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

Abstract

Limited sample size and low sensitivity of currently used functional assays challenge direct analysis of cytotoxic CD8 + T lymphocyte activity to quantify antigen-specific immunity after infection or vaccination. Our flow cytometry-based assay reproducibly detects at least three epitope-specific CD8 + T lymphocytes by their cytolytic function. As exemplified for viral epitopes restricted to the human leukocyte antigen (HLA)-A2, the HLA-A2 + human somatic cell hybrid T2 provided an about 10-fold more sensitive readout as compared to autologous B-lymphoblastoid cells or the human erythroleukemia cell line K562 transfected to express HLA-A2 when used as target cells. We named our assay VITAL-FR assay, referring to Hermans et al. (2004) and indicating the modification of using Far Red (FR) dye instead of CMTMR. Under optimal conditions the VITAL-FR assay proved 30 times more sensitive than the 51chromium-release assay to assess epitope-specific target cell lysis. The high overall sensitivity of the VITAL-FR assay basically depended on the negligible spectral overlap of the emission of a stable Far Red fluorescent reporter with the green tracer for target cell labelling. It also profited from long co-incubation of effector and target cells of up to 72 h, from prior in - vitro culture increasing the frequency of epitope-specific CD8 + T cells and from generic, easily accessible standardized target cells that were used with only 10 3 specific and 10 3 control target cells per individual experimental reaction. Our functional approach with the VITAL-FR assay therefore ideally suits for monitoring CD8 + T cell-mediated cytotoxicity in e.g. vaccination studies with known MHC-restricted immunogenic peptides in scientific and diagnostic applications.