in fl uenza virus A (IMP M1;NCBIaccessionM63527)IMP 58 – 66 (GILGFVFTL)wereexpandedfrom PBMC. Corresponding synthetic peptides were all fromBiosyntan (Berlin, Germany). Autologous dendritic cells wereobtainedfromtheadherentPBMCanddifferentiatedfor4 daysin the presence of 50ng/ml granulocyte-macrophage colony- 57  J. Stanke et al. / Journal of Immunological Methods 360 (2010) 56 – 65  stimulating factor and 1000 U/ml interleukin (IL)-4. Dendriticcells were pulsed in serum free CellGrow DC medium(CellGenix, Freiburg, Germany) with 10 μ  g/ml speci fi c peptidefor 3 h at 37°C and washed thoroughly. Co-cultures of 1×10 7 PBMC and 5×10 5 peptide-loaded autologous dendritic cellsweresetupin6-welltissuecultureplates.Insomeexperimentscultureswithdendriticcellswithoutexogenouspeptideservedas controls. Weekly, cultures were restimulated with freshdendritic cell preparations loaded with the respective speci fi cpeptide as described above. After 14days, cells were stainedwith a CD8-speci fi c monoclonal antibody (clone RPA-T8; BDBiosciences)andHLA-A2/CMVpp65 495 – 503 orHLA-A2/IMP 58 – 66 tetramers. In thesecultures,frequenciesof HLA-A2 tetramer + /CD8 + T cells were in the range of 1.2 – 9.8% and these cell lineswere used as effector cells.  2.4.  51 Chromium-release assay The CRA measures the lysis of radioactively labelled targetcells by natural killer cells or CTL by quantifying releasedradioactivity in culture supernatants (Brunner et al., 1968). Inbrief, 1 – 2×10 6 target cells in fetal bovine serum were loadedwith 300 mCi Na 2 [ 51 Cr]O 4  (Amersham,Freiburg,Germany) for2 hat37°C.Targetcells(5×10 3 )mixedwithtitratednumbersofeffectorTcellsin96-wellV-bottomplateswereincubatedfor4 h at 37°C. Supernatants were harvested by means of aSkatron system (Skatron, Lier, Norway) and counted in agammacounter(LKBWallac,Bromma,Sweden).Percentagesof speci fi c  51 chromium-release were calculated using the formu-la:percentageofspeci fi crelease=(ER  − SR)×100/(MR  − SR),where ER was experimental  51 chromium release, SR thespontaneous  51 chromium release as measured in the superna-tant of 5×10 3 target cells cultured in medium alone, and MR the maximum release after the addition of 100 μ  l 1% Triton X-100 (Sigma). SR had to be lower than 20% MR for results to beincluded into the  fi nal analysis.  2.5. VITAL-FR assay Target cells (1×10 6 ) were incubated with 10 μ  M carbox-y fl uorescein succinimidyl ester (CFSE) or 5 μ  M Far Reddimethyldodecylamine oxide-succinimidyl ester (Far Red;both from Invitrogen) for 5 min in RPMI 1640 at 37 °C. Thereaction was terminated by addition of 20% fetal bovineserum and cells were thoroughly washed with standardculturemedium.Cells stainedwithCFSE wereincubated with10 μ  g/ml of either IMP 58 – 66 , CMV pp65 495 – 503 , or with HIV pol 510 – 518  in RPMI supplemented with 3% FBS and werethoroughly washed before being used as speci fi c target cells.Far Red-stained cells were loaded with 10 μ  g/ml of either thehuman papillomavirus-derived HLA-A2-restricted peptideHPV16 E7 11 – 20  (NCBI accession PPH16; YMLDLQPETT; Bio-syntan) or HIV pol 510 – 518  as control peptides. Effector T cellswere titrated in 96-well V-bottom plates and 1×10 3 CFSE-labelled speci fi c peptide-loaded and 1×10 3 Far Red-labelledcontrol peptide-loaded target cells were added. Wells con-taining the target cells onlyserved as a control. Final volumeswere 200 μ  l of standard culture medium supplemented with10 IU/ml IL-2. Cultures