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The Influence of 1,25-dihydroxyvitamin D3 on the Cross-Priming of Lymphocytic Choriomeningitis Virus Nucleoprotein by Julia Kim A thesis submitted to the Microbiology and Immunology Graduate Program in the Department of Biomedical & Molecular Sciences In conformity with the requirements for the degree of Masters of Science Queen’s University Kingston, Ontario, Canada (August, 2011) Copyright © Julia Kim, 2011 Abstract Biologically active 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) binds the vitamin D receptor (VDR) to exert its effect on target cells. VDR expression is found in a number of immune cells including professional antigen-presenting cells such as dendritic cells. It has been found that the actions of 1,25-(OH)2D3 on the immune system are mainly immunosuppressive. The cross-presentation pathway allows for exogenously derived antigens to be presented by pAPCs on MHC-I molecules to CD8+ T cells. CD8+ T cell activation results in the expansion of epitope-specific T cell populations that confer host protection. These epitopes can be organized into an immunodominance hierarchy. Previous work demonstrated that introducing LCMV-NP via the cross-priming pathway significantly immunodominance hierarchy of a subsequent LCMV infection. alters the Building upon these observations, our study assessed the effects of LCMV-NP cross priming in the presence of a single dose of 1,25-(OH)2D3. Treatment with 1,25-(OH)2D3 was found to have biological effects in our model system. In vitro pAPCs were demonstrated to up-regulate IL-10 and CYP24A1 mRNA, in addition to the transactivation of cellular VDR, as demonstrated by a relocalization to the nuclear region. Mice treated with 1,25-(OH)2D3 were found to produce up-regulated IL-10 and CYP24A1 transcripts. Expression of VDR was increased at both the transcript and protein level. Our results demonstrate that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in this system. Treatment with 1,25-(OH)2D3 did not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigen to epitope-specific CD8+ T cells. Furthermore, 1,25-(OH)2D3 did not alter crosspriming or the establishment of the LCMV immunodominance hierarchy in vivo. By confirming that 1,25-(OH)2D3 does not suppress cross-priming in our model, our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25(OH)2D3 on the outcome of virus infection. Collectively, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with suppressive effects. ii Acknowledgements I have had the privilege of working under the supervision of Dr. Sam Basta over the course of my graduate career. I would like to thank him for his guidance and advice throughout my time in the laboratory. I would also like to express my gratitude to my committee members Dr. Myron Szewczuk and Dr.Elaine Petrof, for their continual support and mentorship throughout this process. I would like to thank Dr. Vanada Gambhir for her dedication and patient teaching throughout. I would also like to thank the members of the Basta Lab: Sarah Siddiqui (who always knows what to do, in any situation), Attiya Alatery, and Rylend Mulder. A special thank you to Alanna Gilmour and to Gareth Jones (the god of cake). Finally, I would like to extend a warm thank you to all the graduate students, past and present, and to the departmental staff. This work was funded by grants from the Natural Sciences and Engineering Research Council of Canada, and Fellowship awards from the Canadian Institute of Health Research, Queen’s University, and the Franklin Bracken Fellowship. iii Table of Contents Abstract ............................................................................................................................................ ii Acknowledgements ......................................................................................................................... iii List of Figures…………………………………………………………………………………….vi List of Abbreviations……………………………………………………………………………viii Chapter 1 Introduction ..................................................................................................................... 1 1.1 The Antigen Presentation Pathway ........................................................................................ 2 1.2 The outcome of cross-presentation events: Induction of tolerance versus priming ............... 3 1.3 LCMV infection model .......................................................................................................... 6 1.4 The immunodominance hierarchy ......................................................................................... 9 1.5 Vitamin D and the immune system ...................................................................................... 10 1.6 The regulation of vitamin D3 in the immune system ........................................................... 15 1.6.1 The conversion of vitamin D3 to 1,25-(OH)2D3 in the immune system ....................... 15 1.7 The effects of 1,25-(OH)2D3 on antigen-presenting cells (APCs) ........................................ 15 1.7.1 Monocytes and macrophages (MΦ) .............................................................................. 15 1.7.2 Dendritic cells (DC) ...................................................................................................... 17 1.7.3 T-lymphocytes .............................................................................................................. 18 1.8 Rationale and Hypothesis .................................................................................................... 19 Chapter 2 Materials and Methods .................................................................................................. 20 2.1 Cells and reagents ................................................................................................................ 20 2.2 Mice ..................................................................................................................................... 20 2.3 Preparation of bone marrow-derived macrophages and dendritic cells ............................... 21 2.4 Induction of cell death ......................................................................................................... 21 2.5 Virus Production & Titration ............................................................................................... 22 2.6 1,25-(OH)2D3 stimulation of immune cells .......................................................................... 23 2.7 Reverse Transcriptase (RT)-PCR......................................................................................... 23 2.8 Western blotting ................................................................................................................... 24 2.9 Fluorescence microscopy ..................................................................................................... 24 2.10 Analysis of VDR expression by flow cytometry ............................................................... 25 2.11 Measurement of T cell activation by intracellular cytokine staining (ICS) and FACS analysis....................................................................................................................................... 25 iv 2.12 Phagocytosis assays ........................................................................................................... 26 2.13 Cross-presentation assay .................................................................................................... 26 2.14 Preparation of NP396 T cells after cross-priming in vivo .................................................. 27 2.15 Evaluation of Immunodominance ...................................................................................... 28 2.16 Culturing of epitope-specific T lymphocytes..................................................................... 28 2.17 Statistics ............................................................................................................................. 29 Chapter 3 Results ........................................................................................................................... 30 3.1 Assessment of 1,25-(OH)2D3 biological effects in pAPCs................................................... 30 3.1.1 The effect of 1,25-(OH)2D3 on gene transcription in pAPCs ........................................ 30 3.1.2 VDR Expression ........................................................................................................... 32 3.2 Biological effects are observed in mice treated with 1,25-(OH)2D3 .................................... 34 3.3 1,25-(OH)2D3 treatment does not alter phagocytosis in pAPCs ........................................... 38 3.4 Investigating the ability of 1,25-(OH)2D3 to affect antigen cross-presentation in vitro ..... 41 3.5 Co-administration of 1,25-(OH)2D3 and LCMV-NP does not significantly affect crosspriming of CD8+ T cells in vivo ................................................................................................ 46 3.6 Cross priming of the LCMV nucleoprotein with 1,25-(OH)2D3 does not affect immunodominance..................................................................................................................... 50 Chapter 4 Discussion ..................................................................................................................... 60 4.1.1 Summary and Conclusions............................................................................................ 67 References ...................................................................................................................................... 69 v List of Figures Figure 1. Major histocompatibility complex (MHC) class I direct- and cross-presentation pathways. ......................................................................................................................................... 4 Figure 2. Exogenous antigen transfer and uptake by non-infected pAPCs in the cross presentation pathway.. .......................................................................................................................................... 5 Figure 3. The “two-signal model” of T cell activation. ................................................................... 7 Figure 4. Simplified diagram of Lymphocytic choriomeningitis virus structure............................. 8 Figure 5. The HEK-NP cell model to study the effects of cross-presentation ............................... 11 Figure 6. A schematic of vitamin D3 metabolism ......................................................................... 13 Figure 7. The effects of 1,25-(OH)2D3 on the regulation of vitamin D responsive genes in target immune cells. ................................................................................................................................. 14 Figure 8. A schematic of the immunomodulatory effects of 1,25-(OH)2D3 on various immune cells.. .............................................................................................................................................. 16 Figure 9. Analysis of 1,25-(OH)2D3 biological effects on pAPCs by RT-PCR. .......................... 31 Figure 10. 1,25-(OH)2D3 treatment does not alter basal VDR expression levels in pAPCs .......... 33 Figure 11. 1,25-(OH)2D3 treatment does not affect VDR expression levels in pAPCs.. ............... 35 Figure 12. 1,25-(OH)2D3 treatment induces localization of VDR to the nuclear region of pAPCs. ....................................................................................................................................................... 36 Figure 13. Detection of VDR localization in pAPCs using different doses of 1,25-(OH)2D3.. ..... 37 Figure 14. Analysis of effects 1,25-(OH)2D3 of gene induction in peritoneal cells of mice by RTPCR ................................................................................................................................................ 39 Figure 15. Analysis of peritoneal cell VDR protein expression is up-regulated in mice treated with 1,25-(OH)2D3 by western blot ................................................................................................ 40 Figure 16. Flow cytometric analysis of the effect of 1,25-(OH)2D3 treatment on phagocytosis by pAPCs. ........................................................................................................................................... 42 Figure 17. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated BMDC. .. 43 Figure 18. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated BMDM...44 Figure 19. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated cell lines…. ....................................................................................................................................................... 45 Figure 20. Cross-presentation of exogenous antigens by pAPCs is not affected by different doses of 1,25-(OH)2D3 treatment ............................................................................................................. 47 vi Figure 21. Co-administration of 1,25-(OH)2D3 and antigen does not affect the initial crosspriming of HEK-NP in vivo ........................................................................................................... 49 Figure 22. Pre-injection and co-administration of 1,25-(OH)2D3 with antigen does not affect cross-priming of HEK-NP in vivo.................................................................................................. 51 Figure 23. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the spleen................... 54 Figure 24. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect number of CD8+ populations in the spleen ..................................................................................................... 55 Figure 25. Pre-injection and co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the spleen. ............................................................................................................................................ 56 Figure 26. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV NP and the LCMV immunodominance hierarchy in the peritoneum. .......................... 58 Figure 27. Pre-injection and co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the peritoneum. .................................................................................................................................... 