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Myeloid-Derived Suppressor Cell Acquisition of Antigen from Dendritic Cells and Induction of Antigen-Specific T Cell

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004686/00001

Material Information

Title: Myeloid-Derived Suppressor Cell Acquisition of Antigen from Dendritic Cells and Induction of Antigen-Specific T Cell
Physical Description: Book
Language: English
Creator: Weber, Hannah
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2012
Publication Date: 2012

Subjects

Subjects / Keywords: Myeloid-Derived Suppressor Cells (MDSC)
Immunology
Cancer
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Myeloid-derived suppressor cell (MDSC)-induced suppression of tumor-specific T cells in an antigen-specific manner occurs in tumor-bearing mice and cancer patients. The mechanism by which MDSC obtain tumor antigens remains to be identified. We sought to elucidate this mechanism and evaluated our hypothesis that dendritic cells (DC) could provide antigens to peripheral lymphoid organ MDSC. We found that during an overnight co-culture DC previously loaded with ovalbumin (OVA) transferred OVA antigen to splenic MDSC, enabling re-isolated MDSC to suppress IFN-? release by activated OVA antigen-specific (OT-I) T cells in an ELISPOT. Addition of soluble factors derived from solid tumors to the co-culture abrogated the need for splenic MDSC to obtain OVA antigen from DC to suppress OT-I T cells. iNOS expression and arginase I expression and activity by splenic MDSC, associated with nonspecific suppression of activated T cells, increased with the presence of tumor-derived factors. This demonstrates that cytokines and growth factors present at the tumor site convert MDSC from antigen-specific to nonspecific suppressors in a dose-dependent manner. This study also demonstrates the ability of splenic MDSC to obtain antigens from DC, providing a potential mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens in vivo. We plan to continue this study and determine the mechanism by which DC transfer antigens to splenic MDSC. Most importantly, we plan to validate our findings in tumor-bearing organisms. Elucidating the mechanism of peripheral lymphoid organ MDSC tumor antigen acquisition would aid in developing treatments that eliminate MDSC immunosuppression and promote anti-tumor immunity.
Statement of Responsibility: by Hannah Weber
Thesis: Thesis (B.A.) -- New College of Florida, 2012
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2012 W3
System ID: NCFE004686:00001

Permanent Link: http://ncf.sobek.ufl.edu/NCFE004686/00001

Material Information

Title: Myeloid-Derived Suppressor Cell Acquisition of Antigen from Dendritic Cells and Induction of Antigen-Specific T Cell
Physical Description: Book
Language: English
Creator: Weber, Hannah
Publisher: New College of Florida
Place of Publication: Sarasota, Fla.
Creation Date: 2012
Publication Date: 2012

Subjects

Subjects / Keywords: Myeloid-Derived Suppressor Cells (MDSC)
Immunology
Cancer
Genre: bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Myeloid-derived suppressor cell (MDSC)-induced suppression of tumor-specific T cells in an antigen-specific manner occurs in tumor-bearing mice and cancer patients. The mechanism by which MDSC obtain tumor antigens remains to be identified. We sought to elucidate this mechanism and evaluated our hypothesis that dendritic cells (DC) could provide antigens to peripheral lymphoid organ MDSC. We found that during an overnight co-culture DC previously loaded with ovalbumin (OVA) transferred OVA antigen to splenic MDSC, enabling re-isolated MDSC to suppress IFN-? release by activated OVA antigen-specific (OT-I) T cells in an ELISPOT. Addition of soluble factors derived from solid tumors to the co-culture abrogated the need for splenic MDSC to obtain OVA antigen from DC to suppress OT-I T cells. iNOS expression and arginase I expression and activity by splenic MDSC, associated with nonspecific suppression of activated T cells, increased with the presence of tumor-derived factors. This demonstrates that cytokines and growth factors present at the tumor site convert MDSC from antigen-specific to nonspecific suppressors in a dose-dependent manner. This study also demonstrates the ability of splenic MDSC to obtain antigens from DC, providing a potential mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens in vivo. We plan to continue this study and determine the mechanism by which DC transfer antigens to splenic MDSC. Most importantly, we plan to validate our findings in tumor-bearing organisms. Elucidating the mechanism of peripheral lymphoid organ MDSC tumor antigen acquisition would aid in developing treatments that eliminate MDSC immunosuppression and promote anti-tumor immunity.
Statement of Responsibility: by Hannah Weber
Thesis: Thesis (B.A.) -- New College of Florida, 2012
Electronic Access: RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE
Bibliography: Includes bibliographical references.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Local: Faculty Sponsor: Walstrom, Katherine

Record Information

Source Institution: New College of Florida
Holding Location: New College of Florida
Rights Management: Applicable rights reserved.
Classification: local - S.T. 2012 W3
System ID: NCFE004686:00001


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MYELOID DERIVED SUPPRESSOR CELL ACQUIS ITION OF ANTIGEN FROM DENDRITIC CELLS AND INDUCTION OF ANTIGEN SPECIFIC T CELL SUPPRESSION BY HANNAH WEBER A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the sponsorship of Dr. Katherine Walstrom Sarasota, Florida May, 2012

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! "" Dedication I dedicate this thesis to my parents Jeff and Linda, for all the love, inspiration, guidance, and support that made this possible.

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! """ Acknowledgements I would like to thank my thesis sponsor and academic a dvisor Dr. Katherine Walstrom and my principal investigator and mentor Dr. Dmitry Gabrilovich for their extensive guidance, support, and advice. I would also like to thank my committee members Dr. Amy Clore and Dr. Paul Scudder for their help and support. I would like to thank Dr. Thomas Condamine for his collaboration, aid, and instruction in this research, as well as Dr. Lily Lu, Dr. Rupal Ramakrishnan, Brianna Lenox, Dr. Alex Corzo, and all other members of the Gabrilovich laboratory. I would also lik e to thank Jodi Kroeger and Kate Shapland of the Moffitt Cancer Center Flow Cytometry Core for use of the flow cytometers.

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! "# Table of Contents Introduction 1 Material and Methods 27 Results 49 Discussion 70 Co nclusion 83 Appendix I: List of Abbreviations 84 Appendix II: Abbreviated Version of Material and Methods 87 Appendix III: Step by Step Protocols 92 References 108

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! List of Figures Figure 1 Structure of the TCR 4 Figure 2 Priming of na•ve T cells by APC. 5 Figure 3 IFN signaling pathway. 9 Figure 4 Tumor microenvironment factors and signaling pathways that promote the accumulation and activation of MDSC. 12 Figure 5 Granulocytic and monocytic MDSC. 16 Figure 6 Chronological overview of the main experi mental set up. 33 Figure 7 Step by step ELISPOT protocol. 35 Figure 8 Analysis of cell surface molecule expression by flow cytometry. 41 Figure 9 Picture of a representative ELISPOT assay showing IFN release by OT I T cells in response to B1 6/Kb OVA cells with or without splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC. 51 Figure 10 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with OVA loaded DC compared to splenic MDSC previously co cultured with unloaded DC. 52 Figure 11 IFN release by OT I T cells in response to B16/Kb OVA cells with or without splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC. 54 Figure 12 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with

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! "# unloaded DC or with OVA loaded DC in the presence of 20% TES, experiment 1. 57 Figure 13 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 20% TES, experiment 2. 58 Figure 14 Suppression of OT I T cell IFN release in respo nse to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 10% TES. 60 Figure 15 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cul tured with unloaded DC or with OVA loaded DC in the presence of 5% TES. 61 Figure 16 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the prese nce of 2.5% TES. 62 Figure 17 Induction of iNOS expression in splenic MDSC by tumor derived factors. 64 Figure 18 Induction of arg I expression in splenic MDSC by tumor derived factors. 65 Figure 19 Induction of arg I activity in spl enic MDSC by tumor derived factors. 66 Figure 20. IFN release by OT I T cells in response to B16/Kb OVA cells with or without splenic MDSC previously co cultured with unloaded

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! "## DC or with OVA loaded DC +/ transwell. 69 Figure 21 Parameters for collecting MDSC from the DC and MDSC co culture by F ACS 99

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! "### MYELOID DERIVED SUPPRESSOR C ELL ACQUISITION OF A NTIGEN FROM DENDRITIC CELLS AND INDUCTION OF ANTIGEN SPECIFIC T CELL SUPPRESSION Hannah Weber New College of Florida, 2012. ABSTRACT Myeloid derived suppressor cell (MDSC) i nduced suppression of tumor specific T cells in an antigen specific manner occurs in tumor bearing mice and cancer patients. The mechanism by which MDSC obtain tumor antigens remains to be identified. We sought to elucidate this mechanism and evaluated o ur hypothesis that dendritic cells (DC) could provide antigens to peripheral lymphoid organ MDSC. We found that during an overnight co culture DC previously loaded with ovalbumin (OVA) transferred OVA antigen to splenic MDSC, enabling re isolated MDSC to suppress IFN release by activated OVA antigen specific (OT I) T cells in an ELISPOT. Addition of soluble factors derived from solid tumors to the co culture abrogated the need for splenic MDSC to obtain OVA antigen from DC to suppress OT I T cells. iNOS expression and arginase I expression and activity by splenic MDSC, associated with nonspecific suppression of activated T cells, increased with the presence of tumor derived factors. This demonstrates that cytokines and growth factors present at the tumor site conve rt MDSC from antigen specific to nonspecific suppressors in a dose dependent manner. This study also demonstrates the ability of splenic MDSC to obtain antigens from DC, providing a potential mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens

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! "# in vivo We plan to continue this study and determine the mechanism by which DC transfer antigens to splenic MDSC. Most importantly, we plan to validate our findings in tumor bearing organisms. Elucidating the mechanism of peripheral lymphoid o rgan MDSC tumor antigen acquisition would aid in developing treatments that eliminate MDSC immunosuppression and promote anti tumor immunity. Dr. Katherine Walstrom Division of Natural Sciences

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! Introduction MDSC: Definition and Importance in Cancer Tumor mediate d suppression of the immune system is a common phenomenon in tumor bearing animals and cancer patients Tumors directly and indirectly suppress immune reacti vity against them through vario us mechanisms, resulting in limited efficacy of many cancer treatments ( Mellman et al. 2011 ) Much c urrent research is focused on elucidating how tumor s inhibit immune responses in order to overcome immune suppression and create therapies that effectively activate anti tumor immunity and promote overall survival in cancer patients ( Mellman et al. 2011 ) The study of MDSC is one particular area of interest with impact on this problem MDSC are a heterogeneous population of immature myeloid cells that acc umulate during various chronic pathological conditions, including sepsis, trauma, and especially cancer ( Youn & Gabrilovich 2010 ). In many types of cancer in both mice and humans, the tumor secretes factors that induce the accumulation and activation of M DSC, which suppress immune responses against the tumor ( Youn & Gabrilovich 2010 ). MDSC mediated inhibition of immune recognition and rejection of the tumor promotes tumor growth and metastasis ( Mauti et al. 2011 ). Conversely, elimination, reprogramming, or differentiation of MDSC in mice and humans has been shown to promote anti tumor immune responses and inhibit tumor progression ( Suzuki et al. 2005 Talmadge 2007 Kerkar et al. 2011, Lee et al. 2011 ). It was initially thought that MDSC could represent a subset of immune cells that play a role in healthy organisms and become subverted during cancer ( Gabrilovich & Nagaraj 2009 ). However, this has been shown not to be the case. MDSC counterparts in

