Evaluating frequency and quality of pathogen-specific T cells

It is generally accepted that enumeration and characterization of antigen-specific T cells provide essential information about potency of the immune response. Here, we report a new technique to determine the frequency and potency of antigen-specific CD8 T cells. The assay measures changes of intracellular Ca 2+ in real time by fluorescent microscopy in individual CD8 T cells responding to cognate peptides. The T cells form continuous monolayer, enabling the cells to present the peptides to each other. This approach allows us to evaluate the kinetics of intracellular Ca 2+ signalling that characterizes the quality of T cell response. We demonstrate the usefulness of the assay examining the frequency and quality of cytomegalovirus-specific CD8 T cells from healthy donor and patient after haploidentical stem cell transplantation. The new assay has a potential to provide essential information determining the status of the immune system, disease morbidity, potency of therapeutic intervention and vaccine efficacy. Characterization of T cell antigen specificity human blood is challenging due to the low clonal frequencies. Here the authors develop a fluorescent microscopy-based method to detect antigen-specific CD8 T cell activation, and apply it to characterize the anti-CMV repertoire.