CCL3 AUGMENTS ANTITUMOR RESPONSES IN
CT26 BY ENHANCING CELLULAR TRAFFICKING
AND INTERFERON-GAMMA EXPRESSION
by
FREDERICK ALLEN
Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of
Philosophy
Dissertation Advisor: Alex Y. Huang, M.D., Ph.D.
Department of Pathology
Immunology Training Program
CASE WESTERN RESERVE UNIVERSITY
January 2018 CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Frederick Allen
Candidate for the Ph.D. degree
Committee Chair
Pamela Wearsch, Ph.D.
Committee Member
Pushpa Pandiyan, Ph.D.
Committee Member
Clive Hamlin, Ph.D.
Committee Member and Advisor
Alex Y. Huang, M.D., Ph.D.
Date of Thesis Defense
November 13, 2017
*We also certify that written approval has been obtained
for any proprietary material contained therein.
2 TABLE OF CONTENTS
Acknowlegment ………………………………………………………………………………………. 10
List of major abbreviations ...... 12
Abstract ...... 13
Chapter I - Introduction to the major tissue, cellular and cytokine components involved in the CCL3-expressing CT26 tumor microenvironment ...... 15
A brief history of immunotherapy ...... 16
The tumor microenvironment – from tumor escape to indirect immunomodulation .... 17
Cytokines ...... 18
Chemokines ...... 19
Macrophage inflammatory protein 1 �, 1�, and rantes ...... 20
CCL3 and CCL4 – Overview ...... 20
CCL3 and CCL4 ...... 21
CCL3 and CCL5 ...... 23
Lymph node ...... 24
Primary and secondary immune organs – general overview ...... 24
Chemokine influence on lymph node cellular trafficking dynamics ...... 25
Interferon-gamma ...... 25
Natural killer cells ...... 26
Dendritic cells ...... 28
Dendritic cells – Overview ...... 28
Dendritic cell maturation ...... 29
3 Chapter II - Unique Transcompartmental Bridge: Antigen-Presenting Cells Sampling across Endothelial and Mucosal Barriers (Review) ...... 31
Abstract ...... 32
Introduction ...... 33
Gut mucosa: apc transepithelial extensions in lamina propria and peyer’s patch ...... 34
Information exchange between LN conduits and follicular DCs (FDCs) ...... 39
LN-resident and migratory DCs interact directly with lymphatic conduits and HEVs 41
CD169+ Macs extend processes from subcapsular sinus into ln follicular zones to assist
in activating the adaptive humoral response ...... 42
Local LN DCs extend dendritic processes into lymphatic fluid to initiate early T cell
activation ...... 43
APC extensions in other anatomical compartments: airway lumen and CNS vessels .. 44
Concluding remarks ...... 46
Chapter III - CCL3 enhances antitumor immune priming in the lymph node via
IFN� with dependency on NK cells ...... 53
Abstract ...... 54
Introduction ...... 55
Materials and methods ...... 57
Results ...... 61
aCCL3 suppresses tumor growth and promotes tumor rejection ...... 61
aCCL3 augments T cell activation by enhancing leukocyte migration to the TDLN 62
aCCL3 bolsters the intracellular production of IFN�+ cells in the TDLN ...... 64
L3TU enhanced leukocyte accumulation is dependent on CCL3 but not NK cells .. 65
4 NK cells, but not CD103+ CD11c+ DCs are important for driving the production of
IFN�-induced chemokines CXCL9 and CXCL10 in the TDLN ...... 66
Exposure of BMDCs to rCCL3 enhances Ag-specific T cell proliferation ...... 66
Discussion ...... 67
Chapter IV - CCL3 augments tumor rejection and enhances CD8+ T cell infiltration through NK and CD103+ dendritic cell recruitment via IFN� ...... 89
Abstract ...... 90
Introduction ...... 91
Material and methods ...... 93
Results ...... 97
CCL3 facilitates tumor rejection via thymic-dependent and thymic-independent
mechanisms ...... 97
CCL3 enhances CD4+ and CD8+ T cell infiltration to the primary tumor site ...... 98
CCL3 recruits NK cells to promote CD103+ DCs infiltration and support T cell
function within the primary tumor ...... 99
Subcutaneously administered rCCL3 significantly slowed tumor growth in
established tumors ...... 101
Discussion ...... 102
Chapter V – Conclusion ...... 126
The significance of IFN� in promoting early and late proinflammatory responses with
L3TU ...... 130
Dendritic cell maturation in the tumor microenvironment ...... 134
5 The interaction of CCL3, interferon-gamma, and CCL5 in the L3TU tumor
microenvironement ...... 137
The dual role of IFN� in tumors ...... 137
Identifying and targeting tumor intrinsic pathways related to IFN� and WNT/�-
catenin, to enhance the constitutive expression CCL3 and tumor rejection ...... 139
Thoughts on the therapeutic application of using high dose recombinant ccl3 ...... 142
Final thoughts ...... 144
Bibliography ...... 145
6 LIST OF FIGURES
Figure 1 - Different models of TE and antigen uptake by LP APC ...... 47
Figure 2 - Overview of LN APC positioning for TE sampling ...... 49
Figure 3 - Activated DCs reside near HEV after migrating from the periphery ...... 50
Figure 4 - LN DCs and Macs extend processes into the medullary sinus to directly sample lymph-borne antigen ...... 51
Figure 5 - Lung DCs sample airway pathogens via TEs ...... 52
Figure 6 - Autologous or recombinant CCL3 slows tumor growth and promotes tumor rejection ...... 73
Figure 7 - aCCL3 expression augments T cell activation by enhancing leukocyte migration to the TDLN ...... 74
Figure 8 - aCCL3 enhances the accumulation of IFN�+ cells in the TDLN ...... 75
Figure 9 - The enhanced accumulation of leukocyte subsets in the L3TU TDLN is negated by blocking CCL3, but enhanced by NK depletion ...... 76
Figure 10 - NK cells, not CD103+ CD11c+ DCs, drive IFN�-induced CXCL9 and
CXCL10 production in the L3TU TDLN ...... 77
Figure 11 - Pretreatment with rCCL3 enhances the capacity of BMDCs to drive OT-I proliferation in vitro ...... 79
Figure 12 - WTTU and L3TU exhibit similar proliferation and PD-L1 expression profiles in vitro ...... 81
Figure 13 - Gross anatomical LN images show enlarged TDLN in the L3TU group compared to the WTTU group ...... 83
Figure 14 - Leukocytes transiently accumulate in NDLNs following L3TU inoculation 84
7 Figure 15 - Comparison of the leukocyte compositions in WTTU and L3TU TDLN cohorts ...... 85
Figure 16 - Quantitative cytokine mRNA profiles in the L3TU TDLN ...... 87
Figure 17 - Efficiency of NK cell depletion in the WTTU and L3TU TDLN cohorts ..... 88
Figure 18 - Autologous CCL3 retards CT26 growth in vivo with partial dependence on both CD8+ T cells and non-T cell sources ...... 108
Figure 19 - L3TU enhances CD4+ and CD8+ T cell infiltrations into the primary tumor site ...... 110
Figure 20 - IFN� levels are sustained in L3TU tumors despite T cell depletions ...... 111
Figure 21 - CCL3 recruits NK cells to drive infiltration of CD103+ DCs and support T cell function at tumor sites ...... 112
Figure 22 - NK cells and CCL3 support inflammation and T cell homing ...... 114
Figure 23 - rCCL3 or irradiated L3TU significantly slows established tumor growth .. 115
Figure 24 - WTTU and L3TU Chemokine production, and growth characteristic of
WTTU and L3TU in vitro ...... 116
Figure 25 - Global cytokine makeup of WTTU and L3TU were dependent on the presence of CD4+ or CD8+ T cells in TME ...... 117
Figure 26 - Intra-tumoral injections of iL3 failed to enhance tumor rejection in vivo ... 119
Figure 27 - Tumor size measurements of L3TU with additional recombinant CCL3 .... 120
Figure 28 - IFN� production in WTTU, L3TU, and L4TU TDLN and primary tumors 121
Figure 29 - Experimental design for tracking NK cell migration in the TDLN ...... 122
Figure 30 - The absolute number of Tregs versus activated CD8 T cells in the TDLN . 123
Figure 31 - The absolute number of CFSE+ BMDCs in the TDLN ...... 124
8 Figure 32 - Proposed experimental design for testing in vivo IFN� suppression of CCL3 in WNT/�-catenin knockout tumors ...... 125
9
ACKNOWLEDGEMENTS
First and foremost, I’d like to thank my mother, Patricia Allen, for always having a patient ear to listen even at the most inconvenient times, and for being a constant source of advice and comfort.
It’s said that it takes a village to raise a child. The same can be said about a Ph.D. student. Christy Kehoe, Melissa Ricco, Valerie Price, thank you for all the administration work you do behind the scenes that go unnoticed. To my thesis committee. Thank you for being understanding and patient with my mistakes and advising me along this path. Dr.
Huang. I could not have been blessed with a better mentor. Thank you for whipping me into shape and teaching me that you can be successful without being selfish. You taught me importance of letting other shine even when it could be you may deserve some of the credit. That speaks volumes about your character. The Huang lab members, past and present: Alex Huang, David Askew, Li-Xin Wang, Francesca Scrimieri, Deb Barkauskas,
Dixon Dorand, Alex Tong, Jay Myers, Saada Eid, Joseph Nthale, Matthew Tsao,
Mohammad Alshebremi, Bryan Benson, Laura Menacol, Candilianne Serrano, Iuliana dit
Bobanga, Hasan Hashem, Anant Vatsayan, Leland Metheny, Yami Huerta, Agne
Petrosiute, Peter Rauhe. We laugh and argue, but don’t let anyone outside the lab say anything bad about us. I could not have asked for a better family. The S.M.D.E.P. leadership, Dr. Robert Haynie, Joseph Williams, Felicite Chatel-Katz, and Joy Williams.
Thank you for letting me be part of an unforgettable experience. I have learned a lot about how to inspire and mentor from your guidance. The leadership of PREP program, past and present: Alison Hall, Joseph Williams, and Sarah O’Keeffe, Paul MacDonald, Diana
10 Ramirez, and Malana Bey. I was a risky investment and you took the risk by taking me in and also continuing to support me. I hope I made the program and the leadership proud.
Henri Brunengraber, Colleen Croniger, Michelle Puchowicz, and Sophie Kochheiser from the CWRU MMPC. You brought me to CWRU so many years ago, and stated me on this journey. It was the joy I got from being under your leadership and watching you work that helped inspire me to take my own journey. To the United States Navy and Marine Corps.
Thank you for showing me that I am capable of being more.
11 LIST OF MAJOR ABBREVIATIONS
APC, pAPC = antigen presenting cell, professional-APC
LN, DLN, TDLN, NDLN = lymph node, draining LN, tumor-DLN, non-DLN
PP = Peyer’s patch
Ag = Antigen
TME = Tumor microenvironment
PTS = Primary tumor site
DC, FDC, TIDC = Dendritic cell, follicular-DC, tumor-infiltrating-DC
TEM = Transendothelial migration
Mac = Macrophage
HEV = High endothelial venule
2PLSM = 2-photon laser-scanning microscopy
TMDs = Trans-M cell dendrites
TE = Transepithelial/transcompartmental extension
SCS = Subcapsular sinus
LP = Lamina propria
EAE = Encephalomyelitis
WTTU, L3TU = Wild-type tumor, CCL3-secreting-TU aCCL3, rCCL3 = Autologous-CCL3, recombinant-CCL3
IC = Immune complexes
12 CCL3 Augments Antitumor Responses in CT26 by Enhancing Cellular
Trafficking and Interferon-Gamma Expression
Abstract
by
FREDERICK ALLEN
Cancer evolves and thrives due to the failures of multiple intracellular and extracellular self-control mechanisms, and as such, often requires a multifaceted approach for complete eradication. An effective vaccine consists of specific, well-developed, and robust CD8+ T cell responses1. However, while inducing tumor reactive CD8+ T cells in vitro or in vivo is achievable, the potential benefits often fail to translate into better patient outcomes2. One of the major difficulties stems from the tumor's ability to induce immune tolerance2,3. Studies have shown that immune cells can be induced to promote tumor growth or regression and chemokines are often integral organizers and modulators of these events2,4. The ability for tumors to maintain a tolerant environment or for immunotherapy to promote strong long-lived antitumor CD8+ T cell responses is dependent on the tumor types and also the chemokines associated within the tumor milieu4-6. Clinical data has shown that increased number of lymph nodes (LNs) infiltrated by tumors directly correlates with poor patient outcomes7,8. Therefore, deciphering the complexities of immune interactions within LNs are essential to understanding how many tumors can redirect or suppress immune responses. Chemokines, such as CCL3, have the ability to attract naïve
CD8+ T cells and modulate their immune responses2,9. CCL3 is also integral in orchestrating nonrandom naïve CD8+ T cell contacts with dendritic cells (DCs) in the draining LNs (DLNs) of vaccinated mice9. These events lead to increased CD8+ T cell-DC
13 surveillance time, decreases effector response time, and enhanced memory T cell generation; all of which are important in building an effective vaccine response.
Furthermore, CCL3 has also been directly implicated in facilitating the clearance of some tumor models6,10-12. These findings suggest possible therapeutic roles for CCL3 in redirecting tumor-tolerant milieus toward a tumor-immunogenic one by conditioning immune cells to induce an inflammatory response to tumor antigens (Ag). The body of work presented here discusses various dynamic cellular responses that occur during Ag capture, and subsequent early and late phase adaptive immune development in the tumor-
DLN (TDLN) and at the primary tumor site (PTS) in response to CCL3 therapy.
14 CHAPTER I
Introduction to the major tissue, cellular and cytokine components involved in the
CCL3-expressing CT26 tumor microenvironment
15 A BRIEF HISTORY OF IMMUNOTHERAPY
The first reported association between the immune system and neoplastic tissues was published by Rudolf Virchow in 186313,14. Virchow showed that chronic inflammation and the development of tissue neoplasia were directly correlated13,14. Then, in the late 19th century, the first documented experiments demonstrating immune cell clearance of neoplastic tissue was shown by a bone sarcoma surgeon named William Coley15. He inoculated patients with an infectious streptococcal organism and noted shrinkage of some tumors. From these experiments, he developed his own serum for which he called, ‘Coley toxins,’ which consisted of endotoxins from heat killed streptococci and Serratia marcescens bacteria15,16. However, his serum resulted in high mortality and did not always work. It would later be discontinued as a mainstream practice and replaced by radiation therapy, which was seen as less dangerous than his bacterial toxin regiment15,16. Never-the- less, Coley’s work ignited a new branch of science in the biomedical community which focuses on understanding how the immune system interacts with neoplastic cells. His discovery and subsequent treatment of patients with an immune-stimulating serum to treat neoplasia, earned him the title, given by many, as the father of tumor immunotherapy15. A few years later, during the early and mid 20th centuries, immunology pioneers like Paul
Ehrlich, Lewis Thomas, and Macfarlane Burnet expounded upon these strategies by observing and reporting that host immune cells could provide direct protection for or against neoplastic tissues16,17. Since then and to this day, basic and clinical tumor immunologists have worked to discover ways of manipulating and boosting immune cells to target and eliminate neoplastic cells. Today the work of these early pioneers has branch
16 into several main compartments of tumor immunology: monoclocal antibody therapies, which are designed to specifically target tumor Ags or immune suppressor cells; non- specific immunotherapies, which are designed to boost the immune system in a nonspecific way; or with tumor vaccines, which are designed to drive specificity and boost the immune system responses overall through the manipulation of key immune cell instigators involved in driving protection or destruction of tumor cells.
THE TUMOR MICROENVIRONMENT – FROM TUMOR ESCAPE TO
INDIRECT IMMUNOMODULATION
Tumor escape mechanisms develop through a process known as
“immunoediting”18. Immunoediting is a process which describes the evolutionary manner of tumor-immune evasion in three steps: elimination, equilibrium, and escape19. Tumor escape mechanisms develop during the equilibrium phase where tumor cells can lay dormant and undergo further mutation that eventually results in a Darwinian process of adaption, where only those tumors that are able to escape immune detection (tolerance) survive18,19. These escape mechanisms involve either hiding from immune cell detection by appearing normal on the surface, or suppressing or converting immune responses through direct or indirect means19. Direct means requires tumor cells to be in contact with the immune cells. Examples of these are program-death ligand-1 (PD-L1) expression on some tumor cells interacting with program death-1 (PD1) receptors proteins on the surface of T cells to drive T cell anergy20. Another example is Vascular cell adhesion protein-1
(VCAM-1), which is expressed on some tumor cells21. VCAM-1 interacts with very late
17 antigen-4 (VLA-4 = CD49d + CD29; �4�1) or α4β7 integrins from tumor-infiltrating immune cells and modulate their differentiation potential or ability to properly infiltrate the tumor node21. While direct modulation or suppression of immune cells can help tumors escape, this method is limited by the number of contacts between tumors and immune cells, as well as the number of suppressive or tolerant-inducing signaling proteins expressed (e.g.
PD-L1, cytotoxic T-lymphocyte-associated protein 4 = CTLA4). However, indirect modulation or suppression of immune cells within the surrounding TME can present a more effective way for tumor cells to ensure their survival, because it can affect local and tumor- infiltrating immune cells on a global scale. Using this method, tumor cells can induce immunomodulatory effects on immune cells by secreting various soluble factors in the
TME that affect immune cell function and migration. These secreted factors in the TME can tip the balance between tumor survival and tumor elimination22,23. Discovering ways to manipulate, boost, or introduce new soluble factors to the TME can directly affect tumor cells functions, drive the infiltration of new unaffected or naïve immune cells, or bolster or renew antitumor immune cells already present in the TME.
CYTOKINES
Cytokines are a broad category of small soluble proteins upregulated by most nucleated cells as a way to target and communicate with specific cell-types and induce particular responses or interactions24. Cytokines can act in autocrine, paracrine, or endocrine fashions24-26. In this way, cytokines are much like hormones. However, hormones are secreted by specialized cells to elicit very specific cellular responses to smaller groups of cells26. Many cytokines have a high degree of receptor-binding overlap with other
18 cytokines and can act synergistically or antagonistically in inducing responses27,28. Some of the types of changes provoked by cytokines include specific cell-to-cell interactions and migrations; induction of particular types of cellular activation and differentiation; or inducing cell death25. Cytokines are broken down into subcategories and grouped according to cell-type or the primary type of response they induce24. For example, a cytokine is called an interleukin (IL) if it involves one leukocyte acting in an autocrine manner or directly on other leukocytes24. Chemokines are cytokines that are released by leukocytes and act on specific cells through an intermediary cell (e.g. fibroblast, endothelial or epithelial cells) to drive chemotaxis25.
CHEMOKINES
Chemokines are the largest subset within the cytokine family. While chemokines are differentiated from other cytokines as strong inducers of cellular chemotaxis, it is important to remember that many chemokines also play important roles in modulating immune cell phenotypes and responses in addition to their chemotactic abilities9,29,30. Chemokines are low molecular weight cytokines (8-12 kDa) that bind to seven-transmembrane herotrimeric
G protein coupled receptors (GPCRs)31,32. Chemokines consists of four main categories depended on the arrangement and location of four conserved cysteine residues and are grouped into four subfamilies based on the arrangement of two cysteine residues located at their N-terminus33. Chemokines fall into either CC, CXC, CX3C or (X)C groups, where
‘X’ symbolizes a non-cysteine residue; ‘()’ a deleted first and third cysteine; and ‘3’ the number of non-cysteine amino acids separating the two N-terminal cysteine bonds33.
19 Chemokines are generally described as either homeostatic or inflammatory33,34. Just as the names imply, homeostatic chemokines primary control leukocyte trafficking during non-inflammatory events (e.g. CCL19 and CCL21 trafficking of lymphocytes), while inflammatory chemokines are primarily associated with the movement of effector leukocytes during inflammation34. Hence, chemokines are not only important for helping immune cells and non-immune cells maintain a stringent cellular makeup in tissues during homeostasis, but they also allow for cells to differentially signal to specific immune cell- types during periods of tissue instability caused by microbial invasion, neoplastic cell formation, or other environmental factors (e.g. radiation, chemical, etc.).
Chemokines continually attract specific immune cells to help maintain homeostasis or, if necessary, drive cell-mediated inflammation to deal with dangers from self- or non-self-
Ags. However, unimpeded chemokine upregulation can help sustain tissue in a chronic inflammatory state24. Chronic inflammation, if left unchecked, could lead to severe negative physiological consequences to the host, such as tumors, autoimmune disorders, and other disease pathologies24.
MACROPHAGE INFLAMMATORY PROTEIN 1 �, 1�, AND RANTES
CCL3 and CCL4 – Overview
Macrophage inflammatory proteins-1 (MIP-1) chemokines are small (8-10 kDa) proteins31,35,36. The discovery of the first murine MIP-1 proteins, MIP-1� and MIP-1�, were first published in 1988 from isolated supernatant of inflamed macrophages that were stimulated with endotoxins, and shown to actively produce inflammation and chemo-
20 attract neutrophils when injected subcutaneously31,35,36. Today the MIP-1 chemokine family in both humans and mice consists of four distinct proteins: MIP-1� (CCL3), MIP-
1� (CCL4), MIP-1� (CCL9 and CCL10), and MIP1� (CCL15)32.