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! # healthy mice and humans, referred to as immature myeloid cells (IMC), form a transient population and quickly differentiate into macrophages, dendritic cells (DC), and neutrophils. They are found at very low percentages in the spleen and peripheral blood, and are virtually absent from lymph nodes ( Gabrilovich & Nagaraj 2009 ). In addition, IMC lack immunosuppressive abilities ( Youn & Gabrilovich 2010 ). MDSC, by contrast, accumulate at tenfold higher numbers in the spleen and peripheral blood and are found in lymph nodes, can remain stable in their immature stat e, and are strongly immunosuppressive ( Gabrilovich & Nagaraj 2009 Youn & Gabrilovich 2010 Lee et al 2011 ). It is this last characteristic that truly sets MDSC apart from IMC: although MDSC share the phenotypic and morphological characteristics of IMC, they are functionally distinct from the latter in their ability to suppress the immune system ( Youn & Gabrilovich 2010 Lee et al. 2011 ). Therefore MDSC do not merely represent an expanded cell compartment that exist s in healthy organisms, but rather form a unique population of cells induced to accumulate and acquire immunosuppressive abilities during cancer and other chronic conditions ( Youn & Gabrilovich 2010 ). Mechanisms of Immunosuppression Although MDSC use a wide variety of mechanisms to suppress the immune system, their primary role is induction of T cell anergy. T cells, a key part of the adaptive immune system, are responsible for targeting infected, pre cancerous, or cancerous cells, aiding in the activation of other immune cells, and regulating the immune response ( Murphy 2012 ). As adaptive immune cells, they are antigen specific each T cell recognizes a different antigen, or peptide derived from a particular pathogen or tumor

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! $ cell referred to as its cognate antigen T cells become anergic w hen they are tolerized to their cognate antigen, or are induced to no longer react against it ( Murphy 2012 ). T cells and APC T cell s recognize their cognate pathogen derived or tumor cell derived antigen by binding to it with their T cell receptor (TCR) ( M urphy 2012 ). The TCR forms a complex with the co receptor CD3 and the homodimeric zeta ( ) chain the latter of which is the main component of the TCR complex responsible for initiating the signal transduction cascade that results in T cell activation after antigen binding (see figure 1) ( Murphy 2012 ). The TCR does not bind free antigen; rat her, T cells recognize antigen ic peptides bound to major histocompatibility complex ( MHC ) molecules. MHC molecules are cell surface glycoproteins that are divided into two types, class I and class II ( Murphy 2012 ). MHC class I, expressed by almost all ce lls in the body, is recognized by T cells that express the cell surface co receptor CD8, referred to as cytotoxic T cells since they directly kill infected cells or tumor cells ( Murphy 2012 ). MHC class II, commonly expressed by antigen presenting cells (A PC), is recognized by T cells that express the cell surface co receptor CD4, referred to as helper T cells since they help stimulate cytotoxic T cells and other immune cells ( Murphy 2012 ). Helper T cells can subdivided into two main classes: T H 1 cells, ch aracterized by their secretion of the cytokines (signaling molecules) interferon gamma (IFN ) and interleukin 2 ( IL 2) which play a key role in viral infections and in antitumor immunity ; and T H 2 cells, characterized by their secretion of the cytokines IL 4 and IL 5, which are usually associated with production of antibodies and responses to pa rasitic infections ( Dunn et al. 2002 Murphy 2012 ).

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! % Figure 1 Structure of the TCR The functional TCR complex is com p osed of the antigen binding TCR # : $ heterodimer associated with four signaling chains (two % one & and one ) collectively called CD 3, which are required for the cell surface expression of the antigen binding chains and for signaling. A homodimer of chains is also associated with the receptor. Taken from Murphy 2012. Regardless of subtype, na•ve T cells, or T cells that have not pr eviously encountered their cognate antigen, need to become activated or primed, in order to recognize and target pathogen infected cells or tumor cells (Murphy 2012) This occurs through interact ion with APC, a specialized type of immune cell thus named because one of their primary roles is to present antigen to and prime na•ve T cells. After priming, na•ve T cells become effector T cells and can then react against pathogen infected cells or tumor cells ( Murphy 2012 ) Priming occurs in a three step fash ion, and all three signals, provided uniquely by APC, are necessary to induce the activation and proliferation of T cells (see figure 2) In fact, T cell binding to cognate antigen without the other signals provided by APC induces T cell anergy and can re sult in apoptosis ( Murphy 2012 ).

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! & During priming, the APC MHC /antigen complex binds to the T cell TCR CD3 complex and CD4 or CD8 co receptor (signal 1, activation) At the same time APC co stimulatory molecules bind T cell co stimulatory receptors (signal 2, expansion), and the APC secrete cytokines that determine the type of effector cell the primed T cell becomes (signal 3, differentiation) ( Murphy 2012 ). For example, interaction between T cell expressed CD28 and AP C expressed CD80 or CD86 induces T cel ls to express IL 2 and the IL 2 receptor (IL 2R) driving proliferation as part of signal 2 For signal 3 APC that secrete IFN and IL 12 induce T H 1 cells, while APC that secrete IL 4 induce T H 2 cells ( Murphy 2012 ) Figure 2. Priming of na•ve T cells by APC Three kinds of signals are involved in the activation of na•ve T cells. TCR (and in this example, CD4 co receptor) bi nding to the MHC/foreign peptide complex expressed by APC transmit s a signal to the T cell that antigen has been encountered (signal 1, arrow 1) Effective T cell activation requires a second signal : for example CD28 on the T cell binds to B7 molecules o n the APC (signal 2, arrow 2), resulting in the increased survival and proliferation of the T cell. For CD4 + T cells in particular, different differentiation pathways produce subsets of effector T cells with different responses, depending on signal 3 (arr ow 3) delivered by the APC. Cytokines are commonly, but not exclusively, involved in directing this differentiation. Taken from Murphy 2012.

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! Common APC include DC, macrophages, and B cells. DC can process the widest variety of antigens and are mainly re sponsible for priming T cells ; macrophages and B cells interact more with CD4+ T cells that have already been primed ( Murphy 2012 ). DC and macrophages are both mature myeloid cells that form part of adaptive immun ity in their capacity as APC but also func tion as innate immune cells. They nonspecifically ingest invading pathogens and infected cells particularly through phagocytosis (afterwards processing pathogenic material into antigens for presentation) and recruit other immune cells to the site of inf ection by secretion of inflammatory cytokines ( Murphy 2012 ). B cells like T cells, are mature lymphoid cells that function as adaptive immune cells Unlike T cells, which recognize antigen bound to MHC molecules on infected cells and tumor cells, B cell s recognize free antigen expressed by extracellular pathogens (like bacteria) or their corresponding toxins (Murphy 2012). B cells secrete antibodies in response to their cognate antigen which bind to and neutralize toxins or opsonize (coat) pathogens, s ignalin g phagocytes to ingest pathogens. Although B cells act as APC, their main function is the secretion of antibodies (Murphy 2012). In cance r, even though APC expressing MHC/ tumor antigen complexes particularly DC, can potentially activate T cells a gainst the tumor, immunosuppressive cells like MDSC have numerous methods of inducing T cell tolerance as described below ( Gabrilovich & Nagaraj 2009, Mellman et al. 2011 ). Depletion of arginine via arginase I and iNOS One of the best characterized mechan isms of MDSC mediated T cell inhibition is the depletion of arginine through high production of arginase I (arg I) and inducible nitric oxide synthase (iNOS) ( Gabrilovich & Nagaraj 2009 ). Arg I and iNOS are enzymes that

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! ( use arginine as a substrate so MDS C in close p roximity to T cells deplete the T cells of arginine ( Zea et al. 2005 Gabrilovich & Nagaraj 2009 ). It was found that remov al of arginine from the local environment of T cells inhibited their expression of cyclin D3 and cyclin dependent kinase 4 (CDK4) ( Rodriguez et al 2007 ). Activated Cyclin/CDK complexes are responsible for phosphorylat ing the retinoblastoma protein which results in the nuclear translocation of E2F transcription factors E2F transcription factors upregulate expression of g enes that are responsible for progression through the cell cycle int o the G 1 and S phases ( Coqueret 2002 ). Therefore depletion of arginine arrests T cells in the G 0 G 1 phase of the cell cycle, preventing proliferation (Rodriguez et al. 2007 ). In additio n, arginine starvation results in impaired expression of the CD3 chain of the TCR complex preventing T cell activation ( Rodriguez et al. 2002 ). Production of NO In addition to the depletion of arginine, upregulation of iNOS also impairs T cell proliferation and activation through the production of nitric oxide (NO ) ( Gabrilovich & Nagaraj 2009 ) NO can diffuse into cells and cause oxidative damage to proteins and DNA ( Harari et al. 2004 ). Beyond direct cellular damage, NO is known to downregulate the expression of MHC class II molecules by APC thereby negatively a ffect ing helper T cell activation ( Harari et al. 2004 ). NO also prevents T cell proliferation and induce s T cell anergy through inhibition of the JAK3/STAT5 pathway ( Bingisser et al. 1998, Gabrilovich & Nagaraj 2009 ). Th e tyrosine kinase JAK3 (Janus kina se 3) is associated with receptor s (such as IL 2 R ) that dimerize upon binding to their ligand (such as IL 2) (Murphy 2012). JAK3 in turn forms a homodimer in which each monomer can phosphorylate and activate the other. The activated homodimer then binds and

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! ) phosphorylates STAT5 (signal transducer and activator of transcription 5), which itself forms a homodimer after activation. STAT5 then undergoes nuclear translocation, acting as a transcription factor to upregulate genes that promote progression throu gh the cell cycle ( Murphy 2012 ). NO has been found to inhibit the phosphorylation of tyrosine residues on JAK3 and STAT5 in T cells, thereby preventing proliferation and arresting T cells in the G 0 G 1 phase of the cell cycle ( Bingisser et al. 1998 ). It wa s recently found that NO produced by iNOS in MDSC chemically induces the nitration of STAT 1 tyrosine residues in T cells (Mundy Bosse et al. 2011) Nitration of STAT1 prevents its ph osphorylation, which, like STAT5 is required to activate it in the JAK/S TAT pathway ( Mundy Bosse et al. 2011 Murphy 2012 ) The nitration of STAT1 was found to correspond to a decreased response of T cells to IFN ultimately suppressing T cell activation ( Mundy Bosse et al. 2011 ). IFN signaling in immune cells IFN is a cytokine produced by macrophages and DC in response to pathogens, and by T cells after activation by APC ( Murphy 2012 ) IFN along with IL 12, sti mulate s helper T cells to differentiate into T H 1 cells ( Dunn et al. 2002, Murphy 2012 ). T H 1 cell produc tion of IFN further creates an autocrine feedback loop that promotes the T H 1 phenotype and inhibits differentiation into T H 2 cells ( Murphy 2012 ). T H 1 cells recruit and help activate cytotoxic T cells, which also secrete IFN ultimately inducing macrop hage activation, upregulation of MHC class I and II expression, and inhibition of viral replication in infected cells ( Schroder et al. 2004 Murphy 2012 ). IFN binds to the IFN receptor ( IFN R ), which then binds JAK1 and JAK2, inducing the autophospho rylation /activation of JAK2. JAK2 then phosphorylates JAK1

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! which in turn phosphorylates IFN R enabling it to bind to STAT1 (Schroder et al. 2004) JAK2 then phosphorylates STAT1, inducing its nuclear translocation to act as a transcription factor (see figure 3) ( Schroder et al. 2004 ). In cells infected by viruses, STAT1 induces the expression of PKR and oligoadenylate synthetase. PKR, once activated by dsRNA produced by viruses, inhibits eIF 2, preventing translation of viral and cellular proteins ( Sc hroder et al. 2004 Murphy 2012 ). Oligoadenylate synthetase activates endoribonuclease, which degrades viral RNA ( Schroder et al. 2004 ). Figure 3. IFN signaling pathway. Originally published in Expert Review in Molecular Medicine by Cambridge University Press (2003). Taken from Alvarez [updated 2005].