59 vii List of Abbreviations Ab antibody Ag antigen ADC antigen donor cell AP-1 activating protein 1 APC antigen presenting cell BFA brefeldin A BMA murine macrophage cell line BMDC bone-marrow derived dendritic cell BMDM bone-marrow derived macrophage CAMP cathelicidin antimicrobial peptide CD cluster of differentiation CFSE carboxyfluorescein succinimidyl ester CTL cytotoxic T lymphocyte CTLA cytotoxic T-lymphocyte antigen CYP cytochrome P450 DC dendritic cell DMEM Dulbecco’s Modified Eagle Medium DNA deoxyribonucleic acid dsRNA double stranded ribonucleic acid EAE experimental autoimmune encephalomyelitis EDTA ethylenediamine tetraacetic acid ER endoplasmic reticulum FACS fluorescence activated cell sorter FBS fetal bovine serum FoxP3 forkhead box P3 GP glycoprotein HEK human embryonic kidney cells HEK-NP human embryonic kidney cells expressing nucleoprotein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPK human primary keratinocytes HRP horseradish peroxidase IFN-γ interferon gamma IL interleukin i.p. intraperitoneal i.v. intravenous LCMV lymphocytic choriomeningitis virus LMP2 lysosomal membrane protein 2 LyUV lysis-UV irradiated MHC major histocompatibility complex MΦ macrophage mDC myeloid dendritic cell MFI mean fluorescence intensity MOI multiplicity of infection viii NFAT nuclear factor of activated T cells NF-κB nuclear factor kappa B NP nucleoprotein OVA ovalbumin pAPC professional antigen presenting cell PBS phosphate buffered saline pDC plasmacytoid dendritic cell pfu plaque forming units RNA ribonucleic acid RPMI Roswell Park Memorial Institute Medium RT-PCR reverse transcriptase- polymerase chain reaction RXR retinoid X receptor SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TAP transporter associated with antigen processing TCR T cell receptor Th1 T-helper 1 cells Th2 T-helper 2 cells TLR toll-like receptor TNF tumour-necrosis factor T-regs T regulatory cells UV ultraviolet VDR vitamin D receptor VDRE vitamin D responsive elements 1,25-(OH)2D3 1,25-dihydroxyvitamin D3, biologically active vitamin D 25-OHD 25-hydroxyvitamin D3 ix Chapter 1 Introduction Several components of the vitamin D endocrine system are expressed within various immune cell types. These components include the vitamin D receptor (VDR), in addition to the enzymes required to convert the inactive precursor, vitamin D3, into biologically active 1,25(OH)2D3. The presence of 1,25-(OH)2D3 has been found to affect the expression of several immune genes, such as tumor-necrosis factor-α (TNF-α) [1], interferon-γ (IFN-γ) [2-3], and interleukin-10 (IL-10) production [4]. Together, these data are suggestive of a physiological role for 1,25-(OH)2D3 in the immune system. In several experimental systems, 1,25-(OH)2D3 has been found to suppress both the adaptive and the innate immune responses. For example, in vitro 1,25-(OH)2D3 strongly inhibits T cell proliferation [3, 5], expression of IL-2 [3, 6-7], and IFN-γ [2-3]. Several innate immune parameters are also inhibited by 1,25-(OH)2D3, for example the inhibition of dendritic cell (DC) differentiation, maturation and immunostimulatory capacity [4, 8-11]. Despite these major inhibitory effects, the innate immune system can also be stimulated by 1,25-(OH)2D3. One study demonstrated that 1,25-(OH)2D3 stimulated human monocyte proliferation in vitro [12]. Furthermore, toll-like receptor (TLR) 2/1 ligation on human monocytes was found to induce 1,25-(OH)2D3 production. Increased levels of 1,25-(OH)2D3 induced cathelicidin production, an anti-microbial peptide, that had direct anti-microbicidal activity against Mycobacterium tuberculosis [11]. Therefore, the known immunomodulatory effects of 1,25-(OH)2D3 are contingent upon the specific parameter of the system being stimulated. Antigen presentation is a process that is carried out by cells such as DCs, and serves to link the innate and adaptive immune systems [13]. Through this process innate immune cells, such as professional antigen presenting cells (pAPCs), are able to control the activation of the adaptive immune response, such as CD8+ T cells. Naïve CD8+ T cells become activated when their receptors recognize antigens presented by pAPCs in the context of major histocompatibility complex class-I (MHC-I) molecules. Upon recognition of target cells, such primed cytotoxic Tlymphocytes (CTLs) are able to limit the spread of virus infection through the lysis of infected 1 cells, thus halting viral propagation in permissive cells. Along with helper CD4+ T cells, CTLs can orchestrate the induction of key cytokines such as the anti-viral IFN- γ and TNF-α, which are required for an optimal immune response [14-16]. CTLs are essential in the specific immune response, such as in the elimination of intracellular pathogens and tumour cells [17-18]. The cross-presentation pathway allows for exogenously-derived antigens to be presented on MHC-I molecules to CTLs [15-16, 19-20]. Cross-presentation is critical in generating antitumour immune responses or in priming CTLs against viruses that are able to inhibit the direct presentation pathway [21-22]. There is no evidence demonstrating the ability of viruses to bypass the cross-presentation pathway and it remains a critical method for overcoming viruses that are able to prevent infected cells from presenting antigens to stimulated naive T cells [16, 23-24]. The cross-presentation pathway should be a focal point of research as it has direct consequences for strategies that target the induction of CTL responses, and has been shown to induce both effector and memory T cell responses [25]. The immunosuppressive effects of 1,25-(OH)2D3 have been well-established on differentiating pAPCs. For example, 1,25-(OH)2D3 impairs the maturation of DC, resulting in decreased surface expression of MHC class II molecules, co-stimulatory molecules (CD40, CD80, CD86), and other maturation-induced surface markers [26]. These pAPCs are critical in the antigen presentation pathway, as they dictate the outcome of T cell activation. Currently, there is no information available on the effects of 1,25-(OH)2D3 on the cross-presentation pathway. 1.1 The Antigen Presentation Pathway All nucleated cells express MHC-I molecules and are capable of presenting antigens to CTLs. However, the “priming” or activation phase of naïve CD8+ T cells requires peptideMHC-I complexes presented by the pAPC, in addition to co-stimulatory signals, such as B7 molecules, CD40, and CD70 [27]. Furthermore, there is a requirement for cytokines such as IL12 and IFN-α [28-30]. The CD8+ T cell priming step can occur via two different mechanisms of antigen presentation: the direct- and cross-presentation pathways. In the direct presentation pathway, the peptides are derived from endogenously synthesized proteins and improperly translated proteins [32-33]. Upon proteasomal degradation, peptides (8-10 amino acids in length) are then transported into the endoplasmic reticulum (ER) 2 via the transporter associated with antigen processing (TAP), and finally through the Golgi complex and loaded onto MHC-I molecules [16]. The bound MHC-I: peptide complexes are transported to the cell surface and presented to CD8+ T cells (Fig. 1). In addition, exogenous antigens can also access the MHC-I antigen presentation pathway through the cross-presentation pathway (Fig. 1) [16, 19-20]. In cross-presentation, several processing mechanisms have been proposed [34] including the canonical model whereby antigens inside the endosomal or phagosomal vesicles are translocated into the cytosol before following the direct presentation route (proteasome/ER/TAP route). pAPCs can internalize exogenous antigens by several mechanisms including phagocytosis, macropinocytosis and receptor-mediated endocytosis. Large particulate materials, such as debris from apoptotic cells, are taken up by phagocytosis and endocytosis. In contrast macropinocytosis results in the uptake of surrounding soluble and small antigens [35-36]. It has been previously reported that apoptotic cells are taken up by phagocytosis while necrotic cell debris enter by macropinocytosis in mouse macrophages [37-38] (Fig. 2). Cell-associated antigen can be derived from different sources such as cellular fragments [39-41], intracellular bacteria [42], virus-infected or tumour cells [43-48], and parasitic infections [49]. Soluble proteins tend to be cross-presented, but with much lower efficiency than cell-associated proteins [50]. Another proposed model deals with organelles optimized for the cross-presentation, such as the phagosome, equipped with the necessary proteases [42, 51-53] to generate MHC class I– peptide complexes from internalized proteins. The antigens are degraded and then loaded onto MHC-I via an undetermined mechanism [40]. Another proposed mechanism involves the transfer of antigen by gap junctions, which are small intracellular pores formed between adjacent cells [54]. As gap junctions are found on a variety of pAPCs, it is suggested that cytosolically processed peptides can be transferred by gap junctions to pAPCs from peripheral infected cells [55]. Regardless of the exact mechanism in play within the cell, the cross-presentation pathway can result in one of two states: cross-tolerance or cross-priming. 1.2 The outcome of cross-presentation events: Induction of tolerance versus priming Inadequate activation of CTLs can result in tolerance, which is an ideal situation for selfantigens. Under normal circumstances cross-presentation of peripheral self- antigens from normal healthy tissue will induce a state of cross-tolerance, characterized by T cell anergy or deletion 3 Figure 1. Major histocompatibility complex (MHC) class I direct- and cross-presentation pathways. Direct presentation results in the loading of endogenously derived antigens from the infected pAPC to CD8+ T cell to prime the T cell for effector functions. Cross-presentation involves the presentation of exogenously derived antigen on non-infected pAPCs to activate CD8+ T cells. Image taken from [19]. 4 Figure 2. Exogenous antigen transfer and uptake by non-infected pAPCs in the cross presentation pathway. The cross-presentation pathway involves the presentation of exogenously derived antigen on a non-infected pAPC to CD8+ T cells. Exogenous antigen transfer from the infected ADC to a non-infected pAPC is the first step in the crosspresentation pathway. Antigen transfer can occur via phagocytosis or endocytosis for large particulate materials, or via macropinocytosis for surrounding soluble and small antigens. Exogenous antigen (8-10 amino acids in length) is processed and presented by the non-infected pAPC in the context of MHC-I. The peptide: MHC-I complex is recognized by T cell receptors on CD8+ T cells, resulting in the generation of a T cell response. 5 [56-57]. However, if immunity is to be induced, signals necessary for T cell activation [58] will be provided by pAPCs leading to clonal expansion, differentiation and establishment of robust memory cells. It has been hypothesized that a T cell response is contingent upon the maturation state of the pAPC involved [59-61]. The “two-signal model” proposes that a T cell response requires two signals: a T cell receptor signal (signal 1), and a co-stimulatory interaction from the pAPC (signal 2) (Fig. 3) [62-63]. An immature DC will lack signal 2 and subsequently induce a state of crosstolerance [62]. The pAPC: T cell receptor interaction is enhanced by stabilizing CD8 co-receptor molecules, and the binding of the CD28 receptor of the T cell to co-stimulatory signals B7.1 and B7.2 from the pAPC [64]. A mature DC will provide both signals necessary for T cell activation leading to crosspriming. Cross-priming is characterized by the clonal expansion and differentiation of naïve CD8+ T cells into their mature antigen-specific forms [64]. A mature CD8+ T cell will have high-affinity to specific antigens, and the ability to release cytokines that induce maturation in neighbouring cells (i.e. IL-2) [65]. The results of the initial cross-presentation events that lead to the priming of naïve CD8+ T cells that will establish the antigen-specific T cell populations that will be initiated upon re-exposure [16, 19], thus conferring beneficial protection for the host. 1.3 LCMV infection model Lymphocytic choriomeningitis virus is the type species of the genus Arenavirus, in the Arenaviridae family. It is a non-cytopathic agent. However, almost all Arenaviridae isolates are associated with rodent-transmitted disease in humans [66]. ambisense single-stranded RNA viruses [66]. Arenaviruses are enveloped, They have a characteristic bi-stranded RNA genome consisting of a large (L) (7.2 Kb), and a small (S) (3.4 Kb) strand (Figure 4). The L RNA codes for the virus RNA-dependent RNA polymerase and a zinc binding protein [67]. The S RNA encodes for viral structural proteins that include the nucleoprotein (NP), and a glycoprotein (GP) precursor. The NP encapsulates the viral nuclear material, and is the most abundant protein in virions and infected cells [66, 68-69]. The GP precursor is processed into two subunits that remain non-covalently linked, forming a receptor that spans the lipid bilayer.GP1 plays a role in binding the virus to target cell receptors whereas GP2 contains the fusion peptide and the transmembrane domain to enter the cell [66-68]. 6 Figure 3. The “two-signal model” of T cell activation [62]. With respect to cross-presentation, after the uptake and processing of exogenous antigen, the initiation of a correct T cell response requires two signals: (1) a T cell receptor signal (with the MHC of the pAPC), and (2) a costimulatory interaction from the pAPC, such as the stabilizing interactions of CD8 co-receptor molecules and the binding of the CD28 receptor of the T cell to co-stimulatory signals B7.1 and B7.2 from the pAPC. If a cell, such as a non-professional APC or an immature pAPC, lacks a signal then a state of anergy or deletion is induced in the T cell. 7 Figure 4. Simplified diagram of Lymphocytic choriomeningitis virus structure. LCMV has a characteristic bi-segmented RNA genome consisting of large (L, 7.2 Kb), and small (S, 3.4 Kb) segments that appear as non-covalently-linked circular nucleocapsids containing NP. The GP1 and GP2 subunits are non covalently linked, forming a receptor that spans the lipid bilayer. The receptor formed by the interaction between GP1 and GP2 is crucial for entry of LCMV into the target cell. 8 The target cell receptor interaction with GP1 and GP2 are crucial for entry of LCMV into the target cell. In our model, we utilize the Lymphocytic choriomeningitis virus (LCMV) WE strain because it is an ideal model to study cell-mediated immunity. LCMV-WE replicates rapidly, allowing for the expansion antigen-specific of CD8+ T cells [70]. Importantly, LCMV-WE does not cause exhaustion of CTLs after antigen stimulation, [70]. In relation to my research, it has been shown that LCMV-NP is cross- presented efficiently when introduced using LCMV-NP transfected HEK293 cells [39, 71-72]. Consequently, LCMV provides us with a very useful model to explore adaptive immune responses. 