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The effects of topical calcipotriol treatment on immune responses to vaccination Bach, Paxton John 2008

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THE EFFECTS OF TOPICAL CALCIPOTRIOL TREATMENT ON IMMUNE RESPONSES TO VACCINATION by Paxton John Bach B.Sc., The University of British Columbia, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2008 © Paxton John Bach, 2008  Abstract: 1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4+CD25+ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8+ T cell priming in response to subcutaneous vaccination, despite the topical coadministration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3+ CD4+ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4+CD25+ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4+ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.  ii  Table of Contents Abstract .............................................................................................................................  ii  Table of Contents .............................................................................................................  iii  List of Figures ...................................................................................................................  vi  Abbreviations ................................................................................................................... vii Acknowledgments ............................................................................................................  ix  1. Introduction 1.1 Structure and immunology of the skin .............................................................  1  1.2 Using the skin immune system to manipulate immune responses ...................  4  1.3 Toll-like receptors ................................................................................... .........  6  1.4 Regulatory T cells ............................................................................................  8  1.5 Ultraviolet radiation induced immunosuppression .......................................... 10 1.6 Vitamin D ........................................................................................................  11  1.7 Topical immunomodulation ............................................................................. 14 1.8 Thesis objectives .............................................................................................. 14 2. Materials and Methods 2.1 Animals ............................................................................................................ 17 2.2 Topical ointment application ........................................................................... 17 2.3 Peptides and proteins ....................................................................................... 17 2.4 Cell isolations and adoptive transfer ................................................................ 18 2.5 Subcutaneous immunization ............................................................................ 18 2.6 Flow cytometry ................................................................................................ 19 2.7 Protein contact hypersensitivity ....................................................................... 20 2.8 Sample collection ............................................................................................. 20 2.9 Quantification of antigen-specific antibody responses .................................... 21 2.10 In vitro Foxp3 induction assay ....................................................................... 22 2.11 Cytokine measurement ................................................................................... 22 2.12 Statistical analysis .......................................................................................... 22 iii  3. Results 3.1 Calcipotriol modulated immunization has potential as a human Treg induction therapy ............................................................................................................... 23 3.1.1 Treatment with topical calcipotriol has no effect on serum calcium levels ...................................................................................................... 23 3.1.2 The effects on calcipotriol on CD8+ T cell responses are largely local .. 24 3.1.3 Calcipotriol induced Tregs are capable of suppressing a delayed-type hypersensitivity response ....................................................................... 26 3.2 Calcipotriol modulates immune responses to subcutaneous immunization ..... 28 3.2.1 Topical calcipotriol inhibits subcutaneous immunization mediated activation of CD8+ T cells ..................................................................... 29 3.2.2 The humoral immune response to subcutaneous immunization is altered by pretreatment with topical calcipotriol .............................................. 33 3.2.3 Treatment with calcipotriol prior to subcutaneous immunization induces transferable tolerance ............................................................................  37  3.3 Effect of topical calcipotriol upon draining lymph node dendritic cell populations .......................................................................................................  41  3.3.1 Calcipotriol decreases the percentage of antigen-bearing dendritic cells within the draining lymph node ............................................................ 42 3.3.2 Function of ex vivo dendritic cells following topical calcipotriol administration and subcutaneous immunization .................................... 45  4. Discussion 4.1 The potential for calcipotriol as a tolerogenic adjuvant ................................... 48 4.2 Humoral effects of calcipotriol on subcutaneous immunization ...................... 51 4.3 Mechanisms of calcipotriol-based immunomodulation .................................... 52 4.4 Differential effects of calcipotriol and betamethasone ..................................... 56 4.5 Applications of calcipotriol modulated subcutaneous immunization .............. 59  iv  4.6 Impact of described work ................................................................................. 62  References .......................................................................................................................... 63 Appendix A ....................................................................................................................... 79  v  List of Figures  Figure 1.  Therapeutic potential of calcipotriol modulated vaccination ..................... 25  Figure 2.  Effect of topical immunomodulation on the phenotype of lymphocytes from the draining lymph nodes following subcutaneous protein immunization .............................................................................................. 31  Figure 3.  Effect of calcipotriol on the CD8+ T cell response to subcutaneous immunization .............................................................................................. 34  Figure 4.  Effect of calcipotriol on the humoral response to subcutaneous immunization .............................................................................................. 36  Figure 5.  Tolerance is transferable from animals treated with calcipotriol and immunized subcutaneously or epicutaneously ........................................... 39  Figure 6.  Foxp3 levels in the draining lymph nodes .................................................. 40  Figure 7.  Dendritic cell phenotype in the draining lymph nodes after subcutaneous immunization .............................................................................................. 44  Figure 8.  Action of ex vivo dendritic cells following subcutaneous immunization ... 46  vi  Abbreviations  APC  antigen presenting cell  BAL  bronchoalveolar lavage  BMV  betamethasone-17-valerate  BSA  bovine serum albumin  CFSE  5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester  CHS  contact hypersensitivity  CpG  cytosine-phosphate-guanosine  CPT  calcipotriol  CTL  cytotoxic T lymphocyte  DC  dendritic cell  DDC  dermal dendritic cell  DLN  draining lymph nodes  ELISA  enzyme-linked immunosorbent assay  FBS  fetal bovine serum  FACS  fluorescence activated cell sorting  Foxp3  forkhead box protein 3  IDO  indoleamine 2,3-deoxygenase  IFNγ  interferon gamma  IL-2  interleukin-2  IL-4  interleukin-4  IL-10  interleukin-10  IL-12  interleukin-12  LC  Langerhans cell  mVDR  membrane-bound vitamin D receptor  ODN  oligodeoxynucleotide  OVA  ovalbumin protein  PAMP  pathogen associated molecular pattern  PBS  phosphate buffer solution  SCI  subcutaneous immunization vii  SIT  allergen-specific immunotherapy  TCI  transcutaneous immunization  TGB-β  transforming growth factor beta  TLR  toll-like receptor  TNF-α  tumour necrosis factor alpha  UV  ultraviolet radiation  VDR  vitamin D receptor  VDRE  vitamin D response element  viii  Acknowledgments  I would like to sincerely thank all of the members of the Dutz lab and my colleagues in CFRI for their input and encouragement throughout the duration of my degree. Particular thanks goes to Dr. Mehran Ghoreishi, with whom I worked closely on all aspects of this project. I am indebted to Dr. Jan Dutz for taking me into his lab and always providing expert guidance, insightful advice, and an enthusiastic opinion. The opportunities I have had in the past two years will have a lasting impact on the rest of my career and I will forever be appreciative. Lastly, the support I have received from my entire family has enabled me to achieve this goal and my gratitude to them extends beyond words. To all of you I say thanks.  ix  1. Introduction  1.1 Structure and immunology of the skin The skin is the largest organ in the human body, and a vital part of the mammalian immune system. It is composed of three layers: the epidermis, the dermis, and the hypodermis. These layers work in concert to protect the organism from pathogens, toxins, and other environmental insults (Young and Heath, 2000).  The epidermis is the outermost layer of the skin and serves as both a physical barrier between a host and its environment as well as the initiation site for many immune responses. It is made up of largely of layers of keratinocytes, interspersed with Langerhans cells, melanocytes, Merkel cells, and lymphocytes. The epidermis can further be divided into five layers, from top to bottom the stratum corneum, the stratum lucidium, the stratum granulosum, the stratum spinosum, and the stratum basale. The stratum corneum lies at the interface between host and environment and is composed of enucleated keratinocytes termed corneocytes, which have accumulated large amounts of the fibrous insoluble protein keratin. These cells are bound together by a cement of lipids, cholesterol and ceramides, forming a semi-impermeable physical barrier to the outside world (Young and Heath, 2000).  While the keratinocytes in the stratum basale are mainly progenitors responsible for replenishing cells lost to desquamation, the keratinocytes in the middle layers of the epidermis play an active role in immune function, and are considered by some to be the  1  primary overseers of the innate immune response in the skin. They express functional toll-like receptors (TLRs) including TLR9 (Lebre et al., 2007), and are capable of antimicrobial peptide secretion as well as cytokine production upon sensation of danger signals (Steinhoff et al., 2001). Interspersed among the keratinocytes lie a subset of dendritic cells (DCs) known as Langerhans cells (LCs). These were once thought to be one of the most potent antigen presenting cells (APCs) in the body due to their ability to prime immune responses in vitro (Dai et al., 1993). Recent literature, however, demonstrates an evolving view on the physiological role of LCs, and suggests they may play an equally important duty in maintaining tolerance to harmless antigens during the steady-state. This is accomplished by the continuous migration of immature or “semimature” tolerogenic LCs to the draining lymph nodes (DLN), where it is thought that they induce tolerance by presenting antigen without secondary signals such as costimulatory molecules or pro-inflammatory cytokines (Romani et al., 2006).  The dermis is the most heterogeneous layer of the skin. It can be further divided into two sections, the papillary region and the reticular region, with the latter being where the vascular and lymphatic vessels, hair follicles, nerve endings, and exocrine glands are located (Young and Heath, 2000). The dermis is where mast cells reside, as well as another subset of DCs known as dermal dendritic cells (DDCs). It was the discovery of these DCs and their importance in mounting an immune response that led to the reevaluation of the physiological role of Langerhans cells. Based on evidence in mouse models that DDCs and not LCs are involved in generating protective responses to herpes simplex virus-2 (Allan et al., 2003) and leishmania (Ritter et al., 2004), as well as the  2  apparently dispensable role of LCs in the generation of contact hypersensitivity responses (Bursch et al., 2007; Fukunaga et al., 2008; Kaplan et al., 2005), it is now believed that under conditions of inflammation the DDCs may be more important for the initial immune response in the cutaneous draining lymph nodes.  The hypodermis is the innermost layer of the skin, also known as the subcutaneous tissue. It is attached to the dermis via collagen and elastin fibers, and is composed primarily of fibroblasts and adipocytes, with a population of resident macrophages. The role of the hypodermis is mainly mechanical as well as the storage of fat, and its role in the immune response is limited (Young and Heath, 2000).  