The characterization of human CCL3 (LD78 or GOS19) and human CCL4 (Act-
774.1) were first published in 1985 and 1988 respectively31. Murine CCL3 and CCL4 are encoded on chromosome 11 and share 68% homologous in their amino acid sequences31.
Human CCL3 and CCL4 reside in close proximity to each other on their respective species chromosome. Human CCL3 and CCL4 are both encoded on chromosome 17 and separated only by 14 kb31. CCL4 has one isotype for mice and two for humans, AT 744.1 and AT
744.2 (AT 744.2 is the non-allelic form)31. Human CCL3 comes in three isoforms, LD78�,
LD78�, and LD78�. LD78� and LD78� are 94% identical and can induce cellular response after binding to their cognate receptors31. However, LD78� is likely a pseudo-chemokine, because it is truncated and no bioactivity has been reported from its binding31. Amongst the three isoforms, LD78� and murine CCL3 share the highest degree of homology at 69% and function similarly in vivo31,32.
CCL3 and CCL4
CCL3 and CCL4 are involved in a wide variety of innate and adaptive immune functions across different organs such as bone, soft tissue, and lymphatics9,31,37. They can be induced in most mature hematopoietic cells with varying degrees of abundance. This is because the receptors that they bind are expressed by a wide range of immune cells. Human and murine chemokines CCL3 and CCL4 both elicit their responses though the chemokine
21 receptor CCR531. CCR5 is constitutively expressed on a variety of cells and cell subsets
(e.g., immature DCs, resting macrophages, monocytes, activated T cells (expect Th2), and some B cell subsets)6. Of all the receptors that CCL3 and CCL4 interacts with, CCR5 is potentially the most heavily researched because of its importance in the field of HIV research. Initial studies with CCL3 and CCL4 were heavily focused on their potentials as
HIV therapies, because CCR5 is a co-receptor used by the human immunodeficiency virus
(HIV) to infect cells38. However, human and murine CCL3 can also bind to CCR1, suggesting a preferential migratory potential between them, despite sharing a high degree of homology31. There is also species difference between murine and human orthologs as well. For example, some reports have shown that only human and murine CCL3 can bind to the receptor CCR339,40, while another report demonstrated that only human CCL4 is capable of binding human CCR341. Lastly, neither murine CCL3 or CCL4 can bind murine
CCL8, but one study reported that, in humans, CCL4 does. However, this remains unclear, because the experimental conclusions reported were all performed in vitro using CCR8
Jurkat stable transfectants and, to date, no other reports have shown this to be the case in vivo42.
CCL3 and CCL4 receptor promiscuity and binding potentials can lead to preferential recruitment and induce different functional outcome in some cases43. Dairaghi et. al, demonstrated this when they showed that CCL4, and not CCL3, was able to induce thymocyte activation and migration44. Moreover, studies comparing CCL3 and CCL4 function in vitro (at concentration of 100 pg/ml) revealed that human CCL4 preferentially attracts naïve CD4+ T cells over activated CD4+ T cells and other lymphocytes; where as
22 human CCL3 attracts more lymphocytes globally as compared to CCL443. In addition, early in vitro evidence also suggest that CCL3 may be a more potent chemo-attractant of both CD8+ T- and B-lymphocytes than CCL443. CCL3 and CCL4 are both actively involved in the recruitment of immune cells during inflammatory responses such as bone reconstruction, wound healing, and guiding CD8+ T cells to CpG and alum immunized
LNs9,32,37. Along with their role in chemotaxis, CCL3 and CCL4 are critically involved in modulating the scope of an immune response. Castellino and colleagues showed that blocking CCL3 and CCL4, or deletion of their common receptor CCR5 on CD8+ T cells resulted not only in reduced contacts with DCs, but also in diminished CD8+ T cell memory pool9. CCL3 also plays a prominent role in a variety of inflammatory pathologies such as arthritis, chronic lymphocytic leukemia, and multiple sclerosis45-47. CCL3 and CCL4 can also work to differentially stimulate or inhibit the upregulation of inflammatory cytokines.
One report showed that macrophages incubated with CCL3 directly enhanced macrophage production of cytokines normally associated with inducing inflammation and cell apoptosis such as TNF�, IL-10, and IL-6. However, CCL4-treated macrophages could not induce macrophages to secret the same cytokines, and in fact they were shown to inhibit CCL3’s ability to stimulate macrophage cytokine production48.
CCL3 and CCL5
Another chemokine that should be considered is RANTES (now referred to as
CCL5). According to the National Center for Biotechnology Information (NCBI), CCL5 is also located on chromosome 11 and 17 in the murine and human genome. Like CCL3,
CCL5 can also bind to CCR3, CCR5, and CCR131,33,49. The role of CCR5 molecules in
23 inflammation is diverse, ranging from chemotaxis, receptor co-stimulation and cell adhesion9,30,50. There are variations in biological expression and responses amongst CCL3 and CCL5 despite shared receptor binding and close homology51,52. Like CCL4, there is data to suggest that CCL5 is a more potent recruiter of immune suppressing Tregs despite both having the capacity to bind to the expressed receptors CCR5 and CCR153,54.
LYMPH NODE
Primary and secondary immune organs – general overview
The immune system consists of primary and secondary immune organs. Primary immune organs include the bone marrow and thymus which are responsible for the development of nearly all hematopoietic cells, and the ‘education’ of thymocytes into naïve
T cells and natural killer T (NKT)55 cells respectively. Secondary immune tissues serve as sentinel conduits designed to attract soluble Ags as well as specific immune cells responsible for producing the bulk of adaptive immune responses. These organs include spleens, Peyer’s patches (PP), and LNs. Each are structurally designed to service specific locations in the body in order to maximize the best responses to Ags. However, this is not to say that each secondary immune organ is only capable of generating responses from the tissues they serve. Each secondary immune organ is equally capable of cultivating antigenic responses that arrive from other sites, if the Ag makes its way to that organ. The spleen is directly connected to major blood vessels and organs though splenic and portal vesicles, and serve as sentinel stations for blood borne Ags56. PP are located along the walls of small intestines and are designed for the sampling of intestinal bacteria that reside within the gut57. LNs are the most numerous immune organs in the body, located in close
24 proximity to epidermal tissues. They serve as sentinel stations for most epithelial-lined organs throughout the body.
Chemokine influence on lymph node cellular trafficking dynamics
The influx of naïve and activated lymphocytes from the peripheral blood into the
LNs occurs via specialized blood vessels called high endothelial venules (HEVs)58,59.
These specialized endothelial cells are lined with unique sialylated glycoproteins, collectively referred to as integrins and selectins, which allow them to select certain immune cells from the peripheral blood and lymph circulation that express the tissue- specific integrin and selectin ligands or receptors (e.g. CD62L and CCR7)59. This process is referred to as leukocyte transendothelial migration (TEM)58. Chemokines are an integral part of this process. The major chemokines integral in the process of TEM are CCL19 and
CCL21. The main chemokine responsible for trafficking within LNs, during steady state conditions once the cells enter the LN parenchyma, are CXCL13, as well as CCL19 and
CCL21. During inflammation, the influx of cells to LNs increase. This is due to transformations in the LN’s architectural matrix as well as enhancements and changes in the presence of local chemokines. During inflammatory condition, there is an upregulation of the homeostatic chemokines CCL19, CCL20, and CXCR4 as well as the presence of inflammatory chemokines such as CCL3, CCL4, CCL5, and others.
INTERFERON-GAMMA
Interferons (IFNs) are part of family of potent cytokines that are upregulated by various immune and non-immune cells alike in response to perceived dangers. IFNs were
25 originally named because of their capacity to inhibit viral infections of cells. The first report on IFNs was published in 1957 by Isaacs and Lindenmann who showed that interferons were expressed by chick chorioallantoic membranes after stimulation from influenza viruses60,61. There are seven types of IFNs found in humans: IFN�, IFN�, IFN�, IFN�,
IFN�, IFN�, IFN�, IFN�, IFN�, and IFN�61. IFNs are broken down into two main categories, type one (I) and type two (II) IFNs. Type-I IFNs consists of all IFNs except for
IFN�62. All type-I IFNs signal though the same type-I receptors and are expressed by a wide variety of cells-types62.
In 1965, Wheelock et. al, was the first the identify the type-II IFN, IFN�. He demonstrated that an IFN�-like virus inhibitor was produced when human leukocytes were incubated with phytohemagglutinin63,64. IFN� is the only type-II IFN and is expressed by a much smaller group of cell-types64. IFN� signals though its own receptor named the IFN� receptor (IFN�R), which consists of two combined subunits, IFN�R1 and IFN�R264. IFN� is upregulated during a variety of innate and adaptive immune responses, and involved in the development and maintenance of many pathologies such as those associated with viral infections, autoimmune disorders, cancers, and others62. When upregulated, IFN� can affect many cellular processes such as migration, Ag presentation, apoptosis, polarization, differentiation, B cell immunoglobulin class switching, adhesion, and proliferation62,64.
NATURAL KILLER CELLS
In the 1970’s several groups of immunologist were on the verge of discovering a new population of innate cells that were killing tumors in a T and B cell independent
26 manner65. These groups independently observed that, when various types of tumor cells were cultured with lymphocytes from unimmunized mice or healthy humans, a natural cytotoxicity to tumor occurred. The first published description of these cytotoxic cells was in 1975 by Rolf Kiessling et. al 66, who coined them ‘Natural Killer’ (NK) cells, and followed closely by Herberman et. al, (also in 1975)67. Then, in the summer of 1981, a senior graduate student of Dr. Kiessling, Klas Kärre, formulated the ‘missing-self’ hypothesis during this thesis and later, with Dr. Ljunggren, published their hypothesis, along with experimental evidence in 199068,69. They showed that murine NK cells cultured with syngeneic tumor cells devoid of MHC-I expression, resulted in tumor killing68,70.
However, this hypothesis did not explain why cells like erythrocytes, which are devoid of
MHC molecules, were not killed by NK cells70. The explanation would come with the discovery that NK cells express receptors that activate or inhibit their activity, and the ligands to these receptors are located on other cells in the body. These discoveries then led to the ‘self-induced’ hypothesis which added to the ‘missing-self’ hypothesis70. In general, this hypothesis states that cells undergoing stress (e.g. viral infection) will either have diminished inhibitory ligands or express activating ligands for NK cells, and the decision for NK cells to kill or ignore a cells is found in the balance between these two states70.
In murine biology, NK cells activity is not as distinct as in humans. However, both retain similar cytolytic and cytokine-producing behaviors71. In the human, NK cells are categorized as cytolytic or high cytokine-producing. Those NK cells that are primary engaged in direct killing of cells are referred to as cytolytic. Cytolytic and IFN�-producing
NK cells are referred to as NKdim and NKbright cells, respectively72. Bright versus dim refers
27 to how intensely they express the surface protein CD56, a neural cell adhesion molecule
(NCAM)70. While these two subsets are commonly referenced as distinct NK subsets, some studies have shown that the functional differences between them may not be as unique as once thought73,74. For example, De Maria et al., showed that, when stimulated, cytolytic
NKdim cells can also release IFN�, but do so at much earlier time points than the IFN�- producing NKbright cells (2-4 hours vs. > 16 hours respectively)73. Also, Wang et. al, further demonstrated that cytolytic NKdim cells can use IFN� and TNF� synergistically to kill tumor cells74.
DENDRITIC CELLS
Dendritic cells – Overview
The first characterizations of DCs were published in 1973, when Ralph Steinman, fascinated by MacFarlane Burnet’s 1959 publication of ‘Clonal Selection Theory of
Acquired Immunity’75 set out on a quest with Zanvil Cohn, to understand how the immune system decides to respond to Ag76. Drs. Steinman and Cohn would go on to describe the morphology of a novel cell that could extend and contract its cytoplasm into large variety of pseudopod configurations (dendrites), that were less adherent than macrophages to splenic fibers, and contained less granulocytic vesicles than macrophages77. The months and years after this ground-breaking discovery lead to more functional assays, by Drs.
Steinman, Cohn, and others, that further defined the DC’s uniqueness and significance in the immune system. Drs. Steinman did indeed fulfil his hopes in defining a major piece of the immunological puzzle, helping us understand more clearly how our body decides to react to Ag, and initiate the clonal expansion of acquired immunity. Today, DCs are
28 recognized as the prime inducers of naïve T cell activation in response to Ag and, therefore, are the prime inducers of cell mediated immunity. To date there are five main types of DCs, conventional, monocyte-derived, and plasmacytoid, CD11b+ DCs and Langerhans78.
Conventional DCs are all derived from CD8+ DCs and encompass those DCs that are CD8+
CD11c+ or are CD8+ CD103+ CD11c+ DCs (CD103+ DCs)78. CD8+ DCs resident primary in lymphoid tissues. CD103 (alpha E beta 7) is an integrin protein that is expressed on DCs and T cells that reside in or home to mucosal tissues78,79.
Dendritic cell maturation
All DCs, regardless of subtype, exist along a spectrum between two phenotypes, immature and mature80. Immature DCs are primarily suited for recognizing and engulfing
Ags that stimulate various extracellular and intracellular toll-like receptors (TLR), intracellular retinoic acid inducible gene I (RIG-1)-like receptors/helicases (RLR) and
Nucleotide Oligomerization receptors (NLR)81-83. Those DCs that are non-lymphoid-organ residents complete their maturation during their migration though afferent lymph vessels and arrive via high endothelial venules (HEV) in LNs and PPs, and the marginal sinus in the white pulp of spleens56,84. This gives migratory and residential mature DCs prime positioning for scanning for incoming T cells that can recognize Ag on MHC molecules presented by DCs.
In order for naïve T cells to become fully activated by DCs they require three district signals85,86. First, is the binding of the T cell receptor (TCR) to a cognate Ag presented on MHC complexes. Second, is stimulation by agonistic (versus inhibitory) co-
29 stimulatory molecules (e.g. CD80, 86). Third, are cytokine signals. Variations in signal strength, availability, and type are determinants of T cell activation and differentiation states85. In addition, stable and prolonged contact between DCs and T cells are also important for the development of fully activated T cells and the formation of memory31,50,87-
91.
The process of DCs maturing entails phenotypic and metabolic changes that are marked by a decrease in Ag phagocytic capabilities, an increase in MHC and co- stimulatory molecules, and a defined cytokine profile92. In addition, there is a decrease in the inflammatory homing receptor, CCR5 and an increase in lymphoid-tissue homing and migratory molecules such as CCR793. Along the DC’s path to maturity can exist various expressions levels of MHC and co-stimulatory molecules, as well as a variety of different types of cytokine or the lack thereof94. These complexities in maturation states grant DCs the flexibility to maintain tolerance during steady state conditions, initiate tolerance to curb inflammatory processes, protect against autoreactive T cells, and drive the activation of adaptive cell-mediated responses during inflammatory events94,95. Similar to the case of T cell activation and differentiation, DCs maturation is shaped by signals received from their external environments94. In order to drive full maturity (defined by the capacity to fully active T cells), DCs need two types of signals. The first signals are ones that trigger pattern recognition receptors94. The second set of signals are from cytokines which can come from local tissue environment (paracrine stimulation) or after upregulation from the DC itself
(autocrine stimulation)94.
30 CHAPTER II
Unique Transcompartmental Bridge: Antigen-Presenting Cells Sampling across
Endothelial and Mucosal Barriers (Review)
Frederick Allen1*, Alexander Tong1*, Alex Y. Huang1,2,3^
Department of 1Pathology and 2Pediatrics, Case Western Reserve University School
of Medicine
3Angie Fowler AYA Cancer Institute / University Hospitals Rainbow Babies &
Children’s Hospital, Cleveland, Ohio 44106, U.S.A.
*Both authors contribute equally to this manuscript
31 ABSTRACT
Potentially harmful pathogens can gain access to tissues and organ systems through body sites that are in direct contact with the outside environment, such as the skin, the gut, and the airway mucosa. Antigen-presenting cells (APCs) represent a bridge between the innate and adaptive immunity, and their capacity for constant immune surveillance and rapid sampling of incoming pathogens and other potentially harmful Ags is central for mounting an effective and robust protective host response. The classical view is that APCs perform this task efficiently within the tissue to sense invading agents intra- compartmentally. However, recent data based on high resolution imaging support an additional transcompartmental surveillance behavior by APC by reaching across intact physical barriers. In this review, we summarize intravital microscopic evidences of APC to sample Ags transcompartmentally at the gut mucosa and other body sites.
32 INTRODUCTION
Our immune system is tasked to develop host defense mechanisms that require speed, sensitivity, and specificity to deal with a vast array of fast evolving pathogens, since the doubling times of bacteria and viruses outpace eukaryotic cell division in most cases.
In this regard, current dogma for the initiation of the adaptive cellular immune responses has centered primarily on the role of tissue-resident DCs and their ability to migrate to the draining lymph nodes (DLNs) and present processed foreign Ags bound to surface major histocompatibility complexes (MHC) to T cells for recognition via cognate T cell receptor
(TCR). However, DC maturation upon pathogen encounter and its subsequent migration to
LN can take hours to days. Upon arrival in the LN, Ag-laden DCs’ presentation to and engagement by T cells can take significant time to mount an effective Ag-specific cellular immune response. Emerging data suggest an alternative route of Ag capture and presentation by DCs and other antigen-presenting cells (APCs) across distinct anatomical compartments. For example, data now suggest that crosstalk between cells in the sinuses and those located within the parenchyma is essential for the maintenance of immune cells during steady (tolerant-inducing) and non-steady (inflammatory-inducing) states in the
LN96-98. Although immune cell transmigration and shuffling of Ags across endothelial and epithelial conduits is not new, the concept that APCs residing in one tissue compartment can extend their processes across intact epithelial and endothelial barriers into sites such as endothelial vessels, LN conduits, airway passages, gut lumen, and central nervous system
(CNS) vasculature without physical transmigration is a growing area of research with potential biological significance. This new appreciation of APC in vivo behavior is made possible due to growing and increasingly sophisticated application of the intravital two-
33 photon laser scanning microscopy (2PLSM) technique in anesthetized mice or intact explanted tissues. Knowledge gained from the growing body of investigations adds to our new understanding of how the immune system mounts meaningful and timely responses to infections and other pathologies and is poised to impact the development of novel vaccine strategies. This review focuses on a presentation of available intravital imaging-based evidence regarding the transcompartmental behavior of DCs and other APCs in various body sites. We will first discuss APC behavior in the gut mucosa, ending with a discussion on the intra-nodal APC behavior in the sentinel LN and other anatomical sites including the airways and the CNS.
GUT MUCOSA: APC TRANSEPITHELIAL EXTENSIONS IN LAMINA
PROPRIA AND PEYER’S PATCH
The gut mucosa is constantly exposed to commensal and potentially pathogenic microbial Ags. Historically, Ag has been thought to be primarily taken up through M cells in the Peyer’s patch (PP) for presentation to APCs on the basolateral side of the mucosal barrier99-101. These APCs were then thought to process and present this Ag in the draining mesenteric LN, thereby initiating a gut-trophic adaptive immune response. However, multiple studies using in situ microscopic evaluation of intestinal lymphoid structures suggest that there may be active surveillance of the brush border via APCs extending dendrites between epithelial cells in order to directly sample luminal Ag. These data reveal that there may be an additional, complex layer of immune surveillance in the form of transepithelial/transcompartmental extension (TE) at the host–pathogen barrier, which allows efficient Ag uptake and processing.
34
For the purposes of this review, we will generally define gut resident DCs as
CD11c+ CX3CR1− CD103+ cells and gut resident macrophages (Macs) as CD11c+
CX3CR1+ CD103− cells, although we recognize that there may be many more nuanced subsets depending on the markers used102-107. Due to the more recent nature of these combinations of markers being used to discriminate the two populations, cell-type identifications from older literature reviewed hereafter will be indicated as clearly as the data allow.
Some of the first studies utilizing in vitro and limited in vivo approaches focused on DC extensions through intact epithelial barrier in the lamina propria (LP)108. The earliest studies used transwell coated with human enterocyte cell line Caco-2 to establish an in vitro model of the gut epithelium. DCs were then seeded on the basolateral side with bacteria added to the luminal side. Electron and confocal microscopy of this system revealed that DCs were able to penetrate the Caco-2 monolayer to directly uptake bacteria.
Complementary immunostaining studies using ligated loops of intestine injected with non- infective Escherichia coli DH5α or infective Salmonella typhimurium showed that some
CD11c+ DCs were able to extend dendrites into the lumen and uptake infective bacteria.
Importantly, these DCs expressed tight junction proteins of their own, suggesting that they were able to maintain brush border microvilli organization via contiguous connection with epithelial cells.