were incubated at 37 °C and resus-pendedbypipettingonceevery24 h.Afterup to72 h,allcellswere collected and immediately assessed by  fl ow cytometry(Fig.1).Theentiretargetcellpopulationwasde fi nedbyalivegate in a forward scatter/side scatter dot plot. Speci fi c targetcells were denoted by regions in Fl-1 (CFSE)/Fl-4 (Far Red)dot plotsanddetectedandenumerated asspeci fi c targetcellsas CFSE + (R3) and control target cells as Far Red + (R2) asshown in Fig. 1. Non- fl uorochrome labelled cells comprisedthe effector cell populations. Peptide-speci fi c lysis wascalculatedfromtheratioR3/R2inculturescontainingde fi nednumbers ( n ) of effector T cells (R3/R2) n  in comparison tocontrol (co) wells without T cells (R3/R2) co  using theformula: 100% − [(R3/R2) n /(R3/R2) co ]×100%. Numbers of epitope-speci fi c effector T cells were calculated from theHLA-A2 tetramer + /CD8 + T cells determined one day prior tothe setup of a VITAL-FR assay.  2.6. Statistical analysis The extent of epitope-speci fi c lysis was determined fromthe variation coef  fi cients and lysis was considered signi fi cantwhen it was higher than the threefold standard deviation of the control wells with the target cells only. For statisticalcomparison,weusedtheSPSSsoftwareforWindows(version15; SPSS, Chicago, IL, USA). 3. Results  3.1. Long-term co-incubation of effector and Far Red-labelledtarget cells determines the sensitivity of the VITAL-FR assay We  fi rst assessed the kinetics of peptide-speci fi c targetcell lysis. To activate and enrich CTL speci fi c for the HLA-A2restricted peptide CMV pp65 495 – 503  PBMC (from CMV-tetramer screened donors) were restimulated once bypeptide-pulsed autologous dendritic cells. Effector cell popu-lations containing at least 2% HLA-A2/CMV pp65 495 – 503 tetramer + CTL were co-incubated with autologous B-LCL orwith HLA-A2 + T2 cells as target cells in VITAL-FR assays(Fig. 2). As with CFSE or CMTMR, staining with Far Redresulted in about 95% target cell viability prior to assay setup.While mean  fl uorescence intensities of CMTMR-labelled cellsdeclined over time eventually overlapping with the negativecontrol population, both CFSE and Far Red remained clearlyseparated (Supplementary Fig. 1).Using autologous CMV pp65 495 – 503 -loaded B-LCL providinga completely matched HLA haplotype of a given donor, 100HLA-A2/CMVpp65 495 – 503 tetramer + effectorcellsrelatingtoaneffector:target ratio of 1:10 mediated about 20% peptide-speci fi c lysis within 4 h (Fig. 2, left panel). Half-maximumlysis was reached after 12 – 14 h. A plateau level of about 90%maximallysisoftargetcellsthatsustainedforatleastupto72 hwasreachedafter24 h.While30peptide-speci fi ceffectorTcellsconferred comparable maximal lysis after 48 h, 10 CMV pp65 495 – 503 -speci fi c CTL were suf  fi cient to reproducibly assesscytolytic function with autologous B-LCL target cells. In parallelexperiments,T2cellsweretestedastargetcellsagainwithCMV pp65 495 – 503  as speci fi c peptide or HIV pol 510 – 518  as controlpeptide (Fig. 2, right panel). In contrast to B-LCL, within 4 h of incubation with 100 speci fi c effector cells a half maximalspeci fi clysisofT2 cellswasreached.Kineticsusing30HLA-A2/CMVpp65 495 – 503 tetramer + CTLandT2astargetcellscomparedto lysis of autologous B-LCL by 100 speci fi c effector cells. Thus, 58  J. Stanke et al. / Journal of Immunological Methods 360 (2010) 56 – 65  T2 cells were advantageous as target cells in this