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MOLECULAR MECHANISMS OF TUMOR DENDRITIC CELL DYSFUNCTION IN CANCER
By
K. C Michael Tang
A thesis submitted in conformity with the partial requirements for the degree of Doctor of Philosophy
Graduate Department of Institute of Medical Science University of Toronto
©Copyright by K.C Michael Tang 2016
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MOLECULAR MECHANISMS OF TUMOR DENDRITIC CELL DYSFUNCTION IN CANCER
K.C Michael Tang
Doctor of Philosophy
Graduate Department of Medical Science University of Toronto, 2016
ABSTRACT
Dendritic cells (DCs) dysfunction in cancer is a well-established
phenomenon that is considered one of the key mechanisms of immune evasion.
Defects in tumor DCs are caused primarily by tumor-derived factors present in the
tumor milieu. However, the mechanisms and the proximal signals that drive this
process remain elusive. This thesis explores the molecular mechanisms that drive
tumor DC dysfunction. In the first sections of this thesis, I showed the critical
importance of tumor-derived toll-like receptor (TLR)-2 ligands in the generation of
immunosuppressive Gr-1+ IL-10-producing human and mouse DCs. TLR2
activation induced two parallel synergistic process that converged to activate
STAT3: stimulation of autocrine IL-6 and IL-10, and upregulation of their respective
cell surface receptors. These processes imprint the capacity of tumor DC in the
milieu to respond to lower concentrations of tumor associated cytokines, therefore
lowering the STAT3 activation threshold. I identified versican as a soluble tumor-
derived factor that ligates with TLR2 to induce DC dysfunction. In the last section of
the thesis, I explored the possibility of TLR2 blockade to enhance the efficacy of
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tumor immunotherapy. TLR2 blockade improved intra-tumor DC immunogenicity.
These functional properties was associated with better anti-tumor immune
responses elicited by a GM-CSF producing whole cell tumor vaccine (GVAX), and
higher rates of proliferation and expansion of adoptively transferred cytotoxic T
cells (CTLs). Overall, the findings presented in this thesis provide a basis of
understanding the molecular mechanisms of DC dysfunction in cancer and
highlight TLR2 as a relevant therapeutic target to improve cancer immunotherapy.
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DEDICATION
To my late father, his courageous battle of cancer inspired me to pursue my studies in cancer research.
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ACKNOLWEDGEMENTS
It takes a village to raise a PhD graduate. The journey from entering into the Cattral lab as an undergraduate summer student to the completion of my doctoral degree has been an exciting roller roaster ride. Reflecting on my growth personally and professionally over the past six years, I couldn’t have produced this piece of work without the unwavering support and encouragement from my mentor, colleagues, friends, and family.
To my mentor and supervisor Dr. Mark Cattral. I am forever grateful for your patience, guidance, support, and countless of opportunities that you have provided me. Thank you for believing in my work and me since the day you accepted me as your student. Thank you for challenging me to become a better scientist and pushing me to be more independent through your constructive criticisms and feedback. Thank you for your encouragement when you guided me through this arduous and convoluted journey of the publication process. I still have a long way to go in building my career, but you have provided me with an excellent start.
To the past and present members of the Cattral lab, thank you for your company. In particular, to Dr. Jun Diao, your technical expertise and your scientific wisdom tremendously inspired me throughout these years. You taught me how to plan and technically carry out many of the experiments described in this thesis. Along with Mark, you also believed in my abilities and pushed me to become more independent. I am also grateful for the friendship we developed over the years. To Ms. Jun Zhao and Mr. Hongtao Gu, I cherish our friendship and conversations when we’re not doing experiments together. Thank you for helping me take care of the mice and lend some helping hands during those ‘big’ experiments.
To my committee members, Drs. Reg Gorczynski, Andras Kapus, and Peter Liu, thank you for your scientific suggestions during our committee meetings. Your positivity and encouragements helped me to complete this degree one meeting at a time.
I would also like to acknowledge past and present members of the Gorczynski and Zhang lab. In particular, Dr. Ismat Khatri, Dr. Karrie Wong, Ms. Camila Balgobin, Dr. Dzana Dervovic, Ms. Paulina Achita, and Dr. Dalam Ly. Thank you for your participation throughout my journey. Other than our scientific discussions, those coffee breaks, lunches, drinking sessions with you are instrumental in keeping my sanity in check. I cherish our friendship outside the lab tremendously. On a more personal note, to my close friends and family, thank you for participating in my personal growth during these golden years. To my mother, who instilled the values of hard work and perseverance in me, I hope I have made you proud. Thank you for your unconditional love, encouragement and support even when I was feeling doubtful at my crossroads. To my sister, Amy, you’ve inspired me through your own successes professionally. To my wonderful partner, Justin,
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who has been my rock in these last challenging couple of years, thank you for tolerating me on my bad days, and lending a set of ears to listen to my frustrations. I look forward to what life has to offer us in the near future and beyond.
I am also grateful for the financial support from the CIHR- Training Program in Regenerative Medicine, CIHR Frederick and Banting and Charles Best Canada Graduate Scholarship, Canadian Cancer Society, and the Institute of Medical Science, University of Toronto.
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CONTRIBUTIONS
I performed and analyzed the majority of experiments described throughout this thesis. Myself, Dr. Jun Diao, and Dr. Mark Cattral, conceived all experiments, and interpreted the results. Ms. Jun Zhao and Mr. Hongtao Gu provided technical assistance in many of the experiments. All other contributions are outlined below.
Chapter 1: Parts of this chapter were taken from this review article: Tang, M., Diao. J., Cattral, M.S. (2016). Molecular mechanisms involved in dendritic cell dysfunction in cancer. Cell. Mol. Life Sci. doi.10.1007/s00018-016-2317-8. I performed the literature search and review, drafted and edited the manuscript. Dr. Jun Diao provided intellectual suggestions. Dr. Mark Cattral drafted and edited the manuscript.
Chapter 3: Parts of this chapter were taken from: Tang, M, Diao, J, Gu, H, Khatri, I, Zhao, J, Cattral, MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):2851-64.
Dr. Peter Liu provided the MyD88-/- mice. Dr. Jun Diao prepared the versican knockdown cell lines used in Figures 3.13 and 3.14. Dr. Ismat Khatri performed the western blot experiment as shown in Figure 3.14. Dr. Catherine O’Brien provided the primary human colon cancer cell lines used in Figure 3.15 and 3.16. We thank Andrea Norgate for helping us prepare human DCs.
Chapters 4 & 5: Parts of this chapter were also taken from: Tang, M, Diao, J, Gu, H, Khatri, I, Zhao, J, Cattral, MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):2851-64.
Dr. Jun Diao prepared the versican knockdown cell lines used in Figure 4.7, and also performed flow cytometry analysis shown in Figures 5.2, 5.3, 5.4, and 5.13.
I thank Drs. Li Zhang and Reg Gorczynski for critical reading of the manuscript, helpful comments, and scientific advices. I would also like to thank the support staffs from the UHN Animal Resource Center as well as the SickKids- UHN Flow Cytometry Facility for their contribution in taking care of the mice and providing assistance in FACS sorting.
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TABLE OF CONTENTS
ABSTRACT ........................................................................................ ii
ACKNOLWEDGEMENTS .................................................................. v
CONTRIBUTIONS ........................................................................... vii
LIST OF FIGURES ............................................................................ xi
LIST OF ABBREVIATIONS ............................................................ xiv
Chapter 1
Introduction and literature review .................................................. 1 1.1- Dendritic cell biology ............................................................................... 2
1.1.1- Dendritic Cells...................................................................................... 2 1.1.2- DC subsets and ontogeny ................................................................... 2 1.1.3- DC maturation, activation and the immune response .......................... 6 1.1.4- Pattern recognition receptors- Toll like receptors & C-type lectins ...... 8 1.1.5- Dendritic cell control of tolerogenic responses .................................. 11
1.2- Dendritic cells in tumor immunology ................................................... 15 1.2.1- Cancer and Immunity ........................................................................ 15 1.2.2- Cancer inflammation .......................................................................... 17 1.2.3- Myeloid cells in tumor microenvironment .......................................... 18 1.2.4- Tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs) ........................................................................... 19 1.2.5- Tumor DC- an overview ..................................................................... 21 1.2.6- Tumor DC heterogeneity and origin of tumor DCs ............................ 22 1.2.7- Tumor CD103+ DCs ........................................................................... 24 1.2.8- DC defects in cancer ......................................................................... 25 1.2.9- Mechanisms of tumor DC dysfunction ............................................... 29
1.3- DCs and Cancer immunotherapy.......................................................... 40 1.3.1- Cancer immunotherapy ..................................................................... 40 1.3.2- Adoptive cell therapy ......................................................................... 41 1.3.3- Chemotherapy and radiation therapy on the immune system ........... 43 1.3.4- DCs and cancer vaccines .................................................................. 44 1.3.5- Checkpoint blockade therapy ............................................................ 46
1.4- Overall objectives and hypotheses ...................................................... 49 Thesis outline ............................................................................................... 49
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Chapter 2
General methods and materials .................................................... 51 Mice ................................................................................................................. 52 Tumor Models.................................................................................................. 52 Cell isolation .................................................................................................... 52 Flow cytometry ................................................................................................ 53 Preparation of conditioned medium ................................................................. 54 Antibodies and reagents .................................................................................. 54 Cytokine assays .............................................................................................. 55 Gene expression analysis ............................................................................... 55 Allogeneic mixed lymphocyte reactions .......................................................... 56 Intracellular staining for phosphorylated STAT3 ............................................. 56 Versican knockdown with lentiviral transduction ............................................. 56 Versican expression by western blotting ......................................................... 57 Intracellular staining for phosphorylated STAT3 ............................................. 57 Generation of human monocyte-derived DC ................................................... 57 GVAX immunization ........................................................................................ 58 Generation of effector OT-I T cells .................................................................. 58 Antitumor CTL responses in vivo .................................................................... 58 Statistical Analysis ........................................................................................... 59
Chapter 3
The role of TLR2 in promoting DC dysfunction in vitro ............. 60 Introduction .................................................................................................... 61 Results ............................................................................................................ 63
Tumors stimulate autocrine production of IL-6 and IL-10 in pre-cDC through TLR2 ............................................................................................................ 63 TCM promotes differentiation of Gr-1+ DC via TLR2 signaling .................... 66 Autocrine IL-6 and IL-10 promotes differentiation of IL-10 producing DC ... 67 TLR2 ligation sensitizes DC to IL-6 and IL-10 stimulation by upregulating IL-6R and IL-10R expression ................................................................................. 72 Upregulation of IL-6R and IL-10R lowers the STAT3 activation threshold .. 75 Tumor-derived versican in TCM induces cDC dysfunction .......................... 77 Human cancers induce DC dysfunction through TLR2 ................................ 80
Discussion ..................................................................................................... 84
Chapter 4
TLR2 signaling drives Tumor DC dysfunction in vivo ............... 86 Introduction .................................................................................................... 87 Results ............................................................................................................ 89
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Tumors in TLR2-/- mice lack Gr-1+cDC ........................................................ 89 Characterizations of tumor DCs in WT and TLR2-/- mice ............................. 92 In vivo effects of tumor-derived versican ..................................................... 95
Discussion ..................................................................................................... 97
Chapter 5
Blockade of TLR2 signaling enhances tumor immunotherapy 100 Introduction .................................................................................................. 101 Results .......................................................................................................... 103
TLR2 deficiency improves T cell responses to anti-cancer vaccine .......... 103 Characterization of tumor DCs in GVAX-treated WT and TLR2-/- mice ..... 105 TLR2 deficiency enhances anti-tumor cytotoxic T cell (CTL) responses ... 107 TLR2 deficiency enhanced proliferation of transferred tumor-antigen specific CTLs .......................................................................................................... 110 TLR2 deficiency enhances therapeutic efficacy of adoptive transfer CTL therapy ....................................................................................................... 111 Therapeutic targeting of TLR2 improves adoptive CTL therapy ................ 112 Anti-TLR2 antibody enhances the therapeutic efficacy of anti-CTLA-4 antibody ................................................................................................................... 115
Discussion ................................................................................................... 118
Chapter 6
General Discussions and Future Directions .............................. 121 Historical perspective on DC dysfunction in cancer ...................................... 122 New model of Tumor DC dysfunction ............................................................ 123 TLR2 blockade as an adjunct therapy ........................................................... 124 Strengths and limitations of the tumor models used ..................................... 126 Tumor DC turnover rate and implications in anti-TLR2 therapy .................... 128 Implications beyond tumor immunity ............................................................. 130 Concluding Remarks ..................................................................................... 133
References .................................................................................... 134
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LIST OF FIGURES Chapter 1
1.1- Current model of DC ontogeny (Pg. 4) 1.2- Model of tumor DC plasticity (Pg. 24) 1.3- Hypothetical model of tumor DC dysfunction (Pg. 50)
Chapter 3
3.1- Tumor conditioned medium stimulate autocrine IL-6 and IL-10 in DCs through TLR2 and MyD88 Pathway (Pg. 64)
3.2- Anti-TLR2 neutralizing antibodies reduce IL-6 and IL-10 production in a dose-dependent manner (Pg. 65)
3.3- TLR2 agonists mimic TCM’s ability to produce IL-6 and IL-10 (Pg. 66)
3.4- Tumor conditioned medium promotes differentiation of immunosuppressive Gr-1+ cDC (Pg. 67)
3.5- Tumor condition medium promotes differentiation of IL-10 Producing through TLR2 (Pg. 68)
3.6- The role of autocrine IL-6 and IL-10 signaling in promoting DC dysfunction (Pg. 70)
3.7- Exogenous IL-6 and IL-10 fail to induce IL-10 producing DC (Pg. 71)
3.8- IL-1β does not play a role in inducing DC dysfunction in vitro (Pg. 72)
3.9- TLR2 ligation regulates IL-6R and IL-10R expression in pre-cDCs (Pg. 73)
3.10- TCM induces IL-6Rα and IL-10Rα even in presence of neutralizing antibody to IL-6 and IL-10 (Pg. 74)
3.11- TLR2 ligation sensitizes DCs to IL-6 and IL-10 signaling (Pg. 76)
3.12- FSL-1 reduces the threshold for STAT3 activation by IL-6 and IL-10 (Pg. 77)
3.13- Versican induces DC dysfunction (Pg. 79)
3.14- Versican expression in LLC cell lines (Pg. 80)
3.15- Versican expression in various human cancer cell lines determined by PCR (Pg. 81)
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3.16- Human cancers induce human DC dysfunction through TLR2 (Pg. 82)
Chapter 4
4.1- Absence of TLR2 and MyD88 blocks the development of Gr-1+ cDCs in tumors (Pg. 90)
4.2- Phenotype of DCs in spleens of B16 melanoma-bearing WT, MyD88-/-, TLR2-/-, TLR4-/- mice (Pg. 91)
4.3- Frequency of Gr-1+CD11b+ cells in B16 melanomas (Pg. 92)
4.4- Phenotypic analysis of tumor and spleen DCs (Pg. 93)
4.5- Superior stimulatory function of tumor cDCs from TLR2-/- mice (Pg. 94)
4.6- Upregulation of IL-6Rα, IL-10R, and STAT3 phosphorylation in sorted DCs (Pg. 95)
4.7- In vivo effects of tumor-derived versican (Pg. 96)
Chapter 5
5.1- TLR2 deficiency improves efficacy of GVAX tumor vaccination (Pg. 103)
5.2- Tumor lymphoid cell analysis in GVAX treated mice (Pg. 104)
5.3- Multi-colour flow cytometry gating strategy for identifying myeloid populations in subcutaneous B16 melanoma (Pg. 105)
5.4- Tumor myeloid cell populations analysis in GVAX treated mice (Pg. 106)
5.5- Characterization of lymph node (LN) DCs in GVAX-treated WT and TLR2-/-
mice (Pg. 108)
5.6- TLR2 deficiency enhances expansion and function of transferred tumor-antigen-specific CTLs (Pg. 109)
5.7- TLR2 deficiency enhances proliferation of adoptively transferred CTLs (Pg. 111)
5.8- Adoptive CTL therapies in WT versus TLR2-/- mice (Pg. 112)
5.9- Anti-TLR2 antibody therapy blocks the generation of Gr-1+ cDC (Pg. 113)
5.10- Anti-TLR2 antibody treatments boost efficacy of adoptive CTL therapy (Pg. 113)
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5.11- TLR2 expression in B16 melanoma (Pg. 114)
5.12- Combination treatment with anti-TLR2 and anti-CTLA-4 antibody enhanced efficacy of GVAX vaccination (Pg. 116)
5.13- Analysis of DCs, T cells, and NK cells in B16 tumors antibody and GVAX treated mice (Pg. 117)
Chapter 6
6.1- New model of DC dysfunction in cancer (Pg. 124)
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LIST OF ABBREVIATIONS
ACT
Adoptive Cell Therapy
Ag
Antigen
AIRE
Autoimmune Regulator
APC
Antigen Presenting Cell
ATP
Adenosine Triphosphate
BATF3
Basic Leucine Zipper Transcription Factor ATF-like 3
BM
Bone Marrow
CAR
Chimeric Antigen Receptor
CCR7
C-C Chemokine Receptor Type 7
CD
Cluster of Differentiation
cDCs
Conventional Dendritic Cells
CDP
Common Dendritic Progenitor
CFSE
Carboxyfluorescein Succinimidyl Ester
CLRs
C-type Lectin Receptors
CR
Complete Response
cpm
Counts Per Minute
CRT
Calreticulin
CSF-1
Colony Stimulating Factor 1
CTLA-4
Cytotoxic T-lymphocyte-Associated Protein 4
CTLs
Cytotoxic T Lymphoctes
DAMPs
Danger Associated Molecular Patterns
DC-d-Ms
Dendritic Cell-derived-Macrophages
DCs
Dendritic Cells
ECM
Extracellular Matrix
ER
Endoplasmic Reticulum
ERK
Extracellular Signal–Regulated Kinases
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FDA
Food & Drug Administration
Flt3L
FMS-like Tyrosine Kinase 3 Ligand
FOXP3
Foxhead Box P3
FPR1
Formyl Peptide Receptor
GAPDH
Glyceraldehyde 3-phosphate dehydrogenase
GM-CSF
Granulocyte Macrophage Colony-Stimulating Factor
GVAX
GM-CSF Vaccine
HIF-1α
Hypoxia Inducing Factor- 1α
HLA
Human Leukocyte Antigen
HMGB1
High Mobility Group Box 1
HSC
Hematopoietic Stem Cells
HSP
Heat Shock Protein
ICD
Immunogenic Cell Death
ICOS
Inducible T-cell Costimulator
ID2
Inhibitor of DNA Binding 2
IDO
Indoleamine-Pyrrole 2,3-Dioxygenase
IECs
Intestinal Epithelial Cells
IFN
Interferon
IgG
Immunogobulin G
IkB
Inhibitor of kappa B
IL
Interleukin
IL-10R
Interleukin-10 Receptor
IL-6R
Interleukin-6 Receptor
iNOS
Inducible Nitric Oxide Synthase
IRAK
Interleukin-1 Receptor-Associated Kinase 1
IRF
Interferon Regulatory Factor
JAK2
Janus Kinase 2
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KO
Knockout
LAG-3
Lymphocyte-activation gene 3
LC
Langerhans Cells
LLC
Lewis Lung Carcinoma
LN
Lymph Node
LPS
Lipopolysaccharide
mAb
Monoclonal Antibody
MAPK
Mitogen-activated protein kinases
MDP
Macrophage and Dendritic Cell Precursor
MDSCs
Myeloid Derived Suppressor Cells
MFI
Mean Fluorescence Intensity
MHC
Major Histocompatibility Complex
MLR
Mixed Lymphocyte Reaction
MMP
Matrix Metalloproteinase
MPS
Mononuclear Phagocyte System
mRNA
messenger RNA
mTECs
Medullary Thymic Epithelial Cells
MyD88
Myeloid differentiation primary response gene 88
NF-kB
Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B cells
NK
Natural Killer
NO
Nitric Oxide
NODs
Nucleotide-Binding Oligomerization Domain Receptors
NSCLC
Non Small Cell Lung Cancer
OR
Objective Response
OVA
Ovalbumin
PAMPs
Pathogen Associated Molecular Patterns
xvii
PBMCs
Peripheral Blood Mononuclear Cells
PD-1
Programmed Death-1
PD-L1, PD-L2 Programmed Death-Ligand 1, 2
pDCs
Plasmacytoid Dendritic Cells
PDGF
Platelet Derived Growth Factor
Pre-cDCs
Precursor- conventional Dendritic Cells
PRRs
Pattern Recognition Receptors
qRT-PCR
Quantitative Reverse Transcription Polymerase Chain Reaction
RALDH
Retinaldehyde Dehydrogenase Type 2
ROS
Reactive Oxygen Species
SD
Standard Deviation
SEM
Standard Error of Mean
shRNA
Short Hairpin RNA
SOCS3
Suppressor of cytokine signalling 3
STAT3
Signal transducer and activator of transcription 3
TAG
Triacylglycerols
TAK1
Transforming Growth Factor β-Activated Kinase 1
TAMs
Tumor Associated Macrophages
TCM
Tumor Conditioned Medium
TCR
T Cell Receptor
TF
Transcription Factor
TIL
Tumor Infiltrating Lymphocytes
Th
T Helper
TIM-3
T-cell Immunoglobulin and Mucin-Domain Containing-3
TIR
Toll-IL-1
TIRAP
TIR-Domain-Containing Adaptor Protein
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TLRs
Toll-Like Receptors
TME
Tumor Microenvironment
TNF
Tumor Necrosis Factor
TRAF6
TNF Receptor Associated Factor 6
Tregs
Regulatory T cells
TRIF
TIR-domain-containing adapter-inducing interferon-β
UPR
Unfolded Protein Response
VEGF
Vascular Endothelial Growth Factor
WT
Wildtype
Zbtb46
Zinc finger and BTB domain containing 46
1
Chapter 1
Introduction and literature review
Parts of this chapter were taken from:
Tang, M., Diao. J., Cattral, M.S. (2016). Molecular mechanisms involved in
dendritic cell dysfunction in cancer. Cell. Mol. Life Sci. doi.10.
1007/s00018-016-2317-8
2
1.1 Dendritic cell biology
1.1.1- Dendritic Cells
Dendritic cells (DCs) comprise a heterogeneous population of professional antigen
presenting cells that initiate and regulate adaptive immune responses (1-3). As sentinels
of the immune system, they play a key role in linking innate and adaptive immunity. DCs
were first discovered in mouse spleen by Ralph Steinman and Zanvil Cohn in 1973, and
named DCs based on their unique dendritic morphology. Subsequent studies, by Steinman
demonstrated that DCs were potent stimulators of T cells in mixed lymphocyte reactions
(MLR) (4). For his pioneering work in DC biology, as well as subsequent efforts to
exploit DCs for immune-based therapies, Steinman was awarded the Nobel Prize in
Physiology or Medicine in 2011.
1.1.2- DC subsets and ontogeny
DCs are broadly categorized into four major categories: 1) conventional or classic
DCs (cDC); 2) interferon-producing plasmacytoid DC (pDC); 3) monocyte-derived DC;
and 4) Langerhans cells in skin. The hallmark function of cDCs is to prime naïve T cells
for adaptive immunity. cDCs are superior to other APCs such as macrophages and B
cells, which was related to their specialized ability to capture, process, and present
antigens on their cell surface (5-7). pDCs produce high amounts of type I interferons
(IFNs) upon recognition of foreign nucleic acids using toll-like receptors (TLRs) 7 and 9,
they (8, 9).
