Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2009.2 [Gene Expression Module v1.5.4]). conventional T cells, Microarray, GSEA == INTRODUCTION == Natural killer T (NKT) cells express both natural killer receptors and semi-invariant T-cell receptors (TCRs). These cells recognize glycolipids presented by CD1d, a MHC class I-like protein expressed by antigen presenting cells, and express limited T cell receptors (TCRs) (1). Upon activation of NKT cells through TCR ligation, NKT cells produce a large amount of various cytokines such as interferon (IFN)-, interleukin (IL)-4, IL-10 and IL-13, which contribute to bridging PR22 innate and adaptive immune responses (2,3). NKT cells are heterogeneous in terms of TCR expression pattern and classified into invariant type I NKT cells expressing a specific Valpha chain of TCR (V14-J18 in mice and V24-JQ in humans) and type II NKT cells that express more diverse TCR (4). Functionally, iNKT cells are activated upon stimulation with alpha-galactosylceramide (-GalCer). Many studies have been focusing on the functions of iNKT cells rather than type II NKT cells. Therefore, iNKT cells have been implicated in in various immune diseases in autoimmunity transplantation, infection, and tumor surveillance (5-8). The iNKT cells express both TCRs and natural killer receptors on cell surface. Therefore, iNKT cells share characters of NK and T cells in terms of functions and expression patterns of various markers. Nevertheless, gene expression pattern of iNKT cells and conventional T cells has not been well characterized so far. To address this, we performed comparative microarray analyses of murine iNKT cells and conventional CD4+CD25-CR-T cells. == MATERIALS AND METHODS == == YL-109 Animals == B6 mice were purchased from the Orient Company (Seoul, Korea). Mice were bred and maintained under specific pathogen-free conditions at the Clinical Research Institute of Seoul National University Hospital (CRISNUH), Seoul, Korea. All animal experimental protocols YL-109 were approved by the Institutional Animal Care and Use Committee (IACUC) at CRISNUH. == Cell preparation == B6 mice were sacrificed to isolate iNKT cells and conventional T cells, and their livers were homogenized, resuspended in RPMI media containing 33% Percoll density gradient medium (GE Healthcare BioScience, Piscataway, NJ), and centrifuged for 15 min at 2,000 rpm at 25. The mononuclear cells (MNCs) were retrieved, washed with PBS, and stained with fluorescein isothiocyanate (FITC)-conjugated mAb specific for CD3 (BD Biosciences, San Jose, CA) and allophycocyanin (APC)-conjugated CD1d/-GalCer tetramer (National Institutes of Health Tetramer Core Facility, Bethesda, MD). The lymph nodes were homogenized, resuspended in PBS, and stained with FITC-conjugated mAb specific for CD3, phycoerythrin (PE)-conjugated mAb specific for CD25 (BD Biosciences, San Jose, CA) and PE-conjugated mAb specific for TCR (BD Biosciences, San Jose, CA). The stained cells were then sorted on a BD FACSAria flow cytometer (Franklin Lakes, NJ). The sorted CD3+CD1d/-GalCer tetramer+iNKT cells and CD3+CD25-TCR-conventional T cells were isolated at >98% purity. == RNA extraction == Total cellular RNA from the sorted NKT cells and conventional T cells was extracted by RNeasy Mini Prep kit (Qiagen, Valencia, CA), as the manufacturer’s instructions, and then used in separate microarray experiments. RNA quality, concentration and purity were assessed by Nano spectrophotometer (Thermospectronics, Rochester, NY). == Labeling and purification == Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cDNA according to the manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo (dT) primer. Second-strand cDNA was synthesized,in vitrotranscribed, and labeled with biotin-NTP. After YL-109 purification, the cDNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA). == Hybridization and data export == 750 ng of YL-109 labeled cDNA samples were hybridized to each mouse-8 expression bead array for 16~18 h at 58, according to the manufacturer’s YL-109 instructions (Illumina Inc., San Diego, USA). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer’s instructions. Array data export processing and analysis was performed using Illumina BeadStudio v3.1.3 (Gene.
