INTERFERON-GAMMA-MEDIATED IMMUNOEVASIVE STATEGIES IN MULTIPLE MYELOMA
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Paul David Ciarlariello
Graduate Program in Molecular Cellular and Developmental Biology
The Ohio State University
2016
Dissertation Committee:
Don M. Benson, Jr., MD, PhD -Advisor
Michael A. Caligiuri, MD
Gregory Lesinski, PhD
Natarajan Muthusamy, DVM, PhD -Advisor
Flavia Pichiorri, PhD
Copyrighted by
Paul David Ciarlariello
2016
Abstract
Natural killer (NK) cells are a major source of interferon-γ (IFN-γ) and may play a key role in innate immunity against multiple myeloma (MM). IFN-γ abrogates MM tumor cell in vitro
proliferation, however, clinical trials of recombinant IFN-γ in patients with MM showed no
benefit. MM cells exhibit strategies designed to evade NK cell surveillance and lysis. Herein,
we provide evidence of a novel means of MM immune evasion in which IFN-γ appears to
play a reciprocal relationship between MM and NK cells. Patients with MM exhibit higher
levels of serum IFN-γ levels than in the healthy setting. NK cells produce IFN-γ in response
to MM cells which express functional IFN-γ receptors. Stimulation with IFN-γ leads to
increased transcription and expression of the inhibitory ligands HLA-E and PD-L1 by MM
cells. This effect may be overcome by interruption of the NKG2A / HLA-E interaction.
Intriguingly, MM cells release extracellular vesicles (EV) which are capable of enhancing
IFN-γ production of NK cell, thus describing a potential cyclic mechanism of perpetual
immune detriment. Taken in total, these results suggest that MM cells subvert the favorable
effects of IFN-γ to attenuate NK cell tumor-directed cytotoxicity.
ii Dedication
To my wife, Nicole, and my baby, Gianluca.
To my parents, Paul and Sue.
I am a scientist - I seek to understand me all of my impurities and evils yet unknown
I am a scientist - I seek to understand me I am an incurable, and nothing else behaves like me
I am a scientist- I seek to understand me [...] the hole I dig is bottomless, but nothing else will set me free
-Guided By Voices
iii Acknowledgments
I would like to thank my advisor Dr. Don M. Benson, Jr. for taking the plunge and adopting me as his first and only graduate research student. Don, you have created a supporting environment in which I could grow as a scientist. If even an ounce of your knowledge, generosity, hospitality, and disposition have rubbed off on me, I will no doubt be a better researcher, father, and overall human being. Thank you, for lending your expertise and shepherding my training. I am forever grateful.
Of course, I would like to acknowledge my co-mentor Dr. Raj Muthusamy for the many constructive conversations which kept me on track to graduate. Raj, curiosities pour out of you and I have had such a rewarding experience discussing my data and its implications. I will always admire your method and approach to properly designed and controlled experiments.
A special thanks to Dr. Michael A. Caligiuri who brought me on to his team in 2007. Mike, you dispense positivity and influence with every word and action to those around you, and I am grateful for your support and guidance throughout the years.
To Dr. Flavia Pichiorri, thank you for the many collaborative efforts and willingness to meet and discuss our mutual findings. I have enjoyed our colorful conversations and your constant excitement for decoding life’s secrets.
To Dr. Greg Lesinski, thank you for your constructive and honest feedback throughout the years. I have always felt at ease in our exchanges, knowing that you were fully present and engaged in my scientific stories.
iv I extend my endless respect and gratitude to my science “mommy”, Dr. Rossana Trotta. Rox,
you make hypothesis-driven methods look deceivingly easy. I did not have a full appreciation of your expertise in experimental design until I was on my own. I think of you when considering experimental controls and especially when I am qualifying the conditions of my results. You have left such an impression on the scientist I have become. Thank you.
I must honor and recognize my lab mate, Dr. Tiffany Hughes for her many supportive roles during my tenure. You have offered your help every day for the past six years and have asked nearly nothing in return. We as a lab were truly lucky to recruit you, and I am confident your many contributions will help rid the world of myeloma, once and for all.
Some of the most rewarding experiences in the lab were moments while mentoring my undergraduates D. Maxwell Banaszak and Rohit Menon. I am glad to have been a part of your experience here at OSU. I know you both are ready to take on the world.
My parents Paul and Sue Ciarlariello have selflessly supported me my entire life. Through their efforts, I have been afforded the free reign of pursuing my passions. Mom and Dad, I am without words as to how incredible my life experience has been. Let this accomplishment symbolize your success as parents. I have been and will always be proud to be your son.
To my love, Nicole Danielle Ciarlariello. You are the most intuitive, thoughtful, and gentle soul I have ever come across and I am lucky to have you by my side. We now embark on new beginnings with our baby boy, Gianluca. I don’t care where we end up, as long as we are together. Thank you for believing in me, and trusting me with your heart.
v
Vita
November 4, 1983 ...... Born, Dayton, Ohio
June 2002 ...... West Carrollton High School
June 2006 ...... B.S. Biochemistry,
The Ohio State University
September 2010 - Present ...... Molecular, Cellular, and Developmental
Biology Graduate School Program,
The Ohio State University
Publications
Harshman, S. W., A. Canella, P. D. Ciarlariello, K. Agarwal, O. E. Branson, A. Rocci, H. Cordero, M. A. Phelps, E. M. Hade, J. A. Dubovsky, A. Palumbo, A. Rosko, J. C. Byrd, C. C. Hofmeister, D. M. Benson, Jr., M. E. Paulaitis, M. A. Freitas and F. Pichiorri (2016). "Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers." J Proteomics.
Briercheck EL, R. Trotta ,L. Chen , A. S. Hartlage, J. P. Cole, T. D. Cole, C. Mao , P. P. Banerjee, H. T. Hsu , E. M. Mace, D. Ciarlariello, B. L. Mundy-Bosse, I. Garcia-Cao, S. D. Scoville, L. Yu, R. Pilarski, W. E. Carson, G. Leone, P. P. Pandolfi, J.Yu, J. S. Orange and M. A. Caligiuri (2015). “PTEN Is a Negative Regulator of NK Cell Cytolytic Function” J Immunol 194(4):1832-40. doi: 10.4049/jimmunol.1401224. Epub 2015 Jan 16.
Collins, S. M., C. E. Bakan, G. D. Swartzel, C. C. Hofmeister, Y. A. Efebera, H. Kwon, G. C. Starling, D. Ciarlariello, S. Bhaskar, E. L. Briercheck, T. Hughes, J. Yu, A. Rice and D. M. Benson, Jr. (2013). "Elotuzumab directly enhances NK cell cytotoxicity against myeloma via CS1 ligation: evidence for augmented NK cell function complementing ADCC." Cancer Immunol Immunother 62(12): 1841-1849.
vi Harshman, S. W., A. Canella, P. D. Ciarlariello, A. Rocci, K. Agarwal, E. M. Smith, T. Talabere, Y. A. Efebera, C. C. Hofmeister, D. M. Benson, Jr., M. E. Paulaitis, M. A. Freitas and F. Pichiorri (2013). "Characterization of multiple myeloma vesicles by label-free relative quantitation." Proteomics 13(20): 3013-3029.
Trotta, R., L. Chen, S. Costinean, S. Josyula, B. L. Mundy-Bosse, D. Ciarlariello, C. Mao, E. L. Briercheck, K. K. McConnell, A. Mishra, L. Yu, C. M. Croce and M. A. Caligiuri (2013). "Overexpression of miR-155 causes expansion, arrest in terminal differentiation and functional activation of mouse natural killer cells." Blood 121(16): 3126-3134.
Trotta, R., L. Chen, D. Ciarlariello, S. Josyula, C. Mao, S. Costinean, L. Yu, J. P. Butchar, S. Tridandapani, C. M. Croce and M. A. Caligiuri (2012). "miR-155 regulates IFN-gamma production in natural killer cells." Blood 119(15): 3478-3485.
Trotta, R., D. Ciarlariello, J. Dal Col, H. Mao, L. Chen, E. Briercheck, J. Yu, J. Zhang, D. Perrotti and M. A. Caligiuri (2011). "The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells." Blood 117(8): 2378-2384.
Costinean, S., S. K. Sandhu, I. M. Pedersen, E. Tili, R. Trotta, D. Perrotti, D. Ciarlariello, P. Neviani, J. Harb, L. R. Kauffman, A. Shidham and C. M. Croce (2009). "Src homology 2 domain-containing inositol-5-phosphatase and CCAAT enhancer-binding protein beta are targeted by miR-155 in B cells of Emicro-MiR-155 transgenic mice." Blood 114(7): 1374- 1382.
Trotta, R., J. Dal Col, J. Yu, D. Ciarlariello, B. Thomas, X. Zhang, J. Allard, 2nd, M. Wei, H. Mao, J. C. Byrd, D. Perrotti and M. A. Caligiuri (2008). "TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells." J Immunol 181(6): 3784-3792.
Trotta, R., D. Ciarlariello, J. Dal Col, J. Allard, 2nd, P. Neviani, R. Santhanam, H. Mao, B. Becknell, J. Yu, A. K. Ferketich, B. Thomas, A. Modi, B. W. Blaser, D. Perrotti and M. A. Caligiuri (2007). "The PP2A inhibitor SET regulates natural killer cell IFN-gamma production." J Exp Med 204(10): 2397-2405.
Fields of Study
Major Field: Molecular, Cellular, and Developmental Biology
vii Table of Contents
Abstract ...... ii
Dedication...... iii
Acknowledgments ...... iv
Vita ...... vi
Publications ...... vi
Table of Contents ...... viii
List of Tables ...... xii
List of Figures...... xiii
List of Abbreviations ...... xv
CHAPTER 1: BACKGROUND...... 1
1.1Multiple Myeloma Biology ...... 1
1.1a Multiple myeloma overview and discovery ...... 1
1.1b Multiple myeloma diversity and progression ...... 3
1.1c Multiple myeloma and plasma cell development ...... 10
1.1d Multiple myeloma emergence and the bone marrow niche ...... 11
1.1e Communication in multiple myeloma including extracellular vesicles ...... 13
viii 1.2 Natural Killer Cell Biology ...... 17
1.2a Natural killer cell effector function overview ...... 17
1.2b The immune synapse and natural killer cell receptor signaling ...... 18
1.2c Major histocompatibility complex class-I biology ...... 22
1.2d Interferons ...... 27
1.2e Interferon-gamma and STAT1 signaling ...... 28
1.3 STAT3 signaling in multiple myeloma ...... 29
CHAPTER 2: INTERFERON-GAMMA LOWERS THE SUSCEPTIBILITY OF
MULTIPLE MYELOMA TO NATURAL KILLER CELL-MEDIATED CYTOTOXICITY
...... 31
2.1 Introduction ...... 31
2.2 Results with Figures ...... 32
NK cells produce IFN-γ in response to MM ...... 32
NK cells produce IFN-γ in response to soluble MM factors ...... 33
IFN-γ increases MM cell surface expression of inhibitory ligands ...... 39
IFN-γ lowers the susceptibility of MM cells to NK cell-mediated cytotoxicity ...... 48
IFN-γ-mediated resistance is differentially recovered by IPH2201 (Anti-NKG2A) ...... 54
CD56Bright rather than CD56Dim NK cells exhibit greater killing against MM cells ...... 56
2.3 Discussion and Summary ...... 61
2.4 Experimental Methods ...... 63
ix Transwell co-cultures ...... 65
Flow cytometry and sorting ...... 65
Real time PCR ...... 66
Western blot analysis ...... 66
ELISA assays ...... 67
Cytotoxicity assays ...... 67
CHAPTER 3: MULTIPLE MYELOMA CELL-DERIVED EXTRACELLULAR VESICLES
ENHANCE IFN-GAMMA PRODUCTION BY NATURAL KILLER CELLS ...... 69
3.1 Introduction ...... 69
3.2 Results and Figures ...... 70
MM EV enhance NK cell-mediated IFN-γ production ...... 70
MM EV contain HLA-E and PD-L1 Inhibitory Ligands ...... 75
MM EV present ligands on their surface ...... 79
3.3 Discussion ...... 81
3.4 Methods ...... 83
Cell lines and NK cell preparations ...... 83
Treatments ...... 84
Extracellular vesicle isolation ...... 84
Flow cytometry ...... 85
EV surface staining and detection by Nanosight ...... 85
x ELISA assays ...... 86
Western blot analysis ...... 86
CHAPTER 4: FUTURE STUDIES AND EXTENDED DISCUSSION ...... 88
4.1 The mechanism for IFN-γ-mediated resistance of U266 to NK cell cytotoxicity ...... 88
4.2 IFN-γ treatment of MM cells to enhance efficacy of proteasome inhibitors ...... 89
4.3 Differential peptide cross-presentation in MM following IFN-γ treatment ...... 89
4.4 Concluding remarks ...... 90
Bibliography ...... 94
xi List of Tables
Table 1: IMWG Criteria for Diagnosing Multiple Myeloma ...... 4
Table 2: Common Chromosomal Abnormalities in Multiple Myeloma ...... 6
Table 3: Attempted Primary NK Cell Cytotoxicity Experiments Involving MM EV ...... 82
xii List of Figures
Figure 1: “Church spire” pattern in MM patient sera ...... 2
Figure 2: Immunoglobulin classes ...... 8
Figure 3: Extracellular vesicle release ...... 16
Figure 4: MHC-I Structure ...... 23
Figure 5: Healthy primary NK cells produce IFN-γ when cultured with MM ...... 33
Figure 6: IFN- γ production in MM-NK cell co-culture does not require cell-cell contact .... 34
Figure 7: MM cells constitutively express IFN-γRs on their surface ...... 36
Figure 8: IFN-γ activates STAT1 and STAT3 in MM cells ...... 37
Figure 9: Transcription of HLA-E and PD-L1 by MM cells after IFN-γ treatment ...... 38
Figure 10: Surface expression of HLA-ABC by MM after IFN-γ treatment ...... 41
Figure 11: Surface expression of HLA-E by MM after IFN-γ treatment ...... 41
Figure 12: Surface expression of PD-L1 by MM after IFN-γ treatment ...... 45
Figure 13: Transcription of HLA-E and PDL-1 by MM cells in contactless co-culture with
NK-92 ...... 45
Figure 14: Surface expression of HLA-E and PD-L1 by MM cells in contactless co-culture with NK92 ...... 47
Figure 15: Viability and susceptibility of MM cells to NK cell cytoxicity after IFN-γ treatment ...... 49
xiii Figure 16: IFN-γ confers resistance of MM cells to NK cell-mediated killing, no stimulation
...... 51
Figure 17: IFN-γ confers partial resistance of MM cells to NK cell-mediated killing, activating conditions ...... 52