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! "+ In T H 1 cells, STAT1 induces the expression of T bet, a transcription factor that activates the g enes for IFN and the IL 12 receptor (IL 12 reinforces T H 1 differentiation and induces expansion via activation of STAT4) and blocks T H 2 differentiation through inhibition of GATA3, a transcription factor that promotes T H 2 differentiation ( Murphy 2012 ). In macrophages, STAT1 upregulates receptor mediated phagocytosis and the production of lysosomal enzymes and the NADPH oxidase system, which produces ROS (reactive oxygen species) to damage pathogens ( Schroder et al. 2004 ). The signaling initiated by IFN ultimately activates helper and cytotoxic T cell s enabling them, in cancer, to r eact against tumor cells. In addition, IFN has been found to have direct anti proliferative and pro apoptotic effects on tumor cells ( Dunn et a l 2002 ). Therefore, MDSC mediated suppression of IFN signaling in T cells via NO production inhibits recognition and rejection of the tumor ( Mundy Bosse et al. 2011 ). Production of PNT and ROS In addition to NO, iNOS also generates superoxide anion in response to arg I consumpti on of arginine (Bronte et al. 2003 ). NO reacts with superoxide anion to form an even more reactive anion, peroxynitrite (PNT) ( Bronte et al. 2003 ). PNT also ca uses oxidative damage to DNA and proteins and induces nitration of tyrosine residues on kinases like JAKs, preventing their phosphorylation and ultimately disrupting T cell activation and proliferation after antigen binding ( Brito et al. 1999 ). It was recently found that intratumorally produced PNT induces nitration of the chemokine (chemotactic cy tokine) CCL2, which normally attracts activated T cells to the tumor. T cells cannot bind to nitrated CCL2, resulting in impaired migration of tumor specific T cells to the site in which they could potentially target tumor cells ( Molon et al. 2011 ). MDSC

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! "" produced PNT also induces nitration of the TCRs of cytotoxic T cells and the MHC molecules of tumor cells. This inhibits T cell activation both by disrupting TCR binding to MHC/antigen complexes on the tumor cell surface, and by preventing intracellular formation of MHC/ tumor antigen comple xes ( Nagaraj et al. 2007 Lu et al. 2011 ). Another well characterized mechanism of MDSC suppression is the release of ROS ( Gabrilovich & Nagaraj 2009 ). ROS, which include superoxide anion and hydrogen peroxide, are pro duced in MDSC primarily by NADPH oxidase (similar to macrophages ) ( Harari et al. 2004 Lu et al. 2011 ). ROS suppress the immune system in many of the same ways as NO: they cause direct oxidative damage and they inhibit T cell activation through the produc tion of PNT after reaction with NO ( Harari et al. 2004 Kusmartsev et al. 2004, Nagaraj et al. 2007 Lu et al. 2011 ). In fact, in some studies it was found that the upregulation of ROS, rather than iNOS, wa s mainly responsible for the production of PNT an d subsequent suppression of activated T cells by MDSC ( Nagaraj et al. 2007 ). Accumulation and Activation Just as MDSC have a variety of ways to suppress the immune system so too does the tumor have a variety of ways to recruit MDSC Tumor cells secrete many different factors, including a number of immunosuppressive and pro inflammatory cytokines that have been implicated in recruiting MDSC (see figure 4 ) ( Ostrand Rosenberg & Sinha 2009 Condamine & Gabrilovich 2011 ). Tumor secreted factors like VEGF (v ascular endothelial growth factor) IL 6, IL 10, GM CSF (granulocyte/macrophage colony stimulating factor), G CSF (granulocyte colony stimulating factor), and M CSF (macrophage colony stimulating factor) are known to inhibit DC maturation via the JAK2/STAT 3 pathway ( Marigo et al. 2010 Condamine & Gabrilovich 2011 )

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! "# The molecular mechanisms behind these factors have been more recently elucidated. For example, it was found that activated STAT3 in MDSC upregulates the expression of NADPH oxidase and the pr oduction of ROS, which, beyond its role in MDSC mechanisms of immunosuppression inhibit s the maturation of myeloid cells ( Kusmartsev & Gabrilovich 2003 Condamine & Gabrilovich 2011 ). In addition, STAT3 activation upregulates C/EBP $ a transcription factor that controls the differentiation and proliferation of myeloid cells ( Marigo et al. 2010, Zhang et al. 2010 ) C/EBP $ and STAT3 induce the expression of c myc in myeloid cells, promoting proliferation through upregulation of CDK 4 ( Zhang et al. 2010 ). C/EBP $ was found not only to induce proliferation and inhibit differentiation of MDSC but also to upregulate iNOS and arg I expression, inducing MDSC immunos uppressive abilities ( Marigo et al. 2010 ). F igure 4 Tumor microenvironme nt factors and signaling pathways that promote the accumulation and activation of MDSC. The many pro inflammatory and immunosuppressive cytokines and growth factors present at the tumor site, such as IL 6, IL 10, G CSF, GM CSF, and VEGF signal through multiple pathways in MDSC. These factors and pathways overlap to form a complex network promoting the expansion and activation of MDSC. For example, STAT3 signaling contributes to increased proliferation, myeloid differentiation, and ROS production. ST AT1 and STAT6 signaling promote production of arg I and iNOS Taken from Gabrilovich & Nagaraj 2009.

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! "$ Although STAT3 is a major regulator of MDSC accumulation and activation, STAT1 also plays a role in MDSC activation. STAT1 signaling in response to IL 1 $ (secreted by the tumor) or IFN (secreted by activated T cells) induces arg I and iNOS expression in MDSC (Kusmartsev & Gabrilovich 2005, Mohavedi et al. 2008 Condamine & Gabrilovich 2011). In addition, STAT5 signaling in MDSC, induced by tumor secret ed GM CSF, was found to confer resistance to a chemotherapeutic drug that blocked their accumulation (Ko et al. 2010). STAT6 signaling in response to IL 4 or IL 13 upregulates arg I production by MDSC (Sinha et al. 2005 Serafini et al. 2006, Serafini et al. 2008). PGE 2 a n inflammatory mediator secreted by many tumor types, is another major inducer of arg I expression in MDSC ( Ocho a et al. 2007 Gabrilovich & Nagaraj 2009 ). PGE 2 is produced from arachidonic acid in a pathway initiated by cyclooxygenase 2 (COX 2) and it binds to the E prostanoid receptors (EP) 1, 2, and 4, all of which are expressed by MDSC ( Rodriguez et al. 2005 Sinha et al. 2007 Gabrilovich & Nagaraj 2009 ). PGE 2 has been found to upregulat e COX 2 expression in DC precursors, resulting in inhibition of differentiation and in production of IL 10, iNOS, and PGE 2 This creates an autocrine feedback loop that induces DC precursors to become MDSC thus promoting MDSC accumulation (Obermajer et al. 2011 a ). PGE 2 also promotes MDSC migration t o the tumor site In a recent study tumor produced PGE 2 upregulate d the expression of the chemokine receptor CXCR4 by MDSC and PGE 2 inhibition blocked tumor production of the corresponding chemokine CXCL12 This resulted in impaired migration of MDSC t oward tumor cells and in impaired MDSC production of COX 2 ( Scotton et al. 2002 Obermajer et al. 2011 b ).

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! "% In several studies, the blocking of EP2 and EP4 and the use of COX 2 inhibitors reduced MDSC production of arg I and iNOS, MDSC accumulation, and MDSC migration to the tumor site. COX 2 inhibitors also decreased the overall ability of MDSC to suppress T cell responses and facilitate tumor progression ( Rodriguez et al. 2005 Sinha et a l 2007 Obermajer et al. 2011a ). Signaling through PGE 2 is therefor e one of the crucial mechanisms by which the tumor induces the expansion, migration, and immunosuppressive abilities of MDSC. It must be emphasized that the recruitment of MDSC in cancer requires two parts: accumulation and activation. Although the signa ls and pathways involved in these processes overlap, the tumor both prevents immature myeloid cells from differentiating, thus inducing their accumulation, and it activates these immature myeloid cells to become immunosuppressive, thus inducing them to bec ome MDSC ( Condamine & Gabrilovich 2011 Gabrilovich et al. 2012 ). Phenotype and Morphology MDSC, as a heterogeneous population of immunosuppressive immature myeloid cells, consist of multiple populations that lack the markers for mature myeloid cells, but otherwise differ in phenotype and morphology ( Youn & Gabrilovich 2010 ). In tumor bearing mice MDSC are broadly defined by co expressi on of the myeloid markers CD11b and Gr 1, and comprise cells of mixed granulocytic and monocytic morphology: that is, leu kocytes with segmented nuclei and cytoplasmic granules, such as neutrophils; and leukocytes with non segmented nuclei, such as macrophages and DC, respectively ( Suzuki et al. 2005 Gabrilovich & Nagaraj 2009 ). Gr 1 consists of two epitopes, Ly 6G and Ly 6 C, and two main subsets of murine MDSC have been identified using these

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! "& epitopes. CD11b + Ly6G + Ly6C low cells have a granulocytic morphology (Ly6G is expressed by granulocytes) and are referred to as G MDSC (granulocytic MDSC) while CD11b + Ly6G Ly6C high cell s are monocytic (Ly6C is expressed by monocytes) and are referred to as M MDSC (monocytic MDSC) ( Youn & Gabrilovich 2010 ). In addition to phenotypic differences, functional differences between G MDSC and M MDSC have been documented but they have generat ed some controversy (see figure 5 ). According to most studies, M MDSC, which have lower Gr 1 expression, are more suppressive than G MDSC, though some studies have found that M MDSC and G MDSC are equally suppressive ( Huang et al. 2006, Movahedi et al. 20 08 Nausch et al. 2008 Youn et al. 2008 Ribechini et al. 2010 ). In addition, the proportions of G MDSC and M MDSC differ according to the tumor model: most tumor types yield more G MDSC than M MDSC in peripheral lymphoid organs, but some produce G MDSC and M MDSC in equal proportion ( Youn et al. 2008 ). At the tumor site, M MDSC have usually been found to be the predominant type of MDSC ( Umemura et al. 2008, Priceman et al. 2010 Youn & Gabrilovich 2010 ). Most studies have found that G MDSC suppress T cell responses via ROS and NADPH oxidase, al though in one study their suppression was at least partially mediated by arg I ( Movahedi et al. 2008, Youn et al. 2008 Peranzoni et al. 2010 ). Most studies have also found that M MDSC primarily suppress T cells via iNOS, in addition to a rg I, and some have implicated COX 2 as well ( Movahedi et al. 2008, Youn et al. 2008 Peranzoni et al. 2010) G MDSC in culture persist in their immature state longer than M MDSC, which tend to differentiate into immunosuppressi ve tumor associated macrophages (TAM) and DC ( Mohavedi et al. 2008, Youn et al. 2008 ).