In terms of skin biology, murine and human skin share similar features and mouse models are a popular approach for studying skin immunology. There are, however, several differences between mice and human skin that must be considered when attempting to translate murine data into human applications. Structurally, mouse skin contains fewer exocrine glands but far more hair follicles than human skin, and melanocytes tend to be restricted to the hair follicles in mice instead of distributed evenly throughout the skin. Mouse skin is also much thinner, for example the mouse stratum corneum averages two to three cells in thickness as opposed to more than ten cell layers in humans, but contains more lipids per dry weight of tissue (Chan, 2004; O'Hagan, 2000). Immunologically, the major difference between the two species is the identity and location of the predominant lymphocyte population. In mice the major skin lymphocytes are of a particular subset known as dendritic epidermal T cells (DETC), which are found  3  in the epidermis and express the γδ TCR chains (Steiner et al., 1988). The function of these T cells is not entirely clear, though they appear to play a role in tumour surveillance (Girardi et al., 2001), wound healing (Jameson et al., 2002), defense against pathogens (Molne et al., 2003), and immunoregulation (Girardi et al., 2002). In humans lymphocytes express the more traditional αβ chains and tend to be located in the dermis (Mestas and Hughes, 2004). Variations in the keratinocytes also exist, for instance human keratinocytes appear more prone to apoptosis (Chaturvedi et al., 2004). Despite the differences between human and murine skin, mice remain the gold-standard model for the early stages of research into new techniques in skin immunotherapy.  1.2 Using the skin immune system to manipulate immune responses Given the multiplicity of immune reactive cells within the skin and the ease of access, the skin is an ideal target for the manipulation of systemic immune responses. Using the immunology of the skin for vaccination is not a novel concept, in fact its immunostimulatory potential was recognized hundreds of years ago when the first historical human vaccine was administered by inoculating small skin wounds with powdered smallpox scabs, a process known as variolation (Belongia and Naleway, 2003). While many of our current vaccines rely on intramuscular injection of antigen, the idea of skin-based vaccines is reemerging. Experimentally, many varieties of skin-based vaccines exist including various combinations of topical, intradermal, or subcutaneous administration of protein, DNA, and even mRNA (Azad and Rojanasakul, 2006; Weide et al., 2008). Systems such as microneedles and biolistic gene guns have been developed in order to bypass the largely impermeable stratum corneum and facilitate delivery of  4  protein antigen or DNA to the target sites (Alarcon et al., 2007; Mumper and Cui, 2003). Skin vaccines already approved for use in humans include an intradermal formulation of the rabies vaccine (Khawplod et al., 2006), as well as the bacille Calmette-Guérin (BCG) vaccine for tuberculosis which can be given both intradermally or percutaneously (Davids et al., 2006). The skin has also been used experimentally in humans as a site of immunization for both hepatitis A (Pancharoen et al., 2005) and B (Ghebrehewet et al., 2008), as well influenza vaccines (Vogt et al., 2008).  The principle of transcutaneous (TCI) and subcutaneous (SCI) immunization with protein antigen is similar to that of traditional intramuscular vaccines. These procedures are intended to deliver antigen to APCs, and are often combined with vaccine adjuvants designed to generate a local immune response. The production of inflammatory mediators then induces the APCs to mature, upregulate co-stimulatory signal expression, and migrate to the draining lymph nodes where they can present the processed antigen and initialize an adaptive immune response. Adjuvants used experimentally in skin vaccines include cholera toxin (Glenn et al., 1998), heat-labile enterotoxin from E. coli (GuerenaBurgueno et al., 2002; Yu et al., 2002), and the synthetic TLR9 agonist unmethylated CpG DNA (Gupta and Cooper, 2008). Another novel approach to the use of the skin immune system is in the indirect modulation of immune responses. This can occur via the topical application of immunoreactive substances onto the skin resulting in local changes that may alter immune responses to antigens administered subcutaneously or intramuscularly. For example the use of the TLR9 agonist CpG, a potent T-helper type 1 (Th1) inducing adjuvant, has been demonstrated to induce a strong cytotoxic T cell  5  (CTL) response when combined with SCI, particularly when the CpG was applied topically over the site of subcutaneous antigen injection. Administering of this particular adjuvant via this route also eliminates undesirable side effects such as splenomegaly and systemic cytokine induction (Najar and Dutz, 2007).  Advantages to skin immunization are broad and include both technical as well as practical benefits. In terms of efficacy, skin-based vaccines demand a greatly reduced volume of protein compared to traditional parenteral vaccines (Kenney et al., 2004), and demonstrate an increased ability to induce CD8+ T cell responses, a property formally available only with live attenuated vaccines for diseases such as measles and rubella (Zinkernagel, 2003). They also offer a viable alternative to subjects who may not respond to standard intramuscular vaccination (Playford et al., 2002). Practical benefits include the potential for increased patient compliance, the elimination of needles and the difficulties associated with sharps disposal, and the feasibility of these vaccines for use in the developing world. Additionally, the skin offers the opportunity to use new adjuvants such as CpG that are not suitable for systemic administration but may provide superior responses than traditional vaccine adjuvants.  1.3 Toll-like receptors The toll-like receptors (TLRs) are a class of membrane-bound receptors capable of initiating innate immune responses. As a group, they form one of the earliest warning systems of pathogen invasion and are integral parts of the host defense system. TLRs function by recognizing a diverse array of components that are conserved across broad  6  ranges of bacteria, viruses or fungi, known as pathogen-associated molecular patterns (PAMPs). There are at least 11 known TLRs in humans and 13 in mice, each recognizing one or more specific PAMPs including double-stranded RNA (a TLR3 ligand), lipopolysaccharide (a TLR4 ligand), and flagellin (a TLR5 ligand) (Uematsu and Akira, 2008). It has been postulated that TLR recognition of endogenous ligands such as heat shock proteins, hyaluronate, or self-DNA may be a contributor to autoimmune disease in humans (Ehlers and Ravetch, 2007).  Structurally, TLRs are composed of a leucine-rich repeat (LRR) region on their extracellular side, and share the intracellular TLR/IL-1 receptor (TIR) domain with the IL-1 receptor. Upon binding with their ligand the TLRs will form hetero or homodimers, recruiting the signaling molecule MyD88 to their intracellular region. This is the beginning of one of several signaling cascades that will ultimately result in production of pro-inflammatory cytokines, chemokines, and the differentiation and maturation of DCs. The only exceptions to this pathway are TLR3, which signals independently of MyD88, and TLR4, which can signal through MyD88 dependant or independent pathways (Appendix A.1) (Uematsu and Akira, 2006).  Unlike many of the TLRs which are located on the cell surface, TLR9 is situated in the endosome and in humans is expressed primarily by B lymphocytes and plasmacytoid DCs, though keratinocytes appear to express lower levels of the receptor as well. It is represented similarly in mice, however lower concentrations can also be found in murine monocytes and myeloid DCs (Krieg, 2006). Upon ligation with unmethylated CpG DNA,  7  TLR9 triggers a signaling pathway that can result in activation of NFκB and production of pro-inflammatory cytokines, or it can promote activation of the IRF family of transcription factors thus promoting IFNα production. Which pathway is chosen appears to depend on the ability of the individual CpG oligonucleotides to form higher order structures, thus affecting their endosomal location (Guiducci, 2006). The success of CpG as an experimental vaccine adjuvant has been largely due to its ability to promote a robust CTL response in immunized animals both alone or in concert with other adjuvants, an effect that is accompanied by long-lasting immune memory (Krieg, 2006).  1.4 Regulatory T cells The existence of a population of “suppressor cells” whose function is to suppress undesirable immune responses was originally proposed in the 1970s, but after a decade of searching they remained elusive and uncharacterized leading to widespread denial of their existence and condemnation of further research. It was not until 1995 when Sakaguchi et al. demonstrated that tolerance was transferable by the purification and injection of CD4+CD25+ T cells that the re-termed regulatory T cells (Tregs) experienced a resurgence in popularity (Sakaguchi et al., 1995). Since then, a variety of cells possessing regulatory activity have been described including CD4+CD25+ natural Tregs, IL-10 producing Tr1 cells, TGF-β producing Th3 cells, and CD8+ T suppressor cells (Beissert et al., 2006).  The natural CD4+CD25+ regulatory T cell is characterized by the expression of the transcription factor forkhead box protein 3 (Foxp3) (Hori, 2003). These cells develop in  8  the thymus and are important mediators of peripheral tolerance. Mutations in Foxp3 lead to fulminant, multi-systemic autoimmunity termed the scurfy phenotype in mice (Zahorsky-Reeves and Wilkinson, 2001) or the immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in humans, illustrating the importance of these cells in maintaining immune homeostasis (Bennett et al., 2001). It is also believed that naïve CD4+ T cells may be induced to transforming into Foxp3 expressing CD4+CD25+ T cells in the periphery given the appropriate stimuli. This has been demonstrated in vitro by the direct stimulation or co-culture of naive CD4+ cells with APCs in the presence of TGF-β (Chen et al., 2003; Luo et al., 2007).  Since their renaissance, Tregs have been a popular research target by immunologists around the world. Due to their unique role in maintaining immune homeostasis, the ability to manipulate these cells with precision would have broad implications for clinical medicine, particularly in fields such as oncology, infectious disease, transplantation, and autoimmunity. Many means of generating Tregs have been demonstrated experimentally such as the oral administration of low doses of peptide (Min et al., 2006), the pharmacologic expansion of Tregs in vitro (Battaglia et al., 2005), or the transfection of naïve CD4+ T cells with Foxp3 (Allan et al., 2008; Chai et al., 2005). While some success has been had with each of these models, as of yet no practical, reliable, and highly specific means for the therapeutic induction of Tregs has been described. A potential mechanism for inducing antigen-specific Tregs is suggested by the tolerogenic effects of ultraviolet (UV) radiation. For example, topical administration of antigen to previously UV irradiated skin results in the generation of a population of antigen-specific  9  CD4+CD25+ Tregs capable of preventing induction of subsequent immune responses (Ghoreishi and Dutz, 2006).  1.5 Ultraviolet radiation induced immunosuppression Long before the existence of Tregs had been widely recognized, it was known that UV light could induce lasting immunosuppression in mice. This was originally ascertained by Margaret Kripke et al., who found that UV induced skin tumours that would prove lethal to irradiated mice could easily be rejected when transplanted onto non-irradiated animals (Kripke, 1974; Kripke, 1977). Moreover, treatment of the recipients with immunosuppressive drugs or low-dose UV radiation prior to transplantation abrogated their ability to eliminate the tumours, indicating that some form of natural immunosuppression was involved (DeFabo and Kripke, 1979; Fisher and Kripke, 1977). Decades of investigation into this phenomenon have still not fully illuminated the underlying mechanisms, but it is clear that at least part of the effect is due to an ability of UV light to promote the generation of antigen-specific Tregs. Co-transfer of T cells along with the skin tumours resulted in an inability of host mice to recognize and defend against the tumour, early evidence for what were then termed “suppressor T cells” (Spellman and Daynes, 1977).  The difficulty in teasing out the mechanisms of UV immunosuppression is attributable to the fact that several distinct forms of suppression can be induced by UV light, often occurring simultaneously. Depending on the dosage of UV radiation given, suppression can occur locally or systemically and can be mediated by regulatory T cells, cytokines, or  10  simply the absence of APCs capable of presenting antigen (Norval, 2006). Additionally, the physiological consequences of UV irradiation of the skin are complex. Effects linked to immunosuppression include triggering the upregulation of RANK ligand (Loser et al., 2006) and release of cytokines such as TNF-α (Corsini et al., 1995; Kock et al., 1990), αMSH (Schiller et al., 2004) and PAF (Barber et al., 1998) by the keratinocytes, histamine release from dermal mast cells (Gilchrest et al., 1981; Hart et al., 1998), generation of cyclobutane pyrimidine dimers and 6,4-photoproducts in the DNA of skin cells (Kripke et al., 1992; Nishigori et al., 1996), and conversion of trans-urocanic acid into its cis conformation (Noonan et al., 1988). The immediate downstream results of these effects are the migration of LCs out of the skin (Kolgen et al., 2002; Toews et al., 1980) as well as the initiation of a cytokine cascade beginning with the prostaglandin PGE2 and resulting in the production of anti-inflammatory cytokines such as IL-4 and IL-10 (Shreedhar et al., 1998). These comprise only a subset of the actions of UV light in the skin, but each has been implicated in some form of UV induced immunosuppression.  