Figure 18: IFN-γ dose-dependent resistance of MM cells to NK cell-mediated cytotoxicity . 53
Figure 19: IPH2201 (Anti-NKG2A) pretreatment of NK cells recovers IFN-γ-mediated resistance ...... 55
Figure 20: Comparing NK cell subsets in healthy and MM peripheral blood ...... 57
Figure 21: Sorted CD56Bright and CD56Dim NK cell cytotoxicity against MM cell line RPMI-
8226 ...... 58
Figure 22: Effect of IPH2201 (Anti-NKG2A) on CD56Bright and CD56Dim NK cytotoxicity against MM cell line RPMI-8226 ...... 60
Figure 23: Extracellular vesicle isolation and prep validation ...... 72
Figure 24: MM cell EV enhance NK cell IFN-γ production ...... 74
Figure 25: Protein profile of EV released from MM cell lines and patient sera ...... 76
Figure 26: HLA-E detected on mass spectrometry study ...... 77
Figure 27: HLA-E and PD-L1 in EV released from MM...... 78
Figure 28: NanoSight NS300 visualization of MM EV and HLA-ABC presentation ...... 80
Figure 29: Graphical Summary of Dissertation Work...... 92
xiv List of Abbreviations aa…………………………………………………………………………………...amino acid ADCC……………………………………...Antibody-Dependent Cell-mediated Cytotoxicity AML………………………………………………………………..Acute Myeloid Leukemia BCR…………………………………………………………………………. B Cell Receptor
β2M……………………………………………………………………..βeta-2-Microglobulin BLIMP1… ……………………………………B-Lymphocyte-Induced Maturation Protein 1 BM…………………………………………………………………..………….Bone Marrow BMSC…………………………………………………………….Bone Marrow Stromal Cell C3…………………………………………………………………Complement Component 3 CD………………………………………………………………..…Cluster of Differentiation CLL………………………………………………………...Chronic Lymphoicytic Leukemia CLP…………..………………………………………………Common Lymphoid Progenitor CRAB………………………...hypercalcemia, renal insufficiency, anemia, lytic bone lesions CXCR5………………………………………………………….CXC-Chemokine Receptor 5 EBF1……………………………………………………………...……..Early B Cell Factor 1 ECM……………………………………………………………..……….Extracellular Matrix ECs…………………………………………………………………………..Endothelial Cells ER…………………………………………………………………….Endoplasmic Reticulum EV………………………………………………………………………Extracellular Vesicles FasL………………………………………………………………………………..Fas Ligand FL………………...………………………………………………………………..Flt3 Ligand FLC…………………………………………………....Free-light-chain (of immunoglobulin) GAS…………………………………………………………….Gamma Activated Sequences HLA……………………..………………………………………..Human Leukocyte Antigen
xv HPC…………………………………………………………...Hematopoietic Progenitor Cell HSC………………………………………………………………....Hematopoietic Stem Cell IFN………..………………………………………………………………………...Interferon Ig..……………………………………………………………..…………….Immunoglobulin IGH……………………………………………………………Immunoglobulin Heavy Chain IL………..…………………………………………………………………………Interleukin IMWG………………………..…………………………International Myeloma Work Group IRF1…………………………………………………………….Interferon Response Factor 1 IRF4…………………………………………………………...Interferon Regulatory Factor 4 IS…………………………………………………………………………….Immune Synapse ITAM…………………………………….Immunoreceptor Tyrosine-Based Activation Motif ITIM……………………………………...Immunoreceptor Tyrosine-Based Inhibitory Motif ITSM………………………………………...Immunoreceptor Tyrosine-Based Switch Motif JAK…………………………………………………………………...Janus Activated Kinase KIR…………………………………………………… Killer Immunoglobulin-like Receptor KL…………………………………………………………………...…………….KIT Ligand LFA-1…………………………………………..Lymphocyte Function-Associated Antigen 1 LPS………………………………………………………………………..Lipopolysaccharide MGUS………..…………………...Monoclonal Gammopathy of Undetermined Significance MHC………………...……………………………………Major Histocompatibility Complex MM………………..………………………….……………………………Multiple Myeloma M-protein…………………………………………………………………Monoclonal protein MTOC……………………………………………………..Microtubule Organizing Complex MVB……………………………………………………………………Multivesicular Bodies MVD……………………………………………………………………..Microvessel Density NCR……………………………………………………………Natural Cytotoxicity Receptor NFκB…………………………………………………………………Nuclear Factor κappa B NK…..…………………………………………………………………..………Natural Killer NKG2…………………………………………………...……………..Natural Killer Group 2 PAX5…………………………………………………………………….Paired Box Protein 5
xvi
PC………..………………………………………………………………………..Plasma Cell PD-1……………………………………………………………………..Programmed Death 1 PD-L1…………………………………………………………...Programmed Death Ligand 1 RANK………………………………………..Receptor Activator of Nuclear Factor Kappa B SDF1………………………………………………………………..Stromal Derived Factor 1 SLAMF7……………………………..Signaling Lymphocytic Activation Molecule Family 7 SLO……………………………………………………………...Secondary Lymphoid Organ SLT……………………………………………………………...Secondary Lymphoid Tissue SMM…………………………………………………..…..….Smoldering Multiple Myeloma SOCS………………………………………………………Suppressor of Cytokine Signaling STAT……………………………………...Signal Transducer and Activator of Transcription TAP………………………………………...Trasnporter Associated with Antigen Processing TBET……………………………………………………………..T-Box Expressed in T Cells TCR…………………………………………………………………………...T Cell Receptor TF………………………………………………….……………………..Transcription Factor TGF………………………………………………….………….Transforming Growth Factor TLR………………………………………………………………………..Toll-Like Receptor TNF……………………………………………………………………Tumor Necrosis Factor TRAIL……………………………………………..TNF-Related Apoptosis-Inducing Ligand VEGF……………………………………………………Vascular Endothelial Growth Factor VLA-4……………………………………………………………………Very Late Antigen 4 XBP1………………………………………………………………..X-Box-Binding Protein 1
xvii
CHAPTER 1: BACKGROUND
1.1Multiple Myeloma Biology
1.1a Multiple myeloma overview and discovery
Multiple Myeloma (MM) is the uncontrolled proliferation of antibody-producing plasma cells
(PC) in the bone marrow (BM) for which there is essentially no cure. MM is the second most common hematological malignancy (after non-Hodgkin lymphoma) in the US and accounts for 1% of all malignant neoplasms and 13% of hematologic malignancies [1] [2]. In 2015, the lifetime risk of developing MM is 0.7% (1 in 143) with an average age range of 65-70 years at diagnosis.
In 1844, the first documented cases of MM in patients Sarah Newbury and Thomas
Alexander McBean were reported. Newbury suffered insufferable pain and multiple bone fractures leading up to her death. Upon McBean’s passing, an autopsy would reveal that his bones had softened and that their content, the marrow, was replaced with a “gelatiniform substance of blood-red colour and of unctuous (greasy) feel”[3]. While examining the urine of McBean, Henry Bence Jones would discover rich protein content, the identity of which would be determined nearly one hundred years later as monoclonal light chains. In 1873, pathologist Johann von Rustizky coined the term "multiple myelomas" upon observing eight separate bone tumors within a deceased patient [4]. With the development of X-ray imaging in the early 1900s, physicians were empowered to assess bone lesions; however therapies
1 were hardly effective. Further, as electrophoretic techniques in the 1940s-1950s were implemented, diagnosing myeloma became easier. MM patient sera have a distinct “church spire” signature attributed to elevated immunoglobulin proteins when compared to healthy sera (Figure 1).To this day, sera protein electrophoresis remains an informative diagnostic for treatment assessment in MM.
Figure 1: “Church spire” pattern in MM patient sera
Figure 1: Cartoon of healthy(left) and MM patient(right) sera electrophoresis on
an agarose gel(top). Proteins separate into albumin α1, α2, β, and γ zones based on
size and charge. Densitometry produces the distinct “church spire” pattern in
(Continue on page 3) 2
(Continued from page 2) myeloma sera (middle, right). Immunofixation (bottom) allows for the Ig subclass
typing of the disease (in this example, IgG Myeloma with κ light chain).
1.1b Multiple myeloma diversity and progression
Despite MM’s prevalence and common symptomatic criteria, the disease is highly variable in
its manifestation. MM is clinically diagnosed at presentation of >10% PCs in the BM or BM
biopsy confirming plasmacytoma, the detection of serum or urinary Monoclonal (M-)protein
at any level, and end-organ damage attributed to plasma cell burden such as hypercalcemia, renal insufficiency, anemia, and/or lytic bone lesions (CRAB criteria, Table 1) [5].
Phenotypic markers of PCs include expression of cluster of differentiation (CD)138, CD38,
and more recently CD319 (SLAMF7, CS1) on their surface [6-8]. Aberrant expression of
CD56 and CD28, but lack of CD19 and CD27 are also commonly observed on MM PCs [9].
3 Table 1: IMWG Criteria for Diagnosing Multiple Myeloma
4 Beyond the surface of the cell, MM is a highly heterogeneous disease, presenting variable
antibody production, chromosomal aberrations, and karyotypic discordance. Like their
healthy PC counterparts, MM PCs clonally expand and selectively produce IgG, IgA, IgM,
IgD, or IgE antibodies (Figure 2), where one isotype dominates during active disease [10].
Although healthy and MM PCs incorporate either Kappa(κ) or Lambda(λ) Ig light chain
components, deviation from a healthy serum κ: λ range of 0.26 to 1.65, indicating clonal
expansion and/or incomplete immunoglobulin formation, is observed in MM [11]. The most common form of MM is IgG(52%) followed by IgA(21%), and free light chain (FLC, 16%)
[12]. Uncommon variants include non-secretory IgD(1%) and rarely IgE(0.01%) [13].
Frequently observed genetic abnormalities in most MM include hyperdiploid and nonhyperdiploid karyotypes. In hyperdiploid MM, a gain of odd chromosomes 3, 5, 7, 9, 11,
15, 19, and 21 is observed, whereas in nonhyperdiploid MM chromosomal translocations of the IgH locus to chromosomes 4, 8, 11, and 16 occur [14-16]. Multivariate studies have refined both risk stratification and prognostic implications of abnormal karyotyping in MM, which are presented in Table 2 [17]. Further, new findings describe heterogeneity within an individual patient’s clonal population of malignant PCs, further complicating efforts to effectively treat MM [18].
5 Table 2: Common Chromosomal Abnormalities in Multiple Myeloma
6 MM is preceded by a premalignant phase known as Monoclonal Gammopathy of
Undetermined Significance (MGUS) [19, 20]. The term MGUS was coined in 1978 after an
extensive 5-year serum study would describe the ambiguous outcomes (some resulting in
death, some completely healthy) of 241 patients whom had all presented initially with
elevated serum M-protein but lacked evidence of advanced disease [21]. MGUS affects 2% and 3% of the population in the United States over the age of 50 and 70, respectively [22].
MGUS is often discovered incidentally during blood evaluations for a variety of clinical
symptoms, including fatigue and forgetfulness [23]. Recently, the International Myeloma
Working Group has refined MGUS criteria to include < 10% of PC in the BM, serum levels
of M-protein < 3 grams per deciliter, and lack of end-organ damage attributed to the plasma
cell burden including CRAB criteria [5]. Although MGUS is considered asymptomatic, some
patients describe neuropathy in their extremities, which is attributed to plasma cell-derived
FLC protein aggregates (amyloidosis) causing systemic microvasculitis [24]. The overall risk
of MGUS progressing to malignancy is ~1% per year, indefinitely [25]. Rather than
treatment, the standard of care for MGUS is active monitoring. Newly diagnosed patients are
recommended to check up every 3-4 months in the first year of MGUS diagnosis and subsequently every 6-12 months if no new symptoms develop [26].
7 Figure 2: Immunoglobulin classes
Figure 2: The prototypic immunoglobulin consists of two heavy chains bound by
disulfide bridges to two light chains (λ or κ). The C-termini of the heavy chains
(Continue on page 9) 8
(Continued from page 8)
make up the base region of the antibody, known as the Fc (Fragmentcrystallizable)
while the N-termini contain variable regions which distinguish the antibody’s
binding affinity, or Fab (Fragmentantigen-binding). The addition of the light chain,
which also contains a variable region, increases the possible binding
combinations the system can generate. Monomers IgG and IgE are free floating
whereas IgD is membrane bound. The Fc regions of both IgA, a dimer, and IgM,
a pentamer, are held together by a J-chain polypeptide.
Emerging from MGUS is an intermediary and indolent phase of MM termed Smoldering
MM (SMM). The smoldering was coined in a 1980 report describing prolonged (>5 years),
elevated, yet stable PC burden in asymptomatic and thus observation-only patients [27].
SMM clinically presents as serum M-protein > 3g/dL or urinary M-protein > 500mg/24hr,
and/or >10% plasma cells present in BM biopsy, but no end organ failure attributed to
CRAB criteria [5]. SMM carries a higher risk of progression to active disease than MGUS.
Indeed, SMM has a 10% per year chance of progressing to malignancy in the first 5 years,
followed by a 3% chance per year for the subsequent 5 years, and ~1% chance for every year
thereafter [28]. Interestingly, SMM patients which present with > 60% BM PC, serum or nonserum-associated FLC ratios > 100, and > 2 focal BM lesions by MRI are at a much greater risk [29, 30]. Such patients carry a 40% chance of progression per year, and
malignancy is nearly inevitable [31].
9 1.1c Multiple myeloma and plasma cell development
The early development of both healthy and MM PCs begin as progenitor hematopoietic stem
cells (HSCs) producing CD34+ common lymphoid progenitor (CLP) cells in the BM. The
commitment to a B cell lineage is driven by cytokines released by stromal cells in the BM
microenvironment, inducing a number of transcription factors (TF) including but not limited
to early B cell factor 1(EBF1), PU.1, E2A, and paired box protein 5 (PAX5) [32].
