Four Color ImmunoSpot ® Assays for Identification of Effector T-Cell Lineages

: Single color IFN-γ ELISPOT assays have evolved as a highly sensitive T cell immune monitoring platform. By detecting individual T cells that secrete IFN-γ in response to antigen exposure, these assays permit the measurement of the frequency of antigen-specific T cells among white blood cells. These assays therefore are well suited to assess clonal expansions, that is, whether a (Th1) T cell response has been induced to an antigen in a test subject. Single color IFN-γ ELISPOT assays are not suited, however, to provide information on the Th2/Th17 quality of the T cell response, nor do they provide insights into the differentiation state of CD8 cells. Recently it has been established that co-expression profiles of IL-2, TNF-α, and granzyme B along with IFN-γ permit to identify CD8 cell subpopulations. Naive CD8 cells, central CD8 memory cells, CD8 terminal effector cells, polyfunctional CD8 cells, stem-cell like CD8 memory cells, dysfunctional- and senescent CD8 cells all differ in the extent they produce these molecules upon antigen re-encounter. We therefore have developed, and introduce here, a four color T cell ELISPOT assay in which the co-expression levels of IFN-γ, IL-2, TNF-α, and granzyme B can be established for individual antigen-specific CD8 cells, thereby identifying the activation/differentiation state of these cells.