Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

Induction of an adaptive, or antigen specific, immune response is critical for eliminating most infections. Pathogen clearance is accomplished primarily, by the actions of CD4+ T cells through their ability to recognize foreign antigens presented at the cell surface by major histocompatibility class II (MHC II) molecules. Consequently, the capacity to regulate expression of MHC molecules is essential to control the adaptive immune response. MHC molecules are regulated at the level of transcription by a master regulator, the class II transcriptional activator, CIITA. Thus, the expression of MHC II is directly related to proper CIITA activity. This thesis focuses on the novel role of S7, an ATPase subunit of the 19S proteasome, in the transcriptional regulation of CIITA and MHC II molecules.

The ubiquitin-proteasome pathway plays vital roles in multiple cellular processes including protein turnover and transcription regulation. The fate of a ubiquitinated protein is determined by the number of ubiquitin molecules added and the site to which they are added. Monoubiquitinated proteins are stabilized and often activated, while polyubiquitinated proteins are rapidly targeted for degradation. Major histocompatibility complex class II (MHC II) molecules are a vital part of the immune response and are responsible for presenting antigens to CD4+ T cells. The class II transactivator (CIITA) is the master regulator of MHC II transcription and has been shown to have increased transactivity when monoubiquitinated. The focus of this thesis is on the impact of ubiquitination on CIITA stability and MHC II gene expression through the identification of an E3 ligase that targets and ubiquitinates CIITA.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

The major histocompatibility complex (MHC) is a genomic region containing genes of immunomodulatory importance. MHC class I genes encode cell-surface glycoproteins that present peptides to circulating T cells, playing a key role in recognition of self and non-self. Studies of MHC loci in vertebrates have examined levels of polymorphism and molecular evolutionary processes generating diversity. The bovine MHC (BoLA) has been associated with disease susceptibility, resistance and progression. To delineate mechanisms by which MHC class I genes evolved to function optimally in a species like cattle, it is necessary to study genomic organization of BoLA to define gene content, and investigate characteristics of expressed class I molecules. This study describes development of a physical map of BoLA class I region derived from screening two BAC libraries, isolating positive clones and confirming gene content, order and chromosomal location through PCR, novel BAC end sequencing techniques, and selected BAC shotgun cloning and/or sequencing and FISH analysis. To date, this is the most complete ordered BAC array encompassing the BoLA class I region from the class III boundary to the extended class I region. Characterization of a frameshift allele exhibiting trans-species polymorphism in Bos and Bison by flow cytometry, real-time RT-PCR, 1D and 2D gel analysis is also described. This frameshift allele encodes an early termination signal within the antigen recognition site (ARS) of exon 3 of the BoLA BSA-Adel class I gene predicting a truncated class I protein that is soluble. An ability to assess MHC diversity in populations and provision of animals with defined MHC haplotypes and genetic content for experimental research is necessary in developing a basis upon which to build functional studies to elucidate associations between haplotype and disease in bovids. The BoLA class I region is immunologically important for disease association studies in an economically important species. This study provides knowledge of gene content and organization within the class I MHC region in cattle, providing a template for more detailed analysis and elucidation of complex disease associations through functional genomics and comparative analysis, as well as evolution of the MHC in bovids to optimize a population’s immune response.

