More than a License to Kill: Immune Regulation of Crohn's Disease by Natural Killer Cell and Its Clinical Implication

Inflammatory bowel disease (IBD) is a group of chronic inflammatory conditions in the gut. A major form of IBD is Crohn's disease (CD). Natural Killer cells are members of the innate immune system mainly known for their cytolytic abilities against infected or tumor cells. They are divided into distinct subsets comprised of licensed and unlicensed cells. NK cells are programmed at a genetic level to express discriminating Killer Immunoglobulin Receptors (KIRs) in humans and Ly49 receptors in mice. The engagement of these receptors by their cognate human leukocyte antigen (HLA) ligands induces differentiation of NK cells into a licensed state, which has functional properties distinct from the unlicensed states. It has been identified that the genetic presence of KIR2DL2/3 in the context of its ligand HLA-C1 is a risk factor for CD, yet the cellular mechanism of this genetic contribution and its contribution to immune-mediated colitis is unknown.We used a co-culture assay to study if peripheral NK cells from CD patients with KIR2DL2/3 and HLA-C1, a genotype permits strong NK cell licensing, have a differential impact on CD4+ T cell proliferation than patients without this genotype. Using co-culture assays, flow cytometry, and a microfluidic platform for cytokine analysis, we assessed the role of KIR-HLA genetics on the immunologic functions of NK cells. To identify if KIR-HLA genetics can be used to predict thiopurine responsiveness, we stratified an independent pediatric inflammatory bowel disease cohort into licensed and unlicensed subsets, and compared the percentages of responders in the two subsets. NK cells from CD patients with strong licensing genotype are more potent in promoting CD4+ T cell proliferation than those from patients with intermediate or low licensing genotype (p<0.005 and p<0.0005 respectively). The effect of licensed NK cell is independent on direct contact. Multiplexed analysis of bulk media and single cell NK cytokine profiles established that licensed NK cells produced higher levels pro-inflammatory cytokines including interferon-γ, tumor necrosis factor-α, and chemokines, including C-C motif ligand-5 and macrophage inflammatory protein-1β. Licensed NK cell supernatant also dramatically promotes the differentiation of TH17 cells, a signature CD4+ T helper subset in CD. Pediatric patients with licensing (strong and intermediate) genotype have a higher responder rate (64.7%) to thiopurines than those with low licensing genotype (20.0%). Using similar methods, we demonstrated that murine licensed NK cells augment CD4+ T cell activation not only by increased secretion of proinflammatory mediators such as IL-6 and TNF-α, but also through contact-dependent mechanism, which is different from the solely cytokine-mediated mechanism seen in human licensed NK cells.In conclusion, NK cell licensing mediated by KIR2DL2/3 and HLA-C1 elicits a novel cytokine program that induces pro-inflammatory CD4+ T cells activation and Th17 cell differentiation, thereby providing a pharmacogenomic tool for predicting responders to thiopurine treatment in inflammatory bowel diseases. Murine and human NK cell licensing share similar features, and its role in immune colitis awaits further investigation