Background: The ability to measure T-cell responses to antigens is proving critical in the field of vaccine\r\ndevelopment and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of\r\ntechnologies exist for this purpose IFNg-ELISpot assays are widely used because of their sensitivity and simplicity.\r\nHowever, ELISpot assays cannot be performed on whole blood, and require relatively large volumes of blood to\r\nyield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay\r\nthat measures antigen-specific T cell responses through changes in monokine gene transcription. The biological\r\namplification of the IFNg signal generated by this assay provides sensitivity comparable to ELISpot, but with the\r\nadvantage that responses can be quantified using small volumes of whole blood.\r\nMethods: Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and\r\nimmunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and\r\ncontrol antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in\r\na TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The\r\ninduction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those\r\nobtained by ELISpot.\r\nResults: Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression\r\nin PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification\r\ngenerated by IFNg-R signaling allows responses to be detected in as little as 25 �µL of whole blood and enables the\r\nassay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.\r\nConclusions: A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation.\r\nAssays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This\r\nassay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when\r\nstorage or transportation is required before processing.
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