3
The precise origin and developmental pathways of cDCs from hematopoietic
progenitors has become clearer in recent years through the identification of new surface
differentiation markers and gene expression analysis (10). With the exception of LCs,
which self-renew mostly in situ, DCs originate from hematopoietic stem cells (HSCs)
residing in the bone marrow (BM) (11). A bipotent progenitor in the BM, called the
monocyte/macrophage and DC precursor (MDP) gives rise to the common DC precursors
(CDPs), which are dedicated to the DC lineage (11-13) (Figure 1.1). CDPs are distinct
from the precursors of monocytes and macrophages (13-19) and differentiate into pDCs,
which complete their development in the BM, and pre-cDCs, an immediate precursor of
cDCs (CD11c+, MHC II-, B220-, expressing the transcription factor Zbtb46) that migrate
rapidly through blood into peripheral lymphoid and non-lymphoid tissues where they
undergo several rounds of cell division (14, 15, 18, 20-22).
4
Figure 1.1- Current model of DC ontogeny. This figure shows the developmental
pathways for DC and monocytes/macrophages in peripheral tissues and tumors. The cell
surface molecules that are used commonly to distinguish these cell populations are shown.
(HSC, hematopoietic stem cell. CMP, common myeloid progenitor. CLP, common
lymphoid progenitor. MDP, monocyte dendritic cell progenitor. CDP, common dendritic
cell progenitor. Pre-cDC, immediate precursor of classical DCs. pDC, plasmacytoid DCs)
Tissue pre-cDCs in the mouse further differentiate into: 1) lymphoid-tissue
resident CD8α+CD11b- and nonlymphoid tissue migratory CD103+ cDCs (23) or 2)
CD11b+ cDC (24)., CD8α+ DCs and CD103+ DCS specialize in cross-presentation of
exogenous antigens on MHC-I molecules to CD8+ T cells (4, 23), whereas CD11b+ cDCs
have a dominant role in presenting endogenous antigens on MHC-II to CD4+ T cells, and
were recently found to induce Th17 responses (25-27). Recent evidence suggests that
5
commitment towards these subsets occurs in the bone marrow (21). Analogous human
counterparts to these populations are CD141+ DCs (also known as BDCA3+ and
thrombomodulin) and CD1c+ DCs (also known as BDCA1+), respectively (12).
Monocytes were once thought to be the main immediate precursors of cDC. This
was a hypothesis generated from studies showing that human CD34+ progenitors or
mouse bone marrow progenitors stimulated in vitro with GM-CSF and IL-4 produce DCs
(28). Similar to pre-cDC-derived DCs, monocyte-derived DC express CD11c, MHC II,
costimulatory molecules and are capable of stimulating T cells. Subsequent studies in
mice showed that monocytes differentiate into cDCs (termed inflammatory DC or Tip-
DC) in vivo within inflamed tissues under inflammatory conditions (29, 30). Furthermore,
monocytes were also shown to contribute to the development of intestinal CD103-CD11b+
DCs (31, 32) and splenic CD11b+CD103+ESAMlo DCs (33) under steady state conditions.
During steady state conditions, DC homeostasis in lymphoid and non-lymphoid
tissues reflects a balance between the influx of new precursors, DC emigration, and cell
death. Steady state DCs in various lymphoid and nonlymphoid tissues display short half-
lives of 3-6 days (10, 34). Flt3 ligand (Flt3L), which is expressed by stromal cells in BM
and lymphoid tissues and by activated T cells, plays an essential role by driving
proliferation and differentiation of Flt3+ bone marrow progenitors and pre-cDCs (17, 35,
36). In mice and humans, overexpression or injection of Flt3L markedly increases the
number of cDCs and pDCs in blood and in lymphoid and non-lymphoid tissues (36-38).
Notch 2 signaling controls differentiation of pre-cDC derived CD11b+ cDC in spleen and
lymphotoxin β receptor signaling regulates CD11b+ spleen cDC development as well as
cDC proliferation (39, 40). Retinoic acid signaling in pre-cDCs influences differentiation
6
and homeostasis of CD11b+CD8α- cDCs in the spleen and CD11b+CD103+cDCs in the
intestine (41).
During acute inflammatory processes, increased expression of
granulocyte/macrophage colony stimulating factor (GM-CSF, also known as CSF-2)
promotes the recruitment of new precursors, augmentation of cDC proliferation in situ,
and differentiation of monocytes into inflammatory DCs (42, 43). Additionally,
inflammation causes egress of tissue DC into lymph nodes by modulating chemokine
receptor expression and altering the structure of regional lymphatics and lymph nodes
(44-46).
Transcription factors (TFs) guide DC development from the hematopoietic
progenitors (47). Some TFs such as Ikaros and PU.1 are critical for all DC as well as
other myeloid populations. Gfi-1, a transcriptional repressor, promotes DC over
macrophage differentiation at the MDP stage. E2-2 expression is absolutely essential for
the development and maintenance of pDC (48, 49), whereas ID2 and the recently
identified TF, Zbtb46, are expressed at high levels in all cDC (20, 22, 50). CD8+ cDC and
CD103+ CD11b- cDC express high levels of IRF8, Batf3, and NFIL3 (23, 51, 52); CD11b+
cDC express high levels of relB and IRF4 (51, 53). These findings in mice have been
shown to be highly relevant to human DC ontogeny. For example, mutation of Gata2 and
IRF8 cause DC deficiency syndromes that increase susceptibility to mycobacterial and
fungal infections (54, 55).
1.1.3- DC maturation, activation and the immune response
7
In the 1980s, Schuler and Steinman discovered that freshly isolated Langerhans
cells (LC), a type of DC found in the epidermis of the skin, expressed high levels of MHC
Class II molecules, but were poor stimulators of T cells responses in MLRs (56). These
cells became potent T cell stimulators in the same MLR assay only when they were
cultured for 2 days in vitro (56). Steinman and colleagues proposed that DCs could exist
in two functional states, immature and mature, with only a mature DC having the abilities
to prime an immune response and drive T-cell clonal expansion and differentiation.
However, the signals that determine whether a DC is in an immature or mature state were
not completely understood until the discovery of toll-like receptors (TLRs) and other
pattern recognition receptors (PRRs) like NODs, RIG-like or C-type lectin receptors. In
the 1990s, Hoffman and colleagues reported that anti-microbial responses in drosophila
were dependent on activation of Toll ligands by receptors previously implicated in
drosophila embryogenesis (57). Shortly afterwards, the mammalian homologue of Toll
was cloned by Medzhitov and Janeway, which was demonstrated to initiate adaptive
immunity (58). Another seminal key finding identified by Beutler’s group showed that
TLR4 is a key molecule that recognizes bacterial lipopolysaccharide (LPS) (59). A variety
of PRRs and their respective ligands have since been identified, and in vivo studies have
confirmed the role of these molecules in promoting DC maturation and activation.
An immune response is largely initiated by signals delivered by PRRs that respond
to evolutionary conserved molecular signatures from microbes known as pattern-
associated molecular patterns (PAMPs) and in some cases, tissue-derived danger signals
known as danger-associated molecular patterns (DAMPs) (60, 61). DCs express a large
repertoire of PRRs (62). In response to signals from the receptors, DCs undergo several
8
phenotypic and functional changes that increased their ability to prime T cell responses
and guide appropriate antigen-specific T cell differentiation. First, upon engagement of
PRRs, DCs immediately increase their capacity to process and load antigen into MHC
molecules (63). Subsequently, DCs alter the expression of chemokine receptors, notably
downregulation of CCR6 and upregulation of CCR7 and CXCR4 to allow homing into
nearby secondary lymphoid organs (64, 65). The engagement within the immunological
synapse between the T-cell receptor on a naïve T cell and the antigen-loaded MHC
molecule on a DC delivers the first activating signal (signal 1) to initiate an immune
response (61). Signaling from the PRRs also upregulates expression of a variety of cell
surface molecules on DCs. These molecules deliver stimulatory signals (signal 2) on T
cells that are necessary for T cell proliferation or survival. These include members of the
B7 family, e.g. B7.1 (CD80), B7.2 (CD86), and inducible costimulatory ligand (ICOS-L).
Another group of costimulatory surface molecules belong to the TNF-receptor family, e.g.
CD40, OX40 and 4-1BB ligand (4-1BBL). The lack of a costimulatory signal often results
in T cell anergy, one of several processes that induce peripheral tolerance. Finally, an
activated DC will also produce a variety of cytokines (signal 3) that act on T cells to
promote differentiation into an effector T cell. For example, IL-12 produced by an
activated DC, instructs the development of type 1 immunity that is characterized by the
development of T-helper 1 (Th1) and cytotoxic T cells (CTL). The coordinated expression
of different molecules, cytokines and receptors that deliver three signals to T cells is a key
feature of DC activation (61).
1.1.4-Pattern recognition receptors- Toll like receptors & C-type lectins
9
Toll-like receptors belong to a group of type-I transmembrane proteins functioning
as PRRs. To date, 10 and 12 functional TLRs have been identified in humans and mice,
respectively (66). These receptors are composed of ectodomains made up of leucine-rich
repeats capable of recognizing a diverse set of PAMPs or DAMPs including: lipoproteins
(TLR1, 2, 6), LPS or HMGB1 (TLR4), flagellin (TLR5), double stranded RNA (TLR3),
single stranded RNA (TLR7, 8) and double stranded DNA (TLR9). Some TLRs are
expressed on the cell surface (TLR1, 2, 6, 4, 5), while others are expressed within the
endosomal compartments (TLR 3, 7, 9) (66).
The cytosolic region of TLRs consists of Toll-IL-1 receptor (TIR) domains, which
mediate downstream cell signaling by recruiting specific TIR-containing adaptor proteins.
All TLRs, except TLR3, signal through myeloid differentiation responses gene 88
(MyD88). TLR3 and TLR4 signal through TIR domain containing adaptor-inducing IFN-
β (TRIF) (66). Additionally, in the case of TLR2 and TLR4, the adaptor protein TIR-
domain-containing adaptor protein (TIRAP) is used to recruit MyD88. The recruitment of
MyD88 commences a series of downstream signaling events that include activation of
several IL-1 receptor associated kinases (IRAKs). IRAKs are capable of associating with
the E3 ubiquitin ligase, TNFR-associated factor-6 (TRAF6) and formation of the
transforming growth factor β-activated kinase 1 (TAK1) complex (66-68). The TAK1
complex phosphorylates the inhibitor of kappa B (IκB)- kinase (IKK)-β resulting in the
degradation of the IκB protein complexes. As a result, the proteolysis of IκB allows for
the nuclear translocation of nuclear factor kappa-B (NF-κB) or mitogen activated protein
kinase (MAPK) for transcription of many genes involving a number of pro-inflammatory
processes including cytokine production(66). For example, stimulation of TLR4 on DCs
10
by LPS, a classic pro-inflamamtory stimulus, leads to the release of IL-1β, IL-6, and
TNF-α. Similar to the MyD88 dependent pathway, TRIF is able to recruit TRAF6 and
IκB complexes, leading to NF-κB activation. Signaling in TLR3, TLR4, TLR7-9 can also
result in the upregulation of interferon regulatory factors (IRF)-3 and IRF7, promoting the
production of type I IFNs (66).
TLR2 is the receptor for the recognition of microbial lipoproteins and is
upregulated in chronic bacterial and viral infections such as Mycobacterium tuberculosis
(MTb) and hepatitis C virus (66, 69). TLR2 is also expressed in B and T cells, in addition
to APCs and other myeloid cells. It is the only receptor that has the unique ability to form
heterodimers with TLR1 or TLR6 to recognize diacylated (e.g. FSL-1, TLR2-6) or
triacylated (Pam3CSK4, TLR2-1) lipoproteins (66). TLR2 also cooperates with dectin-1
to recognize a diverse repertoire of microbial lipoproteins. While activation of most TLRs
promotes pro-inflammatory responses, activation of TLR2 by some microbial products in
DCs promotes tolerogenic responses (70). For example, Lcr, the virulence factor for
Yersinia petis, signals through TLR2-TLR6 on DCs to promote differentiation of
regulatory T cells (71). Mycobacterium tuberculosis also produces a number of
lipoproteins that signal through TLR2 to promote tolerogenic responses that subvert host
immune response (72).
C-type lectin receptors (CLRs) belong to another major class of transmembrane
PRRs that recognize the carbohydrate component of microbial products (73). Like TLRs,
CLRs can activate various signaling pathways that induce DCs to modulate effector T cell
mediated responses. Additionally, CLR signaling interacts with other TLR pathways to
modulate TLR-induced gene expressions (66). One of the best-characterized CLR is
11
dectin-1, which recognizes 1,3-linked β-glucans, such as zymosan. Stimulation of dectin-
1 activates signaling pathways that involve proteins Syk tyrosine kinase and CARD9,
which leads to the production of IL-6, IL-10, and other cytokines (73). Notably, Dectin-1
signaling cooperates with TLR2 to trigger IL-10 production in bone marrow derived DC
(BMDC) and suppresses IL-12p70 production through the sustained activation of the
ERK MAPK signaling pathway (74, 75).
1.1.5- Dendritic cell control of tolerogenic responses
The delicate balance between immunity and tolerance is critical to maintain
health. Breakdown of tolerance leads to autoimmune diseases such as type 1 diabetes,
multiple sclerosis, and inflammatory bowel disease whereas insufficient immunity can
lead to chronic infections and cancer (76). Two main mechanisms of tolerance exist:
central and peripheral. Central tolerance develops mainly in primary lymphoid organs,
where most self-reactive T and B cells are deleted in their early stages of development in
the thymus and bone marrow, respectively (77). Thymic DCs play a critical role in
mediating negative selection of T cells by directly presenting or cross-presenting self-
antigens acquired from medullary thymic epithelial cells (mTECs). mTECs ectopically
express tissue-specific self-antigens through activation of an autoimmune regulator
(AIRE). Clonal deletion in the thymus can also be induced by recirculating peripheral
DCs loaded with self-antigens (78-80).
Potentially auto-reactive thymocytes can evade central tolerance mechanisms and
form part of the T cell repertoire (78). Peripheral tolerance mechanisms are therefore
necessary to prevent the induction of autoimmunity. DCs play a major role in the
12
induction of peripheral tolerance and immune homeostasis through the generation of
regulatory T cells (Tregs) and induction of T cell anergy and deletion (76). The
tolerogenic properties of DCs depend on several factors: maturation status, exposure to
immunosuppressive mediators, anatomical immune privileged sites, and the overall
environmental milieu (81).
At certain anatomical sites, such as those with mucosal surfaces like the gut, lung,
and skin, DCs are responsible for maintaining tolerance to self-antigens, while initiating a
robust immunity to pathogens. In the gut, CD11chiCD103+ migratory DCs transport
intestinal food antigens in the lamina propria to nearby mesenteric lymph notes to induce
naïve CD4+ T cells to become Foxp3+ Tregs through a mechanism dependent on retinoic
acid and TGFβ (82-84). Similarly, CD103+ lung DCs also promote the induction of
tolerance in response to inhaled inert antigens (85). Like the gut and the lungs, the skin is
also continuously exposed to pathogens and harmless antigens (86). Immunity in the skin
is controlled by different DC subsets including Langerhans cells, classical dermal DCs
and langerin+ CD103+ DCs (86). To maintain tolerance to self-tissue antigens in the skin,
langerin+ CD103+ migratory DC have been shown in vivo to promote the development of
antigen specific Tregs (87). In immunologically privileged sites such as the eye,
immature DCs that express low levels of MHC II and costimulatory molecules promote
tolerance (88). Thus, tolerance in various tissues and anatomical sites can be induced by
specific subsets of tissue resident DCs, as well as migratory DCs.
Exposure to immunosuppressive mediators and/or cytokines can also condition
DCs to become tolerogenic (81, 89). These molecules include vitamin A, vitamin D3,
IDO, IL-10 and TGF-β. Several reports have shown that tolerogenic DCs can be
13
generated by exposing immature DCs to low levels of GM-CSF and IL-10, or the
combination of IL-10 and TGF-β (90, 91). Exposing DCs to tumor-derived factors also
modulates their function to promote the development of Tregs (92). Tolerogenic DCs
generated in vitro have been shown to suppress experimental models of autoimmune
diseases such as EAE and type I diabetes (93). In the gut, intestinal epithelial cells (IECs)
secrete anti-inflammatory mediators such as TGF-β and retinoic acid to condition DCs to
a tolerogenic state. Supernatants from cultured IECs promote the development of CD103+
tolerogenic DCs, which inhibited the development of Th1 and Th17 cells (94, 95).
Additionally, stromal cells from the spleen and mesenteric lymph nodes express retinoic
acid metabolic enzymes (RALDH) to induce tolerogenic DCs (96, 97).
One study implicated the ERK-RALDH pathway in promoting DC tolerance.
Here, DCs conditioned with TLR2 ligands such as zymosan express elevated levels of
phosphorylated extracellular-signal regulated kinase (ERK) and mitogen-associated
protein kinase (MAPK) which mediates the induction of RALDH metabolic enzymes,
Raldh1 and Raldh2 (74). The production of retinoic acid via RALDH in DCs promotes
differentiation of Tregs from naïve CD4 T cells. Similarly, specific subsets of DCs in the
intestinal lamina propria also express high levels RALDH, and therefore have the ability
to produce retinoic acid to promote tolerance in the gut (98, 99). Moreover, retinoic acid
also exerts an autocrine effect on DC via retinoid receptors to induce suppressor of
cytokine signaling 3 (SOCS3) expression, which suppresses activation of p38 MAPK and
production of proinflammatory cytokines (74).
In summary, although DCs were once originally recognized as potent stimulators
of the adaptive immunity, DCs also play a major role in maintaining tolerance in
14
homeostatic and pathological conditions. Many of the mechanisms by which DCs
promote tolerance are often exploited by tumors to subvert host immune responses. These
mechanisms will be reviewed in the next section.
15
1.2- Dendritic cells in tumor immunology
1.2.1- Cancer and Immunity
Cancer is driven by multiple genetic mutations of oncogenes and tumor
suppressors to progressively transform healthy cells into highly malignant cell variants
(100). These neoplastic cells display unrestricted cell growth and have lost regulatory
mechanisms that control normal cell division and homeostasis. Hanahan and Weinberg
originally described six hallmarks that are shared by all types of tumors (100): 1) acquired
sustaining growth signals, 2) insensitivity to anti-growth signals, 3) evading apoptotic cell
death, 4) limitless replicating immortality, 5) angiogenesis, and 6) invasion and
metastasis. A decade later, a few more hallmarks have been added to this list, including
evasion of immune destruction and tumor-promoting inflammation (101). This highlights
that tumors are more than a collection of uncontrolled dividing cell variants. Instead,
tumors are heterogeneous, complex, and dynamic, comprised of supportive stromal
elements, neovasculature, and infiltrating immune cells, which collectively form the
tumor microenvironment (TME) (102). It has been suggested that a tumor is similar to a
large lymphoid organ where cancer cells interact with blood and lymphatic vessels,
stromal elements, and immune cells (103). This immune landscape is profoundly
important for prognosis and response to various cancer therapies (104, 105).
Ever since these new hallmarks of cancer are recognized, much work has been
done to understand the dual roles of the immune system in tumor development. On one
hand, immune cells may have cancer-antagonistic effects through cytolytic destruction of
tumor cells (e.g. killing by tumor-specific CTLs). Conversely, immune cells also promote
tumor growth through immunoediting by shaping the immunogenicity of tumors, resulting
16
in the emergence of immune-resistant variants (106). The immunoediting process is
characterized by three distinct phases, ‘elimination’, ‘equilibrium’, and ‘escape’ (107).
In the elimination phase, also known as immunosurveillance, the immune system
seeks to destroy cancer cells. Cancer cells express many types of tumor-associated
antigens (108). DCs in tumors capture tumor antigens and migrate to draining lymph
nodes where they prime and activate tumor-specific T cells (109). Memory and effector
cytotoxic T cell lymphocytes (CTL) return to the tumor to destroy cancer cells. In
addition, several other effector cells such as NK cells and type 1 macrophages are also
activated by proinflammatory cytokines to kill tumor cells through cytotoxic mechanisms.
Evidence suggests that immunosurveillance can sometimes prevent tumor development
and influence the rate of tumor progression (110, 111). The next phase of immunoediting
is ‘equilibrium’, during which rare tumor cells that survived immunosurveillance persist
and are held at bay by the immune system. Immune suppressive mechanisms in the TME
begin to develop, including activation of inhibitory pathways (e.g. CTLA-4, PD-1/PD-L1,
CD200/CD200R), and active suppression by myeloid derived suppressor cells (MDSCs),
tumor-associated macrophages (TAMs), fibroblast stromal cells, and Tregs (112-115).
Additionally, tumor cells at this phase begin to develop non-immunogenic phenotypes
and are selected under immune pressure to further increase resistance to immune attacks
(106). For most patients, natural anti-tumor immune responses fail to control the cancer.
During the last phase of immunoediting, ‘escape’, tumor cells have acquired regulatory
mechanisms that normally control self-tolerance, to evade immune responses and grow
unrestrictedly into aggressive and metastatic tumors.
17
1.2.2- Cancer inflammation
In the 19th century, Rudolf Virchow proposed a possible link between
inflammation and cancer when he noted the presence of leukocytes in tumors (116).
While acute inflammation is often self-limiting, chronic, dysregulated, and unresolved
inflammation is associated with increased risks of cancer. Indeed, chronic inflammation is
featured prominently in many cancers and influences tumor growth, metastasis, treatment
responses, and overall prognosis (101, 116, 117). Only a minority of all cancers is
attributed to inherited germline mutations, the vast majority is associated with somatic
mutations caused by environmental and epigenetic changes such as chronic infections,
obesity, smoking, alcohol consumption and environmental pollutants (102). Many of
these risk factors are associated with some form of chronic inflammation. For example,
persistent Helicobacter pylori infection is strongly associated with gastric cancers and
patients that are chronically infected with hepatitis B virus (HBV) and hepatitis C virus
(HCV) are predisposed to develop hepatocellular carcinoma (HCC) (102).
Chemokines, cytokines, and growth factors in the TME form a cascade of pro-
inflammatory responses, which can act in an autocrine and/or paracrine manner in both
cancer and immune cells (118). Oncogene activation in cancer cells promotes the
activation of proinflammatory transcription factors (such as NF-κB, STAT3, HIF1α)
and subsequently regulates the release of inflammatory cytokines, chemokines (e.g. TNF-
α and IL-6) and enzymes (e.g. COX-2) by cancer and immune cells of the TME (119).
These immune-related mediators recruit additional macrophages, DCs, mast cells and T
18
cells to the tumor inflammatory milieu, and function within the tumor stroma to modulate
immune responses (120).
The drivers of cancer inflammation remain poorly understood. While oncogenic
signals such as STAT3 activation in tumor cells can trigger the release of
proinflammatory cytokines and chemokines, evidence points to the importance of
endogenous “danger signals” released from stressed and dying cancer cells and
components of the extracellular matrix (ECM) that activate PRRs to modulate
inflammation and immunity (121, 122). For example, HMGB1 released from dying
cancer cells can modulate macrophage and DC function by activating TLR4 to release IL-
1 and TNF-α (123). Similarly, versican, a tumor-associated proteoglycan released from
the ECM of cancer cells stimulates myeloid cells through TLR2 to produce IL-6 and
TNF-α that promote metastasis in a murine lung cancer model (124). Additionally, the
intestinal microbiota was recently implicated in influencing cancer progression (125,
126). For example, TLR agonists produced by commensal bacteria in the gut have been
shown to enhance cytotoxic activities of tumor-associated myeloid cells during
chemotherapy (126), or promote systemic tumor inflammation by activating TLR5 to
induce IL-6 production and MDSC mobilization (127).