Development continues as each pro-B cell rearranges its immunoglobulin heavy chain (IgH)
gene segments, whose products pair with a universal surrogate light chain to assemble a
transmembrane IgM capable of binding antigen, the pre-B cell receptor (pre-BCR) [33]. The
pre-B cells phenotypically express CD45, major histocompatibility complex class-II (MHC-
II), CD19, and CD40 on their surface [34, 35]. Recombination-activating genes RAG1 and
RAG2 facilitate combination of 1 of 51 variable(V), 1 of 27 diversity(D) and 1 of 6 joining
(J) gene segments, known as V(D)J recombination, can collectively produce a repertoire of
IgH recognizing > 107 unique antigen specificities [36, 37].
The Pre-B cell, while in the BM, undergoes a crucial test of autoreactivity, by IgM recognition of either cell membrane proteins or soluble factors. Self-reactive events result in clonal deletion (apoptosis) after unsuccessful receptor editing, or anergy. The tolerant naïve
B cell, representing < 3% of all B cells generated, will then exit the BM and home to
CXCL13-rich splenic follicles via CXC-chemokine receptor 5 (CXCR5), CC-chemokine receptor CCR7, and lymph nodes via adhesion molecule JAM-C to await antigen presentation [38-40]. The secondary lymphoid organs (SLO), such as the spleen and lymph nodes, provide a constant supply of circulating antigen.
10
Mature B cell activation occurs when the BCR engages an antigen, either free-floating or facilitated via an antigen-presenting cell (dendritic cell and macrophage). B cell activation can also occur by the T-cell-dependent mechanism of MHC-II peptide presentation and T- cell-independent mechanisms via toll-like receptor (TLR) recognition of lipopolysaccharide
(LPS) and hypomethylated CpG-DNA, or Complement component 3 (C3)-coated antigen which has been extensively reviewed elsewhere [41].
Activated B cells then undergo affinity maturation of the BCR, through further somatic hypermutation, class-switch recombination, and clonal selection allows the B cell with the highest affinity towards the specific antigen to predominate by proliferation. Most clonally expanded B cells will become short-lived effector plasmablasts which immediately secrete antibody against the invading pathogen. Days later, the differentiation of plasmablast to plasma cell initiates through IL-21/STAT3 and CD40L mediated repression of PAX5 [42,
43]. The plasma cell is now committed through the expression of transcription factors B- lymphocyte-induced maturation protein 1 (BLIMP1), interferon regulatory factor 4 (IRF4),
X-box-binding protein 1 (XBP1), and will express cell-binding molecule CD138/Syndecan-1 on its surface [44-47].
1.1d Multiple myeloma emergence and the bone marrow niche
B cells develop in the bone marrow, are affinity-selected in the GCs of SLT and, as plasma cells, egress back to the BM. The cause of PC malignant transformation in MM is currently unknown, but oncogenic events may occur during PC activation in the GC. An early event
11 described in both MGUS and MM is the dysregulation/over expression of the cyclin D gene, a key factor in cell cycle regulation [48]. Chromosomal abnormalities including translocations of the IgH locus during PC class switching in the GC have also been described
[49]. These observations likely serve as primary initiation events to malignancy. Subsequent mutations allow for constitutive activation of the nuclear factor kappa B (NFκB) pathway and somatic activating-mutations of NRAS and KRAS signaling oncoproteins (~15% of MM)
[50]. Plasma cells, malignant or otherwise, are strongly dependent on the BM microenvironment for survival. The homing of PCs to the BM is dependent on the release of chemokine stromal cell-derived factor 1 (SDF1) by BM stromal cells (BMSC) and its interaction with plasma cell-bound receptor CXCR4 [51, 52]. PC adhesion and extravasation to the BM microenvironment is initiated when CD138 binds to fibronectin and type I collagen-rich extracellular matrix (ECM) proteins [53-55]. Further, surface expression and activation of PC integrins, namely lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4), enable the PC to directly interact with osteoblast, osteoclast, stromal, and endothelial cells of the niche [56]. An adhesion event of a MM PC to a BMSC is sufficient to initiate both the MM PC activation of the anti-apoptotic NFκB pathway and
BMSC secretion of key MM survival and growth factor IL-6. In response to IL-6 activation, the MM PCs produce and secrete proangiogenic vascular endothelial growth factor (VEGF) which aids in the restructuring and optimization of the bone marrow niche for long term MM cell growth [57, 58].
12 1.1e Communication in multiple myeloma including extracellular vesicles
The bone marrow niche is a dynamic environment, constantly self-renewing, while
restructuring ECM components and providing hematopoietic support for developing
erythrocytes and immune cells. As MM progresses and tumor burden increases, PCs
outcompete and squelch normal hematopoietic cell processes, thereby altering the makeup
and function of cells, cytokines, and the ECM in the BM.
As discussed in 1.1d, PCs binding to BMSCs, induces proangiogenic factor VEGF and bFGF
[59]. BM angiogenesis and neovascularization, provided by endothelial cells (ECs) and measured by microvessel density (MVD), is increased in MM, but not MGUS [60]. Further, bone resorption and reformation is disrupted in MM, resulting in systemic hypercalcemia and
focused lytic bone lesions [61]. Bone destruction occurs proximal to MM PCs whose surface expression of receptor activator of NF-κB (RANK) ligand binds to RANK on neighboring osteoclast precursor cells, driving their survival, differentiation, and activation (bone
dissolution) [62, 63]. Conversely, osteoblasts, cells responsible for bone reformation, are
developmentally inhibited by MM PC-secreted wnt-signaling antagonist DDK1[64].
MM also influences the cytokine profile, both in the BM and systemically. Age-related changes in systemic cytokine milieu, termed “inflamm-aging”, skews the system to a pro- inflammatory state, whereas patients with end-stage myeloma have a profile that resembles immune senescence [65]. A recent study describes a trend of markedly elevated levels of proinflammatory factors IL-6, IL-8, tumor necrosis factor alpha (TNFα), interferon gamma
(IFN-γ), and IL-10 in the peripheral blood (PB) and IL-6, IL-8, tumor necrosis factor alpha
TNFa, and IL-2 in the BM of MM patients [66]. Pleiotropic transforming growth factor beta
(TGFβ) is released by both PCs and BMSC in MM, which inhibits maturation and effector
13 functions of both the innate and adaptive immune system, namely natural killer (NK) cells, macrophages, T-cells and antigen-presenting dendritic cells [67-73] . Of note, as MM progresses and immunodeficiency develops, ineffective immune surveillance and disease clearance will also leave the body susceptible to infection [74]. Indeed, myeloma patients when compared to healthy age-matched individuals are 7.1-fold and 10-fold more likely to develop a bacterial and viral infections, respectively[75].
Eventually, MM-driven remodeling of the BM will result in PC overcrowding in an unsustainable microenvironment, and subsequent malignant infiltration to a new bone.
Recent findings are beginning to recognize the influential role of extracellular vesicles in facilitating the spread of cancers to the bone. In a healthy setting, the BM is not naturally adapted for metastasis and requires preparation by the invading cancer. In melanoma, the BM premetastatic niche is re-educated by EV released by cancer cells, thereby reorganizing ECM components to facilitate attachment and establishment of a secondary site of malignancy
[76].
Extracellular vesicles (EV) are a collective term for non-organelle-containing, plasma membrane-enveloped packets of subcellular material. EV were originally believed to be cellular debris with little biological significance, until one study described B cell-derived
MHC-II-bearing EVs that were able to induce T-cell response [77]. Two types of EV commonly recognized are exosomes and microvesicles (Figure 3). Exosomes, which range from 50-100nm in diameter, are packaged internally and are released by the Trans-Golgi network from the cell as multivesicular bodies (MVB) [78, 79]. The larger of the EV,
14 microvesicles, which range from 100nm-1000nm in diameter, are generated by the outward blebbing of the cell membrane, and thus contain cargo and surface markers proximal to the membrane [80]. Capable of intercellular communication, EV contain DNA, mRNA, miRNA, and proteins, and can present surface ligands for functional delivery to specific recipient cell types [78, 81]. The collective term of EV is apropos in vesicle studies due to the limitations of successfully separating exosomes and microvesicles during preparation –contemporarily differential ultracentrifugation [82] .
15
Figure 3: Extracellular vesicle release
Figure 3: Exosomes(A) are packaged within multivesicular bodies and released
to the extracellular space. Microvesicles(B) form from outward blebbing of the
cell membrane.
16
1.2 Natural Killer Cell Biology
1.2a Natural killer cell effector function overview
Natural Killer cells are a unique subset of lymphocytes which function at the interface of the
innate and adaptive immune system. Classically, natural killer (NK) cells are defined as large
granular lymphocytes that express surface cluster of differentiation (CD) marker CD56 but
lack CD3, a prototypic T cell marker. Natural killer cells make up 1-6% of total leukocytes
and have a healthy range of 0.08-0.43x106 cells per ml of peripheral blood [83]. NK cells have dual effector cell functions, namely to release recruitment factors and cytokines in response to inflammatory stimuli, and to exhibit cytotoxicity against virally infected and tumor-transformed cells [84]. Differential CD56 expression separates NK cells into two functionally distinct subpopulations, CD56Dim and CD56 Bright [85]. CD56Dim NK cells, considered to be more functionally mature, constitute ~90% of the NK cells circulating in the peripheral blood and are highly cytotoxic. Further, CD56Dim NK cells express an active
CD16 receptor, also known as FCγRIIIA, which recognizes the IgG1 (fragment crystallizable region) Fc region of antibodies, enabling CD56Dim to engage and attack targets coated by
IgG1-isotype antibodies [86]. CD56Bright NK cells constitute ~10% of the peripheral blood
NK and generally lack CD16 [87]. CD56Bright NK cells marshal an immune response by releasing a number of proinflammatory factors.
NK cells develop from bone marrow-derived CD34+ hematopoietic progenitor cell (HPC) to common lymphoid progenitor (CLP) cells that able to give rise to all lymphocyte subsets. NK cells differ from B and T cells due to lack of somatic gene rearrangement in establishing a
17
diverse repertoire of clones. NK cells develop in the bone marrow where BM stromal cells
produce the necessary cytokine/factors including but not limited to FL, KL, IL-7, and IL-15 to drive maturation. NK cell maturation and acquisition of functional competency appears to be site-specific and includes the secondary lymphoid tissues (SLT, i.e. tonsil and lymph nodes) as well as the spleen [84]. The alternative fate and function of tissue-specific NK cells is appreciated in the diverse NK cell populations found in the setting of pregnancy, SLT, BM, and cancer.
NK cells possess a variety of methods to exhibit cytotoxicity against a target. These include natural and antibody-dependent cell-mediated cytotoxicity (ADCC) through engagement of
Fc receptor CD16 and release of lytic granules. NK cells may also utilize members of the tumor necrosis factor (TNF) family TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) to induce caspase-mediated apoptosis on TRAIL receptor and FAS receptor- bearing target cells. Natural and ADCC killing can be measured within 4 hours in vitro while internal apoptotic dispatching by TRAIL and FasL requires at least 6 hours [88]. An NK cell’s preference for killing is established by signals received from its environment and potential target cells discussed in 1.2b.
1.2b The immune synapse and natural killer cell receptor signaling
When an NK cell engages a potential target, the rearrangement of its molecules at the cell surface interface forms the immune synapse (IS). The formation of the IS is not exclusive to
18 modulating cell death, but rather shares an adhesion mechanism for delivering proteins and signaling intermediates for activation and inhibition of recipient cells . In NK cell cytotoxicity, the IS provides a cleft between the NK cell and its target cell for specific and direct delivery of an apoptotic message via cytolytic granules [89]. However, the IS also provides a platform for target cells to present inhibitory ligands upon their surface. The NK cell is required to assess the balance of activating and inhibitory signals in the IS before inducing apoptosis. Interestingly, a single inhibitory event overrides activation, and thus multiple activation events are required to deliver a death message. This balancing mechanic serves two purposes –it has evolved to prevent a trigger-happy immune system while simultaneously promoting both cellular and systemic energy conservation.
The elegant formation of the IS is divided into three discrete stages, which is extensively described and reviewed elsewhere [90]. Briefly, the initiation stage begins as the NK cell, through chemotactic signaling or random surveying, makes early contact and tethers a potential target via members of the selectin family and CD2 [91, 92]. Early commitment to cytotoxicity and firm adhesion to the target is then established by integrins LFA-1 and MAC-
1, and activation of the NK cell through natural cytotoxicity receptor (NCR) members may support further action. In the absence of inhibitory signals, the effector phase describes the F- actin/WASP-dependent polarization and migration of the microtubule organizing complex
(MTOC), docked with many lytic granules containing perforin, granzyme A and granzyme B, towards the IS [93]. As granule contents are released into the synaptic cleft, CD107A- studded granule membrane incorporates into the NK cell surface and provides protection against its own weaponry [94]. In order to keep the neighboring bystander cells from being
19 exposed to lytic factors, the termination phase involves a period of inactivity followed by
disengagement of the NK cell from the target. Intriguingly, NK cells are serial killers and can
re-exhibit IS formation and cytotoxic potential in excess of 5 killings in 60 minutes [95].
These observations showcase the efficacy of the ever-ready NK cell when killing criteria is
properly corroborated.
Activation signals promote an NK cell to mobilize against a target. Multiple overlapping
activation signal intermediates and functional redundancy within SRC-family kinase
members Lck, Fyn, Src, Yes, and Fgr provide an obstacle for determining which case-
specific member is responsible [96]. Engagement of activating receptors bearing cytosolic
immunoreceptor tyrosine-based activation motifs (ITAMs) induces phosphorylation by SRC-
family kinases, pairing of affiliated adapter proteins (i.e. ZAP70, DAP10, DAP12, etc…) and
eventual downstream activation/phosphorylation of VAV1 and PLC-γ2 [97]. Vav1 and PLC-
γ2 signaling are required for calcium mobilization and release of cytolytic granules [98].
ITAM-bearing receptors include the natural cytotoxicity receptor (NCR) family members
NKp30, NKp44, and NKp46. FcγRIIIa(CD16, binds IgG1), SLAM family member
2B4(CD244, binds CD48) as well as lectin-like natural killer cell group 2 (NKG2)family members NKG2C-CD94(binds HLA-E under cellular stress) and NKG2D (binds
MICA/MICB) receptors also potentiate their signals through ITAM [99].
