Validation of Th17 Cell Differentiation from Peripheral Blood CD4+ T Cells Through Assessment of mRNA Expression and Cytokine Secretion using Microplate Reading and Cellular Imaging

Abstract

Abstract CD4+ T cells signal and regulate an immune response to pathogens after interacting with an antigen-MHC complex. This activation causes the cells to differentiate into distinct phenotypic and functional effectors, collectively referred to as T helper cells (Th). One such differentiated T cell subset includes the pro-inflammatory T helper 17 (Th17) cell subset. These cells can be beneficial to the host during infection, as they amplify ongoing inflammation by inducing expression of tumor necrosis factor-α. CD4+ T cells that differentiate into Th17 cells are uniquely characterized by production of the pro-inflammatory cytokine, IL-17. T cells isolated from peripheral blood mononuclear cells and selected for CD4 are differentiated into Th17 cells using a cocktail of specific antibodies and cytokines. The procedure to create and confirm this differentiation can be labor and time intensive. Here we demonstrate a validated, robust method to differentiate peripheral blood CD4+ T cells into functional Th17 cells using a bead-based activation technology. A novel, cell-imaging multi-mode reader can be used to image phenotypic differences between cells exposed to the antibody/cytokine cocktail and control cells as differentiation proceeds. Creation of fully functioning Th17 cells was then confirmed by assessing IL-17F mRNA levels using a fluorescence RNA in situ hybridization assay, in addition to IL-17 secretion using a homogeneous, bead-based immunoassay technology. The reader was able to perform brightfield and fluorescence imaging steps, as well as laser-based excitation for the secretion immunoassay. The combination provides a comprehensive solution for the creation and validation of this important class of helper CD4+ T cells.