1.6 Vitamin D One of the actions of UV irradiation is the generation of biologically active vitamin D. Vitamin D is a lipophilic secosteroid hormone responsible for the maintenance of calcium/phosphorus homeostasis and bone metabolism in the body. Its primary function is to promote calcium and phosphorus uptake in the gut as well as to increase the reabsorption of calcium in the kidneys. It also stimulates osteoclastogenesis, promoting the release of calcium from bone into the blood (Lips, 2006).  11  In recent years it has become apparent that in addition to its role in regulating calcium levels, vitamin D is also a potent immunomodulator. Its actions on the immune system include the ability to prevent DC maturation (Griffin et al., 2001; Penna and Adorini, 2000; Singh et al., 1999), inhibit antigen-specific T cell proliferation (Bhalla et al., 1984), and suppress Th1 immune responses (Mahon et al., 2003; Mattner et al., 2000). Vitamin D has also been implicated in immune tolerance and the generation of Tregs. The development of synthetic vitamin D analogs such as calcipotriol (MC 903) has resulted in compounds that share the local effects of vitamin D but are metabolized rapidly, minimizing any systemic calcemic effects (Kissmeyer and Binderup, 1991). Calcipotriol has been approved for clinical use as an anti-psoriatic due to its ability to inhibit keratinocyte proliferation (Reichrath et al., 1997; Takahashi et al., 2003) and prevent them from producing pro-inflammatory cytokines (Kong et al., 2006; Zhang et al., 1994).  The effects of vitamin D and its analogs are mediated by a nuclear receptor named the vitamin D receptor (VDR), which is a member of the receptor superfamily of steroid/thyroid hormone/retinoic nuclear receptors. The VDR is expressed widely in a variety of mammalian tissues including immune cells such as T lymphocytes, DCs, and monocytes/macrophages (Brennan et al., 1987; Veldman, 2000). Upon ligation with activated vitamin D the VDR forms a heterodimer with the retinoic acid receptor (RXR), allowing recruitment of other co-activator proteins. The complex can then serve directly as a transcription factor, binding to vitamin D response elements (VDRE) in the DNA (Carlberg and Seuter, 2007). A second, non-genomic receptor has been proposed termed the membrane-bound VDR (mVDR), although its existence remains a controversial  12  subject. This putative membrane bound receptor is thought to be responsible for a fastacting response to vitamin D, and has been tentatively identified as the membraneassociated rapid response steroid binding (MARRS) protein (Nemere et al., 1998). Alternatively, non-genomic actions from the classic VDR may also be possible, as it has been noted that some of the rapid actions of vitamin D are lost in VDR knockout osteoblasts (Zanello and Norman, 2004) and that the VDR can associate with lipid raftrich areas of the plasma membrane (Huhtakangas et al., 2004).  In humans vitamin D can be obtained from the diet, or can be synthesized de novo in the skin. The substrate for the biosynthetic pathway is the cholesterol derivative 7dehydrocholesterol (also known as provitamin D), which is converted by UV radiation in the skin into previtamin D3. Previtamin D3 then rapidly and spontaneously isomerizes into vitamin D3 (cholecalciferol) in a heat-dependant process. Finally, vitamin D3 undergoes hydroxylation in the liver by the enzyme 25-hydroxylase into 25,(OH)2D3 (also known as calcidiol), followed by a second hydroxylation in the kidney by 1-αhydroxylase, yielding 1,25-dihydroxyvitamin D3 (1,25(OH)2D3 or calcitriol) the active metabolite of the vitamin (Appendix A.2) (Garrettand Grisham, 2002; Zhu and Okamura, 1995). The critical role of UV radiation in the vitamin D synthesis pathway has raised the possibility that vitamin D may be a mediator of the cutaneous immunosuppressive effects of UV light (Reichrath and Rappl, 2003). This hypothesis is further encouraged by the observation that 1-α-hydroxylase can be produced by macrophages and DCs in the skin (Fritsche et al., 2003; Overbergh et al., 2000), while keratinocytes are capable of  13  producing both hydroxylase enzymes and have all the necessary machinery to generate bioactive 1,25-dihydroxyvitamin D3 (Lehmann et al., 1999; Zehnder et al., 2001).  1.7 Topical immunomodulation Topical immunomodulators are compounds designed to either augment immune responses (compounds such as imiquimod or 5-fluorouracil) or inhibit undesired responses (compounds such as corticosteroids, calcipotriol, or azelaic acid) in the skin. They are commonly used to treat skin cancers, psoriasis, allergic reactions, and the cutaneous manifestations of a host of other diseases. Physicians have been prescribing immunomodulators for decades, however a comprehensive understanding of their mechanisms of action remains uncertain. In particular, the effects of topical immunomodulators on antigen-specific immune responses in the skin have never been documented.  1.8 Thesis objectives The skin is an active immune organ capable of triggering potent immune responses to invading pathogens. The effectiveness of the skin in initializing responses as well as its ease of access have made it an attractive target for manipulating the immune system, and the topical administration of adjuvants is capable of inducing immune responses to not only topical but also subcutaneously injected antigens. UV light has the capability to significantly alter the response to immunization in the skin and induce tolerance, an effect that may be mediated at least in part by the production of vitamin D.  14  Our laboratory has previously described a means of inducing antigen-specific CD4+CD25+ Tregs in C57Bl/6 mice using calcipotriol and TCI. These experiments were all performed using a protocol involving prior topical calcipotriol conditioning of the skin for three days followed by topical co-administration of the prototypic protein antigen ovalbumin (OVA) and CpG oligodeoxynucleotide to tape-stripped skin. This results in a population of Foxp3+ Tregs capable of mediating transferable, antigen-specific tolerance (unpublished data). The mechanism of this process remains unknown.  The goal of my thesis project was to explore the potential and pitfalls of calcipotriol modulated vaccination as a prospective immunomodulatory treatment. Specifically, I investigated the functionality of induced tolerance in suppressing undesirable immune responses, as well as examined potential side effects. I also evaluated the effects of topical calcipotriol on the immune response to subcutaneous immunization. This included determining whether administering the antigen subcutaneously (where it will be processed by dermal or subcutaneous dendritic cells) instead of topically (where LC acquire antigen) affected the generation of Tregs, as well as what consequences topical calcipotriol had on the humoral immune response to SCI. Lastly, I began to elucidate the mechanism through which calcipotriol is acting in order to modulate immune responses to SCI. I compared the effects of calcipotriol to another topical immunosuppressant commonly used by practitioners, the corticosteroid betamethasone-17-valerate (BMV).  Our laboratory has demonstrated that the topical application of an immunomodulator is capable of exerting powerful effects on protein antigen administered subcutaneously  15  (Najar and Dutz, 2007). As a result, we hypothesized that the topical administration of calcipotriol would be capable of significantly altering the immune response to subcutaneous antigen. We expected that these effects would encompass both the cellular and humoral immune responses to SCI, and that topical calcipotriol would also promote the induction of antigen-specific tolerance.  16  2. Materials and Methods  2.1 Animals. Female C57Bl/6 mice were obtained from Charles River Laboratories (Wilmington MA) and housed in the animal facility at the Child and Family Research Institute (CFRI), Vancouver BC. OT-I transgenic mice, which express a T cell receptor specific to ovalbumin residues 257-264 in the context of H2-kb (Hogquist et al., 1994), and OT-II transgenic mice, which express a T cell receptor specific to ovalbumin residues 323-339 in the context of I-Ab (Barnden et al., 1998), were purchased from Jackson Laboratories (Bar Harbour ME) and subsequently bred in house. Experimental animals were between the ages of six to eight weeks, and all experiments were carried out according to institutional animal care protocol requirements with approval from the University of British Columbia Research Ethics Board (Appendix A.3).  2.2 Topical ointment application. Calcipotriol (MC 903, Dovonex, Leo Pharma, Ballerup, Denmark) ointment (0.05%), betamethasone-17-valerate (0.1%), or a plasticized base was applied 50 mg/mouse to shaved dorsal skin of mice for three consecutive days.  2.3 Peptides and proteins. Ovalbumin protein (OVA-V) or bovine serum albumin (BSA, Sigma-Aldrich, Saint Louis MO) were used as immunogens. For tracking DCs OVA conjugated to FITC was used (Invitrogen, Carlsbad CA).  17  2.4 Cell isolations and adoptive transfer. OT-I T cells were isolated from pooled splenocyte and lymphocyte suspensions. The CD8+ population was purified to >90% by positive selection using CD8 microbeads (Miltenyi Biotech, Auburn CA). Cells were then labeled with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE, Molecular Probes, Eugene OR) and 8 x 106 cells were injected into the lateral tail vein of the experimental animals 24-48 hours prior to immunization. Mice were sacrificed three days after immunization and skin draining lymph nodes were harvested for analysis.  For the adoptive transfer of T regulatory cells mice were euthanized five days following tolerization, and suspensions of splenocytes and lymphocytes were produced. CD4+ cells were isolated to a purity >80% by negative selection using mouse CD4+ T cell enrichment kit (Stem Cell Technologies, Vancouver BC). Cells were then labeled with CD4-FITC and CD25-APC (BD Biosciences, San Jose CA) conjugated antibodies and Tregs were purified by fluorescence-activated cell sorting (FACS) (BD FACSAria with BD FACSDiva software, BD Biosciences, San Jose CA) to >97% purity. 7.5 x 105 Tregs were then injected into the lateral tail vein of naïve animals two days prior to immunization.  2.5 Subcutaneous immunization. Protein immunization was performed as previously described twenty-four hours after the final calcipotriol, BMV or vehicle treatment (Najar and Dutz, 2007). Briefly, 100ug OVA-V suspended in 100ul PBS was injected subcutaneously under treated skin of C57Bl/6 mice and 500ug CpG (ODN 1826 5’TCCATGACGTTCCTGACGTT-3’, prepared by the Oligonucleotide Synthesis Facility  18  of the University of British Columbia) suspended in 50ul PBS was applied topically over the treated area after tape-stripping. As a control 500ug of biologically inactive CpG (ODN 1982 5’-TCCAGGACTTCTCTCAGGTT-3’) in 50ul PBS was used. Occlusion of the area with waterproof tape occurred for 48-72 hours after immunization. When indicated animals received a second series of calcipotriol treatments and immunization seven days after the first.  2.6 Flow cytometry. Lymphocyte suspensions were made from draining lymph nodes and were stained using the appropriate fluorescently conjugated antibodies: CD3 (145-2C11), CD4 (GK1.5, RM4-5), CD8 (53-6.7), CD11c (HL3), CD19 (MB19-1), CD69 (H1.2F3), B220 (RA3-6B2), IFNγ (XMG1.2) (BD Biosciences, San Jose CA), CD25 (PC61.5.3), CD80 (RMMP-1), CD86 (RMMP-2), DEC205 (NLDC-145) (Cedarlane, Burlington ON), CD40 (1C10), Foxp3 (F5K-16s) (eBioscience, San Diego CA). Staining was performed in PBS supplemented with 2% fetal calf serum (Invitrogen, Carlsbad CA). Antigenspecific CTL were identified using a PE conjugated Kb-OVA tetramer conjugated to PE made in-house. For intracellular cytokine staining single-cell suspensions from draining lymph nodes were stimulated with PMA and ionomycin and treated with GolgiStop (all from BD Biosciences, San Jose CA) for 4 hours at 36°C. Cells were then washed and stained for surface molecules and fixed using lysis buffer (BD Biosciences, San Jose CA). Following permeabilization using Perm/Wash buffer (BD Biosciences, San Jose CA), cells were stained intracellularly.  19  DC isolation was performed by isolating skin draining lymph nodes twenty-four hours post-immunization and incubating them in collagenase solution (1mg/ml RPMI) for one hour at 36°C. Mononuclear cells were enriched using Ficoll-Hypaque density gradient centrifugation (GE Healthcare Bio-Sciences, Uppsala, Sweden). Data was collected on a BD FACSCalibur (BD Biosciences, San Jose CA) and analysed using FloJo flow cytometry analysis software (Tree Star, Ashland OR).  2.7 Protein contact hypersensitivity. Mice were immunized twice with OVA via TCI over shaved dorsal skin on two consecutive days. Experimental animals were then treated for three days with calcipotriol on the shaved abdomen and immunized on day four with OVA by TCI. Two days later mice were challenged with 50ug OVA and 50ug CpG dissolved in 12.5ul DMSO applied to each side of the right ear. The left ear was challenged with BSA and CpG. Ear thickness was measured with an engineer’s micrometer (Mitutuyo) at twenty-four hour intervals following the challenge and changes in the left ear were subtracted from those on the right.  