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Validation of Th17 Cell Differentiation from Peripheral Blood CD4+ Cells through Assessment of mRNA

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February 03, 2016


Authors: Brad Larson, BioTek Instruments, Inc. Winooski, VT USA; Annegret Taubner, Miltenyi Biotec, Inc. Auburn, CA USA; Dylan Malayter, Affymetrix, Inc. Santa Clara, CA USA; Hilary Sherman, Corning Life Sciences, Kennebunk, ME USA

Miltenyl Biotec     Affymetrix     Corning

Introduction

CD4+ T cells signal and regulate an immune response to pathogens after interacting with an antigen-MHC (major histocompatibility complex). This activation causes the cells to differentiate into distinct phenotypic and functional effectors, collectively referred to as T helper cells (Th), depending on specific cytokine signaling and transcription factors and epigenetic modifications. One such differentiated T cell subset are the pro-inflammatory T helper 17 (Th17) cells. These cells can be beneficial to the host during infection, as they amplify ongoing inflammation by inducing expression of tumor necrosis factor-α (TNF-α). However, uncontrolled activation of Th17 cells are associated with multiple inflammatory and autoimmune disorders including arthritis, primary Sjögren’s syndrome (pSS), multiple sclerosis (MS), and cancer. Naïve CD4+ T cells that are stimulated into Th17 differentiation are uniquely characterized by production of the pro-inflammatory cytokine, IL-17. Research has shown that targeting the IL-17 pathway has attenuated disease severity in preclinical models of autoimmune diseases, which has caused a growing interest in their use as a potential therapeutic target.

The process of attaining viable Th17 cells typically includes multiple steps. CD4+ T cells are isolated from peripheral blood mononuclear cells (PBMCs) and further selected against CD4. Purified cells are then differentiated into Th17 cells using a cocktail of specific antibodies and cytokines. The entire procedure to create and confirm this differentiation can be labor and time intensive.

Here we demonstrate a validated, robust method to differentiate cryopreserved peripheral blood CD4+ T cells into functional Th17 cells using a bead-based activation technology. A novel cell imaging multi-mode reader is used to image phenotypic differences between cells exposed to the antibody/ cytokine cocktail and negative 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 (RNA FiSH) assay, in addition to IL-17 secretion using a homogeneous, bead-based immunoassay technology. The reader previously described performed all 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.

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