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! "' Figure 5 Granulocytic and monocytic MDSC. Possible pathways of MDSC differentiation in cancer. Blue dashed lines = normal myeloid cell differentiation in healthy organisms. Black solid lines = proposed development of MDSC in tumor bearing organisms. HSC = hematopoietic stem cells. CMP = common myeloid progenitor. M = macrophages. DC = dendritic cells. T = T cells. Taken from Youn & Gabrilovich, 2010 The se inconsistencies are amplified when trying to define more specific MDSC subpopulations. Multiple markers expressed by MDSC beyond CD11b and Gr 1 have been found in various tumor models, but none consistently identify a definitive subset, particularly one w ith distinctive immunosuppressive properties ( Peranzoni et al. 2010 ). In addition, unique markers enabling a phenotypic definition of MDSC (in addition to the functional definition) remain elusive: the phenotype of G MDSC is shared by neutrophils, and the phenotype of M MDSC is shared by inflammatory monocytes (short lived monocytes, recruited to sites of inflammation, that quickly differentiate into APC) ( Geissmann et al. 2003, Suzuki et al. 2005, Youn & Gabrilovich 2010 )

PAGE 26

! "( The characterization of human MDSC is even more compl ex Humans do not have a Gr 1 ortholog, so MDSC in cancer patients are much less well defined. Granulocytic and monocytic MDSC subsets with different cell surface proteins have recently been found in different types of cancer, but most studies have not demonstrated distinct or consistent functional differences between them ( Rodriguez et al. 2002, Filipazzi et al. 2007, Rodriguez et al. 2009 Peranzoni et al. 2010, Vuk Pavlovic et al. 2010 ). Hopefully future studies will yield bette r characterization of human MDSC and shed more light on key murine and human MDSC markers (Youn & Gabrilovich 2010). Antigen Specific vs. Nonspecific Suppression Another subject of ongoing debate is whether MDSC mediate antigen specific or nonspecific T cell suppression Tumor bearing mice and cancer patients harboring MDSC do not usually suffer from general immune suppression (except either post chemotherapeutic treatment or in terminal stage of disease), so it was assumed that MDSC only mediate antigen specific suppression ( Nagaraj & Gabrilovich 2008, Solito et al. 2011 ). Most studies have found that murine MDSC suppress antigen activated T cell s but do not inhibit T cells nonspecifically activated by anti CD3 and anti CD28 antibodies or by the mitogen phytohemagglutinin (PHA) ( Brito et al. 1999, Kusmartsev & Gabrilovich 200 5, Nagaraj et al. 2007) This indicates that MDSC require engagement with the TCR through interaction with the MHC/antigen complex for optimal suppression ( Nagaraj et al. 2007 ) Th is corresponds with the necessity of direct cell contact for ROS and PNT to act on surface molecules, like the TCR, expressed by T cells (Suzuki et al. 2005) In contrast, iNOS and arg I could affect T cell proliferation given close proximity between MDSC and T cells, but without direct contact ( Suzuki et al. 2005 ). Ot her

PAGE 27

! ") studies however, have documented the suppression by MDSC of both antigen specific and non specific T cell stimulation ( Kusmartsev et al. 2000 Watanabe et al. 2008 ). Suppression by G MDSC and M MDSC : spleen and tumor After identification of murine G MDSC and M MDSC population s a majority of studies found that G MDSC suppress ed antigen specific T cell responses and M MDSC suppress ed antigen speci fic and non specific responses, although other studies found that neither subset suppressed nonspecific stimulation ( Movahedi et al. 2008 Umemura et al. 2008, Youn et al. 2008 ). As MDSC are known to be present in the peripheral lymphoid organs, namely the spleen, and at the tumor site, a numbe r of later studies focused on exploring differences between these two populations, particularly in the context of G MDSC and M MDSC. Multiple studies found a large proportion of M MDSC at the tumor site indicating that tumor site MDSC mediate d nonspecifi c suppression, in contrast to splenic MDSC (with a large majority of G MDSC), which were thought to mediate antigen specific suppression ( Umemura et al. 2008 Priceman et al. 2010 ). Suppression by splenic MDSC In one early study, splenic MDSC inhibit ed only antigen specific cytotoxic T cell activation, which was dependent on MHC class I expression: blocking MDSC MHC class I molecules resulted in the abrogation of suppression ( Gabrilovich et al. 2001 ). Another later study found that MD SC picked up antige n in vivo and splenic MDSC suppressed antigen specific T cell activation only after being loaded with specific but not control peptide, both in vitro and in vivo Furthermore, this suppression was found to be MHC class I restricted, and splenic MDSC fail ed to suppress anti CD3 and anti CD28 mediated T cell proliferation (Kusmartsev et al. 2005)

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! "* Confirming the findings that splenic MDSC mediated antigen specific suppression, a very recent study found that in murine lung carcinoma, melanoma, lymphoma, an d colon carcinoma tumor models, splenic MDSC suppressed antigen activated cytotoxic T cells both in vivo and in vitro but failed to suppress anti CD3 and anti CD28 mediated T cell activation ( Nagaraj et al. 2012 ). Interestingly, this study also demonstr ated MDSC antigen specific suppression of helper T cell responses mediated by MHC class II expression. MDSC suppression of cytotoxic T cell responses is well documented, while MDSC suppression of helper T cell responses has been inconsistent, with multipl e studies showing that MDSC do not suppress antigen specific activation of helper T cells ( Nagaraj et al. 2012 ). This study indicated that these inconsistencies were due to differences in MDSC expression of MHC class II : o nly MDSC from mice with colon car cinoma had high MHC class II expression and suppressed antigen specific helper T cell activation ( Nagaraj et al. 2012 ). When MHC class II expression was abrogated, so too was MDSC suppression of antigen specific helper T cell activation. Surprisingly, a fter incubation with antigen activated helper T cells, splenic MDSC that expressed MHC class II acquired the ability to suppress nonspecific cytotoxic T cell stimulation. This only occurred when helper T cells were activated by specific antigen, this spec ific antigen was available to MDSC, MDSC had high MHC class II expression and MDSC were able to have direct contact with helper T cells ( Nagaraj et al. 2012 ). Overall t his indicates both that splenic MDSC suppression is antigen specific with regard to cy totoxic and helper T cells and is mediated by MHC class I and class II, respectively, and that the induction of nonspecific suppression by splenic MDSC depends on antigen specific activation of helper T cells and is mediated by MHC class II.

PAGE 29

! #+ The authors fo und that although engagement of MHC class II on MDSC did not upregulate iNOS, arg I, or ROS, it did upregulate COX 2 and PGE 2 expression ( Nagaraj et al. 2012 ). Furthermore, inhibition of COX 2 partially reversed MDSC suppression of antigen specific helper T cell responses and completely reversed their suppression of nonspecific T cell responses. This indicates that nonspecific suppression by MDSC is at least partially mediated by COX 2, in line with the abundance of PGE 2 in the tumor microenvironment and the idea that tumor site MDSC are nonspecific suppressors Although this study did not directly evaluate suppression by G MDSC compared to M MDSC or by splenic MDSC compared to tumor site MDSC, the authors found that splenic M MDSC had higher MHC class II expression than splenic G MDSC (which, as in most studies, comprised a large majority of splenic MDSC), and tumor site MDSC had much higher MHC class II expression than splenic MDSC ( Nagaraj et al. 2012 ). In the context of the ability of helper T cells to induce nonspecific suppression by MDSC through MHC class II, this further reinforces the idea that M MDSC and tumor site MDSC suppress nonspecifically. Comparison of splenic MDSC and tumor site MDSC suppression Other studies have directly compared splenic MDSC to tumor site MDSC According to one study, CD11b+Gr 1+F4/80+ ( where F4/80 is a macrophage marker ) immature macrophages at the tumor site suppressed nonspecific T cell responses, while splenic MDSC did not ( Kusmartsev & Gabrilovich 2005 ). In additi on, MDSC at the tumor site were found to preferentially differentiate into TAM ( Kusmartsev & Gabrilovich 2005 ). A later study confirmed these findings, reporting that MDSC at the tumor site comprised mostly monocytic MDSC that expressed F4/80, had upregul ated

PAGE 30

! #" iNOS and arg I, and suppressed nonspecific T cell responses ( Umemura et al. 2008 ) Therefore, past and current evidence strongly indicates that splenic MDSC, on which most studies have focused and which have been found to be predominantly G MDSC, med iate antigen specific suppression through acquisition of antigen and direct interaction with antigen activated T cells. In contrast, tumor site MDSC, which most studies have found to be predominantly M MDSC, suppress nonspecifically ( Nagaraj & Gabrilovich 2008 ) This is in agreement with findings that, although general immune suppression does not occur in mice or patients with cancer and MDSC T cells in the periphery are mostly able to respond to a variety of tumor unrelated stimuli, while T cells presen t at the tumor site have impaired responses to such stimuli ( Corzo et al. 2010 ). Human studies, due largely to the scarcity of patient samples, have been more challenging not only for phenotypic characterization, but also for antigen specificity studies (S olito et al. 2011). The difficulty of performing human studies means that there are very few that directly evaluate the antigen specificity of human MDSC, or compare G MDSC and M MDSC and peripheral lymphoid organ MDSC and tumor site MDSC, as mouse studie s have done (Solito et al. 2011). Overall there have been conflicting results with regard to the antigen specificity of human MDSC, with data on the subject obtained mostly from studies that focused on either antigen mediated or nonspecific suppression, b ut not on both simultaneously ( Almand et al. 2001 Fricke et al. 2007 Hoechst et al. 2008 Srivastava et al. 2008 Nagaraj et al. 2010 Vuk Pavlovic et al. 2010 ). In contrast to previous studies, one recent study evaluated the suppression by peripheral ly mphoid organ MDSC and tumor site MDSC in mice and humans, in the context of antigen specificity, phenotype, and mechanisms of immunosuppression ( Corzo

PAGE 31

! "" et al. 2010 ). This study confirmed that peripheral lymphoid organ MDSC suppress in an antigen specific m anner mainly via ROS, while tumor site MDSC suppress in a nonspecific manner mainly via iNOS and arg I ( Nagaraj & Gabrilovich 2008 Corzo et al. 2010 ). Specifically, in lymphoma, melanoma, and lung cancer mouse models, tumor site MDSC suppressed antigen s pecific and nonspecific responses via iNOS and arg I, while splenic MDSC only suppressed to a weaker degree, antigen specific responses via NADPH produced ROS ( Corzo et al. 2010 ). However, tu mor site and splenic MDSC did not differ in phenotype or morpho logy, with both containing M MDSC and (predominantly) G MDSC These findings were confirmed in cancer patients: MDSC from the peripheral blood of head and neck cancer patients showed high levels of ROS and low levels of iNOS, while phenotypically identica l MDSC from the tumor showed high levels of iNOS and low levels of ROS (Corzo et al. 2010) In addition, tumor site MDSC but not peripheral blood MDSC, suppressed PHA induced T cell proliferation. The authors confirmed that the induction of antigen speci fic and nonspecific suppression by MDSC was due to the unique environments of the tumor and the peripheral lymphoid organs and not to the presence of inherently phenotypically or morphologically dist inct MDSC subsets at each site ( despite the evidence that they did not diffe r in phenotype or morphology ). Splenic MDSC from donor tumor bearing mice were injected into the spleens or tumors of recipient tumor bearing mice and MDSC suppressive ability was evaluated. Donor MDSC in recipient spleens suppressed a ntigen specific but not nonspecific T cell responses, while donor MDSC in recipient tumor sites suppressed both antigen specific and nonspecific T cell responses (Corzo et al. 2010) In addition, donor MDSC at the recipient tumor site were found to remain at the tumor site

