Figure 1A-D

Aaron Wilson, Scientist II Novartis, Institutes for BioMedical Research

Figure 1. IFC can detect dynamic FOXO1 localization in HuT102 cells treated with an Akt inhibitor or a TCR signaling mimic. HuT102 cells were exposed to media (A), an Akt inhibitor (AktVIII, 10 μM) (B), a TCR signaling mimic, PMA/I (C), or PMA/I + Akti (10 μM) (D), intracellularly stained for FOXO1 and the nucleus (DAPI) and analyzed via IFC. Overlay images confirm that HuT102 cells have nuclear FOXO1 (A), while Akt inhibition increases nuclear FOXO1 (B), PMA/I pushes FOXO1 more cytoplasmic (C) (representative images, 40 × , 1 h), and Akti reverse the PMA effect (representative image, 40 × , 6 h, D). The mean similarity of FOXO1 and DAPI in HuT102 cells was quantified at 0.5, 1, 6, and 24 h (E) and showed that Akt inhibition significantly increases nuclear FOXO1 at all time points (p < .001, p < .01, p < .0005), PMA/I significantly decreases nuclear FOXO1 ( p < .001) at 1 h, while the Akti reverses the PMA/I effect on FOXO1 at 6 and 24 h ( p < .01, p < .0005). Average of three separate experiments. Mean similarity ≥1 (black line) indicates nuclear FOXO1. Error bars depict standard error of the mean.

Figure 1E

Combining two high-content single-cell characterization technologies in a single readout, imaging flow cytometry (IFC) extends the reach of traditional flow cytometry (FC) to obtain single-cell morphological and intracellular localization measurements of desired targets. Used alone, FC can analyze cell surface and intracellular markers, cell supernatants, lysates, and gene expression in a format capable of instantly grouping cell sub-populations to detect even rare cell types among highly heterogeneous populations. In IFC, digital images of each cellular event are collected by magnifying single cells by 20 × , 40 × , or 60 × . Image content is collected by up to 10 fluorescent channels plus bright- and dark-field images. With IFC, cellular phenotypes can be more fully characterized by visualizing the cellular content behind the population distribution plots collected in traditional FC. In a recent article by Golding et al., IFC was used to characterize the intracellular localization of the transcription factor Forkhead box O1 (FOXO1). The authors first establish a model system to monitor FOXO1 in the human cutaneous lymphocyte lymphoma cell line HuT102. By using a classical approach of cellular fractionation and Western blotting, the authors show that this cell model is capable of detecting FOXO1 localization, which when untreated is expected to be located predominantly in the nucleus. Then, by using the TCR agonist, phorbol 12-myristate 13-acetate (PMA)/ionomycin (PMA/I), it is shown that over time FOXO1 expression then becomes shifted to the cytoplasm. This effect can then be reversed by treatment with an Akt VII inhibitor compound (see figure). It is noted that this approach requires at least 3 × 106 cells per condition for an accurate measurement of protein localization, and on its own has no way to resolve a heterogeneous cell population. With these limitations in mind, the authors tested IFC as an alternate and potentially better approach to measure FOXO1 intracellular localization. Here, an Amnis Imagestream Mark II imaging flow cytometer with a 40 × objective was used to collect 10,000 cell events per sample in a time-course experiment. In this test, a live/dead dye to isolate viable cells, DAPI to identify the nucleus, and a CD4 receptor detection antibody were used along with intracellular staining to determine FOXO1 localization. Signal from both DAPI and FOXO1 was used to produce a similarity score by using a log-transformed Pearson’s correlation coefficient calculation, which showed localization of FOXO1 primarily to the nucleus. With this baseline established, PMA/I treatment, AKT VII inhibitor, or both in combination were used to again characterize the modulation FOXO1. A heterogeneous population of human peripheral blood mononuclear cells was then used to observe the effects of PMA/I treatment in distinct CD4 and CD8 cell populations. Taken together, these results show that IFC is a reliable system for the characterization of FOXO1 localization and, compared to other established methods, requires far fewer cells and is capable of locating FOXO1 expression in defined single-cell sub-populations. Noted is the low cell number needed for IFC, which is crucial in such studies of generally lymphopenic patients. This observation suggests that this technology could provide from numerous applications in high-throughput 384/1536 plate-based assays that have restrictively low cell numbers.

* Abstract from J Immunol Methods 2018;454:59–70

While flow cytometry can reliably assess surface and intracellular marker expression within small cell populations, it does not provide any information on protein localization. Several key transcription factors (TF) downstream of lymphocyte surface receptors are regulated by nuclear versus cytoplasmic localization, and one such TF is Forkhead box O1 (FOXO1). FOXO1 integrates antigen-binding, co-receptor activation and metabolic signals in lymphocytes, leading to proliferation and differentiation. Importantly, the nuclear or cytoplasmic localization of FOXO1 is key for gene expression leading to different lymphocyte phenotypes. In effector lymphocytes (Teff), for example, lymphocyte receptor (TCR) signaling leads to an Akt-dependent phosphorylation of FOXO1. Phosphorylated FOXO1 is excluded from the nucleus, promoting proliferation and effector functions. In contrast, nuclear retention of FOXO1 is essential for early and late development of T and B cells and for the thymic development and stability of regulatory T cells. Given the critical role of FOXO1 localization as an indicator and determinant of function, quantification of FOXO1 cellular localization in human lymphocytes can help determine immune cell activation and activity in experimental and clinical scenarios. The standard method used to determine subcellular protein localization is the analysis of nuclear and cytoplasmic protein extracts by Western blotting (WB). However, available techniques, such as WB, are limited by a requirement for a large number of cells and inability to determine FOXO1 localization in individual cells or sub-populations. In contrast, a standardized method using an imaging flow cytometer (IFC) such as the Amnis ImagestreamX Mark II, would provide both qualitative, per-cell localization information, as well as quantitative data on gated sub-populations. To this end, we report the development and optimization of an IFC protocol to examine native FOXO1 localization in human lymphocytes. A human CD4+ lymphocyte line, HuT102, as well as primary human T cells, were assessed for dynamic FOXO1 localization after treatment with a lymphocyte receptor signaling mimic (PMA/ Ionomycin). IFC nuclear translocation analysis permitted us to precisely quantify the alterations over time in nuclear and cytoplasmic localization of native FOXO1 on a per cell basis, including within specific, user-defined sub-populations of cells. For human lymphocytes, using IFC to assess and quantify dynamic FOXO1 localization allows the user to simultaneously study multiple lymphocyte subpopulations as well as to delineate differing effects of dynamic FOXO1 localization that may be lost when other available methods are used.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “ Literature Search and Review ” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, published in the Journal of Immunological Methods titled “Cytometry with a View: Application of Imaging Flow Cytometry“, authors are Hritzo MK, Courneya JP, Golding A.
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