Human Natural Killer Cells Achieve Tolerance by Preferentially Endowing Functional Competence to Inhibitory Killer Ig-Like Receptors Specific for Self-HLA Class I.
Authors: Yu Junli , Pinto-Agnello Clara , Chewning Joseph , Hsu Katharine C. 
Published: October 2019

Abstract
Natural killer (NK) cells are capable of cytotoxic targeting of virally infected cells and some tumor cells. It has been well-demonstrated that NK cells recognize target cells that have down-regulated MHC class I antigen expression (i.e. “missing self recognition”), and that it is the lack of class I engagement of inhibitory receptors such as the killer Ig-like receptors (KIR) that thereby allows NK activation and effector function. How these same inhibitory receptors achieve self-tolerance and simultaneously avoid autoimmunity in humans has not been clear, as more than 60% of individuals have inhibitory KIR for which they lack the HLA ligand. We demonstrate that mature NK cells achieve self-tolerance by preferentially endowing functional competence to the inhibitory KIRs for which they exhibit the cognate HLA ligands. To allow evaluation of inhibitory KIR and avoid interference from potentially class-I recognizing activating KIR, we analyzed NK cells from 10 individuals with various HLA backgrounds, but who were all homozygous for KIR haplotype-A. KIR haplotype-A contains the inhibitory KIR receptors 2DL3, 2DL1, and 3DL1, specific for HLA-Cw3, -Cw4, and -Bw4 ligands respectively, in addition to at most one other activating KIR whose ligand is unknown. Using 6-color staining and flow cytometric analysis of intracellular IFN-γ production, we evaluated the responsiveness of 30 inhibitory KIR-expressing NK subsets following activation with 721.221, a target cell line deficient in class I expression, with 721.221 transfectants expressing HLA-Cw3, -Cw4, or -Bw4 ligands, and with B-lymphocyte cell lines with diverse HLA phenotypes. NK cells exclusively expressing an inhibitory KIR for self-HLA demonstrated increased IFN-γ when coincubated with target cells lacking the cognate HLA ligand, whereas NK cells exclusively expressing an inhibitory KIR for non-self HLA were hyporesponsive to all targets. In all individuals, NK cells expressing inhibitory KIR specific for self-HLA were significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA (p<0.001). All 3 inhibitory KIR (KIR2DL3, 2DL1, and 3DL1) demonstrated capacity for tolerance, with predictable response patterns based on the HLA background of the individual. NK cells lacking all inhibitory NK receptors (KIR−CD94/NKG2A−ILT2−) were identifiable and were markedly hyporesponsive. KIR−CD94/NKG2A+ILT2+ NK cells were also minimally responsive, comparable to the NK subset expressing inhibitory KIR for non-self HLA. These results demonstrate NK tolerance in humans, consistent with the licensing model proposed in mice: NK cells expressing inhibitory KIR to self-HLA are significantly more responsive than NK cells expressing inhibitory KIR for non-self HLA, and are rendered tolerant to self through inhibition upon binding to self-HLA ligands. NK cells expressing inhibitory KIR for non-self HLA are hyporesponsive and therefore rendered tolerant and incapable of autoreactivity when ligand is not engaged.