Inhibitory signals abrogate activation signals and serve to spare target cells from NK cell- mediated death. Engagement of inhibitory receptors results in activation of immunoreceptor tyrosine-based inhibitory motifs (ITIMS) which initiate the reciprocation of activating
20 signals. ITIMS signal through intermediates SHIP-1, SHP1and SHP2 which serve to reverse early activation of concomitant ITAMS and prevent calcium flux by dephosphorylating
VAV1, respectively [100, 101]. ITIM-bearing receptors include killer Ig-like receptors (KIR, bind MHC-I), lectin like NKG2 family member NKG2A-CD94 (Binds HLA-E), and programmed death 1(PD-1) which binds programmed death ligand 1(PD-L1) [102]. KIR and
NKG2A activation phosphorylate Crk, which promotes the disassembly of Crk, CBL-C and
C3G, neutralizing actin reorganization required for cytolytic potential [103]. PD-1 engagement upregulates CBL-b and CBL-C, which in T cells, promote ubiquitin-mediated proteasome dispatching of activating TCR surface receptors [104]. Signaling lymphocytic activation molecule family 7 (SLAMF7 or CS1, self-ligating) contains an immunoreceptor tyrosine-based switch motif (ITSM), which has potential to activate or inhibit downstream signaling. In NK cells, SLAMF7 interacts with CD45 and assembles inhibitory signaling microclusters on the cell surface, which restricts the formation of activating complex assembly. SHIP-1 and its downstream intermediates enable SLAMF7 to inactivate VAV1 and PLC-γ2 [105].
Many therapeutic approaches in MM have been designed to clinically target/neutralize the inhibitory messaging of NK cells, thereby enabling proper clearance of aberrantly presenting
MM cells. Indeed, monoclonal antibodies against CS1(Elotuzumab), inhibitory
KIR(lirilumab), PD-1/PD-L1(BMS-936558/936559), and NKG2A(IPH2201,
AstraZeneca/InnatePharma) are currently on the market or in clinical trials. However, it should be noted that careful consideration for treatment strategies should be made, as the
21 possibility of selectively promoting the rise of more aggressive and resistant MM PC clones
may occur.
1.2c Major histocompatibility complex class-I biology
All jawed vertebrates commonly express major histocompatibility complex (MHC) gene products and their cognate innate and adaptive immune receptors, killer immunoglobulin receptors (KIR) and T-cell receptors (TCR) respectively. Thus the innate immune system and
NK-like cells have been functioning to protect organisms against pathogens and genetic maladies for > 450 million years [106, 107]. MHC genes, known as human leukocyte antigen
(HLA) genes in humans, are clustered on the short arm of chromosome 6 and fall into subclasses MHC-I (classical), MHC-Ib (non-classical), MHC-II based on function. The
general function of all MHC products is to provide a sampling of endogenous and exogenous
peptides, a process termed cross-presentation, to members of the immune system.
MHC-I proteins are either expressed or inducible in all nucleated cells. A functional MHC-I
complex is on the surface of the cell as a heterodimer, consisting of one MHC heavy chain
bound with antigenic peptide and associated with a light chain Beta-2-Microglobulin (β2M)
[108]. The polymorphic 45 kDa MHC-I heavy chain consists of 3 αlpha domains (Figure 4).
The α3 domain comprises a transmembrane domain that non-covalently binds the 12kDa β2M during complex formation, whereas the α1 and α2 domains form the binding cleft for an 8-9 amino acid (aa) long peptide [109, 110].
22
Figure 4: MHC-I Structure
Figure 4: The basic structure of MHC-I, featuring the non-covalent joining of the
alpha chain(blue) with β2M (yellow), and a peptide octamer (red) loaded within
the binding groove. The polymorphic regions within the α1 and α2 domains form
binding grooves of varying specificities towards peptides of 8-10 amino acids in
length. The α3 domain, which contains the membrane-spanning portion, also
serves as a recognition domain to T cells during antigen/peptide presentation.
23
In order to maintain the economy of the ever-changing cell, protein turnover via proteasomes
constantly helps repurpose amino acids to new protein synthesis, all-the-while providing peptides for the process of antigen presentation [111]. Proteins whose function are no longer needed, or are improperly folded/translated are often tagged for degradation by
(poly)ubiquitination [112]. The ubiquitin tag, recognized by the proteasome’s 19s regulatory cap, serves as entry criteria for degradation [113]. Within the giant multicatalytic proteasome
core, multiple rounds of cleavage events fragment the protein and provide an ample library of
peptides for potential MHC loading. To expand the potential antigen pool, longer precursor
peptides are trimmed to 8-9aa in length by peptidase ERAP1 and ERAP2 [114]. The peptides
released from the proteasome are free-floating in the cytosol, only have a half-life of ~10
seconds, and will require the function of the transporter associated with antigen presentation
(TAP-1 and TAP-2) pump to gain access to the endoplasmic reticulum(ER) for incorporation
into the MHC-I heterodimers [115].
The ER lumen is the site of MHC-I peptide loading. Within minutes of translation, the
MHC-I heavy chain associates with β2M, enhancing the binding groove’s affinity for peptides [116]. The MHC-I heterodimers are partially folded and are stabilized by chaperone proteins tapasin, calreticulin, and PDIA3 [117, 118]. The peptide loading complex is completed when tapasin associates with TAP which creates a close junction for efficient peptide loading during peptide translocation to the ER [119]. Bound MHC-I will undergo a proper folding quality control program in the ER prior to transport to the Golgi-apparatus and eventual incorporation into the plasma membrane –reviewed elsewhere [120].
24
The MHC-I (HLA-A, HLA-B, HLA-C) genes are highly polymorphic in distinct regions of the α1 and α2 domains. Although the general structure of the MHC-I is conserved, peptide preference and binding capacity of the 10 aa residue binding groove can differ considerably
[121]. To date, 3356 HLA-A, 4179 HLA-B and 2902 HLA-C alleles have been sequenced and identified [122]. However, an individual genome will only have 2 alleles of each MHC gene represented, one allele inherited from each parent. NK cells, through KIRs, assess the quantitative and qualitative MHC-I self-antigen surface expression of neighboring cells. Lack of MHC-I, or misfolded MHC-I due to cellular or viral stress, will support the activation of the NK cells, and likely induce apoptosis of the target cell. Indeed, the chronic myeloid leukemia-derived cell line K562 completely lacks surface expression of MHC-I and is highly susceptible to natural NK-mediated cytotoxicity [123]. Interestingly, primary MM PCs and some MM cell lines constitutively express MHC-I in abundance (data not shown) [124].
MHC-Ib (HLA-E, HLA-F, and HLA-G) are highly conserved, less polymorphic and provide
a qualitative signal during peptide presentation. To date, 21 HLA-E, 22 HLA-F, and 52
HLA-G alleles have been reported [122]. HLA-E and HLA-G contain 8 and 9 aa binding
groove residues, whereas HLA-F retains only 5aa residues [125, 126]. MHC-Ib members
generally present cell-survival ligands to inhibitory receptors on innate and adaptive immune
cells.
HLA-F remains the least studied and understood member of the MHC-Ib family. Evidence
suggests the interaction of HLA-F tetramers with inhibitory receptor LIR-1 of NK cells, thus
altering the activation threshold of contemporary signals [127]. Shown to associate with free
25 HLA-I heavy chains, HLA-F is proposed to aid in cross-presentation of antigen among activated immune cells [128]. HLA-F expression is tissue restricted to the thymus, spleen, and tonsil where it can be observed on/in NK cells, B cells, T Cells (but not Tregs), and monocytes [129].
In a healthy setting, HLA-G expression is highly restricted to the invading trophoblast in the setting of pregnancy [130]. The embryo requires protection in the decidua as a hemi- allograft, only sharing half of its MHC identity with its mother. HLA-G, on the developing fetus or surrounding it in soluble form, will engage inhibitory receptors LIR and KIR2DL4 on tissue-specific decidual NK and initiate a phase of potent immune tolerance at the fetal- maternal interface that will last until birth [131-133]. Of interest, dysregulation and aberrant expression of HLA-G-bearing cells has been reported in blood cancers, including acute myeloid leukemia(AML) and chronic lymphocytic leukemia (CLL)[134, 135]. Further, the release of soluble HLA-G from cancer cells serves as another dimensional setback to overcome in targeted therapies, as systemic inhibitory signaling may restrict proper immune trafficking to the source.
Unlike other nonclassical MHC-Ib members HLA-G and HLA-F, HLA-E transcriptional expression is not tissue-restricted, and is likely transcribed in all MHC-I-expressing cells
[136]. HLA-E shares the same structure and functional domain distribution with classical
MHC-I. The peptide binding groove of HLA-E has high affinity for its cognate peptides, the
5’-leading strand signal peptides of classical MHC-1 (HLA/B/C) and HLA-G [137, 138].
Upon binding a peptide HLA-E, which is otherwise restricted to ER lumen, is trafficked to
26
the cell surface to present an inhibitory signal to its receptor on NK cells, NKG2A-
CD94(1.2b) [139]. Of note, the affinity for HLA-E to NKG2A is highest when HLA-E
binds/presents the leading peptide of HLA-G [140, 141]. HLA-E’s potent inhibitory
influence was first demonstrated after a xenogenic transfection of human HLA-E to porcine endothelial cells was sufficient in ablating human NK cell xenoreactivity, in vitro [142, 143].
Further, one study describes the protective potential of HLAE on the surface of various cancers including cervical cancer, breast cancer, melanoma, and some leukemias [144].
1.2d Interferons
Interferons were discovered for their anti-proliferative effect on virally infected cells, which in turn, interferes with viral propagation [145, 146]. Interferons are a family of small (<146 aa) naturally occurring yet pleiotropic proteins divided into three classes- Type I-III. Type I interferons include IFN-α and IFN-β, who share some sequence homology, are produced in
response to viral dsDNA detection by DNA sensors RIG-1 and DAI, as well as TLR3 and
TLR4 stimulation via LPS and dsRNA, respectively[147]. IFN-γ, originally dubbed immune
interferon, is the exclusive member of Type II interferon. IFN-γ is produced by NK cells and
T cells commonly after IL-2, IL-15, IL-12/IL18, and Type I IFN stimulation [148]. The least
studied family, Type III interferon members include IFN-λ1 (IL-29) IFN-λ2(IL-28a) and
IFN-λ3(IL-28b), signal through paired IFNλR1/IL10R2 receptor, can augment other IFN
family member responses, and can potently inhibit replication of specific viral families
(HSV-2, ECMV, HBV, HCV) [149-151].
27 In NK cells, IFN-γ is under the transcriptional control of TBET and EOMES where the observed expression of each TF is higher in the cytokine-producing CD56Bright than the cytotoxic CD56Dim cell subset [152]. Some human NK cells constitutively express IFN-γ mRNA transcript in order to expedite their response, and can actively secrete IFN-γ within 30 minutes of activation [153]. IFN-γ can promote TH1 cell differentiation, a subset who produce IFN-γ upon stimulation [154]. IFN-γ can also support inducible Treg differentiation, an immunosuppressive T cell subset that is linked to immune tolerance and suppression in
MM [155]. Unlike perforin and lytic granules who associate with MTOC, IFN-γ secretion facilitated through the endosome recycling [156].
1.2e Interferon-gamma and STAT1 signaling
IFN-γ orchestrates transcriptional control of many gene families and cellular processes.
Extracellular IFN-γ binds surface-expressed IFN-γR1 and/or IFN-γR2 which homodimerize and thereby autophosphorylate their associated tyrosine kinase members janus activated kinase 1 (JAK1) and/or JAK2, respectively [157]. Activated JAKs provide a docking site for signal transducer and activator of transcription family members (STATs). JAK1 phosphorylates the c-terminal tyrosine residue Y701 of STAT1 while JAK2 phosphorylates
Y690 of STAT2 [158, 159]. Both pSTAT1 and pSTAT2 heterodimerize with other STAT proteins and transmigrate to the nucleus where they bind to gamma activated sequences
(GAS) and activate or suppress transcription of >200 downstream target genes such as interferon response factor 1 (IRF1) and suppressor of cytokine signaling (SOCS) genes.
Interestingly, STAT1-mediated activation of T-box Expressed in T cells (TBET) completes a positive feedback loop [160]. Importantly, STAT1 activation induces TAP1/2, the antigen
28 peptide pump required for peptide loading and increased surface expression of MHC-I (1.2c)
[161]. Like MHC-I, IFN-γR1 is expressed or inducible on all nucleated cells. However, IFN-
γR2 expression is restricted to immune cells such as NK cells, CD4+ T helper cells, and dendritic cells after antigen endocytosis during microbial infection [162].
However, JAK-STAT signaling alone is not sufficient to describe the totality of the IFN-γ response. A recent ChIP-Seq based study reports 1441 STAT1-regulated genes, of which only 212 were known IFN-γ regulated genes, and only 194 genes (13.5%) and 42 genes
(2.5%) were upregulated and downregulated in the mRNA transcriptome [163]. The ChIP-
Seq study only describes the influence of IFN-γ as a single agent, underlying the complexity
of STAT1-mediated processes, involving many cooperating initiation factors of specific gene
programs in situ and in vivo.
1.3 STAT3 signaling in multiple myeloma
Recent mathematical modeling describes the crosstalk mechanism between IFN-γ signaling
and the IL-6/STAT3 pathway [164]. IL-6, a key growth factor for MM PC (1.1d), provides a
crucial MM survival signal via STAT3 activation. STAT3 is constitutively activated in some
MM cell lines (U266) and is correlated with resistance to apoptosis through a Bcl-xL-
dependent mechanism [165, 166]. Further, studies have shown in a healthy STAT1-deficient
mouse, IFN-γ can alternatively activate STAT3, which is likely a last-resort act of
resourcefulness to preserve some of the original message through redundant/common STAT
target genes [167]. In human neutrophils, STAT3 activation by IFN-γ has also been reported
[168]. Further, transforming growth factor beta (TGFβ) produced by BMSC in MM can
29 perpetuate the IL-6/STAT3 survival mechanism for MM PC, while simultaneously enacting potent inhibitory effects upon NK cells [169].