1.2.3- Myeloid cells in tumor microenvironment
Myeloid cells in the TME include monocytes, tumor-associated macrophages
(TAMs), immature differentiated myeloid cells (loosely termed myeloid derived
suppressor cells (MDSCs)) and DCs (128). In a recent study, myeloid cells comprised
more than 50% of the CD45+ tumor infiltrating leukocytes in a PyMT, murine
19
spontaneous mammary tumor model (129). Subsets of the tumor myeloid compartment
are distinguishable by a panel of surface markers, which include: MHC II, CD11b, Ly6C,
Ly6G, CD115, CD11c, CD103, CD24 and F4/80 (128). However, the expression of some
of these surface markers is shared between myeloid subsets. For example, TAMs and
tumor DCs both express CD11c and MHC II to an extent, while CD103 is only expressed
by some subsets of DCs (130). Because T cells are the main drivers of tumor immunity,
and in light of the recent advances in targeting T cell activation in the TME (e.g.
checkpoint blockade therapies), understanding the interactions between different myeloid
subsets and tumor specific T cells is pivotal to develop effective cancer treatments (128).
Although myeloid cells as a whole in the TME have been long considered
nonstimulatory or even suppressive (131), it has only been realized that not all myeloid
subsets are equal. Notably, some of these myeloid subsets such as CD103+ DCs are even
considered potent stimulators of tumor immunity. In recent years, advances in tumor
myeloid cells isolation methods, imaging studies and discovery of new markers used in
multi-dimensional flow cytometry have allowed us to visualize, further dissect and
appreciate the tremendous diversity within the tumor myeloid compartment (132-137).
1.2.4- Tumor associated macrophages (TAMs) and myeloid derived suppressor cells (MDSCs)
TAMs are tumor promoting immunosuppressive cells that contribute to
angiogenesis, fibrous stroma formation and metastasis (138). TAMs play key roles in the
TME through tissue remodeling (139). In a TME that is often characterized by low
oxygen tension and acidic conditions, TAMs promote tissue remodeling by generating
trophic signals, removing dying cell debris and secreting VEGF and platelet derived
20
growth factor (PDGF) (140). The molecular mechanisms of TAM-induced
immunosuppression are not yet well defined, but it is likely through the production of
immunosuppressive cytokines and mediators such as IL-10, TGF-β, arginase, and nitric
oxide (NO). For instance, TAMs inhibit T cell responses by expressing coinhibitory
signals such as PD-L1 and producing high levels of arginase, an enzyme that limits the
availability of arginine causing downregulation of the TCR ζ chain (131). A recent study
from Ruffell et al. showed that TAMs secrete high levels of IL-10 to inhibit local IL-12
production by intratumoral DCs, further inhibiting antitumor CTL responses (141).
Reducing the frequency of TAMs by blocking colony-stimulating factor 1 (CSF-1)
significantly impairs tumor growth and enhances tumor immunity (142, 143).
Early studies of the myeloid compartment in tumor-bearing mice revealed that the
myeloid cells are abnormal due to altered myelopoiesis induced by high systemic levels of
tumor-derived factors such as GM-CSF and IL-6 (131). MDSCs belong to loosely defined
population of immature myeloid cells that include precursors of granulocytes,
macrophages, and DCs (139). In mouse models, two common markers in the myeloid
lineage, CD11b and Gr-1, are used to define MDSCs. In humans, MDSCs express high
levels CD33, CD11b, and have low levels or absent expression of HLA-DR. MDSCs are
further dichotomized into granulocytic and monocytic subsets based on expression of two
different Gr-1 epitopes, Ly6G and Ly6C (144). Like TAMs, MDSCs suppress T cell
responses in MLR assays by producing similar immunosuppressive mediators, including
arginase, NO, and other reactive oxygen species. To further enhance immune suppression,
MDSCs also promote the clonal expansion of antigen-specific natural Tregs and stimulate
21
the differentiation of naïve CD4 T cells into inducible Tregs by producing IL-10 and
TGF-β (139, 144).
1.2.5- Tumor DC- an overview
For tumor immunologists, understanding the nature of intra-tumor immune
response and the mechanisms that enable tumor cells to escape immune responses
remains a daunting challenge. Tumor cells produce many different neoantigens, and can
sometimes present these antigens in the context of MHC Class I. However, this process is
often insufficient to generate any lasting anti-tumor immune response characterized by
CTL-mediated killing (128). As part of the immunoediting process, most tumor cells
often partially downregulate surface MHC I Class I/HLA molecule expression (145).
Coupled with the absence of costimulatory molecule expression on tumor cells, antigen
presentation by tumor cells is poor (128, 146). Additionally, tumor-infiltrating myeloid
cells (e.g. tumor-associated macrophage and monocytes) are also poor T cell stimulators.
(132, 147). On the contrary, DCs are superior for their ability to cross- present tumor
antigens and restimulate tumor antigen specific CTLs (134).
One aspect of tumor immunology that has been largely overlooked until recently
is how intratumor DC influence CTL behavior and function. Evidence from infectious
disease models indicates that antigen-experience T cells require cognate interactions with
tissue DC to expand in situ and achieve full effector functions (148-150). Confocal
microscopy and intravital imaging studies of tumors have revealed interactions between
CTL and intra-tumor APCs (134, 151). Recent studies from my lab and others showed
that intra-tumor DCs were the only cells that could stimulate CTL proliferation, at least in
22
vitro (133). Additionally some DC subsets are well equipped to stimulate anti-tumor
immunity and influence tumor progression (128).
In patients with cancer, defective DC function is considered a key cause of
impaired immune responses to tumor-associated antigens (131). Recent efforts to
characterize DCs and myeloid cells in tumors have revealed an unexpected level of
diversity, opening new windows of opportunity to modulate tumor immunity. I will now
discuss recent advances in our understanding of tumor DC heterogeneity and ontogeny,
and factors in the TME that affect DC recruitment, differentiation, and function.
1.2.6- Tumor DC heterogeneity and origin of tumor DCs
Three major subsets of CD11c+ MHC II+ cDCs have been described in the TME:
1) CD11b- CD103+ DCs (BATF3/IR8-dependent), 2) CD11b+ CD103- (IRF4-dependent),
3) CD11b+ CD64+ F4/80+ subset, which maybe more closely aligned with monocytes and
macrophages than DC (132, 136, 152). Collectively, they constitute a relatively minor
population in most tumors, accounting for 5-10% of all myeloid cells (macrophages and
neutrophils predominate in most tumors). pDCs are a rare tumor-infiltrating population.
Pre-cDCs exist in a variety of transplantable tumor models including B16
melanoma, CT26 colon carcinoma, Lewis lung carcinoma (LLC), and EMT6 breast
carcinoma (133). Tumor pre-cDCs are also morphologically, phenotypically, and
functionally indistinguishable from BM and spleen precursors. Adoptive transfer studies
of bone marrow pre-cDCs revealed that tumors recruit pre-cDCs through a CCL3-
dependent mechanism, where they differentiate into proliferating cDCs (153) . Flt3L
therapy promotes expansion of CD103+ DC progenitors (CD11c+MHCII+CD103-CD11b-)
23
and immature CD103+ DCs at the tumor site (136). Monocytes and more primitive bone
marrow progenitors have also been detected in tumors, particular in the setting of
inflammation induced by anthracycline chemotherapeutic agents, and differentiate into
inflammatory DCs (154).
A prior study from my laboratory showed that a small proportion of tumor cDCs
express lower levels of CD11c, MHC II and other co-stimulatory molecules. Additionally,
Gr-1, an epitope that is not normally expressed in differentiated DCs, is expressed in a
small proportion of these tumor DCs (133). The majority of cDCs arise from pre-cDCs in
the tumor and spleen stimulated antigen-specific T cells equally well, and there was no
difference in the stimulatory capacity between sorted endogenous tumor Gr-1- cDCs and
spleen cDCs. This conflicts with much earlier studies of tumor-infiltrating cDCs, which
suggests that all tumor DCs are “paralyzed” and resistant to maturation stimuli (155, 156).
The criteria used to define tumor DCs and different experimental conditions employed
may account for these differences.
Apoptosis of fully differentiated DCs may not necessarily be the end of their life
cycle. Using transgenic mouse model that allowed in vivo tracking of DCs in tumors, my
lab found that immunostimulatory cDCs derived from pre-cDCs can lose their DC
identity to evolve into CD11c-MHC Class II- regulatory macrophages (termed DC-d-Ms)
(Figure 1.2). These cells potently suppressed T cell responses through the production of
immunosuppressive molecules including nitric oxide, arginase, and IL-10 (157). A
relative deficiency of GM-CSF appeared to provide a permissive signal for DC de-
differentiation, as augmenting GM-CSF expression levels in the tumor blocked this
process. These findings highlight the plasticity of DC and suggest that maintenance of DC
24
identity and function depends, at least partly, on cues received from the tumor
microenvironment.
v
Figure 1.2- Model of tumor DC plasticity. Circulating pre-cDC recruited into tumors
differentiate into Gr-1-cDC, which possess the capacity to stimulate proliferation and
expansion of CTLs. Under the influence of the tumor microenvironment, Gr-1- cDCs
generate 1) Gr-1+ cDC, a subpopulation of maturation resistant, IL-10-producing DCs that
induce T cell anergy, and 2) DC-derived-macrophages (DC-d-M) that potently suppress
CTL proliferation by releasing IL-10, arginase, and nitric oxide.
1.2.7- Tumor CD103+ DCs
25
Recent reports have highlighted the importance of tumor CD103+ cDCs in
promoting antitumor immune responses in primary cancers and metastases (132, 141). On
a per cell basis, tumor CD103+ cDCs stimulate naïve and primed tumor-antigen specific T
cells more effectively than tumor CD11b+ cDC, which is attributed to more efficient
cross-presentation machinery and higher expression levels of IL-12 (132). Tumor
CD103+ also specialize in the transport of intact tumor antigens to tumor draining lymph
nodes (136). Targeted reduction/elimination of tumor CD103+ cDCs in BATF3 knockout
mice and in Zbtb46-diphteria toxin receptor (Zbtb46-DTR) transgenic mice attenuated
responses to cancer immunotherapy. Spranger et al reported that active oncogenic
WNT/β-catenin signaling within melanoma cells in BrafV600E/Pten-/-/CAT-STA tumors
inhibited T cell priming by suppressing the CCL4-dependent recruitment of dermal
CD103+ cDCs. Injecting these tumors with poly:IC activated FLT3 ligand-induced bone
marrow-derived DCs restored responses to anti-CTLA-4 and anti-PD-L1 monoclonal
antibodies (158). Similarly, expansion and activation of CD103+ cDC in B16 melanoma
with Flt3L and poly I:C treatment, respectively, enhanced responses to PD-L1 and BRAF
blockade (136). The relevance of these findings to human cancer remains to be clarified.
Notably, the human equivalents of CD103+ DC and CD11b+ DC show less striking
differences in cross-presentation activity (159).
1.2.8- DC defects in cancer
Tumor inflammation resembles a chronic healing wound (102, 160). Elements of
tissue remodeling and wound healing include enhanced or deregulated angiogenesis,
cellular stress, cancer cell death, and accumulation of fibroblasts and regulatory
26
macrophages (161-163). Many of these elements interfere with effective cross
presentation of tumor antigens by DCs. To evade immune surveillance, tumors hijack
normal regulatory mechanisms that regulate self-tolerance (164). Tumor associated
cytokines such as TGF-β and VEGF, which are also associated with angiogenesis and
tissue remodeling, provide potent signals to inhibit immunogenic antigen presentation to
T cells via DCs (129, 163). Indeed, DCs in tumors display multiple defects (164). Tumor-
associated DCs are not only poor cross-presenters of tumor antigens, but they are also
suppressive and tolerogenic towards tumor-antigen specific T cells. DC dysfunction in
cancer is associated with elevated levels of cytokines, including interleukin (IL)-6 and IL-
10, reduced expression of IL-12, and activation of signal transducer and activator of
transcription 3 (STAT3) (156, 165, 166).
The number, phenotype, and function of cDCs can change over time as the tumor
progresses (167, 168). More importantly, tumor progression may be more related to the
phenotype changes of tumor DCs than the extent of tumor DC infiltration. In a
spontaneous model of ovarian cancer, Scarlett et al. detected increased densities of tumor
infiltrating DCs, macrophages, MDSCs, and T cells, as well as a functional switch in DC
from an immunostimulatory to an immunosuppressive phenotype as the tumors grew. In
this model, depletion of DCs at early time points accelerated tumor growth whereas
depletion at later time points led to tumor regression (167). Krempski et al. also showed in
a transplantable ID8 mouse model of peritoneal ovarian cancer that the number of tumor-
infiltrating cDCs correlated with tumor burden. As tumors progress to the advanced
stages, most tumor DCs progressively expressed PD-1, as well as PD-L1, which was
associated with T cell suppression and loss of tumor infiltrating T cells (168). There are
27
some indications in the literature (169, 170) that these observations are relevant in human
cancers. For example, in a study of colorectal cancer, relapsed patients were more likely
to have fewer DCs in their primary tumors. Also, patients experiencing cancer recurrences
have higher densities of immature tumor DCs and lower densities of mature DCs in their
primary tumor sites (170).
Defects in cross-presentation and activation
Antigen-cross-presentation by DCs plays a critical role in the generation of anti-
tumor responses (171-173). DCs can acquire tumor antigens from multiple sources: 1)
apoptotic or necrotic tumor cells (174-176), 2) chaperone proteins such as heat shock
proteins (177, 178), 3) secreted vesicles (e.g. exosomes) from tumor cells (179), 4) gap
junctions that transfer small antigenic protein fragments (180), 5) tumor plasma
membrane fragments (181). In addition, DCs can acquire preformed peptide-MHC Class I
complexes through direct contact with tumor cells, a process resembling trogocytosis
(also known as cross-dressing) (182, 183). Cross presentation of tumors antigens
generally requires stable and high antigen expression levels and tumor cell apoptosis or
necrosis to release the antigens (184). Unfortunately, these conditions are frequently
unmet in untreated cancers, but can be induced with chemotherapy, radiation therapy, and
oncolytic viral therapy.
There are conflicting views in the literature on whether tumor dendritic cells are
dysfunctional in terms of their cross presentation capacities. Evidence from different
studies suggests that DCs from tumor-bearing mice are sometimes able, albeit inefficient
at times, to cross present tumor antigens to CTLs (134, 185-187). Using a transplantable
28
B16 melanoma model and OVA as the model tumor antigen, Stoitnzer et al. showed that
despite the uptake of OVA, CD11c-sorted tumor DCs cannot cross present the antigen to
OVA-specific CD8+ OT-1 and CD4+ OT-II T cells (188). Moreover, a recent study
reported that accumulation of oxidized lipids in tumor DCs blocked cross presentation of
antigens by reducing peptide-MHC Class I complex on the surface (189). Using live cell
imaging, Engeldhdart et al. showed in a spontaneous model of MMTV-PyMT breast
cancer that tumor-antigen bearing DCs engage tumor-antigen specific T cells; however,
these interactions were unable to fully activate T cell effector functions including the
capacity to kill tumor cells (134). Further studies by the same group revealed that CD103+
DCs possesses superior cross-presentation capacities (132).
The outcome of cross presentation depends primarily on the activation status of
tumor DCs (89, 190). Poor DC activation undermines tumor antigen cross presentation,
even though the TME might possess a strong inflammatory signals (184). Antigen
presentation to either CD4+ or CD8+ T cells by immature or inactivated DCs results in a
number of tolerogenic outcomes, including the induction of regulatory CD4+ T cells (89,
191). For example, van Mierlo et al. showed that a robust antitumor CTL response
requires cross-presentation of tumor-derived antigens by fully activated and licensed
CD11c+ DCs. Treatment of tumor-bearing mice with agonistic anti-CD40 monoclonal
antibody or other DC activating agents such as TLR9 agonists led to the influx of large
numbers of T cells that were capable of eradicating established tumors (185).
29
Co-inhibitory and immunosuppressive molecules
PD-1 and its corresponding ligands PD-L1 and PD-L2 negatively regulate T cell
priming and effector functions (192). PD-L1 and PD-1 expression on tumor DCs
correlated with cancer progression in an ovarian cancer model (168). In a melanoma
model, CD103+ DCs from tumor draining lymph nodes express high amounts of PD-L1 as
compared to CD103+ DCs isolated from non draining lymph nodes (136). Antibody
blockade of PD-L1 and PD-1 mitigates DC dysfunction as evident by increased NF-κB
activation, increased IL-12 and IL-1β production and co-stimulatory molecule expression,
and enhanced T cell stimulatory capacity (193, 194).
Tumor DCs also suppress tumor-specific T cells through the release of
immunosuppressive biomolecules such as nitric oxide (NO), arginase I, and Indoleamine
2, 3- dioxygenase (IDO) (164). Arginase I is an enzyme that degrades and lowers the
bioavailability of arginine, an essential amino acid for CD4+ T cell proliferation and
differentiation (192). High expression levels of arginase I promote the accumulation of
reactive oxygen intermediates such as NO, which block CD8+ T cell responses (195).
IDO is a tryptophan-catabolizing enzyme that plays an important role in inducing and
maintaining tolerance. Tumor cells, DCs, and regulatory macrophages can all express
IDO. IDO activates Tregs and create a milieu deficient in tryptophan, another essential
amino acid used by T cells during activation (196-198). Furthermore, IDO triggers the
production of tryptophan metabolites that induce T cell apoptosis and suppress T cell
proliferation (197).
1.2.9- Mechanisms of tumor DC dysfunction
30
Many studies document the presence and accumulation of immature DCs in blood
and tumors in humans and tumor bearing mice (131, 156). The expansion of immature
DCs is often considered to be a systemic process in which tumor-derived factors skew
differentiation of bone marrow progenitors (156). This view is based partly on in vitro
studies of bone marrow cells stimulated with GM-CSF and tumor-conditioned media, and
studies of mice with large tumor burdens. Recent evidence indicates that GM-CSF-
stimulated bone marrow DC are poor representatives of natural DC, and as such, may not
be the best tool for evaluating the effects of cancer (199, 200). Others and my lab have
shown that hematopoiesis and DC development proceeds normally in mice with small
transplantable tumors and spontaneous tumors. And studies of melanoma, breast cancer,
ovarian cancer, and colorectal cancers have shown that while intra-tumor DCs are
immature, DCs at the margins of the tumor are mature (201, 202). Collectively, these
findings suggest that local rather than systemic factors maybe more relevant to DC
dysfunction. Understanding how tumors promote tumor DC dysfunction is expected to
help advance cancer immunotherapy. Some of the factors and their associated
mechanisms of action are reviewed below in this section.
Tumor-associated cytokines: IL-6 & IL-10
IL-6 IL-6 is a pleiotropic inflammatory cytokine that promotes cell growth and is
generally considered as an antiapoptotic factor (203). Within the tumor milieu, both
tumors cells and various populations of infiltrating immune cells produce IL-6, including
DCs, monocytes, and macrophages. IL-6 signals through the IL-6 receptor complex which
is a heterodimer consisting of IL-6Rα and gp130 subunits, the latter of which participates
in IL-6 mediated STAT3 signal transduction (204). Most IL-6 target genes are involved
31
in cell-cycle progression and suppression of apoptosis (205), which underscores the
oncogenic role of IL-6 in tumor progression. Indeed, IL-6 has been documented to be a
factor that promotes tumorigenesis and tumor progression in many animal models (102,
124, 206). In patients with various epithelial and lymphoid cancers, high serum
concentration of IL-6 correlates with poor outcomes and abnormal immune responses
(207, 208). In patients with hepatocellular carcinoma (HCC), low expression of
microRNA-26a in the tumor tissues is associated with elevated IL-6 expression and poor
survival outcomes (209).
IL-6 also plays a key role in modulating tumor immunity via DCs. Although IL-6
is typically considered a pro-inflammatory cytokine, IL-6 dampens immune responses in
some settings (210, 211). Early studies showed that overproduction of IL-6 found in the
sera of multiple myeloma patients was associated with lower absolute numbers of
circulating precursors of myeloid DCs (212). The peripheral blood DCs from these
patients also showed significantly lower expression of HLA-DR, CD40, and CD80, and
impaired stimulation of allogeneic T cell proliferation compared to DCs isolated from
healthy control subjects (75). In vitro, IL-6 restricts the differentiation of DCs from
monocytes or human CD34+ myeloid progenitor cells, while promoting the development
of macrophages with an alternatively activated phenotype typically associated with
wound healing (213, 214). This phenomenon may be related to the ability of IL-6 to
positively regulate expression of the macrophage colony-stimulating factor receptor
(Csf1r) (215). IL-6 also inhibits NF-κB binding activity and lowers the expression of the
chemokine receptor CCR7 in DCs (216). Furthermore, early studies also reported that IL-
32
6 negatively affects LPS-induced DC maturation and suppresses intracellular MHC Class
II expression, affecting subsequent CD4+ T cell responses (210, 217, 218).
Consistent with these findings, my lab reported that IL-6 regulates the
differentiation of Gr-1+ DCs and IL-10 producing DCs, which possess
immunosuppressive properties (133). IL-6-/- tumor-bearing mice had a three-fold decrease
in the frequency of tumor Gr-1+ DCs. The absence of IL-6 restored MHC Class II
expression on tumor DCs. In vitro, adding blocking IL-6 antibody in the tumor-
conditioned medium culture prevented the differentiation of Gr-1+DCs from pre-cDCs
(36). Collectively, these studies confirmed the significant roles of IL-6 in driving tumor
DC dysfunction.
IL-10 Many cells in the TME express IL-10, including Tregs, TAMs, MDSCs, DCs and
cancer cells (219). As a classic anti-inflammatory cytokine, IL-10 inhibits NF-κB
activation to suppress the production of proinflammatory cytokines such as IL-1β and IL-
12 (220, 221), and stimulation of T cells (222, 223). Both tumorigenic and tumor
suppression roles of IL-10 have been reported in the literature. On one hand, IL-10 has
been shown to modulate cancer cell apoptosis and suppress angiogenesis in tumor
regression (224, 225). For example, exogenous IL-10 expression in mammary and ovarian
carcinoma xenografts inhibits tumor growth and metastasis. In that study, it was
suggested that IL-10 downregulates MHC Class I expression, leading to enhanced NK-
cell mediated tumor killing (224). On the other hand, IL-10 may also favor tumor
development, consistent with its ability to activate STAT3, which provides potent
oncogenic signal to tumor cells (206). Indeed, IL-10 autocrine and paracrine signaling
33
promoted tumor cell proliferation and survival in murine B16 melanoma, and two human
primary cultures of stomach adenocarcinoma and glioblastoma multiforme (226).
IL-10 is an immunoregulatory cytokine that inhibits DC maturation and function
(227). It also skews the development of tolerogenic DCs by downregulating antigen
presentation abilities, inhibiting production of proinflammatory cytokines, and actively
suppressing effector T cell responses. Recently, it was shown that IL-10 produced by the
tumor-infiltrating macrophages from a mouse model of mammary carcinoma, inhibited
IL-12 expression by intratumoral DCs (141). The important role of IL-10 in tumor DC
dysfunction was further demonstrated by blocking IL-10 receptor (IL-10R) with a
monoclonal antibody on tumor DCs, which restored IL-12 expression that was necessary
to drive subsequent anti-tumor T cell responses (47).