1.4 The immunodominance hierarchy A critical outcome as a result of T cell priming is establishing the profile of T cell immunodominance hierarchy. Antigen-specific CD8+ T cells can be organized into a hierarchy, in which immunodominant epitopes will cause a set of T cells to expand extensively compared to subdominant epitope-specific T cells [73]. A CTL response could be more effective when generated against a greater number of epitopes, as it is more diverse and leads to a wider number of specific memory CD8+ T cell populations; ultimately this would be more beneficial to the host. Understanding how CD8+ T cells are activated in vivo in virus infections could have applications in the development of a rational vaccine design [15]. Despite its importance, immunodominance is poorly understood at the mechanistic level [74]. The regulation of immunodominance is a key area of research [73]. In one study, following influenza infection, it was found that the immunodominance hierarchy of influenza virus-specific CD8+ T cells remained intact in the absence of either perforin or T helper cells [74]. Furthermore, interference with B7- mediated signalling did not change the establishment of the antiviral CD8+ T cell hierarchy, indicating that co-stimulation of APCs or killing of APCs by CTLs plays only a minor role in establishing the immunodominance hierarchy in this model of infection [74]. Also in influenza infection, immunoproteasomes have been demonstrated to play an important role in the establishment of the CD8+ T cell immunodominance hierarchy [75]. Immunoproteasomes are cytokine-inducible proteasome subunits that are substituted for their constitutively synthesized counterparts [75]. In mice lacking one immunoproteasome subunit (LMP2), it was found that responses to two of the most dominant influenza virus epitopes were 9 inhibited due to decreased generation of these antigenic determinants [75]. In parallel, responses to two subdominant determinants were greatly enhanced which caused an alteration to the CD8+ T cell repertoire [75]. In another study, the role for T regulatory cells (T-regs) in the establishment of immunodominance hierarchies was demonstrated, as they are able to moderate the differences in responses to the antigenic determinants produced during infection [76]. Previous work in the Basta Lab utilized the HEK-NP model to study the effects of LCMVNP cross priming on the LCMV immunodominance hierarchy following viral challenge [77]. Cross-priming was induced using an MHC mismatch model, where MHC-I (HLA) molecules from the injected antigen donor cell (HEK-NP) are incompatible with the T cell receptors on the mouse CD8+ T cells (H-2b). Thus through this system, murine CD8+ T cell priming occurs only via the cross priming pathway (Fig. 5). After cross priming by LCMV-NP, and during virus challenge experiments, it was found that due to the initial cross-priming [77], there was an increase in the magnitude of NP396 epitope specific T cells. It was speculated that initial cross priming resulted in an enhanced ability of NP396-specific clones to expand and outcompete other LCMV epitope specific T cells [77]. Interestingly, CD8+ T cells express the highest levels of VDR, and it has been suggested that they could be a major site of 1,25-(OH)2D3 action [78]. 1,25-(OH)2D3 has been found to reduce CD8+ T cell mediated cytotoxicity[79] and it has been suggested that 1,25-(OH)2D3 may be acting to prevent or suppress over-activation of these cells [80]. Furthermore, all of the components of the vitamin D endocrine system (VDR, 25-hydroxylase, 1α-hydroxylase and 24hydroxylase) are present within immune cells, which suggests an immunomodulatory role for 1,25-(OH)2D3 [26, 78, 81-82]. For our studies, we used the same HEK-NP model to investigate how the presence of 1,25-(OH)2D3 during the initial cross-priming events would influence the activation of epitope specific CD8+ T cells and the immunodominance hierarchy. 1.5 Vitamin D and the immune system The inactive form of vitamin D3 is obtained primarily through photosynthesis in the skin, but can also be derived from food sources such as fortified dairy products [81, 83]. Ultra-violet B light catalyzes the conversion of de novo synthesized 7-dehydrocholesterol into pre-vitamin D3, 10 Cross Presentation Figure 5. The HEK-NP cell model to study the effects of cross-presentation. For the crosspresentation assay, the human antigen donor cells (HEK-NP) MHC class I molecules are xenogenic, and therefore incompatible with T cell receptors on murine cells, therefore eliminating the ability of direct priming by ADCs. Murine T cells are only able to be activated by crosspriming, after antigen transfer of LCMV-NP derived epitopes from ADCs (HEK-NP) to murine professional antigen presenting cells. 11 which undergoes spontaneous isomerisation into vitamin D3 [81, 83]. Vitamin D3 can circulate in the blood upon attachment to a vitamin D3 binding protein (DBP) [82] (Figure 6). In the liver, 25-hydroxylase (CYP27A1) catalyzes the conversion of vitamin D3 to 25- hydroxyvitamin D3 (25-OHD) [81-83]. The second hydroxylation occurs in the proximal tubule cells of the kidney where 25-OHD is targeted by 1α-hydroxylase (CYP27B1), giving rise to active 1,25-(OH)2D3 [81-83] (Figure 6 ). 1,25-(OH)2D3 acts as its own negative feedback regulator, as it potently induces the expression of 24-hydroxylase (CYP24A1) which catabolizes both 25-OHD and 1,25(OH)2D3 into biologically inactive water-soluble metabolites [83]. This negative feedback loop is crucial for avoiding internal excesses of 1,25-(OH)2D3 and subsequent signalling [84-85]. As lipophilic molecules, vitamin D3 metabolites can cross cell membranes [83]. However, only 1,25-(OH)2D3 can exert any biological effects [78, 81, 86], which is due to its ability to bind VDR (Figure 7). Upon binding, the liganded VDR complex undergoes a conformational change that allows it to relocate to the nucleus. After entry into the nucleus, the liganded VDR will hetero-dimerize with another nuclear receptor, the retinoid X receptor (RXR). This complex is able to bind vitamin D3 responsive elements (VDREs) in the promoter region of vitamin D3-responsive genes [84-85]. Binding VDREs enables the recruitment of transcription initiated complexes such as vitamin D receptor interacting proteins, or by the release of corepressors or assembly of co-activators [84]. These interactions will influence the overall rate of RNA polymerase II-mediated transcription of vitamin D responsive genes [4, 87], leading to altered expression patterns (Figure 7). It has been demonstrated that some parameters of the innate immune system are stimulated by 1,25-(OH)2D3. In human monocytes, 1,25-(OH)2D3 can activate the innate immune response, through the up-regulation of anti-microbial peptides in response to TLR 2/1 ligation [11]. In addition, VDR is constitutively expressed in APCs such as MΦ and DCs, and is inducible in activated CD4+ and CD8+ T cells [4, 78, 88]. Furthermore, the presence of 1α-hydroxylase in MΦ and DCs [84-85] results in the production of high local 1,25-(OH)2D3 concentrations, that could have either autocrine or paracrine functions [84-85]. It is feasible to hypothesize that the 1,25-(OH)2D3 immune system will influence the cross-presentation pathway through the effects on key cells involved as highlighted below. 12 Figure 6. A schematic of vitamin D3 metabolism. Vitamin D3 is produced mainly from ultraviolet (UVB) action on the skin but is also acquired from the diet. Vitamin D3 is transported to the liver where it is hydroxylated by 25 hydroxylase (CYP27A1) to 25-OHD. 25-OHD is transported to the kidneys where it is subsequently hydroxylated by 1α hydroxylase (CYP27B1) to become 1,25-(OH)2D3 . In addition, 1,25-(OH)2D3 is its own negative feedback regulator, as vitamin D3 binding to the VDR enhances CYP24A1 expression, which is responsible for the production of inactive water-soluble vitamin D3 metabolites. Schematic is based on image taken from[80]. 13 Figure 7. The effects of 1,25-(OH)2D3 on the regulation of vitamin D responsive genes in target immune cells [83]. As lipophilic molecules, vitamin D3 metabolites can cross cell membranes and translocate easily to the nucleus. However only 1,25-(OH)2D3 can exert any biological effects, as it is able to bind to the nuclear VDR. The 1,25-(OH)2D3 :VDR complex becomes phosphorylated (P), allowing it to translocate to the nucleus. The liganded: VDR complex will hetero-dimerize with another nuclear receptor, the retinoid X receptor (RXR), resulting in the formation of a ligand/VDR/RXR complex that is able to bind VDREs in the promoter region of vitamin D3-responsive genes. Binding VDREs enables the recruitment of transcription-initiated complexes such as vitamin D receptor interacting proteins by the release of co-repressors or assembly of co-activators. These interactions will influence the overall rate of RNA polymerase II-mediated transcription of vitamin D3 responsive genes, leading to alterations in expression patterns. 14 1.6 The regulation of vitamin D3 in the immune system 1.6.1 The conversion of vitamin D3 to 1,25-(OH)2D3 in the immune system Although the essential components of the vitamin D3 endocrine system are present within immune cells, they are differentially regulated when compared to the classical endocrine system. For example, the 1 α-hydroxylase found in immune cells (Figure 8), such as MΦ, can be regulated by IFN-γ and TLR agonists [89-95]. In addition, CYP24A1 is expressed in immune cells [96-98]. CYP24A1 catabolizes the initial steps of the breakdown of 1,25-(OH)2D3 [83] (Figure 8), and its expression is potently induced by 1,25-(OH)2D3 [83]. This negative feedback loop is crucial for avoiding internal excesses of the active hormone [84-85]. As previously mentioned, VDR is highly expressed in immune cells. A mechanism by which 1,25-(OH)2D3 exerts most of its immune effects is through the interference with signalling by other transcription factors [26]. Liganded VDR complexes interfere with the signalling of the nuclear factor of activated T cells (NFAT), nuclear factor kappa beta (NF-kB), and activating protein-1 (AP-1), which are all critical in the regulation of immunomodulatory genes [99-101]. The dysregulation of these important transcription factors have been suggested to be involved in the pathogenesis of different autoimmune diseases[102]. Both the broad range of VDR expression and the diverse ways 1,25-(OH)2D3 interferes with gene expression can explain its wide spectrum of activities and pleiotropic effects [84]. 1.7 The effects of 1,25-(OH)2D3 on antigen-presenting cells (APCs) 1.7.1 Monocytes and macrophages (MΦ) As APCs, monocytes and MΦ can serve as a link between the adaptive and innate immune system. Monocytes and MΦ can elicit innate immune responses against various infectious agents upon recognition by their PRRs (i.e. TLRs) [103-104]. Interestingly, 1,25(OH)2D3 and TLR2/1 ligation are able to induce the expression of human cathelicidin antimicrobial peptide (CAMP) in monocytes [105-108]. TLR 2/1 ligation resulted in the upregulation of both 1α-hydroxylase, VDR, and 1,25-(OH)2D3 production [82, 109-110]. At later stages of infection (after 72 h), 1,25-(OH)2D3 suppresses the expression of TLR2 and TLR4 in human monocytes in a VDR-dependent mechanism[111]. This could represent a negative feedback mechanism to prevent excessive TLR-activation and subsequent sepsis [26]. It was also 15 Figure 8. A schematic of the immunomodulatory effects of 1,25-(OH)2D3 on various immune cells. Upon administration of 1,25-(OH)2D3 , it was found that DCs (APCs) surface expression of MHCII-Ag complexes and co-stimulatory molecules were inhibited. As a result of inhibited IL-12 secretion, T cell population were polarized towards a Th2 phenotype. T cells were directly affected by 1,25-(OH)2D3 administration with the generation of T-regs and the Th2 cytokines. Th1 lymphocytes and cytokine production is inhibited by Th2 and T-regs. Image taken from [85]. 16 found that VDR is down-regulated in MΦ at later stages of immune activation [8, 112]. Interestingly, MΦ subjected to 1,25-(OH)2D3 attain enhanced expression of the pro-inflammatory cytokine TNF-α [1]. The increases in TNF-α occurred in monocytes, MΦ, and in immature cells such as bone marrow cells [1]. However, cytokine production was decreased in mature cells such as peripheral blood mononuclear cells [113]. It has been reported that an increase in TNF- α production by bone marrow cells was partially mediated by direct binding of liganded-VDR-RXR complex to a VDRE in the promoter region of the gene [1]. In contrast, a negative VDRE was found in the promoter of the IFN-γ gene, suggesting that IFN-γ production is inhibited by the binding of liganded VDR-RXR complexes [114]. Importantly, the effects of 1,25-(OH)2D3 on APCs appear to be strongly contingent upon maturation status. 1.7.2 Dendritic cells (DC) The most pronounced effect of 1,25-(OH)2D3 has been demonstrated in APCs, especially DCs [26] (Figure 8). Multiple in vitro studies have demonstrated that 1,25-(OH)2D3 potently inhibits DC differentiation from either human peripheral blood monocytes or murine bone marrow cells in a VDR-dependent mechanism [9-10, 115-119]. 1,25-(OH)2D3 treatment can redirect differentiated DCs toward a completely unrelated CD14+ cell type [26]. The maturation process of DCs is seriously impaired, resulting in decreased surface expression of MHC class II, co-stimulatory molecules (CD40, CD80, CD86), and other maturation-induced surface markers [26]. Furthermore, in the presence of 1,25-(OH)2D3, differentiating DCs were unable to migrate successfully in response to lymph node homing chemokines, although the expression of the reciprocal chemokine receptors was unaffected [120]. In addition, 1,25-(OH)2D3 alters the cytokine expression pattern of DCs, with strong inhibition of IL-12 and a parallel increase in IL10 expression [9, 119]. This altered cytokine profile is supportive of cells with a tolerogenic or regulatory phenotype [9-10, 115-119, 121-122]. As the most potent among APCs, the effect of 1,25-(OH)2D3 on DCs has a strong influence on T cell behaviour downstream. Moreover, 1,25(OH)2D3 gives rise to tolerogenic DCs, which contributes to the induction of regulatory T cells, T cell anergy and apoptosis[123]. This novel mechanism requires further investigation, and suggests the possibility that DCs can be manipulated and exploited to support a more antiinflammatory or tolerogenic response in vivo [124]. 17 1.7.3 T-lymphocytes 1,25-(OH)2D3 can act both directly and indirectly on T-lymphocytes (Figure 8). The indirect actions on T cells are mediated by APCs. For example, when either naive or committed auto-reactive T cells were cultured with 1,25-(OH)2D3 -modulated DCs, both subsets demonstrated complete hypo-responsiveness, characterized by decreased proliferation and IFN-γ secretion [9, 119]. 1,25-(OH)2D3 can also have direct, pronounced inhibitory effects on T cell proliferation [3-4, 125]. It was found that 1,25-(OH)2D3 can directly inhibit the expression of several critical T cell cytokines such as IL-2 [3, 6-7] and IFN-γ [2]. The decreases in IL-2 and IFN-γ are mediated by the binding of VDR complexes to VDREs in the promoter regions of each gene [114, 126]. Overall, 1,25-(OH)2D3 blocks Th1 induction cytokines (IFN-γ) while promoting Th2 responses (IL-4) [4, 125], which is mediated both indirectly by decreased IFN-γ, and directly by enhanced IL-4 [4] and IL-10 secretion[122]. Th1 cytokines provide help for MΦ and cellular immune responses, while Th2 cytokines help B cell class switching and antibody production [127]. The 1,25-(OH)2D3 mediated up-regulation of IL-4, and subsequent protective effects in autoimmunity, were confirmed in vivo when 1,25-(OH)2D3 induced an auto-antigen specific Th1 to Th2 shift [128]. Thus, 1,25-(OH)2D3 indirectly induces CD4+ T cells to polarize towards a Th2 phenotype, attenuating autoimmunity and graft rejection [4, 125]. More recently, the potential effects of 1,25-(OH)2D3 on the expression of inflammatory cytokines and regulatory markers in T cells were studied [129]. It was found that the administration of 1,25-(OH)2D3 down regulated the production of effector cytokines such as IL17, a hallmark of Th17 effector responses [129-130]. Interestingly, 1,25-(OH)2D3 treated T cells adopted the phenotypic and functional properties of adaptive regulatory T cells (T-regs), expressing high levels of the markers CTLA-4 and FoxP3 [129], and with suppressive function [131]. Seemingly, the immunomodulatory effect of 1,25-(OH)2D3 appears to be mainly immunosuppressive. Its effects on both the innate and adaptive immune system demonstrate an ability to gear the immune response towards a more regulatory nature. However, under physiological conditions a balance of the immunoregulatory effects may exist, in which 1,25(OH)2D3 may function to facilitate rather than inhibit element of normal immune response [13]. 