30 CHAPTER 2: INTERFERON-GAMMA LOWERS THE SUSCEPTIBILITY OF MULTIPLE MYELOMA TO NATURAL KILLER CELL-MEDIATED CYTOTOXICITY
2.1 Introduction
Natural killer (NK) cells are large granular lymphocytes that comprise a key cellular subset of the innate immune response. Capable of lysing virally-infected and malignant targets independent of co-stimulatory signals and gene rearrangement events, NK cells are also a major source of potent, immunomodulatory cytokines which can marshal and guide the immune response. Among these, NK cells are a principal source of interferon-γ (IFN-γ) which serves to augment immune function, induce Th1 polarization and recruit immune cells to inflammatory sites [170].
NK cells appear to play an important role in the immune response to multiple myeloma
(MM). However, MM cells utilize strategies to evade detection and lysis by immune cells.
The specific mechanisms responsible for these immunoevasive events are incompletely understood. Herein, we provide new evidence supporting a role for IFN-γ in mediating MM resistance to NK cell surveillance and killing. NK cells produce IFN-γ when exposed to MM cells; however, MM cells express functional IFN-γ receptors and, via STAT1 and STAT3 signaling, mediate transcription of HLA-E, an inhibitory ligand of the NK receptor NKG2A
31 [171]. HLA-E in turn suppresses NK cell-mediated cytotoxicity against MM targets through
interaction with the inhibitory receptor NKG2A, which appears to be overexpressed on NK
cells in MM (unpublished observations). This immunosuppressive axis may be overcome
with the use of an anti-NKG2A antibody to interrupt this receptor/ligand interaction.
Collectively, these data provide novel insight into the means by which MM cells may subvert
signaling in the immune system to achieve NK-cell directed immunoevasion; furthermore, these findings highlight new considerations for MM treatment strategies. In combination with the observation of NKG2A overexpression on NK cells in MM, these findings implicate this antigen as a potential novel target for checkpoint inhibitor immunotherapy in MM, as well.
2.2 Results with Figures
NK cells produce IFN-γ in response to MM
We have previously observed that MM patient-derived NK cells secrete IFN-γ in response to autologous, primary MM tumor cells [172]. Numerous studies have indicated abnormal NK cell effector functions in the setting of MM, so we verified this effect utilizing healthy donor
NK cells against U266 MM cell line targets (Figure 5, A) as well as primary MM tumor cells
(Figure 5, B).
32 Figure 5: Healthy primary NK cells produce IFN-γ when cultured with MM
Figure 5: ELISpot assays describe spontaneous IFN-γ production by primary
enriched NK cells co-cultured 10 NK cells: 1MM cell for 24 hrs. Spontaneous
IFN- γ production by primary NK cells was observed when co-cultured with
either the MM cell line U266 (A) or primary CD138+ purified MM PC (B).
NK cells produce IFN-γ in response to soluble MM factors
Interestingly, NK cell IFN-γ production does not require direct NK cell: MM cell contact. To
assess the contribution of soluble factors to IFN-γ induction, a porous membrane which
restricts cell migration, thus preventing NK cell:MM cell contact, was used (Figure 13, A). In
this contactless co-culture system, when challenged with the RPMI-8226 MM cell line
(Figure 6, A) as well as primary, CD138+ MM tumor cells (Figure 6, B) at 24hrs, IFN-γ production by the NK cell line, NK-92, increased as a function of the quantity of MM target cells present.
33 Figure 6: IFN- γ production in MM-NK cell co-culture does not require cell-cell contact
* *
**
Figure 6: Either MM cell line RPMI-8226(A) or primary CD138+ MM PCs(B)
were co-cultured with NK cell line NK92 for 24hrs in contact-restrictive
conditions, while allowing the passing and exchange of soluble factors under
400nm in diameter. Supernatants from each co-culture were harvested and
relative IFN-γ production was assessed by Enzyme-linked Immunosorbent Assay
(ELISA). NK92 cells made significantly more IFN-γ when contactless co-
cultured with MM cell line RPMI-8226 (A, P-value <0.001) and primary plasma
cells (B, both P-values < 0.003) at MM:NK cell ratios of 4:1 and 2:1(B only). Of
note, as the MM:NK cell ratio increased, IFN-γ output was amplified.
34 MM cells express functional IFN-γ receptors
Elevated IFN-γ levels are a systemic factor in MM, and our novel finding of MM-induced
IFN-γ production led us to assess the sensitivity of MM cells to IFN-γ. In normal culture conditions (i.e. RPMI media supplemented with 10% serum), MM cell lines RPMI-8, A) and
U266 (, B) constitutively expressed both IFN-γR1 and IFN-γR2 on their surface and expression was not altered after treatment with IFN-γ (data not shown, 48hr). In accordance with the canonical IFN-γ signaling pathway, a time course treatment of RPMI-8226 (Figure
8, A) and U266 (Figure 8, B) cells with IFN-γ revealed early and sustained activation/phosphorylation of signal transducer and activator of transcription 1
(STAT1,Y701) protein, confirming the prior observation by liu, et al [173]. With the recent discovery that IFN-γ can alternatively activate/phosphorylate STAT3, we assessed and observed early, though unsustained, activation/phosphorylation of STAT3(Y705) by IFN-γ
(Figure 8A/B) [167]. STAT1 regulates many genes (>1400 known) and robustly drives gene transcription and surface expression of MHC-Ia molecules. However, STAT1 has also been reported to upregulate the inhibitory ligand PD-L1 on MM cells [173]. The promoter regions of PD-L1 and the inhibitory MHC-Ib ligand HLA-E are studded with STAT1 and STAT1/3/5 binding sites (Figure 9A), respectively. We next tested whether IFN-γ treatment was sufficient to drive gene expression of the inhibitory ligand HLA-E while verifying PD-L1 gene upregulation. Indeed, both HLA-E (Figure 9, B left) and PD-L1(Figure 9, B right) gene transcription is enhanced after (48hr) treatment with IFN-γ, but not by the MM cell proliferation factor IL-10 in both normal and serum-starved culture conditions.
35 Figure 7: MM cells constitutively express IFN-γRs on their surface
RPMI-8226 A.
U266
B.
IFN-γ R1 IFN-γ R1
Figure 7: MM cell lines RPMI-8226 and U266 were maintained in normal
culture conditions of RPMI media supplemented with 10% fetal bovine serum, 2
mM Glutamax (glutamine) and antibiotic/antimycotic added. Flow cytometric
analysis of IFN-γR1 and IFN-γR2 on MM cell lines RPMI-8226 (Top) and U266
(Bottom) shows constitutive expression of both IFN-γR1 and IFN-γR2 in both
cell lines. Of note, 48hr IFN-γ treatment of either cell line showed no change in
IFN-γR1 and IFN-γR2 surface expression (data not shown).
36 Figure 8: IFN-γ activates STAT1 and STAT3 in MM cells
Figure 8: A two hour time course and assessment of IFN-γ early activation of
STAT1 and STAT3 on MM cell lines RPMI-8226 (A) and U266 (B). pSTAT3
depicts the assessment of tyrosine 705 (Y705). GAPDH serves as a loading
control and neither total STAT1 nor total STAT3 protein levels were affected by
IFN-γ treatment. Due to labeling and label-stripping restrictions, both A and B
represent two separate western blots of the same protein lysates with loading
equality verified by uniform GAPDH signal.
37 Figure 9: Transcription of HLA-E and PD-L1 by MM cells after IFN-γ treatment
Figure 9: A database search(SABiosciences) provides a profile of the promoter
regions of both PD-L1 and HLA-E (CD274 and HLAE genes) which contain
multiple STAT binding sites (A). Treatment of MM cell line RPMI-8226 with
IFN-γ for 48hrs induced both HLA-E (B, left, P< 0.002) and PD-L1(B, right,
P<0.02) gene transcripts. HLA-E and PD-L1 transcription was not altered by
serum-starvation (light grey) or treatment with the MM cell proliferation factor
IL-10.
38 IFN-γ increases MM cell surface expression of inhibitory ligands
The biological readout for IFN-γ signaling is the cross presentation of potentially antigenic peptides through MHC-I. We assessed dose response effects of IFN-γ treatment on surface expression of HLA-ABC(Figure 10), HLA-E(Figure 11) and PD-L1(Figure 12) in MM cell lines, as well as K562 cells, which lack MHC-I. Interestingly, although the MM cell lines
U266 and OPM2 also lacked MHC-I on their surface, both cell lines still showed a dose- dependent increase in expression of all markers tested. Further, the RPMI-8226 cell line had the most robust measured response with regards to upregulating HLA-E and PD-L1, thus was chosen to serve as a model cell line for future functional assays. When RPMI-8226 cells were co-cultured with NK-92 cells in a contactless system for 48 hours in IFN-γ-secreting conditions, the RPMI-8226 showed a marked increase in the inhibitory ligands HLA-E and
PD-L1 gene transcripts (Figure 13). As expected, in the same contactless co-culture setup, increased surface expression of both HLA-E and PD-L1 was observed (Figure 14).
.
39
(Continue on page 41) on (Continue
γ treatment - ABC by MM after IFN after MM by ABC - Surface expression of HLA Figure 10:
40 (Continued from page 40)
Figure 10: Surface expression of HLA-ABC by MM after IFN-γ treatment
Flow cytometric assessment of MHC-I (HLA-ABC) on MM cell lines after
culture in normal conditions (No IFN-γ, grey line), or after low dose (1ng/ml ,
black line) and high dose (10ng/ml, filled in) IFN-γ treatment. Note that K562,
U266, and OPM2 cells lack MHC-I surface expression following culture in
normal conditions.
41
(Continue on page 43) on (Continue t γ treatmen γ - E by MM after IFN MM after E by - Surface expression of HLA Figure 11:
42 (Continued from page 42)
Figure 11: Surface expression of HLA-E by MM after IFN-γ treatment
Flow cytometric assessment of inhibitory ligand HLA-E (MHC-1b) on MM cell lines in normal culture conditions (No IFN-γ, grey line), or after low dose
(1ng/ml , black line) and high dose (10ng/ml, filled in) IFN-γ treatment. A stepwise, dose-dependent increase in surface expression of HLA-E was observed for all cell lines.
.
43
(Continue on page 45 ) on (Continue t γ treatmen - L1 by MM after IFN MM after by L1 - Surface expression of PD Figure 12:
44 (Continued from page 44)
Figure 12: Surface expression of PD-L1 by MM after IFN-γ treatment
Flow cytometric assessment of inhibitory ligand PD-L1(CD274) on MM cell lines in normal culture conditions (No IFN-γ, grey line), or after low dose
(1ng/ml , black line) and high dose (10ng/ml, filled in) IFN-γ treatment. The induction of PD-L1 surface expression was detected in all cell lines following treatment with IFN-γ(10ng/ml); the marginal shift in positive PD-L1 expression may be attributed to inefficient epitope binding.
45 Figure 13: Transcription of HLA-E and PDL-1 by MM cells in contactless co-culture
with NK-92
A. RPMI-8226 400nm porous membrane NK-92 Soluble Factors
B. RPMI-8226 RPMI-8226 * *
Figure 13: Contactless co-culture (schematic, A) of NK-like cell line NK92 with
MM cell line with RPMI-8226 in IFN-γ-producing conditions (24 hrs) increases
RPMI-8226 cells’ transcription of both HLA-E (B, left, P <0.001) and PD-L1 (B,
right, P < 0.001) genes. HLA-E shows an increased trend of transcription as the
MM:NK cell ratio also increased. The opposite can be said of PD-L1, but in the
higher populated conditions, the observed effect may be at least partially
attributed to approaching conditions of nutrient depletion.
46 Figure 14: Surface expression of HLA-E and PD-L1 by MM cells in contactless co- culture with NK92
IL-2 Stimulation
Figure 14: Flow cytometric analysis of 48hr contactless co-cultured MM cell line
RPMI-8226 with the NK-like cell line NK92 + IFN-γ-producing(IL-2)
conditions. Although a slight increase in surface expression of HLA-E on RPMI-
8226 cells was observed in the unstimulated control sample(2nd row, right,
23.21%, MFI 2549 vs 1419), both PD-L1(3rd row, middle, 26.09%, MFI 2767 vs
994) and HLA-E(3rd row, right, 59.79%, MFI 4361 vs 1419) expression was
greatest at 1:1 NK:MM cell ratio in IFN-γ-producing(IL-2) conditions.
47 IFN-γ lowers the susceptibility of MM cells to NK cell-mediated cytotoxicity
Next we assessed the direct effects of IFN-γ treatment on MM cell viability and
proliferation. The proliferation rate of all MM cell lines tested at 48hrs by MTS
assay (Figure 15, A), as well as viability (data not shown), remained unchanged.
With no observed direct toxicity, we proceeded to assess the effect of IFN-γ on
the MM cell’s susceptibility to NK cell-mediated lysis. MM cell lines U266
(Figure 15, B and C) and RPMI-8226 (Figure 15, D and E) treated with IFN-γ
were less susceptible to primary enriched NK cell-mediated cytotoxicity. IFN-γ
treatment conferred resistance of MM cell lines to enriched primary NK-cell
mediated natural (Figure 16) and IL-2-activated cytotoxicity (Figure 17). Further,
differential sensitivity to IFN-γ and resistance to NK cell-mediated lysis was
observed in RPMI-8226 and U266 cell lines (Figure 18).
48 Figure 15: Viability and susceptibility of MM cells to NK cell cytoxicity after IFN-γ treatment A.
B. U266 C. U266
* *
D. E. RPMI-8226 RPMI-8226
*
*
(Continue on page 50) 49 (Continued from page 49) Figure 15: High dose IFN-γ (10ng/ml) treatment of MM cell lines did not affect viability or proliferation as measured by MTS assay at 48hrs (A). IFN-γ
treatment (10ng/ml, 48hrs) of U266 and RPMI-8226 confers their resistance to
primary enriched NK cell-mediated natural cytotoxicity in both NK-unstimulated
(B, P-value < 0.001 and D, P-value < 0.026 respectively) and NK-activated
conditions (IL-2, 24hrs, C, P-value <0.01 and E, P-value <0.001, respectively).
Data depicts a standard 51Cr release assay and error bars depict the standard deviation of 3 technical replicates.