VEGF Vascular endothelial growth factor (VEGF) is a secreted multifunctional signaling
molecule produced by most solid tumors and is responsible for the formation of
neovasculature that supports tumor growth and development (228). Signaling by VEGF
also induces proliferation of nearby endothelial cells in the tumor milieu. Elevated serum
level of VEGF is correlated with poor prognosis and survival outcomes in patients with
various cancers (228, 229). VEGF exerts its effects by binding to high affinity membrane
tyrosine kinase receptors, VEGFR-1, 2, 3, expressed mostly on endothelial cells and few
populations of hematopoietic cells, including DCs. Engagement of VEGFR-1/2 by VEGF
induces activation of the serine/threonine MAPK kinase signaling cascade (228).
However, the involvement STAT3 proteins in VEGF signaling pathway has also been
reported. In fact, constitutive STAT3 activation upregulates VEGF expression to promote
tumor angiogenesis (230).
34
Early studies showed that VEGF inhibits DC differentiation and maturation in
vitro through a NF-κB dependent pathway. Addition of neutralizing VEGF-specific
monoclonal antibodies restored normal DC maturation and function in response to various
stimuli (231, 232). These initial findings were confirmed in vivo. Administration of
recombinant VEGF at physiologically equivalent levels to tumor-free mice resulted in
impaired DC development and accumulation of immature Gr-1+ myeloid cells (loosely
termed MDSCs). Treatment of tumor-bearing mice using neutralizing VEGF antibodies
increased the number of spleen and lymph node DCs and improved DC function (233).
Furthermore, VEGF neutralizing antibodies also improved anti-tumor CTL response in
mouse tumor immunotherapy models by improving endogenous DC function (234, 235).
STAT3 activation
Signaling of IL-6, IL-10, and VEGF involve downstream STAT3 activation as a
common mechanism to induce DC dysfunction in tumors (166). Signal transducer and
activator of transcription (STAT) 3 is a member of the STAT family of signal transducer
proteins that is activated by tyrosine phosphorylation cascades in response to cytokines
and growth factors. Ligation of cytokines or growth factors to its cognate cell surface
receptors (e.g. IL-6Rα, IL-10Rα) promotes the phosphorylation of downstream Janus
Kinase 2 (JAK2) enzymes to recruit the cytoplasmic STAT3 protein to the gp130 subunit
to be activated. Then, STAT3 becomes activated after phosphorylation of tyrosine 705
(Y705). Activated STAT3 translocates to the nucleus, where it binds to the promoter
region of genes involving in cell growth, angiogenesis, and immune function (236). In
normal cells under physiological conditions, STAT3 activation is rapid and transient
35
because negatively regulatory proteins such as suppressor of cytokine signaling (SOCS)
and protein inhibitor of activated STAT (PIAS) inactivate the signaling cascade (237).
Constitutive activation of STAT3 occurs in many cancers, which correlates with
accelerated progression and poor outcomes (238). Constitutive STAT3 activation is
reported in both tumor cells and tumor infiltrating immune cells, including DCs. When
STAT3 is hyperactivated in tumor cells, it promotes oncogenesis by enhancing tumor cell
proliferation, survival and invasion (239). STAT3 hyperactivation up-regulates the
expression of anti-apoptotic proteins such as BCL-XL, MCL1, cyclin D1 and MYC,
thereby promoting tumor cell survival. STAT3 activation in tumor cells also promotes
angiogenesis in the TME by inducing the expression of many pro-angiogenic factors such
as HIF-α, VEGF, MMP-2, and MMP-9 (230). In addition to STAT3’s intrinsic oncogenic
activities, STAT3 hyperactivation contributes to tumor progression by enhancing tumor
inflammation and hampering anti-tumor immunity. STAT3 activation mediates cancer
cell-initiated immune evasion signals in various immune cells.
STAT3 activation in DC has been linked to defects in differentiation, maturation,
and function (165, 166, 236, 240). An early study showed that tumour-derived factors
inhibit the differentiation of mature DCs by inducing STAT3 signaling in bone marrow-
derived cells (165). Later studies suggest that constitutive STAT3 activation in DCs
inhibits its maturation (240). Constitutive STAT3 activation in DCs is also linked to lower
MHC II and costimulatory molecules expression. Both the intensity and duration of
STAT3 signaling determines whether DC dysfunction develops (241, 242).
Pharmacologic inhibitors and genetic ablation of STAT3 signaling restores maturation
response to inflammatory stimuli in DCs incubated in tumor-conditioned medium. In a
36
study using the Cre-loxP system to delete STAT3 in hematopoietic cells, tumor-
infiltrating DC from STAT3-/- mice expressed higher levels of surface MHC Class II,
CD80, CD86, and produced more IL-12, which was associated with better anti-tumor
immune responses (243). Furthermore, STAT3-depleted DCs are more resistant to the
effects of tumor-derived factors by becoming more potent T cell stimulators and retaining
a high capacity to induce IL-12, IFN-γ type Th1 immune response (244).
37
TIM-3
T cell Ig and mucin domain (TIM-3), a receptor for galectin-9, was identified
initially as a negative regulator of Th1 immunity (245), but is also expressed by myeloid
cells, including DCs (246). Chiba et al. reported that tumor-derived factors upregulate
TIM-3 expression in tumor DC (247). TIM-3 plays dual roles in antitumor immunity.
Under some conditions, the interactions between galectin-9 and TIM-3 promote DC
maturation and cross priming of tumor specific T cells (247, 248). By contrast, the
interaction of TIM-3 with HMGB1, a classic DAMP commonly present in high amounts
in the TME, prevents tumor DCs from sensing tumor-derived nucleic acid danger signals
released from dying tumor cells, which suppresses type 1 interferon- and IL-12-mediated
anti-tumor responses (247). Furthermore, others have shown that ligating TIM-3 on bone-
marrow-derived and spleen DCs with cross-linking antibodies activates Bruton’s tyrosine
kinase (BTK) and c-SRC, which inhibit DC activation and maturation by blocking the
NF-κB signaling pathway (249).
Lipids
Factors that regulate the uptake, storage, and metabolism of oxidized lipid
molecules in DCs have also been implicated to cause DC dysfunction in cancer (250).
Intracellular fat and glycogen accumulates in DCs with exposure to maturation stimuli in
vitro, and occurs during normal development in lymphoid tissues (251). Several reports
suggest that accumulation of oxidized lipids, especially triglycerides (triacylglycerols,
TAG), causes dysfunction and shortens the lifespan of DCs (189, 252-255). CD11c+ DCs
from mouse EL-4 lymphoma, CT-26, and B16-F10 tumors, and in some cancer patients
38
exhibit elevated TAG levels (254). Bone-marrow derived DCs cultured in the presence of
tumor-conditioned explants were found to have elevated levels of TAGs, suggesting that
tumor-derived factors induce lipid accumulation in DCs (254). Tumor-derived factors
regulate the expression levels of scavenger receptor A (SRA1, CD204, MSR1) on DCs,
liproprotein lipase (LPL) and fatty acid binding protein 4 (FABP4) (252, 254, 255). As
compared to normal DCs from tumor-free mice, lipid-laden DCs stimulated T cell poorly
because of defects in antigen cross-presentation and increased production of IL-10 (189,
254).
Endoplasmic Reticulum (ER) stress response
Tumors adapt to hypoxia, nutrient deficiency, and oxidative stress by triggering an
endoplasmic reticulum (ER) stress response, also known as unfolded protein response
(UPR) (152, 256-258). Cubillos-Ruiz et al. demonstrated recently that the TME also
induces an ER stress response in DC that is mediated by spliced transcription factor XBP1
(253). Although the ER stress response contributes to normal DC development and
survival (259, 260), constitutive XBP1 activation in tumor DCs induced abnormal
accumulation of oxidized lipids by targeting multiple triglyceride biosynthetic genes
(253). Downstream of XBP1 activation, the accumulation of oxidized lipids in tumor DCs
reduced antigen presentation capacity by decreasing cell surface expression of peptide-
MHC complexes. Reactive oxygen species generated reactive lipid peroxidation
byproducts (e.g. aldehyde 4-hydroxy-trans-2-nonenal (4-HNE)) that sustained XBP1
activation. Notably, lipid accumulation induced by this pathway operates independently
of scavenger receptors involved in the uptake of extraceullular lipids. Inactivation of
39
XBP1 in tumor DC improved anti-tumor T cell immune responses and the control of
tumor growth in an ovarian cancer model.
Gangliosides
Gangliosides are sialic-acid containing glyosphigolipids that are expressed
endogenously in the plasma membrane of all vertebrate cells. Gangliosides shed from
various tumor have been shown to suppress anti-tumor immune responses by affecting T
cells, NK cells, and DCs (261, 262). Gangliosides derived from neuroblastoma and
melanoma inhibit DC differentiation from human nomonocytes and mouse bone marrow
cells (263, 264). Exposure of bone marrow-derived DCs to GM1 ganglioside inhibited
maturation and up-regulation of co-stimulatory molecules CD80 and CD86, reducing their
capacity to prime naïve T cells. T cells primed with per-treated ganglioside DCs also
produced significantly less IFN-γ and IL-2 upon re-stimulation (261, 264).
40
1.3- DCs and Cancer immunotherapy
1.3.1- Cancer immunotherapy
Activating the immune system to achieve therapeutic benefits in cancer has long
been a goal in oncology (265). The manipulation of the immune system for cancer
therapeutic effects dates back to ‘Coley’s toxins’ in the 1890s. The idea was to stimulate
‘antibacterial phagocytes’ (e.g. macrophages) using inactivated mixtures of S. pyogenes
and S. marcescens to kill bystander tumor cells. Some significant responses were
recorded, but successes were sporadic and difficult to reproduce (265). Today, it is
evident that immunity plays a critical role in tumor growth and cancer progression. Our
knowledge of the role of the immune system in cancer has led to recent successes of
several proof-of-concept clinical trials. Notably, immunotherapies using checkpoint
blockade inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) and adoptive cell therapy
(ACT) using expanded tumor infiltrating lymphocytes (TILs) or chimeric antigen receptor
(CAR) T cells have gained tremendous prominence in treating cancer patients with more
immunogenic tumors, in particular metastatic melanoma (266-269). However, only
subsets of patients show robust response to these immunotherapies, which warrant further
explorations into the immune mechanisms of cancer immunotherapies in order to improve
clinical efficacy.
The success of cancer immunotherapy requires a multipronged approach. While a
major goal of immunotherapy is to expand the number of functional tumor antigen-
specific CTLs (270), tumor immune suppressive mechanisms consistently hinder
antitumor immunity (265). Anti-tumor immune responses elicited by ACT and active
41
vaccination approaches rely on interactions between CTLs and the TME. Furthermore,
recent evidence strongly suggests that secondary antigen presentation by DCs in the
tumor modulates the number, duration and quality of CTLs (132, 148). Unfortunately, due
to the immunosuppressive nature of the TME, tumor DCs are often dysfunctional,
resulting in inefficient tumor CTL activities. Additionally, tumors evade immune attack
through many mechanisms, including the activation of checkpoint regulatory pathways
through CTLA-4 and PD-1/PD-L1, and active suppression by MDSCs, TAMs, and Tregs
(112-115). Therefore, an ideal immunotherapy approach must also address these issues
simultaneously.
1.3.2- Adoptive cell therapy
Pioneered by Rosenberg and colleagues, adoptive cell therapy (ACT) is a highly
personalized cancer therapy that involves ex vivo (usually autologous) expansion of
tumor-specific T cells (108). The resected tumor specimen is digested into a single-cell
suspension or divided into multiple tumor fragments that are individually grown in IL-2
(up to 6000 IU/mL). TILs overgrow and destroy tumor cells within 2 to 3 weeks, and
generate cultures of T cells that can be tested for tumor recognition reactivity. These
cultures are then rapidly expanded (up to 1011 T cells per infusion) in the presence of
irradiated feeder lymphocytes, OKT3 (mAb to CD3), and high doses of IL-2. By
approximately 5 to 6 weeks from the initial resection, these T cells can be collected for
infusion into patients, who typically also undergo lymphodepletion using a
nonmyeloblative chemotherapy regimen (271). Since the original clinical study reported
by Rosenberg and colleagues at the NIH in 1988 (272), between 35-55% of melanoma
patients have shown objective response (OR) to TIL-ACT therapy (271). Impressively,
42
during a clinical study reported in 2011, 20% of recruited patients (n=93) with advanced
metastatic melanoma achieved complete response (CR) beyond 5 years (269).
Despite reports of these impressive statistics, the exact specificities of the infused
T-cells remain unknown. A major limiting factor for the success of ACT in humans is the
identification of antigens that are selectively expressed on cancer cells, but not in normal
tissues (271). Several classes of tumor-specific Ags are defined that could potentially be
used as targets for TILs, including: 1) overexpressed and differentiation antigens (e.g.
MART-1 and gp100); 2) cancer-testis antigens expressed on tumor specific cells (NY-
ESO-1); and 3) neoantigens, mutated antigens that are truly tumor-specific (273). As
compared to the other two classes of Ags, TILs that recognize tumor-specific neoantigens
are more desirable to reduce toxicities to healthy tissues (273). In recent years, exome
sequencing enabled the identification of tumor-specific mutations and to engineer TILs
that recognize neoantigens (271). Knowledge of these mutations can be used to synthesize
‘minigenes’ or polypeptides that can be expressed by a patient’s autologous APCs,
processed and presented in the context of the patient’s HLA. Coculture of autologous T
cells with these APCs ex vivo allows selection of T cells that recognize individual
mutations based on the expression of activation markers (e.g. 4-1BB on CD8+ T cells) that
are upregulated when they recognize their cognate target antigen. Using FACS, these T
cells are purified before expansion and reinfusion into the patient (271). Using this
approach, neoepitope specificities were successfully identified in the isolated TILs from
melanoma patients, although the anti-tumor responses in vivo were not measured and
examined in these studies (274, 275).
43
Another breakthrough in immunotherapy related to ACT is the development of T
cells expressing CARs. CAR-transduced autologous T cells are engineered to express
TCRs that recognize specific tumor antigens (271). These TCRs are engineered by linking
the variable regions of the antibody heavy and light chains to intracellular chains such as
CD3ζ, often coupled with costimulation domains such as CD28 and 4-1BB. The
advantage of this approach is that CARs provide non-MHC-restricted recognition of cell
surface antigens such as CD19, and can be transduced easily into autologous T cells using
viral vectors (271). Similar to TIL-ACT, CAR-transduced T cells are then expanded
vigorously ex vivo before infusing into the patients. Ongoing clinical trials using
engineered CAR T cells to treat leukemia and lymphoma patients showed promising
results, with the first successful clinical application of anti-CD19 CAR gene therapy in a
lymphoma patient (276). Administration of autologous T cells expressing anti-CD19 CAR
resulted in dramatic tumor regression and prolonged B cell depletion (with a follow up of
39 weeks) (276). Since then, multiple groups have designed several anti-CD19 CARs that
express different costimulation domains to effectively treat follicular lymphoma, large-
cell lymphoma, and chronic lymphocytic leukemia (CLL) and acute lymphocytic
leukemia (ALL) (267, 277-279).
1.3.3- Chemotherapy and radiation therapy on the immune system
Immunogenic cell death (ICD) is a recognized benefit of conventional cancer
therapies. It is characterized by the release of DAMPs molecules such as calreticulin
(CRT), heat shock proteins (Hsp70/Hsp90), ATPs, and HMGB1 in response to
chemotherapeutic agents (e.g. anthracyclines, oxaliplatin) or radiotherapy (280). The
immune response stimulated by chemotherapy requires DCs to engulf, process, and cross-
44
present antigens from dying tumor cells to tumor-specific CD8+ T cells. During ICD,
cancer cells undergoing endoplasmic reticulum stress and autophagy expose CRT on the
outer leaflet of the plasma membrane, and secrete ATP during apoptosis. Additionally, the
nuclear protein HMGB1 is released as the cancer cells’ membranes become permeabilized
during necrosis. Certain DAMPs (CRT, ATP, HMGB1) released by cancer cells through
ICD serve as proinflammatory stimuli by ligating to CD91, P2PX7 (a purigenic receptor),
and TLR4 on DCs respectively. These molecules facilitate the recruitment of DCs to the
tumor bed (stimulated by ATP), engulfment of tumor antigens by DCs (stimulated by
CRT), and optimal antigen presentation to T cells (facilitated by HMGB1 stimulation of
TLR4). In a recent report by Vacchelli et al, the homing of DCs to the tumor bed after
chemotherapy also requires the expression of formyl peptide receptor 1 (FPR1) on DCs
(46). The release of its ligand by tumor cells, annexin-1, promoted stable interactions
between chemotherapy treated dying tumor cells and DCs (281). Together, these
processes contribute to DC maturation, resulting in the release of IL-1β and IL-12,
triggering IL-17-dependent and IFN-γ mediated immune response involving tumor-
specific CTLs and γδ T cells directed to eradicate cancer cells after chemotherapy.
1.3.4- DCs and cancer vaccines
Therapeutic cancer vaccines are designed to generate protective tumor-specific
CTLs and long-lived memory CD8+ T cells (282). Accordingly, many vaccine strategies
and protocols have been developed with these goals in mind, these strategies include:
vaccination with tumor-associated peptides or whole tumor proteins/lysates, transfer of
peptide-pulsed immunogenic APCs, infection with recombinant viruses, and transfer of
engineered tumor cells expressing GM-CSF or FLT3L (265). During the preparation of
45
tumor vaccines, DCs play a central role by capturing, processing, and presenting tumor
antigens to T cells (184). Activated (matured), antigen loaded DCs are desired when
initiating the differentiation of tumor antigen specific T cells to effector T cells.
One of these tumor vaccine preparation approaches involves transducing
autologous or allogeneic irradiated tumor cells to stably express GM-CSF or FLT3L,
which attracts and promote the development and activation of DCs (283). The resulting
vaccines, GVAX (GM-CSF- transduced) and FVAX (FLT3L- transduced), have shown
some successes in early stages of clinical trials for pancreatic, melanoma and prostate
cancer, however, some have failed in phase III stage due to lack of clinical efficacy (284-
286). This failure might reflect the low immunogenicity elicited by these approaches or
the inability of allogeneic tumor cell lines to represent the broad spectrum of tumor
antigens, including shared or neoepitopes, in melanoma and prostate cancers (265).
Other than whole tumor cell/ peptide based vaccines, DC based vaccines are of
considerable interest. In this approach, DCs are isolated from a cancer patient and loaded
with selected antigens ex vivo, such as peptides or tumor cell lysates, matured and
activated with TLR ligands, and the reinfused back into the patient to induce effective
tumor-specific T cell expansion (282). One of the earliest trials of DC based vaccines
used CD14+ monocytes harvested from patient’s PBMCs, and then cultured in the
presence of GM-CSF and IL-4 to produce DCs (287, 288). Few studies also suggest that
substituting IL-4 with IL-15 during the differentiation process may generate DCs that are
better primed for CD8+ T cell activation (289, 290). Another method for generating DCs
involves leukapharesis and enrichment of CD34+ hematopoietic progenitors, which can
then be cultured in the presence of GM-CSF, FLT3L and matured with TNF-α (291). This
46
results in a mixture of monocyte-derived DCs and a subset resembling Langerhans cells.
In clinical trials for melanoma patients, DCs generated from CD34+ progenitors generated
IFN-γ producing T cells in vivo and elicited tumor-antigen specific tetramer responses
(291, 292).
1.3.5- Checkpoint blockade therapy
CTLA-4 and PD-1 are examples of inhibitory molecules expressed by activated T
cells (293). Upon engagement of TCR with cognate antigens, activation of the CTLA-4 or
PD-1 pathway modulates T cell activation by inducing TCR stop signals (294), to
downregulate adaptive immune responses. Recent advances in developing blocking
antibodies that directly target these inhibitory molecules have shown promising results
(295). Ipilimumab (anti-CTLA-4) and nivolumab or pembrolizumab (anti-PD-1) form a
new class of cancer immunomodulatory treatments (293). These antibodies have elicited
durable clinical responses, and in a fraction of patients, long term remissions where
patients exhibit no clinical signs of cancer (268, 295, 296).
The anti-CTLA-4 antibody was the first checkpoint inhibitor approved by the
FDA for clinical use in advanced melanoma patients (295). Durable responses with
ipilumumab are rather limited, but when used in combination with nivolumab, the
responses are better with either agent alone (296, 297). The rationale of using anti-CTLA-
4 is to block its interaction with B7 molecules to allow persistent anti-tumor T cell
responses (298). The clinical benefits of ipilimumab correlate with the mutation load in
the tumor genome as well as the number of tumor neoantigens (299, 300). In a recent
study reported by Kvistborg et al., CTLA-4 blockade allows T cells to react to new
47
melanoma associated antigens, which broadens the anti-tumor CD8+ T cell repertoire
(301). After CTLA-4 treatment, the patient’s anti-tumor CD8+ T cells not only recognized
neoantigens, but also melanoma-associated shared self-antigens as well, suggesting that
CTLA-4 blockade may break tolerance to these self-antigens expressed by melanoma
cells (301). The mechanisms of how CTLA-4 blockade can enhance antigen CD8+ T cell
responses are unclear. But it is hypothesized that CTLA-4 blockade allows CD8+ T cells
to be more responsive to tumor antigens by lowering the activation threshold, and/or
deplete Tregs and inhibit Treg activities (302, 303). Since Tregs can potentially suppress
the DC function in the TME (304), blocking CTLA-4 might enhance cross-presentation of
tumor antigens.
PD-1 is expressed by activated T cells, while its ligand, PD-L1 is expressed on
APCs, epithelial, endothelial, and tumor cells in response to IFN-γ (305). Unlike CTLA-4,
the function of PD-1 does not inhibit costimulation, but rather interferes with downstream
TCR signaling pathway (306). Since PD-L1 can be expressed on many cell types other
than T cells, this suggests that rather than functioning early in T cell activation, activation
of PD-1/PD-L1 pathway prevents other cells from T cell attack later during an immune
response (298). Although both CTLA-4 and PD-1 are both negative regulators of T cell
activation, due to the timing of activation and mechanistic differences, blocking PD-1
pathway offers distinct advantages compared to CTLA-4 blockade, which might explain
why it is advantageous to offer combination antibodies to cancer patients. Clinical data
regarding PD-1/PD-L1 blockade showed promising benefits in patients with non-small
cell lung cancer (NSCLC), advanced melanoma, renal cell carcinoma, and colorectal
carcinoma (268). Positive response to PD-L1 blockade appeared more likely in patients
48
with constitutive PD-L1 expression in the tumors cells, as well as tumor-infiltrating
immune cells (268). Because PD-L1 expression is inducible by signals derived from
CD8+ T cells, such as IFN-γ, PD-L1 expression in some cases is driven by tumor
infiltrating CD8+ T cells and may predict antitumor T cell responses, suggesting that PD-
L1 expression levels could be used as a surrogate marker to predict patients who are most
likely to response to anti-PD-1 therapy (307).
49
1.4- Overall objectives and hypotheses
Growing evidence strongly supports an important role of DCs in tumor immunity.