35 Subsequently, Niess et al. used living intestinal explants to identify CX3CR1+
APCs specifically in the terminal ileum extending dendrites into the bowel lumen109. This explant system revealed that the number of LP APCs extending dendrites per villus ranged from ~1–2 in the non-inflamed setting to ~4 in the Salmonella infected setting. Here, LP
APCs were dependent on recognition of CX3CL1 on intestinal epithelial cells (IECs) for their function, as only CX3CR1GFP/WT (functional heterozygous) but not
CX3CR1GFP/GFP (knockout homozygous) animals were able to extend luminal dendrites.
Other groups adapted the intestinal explant technique for 2PLSM visualizing the luminal side of the LP. Chieppa et al. everted clips of small intestine from various reporter mice to expose the lumen for 2PLSM110. They showed that LP APCs project extensions into gut lumen with either finger-like projections or a distinct balloon body (BB) shape in order to sample pathogenic S. typhimurium. As the BB projections were not visualized in other explant preparations, the authors acknowledged that it could be an artifact secondary to removal of the mucus layer, although other data suggest that these could be APCs beginning to unidirectionally transmigrate into the gut lumen in a flagellin-dependent manner in order to participate in pathogen exclusion111. Significantly, in contrast to previous studies, Chieppa et al. found that CD11c+/MHC-II+ APC extensions into lumen were visualized in all parts of the small intestine LP at densities similar to previous reports
(~1–5 DCs per villus), raising the question whether this mode of Ag uptake could be more universally distributed than previously described109,110. A later study by Hapfelmeier et al. examined specifically the large intestine LP DCs using the same explant approach and failed to identify any TEs in the cecum112. However, cecal LP DCs were still found to be
36 critical for facilitating the early phase of S. typhimurium infection, and the authors did not rule out the possibility that rare LP DC sampling events not captured by imaging could drive Salmonella epithelial translocation.
The ability of gut APCs to extend TEs into lumen is not restricted to LP APCs.
Lelouard et al. used 2PLSM of PP explants to identify highly mobile PP CD11c+ APCs extending dendrites through M-cell specific pores113. The number of APCs with trans-M cell dendrites (TMDs) was estimated to be 35 – 125 per PP, and these TMDs could actively retract back into the sub-epithelial dome of the PP after sampling the lumen. Consistent with LP APC studies, PP APCs were able to uptake S. typhimurium through TMDs in the early hours of infection.
All of the explant studies discussed above involve significant trauma to the intestinal explant (lengthwise incision followed by multiple washes) or ligated loops.
Therefore, more recent studies have taken a relatively less invasive, intravital approach to visualizing LP APCs114,115. Laboratories have accomplished this by making a small abdominal incision, drawing out a small portion of the small intestine and securing this to a coverslip by epoxy or by the mouse’s own weight for imaging on an inverted microscope.
Using a serosal 2PLSM imaging approach, McDole et al. showed that goblet cells deliver low molecular weight Ag to CD103+ DCs in the small intestine, whereas CX3CR1+ Macs may directly sample higher molecular weight Ag via extensions into gut lumen114. In a similar fashion through a mucosal imaging approach, Xu et al. demonstrated that CX3CR1+
Macs project into LP in vivo, although these Macs are largely sessile in the uninflamed
37 setting and do not appear to migrate away from the epithelium115. In contrast, a subsequent intravital study by Farache et al. failed to demonstrate CX3CR1+ Macs exhibiting TEs even under pathogenic bacterial stimulation. Instead, CD103+CX3CR1− DCs were recruited to the epithelium by S. typhimurium, thereafter extending TEs to directly sample and capture luminal bacteria116.
While there is strong evidence for APC TEs based on the above imaging studies, there are contrasting data questioning the universality of these observations. Ligated small intestinal loop studies using a BALB/c mouse background strain and a model fungal pathogen failed to show the same dendrite formation117. Depletion of CD11c+ cells still allowed for fungal pathogen translocation into LP in this in vitro infection model, suggesting that LP DCs or Macs were not critical for pathogen uptake. A comparison of
CX3CR1 reporter mice on BALB/c, C57BL/6, and F1 backgrounds revealed that as-of-yet undefined genetic factors in BALB/c mice may be responsible for the absence of TEs originally identified in C57BL/6 strains. The outstanding question remains whether these
APC extensions are relevant in human mucosal surface surveillance.
Aside from mouse strain dependence, is there evidence for functional consequences of pathogen/Ag uptake by LP APCs via TEs? Schulz et al. have addressed this in studies elucidating the function of two major LP APC subpopulations, showing that the CX3CR1+
Macs population comprises non-migratory cells with limited capacity to prime naive T cells118. In contrast, CD103+ LP DCs are fully capable of migrating to draining mesenteric
LN and activating T cells. Anatomically, CX3CR1+ Macs were found adjacent to gut
38 epithelia whereas CD103+ DCs were closer to the villus core. Previous data suggest that while CX3CR1+ Macs can efficiently uptake soluble Ag, CD103+ DCs do not share this ability but appear to be able to directly capture bacteria116,119. These data suggest that although CX3CR1+ “resident” LP Macs can uptake bacteria or Ag in the LP, they likely then pass it on to CD103+ “migratory” DCs, which then travel to the mesenteric LN for T cell activation. This model has been corroborated by Mazzini et al., who showed that
CX3CR1+ Macs efficiently uptake Ag for transfer to CD103+ DCs in a process that is dependent on connexin 43120. Taken together, multiple avenues have been described for
Ag uptake by LP DCs leading to Ag-specific T cell activation in the draining mesenteric
LN: direct uptake of translocated pathogen/Ag, transfer of Ag from goblet cells or LP
Macs, and direct sampling of pathogens via TEs (Fig. 1). Gut APC TEs may therefore play important roles in initiating downstream gut-trophic adaptive immune responses, although further work is needed to better quantify TE functional contribution to the differential recognition and control of commensal and pathogenic microbiomes.
INFORMATION EXCHANGE BETWEEN LN CONDUITS AND FOLLICULAR
DCS (FDCS)
Transepithelial/transcompartmental extension behavior of DC and Macs is not unique to those found in the gut mucosa, as similar behavior can be found in secondary lymphoid organs. The LN is a highly specialized immune organ designed for the organization and fine tuning of immune cells in order to promote rapid and robust adaptive cell-mediated and humoral responses toward foreign insults. Ag immigration into the LN parenchyma is done in an organized fashion directed mainly by APCs within the
39 subcapsular sinus (SCS), medulla, and stromal cells that line the SCS and insulate the conduits in the LN. In 1965, Nossal and colleagues were the first to show that low molecular weight Ags could cross the vasculature of the LN capsule and contact immune cells within121. They subcutaneously injected 125I-labeled intact flagella into the hind footpads of rats and then isolated the DLNs for scintillation counting121. Two things pertaining to the LN follicles were observed. First, they observed a more rapid uptake of
125I-labeled flagella into LN follicles of rats that were previously immunized with unlabeled flagella121. Second, the 125I-labeled flagella began to appear in cortical LN follicles within 30 min after injections and were retained for a week afterward121. Nossal and colleagues attributed this appearance of 125I-labeled flagella in the follicles to what they called macrophage fibrils, which were also enhanced by opsonization of the 125I- labeled flagella bound by antibodies121. In 1975, Arthur Anderson first introduced the concept that LNs contained traversing conduits through which Ags can travel. He and colleagues showed that low molecular weight substances could reach high endothelial venules (HEVs) in the LN paracortex as soon as 1 min after injection122. Since then, studies of Ag transport within these conduits have blossomed, and they have revealed a complex filtering system in which specific immune cells, foreign- and self-Ags, and cytokines are shuffled, organized, and selected for entry into the LN parenchyma. Today, we identified these macrophage fibrils described by Nossal to be FDCs, which reside within B cell follicles. FDCs function by maintaining and organizing the B cell follicular zone123 and aiding in the development of B cell germinal centers97 through the release of cytokines and the presentation of unprocessed Ags to B cells in order to promote somatic hypermutation.
40 FDCs capture unprocessed, opsonized immune complexes (ICs)124,125, and free- form low molecular weight Ags [<70 kDa (5.5 nm)]126,127 for presentation to B cells through complement receptors 1 (CD21), 2 (CD35), and FcγRIIb128-130. FDC capture of large ICs come primarily through B cell-Ag transference (Fig. 2)131-133, which can, in theory, be retained for months to years134. However, the transport of small Ags from B cells to FDCs does not happen as efficiently as the transfer of larger ICs133. In 2009, one study provided direct evidence that small Ags accumulating in the B cell follicles gained access by gaps between the fibroreticular stromal cells (FSC) network that are prominent in LN conduits within the B cell follicular zones133. Adding to this, Bajénoff and Germain showed in 2009 that FDCs occupy a large portion of these gaps between FSCs, and FDCs are found to be in direct contact with LN conduits within the B cell follicles126. To date, it is still unclear whether FDCs can capture Ag from within LN conduits by direct sampling or whether these low molecular weight Ags diffuse into the follicles and are subsequently captured by FDCs134.
LN-RESIDENT AND MIGRATORY DCS INTERACT DIRECTLY WITH
LYMPHATIC CONDUITS AND HEVS
The concept that resident DCs in the DLN can directly sample Ags coming from the lymph fluid was first documented by Manickasingham and Caetano Reis e Sousa135 in
2000. They showed that before migratory DCs arrive at the DLNs, CD8α+/CD11c+ LN- resident DCs take up exogenous Ags delivered to the LN via the lymphatic fluid135,136. In
2003, Itano and colleagues showed that these resident DCs are primarily composed of
MHC-IIhigh/CD11c+/CD11b+/CD205int/CD8αlow/B220− interstitial DCs found in all LN
41 types from the afferent ducts and MHC-IIhigh/CD11c+/CD11b+/CD205high/CD8αint/B220− epidermal Langerhans DCs, which migrate exclusively from the skin to DLNs137. Sixt and colleagues provided direct evidence that immature resident myeloid (CD11b+/CD11c+)
DCs are found to reside embedded within the cell layer of the FSC network located within the LN parenchyma and can make direct contact with the LN conduits though the expression of β1 integrins and the binding of laminin-10 and fibronectin on conduit fibers96
(Fig. 2). They also showed these DCs to be distinct from immigrating mature DCs, which do not associate with LN conduits but instead associate predominantly near HEVs58,96, where they can be observed to exhibit TE behavior into the HEVs (Fig. 3; unpublished data). However, as in the case of FDC association with LN conduits, it is unclear how Ag is physically retrieved from the conduits by resident DCs, or whether such association licenses the DCs to exhibit TE behavior into the lymph fluid. Similarly, future studies are needed to establish immunological significance of trans-HEV dendritic extensions by DCs for immune cell recruitment or defense against blood-borne pathogens.
CD169+ MACS EXTEND PROCESSES FROM SUBCAPSULAR SINUS INTO LN
FOLLICULAR ZONES TO ASSIST IN ACTIVATING THE ADAPTIVE
HUMORAL RESPONSE
Resident LN Macs can be divided into three main subsets based on their LN anatomical locations: SCS Macs, medullary sinus Macs, and medullary cord Macs138. They originate from the bone marrow and can migrate to the LN under inflammatory and steady- state conditions by way of the afferent ducts139. The function of each Mac population depends on the cues received from the surrounding LN microenvironment. Macs migrate
42 to the LN by way of the afferent lymphatic duct; however, unlike DCs, they do not enter the LN parenchyma with the exception of a small subset of Macs132,138. While some Macs can transmigrate across the subcapsular floor into the LN follicles or the medullary cords, some Macs are embedded within the capsular linings of their respective areas and deliver
Ags across compartments to the LN parenchyma132,140,141. How exactly LN-bound Macs find themselves bound across the epithelial layers between the LN parenchyma and SCS remains to be fully explored, but evidence points toward chemokine signals, such as
Lymphotoxin alpha-1/beta-2 (LTα1β2), secreted by B cells and other stimulating factors found within the afferent lymphatic ducts and around the LN follicles138. Transluminal resident Macs can deliver both opsonized Ag in the form of ICs to non-cognate B cells by way of complement receptors 1 and 2 on the B cells for delivery to FDCs132,140 and processed Ags for delivery to DCs and T cells in the inter-follicular regions of the LN (Fig.
2)138. Therefore, intra-parenchymal Macs represent the first line of encounter against lymph-borne Ags and are capable of initiating the adaptive humoral immune responses.
LOCAL LN DCS EXTEND DENDRITIC PROCESSES INTO LYMPHATIC
FLUID TO INITIATE EARLY T CELL ACTIVATION
While Macs are observed to reach across the endothelial wall of the LN SCS and medullary sinus floors, migrating DCs have been implicated to induce structural changes of the SCS floor from within the afferent lymphatic duct to facilitate their transmigration across the SCS floor into LN parenchyma142,143. However, a recent study conducted by
Gerner and colleagues revealed that DCs not only can transmigrate across the SCS but can also become embedded in the medullary sinus as resident DCs, extending their processes
43 through intact LN sinus endothelium to sample and process Ags directly from the lymphatic fluid. In turn, this leads to the activation of T cells without the aid of migratory
DC from the periphery (Fig. 2 and 4)144. Using real-time in vivo analysis through a combination of histocytometry, 2PLSM, and 3D LN imaging, Gerner and colleagues identified a subpopulation of DCs that are CD169−/CD11b+/CD11c+ and that transverse the LN medullary sinus. Using >70 kDa labeled proteins (to ensure that they cannot passively diffuse into the LN conduits) and both live and attenuated bacteria, they provided real-time data to demonstrate that these LN-resident DCs can indeed take up the Ags directly. Taking this a step further, they immunized OT-I and OT-II containing animals with conjugated OVA proteins with 1 µm microspheres to show OT-I and OT-II cell clustering around OVA beads-engulfed DC within 8 h. Additional experiments were performed to show that resident, not migratory, DCs accounted for this DC population.
APC EXTENSIONS IN OTHER ANATOMICAL COMPARTMENTS: AIRWAY
LUMEN AND CNS VESSELS
Besides the gut mucosa and LN, DCs and Macs in other anatomical sites have been observed to exhibit transcompartmental reach across intact physical barriers (Fig. 5). In
2006, two separate groups showed evidence in tracheal tissue explant and sections that
CD103+ mucosal DCs expressed langerin and tight junction proteins that allowed them to migrate across the airway epithelia145 and extend processes into the airway lumen to sample
Ag145,146, which is consistent with findings of the same type of phenomenon found in the gut as discussed above. This was later corroborated by other studies using 2PLSM147-149.
This transluminal scanning behavior in the airway was shown to be directly tied to TLR4
44 signaling on the airway epithelial cells150. However, despite the evidence that mucosa DCs can extend these processes into the airway lumen, the biological significance and contribution of this particular finding remains controversial. Zoltán Veres and colleagues used CD11c-enhanced yellow fluorescent protein transgenic mice to demonstrate both Ag capture and subsequent migratory behavior of airway mucosa DC populations148. The movements of DCs were visualized in both ex vivo and in vivo preparations using 2PLSM.
They showed that these DCs are able to process Ag, migrate to DLNs, and present to T cells; however, despite the ability of Ags to cross the airway epithelium and be taken up by CD11c+ alveolar macrophages in the lung parenchyma, visual evidence for airway DC’s ability to directly capture Ag through transepithelial extensions into the airway was documented only twice in multiple imaging experiments148. The authors emphasize that these airway DC TE events were indeed very rare, making the functional relevance of these events in the airway difficult to discern148. In stark contrast to the gut microenvironment, the CNS represents a unique, immune-privileged site wherein the resident APCs consist of microglia and perivascular/meningeal Macs151,152. As an area of the body that is sealed off by the blood–brain barrier, the CNS has historically not been thought of as a significant site of homeostatic Ag surveillance. Recently, however, it has been shown by intravital
2PLSM in the uninflamed CNS that CX3CR1+ APCs are able to extend dendrites into vessels to directly sample for Ag in the bloodstream153. Interestingly, the number of these extensions per millimeter square of vessel wall significantly increases in the setting of
EAE, while the numbers remain the same in the settings of dorsal column crush injury and intracranial tumor. These results suggest that inflammatory signals can drive intraluminal extensions of CNS APCs, which could then prime adaptive immune responses in an
45 efficient in situ manner. The functional implication of this intravascular extension behavior by CNS resident CX3CR1+ APC remains to be fully elucidated.
CONCLUDING REMARKS
Antigen-presenting cells are essential coordinators of adaptive immunity. At various anatomical sites, such as mucosal, vascular, and lymphatic interphases, they function as critical sentinels, constantly sensing the tissue microenvironment for information to help determine host response. Emerging bodies of evidence suggest a different view of their in vivo behavior than previously thought. Rather than positioning at mucosal, vascular, and lymphatic interphases with another tissue compartment passively awaiting encounters with invading pathogens, intravital imaging data provide a highly dynamic view of these important immune cells actively reaching across intact and separate anatomical compartments to survey potential offending agents on the other side of the physical barrier. More studies are required in the future to fully understand the functional implication of this common observation of APCs for potential therapeutic exploitation of this intriguing in vivo behavior.
46 Figure 1
Figure 1. Different models of TE and antigen uptake by LP APC
LP APCs adjacent to the villus epithelia extend finger-like projections or balloon bodies
(BB) directly into the gut lumen to sample microbes. Both CD11c+CX3CR1+CD103- Macs and CD11c+CX3CR1-CD103+ DCs have been shown to push TEs into intestinal lumen to directly interact with pathogens. CD11c+MHC-II+ DCs have been visualized to extend BBs into the lumen in some explant preparations. Tight junction proteins expressed by LP APCs
47 allow preservation of villus epithelia organization. CD103+ DCs closer to the villus core may receive Ag from goblet cells and/or CX3CR1+ Macs, or may directly sample translocated pathogens/Ags themselves before migrating to the draining mesenteric LN.
CD11c+CX3CR1+MHC-II+CD11b- phagocytes have also been shown to uni-directionally translocate into the lumen to participate in pathogen exclusion.
48 Figure 2
Figure 2. Overview of LN APC positioning for TE sampling
A cartoon of the LN depicting the various APC extensions into or in direct contact with the subscapular sinus, medullary sinus, B cell zone LN conduits, high endothelial venules, and paracortical zone LN conduits (adapted from Girard et al.43).
49 Figure 3
Figure 3. Activated DCs reside near HEV after migrating from the periphery
Single 0.5 µm optical section from an intravital 2PLSM experiment showing the peri- vascular juxtaposition of migrating DC (blue) from the periphery and the high endothelial venule (HEV; light green) in the LN. Newly i.v. injected T cells (deep bright green) can be seen attaching to the luminal wall as well as migrating in the LN parenchyma through the
HEV structure (A.Y.H., unpublished result).
50 Figure 4
Figure 4. LN DCs and Macs extend processes into the medullary sinus to directly sample lymph-borne antigen
An expanded cartoon of the LN medullary sinus from Fig. 2, depicting DCs, subcapsular and medullary sinus macrophages embedded into the medullary sinus floor and contacting naïve T cells (adapted from Gerner et al.50).
51
Figure 5
Figure 5. Lung DCs sample airway pathogens via TEs
A cartoon of the airway leading into the alveolar space, showing alveolar tissue-resident
DCs extending their processes into the air canal between tight junctions of the alveolar epithelial cells to contact pathogens under steady-state conditions (adapted from Hammad et al.60).
52 CHAPTER III
CCL3 enhances antitumor immune priming in the lymph node via IFN� with
dependency on NK cells
Frederick Allen1, Peter Rauhe2, David Askew2, Alexander A. Tong1, Joseph Nthale2,
Saada Eid2, Jay Myers2, Caryn Tong2, Alex Y. Huang1,2,3,4*
1Department of Pathology, Case Western Reserve University School of Medicine,
Cleveland, OH 44106 USA
2Department of Pediatrics, Case Western Reserve University School of Medicine,
Cleveland, OH 44106 USA
3Angie Fowler AYA Cancer Institute, UH Rainbow Babies & Children’s Hospital,
Cleveland, OH 44106 USA
4Case Comprehensive Cancer Center, Cleveland, OH 44106 USA
Keywords: CCL3, natural killer cells, CD103+ dendritic cells, Lymphocytes, interferon- gamma, Lymph node
53 ABSTRACT
LNs play a critical role in tumor cell survival outside of the primary tumor sites and dictate overall clinical response in many tumor types154,155. Previously, we and others have demonstrated that CCL3 plays an essential role in orchestrating T cell – APC encounters in the draining LN following vaccination, and such interactions enhance the magnitude of the memory T cell pool9,30,156. In the current study, we investigate the cellular responses in the tumor draining LNs (TDLN) of a CCL3-secreting CT26 colon tumor (L3TU) as compared to wild-type tumor (WTTU) during the priming phase of an antitumor response
(≤10-days). In comparison to WTTU, inoculation of L3TU resulted in suppressed tumor growth, a phenomenon that is accompanied by altered in vivo inflammatory responses on several fronts. Autologous CCL3 (aCCL3) secretion by L3TU bolstered the recruitment of
T- and B-lymphocytes, tissue-migratory CD103+ DCs, and CD49b+ NK cells, resulting in significant increases in the differentiation and activation of multiple Interferon-gamma
(IFN�)-producing leukocytes in the TDLN. During this early phase of immune priming,
NK cells constitute the major producers of IFN� in the TDLN. CCL3 also enhances CD8+
T cell proliferation and differentiation by augmenting DC capacity to drive T cell activation in the TDLN. Our results revealed that CCL3-dependent IFN� production and CCL3- induced DC maturation drive the priming of effective antitumor immunity in the TDLN.
