Download The Influence of 1,25-dihydroxyvitamin D on the Cross-Priming of Lymphocytic

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]. Thus, in order to fully delineate the in vivo effects of the
vitamin D3 system on the immune response, future work should attempt to investigate VDR
deficiency restricted to individual cell types [85], such as DC, MΦ or T cells. Collectively, our
study helps to expand the understanding of the physiological role played by 1,25-(OH)2D3 in the
regulation of the adaptive immune response.
68
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