Using the Autofluorescence Finder on the Sony ID7000TM Spectral Cell Analyzer to Identify and Unmix Multiple Highly Autofluorescent Murine Lung Populations.

Autofluorescence (AF) is a feature of all cell types, though some have more than others. In tissues with complex heterogeneous cellularity, AF is frequently a source of high background, masking faint fluorescent signals and reducing the available dynamic range of detectors for detecting fluorescence signals from markers of interest in a flow cytometry panel. Pulmonary flow cytometry presents unique challenges because lung cells are heterogeneous and contain varying amounts of high AF. The goal of this study was to demonstrate how a novel AF Finder tool on the Sony ID7000™ Spectral Cell Analyzer can be used to identify and screen multiple AF subsets in complex highly AF tissues like murine lungs. In lung single cell suspensions, the AF Finder tool identified four distinct AF spectra from six highly AF subsets. The subtraction of these distinct AF spectra resulted in a resolution increase by several log decades in several fluorescent channels. The major immune and lung tissue resident cells in a murine model of asthma were easily identified in a multi-color panel using AF subtraction. The findings demonstrate the practicality of the AF Finder tool, particularly when analyzing samples with multiple AF populations of varying intensities, in order to reduce fluorescence background and increase signal resolution in spectral flow cytometry.

Cdc6 knockdown inhibits human neuroblastoma cell proliferation.

The cell division controller Cdc6 plays a central role in the initiation of DNA replication. It was found that elevated levels of Cdc6 were present in many of human cancer cells, and the accumulation of Cdc6 is required for cell proliferation. In this study, we have investigated the control of Cdc6 expression and its effect on cell proliferation and death in human neuroblastoma cells. Elevated levels of Cdc6 are found in the LA-N-2, CHLA255, and other cell lines that grow fast. Cdc6 knockdown via a Cdc6 short hairpin RNA lentivirus causes the accumulation of sub-G1 populations with the decrease of S contents in the LA-N-2 and CHLA255 cells. Expression profile from the selected genes shows the reduction of cyclin E, cyclin A, and Cdc25C, with a boosted increase of the CDK inhibitor p27Kip1, indicating the suppression of tumor cell proliferation. Further, Cdc6 knockdown causes the increase of pro-apoptotic Bax accompanied with the decrease of anti-apoptotic Bcl-2, resulting in the increased cell death. Furthermore, Cdc6 knockdown causes a sharp reduction of tumor suppressor protein p53, and Cdc6 overexpression renders a boosted p53 expression; and this regulation is at p53 posttranscriptional level. Our study indicates that human Cdc6 functions in several pathways to control the cell proliferation and the cell death.

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.

Functional identification of kinases essential for T-cell activation through a genetic suppression screen.

Activation of T-cells by antigens initiates a complex series of signal-transduction events that are critical for immune responses. While kinases are key mediators of signal transduction networks, several of which have been well characterized in T-cell activation, the functional roles of other kinases remain poorly defined. To address this deficiency, we developed a genetic screen to survey the functional roles of kinases in antigen mediated T-cell activation. A retroviral library was constructed that expressed genetic suppressor elements (GSEs) comprised of peptides and antisense nucleotides derived from kinase cDNAs including members of the STE, CAMK, AGC, CMGC, RGC, TK, TKL, Atypical, and Lipid kinase groups. The retroviral library was expressed in Jurkat T-cells and analyzed for their effect on T-cell activation as monitored by CD69 expression. Jurkat cells were activated by antigen presenting cells treated with superantigen, and sorted for a CD69 negative phenotype by flow cytometry. We identified 19 protein kinases that were previously implicated in T-cell signaling processes and 12 kinases that were not previously linked to T-cell activation. To further validate our approach, we characterized the role of the protein kinase MAP4K4 that was identified in the screen. siRNA studies showed a role for MAP4K4 in antigen mediated T-cell responses in Jurkat and primary T-cells. In addition, by analyzing multiple promoter elements using reporter assays, we have shown that MAP4K4 is implicated in the activation of the TNF-alpha promoter. Our results suggest that this methodology could be used to survey the function of the entire kinome in T-cell activation.

Distinct homeostatic requirements of CD4+ and CD4- subsets of Valpha24-invariant natural killer T cells in humans.

CD1d-restricted Valpha24-invariant natural killer T cells (iNKTs) are important in immunoregulation. CD4(+) and CD4(-) iNKTs develop with similar frequencies in murine thymus and depend on interleukin-15 (IL-15) in periphery. However, homeostatic requirements of iNKTs have not been analyzed in humans. We evaluated thymic production, peripheral dynamics, and functional maturation of human iNKTs. CD4(+) subset comprises 90% of iNKTs in mature thymocytes and cord blood (CB) but only 40% in adult blood. Using T-cell receptor excision circle (TREC) analysis, we directly measured in vivo replicative history of CD4(+) and CD4(-) iNKT cells. Compared to CD4(+), CD4(-) iNKTs contain fewer TRECs, express higher levels of IL-2Rbeta, and proliferate with higher rate in response to IL-15. In contrast, CD4(+) cells express higher levels of IL-7Ralpha and better respond to IL-7. Neither thymic nor CB iNKTs are able to produce cytokines unless they are induced to proliferate. Therefore, unlike in the mouse, human CD4(+) iNKTs are mainly supported by thymic output and limited peripheral expansion, whereas CD4(-) cells undergo extensive peripheral expansion, and both subsets develop their functions in periphery. These findings reveal important differences in homeostatic requirements and functional maturation between murine and human iNKTs that are to be considered for clinical purposes.