Development of an MHC class I Ld-restricted PSA peptide-loaded tetramer for detection of PSA-specific CD8+ T cells in the mouse

Abstract

ObjectivesWe set out to develop a prostate specific antigen (PSA) peptide-loaded tetramer for enumeration of PSA-specific CD8+ T cells in the Balb/c mouse model.MethodsA candidate MHC class I PSA peptide (HPQKVTKFML188–197) was selected based on its ability to restimulate PSA-specific CD8+ T cells to secrete IFN-γ in our assays. Next, H-2Ld-restricted peptide-loaded and fluorescently labeled tetramers were produced in conjunction with the NIH Tetramer Core Facility. This tetramer was then tested for staining specificity and optimized for detection of PSA-specific CD8+ T cells induced by our PSA-encoding adenovirus tumor vaccine.ResultsThe MHC class I PSA peptide demonstrated successful restimulation of CD8+ T cells isolated from mice previously vaccinated with a PSA-encoding adenovirus tumor vaccine, with no restimulation observed in control vaccinated mice. The peptide-loaded H-2Ld tetramer exhibited the desired binding specificity and allowed for detection and frequency determination of PSA-specific CD8+ T cells by flow cytometry.ConclusionsWe have successfully designed and validated a PSA peptide tetramer for use in the Balb/c mouse model that can be used to test PSA-based prostate cancer vaccines. Until now, PSA-specific CD8+ T cells in the mouse have only been detectable via cytotoxic T lymphocyte (CTL) assays or intracellular cytokine staining, which primarily assess Ag-specific functional activity, not their absolute number. This research tool provides laboratories the ability to directly quantitate CD8+ T cells elicited by PSA-specific immunotherapies and cancer vaccines that are tested in mouse models.