Although cancer-associated inflammation is a well-recognized cause of DC dysfunction,
the drivers of this process remain poorly understood. The overall objective of this study is
to understand the molecular mechanisms and to identify the proximal signals from the
TME that cause tumor DC dysfunction. I will also highlight potential therapeutic targets
to enhance cancer immunotherapy by improving tumor DC function. Based on the current
understanding of tumor DC development and dysfunction, I hypothesized that:
1. Tumor-derived factors provide the proximal signals in the TME (e.g. TLR
and IL-6 signaling) that induce aberrant differentiation of tumor DCs
(Figure 1.3)
2. Tumor- induced differentiation of immunosuppressive tumor DCs is a key
determinant of tumor immunity
3. Blocking signals that induce tumor DC differentiation may provide a novel
therapeutic strategy to improve cancer immunotherapy
Thesis outline
In chapter 3, I will provide data from in vitro experiments that strongly support the
role of TLR2 in causing tumor DC dysfunction. I will also explain how TLR2 activation
regulates signaling of IL-6 and IL-10 in tumor DCs. In chapter 4, I will describe
translational studies using mouse transplantable in vivo tumor models and human
monocyte-derived DC differentiation models to validate the mechanisms proposed in
50
chapter 3. Finally, in chapter 5, I will show that TLR2 is a relevant therapeutic target to
improve cancer immunotherapy.
Figure 1.3- Hypothetical model of tumor DC dysfunction in cancer. Tumor-dervied
factors released from the tumor stimulates PRRs on surface of DCs to cause the release of
tumor associated cytokines such as IL-6, that regulates the development of tumor DC.
51
Chapter 2
General methods and materials
52
Mice
Male C57BL/6, BALB/c, TLR2-/-, TLR4-/-, and OT-1 mice and C57BL/6.SJL (CD45.1)
congenic mice were purchased from The Jackson Laboratory or Taconic Farms and bred
in our animal facility. MyD88-/- mice were provided by Dr. Peter Liu (University of
Toronto). Mice were maintained in pathogen free conditions in accordance with
University Health Network Animal Research Committee of University Health Network
reviewed and approved the studies. (Chapters 3-5)
Tumor Models
B16-F10 melanoma (B16), LLC, and human cancer cell lines were purchased from the
American Type Culture Collection (ATCC). B16-OVA was kindly provided by R.W.
Dutton at the Trudeau Institute. Primary colon cancer cells were kindly provided by C.
O’Brien (Ontario Cancer Institute).
To establish the tumors, 0.5-1 x 106 B16 or B16-OVA tumor cells in 100 µl PBS were
injected subcutaneously (s.c.) into the flank of C57BL/6 mice. Tumor dimensions were
measured with calipers, and tumor size was calculated by multiplying 2/3 length and
width. (Chapters 3-5)
Cell isolation
Tumors and spleens from B16 tumor-bearing animals were minced, digested with
collagenase and DNase-I for 0.5h at 37oC, and incubated in PBS containing 2mM EDTA
and 5% FCS for 10 min at room temperature. Mononuclear cells were isolated by
Lympholyte-M (Cedar Lane) or Nycodenz (Axis-Shield) density gradient centrifugation
53
and further enriched for CD11c+ cells by positive selection using MACS (Miltenyi
Biotec) and CD11c+-immunomagnetic beads. Cells were stained with anti-I-Ab or anti-I-
A/I-E (Allophycocyanin-780), anti-CD11c (FITC or allophycocyanin), and anti-lineage
markers (anti-CD3-, anti-CD19, anti-B220, anti-Gr-1, and anti-CD49b). Desired
populations were sorted on a BD FACS Aria II using FACS Diva acquisition and analysis
software (BD, San Jose, CA). We obtained pre-cDC from spleens of C57BL/6 mice
inoculated with Flt3L-producing B16 melanoma. Mononuclear cells isolated by
Nycodenz density gradient centrifugation were negatively selected for lineage markers
(CD3, CD19, B220, CD49b, Gr-1) and MHC II using biotin-conjugated antibodies and
streptavidin-immunomagenetic beads, then further enriched for CD11c+ cells by positive
selection using CD11c+-immunomagnetic beads. The purity of the cell populations used
were routinely >95% based on reanalyzed samples. Isolated cells were cultured in 96-
well plates in 200 µL RPMI 1640 supplemented with 10% FBS, 50 µM 2-
mercaptoethanol, 1 mM sodium pyruvate, 10 mM non-essential amino acids, 50 units/mL
penicillin and 50 µg/mL streptomycin (complete medium) with or without cytokines as
indicated (Chapters 3-5)
Flow cytometry
Cell suspensions were preincubated with anti-CD16/32 to block Fc receptors and then
were washed and incubated with the indicated monoclonal antibody (mAb) conjugates for
30 min at 4o C in a final volume of 100 ml PBS containing 0.5% BSA and 2mM EDTA.
In all experiments, appropriate control isotype matched Abs were included to determine
the level of background staining. (Chapters 3-5)
54
For intracellular cytokine detection, surface antibody-labeled cells were fixed,
permeabilized, and stained with anti-cytokine according to the instructions from the BD
cytofix/Cytoperm Kit (BD Biosciences).
Preparation of conditioned medium
Cancer cell lines, S17 cells (a kind gift from K. Dorshkind, David Geffen School of
Medicine at UCLA, Los Angeles, CA), and human spleen-derived fibroblasts were
cultured at 90% confluence in serum free medium for 20-24 hr. Supernatants were
filtered through a 0.22-µm filter, stored at -80°C, and thawed immediately before use. All
conditioned medium was confirmed to be free of mycoplasma by the Department of
Microbiology, Hospital for Sick Children, Toronto, Canada. Mouse pre-cDC and human
DC were cultured in 50% conditioned medium for 1-3 d. In some murine experiments,
cytokines (10 ng/mL) and neutralizing anti-cytokine (10 µg/ml), anti-TLR2 (100 ng/ml)
were added to the culture medium. For human DC experiments described in chapter 3,
anti-TLR2 antibodies were used at a concentration of 10 µg/ml. (Chapters 3, 4)
Antibodies and reagents
Anti-CD11c (clone HL3), I-Ab (KH84, 25-9-17), I-A/I-E (MF/114.15.2), CD3 (17A2),
CD8, CD19 (1D3), CD49b (pan-NK, DX5), Gr-1 (RB6-8C5), CD11b (M1/70), B220
(RA3 6B2), CD45 (30-F11), CD45.1 (A20), CD40 (3/23), CD80 (16-10A1), Ly6C
(AL21), IFNγ (XMG1.2), CD69 (H1.2F3), IL-6R (D7715A7), STAT3-pY705 (4/P-
STAT3) were purchased from BD Pharmingen. Anti-IL-10R (1B1.3a) and anti-IL-12
(C17.8) were purchased from eBioscience. Anti-IL-6 (MP5-20F3), anti-IL-10
55
(JES052A5), and anti-human TLR2 (Clone 383936) were purchased from R&D. Anti-
mouse TLR2 (T2.5) antibody was purchased from Invivogen. Anti-mouse versican
(EPR12277) and anti-mouse HMBB1 (4C3) antibodies were purchased from Abcam.
These antibodies were unlabelled or conjugated to fluorescein isothiocyanate (FITC),
phycoerythrin (PE), allophycocyanin (APC), Pe-PC7 or biotin as indicated. Biotinylated
antibodies were revealed with FITC, PE, APC, Tex-red, PC5 or PC7.
Murine GM-CSF and FLT3L were purchased from BD Pharmingen. Human IL-4
and GM-CSF were purchased from R&D. Pam3CSK4 and FSL-1 were purchased from
Invivogen.
Cytokine assays
IL-6, IL-1β, IL-10, and IL-12p70 concentrations were measured by ELISA with
commercial kits (OptEIATM, BD Biosciences). Notably pre-cDCs do not require pre-
treatment with a caspase activator to produce IL-1β (He et al., 2013). (Chapters 3-5)
Gene expression analysis
Total cell RNA was prepared DC with the RNAeasy kit (QIAGEN), followed by first-
strand cDNA synthesis suing M-MLV Reverse Transcriptase (Invitrogen). Quantitative
PCR for IL-6R, IL-10R, and STAT3 gene expression was performed using the
LightCycler 480 System (Roche) according to standard manufacturer protocols. Mean
relative gene expression from 3 triplicate measurements was calculated using 2-ΔΔ
CT
method and normalized to GAPDH expression. Primer pairs for IL-6Rα, IL-10Rα,
STAT3, versican, and GAPDH respectively were 5’- atcctctggaaccccacac- 3’/3’-
56
gaactttcgtactgatcctcgtg- 5’; 5’-aagcccttcctatgtgtggtt- 3’/3’-tggtttgggataggtttcca- 5’; 5’ –
gttcctggcaccttggatt- 3’/3’- caacgtggcatgtgactctt- 5’; 5’- tcctgattggcattagtgaaga- 3’/3’-
tttgttttgcagagatcaggtc- 5’; 5’- cggtgctgagtatgttgtgg- 3’/ 3’- ttttggctccaccccttc- 5’.
(Chapters 3, 4)
Allogeneic mixed lymphocyte reactions
Graded numbers of stimulator cells were seeded in triplicate with responder BALB/c
spleen cells (1 x 105/well), and cultured for 3 days at 37°C. Cells were pulsed with 1 µCi
of 3H- thymidine 16h before harvest, and collected onto glass fibre filtres; 3H- thymidine
incorporation was quantified using a scintillation counter. For human DC experiments,
naïve allogeneic lymphocytes were obtained from an unrelated healthy subject.
(Chapters 3, 4)
Intracellular staining for phosphorylated STAT3
Unstimulated control and TCM-treated spleen pre-cDC were washed twice with PBS and
cultured for an additional 4 hours in complete medium to reduce background
phosphorylated STAT3 expression. DCs were recovered, stimulated with graded
concentrations (1-200 ng/ml) of IL-6 of IL-10 for 30 min at 37°C, fixed and stained for
CD11c and MHC II, and permeabilized and stained for phosphorylated STAT3. (Chapter
3)
Versican knockdown with lentiviral transduction
Versican shRNA (SHCLNG-NM_004385) and control shRNA (SHC016) constructs were
purchased from Sigma-Aldrich and packaged into lentivirus vectors in our laboratory.
57
LLC and B16 cells were incubated with lentivirus culture supernatants containing
polybrene (10 µg/ml) for 48 hr and selected in the presence of puromycin (1 mg/ml;
Invivogen). Knockdown of versican expression was verified by quantitiative real-time
PCR and western blot. (Chapters 3, 4)
Versican expression by western blotting
LLC cells transduced with versican- and control-shRNA were lysed in RIPA buffer
(containing protease inhibitor cocktail) and 40C and ran on SDS-PAGE gels. Protein
concentrations were determined using QuickStartTM Bradford Protein Assay (BioRad). A
total of 75 µg of protein per lane were loaded on 10% SDS-PAGE gels. After transfer
onto nitrocellulose membranes, blots were then probed with 2 µg/ml of rabbit polycloncal
anti-versican (Clone ab19345, Abcam) and anti-rabbit HRP antibody at 1:3000 to detect
the indicated protein. Rabbit anti-beta actin IgG (Clone CL2810AP, Cedarlane) was used
as loading control. Blots were developed using an ECL Western blot detection kit (Fisher
Scientific). (Chapter 3)
Intracellular staining for phosphorylated STAT3
DCs sorted from B16 tumors and spleen of WT and TLR2-/- mice were fixed and stained
for CD11c and MHC II, and permeabilized and stained for phosphorylated STAT3.
(Chapter 4)
Generation of human monocyte-derived DC
PBMCs were isolated from fresh blood samples using Ficoll-Paque and resuspended in 5
mL of AIM V media, and allowed to adhere to the surface of T25 flask for 2 hours. Non-
58
adherent cells were then removed by gently washing twice with media, and the remaining
adherent cells were maintained in 5 mL AIM V media supplemented with GM-CSF (1
ng/ml) and IL-4 (10 ng/ml) for 7 days. (Chapter 4)
GVAX immunization
GVAX immunization was performed, as described (308). GM-CSF producing B16 cells
were generated with a retrovirus encoding murine GM-CSF, as described (153).
Supernatants from 24 hour-cultured B16-GM-CSF cells (0.5×106 in 4 ml medium)
contained ∼3000 pg/mL of GM-CSF. Mice were injected with 2.5 × 104 B16-F10
melanoma (B16) cells subcutaneously in the left flank. On the same day, they were
immunized with 1 × 106 irradiated GM-CSF producing B16 cells injected subcutaneously
in the right flank, which was repeated on days 3 and 6. (Chapter 5)
Generation of effector OT-I T cells
OT-I C57BL/6.SJL (CD45.1) transgenic mice were injected i.p. with 1 x 109 viral
particles of the adenovirus vector encoding OVA. Effector CD8+ T cells were isolated 3
days later by positive selection using CD8 magnetic beads (Miltenyi). (Chapter 5)
Antitumor CTL responses in vivo
Effector CD45.1 OT-I cells (1-5 x 106) were labeled with CFSE and injected i.v. into
TLR2-/- and wild-type CD45.2 mice bearing 0.5-1 cm diameter B16-OVA or B16 tumors.
Three days later, the tumors were recovered to assess the frequency and proliferation in
tumors and lymphoid tissues by flow cytometry. Intracellular IFN-γ production in OT-I
CTLs was assessed after restimulation with SIINFEKL peptide in vitro. For tumor
59
survival studies, 1 x 107 OT-I CTLs were injected into mice 3 days after tumor
inoculation; mice were killed when tumors exceeded 1 cm2. (Chapter 5)
Statistical Analysis
Results are expressed as means ± SEM and mean ± SD. Data were analyzed by Student’s
t test (parametric data), Mann-Whitney test (non-parametric data), two-way ANOVA, and
the long-rank test. The analyses were performed with the GraphPad Prism statistical
program. p values < 0.05 were considered significant.
60
Chapter 3
The role of TLR2 in promoting DC dysfunction in vitro
Contributions
Michael Tang, Jun Diao, Hongtao Gu, Ismat Khatri, Jun Zhao,
and Mark Cattral
Parts of this chapter were taken from:
Tang M, Diao J, Gu H, Khatri I, Zhao J, Cattral MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating
IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):2851-64.
61
Introduction
Inflammation is now widely accepted as one of the hallmarks of cancer (101).
Infiltrating leukocytes, mesenchymal stromal cells, extracellular matrix (ECM), and
various cytokines and growth factors collectively form a tumor microenvironment (TME)
that is heterogeneous, complex and dynamic (120). The composition of the TME
influences tumor growth, metastasis, cancer progression and prognosis (105). The drivers
of inflammation in cancer remain poorly understood, although evidence points to the
importance of endogenous “danger signals” released from stressed and dying cancer cells
and components of the TCM that activate PRRs such as TLRs and NOD-like receptors
and downstream signaling cascades (121, 122). In addition, TLR agonists produced by
commensal bacterial in the gut have been shown recently to enhance cytotoxic activity of
tumor-associated myeloid cells during chemotherapy (126).
The immune responses to tumor antigens play a critical role in controlling tumor
growth and progression. As the primary APCs of the immune system, DCs provide a key
link between innate and adaptive immunity (7). In tumor immunity, DCs modulate
antitumor responses by regulating the magnitude and duration of infiltrating CTL
responses (128). Unfortunately, due to the immunosuppressive nature of the TME, as well
as the inherent plasticity of DCs, tumor DCs are often dysfunctional, a phenomenon that
contributes to immune evasion (131).
DC dysfunction in cancer is strongly associated with elevated expression levels of
IL-6 and IL-10, reduced expression of IL-12, and activation of STAT3 (131, 166).
Efforts to further define the mechanisms and their relationship to cancer-associated
62
inflammation have been hampered by the poor understanding of tumor DC ontogeny and
their evolution within the TME.. Previous work from my lab showed that tumors recruit
circulating pre-cDCs through a CCL3-dependent mechanism, where they differentiate
into proliferating cDCs (153). We showed that the TME drives immunostimulatory Gr-1-
cDCs to become a functionally defective Gr-1+ cDC subpopulation that induces T cell
tolerance (133). Further studies supported the role of IL-6 in regulating this aberrant DC
differentiation process (133), although the proximal signals remain unclear . I
hypothesized that tumor-derived factors drive tumor DC dysfunction by activating TLRs
and down-stream signaling cascades.
63
Results
Tumors stimulate autocrine production of IL-6 and IL-10 in pre-cDC through TLR2
Cancer has no apparent adverse effects on circulating pre-cDCs (153), at least for
tumors that are less than 1cm in diameter, suggesting that tumor DC dysfunction is
initiated by direct exposure to the tumor microenvironment. We hypothesized that the
immediate cytokine response of pre-cDC to tumor-conditioned medium (TCM) might
serve as a screening tool to investigate whether TLR signaling regulates cDC
differentiation in cancer. All TLRs, except TLR3, signal through the adaptor molecule
myeloid differentiation factor 88 (MyD88) (66). We first cultured WT and MyD88-
deficient spleen pre-cDC in the presence of TCM generated from Lewis lung carcinoma
(LLC) cell line, and measured IL-6 and IL-10 concentrations in culture supernatants 24
hours later. We examined these cytokines because of their known association with tumor
DC dysfunction. We found that WT pre-cDC, but not MyD88-deficient spleen pre-cDC,
produced both IL-6 and IL-10; neither cytokine was detectable in the TCM alone (Figure
3.1A). We also obtained similar results using TCM generated from B16 melanoma. These
results suggested that the MyD88-dependent signaling pathway is involved in the
production of IL-6 and IL-10 from TCM- simulated pre-cDCs.
TLR2 and TLR4 are well-recognized receptors for danger-associated molecular
patterns (DAMPs) in the tumor microenvironment (309). Therefore, we investigated the
response of spleen pre-cDC from TLR2-/- and TLR4-/- mice to TCM. TCM failed to
stimulate IL-6 and IL-10 production in TLR2-/- pre-cDC, whereas pre-cDC from TLR4-/-
mice produced both cytokines at levels similar to WT control (Figure 3.1B). As expected,
64
control S17 fibroblast-conditioned medium (S17) stimulated low levels of IL-6 and IL-10
in WT, TLR2-/-, TLR4-/- pre-cDC.
Figure 3.1 TCM stimulates autocrine IL-6 and IL-10 in DCs through TLR2 and
MyD88. A) IL-6 and IL-10 production, as measured by ELISA from WT and MyD88-/-
mice pre-cDCs stimulated with TCM for 24 hr. B) IL-6 and IL-10 production by WT,
TLR2-/-, and TLR4-/- mice pre-cDCs incubated in TCM and S17 conditioned medium
(S17) for 24 hr.
We also considered whether a cell-intrinsic defect accounted for the inability of
TCM to stimulation IL-6 and IL-10 production in TLR2-/- pre-cDC. However, when I
added neutralizing anti-TLR2 antibodies in the TCM culture, they inhibited cytokine
production in WT pre-cDC in a dose-dependent manner (Figure 3.2).
65
Figure 3.2 Anti- TLR2 neutralizing antibodies reduce autocrine IL-6 and IL-10 in a
dose-dependent manner. IL-6 and IL-10 production by WT pre-cDCs cultured in TCM
with neutralizing TLR2 mAb or immunoglobulin G1 (IgG1) control antibody for 24 hr.
TLR2 forms heterodimers between TLR1 or TLR6 to recognize a wide range of
triacylated (e.g. Pam3CSK4) or diacylated (e.g. synthetic FSL-1) lipoproteins,
respectively. Both FSL-1 and Pam3CSK4 mimicked TCM in their ability to stimulate IL-
6 and IL-10 in WT pre-cDC, whereas LPS only stimulated IL-6 production (Figure 3.3).
Collectively, these findings indicated the tumor-derived factors stimulated production of
autocrine IL-6 and IL-10 in pre-cDC through a TLR2/MyD88-dependent signaling
pathway.
66
Figure 3.3 TLR2 agonists mimic TCM’s ability to produce IL-6 and IL-10. IL-6 and
IL-10 production by WT pre-cDCs cultured in TCM, FSL-1 (100 ng/ml), Pam3CSK4
(100 ng/ml), and LPS (1 µg/ml) for 24 hr.
*Figures 3.1-3.3: Results are displayed as mean ± SEM and are representative of more
than three independent experiments. *p<0.05, **p<0.005, *** p<0.001, determined by
Student’s t test.
TCM promotes differentiation of Gr-1+ DC via TLR2 signaling
We previously reported that under the influence of tumor milieu, some pre-cDCs
differentiate into a subpopulation of immunosuppressive cDC that can be distinguished by
the Gr-1 cell surface marker. To further characterize the role of TLR2 in Gr-1+cDC
differentiation, we sorted splenic pre-cDC from WT and TLR2-/- mice and cultured them
in TCM, S17 conditioned medium (SCM), and culture medium containing GM-CSF. At
48 hours, we found that 10-15% of WT cDC cultured in the presence of TCM expressed
Gr-1 de novo, whereas most TLR2-/- cDC remained Gr-1 negative. Pre-cDC cultured in
the presence of SCM or GM-CSF generated few Gr-1+cDCs regardless of TLR2
67
expression (Figure 3.4A). To test the immunostimulatory capacities of these cells, we
recovered the cells at 72 hrs and placed them in mixed allogeneic lymphocyte reactions.
We found that TLR2-/- cDC from TCM cultures stimulated higher levels of proliferation
compared to the WT counterparts (Figure 3.4B). Therefore, TCM induced the generation
of Gr-1+cDC via TLR2.
Figure 3.4 Tumor conditioned medium promotes differentiation of
immunosuppressive Gr-1+ cDC. A) Pre-cDCs from wildtype and TLR2-/- mice were
incubated with TCM, S17 conditioned medium (SCM), and GM-CSF (1000 U/ml) for 48
hr. Frequency of Gr-1+ CD11c+ MHC II+ cells were measured by flow cytometry. B)
Stimulation capacity of DCs recovered at 72 hr in mixed lymphocyte reactions. Results
are displayed as mean ± SEM, and are representative of >3 independent experiments.
*p<0.05, **p<0.005, determined by Student’s t test.
Autocrine IL-6 and IL-10 promotes differentiation of IL-10 producing DC
We found that the Gr-1 cell surface marker was not stable in vitro. We therefore
chose to use IL-10 production as a read-out for DC dysfunction in the subsequent
experiments. Notably, previous studies have shown that in contrast with spleen DC, tumor
DCs preferentially produce IL-10 rather than IL-1β and IL-12p70 when stimulated with
68
LPS. To determine whether TCM induces this aberrant response in pre-cDC, we extended
the culture period to 72 hours, recovered and washed the cells extensively, and stimulated
them with LPS overnight. WT pre-cDCs pretreated with TCM, but not SCM, produced
large quantities of IL-10. TLR2 deficiency blocked this response and increased their
capacity to produce IL-1β and IL-12p70 (Figure 3.5A). Pre-cDCs pretreated with TLR2
ligands, FSL-1 and Pam3CSK4 also produced high amounts of IL-10 as compared to
those cells pretreated with LPS (Figure 3.5B), whereas adding anti-TLR2 neutralizing
antibodies to TCM blocked IL-10 production (Figure 3.5C)
69
Figure 3.5 Tumor conditioned medium promotes differentiation of IL-10 Producing
through TLR2. A) Production of IL-10, IL-1β, and IL-12p70 by WT and TLR2-/- pre-
cDCs pretreated with TCM or S17 for 72 hr, then recovered, washed and recultured for 18
hr B) IL-10 production by WT pre-cDCs pretreated with TCM, FSL-1, Pam3CSK4, and
LPS for 72 hr. C) IL-10 production by WT pre-cDCs pretreated with TCM with orwithout
added anti-TLR2 antibodies (25-100 ng/ml) for 72 hr.