18 1.8 Rationale and Hypothesis The regulated presence of 1,25-(OH)2D3 in immune cells demonstrates a critical role for the VDR-vitamin D3 system in immune function. As previously discussed, it has been established that 1,25-(OH)2D3 can suppresses the adaptive and innate immune system [4, 8-11]. In contrast, it has also been demonstrated that some parameters of the innate immune system are stimulated by 1,25-(OH)2D3 [11]. Together these data could lead to the hypothesis that 1,25(OH)2D3 treatment would support a more tolerogenic immune state, due to down-regulation of antigen-presentation events. Thus, the purpose of our studies was to delineate whether the 1,25(OH)2D3 would down-regulate cross-presentation and cross-priming . Previous work in the Basta lab demonstrated that the introduction of the LCMV-NP via the cross-priming pathway alters the immunodominance hierarchy of an LCMV infection significantly. Building upon these observations, our study utilized the HEK-NP model to study the effects of LCMV-NP cross priming in the presence of 1,25-(OH)2D3. For our study, we utilized a single low dose (100 ng) of 1,25-(OH)2D3, that is similar to previous reports [132]. We used this dose to limit the effects to the initial antigen cross-priming events that occur within the first 24 h; if we were to prolong exposure it could be argued that 1,25-(OH)2D3 would be acting directly at the T cell level. It is important to delineate how these cross-priming events are regulated, and how the presence of exogenous molecules such as 1,25(OH)2D3 can influence this outcome. 19 Chapter 2 Materials and Methods 2.1 Cells and reagents Cell culture media, Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute Medium (RPMI-1640), were purchased from Invitrogen (ON, Canada). Fetal Bovine Serum (FBS) was purchased from HyClone (Fisher Scientific, ON, Canada). The Human Embryonic Kidney (HEK293) cell line [ATCC, Manassas, USA] was cultured in DMEM containing 10% FBS. The HEK-NP cell line is derived from HEK293 cells that are stably transfected with a plasmid encoding the LCMV nucleoprotein (HEK-NP cells) as well as a selection plasmid for puromycin resistance [39]. HEK-NP cells were cultured in DMEM containing 10% FBS and 2.5 µg/mL puromycin; the puromycin was prepared from 25 mg powder dissolved in H2O [Sigma-Aldrich, Oakville] to select for LCMV-NP expression. The cell lines DC 2.4 and BMA, a murine dendritic and macrophage cell line respectively, were a gift from Dr. K. Rock (University of Massachusetts Medical School, Worcester, MA) and were used as previously described [139]. The MC57 mouse fibrosarcoma cells [ATCC, Manassas, USA] was used to titrate the LCMV-WE. BMA, DC2.4, and MC57 cells were cultured in RPMI containing 5% FBS. Cell lines were grown in polystyrene flasks [Sarstedt, Montreal; Nunc, Rochester, NY; CellStar Greiner Bio-One,Germany] at 37°C in a CO2 humidified incubator. Cells were passaged or collected for further experimentation with 0.5% trypsin/sterile PBS solution when 80% confluent [Invitrogen, Burlington]. Viable cells were counted by staining with 0.3% (v/v) trypan blue exclusion method [Sigma-Aldrich, Oakville] (2% EDTA v/v [Fisher Scientific, Whitby]/PBS), after which unstained, or viable, cells were counted with a haemocytometer [American Optical, Buffalo, NY]. 2.2 Mice b C57BL/6 (H-2 ) mice were obtained from Charles River (St Constant QC, Canada) and were used in accordance with the guidelines of the Canadian Council of Animal Use. 20 Experimental protocols were approved by the Queen’s University Animal Care Committee (UACC). Animals were used for experimentation at 6-8 weeks of age. 2.3 Preparation of bone marrow-derived macrophages and dendritic cells Femurs and tibias from 6-8 weeks old C57BL/6 mice were collected and the marrow was flushed with PBS using a 26 G 3/8 sterile needle [Becton-Dickinson, Rutherford, NJ]. Bone marrow cells were washed twice with sterile PBS, and red blood cells lysed by re-suspending in lysis buffer (1.66% w/v ammonium chloride) for 5 min. Bone marrow cells were re-washed with sterile PBS and debris removed by passing the cell suspension over a metal sieve. Approximately 4-6 x 107 cells were obtained per mouse. Bone marrow derived macrophages (BMDM) were generated as previously described [71, 133]. Briefly, cells were cultured in 6-well tissue culture plates (3-5 x 106 cells/ well) in RPMI media containing 10% FBS in addition to 20% of L929 supernatant as a source of M-CSF [71]. Non-adherent cells were removed and fresh medium added after 3 days. This process was repeated every 2 days until day 7 when cells were harvested with 1x Trypsin-EDTA and used for experimentation. Bone marrow derived dendritic cells (BMDC) were generated as previously described [134] with slight modifications [71]. Briefly, bone marrow cells were cultured in 6-well tissue culture plates (3-5 x 106 cells/ well) in RPMI medium containing 10 % FBS, supplemented with 50 μM 2ME (Bioshop Canada Inc., Burlington, Ontario), 10 ng/ml GM-CSF (Cedarlane, Ontario, Canada), and gentamicin 3 μg/ml (Invitrogen, Ontario, Canada). Every 2 days, 2 mL of medium was removed and replenished with fresh medium. On day 6, non-adherent cells were transferred to a new 6-well plate and left for 24 hours at 37°C before using in the assays. The loosely adherent cells (highly enriched CD11c+ MHC-II+ BM-DC) were harvested and used for experimentation. 2.4 Induction of cell death To induce cell death, HEK293 or HEK-NP cells were snap frozen in liquid nitrogen (to cause lysis) and then treated with a CL-1000M UV cross-linker at a radiation intensity of 200,000 µJ/cm2 (maximum intensity) [Ultraviolet Products, Upland, CA] for 5 minutes. It has been 21 previously demonstrated that UV light activates the Fas pathway, a potent mediator of apoptotic cell death [135]. After irradiation, cells were re-suspended in sterile DMEM to the appropriate concentration for each experiment. 2.5 Virus Production & Titration The L929 fibroblast cell line [ATCC] was used to propagate LCMV-WE [obtained from F. Lehmann-Grube, Hamburg, Germany]. Briefly, L929 cells were allowed to grow to approximately 80% confluency (T150) flasks with DMEM containing 5% FBS. Medium was removed, and L929 cells were infected with LCMV-WE at a multiplicity of infection (MOI) of 0.01 in 5 mL of fresh medium. Flasks were incubated for 1 h at room temperature on a shaker, after which 20 mL of fresh medium was added. Flasks were incubated for 48 hours at 37°C. Supernatant containing virus was collected and aliquoted on ice at 48 and 72 hours. Stocks were kept frozen at -80°C for further use. Virus stocks were titrated using a murine fibroblast MC57 cell line [ATCC] [136]. Briefly, in a 24-well plate MC57 cells and various dilutions of the initial virus stock were incubated for 48 hours with an agarose overlay at 37°C to allow for virus plaques to form. Cells were fixed with 4% formalin-PBS for 30 min [Sigma-Aldrich, Oakville] and permeabilized with 1% Triton X-100 solution [Fisher Scientific, New Jersey] for 20 min. Cells were stained for LCMV-NP using a rat anti-LCMV-NP antibody [136], followed by a secondary peroxidaseconjugated Affinipure goat anti-rat IgG (H+L) antibody (Lot number 67165) [Cedar Lane, Hornby]. A substrate containing 12.5ml stock A (0.2M Na2 HPO4 2H2O) [Sigma-Aldrich, Oakville], 12.5ml stock B (0.1M Citric Acid) [J. T. Baker Chemical Co., Phillipsburgnm], 25ml ddH2O, 20mg Ortho-Phenylendiamin tablet [Sigma-Aldrich, Oakville], and 50μl 30% H2O2 [Sigma-Aldrich, Oakville] was added and incubated at room temperature until a colour reaction occurred, such that plaques could be visualized. Plaques were then counted for quantification of virus titer. 22 2.6 1,25-(OH)2D3 stimulation of immune cells 1,25-(OH)2D3 was purchased from Cayman Chemical Company [Ann Arbor, MI]. The crystalline solid was resuspended in minimal DMSO and PBS, and kept as a 10 mg/mL stock until further use. 1,25-(OH)2D3 was used at a final concentration of 1 ng/mL equivalent to 2.5nM. In general pAPCs, DC2.4, BMA, BMDC or BMDM (1-5 x 105 cells/well), were stimulated with 1,25-(OH)2D3 (1 ng/mL) for 6 h or 24 h in 96-well round-bottom culture plates (Corning Inc.). Dose response experiments for 1,25-(OH)2D3 were in the range of (0-100 ng/mL). For in vivo experimentation, C57BL/6 mice, were injected with or without 1,25-(OH)2D3 (100 ng/mouse) similar to previous reports [132] via intraperitoneal route. After the appropriate time course (0-24 h), mice were sacrificed and peritoneal lavages were collected under sterile conditions. For peritoneal lavages, abdominal cavities were flushed with 10 mL of sterile PBS using a 10mL syringe and 22G1 needle [Becton Dickinson, Franklin Lakes, NJ]. 2.7 Reverse Transcriptase (RT)-PCR Cultured pAPCs cells were pre-treated with 1,25-(OH)2D3 (0.2 µM in 5% RPMI). For in vivo RT-PCR experimentation, mice were treated with 1,25-(OH)2D3 (100 ng/mouse) or PBS alone (control) via intraperitoneal route. After the appropriate time course (0-24 h), mice were sacrificed and peritoneal lavages were collected under sterile conditions. Collected cells were pelleted and RNA extracted as described below. Total RNA was isolated from collected samples using TRI reagent (Sigma-Aldrich, Oakville, ON), and reverse transcribed using a RT master mix (M-MuLV RT, random primers, dNTPs, RNase inhibitor) prepared with reagents obtained from New England Biolabs (Ipswich, MA). PCR was performed using Taq 5X Master Mix (New England Biolabs) and the following primers obtained from Integrated DNA Technologies (Coraville, Iowa): 18S rRNA F: 5’-AAACGGC TACCACATCCAAG-3’, R: 5’-CCTCCAATGGATCCTCGTTA-3’; VDR rRNA F: 5’GTGCGGAT TTCCTTTGTGAT-3’, R: 5’-ATTGTCTTCGCTAGAGCCCA-3’; CYP24a1 rRNA F: 5’-CCGCAT CACCAAGGAGAACC-3’, R: 5’-TAGCTCCCTGTACTTACGTC-3’; IL-10 rRNA F: 5’-ATT TGA ATT CCC TGG GTG AGA A-3’, R: 5’-ACA CCT TGG TCT AGC TTA TTA A-3’. PCR assay cycles and annealing temperatures were as previously described [137]. The amplified products were resolved by electrophoresis on 1.2% agarose gels 23 and visualized by UV detection of ethidium bromide intercalation on the AlphaInnotech HD2 Imager (AlphaInnotech). 2.8 Western blotting Cell pellets (4.0 x 106) were lysed with 300 µl of lysis buffer (50 mM HEPES (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 1% Triton X-100, and 10% glycerol) on ice for 10 min. Cell lysates were boiled at a 1:1 ratio with 2x reducing Laemmli sample buffer for 5 min at 95°C, and separated by SDS-PAGE (12% gel). Proteins were blotted onto PVDF membranes (Pall Corporation). Membranes were blocked in PBS containing 0.2% Tween 20 and 5% BSA for 1 h. Membranes were washed three times in 1xTBST before being incubated with the rabbit anti-VDR antibody at 0.2 μg/mL (Santa Cruz Biotechnology, # SC-9164) in PBS containing 0.2% Tween 20 and 5% BSA overnight at 4°C, washed three times in PBS/0.2% Tween 20, and then incubated with HRP-conjugated Ab (1 μg/ml) in PBS/5%BSA; Dako Cytomation). Western blots were detected by enhanced chemiluminescence with Amersham’s ECL Advance kit (Amersham, Piscataway, NJ). 2.9 Fluorescence microscopy For microscopic work, either BMA or DC2.4 cells (1x 105) were seeded on sterile Fisherbrand microscope coverslips [Fisher Scientific, Whitby] in the wells of a 24-well flat bottom tissue culture plate [VWR, Mississauga]. Cells were allowed to adhere before treating with 1,25(OH)2D3 (1 ng/mL) over a 24 h time-course. After treatment, cells were subsequently fixed in 1% paraformaldehyde for 45 minutes, then permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature. Coverslips were washed twice with PBS and incubated with antirabbit VDR antibody at 2.8 μg/ml (Santa Cruz Biotechnology) overnight at 4 °C. After adequate washing with PBS, coverslips were then incubated with AlexaFluor488 anti-rabbit Ab (Molecular Probes, USA) at 1.0 μg/ml. Coverslips were washed twice with PBS and then examined using fluorescence microscopy (Leica DM IRE2, Germany) at 60X magnification. acquired using the Leica DFC340 digital camera. 24 Images were 2.10 Analysis of VDR expression by flow cytometry For these experiments, either BMA or DC2.4 cells (1x 107) were seeded in 12-well tissue culture plates, and allowed to adhere before treating with 1,25(OH)2D3 (1 ng/mL) over a 48 h time-course. Cells were subsequently fixed in 1% paraformaldehyde for 45 minutes then permeabilized with 0.2% Triton X for 10 minutes at room temperature. Coverslips were washed twice with PBS and incubated with anti-rabbit VDR antibody at 2.8 μg/ml concentration (Santa Cruz Biotechnology) overnight at 4 °C. Cells were then incubated with 1.0 μg/ml AlexaFluor488 anti-rabbit Ab (Molecular Probes, USA). Cells were washed and resuspended in ~ 400 μL FACS buffer (0.8g NaCl, 0.02g KCl, 0.115g Na2HPO2, 0.013g NaN3 in 100 mL) and subsequently acquired with an Epics XL-MCL flow cytometer [Beckman Coulter, Miami, FL]. 2.11 Measurement of T cell activation by intracellular cytokine staining (ICS) and FACS analysis For ex vivo experimentation, the cell suspensions from tissues were divided into wells of a 96 well tissue culture plate as needed and antigen donor cells (ADC) (macrophages, dendritic cells, or splenocytes) were added, at the appropriate ratio as outlined in each section below, pulsed with an LCMV epitope (107M); either NP396-404 (FQPQNGQFI), NP205-212 (YTVKYPNL), GP33-41 (KAVYNFATC), or GP276-286 (SGVENPGGYCL) from CPC Scientific (San Jose, CA) and incubated with tissue cells at 37°C in the presence of Brefeldin A (10 µg/mL) for restimulation. To specifically study the activation of CD8+ T cells, intracellular cytokine staining (ICS) was restricted to the detection of IFN-γ (produced upon activation) in CD8+ T cells. Activated CTLs from the in vivo experiments were placed in a 96-well tissue culture plate and stained with PE-Cy5 anti-mouse CD8a (Ly-2) antigen monoclonal antibody [Cedar Lane, Hornby] (50μl, 1 μg/mL in PBS) on ice and incubated in the dark for 30 minutes. The samples were subsequently fixed with 1% paraformaldehyde (50 µl), washed twice, and stained with a FITC-conjugated rat monoclonal anti-mouse IFN-γ antibody anti-body (1 μg/mL in PBS) (Lot number 0604) [Caltag Laboratories, Burlingame, CA] diluted in 0.1 % saponin in PBS [Sigma-Aldrich, Oakville] to permeabilize overnight (4°C). Next day, cells were washed and harvested in ~400 μl FACS buffer. Cell acquisition for staining and analysis were complete by flow cytometry. 25 All data from processed samples were acquired on a Beckman Coulter Epics XL-MCL flow cytometer, with a 488 nm argon laser, and analyzed with the Expo 32 Advanced Digital Compensation Software package [Beckman Coulter, Miami, FL]. 2.12 Phagocytosis assays The pAPCs, DC2.4, BMA, BMDC or BMDM, were seeded (1x105 cells/well) in roundbottom 96-well plates. Cells were allowed to adhere before treating with 1,25-(OH)2D3 (1, 10, 100 ng/mL) over a 24 h time-course. pAPCs were co-cultured with antigen donor cells (ADCs) at a ratio of 3:1 (ADCs: pAPCs). For these experiments, HEK293 cells served as ADCs. HEK293 cells were labelled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE) according to manufacturer’s instructions (Invitrogen). Briefly, HEK293 cells were harvested and re-suspended in PBS followed by incubation with CFSE dye (0.2 μg/ml) for 15 min at 37 C. Cells were washed twice and re-suspended in fresh DMEM media containing 5% FBS. CFSE-labelled HEK293 cells were lysed and UV irradiated (LyUV), causing the cells to undergo death [39]. Pre-treated pAPCs and LyUV treated ADCs were co-cultured for 1 hr at 37 C, after which pAPCs were labelled with an anti-mouse H-2Kb R-PE antibody for 15 min at room temperature. This allowed pAPCs to be distinguish from ADCs (CFSE-labelled only). Cells were analyzed by flow cytometry and the percentage of phagocytosis was calculated based on the number of double positive macrophages that indicated uptake of the CFSE-labeled LyUV-treated HEK cells. Briefly, cells were washed and resuspended in ~ 400 μL FACS buffer (0.8g NaCl, 0.02g KCl, 0.115g Na2HPO2, 0.013g NaN3 in 100ml) and subsequently acquired with an Epics XL-MCL flow cytometer [Beckman Coulter, Miami, FL]. 