2.8 Sample collection. Serum. Blood samples were taken from the submandibular vein at fourteen and twenty-one days post immunization. Serum was collected and stored at -20°C until tested by ELISA. Bronchoalveolar lavage (BAL). A catheter was inserted into the trachea of the mouse and lungs were flushed once with 1mL of PBS. Samples were then centrifuged and supernatants were collected and stored at -20°C.  20  Vaginal lavage (VL). Samples were obtained by repeated flushing of the vagina with 50ul PBS. Samples were then centrifuged and supernatants were collected and stored at 20°C. Faecal samples. Three fresh faecal pellets were obtained from each mouse and vortexed vigorously in 400ul of PBS. Samples were then left at room temperature for thirty minutes prior to centrifugation. Supernatants were collected and stored at -20°C until analysis.  2.9 Quantification of antigen-specific antibody responses. Serum, BAL, VL and faecal samples were measured for OVA-specific IgG1, IgG2a and IgA titers by indirect ELISA. Briefly, OVA peptide was diluted in carbonate/bicarbonate buffer (1M NaHCO3, 0.33M Na2CO3) to a concentration of 30ug/ml and was dispensed into 96-well plates, which were incubated overnight at 4°C. Plates were then washed three times with ELISA buffer (PBS + 1% BSA + 0.001% Tween 20) and blocked with PBS containing 2% BSA for 2 hours at 37°C. After a second series of washes, samples were diluted in ELISA buffer and added to the plates for a two hour incubation at 37°C. Following incubation, plates were washed and an optimal dilution of detection antibody (horseradish peroxidaseconjugated goat anti-mouse IgA, rabbit anti-mouse IgG1 [Zymed Laboratories, San Francisco CA], or goat anti-mouse IgG2a [AbD Serotec, Raleigh NC] OVA-specific antibody) was added. After a final two hour incubation and series of washes the ELISAs were developed using TMB substrate and 2N H2SO4 as a stop solution. Plates were read at 450nm.  21  2.10 In vitro Foxp3 induction assay. OT-II CD4+ T cells were extracted from a single cell suspension of pooled lymphoid organs using a mouse CD4+ T cell enrichment kit (Stem Cell Technologies, Vancouver BC) and stained with the cell tracker Oregon Green 488 (Invitrogen, Carlsbad CA). Skin draining lymph nodes were taken twenty-four hours post-immunization from mice treated with or without calcipotriol and immunized via SCI. DCs were enriched to >60% using a CD11c positive selection kit (Miltenyi Biotech, Auburn CA). 105 CD4+ cells suspended in 100ul complete media were added per well along with DCs at 2:1, 5:1 and 10:1 ratios for a total volume of 150ul. Cells were incubated at 37°C for 96 hours prior to analysis by flow cytometry. Supernatant was taken at 96 hours and stored at -20°C. Data shown corresponds with a 2:1 CD4+ T cell to DC ratio.  2.11 Cytokine measurement. IL-10 and TGF-β were measured in supernatant using a capture ELISA (eBioscience, San Diego CA) according to the manufacturers instructions.  2.12 Statistical analysis. All data is presented as mean values ± SEM. Groups were compared using two-tailed Student’s t test and graphs are displayed using Prism 3 (GraphPad Software, San Diego CA).  22  3. Results  3.1 Calcipotriol modulated immunization has potential as a human Treg induction therapy. Current experimental Treg induction protocols are plagued with obstacles such as efficacy, specificity, and technical difficulty. Despite these hurdles however, the promise of a means of manipulating tolerance remains a highly sought after goal. We have shown previously (manuscript under revision) that calcipotriol treatment prior to TCI with OVA plus CpG results in a population of functional, antigen-specific CD4+CD25+ Tregs. From a therapeutic standpoint, advantages to combining calcipotriol with a CpG-based skin vaccine include the fact that calcipotriol is already approved for human use with an excellent safety profile (Scott et al., 2001), while CpG is currently in human trials as an immune response modifier (VaxImmune®, ProMune®, Coley Pharmaceutical Group Inc.). Furthermore, the importance of a TLR9 ligand in our paradigm has yet to be evaluated and it is possible that other adjuvants that have been used experimentally in vaccines such as the human approved TLR7 agonist imiquimod would prove effective as well (Zhang and Matlashewski, 2008). Nonetheless, we have begun early work to assess the strengths and limitations of topical calcipotriol induced antigen-specific Tregs in terms of their therapeutic potential.  3.1.1 Treatment with topical calcipotriol has no effect on serum calcium levels Oral vitamin D may be used in the treatment of experimental models of autoimmunity such as type I diabetes (Mathieu et al., 1994), experimental autoimmune  23  encephalomyelitis (EAE) (Lemire and Archer, 1991), and rheumatoid arthritis (Cantorna et al., 1998). The effectiveness of oral vitamin D in these models however has been offset by the observed side effect of hypercalcemia and its accompanying complications (Lemire and Archer, 1991; Mathieu et al., 1994). The use of topical vitamin D in man has also been reported to have negative calcitropic effects (Li et al., 2006), however reports of hypercalcemia in patients following calcipotriol treatment are very rare and most occur in patients exceeding the maximum recommended dose of 100g/week (Scott et al., 2001). To examine whether our tolerogenic TCI protocol may disrupt the calcium homeostasis of our mice we measured the concentration of calcium in the serum after three days of treatment with 50mg of topical calcipotriol and found no differences between treated and control groups (Fig. 1A). Limiting negative calcemic effects through topical application is clearly an advantage for our paradigm that is furthered by the short half-life of calcipotriol compared to vitamin D.  3.1.2 The effects of calcipotriol on CD8+ T cells responses are largely local A major advantage of Treg induction over pharmacologic immunosuppression is specificity, thus effective protocols should ideally avoid generalized immune effects. While mild systemic complications may be acceptable, the inadvertent induction of pathogen-specific tolerance would be disastrous. In humans there have never been reports of hematologic or biochemical changes in response to calcipotriol treatment, with most side effects reported as localized lesional or perilesional irritation, skin rashes and/or irritation, and worsening of psoriasis (Scott et al., 2001).  24  C 2  1  Untolerized Tolerized  8  3  Ear swelling (um)  Serum calcium (mmol/L)  A  6  ∗  4 2 0  0  Basal  0  Calcipotriol  24  48  72  96  Time (hrs)  B  ∗∗  ∗∗  Day 1-3  Dorsal/abdominal CPT  Day 3  OT-I AT  Day 4  Dorsal TCI  Day 7  Analyse DLN  % compared to +ve  100 75  CPT/TCI same site  50  CPT/TCI different site  25 0  CFSE  IFNγ  Figure 1. Therapeutic potential of calcipotriol modulated vaccination. (A) Serum calcium concentrations taken 24 hours following the third day of treatment with 50mg calcipotriol. Data is representative of 2 experiments. (B) Mice were treated dorsally or ventrally with calcipotriol (CPT) and all groups immunized dorsally. Proliferation and IFNγ production by OT-I CD8+ T cells was measured in DLN three days post-TCI. (C) Mice were sensitized to OVA by TCI twice, 2 days apart. One group was then tolerized by calcipotriol + TCI. Ear swelling upon OVA challenge was compared against a non-tolerized group. Data is representative of 3 experiments (n = 9-10). *p < 0.05, **p < 0.01.  25  In order to ensure antigen-specificity with our paradigm, it was important to assess the ability of calcipotriol treatment to induce a local tolerogenic environment only. We approached this question using an in vivo proliferation assay following the adoptive transfer of antigen-specific CD8+ T cells into mice (Lyons and Parish, 1994). Topical coadministration of CpG with the model antigen OVA results in the priming of CD8+ T cells to this antigen in the DLN (Najar and Dutz, 2007). Calcipotriol was applied topically for three days on shaved dorsal or abdominal skin. On day four all animals were immunized by TCI on the dorsal skin. After seventy-two hours DLN were harvested and proliferation was compared between the two groups. We found that abdominally applied calcipotriol had no effect on the proliferation of OT-I CD8+ T cells in response to dorsal immunization (Fig. 1B). In contrast, pre-application of calcipotriol to the site of subsequent immunization virtually abolished antigen-specific CD8+ T cell priming (as detected by proliferation). When proliferating cells were analysed for production of IFNγ calcipotriol treatment at the site of immunization was found to have strong inhibitory effects on CTL activation. It is worth noting that abdominal treatment with calcipotriol had a slight but statistically significant inhibitory effect on IFNγ production in proliferating cells as well, indicating minor systemic effects from our treatment (data not shown).  3.1.3 Calcipotriol induced Tregs are capable of suppressing a delayed-type hypersensitivity response A major asset for a Treg therapy would be the ability to suppress the elicitation of an established immune response. Without this effect application of the protocol would be  26  limited to prophylaxis, constrained by having to identify high-risk candidates prior to the manifestation of their disease. Previous work with UV induced Tregs has indicated that they are limited in their abilities to suppress established hapten-mediated inflammation and are only effective if an animal is tolerized before the initial sensitization has occurred (Schwarz et al., 2004). This appears to be due to their location in the body rather than suppressive abilities, as direct injection of Tregs into the tissue at the elicitation site allowed them to suppress contact hypersensitivity (CHS). This was explained by the observation that UV induced Tregs express CD62L and are sequestered in the secondary lymphoid organs, where they are able to prevent a primary immune response from developing but are isolated from the areas where elicitation of CHS responses occurs (Schwarz et al., 2004).  We used an ear-swelling model of delayed-type hypersensitivity to examine the ability of our Tregs to suppress CHS responses. Animals were sensitized against OVA by TCI, after which one group was tolerized using calcipotriol treatment and TCI. OVA plus CpG was then applied to the right ear and swelling was compared to the opposite ear, which was treated with a control protein (BSA) and CpG. We found that our therapy significantly reduced ear swelling in tolerized animals despite the treatment occurring post-sensitization (Fig. 1C). It remains to be determined whether this is occurring due to the action of antigen-specific Tregs, however these results are promising for the development of a therapeutic intervention for undesired immune reactions.  27  3.2 Calcipotriol modulates immune responses to subcutaneous immunization The immunomodulatory potential of vitamin D and its analogues is well described. It has been shown in vitro and in vivo to inhibit Th1 immune responses (Imazeki et al., 2006; Mattner et al., 2000), and under some conditions is capable of skewing responses towards a Th2 phenotype (Boonstra et al., 2001; Jirapongsananuruk et al., 2000). Incorporation of vitamin D directly into vaccines has been demonstrated to promote mucosal immunity in immunized mice (Daynes et al., 1996a; Ivanov et al., 2006) and pigs (Van Der Stede et al., 2004). Additionally, it has been implicated in tolerance induction in several models of autoimmunity and is known to promote Treg induction alone or when combined with other immunosuppressive drugs (Gregori et al., 2001; Gregori et al., 2002; Mathieu et al., 1994). While we have demonstrated the ability of calcipotriol to modulate immune responses to topically applied antigen during TCI, we asked whether a similar effect occurs when protein is injected subcutaneously. This is an important question because of the advantages of subcutaneous antigen delivery in circumventing the skin barrier and in potentially decreasing the required amount of protein antigen. Additionally, should topical calcipotriol treatment modulate responses to subcutaneous antigen, it may also provide some mechanistic insight into the cell types responsible for tolerance induction. It has been well described that treatment of skin with topical calcipotriol results in the depletion of epidermal residing LCs, potentially attenuating or abrogating the effects of topical vaccines (Dam et al., 1996). In order to control for these effects we compared calcipotriol to another compound known to induce apoptosis and decrease the number of LCs in the skin, the topical corticosteroid BMV (Ashworth et al., 1988; Hoetzenecker et al., 2004).  28  3.2.1 Topical calcipotriol inhibits subcutaneous immunization mediated activation of CD8+ T cells The ability of vitamin D to inhibit the production of Th1 cytokines is powerful. In culture, this can occur both via the suppression of maturation and inhibition of proinflammatory mediators from human or mouse DCs (Matsuzaki et al., 2006; Penna and Adorini, 2000). Vitamin D has also been shown to have direct Th2 polarizing effects on murine CD4+ T cells (Mahon et al., 2003). CD8+ T lymphocytes are also likely targets of vitamin, as they have the highest concentration of VDR amongst all lymphocytes, though its effects on these cells are not as well understood (Veldman, 2000). One of the earliest hallmarks distinguishing the ability of vitamin D to skew the Th1/Th2 balance is its ability to potently suppress IL-2 (Alroy et al., 1995) and IFNγ production (Cippitelli and Santoni, 1998) from lymphocytes. In addition, in vitro vitamin D has been shown to inhibit secretion of IL-12p40 (D'Ambrosio et al., 1998) from murine macrophages while upregulating production of IL-4 from CD4+ T cells (Boonstra et al., 2001). The exact nature of its ability to enhance Th2 effects remains murky however, since a conflicting report has found that vitamin D may suppress IL-4 secretion from naive CD4+ T cells, while having no effect whatsoever on production from activated CD62L+CD4 T cells (Staeva-Vieira and Freedman, 2002). Regardless, in all instances IFNγ is inhibited, thus encouraging a Th2 response.  