The immune recognition receptor complex NKG2D-DAP10 on natural killer cells is stimulated by specific ligands carried on virus-infected and malignant cells. Because DAP10 does not have an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail, its ability to trigger killing has been debated. Here we show that a crucial Tyr-Ile-Asn-Met amino acid motif in the cytoplasmic tail of DAP10 couples receptor stimulation to the downstream activation of phosphatidylinositol 3-kinase, Vav1, Rho family GTPases and phospholipase C. Unlike that of ITAM-containing receptors, the activation of NKG2D-DAP10 proceeds independently of Syk family protein tyrosine kinases. Yet the signals initiated by NKG2D-DAP10 are fully capable of inducing killing. Our findings identify a previously unknown mechanism by which receptor complexes that lack ITAM motifs can trigger lymphocyte activation.
NKG2D is an important immunosurveillance receptor that responds to stress-induced ligand expression on tumors and virus-infected cells. Human natural killer cells express NKG2D and require the transmembrane adaptor DAP10 to initiate their full cytotoxic activation. However, DAP10 has no immunoreceptor tyrosine-based activation motif and thus the mechanism of recruiting 'downstream' effector proteins is unclear. We show here that binding of the p85 subunit of phosphatidylinositol-3- kinase to DAP10 could not by itself trigger cell-mediated cytotoxicity and that binding of an intermediate consisting of the DAP10 binding partner Grb2 and the effector molecule Vav1 (Grb2-Vav1) to DAP10 was sufficient to initiate tyrosine-phosphorylation events. For full calcium release and cytotoxicity to occur, both Grb2-Vav1 and p85 had to bind to DAP10. These findings identify a previously unknown mechanism by which NKG2D-DAP10 mediates cytotoxicity and provides a framework for evaluating activation by other receptor complexes that lack immunoreceptor tyrosine-based activation motifs.
Central nervous system-infiltrating CD8+ T cells are potential mediators of neuropathology in models of multiple sclerosis induced by Theiler's murine encephalomyelitis virus (TMEV) infection. C57BL/6 mice mount a vigorous cytotoxic T lymphocyte (CTL) response against the immunodominant virus peptide VP2121-130 and clear TMEV infection. Interferon-g (IFN-g)R-/- mice also mount a strong CTL response against the VP2121-130 epitope, but because of genetic deficiencies in critical IFN-g signaling pathways, they do not clear TMEV infection and develop prominent neurological deficits within 6 wk. This pronounced disease process, coupled with a defined CTL response, provides an ideal model for evaluating the importance of antiviral CTL activity in the development of severe demyelination and loss of motor neuron function. By administering the VP2121-130 peptide before and during TMEV infection, 99% of the VP2121-130-specific CD8+ T cell response was inhibited. No decrease in virus infection was observed. Peptide treatment did result in significantly less motor dysfunction, even when no differences in levels of demyelination were observed. Although most investigators focus on the role of CD4+ T cells in demyelinating disease, these studies are the first to demonstrate a clear contribution of antiviral CD8+ T cells in neurological injury in a chronic-progressive model of multiple sclerosis.
The two isoforms of phospholipase C (PLC)-γ couple immune recognition receptors to important calcium- and protein kinase C-dependent cellular functions. It has been assumed that PLC-γ1 and PLC-γ2 have redundant functions and that the receptors can use whichever PLC-γ isoform is preferentially expressed in a cell of a given hemopoietic lineage. In this study, we demonstrate that ITAM-containing immune recognition receptors can use either PLC-γ1 or PLC-γ2, whereas the novel NK cell-activating receptor NKG2D preferentially couples to PLC-γ2. Experimental models evaluating signals from either endogenous receptors (FcR vs NKG2D-DAP10) or ectopically expressed chimeric receptors (with ITAM-containing cytoplasmic tails vs DAP10-containing cytoplasmic tails) demonstrate that PLC-γ1 and PLC-γ2 both regulate the functions of ITAM-containing receptors, whereas only PLC-γ2 regulates the function of DAP10-coupled receptors. These data suggest that specific immune recognition receptors can differentially couple to the two isoforms of PLC-γ. More broadly, these observations reveal a basis for selectively targeting the functions initiated by distinct immune recognition receptors.
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