title: A whole blood monokine-based reporter assay provides a sensitive and robust measurement of the antigen-specific T cell response.
abstract: BACKGROUND
  The ability to measure T-cell responses to antigens is proving critical in the field of vaccine development and for understanding immunity to pathogens, allergens and self-antigens. Although a variety of technologies exist for this purpose IFNγ-ELISpot assays are widely used because of their sensitivity and simplicity. However, ELISpots cannot be performed on whole blood, and require relatively large volumes of blood to yield sufficient numbers of peripheral blood mononuclear cells. To address these deficiencies, we describe an assay that measures antigen-specific T cell responses through changes in monokine gene transcription. The biological amplification of the IFNγ signal generated by this assay provides sensitivity comparable to ELISpot, but with the advantage that responses can be quantified using small volumes of whole blood.
  
  
  METHODS
  Whole blood or peripheral blood mononuclear cells (PBMCs) from healthy controls and immunosuppressed recipients of solid organ transplants were incubated with peptide pools covering viral and control antigens or mitogen for 20 hours. Total RNA was extracted and reverse transcribed before amplification in a TaqMan qPCR reaction using primers and probes specific for MIG (CXCL9), IP-10 (CXCL10) and HPRT. The induction of MIG and IP-10 in response to stimuli was analysed and the results were compared with those obtained by ELISpot.
  
  
  RESULTS
  Antigen-specific T cell responses can be measured through the induction of MIG or IP-10 gene expression in PBMCs or whole blood with results comparable to those achieved in ELISpot assays. The biological amplification generated by IFNγ-R signaling allows responses to be detected in as little as 25 uL of whole blood and enables the assay to retain sensitivity despite storage of samples for up to 48 hours prior to processing.
  
  
  CONCLUSIONS
  A monokine-based reporter assay provides a sensitive measure of antigen-specific T cell activation. Assays can be performed on small volumes of whole blood and remain accurate despite delays in processing. This assay may be a useful tool for studying T cell responses, particularly when samples are limited in quantity or when storage or transportation are required before processing.
authors.affiliation: richard.cornall@ndm.ox.ac.uk
authors.forename: Aron
authors.initials: A
authors.lastname: Chakera
authors.forename: Sophia C
authors.initials: SC
authors.lastname: Bennett
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Cornall
mesh_terms: D002478:Cells, Cultured; D058501:Enzyme-Linked Immunospot Assay / Q000379:methods*; D006801:Humans; D007165:Immunosuppression Therapy; D007371:Interferon-gamma / Q000494:pharmacology; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D018655:Lymphocyte Count; D015846:Monokines / Q000097:blood*; D018121:Receptors, Cytokine / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D037182:T-Cell Antigen Receptor Specificity / Q000276:immunology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology*
chemical_list: C104577:IP10-Mig receptor; D015846:Monokines; D018121:Receptors, Cytokine; D011994:Recombinant Proteins; D007371:Interferon-gamma