Normalizing ELISPOT responses to T-cell counts: a novel approach for quantification of HCMV-specific CD4(+) and CD8(+) T-cell responses in kidney transplant recipients

Background: Human cytomegalovirus(HCMV)isthemostcommonopportunisticvirusinfectioninsolid organ transplantrecipients.TheanalysisofHCMV-specificT-cellimmunityafterorgantransplantisof relevant clinicalinterest. Objectives: To analyzeHCMV-specificCD4+ and CD8+ T-cell responsesinhealthysubjectsandkidney transplant recipients(KTR). Study design: HCMV-specific T-cellresponseswereevaluatedbyinterferon- (IFN-) enzyme-linked immunospot (ELISPOT)usingoverlapping15-merpeptidepoolsofimmediateearly(IE)-1,IE-2,phos- phoprotein 65(pp65)(forstimulationofbothCD4+ and CD8+ T-cell responses)andapoolof34short peptides (8–12aminoacidsinlength,forstimulationofCD8+ T-cell responses).ELISPOTresultswere normalized toT-cellsubsetcountsandtheircorrelationswithareporteddendriticcell(DC)-basedassay, which simultaneouslyquantifiesHCMV-specificCD4+ and CD8+ T-cell responses,wereanalyzed. Results: HCMV-seropositive KTRshowedhigherELISPOTresponsescomparedtoHCMV-seropositive healthy subjects.IE-1andpp65ELISPOTresponsesweremediatedmainlybyCD8+ T-cells and,toa lesser extent,CD4+ T cells;IE-2peptidesappeartostimulateCD56+ cells (naturalkillercells).InHCMV- seropositive healthysubjects,ELISPOTresults(expressedeitherasnetspots/millioncellsornormalized to thecorrespondingT-cellcount)significantlycorrelatedwiththeDCassay.However,inHMCV- seropositive KTR,onlynormalizedELISPOTresponsestooverlapping15-merpeptidepoolssignificantly correlated withDC-assayresponses. Conclusions: The normalizedELISPOTrepresentsanovelandsimpleapproachforquantifyingandmoni- toring HCMV-specificCD4+ and CD8+ T-cell responsesinKTR