Natural killer T-cell characterization through gene expression profiling

Natural killer T (NKT) cells are a distinct lymphocyte lineage thought to operate primarily at the interface between the innate and adaptive immune response. Yet, their unique role in the immune system remains elusive. Whilst NKT cells show high similarities to other cells of the innate and adaptive immune system, they express unique functional features such as rapid, concomitant production of Th1 and Th2 cytokines upon TCR ligation. In order to portray gene expression of NKT cells and to analyze their complete functional potential, we performed comparative microarray analyses of naive NKT cells, naive NK cells as well as naive conventional CD4+ T cells (Th, CD4+CD25–) and naive regulatory CD4+CD25+ T cells (Treg ). Furthermore, we compared the gene expression profiles of naive versus alpha-galactosylceramide activated NKT cells to elucidate the gene set rapidly expressed upon activation. We describe profound gene expression differences between the different cell types as well as between naive and activated NKT cells allowing the identification of a unique gene expression profile of NKT cells. In addition to known NKT cell specific markers, a high number of genes were expressed and detected which had not been attributed to NKT cells. Notably, our analyses reveals that NKT cells are not only of Th1 and Th2 type but also fulfil criteria of Th17 cells. Hence, our data provide new insight into the genetic décor of NKT cells which will facilitate a better understanding of their versatile role during the immune response. Keywords: NKT, NK, Th and Treg cell type comparison Microarray experiments were done as two-color hybridizations. Total RNA was extracted from single-cell suspensions. An amount of 4 µg total RNA was reverse transcribed with an oligo-dT-T7-promotor primer by a fluorescent linear amplification reaction (Agilent Technologies) and cDNA was labeled either with Cyanine 3-CTP and Cyanine 5-CTP (NEB Life Science Products, Ipswich, MA, USAFrankfurt, Germany) in a T7 polymerase amplification reaction according to the supplier’s protocol. In order to compensate specific effects of the dyes and to ensure statistically relevant data analysis, a color- swap dye-reversal was performed [Churchill, 2002 #100]. The RNA samples were labeled vice versa with the two fluorescent dyes (fluorescence reversal). After precipitation, purification and quantification with a Nanodrop ND-1000 spectrophotometer (Kisker), 1.25 µg of each labeled cRNA was mixed, fragmented and hybridized to an 8.4 K custom-made mouse array (AMADID 010646) according to the supplier`s protocol (Agilent Technologies). Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). Features were extracted with an image analysis tool version A.6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmics platform Resolver Version 5.0. Ratio profiles were combined in an error-weighted fashion with Resolver to create ratio experiments. A two fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis set highly significant (P-value > 0.05), robust and reproducible