50 Figure 16: IFN-γ lowers the susceptibility of MM cells to NK cell-mediated killing, no stimulation
* *
* *
Figure 16: IFN-γ treatment (10ng/ml, 48hrs) of MM cell lines confers their
resistance to primary enriched NK cell-mediated natural cytotoxicity in NK-
unstimulated conditions. NK cells killed cell lines L363, MM1.S, OPM2 and
U266 significantly less when the MM cell lines were pretreated with IFN-γ for
48hrs (All P-values < 0.01). Note, the values and scale for H929 and RPMI-8226
data were low. Data represents 1 of 3 donors/experiments with similar trends.
Data depicts a standard 51Cr release assay and error bars depict standard deviation
of technical triplicates.
51 Figure 17: IFN-γ confers partial resistance of MM cells to NK cell-mediated
killing, activating conditions
* *
*
Figure 17: IFN-γ treatment (10ng/ml, 48hrs) of MM cell lines confers their resistance to primary enriched NK cell-mediated natural cytotoxicity in NK- activated (IL-2, 24hrs) conditions. NK cells activated with IL-2 killed cell lines
H929,MM1.S and RPMI-8226 significantly less when the MM cell lines were pretreated with IFN-γ for 48hrs (All P-values < 0.005). Also, the observed resistance of MM cell lines OPM2, U266, and L363 to NK cell-mediated killing
(Figure 16 data) was overcome in NK cell-activated conditions. Data represents 1
of 3 donors/experiments with similar trends. Data depicts a standard 51Cr release
assay and error bars depict standard deviation of technical triplicates
52 Figure 18: IFN-γ dose-dependent resistance of MM cells to NK cell-mediated cytotoxicity
Figure 18: MM cell lines were treated with a low dose of IFN-γ (1ng/ml) or a high dose of IFN-γ (10ng/ml) for 48hrs. MM cell lines U266 and RPMI-8226 exhibit differential sensitivity to IFN-γ treatment doses 1ng/ml and 10ng/ml. The data suggests that U266(Left) cells require higher amounts of IFN-γ in order to gain protection from NK cell-mediated lysis. The RPMI-8226(Right) cells show a measurable resistance to NK cell-mediated lysis at the lower dose(1ng/ml), and resistance is increased as the dose escalates(10ng/ml). Data depicts a standard
51Chromium release assay and error bars depict the standard deviation of technical triplicates.
53 IFN-γ-mediated resistance is differentially recovered by IPH2201 (Anti-NKG2A)
IPH2201 is a humanized IgG4 (non-depleting) anti-NKG2A antibody which sterically blocks the potential for inhibitory ITIM signaling in the NK cell. Thus IPH2201 serves as an apropos tool to assess HLA-E’s part in mediating MM cell resistance to NK-mediated cytotoxicity. Primary enriched NK cells pretreated with IPH2201 were able to overcome
RPMI-8226 (Figure 19, A and B) cells’ resistance to NK-mediated cytotoxicity, but oddly, the same was not true for the U266 cell line (Figure 19, C and D). Further, IFN-γ treated
U266 remained resistant to NK-mediated killing after blocking TRAIL (data not shown) or
MHC-I entirely (data not shown).
54 Figure 19: IPH2201 (Anti-NKG2A) pretreatment of NK cells recovers IFN-
γ-mediated resistance
A. B. * *
C. D. n.s. n.s. n.s.
n.s.
Figure 19: Primary enriched NK cells pretreated + IL-2 or + IL-15 for 24hrs
were given a two hour pretreatment of anti-NKG2A antibody IPH2201(2ug/ml).
IPH2201 reverses IFN-γ-mediated resistance of MM cell line RPMI-8226 to
primary NK cell-mediated cytotoxicity in IL-2(A, P-value <0.025) and IL-15(B,
P-value<0.025)-activated conditions. Interestingly, reversal of IFN-γ-mediated resistance of MM cell line U266(B) to NK cell killing by IPH2201 was not (Continue on page 56) 55 (Continued from page 55) observed in unstimulated(C) and IL-2-activated(D) conditions. Data represents 1
of 3 donors/experiments with similar trends. Data depicts a standard 51Cr release
assay and error bars depict the standard deviation of technical triplicates.
CD56Bright rather than CD56Dim NK cells exhibit greater killing against MM cells
When compared to healthy PB, NK subpopulations in the PB of MM patients
display an over-representation of CD56BrightCD94+ cells (Figure 20). Our group
has previously observed an abundance of NKG2A/CD94 on the surface of NK
cells from MM patients. As nearly all healthy CD56Bright but only ~50% of the
CD56Dim NK cells express CD94/NKG2A, we separated the two subpopulations
to see if they have a differential response to IPH2201. Impressively, CD56Bright
NK cells (Figure 21, left) exhibit greater cytotoxicity than do CD56Dim NK cells
(Figure 21, Right) against RPMI-8226 cells. Further, IPH2201 treatment of
CD56Bright NK cells (Figure 22) reestablished killing potential against an IFN-γ-
responding MM target.
56 Figure 20: Comparing NK cell subsets in healthy and MM peripheral blood
Figure 20: Peripheral blood mononuclear cells were isolated from three healthy
donors (Leukopacks, Red Cross) and three random MM patients. Surface
expression of prototypic NK cell markers CD56 and CD94 of each was assessed.
+ + Flow cytometric analysis excluding CD3 and CD138 populations in the
PB(excludes T cells and MM PCs , respectively) from three healthy donors (top)
and three MM patients(bottom). Graphs depict NK populations via markers CD56
(Y-axis) and CD94(X-axis). The CD56/CD94 profile which appears uniform in
the healthy donor samples is not represented in the patient samples. Both CD56+
and CD94+ markers are highly expressed and variably over-represented in the
MM samples.
57 Figure 21: Sorted CD56Bright and CD56Dim NK cell cytotoxicity against MM cell line RPMI-8226
*
Figure 21: Healthy primary NK cells were enriched, labeled for CD56
expression and FACS sorted/separated into CD56Bright and CD56Dim subset
populations. Both CD56Bright and CD56Dim NK cells were pretreated for 24 hrs
with IL-2 and their cytotoxic potential was assessed on the target MM cell line
RPMI-8226 + 48hr pretreatment with IFN-γ (10ng/ml). In IL-2 activating
conditions, CD56Bright NK cells exhibit greater cytotoxicity than CD56Dim NK cells towards MM cell line RPMI-8226 ( P-value < 0.001). Of note, both
(Continue on page 59) 58 (Continued from page 58) CD56Bright and CD56Dim NK cells exhibit undetectable amounts of killing toward
RPMI-8226 without stimulation/activation with IL-2 (data not shown). Graph depicts 1 of 3 experiments with similar trends. Data depicts a standard
51Chromium release assay and error bars depict the standard deviation of
technical triplicates.
59 Figure 22: Effect of IPH2201 (Anti-NKG2A) on CD56Bright and CD56Dim NK cytotoxicity against MM cell line RPMI-8226
* **
Figure 22: Two hour pretreatment of activated(IL-2, 24hrs) CD56Bright NK cells
with IPH2201(2ug/ml) significantly enhances cytotoxic potential against
untreated RPMI-8226 (P-value <0.003). In the same activating conditions,
IPH2201 significantly enhances CD56Bright NK cell-mediated cytotoxicity against
IFN-γ-treated RPMI-8226 target (P-value <0.025). The killing capacity of
IPH2201-pretreated CD56Bright NK cells is comparable regardless of target cell
pretreatment. Of interest, killing was successfully measured at E:T ratios less than
1. Killing for the CD56Dim for all conditions was below the threshold of
detection(data not shown).
60 2.3 Discussion and Summary
The body’s systemic cytokine profile skews towards proinflammatory (i.e. IFN-γ) conditions
with aging, a paradox termed inflamm-aging [174]. However, as MM manifests, the cytokine
milieu changes and resembles an immunosuppressive profile [65]. IFN-γ contributes to
differentiation of regulatory T cells, which are an immunosuppressive population often found
in elevated quantities during MM [175]. NK cells have altered and often muted effector
response in MM, and thus we tested if healthy NK cells would similarly respond in a MM co-
culture. Our data extends the observed phenomenon of spontaneous NK cell-mediated IFN-γ
production in the presence of MM cells to a healthy setting. IFN-γ production did not require
cell:cell contact, implying that soluble factors and extracellular vesicles may play a role
(Chapter 3). Although mathematical models have implicated the association and crosstalk of
IFN-γ with the IL-6/STAT3 networks, we are the first to report that IFN-γ alternatively
activates STAT3 in MM cells [164]. These observations taken together imply a mechanism in
early MM disease development that may perpetuate STAT3 activation and aid in the survival
of MM PCs.
We have also demonstrated that MM cells respond to IFN-γ by upregulating inhibitory
ligands on their surface. Gao, et al describe the mechanism of MM resistance to NK-
mediated lysis to be HLA-I-dependent, which may be true; however, it should be noted that
the authors of this study used a method of acid treatment that dissociated the MHC-I:β2M, and likely rendered all MHC-I unrecognizable[124]. Others have proposed that STAT1 is involved in MM cell immune evasion through the PD-1/PD-L1 axis [176]. HLA-E
61 expression has also been implied; however a mechanism for its expression on MM cells was
not determined [177].
HLA-E is classically recognized as an inhibitory ligand, however, a cellular stress
mechanism has also been described that blocks HLA-E’s affinity for NKG2A. This
subversion can occur when heat shock protein 60 (HSP60)-derived peptides compete with
MHC-I leading strand peptides for the HLA-E binding groove [178]. These ambiguities in
HLA-E function, especially under control of pro-inflammatory response element IFN-γ,
required further study. Our data indicate that both basal and induced HLA-E expression and
its inhibitory influence on NK-mediated lysis of RPMI-8226 can be overcome with the novel
anti-NKG2A antibody, IPH2201. A previous report also describes RPMI-8226’s natural resistance to NK-mediated lysis is in part dependent on NKp30 activation [179]. Further, the overexpression of IFN-γ/STAT-affiliated genes contributes to RPMI-8226’s resistance to
both IR and doxorubicin therapies [180]. These collective observations provide a rationale
for combined therapeutic approaches. However, our functional blocking data also implicates
IFN-γ in affording U266 cells an elusive and opportunistic resistance to NK cell
mobilization, independent of HLA-E, TRAIL, or MHC-I expression.
The accepted understanding of NK cell biology and maturation traditionally designates the
CD56Bright NK subset as a less cytotoxic effector to its CD56Dim counterpart. However, we present a scenario in which CD56Bright NK cells exhibit greater killing potential than CD56Dim
NK cells against the MM cell line RPMI-8226. Further, CD56Bright NK cells, even at low
NK:MM ratios, are capable of producing measurable killing while CD56Dim NK cells did not
62 (Figure 22 subtext). Perhaps the over-representation of CD56Bright NK cells in MM is not a consequence of dysregulated NK cell maturation, but rather describes a regulatory albeit incomplete response of NK cells towards malignancy. Our data may describe a previously unrecognized role for CD56Bright NK cells in the natural detection and clearance of MM
tumor cells and their precursors.
2.4 Experimental Methods
Cell lines and NK cell preparations
The multiple myeloma cell lines RPMI 8226 and U266 (ATCC, Manassas, Virginia, USA)
were maintained in normal culturing conditions; in RPMI 1640 media, supplemented with
10% Fetal Bovine Serum, 2mM GLUTIMAX®, and 2mM antibiotic/antimycotic (Life
Technologies, Carlsbad, California, USA). The human IL-2-dependent natural killer cell line
NK-92 (ATCC) was maintained in culture in RPMI 1640 media supplemented with 20%
FBS, 2 mM GLUTIMAX®, 2mM antibiotic/antimycotic, and 150 IU/ml recombinant human
IL-2 (catalog Ro 23-6019, Hoffmann-La Roche, Basel, Switzerland). Primary natural killer
cells were enriched from healthy donor sourced leukocytes (American Red Cross, Columbus,
Ohio, USA) by addition of RosetteSep™ Human NK Enrichment Cocktail (Catalog 15065,
StemCell Technologies, Vancouver, British Columbia, Canada) and density centrifugation on
Ficoll-Paque™PLUS gradient (Catalog 17-440-03, GE Healthcare Life Sciences). Residual
red blood cells were lysed using Ammonium Chloride Solution (Catalog 07850, StemCell
Technologies). In all instances, an enrichment of >75% CD56+ cells was achieved.
63 Treatments
When indicated, primary NK or NK-92 cells were treated with either 150 IU/ml of recombinant human IL-2 or recombinant human IL-15 (Catalog 130-093-955, Miltenyi
Biotec, San Diego, California, USA). MM cell lines were treated with 1ng/ml recombinant human IFN-γ (Catalog RIFNG50, Thermo Scientific) for at least 24 hours. Primary enriched
NK cells were treated with humanized IgG4 Anti-NKG2A antibody (IPH-2201, Innate
Pharma S.A., Marseille, France) or isotype control at a final concentration of 2ug/ml 4 hours prior to and throughout 51Cr release assays.
ELISPOT assays
ELISPOT experiments were conducted using MultiScreen 96-well plates (Millipore) as described previously [181]to measure NK-cell IFN-γ production. Spots were counted using an Immunospot Imaging Analyzer (Cellular Technology).
MTS assay
20,000 cells in 200ul (100,000/ml) were plated in a 96-well round bottom plate at day 0 in
10% FBS supplemented RPMI + 10ng/ml IFN-γ. At 24 and 48 hrs, cells were resuspended by pipette and 100ul transferred to a 96-well flat bottom plate. Subsequently 25ul of MTS agent
(Promega) was added and absorbance readings at 490nm were taken at 30’, 60’ 120’ and
240’. Values reflect the Abs490nm of sample minus that of a media-only control.
64 Transwell co-cultures
Contactless co-culture of MM and NK cells was achieved by placing 0.4 μm pore size
transwell inserts (Catalogs 3472 and 3450, Corning, Tewksbury, Massachusetts, USA) into
the wells of a culture plate. When used, NK-92 cells were washed 2x with PBS and starved
from IL-2 for 24 hours prior to co-culture.
Flow cytometry and sorting
For analysis of multiple myeloma cell surface expression, we used the following antibodies:
HLA-E-PE (Catalog 12-9953, clone 3D12HLA-E, eBioscience, Inc., San Diego, California,
USA), PD-L1-PE (Catalog 12-5983, clone MIH1, eBioscience, Inc.) IFNγR1-PE (Catalog
FAB673P, clone 92101, R&D Systems) IFNγR2-APC (Catalog FAB773, R&D Systems).