The class II Transactivator (CIITA) is the master regulator for Major Histocompatibility Class II (MHC II) molecules. CIITA is dynamically regulated by a series of Posttranslational Modifications (PTMs). CIITA is responsible for initiating transcription of MHC II genes, thus allowing peptides derived from extracellular antigens to be presented to CD4+ T cells. CIITA’s PTMs are necessary for regulation of CIITA’s location, activity, and stability. Our work identifies the kinase complex ERK1/2 as being responsible for phosphorylating the previously identified regulatory site, serine (S) 280 on CIITA. Phosphorylation by ERK1/2 of CIITA S280 leads to increased levels of CIITA mono-ubiquitination and overall increases in MHC II activity. We further identify a novel ubiquitin modification on CIITA, lysine (K) 63 linked ubiquitination poly ubiquitination. Our data shows novel crosstalk between K63 ubiquitination and ERK1/2 phosphorylation. K63 ubiquitinated CIITA is concentrated to the cytoplasm, and upon phosphorylation by ERK1/2, CIITA translocates to the nucleus, thus demonstrating that CIITA’s location and activity is regulated through PTM crosstalk. While ubiquitination has been shown to be a critical PTM in the regulation of CIITA, the enzyme(s) mediating this important modification remained to be elucidated. Previous reports implicating the histone acetyltransferase (HAT), pCAF as an ubiquitin E3 ligase were intriguing, as pCAF is also known to participate in the acetylation of both histones at the MHC II promoter and in acetylation of CIITA. We now identify novel roles for pCAF in the regulation of CIITA. We show pCAF acts as an E3 ligase, mediating mono, K63, and K48 linked ubiquitination of CIITA. We therefore demonstrate an additional substrate for the “dual acting” enzyme, pCAF. In sum, our observations identify enzymes involved in both the phosphorylation and ubiquitination of key residues of CIITA, which ultimately regulate CIITA activity. Together our observations contribute to knowledge of CIITA’s growing network of PTMs and their role in regulating the adaptive immune response, and will allow for development of novel therapies to target dysregulated CIITA activity during adaptive immune responses.