54 INTRODUCTION
Immunogenic tumors are capable of triggering robust antitumor immune responses.
However, intrinsic and extrinsic factors such as tumor cell growth rates and immune- suppressing factors protect tumors from effective immune elimination18. The balance between immune-mediated tumor suppression and evasion depends on many factors including the timing, robustness, and types of responding immune cells within specific contexts of the local tissue microenvironments. Secondary lymphoid organs such as LNs play an important role in the development of these mediating factors. LNs are specialized sentinel stations designed to promote timely cellular interactions, disseminate antigenic information, and formulate adaptive responses that help to maintain tissue homeostasis.
The importance of LNs in the spread of cancer is evident in the clinical environment where studies have reported that 80% of all tumor metastasis occur though LNs and that LN metastasis correlates negatively with clinical outcomes157. Despite the important role LNs play in tumor progression, many of the current mechanistic insights of how immune cells respond in a tumor microenvironment (TME) come primarily from interrogations of cellular events that occur in the primary tumor sites. Studying immune interactions in the
TDLN, not just in the primary tumor site, can provide important clues to local and global immune responses toward disseminating tumors158,159.
Chemokine-based immunotherapy has been studied as a means to modulate and bolster the development of anti-tumor immunity12,160. Chemokines are cytokines that function primarily as chemoattractants and help maintain specific immune microenvironments by coordinating various immune cell-cell interactions in a specific
55 spatio-temporal manner. For example, we and others have demonstrated a crucial role for
CCL3 and CCL4 in maximizing chance encounters between naïve CCR5+ CD8+ T cells and DCs that undergo productive interactions with antigen (Ag)-specific CD4+ or CD8+ T cells in the LN draining vaccine sites 9,30,156. Furthermore, CCL3 has been shown to be required for maximal helper CD4+ T cell-dependent memory CD8+ T cell generation in the
DLN during the initial T cell priming phase9. In the current study, we examined whether the chemoattractant and cytokine functions of CCL3 in the TDLN could be leveraged to improve immune responses to a murine colon cancer model by engineering tumors to secrete CCL3.
The cognate receptors for CCL3 include CCR5 and CCR1 in both mice and humans, and CCR3 in humans alone32. Despite the differences of CCL3 isoforms and receptor binding between mice and humans, both species-specific CCL3 isoforms have been shown to eliminate tumors in mouse models31. We hypothesize that the continuous presence of autologous tumor-derived CCL3 (aCCL3) in the TME will lead to the generation of a greater antitumor cellular response in the TDLN. Here, we employed CT26, a highly immunogenic murine colon tumor derived from Balb/c mice and expresses the immune-dominant Ag, AH-1, presented on the H2-Ld haplotype to CD8+ T cells161,162. We transfected wild-type (WTTU) CT26 to stably secrete CCL3 (L3TU) and examine early cellular immigration of leukocytes to the TDLN. We show that CCL3 helps to maximize inflammatory responses in the TDLN in two parallel steps. Firstly, we show that CCL3 increases the chance encounters of Ag-specific T cells with professional Ag-presenting cells (pAPCs) though the enhanced recruitment of CD103+ CD11c+ DCs and T cells in the
56 TDLN. Secondly, CCL3 simultaneously enhances the global pool of interferon-gamma
(IFN�) in TDLN primarily through the mobilization of IFN�-producing natural NK cells.
Furthermore, we show that DCs exposed to recombinant CCL3 (rCCL3) exhibit enhanced
Ag-presentation capacity to drive greater CD8+ T cell proliferation in vitro, thus demonstrating an alternative biological function of CCL3 from its classic cellular recruitment function to influence DC maturation.
MATERIALS AND METHODS
Mice
Mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or Taconic.
We used 8- to 12-week-old male and female wild-type Balb/c, C57/BL6J, or OT-I TCR transgenic mice on the RAG-1 knockout background. All animals were housed and handled according to National Institutes of Health institutional guidelines under an approved protocol by Case Western Reserve University Institutional Animal Care and Use
Committee (No. 2012-0126 and 2015-0118).
Tumor and LN Measurements
For tumor measurements, mice were injected s.c. with 1x106 of each tumor construct alone or mixed at 1:1 ratio (5x105 each) in the left flank, and tumors were measured twice weekly. Tumor growth was measured using electronic calipers. The formula, V = � x D x d2, was used to calculate tumor volumes 163, where “D” is the largest diameter and “d” is the smallest diameter. Mice were sacrificed between days 21-50, depending on the experimental setup.
57
For gross LN measurements, mice received s.c. footpad injections of tumor cells
(1x106), and the popliteal TDLN and NDLN were removed on days 5 or 7. High-resolution pictures were taken of TDLN and NDLN from each group (NI, WTTU, L3TU,
WTTU+�Asialo-GM1, or L3TU+�Asialo-GM1) prior to FACS analysis. The large and small diameters of the LNs were measured in Adobe Illustrator software. The formula, V
= � x D x d2, was used to calculate LN volumes163. In order to normalize measurements from different photo sessions, each experiment was divided by the average of the WTTU group within the same experiment, and the relative fold-change was calculated.
Flow Cytometry Analysis
Antibodies were purchased from eBioscience, BD Pharmingen, or BioLegend and are as follows: rat anti-mouse CD4 FITC and PE (GK1.5), APC (RM4-5); CD8a FITC, PE, and APC (53-6.7); CD19 FITC, PE, and APC (1D3); CD11c FITC, PE, and APC (N418);
CD49b PE and APC (DX5); CD3 PE and APC (145-2C11); CD103 FITC (2E7); PD-L1
PE and APC (10F.9G2); CD69 PE and APC (H1.2F3); and IFN� APC (XMG1.2). Mice received s.c. footpad injections of either tumor constructs and the popliteal TDLN and
NDLNs were removed 1, 3, 5, 7, or 10 days later. LNs were made into single-cell suspensions with ice-cold FACS buffer (0.5% FBS and 0.5% EDTA in 1x sterile PBS).
For surface staining, unlabeled rat anti-mouse blocking Fc antibody was applied for 30 minutes on ice followed by primary antibody staining for 30 minutes on ice and protected from light. Cell viability test was conducted using 7-AAD (Biolegend), which was added
20 minutes into the primary staining. The samples were then washed twice with ice-cold
58 FACS buffer and analyzed on an Accuri C6 flow cytometer. For intracellular IFN� staining, cells were plated with 1 µl / ml of GlogiStop (eBioscience) for 6-hours on a 96 well plate pre-coated with unlabeled anti-CD3 at 1 µg / ml for a total of 90 minutes at 37oc prior to membrane permeabilization with Cytofix/Cytoperm (BD Biosciences), followed by staining with anti-IFN� antibody. Analysis was performed using Accuri C6 and FlowJo software.
NK-depletion and CCL3 blocking
For NK cell deletion, mice received intraperitoneal (I.P.) injections of 50 µg of
�Asialo-GM1 (Poly21460) in 100 µl of 1x PBS. One day later, mice received s.c. footpad injections of 1x106 tumor cells. 5 days later, the popliteal TDLN and NDLN were removed for FACS analysis. For CCL3 neutralization, mice received I.P. injections of anti-CCL3 antibodies (50 µg / mouse; R&D System) concurrently with 1x106 WTTU or L3TU. Anti-
CCL3 antibody injection was repeated again 48 hours later. The popliteal TDLN and
NDLN were removed 5 days later for FACS analysis.
ELISA
o WTTU or L3TU were incubated for 24 hours at 37 C in 95/5% O2/CO2 in 1 ml of complete media (RPMI 1640 with 10% fetal bovine serum, 1% HEPES, 1% non-essential amino acids, and 1% streptomycin). The spent media from in vitro cultures or serum samples obtained from tumor-bearing mice were quantified for CCL3 protein contents by
ELISA in accordance to the manufacture’s protocol (R&D systems, MMA00).
59 CT26 transfection
CT26 tumor cells were stably transfected with a PCDNA3.1 plasmid vector that contains the mouse CCL3 cDNA and maintained under Hygromycin (150 µg / ml) selection.
OT-I proliferation assay
Bone marrows from C57BL6 mice were isolated and BMDCs were generated in complete media at 3x106 cells / 3 ml / well in 6 well tissue culture plates supplemented with 15 ng / ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng / ml Interleukin-4 (IL-4) on days 0, 3, and 5. On day 3, the media was removed and fresh media plus cytokines were added at 3 ml / well. On day 5, the cultures were replaced with fresh media plus cytokines. On day 7, non-adherent immature BMDCs were collected, washed with complete media and plated in a 6-well tissue culture plate with or without 100 ng / ml of CCL3 for 24 hours. BMDCs were then collected and pulsed at 37OC in 95/5%
O2/CO2 with various doses of SIINFEKL-peptide for 1 hour, washed and cultured in triplicates with 100,000 CFSE-labeled (1 uM) naïve OT-I cells (at DC-to-OT-I ratio of 1:5)
O at 37 C in 95/5% O2/CO2 for 72 hours. The percent of CFSE-dilution peaks, relative to non-pulsed and cultured BMDCs and OT-I cells, was calculated using FACS.
Quantitative RT-PCR analysis
Total LN mRNA was isolated using TRIzol reagent in accordance with the manufacturer’s protocol (Gibco BRL, Carlsbad, CA) and purified using an IllustraTM
RNAspin Mini Kit (GE Healthcare Life Sciences). RNA quality was assessed by
60 spectrophotometer absorption at 260/280 nm using the NanoDrop2000 spectrophotometer.
RNA was converted to cDNA using EasyScriptTM Reverse Transcriptase protocol consisting of 200 U/µl Moloney murine leukemia virus reverse transcriptase incubated for
60-minutes at 42OC in the presence of 50mM Tris-HCl (pH 8.3), 100 mM NaCl, 0.1 mM
EDTA, 5 mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol, 10 uM of oligo (dT), 10 mM
29-deoxynucleoside 59-triphosphate, and 40 U/ul recombinant RNase inhibitor (Lamda
BIOTECH, St. Louis, MO). cDNA was amplified in the presence of FAM-labeled gene- specific primers and Bullseye EvaGreen qPCR Mastermix (MIDSCITM; Saint Louis, MO) in a 96 well microtiter plate using the ABI Prism 7300 sequence detection system (Applied
Biosystems). Each PCR reaction was performed in triplicate and compared to WTTU.
Relative levels of mRNA were determined using the cycle threshold (Ct). The gene expression was standardized according to cytochrome-c (CyC) expression within the
TDLN. In order to compare the Ct values between target genes we normalized each Ct to
-(Target gene – CyC - target normalizer) the average of the WTTU Ct using the following equation: 2^ .
Statistical analysis
Significance analyses were performed using the standard t-test. Figures 6 and 7 were displayed as standard error of mean for ease of visualization. Standard deviations were shown for other figures.
RESULTS
aCCL3 suppresses tumor growth and promotes tumor rejection
61 We first constructed L3TU by stably transfecting WTTU CT26 with plasmids containing murine CCL3. No significant differences in the growth kinetics between WTTU and L3TU were observed in vitro (Figure 12A). Enzyme-linked immunosorbent assay
(ELISA) analysis revealed that L3TU produced ~350 pg / ml of CCL3 per 1x106 tumor cells in vitro over 24 hours, whereas WTTU failed to secrete any detectable CCL3 (Figure
12B). Serum obtained from mice 7 days after 1x106 L3TU inoculation contained ~150 pg
/ ml CCL3, while serum from non-injected (NI) and WTTU-injected mice had negligible
(<8 pg / ml) CCL3 (Figure 12C). Both L3TU and WTTU expressed similar surface
Programmed Death-Ligand 1 (PD-L1) at baseline and following IFN� stimulation in vitro
(Figure 12D-E). Next, we measured the tumor growth behavior in naïve Balb/c mice inoculated subcutaneously (s.c.) with 1x106 WTTU, L3TU, or WTTU + L3TU mixture at
1:1 ratio (5x105 each) in the flank. While WTTU grew aggressively in 100% of the recipient mice and became measureable by day 7 with an average tumor volume of ~12,000 mm3 after 3 weeks, 30% of mice in L3TU group and 20% of mice in the WTTU + L3TU group completely rejected the tumors with the remaining clinically evident tumor sizes being 25 and 6 fold smaller than WTTU, respectively (Fig. 6A-B). A 20-fold reduction in the L3TU inoculum (5x105 cells) resulted in a 40% tumor-free incidence (Fig. 6C-D). As expected, mice that rejected primary L3TU tumors were capable of rejecting subsequent challenge with a lethal dose of WTTU, demonstrating the successful generation of anti- tumor immune memory (data not shown).
aCCL3 augments T cell activation by enhancing leukocyte migration to the TDLN
62 Studies by Gretz and colleagues showed that recombinant CCL3 (rCCL3) administered s.c. in the footpad could readily drain though the afferent lymphatic ducts to associate within the high endothelial venules (HEVs)164. Therefore, we examine how
CCL3 produced by L3TU influence cellular traffic to the tumor draining LN (TDLN) during the early phase (≤10 days) of immune priming following tumor inoculation. We examined the TDLN cellularity of CD4+ T cell, CD8+ T cells, CD19+ B cells, CD11c+ pAPCs, and CD49b+ cells. Both WTTU and L3TU inoculation led to significant increases in CD4+, CD8+, CD19+, CD11c+, and CD49b+ leukocytes in the TDLN compared to NI group (Fig. 7). However, TDLNs draining L3TU showed 2 to 6 fold increases in total leukocyte sub-populations compared to WTTU TDLNs, with the enhancement of leukocyte accumulation reaching statistical significance after 3 days and peaking on days
5 and 7 (Fig. 7A-E). These changes were accompanied by gross anatomical differences in
LN sizes (Figure 13). These striking changes were not caused solely by CCL3 alone, as mice receiving direct s.c. injections of rCCL3 showed only transient (<2 days) changes in leukocyte accumulation in TDLN that were similar to PBS controls (Fig. 7H). With the exception of CD4+ T cells, leukocyte numbers in both WTTU and L3TU TDLN returned to NI levels by day 10. Next, we interrogated whether the significant increases in T cell numbers correlated with changes in the T cell activation status. Mice were injected similarly as above, and both TDLN and contralateral non-draining lymph nodes (NDLNs) were removed on days 1, 3, 5, and 7 for CD69 expression assessment by flow cytometry
(Fig. 7F-G). In both CD4+ and CD8+ T cell subsets, we detected greater numbers of CD69+
T cells in L3TU TDLN. The number of CD69+ T cells began to dissipate after day 5.
Interestingly, we also observed a significant and reproducible increase in leukocyte
63 accumulation in the NDLNs on day 5, suggesting a systemic effect of CCL3 on immune cell mobilization and trafficking (Figure 14). However, this effect was transient and quickly dissipated after day 5, and the transient cellularity increase was not accompanied by CD69+ T cell activation (Figure 14F-G), suggesting a requirement for the presence of tumor cells to sustain the accumulation of CD69+ T cell subset in TDLN. Despite differences in absolute cellularity of TDLN and NDLNs between WTTU and L3TU, the overall cellular compositions were similar between the two tumors. Normally, CD4+ T cells predominates amongst LN leukocytes in the naive mouse; however, B cells became the most prominent population proportionally within TDLN 3 days after tumor injections, and the trend continued until after day 7 when the relative composition returned to baseline
(Figure 15). The rate of composition reversal by day 10 in TDLN and NDLN between
CD4+ T cells and B cells appeared be more dramatic in L3TU compared to WTTU.
aCCL3 bolsters the intracellular production of IFN�+ cells in the TDLN
Next, we asked whether the enhanced number of activated CD69+ T cells would correlate with an anti-tumorigenic milieu in TDLN. To address this, following tumor inoculation we measured global cytokine mRNA differences, including TGF�, TNF�, IL-
10 and IFN�, between WTTU and L3TU groups on day 5, the time point where we observed the greatest differences in cellular accumulation and CD69 positivity. While the average transcript levels of TGF�, TNF�, and IL-10 in TDLN were similar between L3TU and WTTU, TDLN in L3TU contained a ~2.5-fold greater IFN� mRNA transcript level as compared to that in WTTU (Figure 16). We then examined intracellular IFN� content among the cells found within TDLN on day 5. With the exception of CD8+ T cells, all other
64 cell-types analyzed contained significantly elevated numbers of cells expressing intracellular IFN� in L3TU TDLN (Fig. 8). This finding was further confirmed by
ELISPOT analysis (data not shown).
L3TU enhanced leukocyte accumulation is dependent on CCL3 but not NK cells
By day 5, CD49b+ cells not only make up the cell-type with the most significant difference between WTTU and L3TU, but they also constituted the most statistically significant IFN�+ population differences (Fig. 7E, 8F). CD49b (VLA-2) is an integrin protein expressed on NK cells, and a subpopulation of NK T (NKT), CD4+ and CD8+ cells165,166. NK, NKT, and CD8+ cells have been associated with tumor rejection and IFN� upregulation after stimulation167-169. Recently, CD49b+ CD4+ cells were defined as a subpopulation of regulatory T cells that produced IL-10 in response to stimulation rather than IFN�170. We hypothesized that the majority of the global IFN� production within
TDLN on day 5 is derived from activated CD49b+ CD3- NK cells, which express CCR5 and could potentially be recruited directly by CCL3 to the TDLN169. Under homeostatic conditions, NK cells represent a small population (≤1%) in the LN but can accumulate and supply significant IFN� to drive T cell activation and differentiation upon stimulation
(Figure 17)171. We examined the dependence of cellular accumulation in the L3TU TDLN on CCL3 and NK cells by administering anti-CCL3-neutralizing monoclonal Abs (mAbs) or NK-depleting �Asialo-GM1 antibody prior to footpad inoculation with WTTU or L3TU
(Figure 17). We then examined the cellularity of TDLN and NDLN on day 5 as previously described (Fig. 9). Blocking CCL3 resulted in diminished cellular accumulation in both
TDLN and NDLN (Fig. 9 and data not shown). While NK cell depletion over the course
65 of 5 days was associated with a decrease in global IFN� expression in TDLN (Figure 16), however, counter to expectation, NK cell depletion also potentiated the enhanced cellular accumulation observed with aCCL3 (Fig. 9).
NK cells, but not CD103+ CD11c+ DCs are important for driving the production of
IFN�-induced chemokines CXCL9 and CXCL10 in the TDLN
IFN� expression at primary tumor sites can drive DC maturation and rapid migration to DLN for Ag presentation172. IFN� has also been shown to induce the production of CXCL9 and CXCL10, which can aid in tumor rejection through the recruitment of activated CD8+ T cells and IFN�-secreting Th1 cells to the primary tumor sites173-175. Recently, studies have shown that CD103+ CD11c+ DCs, a subpopulation of dermal- and gut-resident pAPCs, respond to tumor-derived CCL4 and are the chief cells that produce CXCL9 and CXCL10 to recruit tumor-infiltrating T cells176,177. Indeed, we observed an accumulation of CD103+ CD11c+ DCs in L3TU TDLN, and such enhanced accumulation was dependent on CCL3, not NK cells or associated IFN� (Fig. 10A-B;
Figure 16). However, we show that TDLN NK cells are crucial for CXCL9 and CXCL10 production on day 5 (Fig. 10C-D), as NK depletion resulted in a dramatic decrease in the mRNA transcripts of both chemokines.
Exposure of BMDCs to rCCL3 enhances Ag-specific T cell proliferation
Finally, we examined the immune-modulatory effects of CCL3 on pAPC function.
Several reports have observed the direct modulatory effect of CCL3 on pAPC function.
Watanabe and colleagues showed that macrophages pretreated with CCL3 exhibit
66 strengthened adhesion to osteoblasts leading to the formation of pre-osteoclast cells in vitro, an important step in the process of bone reabsorption178. Previously, we showed that blocking CCL3 (and its paralog CCL4) in vivo decreased CD8+ T cell and DC contacts in the vaccine-draining LN and diminishes the magnitude of the overall CD8+ T cell memory pool156. In two separate studies, Jaehyung and colleagues showed that pretreatment of DCs with CCL3 in combination with CCL19 and LPS stimulation led to enhanced OVA- specific CD4+ (OT-II) T cell proliferation in vitro. To address a potential functional modulatory role of CCL3 on pAPC, we pretreated bone marrow-derived DCs (BMDCs) from C57BL/6 mice with rCCL3 for 24 hours, then co-cultured them with OT-I T cells for
3 days with and without Ags in vitro. CCL3-conditioned BMDC displayed a significantly enhanced capacity to drive OT-I proliferation when pulsed with SIINFEKL peptides at doses of 0.1 to 10 µg / ml (Fig. 11A). Interestingly, CCL3-conditioned BMDC also exhibited enhanced cross-presentation capacity to drive OT-I proliferation when cultured with whole OVA-coupled beads, especially at low Ag doses of 0.1 to 1 ng / ml (Fig. 11B).