Appropriately labeled isotypes were used as controls. All samples were run on an LSRII flow
cytometer (BD Biosciences) and further analyses and graphics were generated using the
FlowJo v7.5.5 Software (FlowJo, LLC, Ashland, Oregon, USA). FACS sorting of CD3-
CD56bright and CD3-CD56dim NK cell populations were performed on a BD FACS ARIA II
system (BD Biosciences). Briefly, primary enriched NK cell preparations were labeled with
CD3-FITC (Catalog 555339, clone HIT3a, BD Biosciences, San Jose, California, USA) and
CD56-APC (Catalog IM2474, clone N901, Beckman Coulter, Pasadena, California, USA) antibodies and Sytox Blue viability dye (Catalog S34587, Life Technologies). Gates were strictly set on distinct target populations, and purity of viable sorted populations was > 98% before proceeding.
65 Real time PCR
Cells from each condition were washed 2x with PBS before lysis in P1 Buffer from the
Qiagen RNeasy kit (Qiagen, Valencia, California, USA) and RNA processing was carried out
according to manufacturer’s instructions. RNA was quantified using a NanoDrop 2000
(Thermo Fisher Scientific, Waltham, Massachusetts, USA) instrument. Samples with
acceptable Abs260/280 and Abs260/230 values > 1.9 were used for further analysis. For each sample, cDNA was generated from 1ug RNA template with M-MLV Reverse Transcriptase reagents (Catalog 28025, Thermo Fisher Scientific) and 10ng cDNA template was loaded uniformly for each real-time PCR reaction. Both HLA-E and PD-L1 primer/probes were
purchased as TaqMan® gene expression Assays (Catalog Hs03045171_m1 and
Hs01125301_m1, Life Technologies, Grand Island, New York, USA) and ribosomal 18s was
used as a housekeeping gene to normalize results. Real-Time PCR was carried out on an
ABI7900HT system with fast cycling conditions standard for the TaqMan® FAST Advanced
Reaction mix (Catalog 4444556, Life Technologies) and after threshold and baseline was
established using SDS 3.0.1 Software (Life Technologies), expression was determined by the
2-ΔΔCT method of calculation. Values represent the mean of technical triplicates + SEM.
Western blot analysis
Cells were harvested, washed once with ice-cold PBS, and lysed (108 cells/ml) in RIPA
buffer: 0.15 M NaCl, 1% Nonidet P-40, 0.1% SDS, 50 mM Tris (pH 8.0), supplemented with
Halt™ Protease and Phosphatase Inhibitor Cocktail (Catalog 78440, Life Technologies) as
described [182].Western blotting was performed according to previously published protocols
[183] and Ab-reactive proteins were detected with HRP-labeled donkey anti-rabbit (Catalog
66 NA934V, GE Healthcare Life Sciences, Pittsburgh Pennsylvania, USA) and sheep anti-
mouse antibodies (Catalog NA931V, GE Healthcare Life Sciences), and visualized using
ECL western blotting substrate (Catalog RPN2106, GE Healthcare Life Sciences). Proteins
were analyzed in 4–15% SDS-PAGE or any kD TGX ready-gels (Catalogs 456-1084 and
456-9034, Bio-Rad Laboratories, Hercules California, USA) using reducing conditions.
Primary antibodies used include Phospho-Stat1Tyr701 (Catalog #9171), Phospho-Stat2Tyr690
(Catalog #4441), Phospho-Stat3Tyr705 (Catalog #9145, Clone D3A7), Stat1 (Catalog #9172), and Stat3 (Catalog #9132, all Cell Signaling Technologies, Danvers Massachusetts, USA).
ELISA assays
To quantify the amount of human IFN-γ secreted by NK cells, an ELISA using capture mAb
(Catalog M700a, clone 2G1, Thermo Fisher Scientific) and detection biotin-conjugated mAb
(Catalog M701B, clone B133.5, Thermo Fisher Scientific) was performed as previously described in [184]. Graphed values represent the mean of technical triplicates + standard
deviation.
Cytotoxicity assays
Natural cytotoxicity was assessed by standard 51Chromium release assay as previously described [69]. Briefly, effector cells treated or stimulated for 4 hours, 12 hours, or overnight, as indicated, were plated and serially diluted. Target cells were labeled with 50uCi 51Cr for
75 minutes, washed repeatedly and plated. Cell-free supernatants were harvested after a 4-
hour incubation and transferred to scintillation vials and radioactivity was counted (CPM) on
an LS6500 Scintillation Counter (Beckman Coulter). A positive control of 5% SDS detergent
67 (+ 51Cr-labeled target cells) for maximum 51Cr release and a negative control of media-alone
(+ 51Cr-labeled target cells) for spontaneous 51Cr release were included. The %51Cr release in
each sample was directly proportional to the % of target cells lysed. Percentage of targets
lysed was calculated by (CPMsample – CPMspontaneous)/(CPMmaximum -CPMspontaneous) x 100.
Graphed values represent the mean of technical triplicates + Standard Deviation.
68 CHAPTER 3: MULTIPLE MYELOMA CELL-DERIVED EXTRACELLULAR VESICLES ENHANCE IFN-GAMMA PRODUCTION BY NATURAL KILLER CELLS
3.1 Introduction
Multiple myeloma (MM) is an incurable hematologic manifestation of the bone marrow
(BM). After homing and adhesion to bone marrow stromal cells (BMSC), malignant plasma
cell (PC) survival is largely dependent on and growth factors (i.e. IL-6, IL-10) provided by
the bone marrow microenvironment (BMME). Although MM cell communication in the
BMME has been demonstrated to include cell:cell contact and the exchange of soluble
factors, contemporary reports are beginning to describe the various roles of extracellular
vesicles (EV) in MM progression.
EV are membrane-encapsulated organelle-free subcellular material containing mRNA, DNA,
protein, and are capable of presenting antigen and delivery of contents to cell-specific targets
[78, 81]. Upon their discovery in the 1940s, EV were regarded as cellular debris. Although
detected in a variety of settings, including viral infection and tumor release, EV functionality
remained elusive until 1996 when Raposo, et al discovered that EV isolated from EBV-
transformed B cells were antigenic [77]. The report that figuratively opened the flood gates to current EV studies in cancer described how a mutant oncogenic receptor (EGFRVIII) on EV
69 could be transferred to recipient cells and initiate their malignant transformation [185]. With
regard to MM, new studies implicate EV functionality in promoting angiogenesis, driving
osteoclast differentiation, and activating myeloid-derived suppressor cells in the BMME
[186-188]. Interestingly, BMSC EV have been reported to promote MM progression while providing drug resistance to the protease inhibitor bortezomib [189]. The complexity and elegance of EV biology is only beginning to be understood and appreciated.
Our findings implicate MM EV as potential mediators of the natural killer (NK) cell pro- inflammatory response. Upon treatment with MM EV, NK cells show enhanced IFN-γ secretion. In reciprocation, IFN-γ induces the expression of inhibitory ligands HLA-E and
PD-L1 on the surface of MM cells (2.2 , Figure 11, Figure 12). IFN-γ-treated MM cells
release EV bearing MHC-I, HLA-E and PD-L1, which may possess inhibitory potential to
their cognate ligands on both NK cells and T cells of the innate and adaptive immune
systems, respectively. Altogether, our findings may describe a previously unrecognized
mechanism of immune evasion both initiated and maintained by MM EV.
3.2 Results and Figures
MM EV enhance NK cell-mediated IFN-γ production
Previously, our lab observed that spontaneous release of IFN-γ by NK cells in co-culture with
MM cells does not require contact (Figure 6, Chapter 2). The transwell membrane used in
that assay had a pore size of 400nm, which is roughly the average diameter of reported EV
sizes and includes all exosomes (50-100nm) but restricts some microvesicles (100-1000nm).
In lieu of the many soluble factors present in the co-culture, we focused our efforts on the
70 potential contribution of MM EV to the observed effect. We adopted a differential centrifugation protocol originally described by Thery, et al (Figure 23, A) and validated the
MM EV prep fractions by flow cytometry to ensure optimal yield (Figure 23, B) [82].
Functionally, MM cell-derived EV were sufficient to enhance NK cell-mediated IFN-γ production in IL-2-activated conditions (Figure 24).
71 Figure 23: Extracellular vesicle isolation and prep validation A.
B.
Figure 23: The protocol for isolating EV by differential ultracentrifugation (A)
including the spin forces and pellet contents. Briefly, cells are spun out and
discarded with a 300 x g (x gravitational force). Subsequently, apoptotic bodies
and cellular debris including organelle-containing vesicles is precipitated and
discarded in the 2000 x g and 10,000 x g spins. Isolation of EV occurs with
100,000 x g spin, when pellets are resuspended, washed, and pooled into a single
prep tube to precipitate and uniformly distribute for multiple readouts/treatments.
Visualization of EV preparation (B) from the MM cell line U266 on an LSRII (Continue on page 73) 72 (Continued from page 72) flow cytometer using sizing gates generated from standardized polystyrene beads of known sizes (100 nm, 200nm, 1000nm, and 2000nm in diameter). Note that
PBS, EV-Free Media, and EV Wash Step serve as control samples to validate prep purity and yield. Of note, our diluted EV prep generated 108,940 events in
30 seconds whereas the other fractions (PBS alone, EV-Free Media, and EV wash step) all generated <9000 events.
73 Figure 24: MM cell EV enhance NK cell IFN-γ production
A. * B. *
Figure 24: NK-92 cells were starved for 24 hours from growth factor IL-2 prior
to a 24hr re-stimulation of IL-2 + EV isolated from MM cell lines U266(A) and
RPMI-8226(B). In IL-2 NK cell-activated conditions, both U266 (A, P-value
=0.044) and RPMI-8226 (B, P-value<0.01) cell line-derived EV significantly
enhanced NK-92 cell IFN-γ production vs EV-free media control. Cell free
supernatants were harvested and used in an ELISA for hIFN-γ detection. Error
bars represent the standard deviation of technical replicates. Ratios represent the
EV produced by X number of MM cells (calculated by known number of cells
from which the EV were derived and the known volume of EV prep).
74 MM EV contain HLA-E and PD-L1 Inhibitory Ligands
We were the first to characterize the protein content of EV from MM cell lines and reported
an abundance of MHC-I within the EV [190]. We isolated EV from the PB and BM of MM
patients and healthy donors (PB only) and observed that their EV contained a full range of
detectable proteins ranging from 12~200 kDa (Figure 25, A). Further, MHC- I and β2M were elevated in the EV from MM but not from healthy PB (Figure 25, B). The mass spectrometry profile of identified MHC-1/HLA-I spectral hits in Harshman, et al. (Figure 26, A) showed a single identified unique peptide signature for the inhibitory ligand HLA-E (Figure 26, B)
[190]. Paired with our data regarding the upregulation of HLA-E and PD-L1 expression by
IFN-γ, we assessed if either inhibitory ligand was present in the EV and if increased cellular
expression of either would be translated to the vesicles. Indeed, EV derived from the MM
cell line RPMI-8226 contained both HLA-E and PD-L1 proteins (Figure 27). Further,
treatment of RPMI-8226 cells with IFN-γ increased the amount of detectable HLA-E and
PD-L1 in the EV fraction (Figure 27, right).
75 Figure 25: Protein profile of EV released from MM cell lines and patient
sera
Figure 25: PonceauS staining of EV proteins on a nitrocellulose membrane (A) reveals proteins of many sizes in healthy PB (lanes 2-4), MM PB (5-10) and MM
BM (Lane 11) when compared to MW ladder (lane 1). Western blot analysis of
MHC-I and β2M (B) describes increased expression in the MM samples, but not in the healthy PB. Impressive expression of both MHC-I and β2M was observed
in the MM BM sample, but the study lacks a healthy BM sample for baseline
comparison. Due to biological variability, the uniform signal from PonceauS
(total protein) staining serves as the loading control.
76 Figure 26: HLA-E detected on mass spectrometry study
Figure 26: All HLA unique spectral identities and heat map of expression in MM
cell lines and their EV (A) imply enrichment of HLA in the EV. A single
spectrum count for MHC-Ib member and inhibitory ligand HLA-E was also
detected (B).
Data was adapted from [190] with permission from the authors: Harshman, S. W., Canella, A., Ciarlariello, P. D., Rocci, A., Agarwal, K., Smith, E. M., Talabere, T., Efebera, Y. A., Hofmeister, C. C., Benson, D. M., Paulaitis, M. E., Freitas, M. A. and Pichiorri, F. (2013), Characterization of multiple myeloma vesicles by label-free relative quantitation. Proteomics, 13: 3013–3029. doi:10.1002/pmic.201300142 77 Figure 27: HLA-E and PD-L1 in EV released from MM
Figure 27: MM cell line RPMI-8226 was either placed in normal culture
conditions or starved from serum for 48hrs + IFN-γ (10ng/ml). Serum-starvation
is required due to the possibility of cross contamination of vesicles naturally
present in the serum. Whole cell lysates and EV isolated from the serum-starved
cells were assessed for inhibitory ligands HLA-E and PD-L1, as well as Lamin
B1 (nuclear protein absent in EV) and GAPDH (loading control). Both HLA-E
and PD-L1 proteins are detected on the EV fractions, and enhancement of their
signals are observed when RPMI-8226 were pretreated with IFN-γ) Note the
enrichment of PD-L1 in the EV when compared to the global cell lysates.
78 MM EV present ligands on their surface
To determine the orientation of membrane proteins of the EV, we chose to fluorescently
label RPMI-8226 EV preps with a pan-HLA-I antibody and visualized the EV using the
NanoSight NS300 particle tracking system (Figure 28). Detected EV particles in the fluorescent channel for both untreated(Figure 28, top right) and IFN-γ-treated conditions(Figure 28, bottom right) imply orientation of surface ligands HLA-ABC, PD-L1
and HLA-E on MM EV are likely presenting outward, and have the capacity to engage their cognate ligands on recipient cells.
79 Figure 28: NanoSight NS300 visualization of MM EV and HLA-ABC presentation
Figure 28 Simultaneous visualization, quantification and size distribution of
RPMI-8226 EV in untreated (top left) and IFN-γ-treated (10 ng/ml, bottom left) conditions. Fluorescently labeled MHC-I (HLA-ABC)-bearing EV are detected in both in untreated (top right) and IFN-γ-treated (10 ng/ml, bottom right) conditions.