2.13 Cross-presentation assay For these experiments, pAPCs, either DC2.4 or BMA, were seeded (1.0 x 105 cells/well) and pretreated with various doses of 1,25(OH)2D3 (1, 10, 100 ng/mL) for 6 or 24 h in a 96 well plate as previously described (Section 2.4). Pre-treated pAPCs were co-incubated with ADCs (in this protocol LyUV treated HEK-NP cells) at a ratio of 1:5 for 18 h. This time period allowed for 26 exogenous antigen (LyUV HEK-NP) uptake, processing and presentation on MHC class I. As a readout, the activation of NP396-specific T cell lines was measured (see section 2.11). This allowed us to determine how antigen was presented on the surface of pAPCs due to the presence of 1,25-(OH)2D3 in culture. For generating peptide-specific T cell lines see section 2.16. 2.14 Preparation of NP396 T cells after cross-priming in vivo Briefly, 6-8 week old C57BL/6 mice, were injected with HEK-NP cells (~7x106 cells) with or without 1,25(OH)2D3 (0.1 μg/mouse) in PBS (900 μl total volume) via intraperitoneal route (using 22G1 needle and 3 mL syringe from [Becton Dickinson, Franklin Lakes, NJ]). In another set of experiments, mice were pre-treated with 1,25(OH)2D3 24 hours before the initiation of cross-priming with HEK-NP with or without 1,25(OH)2D3. After 7 days, mice were sacrificed under sterile conditions and spleens removed and transferred to 15 mL polypropylene screw cap, conical tubes [Sarstedt, Newton, NC] in 10 mL of sterile PBS. Spleens were homogenized by grinding tissues on a metal sieve with the backside of a syringe plunger in a sterile petri dish. Homogenates were centrifuged and re-suspended in RPMI media containing 10% FBS in a new 15 mL polypropylene screw cap, conical tubes [Sarstedt, Newton, NC]. Lymphocytes were purified by Isopaque Ficoll-gradient centrifugation by adding 2.0 mL of lymphocyte separation medium [Fisher Scientific, Whitby] underneath the cell suspension and centrifuging (1200rpm, 14min, low break) (Williams and Bevan 2007). The interface was carefully removed and placed in a fresh 15 mL tube, washed with sterile PBS and centrifuged to pellet. Cells were re-suspended in CTL-medium (RPMI, 10% FBS, 45μl IL-2 supernatant, 50μM 2-mercaptoethanol [BioShop, Burlington], 3 μg/ml gentamycin [Invitrogen, Burlington]). To re-stimulate NP396-specific T cells, APCs (DC2.4 or BMAs) were pulsed with NP396 specific peptide and gamma irradiated (4000-5000 rads) to inhibit further cell division. These treated antigen donor cells were then added to each well of splenocytes (at a ratio of 10 splenocytes: 1 APC) in a 6-well culture plate. The cells were incubated at 37°C for 5-6 days. After 5-6 days, the cells were collected and again separated by Isopaque Ficoll centrifugation, and re-suspended in the wells of a 24-well plate for 2 additional days. After these 2 days, the cells were ready for incubation with pAPCs at a ratio of 10 tissue cells: 1 pAPC pulsed with the NP396 synthetic peptide in the presence of BFA (10 μg/ml) for 4 27 hours at 37C, before staining with FITC-labeled antibody specific for IFN-γ (a specific marker for T cell activation) and PE-Cy5-labeled anti-CD8 antibody (a specific marker for cytotoxic T cells). 2.15 Evaluation of Immunodominance Briefly, 6-8 week old C57BL/6 mice, were injected with HEK-NP cells with or without 1,25(OH)2D3 (100 ng/mouse) via i.p. route. After 7 days, mice were infected with LCMV-WE intravenously (i.v.) (200 pfu / in 200 ul using a 26G 5/8 needle and 1 mL syringe) [Becton Dickinson, Franklin Lakes, NJ]). After an additional 7 days, mice were sacrificed and murine cells were obtained from peritoneal lavage and spleens. In another set of experiments, mice were pre-treated with 1,25(OH)2D3 24 hours before the initiation of cross-priming with HEK-NP with or without 1,25(OH)2D3. Spleens were homogenized and red blood cells were lysed with lysis buffer (1.66% w/v ammonium chloride) for 5 min. Cells were washed and incubated with virus peptide pulsedpAPCs in a peptide restimulation assay to detect IFN-γ cytokine production (see section 2.11), at a ratio of 10:1 (splenic cells: pAPCs) or 3:1 (lavage cells: pAPCs). All experiments using mice were repeated at least three times using three mice for each condition. 2.16 Culturing of epitope-specific T lymphocytes To generate memory CTLs, 6-8 week old C57BL/6 mice, were injected with LCMV-WE intravenously (200 pfu) as previously described [71]. After an additional 4 weeks, mice were sacrificed and spleens harvested under sterile conditions. Spleens were homogenized, and the homogenates were centrifuged and re-suspended in RPMI media containing 10% FBS. Lymphocytes were purified by Isopaque Ficoll-gradient centrifugation and cells were resuspended in CTL-medium (RPMI, 10% FBS, 45μl IL-2 supernatant, 50μM 2-mercaptoethanol [BioShop, Burlington], 3 μg/ml gentamycin [Invitrogen, Burlington]). To restimulate and expand the NP396-specific memory T cells, antigen-presenting cells (DCs) were pulsed with NP396 synthetic peptide and gamma irradiated (4000-5000 rads) to inhibit further cell division. These treated antigen donor cells were then added to splenocytes (at a ratio of 10 splenocytes: 1 APC) in a 6-well culture plate. The cells were incubated at 37°C for 5-6 28 days. Then the cells were collected, dead cells were removed by Isopaque Ficoll centrifugation, and live cells were cultured for 2 additional days. Tests were performed at this time point where T cells were incubated with APCs (at a ratio of 1 tissue cells: 1 APC). The APCs were pre-pulsed with NP396 peptide, and then 10μg/ml BFA was added for 3 hours. CD8+ T cell activation was measured by ICS followed by FACS analysis (see section 2.11). 2.17 Statistics Statistics were completed using Microsoft Office Excel 2007 software. Paired, two-tailed t tests were performed and differences in results between treatment conditions were deemed significant when p < 0.05. 29 Chapter 3 Results We approached this study based on multiple reports suggesting that 1,25-(OH)2D3 suppresses key immune cells, such as DCs, and thus would likely inhibit cross-presentation [124]. The role of cross-priming in regulating the immunodominance hierarchy has been previously reported by utilizing HEK-NP cells to study cross priming of LCMV-NP [77]. The current study attempted to build upon these observations, by delineating the role of 1,25-(OH)2D3 on the ability of pAPCs to cross prime CD8+ T cells. Specifically, the effects on exogenous antigen uptake, cross-presentation, cross-priming, and the immunodominance hierarchy were addressed as described below. 3.1 Assessment of 1,25-(OH)2D3 biological effects in pAPCs 3.1.1 The effect of 1,25-(OH)2D3 on gene transcription in pAPCs It has been reported that 1,25-(OH)2D3 mediates its effects through VDR, which is constitutively expressed in target cells such as pAPCs [83]. Thus initially we assessed the effects of 1,25-(OH)2D3 on VDR expression. For these experiments the pAPC cell lines (DC2.4 or BMA) or primary cells (BMDC or BMDM) were stimulated with 1,25-(OH)2D3 (1 ng/mL) for 6 h or 24 h . Post-stimulation, total RNA was isolated and VDR transcript levels were amplified. RT-PCR analysis revealed a detectable basal expression of VDR mRNA in untreated pAPCs (Fig.9) and that 1,25-(OH)2D3 treatment did not alter VDR mRNA transcript levels over the 48 h time-course in either DC2.4 or BMA (Fig. 9a). Comparable results were found for BMDC and BMDM cells over the 24 h time-course (Fig. 9 b). For further confirmation of 1,25-(OH)2D3 biological activity, CYP24A1 expression was measured. CYP24A1 is an enzyme that catabolises the initial degradation of 1,25-(OH)2D3, leading to the production of inactive metabolites [83]. For these experiments, cell lines were stimulated with 1,25-(OH)2D3 for either 48 h (DC2.4 or BMA) or 24 h (BMDC or BMDM). After stimulation, total RNA was isolated and CYP24A1 transcript levels were amplified using specific primers. Our results indicated that CYP24A1 mRNA is not expressed in untreated pAPCs 30 Figure 9. Analysis of 1,25-(OH)2D3 biological effects on pAPCs by RT-PCR. (A) Cells lines (DC2.4 and BMA) or (B) primary cells (BMDC and BMDM) were cultured in the presence of 1,25-(OH)2D3 (1 ng/mL) over a (A) 48 h or (B) 24 h time-course. Samples were tested for the induction of, VDR, CYP24A1 and IL-10; 18S was employed as a control. The PCR products were visualized on 1.2% agarose gels and ethidium bromide stained. One experiment representative of three is shown. 31 (Fig 9a, b). In DC2.4 cells, CYP24A1 transcript expression was induced at 48 h post-stimulation (Fig. 9a). Induction was also detected at 24 h post-stimulation in primary BMDC cells (Fig. 9b). Interestingly, CYP24A1 was undetectable in both BMA (Fig. 9a) and BMDM cells (Fig. 9b) over the course of 1,25-(OH)2D3 stimulation. In addition, it was reported that IL-10 production increases upon 1,25-(OH)2D3 stimulation [4]. As an additional measure of 1,25-(OH)2D3 activity on the immune system, the production of IL-10 in pAPCs was assessed. pAPCs were stimulated as outlined above, total RNA was isolated, and IL-10 transcript levels were amplified using specific primers. Results indicated that IL-10 transcripts were undetectable in untreated DC2.4 (Fig. 9a), BMDC (Fig. 9b), and BMDM cells (Fig. 9b). In contrast basal levels of IL-10 transcript expression was detected in BMA cells (Fig 9a). At 24 h post-stimulation, IL-10 transcript induction was found in DC2.4 cells (Fig. 9a). In treated BMA cells, basal IL-10 transcripts were increased as a result of treatment 24 h post stimulation (Fig. 9a). At 6 h post-stimulation, IL-10 transcript expression was induced in both BMDC and BMDM cells (Fig. 9b), with the levels of induction remaining detectable at 24 h. Thus, we were able to conclude that 1,25-(OH)2D3 was biologically active in our experimental systems. 3.1.2 VDR Expression Next, the effects of 1,25-(OH)2D3 treatment on VDR protein expression were assessed. For these experiments, either DC2.4 or BMA cells were stimulated with 1,25-(OH)2D3 over a 24 h time-course as previously described. Post treatment cell were lysed and samples separated by SDS-PAGE. Membranes were probed with an anti-VDR antibody, followed by an HRP-labelled secondary antibody. Total cellular VDR expression was detected by chemiluminescence. In accordance with our previous results, untreated cells demonstrated basal VDR protein expression. It was found that these VDR protein levels remained unaltered over the entire 24 h time-course of 1,25-(OH)2D3 stimulation (Fig. 10). In addition, VDR protein expression was also assessed by FACS analysis. For these experiments either DC2.4 or BMA cells were treated with 1,25-(OH)2D3 over a 24 h time course. Cells were subsequently fixed and permeabilized, before being incubated with an anti- VDR antibody. Cells were incubated with an appropriate labelled secondary antibody and samples 32 Figure 10. 1,25-(OH)2D3 treatment does not alter basal VDR expression levels in pAPCs. (A) Murine dendritic (DC2. 4) and macrophage (BMA) cells were treated with 1,25-(OH)2D3 (1 ng/ml) over of 24 h time course. Cell lysates were separated by SDS-PAGE, and membranes probed with a rabbit anti-VDR antibody, followed by incubation with an HRP-conjugated antibody. Total cellular VDR expression was detected by chemiluminescence. Data is representative of two experiments. 33 acquired with an Epics XL-MCL flow cytometer . In concurrence with previous results, FACS analysis demonstrated a basal level of VDR protein in the DC2.4 and BMA cells. As a more quantitative method, FACS data analysis revealed a similar expression of VDR in both cell lines. BMA cells were found to have a slightly higher VDR expression than DC2.4, although the difference was not found to be statistically significant. Furthermore, it was re-confirmed that VDR protein expression remained unaffected by 1,25-(OH)2D3 treatment in both DC2.4 and BMA cells (Fig. 11). The effects of 1,25-(OH)2D3 on induced changes in the localization of VDR was also assessed. For these experiments, either DC2.4 or BMA cells were seeded on sterile microscope coverslips and treated with 1,25-(OH)2D3 for 24h . After treatment cells were fixed, permeabilized, incubated with an anti-rabbit VDR antibody, followed by an appropriately labelled secondary antibody. Cells were examined using a fluorescence microscopy at 60X magnification. Images were acquired using the Leica DFC340 digital camera. Upon assessment, it was re-confirmed that both DC2.4 and BMA cells have high constitutive expression of VDR. Images revealed that the immunofluorescent signal was located mainly in the peri-nuclear area in both cell types (Fig. 12). At 6 h post-treatment, it was found that the VDR protein became more abundant in the nuclear region (Fig. 12). This alteration in VDR location was found to decrease at 24 h, when the signal resembles the untreated cells. These results were also reproducible at different doses (1-100 ng/mL) (Fig. 13). In conclusion, 1,25-(OH)2D3 has biological effects in vitro, as it causes VDR to localize to a more nuclear location in pAPCs. Ultimately, this would allow for the VDR:ligand complex to interact with and alter the expression of responsive genes such as CYP24A1 or IL-10. 3.2 Biological effects are observed in mice treated with 1,25-(OH)2D3 The next set of experiments assessed the effects of 1,25-(OH)2D3 treatment in vivo. Administration was through the injection of 1,25-(OH)2D3 (100 ng) in the peritoneal cavity of C57B/6 mice. Specifically, this route was chosen as we induce cross priming of LCMV-NP by the administration of HEK-NP cells by this route. 34 Figure 11. 1,25-(OH)2D3 treatment does not affect VDR expression levels in pAPCs. Intracellular staining was performed to evaluated the VDR expression in (A) DC2.4 and (B) BMA after treatment with 1,25-(OH)2D3 (1 ng/mL) over a 24 hour time course. An anti-rabbit VDR antibody was used to bind VDR, and a secondary AlexaFluor488 antibody was used to visualize the primary antibody staining. Cells were acquired by FACS and mean fluorescence intensity (MFI) was recorded. Negative controls were cells stained with secondary antibody alone. (C) Graphical representation of data obtained and analyzed from (A) and (B). One experiment representative of three is shown. 35 Figure 12. 1,25-(OH)2D3 treatment induces localization of VDR to the nuclear region of pAPCs. Intracellular staining was performed to evaluated the VDR expression in (A) DC2.4 and (B) BMA cells after treatment with 1,25-(OH)2D3 (1 ng/mL) over a 24 hour time course. An antiVDR antibody was used to bind cellular VDR. A secondary AlexaFluor488 antibody was used to visualize the primary antibody staining. Antibody staining was assessed by fluorescence microscopy. Control cells were stained with secondary antibody alone. The images were acquired with a Magnification 60X. One experiment representative of three is shown. 