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CD46 is a ubiquitously expressed transmembrane protein in humans with a role in immune homeostasis. Originally identified as a complement regulator, CD46 has since been regarded a receptor for several pathogens and most recently, described as a T cell costimulatory molecule. Its coligation with CD3 and consequent cleavage from the T cell surface serves as a costimulatory stimulus for T cell activation. In addition, in the presence of IL-2, CD46 induces Tr1 cell differentiation which is characterised by low IFN-γ and high IL-10 secretion. CD3/CD46-induced Tr1 differentiation is defective in patients with MS, rheumatoid arthritis and asthma, highlighting the need to investigate the mechanisms involved in the regulation of the CD46 pathway. CD46 is a highly glycosylated protein with three N-glycosylation sites in the short consensus repeats and multiple O-glycosylation sites in the STP region. Previous data from the lab have shown that CD3 activation causes a change in CD46 glycosylation. Herein, I convey that this change is more pronounced in memory than naive CD4+ T cells and is mainly due to changes in CD46 O-glycosylation. Furthermore, these changes are required for the T cell responses triggered by CD46 costimulation including T cell activation and Tr1 differentiation. Interestingly, CD46 is recruited to the immunological synapse formed between a T cell and an antigen presenting cell and I illustrate that the STP region is needed for this also. These data suggest that the glycosylation status of CD46 regulates its function. In MS, vitamin D deficiency is considered to be a significant risk factor and many patients take vitamin D supplement to help manage their condition. Herein, I report that treatment of healthy and MS CD4+ T cells with vitamin D does not prevent T cell activation but it decreases adhesion molecule expression. Moreover, vitamin D supplementation in MS enhances CD46 cleavage. Therefore, vitamin D also plays a role in the regulation of the CD46 pathway and it would be interesting to investigate whether vitamin D affects CD46 glycosylation. During my MSc, I showed that a recombinant protein derived from adenovirus serotype 35 (which naturally binds CD46) known as Ad35K++ controls the CD46 pathway in CD4+ T cells. Lymphoma cells treated with Ad35K++ in combination with monoclonal antibody therapy rituximab have demonstrated increased sensitivity to rituximab and prove that virus-derived recombinant proteins that target CD46 have therapeutic potential. Considering the key role of CD46 as a T cell costimulatory molecule, I have investigated the effects of Ad35K++ on the CD46 pathway following its use in vivo and confirm CD46 is still cleaved from the cell surface and the cells still become activated. Overall these results provide insight into the mechanisms involved in the regulation of the CD46 pathway and highlight how it can be manipulated for therapeutic use.

Atopic dermatitis (AD) is a common, chronic relapsing inflammatory skin disease associated with cutaneous hyper-reactivity to environmental triggers that are innocuous to normal nonatopic individuals. AD affects 10% to 15% of children and 2% to 10% of adults in industrialized countries. There has been increasing interest in this disease triggered by its increasing prevalence in western societies and its contribution to the increasing health care costs. Yet, the underlying pathophysiologic and genetic mechanisms leading to the manifestation of AD are not clear. AD results from a complex interplay between environmental triggers, susceptibility genes including mutations in the keratinocyte protein filaggrin and altered immune responses resulting in allergic CD4+ T cell (Th2) immunity to epidermally encountered antigens. Regulatory T cells (Tregs) play an important role in controlling responsiveness to self-antigens and preventing autoimmune diseases, as well as in limiting inflammatory responses during inflammation and infection. Currently, studies investigating the number and function of Tregs in patients with AD have shown controversial results. It has been long established that symptoms of AD improve on exposure to sunlight. Narrowband UVB (NB-UVB) phototherapy is a common treatment modality for a variety of skin diseases. Considering the adverse effects for systemic treatment for severe adult AD, phototherapy, especially NB-UVB phototherapy may be a more practical long-term treatment. However, approximately 50% of patients over an 8-week treatment course do not improve after NB-UVB phototherapy. Therefore, it is important to identify characteristics of AD patients to determine whether they will respond to phototherapy and to avoid adverse effects for unresponsive patients. UVB exposure has also been associated with induction of Tregs in mice and increasing their numbers and/or functional capacity may offer benefit to patients with chronic AD. Active vitamin D (1,25(OH)2D3), one of the factors induced by UV-B radiation induces Tregs and is suggested to contribute to the suppressive effect of NB-UVB phototherapy. However, UV radiation could also have beneficial effects through other pathways known to affect immunoregulation. UVB exposure upregulates production of nitric oxide (NO) in the skin which also affects immune cell function. The protein filaggrin is broken down in differentiating keratinocytes to form the natural moisturizer of the skin. The gene encoding filaggrin (FLG) has been shown to be a major predisposing factor for AD. A key breakdown product is urocanic acid (UCA) which also acts as a natural sunscreen and undergoes trans-cis isomerisation on exposure to UV-B. Cis-UCA is known to modulate immune responses, however, the mechanisms of its action remain elusive. The production of all three compounds, vitamin D, cis-UCA and NO might all increase in the circulation of patients undergoing UVB