We next investigated the role of TCM-induced IL-6 and IL-10 in this process. We
found that blocking IL-6 or IL-10 with neutralizing antibodies significantly reduced the
capacity of TCM to induce the generation of IL-10 producing DCs from WT pre-cDC
(Figure 3.6A). The simultaneous blockade of both cytokines worked synergistically to
inhibit IL-10 production. These findings suggested that paracrine stimulation with IL-10
and IL-6 might be sufficient to induce the generation of immunosuppressive DCs.
However, exogenous IL-6 and IL-10 failed to restore IL-10 production completely when
TLR2 was blocked with neutralizing antibodies or was genetically deficient (Figures
3.6B and C), even though the concentration of these cytokines was 2- to 3- fold higher
than those produced by WT pre-cDCs in TCM. Furthermore, adding IL-6 and IL-10 to
WT pre-cDCs cultured in Flt3 ligand or S17 medium for 72 hr failed to generate IL-10
producing DCs (Figure 3.7A and B). Collectively, these findings suggested that TLR2
signaling engages mechanisms beyond stimulating autocrine IL-6 and IL-10 to promote
DC dysfunction.
70
Figure 3.6 The role of autocrine IL-6 and IL-10 signaling in promoting DC
dysfunction. A) IL-10 production by WT pre-cDCs pretreated with TCM in the presence
of absence of blocking antibodies to IL-6 (10 µg/ml) and IL-10 (10 µg/ml) for 72 hr. B) IL-
10 production by WT pre-cDCs pretreated with TCM in the presence or absence of
blocking antibodies to TLR2 (100 ng/ml) and exogenous IL-6 and IL-10 (both 1 µg/ml) in
the primary culture for 72 hr. C) IL-10 production by WT and TLR2-/- pre-cDCs pretreated
with TCM in the presence or absence of exogenous IL-6 and IL-10 for 72 hr. Results are
displayed as mean ± SEM. **p < 0.005; ***p < 0.001.
71
Figure 3.7 Exogenous IL-6 and IL-10 fail to induce IL-10 producing DC. Pre-cDC
were cultured in medium containing Flt3L (A) or S17 fibroblast-conditioned medium (B)
for 72 hr with or without the addition of IL-6, IL-10, or FSL-1 (positive control). At the end
of the culture period, the cells were recovered, washed, and incubated in fresh complete
medium with LPS for 18 hr. The concentration of IL-10 in the supernatant was measured
by ELISA. Data is displayed as mean ± SEM and is representative of 1 or 3 independent
experiments.
We found that TCM stimulates the release of IL-1β from pre-cDCs during the first
24 hours of culture (Figure 3.8A). To investigate whether IL-1β contributes to the
development of DC dysfunction, we added neutralizing anti-IL-1β antibody to TCM.
Blocking IL-1β had no significant effect on IL-10 and IL-6 production at 24 hrs, and
failed to prevent differentiation of IL-10-producing DCs at 72 hrs (Figures 3.8B and
3.8C). These data indicate that IL-1β does not play a role in DC dysfunction.
72
Figure 3.8 IL-1β does not play a role in inducing DC dysfunction in vitro. A) IL-1β
production from WT mice pre-cDCs stimulated with TCM for 24 hr. B) IL-6 and IL-10
production at 24 hr by WT pre-cDCs cultured in TCM in the presence or absence of
neutralizing IL-1β mAb (10 µg/ml). C) IL-10 production at 72 hr by WT pre-cDCs
pretreated with TCM in the presence or absence of blocking antibodies to IL-1β (10
µg/ml). Results displayed as mean ± SEM ***p <0.001 by student’s t test.
TLR2 ligation sensitizes DC to IL-6 and IL-10 stimulation by upregulating IL-6R
and IL-10R expression
We hypothesized that TLR2 signaling might increase the sensitivity of pre-cDCs
to IL-6 and IL-10 to promote DC dysfunction. To test this idea, we first measured the
expression levels of IL-6Rα and IL-10α, the cytokine binding component of these
receptors. As compared to the unstimulated controls, WT pre-cDC stimulated with TCM
and FSL-1 increased cell surface expression of both IL-6Rα and IL-10Rα, whereas
incubation with S17 and stimulation of LPS reduced expression levels slightly (Figure
3.8A & B). Induction of IL-6Rα and IL-10Rα mRNA, as measured by quantitative real-
TCMSCM
GM-CSF
0
500
1000
1500IL
-1β
(pg/m
l)***
0
200
400
600
800 IL-6IL-10
TCM
IL-1β Ab+ +
+-
Cyt
okin
e le
vels
(pg/
ml)
0
500
1000
1500
2000
TCM
IL-1β Ab+ +
+-
IL-1
0 (p
g/m
l)
A B C
73
time PCR, accompanied IL-6Rα and IL-10Rα upregulation (Figure 3.9C). Adding anti-
TLR2 antibodies to TCM cultures blocked these responses completely (Figure 3.9C).
Figure 3.9 TLR2 ligation regulates IL-6R and IL-10R expression in pre-cDCs A-B)
Mean fluorescence intensity (MFI) of surface IL-6Rα or IL-10Rα expression by
unstimulated DCs or DCs cultured in TCM, with or without neutralizing anti-TLR2
antibodies (100 ng/ml), S17, FSL-1, and LPS for 18 hr. C) Real-time PCR analysis of IL-
6Rα (il6ra) and IL-10Rα (il10ra) mRNA in DCs. All values were normalized to gapdh
expression, and fold changes are relative to unstimualted control. Results are displayed as
mean ± SEM. **p < 0.005, ***p < 0.001 by Student’s t test.
Since TCM stimulated IL-6 and IL-10 production in pre-cDC, we speculated that
IL-6Rα and IL-10Rα expression levels were being self-regulated by their respective
cytokine. However, we found that TCM stimulation continued to induce IL-6Rα and IL-
10Rα mRNA and surface receptor expression despite the presence of neutralizing IL-6
and IL-10 antibodies (Figure 3.10). Therefore, these findings suggests that TLR2
74
signaling induced IL-6Rα and IL-10Rα expression independent of its effect on autocrine
IL-6 and IL-10 production and signaling.
Figure 3.10 TCM induces IL-6Rα and IL-10Rα even in presence of neutralizing
antibody to IL-6 and IL-10. Pre-cDC were cultured for 24 hours under the indicated
conditions and analyzed for IL-6Rα and IL-10Rα expression by flow cytometry (left
panel) and qRT-PCR (right panel). A) IL-6Rα expression in the presence or absence of
TCM and anti-IL-6 and anti-IL-10 antibodies. B) IL-10Rα expression in the presence or
absence of TCM and anti-IL-6 and anti-IL-10 antibodies. qRT-PCR values were
normalized to gapdh expression and fold changes are relative to unstimulated control.
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Data are representative of 3 independent experiments, Results are expressed as mean ±
SEM *p < 0.05, *** p < 0.001, determined by Student’s t-test.
Upregulation of IL-6R and IL-10R lowers the STAT3 activation threshold
STAT3 is the main downstream molecular target for IL-6R and IL-10R signaling.
Engagement of IL-6 and IL-10 with its respective cytokine receptors activates STAT3 via
tyrosine phosphorylation. STAT3 phosphorylation promotes IL-10 production while
inhibiting IL-12 production in DCs. To directly test the functional relevance of increased
IL-6Rα and IL-10Rα, we assessed phosphorylated STAT3 levels (pSTAT3) by
phosphoflow-STAT3 assays in response to graded concentrations of IL-6 and IL-10.
Spleen pre-cDCs from WT mice were cultured in TCM or control medium for 24 hours,
recovered and rested cells for 4 hours, and then stimulated with IL-6 and IL-10 for 30
min. Before cytokine stimulation, pre-cDC cultured with TCM and control medium
exhibited similar basal levels of pSTAT3. The threshold IL-6 and IL-10 concentrations
that induced STAT3 phosphorylation was about 50-fold lower for pre-cDC incubated with
TCM as compared to control (1 ng/ml vs. 50 ng/ml) (Figure 3.11A). pSTAT3 expression
peaked at IL-10 and IL-6 concentrations of 100 ng/ml for both unstimulated and TCM-
pretreated DC; however, at peak cytokine concentrations, TCM pre-treated DC achieved
higher STAT3 phosphorylation than unstimulated cells. STAT3 transcription levels in
pre-cDC pre-treated with TCM remained similar to control, further supporting the
hypothesis that increased pSTAT3 levels resulted from increased IL-6 and IL-10 receptor
signaling (Figure 3.11B).
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Figure 3.11 TLR2 ligation sensitizes DCs to IL-6 and IL-10 signaling. A) Intracellular
expression of phosphorylated STAT3 (STAT3-pY705) by DCs that were pretreated with
TCM or left untreated for 18 hr then stimulated with the indicated concentrations of IL-6
and IL-10 for 30 min. B) STAT3 mRNA expression determined by real-time PCR of DCs
for 18 hr with or without TCM.
Consistent with these findings, the increased expression levels of IL-6R and IL-
10R in pre-cDCs pre-treated with FSL-1 reduced the concentrations of IL-6 and IL-10
needed to activate STAT3 (Figure 3.12). Collectively, these data indicate that TLR2
activation by soluble tumor-derived factors in TCM promotes the differentiation of IL-10
producing DCs by increasing their sensitivity to IL-6 and IL-10 stimulation.
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Figure 3.12 FSL-1 reduces the threshold for STAT3 activation by IL-6 and IL-10.
Spleen pre-cDC from WT mice were cultured in FSL-1 or control medium for 18 hr,
recovered, and rested for 4 hr, then stimulated with the indicated concentrations of IL-6
and IL-10 for 30 min.
Tumor-derived versican in TCM induces cDC dysfunction
Versican is an ECM proteoglycan upregulated in many cancers. It has been shown
to stimulate macrophages to secrete TNF and IL-6 through TLR2 (124). We speculated
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that versican may trigger DC dysfunction. To test this hypothesis, we added anti-versican
neutralizing antibodies to TCM cultures. Neutralizing versican antibody reduced IL-6 and
IL-10 secretion by pre-cDCs at 24 hr, whereas the addition of antibodies to HMGB1, a
pro-inflammatory TLR4 ligand released by dying cancer cells, inhibited neither IL-6 nor
IL-10 secretion (Figure 3.13A). To further investigate the role of versican, we generated
TCM from LLC cells transduced with versican and control short hairpin RNA (shRNA)
(Figure 3.14A & B). Versican knockdown decreased the capacity of TCM to stimulate
IL-6 and IL-10 in pre-cDCs at 24 hr (Figure 3.13B); and cells recovered and re-
stimulated with LPS at 72 hr showed a lower and higher capacity to produce IL-10 and
IL-12p70, respectively. These results mirrored those obtained with the addition of anti-
TLR2 antibodies to control shRNA TCM (Figure 3.13C), supporting the view that
versican is a key TLR2 ligand in LLC TCM.
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Figure 3.13 Versican induces DC dysfunction. A) IL-6 and IL-10 production by pre-
cDC incubated with or without TCM for 24 hr in the presence or absence of neutralizing
anti-versican (1 µg/ml), anti-HMGB1 (1 µg/ml) and control IgG antibodies. B) IL-6 and
IL-10 production by pre-cDC incubated for 24 hr with TCM derived with LLC transduced
with versican- or control shRNA with or without anti-TLR2 antibodies. C) Production of
IL-10 and IL-12p70 by pre-cDC pretreated with TCM derived from LLC transduced with
versican or control-shRNA with or without anti-TLR2 antibodies for 72 hr, then
recovered, washed, and re-cultured for 18 hr in fresh complete medium containing LPS
(10 ng/ml).
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Figure 3.14 Versican expression in LLC cell lines. Versican expression in LLC
transduced with control or versican shRNA lentivirus analysed by A) qRT-PCR and B)
western blotting
Human cancers induce DC dysfunction through TLR2
We next addressed whether TLR2 affects the response of human DCs to human
cancers. We screened a variety of human cancer cell lines for versican expression by
qPCR. We found that H-125 (lung cancer), HepG2 (liver cancer), and Colon 328 (primary
colon cancer) express versican (Figure 3.15).
We measured IL-6 and IL-10 production by monocyte-derived human DC
incubated for 24 hr in conditioned medium generated from lung (H-125) and liver
(HepG2) cancer cell lines, primary colon cancer cells (Colon 328), and control human
spleen fibroblasts (FCM). As compared to unstimulated control, TCM from all three
cancers stimulated autocrine production of IL-6 and IL-10 (Figure 3.16A), which was
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inhibited by the addition of anti-TLR2 antibodies to the culture medium (Figure 3.16B).
FCM stimulated no IL-10 and little IL-6, which was blocked by anti-TLR2 antibodies.
Further analysis of the effects of H-125 TCM on human DCs showed that it increased the
surface expression of IL-6Rα and IL-10Rα, as well as the mRNA levels for these
receptors (Figure 3.16C & D).
We also found that H-125 downregulated cell-surface expression of HLA-DR
(analogous to MHC II expression in mice) and CD86 (Figure 3.16E) and induced the
differentiation of IL-10-producing DCs, as detected by LPS stimulation in secondary
cultures (Figure 3.16F). Anti-TLR2 antibodies blocked these changes and restored the
production of IL-1β and IL-12p70 to levels similar to those produced by DCs that were
maintained in GM-CSF. Furthermore, anti-TLR2 antibodies improved their stimulatory
capacity in mixed allogeneic lymphocyte reactions (Figure 3.16G). Collectively, these
data indicate that the mechanisms causing DC dysfunction in mice also operate in human
DCs.
Figure 3.15 Versican expression in various human cancer cell lines determined by PCR
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Figure 3.16 Human cancers induce human DC dysfunction through TLR2. A) IL-6
and IL-10 production by human DC unstimulated (US) or cultured for 24 hr in
conditioned medium derived from human spleen fibroblasts (FCM), NCI-H125, HepG2,
and Colon328. B) IL-6 and IL-10 production by human DC under the same conditions as
in (A) in the presence of anti-TLR2 (10 µg/ml) blocking antibodies or control IgG2b
antibodies. Results displayed as mean ± SEM, and are representative of 3 independent
experiments. ***P<0.001, assessed by unpaired T-tests. C) Surface expression of IL-6Rα
and IL-10Rα at 24 h human DC unstimulated (US) or cultured for 24 hr in conditioned
medium derived from NCI-H125. *P<0.05 D) IL-6Rα and IL-10Rα mRNA expression at
24 hr determined by qRT- PCR. Fold change is relative to unstimulated control. E) Flow
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cytometric analysis of HLA-DR and CD86 expression of human DCs cultured for 48 hr in
GM-CSF, FCM, or NCI-H125 TCM with or without anti-TLR2 antibodies. F) IL-10, IL-
1β, and IL-12p70 production by human DC pretreated with NCI-HI25 TCM and FCM or
left in GM-CSF for 72 h, then washed and restimulated with LPS for 18 hr. Results
displayed as mean ± SEM and are representative of 3 independent experiments. *P<0.05
**P<0.005 ***P<0.001, assessed by unpaired T-tests. G) Stimulation capacity of human
DC cultured as in (E) in mixed allogeneic lymphocytes reactions. Results displayed as
mean ± SEM, and are representative of 3 independent experiments. *P<0.05 **P<0.005
***P<0.001, assessed by unpaired T-tests.
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Discussion
In this chapter, we show that TLR2 activation is a critical proximal signal that
promotes the generation of immunosuppressive DC from pre-cDCs and human monocyte-
derived DCs. TLR2 ligation not only increased the secretion of IL-10 and IL-6, but also
upregulated the cell surface expression of the receptors for these cytokines, which
lowered the threshold concentration, required to activate STAT3. This feed-forward
amplification loop, reprogrammed DCs to produce high amounts of IL-10, rather than IL-
12 and IL-1β when stimulated with LPS, a classic proinflammatory stimulus. These DCs
stimulated allogeneic lymphocytes less effectively in mixed lymphocyte cultures. We also
found that versican is a tumor-derived factor in TCM that activates TLR2.
Earlier studies established a linkage between STAT3, tumorogenesis, and
impairment of anti-tumor immunity (310). Elevated STAT3 activity in tumor DC is
ascribed mostly to paracrine stimulation by tumor-derived cytokines such as IL-6, IL-10
and VEGF(240, 311, 312), although macrophages and other tumor-infiltrating cell
populations can also produce these cytokines (313). Our study reveals the previously
unrecognized role of autocrine cytokines induced by TCM in causing DC dysfunction.
We found that both IL-6 and IL-10 were key to this process, and behaved synergistically.
Although typically considered a pro-inflammatory cytokine, IL-6 dampens immune
responses in some settings (210, 211). TCM also stimulated the release of IL-1β from pre-
cDC; however, neutralizing anti-IL-1β failed to prevent the subsequent differentiation of
IL-10-producing DC. Although autocrine IL-6 and IL-10 mediated DC dysfunction in
vitro, the relative importance of autocrine versus paracrine signaling in vivo remains
unclear.
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Previous studies have shown that the intensity and duration of STAT3 signaling
governs whether an anti-inflammatory signal is delivered to DCs (241, 242). We show
that IL-6R and IL-10R expression levels calibrate the sensitivity of DCs to IL-6 and IL-10
based on the ability to activate STAT3. TLR2 activation upregulates the expression levels
of IL-6R and IL-10R independently of IL-6 and IL-10 signaling. In the absence of
receptor upregulation, the capacity of paracrine IL-6 and IL-10 to induce STAT3 and DC
dysfunction was diminished markedly. Therefore, TLR2 ligation imprints the capacity to
respond to lower concentrations of these cytokines, which helps explain the relative tumor
specificity of DC dysfunction. These findings also suggest that cytokine receptor
expression levels, rather than the source of the cytokines per se, may be the ultimate
arbiter of DC fate and function in vivo. In support of this theory, a recent study found
that IL-10R expression was absolutely required for intestinal macrophages to prevent
spontaneous colitis whereas autocrine IL-10 was dispensable (314).
Human and mouse tumors produce a variety of TLR2 ligands including versican,
laminin-β1, procollagen III-α1, Hsp60, and Hsp72 (124, 315, 316). The microbiome also
generates TLR2 ligands that could potentially influence the tumor microenvironment and
tumor progression (317). This panoply of TLR2 ligands suggests that targeting the
receptor rather than individual ligands may be a more effective strategy to modulate anti-
tumor immunity. The tumor microenvironment also produces immunostimulatory
molecules such as HMGB1 (a TLR4 ligand) and adenosine triphosphate (309, 318).
How tumor DC respond to ostensibly conflicting signals merits further investigation. Our
findings raise the possibility that treatments aimed to stimulate the release TLR4 ligands
in tumors may augment IL-10 production from “re-programmed” DC.
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Chapter 4
TLR2 signaling drives Tumor DC dysfunction in vivo
Contributions
Michael Tang, Jun Diao, Hongtao Gu, Jun Zhao, and Mark Cattral
Parts of this chapter were taken from:
Tang M, Diao J, Gu H, Khatri I, Zhao J, Cattral MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating
IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):2851-64.
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Introduction
DCs in tumors display multiple defects. Many studies in the past have suggested
that tumor-associated DCs are not only poor presenters of tumor antigens, but are also
suppressive and induce tolerance to tumor-antigen specific T cells (131). Several
attributes of tumor DCs explain their suppressive activities, resistance to maturation
stimuli, constitutive production of IL-10, and low levels of costimulatory molecules and
MHC II. Previous study from my lab has shown that all of these attributes hold true in
immunosuppressive subpopulation of Gr-1+ cDCs (133).
Gr-1+ cDCs in tumors differ from Gr-1+ MDSCs (133), a loosely defined
population of immature myeloid cells consists of neutrophils, macrophages and
monocytes that accumulate systemically in tumors, bone marrow and lymphoid tissues.
Not only do MDSCs lack classic DC markers, CD11c and MHC II, MDSCs suppress
MLR by producing immunosuppressive mediators, including arginase, NO and other
metabolites (131). Although these mechanisms are helpful to explain the suppressive
activities of MDSCs, it remains to be determined whether suppression by MDSCs has any
in vivo relevance (128). By contrast, studies from my lab and others have shown that
tumor DCs, both Gr-1+ and Gr-1- cDCs can modulate antitumor immune responses by
regulating the duration of infiltrating CTL responses in vivo (132-134).
DC dysfunction in tumor is primarily caused by tumor-derived factors present in
the TME. Many studies reported the presence of immature DCs in the blood, lymphoid
tissues and tumors of tumor-bearing mice and concluded that tumors cause systemic
defects in DCs (131, 156). My lab reported that some DCs in small transplantable and
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spontaneous tumors are phenotypically indistinguishable compared to DCs from the
lymphoid tissues (133). Additionally, confocal studies have shown that DCs at the
margins of the tumor are mature, and capable of cross-presenting tumor antigens (134).
Therefore, these findings suggest that the TME induces DC dysfunction in vivo locally,
rather than systemically.
Having established that TLR2 activation plays a role in mouse and human tumor
DC dysfunction in vitro, I will now examine whether these mechanisms have any
relevance in vivo. Previous work from my lab showed that blocking the development of
Gr-1+ cDCs by inhibiting IL-6 signaling enhanced intratumor expansion of CTLs,
resulting in suppression of tumor growth (133). Based on these findings, I hypothesize
that TLR2 deficiency will reduce the frequency of Gr-1+ IL-10 producing DCs and
improve immunogenicity of tumor DCs in murine tumors.
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Results
Tumors in TLR2-/- mice lack Gr-1+cDC
We reported that tumors recruit pre-cDC, where they generate functionally normal
immunostimulatory cDC that undergo several rounds of cell division (153); however,
under the influence of the cancer milieu, some of these cells differentiated into a
subpopulation of immunosuppressive cDC that could be distinguished by the cell surface
marker Gr-1 (133). To investigate whether TLR2 signaling regulates tumor Gr-1+
differentiation, we first characterized cDC populations in subcutaneous B16 melanoma
and LLC tumors from wildtype (WT) and MyD88-deficient mice. Mononuclear cells
isolated from tumors and spleens of tumor-bearing mice were stained with antibodies to
lineage markers (CD3, CD10, CD49b, B220), CD11c, and MHC Class II (MHC II). We
defined Gr-1+cDC in the Lineage negative population based on co-expresion of CD11c,
MHC II and Gr-1. Consistent with previous studies from our lab, Gr-1+cDC constituted
20-50% of tumor cDC in WT mice, whereas this subset was undetectable in B16 tumors
from MyD88-/- mice (Figure 4.1A & B). TLR2 and TLR4 are recognized receptors for
danger signals that exist in the tumor microenvironment. Therefore, we examined the DC
composition in B16 tumors from TLR2-/- and TLR4-/- mice. Tumors from TLR2-/- mice
contained a low frequency of Gr-1+cDC whereas the frequency of Gr-1+cDC in TLR4-/-
mice was similar to WT control (Figure 4.1 and B). Similar observations in LLC tumor
indicated that Gr-1+cDC development occurred independently of tumor type.
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Figure 4.1 Absence of TLR2 and MyD88 blocks the development of Gr-1+ cDCs in
tumors. A) Phenotypic analysis of DCs in B16 melanomas from wildtype (WT), MyD88-
/-, TLR2-/-, TLR4-/- mice. Numbers in the dot plots indicates the frequency of Gr-
1+CD11c+MHCII+ DCs B) Frequency of Gr-1+ cDCs in B16 tumors from WT, MyD88-/-,
TLR2-/-, TLR4-/- mice. Each symbol represents an individual mouse. Results are
representative of 3 independent experiments with 3-5 mice per group.