DISCUSSION
Recent studies in melanoma implicate that tumor cells modulate intra-tumoral T cell density in part by regulating inflammatory chemokine productions in the TME via a
�-catenin-dependent mechanism176,179. In particular, Spranger et al. showed melanoma tumor cells that harbor genetic alterations in the �-catenin pathway could up-regulate inflammatory chemokine, CCL4, which attracts dermal-resident CD103+ DCs. The
CD103+ DCs elevated CXCL9 and CXCL10 in order to further attract T cells to infiltrate the tumor, and deletion of CCL4 in the melanoma cells abrogated T cell infiltration176.
67 Although these studies were primarily focused on CCL4, the production of CCL3 was also significantly increased in their tumor system176. Comparable to these findings, our CT26 colon tumor model does not produce detectable amount of CCL3. Enhancing the production of CCL3 in CT26 by genetic manipulation resulted in significant slowing and ultimate eradication of tumors in a significant fraction of naïve recipient mice. The exact cellular mechanism mediating primary L3TU rejection is the subject of an ongoing parallel study. In the current study, we aimed to distinguish the effect of CCL3 on early anti-tumor immune priming in the LN from the later adaptive immune responses at the primary tumor site. We did this by assessing CCL3’s effect on global immune cell trafficking and inflammatory changes in the TDLN. Indeed, we observed an increased cellularity in overall
CD11c+ and CD103+ subpopulation of DCs in the TDLN in both WTTU and L3TU as compared to that in NI mice. While there was a slight enhancement of the DC populations in the WTTU TDLN, the magnitude of total and CD103+ subset of CD11c+ DC accumulation was 2.7-fold and 2-fold less than the accumulation in the L3TU TDLN, respectively (Fig. 10A, B). CCL3 was responsible for the increased accumulation of total and CD103+ subset of CD11c+ cells in L3TU TDLN, as the administration of anti-CCL3 neutralizing antibody abrogated this local LN accumulation (Fig. 10A, B). Similar to the observation reported by Spranger et al., we measured a significant increase in the production of CXCL9 and CXCL10 in L3TU TDLN. However, the production of CXCL9 and CXCL10 was disrupted in the L3TU group following NK cell depletion (Fig. 10C, D) in conjunction with IFN� suggesting that IFN� plays an important role for promoting
CXCL9 and CXCL10 contents in L3TU TDLN. Additional experiments will be required to elucidate this further.
68
The presence of IFN�-producing cells in the primary tumor mass is inversely correlated with tumor growth180. Early presence of IFN� in the TME favors development of activated DCs and T cells with an inflammatory phenotype. Both CCL3 and IFN� are implicated in endowing DCs the ability to polarize towards the establishment of type-1 inflammatory responses in T cells, CD8+ T cell proliferation, and immune memory generation9,173,181-184. Interestingly, while a greater number of CD8+ T cells expressed
CD69 in the L3TU groups on day 5, IFN� production by these CD8+ T cells was not significantly different than those found in WTTU TDLN or NI LN, suggesting that the full activation and effector function acquisition of CD8+ T cells in L3TU TDLN occurred at a later time-point beyond day 5 (Fig. 7G, 3C). An unexpected finding was that, compared the WTTU TDLN and NI LN, only L3TU TDLN contained significantly elevated IFN�+
B cells on day 5. A study by Bao et al. showed that a subpopulation of innate secondary- lymphoid-resident B cells could drive the activation of macrophages through IFN�185.
These IFN�-expressing innate B cells were shown to accumulate in secondary lymphoid organs as early as 3 days following bacterial or LPS challenge, similar to our current observation with aCCL3185. These IFN�+ B cells were the second most abundant early source of IFN�+ cells in the L3TU TDLN (Fig. 8D)186, suggesting that CCL3 may contribute directly or indirectly to the development or accumulation of this particular B cell subset. The significance of these IFN�+ B cells in the antitumor responses remains to be fully explored.
69 In the present study, CD49b+ NK cells represented the most significant source of
IFN� in the L3TU on day 5, as depletion of this immune subset diminished the global IFN� production in the TDLN (Figure 16). Previous reports showed that the major source of
CXCL9 and CXCL10 within the LN comes from LN-endothelial cells and DCs, respectively187. NK cells are an important source of early IFN� for LN DCs, and they are recruited to the DLNs by mature DCs in a CCR7-independent and CXCR3-dependent manner though DC production of CXCL10173. Therefore, it was not surprising to observe a significant decrease in IFN� production following NK depletion. We expected that the presence of IFN�+ NK cells might synergistically increase the production of CXCL9 and
CXCL10 in the TDLN. Indeed, depleting NK cells as a major provider of IFN� resulted in a global decrease of CXCL9 and CXCL10, despite the apparent CCL3-driven increases in
CD103+ CD11c+ DCs to the TDLN (Fig. 10). A surprising observation in our study was the observed additional enrichment in the overall cellular recruitment in the L3TU group after NK depletion (Figs. 9, 10). However, two separate studies have reported similar phenomenon with neutrophil recruitment to the DLNs of immunized mice188,189. CCL3 has also been shown to contribute to tissue-homing of neutrophils during microbial infections190. In addition, CCL3 expression can be suppressed by IFN� to auto-regulate inflammatory responses in tissues191. In our system where CCL3 is continuous elevated in
L3TU (Figure 12C), it is plausible that the depletion of NK cells - the major source of IFN�
- may further enhance the recruitment capacity of CCL3 in L3TU TDLN by removing the
IFN�-mediated suppressive mechanism on local LN immune cell populations. The exact mechanisms and the associated immune effector function in NK-depleted L3TU TDLN remains to be explored.
70
We show that s.c. administration of PBS alone can briefly enhance leukocyte migration patterns temporarily and locally through transient increases in the interstitial pressure in the DLN. Ultimately, however, the DLN responds quickly by returning to homeostasis within 24-48 hours (Fig. 7). The s.c. administration of rCCL3 alone was also not enough to induce a sustained increase in leukocyte traffic to the LN (Fig. 7). This observation agrees with published literature showing that CCL3 could drive T cell emigration from peripheral blood to tissues only under the influence of immunogens such as dinitrofluorobenzene192. Our data suggest that factors derived from tumor cells are also critical for CCL3-induced TDLN accumulation of leukocytes.
While enhancing T cell recruitment can increase the efficiency of their scanning of potential cognate Ag on DCs, delivery of relevant Ag to TDLN is just as vital for eliciting robust T cell responses. Cytokines such as CCL3 can begin to affect cellular responses within the TDLN early in this process before metastasizing tumors or skin migratory DCs could directly influence the immune responses186. The current dogma dictates that DC migration to the DLN from tissues occurs in a CCR7-dependent manner. Interestingly, however, we show that DC migration to the TDLN is enhanced by aCCL3 (Fig. 10)176,193.
Furthermore, aCCL3 modulates DC’s functional ability to induce T cell proliferation in vitro. Jaehyung and colleagues showed that CCL3-exposed DCs enhance OT-II proliferative capabilities in an Ag-specific manner, but only after co-stimulation with
CCL19 and LPS194, which maintain exposed DCs in a semi-mature state to allow for greater
Ag uptake and subsequent loading onto MHC molecules for presentation to T cells.
71 Yanagawa and Onoe observed that short-term (1 hour) exposure of DCs to CCL3 could directly activate the endocytotic pathway in immature DCs, suggesting that the initial uptake of Ag by DCs could be enhanced in the short term195. However, we failed to detect any augmentation of MHC, CD40, CD80 or CD86 expression on the surface of DCs after
24 hours of exposure to CCL3 alone (data not shown) as an explanation for the observed enhanced T cell proliferation. Furthermore, we also did not detect any significant changes in intracellular fluorescence intensity when we exposed 24-hour CCL3-cultured DCs to fluorescently labeled Latex-OVA beads to detect differences in Ag uptake (data not shown). While we cannot account for the enhancement in Ag-uptake with short-term exposure of DCs to CCL3, our data suggest that prolonged exposure of DCs to CCL3 may facilitate enhanced processing rather than uptake of Ag.
Taken together, our data implicate a direct immune modulating effect of CCL3 in the TDLN through the accumulation of IFN� NK cells and CD103+ DCs, enhanced production of CXCL9 and CXCL10, improved Ag presentation and stimulation capacity of DCs, and improved T and B lymphocytes activation. Our current data further support the exploration of CCL3 as an adjuvant for enhancing antitumor immune response.
72 Figure 6
Figure 6. Autologous or recombinant CCL3 slows tumor growth and promotes tumor rejection
A) Tumor growth kinetics of 1x106 tumor cells injected s.c. of WTTU (n=10), L3TU
(n=10), or WTTU+L3TU mixed at a ratio of 1:1 (n=5). Experiments were repeated 3 times.
B) Kaplan-Meier graph showing overall tumor incidence of A. C) Tumor growth kinetics of WTTU (n=10) and L3TU (n=10) after s.c. injections of 5x105 tumor cells. D) Kaplan-
Meier graph showing overall tumor incidence of C. Not significant (ns), p > 0.05; *, p =
0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p < 0.0001.
73 Figure 7
Figure 7. aCCL3 expression augments T cell activation by enhancing leukocyte migration to the TDLN
A-E) Cellular accumulations of specific immune subtypes in the TDLN are shown over the course of 10 days following tumor inoculation. F-G) Upregulation of the early activation marker, CD69, on T cells over the first 7 days following tumor inoculation. H) Total cellularity changes in the popliteal LN of naive mice in the first 48 hours following a footpad injection of recombinant CCL3 (rCCL3; 100ng / mouse). For A-G, N=3 to 7 mice for each day. Experiments were repeated 2-3 times for each data point. H represents 2 biological repeats with N=2 mice in each time point. Not significant (ns), p > 0.05; *, p =
0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p < 0.0001.
74 Figure 8
Figure 8. aCCL3 enhances the accumulation of IFN�+ cells in the TDLN
Absolute numbers of IFN�+ immune cell subsets in the TDLN on day 5 following tumor inoculations were measured by FACS. N=4 to 10 mice per group with 2 repeats. Not significant (ns), p > 0.05; *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to
0.001; ****, p < 0.0001.
75 Figure 9
Figure 9. The enhanced accumulation of leukocyte subsets in the L3TU TDLN is negated by blocking CCL3, but enhanced by NK depletion
A-C) Absolute numbers of immune cell subsets in the TDLN 5 days post-tumor injection of mice with and without the anti-CCL3 blocking Ab or NK cell (anti-Asialo-GM1) depletion Ab. N=3 mice per cohort. NI (circles), WTTU (squares), and L3TU (triangles) cohorts were conducted concurrently along with the CCL3-blocking and NK depletion cohorts. IgG1a antibody was administered to WTTU, and L3TU cohorts as controls. Not significant (ns), p > 0.05; *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to
0.001; ****, p < 0.0001.
76 Figure 10
Figure 10. NK cells, not CD103+ CD11c+ DCs, drive IFN�-induced CXCL9 and
CXCL10 production in the L3TU TDLN
A-B) Absolute total and CD103+ subset of CD11c+ cell numbers in the TDLN 5-days post- tumor injections were numerated by FACS. N=3 mice per cohort. NI, WTTU, and L3TU injection groups were conducted simultaneously with each depletion experiment and combined into the present graphs. IgG1a antibody was administered to NI, WTTU, and
L3TU cohorts as for CCL3 blockade or NK depletion. C-D) Fold change of CXCL9 and
CXCL10 mRNA expression in the L3TU TDLN compared to that of WTTU group. LNs
77 from 2-3 mice in the NI group were pooled for analysis due to low mRNA content. Not significant (ns), p > 0.05; *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to
0.001; ****, p < 0.0001.
78 Figure 11
Figure 11. Pretreatment with rCCL3 enhances the capacity of BMDCs to drive OT-
I proliferation in vitro
A) Day 7 BMDCs were cultured for 24 hours in the presence or absence of CCL3 (100 ng
/ ml) or LPS (100 ng / ml), then washed and pulsed with the indicated concentration of
SIINFEK peptide, and plated in the presence of CFSE-labeled (1 uM) naive OT-I T cells at 1:5 BMDC-to-T cells ratio for 72 hours. A total of 3 independent experiments were performed. B) Day 7 BMDCs were cultured for 24 hours with media, 100 ng / ml rCCL3
79 or LPS (100 ng / ml), then washed and incubated with OVA-latex beads at varying concentrations with CFSE-labeled (1 uM) naïve OT-I T cells at 1:5 BMDC-to-T cells ratio for 72 hours. C-D) Quantification of results in A and B. Each bar was calculated after subtracting the background CFSE dilution in the absence of added Ag. Each graph is representative of 3-4 experimental replicates. Not significant (ns), p > 0.05; *, p = 0.01 to
0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p < 0.0001.
80 Figure 12
Figure 12. WTTU and L3TU exhibit similar proliferation and PD-L1 expression profiles in vitro
A) In vitro proliferation assay measuring the growth kinetics between WTTU and L3TU.
B) ELISA of CCL3 proteins secreted from tumor cultures in vitro. C) CCL3 ELISA of serum from control or mice 7 days after inoculation with WTTU or L3TU. D) Mean fluorescence intensity (MFI) of PD-L1 expressed on tumor cells before and after 24 hour
IFN� stimulation in vitro. E) Percent of WTTU and L3TU cells that express PD-L1 before
81 and after 24 hour IFN�-stimulation in vitro. Not significant (ns), p > 0.05; *, p = 0.01 to
0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p < 0.0001.
82 Figure 13
Figure 13. Gross anatomical LN images show enlarged TDLN in the L3TU group compared to the WTTU group
Gross photographs were taken of the popliteal LN of NI, TDLN and NDLN of WTTU,
L3TU, WTTU+�Asialo-GM1, and L3TU+�Asialo-GM1 cohorts 5 days (A) or 7 days (B) following tumor inoculations. Measurements of LN sizes are depicted below the photographs. Not significant (ns), p > 0.05; *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p < 0.0001.
83 Figure 14
Figure 14. Leukocytes transiently accumulate in NDLNs following L3TU inoculation
A-E) Cellular accumulations in the NDLNs are shown for the 10 days following tumor inoculation. F-G) Accumulations of CD69+ T cell subsets were enumerated using FACS over 7 days following tumor inoculation. N=2 to 7 mice per cohort per day. Not significant
(ns), p > 0.05; *, p = 0.01 to 0.05; **, p = 0.001 to 0.01; ***, p = 0.0001 to 0.001; ****, p
< 0.0001.
84 Figure 15
Figure 15. Comparison of the leukocyte compositions in WTTU and L3TU TDLN cohorts
Percent composition of various immune subsets in DLN (A, B) and NDLN (C, D) were enumerated using FACS during the first 10 days following WTTU (A, C) and L3TU (B,
D) inoculation. Calculations were based on experiments in Figure 2. Percent composition for each day was calculated using the ratio between the average absolute cell numbers of
CD4+, CD8+, CD19+, CD11c+ or CD49b+ cells and the total cellularity and multiplied by
85 100. The baseline represents the average absolute number values calculated from NI mice for each cell subset.
86 Figure 16
Figure 16. Quantitative cytokine mRNA profiles in the L3TU TDLN
A) Various cytokine mRNA content within L3TU TDLN was analyzed 5 days following tumor inoculation. The graph shows fold changes between each L3TU replicate and that of
WTTU. Each individual symbol represents a biological replicate. TGF� analysis was repeated 3 times; TNF�, 2 times; IL-10, once; IFN� 6 times; and IFN� with �Asialo-GM1, once.
87 Figure 17
Figure 17. Efficiency of NK cell depletion in the WTTU and L3TU TDLN cohorts
Representative FACS analysis of NI, WTTU, or L3TU TDLNs 5 days following tumor inoculation. N = 3 mice per cohort.
88 CHAPTER IV
CCL3 augments tumor rejection and enhances CD8+ T cell infiltration through NK
and CD103+ dendritic cell recruitment via IFN�
Frederick Allen1*, Iuliana D. Bobanga2*, Peter Rauhe3, Deborah Barkauskas3,
Nathan Teich3, Caryn Tong3, Jay Myers3, Alex Y. Huang1,3,4,5
Departments of 1Pathology, 2Surgery and 3Pediatrics, Case Western Reserve
University School of Medicine; 4Angie Fowler AYA Cancer Institute, UH Rainbow
Babies & Children’s Hospital; 5Case Comprehensive Cancer Center, Cleveland, OH
44106 USA
89 ABSTRACT
Inflammatory chemokines are critical contributors in attracting relevant immune cells to the TME and driving cellular interactions and molecular signaling cascades that dictate the ultimate outcome of host anti-tumor immune response. Therefore, rational application of chemokines in a spatial-temporal dependent manner may constitute an attractive adjuvant in immunotherapeutic approaches against cancer. Existing data suggest that the macrophage inflammatory protein (MIP)-1 family, consisting of CCL3 (MIP-1�),
CCL4 (MIP-1�), and CCL5 (RANTES), can be major determinant of immune cellular infiltration in certain tumors through their direct recruitment of APCs, including DCs to the tumor site. In this study, we examined how CCL3 in a murine colon TME, CT26, enhances antitumor immunity. We identified NK cells as a major lymphocyte subtype that is preferentially recruited to the CCL3-rich tumor site. NK cells contribute to the overall
IFN� content, CD103+ DC accumulation, and augment the production of chemokines
CXCL9 and CXCL10 for enhanced T cell recruitment. We further demonstrate that both soluble CCL3 and CCL3-secreting irradiated tumor vaccine can effectively halt the progression of established tumors in a spatial-dependent manner. Our finding implies an important contribution of NK in the CCL3 - CD103+ DC - CXCL9/10 signaling axis in determining tumor immune landscape within the TME.
90 INTRODUCTION
Tumor intrinsic and extrinsic factors within the tissue microenvironment dictate the ultimate function, timing and robustness of local and systemic host immune responses, thereby tipping the balance between effective tumor elimination and immune escape 18.
Chemokine content within the TME constitutes one of the important factors contributing to the orchestration and modulation of cellular trafficking, interaction and delivery of effector function among relevant immune cells and tumor cells12,160. The effect of such chemokine networks on the intensity of tumor-infiltrating lymphocytes (TIL) within TME is distinct from that arising from tumor-intrinsic mutational load, which is thought to correlate with the spectrum of antigenic peptides and responding effector cells. In support of this view, recent studies in melanoma implicate that tumors modulate intra-tumoral T cell density in part by regulating inflammatory chemokine productions in the TME via a
WNT/�-catenin-dependent mechanism 176,179. In these studies, melanoma tumor cells that harbor genetic alterations in the WNT/b-catenin pathway could up-regulate inflammatory chemokine, CCL4, which attract dermal-resident CD103+ dendritic cells (DCs). The
CD103+ DCs elevated the production of CXCL9 and CXCL10, resulting in attracting T cells to infiltrate the tumor 176. Although these studies were primarily focused on CCL4, the production of CCL3 was also significantly increased in their tumor system176. We and other investigators have previously reported a critical role for both CCL3 and CCL4 in maximizing Ag scanning by naïve CCR5+ CD8+ T cells on activated DCs undergoing Ag- specific interactions with CD4+ or CD8+ T cells in the vaccine-draining LN. These interactions are a necessary process for maximal helper CD4+ T cell-dependent memory
CD8+ T cell generation 9,30,196.
91
For these and other reasons, CCL3 has been demonstrated to diminish tumor growth in mouse models 31. In particular, reports have shown that irradiated wild type CT26 tumors
(WTTU) and those engineered to secrete CCL3 (L3TU) can be used as vaccines to reduce growth under some circumstances 12,160. In another study, CCL3 was shown to enhance the abscopal effect following local radiation 197. These studies were followed by a clinical trial utilizing CCL3 as an immune adjuvant (clinicaltrials.gov: NCT01441115).
The afore-mentioned studies were focused primarily on evaluating the function of the adaptive immune responses that mediate the primary tumor rejection, but the molecular and cellular mechanisms by which CCL3 exerts its affect was not described in detail. In this current study, we examined the local and systemic effects of CCL3 on cellular and cytokine composition within TME. We employed CT26, a highly immunogenic murine colon tumor derived from BALB/c mice 161,162, and compared the host immune responses between CCL3-negative wild-type (WTTU) CT26 and CT26 engineered to constitutively secrete CCL3 (L3TU). We hypothesize that tumor-derived CCL3 could maximize inflammatory responses in the TME by soliciting the recruitment of innate immune cells, particularly NK cells, that may contribute to the subsequent recruitment of CD103+ DC and T cells as described by others 176,179. We demonstrate that CCL3 production by CT26 significantly slowed in vivo tumor growth, a process partially driven by CCL3-dependent accumulation of NK cells that supply the critical IFN� in the TME. In turn, enhanced NK and IFN� accumulation resulted in increased CXCL9 and CXLC10 production as well as
CD103+ CD11c+ DCs (CD103+ DCs) to the primary tumor site (PTS). Finally, we
92 demonstrate therapeutic efficacy of recombinant CCL3 (rCCL3) and irradiated whole-cell
L3TU vaccine in blunting established CT26 tumor growth in a site-dependent manner.