36 Figure 13. Detection of VDR localization in pAPCs using different doses of 1,25-(OH)2D3. Intracellular staining was performed to evaluated the VDR expression in (A) DC2.4 and (B) BMA cells after treatment with various doses of 1,25-(OH)2D3 (1, 10, or 100 ng/mL) over a 24 hour time course. An anti-VDR antibody was used to bind cellular VDR. A secondary AlexaFluor488 antibody was used to visualize the primary antibody staining. Antibody staining was assessed by fluorescence microscopy using magnification 60X. Control cells were stained with secondary antibody alone, with no detectable signals as seen in Figure 12. One experiment representative of three is shown. 37 After 6 or 24 h post-injection, peritoneal macrophages were isolated by lavage and samples from mice in each treatment group (n=3) were pooled. Total RNA was extracted and samples were tested for the induction of VDR, CYP24A1 and IL-10. Low basal levels of VDR were detected in untreated mice, with levels found to increase 6 h after 1,25-(OH)2D3 treatment. Interestingly, IL-10 transcripts were found in untreated mice. Upon analysis, 1,25-(OH)2D3 was found to increase IL-10 expression at 6 h post treatment (Fig. 14). In addition, we detected CYP24A1 induction after 24 h of 1,25-(OH)2D3 treatment (Fig. 14). The induction of CYP24A1 indicates that cells induce this enzyme to degrade the 1,25-(OH)2D3 that we introduced into the peritoneum. We also tested for protein expression in peritoneal cells. Red blood cells were lysed, and proteins were separated by SDS-PAGE. Membranes were probed with rabbit anti-VDR antibody, followed by an HRP-conjugated antibody. Total cellular VDR expression was detected by ECL and visualized. As a positive control, human primary keratinocyte (HPK) cell line was used, provided by Dr. Glenville Jones. Keratinocytes are known to express VDR and respond to 1,25(OH)2D3 [138]. Untreated mice demonstrated detectable levels of VDR protein expression. Interestingly, 1,25-(OH)2D3 administration elevated VDR expression levels in the peritoneum 24 h post administration (Fig. 15). After establishing the biological activity of 1,25-(OH)2D3 treatment, we wanted to assess its effects on the cross-presentation pathway in vitro and in vivo. 3.3 1,25-(OH)2D3 treatment does not alter phagocytosis in pAPCs Our next set of experiments began to investigate the in vitro effects on cross-presentation. The uptake of exogenous antigen by pAPCs is the first step in the cross-presentation pathway, so the effects of 1,25-(OH)2D3 treatment on the ability of pAPCs to phagocytose antigen donor cells (ADCs) were assessed. Briefly, pAPCs were pre-treated with 1,25-(OH)2D3 (0-100 ng/mL) over a 24 h timecourse as previously described. ADCs, for this assay HEK293 cells, were labelled with the fluorescent marker carboxyfluorescein diacetate succinimidyl ester (CFSE), after which they were lysed and UV irradiated (LyUV). This treatment has been shown to induce programmed cell death [39]. Pre-treated pAPCs and labelled LyUV ADCs were co-cultured, after which pAPCs were labelled with an anti-mouse H-2Kb-PE antibody and analyzed by FACS. For FACS 38 Figure 14. Analysis of effects 1,25-(OH)2D3 of gene induction in peritoneal cells of mice by RT-PCR. Mice were injected via intraperitoneal route with 0.1 µg 1,25-(OH)2D3, or PBS (untreated). After 6 or 24 hours, peritoneal macrophages were isolated by lavage and samples from 3 mice pooled for each treatment group. Cells were tested for the induction of VDR, CYP24A1 and IL-10. 18S served as a control for equal loading. The PCR products were visualized on 1.2% agarose gels and ethidium bromide stained. One experiment representative of three is shown. 39 Figure 15. Analysis of peritoneal cell VDR protein expression is up-regulated in mice treated with 1,25-(OH)2D3 by western blot. (A) C57B/L mice were injected i.p. with 0.1 µg 1,25-(OH)2D3 (in PBS) or PBS alone (control); three mice were injected per condition. After 24 hours, peritoneal cells were harvested by lavage, red blood cells lysed, and protein extracted and quantified. For each sample, two amounts of sample were loaded (10 and 20 µg). Cell lysates were separated by SDS-PAGE, and membranes probed with a rabbit anti-VDR antibody, followed by incubation with an HRP-conjugated antibody. VDR expression was detected by chemiluminescence. As a positive control (+) , HPK cells were used. (B) Densitometry analysis, corrected for background. One experiment representative of two is shown. 40 analysis, a gate (based on size and granularity) was drawn to incorporate all live cells (Fig. 16a). A gate was subsequently drawn on H-2Kb positive cells, therefore isolating the pAPC population (Fig. 16b). As a negative control, labelled pAPCs and CFSE-labelled ADCs were mixed and read immediately; thus no phagocytosis was able to occur (Fig. 16c). The percentage of phagocytosis was calculated based on the number of double positive cells, indicating pAPCs that had phagocytosed CFSE-labelled LyUV-treated HEK cells after 1 h of co-incubation (Fig. 16d). Untreated pAPC samples demonstrated a high rate of phagocytosis for all pAPCs (~60-75% double positive) (Fig 17-19). Subsequent data was normalized to untreated samples. Interestingly, the rate of phagocytosis remained unaffected by 1,25-(OH)2D3, independent of both dose and kinetic time points for BMDC (Fig. 17), BMDM (Fig. 18) and both DC2.4 and BMA (Fig.19). Our results reconfirm the observation that BMDC and BMDM are able to phagocytosis exogenous antigen at comparable levels, as previously reported [71]. Taken together, our results excluded any potential immunosuppressive effects on the cross-presentation pathway by 1,25(OH)2D3 at the stage of antigen uptake. 3.4 Investigating the ability of 1,25-(OH)2D3 to affect antigen cross-presentation in vitro After exogenous uptake, antigen is processed and presented to CD8+ T cells in the context of MHC-I molecules. Thus, we wanted to delineate whether 1,25-(OH)2D3 treatment affects the processing and subsequent cross-presentation of exogenous antigen in vitro to CD8+ T cells. For this assay, HEK-NP served as ADCs and were cross presented by pAPCs to NP396 specific CD8+ T cells. Either DC2.4 or BMA cells were pre-treated with 1,25-(OH)2D3 for 24 h. Pre-treated pAPCs were incubated with LyUV treated HEK-NP cells at a ratio of 5:1 for 18 h, allowing for exogenous antigen uptake, processing and presentation on MHC-I molecules. As a readout, the activation of NP396-specific T cell lines was measured to determine how antigen is presented on the surface of pAPCs due to the presence of 1,25-(OH)2D3. Effector T cells were incubated with pAPCs at a ratio of 1:1 in the presence of brefeldin A (10 μg/ml). To specifically study the activation of CD8+ T cells, intracellular cytokine staining (ICS) was restricted to the detection of IFN-γ (produced upon activation) in CD8+ T cells. CD8+ T cells that were stimulated by antigen cross-presented by untreated DC2.4 and BMA cells had comparable levels of activation (~35% IFN-γ + cells). It was found that these levels of activation were unaffected 41 Figure 16. Flow cytometric analysis of the effect of 1,25-(OH)2D3 treatment on phagocytosis by pAPCs. (A) A gate is drawn on un-gated cells, based on forward scatter (granularity) and side scatter (size), to attain all live cells for analysis. (B) A subsequent gate is drawn to isolate pAPCs, or those staining positive for MHC-I. Based on a negative control, or pAPCs that have not been allowed to phagocytose CFSE-labelled ADCs (LyUV HEK cells), a gate is drawn (C) to determine background fluorescence. Based on gate C, the percentage of double positive (MHCI+CFSE+) cells are able to be determined (D) , reflective of the amount of phagocytosis by pAPCs. 42 Figure 17. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated BMDC. BMDC were pre-treated for either 6 or 24 h with 1,25-(OH)2D3 at various concentrations (1, 10 100 ng/mL as indicated on x-axis). For this assay, LyUV HEK293 cells served as ADCs. Pretreated pAPCs and ADCs were co-cultured for 1 hr at 37 C, after which pAPCs were labelled with an anti-mouse H-2Kb R-PE antibody, allowing for pAPCs to be distinguish from ADCs. Cells were analyzed by flow cytometry and the percentage of phagocytosis was calculated based on the number of double positive macrophages that indicated uptake of the CFSE-labeled LyUVtreated HEK cells. (A) Histogram representative of one experiment. The percentage of phagocytosis by untreated BMDC is indicated by the dotted line. Data are representative of one experiment. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). (B) Data are normalized to untreated BMDC for all experiments (n=9). 43 Figure 18. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated BMDM. BMDM were pre-treated for either 6 or 24 h with 1,25-(OH)2D3 at various concentrations (1, 10 100 ng/mL as indicated on x-axis). For this assay, LyUV HEK293 cells served as ADCs. Pre-treated pAPCs and ADCs were co-cultured for 1 hr at 37 C, after which pAPCs were labelled with an anti-mouse H-2Kb R-PE antibody, allowing for pAPCs to be distinguish from ADCs. Cells were analyzed by flow cytometry and the percentage of phagocytosis was calculated based on the number of double positive macrophages that indicated uptake of the CFSE-labeled LyUV-treated HEK cells. (A) Histogram representative of one experiment. The percentage of phagocytosis by untreated BMDM is indicated by the dotted line. Data are representative of one experiment. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). (B) Data are normalized to untreated BMDM for all experiments (n=9). 44 Figure 19. Phagocytosis of exogenous antigen is not affected in 1,25-(OH)2D3 treated cell lines. pAPCs, either DC2.4 (A), BMA (B) were pre-treated for either 6 or 24 h with 1,25(OH)2D3 at various concentrations (0-100 ng/mL as indicated on x-axis). For this assay, LyUV HEK293 cells served as ADCs. Pre-treated pAPCs and ADCs were co-cultured for 1 hr at 37 C, after which pAPCs were labelled with an anti-mouse H-2Kb R-PE antibody, allowing for pAPCs to be distinguish from ADCs. Cells were analyzed by flow cytometry and the percentage of phagocytosis was calculated based on the number of double positive macrophages that indicated uptake of the CFSE-labeled LyUV-treated HEK cells. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). (A) Data are normalized to untreated DC2.4 and (B) BMA cells (n=9). 45 by 1,25-(OH)2D3 treatment (Fig. 20a). The levels of activation remained unchanged when comparing untreated and treated cells for all doses tested (Fig. 20a). As a positive control, a separate set of pAPCs were pulsed with synthetic NP396 peptide prior to incubation with effector T cells. Effector CD8+ T cells demonstrated high levels of activation upon exposure to the synthetic peptide (~70-80 % IFN-γ positive) (Fig. 20 b). It was found that 1,25-(OH)2D3 treatment did not affect the ability of either DC2.4 or BMA cells to activate T cells when presenting synthetic NP396 peptide (Fig. 20 b). These results demonstrate that 1,25-(OH)2D3 does not affect the levels of MHC-I expression on pAPC cellular surfaces. Thus we could conclude that antigen cross-presentation by pAPCs is not affected by 1,25(OH)2D3 treatment in vitro. 3.5 Co-administration of 1,25-(OH)2D3 and LCMV-NP does not significantly affect cross-priming of CD8+ T cells in vivo In parallel to our in vitro investigation, the impacts of 1,25-(OH)2D3 in our in vivo models were assessed. As physiological activity of the active hormone was already delineated, the effects of 1,25-(OH)2D3 on cross-priming were subsequently investigated. Cross priming was induced through injection of 107 HEK-NP cells into the peritoneal cavity of mice. A single dose of 1,25-(OH)2D3 (100 ng per mouse) was co-administered with antigen. On day 7 lymphocytes were isolated, and NP396-specific T cells expanded in vitro by incubation with synthetic NP396-404 peptide pulsed pAPCs. To test for activation, T cells were restimulated by incubating with NP396-404 peptide-pulsed pAPCs in the presence of BFA (10 μg/ml) for 4 hours. Following peptide restimulation, cells were stained for CD8 and IFNγ, and samples were acquired and analyzed by FACS. This allowed us to analyze the in vivo effects of 1,25(OH)2D3 on CD8+ T cell activation due to cross priming. Contrary to our hypothesis, it was found that initial cross priming events were unaffected by 1,25-(OH)2D3 co-administration. As shown in Figure 21, we found that untreated mice (~45% IFN-γ+) and 1,25-(OH)2D3 treated mice (~50% IFN-γ+) generated NP396 specific T cells that had comparable levels of activation. In another set of experiments, mice were injected with a single dose of 1,25-(OH)2D3 24 hours before the initiation of cross-priming. Mice that were pretreated with 1,25-(OH)2D3 received another dose of the hormone co-administered with HEK-NP, 46 Figure 20. Cross-presentation of exogenous antigens by pAPCs is not affected by different doses of 1,25-(OH)2D3 treatment. (A) pAPCs, either DC2.4 or BMA, were treated with various doses of 1,25(OH)2D3 (0-100 ng/mL) for 24 h as indicated on the x-axis. Pre-treated pAPCs were co-incubated with ADCs (LyUV treated HEK-NP cells) for 18 h. This time period allowed for exogenous antigen (LyUV HEK-NP) uptake, processing and presentation on MHC class I. As a readout, the activation of NP396-specific T cell lines was measured. To quantify T cell activation, IFN-γ production was measured by ICS assays and analyzed by FACS. (B) As a positive control, a separate set of pAPCs were also pulsed with synthetic NP396 peptide for 1 h prior to incubation with NP396-specific T cells. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). 47 48 Figure 21. Co-administration of 1,25-(OH)2D3 and antigen does not affect the initial crosspriming of HEK-NP in vivo. C57BL/6 mice were injected with A) HEK-NP cells or HEK-NP cells (107) with 1,25-(OH)2D3 (100 ng) via intraperitoneal route. After 7 days, lymphocytes were obtained and NP396-specific T cells re-stimulated. To quantify T cell activation, IFN-γ production was measured by ICS assays and analyzed by FACS. (B) Histogram representation of raw FACS data, and (C) normalized data. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment 49 and lymphocytes were prepared and tested as described above. Again, we found that T cells generated from both groups demonstrated comparable levels of activation (~45% % IFN-γ+) (Fig. 22). Taken together, we can conclude that 1,25-(OH)2D3 treatment does not affect cross-priming in vivo in our model of experimentation. 