The Major Histocompatibility Complex in Rabbits

Parasites impose strong selective forces on natural populations, creating a unique opportunity to investigate evolutionary processes as they take place. The major histocompatibility complex (MHC) is a key component of the immune system’s response to both fight disease and maintain health. Present in all jawed vertebrates, the MHC has played a key role in immunocompetence for over 400 million years. Its primary function in adaptive immunity is to produce cell surface glycoproteins that bind intracellularly or extracellularly derived peptides and display these epitopes for T-cell recognition, thereby facilitating the elimination of pathogens or the establishment of self-tolerance. The MHC is traditionally classified into three classes termed MHC-I, MHC-II, and MHC-III. MHC-I molecules are expressed in all nucleated cells and present intracellular peptides to CD8+ T-cells, whereas MHC-II molecules are expressed mainly in antigen presenting cells (APCs) presenting extracellular peptides to CD4+ T-cells. The genetic diversity of MHC molecules is exhibited by their polymorphism, particularly in their antigen-binding regions. This polymorphism is maintained by mechanisms of balancing selection, such as heterozygote advantage and rare allele advantage. Such diversity is crucial for populations to adapt to evolving pathogenic pressures, as evidenced by the coevolution of the myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus). The European rabbit, originating from the Iberian Peninsula, has been introduced worldwide, often becoming a pest. MYXV causes myxomatosis, a lethal disease for European rabbits, and was used as a biological control to manage the invasive rabbit populations. Following the release of the virus in Australia in 1950, and later in Europe, outstanding mortality rates were observed initially. However, in a short time, both virus virulence decreased, and rabbit resistance increased, demonstrating an evolutionary arms race. A key focus in this evolutionary battle has been the MHC region, which showed the most significant genetic changes. However, little is known about the MHC region in lagomorphs, requiring further investigation. In this PhD thesis, we started by characterising the Major Histocompatibility Complex (MHC) class II genes in rabbits (Oryctolagus cuniculus). Full-length transcripts were sequenced using PacBio technology, and gene models were curated for accuracy. Thirteen MHC-II candidate genes were identified and annotated. Phylogenetic analyses confirmed the orthology between rabbit, human, and mice MHC-II genes, indicating a simple MHC-II system where all the major genes except Orcu-DRB are present in a single copy. Specifically, we found five Orcu-DRB paralogs where -DRB1, -DRB2, and -DRB3 possessed the classical MHC-II characteristics whereas -DRB4, and -DRB5 were pseudogenes. Further analyses focused on genetic diversity across MHC-II genes, exhibiting high nucleotide diversity and trans-species polymorphisms particularly within the peptide binding region in classical MHC-II genes Orcu-DQA, Orcu-DQB, Orcu-DRB indicative of balancing selection. Interestingly, the Orcu-DP genes were practically monomorphic, whereas the Orcu-DQ genes were the most polymorphic. In contrast, non-classical MHC-II genes (Orcu-DM, Orcu-DO) exhibited lower polymorphism, consistent with purifying selection. To understand how those genetic sequences impacted the structure and thereby potentially the function of the MHC molecules, we focused on homology modelling and model predictions. We began by investigating the hydrogen bonds and salt bridges in the peptide binding regions of MHC molecules in humans, rabbits, and hares. Using crystal structures of human MHC-II molecules, we analysed interactions with bound peptides, identifying key binding residues. Electrostatic potential maps were generated using the Adaptive Poisson-Boltzmann Solver (APBS), visualising charge distribution differences in the peptide binding clefts among species. Polymorphisms were traced in the peptide binding regions of classical MHC-II molecules (DP, DQ, DR) and non-classical MHC-II molecules (DM, DO). Additionally, AlphaFold2 models were constructed for the MHC-II molecules of rabbits and hares to compare with human crystal structures and to examine polymorphisms within each species for each gene. For DP molecules, the residues which were predicted to affect peptide binding were found to be monomorphic, however they retained all structurally important residues and structures of a classical MHC-II molecule. The DQ molecules, in contrast, were highly polymorphic, with substantial variation observed within the peptide binding region, as well as variation in the DM-binding region which is typically highly conserved across species. Among DRB molecules, Orcu-DRB1 showed the highest polymorphism, while -DRB2 and -DRB3 exhibited moderate polymorphism but all possessed the same structural motifs as a classical MHC molecule. Non-classical MHC-II molecules (DM, DO), were also analysed structurally, showing conservation of key functional sites. These findings provide an understanding of the structure of MHC-II molecules across lagomorphs, indicating implications for antigen presentation and immune response in rabbits and hares. We then characterised the classical Major Histocompatibility Complex Class I (MHC-I) genes in O. cuniculus. We began by focusing on the identification and analysis of protein motifs and domains. We utilised domain annotation and protein alignment methods, conducted gene ontology analysis to link identified genes with relevant biological processes and molecular functions. We collected modern tissue samples from various locations from the United Kingdom, and Australia and preserved them for sequencing to obtain high-quality long read sequencing data. We analysed tissue-specific expression of MHC-I genes, identifying the distribution and expression levels across different tissues. Finally, we investigated the genetic diversity and polymorphisms present in the rabbit MHC-I genes. Our findings indicated that genes Orcu-U1 and Orcu-U2 aligned with classical MHC-I characteristics, whereas genes -U3, -U4, -U5, -U6, -U7, -U8, and -U9 did not possess those classical characteristics, suggesting divergence from classical MHC-I functionality. We also measured allele-specific expression to understand genetic variation affecting gene transcription. These results advance our understanding of MHC-I and MHC-II gene diversity and its implications for immune function in lagomorphs.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Lirintzi, Marina

Advisor dc:contributor.advisor

• Jiggins, Francis

• MHC
• Major Histocompatibility Complex
• Oryctolagus Cuniculus
• Rabbits

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/10edb1e7-4e8c-49f8-b0b1-f104ebc86a16/download
• http://purl.org/NET/rdflicense/allrightsreserved

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.119407

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/385997