The koala (Phascolarctos cinereus) is an iconic Australian marsupial that is the sole extant representative of the Family Phascolarctidae and forms part of the mammalian fauna in four Australian States and one Territory. There has been a significant decline in the range and distribution of the koala since European settlement, with habitat loss and its associated threats, including vehicle strikes, dog attacks and disease, continuing to reduce population numbers. Koalas provide the tourism industry and the Australian economy with revenue up to $1 billion per annum with 80 zoos and wildlife parks around the country displaying koalas. Koalas were introduced to a number of islands in southern Australia the early 20th century, in an attempt to ‘save’ them from near extinction due to habitat loss, hunting and disease. While populations on Queensland islands remain stable and require no management intervention, introduced populations on southern Australian islands show extremely low levels of genetic diversity yet have grown substantially and require constant monitoring and management to overcome overabundance and starvation associated with defoliation of food trees. The only known extant, naturally occurring, island population of koalas is on North Stradbroke Island in Southeast Queensland’s Moreton Bay. Infection with bacteria of the Family Chlamydiacae is at high prevalence in most koala populations but the resulting diseases are generally of low prevalence in koalas in Southeast Queensland and New South Wales. The role of the koala retrovirus (KoRV) in relation to infectious diseases is not fully understood, but it has been identified from all northern koala populations and the majority of southern koala populations though, like chlamydial infection, it is not usually associated with high disease prevalence. The major histocompatability complex (MHC) is a gene family in mammals that is vital for its role in disease resistance and so may play a role in disease susceptibility in koalas. This study provided detailed information about the genetic diversity and population structuring of the mainland koala population in Southeast Queensland, which is amongst the most threatened of koala populations because of anthropogenic disturbance. Microsatellite genotyping of 512 samples and mitochondrial DNA sequencing of a subset of 77 samples collected from sick, injured and dead koalas showed that in Southeast Queensland koalas had high microsatellite diversity (12.8 alleles/locus, He = 0.81) and at least 16 mitochondrial haplotypes. Population genetic analysis revealed six genetically differentiated clusters consistent with the existence of biogeographic and anthropogenic barriers like rivers and highways. The Koala Coast, an area in Southeast Queensland that provides habitat for one of the most significant natural koala populations in Australia was found to be genetically distinct from adjacent areas; the rapid decline of this population means it meets the criteria for classification as “Endangered Wildlife” under Queensland’s Nature Conservation Act 1992. Climatic patterns of warming and cooling that produced the disappearance and reappearance of rainforests is likely to have affected long term koala distribution in coastal eastern Australia – expansion into drier forests when they were available and restriction to refugia during times of rainforest dominance. The koala population in Northeast New South Wales, which has conventionally been classified as a different subspecies to the koalas in Southeast Queensland, has colonised an extensive part of the area since the destruction of the “Big Scrub” rainforest. This forest occupied about 75,000 ha before European settlement and would almost certainly have represented a natural barrier to the movement of koalas as it was unsuitable habitat due to its lack of suitable food trees. Population structuring analysis identified two major genetic clusters in the region, the northernmost of which was less differentiated from koalas sampled on the Gold Coast in Southeast Queensland than the southernmost cluster. The southern cluster, largely found around the city of Lismore, had significantly lower diversity than the northern cluster, consistent with a southward radiating colonisation pathway. Ninety-six percent (96%) of koalas in Northeast New South Wales shared the same mitochondrial DNA haplotype, which was the most common haplotype in Southeast Queensland. There was, therefore, little evidence to support the subspecies delineation of coastal koalas in Southeast Queensland and Northeast New South Wales. From a comparison between wild, mainland populations and two captive koala colonies in Southeast Queensland, genetic diversity in 106 captive koalas was found to be similar to the local wild populations (Dreamworld 9.2 alleles/locus, He = 0.75, Currumbin Wildlife Sanctuary 10.2 alleles/locus, He = 0.80). Microsatellite alleles in captive koalas were, in general, representative of the local wild populations and of similar frequencies, but the captive koalas possessed mitochondrial DNA haplotypes that were not found in the local wild koalas. Some of the original founders of the captive populations were not from Southeast Queensland so these different mitochondrial haplotypes are likely remnants of the koala genotypes at the original founders’ locations. The preservation of habitat and reduction of consequential threats to the koala’s long term survival in the wild are the most fundamental aspects of koala conservation in this region. However, the successful preservation of high genetic diversity at neutral loci in the captive colonies studied indicates that ex situ measures incorporating a captive breeding program could potentially be used as reservoirs for important genetic material should such an approach become necessary in the context of the drastic and accelerating decline towards local extinction of key Southeast Queensland koala populations. Urgent action would be essential to obtain a representative sample of current genetic diversity (both genomic and mitochondrial), given the drastic rate of decline. Island populations of organisms are expected to show reduced genetic diversity compared to their mainland counterparts because of factors such as founder effects and the increased susceptibility of small populations to the effects of genetic drift. A comparison of genetic diversity was made between the naturally occurring North Stradbroke Island koala population in Southeast Queensland and the introduced populations on St Bees, Brampton, Newry and Rabbit Islands in central Queensland with the mainland populations. As expected, the island populations had lower genetic diversity than mainland populations, however, the introduced St Bees Island population had higher mean number of alleles and expected heterozygosity (5.7 alleles/locus, He = 0.67) than the naturally occurring population on North Stradbroke Island (3.7 alleles/locus, He = 0.55). Anecdotal evidence suggests that the koala populations on Brampton, Newry and Rabbit Islands off the central Queensland coast were established by the unauthorised transfer of koalas from St Bees Island. Mitochondrial DNA haplotype analysis supports this claim however, the most likely explanations for the presence of at least four microsatellite alleles in the Brampton, Newry and Rabbit Island populations that were not found in St Bees Island koalas are that either there were additional introductions from elsewhere or that koalas were already present on these islands prior to the transfers from St Bees Island. A study on the prevalence of chlamydial and retroviral infection in captive and wild koalas showed that 100% of koalas in this study had retroviral and chlamydial infection in at least one tested swab site. None of the captive animals showed clinical signs of disease despite the high prevalence of infection. Signs of disease were evident in some wild koalas, which, in some cases, was the reason for their admission to Moggill Koala Hospital for treatment. In an attempt to understand the immunological mechanisms underlying the apparent disease resistance of koalas which usually limits latent infection progressing to clinical disease states, the MHC genes of koalas were investigated. Low variation at MHC loci is thought to increase susceptibility to infectious diseases because fewer foreign antigens are recognised. The first class II sequences identified from koalas are presented here and showed variation. Variation was also detected at exon 3 of class I, with evidence for at least three class I loci. The koala is a unique Australian marsupial that makes a nationally significant contribution to the Australian economy annually. The results of this study have important conservation and management implications for koala populations in Southeast Queensland and Northeast New South Wales - particularly declining populations in Southeast Queensland - as well as being informative on other aspects of the species’ biology and for mammalian conservation genetics, generally.