Imaging flow cytometry enhances a new model of dynamic FOXO1 nuclear localization in human CD4 T cells

Abstract

Abstract The forkhead box O (FOXO) family contains transcription factors critical to differentiation and homeostasis in T cells. FOXO1, acts as a key integrator of proliferation and differentiation signals in both effector T cells (Teff) and regulatory T cells (Treg). In Teff, when the phosphoinositide 3-kinase (PI3K)/Akt pathway is activated downstream of T cell receptor (TCR) signaling and co-stimulation, Akt phosphorylates FOXO1 and excludes it from the nucleus. This promotes T cell proliferation and effector functions. In contrast, nuclear FOXO1 is essential for Treg thymic development and peripheral stability. Multiple studies have examined the effects of FOXO1 deficiency or overexpression in T cells, but to date, few have been able to directly examine the dynamics of the native FOXO1 signaling in human T cells. The accepted method of examining FOXO1 signaling dynamics is nuclear/cytoplasmic protein extraction and subsequent western blotting (WB). In order to assess native FOXO1 signaling dynamics, we obtained the human CD4+ T cell line, HuT102. Using the classic WB approach, we demonstrate that HuT102 cells have more nuclear FOXO1 compared to a similar CD4+ T cell line, HuT78. However, WB does not allow us to examine FOXO1 signaling dynamics in subpopulations. To answer this question, we developed a staining protocol to assess FOXO1 nuclear translocation in HuT102 cells using imaging flow cytometry on an Amnis Imagestream MKII. We show that addition of PI3K and Akt inhibitors increases nuclear FOXO1, while TCR signaling mimics such as PMA/ionomycin and anti-CD3/CD28 decrease nuclear FOXO1 kinetically. Using this protocol, we are now able to directly examine native FOXO1 signaling dynamics in human CD4+ T cells.