The spleens of tumor-bearing WT, TLR2-/-, TLR4-/-, and MyD88-/- mice contained
trivial numbers of Gr-1+ cDC, which underscored the importance of the local tumor
milieu in their development (Figure 4.2).
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Figure 4.2 Phenotype of DCs in spleens of B16 melanoma-bearing WT, MyD88-/-,
TLR2-/-, TLR4-/- mice. Flow cytometric analysis of the indicated cell surface markers.
Numbers in the dot plots indicates the frequency of Lin-Gr-1+CD11c+MHC II+cDC
We wondered whether the absence of tumor Gr-1+ cDC in MyD88-/- and TLR2-/-
mice represented an intrinsic defect of Gr-1 expression; however this seemed unlikely
because MyD88 and TLR2deficiency had no effect on the frequency of Gr-1+ CD11c-
CD11b+ myeloid-derived suppressor cells (MDSC) in tumors (Figure 4.3) and Gr-1+
granulocytes in spleen.
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Figure 4.3 Frequency of Gr-1+CD11b+ cells in B16 melanomas. B16 melanomas were
recovered from WT, MyD88-/-, TLR2-/-, TLR4-/- mice and analyzed by flow cytometry for
the presence of Lin-CD11b+Gr-1+CD11c-MHC II- cells. Each symbol represents an
individual mouse. The horizontal line indicates the mean percentage. Results are
representative of 3 independent experiments with 3-5 mice per group.
Collectively, these findings, along with the in vitro results from the previous
chapter, indicated that the TLR2-MyD88 signaling pathway was involved in the
development of Gr-1+cDC.
Characterizations of tumor DCs in WT and TLR2-/- mice
Gr-1+ cDC in tumors possess immunosuppressive properties, mediated in part
through the production of IL-10, that inhibit lymphocyte activation and proliferation. We
found that tumor cDC in TLR2-/- mice expressed more cell surface MHC II, CD40, and
CD86 markers than those from WT mice, consistent with a higher state of maturation or
activation (Figure 4.4).
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Figure 4.4 Phenotypic analysis of tumor and spleen DCs. Tumor and spleen Lin-
CD11c+ MHC II+ DCs from WT and TLR2-/- mice were analyzed by flow cytometry for
the indicated cell surface molecules. MFI, mean fluorescence intensity. Results are
representative of two independent experiments with 3-4 mice per group. * p< 0.05, ***
p< 0.001 by student’s t test.
We speculated that the low frequency of Gr-1+cDC in tumors from TLR2-/- mice
would enhance tumor DC immunogenicity. To test the function of tumor DCs from WT
and TLR2-/- mice, we sorted cDC from tumors and the spleen, and compared their ability
to produce IL-10 and IL-1β and stimulate allogeneic lymphocytes. Spleen DC from both
WT and TLR2-/- mice produced high and low amounts of IL-1β and IL-10, respectively.
WT tumor DCs produced high amounts of IL-10 but no IL-1β, whereas tumor cDCs from
TLR2-/- mice produced IL-10 and IL-1β at levels similar to that of spleen cDCs (Figure
4.5A). Tumor cDCs from TLR2-/- mice also stimulated proliferation of allogeneic
94
lymphocytes more effectively than those from WT mice (Figure 4.5B). Thus, the
reduction of tumor Gr-1+ cDC in TLR2-/- mice improved tumor DC function
Figure 4.5 Superior stimulatory function of tumor cDCs from TLR2-/- mice. A)
Production of IL-10 and IL-1β by sorted tumor and spleen DCs after overnight culture in
LPS (1 µg/ml). B) Stimulation capacity of sorted tumor and spleen DCs from WT and
TLR2-/- mice in mixed allogeneic lymphocyte reactions. Results are expressed as mean
counts per minutes (cpm) x 103 ± SEM, *p< 0.05, ** p<0.005, *** p< 0.001, determined
by student’s t test.
In chapter 3, we showed that direct TLR2 stimulation with TCM and FSL-1
sensitized pre-cDC to IL-6 and IL-10 stimulation by upregulating the expression levels of
IL-6Rα and IL-10Rα, respectively. To determine whether this mechanism is operational
in vivo, I analyzed the expression levels of IL-6Rα, IL-10Rα, and phosphorylated STAT3
in cDC isolated from spleens and tumors of mice bearing subcutaneous B16 melanoma.
WT tumor cDC displayed increased expression levels of both cytokine receptors and
phosphorylated STAT3 as compared to tumor DC from TLR2-/- mice (Figure 4.6A & B).
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Spleen DC from tumor bearing WT and TLR2-/- mice expressed similar low levels of IL-
6Ra, IL-10R, and pSTAT3 (Figure 4.6A & B). Collectively, these findings indicated that
TLR2 deficiency inhibits the ability of the tumor milieu to upregulate IL-6Rα and IL-
10Rα, activate STAT3, and impair the function of tumor cDCs. By contrast, spleen DCs
in TLR2-/- mice remain similar to those in WT mice, which further supports the view that
DC dysfunction in B16-bearing mice occurs predominately in the cancer.
Figure 4.6 Upregulation of IL-6Rα, IL-10R, and STAT3 phosphorylation in sorted
DCs. A) Representative flow cytometric plots showing expression of IL-6Rα, IL-10Rα,
and STAT3-pY705 by DCs sorted from B16 tumors, and spleens of WT (red line) and
TLR2-/- (blue line) mice. B) Bar graphs show MFI of IL-6Rα, IL-10Rα, and STAT3-
pY705 by tumor DCs (n= 3-5 mice per group) **p <0.005, ***p <0.001, determined by
student’s t test.
In vivo effects of tumor-derived versican
Next, we analyzed the in vivo effect of tumor-derived versican on tumor DCs. LLC
transduced with versican-shRNA and control shRNA were inoculated into B6 mice
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subcutaneously. We detected 4-fold higher expression levels of intracellular IL-12p40 in
DCs isolated from versican knockdown tumors as compared to those from control tumors
(Figure 4.7). These findings further support that tumor-derived versican promote tumor
DC dysfunction.
Figure 4.7 In vivo effects of tumor-derived versican. Intracellular expression of IL-
12p40 in DC isolated from subcutaneous versican- and control-shRNA LLC tumors. Data
is displayed as mean ± SD.)
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Discussion
The number and function of tumor-infiltrating DCs reflect the interaction of
several processes: recruitment, differentiation, death, and migration. We have shown that
tumors recruit pre-cDC that are morphologically, phenotypically, and functionally
indistinguishable from those that are found in lymphoid tissues (153). As with other
populations within the myeloid lineage, tremendous plasticity exists among DCs, which
enables the TME to shape their fate, resulting in a continuum of functionality. For
example, tumor Gr-1-cDC exhibit normal functional attributes as compared to spleen cDC
from non-tumor bearing mice (153). Gr-1+ DCs offer a convenient readout of DC
dysfunction in the tumors. However, the frequency of this population may under represent
the true extent of DC impairment. Indeed, cDCs can evolve into regulatory macrophages
(DC-d-M) under the tumor milieu (157). Whether Gr-1+ cDCs represent an intermediate
population from immunostimulatory Gr-1-cDC and DC-d-M remains unclear. A human
equivalent for Gr-1 has yet to be identified. Our findings suggest that elevated expression
levels of IL-10R and/or IL-6R might serve as an alternative surrogate signature for tumor
DC dysfunction.
Whether STAT3 delivers an anti-inflammatory signal to DC depends on the
intensity and duration of STAT3 signaling (241, 242). Another study implicated
sphingosine-1-phosphate receptor-1 (S1PR1), a G protein-coupled receptor for
sphingosine-1 phosphate, in promoting the persistence of STAT3 activity in tumor DC
(312). In our study, TLR2 deficiency abolished increased expression levels of IL-6Rα, IL-
10Rα and STAT3 in tumor DC, confirming its critical role in vivo. Interestingly, STAT3
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has also been shown to regulate TLR2 expression (319), which may further amplify DC
dysfunction in cancer.
Previous adoptive transfer experiments established that the Gr-1+ cDCs belong to
the DC lineage (133). A recent study of the tumor-infiltrating myeloid cells in a
spontaneous mouse model of breast cancer indicated that cells expressing the classic DC
markers, CD11c and MHC II, were monocyte-derived macrophages based on gene
transcriptional profiling (129). This finding, and others (12, 320, 321), highlights the
limitations of using cell surface markers alone to assign cell lineage origin and make
inferences about functional attributes (11, 12, 128). We cannot exclude the possibility that
reduced frequency of Gr-1+ cDC in B16 and LLC tumors from TLR2-/- and MyD88-/- mice
may also represent an effect in monocytes. Further studies are required to clarify whether
the lineage origin of DC influences their behavior and function in the TME.
TCM from versican-expressing tumor cell lines induced IL-6/IL-10 production
from pre-cDCs and generation of immunosuppressive DCs (Chapter 3, Figures 3.13 &
3.16). Considering that the inhibitory effect of anti-versican antibody was partial in the in
vitro experiments, we extended these studies and compared IL-12 production in tumor
DCs from versican-knockdown tumors versus control tumors to determine the in vivo
relevance of versican. Our results show that tumor DC versican-knockdown tumors
express more IL-12, suggesting that versican contributes to DC dysfunction in vivo. Our
studies are consistent with the pro-tumor effects of versican reported a study by Kim et
al. (124). Notably, in that study, versican was thought to promote tumor metastases by
simulating macrophages to produce pro-inflammatory cytokines. Our suggest that DC
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dysfunction resulting in impaired adaptive immunity may be another explanation for their
findings (124).
In summary, studies from this chapter provided further validates that TLR2
activation induces tumor DC dysfunction. Indeed, the mechanisms that I proposed in
chapter 3 are relevant in vivo. Blockade of TLR2 enhanced DC immunogenicity in murine
tumors, as characterized by increased co-stimulatory molecule expression, low levels of
IL-10 secretion, reduced IL-6R and IL-10R expression, and reduced STAT3
phosphorylation. These properties suggest that blocking TLR2 on DCs may enhance anti-
tumor immunity.
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Chapter 5
Blockade of TLR2 signaling enhances tumor immunotherapy
Contributions
Michael Tang, Jun Diao, Hongtao Gu, Jun Zhao, and Mark Cattral
Parts of this chapter were taken from:
Tang M, Diao J, Gu H, Khatri I, Zhao J, Cattral MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating
IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):2851-64.
101
Introduction
Immunotherapy offers great potential for long-term cure and survival for cancer
patients (322). Advances in understanding T cell biology has led to new approaches to
increase the efficacy of immunotherapy, including adoptive cell therapy, cancer vaccines
(e.g. irradiated tumor cell vaccine expressing GM-CSF, GVAX), and checkpoint blockade
inhibitors (e.g. anti- CTLA-4/CD28, anti- PD1/PD-L1) (265). Although spectacular
responses have been reported, these therapies are not consistently effective, and many
patients derive little or no benefit (108).
A major goal of immunotherapy is to achieve sufficient numbers of functional
tumor-antigen specific CTLs and to prolong the duration of their antitumor responses in
the tumor milieu (270, 322). Antigen-specific CTLs require cognate interactions with
tissue DCs for local expansion and acquiring effector functions (149). Furthermore,
intratumor DCs are capable of stimulating effector and memory T cells in situ, therefore
enhancing T cell expansion and effector cell differentiation (132-134). While tumor cells
and stromal cells can sometimes present tumor-derived antigens, and serve as targets for
CTLs, DCs are especially equipped to participate in the cross-presentation of “weak” and
less immunogenic tumor-derived antigens (128). Additionally, recent studies from my lab
and others showed that intra-tumor DCs were the only cells that could effectively
stimulate CTL proliferation, at least in vitro. These findings strongly suggest that
manipulating tumor DC may be a helpful approach to improve responses to cancer
immunotherapy.
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Prior studies from our lab and others report that defective DC differentiation in the
tumor milieu contributes to the impairment of CTL function (133). Having established in
the last chapters that tumors induce DC dysfunction by activating TLR2, I hypothesize
that blocking TLR2 signaling can reverse tumor DC dysfunction and improve anti-tumor
CTL responses.
103
Results
TLR2 deficiency improves T cell responses to anti-cancer vaccine
To test whether improved function of tumor cDCs from blocking TLR2 signaling
would enhance anti-tumor immunity, we compared the efficacy of an irradiated B16
melanoma vaccine engineered to express GM-CSF (GVAX) in WT and TLR2-/- mice
bearing established B16 melanoma. Tumor growth rates were similar in unvaccinated WT
and TLR2-/- mice, consistent with the poor immunogenicity of B16 melanoma (323). In
agreement with previous reports, GVAX alone did not have an effect on tumor growth in
WT mice (308). By contrast, GVAX suppressed tumor growth significantly in TLR2-/-
mice (Figure 5.1).
Figure 5.1 TLR2 deficiency improves efficacy of GVAX tumor vaccination. Relative
growth of subcutaneous B16 melanoma in treated and GVAX treated WT and TLR2-/-
mice. GVAX treatment schedule is indicated in the schematic on the left. Results are
displayed as mean ± SEM with 13 mice per group. *p <0.05, by unpaired Mann-Whitney
test.
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Flow cytometric analysis of the B16 melanomas revealed that GVAX
immunization in TLR2-/- mice was associated with a significant increase in the frequency
of CD3+ T cells, CD8+ T cells, and CD4+ T cells, whereas the frequencies of these
populations were similar in vaccinated and unvaccinated WT mice (Figure 5.2). There
were no significant differences in the frequencies of Foxp3+ Tregs and γδ T cells after
GVAX in WT or TLR2-/- mice. Furthermore, the frequency of NK cells was higher in
vaccinated TLR2-/- mice than WT mice (Figure 5.2). Analysis of the corresponding
lymphoid populations in spleen revealed no differences in the 4 groups of mice, indicating
that the absence of TLR2 signaling did not have a systemic effect in the lymphoid
compartment.
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Figure 5.2 Tumor lymphoid cell analysis in GVAX treated mice. Flow cytometric
quantitation of lymphoid cell populations in B16 melanoma. Results are displayed as
mean ± SEM. *p <0.05; **p< 0.005, by unpaired Mann-Whitney test; NS, not significant
Characterization of tumor DCs in GVAX-treated WT and TLR2-/- mice
Next, we analyzed tumor DC subsets and other myeloid populations. Recent
reports indicate that the frequency of intra-tumor CD103+cDC, which is primarily
responsible for cross-presentation of tumor-derived antigens to T cells, may enhance the
efficacy of anti-tumor immune response and affect tumor growth. We assessed the
frequency of CD11chiMHC IIhi cDC subpopulations in the B16 tumors of WT and TLR2-/-
mice according to the following gating schema – DC1 (CD103+CD11b-), DC2 (CD103-
CD11b+CD64-F4/80-), and DC3 (CD103- CD11b+CD64+ F4/80+) (Figure 5.3).
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Figure 5.3 Multi-colour flow cytometry gating strategy for identifying myeloid
populations in subcutaneous B16 melanoma. Side scatter (SSC) and surface markers
are indicated on the axes of the dot-plots; numbers indicate percentage of cells; and
arrows indicate direction of subgating. Monocytes (Mo) and macrophages (Mac) were not
separated.
This analysis showed no differences in the frequency of these DC subsets in the
tumors of WT and TLR2-/- mice (Figure 5.4). We did not compare the frequency of pDC
because they exist in trace numbers in B16 melanoma. Tumor-infiltrating macrophages
and neutrophils can both support and suppress anti-tumor T cell responses. We detected
no difference in the frequency of macrophages/monocyte (F4/80+CD11b+MHCII-) and
neutrophils (Ly6G+).
Figure 5.4 Tumor myeloid cell populations analysis in GVAX treated mice. Flow
cytometric quantitation of DCs, neutrophils, and monocyte/macrophages in B16 melanoma
and spleen. Refer to chapter 4, Figure 4.4, for gating strategies of DC subsets (DC1, DC2,
DC3). Results are displayed as mean ± SEM.
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TLR2 deficiency enhances anti-tumor cytotoxic T cell (CTL) responses
T cell priming in lymphoid tissues and cognate interactions with intra-tumor DCs
contribute to T cell expansion in tumors (132, 133, 154, 324). Since the function and
frequencies of spleen DCs were similar in WT and TLR2-/- mice (Chapter 4, Figures 4.6-
4.7), it seemed unlikely that differences in T cell priming would account for the increased
frequency of intra-tumor T cells. Further examination of the lymphoid DCs in GVAX-
treated mice revealed no difference in the frequency of resident and migrated lymph node
DCs (Figure 5.5A); surface expression levels of CD40, CD80, and CD86 (Figure 5.5B);
or T cell stimulation capacity (Figure 5.5C).
To eliminate the possibility that TLR2 signaling might affect T cell survival and
proliferation, and to facilitate the study of tumor-specific CTL responses in situ, we
injected antigen-activated CD45.1 OT-I CTLs intravenously into CD45.2 WT and TLR2-/-
mice bearing established B16 (control) or B16 tumors expressing ovalbumin (B16-OVA)
and recovered the tumors 3 days later, when they were ~1cm in diameter, for analysis. As
expected, B16 tumors contained few OT-I CTLs. B16-OVA tumors from TLR2-/- mice
contained a 5- to 10- fold higher frequency of OT-I CTLs than those from WT mice
(Figure 5.6A and B). Almost 50% of these cells expressed IFN-γ after restimulation
with SIINFEKL peptides as compared to <10% in WT mice (Figure 5.6C), consistent
with more robust effector activity. The spleen and non-draining lymph nodes of tumor-
bearing TLR2-/- and WT mice contained a low frequency of OT-I CTL, regardless of
tumor OVA expression.
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Figure 5.5 Characterization of lymph node (LN) DCs in GVAX-treated WT and
TLR2-/- mice. Skin-draining LNs were recovered and processed 3 days after GVAX
treatment. A) Gating strategy for identifying migrated and resident DC among lineage
negative (CD3, CD19, B220, CD49b, Ly6G) LN cells. Bar graphs show the percentage of
DC in the Lin- population (left) and the percentage of CD103+ DC among migrated,
resident and total LN DC (right). B) Expression of CD40, CD80, CD86 by LN DC.
Dotted black line indicates background florescence with isotype control Ab. Results
expressed in mean ± SD, 4 mice in each group. C) Stimulation of naïve OT-I T cells by
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LN DC incubated with or without OVA for 8 hrs. T cell activation was assessed y CD86
expression at 12 hrs and T cell proliferation was assessed by CFSE dilution at 72 hrs.
Results are representative of 3 independent experiments.
Figure 5.6 TLR2 deficiency enhances expansion and function of transferred tumor-
antigen-specific CTLs A and B) Frequency of OT-I CTLs in B16 and B16-OVA tumors
3 days after adoptive transfer of CD45.1 OT-1 CTL into WT and TLR2-/- mice. Numbers
in the dot plots indicate percentage of CD45.1+ CD8+ cells in B16 and B16-OVA tumors.
C) Frequency of CD45.1+IFN- γ+CD8+ T cells in B16 and B16-OVA tumors of WT or
TLR2-/- mice. Results in B and C are displayed as mean ± SEM. **p < 0.005.
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TLR2 deficiency enhanced proliferation of transferred tumor-antigen specific CTLs
Tumor DCs possess the unique ability to stimulate proliferation of tumor-antigen-
specific CTLs, even in the presence of other tumor-infiltrating cells, including MDSCs,
monocytes, and macrophages (132, 133). Since the frequency of CTLs in lymphoid
tissues was similar in WT and TLR2-/- mice, we speculated that the higher intra-tumor
CTL frequency in TLR2-/- mice was due to increased CTL proliferation within the tumor
(325). To monitor cell division, we injected CFSE-labeled OT-I CTL into tumor-bearing
mice and assessed tumors and lymphoid tissues 5 days later. OT-I CTL divided more
extensively in B16-OVA tumors from TLR2-/- mice than WT mice, as measured by CFSE
dilution (Figure 5.7). In both WT and TLR2-/- mice, low levels of OT-I CTL
proliferation occurred in B16-OVA tumor-draining lymph nodes whereas non-draining
lymph nodes and spleen contained no divided OT-I CTL. In the absence of OVA, OT-I
CTL did not divide in any of the tissues examined (Figure 5.7). These findings support
the view that CTL proliferation and acquisition of effector functions within the tumor
regulate anti-tumor T cell responses in TLR2-/- mice.
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Figure 5.7 TLR2 deficiency enhances proliferation of adoptively transferred CTLs.
CFSE dilution of CD45.1 OT-I CTL analyzed 5 days after transfer into WT and TLR2-/-
mice bearing B16 or B15-OVA tumors. dLN, draining lymph nodes.. ***p <0.001.
TLR2 deficiency enhances therapeutic efficacy of adoptive transfer CTL therapy
To evaluate whether the increased intra-tumor frequency of CTL in TLR2-/- mice
affects tumor growth and survival, we transferred OT-I CTL into TLR2-/- and WT mice 3
days after B16-OVA or B16 inoculation. Analysis of tumor growth rates and animal
survival revealed enhanced efficacy with significantly better anti-tumor responses to OT-I
CTL therapy in TLR2-/- mice compared to WT mice bearing B16-OVA tumors. Without
CTL therapy, tumors grew at similar rates in WT and TLR2-/-mice (Figure 5.8).
Collectively, these data suggest that the improved function of tumor cDC and reduced
intra-tumor frequency of Gr-1+ cDC in TLR2-/- mice enhances the expansion and
functional activity of adoptively transferred CTL.
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Figure 5.8 Adoptive CTL therapy in WT versus TLR2-/- mice. (Far Left) Schedule of
tumor inoculation and adoptive CTL therapy; WT and TLR2-/- mice were inoculated with
0.5 x 106 B16 or B16-OVA cells. (Middle and Far Right) Graphs show mean tumor
growth and Kaplan-Meier tumor-free survival (Significance was determined by the Long-
rank test).
Therapeutic targeting of TLR2 improves adoptive CTL therapy
We next determined whether TLR2 could be targeted with neutralizing antibodies
to suppress Gr-1+ cDC development. Two injections of anti-TLR2 antibody over 3 days,
but not the control antibody, markedly reduced the intra-tumor frequency of Gr-1+cDC
(Figure 5.9 A&B). To determine whether this approach of blocking TLR2 would enhance
adoptive CTL therapy, WT mice bearing B16-OVA tumors received 3 intravenous
injections of anti-TLR2 or control antibodies and OT-I CTL (Figure 5.10). Mice treated
with this short course of anti-TLR2 antibody therapy showed significantly lower rates of
tumor growth and longer survival as compared to mice receiving control antibodies Thus,
targeting TLR2 with blocking antibodies reduced the frequency of immunosuppressive
Gr-1+cDC and increased the efficacy of adoptive CTL therapy.
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Figure 5.9 Anti-TLR2 antibody therapy blocks the generation of Gr-1+ cDC. A)
Phenotypic analysis of DC in B16 melanoma from WT mice treated with anti-TLR2 or
control antibodies at days -3 and -1 relative to tumor recovery. B) Dot plot representing
the frequencies of Gr-1+ cDC in B16 melanoma as in (A). Each symbol represents an
individual mouse. Results are representative of two independent experiments. *** p<
0.001 by Student’s T-test
Figure 5.10 Anti-TLR2 antibody treatments boost efficacy of adoptive CTL therapy.
(Far left) Treatment schedule of α-TLR2/ IgG antibody and OT-I CTLs after B16-OVA
tumor inoculation. (Middle and Far Right) Graphs show mean tumor growth and Kaplan-
Meier tumor-free survival (Significance was determined by the Long-rank test)
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Published reports indicates that B16 melanoma express TLR2 by qPCR, which
raised the possibility that anti-TLR2 antibodies might influence their behavior directly.