MATERIAL AND METHODS
Mice
BALB/c mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or bred in house. Both male and female 8- to 12-week-old BALB/c mice were used for all experiments. Mice were housed and handled according to National Institutes of Health institutional guidelines under approved protocols by Case Western Reserve University
Institutional Animal Care and Use Committee (No. 2012-0126 and 2015-0118).
CT26 transfection, maintenance, and use
In order to create CCL3-secreting CT26 (L3TU), CT26 (WTTU) tumor cells
(purchased from ATCC®) were stably transfected with a PCDNA3.1 plasmid vector that encodes mouse CCL3 under the CMV promoter and maintained under Hygromycin (150
µg / ml) selection. Before storage, cells are tested for microplasm contamination and stored as passage zero for experimental use later. Cells are stored in 10% dimethyl sulfoxide
(DMSO) and 90% complete media (RPMI 1640 with 10% FBS, 1% HEPES, 1% non- essential amino acids, and 1% penicillin and streptomycin) in liquid nitrogen tanks when not in use. Cells are thawed (passage zero) and used for experiments [3-12 passages (~4-6 weeks)] after in vitro growth is stabilized (~3 passages). During use, cells are incubated at
o 37 C with 95%O2 and 5% CO2 infusion. Cell viability is measured using Trypan blue dye.
Tumor cells are used for experiments only if cell viability (number of cells alive/number
93 of cells dead) is above 90% after counting. Prior to in vivo use, cells are washed with aqueous amounts of 1x PBS three times.
Tumor Measurements
For tumor measurements, mice were injected s.c. with either WTTU or L3TU in the left flank. Tumors were inspected, palpated, and measured using electronic calipers twice weekly. Tumor volumes were calculated according to the formula, V = � x D x d2, where “D” is the larger diameter and “d” is the smaller diameter 163.
Tumor Isolation and tissue preparation for flow cytometry and qPCR analyses
Tumor masses were excised, weighed for comparison against volume measurements, and finely chopped with a razor blade. The tumor was then stirred thoroughly to mix cells into a uniform heterogeneous cell mixture and a small portion is then removed for qPCR analysis. The remaining sample was placed into a conical tube containing FACS buffer (0.5% fetal bovine serum (FBS) and 0.5% of pH 8 EDTA in 1x sterile PBS), collagenase D and DNase-1, then incubated at 37° C in a mixer. The mixture was then passed through a 40 µm strainer twice and prepared for antibody staining and analysis by fluorescence-activated cell sorting (FACS).
Flow Cytometry, recombinant CCL3, and immunofluoresence materials and supplies
Antibodies for flow cytometry and immunofluorescence staining (IF) were purchased from eBioscience, BD Pharmingen, or BioLegend and included the following: rat anti-mouse CD4 FITC and PE (GK1.5), APC (RM4-5); CD8a FITC, PE, and APC (53-
94 6.7); CD11c FITC, PE, and APC (N418); CD49b PE and APC (DX5); CD3 PE and APC
(145-2C11); CD103 FITC (2E7). Analysis was performed using an Accuri C6 and FlowJo software against isotype controls and fluorescence minus one techniques. Recombinant murine CCL3 was purchased from PeproTech.
Tumor growth and antibody depletion experiments
For immune cell depletion studies, mice were injected with 100 µl of HBSS
(control), 50 µg of �Asialo-GM1 (Poly21460), 75 µg anti-CD4 (GK1.5), 50 µg anti-CD8
(2.43), or both GK1.5 and 2.43 neutralizing antibodies given intraperitoneally to BALB/c mice on days -3, -1, 1, and then twice weekly thereafter. 1x106 WTTU, L3TU, or
WTTU+L3TU cells were injected subcutaneously into the left flank on day 0. Tumor volumes were measured twice weekly as described above.
ELISA
6 o 1x10 WTTU or L3TU were incubated for 24 hours at 37 C in 5% CO2 in 1 ml of complete media (RPMI 1640 with 10% FBS, 1% HEPES, 1% non-essential amino acids, and 1% penicillin and streptomycin). The spent culture media or serum samples obtained from tumor-bearing mice were quantified for CCL3, CCL4 or CCL5 protein contents by
ELISA in accordance with the manufacture’s protocol (R&D systems, MMA00).
IFN� ELISPOT
Single cell suspensions were made from harvested tumors at indicated times following tumor inoculation. Tumor infiltrating immune cells were isolated from tumor cells using
95 Ficoll separation protocol. Immune cells were plated on IFN�-coated plates in biological duplicates and IFN� ELISPOT assays were performed as follows: Day 1, 96 well plates were coated with 1µg/ml of primary IFN� overnight at 4oC. Day 2, 1x106 immune cell were added to the well along with 0.5x106 irradiate (2000 Rads) AH1-peptide-pulsed
o o splenocytes (1µg/ml peptide for 1 hour at 37 C and 5% CO2) and incubated at 37 C and
5% CO2 for 24 hours. On day 3, biotinylated IFN� secondary antibody (4 µg/ml) was added to each welI and incubated overnight at 4oC, and 100ul/well of alkaline phosphatase strepavertin was added to each well in a humidity bag and incubate in the dark for 20 minutes or less (depending on the intensity of the forming spots). After drying, the plates were analyzed using an automatic C.T.L. ELISPOT plate reader.
Quantitative RT-PCR analysis
Total LN mRNA was isolated using TRIzol reagent in accordance with the manufacturer’s protocol (Gibco BRL, Carlsbad, CA) and purified using an IllustraTM
RNAspin Mini Kit (GE Healthcare Life Sciences). RNA quality was assessed by spectrophotometer absorption at 260/280 nm using the NanoDrop2000 spectrophotometer.
RNA was converted to cDNA using the EasyScriptTM Reverse Transcriptase protocol consisting of 200 U/µl Moloney murine leukemia virus reverse transcriptase incubated for
60 minutes at 42OC in the presence of 50mM Tris-HCl (pH 8.3), 100 mM NaCl, 0.1 mM
EDTA, 5 mM DTT, 0.1% Triton X-100, 50% (v/v) glycerol, 10 µM of oligo (dT), 10 mM
29-deoxynucleoside 59-triphosphate, and 40 U/µl recombinant RNase inhibitor (Lamda
BIOTECH, St. Louis, MO). cDNA was amplified in the presence of FAM-labeled gene- specific primers and Bullseye EvaGreen qPCR Mastermix (MIDSCITM; Saint Louis, MO)
96 in a 96 well microtiter plate using the ABI Prism 7300 sequence detection system (Applied
Biosystems). Each PCR reaction was performed in triplicate and compared to WTTU.
Relative levels of mRNA were determined using the cycle threshold (Ct). The gene expression was standardized to cytochrome-c (CyC) expression within the tumor draining
LN (TDLN). In order to compare the Ct values between target genes we normalized each
-(Target gene – CyC - target Ct to the average of the WTTU Ct using the following equation: 2^ normalizer).
Statistical analysis
Statistical analyses were performed using the standard one tailed unpaired t-test.
All data presented as either +/- standard error of the mean (Figures 18 only) or standard deviation.
RESULTS
CCL3 facilitates tumor rejection via thymic-dependent and thymic-independent
mechanisms
In order to examine how conventional T cell responses affect the growth of aggressive murine colon tumor CT26 (WTTU) and CT26 engineered to secrete CCL3
(L3TU), we measured tumor growth rates in both athymic nude and immunocompetent mice. At baseline, WTTU secretes CCL5 but not CCL3 and CCL4 (Figure 24A) 198,199.
L3TU produces similar levels of CCL4 and CCL5 as WTTU, with CCL3 production at an average of 1000 pg (ranges ~350-1300 pg / 1x106 cells / ml in 24 hours) as measured from
3 independent studies. Similar to tumor growth rates in vitro (Figure 24B), WTTU and
97 L3TU grew at a similar rate in vivo in athymic nude mice (Fig. 18A), suggesting that introducing CCL3 into CT26 did not cause an intrinsic growth defect. Interestingly, L3TU grew significantly slower than WTTU over the course of 3 weeks in BALB/c mice (Fig.
18B). The depletion of CD4+ cells did not significantly affect the growth of either L3TU or WTTU (Fig. 18C). However, the depletion of CD8+ T cells alone or in conjunction with
CD4+ T cells depletion resulted in a rapid tumor progression in WTTU, with tumor sizes approaching that in athymic nude mice. Although L3TU tumor sizes were also significantly increased in BALB/c mice depleted of CD8+ T cells alone or both CD4+ T cells and CD8+
T cell (Fig. 18B, 18D, 18E), the resulting tumors were smaller than those in athymic nude mice, suggesting an additional, non-CD4+/CD8+ T cell-dependent mechanism that is partially responsible for suppressing L3TU growth in vivo.
CCL3 enhances CD4+ and CD8+ T cell infiltration to the primary tumor site
As the presence of CCL3 enhanced CD4+ and CD8+ T cell-dependent rejection of
CT26, we sought to verify whether the presence of CCL3 promoted the infiltration of these
T cell subsets into the PTS. Immunofluorescence (IF) analysis of tumor tissue sections at
21 days post inoculation revealed a significant increase in both CD4+ and CD8+ T cell infiltrations in the L3TU TME as compared to WTTU (Fig. 19A-B). Interestingly, the degree of T cell infiltration was also inversely correlated with tumor size (Fig. 19C-D), further strengthening the association between the intensity of T cell infiltration and tumor growth kinetics.
98 To further understand the molecular mechanisms affecting CCL3-promoted T cell accumulation in the TME, we analyzed cytokine and chemokine contents by qPCR 200.
Compared to WTTU, L3TU TME expressed similar levels of TGF� and TNF� (0.9- and
1.2-fold, respectively; Fig. 3A). However, the amount of IL-10, IFN� CXCL9 and
CXCL10 mRNAs were upregulated in the L3TU (1.8-, 3.2-, 2.9- and 1.6-fold, respectively;
Fig. 3A). While the relative levels of CXCL9 mRNA were higher in L3TU, the overall mRNA abundance was low in both WTTU and L3TU TME relative to the other cytokines and chemokines. Next, we analyzed the contribution of CD4+ and CD8+ T cell subsets to the abundance of the cytokine mRNA contents. Depletion of CD4+ and CD8+ T cells in the
L3TU resulted in an even more profound reduction in the overall TNF� content as compared to TNF� levels in WTTU after depletion of these T cell subsets (~5-fold; Fig.
14B, 14C, 14D and Figure 25), supporting the notion that the source of TNF� comes primarily from the T cell subsets with CCL3-enhanced CT26 tumor rejection. Indeed,
CD4+ T cell depletion did not reduce TNF� abundance in the WTTU CD4+ depletion group, and CD8+ depletion or CD4+/CD8+ double depletion reduced TNF� only by ~50% in the WTTU TME (Figure 25A, 25C, 25E). Next, we examined the effect of CD4+ and
CD8+ T cell subsets on TME IFN� levels. Both CD4+ and CD8+ T cells clearly contributed to the IFN� content of the L3TU TME (Figure 25B, 25D, 25F). However, despite a reduction in TNF�, IL10 and CXCL9 contents in L3TU TME relative to WTTU TME, depletion of these two T cell subsets failed to completely abrogate the total IFN� content associated with CCL3 (Fig. 20D).
CCL3 recruits NK cells to promote CD103+ DCs infiltration and support T cell
99 function within the primary tumor
NK cells can serve as a predominant source of IFN� 201,202 and CD103+ DCs have been identified as a major source of CCL4-driven CXCL9 and CXCL10 production within
TMEs 176. Therefore, we examined the relative abundance of these important cell populations and their contributions to the differences observed between L3TU and WTTU that were not accounted for by the presence of CD4+ and CD8+ T cells (Fig. 20; Figure 8).
A nearly 3-fold increase in the number of NK cells were observed within L3TU TME, and
NK cell numbers correlated inversely with the tumor size (Fig. 21A, 21B). Similarly,
CD103+ DCs were found to be increased ~3-fold in L3TU relative to WTTU, with a similar inverse correlation between the density of CD103+ DCs and tumor size (Fig. 21C, 21D).
In contrast, macrophage and neutrophil accumulation was not altered in the presence of
CCL3 within TME (data not shown). Although the accumulation of CD103+ DC has been linked directly to tumor production of CCL4176, we wished to determine whether the presence of NK cells and their associated IFN� production could affect local CD103+ DC accumulation, as IFN� has been shown to induce other immune cells to upregulate CXCL9 and CXCL10, chemokines which are important for directing the homing of DCs and activated T cells to the TME 179,203,204. Indeed, depletion of NK cells resulted in a dramatic reduction in the number of CD103+ DC within the L3TU TME to a level similar to WTTU
(Fig. 4C). Interestingly, depletion of NK cells also reduced the numbers of CD4+ and CD8+
T cells within L3TU and trended towards a larger overall tumor size (Fig. 21E, 21F).
Next, we assessed NK contribution to the global production of cytokines and chemokines within the TME. Depletion of NK cells lead to a reduction in IFN� and TNF�
100 mRNA in both the WTTU and L3TU TME (Fig. 22A, 22B). NK depletion also resulted in an increase of TGF� mRNA by 1.3- and 1.6-fold in WTTU and L3TU, respectively.
Overall, removal of NK cells normalized the levels of TGF�, IL10, TNF� and CXCL9 in
L3TU to that in WTTU (Fig. 22C). IFN� and CXCL10 remained elevated in L3TU relative to WTTU after NK depletion, suggesting additional sources, such as T cells, further contributed to the increased immune infiltration in L3TU (Fig. 20D; Fig. 16C).
Subcutaneously administered rCCL3 significantly slowed tumor growth in
established tumors
To test the antitumor efficacy of CCL3 as a therapy, we performed two immunotherapeutic approaches to treat established CT26. First, we used irradiated L3TU whole cell tumor vaccines to treat established WTTU in vivo. Disappointingly, direct intra- tumoral injections of either irradiated WTTU (iWT) or L3TU (iL3) tumor cell starting on day 7 failed to significantly slow the aggressive WTTU growth in vivo (Figure 26). To avoid the tolerizing local LN microenvironment into which both established WTTU tumor and iL3 vaccines drain, we investigated whether administering irradiated tumor vaccine at a distal body site could potentially avoid the immune suppressing factors expressed by established tumor and to unmask the inflammatory effector functions of CCL3. Following injection of WTTU in the left flank, we waited 7 days and inoculated in the contralateral footpad twice weekly with 1X106 iWT (WT/iWT) or iL3 (WT/iL3). Interestingly, the
WT/iL3 protocol significantly stunted tumor growth compared to WT/iWT or WTTU alone, even though the effect was not as robust as L3TU tumor alone (Fig. 23A), suggesting
101 measurable efficacy of irradiated L3TU as a vaccine in curbing WTTU growth when such vaccines were given at a site other than the primary WTTU site.
Next, we tested the efficacy of recombinant CCL3 (rCCL3) on established WTTU or L3TU tumor sites using high-dose (100 ng/dose) bolus s.c. administrations 31,48. After injecting WTTU or L3TU in the left flank on day 0, mice received s.c. injections of rCCL3 starting on day 7 in either the ipsilateral footpad (WTR7_ipsi), contralateral footpad
(WTR7_contra or L3R7_contra), or intra-tumorally (WTR7_IT or L3R7_IT). For comparison, additional cohorts of mice bearing WTTU or L3TU were injected with PBS starting on day 7, or inoculated with 1:1 mixture of live WTTU+L3TU on day 0.
Interestingly, intra-tumoral injections of rCCL3 starting on day 7 failed to slow WTTU growth and in some of the mice even facilitated tumor growth when compared to WTTU group alone (Fig. 23B; data not shown). However, rCCL3 administered distally either in the ipsilateral or contralateral footpad starting on day 7 resulted in a significant decrease in the overall WTTU tumor growth similar to 1:1 live WTTU+L3TU tumor mixed (Fig. 23B).
Although no significant differences were observed between L3R7_contra and L3R7_IT, these tumors were smaller when compared to L3TU alone (Fig. 23B, 27), suggesting a differential effect of CCL3 in the anti-tumor immune priming when administered on day 0 versus day 7.
DISCUSSION
Colon cancer and melanoma have been associated with suppressed expressions of
MIP-1 family of proteins that are typically upregulated by their respective normal tissue
102 counterparts under stressed conditions 205. CCL3, CCL4 and CCL5 are members of the
MIP-1 family, share a high degree of homology, and bind to CCR5 (all) and CCR1 (CCL3 and CCL5) on many cell types including immature DCs and T cells in both humans and mice 31,196. CCL3-transfected melanoma tumors can recruit adoptively transferred, Ag- primed bone marrow derived DCs (BMDCs) to PTS and drive tumor rejection 12. CCL3 and CCL4 have also been implicated in directing CD8+ T cell infiltration into primary tumor sites in melanoma and colon cancers 176,179. In particular, Spranger and colleagues demonstrated how melanoma tumors could prevent CD103+ DC infiltration though active suppression of CCL4 in a WNT/�-catenin-dependent pathway 176. Alterations in the
WNT/�-catenin pathway up-regulate CCL4 in melanoma, resulting in the recruitment of dermal-resident CD103+ DC to the PTS with subsequent migration to the tumor DLN
(TDLN) for anti-tumor T cell priming176. In this paradigm, CCL4/CCL3-enhanced recruitment of CD103+ DCs are responsible for the production and release of CXCL9 and
CXCL10, which facilitate subsequent accumulation of effector T cells to the tumor site. As the WNT/�-catenin pathway is also active in CT26206, similar molecular and cellular interplay may be at work in our current study. While multiple reports have suggested that an active WNT/�-catenin pathway suppresses the activation of CCL3 and CCL4 in some immune and tumor cell types176,207,208, it can also drive the production of CCL5 in other tumor models209. Indeed, despite evidence of baseline CCL5 production (Figure 24), engineered CCL3 over-expression by CT26 recapitulated similar findings as described by prior studies, resulting in both enhanced homing of CD103+ DCs and T cell infiltration into the primary tumors12,176. However, our current study further revealed an additional critical component in the CCL3-dependent cellular recruitment paradigm by identifying NK cells
103 as a major contributor in blunting CT26 growth via production of IFN� within the TME.
Our NK cell findings add another player in the CCL3/4-directed, CD103+ DC-driven,
CXCL9/CXCL10-induced mechanism of immune infiltration into tumors176, and recapitulates previous report showing IFN� as being involved in the recruitment of CD103+
DCs204.
We compared the global mRNA expressions of TNF�, IFN�, TGF�, and IL-10, as well as CXCL9 and CXCL10, which can be induced by IFN� and are associated with improved prognosis in patients210-212. We showed that NK cells are critical for enhancing
CD103+ DCs recruitment and augmenting proper effector function of T cells. We also identified IFN� as contributing to CD103+ DC recruitment in L3TU, which correlated with the CCL3-directed homing of NK cells into the tumor sites (Fig. 21, 22). Depletion of NK cells in L3TU not only significantly reduced CD103+ DC accumulation, but also decreased the abundance of IFN� at the PTS, supporting the view that NK cells are important sources of IFN�. Although T cells constitute another major source of IFN� in the tumor rejection process, our data is further supported by previous studies using IFN�-deficient T cells to demonstrate that NK cells could supply much of the IFN� needed in effective tumor rejection201. However, NK cells also are dependent on T cells for full activation, thus underscoring the existing crosstalk between innate and adaptive anti-tumor responses201.
The presence of IFN� further prompts the production of CXCL9 and CXCL10 from the surrounding epithelial and immune cells, thereby augmenting T cell infiltration and enhancing DC accumulation though CXCR3179,213. Our study further demonstrated the functional importance of this molecular crosstalk by observing a drastic decrease in IFN�
104 and associated CXCL9 and CXCL10 levels after NK depletion, resulting in diminished T cell infiltration and accelerated tumor growth. Furthermore, our observation agrees with previous findings that the presence of IFN�-producing cells in the primary tumor mass is inversely correlated with tumor growth180, and that IFN� early in the TME activates DCs and T cells towards inflammatory phenotypes. Both CCL3 and IFN� were implicated in endowing DCs the ability to polarize T cell inflammatory responses, proliferation, and immune memory generation9,173,181-184.
Previous studies have attributed CCL3-promoted CT26 tumor rejection to increased macrophage and neutrophil infiltration at the PTS within the first 5 days following tumor inoculation as evidenced by histological examination160. However, our day 21 analysis of L3TU tumors by flow cytometry did not find such observation. The reason for this discrepancy is not entirely clear. One possibility is that the numbers of macrophages and neutrophils may have equilibrated between WTTU and L3TU by day 21.
Another possibility could be the differences in the function, not the relative abundance, of these cells in the two different PTS. Ultimately, CT26 rejection is most effectively controlled by CD8+ T cells, which recognize the immuno-dominant Ag, AH1, on the H2-
Ld haplotype162. Therefore, it was not a surprise that depletion of CD8+ T cells resulted in significant accelerated tumor growth in both WTTU and L3TU. CD4+ T cells have also been shown to modulate CT26 rejection 214. Indeed, depletion of CD4+ T cells results in a significantly accelerated L3TU tumor growth when performed concurrently with CD8+ T cell depletion (Fig. 18E). These effects are likely linked to increased cytokine production associated with these T cell populations in the L3TU TME (Figure 25F).
