Natural killer T-cell characterization through gene expression profiling: an account of versatility bridging T helper type 1 (Th1), Th2 and Th17 immune responses

Abstract

Natural killer T (NKT) cells constitute a distinct lymphocyte lineage at the interface between innate and adaptive immunity, yet their role in the immune response remains elusive. Whilst NKT cells share features with other conventional T lymphocytes, they are unique in their rapid, concomitant production of T helper type 1 (Th1) and Th2 cytokines upon T-cell receptor (TCR) ligation. In order to characterize the gene expression of NKT cells, we performed comparative microarray analyses of murine resting NKT cells, natural killer (NK) cells and naïve conventional CD4+ T helper (Th) and regulatory T cells (Treg). We then compared the gene expression profiles of resting and alpha-galactosylceramide (alphaGalCer)-activated NKT cells to elucidate the gene expression signature upon activation. We describe here profound differences in gene expression among the various cell types and the identification of a unique NKT cell gene expression profile. In addition to known NKT cell-specific markers, many genes were expressed in NKT cells that had not been attributed to this population before. NKT cells share features not only with Th1 and Th2 cells but also with Th17 cells. Our data provide new insights into the functional competence of NKT cells which will facilitate a better understanding of their versatile role during immune responses.

Figure 1

Venn diagrams of microarray results for natural killer T (NKT) cells compared with natural killer (NK) cells, NKT cells compared with CD4⁺ CD25^–[conventional T helper (Th)] cells, and NKT cells compared with CD4⁺ CD25⁺ cells [T regulatory cells (Treg)] from spleens of C57BL/6 mice. NKT cell RNA was used in all comparisons as a common reference. (a) A summary of the genes that are differentially expressed in the cell types described. (b) Up-regulated genes: up-regulated in all other cell types compared with NKT cells. (c) Down-regulated genes: down-regulated in all other cell types compared with NKT cells, i.e. up-regulated genes in NKT cells. Roman numerals represent the Venn diagram subsets, and Arabic numerals the number of genes: grey, results from the first experimental set; black, results from the second experimental set; italic, intersection of genes from both experiments that were up- or down-regulated in all other cell types compared with NKT cells. Accession numbers and fold change expression differences for listed genes are available in the online Supplementary material (Table S1). Numbers after the summation sign represent the intersection of differentially regulated genes of the first (grey) and second (black) experimental set. The upper number represents the intersection of differentially expressed genes in both experimental sets.

Figure 3

Genes with up-regulated expression in alpha-galactosylceramide (αGalCer)-activated natural killer T (NKT) cells compared with resting NKT cells. Fold change differences for genes found in two independent microarray analyses are shown. Grey bars, genes belonging to the group of cytokines and chemokines; black bars, genes belong to the group of cell cycle control and apoptosis-related genes. c-rel, cellular reticuloendotheliosis oncogene; EGR, early growth response; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; Jun-B, Jun-B oncogene; L-CCR, Lipopolysaccharide-inducible CC Chemokine Receptor; LIGHT, homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes; MIP, macrophage inflammatory protein; TNF, tumour necrosis factor.

Figure 2

Real-time quantitative polymerase chain reaction (qPCR) for selected genes identified by microarray analyses. Differences in gene expression are shown as delta-delta cycle threshold (ct). (a) Gene expression in natural killer T (NKT) cells versus conventional T helper (Th) cells. (b) Gene expression in NKT cells versus regulatory T cells (Treg). (c) Gene expression in NKT cells versus natural killer (NK) cells. (d) Gene expression in resting versus alpha-galactosylceramide (αGalCer)-activated NKT cells. Real-time qPCR was performed twice in triplicate and delta ct values were normalized to internal GAPDH expression. ATAC, activation-induced, T-cell-derived and chemokine-related; BLK, B-cell lymphocyte kinase; FasL, Fas ligand; IL, interleukin; KLF, Kruppel-like factor.