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! #$ and preferentially differentiate into CD11b+F4/80+ TAM, while MDSC in the recipient spleen remained in the spleen, maintained their immature state for longer an d subsequently differentiated equally into macrophages and DC The authors found that tumor site MDSC nonspecific suppression and differentiation into TAM was governed by the hypoxic tumor microenvironment, which induced expression of the transcription factor hypoxia inducible factor 1alpha (HIF 1 # ) in MDSC (Corzo et al. 2010) Antigen acqu i sition by peripheral lymphoid organ MDSC? Although this study provided solid, side by side evidence for antigen specific suppression mediated by peripheral lymphoid organ MDSC and nonspecific suppression me diate d by tumor site MDSC, it introduced another complication. It was initially thought that peripheral lymphoid organ MDSC induced tumor specific T cell suppression by traveling to the tumor site obtaining tumor antigens, and traveling back to peripheral lym phoid organs where they interacted with tumor specific T cells and induced tumor specific T cell anergy ( Kusmartsev et al. 2005 ). This recent study, however, showed that MDSC directed to the tumor site remain there, maintain nonspecific suppression, and differentiate into TAM, while peripheral lymphoid organ MDSC remain in their respective compartment and maintain antigen specific suppression and an immature state ( Corzo et al. 2010 ). Th ese results impl y that although peripheral lymphoid organ MDSC supp ress immune responses specifically against tumor antigens, they do not obtain tumor antigens directly from the tumor site. Although it is possible MDSC could pick up soluble tumor antigens in the periphery, the availability of soluble tumor antigens would likely be far to o low to allow this A more likely scenario is that MDSC obtain tumor antigens from

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! #% another immune cell type capable of traveling to the tumor site and peripheral lymphoid organs and that has a strong capacity for obtaining and presenting antigen s that is, professional APC. The strongest candidate would be DC. DC as APC DC that are mainly responsible for antigen presentation are termed conventional DC (cDC) which are referred to here as DC O nce fully differentiated in the bone marrow, DC travel to the peripheral tissues ( Gabrilovich et al. 2012 Murphy 2012 ). There they sample antigens through phagocytosis and macropinocytosis (Murphy 2012). When they encounter certain molecular patterns commonly expressed by foreign pathogens (calle d pathogen associated molecular patterns (PAMPs)), they are activated and become fully mature ( Gabrilovich et al. 2012, Murphy 2012 ). PAMPs bind to toll like receptors which activate multiple signaling pathways in DC. Ultimately this induces the product ion of cytokines and the expression of co stimulatory molecules ( Murphy 2012 ). In addition, the expression of MHC class I and II molecules is upregulated, so that antigens derived both intracellularly and extracellularly can be loaded onto MHC molecules a nd effectively presented to T cells ( Murphy 2012 ). The DC are now licensed, or equipped to activate na•ve T cells Mature DC are directed to peripheral lymphoid organs, namely the lymph nodes, by the binding of the chemokines CCL19 and CCL21 to the rec eptor CCR7, which is upregulated on DC upon maturation ( Murphy 2012 ) Once in the lymph nodes, DC are further stimulated to express MHC and co stimulatory molecules and the chemokine CCL18, which attracts na•ve T cells (Murphy 2012). Then DC encounter na• ve T cells, which they prime to become effector cells

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! #& Although DC in healthy individuals undergo maturation and act as effective APC in the above fashion in many cases DC in cancer have been shown to be defective in activating immune responses. Part o f this stems from the subversion of myelopoiesis and recruitment of MDSC by the tumor and subsequent shortage of mature DC ( Gabrilovich et al. 2012 ). The tumor also produces factors many of which play a role in recruiting MDSC, that prevent mature DC bot h in the periphery and at the tumor site from becoming activated ( Gabrilovich et al. 2012 ). Furthermore, DC at the tumor site are often induced to become immunosuppressive themselves ( Gabrilovich et al. 2012 ). Mechanisms of Antigen A cquisition by MDSC De spite their dysfunction in activating T cells, DC in cancer can present antigen, and could provide antigen to MDSC ( Orange et al. 2003 ) MDSC not only have been shown to be expanded in lymph nodes during cancer, but they also have been found to be in clos e proximity to and interact with DC in lymph nodes ( Nagaraj & Gabrilovich 2008 unpublished data from Lu and Gabrilovich). In our study we evaluated the ability of DC to provide antigens to splenic MDSC and enable MDSC to suppress antigen activated T cell s Our in vitro experiments indicate that murine splenic MDSC, when co cultured with antigen loaded DC, obtain antigen from DC and subsequently suppress antigen specific T cell responses. This supports the hypothesis that in tumor bearing organisms DC co uld be the source of tumor antigens for peripheral lymphoid organ MDSC, enabling MDSC to induce tumor antigen specific immune suppression. Furthermore, we found that in the presence of increasing amounts of tumor derived factors, splenic MDSC no longer n eeded antigen obtained from DC in order to suppress antigen activated T cells. MDSC production of arg I and expression of the

PAGE 35

! #' genes encoding iNOS and arg I, all associated with nonspecific suppression of activated T cells also increased with the amount o f tumor derived factors present This is the first time that the dependence of nonspecific suppression by MDSC on tumor site cytokines and growth factors such that they are converted from antigen specific to nonspecific suppressors in a dose dependent ma nner has been directly demonstrated. Although the interactions between DC and MDSC are not very well documented, recent studies indicate that MDSC and DC undergo crosstalk in tumor bearing organisms, resulting in dysfunctional DC maturation and in the re duced ability of DC to activate T cells ( Ostrand Rosenberg et al. 2012 ). However, the reciprocal effect of DC on MDSC is not well documented and the potential of DC to provide antigen to peripheral lymphoid organ MDSC has, to our knowledge, never been pr eviously tested. Furthermore, there is no published study critically evaluating the mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens. This is the first time that the ability of splenic MDSC to obtain antigen from DC has been demons trated, providing a potential mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens in vivo However, i t is unclea r how and in what form antigen is transferred to MDSC from DC. DC could transfer an antigenic peptide a partial protein or the MHC class I / antigen complex to MDSC. These processes may require direct cell contact or they may be mediated indirectly It is also possible that peripheral lymphoid organ MDSC could obtain antigens from another source besides DC, namely macroph ages. W e plan to evaluate these possibilities to further define antigen acquisition by peripheral lymphoid organ MDSC and, most importantly, to validate our finding in tumor bearing organisms.

PAGE 36

! #( Material and Methods Experimental Outline/Set U p The object ive of this study is to investigate the mechanism by which peripheral lymphoid organ MDSC obtain tumor antigen s which enables them to suppress activated tumor antigen specific T cells. Most data t o date indicate that MDSC from peripheral lymphoid organs suppress antigen specific T cell activation in a system where MDSC have access to antigen, but they do not suppress non specific T cell activation. This is in contrast to tumor site MDSC, which have been shown to suppress T cell reactivity in a nonspecifi c manner. However, the mechanism of antigen acquisition by peripheral lymphoid organ MDSC in vivo is currently unknown. In order to test the mechanism of MDSC antigen acquisition it is necessary to have a reliable and consistent system of antigen specifi c T cell activation. We decided to use, as an antigen specific stimulus, the B16/Kb OVA cell line, which is the B16F10 cell line (a murine melanoma cell line) transfected to express the fusion MHC class I/SIINFEKL peptide complex. SIINFEKL is an 8 amino acid long peptide derived from OVA (ovalbumin, an immunogenic chicken egg white protein). T cells generated by OT I transgenic mice are specific for the SIINKFEKL/MHC class I complex (see Mouse Models pp. 35 37). Therefore, OT I cytotoxic T cells will se crete IFN in response to B16/Kb OVA cells (but not in response to B16F10 cells). The amount of IFN release, and therefore the level of T cell activation, can be quantified in an ELISPOT (see ELISPOT pp. 34 35). This provides a solid model of antigen specific activation that can be used to explore antigen acquisition and antigen specific suppression by murine splenic MDSC (representative of peripheral lymphoid organ MDSC), since MDSC in this system cannot

PAGE 37

! #) obtain antigen unless they are previously exposed to it. In contrast to previous experimental systems, free peptide is not available to MDSC: the only source of SIINFEKL is bound to MHC class I molecules on tumor cells, which, since they are not APC, cannot provide it to MDSC The amount of IFN released by T cells in the absence of MDSC and in the presence of MDSC with no antige n exposure, MDSC directly pre exposed to antigen, or MDSC indirectly pre e xposed to antigen can be compa red to evaluate MDSC suppression of T cell activation Therefor e this system can be used to test possible mechanisms of antigen acquisition by MDSC. O ur hypothesis is that peripheral lymphoid organ MDSC obtain antigen from DC so we tested this by co culturing DC, pre loaded with OVA, with splenic MDSC. Essentially, DC are incubated overnight with OVA, which they then process and could present to MDSC. The DC are then cultured with splenic MDSC overnight, and the next day the DC and MDSC are separated from each other by flow cytometric sorting to isolate the MDSC (s ee Flow Cytometry pp. 38 43). For comparison, DC are kept overnight with no exposure to OVA, and are then co cultured with splenic MDSC overnight and separated the next day. If DC and MDSC interact and DC transfer antigen to MDSC, then the splenic MDSC co cultured overnight with OVA loaded DC should have OVA antigen transferred to them (whether as SIINFEKL, partial OVA protein, or SIINFEKL bound to MHC class I), resulting in MDSC expression of the MHC class I/SIINFEKL complex, and they should be able to su ppress IFN release by B16/Kb OVA activated OT I T cells. For comparison, splenic MDSC co cultured overnight with unloaded DC (not incubated with OVA protein) are added to OT I T cells and B16/Kb OVA cells. These

PAGE 38

! #* MDSC, since they were never exposed to O VA, should show negligible suppression of T cell activation. For comparison (if cell numbers permit) splenic MDSC cultured alone and splenic MDSC cultured alone with OVA added to the overnight culture would be used. MDSC cultured alone, not exposed to an tigen, would be expected to show negligible suppression while MDSC cultured alone with OVA should be directly loaded with OVA antigen and would be expected to show significant suppression. After our results indicated that splenic MDSC did obtain OVA anti gen from DC during the overnight co culture, we evaluated the possible mechanisms by which DC transfer antigen to MDSC. One mechanism we tested was the transfer of antigen by exosomes. It is known that DC secrete these vesicles, which contain MHC molecul es and can be loaded with antigen, so it seemed possible that DC could transfer the MHC class I/SIINFEKL complex to MDSC via exosome secretion. We evaluated this by separating OVA loaded DC and MDSC during the co culture by a transwell, which is permeable to soluble factors, including exosomes, but would prevent close cell contact between DC and MDSC, since the cells are too large to pass through. However, thin intercellular connections like tunneling nanotubes (TNT) could pass through the transwell, enab ling long range cell contact and potential antigen transfer via TNT, though transfer of antigen between cells via TNT has not been definitively demonstrated in vitro or in vivo Therefore, use of a transwell during the co culture would not conclusively sh ow that antigen transfer occurs by exosomes, but this would indicate whether it is plausible or whether close cell contact is needed for antigen to transfer from DC to MDSC. The DC used in this system were generated in vitro from bone marrow precursors. T his provided a simple and straightforward way to obtain DC in large enough numbers to