105
Chemokine-based immunotherapy has been investigated as a means to modulate anti-tumor immunity12,160. We provided exciting data demonstrating benefits in translational application of using iL3 vaccine or bolus rCCL3 therapy to significantly blunt the growth of established CT26 (Fig. 23, Figure 27). Despite reports supporting the benefits of intra-tumoral administration of cytokines as therapies215, we failed to observe notable effects of intra-tumoral iL3 or rCCL3 administration on slowing the growth of established
WTTU. The exact mechanism for the failure of this approach is unclear. One possibility could be that day 7 WTTU TME and draining LN may be too immune tolerant to benefit from the therapeutic effects of iL3 or rCCL3. Most interestingly, however, we observed that contralateral or ipsilateral s.c. injections of iL3 and rCCL3 distal to the tumor site could efficiently blunt established CT26 growth. We hypothesize that that CCL3 administered at a distal site can either robustly stimulate immune cells within DLNs that are not exposed to the immediate suppressive factor derived from the tumor, or efficiently cause the mobilization of cells from the bone marrow to migrate into PTS or the LN. Supporting this hypothesis, CCL3 has been shown to enhance the release of innate responders such as NK cells and DCs from the marrow to the blood where they can home to tissues216,217. We are currently examining this fascinating aspect of CCL3-induced immune activation in the draining LN early in the therapeutic response.
In summary, we examined CCL3 contribution in facilitating effective cellular crosstalk between the innate and adaptive anti-tumor immune responses. We provided new insights into how this crosstalk supports the accumulation and effector function of T cells,
106 NK cells and CD103+ DCs at the PTS. We also demonstrated the efficacy of CCL3 as a modulating immunotherapeutic for the treatment of established tumors. NK cells supporting T cell infiltration and function is a phenomenon that goes beyond mouse tumor models. NK cells involvement in the stepwise infiltration of lymphocytes has also been reported in human colon cancers218. In addition, CCL3 upregulation by IFN�-producing
NK cells has been implicated as an important component in the clinical efficacy of the
FDA-approved monoclonal antibody, trastuzumab, for the rejection of HER2+ breast cancers219.
Although our study focuses exclusively on CCL3 which showed a pronounced effect on CT26 rejection in vivo, similar contribution to tumor rejection has been ascribed to CCL4 in melanoma176. Indeed, CCL4-secreting CT26 (L4TU) also resulted in a significant production of IFN� at the tumor site compared to WTTU (Figure 28). However, compared to L4TU, L3TU resulted in a more rapid rejection (data not shown). Future studies will exam the mechanisms of s.c. administered CCL3 in mobilizing marrow- derived immune cells to facilitate tumor rejection. Taken together, our current data further support the exploration of CCL3 as an adjuvant for enhancing antitumor immune response.
107 Figure 18
Figure 18: Autologous CCL3 retards CT26 growth in vivo with partial dependence on both CD8+ T cells and non-T cell sources
Mice were injected with 1x106 WTTU (square) or L3TU (triangle) tumor cells in the left flank. Average tumor growths were shown in BALB/c athymic mice (A), mice without antibody depletion (B), CD4+ depletion antibody (C), CD8+ depletion antibody (D), or in
CD4+ and CD8+ double depleted mice (E). N=12 mice/cohort for WTTU group and n = 15 mice/cohort for L3TU group. Data shown are combined results of 3 independent
108 experiments. Black stars (*) compare WTTU and L3TU groups within each graph. Gray dashed (----) and dotted (….) lines compare the growth shift of each graph to no depletion
WTTU and L3TU in figure 1B. Gray stars (*) compare the significance between the growth shifts of the gray dashed and dotted lines compared to no depletion WTTU and L3TU in figure 1B. NS: not significant; *, P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P = 0.0001 to 0.001; ****, P < 0.0001. Error bars are shown as standard error of mean (SEM).
109 Figure 19
Figure 19: L3TU enhances CD4+ and CD8+ T cell infiltrations into the primary tumor site
Mice were injected with 1x106 WTTU or L3TU cells in the left flank. At day 21, mice were euthanized and only visible tumors were removed for FACS and IF analyses. A,
Representative IF images of GFP-labeled tumors (green), CD4+ T cells (purple) and CD8+
T cells (red). B, Quantification of IF showing T cell number per 2500 um2. Shown are data obtained from a total of 4 tumors with 16 sections per tumor and 3 areas per section. C-D,
Correlation of CD4+ or CD8+ T cell numbers per 1x105 total cells by FACS versus WTTU
(circle) or L3TU (triangle) tumor volume; N=5 experiments with 5 biological replicates for each group. ****, P < 0.0001. Error bars are shown as standard deviation (SD).
110 Figure 20
Figure 20: IFN� levels are sustained in L3TU tumors despite T cell depletions
Mice were injected with 1x106 WTTU or L3TU cells in the left flank. On day 21, mice were euthanized and tumors removed for qPCR analysis of TGF�, IL-10, FOXp3, TNF�,
IFN�, CXCL9, and CXCL10. A, Expressions of various cytokine mRNAs in L3TU compared to WTTU. B-D, Relative cytokine mRNA levels of L3TU compared to WTTU in animals depleted of CD4+ cells (L3�CD4 or WT�CD4), CD8+ cells (L3�CD8 or
WT�CD8), or both CD4+ and CD8+ cells (L3�CD4+�CD8 or WT�CD4+�CD8). Each symbol represents an individual animal. The expressions of TNF� and CXCL9 were below detection levels in some of the groups. Data represent a compilation of 3 independent experiments. ND: Not detected.
111
Figure 21
Figure 21: CCL3 recruits NK cells to drive infiltration of CD103+ DCs and support T cell function at tumor sites
Mice were injected with 1x106 WTTU (circles) or L3TU (triangles) cells in the left flank.
At day 21, only visible tumor masses were removed for FACS analysis. A, Enumeration
112 of NK cells (CD49+ CD3-). N = 5 biological replicates. B, Correlation of data in A with individual tumor volume. C, Enumeration of dermal-resident (CD103+ CD11c+) DC subset in tumor masses with or without NK cell depletion (WT�GM1 or L3�GM1). D,
Correlation of data in C with individual tumor volume. E, Correlation of CD4+ T cell density in individual tumor mass with (black) or without (white) NK cell depletion. F,
Correlation of CD8+ T cell density in individual tumor mass with (black) or without (white)
NK cell depletion. N=2 independent experiments with 5 biological replicates each. **, P
= 0.001 to 0.01. Error bars denote standard deviation (SD).
113 Figure 22
Figure 22: NK cells and CCL3 support inflammation and T cell homing
Mice were injected with 1x106 WTTU or L3TU cells in the left flank. At day 21, tumors were analyzed by qPCR for TGF�, IL-10, TNF�, IFN�, CXCL9, and CXCL10. A,
Relative change in cytokine expression in WTTU mass with or without NK cell depletion.
B, Relative change in cytokine expression in L3TU tumor mass with or without NK cell depletion. C, Relative changes in cytokine production by L3TU and WTTU with NK cell depletion. Each symbol represents an individual animal.
114 Figure 23
Figure 23: rCCL3 or irradiated L3TU significantly slows established tumor growth
Mice were injected with 1x106 WTTU cells in the left flank. 7 days later mice were injected in the footpad or intra-tumorally (i.t.) with whole tumor lysate from lethally irradiated
WTTU (iWT) or L3TU (iL3), or recombinant CCL3 (rCCL3; 100 ng). A, WTTU tumor growth in mice that received whole cell lysates of iWT or iL3. B, Tumor growth in mice that received either i.t. or s.c. injections of rCCL3. Subcutaneous injections were administered either ipsilaterally (ipsi) or contralaterally (contra) relative to WTTU 7 days post tumor injection (R7). Data shown as compilation of 2 independent experiments with n = 5-10 mice per group. NS: not significant; *, P = 0.01 to 0.05; **, P = 0.001 to 0.01;
***, P = 0.0001 to 0.001; ****, P < 0.0001. Standard error is shown as SD.
115 Figure 24
In vitro proliferation assay
1×107 WTTU 8×106 L3TU
6×106
4×106
2×106 Number of live cells
0 0 4 6 Days
Figure 24: WTTU and L3TU Chemokine production, and growth characteristic of
WTTU and L3TU in vitro
A, 1x106 cells of either WTTU or L3TU were cultured in serum-free media for 24 hours and supernatant were assayed for CCL3, CCL4 and CCL5 by ELISA. Assays were performed in triplicates. Results were shown as an average of 3 independent experiments.
B, Total live cell counts of WTTU and L3TU over 6 days in vitro. Assays were performed in triplicates. Results were shown as an average of 4 independent experiments. Error bars were shown as standard deviations. ND: not detected.
116 Figure 25
Figure 25: Global cytokine makeup of WTTU and L3TU were dependent on the presence of CD4+ or CD8+ T cells in TME
Mice were injected with 1x106 WTTU or L3TU cells in the left flank in the presence or absence of CD4+ T cells, CD8+ T cells or both. At day 21, mice were euthanized and tumors removed for qPCR analysis of, interleukin-10 (IL-10), tumor necrosis factor-� (TNF�), interferon-� (IFN�), CXCL9, and CXCL10. A, Relative cytokine mRNA levels of WTTU tumors with (WT�CD4) or without (WTTU) CD4+ T cell depletion. B, Relative cytokine mRNA levels of L3TU tumors with (L3�CD4) or without (L3TU) CD4+ T cell depletion.
C, Relative cytokine mRNA levels of WTTU tumors with (WT�CD8) or without (WTTU)
CD8+ T cell depletion. D, Relative cytokine mRNA levels of L3TU tumors with (L3�CD8)
117 or without (L3TU) CD8+ T cell depletion. E, Relative cytokine mRNA levels of WTTU tumors with (WT�CD4+�CD8) or without (WTTU) depletion of both T cell subsets. F,
Relative cytokine mRNA levels of L3TU tumors with (L3�CD4+�CD8) or without
(L3TU) depletion of both T cell subsets. Each symbol represents an individual animal. The expressions of TNF� and CXCL9 were below detection levels in some depletion groups with L3TU. Data represent a compilation of 3 independent experiments. ND: not detected.
118 Figure 26
Figure 26: Intra-tumoral injections of iL3 failed to enhance tumor rejection in vivo
Mice were injected with 1x106 WTTU (WT) in the left flank followed by intra-tumoral
(i.t.) injections of 1x106 iWT or iL3 on day 4 and twice weekly thereafter. NS: not significant.
119 Figure 27
Figure 27: Tumor size measurements of L3TU with additional recombinant CCL3
Distribution of individual tumor sizes in Figure 6B from mice that were inoculated with
L3TU only, L3TU with contralateral rCCL3 on day 7 (L3R7_contra), and L3TU with intra- tumoral rCCL3 on day 7 (L3R7_IT).
120 Figure 28
Figure 28: IFN� production in WTTU, L3TU, and L4TU TDLN and primary tumors
Mice were injected with PBS or 1x106 of WTTU, L3TU or L4TU. 14 days later tumors were harvested for ELISPOT analysis of IFN� production. Results from the ELISPOT assays were quantified as the number of spots detected per milligram (mg) of tumor sections. N = 2 mice/group. *, P = 0.01 to 0.05. Standard error is shown as SD.
121 Figure 29
A F) Time course FACS A) Block egress of TDLN and NDLN with FTY720
Time course 24 hrs
E) Stop B) s.c. footpad injection Mice FTY720 of WTTU or L3TU
24 hrs 48 hrs
24 hrs D) Block ingress C) CFSE-labeled with anti-CD62L NK cells
Figure 29: Experimental design for tracking NK cell migration in the TDLN
A, injection mice with FTY720. B, 24 hours later, give s.c. footpad injections of WTTU or
L3TU. C, 48 hours later inject CFSE-labeled NK cells i.v. D, 24 hours later stop ingress with anti CD62L antibodies. E, 24-hour later stop FTY720. F, conduct FACS analysis over various time points to assess egress efficiency of NK cell over time.
122 Figure 30
A Tregs B Tregs 30000 300000 * NS NS
20000 200000 *
10000 100000 Absolute Number Absolute Number
0 0 DAY 5 DAY 10 C CD8 CXCR3 CD8 CXCR3 3000 NS (P= 0.09) 3000 NI 2000 WTTU 2000 L3TU 1000 Absolute Number
10000 Day 5 Absolute Number Figure 30: The absolute number of Tregs versus activated CD8 T cells in the TDLN 0 Day 5 FOXp3-GFP BALB/c mice were injected with 1x106 WTTU or L3TU cells in the footpad.
Draining pLNs were harvested from each mouse on day 5 or day 10 as indicated, then analyzed by FACS for expressions of GFP to indicated Treg cells (A and B), or the expressions of CXCR3 and CD8 to indicated activated CD8 T cells (C). n = 3 mice per group. A= one experiment. B= two experiment. C= one experiment. NS: not significant; *,
P = 0.01 to 0.05; **, P = 0.001 to 0.01; ***, P = 0.0001 to 0.001; ****, P < 0.0001. Standard error is shown as SD.
123 Figure 31
A CFSE+ in TDLN 10000 NS (P= 0.08) 8000
6000
4000
Absolute Number 2000
0 WTTU L3TU
Figure 31: The absolute number of CFSE+ BMDCs in the TDLN
BALB/c mice were injected with 1x106 WTTU or L3TU cells in the footpad, followed by
1x106 intra-tumor injections of CFSE-labeled BMDCs 48 hours later. The draining pLNs were harvested 24 hours after BMDCs injections and analyzed by FACS for CFSE expression. A= 2 experiments. NS: not significant; *, P = 0.01 to 0.05; **, P = 0.001 to
0.01; ***, P = 0.0001 to 0.001; ****, P < 0.0001. Standard error is shown as SD.
124 Figure 32
A IFN"
STAT-1
Active in various cell lines NOS2 induction
p38MAPK activation Wnt/!-catenin ATF3 induction
CCL3 and CCL4 suppression
Active in various Adopted from Chandrasekar et. al cell lines
B
! IFN!R-/- = IFN response from = CCL3 serum levels tumor burdened mice
IFN!-/- = IFN! response from = CCL3 serum levels tumor burdened mice
WT = IFN! response from = CCL3 serum levels tumor burdened mice
Figure 32: Proposed experimental design for testing in vivo IFN� suppression of
CCL3 in WNT/�-catenin knockout tumors
A, a simplified schematic showing the possible signaling pathway in which IFN�, WNT/�- catenin, and CCL3 might intersect one another176,191,220. B, proposed outcome after injection of IFN�R-/-, IFN�-/- or WT mice with WNT/�-catenin knockout tumors.
125 CHAPTER V – Conclusion
126
HIGHLIGHTING THE NOVELTIES
Like other cytokine and chemokine therapies under study, CCL3 has also been directly implicated as a potential therapy for facilitating the clearance of many tumor models, including CT266,10-12,23,48,160,221. However, there has been little data telling a more complete story of how such therapies shape the microenvironment of both the PTS and the
TDLNs, which are ultimately responsible for determining how the adaptive immune system will react to tumors. This is clinically relevant, because tumor survival in native or non-native (cancer) tissues depends on their capacity to hide from, suppress, or convert immune effector functions in LNs154,155,157,159. The early dissemination of TAAs and tumor associated cytokines (e.g. TGF�, IL-10, CCL5) to TDLNs play an important part in whether to promote or suppress inflammatory responses22,198,222,223.
In chapter 2, we reviewed collections of work that discussed how low-weight proteins, like chemokines and other small Ags (<70 kDa), can readily disseminate to DLNs and initiate early immune responses. In this chapter, we also reviewed published data on unique ways in which APCs can capture Ags across tissue compartments by either extending their dendrites from one tissue to another or becoming embedded in the outer capsule of tissue compartments. Early events such as these are important, because they can increase the adaptive response time and help start a chain of complex events that lead to
LN architectural remodeling, changes in cellular dynamics, and shape immune cell effector responses.
127 In chapters 3 and 4, we expounded on chapter 2 by incorporating these insights on early Ag and chemokine propagation to the tumor system with implications towards immunotherapeutic interventions. We showed how the early dissemination of TAAs and
CCL3 in LNs play a significant part in amplifying proinflammatory cellular responses in the TDLN and PTS. First, we demonstrated how CCL3’s presence in the TME augments cellular trafficking and effector responses during the early stages of T cell priming in the
TDLN. Then we showed how the maintenance of these events are closely mirrored at the
PTS and preserved over a longer period of time. Specifically, we reported mirrored changes in the TDLN and PTS that lead to enhanced T and B lymphocyte, NK cell, and DC ingress.
Rapid changes occurred in both the composition and total number of cells in the TDLN of
L3TU and at the PTS, ultimately driving global increases in proinflammatory cytokines, like IFN� and TNF�, and proinflammatory chemokines, like CXCL9 and CXCL10; all of which are historically associated with antitumor responses. The novel cellular component integral to both the early and late proinflammatory responses are IFN�-producing NK cells.
CCL3 EFFECTS ON CELLULAR TRAFFICKING IN THE TUMOR-DRAINING
LYMPH NODE OF L3TU
Interrogating CCL3 effects in the TDLN during T cell priming sparked several questions regarding CCL3’s effects on LN trafficking. We will consider two of them here.
The first question considers how naïve T cell ingress under the control of CCL3 in L3TU.
This one has already been answered though other studies conducted in our lab. The second question remains unclear. This one considers whether CCL3 might also be involved in opposing immune cell egress in L3TU.
128
CCL3 is largely produced during the early stages of the inflammation process in tissues to attract CCR5+ and CCR1+ cells to sites of insult (e.g. NK cells, macrophages, monocytes, DCs, T cell)9,48,164,169,224,225. Within inflamed LN, CCL3 can induce TEM across HEVs, and draw T cells from peripheral circulation226. In 2006, two studies reported on CCL3’s migratory behavior within inflamed LNs. The showed that CCL3 guided naïve
T cell and enhanced their contacts with mature DCs within inflamed LNs9,30. In addition, these contacts were demonstrated as an important component for the development of memory CD8+ T cells9. However, before this report it was widely believed that CCL3 (and
CCL4) could only act on activated T cells, because they constitutively expressed cognate receptors only after activation. This conundrum was solved in our lab in 2016 by Askew et. al, when we demonstrated that some naïve T cell populations transiently express CCR5 at the initial time of entrance from HEVs into the LN parenchyma156.
The evidence shown by our lab validates a mechanism by which CCL3 can enhanced LN ingress during inflammation. However, what remains unclear is whether
CCL3 is also involved in the process of LN egress under conditions of inflammation caused by tumor invasion. Sphingosine-1-phosphate (S1P), which signals though the receptor
S1PR on T- and B-lymphocytes227, and NK cells228 is a primarily responsible for cellular egression from LNs to peripheral tissues. Notably, a review on NK cell trafficking showed that S1P and CCL3 take part in a dual effort to drive NK cell egression from the bone marrow to the peripheral circulation216,229. Whether this particular role of CCL3 is exclusive to NK cells in the BM is uncertain. In the BM, CCL3 works alongside SP1 to stimulate egress229. However, in inflamed LNs, CCL3 works to guide and maintain T cells
129 contacts with mature DCs,9 while S1P promotes cellular egress by creating a concentration gradient with the peripheral circulation to draw S1PR+ cells (e.g. T and NK cells) out of
LNs227,228. This suggest the possibility that CCL3 and S1P might have competing roles on cells that constitutively express CCR1 or CCR5, such as activated T cells and NK cells.
Under normal development of inflammation, CCL3 is releases at the beginning of the process and downregulated later in order for tissues to return to steady-state. In our system with L3TU, CCL3 is continuously present in the TME during the process of invasion by tumors and TAAs. In this context, it is possible that CCL3 contends with S1P for attention.
Figure 29 is a diagram of a proposed experiment to test CCL3 effects on LN egress. This method can be done to assess the egress potential of CD4+, CD8+, and CD19+ lymphocytes as well.
THE SIGNIFICANCE OF IFN� IN PROMOTING EARLY AND LATE
PROINFLAMMATORY RESPONSES WITH L3TU
In order for tumors to escape immune detection it must first hide or convert immune cells from innate immune system. The expression of inhibitory or lack of activating ligands on tumor cells play an important part in helping tumors escape NK cell killing70. NK cells exhibit their effector functions though cell-to-cell engagements by receptor-ligand mediated cytolytic killing, or though the release of inflammatory cytokines, such as IFN�.
IFN� can directly inhibit tumor growth and add support to cell-mediated tumor killing by enhancing the upregulation of MHC on some tumors and making tumor Ags more visible to detection62. Conversely, IFN� can protect tumors from NK cell contact-dependent killing by enhancing the expression of MHC molecules on tumors and inhibiting NK
130 cells62. This is because MHC proteins are also inhibitory ligands that can suppress NK cells effector functions230. However, NK cells can continue to support tumor clearance without the need for direct interactions with tumor cells. For example, cytokines produced by NK cells can drive DC maturation and direct their effector functions toward the induction of inflammatory T cell responses172. In addition, cytokines produced by NK cells can also support T cell effector functions during tumor clearance as well172,231. This work focuses on how CCL3 navigates the TME of the TDLN and PTS to enhance NK cell-derived IFN�.
