RESUMEN

Natural killer (NK) cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization were studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without the expression of single-chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at the nanometer scale, leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D, consequently impairing Ly49A-mediated inhibition. Thus, our data shows that NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Our results together suggest that the balance of NK cell signals and NK cell responses is determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

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RESUMEN

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between ß-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems.

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RESUMEN

Natural killer (NK) cells are essential for killing transformed and virally infected cells. To prevent auto-reactivity, NK cell activation is inhibited by inhibitory receptors that activate the tyrosine phosphatase SHP-1, which dephosphorylates signaling molecules crucial for NK cell activation. Initially, only a single SHP-1 substrate was identified in NK cells, the GEF VAV1. We recently demonstrated that under inhibitory conditions, LAT, PLCγ1 and PLCγ2 serve as novel SHP-1 substrates in NK cells. Furthermore, we showed that during NK cell inhibition, LAT is ubiquitylated by c-Cbl and Cbl-b, leading to its proteasomal degradation, abolishing NK cell cytotoxicity. Here, we address the mechanism through which the Cbl proteins are activated following inhibitory receptor engagement. We demonstrate that during NK cell inhibition, the expression level of the Cbl proteins significantly increases. These data suggest that inhibitory KIR receptors regulate the stability of the Cbl proteins, thereby enabling Cbl-mediated inhibition of NK cell cytotoxicity.

RESUMEN

Imaging technology has undergone rapid growth with the development of super resolution microscopy, which enables resolution below the diffraction barrier of light (~200 nm). In addition, new techniques for single molecule imaging are being added to the cell biologist's arsenal. Immunologists have exploited these techniques to advance understanding of NK biology, particularly that of the immune synapse. The immune synapse's relatively small size and complex architecture combined with its exquisitely controlled signaling milieu have made it a challenge to visualize. In this review we highlight and discuss new insights into NK cell immune synapse formation and regulation revealed by cutting edge imaging techniques, including super-resolution microscopy, high-resolution total internal reflection microscopy, and Förster resonance energy transfer.