Developing a System to Determine Proper Peptide Binding in Class II Major Histocompatability Complex Molecules

Abstract

The T-cell compartment of the adaptive immune system executes a critical role in human health by taking advantage of combinatorial diversity to recognize virtually any antigen. Upon infection, dendritic cells (DCs) use major histocompatibility complex (MHC) molecules to present antigenic peptides to T-cell receptors (TCR) on T-cells. TCR are then able to recognize the composite surface of peptide- MHC (pMHC) complexes and pass this information into the T-cell, thus linking the innate and adaptive immune systems. CD4⁺ T-cells then play a fundamental role in directing and regulating the immune response to a pathogen. A significant decline in immune system function and an increased susceptibility to infections from pathogens such as West Nile Virus (WNV) is seen upon aging. We hypothesized that this may be due to a reduced frequency of CD4⁺ T-cells that are specific for, and capable of responding to, WNV. To quantify these frequencies we have developed pMHC II tetramers, a powerful tool for characterizing the T-cell repertoire and one that has been a challenge to produce. The development of these tetramers hinges on the ability of the peptide fragment to bind properly to the MHC molecule. It is therefore beneficial to have a system for optimizing peptide selection prior to beginning the elaborate tetramer building process. We have engineered retroviral constructs encoding IAᵇ α and β chains, and we plan on using these constructs to stably transduce M12 cells. We will use cell surface expression of IAᵇ molecules as readout for proper pMHC II complex folding. The goal of the project is to develop a cost-effective system to optimize binding of a variety of peptides to MHC II molecule to aid the development of pMHC II tetramers.