Estimating Point and Interval Frequency of Antigen-Specific CD4+ T Cells Based on Short In Vitro Expansion and Improved Poisson Distribution Analysis 英文参考文献.docVIP

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Estimating Point and Interval Frequency of Antigen-Specific CD4+ T Cells Based on Short In Vitro Expansion and Improved Poisson Distribution Analysis 英文参考文献.doc

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EstimatingPointandIntervalFrequencyofAntigen-SpecificCD4TCellsBasedonShortInVitroExpansionandImprovedPoissonDistributionAnalysis英文参考文献

EstimatingPointandIntervalFrequencyofAntigen- SpecificCD4 TCellsBasedonShortInVitroExpansion andImprovedPoissonDistributionAnalysis + GiuliaDiLullo1,2,FrancescaIeva3,RenatoLonghi4,AnnaMariaPaganoni3,MariaPiaProtti1,2* 1TumorImmunologyUnit,SanRaffaeleScientificInstitute,Milan,Italy,2DivisionofImmunology,TransplantationandInfectiousDiseases,SanRaffaeleScientificInstitute, Milan, Italy, 3Laboratorty for Modeling and Scientific Computing (MOX), Dipartimento di Matematica, Politecnico di Milano, Milan, Italy, 4Consiglio Nazionale delle Ricerche(CNR),IstitutodiChimicadelRiconoscimentoMolecolare,Milan,Italy Abstract Background:Knowledgeofantigen-specificCD4+Tcellsfrequenciesispivotaltothechoiceoftheantigentobeusedin anti-viralandanti-tumorvaccinationproceduresandformonitoringofimmuneresponses.Methodsthatemploysmallcell numbersfrompatientsamples,areeasytoperformanddonotrequirecomplextechniques/instrumentationsandtherefore standardizationaredesirable. Methodology/PrincipalFindings:PurifiedbloodCD4+Tcellsfromhealthydonorswereculturedwithautologousantigen presenting cells in several replicate wells in equal numbers in the absence (un-stimulated wells) or in the presence of synthetic peptides correspondingtoviralantigens promiscuous HLA-DR epitopes(antigen-stimulated wells). Atday 7 of culturelowdoseIL-2wasaddedandatday14IFN-candIL-5releaseinthesupernatantwasmeasured.Astatisticalanalysis + approach,basedonPoissondistribution,wasthenimplementedtocalculatethefrequencyofviral-specificCD4 Tcells.We firstdeterminedapatient-specificexceptionalitythresholdofcytokinereleaseintheun-stimulatedwellsandthen,basedon this threshold, we counted the inactive/active wells within the antigen-stimulated wells. This number, along with the numberofcellsperwell,allowedthepointandintervalestimatesoffrequencies.Aready-to-useExcelworksheettemplate withautomaticcalculationsforfrequenciesestimatewasdevelopedandisprovidedasasupplementalfile(TableS9). Conclusions/Significance: We report a simple