3.6 Cross priming of the LCMV nucleoprotein with 1,25-(OH)2D3 does not affect immunodominance We next investigated the effects of 1,25-(OH)2D3 treatment on the immunodominance hierarchy. For these experiments, cross priming was induced through injection of HEK-NP cells in the peritoneal cavity of mice. A single dose of 1,25-(OH)2D3 was co-administered with HEKNP (100 ng per mouse). By injecting one dose, the range of 1,25-(OH)2D3 effects in vivo are limited to the cross-priming events that occur within the first 24 h. In another set of experiments, mice were injected with a single dose of 1,25-(OH)2D3 24 hours before the initiation of crosspriming. Mice that were pre-treated with 1,25-(OH)2D3 received another dose of the hormone, co-administered with HEK-NP. On day 7, 200 pfu LCMV-WE was injected intravenously. Since the LCMV-NP can only be cross presented to murine CD8+ T cells, changes in the immunodominance hierarchy compared to normal LCMV infections have been found to be consequence of cross priming [77]. On day 14, mice were sacrificed and spleens were collected and homogenized; peritoneal lavages were also performed. Next, spleen cells and peritoneal cells were activated ex vivo by stimulation with peptide-loaded pAPCs using optimal concentrations -7 (10 M) of four major dominant synthetic viral peptides of LCMV (either NP 396-404, NP205-212, GP33-41, or GP276-286). Following peptide restimulation, cells were stained for CD8 expression and intracellular cytokine staining was performed for IFN-γ. Samples were acquired and analyzed by FACS, allowing us to analyze the in vivo effects of 1,25-(OH)2D3 on CD8+ T cell activation when cross priming precedes viral infection. As previously reported, in a normal virus infection the immunodominance hierarchies follow: GP33-41 > NP396-404 > NP205-212 > GP276-286. Additionally, it was found that when cross priming with LCMV-NP precedes LCMV infection, a shift in the hierarchy was discovered, following: NP396-404 > NP205-212 = GP33-41> GP276-286 [77]. Contrary to our hypothesis, it was found 50 Figure 22. Pre-injection and co-administration of 1,25-(OH)2D3 with antigen does not affect cross-priming of HEK-NP in vivo. C57BL/6 mice, were injected with 1,25-(OH)2D3 (100 ng) via intraperitoneal route 24 h before the initiation of cross-priming. After 24 h, untreated mice were injected A) HEK-NP cells, while mice that received 1,25-(OH)2D3 prior, were injected with HEK-NP cells (107) with 1,25-(OH)2D3 (100 ng) via intraperitoneal route. After 7 days, mice were sacrificed and spleens were homogenized. Lymphocytes were purified and NP396-specific T cells restimulated. To quantify T cell activation, IFN-γ production was measured by ICS assays and analyzed by FACS. Histogram representation of raw FACS data. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment. 51 that initial cross priming events were unaffected by 1,25-(OH)2D3 co-administration. This was concluded since the hierarchy had the same structure, following: NP396-404 > NP205-212 = GP33-41> GP276-286. Statistical analysis of shifts in epitope-specific CD8+ T cell activation revealed there were no significant differences in the immunodominance hierarchy when cross-priming was induced with LCMV-NP and 1,25-(OH)2D3 or LCMV-NP alone (Fig. 23) Analysis of the spleen revealed no changes in the number of CD8+ population (Fig. 24). Similar results were observed when mice received an additional dose of 1,25-(OH)2D3 24 h before the initiation of crosspriming with HEK-NP (Fig. 25). Again, no significant alterations to the immunodominance hierarchy were observed. The immunodominance hierarchy was also assessed in the peritoneum, the site of crosspriming. Data re-confirmed that when cross priming is followed by viral infection, immunodominance shifts in the peritoneal cavity are more defined compared to the spleen. The strong NP396-404 T cell response was due to the direct injection of NP antigen into this anatomical compartment [77]. Thus the CD8+ T cell immune response to different epitopes in a viral infection can differ depending on the anatomical location [77]. For these studies, there was an observed increase in the percentage of activated NP396-specific CD8+ T cells, in addition to increases for the epitopes NP205 and GP33 when compared to a normal LCMV infection [77]. Our results indicate that co-administration of a single dose of antigen and 1,25-(OH)2D3 did not alter the immunodominance hierarchy observed in the peritoneum when cross-priming normally precedes LCMV infection (Fig. 26). There were observed decreases in the activation of subdominant epitope specific CD8+ T cells (NP205, GP33, GP276). However these results were not found to be significant compared to untreated mice. Similar results were observed when mice received an additional dose of 1,25-(OH)2D3 24 h before the initiation of cross-priming with HEK-NP (Fig.27). Again, no significant alterations to the immunodominance hierarchy were observed. We can therefore conclude that, contrary to our hypothesis, cross priming with a single dose of 1,25-(OH)2D3 and antigen does not alter the immunodominance hierarchy when compared to the untreated condition. Collectively our results imply that cross priming was not affected by 1,25-(OH)2D3. 52 53 Figure 23. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect crosspriming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the spleen. Mice were primed i.p. with A) HEK-NP cells or HEK-NP cells with 1,25-(OH)2D3 (100 ng) and 7 days later infected with 200 pfu LCMV-WE (i.v.). Spleens were collected on day 14 and assessed for % activated NP396, NP205, GP33, or GP276-specific CD8+ T cells by ICS, and FACS analysis directly ex vivo. Dot plots have been gated for CD8 positive cells. Plots are representative data from one experiment. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment . (B) Histogram representation of rawdata. 54 Figure 24. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect number of CD8+ populations in the spleen. (A) Number of CD8+ splenocytes found in spleens. Mice were primed i.p. with A) HEK-NP cells or HEK-NP cells with 1,25-(OH)2D3 (100 ng) and 7 days later infected with 200 pfu LCMV-WE (i.v.). Spleens were collected on day 14 and assessed for total numbers and % CD8+ T cells by staining with anti-mouse CD8 antibody and FACS analysis directly ex vivo. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment. 55 Figure 25. Pre-injection and co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the spleen. C57BL/6 mice, were injected with 1,25-(OH)2D3 (100 ng) via intraperitoneal route 24 h before the initiation of cross-priming. After 24 h, untreated mice were injected A) HEK-NP cells, while mice that received 1,25-(OH)2D3 prior, were injected with HEK-NP cells (107) with 1,25-(OH)2D3 (100 ng) via intraperitoneal route. 7 days later mice were infected with 200 pfu LCMV-WE (i.v.). Spleens were collected on day 14 and assessed for % activated NP396, NP205, GP33, or GP276-specific CD8+ T cells by staining with anti-mouse CD8 antibody, ICS for IFN-γ (using FITC-conjugated rat anti-mouse IFNγ antibody), and FACS analysis directly ex vivo. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment. 56 57 Figure 26. Co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect crosspriming of LCMV NP and the LCMV immunodominance hierarchy in the peritoneum. Mice were primed i.p. with A) HEK-NP cells or HEK-NP cells with 1,25-(OH)2D3 (100 ng) and 7 days later infected with 200 pfu LCMV-WE (i.v.). Peritoneal cells were collected on day 14 and assessed for % activated NP396, NP205, GP33, or GP276-specific CD8+ T cells by staining with anti-mouse CD8 antibody, ICS for IFN-γ (using FITC-conjugated rat anti-mouse IFN-γ antibody), and FACS analysis directly ex vivo. Dot plots have been gated for CD8 positive cells. Plots are representative data from one experiment. Three mice were used for each experiment (n=3) and these experiments were repeated three times. (B) CD8+ percentage in peritoneum were unaffected by co-administration of 1,25-(OH)2D3 and HEK-NP. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment. 58 Figure 27. Pre-injection and co-administration of 1,25-(OH)2D3 and HEK-NP in vivo does not affect cross-priming of LCMV nucleoprotein and the LCMV immunodominance hierarchy in the peritoneum. Mice were injected with 1,25-(OH)2D3 (100 ng) via intraperitoneal route 24 h before the initiation of cross-priming. After 24 h, untreated mice were injected A) HEK-NP cells, while mice that received 1,25-(OH)2D3 prior, were injected with HEK-NP cells (107) with 1,25-(OH)2D3 (100 ng) via intraperitoneal route. 7 days later infected with 200 pfu LCMV-WE (i.v.). Peritoneal cells were collected on day 14 and assessed for % activated NP396, NP205, GP33, or GP276-specific CD8+ T cells by staining with anti-mouse CD8 antibody, ICS for IFN-γ (using FITC-conjugated rat anti-mouse IFNγ antibody), and FACS analysis directly ex vivo. Experiments were repeated three times independently, and error bars represent the standard error of deviation from one experiment (n=3). Three mice were used per condition per experiment. 59 Chapter 4 Discussion The cross-presentation pathway involves the presentation of exogenous antigens by pAPCs to CD8+ T cells. Since 1,25-(OH)2D3 has been demonstrated to adversely affect key cells of the pathway, it is possible that cross-presentation can be manipulated with 1,25-(OH)2D3 to support a more tolerogenic response in vivo [124]. However, there is currently no information available on the effects of 1,25-(OH)2D3 on the cross-presentation pathway. It has been suggested that 1,25-(OH)2D3 modulates the immune response leading to immunosuppression [4]. Many in vitro studies have demonstrated that 1,25-(OH)2D3 is potently immunosuppressive when target cells undergo differentiation in the presence of the active sterol. For example, BMDCs differentiated in the presence of 1,25-(OH)2D3 had inhibited maturation, leading to low expression of co-stimulatory molecules and decreased pro-inflammatory cytokine production [4]. Thus, a major factor we focused on in the current study was the effects of 1,25(OH)2D3 on mature pAPCs, with respect to their ability to cross-prime CD8+ T cells. On the other hand, it was found that TLR2/1 ligation induced the expression of VDR and CYP27A1, which is the enzyme that converts 25-OHD into 1,25-(OH)2D3 [11]. The expression of CYP27A1 lead to a subsequent increase in 1,25-(OH)2D3 production in human MΦs [11]. The cellular production of 1,25-(OH)2D3 was found to induce the antimicrobial peptide cathelicidin, that had direct action against intracellular Mycobacterium tuberculosis [11]. Therefore the induction of an innate immune response can be influenced by presence of the active hormone. In general, the diverse expression of VDR is suggestive of widespread immunological functions [26]. However, the factors that dictate whether 1,25-(OH)2D3 stimulation results in suppressive or stimulatory effects remain to be elucidated. Thus, understanding the exact role of 1,25-(OH)2D3 is important due to the overwhelming evidence of its immunosuppressive effects on individual cells types in vitro. Initially, our study assessed the effects of 1,25-(OH)2D3 on target cells through its interactions at the VDR level. Interestingly, constitutive VDR mRNA and protein expression has 60 been demonstrated in monocytes [112]. However, VDR was down-regulated when the monocytes matured into MΦs [112]. Maturing cells were found to produce gradually increasing amounts of 1,25-(OH)2D3, which was enhanced approximately twofold by IFN-γ stimulation at all stages of differentiation [112]. This study provides evidence of a regulatory role for 1,25(OH)2D3 in the maturation process of monocyte-dervied MΦs. Interestingly VDR protein was upregulated by 1,25-(OH)2D3 stimulation in monocytes and, to a lesser degree, in MΦ. However, VDR mRNA concentrations were not influenced by 1,25-(OH)2D3 [112]. Thus, cells of the monocyte/MΦ lineage possess small amounts of VDR that are not affected by activation but are increased by treatment with 1,25-(OH)2D3 [78]. Several reports have supported this observation by demonstrating that VDR expression levels vary with the differentiation state of monocytes [98, 112]. In our study, we found no significant changes in either VDR protein or mRNA levels when testing the effects of 1,25-(OH)2D3 treatment on pAPCs. It is possible that the maturation state of these cell lines, for example the DC2.4 has been shown to be in a semi-mature state [139], resulted in different responses to those previously observed [98, 112]. However, it has been demonstrated in vitro that MΦ treated with 1,25-(OH)2D3 did not display any changes in VDR expression levels [140]. Similarly, mature human monocyte derived-DCs are less sensitive to 1,25-(OH)2D3 treatment when compared to progenitor cells differentiated in the presence of the active hormone [141]. Furthermore, DCs differentiated in the presence of 1,25-(OH)2D3 were unable to be activated by TLR stimulation, remaining in an immature state, characterized by lower expression of MHC-II and co-stimulatory molecules [141]. Therefore, in vitro, the influence of 1,25-(OH)2D3 on VDR could be dependent on the maturation or activation state of the cells [140]. It has been shown that MΦ subjected to 1,25-(OH)2D3 attain enhanced expression of the pro-inflammatory cytokine TNF-α [1]. However, the increases occurred in monocytes, MΦ, and in immature cells such as bone marrow cells, while production was decreased in mature cells such as peripheral blood mononuclear cells [1]. This is another example of how the effects of 1,25-(OH)2D3 are contingent upon the maturation state of the target cell. As previously stated, 1,25-(OH)2D3 stimulation induces IL-10 production in immune cells [4] such as DCs. In addition to enhancing IL-10, 1,25-(OH)2D3 decreases IL-12, while promoting DC apoptosis [4]. Collectively, the effects of 1,25-(OH)2D3 inhibit DC-dependent T 61 cell activation. Furthermore, the production of IL-10 is essential in the 1,25-(OH)2D3 mediated inhibition of experimental autoimmune encephalomyelitis (EAE) [142]. 1,25-(OH)2D3 was unable to prevent the manifestation of EAE in IL-10 or IL-10 receptor knock-out mice, demonstrating that it may be enhancing an IL-10 mediated anti-inflammatory loop [142]. We were able to confirm the up-regulation of IL-10 mRNA expression in pAPCs when treated with 1,25-(OH)2D3 over a 24 h time-course. Thus, it was anticipated that the suppressive effects of 1,25-(OH)2D3 on DC-dependent T cell cross-priming would be observed. CYP24A1 expression was also measured upon 1,25-(OH)2D3 stimulation. The catabolism of 1,25-(OH)2D3 is catalyzed by CYP24A1, with its expression being potently induced by 1,25-(OH)2D3 [83]. This negative feedback loop is crucial for avoiding the accumulation of internal excesses of 1,25-(OH)2D3 [84-85]. In human DCs the induction of CYP24A1 mRNA levels was observed after stimulation with 1,25-(OH)2D3 [143]. In our study, when measuring CYP24A1 transcript levels in treated pAPCs, it was found that treatment induced expression in DCs, however not in MΦ. This may be related to the expression profiles of CYP24A1 in different MΦ populations, as well their activation state [140]. For example in other reports, undifferentiated monocytes were found to be highly susceptible to 1,25-(OH)2D3 mediated CYP24A1 induction [13, 26] whereas activated or differentiated MΦ were resistant to induction of CYP24A1 [144]. This was due to the reduced binding of VDR/RXR complexes to VDRE in the promoter, which resulted in decreased CYP24A1 activation [144]. An important deviation of our model from published reports of the immunosuppressive effects is the absence of 1,25-(OH)2D3 during pAPC differentiation, reiterating the fact that the time of target cell exposure is critical. Although we observed several biological effects after 1,25-(OH)2D3 treatment of pAPCs, we did not record any significant changes in the phagocytosis of cell associated antigens in vitro. We examined this function because uptake of exogenous antigen is a critical step in the cross-presentation pathway. It was surprising that no immediate effects were observed, as in other models it was shown that that treatment of monocytes with 1,25-(OH)2D3 enhanced the immunoglobulin- and complement-dependent phagocytosis in a dose- and time-dependent manner [145]. However, this model investigated the effects of 1,25-(OH)2D3 and IL-4 on the differentiation and activation of human blood monocytes [145]. It was found that 1,25-(OH)2D3 inhibits MHC-II expression as well as accessory