Optimization and validation of a robust human T-cell culture method for monitoring phenotypic and polyfunctional antigen-specific CD4 and CD8 T-cell responses.

Abstract

Monitoring cellular immune responses is one prerequisite for the rational development of cancer vaccines. We describe an extensive effort to optimize and validate quantitatively an in vitro T-cell culture method by determining the phenotype and function of both CD4(+) and CD8(+) T cells, including measurement of the phenotype markers CCR7, CD45RA, CD28 and CD27 and the functional markers interferon (IFN)-gamma, interleukin (IL)-2, macrophage inflammatory protein (MIP)-1beta, tumor necrosis factor (TNF)-alpha and CD107a. Autologous peripheral blood mononuclear cells (PBMC) were potent stimulators that expanded antigen (Ag)-specific CD8(+) T cells during short-term culture with the addition of IL-2 and IL-15 cytokines. Polyfunctional Ag-specific CD4(+) and CD8(+) T cells were detectable using this method. Our culture system represents a robust human T-cell culture protocol that permits phenotypic, quantitative and qualitative evaluation of vaccine-induced CD4(+) and CD8(+) T-cell responses.