Calcipotriol shares the anti-inflammatory attributes of vitamin D, and is used as a topical anti-psoriatic. The effects of calcipotriol in treating psoriasis are likely due primarily to its anti-proliferative effects on human keratinocytes (Reichrath et al., 1997; Takahashi et  29  al., 2003), with the drug’s ability to suppress secretion of pro-inflammatory cytokines such as IL-1, IL-6, IL-8, and IL-18 from both human and murine keratinocyte also playing a role (Kong et al., 2006; Zhang et al., 1994). Since our model of subcutaneous vaccination relies on the pro-inflammatory capabilities of a Th1 promoting adjuvant in the skin, we asked what effects calcipotriol treatment would have on SCI.  To evaluate the effects of calcipotriol on SCI, initially we began by phenotyping the skin DLN seventy-two hours post-immunization (using CpG as a topical adjuvant administered at the time of subcutaneous antigen delivery) with or without prior calcipotriol or BMV treatment (Fig. 2A). We used an inactive DNA oligodeoxynucleotide (ODN) as a control to assess the effect of CpG. When assessing the number of cells present in the DLN post-immunization, we found no appreciable difference in cellularity between calcipotriol treated and untreated immunized animals, whereas pretreatment with BMV resulted in a significant decrease in cell number in response to SCI. CpG resulted in a robust increase in lymphocyte numbers compared to ODN in both vitamin D treated and untreated groups, indicating the importance of the adjuvant in promoting lymphocyte proliferation and consistent with previous observations (Najar and Dutz, 2007). The small increase we did see in groups immunized with ODN is likely attributable to the mild inflammation caused by the tape-stripping process (Nishijima et al., 1997). Immunophenotyping of the B cell, CD4+ and CD8+ subsets of lymphocytes in the DLN, demonstrated no difference in proportions or absolute numbers between calcipotriol treated and control animals. Once more, lymphocyte expansion was CpG dependant (Fig. 2B).  30  A  B  *  15  DLN cell count (x10^6)  50 40 30 20 10  10  ns  ns  ns  ns  ns  5  0 Na ive  B cells  CD4 T cells  ive Na  N  OD T+  OD  1.0 0.5  ive  OD  Na  N  0.0 N  ive  CD25  1.5  T+  T+  Na  N OD  N OD CP  V+ BM  CP T  +  Cp G  Cp G  0  2.0  OD  5  2.5  CP  10  3.0  Cp G  CD8+ T cells  15  *  Cp G  CD8+ cells expressing CD25 (%)  *  CP  T+ CP  CP  3.5  20  Cp G  Cp G  ive  N OD  Na  N OD  T+  V+  Cp G  Cp G T+  CP  ***  *  25  N  0  CD25  Cp G  0  5  Cp G  2  V+  4  10  BM  6  V+  8  15  BM  CD4+ T cells  10  Cp G  CD8+ cells expressing CD69 (%)  CD8+ T cells  CD4+ cells expressing CD25 (%)  12  Cp G  CD69  20  14  BM  CD4+ T cells  CD4+ cells expressing CD69 (%)  * 16  T+  C  CD69  CD8 T cells  CP T+  OD N  OD N  Cp G  Cp G  BM V+  CP T+  Cp G  0  CP  DLN cell count (x10^6)  60  CpG CPT + CpG BMV + CpG ODN CPT + ODN Naive  ns  Figure 2. Effect of topical imunomodulation on the phenotype of lymphocytes from the draining lymph nodes following subcutaneous protein immunization. (A) Cell numbers in the draining lymph nodes 72 hours post-immunization with OVA + CpG or inert ODN. Where indicated animals were pretreated with 50ug of calcipotriol (CPT) ointment or betamethasone-17-valerate (BMV). Data is representative of 7 experiments. (B) The distribution of individual subsets of lymphocytes (n = 4-14). (C) Percentage of CD4+ and CD8+ T cells expressing the early activation markers CD69 and CD25 (grey line= naive T cells, solid black = CpG only, dashed line = CPT + CpG). Data is representative of 6 experiments. * p < 0.05.  31  Expression of CD69 (a contributor to T cell activation) and CD25 (the alpha chain of the IL-2 receptor) are markers of early activation in T cells (Cotner et al., 1983; Testi et al., 1989). Our next step was to analyse the expression of these markers by CD4+ and CD8+ T cells in the skin DLN after immunization as a more precise measure of lymphocyte activation status. We found that CD8+ T cells in calcipotriol treated animals showed a significant decrease in expression of the activation markers CD69 and CD25 three days post-immunization (Fig. 2C). When CpG was removed we saw decreased CD69 levels in all CD8+ T cells, however again there was a discrepancy in CD69 expression between calcipotriol treated and untreated animals. While immunization with or without CpG resulted in an increase in activation markers in CD4+ T cells, no differences were observable between calcipotriol treated and untreated groups. These observations were in contrast to the BMV treated animals who, despite having lower whole numbers of lymphocytes, showed no decrease in the percentage of CD4+ or CD8+ T cells expressing activation markers compared to the control group, and in fact expressed higher levels of CD69 in CD8+ T cells.  Topical application of the TLR9 agonist CpG at the time of SCI generates an antigenspecific CTL response to the subcutaneously administered antigen quantifiable by class I MHC-peptide complex tetramer staining. Since calcipotriol pre-treatment of the skin appeared to selectively inhibit the activation of CD8+ T cells following SCI, we asked whether it had the ability to prevent the induction of an antigen-specific CTL response in the peripheral blood of calcipotriol treated animals after immunization. Experimental animals were primed and boosted one week apart by SCI, with or without  32  calcipotriol/BMV treatment preceding each immunization. Three days after the second immunization peripheral blood was taken and stained with a Kb-OVA tetramer to look for the presence of OVA-specific CD8+ T cells. Both calcipotriol and BMV treated groups showed a significant decrease in the numbers of circulating antigen-specific CD8+ T cells compared to animals treated with a vehicle ointment, indicating an impaired Th1 response (Fig. 3A). The decreased number of circulating antigen-specific CD8+ T cells could be a result of decreased proliferation of these cells, decreased survival, or altered trafficking. To determine if calcipotriol affects the priming and proliferation of CD8+ T cells we next performed an in vivo proliferation assay. Animals were adoptively transferred with 8x106 CFSE-labeled transgenic OT-I CD8+ T cells and the experimental group was then treated for three days with calcipotriol. Seventy-two hours after the mice were immunized via SCI, the lymph nodes were harvested for analysis. We found that calcipotriol pretreatment significantly inhibited proliferation of the transgenic T cells three days post-immunization, confirming calcipotriol’s ability to suppress CD8+ T cell priming to subcutaneously administered antigen (Fig. 3B).  3.2.2 The humoral immune response to subcutaneous immunization is altered by pretreatment with topical calcipotriol It has been reported that vitamin D can be incorporated into subcutaneous vaccines to promote the generation of a mucosal immune response in mice, characterized by high levels of IgA and IgG1 and decreased IgG2a production (Enioutina et al., 1999). The effect mimics the result of vaccination over skin that had been irradiated by UVB light (Enioutina et al., 2002), and appears to be due to altered trafficking of antigen-presenting  33  A  *  Naive  Calcipotriol  0.09  3  2  Vehicle 1.73  0.60  Kb-OVA  OVA-specific CTLs (%)  4  1  CD8  Naive 1.05  CFSE  ive Na  Cp G + V  100  Vehicle 90.6  Calcipotriol 31.8  OT-I proliferation (%)  B  ***  BM  Ve h  icl  CP  e+  T+  Cp G  Cp G  0  75 50 25 0  Vehicle  Calcipotriol  Figure 3. Effect of calcipotriol on the CD8+ T cell response to subcutaneous immunization. (A) Mice were immunized twice seven days apart. Indicated groups received calcipotriol or BMV treatment on 3 consecutive days before each immunization. Peripheral blood was taken and stained for OVA- specific CTLs using a Kb-OVA tetramer 3 days after the second immunization. Data is representative of 4 experiments. (B) OVA-specific OT-I CD8+ cells were CFSE labeled and adoptively transferred into naive mice 48 hours prior to immunization with or without calcipotriol pretreatment. Proliferation of labeled cells was measured 72 hours later. * p < 0.05, *** p < 0.001.  34  DCs (Enioutina et al., 2007b). Interestingly it has been shown that immunization with TLR3/TLR4 ligands as adjuvants can achieve a similar result due to their ability to upregulate 1-α-hydroxylase production in DCs, an effect that is not shared with TLR9 agonists such as CpG (Enioutina et al., 2007a). Our combination of calcipotriol treatment and CpG as an adjuvant appeared to have no negative effects on CD4+ T cell activation so we decided to measure the ability of vaccine to promote production of antigen-specific IgA, IgG1 and IgG2a antibodies in the blood as well as various secretions from our mice.  Animals were immunized twice, seven days apart, with CpG or ODN and with or without three days of calcipotriol or BMV treatment. Three weeks after the second immunization serum, BAL, VL, and faecal samples were taken and analysed by ELISA for the presence of OVA-specific antibodies. We found that topical calcipotriol treatment prior to subcutaneous antigen injection resulted in an increase in antigen-specific IgG1 concentration in the serum (Fig. 4). Topical CpG administration induced the production of OVA-specific IgG2a, however this effect was completely and dramatically blocked by calcipotriol pretreatment. Although co-administration of antigen and vitamin D has been reported to affect antigen-specific IgA production, we did not detect any effects on antigen-specific IgA levels. The effects of calcipotriol were in contrast to BMV treatment, which resulted in attenuated levels of IgA and IgG1 while having no significant effects on IgG2a production. Antibody profiles in the BAL, VL and faecal samples were comparable for each treatment group to the respective profiles in the serum (Appendix A.4). The results of this experiment indicate a skewing towards Th2 immunity, though we did not observe a direct enhancement of mucosal (vaginal, fecal, or  35  OVA-specific IgA **  Optical Density  1.0 0.8 0.6 0.4 0.2  ive Na  CP  T+  OD N  OD N  Cp G  BM V+  CP  T+  Cp G  Cp G  0.0  OVA-specific IgG1 1.3  Optical Density  *  *  **  1.1 0.9 0.7 0.5 0.3 0.1  ive  OD T+ CP  Na  N  N OD  Cp G V+  BM  CP  T+  Cp G  Cp G  -0.1  OVA-specific IgG2a ***  ***  Optical Density  1.1 0.9 0.7 0.5 0.3 0.1  Na ive  OD N  CP T+  OD N  Cp G V+  BM  CP  T+  Cp G  Cp G  -0.1  Figure 4. Effect of calcipotriol on the humoral response to subcutaneous immunization. Mice were immunized twice 7 days apart with OVA + CpG or ODN. Calcipotriol or BMV treatment preceded both immunizations when indicated. 21 days after the second immunization serum, vaginal lavage, faecal pellets, and bronchoalveolar lavage samples were analysed for OVA-specific IgA, IgG1 and IgG2a antibody titers by indirect ELISA. Data shown is from serum samples. * p < 0.05, ** p < 0.01, *** p < 0.001. 36  pulmonary antibody levels, or changes in IgA titer) immunity as noted by others. This may indicate that either pretreatment may not be optimal for modulating the response compared to incorporation directly into the vaccine, or that calcipotriol is not as potent as vitamin D in affecting humoral responses. We were unable to detect measurable levels of IgE antibodies in any samples, a potentially important finding for any prospective therapeutic applications.  3.2.3 Treatment with calcipotriol prior to subcutaneous immunization induces transferable tolerance It is well described that immunization or hapten sensitization over UV treated skin results in the generation of a population of antigen-specific Tregs (Schwarz, 2007). We have demonstrated that CD4+CD25+ cells from UV tolerized animals can mediate transferable tolerance in an in vivo proliferation assay, while CD4+ cells transferred from animals who have received UV irradiation or OVA immunization alone display no suppressive ability (Ghoreishi and Dutz, 2006). A recent report has stated that topical vitamin D alone can increase the functional activity of CD4+CD25+ Treg cells in the skin DLN, and improve their responsiveness to a secondary stimulus (Gorman et al., 2007). It has been previously shown by our lab that TCI over calcipotriol treated skin can generate a population of Tregs with a similar phenotype as those induced by UV (unpublished data), but while it is clear that calcipotriol treatment affects the ability of SCI to generate a CTL response, its ability to promote Treg induction is unknown.  37  To look for the induction of functional tolerance in calcipotriol tolerized mice, we measured the ability of CD4+CD25+ T cells from treated mice to suppress OT-I CD8+ cell proliferation in vivo. Animals treated with calcipotriol were immunized subcutaneously or epicutaneously. Three days post-immunization they were sacrificed and their lymphoid organs pooled. CD4+CD25+ cells were sorted to high (>97%) purity using a fluorescence conjugated cell sorter, and 7.5x105 cells were transferred into naïve mice. The naive mice were also co-transferred with 8x106 CFSE-labeled CD8+ OT-I cells, and immunized via TCI twenty-four hours later. Three days after immunization, skin DLN were harvested and analysed by flow cytometry for OT-I cell proliferation (Fig. 5A). We found that the co-transfer of CD4+CD25+ cells from either subcutaneously (sc Tr) or epicutaneously (ec Tr) immunized calcipotriol treated mice was capable of significantly inhibiting proliferation of the transgenic CD8+ cells (Fig. 5B). Additionally, IFNγ production appears inhibited, indicating a decrease in the differentiation of CTLs. This demonstrates that calcipotriol is capable of inducing Tregs to subcutaneous immunization, albeit perhaps less effectively than to TCI. Alternatively, our doses of antigen in the subcutaneous vaccine may require optimization in order to be as effective as TCI. As these experiments were not performed with an irrelevant antigen control, it remains to be seen whether these SCI Tregs exhibit the ability to suppress immune responses in an antigen-specific manner.  