However, we found that, in contrast with splenocytes, B16 melanoma cells neither
express TLR on their cell surface nor produce IL-6 and IL-10 when stimulated with TLR2
ligands, Pam3CSK3 or FSL-1 (Figure 5.11). Consistent with these findings, anti-TLR2
therapy alone had no effect on tumor growth or survival (Figure 5.10). Therefore, the
benefit of blocking TLR2 signaling does not depend on TLR2 expression in B16
melanoma.
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Figure 5.11 TLR2 expression in B16 melanoma. A) Surface expression of TLR2 (blue
histogram) in B16 melanoma and spleen cells (positive control) by flow cytometry. Grey
histogram represents the isotype control. B) IL-6 and IL-10 concentrations in culture
supernatants of B16 melanoma cells and spleen cells unstimulated or stimulated with
FSL-1 (100 ng/ml) and Pam3CSK4 (100 ng/ml) for 24 hrs. Data are presented as mean ±
SD.
Anti-TLR2 antibody enhances the therapeutic efficacy of anti-CTLA-4 antibody
α-CTLA-4 antibody has been actively explored clinically in melanoma patients
(295). Although the success rates of immunotherapy using α-CTLA-4 continue to
improve, it is not consistently effectively (108). The studies presented so far suggest that
targeting TLR2 to prevent tumor DC dysfunction may be a promising adjunct to improve
the results of α-CTLA-4 treatments. We initiated preliminary studies to explore whether
α-TLR2 in combination with α-CTLA-4 can enhance the efficacy of GVAX treatments in
mice bearing B16 melanoma. Analysis of tumor growth rates revealed enhanced efficacy
of GVAX treatments in mice treated with both α-TLR2 and α-CTLA-4 antibodies
compared to either antibody alone (Figure 5.12). Compared to mice treated with α-
CTLA-4 alone, CD11b+ cDCs isolated from B16 tumors treated with both antibodies
displayed reduced expression levels of IL-10R, as well as increased CD80 expression
(Figure 5.13A). This was accompanied by a slight increase in tumor CD8+ T cell
infiltration and enhanced CD8+/CD4+ ratio (Figures 5.13 B&C). Thus, these studies
suggest that the combination of α-TLR2 and α-CTLA-4 antibodies might be more
effective than either antibody alone in inhibiting the growth of B16 melanoma.
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Figure 5.12 Combination treatment with anti-TLR2 and anti-CTLA-4 antibody
enhanced efficacy of GVAX vaccination. (Left) Treatment schedule of anti-TLR2, anti-
CTLA-4 antibody (blue arrows), and GVAX (red arrows) after B16 tumor inoculation.
(Right) Graph shows mean tumor growth in each group of animals, n=5 per group. * p <0.05
by unpaired Mann-Whitney test.
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Figure 5.13 Analysis of DCs, T cells, and NK cells in B16 tumors antibody and
GVAX treated mice. A) Gating strategy and CD80/IL-10R expression of CD11b+ DCs in
B16 tumors of treated and untreated mice. B) Absolute cell number of CD8, CD4 T cells,
and CD49 NK cells in treated or untreated B16 tumors. C) Ratio of CD8/CD4 T cells in
B16 tumors.
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Discussion
Studies from chapters 3 and 4 showed that tumor DCs from TLR2-/- exhibit
several key attributes of effective antigen presenting cells: 1) high expression levels of
MHC II and co-stimulatory molecules; 2) expression of pro-inflammatory IL-1β and IL-
12p70, rather than IL-10; 3) potent stimulation of allogeneic lymphocytes. These
functional properties correlated with better anti-tumor T cell responses elicited by a
GVAX vaccine, and higher rates of proliferation and expression of IFN-γ in adoptively
transferred OT-1 CTL. Since other tumor infiltrating immune cells also express TLR2, we
cannot exclude that the absence of TLR2 signaling in these cells also influence anti-tumor
immune responses. It is noteworthy, however, that growing evidence points to a unique
role of tumor DCs in regulating the proliferation, expansion, and function of tumor-
infiltrating CTLs through cross-presentation of tumor antigens (128, 132, 133, 154). This
role for tumor DCs aligns well with the function of tissue DCs in restimulating effector
and memory T cells in situ (149, 150).
A study from Geng et al. showed that TLR2 signaling can increase CD8+ T cell
effector activity and survival and reduce the suppressive function of Tregs (326). In our
study, GVAX immunization of TLR2-/- mice resulted in higher frequencies of intra-tumor
CD8+ T cells and no difference in the frequency of Foxp3+ Tregs, as compared to those in
WT mice. We propose that the immunologic benefit of blocking TLR2 signaling in tumor
DCs in B16 melanoma outweighs the loss of TLR2 signaling in T cells. This dynamic
may vary in different tumor models, which may explain why TLR2 agonists have been
reported to both promote and inhibit tumor growth (327, 328). In a mouse model of
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gastric cancer, TLR2 signaling directly contributes to tumorigenesis and tumor cell
proliferation by activating STAT3 (319). In our study, we did not detect cell surface
expression of TLR2 or a functional response to TLR2 agonists in B16 melanoma cells.
Recent reports suggest that tumor CD103+ DCs are especially equipped to promote
anti-tumor immunity. CD103+ DCs, which are ontogenetically linked to spleen CD8+ DC,
express higher levels of IL-12 and stimulate T cells more effectively than tumor CD11b+
DC (23). Indirect or direct inhibition of tumor-derived IL-10 using anti-CSF and anti-IL-
10R antibodies, respectively, increased the density of tumor CD103+ DCs, which
correlated with a better response to the chemotherapeutic agent, paclitaxel, in a
spontaneous breast cancer model (141). In our study, TLR2 deficiency had no specific
effect on the frequencies of cDC subpopulations in treated and untreated GVAX groups;
and all populations displayed a similar increase in the expression levels of MHC II and
co-stimulatory molecules as compared to tumor DC from WT mice. Although CD11b+
DCs are less effective antigen-presenting cells than CD103+ DCs on a per cell basis (11),
they have a substantial numerical advantage in most tumors, and are likely important
contributors to anti-tumor immunity. Indeed, another study reported that depletion of
tumor CD11b+ DC abolished the efficacy of chemotherapy-induced anti-tumor T cell
response (154).
Preliminary studies showed that the combination of α-TLR2 and α-CTLA-4
antibodies is more effective than either antibody alone in inhibiting the growth of B16
melanoma. The combination of both antibodies also seems to improve DC
immunogenicity (e.g. increased in CD80 expression, and reduced IL-10R expression).
These results are promising and suggest that α-TLR2 can be potentially used clinically as
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an adjunct therapy when used in combinations with other immunotherapy strategies.
While blockade of CTLA-4 allows persistent anti-tumor T cell responses by inhibiting
TCR stop signals (298), α-TLR2 therapy offers another strategy by improving DC
immunogenicity and thus, increasing the frequency and duration of anti-tumor CTL
responses.
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Chapter 6
General Discussions and Future Directions
122
Historical perspective on DC dysfunction in cancer
Prior to my graduate studies, there was great interest in the tumor immunology
field to understand the relationship between cancer inflammation, the nature and
composition of inflammatory infiltrates, and cancer-mediated immune evasion. As the
primary APC, DC dysfunction is considered a key cause of impaired immune responses to
tumor antigens (329). The accumulation of immature DCs in the blood and tumors of
patients and tumor-bearing mice were thought to be a dominant mechanism of DC
dysfunction (156). A variety of tumor-derived factors such as IL-6, VEGF, lipids, and
gangliosides, and their downstream signaling mechanism (e.g. STAT3 activation) were
implicated in skewing the development of bone marrow progenitors to cause DC
dysfunction (131, 156). Further, many embraced the view that DC dysfunction is a
systemic process and that all tumor DCs were functionally defective. This view was based
partly on studies that used in vitro GM-CSF-stimulated bone marrow derived DCs, which
we now know are poor representatives of natural in vivo DCs (199, 200), may not be the
best tool to evaluate the effects of cancer on DCs. Efforts to further define the
mechanisms of tumor DC dysfunction were hampered by a poor understanding of tumor
DC ontogeny, how DCs behave under the direct influence of the tumor milieu, and how
DCs regulate antitumor CTL responses.
Recent studies from our lab clarified how DCs develop under the influence of the
TME and their role in regulating antitumor CTL responses (133, 153). Tumors recruit
circulating pre-cDCs through CCL3, and induce differentiation of immunosuppressive
Gr-1+ cDC, a localized process mediated by the inflammatory cytokine milieu in tumors,
particularly IL-6. Inhibiting the development of Gr-1+ cDC enhanced the expansion of
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tumor-specific CTLs and improved antitumor responses. Since these studies were
published, growing evidence supports a unique role of tumor DCs in regulating the
proliferation, expansion, and function of CTLs and antitumor immune response (128, 132,
133, 154).
New model of Tumor DC dysfunction
Having established that the TME directly cause DC dysfunction locally in the
tumor, an outstanding question is how tumor-derived factors drive this process. The
studies presented in this thesis addressed this question, and provided further mechanistic
insights (Figure 6.1). I showed that TLR2 ligation contributes to DC dysfunction. TLR2
ligation not only triggered the release of autocrine IL-10 and IL-6, but also led to
sustained elevation of cell surface cytokine receptors, IL-6R and IL-10R. As presented in
chapter 3, the upregulation of these receptors decreased the threshold IL-6 and IL-10
concentration required to activate STAT3. Therefore, TLR2 ligation imprints the capacity
of DC to respond to lower concentrations of these cytokines, which explains, at least
partly, why tumor DC dysfunction is a local rather than a systemic process. Additionally,
I found that versican, a ECM glycoprotein that is overexpressed in many cancer, is a key
TLR2 ligand that induced DC dysfunction. Our new model of tumor DC dysfunction
integrates several molecular mechanisms described previously in the literature, namely,
IL-6/IL-10 in the TME and STAT3 activation (Figure 6.1). Overall, these studies
advanced our understanding of DC dysfunction in cancer and revealed key molecules that
could potentially serve as therapeutic targets to improve DC immunogenicity and cancer
immunotherapy.
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Figure 6.1- Model of DC dysfunction in Cancer. Tumor-derived versican stimulates
pre-cDC through TLR2, resulting in the secretion of autocrine IL-6 and IL-10, and
upregulation of cell surface IL-6R and IL-10R expression. These processes converge to
activate STAT3, which leads to the generation of DC that preferentially produce anti-
inflammatory IL-10, rather than pro-inflammatory IL-1β and IL-12.
TLR2 blockade as an adjunct therapy
Effective immunotherapy requires overcoming tumor immunosuppression and
achieving a high number of functional CTLs to destroy tumors. Tumor
immunosuppressive factors include the presence of Tregs, TAMs, MDSCs and
immunosuppressive DCs, and activation of negative regulatory pathways such as CTLA-4
and PD-1, which prematurely terminates CTL activities (164). Other than targeting the
immunosuppressive cells in the tumor milieu, the focus of many current immunotherapy
strategies is to target regulatory inhibitory pathways that hinder CTL activation and
antitumor responses (266, 268, 295, 298). For example, the combination of anti-CTLA-4
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antibody ipilimumab and anti-PD-1 nivolumab, showed high efficacy in patients with
advanced metastatic melanoma (296). Despite forward progress in developing these
checkpoint inhibitors, significant toxicities related to these drugs have been reported,
especially in patients treated with anti-CTLA-4. These toxicities are part of a unique
spectrum of side effects termed immune-related adverse events (irAEs). irAEs are
inflammatory side effects (e.g. diarrhea/colitis, rash and mucosal irritations,
hepatoxicities, autoimmune thyroid syndrome) that arise from general immunological
enhancements, and can affect multiple organ systems (330). A recent clinical study
reported that 85% of melanoma patients (n=298) treated with ipilimumab experienced
irAEs, and 19% of these patients, had to discontinue therapy (331).
The work in this thesis identified TLR2 as a target to overcome immune
suppression, in particular DC dysfunction. In some ways, the induction of DC tolerance
through the TLR2 signaling pathway is analogous to other negative regulatory pathways
in T cells. Recent evidence suggests that CTLs may need to be restimulated in the TME
by tumor DCs in order to expand and acquire full effector functions (133). My work
suggests that blocking TLR2 restores the normal immunostimulatory activity of tumor
DCs. Therefore, anti-TLR2 therapy can potentially used in the clinic to improve
immunotherapy. A phase 1 study investigating the safety, tolerability, pharmacokinetics
and pharmacodynamics of OPN-305, a humanized anti-TLR2 mAb, revealed that the
antibody is generally well tolerated in healthy subjects across all doses (332).
While checkpoint blockade inhibitor therapy has delivered promising results, only
a fraction of patients derive a benefit. It appears that patients with immunogenic tumors
have had the best response to this therapy. Immunogenic tumors are characterized by the
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presence of CD8+ T cells expressing high levels CD45RO and granzyme B, as well as
tumor cells expressing PD-L1 (105, 297). For patients with nonimmunogenic tumors,
disruption of inhibitory networks may be complemented by strategies to activate and
enhance the antitumor immune responses (297). These include adoptive transfer of CAR-
TILs, inducing ICD with chemotherapy or radiation therapy, oncolytic viral therapy, and
cancer vaccines that mobilize rapid expansion of effector and memory T cell subsets (136,
297, 333). Similar to the checkpoint blockade antibodies, I envision that anti-TLR2
therapy could be used as an adjunct to the many current immunotherapy strategies or
conventional therapies to activate the immune system in cancer patients. The therapeutic
benefits observed with the co-administration of anti-TLR2 antibody and other checkpoint
blockade immunomodulatory agents can be enhanced by combining them with any of the
strategies mentioned above, particular with those that can achieve high numbers of
functional CTLs in the cancer.
Strengths and limitations of the tumor models used
The transplantable syngeneic tumor models, Lewis lung carcinoma (LLC) and
B16 melanoma, were used to study tumor DC impairment. These model systems are
attractive because of their simplicity, reproducibility, consistency, and ease of monitoring
(323). The rapid formation of tumors also allows the study of many treatment groups
simultaneously. Insights gained from these models have helped advance our
understanding shared and tumor-specific antigens, intrinsic immunogenicity of tumor
cells, and the generation of antitumor immune responses by the host (323), and the
development of immunotherapy in humans (e.g. Allison’s work with anti-CTLA-4).
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These models allowed us to evaluate the efficacy of the GVAX vaccine in murine tumor
models, as well as adoptive CTL therapy, in the absence or blockade of TLR2.
A potential drawback of using these transplantable tumor models is that the
antitumor effects of these therapies is highly dependent on the number of tumor cells
initially implanted, which may not recapitulate the heterogeneity of human tumors.
Furthermore, the transplantation process involves the injection of a large number of tumor
cells all at once, which can cause acute localized tissue inflammatory responses and
damage, potentially introducing a confounding variable when evaluating the effects of the
tumor milieu on DC dysfunction.
The enforced expression of model antigens such as ovalbumin on B16 cells
facilitated our investigations of tumor-specific CTL responses in situ. This tumor model
allowed us to monitor and track T cells that express transgenic TCRs specific for OVA.
Some argue that these model systems are too artificial and may overestimate the effects of
immune therapies such as TLR2 blockade on antitumor responses. To address this
criticism, we also studied the effects of TLR2 blockade with GVAX, a clinically relevant
vaccination therapy for cancer.
GVAX is a GM-CSF gene-transduced tumor cell vaccine. In the original
preclinical study published in 1993, Dranoff and colleagues demonstrated that GVAX
provided potent, long lasting, antitumor immunity in mice (283). Subcutaneous injection
of GVAX stimulates an intense inflammatory reaction characterized by infiltration of
DCs, macrophages, and granulocytes. Extensive preclinical studies showed that forced
expression of GM-CSF on irradiated nonimmunogenic tumor cells (e.g. B16-F10) also
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enhanced the recruitment and activation of DCs and other APCs in the TME, which
subsequently activate T cells to mediate tumor regression (334). Further enhancement of
antitumor response was demonstrated when GVAX is combined with anti-CTLA-4 (308)
or other DC activating agents such as CD40L (335). Despite these impressive antitumor
effects in preclinical models, high variability within the GVAX treatment groups
continues to be problem, as we observed in our preliminary studies of α-TLR2 in
combination with α-CTLA-4 (Figures 5.12 & 5.13). The success of GVAX depends on a
number of variables such as the number of functional CTLs generated (285), the amount
of GM-CSF secreted by the vaccine, the timing of delivery of GVAX relative to tumor
cell inoculation, and the rate of tumor growth (334).
A major limitation of our transplantable tumor models is that they do not fully
recapitulate the complex tumor microenvironment seen in human cancers (323).
Alternative models include spontaneous tumor models (e.g. driven by constitutive
oncogenes such as KRAS) and humanized tumor models (e.g. human tumor xenografts
implanted in NOD-SCID immunodeficient that have been reconstituted with human
immune cells). Unfortunately, these models also have limitations including the long time
period for tumor development and evaluation of therapeutic responses. They are also
more costly and labor-intensive due to the complexity of generating and maintaining these
mice.
Tumor DC turnover rate and implications in anti-TLR2 therapy
129
In chapter 5, we explored the possibility of targeting TLR2 using antagonistic
TLR2 antibodies to reverse DC dysfunction and determined that the intra-tumor
frequency of the Gr-1+ cDC population is markedly reduced. However, these studies
revealed that the effect of a single dose of anti-TLR2 antibody (100 µg) on tumor DC
function and phenotype is often lost after 3 days. For example, Gr-1 expression on
intratumor DC was recovered after discontinuation of anti-TLR2 antibody treatments. We
considered two possibilities that may explain these results: 1) Degradation of anti-TLR2
antibodies in vivo after intravenous injections, and 2) High tumor DC turnover rate
requiring higher doses of anti-TLR2 antibodies. We, and others confirmed that a single
intravenous injection of anti-TLR2 antibodies (1 mg/kg) effectively inhibited Pam3CSK4-
induced cytokine secretion in recovered splenocytes for at least 2 days (336).
Furthermore, pharmacodynamics analysis by another study revealed that anti-TLR2
antibody has a half-life of 8-9 days and it is functional over a period of 14 days (337).
Thus, it is unlikely that the effects of TLR2 are lost due to antibodies degradation. With
regards to the second possibility, the short lifespan of intratumor DC can potentially
require more anti-TLR2 antibodies to block DC dysfunction. Homeostasis of
nonlymphoid tissue DCs (including tumor DC in situ) is rapid and requires continuous
replenishment of circulating pre-cDCs (14, 15, 34, 153). Unpublished data from our lab
showed that the entire tumor DC population turns over in a short span of time,
approximately 2-3 days. This high rate of DC turnover explains partly why the effects of a
single dose of anti-TLR2 antibody treatment on tumor DC phenotype and function are lost
after a few days. Therefore, this would suggest that TLR2 antibody must be given
frequently.
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Implications beyond tumor immunity
The ability to alter and evade host immune responses by inducing DC dysfunction
is crucial for the survival and persistence of cancer cells, as well as pathogenic microbes
that induce chronic infections (338). Some microbes, such as Helicobacter pylori, have
evolved immune evasion mechanisms that thwart host responses, which results in chronic
infections and microbe persistence. Like infections, inflammation affects tumor
progression from their early initiation, dissemination, and eventually death of the host
(102). A major theme in this thesis is how chronic inflammation contributes to immune
evasion in the context of cancer. Our discovery of TLR2 as a critical proximal signal that
trigger tumor DC dysfunction has broad implications in chronic infections and tolerance
induction by commensals.
In the first section of the thesis, we reported that the TLR2 ligands, FSL-1 and
Pam3CSK4 mimicked TCM in their ability to promote differentiation of IL-10-producing
DCs. These ligands are synthetic lipoproteins resembling microbial fragments derived
from various strains of Mycoplasma bacteria. In fact, there was an early concern that
contamination of TCM with Mycoplasma and/or endotoxins may have accounted for the
effects I observed in the in vitro studies. Indeed, some Mycoplasma strains activate
myeloid cells to produce proinflammatory cytokines such as IL-1 and IL-6 (339), which is
involved in TCM-induced DC dysfunction. To address this concern, samples of TCM
derived from various tumor cell lines were confirmed to be free of Mycoplasma and
endotoxins by the microbiology laboratory at Hospital for Sick Children. In addition, my
results from the in vivo studies further validated that the TME induced tumor DC
dysfunction through TLR2.
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The findings from the in vitro experiment using synthetic TLR2 ligands broadly
suggest that certain microbes also stimulate TLR2 pathway to establish immune tolerance.
For example, polysaccharide A (PSA) secreted from the gut commensal Bacteroides
fragilis signals through the TLR2 pathway on Foxp3+ Tregs to promote immunologic
tolerance and commensal colonization (340). Another recent study showed that PSA
stimulates TLR2 on pDCs to augment IL-10 production in Tregs via cognate interactions
between pDCs and CD4+ T cells (341). Additionally, a number of lipoproteins secreted by
Mycobacterium tuberculosis (e.g. LprA, LpqH, LprG) function as TLR2 agonists that
modulate APC functions through inhibiting MHC class II expression (342).
Polymorphisms in the human TLR2 gene are also associated with enhanced susceptibility
to leprosy and tuberculosis (343, 344).
With regards to pathogenic microbes in chronic infections, several studies indicate
that H. pylori infection impairs DC function and promotes the development of tolerogenic
DCs both in vitro coculture system and in vivo murine models, as means to subvert host
adaptive immune response (345-347). Another recent study that I collaborated on showed
that H. pylori inhibit cDC maturation through autocrine IL-10-mediated STAT3 activation
(348). However, the proximal signals activated on DCs were not defined in that study.
Whether TLR2 also plays a role in inducing DC tolerance to H. pylori merits further
investigation. Interestingly, chronic H. pylori infections have been implicated in the
carcinogenesis of gastric cancers (349). The discovery of tolerogenic DCs induced by
chronic H. pylori infections suggests that this mechanism may contribute to decreased DC
tumor immunosurveillance (348). Given the parallels between immunity to chronic
infection and cancer, the findings presented in this thesis suggest that certain pathogenic
132
microbes (e.g. H. pylori) exploit similar molecular mechanisms, namely TLR2 activation,
to subvert host immune responses by inducing DC tolerance and develop chronic
infections.
133
Concluding Remarks
The objectives of this thesis were to identify the proximal signals that cause DC
dysfunction in cancer, to investigate the mechanisms of DC dysfunction associated with
TLR2 activation, and to evaluate whether TLR2 blockade can enhance immunotherapy by
improving intra-tumor DC immunogenicity. We demonstrated that TLR2 signaling
triggered by tumor-derived factors in the cancer milieu induces DC dysfunction in vitro
and in vivo. Additionally, we showed that TLR2 activation lowered the sensitivity of DCs
to respond to IL-6 and IL-10 by upregulating their respective cytokine receptors, which
partly explains why DC dysfunction occurs locally in the tumor milieu rather than
systemically. These findings warrant further investigations of TLR2 blockade as a means
to reverse DC dysfunction in cancer, and improve DC immunogenicity. To our current
knowledge, this is a new mechanism proposed to explain how cancer inflammation (e.g.
upregulation of tumor-derived factors and tumor associated cytokines) can contribute to
immune evasion. We envision TLR2 blockade as a practical adjunct therapy to current
immunotherapy strategies such as checkpoint blockade, adoptive cell therapy, and cancer
vaccines.
134
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