A reproducible method for the enumeration of functional (cytokine producing) versus non‐functional peptide‐specific cytotoxic T lymphocytes in human peripheral blood

Summary One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme‐linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample‐specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen‐presenting cell platform for flow cytometry‐based quantification of the frequency and activation status of peptide‐specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)‐A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine‐producing) and inactive tetramer‐positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high‐throughput applications and automation. The utility and application of this method are discussed.