80 3.3 Discussion
In this study, we demonstrated that we can reproducibly isolate EV from MM cell lines and patient sera and further study their contents, properties of extracellular-presentation and biological functionality. Further, our data implicate a previously unrecognized form of communication between MM PC and the proximal BMME and may extend systemically. Of note, PD-L1 appears to be selectively loaded on the EV of MM for release (Figure 21). These data indicate that circulating EV may saturate the PB with inhibitory messaging specific to
NK and T cell responses. We attempted, extensively, to capture the potential inhibitory effect of HLA-E and PD-L1-bearing vesicles on NK cell cytoxicity but could not reproducibly confirm either inhibitory or activatory influence (Data not shown). Conditions attempted are summarized in Table 3. We speculate that the over-abundance of HLA-ABC represented in the EV may out-compete the conveyance of potential inhibitory signaling provided by HLA-E and PD-L1. Further, paracrine and autrocrine factors in vivo may contribute to the specific cargo and surface landscape of the MM EV, thus an assessment of
MM EV and NK cell cytotoxic influence in an autologous setting would best resolve these inquiries.
81 Table 3: Attempted Primary NK Cell Cytotoxicity Experiments Involving MM EV 82
Our data also present a counter-intuitive observation in that EV laden with known NK and T cell inhibitory receptors were directly able to enhance the NK cell cytokine response.
Although demonstrated in an allogeneic system with EV from cell lines, our previous data showing the spontaneous release of IFN-γ by autologous NK cells in culture with MM PC suggests the phenomenon may be physiologically relevant. Limited quantities of EV source material (i.e. patient blood and bone marrow) and availability of autologously paired samples will remain the biggest barriers to overcome for progress in future studies.
Interestingly, our data supports a multifunctional role for EV in MM. Serum starvation of
RPMI-8226 increases both the output and angiogenic potential (via delivery of miR-135b) of its EV [186, 191]. We may be describing a phenomenon whereby MM cells release EV to simultaneously subvert NK cell recognition and induce IFN-γ release for beneficial ECM remodeling in the BMME.
3.4 Methods
Cell lines and NK cell preparations
The multiple myeloma cell lines RPMI 8226 and U266 (ATCC, Manassas, Virginia, USA) were maintained in RPMI 1640 media, supplemented with 10% Fetal Bovine Serum,
2mM GLUTIMAX®, and 2mM antibiotic/antimycotic (All Life Technologies). The human
IL-2-dependent natural killer cell line NK-92 (ATCC) was maintained in culture in RPMI
1640 media supplemented with 20% FBS 2 mM GLUTIMAX®, 2mM antibiotic/antimycotic,
83 and 150 IU/ml recombinant human IL-2 (catalog Ro 23-6019, Hoffmann-La Roche, Basel,
Switzerland). Primary natural killer cells were enriched from healthy donor sourced
leukocytes (American Red Cross, Columbus, Ohio, USA) by addition of RosetteSep™
Human NK Enrichment Cocktail (Catalog 15065, StemCell Technologies, Vancouver,
British Columbia, Canada) and density centrifugation on Ficoll-Paque™PLUS gradient
(Catalog 17-440-03, GE Healthcare Life Sciences). Residual red blood cells were lysed using
Ammonium Chloride Solution (Catalog 07850, StemCell Technologies). In all instances, an
enrichment of >75% CD56+ cells was achieved.
Treatments
NK-92 cells were treated with 150 IU/ml of recombinant human IL-2 in the presence of EV derived from MM cell lines U266 and RPMI-8226 for 24hrs. Extracellular vesicle dose ratios represent the amount of EV generated by the number of MM cells to the number of NK cells plated. A cell’s amount of EV is directly proportional to the known concentration of cells plated and the harvested volume. EV treatments ranged from 4 to 24 hours as indicated for each assay.
Extracellular vesicle isolation
Isolation of cell line-derived extracellular vesicles has been previously described by Théry, et al [82]. and performed in Harshman, et al [190]. Concisely, differential centrifugation was used to isolate cells (300 x g), cellular debris (2000 x g), and apoptotic bodies (10,000 x g) for removal, and the remaining supernatant was used to pellet EVs (100,000 x g). EV pellets
84 were washed/pooled with PBS and centrifuged one more time (100,000 x g), after which
supernatants were discarded and EV pellets resuspended in application-dependent buffer.
Flow cytometry
Visualization of purified EV preps was carried out on an LSR II flow Cytometer (BD
Biosciences, San Jose, California, USA). Logarithmic scaling for both FSC and SSC
channels was required for proper separation of microparticles. To compare the contents of
each fraction of EV prep, uniform acquisition of all events for a set 30” period was used. To
determine the size distribution of EV present, EV acquisition events were compared to
standardized polystyrene beads of known 0.1 um, 0.2 um, 1.0 um and 2.0 um in diameters
(Spherotech, Lake forest, Illinois, USA).
EV surface staining and detection by Nanosight
EV surface labeling with antibodies of HLA-ABC (Catalog 561349, clone G46-2.6, BD
Pharmingen) and isotype control (Catalog 557872, clone MOPC-21, BD Pharmingen)
conjugated to PE-Cy7 fluorochrome. For each sample, EVs were prepared from a starting
volume of 107 cells’ worth of vesicles. EV pellets were resuspended in a small volume of
PBS before equal distribution for antibody labeling and placed on ice for 30 mins. PBS was
added to block the antibody to a dilution of 200x the labeling volume (10 ml wash added to
50ul labeling volume), mixed thoroughly by inversion and vortexing before being loaded into the Nanosight Syringe Pump for automated injection and analysis with the NS300 system
(Malvern Instruments Ltd, Malvern, UK) fitted with a laser diode of 532 nm. To detect PE-
Cy7 fluorescently labeled EVs, the camera channel was switched to detection of long pass
85 570nm filter. Both total EV and fluorescent EV populations were quantified and size distribution profile determined within the same tube by consecutive 5 x 60” video series recordings at 60 frames per second throughout an automated and continuous sample flow stream. All graphics and analyses were processed using Nanoparticle Tracking Analysis
Software 3.0 (Malvern Instruments Ltd).
ELISA assays
To quantify the amount of human IFN-γ released by NK cells, an ELISA using capture mAb
(Catalog M700a, clone 2G1, Thermo Fisher Scientific) and a biotin-conjugated detection mAb (Catalog M701B, clone B133.5, Thermo Fisher Scientific) previously described in
(source) was used. Graphed values represent the means of technical triplicates + standard deviation. Extracellular vesicle dose ratios represent the amount of EV generated by the number of MM cells to the number of NK cells plated. A cell’s amount of EV is directly proportional to the known concentration of cells plated and the harvested volume. EV treatments ranged from 4 to 24 hours as indicated for each assay.
Western blot analysis
Cells or EV were harvested, washed once with ice-cold PBS, and lysed (108 cells/ml or 25ul total per EV pellet) in RIPA buffer: 0.15 M NaCl, 1% Nonidet P-40, 0.1% SDS, 50 mM Tris
(pH 8.0), supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Catalog
78440, Life Technologies) as described [182]. Western blotting was performed according to previously published protocols [183], and Ab-reactive proteins were detected with HRP- labeled donkey anti-rabbit (Catalog NA934V, GE Healthcare Life Sciences, Pittsburgh
86 Pennsylvania, USA) and sheep anti-mouse (Catalog NA931V, GE Healthcare Life Sciences)
and ECL (Catalog RPN2106, GE Healthcare Life Sciences). Proteins were analyzed in 4–
15% SDS-PAGE or any kD TGX ready-gels (Catalogs 456-1084 and 456-9034, Bio-Rad
Laboratories, Hercules California, USA) using reducing conditions. Primary antibodies used includeGAPDH (Catalog ab8245, Abcam, Cambridge Massachusetts, USA), PD-L1 (Catalog
ab174838, Abcam), and HLA-E (Catalog ab2216, Abcam), and Lamin B1(Abcam).
87 CHAPTER 4: FUTURE STUDIES AND EXTENDED DISCUSSION
4.1 The mechanism for IFN-γ-mediated resistance of U266 to NK cell cytotoxicity
We have shown that the MM cell line U266 cells resist NK cell-mediated natural cytotoxicity upon treatment with IFN-γ (Figure 15 B and C, Figure 16) and that the mechanism does not involve HLA-E (Figure 19, C and D), MHC-I (data not shown) over-expression, and
CD107A (data not shown). Further, healthy primary NK cells express little, if any, PD-1 on their surface, supporting that PD-L1 is likely not involved in the mechanism of action [192,
193]. We hypothesize that the mechanism of resistance is mediated by cell surface ligands which are up-regulated and present in response to IFN-γ. To refine the candidates mediating this phenomenon, I plan to prepare surface-protein extracts from U266 cells + IFN-γ and perform a 2D Western blot, first separating the proteins by isoelectric focusing, followed by a second separation by size. Our hope is that the differential protein profile will provide three observable groups when blots are overlaid – unchanged with treatment, up-regulated with
IFN-γ treatment and down-regulated with IFN-γ treatment. Candidate surface protein signals up-regulated by IFN-γ will be identified using mass spectrometry, and their contribution to
IFN-γ-mediated resistance will be validated using neutralizing antibodies (if available).
Moreover, our proposed method may lead to the discovery of a novel surface protein, or implicate a novel role for an already identified protein in MM cell resistance to NK-mediated
88 cytoxicity. Of course, we would likely extend this study to EV lysates to see if any candidate
inhibitory ligands are released either spontaneously or in response to IFN-γ.
4.2 IFN-γ treatment of MM cells to enhance efficacy of proteasome inhibitors
Long term exposure of MM cells to IFN-γ decreases their proliferation [194, 195]. Cell cycling is halted while the immune system assesses MM cells for stress and potentially antigenic endogenous peptides. We observed that our MM cells robustly respond to short term IFN-γ treatment by newly synthesizing proteins with little effect on proliferation capacity. Further, treatment of MM cells with the irreversible proteasome inhibitor bortezomib down-regulates their MHC-I expression and increases their susceptibility to NK cell-mediated lysis [196]. We hypothesize that the pretreatment of MM cells with IFN-γ and subsequent treatment with either proteasome inhibitor bortezomib or carfilzomib may enhance their susceptibility to direct and indirect (NK-cell mediated) cytotoxicity. These data may indicate that we can abrogate the otherwise advantageous response of MM cells to IFN-
γ; however, the therapeutic translation for this data may be moot in that IFN-γ is not a tolerable treatment in the clinical setting, and is associated with a variety of flu-like symptoms including fever, headache, and diarrhea.
4.3 Differential peptide cross-presentation in MM following IFN-γ treatment
Our understanding of HLA-E peptide presentation is still evolving. Classically, HLA-E presents leading strand peptides of MHC-1a. Recently, a newly discovered peptide repertoire of which all members are capable of presentation via HLA-E has been reported [197]. This peptidome, consisting of 36 peptides ranging from 7-16 aa in length, represents a paradigm
89 shift in HLA-E functionality. Even more bizarre is the discovery that the proteasome during
protein degradation can splice peptides whose product sequences do not seem to reflect
genomically possible combinations [198]. Although this process was not studied in the
myeloma cell per se, it hints toward the possibility of identifying specific and novel peptide
sequences within the MM peptidome. These peptides may serve as biomarkers, or better yet,
novel therapeutic targets. I propose to characterize the peptides presented on MM cells +
IFN-γ treatment. The method uses an acid wash which detaches β2M from all MHC proteins,
and therefore the pool of peptides will represent all MHC (Ia, Ib, and II)-bound peptides.
Peptides will be separated by size via HPLC and sequenced using mass spectrometry.
Perhaps the most informative data from this proposed study would be to assess if the peptides
loaded upon MHC molecules within the vesicles, show a difference in distribution and
character. This may highlight a selective mechanism in MM cells in order to release
unfavorable peptides in EV while maintaining a less suspect cell surface.
4.4 Concluding remarks
In this thesis, for the first time MM, we have provided evidence of an alternative role of IFN-
γ in activating STAT1 and STAT3 which facilitates the increased surface expression of PD-
L1 and HLA-E, and their eventual incorporation and release on extracellular vesicles. If anything, the phenomenon described herein should provide considerations towards cancer therapies and their targets. To elaborate, if an MM patient’s system is saturated with HLA-E and PD-L1-bearing vesicles, then therapeutic dosing would likely need reconsideration to account for the non-active binding of these agents. Further, this report provides evidence that perhaps targeting the inhibitory receptors towards these ligands may be more effective.
90 We also present a potential and previously unrecognized role for CD56Bright NK cells as potent cytotoxic effector cells towards an MM target, perhaps alluding to a regulatory mechanism of NK cells in detecting early malignant transformation.
Lastly, we describe a reciprocating communication between NK cells and Myeloma cells summarized in Figure 29, which is previously unreported, and requires more investigation to determine the extent of the biological functionality of MM EV on the innate immune system.
91 Figure 29: Graphical Summary of Dissertation Work
Figure 29 (Continue on page 93) 92 (Continued from page 92)
Figure 29: Graphical Summary of Dissertation Work
We observed spontaneous IFN-γ production by NK cells when co-cultured with
MM PC (Figure 5) , the effect does not require cell:cell contact (Figure 6), and
later discover that MM-derived EV were sufficient to enhance NK cell IFN-γ
release (Figure 24). These observations may describe a perpetuating cycle in
which MM cells benefit from the constant release of IFN-γ and other associated
factors. Further, MM Cells have functional IFN- γRs (Figure 7) which signal
through STAT1 and STAT3 (Figure 8) to increase expression of many
genes/proteins including MHC-I (Figure 10), and inhibitory ligands HLA-E
(Figure 11,Figure 13) and PD-L1 (Figure 12,Figure 13). Treatment of MM cells
with IFN-γ allows measurable protection from NK cell mediated lysis (Figure 16,
Figure 17, Figure 18), which is partially dependent by HLA-E:NKG2A
interaction in the RPMI-8226 cell line (Figure 19,Figure 22), but not for U266
(Figure 19). Myeloma cells also respond to IFN-γ releasing vesicles with
membrane bound HLA-E and PD-L1 (Figure 26, Figure 27, Figure 28) whose
biological functionality are subject to future studies.
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