With this work, we show that the removal of NK cells from L3TU also diminishes a significant portion of IFN� from the TME. However, further studies should to be conducted to directly correlate the increase presence of IFN� in the TDLN to assess changes in T cell phenotypes in L3TU versus WTTU. This would expand our understanding of the significance of having an enhanced presence of IFN� at the PTS and in the TDLN during
T cell priming.
Assessing the importance of NK cell supplied IFN� on T cell phenotypes in the
primary tumor
The interrogation of T cell phenotypes at the PTS is more straight forward than assessing ones migrating in the TDLN. Tumor can be extracted from mice after 21 days post tumor injection with WTTU or L3TU and those that have been depleted of NK cells.
All T cells that infiltration the primary tumor are likely to be activated and express high
CXCR3179. Immune cells should be isolated from the tumors and assessed using FACS for molecules that define the cells as either Treg and Th1 for CD4+ cells, or expression proteins that define whether a CD8+ T cells is exhausted or active. IFN� should promote more Th1
131 differentiation in the TDLN and therefore we would expect to see more Th1 responses at the PTS from CD4+ T cells. Exhausted CD8+ T cells show limited to no production of IFN� production232.
Assessing the importance of NK cell supplied IFN� on T cell phenotypes in the
tumor draining lymph node
The continuous migration of immune cells moving in and out of the TDLN need to be considered when interrogating changes in the TDLN. The first step would be to administer NK blocking antibodies followed one day later by antibodies to block immune cell egress. Using the S1P blocking antibody Fingolimod (FTY720) prior to tumor injection would allow the capture of incoming cells, but block their exiting from LNs. WTTU and
L3TU should then be administered to mice with and without NK cell depletion via s.c. footpad injection 24 hours later. Next the decision of when to stop blocking should be assessed and what type of analysis to use. Considering that our data shows that T cells leave the TDLN between 7 and 10 days (Fig. 7), FTY720 should be administered before
10 days in order to maximize the capture of exiting lymphocytes. It may be best to extract
TDLNs at day 7. This is because on day 10, we have unpublished data to suggest that there is a significant population of Treg cells remaining in the L3TU compared to WTTU (Fig.
30B). In addition, we show that on day 5, before the rapid egression, activated CXCR3+
CD8+ T cells are treading toward significance from WTTU (Fig. 30C; P= 0.09) while there is no difference between the tumor types for Tregs on the same day (Fig. 30A). This would suggest the possibility, that sometime after day 5 and before egress on day 10, fully
132 activated CD8+ T cells are accumulating in the TDLN in significant amounts, and the time to catch them would be before day 10 but after day 5.
On day 7, the TDLNs should be extracted and FACS analysis performed to assess the differences in T cell phenotypes between WTTU and L3TU with and without NK depletion. For CD4 T cells, this should consist of protein expression patterns that describe
Tregs or Th1 phenotypes. For Tregs, use CD3, CD25, CD4, FOXp3, and TGF� monoclonal antibodies. For Th1, use CD3, CXCR3, CD4, and IFN� monoclonal antibodies. TGF� and IFN� may not be necessary for defining Tregs and Th1 phenotypes, but it will give information regarding whether the aCCL3 affects the function of cytokines in vivo. For CD8+ T cells, use CD3, CD8, CXCR3, and IFN�. CXCR3 is constitutively expressed on activated CD8+ T cells while IFN� will reveal information about the functionality of CD8+ T cells.
CONSIDERING THE INTERPLAY BETWEEN CCL3, CCL5, AND NK
CELL RESPONSE IN L3TU
Human and mice NK cells both express the ligands CCR1 and CCR5233,234.
Therefore, considerations should also be given to CCL5, which is expressed in the CT26 model (Figure 24A)199,223. We show that NK cells are present in the TME of WTTU TDLN and PTS (Figures 7E, 21A). Conversely, in comparison to NI, WTTU NK cells are not a significant source of IFN� during T cell priming (Figure 8A, 8F). However, CT26 cells are susceptible to NK cell killing under some settings,235 which can attract and activate NK cells in vivo169,236. In this context, CCL5, along with CCL3, should drive CT26 rejection.
Indeed, NK cells do provide some protection in the WTTU setting without CCL3 (Figure
133 15). It could be very true that the addition of CCL3 in CT26 TME acts synergistically with
CCL5. It could also be true that CCL3 and CCL5 bind to the same receptors, but have different affinities and preferential recruitment of immune cell subsets that could influence the effector potential of NK cells in the TME. Lastly, CCL3 and CCL5 binding may induce different biological responses in some cases as well. Caution should still be given when making assumptions about chemokines binding to the same receptors binding leading to the same outcomes. For example, CCL3 and CCL5 can both attract Tregs53 and NK cells169 in vivo, but there is evidence which suggest they may enact different biological and migratory affects237. Other reports have suggested that CCL5 is upregulated in some cancers such as melanoma,53 while CCL3 and CCL4 are suppressed in others176,238.
Conversely, the upregulation of CCL3 has been observed as a driver of melanoma rejection in some studies as well179,238. Lastly, CCL5 has been shown to be a more potent recruiter than CCL3 for Tregs53,54 while CCL3 had greater effect over NK cytolytic and chemokine upregulation236.
DENDRITIC CELL MATURATION IN THE TUMOR MICROENVIRONMENT
Ag recognition is a critical component for any tumor immunotherapy to be successful. The interactions between DC and TAAs put them at the crossroads of driving tolerant or antitumor adaptive immune responses in many tumor models94,239-241. In addition, tumors escape mechanism that shelter them from NK cell cytotoxicity, must also avoid alerting DCs to the presence of danger. Tumors can also evade immune detection from adaptive immune responses by hiding from or altering tumor-infiltrating DCs (TIDC).
Some tumors express TAAs, but also release suppressive cytokines or directly interact with
134 DCs to trigger tolerance242. Cytokine and direct cell-to-cell contact can interfere with DC maturation and keep them in an immature state which will favor the induction of tolerance242. Tumors can also cause dysfunction in DC motility and Ag processing, keeping
DCs immobile at the PTS, suppressing endocytosis to keep them immature, or inducing
DC apoptosis94. Therefore, therapeutic efforts to drive DCs to promote antitumor adaptive responses will depend on whether there are TAAs that DCs can recognize, and a presence of appropriate inflammatory cytokines.
Future experiments using L3TUs should consider the direct effects on DC maturation in the TDLN and the PTS. Increases in mature DCs are the bases for many tumor immunotherapies23. The use of constitutively expressing CCL3 or the continuous infusion of CCL3 may have therapeutic benefits as a natural maturing agent for TIDCs.
Our work and others have shown that CCL3 in the TME may be able to aid or enhance the
DCs maturation process (Figure 11)194,243. In vitro studies have suggested that DCs pretreated with CCL3 will lead to enhanced Ag uptake by maintaining DCs in a semi- mature phenotype194,243. After stimulating with lipopolysaccharide (LPS) these DCs demonstrated large increases in CD4+ T cells proliferation compared to untreated194,243. In addition to in vitro data on DCs pretreated with CCL3 there is indirect evidence in our present work to suggest this that DC maturation may be enhanced in vivo. Without inflammation or some stimulating factor, rCCL3 alone does not have apparent or lasting influences on DLNs as it relates to cellular trafficking (Figure 7H). Contrasting this with the addition of CT26, we show enhanced DC migration to TDLNs and subsequent increases in T cell activation, suggesting that DCs are maturing under these conditions.
135 This has yet to be tested in vivo. There are a few ways to assess this in vivo question. The most direct way would be to isolate CD11c+ cells from the PTS and the TDLNs, then conduct FACS analysis. The first step in both the TDLN and at the PTS should be to assess whether the DCs maturation is suppress or not. The most direct way would be to analyzed
DCs for the expressions of CD83, which is expressed exclusively on mature DCs244,245.
Some reports have shown that tumors can suppress the expression of CD83 on various DC populations, and this is also correlated with lower expressions of MHC and co-stimulatory molecules246. Since DCs exist across a range of maturity states, analysis of MHC molecules, MHC-I and MHC-II, and co-stimulatory molecules CD80, CD86, and CD40 should also be done. Lastly, we have unpublished data to suggest that B220+ DC and CD8+
CD103- DCs are also accumulating in greater quantities in the L3TU injected mice compared to WTTU (data not shown). B220+ DC typically enter LNs from the peripheral circulation and CD8+ CD103- DCs are typically residential DCs accumulate directly from the BM247. Therefore, a further breakdown of the types of DCs accumulating in the TDLN across days during T cell priming will also broaden our understanding of the dynamic changes occurring in the TDLN. This is important, because different DC sub-types can result in different biological consequences. Keeping a whole picture analysis in mind when conducting analysis of DC maturity status in vivo is also important. This is because a false assessment between DC maturation in the TDLN and at the PTS could occur if considerations are not taken regarding the CCL3, CCR1, and CCR5. CCL3 will attract
CCR1 and CCR5 positive cells DCs which will initially have a more immature phenotype.
In fact, we have preliminary data to suggest this is the case within the TDLN around day 7
(data not shown, unpublished). Without considering this implication in the context of
136 proinflammatory cytokines, it could be assumed that more immature DCs exist in the TME of L3TU than WTTU, and therefore more tolerant responses should be found in the L3TU system. This of course is likely not the case in a system were TAAs are captured by DCs in an NK-rich environment with enhanced IFN� presence.
We also have unpublished data that suggest the possibility that DCs at the PTS may more readily migrate to the TDLNs (Fig. 31). We administered intra-tumor injections of
CFSE-labeled BMDCs into established WTTU and L3TU. Then 24 hours later we analyzed their accumulation into the TDLN. The data suggests that L3TU may enhance DC migration from the PTS to the TDLN. However, repeated experiments should be conducted to confirm this observation. We also assessed status of the LN-homing receptor, CCR7, by
FACs after pretreatment with CCL3 (data not shown), but our way of analysis proved to be problematic. This could be due to rapid internalization of CCR7 after binding, and the answer to this question remains unclear. It may be more appropriate to analyze CCR7 by intra-cellular staining or qPCR if a follow experiment is pursued.
THE INTERACTION OF CCL3, INTERFERON-GAMMA, AND CCL5 IN THE
L3TU TUMOR MICROENVIRONEMENT
The dual role of IFN� in tumors
Early studies in mice using recombinant IFN� showed much promise as a therapy for eliminating tumors. The development of IFN�R knockout mice brought a lot of excitement to the field of tumor immunology. In vivo studies revealed that some tumors,
137 normally rejected in WT, grew unimpeded in mice that were unresponsive to IFN�62. In addition, IFN� was also shown to have anti-proliferative effects on some tumor models and enhance tumor immunogenicity by augmenting the upregulation of MHC-I and –II expressions leading the enhance T cell recognition62. Lastly, many studies have confirmed that IFN� can also drive the differentiation of CD4+ T cells toward a Th1-phenotype 248.
Th1 cells are also potent producers of IFN� and are heavily correlated with antitumor responses248,249. The observations recorded from these types of studies were the bases of what led to clinical trials to treat several different types of tumor62. However, despite hopes for using IFN� as an adjuvant to stimulate tumor apoptosis and antitumor immune responses, the efficacy of recombinant IFN� in clinical trials resulted in mixed reviews, and in some reports, actually aiding in tumor survival62. This is because IFN�, can play a dual role in the development of the inflammatory response. For example, clinical trials using recombinant IFN� in bladder and ovarian cancers led to tumor rejection, while in melanoma tumors, there had no discernable effects or it resulted in a greater tumor growth62. IFN� upregulation can augment the expression of MHC molecules on tumors but this can the dual effect of neutralizing from NK cell effector function, but allowing increase recognition of TAAs by effector T cells. However, in some cases, IFN� may also be able to hide from effect T cells in the same manner by decreasing recognition of tumor Ags. For example, in the context of CT26 tumors, IFN� has been shown to upregulate MHC-I, but also downregulate the expression of AH-1, thereby hiding the immunodominant tumor Ag from CD8+ T cell recognition250. IFN� can also promote the expression indoleamine 2,3- dioxygenase (IDO) in some tumors leading to the recruitment of regulatory T cells (Tregs) and promoting tumor survival62,251. However, despite having pro-tumorogenic roles, IFN�
138 remains one of the most potent initiator of the inflammatory response. These pro- tumorogenic roles (e.g. MHC upregulation, Treg induction) can be viewed as a way for the immune system to self-regulate and keep uncontrolled immune responses in check. Finding ways to elicit the proinflammatory responses of IFN� while avoiding some of the self- regulating mechanisms by IFN� may help improve future applications of IFN� as a tumor immune therapy.
Identifying and targeting tumor intrinsic pathways related to IFN� and WNT/�-
catenin, to enhance the constitutive expression CCL3 and tumor rejection
There are also instances where IFN� and CCL3 can act in a paracrine and autocrine fashion to induce the expression of the other. IFN� can enhance the presence of CCL3 in inflamed tissues and lymphoid organs through the polarization or activation of various cell- types (e.g. DCs, macrophages, Th1 cells, NK cells)252. CCL3 can in turn promotes IFN� expression though immune modulation and activation of some cell-types, such as macrophages and NK cells, resulting in enhanced production of cytokines like IFN�48,178.
In addition, CCL3 can increase contacts between NK cells and DCs in TDLNs and activate
NK antitumor responses253. IFN� and CCL3 can also act sequentially to drive inflammatory responses. For example, IFN� can drive the upregulation of CCL3, which in-turn enhances or initiate the recruitment of neutrophils and myeloid cells to sites of inflammation,190,254. However, IFN� may also act as a suppressor of CCL3 expression as well191. Some reports using IFN� and IFN�R knockout mice showed that IFN� also attenuates neutrophils and myeloid cells infiltration255,256. The mechanism may be due to
IFN� downregulation of CCL3 transcription191. This report demonstrates how IFN�
139 signaling could downregulate the expression of CCL3 and CCL4, in a STAT1 directed manner, in adherent peritoneal exudate cells (APEC, macrophages of the gut), bone marrow–derived macrophages, and in IFN�-knockout mice infected with Serovar
Typhimurium191. Noteworthy in this report is the mention that this downregulation by IFN� was a delayed response, suggesting that IFN� suppresses CCL3 and CCL4 in a temporal manner possibly as a way to regulate inflammatory responses191.
WNT/�-catenin and IFN� also show crossover as well in some tumor systems.
IFN� has been demonstrated to be a strong repressor of the �-catenin pathway in hepatocellular carcinoma cells in a STAT3 directed manner257. This suggest that IFN� may directly inhibit �-catenin signally via STAT3 pathways, but also suppress CCL3 and CCL4 upregulation though STAT 1 directed pathways191,257.
A question to consider with this information is whether tumors that can constitutively expressed CCL3 rebuffs IFN� regulation, but allow IFN� proinflammatory responses? Tumors transfected or transduced with CCL3 will not upregulate CCL3 though natural mechanisms, and would not be subject to normal IFN� regulation so these types of tumor would not be a good model to identify intrinsic tumor pathways. Tumors that do not express CCL3 normally, but have an active WNT/�-catenin pathway might allow for a direct pathway to create an inducible system to test for IFN� suppression of CCL3 in tumors. In vitro studies using unstimulated APECs demonstrated how IFN� can drive the repression of CCL3 in though STAT-1 activation leading to further downstream activation of p38MAPCK and subsequent ATF3 injection (Fig. 32A). Several reports have shown
140 that ATF3 induction suppresses CCL3 and CCL4 upregulation (Fig. 32A)176,191. WNT/�- catenin signaling has been associated with the activation of p38MAPK activation, subsequent ATF3 induction, and the suppression of CCL3 and CCL4 (Fig 32A)191. It stands to reason that knocking out WNT/�-catenin in a similar fashion as Spranger et. al, would allow for the expression of CCL3 in some tumor systems, and IFN� to be used as an inducible repressor of CCL3 along the same pathway176. However, CT26 from our system may not be the best model to test this inducible system. Genomic reports on CT26 show the WNT/�-catenin pathway is also active in these tumors206. IFN� suppression CCL3 and
CCL4 also involves the induction of nitric oxygen synthase-2 (NOS2), which is not produced by CT26258. Nitric oxide (NO) has been the subject of lot of studies because of their dual role in tumorigenesis259. Many tumors produced NO and are subject to IFN� stress in vitro, but in vivo they have evolved other mechanisms to survive259. The goal would be to find a tumor cell line that can be stimulated by IFN� along the IFN�/NO2 pathway that would also drive CCL3 suppression. These tumors must also have an intact
WNT/�-catenin pathway which can be knocked out without affecting growth or cell viability. There are many tumor cell lines that express various components of these. One such tumor cell line might be the colon tumor HT-29. These tumors can evade immune rejection, have intact WNT/�-catenin pathways, and can be stimulated by IFN� to produce
NO259-261. The first step would be to knockout the WNT/�-catenin pathway and see if this results in the expression of CCL3. Next would be to test the suppression of CCL3 transcription, via qPCR, to verify whether IFN� can suppress CCL3 expression despite
WNT/�-catenin deletion. Last would be to design a strategy to test in vivo. A schematic of this design and hypothesized outcome is listed in figure 32B. The readout from this
141 experiment would be serum detection CCL3 by ELISA. The hypothesis would be that tumors growing in IFN� knockout mice (IFN�-/-) mice would have the highest concentration serum CCL3, because there would be no endogenous IFN� to suppress
CCL3 transcription in the tumors. IFN�R-/- would have the lowest concentration of serum
CCL3, because IFN� upregulation by endogenous population would increase unchecked due to the lack of sensitivity by the endogenous population of IFN�R-/- mice. WT mice would be somewhere in the middle because IFN� would be upregulated normally as compared to IFN�R-/- mice with tumors.
The data from these experiments would not only confirm the findings from
Spranger et. al,176 in other tumor models but potentially it could be used to reveal other intrinsic targets that can targeted along the interphase between the WNT/�-catenin and
IFN� signaling pathways.
THOUGHTS ON THE THERAPEUTIC APPLICATION OF USING HIGH DOSE
RECOMBINANT CCL3
In chapter 3 we discussed the importance for considering the delivery route of recombinant CCL3 therapy. We show that high dose bolus injections of rCCL3 distally from the PTS resulted in slowing tumor progression (Figure 17). Another consideration should be given to the actual biological activity of CCL3 in vivo. Reports show that CCL3 at concentrations less than 100 ng/ml CCL3 exists in its monomeric form. However, for our rCCL3 therapies, we use concentration equal to 100 ng/ml for s.c. injections31. At
142 concentrations equal or greater than 100 ng/ml, CCL3 forms reversible dimers and other various dodecamer (>100 µg/ml) aggregates31. However, self-aggregation of CCL3 does not block their bioactivity31. The ability to dimerize and oligermorize in vivo can protect some proteins, such as chemokines, from enzyme degradation, thereby protecting their function262. For this reason, concentrations equal to or greater than 100 ng/ml of CCL3 may need to be considered in the context of delivery in the clinical setting.
The formation of aggregates may have different biological consequences. The formation of aggregates in vivo with CCL3 does not affect its biological function, however, it may affect its ability to diffuse readily though tissues and therefore have relevance in clinical applications. For this reason, in some clinical applications, the formation aggregates may not be favorable. It is unknown whether aggregate formations of cytokine or chemokine protein infusion has negative or positive consequents on biological efficacy.
Finally, we show two different ways in which to effectively deliver high dose
CCL3. The first is to use constitutively CCL3 from irradiated tumors. The second would be to deliver rCCL3 using an approved drug delivery method for long-term dose delivery263.
143
FINAL THOUGHTS
CCL3 is like most immune cells, cytokines, and chemokines, in that its role in tumor system is complex, with effector functions that drive immune suppression or inflammation. These complexities must be taken into account when designing any therapy, including chemokine therapies. An assessment of a tissue’s microenvironment (e.g. lung, colon) and a tumor biopsy to evaluate the kinds of chemokines that are upregulated by the tumor may be critical to figuring out what type of chemokine or chemokine-combination therapy to use. For example, breast cancers that have metastasized to the lung correlates
CCL3 with tumor progression264 while melanoma rejection is achievable in a CCL3 directed manner12,179. These considerations are also important with tumors of the same tissue origin. For example, some studies have reported the presence of CCL3 in human colon cancer aids in tumor survival,265 however, others have correlated CCL3 with colon cancer patients with favorable outcomes266.
The data presented here with CCL3 is not meant to convey it as a cure for cancer.
This project incorporates the use of inflammatory chemokines in the TDLN microenvironment to provide a rationale for incorporating these chemokines to enhance immunotherapeutic efficacy against cancer. It is precisely CCL3’s unique involvement in the early stages of the adaptive inflammatory process, and its specificity to drive the immune response toward inflammation in the TDLN and the PTS, that makes it an attractive candidate for research in tumor immunology.
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