Regulation of immune responses by inhibitory receptors

National Institute Of Allergy And Infectious Diseases

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Abstract

Killer cell immunoglobulin-like receptors (KIR) that bind HLA-C block activation of human natural killer (NK) cells. This inhibition serves to protect healthy cells, which express HLA-C. At the same time, inhibition by KIR maintains NK cells in a state of high responsiveness to subsequent activation signals. This feature has been referred to as licensing of NK cells. Licensed NK cells are primed to react strongly to missing self and kill cells that do not express HLA-C. The inhibitory function of KIR is dependent on zinc. We have reported that zinc induces the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy. KIR filaments formed spontaneously, without addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C+ cells. To test how zinc is delivered to KIR at inhibitory immunological synapses, live imaging was performed to visualize 'free' zinc in NK cells and to follow the localization and movement of zinc transporters. The transporter ZnT1 is the main zinc channel at the plasma membrane for extrusion of zinc out of cells. A ZnT1 tagged with a fluorescent protein that was expressed in an NK cell line remained evenly distributed at the plasma membrane of activating immunological synapses between NK and target cells. Strikingly, ZnT1 polarized and accumulated at inhibitory immunological synapses with HLA-C+ target cells, suggesting that it delivers the zinc required for inhibitory KIR function. The inhibitory signal transmitted by KIR occurs by recruitment of tyrosine phosphatase SHP-1 to two phosphorylated tyrosines in the cytoplasmic tail of KIR. Dephosphorylation of tyrosine-phosphorylated substrates by SHP-1 blocks activation signals. SHP-1 has two tyrosine residues of its own in the C-terminal tail, which are phosphorylated upon SHP-1 recruitment by inhibitory receptors. An interactome of SHP-1 was determined by mass spectrometry during activation and inhibition of NK cells. This proteomics approach revealed several proteins that were selectively associated with SHP-1 during inhibition but not with a SHP-1 mutant with phenylalanine substituted for the two C-terminal tyrosines. One of the proteins identified is a scavenger of reactive oxygen, which helps reduce hydrogen peroxide (H2O2). Imaging experiments have confirmed that this scavenger is recruited at inhibitory immunological synapses. These results showed that inhibitory signaling by KIR includes local protection from reactive oxygen.