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

Abstract

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. Naïve 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.

Natural T cell autoreactivity to melanoma antigens: clonally expanded melanoma-antigen specific CD8 + memory T cells can be detected in healthy humans

“…Ongoing efforts to characterize antigen-specific CD8 + T cells primarily focus on defining their surface phenotype, and in turn, seek to detail their activation/differentiation status. In the present study, we rely on a novel four-color ImmunoSpot® analysis approach to similarly define the effector capabilities of antigen-specific T cell subpopulations [ 14 ].…”

“…Third, we perform four-color ELISPOT assays to simultaneously measure IFN-γ, tumor necrosis factor alpha (TNF-α), IL-2, and GzB production. Collectively, the cytokine secretion profile of individual MA-specific CD8 + T cells defines their subpopulation lineage [ 14 ]. Finally, we measure the affinity [ 16 ] of MA-specific CD8 + T cells.…”

Detection of Antigen-Specific T Cell Lineages and Effector Functions Based on Secretory Signature

“…As shown in Table 2, many of the CD8 + T cell subpopulations described above can be identified/ segregated based on their secretory signature through measurement of IFN-γ, TNF-α, IL-2 and GzB production following antigen challenge. Since these subpopulations occur in rather low frequencies, four-color ImmunoSpot® assays particularly lend themselves to dissecting the expression/co-expression pattern of these analytes at single-cell resolution 64 . For the accurate identification of these distinct CD8 + subpopulations, it is critical that each analyte is measured without signal overlap.…”

CERI, CEFX, and CPI: largely improved positive controls for testing antigen-specific T cell function in PBMC compared to CEF

“…In this way, the frequency of antigen-specific T cells, and thus the magnitude of antigen-specific T cell immunity, can be established [13]. Measurements of multiple cytokines simultaneously, either in double-color ELISPOT or multi-color FluoroSpot assays, can also define the effector lineage(s) of the antigen-specific T cells [14]. As is the case for any functional T cell assay, ELISPOT assays are also critically dependent on the functionality of the T cells and APC being preserved after storage/shipment of the blood, isolation of PBMC, and freezing and thawing of the cells before the actual test is performed [15].…”