PAGE 39

! $+ use for the co culture with MDSC for preliminary experiments. However, although these DC would be useful to verify DC transfer of antigen to MDSC as proof of concept for preliminary experiments, these DC would not be exposed to the same conditions as those in tumor bearing organisms. To create a more biologically relevant system that introduced the type of tumor derived factors to which DC and MDSC in cancer would be exp osed, after the preliminary experiments, we generated TES (tumor explant supernatant) from tumor bearing mice and introduced it into the DC culture and into the DC and MDSC overnight co cultures. Chronological O verview For this experiment, bone marrow cell s taken from GFP B6 mice (see Mouse Models pp. 35 37) were cultured for 6 days with GM C SF and IL 4 in order to promote the differentiation of DC (see figure 6). In the experiments with TES, TES was added to the DC culture starting on day 1 so that the me dia comprised 20% TES, in order to create a better mimic of the environment to which the DC would be exposed in cancer. GFP B6 mice are non tumor bearing mice that should constitutively express GFP in all their cells. This means that bone marrow cells cu ltured from them were GFP+ (fluorescent green), which was used during cell sorting to distinguish them from MDSC. On day 5 OVA protein was added to half of the DC culture and left overnight so that the DC could obtain and process OVA; the other half of th e DC culture had no OVA protein added. On day 6 CD11c+ DC (CD11c is a DC marker and this eliminated undifferentiated cells/non DC) were isolated from the OVA loaded and unloaded DC subsets separately, and each subset was co cultured overnight with Gr 1+ M DSC isolated on day 6 from an EL 4 tumor bearing mouse spleen ( EL 4 is a murine lymphoma cell line, and it is injected into B6

PAGE 40

! $" mice to create EL 4 tumor bearing mice ) (see Magnetic Isolation p. 43). For the transwell experiments evaluating the possibility of antigen transfer by exosomes, an extra condition was added where a 0.4 m transwell was inserted between the MDSC and OVA loaded DC in the co culture. GM CSF was added to all overnight co cultures to sustain MDSC and DC survival. For the TES experiments, TES was added to the overnight co culture so that the media comprised 2.5%, 5%, 10%, or 20% TES again to better mimic the environment in which DC and MDSC would interact in cancer. 1 2 On day 7 the cells were collected and sorted using flow cytometry to separate the DC and MDSC from each other and to collect the MDSC fra ction (see figure 6 and Flow Cytometry pp. 38 43). MDSC were identified as GF P CD11c Gr 1+CD11b+ cells; DC were identified as CD11c+GFP+ cells ( see Appendix III, Figure 21, p. 99 for details). All subsets sorted were as follows: splenic MDSC co cultured overnight with unloaded !!!!!!!!!!!!!!!!!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!!!!!!!! By adding OVA to the DC culture on day 5 and then isolating CD11c+ DC on day 6 before c o culture with MDSC, we ensured that any soluble OVA left in the DC culture after loading was thoroughly washed away, so that the only source of OVA for MDSC during the co culture was OVA loaded DC. Before, during, and after the isolation of CD11c+ DC the cells were washed several times each. Likewise, all MDSC subsets, including MDSC cultured alone with OVA, were washed thoroughly before and after cell sorting so that no soluble OVA would be present during the ELISPOT cell incubation period. In additio n, B16/Kb OVA cells were thoroughly washed before and after irradiation to ensure that live cells expressing intact MHC class I/SIINFEKL on their surface were added to the ELISPOT. # 20% TES was originally used for the overnight DC and MDSC co culture, bu t it was noted that this high a percentage of TES converted splenic MDSC to nonspecific suppressors. In the periphery the concentration of tumor derived factors would most likely be much lower, since splenic MDSC suppress activated T cells in an antigen s pecific manner in vitro and in vivo Therefore we decreased the percentage of TES to 10%, which still resulted in nonspecific suppression. We decided to do a titration to yield the percentage of TES that corresponded to the conversion of splenic MDSC fro m antigen specific to nonspecific suppressors, to both determine the amount of TES necessary to best mimic the environment of the peripheral lymphoid organs, and to directly demonstrate the conversion of splenic MDSC from antigen specific to nonspecific su ppressors by tumor derived factors.

PAGE 41

! $# DC; splenic MDSC co cultured overnight with OVA loaded DC; splenic MDSC cultured overnight with OVA loaded DC separated by transwell; splenic MDSC cultured overnight alone; and splenic MDSC cultured overnight alone with OVA. On day 7 CD8+ T cells were isolated from an OT I mouse spleen (see Magnetic Isolation p. 4 3 ). B16 /Kb OVA cells and B16F10 cells were collected and irradiated at 60 Gy so that once seeded in the ELISPOT they did not proliferate, deplete the media, and/or over st imulate the T cells (60 Gy was previously determined to be the ideal radiation dose for this). On day 7, after all the cells were collected, OT I T cells were mixed in the ELISPOT with the antigen specific stimulus, the B16/Kb OVA cells (or B16F10 cells a s a negative control). OT I T cells and B16/Kb OVA cells were mixed with splenic MDSC co cultured overnight with unloaded DC, with splenic MDSC co cultured overnight with OVA loaded DC, with splenic MDSC co cultured overnight with OVA loaded DC separated by transwell, with splenic MDSC cultured overnight alone, or with splenic MDSC cultured overnight alone with OVA (see figure 6). After approximately 42 hours incubation, IFN release by OT I T cells under each condition was determined. The number of spo ts per well representing IFN release was compared (with each condition performed in triplicate) and the two tailed student's t test (with Welch's correction for significantly different variances) was used to determine statistically significant difference s in IFN release among the different conditions, with p<0.05 reaching statistical significance. T cells with B16/Kb OVA cells provided a positive control, giving the highest amount of IFN release without any suppression. T cells were incubated alone o r with B16F10 cells (with or without splenic MDSC) in the ELISPOT as a negative control. T

PAGE 42

! $$ cells by themselves or with B16F10 cells, with or without splenic MDSC, should be un stimulated and should release a negligible amount of IFN (after it was establ ished that OT I T cells did not release IFN in response to B16F10 cells with or without MDSC, T cells alone were used as the negative control). If splenic MDSC cultured under certain conditions (i.e., with OVA loaded DC) are suppressive, then their addi tion to the OT I T cells and B16/Kb OVA cells should result in a significant decrease in the amount of IFN release. If splenic MDSC cultured under certain conditions (i.e., with unloaded DC) are not suppressive, then their addition to the OT I T cells a nd B16/Kb OVA cells should not result in a significant decrease in the amount of IFN release. Figure 6. Chronological overview of the main experimental set up. In a separate experiment, we isolated MDSC from an EL 4 tu mor bearing mouse spleen and cultured the MDSC overnight with various amounts of TES, from 0% to 10% Generate DC (20% TES) Isolate OT I T cells Collect B16/Kb OVA cells Isolate bone marrow Isolate splenic MDSC Develop ELISPOT Add MDSC no MDSC Incubate in ELISPOT unloaded DC (unl. DC) OVA DC Isolate MDSC Add unl. DC Add OVA DC Co culture overnight (TES) add OVA overnight no OVA overnight Isolate differentiated DC Add B16/Kb OVA cells Generate DC (20% TES) Isolate OT I T cells Collect B16/Kb OVA cells Isolate bone marrow Isolate splenic MDSC Develop ELISPOT Add MDSC no MDSC Incubate in ELISPOT unloaded DC (unl. DC) OVA DC Isolate MDSC Add unl. DC Add OVA DC Co culture overnight (TES) add OVA overnight no OVA overnight Isolate differenti ated DC Add B16/Kb OVA cells

PAGE 43

! $% TES. The next day we collected the cell supernatant and lysate for each condition and evaluated NO production and arg I activity (see Measurement of NO Pr oduction p. 44 and Measurement of Arg I Activity pp. 44 45). We also evaluated iNOS and arg I expression for each condition (see Determination of iNOS and Arg I Expression pp. 45 48). Since iNOS and arg I are produced by tumor site MDSC and are associate d with nonspecific suppression of activated T cells, we studied their activity as the amount of TES increased to verify that it followed the trend of nonspecific suppression ELISPOT (Information taken from Mabtech[date u n known] and from Millipore[date u nknown]. ) An ELISPOT (enzyme linked immunosorbent spot assay) is designed to visualize IFN release by T cells in response to a stimulus one colored spot appears in an ELISPOT for each IFN secreting T cell, enabling quantification of IFN release and sensitive detection of T cell activation. An ELISPOT plate has a membrane that binds to a primary anti IFN antibody (figure 7, step 1). After coating the plate with primary antibody, T cells and stimuli are added to the plate and incubated. Activated T cells release IFN which binds to the primary antibody (figure 7, step 2). The cells are then washed off, leaving bound IFN primary antibody complex. A secondary antibody is added which binds to a different epitope of IFN creating a primary ant ibody IFN secondary antibody "sandwich" (figure 7, step 3). The secondary antibody is conjugated to biotin. Horseradish peroxidase conjugated to streptavidin (HRP streptavidin) is then added (figure 7, step 4). Streptavidin binds very strongly to biot in. In the last step, the HRP substrate TMB is added, which is cleaved by HRP to form a blue colored precipitate (figure 7, step 5).

PAGE 44

! $& Figure 7 Step by step ELISPOT protocol. 1. Coat membrane with antibodies. Add immune cells and incubate. 2. Respond ing cells produce cytokines. The cytokine of interest is then bound by the antibody. 3. Wash to remove cells. Add biotinylated antibodies which bind to the cytokine antibody complex. 4. Add strept avidin enzyme conjugate. 5. Add enzyme substrate and each re sponding cell will result in one spot. Taken from Millipore[date unknown]. Since any unbound reagent is washed after each step, the substrate is cleaved to form a colored precipitate only in the presence of HRP streptavidin bound to biotinylated secondar y antibody bound to IFN bound to primary antibody bound to the plate. Therefore, the appearance of blue spots should correspond to IFN released by T cells during incubation, and the number of blue spots per well can be used to compare T cell activation under different condit ions. An ELISPOT reader takes pictures of each well and has software that calculates the number of spots in each well. Mouse Models Mice are excellent model organisms for studying human diseases, particularly cancer. As mammals, they are genetically and physiologically one of the closest model organisms to humans: 99% of their genes have human homologs, and they have approximately the same number of genes as humans (Frese & Tuveson 2007, Griffiths et al. 2008). Mice ca n be genetically manipulated to prov ide a faithful mimic for the

PAGE 45

! $' development of different cancers in humans (Frese & Tuveson 2007, eMICE[date unknown] ) In addition, they can be used to study the behavior and function of immune cells ( Murphy 2012 ). Mice are especially valuable because the y can be inbred over multiple generations to maintain a genetically identical stock that provides a consistent standard model, enabling comparison of results among different laboratories studying the same disease with the same mouse model (eMICE[date unkn own]). Transgenic mice are one of the most commonly used models in these areas. Transgenic mice are created either by direct injection of cDNA constructs into fertilized mouse oocytes, or by lentiviral transduction into mouse embryonic stem cells. Althou gh the transgene inserts randomly into the genome, its expression and location can be tracked by fusion of the transgene with a reporter gene (such as GFP), and its expression can be controlled using exogenous ligands that bind to the promoter of the trans gene, which can either induce or suppress expression (Frese & Tuveson 2007, Griffiths et al. 2008). Transgenic mice have been used, for example, to evaluate the functions of TCRs and B cell receptors in lymphocyte development (Murphy 2012). One particula r mouse model widely used in immunology is the OT I (originally OVA TCR I or OVA specific class I restricted TCR) transgenic mouse (Clarke et al. 2000). These are C57BL/6 mice (a strain commonly referred to as Black 6 or B6) that contain transgenes encod ing the alpha and beta chains of the TCR ( JAX Mice Database: C57BL/6 Tg(TcraTcrb)1100Mjb/J [date unknown). (B6 mice are a commonly used inbred strain of mice and have a wide variety of purposes as a "general purpose" strain, as a "background" strain, a nd as the basis for the creation of many different transgenic strains ( JAX Mice Database: C57BL/6J [date unknown]).) These two transgenes

