Innate Cells with Adaptive Specificity: Bond Kinetics of NK Cell Inhibitory Receptor Ly49C

Natural killer (NK) cells function as cytotoxic cells towards virally-infected and tumor cells. They are also important producers of cytokines to orchestrate immune response. These functions are regulated through a balance of activating and inhibitory receptor signals in the presence of stress- and self-signals. When virally infected or malignant cells downregulate expression of MHC class I (MHC-I), inhibitory receptors on NK cells are no longer engaged, inducing NK cell function. Despite the idea that NK cells are based on such innate recognition mechanisms, NK cells demonstrate different target cellular lysis when presented with different peptides such as SIINFEKL from ovalbumin (OVA) or RGYVYGQGL from Vesicular Stomatitis Virus (VSV) on MHC-I. This differential effector function occurs in the complete absence of direct contacts between peptide and Ly49C and the basis for this is unknown. The overall goal of the proposed work is to determine how Ly49C imparts these different NK cell functions with a view to developing strategies to manipulate NK cells. Antigen-dependent NK cell recognition via modulation of inhibitory receptor recognition is a paradigm shifting concept and may impart NK cell trained immunity. The Ly49 (mouse) family of inhibitory receptors in mice have been well characterized with Ly49C being the dominant player in NK cell inhibition and licensing. Ly49C binds to MHC-I H-2Kb to signal self and prevent the breakdown in tolerance. Ly49C binds H-2Kb underneath the peptide-binding domain alongside the α3 helix domain with no direct contacts to the H-2Kb loaded peptide. As an innate germline encoded receptor, non-specific binding to the constant region of MHC-I would be consistent with the nature of innate immunity. However, Ly49C-peptide-H-2Kb interaction results in different biological activity depending on the peptide. This project is based on published and preliminary data showing: 1) no peptide-dependent structural differences with peptides bound to H-2Kb or when the entire Ly49C-peptide-H-2Kb interacts; 2) mechanosensing measures such as two dimensional bond lifetime under force can dictate differential cell responses and, 3) our preliminary data demonstrating that mechanosensing properties of inhibitory receptor Ly49C and its interaction with peptide-H-2Kb is different depending on the peptide. My working hypothesis is that the bond lifetime as it occurs at the membrane allows Ly49C to distinguish different peptides loaded into MHC. I will test my working hypothesis under the following 2 specific aims: Aim 1. Demonstrate that Ly49C bond lifetime with peptide:MHC molecules is a key feature for NK cell function. Aim 2. To delineate critical amino acids of peptide for Ly49C function. At the completion of the proposed work I expect to define how NK cell trained immunity occurs in an antigen specific manner. This information could be used for rationally designed engineering for the treatment of cancers that can be destroyed by NK cells or infectious diseases where peptide priming of NK cells could lead to trained NK cells that could better fight infectious diseases. Project Number: 1F32AI191473-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jacob Kisiolek | Institution: UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH, SALT LAKE CITY, UT | Award Amount: $78,040 | Activity Code: F32 | Study Section: Special Emphasis Panel[ZRG1 F07B-G (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/1F32AI19147301