phototherapy. While the immunomodulatory effect of Vitamin D is well described, cis-UCA and NO may also affect the behaviour of T lymphocytes systemically. Therefore, I investigated the effect of NO and cis-UCA on the phenotype and function of CD4+T cells and monocyte-derived dendritic cells (Mo-DCs) derived from peripheral blood mononuclear cells (PBMCs) from healthy volunteers. I also investigated the correlation between plasma concentration of 25(OH) vitamin D and nitrate, FLG genotype, circulating Tregs and clinical efficacy of NB-UVB phototherapy. My results showed that NO did not affect the phenotype of human mo-DCs and directly affected peripheral CD4+ T cells by inducing functional CD25+Foxp3+CD127-Tregs from CD4+CD25lo/- effector T cells. Moreover, NO increased expression of the of skin homing marker CLA on these Tregs, suggesting an increased ability of NO-induced Tregs to migrate to the skin. These NO-induced CD25+Foxp3+CD127-Tregs had immunosuppressive functions and inhibited autologous CD4+ T cell proliferation. Cytokines, at least IL-10, secreted by NO-treated CD4+ T cells were not sufficient for the suppressive function of NOinduced Foxp3+Tregs. The immune regulatory function of NO-induced Fopx3+Tregs required cell-cell contact and was mediated by membrane bound TGFβ and PD-1/PD-L1 but not CTLA-4. Results also showed that cis-UCA might have both pro- and anti-inflammatory effects. Cis- UCA significantly decreased the proportion of CD25hi Foxp3+ cells from activated CD4+ T cells. It also decreased the expression of vitamin D receptor in CD4+ T cells which may interfere with the immune regulatory function of vitamin D. These results suggested that there might be a fine balance between UV-induced anti-inflammatory molecules’ effect on CD4+ T cells. However, Cis-UCA also modulated CD4+ T cell directly by decreasing CD4+ T cell proliferation, decreasing phosphorylation of ERK after TCR activation, enhancing immune suppressive cytokines secretion, and inhibiting the percentage of CLA+CD4+T cells suggesting a decreased ability to migrate to the skin, . Cis-UCA also affected the phenotype and function of antigen presenting cells by decreasing the expression of HLA-DR, CD86 and CD40 on immature mo-DCs, which led to increased proportion of CD25+Foxp3+CD127- T cells when co-cultured with allogenic CD4+ T cells. Results generated from the clinical study in which all 29 patients got better after phototherapy suggested although circulating 25 (OH) vitamin D concentration was significantly increased after NB-UVB phototherapy, the change of circulating 25 (OH) vitamin D concentration did not correlate with disease improvement. This suggests that vitamin D is not the only pathway involved and that other molecules contribute to UVB-induced immune-regulation. The data also show that of the levels of circulating nitrate and the FLG genotype did not correlate with improvement / change with phototherapy. However, the expression of CD69 and CLA on circulating CD4+ T cells was decreased after treatment suggesting that UVB affected T cell activation and migration to the skin, and their importance in determining clinical responses requires further investigation. Taken together, the results from my study provide evidence that vitamin D is not the only molecule responsible for the beneficial effect of NB-UVB phototherapy. NO and cis-UCA may down-regulate immune responses by affecting human peripheral CD4+ T cells and mo- DCs phenotype and function. A further understanding of the effect of NO and cis-UCA on skin resident immune cells will provide more insights for narrowing NB-UVB phototherapy which will help to select patients that most likely to benefit from a mechanism-based treatment.

CD46 is a ubiquitously expressed transmembrane molecule and has an important role in the innate and adaptive immune system. CD46 was originally identified as a complement receptor that protects cells from autologous attack. However, CD46’s immunological profile is ever expanding and more recently it was identified as a T cell costimulatory molecule. Notably, in the presence of IL-2, CD46 can induce a Tr1-like phenotype that is characterized by the secretion of large amounts of the potent anti-inflammatory cytokine, IL-10. Defects in CD46- induced IL-10 secretion have been identified in multiple sclerosis, asthma and rheumatoid arthritis. Despite CD46’s promiscuous nature in immune responses there is little known about its underlying processing and signalling pathways. Herein, I report that CD46 expression and processing are important for regulating T cell anti-inflammatory responses and activation. Cyt1 but not Cyt2 promotes an increased ratio IL-10+ cells compared to cells that secrete both Il-10 and IFNγ. Upon CD46 costimulation, proteolytic cleavage of CD46 occurs at the surface and intracellularly with the subsequent release of a functional intracellular domain (ICD). As a result of alternative splicing, there are two main cytoplasmic isoforms of CD46, both of which release ICDs, Cyt1 and Cyt2. It is shown that the smaller Cyt1 ICD fragment facilitates T cell activation, whereas, Cyt2 promotes T cell activation when expressed in an uncleaved form. As the expression and cleavage of CD46 is important for regulating T cell function, I went on to identify factors that can regulate CD46 cleavage. Herein, it is demonstrated that T cell activation by the T cell receptor (TCR) acts as a major regulator of CD46 cleavage and expression, emphasizing the inherent role of CD46 in T cell activation. TCR stimulation also modulates CD46 glycosylation, which may effect CD46 expression and T cell phenotype. Importantly, I have identified a dysregulated expression of CD46 in a preliminary cohort of RRMS patients. It will be interesting to examine if aberrant CD46 glycosylation or cleavage accounts for its altered expression levels and impaired IL-10 secretion in RRMS patients.