PAGE 46

! $( together encode a TCR that is specific for the complex between an MHC class I molecule (corresponding to cytotoxic T cells) and the OVA derived peptide SIINFEKL (J AX Mice Database: C57BL/6 Tg(TcraTcrb)1100Mjb/J [date unknown]) Therefore, the cytotoxic T cells from OT I mice should recognize the MHC class I/SIINFEKL complex and specifically target any cells expressing it. OT I T cells have been used to study, among many other things, the interactions between T cells and tumor cells (Clarke et al. 2000, Schuler & Blankenstein 2003). This has been made possible by the development of tumor cell lines expressing SIINFEKL bound to MHC class I, which should therefore activate OT I T cells (Schuler & Blankenstein 2003). One such cell line is B16/Kb OVA (Schuler & Blankenstein 2003). Although optimal activation of OT I T cells requires co stimulation by APC, recognition of S IINFEKL/MHC class I complexes by the TCRs of OT I CD8+ T cells does induce observable stimulation in vitro ( Schuler & Blankenstein 2003 Sompayrac 2003). Therefore, OT I CD8+ T cells and B16 /Kb OVA cells provide a reliable system used in this study, with which to evaluate antigen specific T cell activation and investigate external factors that could affect antigen specific T cell responses. Overall, m ouse models are widely used in current biomedical research have provided great insight into the workings of the human body during health and disease, and have been useful in testing a variety of therapeutic treatments ( Frese and Tuveson 2007, Murphy 2012, eMICE[date unknown] ). New strategies are being used to develop even more advanced mouse models and to provide further insight into the workings of the immune system ( Chow et al. 2011 ).

PAGE 47

! $) Flow Cytometry (Information taken from Murphy 2012 and from Invitrogen's Flow Cytometry Resource Center. [date unkn own] ) Flow cytometry combines optics, fluidics, and ele ctronics to analyze and/or separate cell subsets. It is a sensitive technique that can be used on small or large cell samples to analyze multiple parameters, in particular cell phenotype, and to collect different cell subpopulations. For example, flow cy tometry has been used to compare G MDSC and M MDSC phenotypes and to isolate DC subsets from lymph nodes. In the first step of flow cytometric analysis, the total cell population is treated with an antibody specific for a surface molecule (commonly a cell receptor) or intracellular molecule expressed by the cell subpopulation of interest (see figure 8). The antibody is conjugated to a fluorophore that fluoresces when excited by light in a certain wavelength range. Upon excitation, the fluorophore absorbs the energy and reaches a high and unstable energy state. It quickly releases some of this energy as heat in collisions with surrounding solvent molecules to reach a more stable, but still excited, energy state. It then releases the rest of the energy as emitted light, or fluorescence, to return to the ground state. Since it "loses" some of its energy before reaching the ground state, the emitted light is of lower energy and longer wavelength than the excited light, so the excitation and emission spectra of the fluorophore are distinct. (This shift in excitation and emission spectra is called the Stokes Shift.) Although each fluorophore can be excited by light in a range of wavelengths (the excitation spectrum), maximum absorption occurs at a specific wa velength within the spectrum, called the excitation maximum. The fluorophore also emits light in a range of wavelengths, giving rise to the

PAGE 48

! $* emission spectrum, with the intensity of emitted light peaking at the emission maximum. After labeling with the flu orophore conjugated antibody, the total cell population is suspended in a small volume and placed into the flow cytometer through a nozzle. The small volume of cells is injected into a stream of a larger volume of saline or sheath fluid, forcing the cells into a stream of singly spaced cells (see figure 8). This process is referred to as hydrodynamic focusing, and it enables the cells to pass through a series of laser beams in the flow cytometer one cell at a time, so that each cell can be detected and an alyzed individually. In the flow cytometer, each cell passes through multiple lasers corresponding to different wavelengths to cover the spectrum of visible light (see figure 8). Each cell refracts light at multiple angles as it passes through a laser. The larger the cell, the more light is scattered in the forward direction at a low angle, so the amount of forward scatter reflects the size of the cell. The more granular, or the more structurally complex the cell, the more light is scattered to the side at large angles, so the amount of side scatter reflects the granularity of the cell. If a cell is labeled with the antibody, when it reaches a laser corresponding to the excitation spectrum of the fluorophore, it emits fluorescence. To ensure that each light source provides a short excitation range, lasers are optimized so that their spectra are close to the excitation maximum of commonly used fluorophores. In addition, e xcitation bandpass filters are used to block out light in unwanted wavelengths. F low cytometers contain multiple photomultiplier tubes that act as sensitive detectors to collect forward scatter, side scatter, and multiple fluorescent signals separately (see figure 8). A detector placed in front of the cell's path collects forward scat ter, while a detector placed perpendicular to the cell's path collects side scatter. The

PAGE 49

! %+ fluorescence emitted from the labeled cell is also collected perpendicular to the cell's path. Multiple detectors are placed with mirrors and bandpass filters in fro nt of them to direct specific wavelength ranges to specific detectors. Since different fluorophores emit light of different wavelengths, multiple fluorophore signals can be distinguished and cells can be tracked by the expression of multiple molecules sim ultaneously. The detectors convert the scattered and emitted light into electronic signals as voltage proportional to the intensity of the scattered and emitted light, which are transmitted to a computer and can be quantified and analyzed using flow cytom etry software (see figure 8) Even though multiple lasers, detectors, mirrors, and filters can be used to distinguish the fluorescence due to multiple fluorophores, there is often spectral overlap of the emission wavelengths for different fluorophores us ed together. Although bandpass filters are optimized to promote narrow detection of the emission maximum for their corresponding target fluorophore, because of spectral overlap, each detector will still collect some emitted light due to a different non ta rget fluorophore. Compensation is used to remedy this problem. In addition to the total sample stained with all antibodies, aliquots of the cell sample are stained with each individual fluorophore labeled antibody separately. From each single stain aliq uot the amount of fluorescence from a fluorophore that is collected by the non corresponding detector is determined. Based on this the percentage of fluorescence in the total sample due to that non target fluorophore collected in a non corresponding det ector is subtracted from the fluorescence in that same channel due to the target fluorophore. This ensures that the fluorescence collected in each detector is attributable to one fluorophore, so that expression of each molecule can be accurately determine d.

PAGE 50

! %" Figure 8 Analysis of cell surface molecule expression by flow cytometry. Cells are labeled with fluorescent dyes coupled to antibodies specific for cell surface antigens (top panel). The cells are forced through a nozzle in a single cell stream t hat passes through a laser beam (second panel). Photomultiplier tubes detect the scattering of light (yielding cell size and granularity), and emissions from the fluorescent dyes. This information is analyzed by CPU. By analyzing many cells like this, t he number of cells with specific characteristics can be counted and the levels of expression of various molecules can be measured. The lower part of the figure shows how these data are represented. When analyzing the expression of one molecule, the data are usually displayed as a histogram (left panels). Histograms show the distribution of cells expressing a single measured parameter. When two or more parameters are measured, different two color plots can be used to display the data (right panels). All four plots represent the same data. The horizontal axis represents the fluorescence intensity due to one surface molecule, and the vertical axis represents the fluorescence intensity due to a different surface molecule. Taken from Murphy 2012.

PAGE 51

! %# After comp ensation, the fluorescence due to the expression of each molecule is quantified with flow cytometry software. Cell subpopulations can be selected based on size and granularity, and the expression of multiple fluorescent markers, as well as intracellular p roduction of different molecules, can be analyzed. In addition, cell populations can be separated by specialized flow cytometers in a process called fluorescence activated cell sorting (FACS). In FACS, as each cell passes through the lasers, the signals sent to the computer with information on size, granularity, and molecule expression are used to generate an electric charge, which is transmitted through the nozzle at the exact time that the stream of cells breaks up into droplets of individual cells. T he instrument parameters are set so that each cell corresponding to the subset to be collected is given an electric charge and is deflected as the stream of cells then passes between oppositely charged plates, enabling separation and collection of only the se charged cells of interest. Cells that do not meet the parameters remain uncharged and un deflected, and can be collected separately or discarded. Therefore flow cytometry is a useful tool not only for analysis, but also for isolation of cells to furth er use in various assays. In this study, FACS as detailed above was used to separate Gr 1+CD11b+ MDSC from CD11c+(GFP+) DC in order to collect the MDSC after overnight co culture. The mixed DC and MDSC samples were labeled with antibodies targeting the a bove markers and conjugated to three different fluorophores: PE (phycoerythrin), PE Cy 7, and APC D API was also used to differentiate between live cells and dead cells, so that only live MDSC were collected. DAPI is a fluorescent dye that binds to nucle ic acids and stains

PAGE 52

! %$ nuclei blue violet It cannot cross the plasma membrane of live cells so dead cells are stained violet, while live cells are unstained. Magnetic Isolation (Information taken from Miltenyi Biotec [date unknown]. Cell subsets expressin g specific surface markers can also be separated by magnetic isolation. This is especially useful for quick isolation of a large subpopulation of cell s that express a distinct marker. The total cell population is labeled with an antibody directed against the marker of interest. This antibody is conjugated to biotin. Magnetic microbeads conjugated to streptavidin are then added to the cells. Since streptavidin strongly binds to biotin, the microbeads become indirectly attached to the subpopulation of in terest. The total cell population is then passed through a column placed in a MACS separator, which is essentially a strong magnet. The column contains ferromagnetic spheres that enhance the magnetic field. The magnetic microbeads attached to the cells of interest are attracted to the magnetic field and are kept in the column. Unlabeled cells pass through the column with several washes of elution buffer. The column is then removed from the magnetic separator, and the cells retained in the column are el uted by washing the column with elution buffer and plunging the cells through. This method was used to isolate Gr 1+ MDSC from the spleen of an EL 4 tumor bearing mouse and to isolate CD11c+ DC from the DC culture on day 6, and to isolate CD8+ T cells fro m an OT I mouse spleen on day 7.

