Dissecting the functional impact of natural killer cell receptor variation in cattle.

Challenges to the health of an organism are met by the immune system. These challenges can arise from external sources, such as bacteria, viruses, or allergens as well as internal sources such as tumours. Meeting all these challenges is a difficult task and has resulted in a highly complex immune system. Pathogenic invaders are constantly changing in an attempt to avoid immune detection. This in turn drives the immune system, fostering a repetitive cycle of change and adaptation for both the host and pathogen. Diverse receptors expressed on a variety of immune cells are an essential part of this host adaptation. A significant proportion (~5 %) of mammalian genomes is dedicated to the immune system, particularly those immune receptors which form gene clusters and families. Within these families mutation and germ-line recombination creates a source for new genes and new variants of old genes. In fact, immune related genes have diversified more than any other type in mammalian genomes. A distinct set of variable and diverse receptors are expressed on natural killer (NK) cells. NK cells are one of the first responders to pathogens. The magnitude of the NK response subsequently directs how the rest of the immune system responds. NK cells are controlled by some of the most diverse immune receptors identified and are especially important in antiviral immunity. Amongst mammals, cattle have the most diverse NK cell receptor system so far identified. As this system evolved to combat infection, it is essential to examine this system in cattle to understand how these economically crucial animals fight infection. This knowledge will enable applied studies to improve vaccines and breed for disease resistance.
This proposal will use information from the cattle genome and high-throughput DNA sequencing technology to completely characterise the NK cell receptor gene families in six Holstein-Friesian dairy cattle. This is an economically important breed in the UK and worldwide. By comparing several animals within this breed we can determine the extent of NK cell receptor diversity. Using knowledge of these receptors, we will determine the molecules that they interact with on infected cells to control NK cell function. This will determine if animals that have dissimilar NK cell receptors are likely to respond differently to infection. Finally, we will infect particular cattle cells that are known to activate NK cells and in turn become activated by NK cells. These will be infected with a known economically important cattle pathogen, bovine herpes virus-1. We will then incubate these infected cells with NK cells that express different NK cell receptor genes. By measuring how these NK cells function and how the infected cells respond, it will be possible to determine if NK cell variation causes different immune responses.
Knowledge of how NK cells respond to virus infection depending on their receptors is essential. This fundamental research will allow future examination of how individual animals vary in their response to individual pathogens and vaccination. This will create opportunities to increase the frequency of beneficial genes through genomic selection breeding strategies and more accurately measure how effective vaccines protect animals from pathogen challenge.

Natural killer (NK) cells are lymphocytes of the innate immune system that recognise and respond to infected or transformed cells, and through interaction with antigen presenting cells help control the adaptive immune system. These innate responses are crucial for survival as humans lacking functional NK cells succumb to overwhelming viral infection, despite possessing a functional adaptive immune system. NK cells are controlled by a diverse receptor repertoire that has evolved independently in different mammalian lineages. Cattle have a unique and highly diverse NK cell receptor system that has been barely studied.

The pressure to diversify NK cell receptors is attributed to interactions between the host and intracellular pathogens. This is illustrated by the several viruses that have developed strategies to avoid or manipulate the rodent and primate NK cell response. It is therefore likely that extraordinary diversity of cattle NK cell receptors has also been driven by pathogens. By comparing the NK cell receptor gene content with a cattle breed we will determine how diverse this control system is, which receptors are polymorphic and those likely to be functional. This will lead to in vitro functional analysis of these receptors to identify their natural ligands. The research will culminate in an examination of how NK cells and the accessory cells they interact with respond to viral infection, using an in vitro model with a ubiquitous and economically important cattle pathogen. The ultimate goal of this research is to discover the consequence of NK cell receptor variation between individual cattle depending on receptor and ligand genotype. This knowledge is essential to fully appreciate the role of NK cells to specific pathogen challenge and vaccination, to identify beneficial genes and fully examine vaccine efficacy.

Despite a pressing need, natural killer (NK) cell research in livestock has been limited. By deciphering the complex genetics behind cattle NK cell function, this project will stimulate and enhance livestock NK cell research. For the first time, genotype can be utilised when examining the crucial role of NK cells in ruminant immune responses to infection and vaccination. This can be translated into beneficial associations to direct breeding strategies and identify novel targets for vaccines and adjuvants. Understanding how the immune system reacts to pathogens is fundamental to the development of new and more effective vaccines; one of the most rapidly growing segments of the pharmaceutical industry. Without knowing how genetic variation impacts the immune response, such experiments are impossible to fully interpret.
The fine resolution and extensive approach proposed will place cattle alongside rodents and primates in our capability to use animals to test theories of NK cell control and function. By using the IAH genetically defined MHC cattle, we will also increase the impact and usefulness of this BBSRC resource for future research. Together, this resource and research will place the IAH at the front of an emerging and fundamental field of livestock research. This will produce extensive future impact by stimulating research projects in the UK and beyond.
Improving the cattle genome assembly will be significant value for comparative genomics. Post genomic technologies, especially SNP genotyping strategies, can exploit these regions of extensive natural variation for the first time. Therefore, resolving and characterising these complex regions could have impact upon the UK and global biosciences. This will enable research and development money to be sought to develop post-genomic techniques to analyse NK cell immune function in entire herds and across breeds.
This proposal will place recent primate and rodent research into a wider evolutionary and functional perspective. In addition, higher primates and cattle are the only species discovered that have expanded the killer immunoglobulin-like receptor genes. This research presents an opportunity to use cattle as a model system to test theories of NK cell receptor function as part of the 'one health' agenda. Through directed breeding, cattle present a unique opportunity to explore the development and function of NK cells not possible in humans and in a more relevant system than rodents.
This research will produce a highly skilled cross disciplinary researcher with unparalleled knowledge in the functional genetics of cattle NK cells. Producing such skilled researchers with expertise in animal immunology is of significant benefit to the UK academic and non-academic communities.
Ultimately, this research will facilitate the applied research necessary to translate genotype into in vivo function and the impact of NK cells on the immune response to pathogens. This will improve the information farmers and breeders use to make decisions on herd management and genetic structure. This could have enormous beneficial impact in reducing the cost of disease management and increasing sustainability. Any advancement in understanding disease resistance will be of benefit by improving productivity and hence wealth creation. As part of improving food security this research will have a beneficial impact on UK society in general and ultimately the rest of the world. Any effect on reducing the burden of disease in these countries will have a major beneficial effect on social welfare, wealth creation through development of livestock industries and the removal of barriers to trade. As such, this project directly addresses BBSRC strategic priority areas in Food Security and therefore contributes to meeting its targets. This project also facilitates data sharing within the animal genetics community, and several other bioscience areas, to facilitate global research within the food security agenda.