A characteristic of tolerance generated by calcipotriol treatment prior to TCI is an increase in the percentage of Foxp3+ CD4+ T cells in the skin DLN (Fig. 6A). This increase is an additive result of both calcipotriol treatment in combination with TCI, as  38  B SCI  Day 7 sc Tr + SCI  Tr +  Na  I  I ec I SC Tr +  CFSE  sc  Analyse DLN  SC  OVA + CpG TCI  ive  OT-I AT  Day 6  Na  Treg AT  Day 3  I  Day 2  I  1.21  90 80 70 60 50 40 30 20 10 0  SC  Naive  IFNg producing OT-I cells (%)  sc  Recipient  Day 1  SC  24.9  ive  0  Treg isolation  SC  OVA + CpG SCI/TCI  25  Tr +  CPT  50  I  Day 4  75  SC  Day 1-3  OT-I proliferation (%)  96.9  **  *  100  Tr +  Donor  ec  A  Figure 5. Tolerance is transferable from animals treated with calcipotriol and immunized subcutaneously or epicutaneously. (A) Mice were treated with 3 days calcipotriol and immunized with OVA + CpG via SCI or TCI. After 3 days CD4+CD25+ cells were isolated to high purity (>98%) and 7.5x10^5 were adoptively transferred into recipients (sc Tr = Tregs from SCI animals, ec Tr = Tregs from TCI animals) along with 8x10^6 CFSE labeled OT-I cells. Animals were then immunized with OVA + CpG and 3 days later DLN were harvested for analysis. (B) Proliferation and IFNγ production by labeled OT-I cells measured by FACS. Proliferation data is representative of 3 experiments. *p < 0.05, **p < 0.01.  39  D  ** 15.0  Foxp3+ CD4 T cells (%)  12.5 10.0 7.5 5.0 2.5  5  CP  ive  N OD T+  Na  ive Na  C  N CP  0  T+  Naive  N  CPT  OD  2.5  1  T+  5.0  CP  7.5  2  N  10.0  OD  12.5  *  3  Cp G  CD4+Foxp3+ T cells (x10^6)  15.0  OD  T+ CP  + CP T  E  *  0.0  Cp G  Cp G  Cp G  Cp G  ive Na  B Foxp3+ CD4 T cells (%)  10  0  0.0  ***  45 35 25 15  ive Na  N OD +  OD N  CP T  CP T  +  Cp G  5  Cp G  CD4+ T cells in DLN (%)  15  Cp G  Foxp3+ CD4 T cells (x10^6)  A  Figure 6. Foxp3 levels in the draining lymph nodes. (A) Percentage of Foxp3+ CD4 T cells in the DLN of immunized animals five days post-TCI.Data shown is representative of 2 experiments. (B) Percentage of Foxp3+ CD4 T cells in the DLN after 3 days treatment with calcipotriol. (C) Percentage of CD4+ T cells in the DLN of immunized animals five days post-SCI. Data shown is representative of 2 experiments. (D) Percentage of Foxp3+ CD4 T cells in DLN of animals five days post-SCI. Data shown is representative of 2 experiments. (E) Total number of Foxp3+ CD4 T cells 5 days post-SCI. Data shown is representative of 2 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.  40  three days of calcipotriol treatment alone has a very minor effect on Foxp3 levels in the DLN (Fig. 6B). We asked whether the same result was observable using the subcutaneous method of antigen administration. Mice were treated with calcipotriol for three days, and on day four immunized. Five days post-immunization DLN were harvested and analysed by flow cytometry for Foxp3 levels in the CD4+ T cells. We found that after five days SCI in combination with calcipotriol increased the total percentage of CD4+ cells in the DLN when compared to animals immunized only (Fig. 6C). However, within the CD4+ T cell subset itself the levels of Foxp3 were approximately equal (Fig. 6D). Additionally, the lymph nodes of non-calcipotriol treated animals were slightly larger at day five, resulting in an elevated but comparable total number of CD4+Foxp3+ T cells in both CpG immunized groups (Fig. 6E).  3.3 Effect of topical calcipotriol upon draining lymph node dendritic cell populations Vitamin D and its analogues are known to affect the maturation and cytokine profile of murine DCs in vitro and in vivo by inducing a persistent state of immaturity, characterized by low levels of co-stimulatory marker and MHC II expression (Griffin et al., 2001). This is accomplished at least in part by VDRE located in the promoter regions of the NFκB protein RelB, regulating its transcription (Griffin et al., 2007). The end result is the generation of tolerogenic DCs capable of inducing Tregs, although its effects on LCs are more complex and include not only the downregulation of MHC II and costimulatory markers, but enhancement of IL-6, IL-12p40, and Th1 chemokine secretion (Fujita et al., 2007). Since the results of our subcutaneous vaccine are altered by  41  calcipotriol pretreatment, we wished to explore whether there was a direct DC dependant mechanism for this effect.  3.3.1 Calcipotriol decreases the percentage of antigen-bearing dendritic cells within the draining lymph node Subcutaneous immunization makes use of the immune cells concentrated in the skin in order to achieve a strong CTL response and lasting protection against pathogens. It is well known however that calcipotriol treatment depletes LCs from the epidermis, potentially inhibiting the ability of SCI to work via the elimination of APCs (Dam et al., 1996). Additionally, the requirement of CpG or a similar adjuvant to generate a response to SCI alludes to the importance of DC maturation, and it is clear that calcipotriol can both inhibit this process as well as suppress CTL priming. It is unknown whether calcipotriol is exerting these effects by altering the maturation/differentiation of DCs, depleting them from the skin, or is acting through other cell types entirely.  Following SCI, antigen is concentrated within dendritic cells expressing a phenotype consistent with DDCs (Najar and Dutz, 2007). In order to assess whether these are the primary DCs that capture antigen in our calcipotriol-based paradigm, we made use of fluorescently conjugated OVA protein to identify and characterize the APCs carrying OVA in the DLN. Animals were immunized via SCI with or without calcipotriol or BMV treatment using OVA-FITC as the protein antigen. We found that twenty-four hours postimmunization there was a significantly decreased percentage of OVA-FITC+ DCs present in the skin DLN of calcipotriol or BMV treated animals, compatible with the ability of  42  these compounds to deplete DCs from the skin prior to immunization (Fig. 7A). In all treatment groups we found that the majority of OVA-FITC+ APCs in the DLN had a CD8-DEC205-B220- phenotype, consistent with the profile of DDCs (Fig. 7B). It has been reported that Vitamin D alters the migration of DCs and allows them to bypass sequestration in the DLN and direct them to the mesenteric lymphoid organs (Enioutina et al., 2007b), however we were unable to detect significant numbers of OVA-FITC+ APCs in the spleen or mesenteric lymph nodes (data not shown). A failure of APCs to mature fully and upregulate co-stimulatory markers results in what is generally accepted as a tolerogenic phenotype, capable of Treg induction (Roncarolo et al., 2001). In order to determine whether this was occurring in our system and perhaps responsible for antigenspecific Treg induction/activation, we analysed the OVA-FITC+ and OVA-FITC- DCs for expression of the co-stimulatory markers CD40 and CD86. Interestingly, expression of these markers was not decreased in calcipotriol treated animals, in fact in the case of CD40 it appears to be increased in FITC+ DCs (Fig. 7C). This is in direct contrast with calcipotriol’s reported effects on DCs in vitro, however it may be attributable to the mild local skin irritation caused by upregulation of TSLP upon topical calcipotriol treatment (Li et al., 2006). Also of note is the decrease in co-stimulatory marker expression by DCs from BMV treated animals. This may explain the significant decrease in cellularity in the DLN of these animals post-immunization.  43  A  B  * 0.045  CpG  0.19  0.41  7.5  CD8  5.0 CD11c  OVA+ CD11c cells (%)  CPT + CpG  DEC205  Naive  10.0  2.5  OVA-FITC  B220  Na ive  BM  CD8  C  *  5 4 3 2 1  *  2.5 2.0 1.5 1.0 0.5  V+  Cp G BM  T+ CP  *  7.5  CD86 MFI (fold-increase)  CD86 MFI (fold-increase)  Cp G  Cp G  CP  BM  T+  V+  Cp G  Cp G  **  30  Cp G  0.0  0  20  10  5.0  2.5  V+  Cp G T+ CP  Cp G V+ BM  CP  T+  Cp G  Cp G  Cp G  0.0  0  Cp G  CD40  CD86  OVA- DCs CD40 MFI (fold-increase)  CD40 MFI (fold-increase)  OVA+ DCs 6  BM  V+  Cp G  Cp G CP T  +  Cp G  0.0  Figure 7. Dendritic cell phenotype in the draining lymph nodes after subcutaneous immunization. (A) Mice were immunized with OVA-V protein conjugated to FITC plus CpG, with calcipotriol or BMV treatment when indicated. 24 hours post-immunization DLN were harvested and analysed for percentage of CD11c+ cells presenting OVA-FITC. (B) Phenotype of OVA presenting CD11c+ cells. Gates were drawn according to total CD11c+ cells. (C) Expression of the co-stimulatory markers CD40 and CD86 on OVA-bearing (OVA+) and non-bearing (OVA-) DCs was measured in the DLN 24 hours post-immunization. Data is expressed as the foldincrease in MFI of indicated subsets compared to the MFI of total CD11c+ cells in a naive animal (grey line= naive T cells, solid black = CpG only, dashed line = CPT + CpG). *p < 0.05, **p < 0.01.  44  3.3.2 Function of ex vivo dendritic cells following topical calcipotriol administration and subcutaneous immunization We have shown that topical calcipotriol inhibits CTL priming to SCI, while having modulatory effects on the humoral response to immunization. Furthermore, calcipotriol plus SCI induces a population of CD4+CD25+ cells capable of mediating transferable tolerance. The mechanism through which calcipotriol is exerting these effects is unknown, however the literature on UV induced immunosuppression suggests that a DC dependant mechanism is involved in Treg induction. A current hypothesis is that DNA damage caused by UV light results in DCs that take up antigen but are incapable of maturation, leading to the generation of Tregs in the DLN. While vitamin D does not appear to be arresting the maturation or differentiation of DCs in our protocol as measured by surface markers, this does not rule out these DCs from being tolerogenic in nature (Menges et al., 2002; Sporri and Reis e Sousa, 2005).  To assess the ability of antigen-presenting DCs to activate T cells in our model we examined their ability to induce proliferation of OT-II CD4+ T cells in vitro. Animals were immunized by TCI with or without calcipotriol treatment and twenty-four hours post-immunization CD11c+ cells were enriched from pooled DLN suspensions to >60% concentration (Fig. 8A). These DCs from OVA-immunized animals were then cultured with CFSE labeled naïve OT-II T cells for 96 hours after which their proliferation and Foxp3 expression was measured. We found that the DCs from calcipotriol treated animals were capable inducing proliferation in the OT-II cells, but not to the same extent as DCs from untreated animals (Fig. 8B). This may be attributable to a decreased ability  45  A 68.8  CD11c  ***  B  OVA + CpG  25.5  CPT + OVA + CpG  CPT + BSA + CpG  1.69  12.3  OT-II proliferation (%)  30  Oregon Green  *** 20  10  0  C  OVA + CpG  FoxP3  11.0  CD4  CPT + OVA + CpG 12.3  CPT + BSA + CpG 7.3  Foxp3+ CD4 OT-II cells (%)  A OV  +  G Cp T+  A OV  +  G Cp T+  BS  G Cp  CP  CP  14  A  +  12 10 8 6 4 2 0  A OV  +  G Cp CP  T+  A OV  +  G Cp  A  + PT  BS  +  G Cp  C  Figure 8. Action of ex vivo dendritic cells following subcutaneous immunization. (A) CD11c+ cells from DLN of animals after were enriched (>60%) 24 hours following SCI with BSA or OVA with or without calcipotriol pretreatment. (B) DCs were co-cultured with Oregon Green labeled CD4+ cells from a naive OT-II mouse at a 1:2 ratio. After 96 hours incubation at 36 degrees the OT-II cells were analysed for proliferation. (C) Foxp3 expression in OT-II cells post-culture. ***p < 0.001.  46  to stimulate proliferation, however it is more likely related to the decreased percentage of OVA-presenting DCs in the lymph nodes of calcipotriol treated animals. These DCs do not appear capable of increasing Foxp3 expression in the OT-II CD4+ T cells, a result indicating that Treg induction may not be occurring though a DC dependant mechanism (Fig. 8C). No IL-10 or TGF-β was detectable in the culture supernatants, two cytokines with known tolerogenic properties.  