Understanding antigen processing in chickens using genome editing technology
Creators: name: Wise, Daniel
Publisher: name: Apollo - University of Cambridge Repository
Year: 2019
Subjects: MHC
antigen processing
antigen presentation
chicken
CRISPR-Cas9

description: The major histocompatibility complex (MHC) plays crucial roles in many biological processes, most especially disease resistance. Classical MHC molecules are extremely polymorphic and encode cell surface glycoproteins that present antigenic peptides to T lymphocytes of the immune system. Several other molecules are involved in loading and optimising the peptides for the two major groups of classical MHC molecules, including the transporter associated with antigen processing (TAP) for MHCI molecules, and the dedicated chaperone DM for the MHCII molecules. In mammals, these critical antigen loading molecules have limited polymorphism, providing peptides for a multigene family of well-expressed classical MHC molecules. Outside of mammals, the chicken is the only system in which peptide loading and presentation has been studied in molecular detail. Compared to mammals, the chicken MHC is small, simple and arranged differently. Chickens have two classical MHCI genes but strongly express only one at the RNA and protein levels which presents the majority of peptides. This phenomenon is apparently due to co-evolution with the polymorphic TAP genes, among other peptide loading associated genes. The exact polymorphic TAP residues involved in peptide loading of chicken MHCI remain unclear. For the class II system, there are two MHCII B genes and two DMB genes, but only BLB2 and DMB2 are expressed at high levels in haemopoietic cells. One hypothesis is that each MHCII B gene co-evolves with one of the two DMB genes, resulting in differential expression, but the interactions and importance of each DM gene are unknown. In this thesis, the genome editing approach of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 was used to begin to understand the peptide loading systems in the chicken. For the first time, the CRISPR-Cas9 system was optimised and implemented in avian cell lines, beginning with the creation of MHCI and TAP gene knock-out (KO) chicken cell lines. TAP-dependent peptide transport was assessed in each line allowing for the first steps to determine the critical peptide binding residues in chicken TAP transport. To understand the role of DM in chickens, KO cell lines for BLB and DM genes were created and analysed at the RNA and protein levels, additionally the peptide repertoire was determined by immunopeptidomics. Striking and unexpected changes in MHCII expression were seen by deleting different components of MHCII presentation. Initial findings suggest the chicken MHCII system utilises two DM chaperones for effective MHCII expression, stability and peptide presentation. The work in this thesis provides some of the first detailed insights into the peptide loading systems for MHC molecules in an organism outside of mammals and provides the basis for genome editing in primordial germ cells (PGCs), eventually allowing analysis of MHC presentation in vivo for improved understanding of pathogen resistance and vaccine responses.
description: Medical research council (MRC)