Transcriptional activation of Major Histocompatability Complex (MHC) I and II molecules by the cytokine interferon gamma (IFN-g) is a key step in cell-mediated immunity against pathogens and tumors. Following IFN-g induction, JAK/STAT signaling triggers activation of MHC genes. Recent evidence suggests suppression of MHC I and II expression on multiple tumor types plays important roles in tumor immunoevasion. One such tumor is malignant melanoma, the leading cause of skin cancer related deaths. Despite awareness of MHC expression defects, the molecular mechanisms by which melanoma cells suppress MHC and escape from immune-mediated destruction remain unknown. Here we analyze dysregulation of the JAK/STAT pathway and its role in suppression of MHC II in melanoma cell lines at the Radial Growth Phase (RGP), the Vertical Growth Phase (VGP) and the Metastatic Phase (MET). RGP and VGP cells express both MHC II and the MHC master regulator, the Class II Transactivator (CIITA). MET cells lack not only MHC II and CIITA, but also both STAT 1 and the STAT 1 coactivator, the Interferon Response Factor (IRF) 1. Our studies have implicated that the suppression of MHCII on the cell surface of metastatic melanoma is due to silencing at the level of STAT1 transcription. Furthermore, we determined that silencing of STAT1 is, in part, due to hemi-methylation of the STAT1 promoter.

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Type 2 (Th2) immune responses are required for immune defence against helminths, but can also have pathogenic effects in allergic conditions. This thesis examined two factors which may influence Th2 immunity at a cellular and molecular level: cross-talk between Natural Killer (NK) cells and dendritic cells (DCs) and the cell surface organisation of DCs. Cross-talk between NK cells and DCs is well-established to impact Th1 responses against tumours and infection; however the influence of this interaction during Th2 inflammation is unknown. To investigate this, human monocyte-derived DCs were stimulated in vitro with different pathogen-associated molecules; LPS or Poly(I:C) which polarise a Th1 response, or soluble egg antigen (SEA) from the helminth worm Schistosoma mansoni, a potent Th2-inducing antigen. These cells were then combined with autologous NK cells. Confocal microscopy showed polarisation of the NK cell microtubule organising centre (MTOC) and accumulation of LFA-1 at contacts between NK cells and immature or Th2-polarising DCs, but not Th1-polarising DCs, indicative of the assembly of an activating immune synapse. NK cells did not lyse DCs treated with LPS or Poly(I:C), but degranulated to and lysed both immature DCs and Th2 polarising DCs. Antibody blockade of NK cell activating receptors NKp30 and DNAM-1 prevented this lysis. Furthermore, depletion of NK cells in mice which were then transferred with Th2 polarising DCs led to an enhanced Th2 recall response. Thus, these data indicate a previously unrecognised role of NK cell cytotoxicity in restricting the pool of DCs involved in Th2 immune responses. Secondly, this thesis investigated the nanoscale organisation of MHC-II on the surface of Th1 and Th2 polarising DCs using ground state depletion super-resolution microscopy. MHC-II was relatively homogenously distributed across the membrane with no significant changes in clustering between immature, Th1 and Th2 polarising DCs. In contrast, imaging CD74, which can mediate internalisation of MHC-II, revealed increased expression and a more homogenous distribution of this receptor on the surface of Th2-polarising DCs compared to Th1-polarising DCs. These data suggest that changes in the clustering of CD74 could modulate MHC-II surface expression during Th2 responses. Overall, the results in this thesis indicate that both molecular and cellular level modulation of DC function contribute to the development of Th2 responses.

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>

<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 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.

Results of recent studies indicate that dendritic cells are capable of transporting commensal intestinal bacteria into the mammary glands, which ultimately leads to their occurrence in breast milk. We have therefore decided to evaluate the phenotype of immunologically relevant antigen presenting cells (APCs) present in the mammary glands and the small intestine, respectively and perform a comparison study. We also studied plasticity of these populations during lactation. In situ immunodetection and flow cytometry methods were used to determine phenotype. We succeeded in optimising the methods for preparation of samples for flow cytometry and microscopy. We thoroughly tested protocols for 3D visualisation of APC populations and quantitative image analysis for correlation with flow cytometry, further optimization is nevertheless needed. We found out that during lactation large numbers of MHC II+ cells cluster around the alveoli and milk ducts. These cells are of a distinctly dendritic shape and their phenotype does not correspond to the APCs in the surrounding tissue. A pronounced increase of APC cells in the mammary glands between the fourth and sixth days of lactation was observed, with the majority of these cells expressing the CD103 antigen typical for cell populations of immune cells of the...

Presentation of antigenic peptides on MHC-II molecules is essential for induction of tolerance to self and for effective immunity against foreign pathogens. The non- classical MHC-II molecule DO (HLA-DO in humans, H2-O in mice) functions in selection of MHC-II epitopes by competitively inhibiting the peptide exchange factor DM. Previous studies have suggested a role for DO in development of autoimmunity and in the immune response to retroviral infection, presumably via modulation of the MHC-II peptidome, but the precise effect of DO has been difficult to discern. Through characterization of the full spectrum of peptides from DO-sufficient and DO-deficient cells, we demonstrate that DO functions to broaden the diversity of peptide species presented on MHC-II. DO is regulated differently from other components of the MHC-II processing machinery, with expression limited to B cell and dendritic cell subsets, as well as thymic epithelial cells, suggesting a role for DO in mediating central tolerance. In a mouse model lacking DO, we show that selection of T regulatory cells (Tregs) is increased and that DO- deficient Tregs are more activated and exert greater suppressive capacity. Despite augmented Treg function, mice lacking DO display enhanced susceptibility to autoimmunity, with altered germinal center (GC) Tregs and B cells indicative of an aberrant GC reaction. These data suggest that DO expression serves to fine-tune the immunopeptidome in order to promote self-tolerance to a wide spectrum of epitopes and to select a Treg population with appropriate specificity for self- antigens.