47  4. Discussion  This thesis was based upon the hypothesis that topical pretreatment of the skin with the synthetic vitamin D analog calcipotriol would alter the immune response to subcutaneously administered antigen. Our experimental observations in a mouse model indicate that the drug does have activity on the immune response to subcutaneous immunization, and that the effects are manifold. This study is the first to report the induction of transferable tolerance by SCI under calcipotriol treated skin. It also confirms data indicating that vitamin D or its analogs can affect immune responses to vaccines and promote mucosal immunity, and demonstrates that this effect is powerful enough to overcome the effects of the potent Th1 adjuvant CpG. These effects are specific to calcipotriol as the topical corticosteroid BMV simply suppresses the immune responses to SCI.  4.1 The potential for calcipotriol as a tolerogenic adjuvant Background The development of a viable method for the induction of functional antigen-specific Tregs would have a profound impact on clinical medicine. In particular, Treg based treatments for autoimmunity could result in a paradigm shift in our approach towards millions of patients suffering from autoimmune diseases who are currently limited to pharmacologic immunosuppression. Experimental protocols have been devised to induce Tregs including the in vitro generation of CD4+CD25+ Tregs from naïve T cells (Allan et al., 2008), oral administration of low doses of antigen (Min et al., 2006), or the use of  48  ultraviolet radiation (Ghoreishi and Dutz, 2006). Unfortunately these techniques are limited by their specificity, practicality, and safety in terms of clinical application. The concept of immunosuppressive or tolerogenic adjuvants has recently been proposed, based on the principle that anti-inflammatory compounds may be capable of creating a local environment favouring the development of immune tolerance. Indeed, the successful prevention of diabetes onset was accomplished by using co-injection of an insulin-derived peptide and the corticosteroid dexamethasone in the footpad of a NOD mouse (Kang et al., 2008). Vitamin D has also been suggested as an adjuvant candidate based on its activity in a murine asthma model where it was combined with dexamethasone to potentiate a sublingual asthma therapy (Van Overtvelt et al., 2008). Improvement was associated with the generation of a population of CD4+CD25+Foxp3+ Tregs.  Calcipotriol based Treg induction A central question remaining unanswered after this work is whether calcipotriol modulated SCI is capable of inducing functional, antigen-specific Tregs. Previous work in our lab has demonstrated convincingly that calcipotriol plus TCI does induce CD4+CD25+ Tregs specific to the applied antigen (unpublished data). This combination also results in an increase in Foxp3 expression in the CD4+ cells of the DLN, an observation we have not seen with SCI. Nonetheless, we did observe transferable tolerance with our paradigm, although the specificity is still unknown. Should it become apparent that we are not inducing a population of antigen-specific Tregs as we do with TCI, there are two possible explanations for why that may be. The first is that topical  49  antigen delivery may be required for Treg induction. Injection of the antigen subcutaneously may bypass the LCs and thus circumvent their tolerogenic abilities, or allow the activated DDCs to supersede the effects of the tolerogenic LCs. Indeed the latter is particularly plausible since DDCs are known to migrate to the DLN much more rapidly than LCs (Kissenpfennig et al., 2005). The second possibility is one of antigen dose. In oral tolerance models, the induction of Tregs is highly sensitive to the dosage of antigen. While low doses of antigen result in the generation of antigen-specific Tregs, a higher dose results in T cell anergy and passive tolerance. Since it is difficult to precisely control oral dosage, this balancing act is one of the central pitfalls to this mode of tolerance induction. Topical antigen administration shares this confound because of variable antigen penetration, while in contrast subcutaneous antigen injection offers the ability to make precise dosage adjustments. In our paradigm we have used a subcutaneous dosage that has proved efficacious in inducing a CTL response to SCI, but it may be that this is not optimal for tolerance induction. This brings us to one of the central barriers we have encountered in pursuing this work: the development of a means of directly enumerating antigen-specific Tregs. With the development of a technique to quantify the induction of Tregs, the appropriate dose-response experiments could be easily performed. Potential approaches to this problem include the use of class II OVAtetramers, adoptive transfer of traceable OVA-specific OT-II CD4+ T cells, or transgenic Foxp3 reporter mice with antigen-specific T cells.  50  4.2 Humoral effects of calcipotriol on subcutaneous immunization Background Vitamin D incorporated into mouse vaccines promotes a mucosal immune response characterized by IgA and IgG1 production and the increased concentration of antibodies in secretions (Enioutina et al., 1999). This has been explained by a change in the cytokines in the DLN post-immunization characterized as increased IL-4, IL-5 and IL-10 as well as decreased IL-2 and IFNγ (Daynes and Araneo, 1994). An alteration in DC trafficking allowing antigen-presenting DCs to migrate directly to the Peyer’s patches (Enioutina et al., 2007b) and increased plasma cell homing to the lamina propria of the intestine and the lungs has also been observed (Daynes et al., 1996a). The advantages to the increased secretion of immunoglobulins are well established, and include a decrease in colonization of host mucosal surfaces by microorganisms (Harabuchi et al., 1994; Rice et al., 1997) as well as the neutralization of viruses and bacterial toxins within the gut and mucosa (Keren et al., 1989; Parr and Parr, 1997; Ruggeri et al., 1998). IgA also has the benefit of carrying out its actions without inducing inflammation. It was long thought that achieving an increased mucosal defense demanded administration of antigen at a site associated with mucosal membrane surfaces, often minimizing serum antibody responses, thus an adjuvant that has the capabilities to promote both arms of the humoral immune responses is highly desirable.  Calcipotriol plus SCI alters antigen-specific antibody production Pretreatment with calcipotriol was found to have slight adjuvant effects on the humoral response to SCI (refer to Fig. 4). These were characterized by a small increase in IgG1  51  titers and an almost complete reduction in IgG2a. This result was uniform across serum, BAL, VL and faecal samples and is consistent with the effects of vitamin D on vaccines in the literature, although seemingly less potent. The attenuated response may be due to the use of calcipotriol in place of vitamin D, a result of pretreatment rather than direct incorporation into vaccines, or it may be attributable to the adjuvant that was used in these experiments. Indeed the effects calcipotriol has in spite of the strength of this Th1promoting adjuvant are a testament to its immunomodulatory capacity, especially since TLR9 stimulation is known to promote recombination to the IgG2a isotype directly in B cells (Jegerlehner et al., 2007). Since no dose-response analysis was performed for our pretreatment regimen, adjustments to the dosage and timing would likely be able to optimize the protocol to increase its therapeutic value. A major advantage to calcipotriol pretreatment in combination with existing vaccines is that no reformulation is required and both components are already established as safe for human use. It remains to be seen however whether a human application is feasible, since while vitamin D based vaccine modulation has been demonstrated in mice and in pigs, it has been reported that the coadministration of vitamin D and the influenza vaccine had no mucosal benefits in human volunteers (Kriesel and Spruance, 1999).  4.3 Mechanisms of calcipotriol-based immunomodulation Effects of calcipotriol on dendritic cells An unexpected result of this study was the apparent phenotypic similarity of DCs in all immunized animals, regardless of treatment. Across all groups, these appeared to be DDCs based on the lack of CD8, B220 and DEC205 expression, however conclusive  52  identification will require examining expression of the LC marker Langerin (CD207) as well as any of the markers CD11b, Ep-CAM, Dectin-1 or CD103 that distinguish the Langerin+ subpopulation of DDCs (Bursch et al., 2007). While the numbers of antigenbearing DCs were not identical between calcipotriol treated and untreated animals, in all cases they appeared to be expressing adequate amounts of antigen and co-stimulation (refer to Fig. 7). It has been shown, however, that high expression of co-stimulatory markers does not preclude tolerogenic activity in DCs, thus in light of our transferable tolerance data we cannot say conclusively one way or the other until the profiles of the two groups of DCs are more carefully assessed (Menges et al., 2002; Sporri and Reis e Sousa, 2005). IL-10 and TGF-β production have been implicated in many models of tolerance induction (Rutella et al., 2006) leading us to examine cytokine release in the supernatants of DC-T cell co-cultures. We were unable to find detectable levels of these cytokines, however their production has been linked to vitamin D and they certainly warrant further examination (Haugen et al., 1996; Imazeki et al., 2006). The commercial availability of antibodies for the inhibitory ILT markers that denote DCs with tolerogenic capabilities (Manavalan et al., 2003) may also reveal significant differences in antigenbearing DCs in calcipotriol treated pretreated animals. ILT3 expression in particular has been linked to vitamin D treatment, although it appears unnecessary for tolerance induction itself (Penna et al., 2005). With these factors remaining unknown it is thus premature to discard the tolerogenic DC hypothesis.  Another set of experiments that could be highly informative, but would necessitate the enumeration of antigen-specific Tregs, would be to address the questions of DC maturity  53  and the local versus systemic immune effects of topical calcipotriol treatment. We have shown here that abdominal treatment has little effect on the proliferation of transgenic CD8+ T cells in response to a dorsal immunization. This implies that calcipotriol is having a predominantly local immunosuppressive effect in the skin. A recent publication has stated that topical vitamin D does not affect numbers but increases the responsiveness of Tregs in the DLN to expanding upon receiving a second stimulus (Gorman et al., 2007). This could suggest that calcipotriol treatment in our model is promoting Treg induction by having a direct effect on the lymph nodes and priming the Tregs to response preferentially to a subsequent vaccination, a hypothesis that would explain how tolerance might occur despite a relatively mature phenotype in antigen carrying DCs. The fact that calcipotriol has a seemingly local effect in the skin disputes this idea, unless the mechanisms behind inhibition of CD8+ T cell expansion and Treg induction are uncoupled. It is conceivable that the inhibition seen in our in vivo proliferation assay is a direct result of depletion of APCs in the skin and/or subtler changes to their cytokine profiles, while Treg induction is a result of a calcipotriol-altered environment in the DLN affecting the T cell response to the remaining mature DCs and skewing towards a tolerogenic response. This question could easily be addressed with an effective means of measuring antigen-specific Treg induction and further comparison of dorsal versus abdominal calcipotriol application. An alternative approach would be to measure the ability of DCs enriched from immunized animals to induce proliferation of OT-I CD8+ T cells ex vivo. Conversely, the proliferative ability of CD8+ T cells taken from naïve OT-I animals treated with calcipotriol could be examined in vitro as well.  54  The role of CpG Topical calcipotriol is shown here to prevent the priming of CD8+ T cells in response to CpG adjuvant stimulation and to prevent CpG induced IgG2a production. Topical calcipotriol also induces functional regulatory T cells to subcutaneous antigen when topical CpG is administered. However, the importance of TLR9 agonism to the generation of Tregs in our protocol is not yet clear. CpG is an extremely effective adjuvant for traditional subcutaneous vaccines, however it has also been linked to tolerance in certain situations. Specifically, high-dose intravenous administration of CpG to mice has been shown to upregulate the immunosuppressive tryptophan-degrading enzyme indoleamine 2,3-deoxygenase (IDO) in the CD19+ DCs of the spleen (Mellor et al., 2005). It was published that this occurs through a type I interferon dependant process, however a conflicting report states a similar observation dependant on TLR9 but not type I or type II interferons (Wingender et al., 2006). Regardless, this is likely a counterregulatory mechanism designed to prevent excessive immune responses. While it seems that an adjuvant is not required for our vaccine to exert its effects on humoral immunity, it is not yet known whether the same holds true for tolerance induction. Furthermore, if an adjuvant is found to be an important part of the process it will be interesting to see whether CpG and TLR9 themselves play an active role in Treg generation or if substitution with other TLR agonists can achieve the same effects.  VDR or mVDR The nuclear VDR is widely expressed in leukocytes and plays an important role in downregulating transcription of NFκB subunits and cytokines such as IL-2, IL-12 and  55  IFNγ (Alroy et al., 1995; Cippitelli and Santoni, 1998; D'Ambrosio et al., 1998). Whether the effects of calcipotriol are functioning through this canonical VDR or the hypothesized mVDR in these experiments is unknown. As it is as of yet uncharacterized, the existence of the putative mVDR remains contentious, however strong evidence supporting some type of membrane-bound receptor comes from observations that certain cis-locked conformations of vitamin D are capable of affecting cells in a strictly non-genomic fashion (Norman et al., 2001; Norman et al., 1999). It is thought that these effects are mediated by the mVDR, and some believe that many of the immunological results of vitamin D treatment function through this pathway. It would thus be interesting to perform a series of Treg induction experiments using one of these analogs in place of calcipotriol.  