activity of monocytes. However when IL-4 and 62 1,25-(OH)2D3 are co-administered, the effects of 1,25-(OH)2D3 are reverted by IL-4, suggesting that depending on defined stimuli, monocyte development can be altered based upon the exogenous messengers present [145]. Although efficient presentation of exogenous antigens was originally attributed to MΦ [146], it is now clear that such function can be achieved by different bone marrow-derived APCs including, spleen-derived MΦ [147] and DCs [148-150]. Interestingly, it appears both spleen and bone marrow-derived MΦ down regulate their ability to cross present cell associated antigens during differentiation [147]. We did not monitor the effects of 1,25-(OH)2D3 during differentiation of pAPCs. Since the immunosuppressive effects of 1,25-(OH)2D3 have been reported during the differentiation of BMDCs, future work should attempt to study how phagocytosis is affected when pAPCs are differentiated in the presence of the active sterol. The most pronounced effect of 1,25-(OH)2D3 has been demonstrated in pAPCs, especially DCs [26]. Multiple in vitro studies have demonstrated that 1,25-(OH)2D3 potently inhibits DC differentiation from either human peripheral blood monocytes or murine bone marrow cells in a VDR-dependent mechanism [9-10, 115-119]. Cross-priming of CD8+ T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs [151]. The maturation process of DCs is seriously impaired by 1,25-(OH)2D3 , as they have decreased surface expression of MHC class II, and co-stimulatory molecules (CD40, CD80, CD86) [26]. This would hypothetically block priming of naïve T cells. Analysis of the effects of 1,25-(OH)2D3 demonstrated a capacity to modulate cytokine and chemokine production in DCs [143]. In myeloid DCs (mDCs), the production of IL-12 (signature cytokine), MHCII expression and CCR7 (a key regulator of DC migration to secondary lymphoid organs) expression were markedly inhibited [143]. As all of these molecules are controlled by NF-kB signal transduction pathway, it is suggested that 1,25-(OH)2D3 targets NFkB in certain DC populations [143] . In concurrence, the effects were not observed in plasmacytoid DC (pDC) [143, 152]. This differential capacity to respond was not due to diverse VDR-expression or VDR-dependent signal transduction [153] as both mDCs and pDCs express similar levels of VDR and responded equally well to VDR ligation [154-155]. It is suggested that since pDCs may represent naturally occurring regulatory DCs [61, 156], the administration of 1,25-(OH)2D3 would leave their innate functions unmodified [154-155]. 63 As previously mentioned, 1,25-(OH)2D3 alters the cytokine expression pattern of DCs in vitro, with strong increase in IL-10 expression [9, 119] supportive of cells with a tolerogenic or regulatory phenotype [9-10, 115-119, 121-122]. Such tolerogenic DCs, contribute to the induction of regulatory T cells, T cell anergy and apoptosis [123]. Furthermore, 1,25-(OH)2D3 was found to significantly influence the functional differentiation of BMDCs by down-regulation of co-stimulatory molecules. [157]. 1,25-(OH)2D3 also significantly lowered IFN-γ and IFN-β mRNA expression, both of which are essential for cytotoxic T lymphocyte induction [157]. This inhibition could explain the failure in the induction of immunity by 1,25-(OH)2D3 -treated BMDC [157]. Thus, 1,25-(OH)2D3 inhibited the differentiation of functionally competent BMDC during the early phase of differentiation but not during the late differentiation period [157]. Accordingly if 1,25-(OH)2D3 acted in a similar suppressive manner in our system, we had expected to see strong suppression on cross-presentation. However, using our experimental model we found that treatment of pAPCs with 1,25-(OH)2D3 did not affect their ability to crosspresent exogenous antigen to T cells in vitro. As we had established that there was no effect on exogenous antigen uptake, we were able to determine through these experiments that the ability to process and present antigen in the context of MHC-I molecules was also unaffected in vitro. Taken together, and in view of published data in other models, our results support the observation that the in vitro immunomodulatory effects of 1,25-(OH)2D3 are contingent upon the maturation state of the cells. It could be stated that precursor cells differentiating in a microenvironment with high local 1,25-(OH)2D3 will be predisposed to be tolerogenic or regulatory. This could be due to the fact that overproduction of 1,25-(OH)2D3 is often indicative of disease, as seen in chronic granulomatous diseases [158]. In vitro, pAPCs treated with 1,25-(OH)2D3 did not display any changes in VDR expression levels, most likely because of the maturation state of the cells [140]. To investigate these effects in vivo, we introduced a single dose of 1,25-(OH)2D3 via i.p. route. By using a single low dose, we reduced the manifestation of the potentially detrimental effects of 1,25-(OH)2D3, which can include symptoms such as hypercalcemia and bone de-mineralization associated with chronic exposure [83]. When mice were injected with 1,25-(OH)2D3 i.p., we detected an increase in VDR mRNA transcript levels 6 hr post treatment. In conjunction with these observations, an increase in VDR protein level was observed 24 hours post injection in peritoneal cells, which are mainly composed of MΦ. Thus, 1,25-(OH)2D3 was found to increase the transcriptional 64 expression of VDR mRNA, subsequently followed by the translational production of protein. This indicates that 1,25-(OH)2D3 has biological effects in the anatomical location where the crosspresentation of antigens occurs. In one study, recombinant VDRs were incubated with skin lysates in the presence or absence of 1,25-(OH)2D3 [159]. It was found that the presence of 1,25-(OH)2D3 substantially protected VDRs against degradation and that there was a subsequent increase in VDR protein [159]. Thus, the presence of 1,25-(OH)2D3 in the peritoneum could protect immune cell VDR from degradation, resulting in the observed elevated levels post treatment. As further confirmation of 1,25-(OH)2D3 effects in vivo, it was found that treatment caused the upregulation of CYP24A1 transcripts, which as previously discussed, is regulated by vitamin D. In addition, there was increased IL-10 expression at 6 h post treatment which agrees with a previous report [4]. In another study, 1,25-(OH)2D3 modulated DCs induced alloreactive CD4+ cells to secrete less IFN-γ upon restimulation [9]. The inhibition of DC differentiation and maturation may explain the immunosuppressive activity of 1,25-(OH)2D3 [9]. T cells have been found to express VDR, whose levels are increased when treated with 1,25-(OH)2D3 [78]. Interestingly, CD8+ T cells express the highest levels of VDR, and it has been suggested that they could be a major site of 1,25-(OH)2D3 action [78]. 1,25-(OH)2D3 has been found to reduce CD8+ T cell mediated cytotoxicity [79] suggesting that 1,25-(OH)2D3 may be acting to prevent or suppress overactivation of these cells [80]. We furthered the understanding of how 1,25-(OH)2D3 indirectly modulates CD8+ T cells through its mediating effects on pAPCs and cross-priming. The role of cross-priming in regulating CD8+ T cell immunodominance has previously been elucidated [77]. For our studies, we used the same model to investigate how the presence of 1,25-(OH)2D3 during the initial cross-priming events in vivo would influence the activation of epitope specific CD8+ T cells and the immunodominance hierarchy. Cross-priming was induced through a xenogenic system in which CD8+ T cell priming can occur only via the cross priming pathway. By using a single low dose, the range of 1,25-(OH)2D3 effects in vivo would hypothetically be limited to the initial cross-priming events that occur within the first 24 h. Arguably, if we were to prolong exposure to the substance through multiple doses both pre and post initiation of cross-priming, the effects could be a result of direct actions on T cells. In addition, utilizing higher doses would increase the risk of detrimental effects such as hypercalcemia [132]. Thus, although our chosen dose was able to elicit biological effects, we 65 have shown that they do not elicit immunosuppression or detrimental effects associated with over signalling due to 1,25-(OH)2D3. Our results demonstrated that the presence of 1,25-(OH)2D3 during the initial cross-priming events did not alter the effector function of the immunodominant NP396-specific CD8+ T cells. This was contrary to our hypothesis, as we had predicted that the presence of 1,25-(OH)2D3 would inhibit the ability of pAPCs to cross-prime CD8+ T cells. Furthermore, priming of mice with 1,25-(OH)2D3 24 hours prior to the initiation of cross-priming did not affect T cell activation. In a model of chronic retroviral infection with a LPBM5 virus, it was found that the number of monocyte/MΦ expressing VDR were markedly increased [89]. The retroviral infection was found to alter the local production of 1,25-(OH)2D3 in the spleen. It did not alter this production in monocyte/MΦ, but increased production in isolated T cells [89]. Thus, chronic retroviral infection alters both the local vitamin D metabolism and VDR expression by immune cells in mice. These findings could suggest possible local interactions between 1,25-(OH)2D3 and the generation of immune responses during viral infection [89]. The interactions between 1,25-(OH)2D3 and viral infection were studied in our model by comparing the profile of the LCMV immunodominance hierarchy. To investigate this phenomenon, mice were challenged with the LCMV after the induction of cross-priming with LCMV-NP. One week post challenge, shifts in the LCMV immunodominance hierarchy were assessed. It was previously found that after cross priming with LCMV-NP, the LCMV NP396 epitope completely dominated the immune response, followed by NP205, GP33, and GP276 [77]. This was shifted from the typical LCMV immunodominance hierarchy which follows: GP 33> NP396 > GP276, NP205. Our study found that 1,25-(OH)2D3 does not affect the initial crosspriming events, nor does it affect the outcome of the immunodominance hierarchy. There was a slight but insignificant decrease in the activation level of NP396-specific CD8+ T cells in the spleen from mice that received 1,25-(OH)2D3. This was contrary to our hypothesis, as it was thought that the presence of 1,25-(OH)2D3 would inhibit the ability of pAPCs to cross-prime CD8+ T cells. Thus, we expected that the initiation of a virus challenge would result in the generation of a typical LCMV immunodominance hierarchy (GP 33> NP396 > GP276, NP205), or perhaps a lower activation profile for NP396-specific CTLs. Immunodominance was also assessed at the site of antigen entry. In our model this was the peritoneum of the mouse, as HEK-NP cells were injected by intraperitoneal route. As 66 previously reported, in a Respiratory Syncytial Virus model, CD8+ T cell immunodominance responses to infection were more pronounced at the location of antigen entry versus the systemic compartment [97]. In concurrence with previous studies [77], it was found that the immunodominance hierarchy was more pronounced at the site of antigen entry. Previous work found that the percentage of responding CD8+ T cells specific for the epitopes GP33 and NP205 also increase in the peritoneal cavity after cross priming, whereas in the spleen this is not observed [77]. The initial recruitment of resources, such as cytokines (IL-2, IL-6) or CD4+ T cells, to the peritoneal cavity after cross-priming events could generate a microenvironment that is favourable towards the generation of robust CD8+ T cell response to other epitopes upon viral challenge [77]. Upon assessment of the immunodominance hierarchy, in the peritoneal cavity, it was found that the profile was not altered. However, there were observed decreases in the activation of peritoneal subdominant-epitope specific CD8+ T cells when mice received 1,25(OH)2D3, although these results were not found to be significant. Interestingly, in another study utilizing mDCs in vivo, it was found that 1,25-(OH)2D3 influenced the trafficking of fully differentiated immature DCs [160]. DCs treated with 1,25(OH)2D3 gained the capacity to bypass sequestration in draining lymph node and traffic to Peyer’s patches [160]. The effects were found to be temporary, perhaps due to the short half-life of the active hormone. However 1,25(OH)2D3 did not affect antigen processing or peptide presentation to CD4+ T cells [160]. This study is supportive of our observations with regard to MHC-I antigen processing, and thus it is possible that the presence of 1,25-(OH)2D3 results in alterations to the trafficking patterns of CD8+ T cells. Furthermore, DCs differentiated in the presence of 1,25-(OH)2D3 were compromised in antigen presentation, while manifesting clear alterations to their trafficking properties in vivo [160]. This study suggests that 1,25-(OH)2D3 could be important in controlling the types of immune effector responses elicited subsequent to either infection or vaccination. 4.1.1 Summary and Conclusions Taken together, our results provide evidence that a single dose of 1,25-(OH)2D3 does not affect the cross-priming pathway in vitro or in vivo. We found that 1,25-(OH)2D3 does not influence the ability of differentiated pAPCs to phagocytose or cross-present exogenous antigens 67 in our model. We also confirmed that 1,25-(OH)2D3 does not affect cross-priming events, as there were no changes observed in the LCMV immunodominance hierarchy that is shaped by LCMV-NP cross-priming in vivo. Treatment with 1,25-(OH)2D3 was observed to decrease the activation of peritoneal subdominant-epitope specific CD8+ T cells, however these results were not found to be significant. Our study helps to expand the understanding of the immunomodulatory role of exogenous 1,25-(OH)2D3 on the outcome of virus infection. Critically, our data supports the observation that the role of 1,25-(OH)2D3 in the immune system is not always associated with potently immunosuppressive effects. Our data does reiterate the observation that 1,25-(OH)2D3 is not as immunosuppressive as we are lead to believe. Several caveats need to be addressed in the current study, as the lack of immunosuppressive effects observed could be attributed to multiple factors. It is important to consider the amount of 1,25-(OH)2D3 administered. Due to the short half-life of the active sterol, it is possible that effects were not seen due to insufficient stimulation. The dosing schedule of 1,25-(OH)2D3 could be modified in future experiments to attempt to maximize the amounts administered, without inducing toxicity. For example, in a model of EAE, administration of 0.1/~g (5pg/kg/2 days) 1,25-(OH)2D3 i.p. every other day, starting 3 days prior to and for up to 15 days (subsequently up to 5 days) post-immunization, significantly prevented the expression of disease [25]. Alternatively, in lieu of 1,25-(OH)2D3, future studies could administer synthetic analogs such as the VDR modulator compound A, that has been demonstrate to inhibit the induction and progress of EAE in mice without inducing hypercalcemia in vivo [161]. Despite the hypothesized role of 1,25-(OH)2D3 in the maintenance of immune homeostasis, VDR knockout mice have been found to have a normal composition of immune cells [85]. In addition, VDR deficient mice are able to reject allogenic and xenogenic transplants comparatively to wild type mice [85]. 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