setting.Emphasizingtheimpactofthedurationoftheco-cultureonthesensitivity of the VITAL-FR assay, as few as 1 – 3 CMV pp65 495 – 503 -speci fi cCTLreproduciblycausedaround20%speci fi clysisof T2 target cells after 72 h.  3.2. The VITAL-FR assay is highly speci  fi c for activated CTL and for the cognate peptide epitope During the incubation period of several days there is apotential risk for unspeci fi c lysis or intra-assay activation of T cells. To excludethis wetested thepeptide speci fi city oftheVITAL-FR assay. We fi rst compared freshly isolated PBMC andPBMC of the same donor that were stimulated twice withCMV pp65 495 – 503  peptide-loaded autologous dendritic cells.Frequencies of CMV pp65 495 – 503  speci fi c CTL were 0.03% or2.2%,respectively,accordingtoHLA-A2tetrameranalysis.After72h of incubation in the VITAL-FR experiment, target cellratios were measured and speci fi c lysis was calculated(Fig. 3A). For wells containing about 300 restimulated effectorcells, average speci fi c target cell lysis was approximately 50%.About 1000 effector cells, corresponding to 20 – 30 HLA-A2/CMV pp65 495 – 503  tetramer + CTL conferred maximal lysis. Incontrast, unstimulated PBMC of the same donor did not showlysis even with 3000 PBMC (corresponding to approximately1 CMVpp65 495 – 503  tetramer + CTL   ex vivo ) per well, after 72hincubation time. Therefore, unspeci fi c lysis or intra-assaypriming of speci fi c CTL was not observed. Next we analyzedpeptide-speci fi c lysis. PBMC stimulated twice with autologousdendritic cells that were pulsed with the IMP 58 – 66  peptidewere used as effector cells. These cell populations eventuallycontained 5.2% HLA-A2/IMP 58 – 66  tetramer + CTL. CFSE-labelled Fig.1. Flowcytometric readout oftarget celllysisby VITAL-FRassay.Speci fi ctarget cellswerestained withCFSEand loaded withanantigenic peptide.Forcontrol,cells of the same line were labelled with Far Red and loaded with the respective control peptide. Mixtures of 10 3 speci fi c and 10 3 control target cells wereincubatedeitheraloneorinthepresenceoftitratedCTLnumbers.After72 htargetcellswereacquiredinalivegateby fl owcytometry(A).RatiosofFarRed + (R2)and CFSE + (R3) labelled target cell numbers were directly determined and their relative amount de fi ned the lysis within individual cultures (B). Epitope-speci fi cCTL-mediated target cell lysis was calculated in comparison to control cultures without CTL. Fig.2. Kineticofspeci fi clysisofautologous B-LCLandT2targetcells.AutologousB-LCL(A)orT2cells(B)werelabelledwithCFSEorFarRedtobeusedastargetorcontrolcells,respectively.CellswereloadedwithCMVpp65 495 – 503 asspeci fi corHIVpol 510 – 518 ascontrolpeptide.Speci fi candcontroltargetcellsweremixedwithT cell lines containing the indicated numbers of HLA-A2/CMV pp65 495 – 503 -tetramer + CD8 + T cells. Target and effector cells were incubated for 4 h to 72 h andepitope-speci fi c lysis was assessed by  fl ow cytometry. Representative for three independent donors. Mean values±SD of triplicate determinations.59  J. Stanke et al. / Journal of Immunological Methods 360 (2010) 56 – 65  T2 cells were loaded with the IMP 58 – 66  peptide, and Far Red-labelled T2cells were loaded with HIV pol 510 – 518  and used in aVITAL-FRassay.About100prestimulatedeffectorcellscausedadistinct speci fi c lysis of the peptide-matched target cells(Fig. 3B). To further control for epitope speci fi city, a secondset of target cells using CFSE/HPV16 E7 11 – 20  as an unrelatedepitope and Far Red/HIV pol 510 – 518  again as a control epitopewas tested, with the same IMP 58 – 66 -speci fi c effector cellpopulation. T2 cells loaded with the irrelevant HLA-A2-restricted peptide HPV16 E7 11 – 20  as potential target cells didnot show any relative reduction to HIV pol 510 – 518  loaded T2controltargetcellsevenwith3000 PBMCadded.These fi ndingscon fi rmed the high sensitivity and speci fi city of the VITAL-FR assay even if using HLA-A2 matched allogeneic target cells.  