4.4 Differential effects of calcipotriol and betamethasone Background While both vitamin D analogs and corticosteroids are used frequently in practice, a comprehensive understanding of their exact mechanisms remains elusive. The comparison between topical calcipotriol and BMV treatment on antigen-specific cutaneous immune responses is another unique aspect of this work. The actions of topical calcipotriol and betamethasone have been compared in the past with regards to their abilities to affect CHS, where it was described that calcipotriol treatment at the elicitation site prior to a challenge boosted the CHS response, while betamethasone diminished it. These authors concluded that these results indicate the clinical effects of calcipotriol are based on a different mechanism than those of betamethasone (Garrigue et al., 1993). We  56  now know that both vitamin D and corticosteroids suppress pro-inflammatory signals such as IL-12 from DCs (D'Ambrosio et al., 1998; Elenkov et al., 1996), although unlike the VDR the glucocorticoid receptor (GR) can function via transcriptional effects or by interacting directly with other transcription factors including NFκB, AP-1, and STAT proteins (Jonat et al., 1990; Scheinman et al., 1995). Corticosteroids skew the Th1/Th2 balance in a similar manner as vitamin D by upregulating IL-4, IL-10 and IL-13 production from T cells through their effects on APCs or by acting on the lymphocytes directly (Blotta et al., 1997; Ramirez et al., 1996). These compounds are also capable of affecting DC differentiation by restricting their ability to fully mature (Moser et al., 1995; Piemonti et al., 1999). In many ways these two drugs share similar effects on immune cells, however a closer comparison of their effects on DCs offers an explanation for their discrepant results in our experiments. Dexamethasone (another popular topical corticosteroid) has previously been compared to vitamin D in its ability to prevent DC maturation. While dexamethasone had strong suppressive effects on production of Th1 cytokines, its ability to suppress DC stimulatory function was modest. This was in stark contrast to vitamin D, which exhibited potent inhibition of DC stimulatory capabilities while in fact upregulating production of the Th1 chemokines MCP-1 and MIP-1α (Xing et al., 2002). When DCs were treated with both compounds the effects were additive and general DC function was significantly inhibited, possibly explaining why combination therapies using calcipotriol and corticosteroids have become popular for the control of psoriasis (Del Rosso Do, 2006). A second difference that may play a role in our model is the relationship between these two compounds and TLRs. While TLRs and vitamin D have been shown to coordinate with each other to promote innate immunity in the skin  57  (Lin et al., 2002; Liu et al., 2006; Schauber et al., 2007), glucocorticoids appear capable of inhibiting TLR signaling at multiple levels (Chinenov and Rogatsky, 2007). Since our model is based on topical immunomodulation with a TLR agonist, these may be important differences.  Results Our results indicated similar suppressive effects from both calcipotriol and BMV on the CTL response. However, while calcipotriol pretreatment has slight adjuvant effects on the humoral response in line with the actions of vitamin D in the literature, BMV appears to suppress antibody production as well rather than skew towards Th2. In fact, the only immunoglobulin that was not significantly reduced by topical BMV treatment was IgG2a, an isotype generally promoted by Th1 cytokines (Belardelli, 1995). An explanation for the effects of BMV may be the significant reduction in both number of antigenpresenting DCs found in the DLN, as well as their co-stimulatory marker expression (refer to Fig 7). The reduction in number may be due at least in part to the potent ability of BMV to deplete APCs in the skin, although this is a property that is also shared with calcipotriol. Dosage may also be a factor, however in keeping with the described properties of corticosteroids, the difference may in fact be due to the local suppression of Th1 cytokines. Vitamin D seems to promote certain Th1 chemokines, perhaps explaining why the number of responding DCs in calcipotriol treated animals is much higher than in the BMV group. This may also partly explain why BMV had a strong suppressive effect on the cellularity of the DLN post-immunization, while calcipotriol application had no effect on cell numbers after three days. These results imply that the actions of BMV on  58  immune responses in the skin are not Th2 skewing but generally immunosuppressive, while calcipotriol may be best described as an immunomodulator.  4.5 Applications of calcipotriol modulated subcutaneous immunization Treg induction Our model uses calcipotriol as a tolerogenic adjuvant and shares the advantages of noninvasiveness and specificity with the above examples. Whether the Tregs generated in our experiments are functional and capable of suppressing physiological immune reactions (as demonstrated for the Tregs induced by TCI following calcipotriol treatment) remains to be seen, however our results with calcipotriol and TCI suppressing CHS are promising. A potentially exciting advantage to our model may be the ability to interrupt established immune responses. As previously stated, this had been a central difficulty with UV Tregs due to their patterns of migration (Schwarz et al., 2004). An explanation for why our Tregs may be better at suppressing CHS in the skin may lie in the ability of vitamin D conditioned DCs to imprint T cells to return to the skin. It appears that these DCs have the ability to upregulate CCR10 in responding CD4+ cells allowing them to migrate to the skin-specific chemokine CCL27 secreted by keratinocytes (Sigmundsdottir et al., 2007). Additionally, downregulation of gut-homing receptors α4β7 and CCR9 was also reported, which may suggest that different immunosuppressive adjuvants will be required for different immune disorders. In contrast to these experiments however, it has also been noted that both vitamins A and D are capable of downregulation of cutaneous lymphocyte antigen (CLA) in CD4+ T cells, and decreased skin infiltration by effectors (Yamanaka et al., 2007). In this report vitamin A upregulated α4β7 and promoted gut  59  homing, while vitamin D had no effect on this receptor. Once we have developed a method of identifying antigen-specific Tregs in our model, the characterization of their migratory markers may clarify this issue.  Allergen-specific immunotherapy The manipulation of immune activity through the use of calcipotriol and SCI may have a potentially exciting application in humans because of the precedent for this type of antigen administration in allergen-specific immunotherapy (SIT). While temporary alleviation of allergic symptoms is achievable through the use of antihistamines, antileukotrienes, and corticosteroids, SIT is currently the only long-term therapy available for severe allergies. The disadvantage to this treatment option is that it involves a series of subcutaneous allergen injections that is often spread out over a period of months or years. The mechanism through which this therapy functions is still unknown, but there are two predominant theories. The first is that it works by promoting the production of “blocking” IgG antibodies that are able to prevent the interaction of allergens with effector cell-bound IgE antibodies, thus preventing degranulation by mast cells and basophils (Lichtenstein et al., 1966). The second theory, one that is currently gaining favour, is that it induces the generation of a population of allergen-specific Tregs (Verhagen et al., 2006). The major disadvantage to the treatments is the frequency and duration of injections necessary to achieve long-term improvement in allergy patients, with some patients requiring hundreds of injections over a period of years to attain any permanent benefits. We have demonstrated in this study that topical calcipotriol promotes B cell immunoglobulin class-switching towards IgA and IgG1 isotypes. The increased  60  secretion of these antibody classes may have beneficial effects in allergy patients beyond preventing further IgE production, due to the blocking effect of these antibodies. Moreover, the increased secretion of these antibodies could promote neutralization of allergens before they even enter the body. We have found no detectable levels of IgE, although interestingly the literature reports that vitamin D can have both positive and negative influences on the production of this class of antibody (Heine et al., 2002; Matheu et al., 2003). Our paradigm also may be capable of inducing Tregs, which could simultaneously prevent undesired inflammation and T cell proliferation. It is feasible that pretreatment with calcipotriol or other vitamin D analogs may be able to increase the efficacy of SIT treatment and improve the results of the therapy.  Vitamin D based treatments for cutaneous infections A final consequence of this work may be to engender caution in researchers proposing novel applications for vitamin D based immunotherapy. In the past few years a great deal of research has centered on how vitamin D and its analogs may be used to manipulate immune activity to improve host defense. The idea that vitamin D promotes mucosal immunity when it is incorporated into vaccines was previously mentioned (Daynes et al., 1996b). It has also been observed that vitamin D can promote TLR2 activity as well as cathelicidin and defensin2 production in the skin (Liu et al., 2006; Schauber et al., 2007; Wang et al., 2004). These authors have suggested that topical vitamin D may be useful in treating infections in the skin from agents such as Mycobacterium tuberculosis. Our results do not dispute that vitamin D may aid in resolving infections, and support its ability to induce mucosal antibodies, however examining these proposals to use vitamin  61  D therapeutically in light of its tolerogenic capabilities raises a major concern. The inadvertent induction of Tregs and immune tolerance may be a possible complication to both of these applications that would have disastrous consequences for patients, potentially leading to a vastly reduced ability to defend versus certain pathogens.  4.6 Impact of described work In summary, we have shown that topical calcipotriol is a potent immunomodulator capable of altering the Th1/Th2 balance despite the strong Th1 bias of our vaccine adjuvant, the TLR9 agonist CpG. In psoriasis, immune responses are thought to be generated in part following IFNα production by plasmacytoid DCs activated through TLR9 recognition of self-DNA (Lande et al., 2007). Our observation that calcipotriol treatment largely inhibits the CTL inducing effect of TLR9 activation may give some insight into another putative mechanism through which calcipotriol asserts its antipsoriatic activities. Lastly, we have begun to describe the mechanism behind an easy, effective, and relatively non-invasive means of inducing Tregs in the form of calcipotriol modulated vaccination. This is a therapy that could be easily translated to the clinic either as an improvement on existing treatment protocols, or as a modality all of its own.  62  References Alarcon, J. B., Hartley, A. W., Harvey, N. G. and Mikszta, J. A. (2007) Preclinical evaluation of microneedle technology for intradermal delivery of influenza vaccines. Clin Vaccine Immunol 14, 375-81. Allan, R. S., Smith, C. M., Belz, G. 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The original figure can be found here: Uematsu, S. and Akira, S. (2006) Toll-like receptors and innate immunity. J Mol Med 84, 712-725.  79  A.2 The vitamin D biosynthestic pathway.  Appendix A.2 has been removed due to copyright restrictions. The information removed is a figure summarizing the sequence of events occurring during the synthesis of vitamin D in humans. The original figure can be found here: Garrett, R. H. and Grisham, C. M. (2002) Biochemistry, Harcourt College Publishers, Orlando, FL.  80  A.3 University of British Columbia Research Ethics Board Certificate of Approval  81  82  A.4 Mean optical density of OVA-specific immunoglobulin ELISAs in various samples - Values refer to the mean +/- standard deviation of 5 mice per treatment group. Serum, BAL, VL and faecal samples were taken from each mouse. IgA OVA+CpG CPT+OVA+CpG BMV+OVA+CpG OVA+ODN CPT+OVA+ODN Naïve  IgG1 OVA+CpG CPT+OVA+CpG BMV+OVA+CpG OVA+ODN CPT+OVA+ODN Naïve  IgG2a OVA+CpG CPT+OVA+CpG BMV+OVA+CpG OVA+ODN CPT+OVA+ODN Naïve  Serum 0.876 +/- 0.092 0.846 +/- 0.042 0.580 +/- 0.155 0.895 +/- 0.076 0.868 +/- 0.049 0.108 +/- 0.008  1.100 1.110 0.587 1.039 1.174 0.156  BAL +/- 0.201 +/- 0.199 +/- 0.248 +/- 0.264 +/- 0.354 +/- 0.034  Serum BAL 0.983 +/- 0.102 0.803 +/- 0.135 1.111 +/- 0.121 0.973 +/- 0.160 0.621 +/- 0.290 0.436 +/- 0.233 0.974 +/- 0.089 0.781 +/- 0.137 1.028 +/- 0.108 0.931 +/- 0.204 -0.0173 +/- 0.012 -0.006 +/- 0.005  Serum 0.933 +/- 0.225 0.040 +/- 0.039 0.885 +/- 0.256 0.076 +/- 0.088 0.045 +/- 0.092 -0.023 +/- 0.007  BAL 0.640 +/- 0.371 0.011 +/- 0.017 0.465 +/- 0.515 0.005 +/- 0.022 -0.002 +/- 0.016 0.006 +/- 0.008  0.332 0.129 0.082 0.274 0.155 0.077  VL +/+/+/+/+/+/-  0.232 0.062 0.059 0.233 0.079 0.032  Faecal sample 0.320 +/- 0.303 0.593 +/- 0.288 0.093 +/- 0.209 0.307 +/- 0.183 1.000 +/- 0.642 0.337 +/- 0.225  0.300 0.190 0.098 0.596 0.174 0.001  VL +/+/+/+/+/+/-  0.529 0.148 0.132 0.591 0.155 0.010  Faecal sample 0.265 +/- 0.073 0.308 +/- 0.225 0.007 +/- 0.025 0.148 +/- 0.091 0.182 +/- 0.098 0.004 +/- 0.010  VL 0.037 +/- 0.010 0.015 +/- 0.008 -0.006 +/- 0.022 0.006 +/- 0.011 0.002 +/- 0.010 -0.008 +/- 0.006  Faecal sample 0.015 +/- 0.020 0.023 +/- 0.009 -0.018 +/- 0.009 -0.014 +/- 0.024 -0.007 +/- 0.005 -0.021 +/- 0.015  * values not normalized between different sample groups.  83  

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