The NKG2D receptor: immunobiology and clinical implications.

NK cells are critical components of our immune system functioning, in part, to recognize and then eradicate virally infected or tumorigenic cells without previous sensitization. One of the best-characterized activating receptors expressed on NK cells is the NKG2D receptor, which is capable of transmitting co-stimulatory signals by subsets of T cells. Viruses and tumors have evolved strategies to evade NKG2D-mediated immune recognition thus highlighting the importance of this receptor in immunity. This review will focus on the structure of NKG2D and its interaction with its diverse array of ligands, as well as highlighting current knowledge regarding NKG2D signal transduction and biological mechanisms that govern its cell surface expression. The impact that NKG2D has in disease pathologies is also assessed.

The inhibitory leukocyte-associated Ig-like receptor-1 (LAIR-1) is expressed at high levels by human naive T cells and inhibits TCR mediated activation.

Human leukocyte-associated Ig-like receptor-1 (LAIR-1) is a transmembrane glycoprotein with a single extracellular Ig-like domain and a cytoplasmic tail containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs). It is constitutively expressed on the majority of human mononuclear leukocytes and functions as an inhibitory receptor. In this study, we show that freshly isolated peripheral blood T cells are heterogeneous in their expression levels of LAIR-1. We have found that naive T cells express the highest levels of LAIR-1, even more than memory cells. The cross-linking of LAIR-1 inhibits T cell receptor (TCR) mediated signals in freshly isolated human naive T cells and whole populations of CD4+ or CD8+ T cells. TCR cross-linking increased cell surface expression of LAIR-1 in a process that requires p38 MAP kinase and ERK signaling. Altogether, these results indicate that LAIR-1 is capable of negatively regulating T cell functions, and its high level of expression by naive T cells suggests that it may function at an early stage in the development of an immune response.

Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells.

Natural killer (NK) cells express receptors that are specific for MHC class I molecules. These receptors play a crucial role in regulating the lytic and cytokine expression capabilities of NK cells. In humans, three distinct families of genes have been defined that encode for receptors of HLA class I molecules. The first family identified consists of type I transmembrane molecules belonging to the immunoglobulin (Ig) superfamily and are called killer cell Ig-like receptors (KIR). A second group of receptors belonging to the Ig superfamily, named ILT (for immunoglobulin like transcripts), has more recently been described. ILTs are expressed mainly on B, T and myeloid cells, but some members of this group are also expressed on NK cells. They are also referred to as LIRs (for leukocyte Ig-like receptor) and MIRs (for macrophage Ig-like receptor). The ligands for the KIR and some of the ILT receptors include classical (class Ia) HLA class I molecules, as well as the nonclassical (class Ib) HLA-G molecule. The third family of HLA class I receptors are C-type lectin family members and are composed of heterodimers of CD94 covalently associated with a member of the NKG2 family of molecules. The ligand for most members is the nonclassical class I molecule HLA-E. NKG2D, a member of the NKG2 family, is expressed as a homodimer, along with the adaptor molecule DAP10. The ligands of NKG2D include the human class I like molecules MICA and MICB, and the recently described ULBPs. Each of these three families of receptors has individual members that can recognize identical or similar ligands yet signal for activation or inhibition of cellular functions. This dichotomy correlates with particular structural features present in the transmembrane and intracytoplasmic portions of these molecules. In this review we will discuss the molecular structure, specificity, cellular expression patterns, and function of these HLA class I receptors, as well as the chromosomal location and genetic organization.

Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system.

Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities.

NKG2D is a costimulatory receptor for human naive CD8+ T cells.

In humans, all alpha beta CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-gamma and TNF-alpha. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.