3.3. Standardized HLA-A2 + allogeneic target cells allow for highly sensitive, effector T cell dose-dependent assessment of CTL function So far we had demonstrated suitability of autologous B-LCL and T2 cells as generic target cells in the VITAL-FR assay.Anotherpotentiallygenerictargetcelltypecould beK562cellstransfected to express individual HLA alleles. Therefore wetested K562-A2 ectopically expressing HLA-A*0201 (Fig. 4A).Parental K562 and HLA-A2 transfected K562 cells were pulsedwith the CMV pp65 495 – 503  peptide and incubated with a CMV pp65 495 – 503 peptidepre-activatedTcelllinehavingatetramer-reactive CTL frequency of 7%. Peptide-loaded K562-A2 cellswere signi fi cantly lysed by addition of 10 CMV pp65 495 – 503 -speci fi cCTL.ParentalK562usedascontroltargetcellswerenotlysed even with increasing CTL numbers demonstrating highspeci fi city of the lytic activity for HLA restriction. This alsodemonstrates that activity of natural killer cells did notcompromise the result of the VITAL-FR assay even after threedays of incubation.InordertovalidatethequalityofautologousB-LCL,K562-A2,andT2astargetcellswedirectlycomparedtheirspeci fi clysisinthe presence of CMV pp65 495 – 503 -prestimulated CTL in a 72 hassay (Fig. 4B). T2 cells were signi fi cantly better lysed at lowereffector cell numbers than K562-A2 or B-LCL. In most Fig. 3.  Speci fi city of the VITAL-FR assay. (A) PBMC were sensitized for 14daysbyautologousdendriticcellspulsedwiththeCMVpp65 495 – 503 peptide.T2cellslabelledwith CFSEor Far Red were loadedwithCMV pp65 495 – 503  as speci fi c orHIV pol 510 – 518 ascontrol peptide.(A) Pre-sensitizedand freshly isolatedPBMCofthesamedonorwereusedaseffectorTcellpopulationsandresultsacquiredafter 72h. (B) PBMC were pre-sensitized for 14days with IMP 58 – 66  pulsedautologous dendritic cells. T2 target cells were labelled with CFSE and loadedwith IMP 58 – 66  or with HPV16 E7 11 – 20  peptides. Far Red-stained control targetcellswerepulsedwiththeHIVpol 510 – 518 peptide.Speci fi clysiswasdeterminedby fl owcytometryafter72h.Representativeoftwo independentexperiments.Mean values±SD of triplicate determinations. Fig. 4.  SensitivityoftheVITAL-FRassayusingautologousB-LCL,T2,andK562-A2target cells. Speci fi c target cells were labelled with CFSE and loaded with CMV pp65 495 – 503  while control target cells were labelled with Far Red and the HIV pol 510 – 518  peptide. Target cells and titrated numbers of effector cells wereincubated for 72h. (A) HLA negative K562 and HLA-A2 + K562-A2 cells wereused as target cells. Effector T cells contained 5.2% HLA-A2/CMV pp65 495 – 503 tetramer + CD8 + Tcells.(B)HLA-A2 + K562-A2,B-LCL,andT2cellsweredirectlycomparedastargetcells.EffectorTcellscontained9.9%HLA-A2/CMVpp65 495 – 503 tetramer + CD8 + Tcells.Onerepresentativeoffourindependentdonorsisshown.Mean values±SD of triplicate determinations.60  J. Stanke et al. / Journal of Immunological Methods 360 (2010) 56 – 65
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