PAGE 53

! %% Measurement of NO Production (Information taken from Invitrogen's Griess Reagent Kit, for nitrite quantification. [date unknown]). NO produced by cells, such as MDSC, is oxidized to form nitrite and can be collected in t he cell supernatant. Nitrite reacts with the Griess reagent, a mixture of sulfanilamide and N (1 naphthyl)ethyl enediamine dissolved in phosphoric acid and water, to form a purple dye. Sulfanilamide reacts with nitrite under acidic conditions to form a di azonium salt, which then then couples to N (1 naphthyl)ethyl enediamine, resulting in the formation of a purple azo dye. This dye absorbs strongly at 550 nm, so the presence of NO/nitrite in the cell supernatant can be detected as the concentration of the purple dye that forms, as compared to standards of sodium nitrite at known concentrations. This then yields the level of iNOS activity. Measurement of Arg I Activity (Information taken from Bioassay System[date unknown] and from Bio Rad[date unknown]). Arg I catalyzes the reaction of L arginine and water to produce urea and L ornithine, so the amount of urea produced by cells, such as MDSC, reflects intracellular arginase I activity. Arg I is retained in the cells, so cells are lysed with the detergent Triton X 100 and the lysate is collected. Some of the lysate is used to quantify the total amount of protein present by adding Coomassie Brilliant Blue G 250 dye to it. This dye can take three forms cationic, anionic, or uncharged and each form corre sponds to a different color red, blue, or green, respectively. Under acidic conditions, the dye is doubly protonated and cationic, so it appears red. When mixed with proteins, the dye binds to them and takes on the unprotonated anionic form (which is mo re stable), so it

PAGE 54

! %& turns blue. In this form it absorbs strongly at 595 nm, so the absorbance of the cell lysate at 595 nm can be used to quantify the total amount of protein in each sample. Serial dilutions of bovine serum albumin at known concentrations are used as standards. The total amount of protein can then be compared to the amount of urea in each sample to determine the arg I activity relative to the total amount of protein. For the arg I activity assay Tris HCl and magnesium chloride are added to the cell lysate and the mixture is heated at 56 ¡C to activate arg I. Arginine is then added and the mixture is heated at 37¡C for 1 hour to allow arginine hydrolysis to occur. A mixture of strong acids is then added to denature arg I and stop the react ion. # isonitrosopropiophenone is subsequently added and the mixture is heated at 95 ¡C. # isonitrosopropiophenone, when heated in acid, reacts with urea produced by arginine hydrolysis to form a pink dye, which absorbs strongly at 540 nm. Serial dilutions of urea at known concentrations are prepared and reacted with # isonitrosopropiophenone under the same conditions as the samples. The absorbance of each sample is measured at 540 nm and compared to the urea standards to determine arg I activity. Determinati on of iNOS and Arg I Expression RNA is extracted from the cells and then reverse transcribed into total cDNA. Real time PCR is then performed on the cDNA mixture to detect the levels of expression for the genes encoding iNOS (Nos2) and arg I (ArgI). RNA e xtraction (Information taken from Omega Bio Tek[date unknown]). Cells are collected and lysed in a buffer containing guanidine thiocyanate, which lyses cells and denatures/inactivates RNases. Ethanol is added to the lysate and the

PAGE 55

! %' mixture is added to a s pecialized column which, under proper binding conditions (in the presence of ethanol), binds specifically to RNA. A series of buffer washes are applied to the column and the flow through is discarded, allowing for removal of cellular debris and other cont aminants from the RNA bound to the column. The RNA is finally eluted from the column in RNase free water. Reverse t ranscription (Information taken from Invitrogen's High Capacity cDNA Reverse Transcription Kit. [date unknown]). After the RNA is purified, it is reverse transcribed into total cDNA. The total RNA sample is added to a mixture comprising random primers, dNTPs, RT buffer, DI water, RNase inhibitor, and the reverse transcriptase MMLV. This allows for reverse transcription of all the RNA present in the sample. The samples are added to a thermocycler, where the reverse transcriptase is activated at 25 ¡C for 10 minutes, the RNA is reverse transcribed into cDNA at 37 ¡C for 2 hours, the reaction is stopped by inactivation of the reverse transcript ase at 75 ¡C for 5 minutes, and the resultant cDNA samples are preserved at 4 ¡C until ready for freezing/use. Real t ime PCR (Information taken from Invitrogen's Real Time PCR Learning Area. [date unknown]). Once total cDNA is obtained, real time PCR can be performed to determine the expression of specific genes (i.e. those for iNOS and arg I) by amplification of the cDNA corresponding to those genes and concurrent quantification of the amplification. For each gene to be tested, the total cDNA is added to a mixture that contains: a Taqman oligonucleotide probe, forward primer, and reverse primer mix; master mix with AmpliTaq Gold DNA polymerase; and DI water. The samples are then added to a real

PAGE 56

! %( time PCR thermocycler, where the DNA polymerase is activated at 95 ¡C for 10 minutes and the cDNA corresponding to the gene of interest is amplified during 40 cycles: 15 sec onds at 95 ¡C for denaturation, and 5 min at 75 ¡C for annealing and extension. During real time PCR, the Taqman probe binds to its correspond ing gene (i.e. Nos2 or ArgI) downstream of one of the primers. The probe is attached to a fluorescent reporter dye at the 5' end and a non fluorescent quencher dye attached to the 3' end. The quencher dye absorbs any fluorescence emitted by the reporter dye through FRET, inhibiting detectable fluorescence. If the gene is present in the cDNA mixture, the primer and probe bind to its complementary sequence. When DNA polymerase extends the primer, it runs into the probe and cleaves the probe by its 5' exon uclease activity. This removes the reporter dye from the quencher dye, resulting in significant fluorescence emission (with light from the real time PCR instrument as an excitation source). This also separates the probe from the cDNA so that the primer is fully extended and amplification can continue unhindered. The more cDNA corresponding to the gene of interest there is in a sample, the higher the amplification, the more fluorescent dye molecules cleaved, and the higher the fluorescence detected by th e real time PCR instrument. This enables the determination of gene expression based on fluorescence intensity. Fluorescence is detected as the annealing and extension occur, allowing for quantification of fluorescence, and therefore quantification of amp lification, to occur as the cDNA is being amplified (i.e. in real time). Once the reaction is finished, real time PCR software can be used to determine the Ct value for each sample, or the cycle number at the which the fluorescence intensity reaches above a background value/threshold the higher the gene expression and

PAGE 57

! %) fluorescence, the lower the Ct value. This allows for sensitive detection and quantification of a significant increase in fluorescence and target gene expression. Gene expression is report ed as fold change in expression relative to a baseline sample, normalized to the expression of an endogenous control in each sample. The formula used is : fold change in expression = 2 '' Ct For example, fold change in arg I expression for MDSC cultured in 10% TES compared to MDSC cultured in 0% TES (baseline), with $ actin as an endogenous control = 2 [{Ct of argI(10%TES) Ct of $ actin(10%TES)} {Ct of argI(0%) $ actin(0%)}]

PAGE 58

! %* Results Splenic MDSC Obtain Antigen from DC and Suppress IFN Release by Antigen Specific T Cells To evaluate the transfer of antigen from DC to MDSC we chose to use the immunogenic protein ovalbumin (OVA). In the process of cross presentatio n, DC exposed to extracellular foreign proteins ingest and process them into peptides then loaded onto MHC class I molecules, which are exported to the cell surface to be presented to cytotoxic T cells (Murphy 2012 ) DC are also known to be able to transf er MHC class I/antigen complexes, as well as free antigen, among themselves (AndrŽ et al. 2004, Saccheri et al. 2010). We hypothesized that bone marrow derived DC in their capacity as APC, would process OVA overnight. Isolated CD11c+ differentiated DC w ould then transfer OVA in some form (as a n MHC class I/peptide complex, free peptide, or partial protein) to isolated Gr 1+ splenic MDSC, during overnight co culture. After the co culture, splenic MDSC would be loaded with either DC derived MHC class I c omplexed with SIINFEKL (immunogenic peptide derived from OVA), or endogenous MHC class I complexed with SIINFEKL. This would enable MDSC isolated from the co culture to engage the TCR of OT I T cells specific for the MHC class I/SIINFEKL complex and damag e the T cells through release of ROS, etc. This would prevent OT I T cells from reacting against B16/Kb OVA tumor cells that express the MHC class I/SIINFEKL complex, resulting in suppression of T cell IFN release in an ELISPOT. Suppression of T cell a ctivation by splenic MDSC co cultured with OVA loaded DC would indicate that DC could potentially transfer tumor antigens to peripheral lymphoid organ MDSC in vivo

PAGE 59

! &+ providing the source of tumor antigens for peripheral lymphoid organ MDSC that mediate supp ression of tumor specific T cell immunity. The response of OT I T cells to B16/Kb OVA cells was robust in all cases and generated 100 to 200 spots of IFN release per well in four ELISPOT s performed (see figure 9 for sample ELISPOT picture). The response of OT I T cells alone o r to B16F10 cells with or without MDSC generated fewer than 25 spots per well in all cases, indicating that the response of OT I T cells was specific for the MHC class I/SIINFEKL complex expressed by B16/Kb OVA cells. In concordance with our hypothesis, we found that, at a 1:1 ratio MDSC: T cells, splenic MDSC co cultured with DC previously exposed to OVA exhibited strong suppressio n OT I T cell IFN release, as evidenced by the consistent and significant decrease in the number of spots per well with these MDSC added (see figure 10). In contrast, splenic MDSC co cultured with DC that had not been exposed to OVA showed weak and insignificant suppress ion of OT I T cell IFN release at a 1:1 ratio MDSC: T cells (see figure 10). Furthermore, there was a consistent and significant difference in suppression between MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC, at a 1:1 ratio MDSC: T cells (see figure 10). At a 1:3 ratio MDSC: T cells, the suppression by MDSC co cultured with OVA loaded DC was abrogated there was no significant difference in IFN release compared to T cells with B16/Kb OVA cells (see figure 10). However, th ere was a significant difference in IFN release in the presence of MDSC co cultured with OVA loaded DC compared to IFN release in the presence of MDSC co cultured with unloaded DC at 1:3 MDSC co cultured with unloaded DC not only failed to exhibit ev en slight suppression, they also stimulated IFN release (see figure 10 and Discussion ).

PAGE 60

! &" Figure 9 Picture of a representative ELISPOT showing IFN release by OT I T cells in response to B16/Kb OVA cells with or without splenic MDSC previously co culture d with unloaded DC or with OVA loaded DC. T cell release of IFN was quantified as the number of spots seen in each well. Each condition, as in every experiment, was performed in at least triplicate, except when limited cell number dictated use of du plicates. OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 15,000 per well (top row), yielding a robust response of approximately 150 spots per well. Splenic MDSC that had been co cu ltured with unloaded DC (second row) or with OVA loaded DC (third row) were added to each well, at either 100,000 per well (1:1 MDSC: T cells) or 33,333 per well (1:3 MDSC: T cells). A clear decrease in the number of spots per well can be seen when MDSC c o cultured with OVA loade d DC were added, 1:1. A slight decrease in the number of spots can be seen when MDSC co cultured with unloaded DC were added, 1:1. The number of spots with all MDSC subsets at 1:3 appears to be restored at least to the level of T cells with B16/ Kb OVA cells. T cells alone were added to each well at 100,000 per well (bottom row), yielding fewer than 25 spots per well and corresponding to negligible IFN release by unstimulated T cells.

PAGE 61

! &# Figure 10 Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with OVA loaded DC compared to splenic MDSC previously co cultured with unloaded DC. T cell release of IFN was quantified as the number of spots per well. Four separate experiments are reported. For every experiment each condition was performed in at least triplicate, except when limited cell number dictated use of duplicates. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asterisks denote p<0.001. Black lines bridge two conditions that were compa red for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000, 15,000, or 30,000 per well, yielding a robust response between 100 and 200 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well, at 100,000 per well (1:1 MDSC: T cells) or at 33,333 per well (1:3 MDSC: T cells). The number of MDSC that could be collected determined if one or both conditions were used in each experiment MDSC co cultured with unloaded DC yielded a small and insignificant suppression of IFN release at 1:1 (dark green, p =0.1809) and a significant increase in IFN release at 1:3 (light green, p =0.0407). MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:1 (dark orange, p =0.0103) and an insigni ficant decrease in IFN release at 1:3 (light orange, p =0.4685). There was a significant difference in IFN release by T cells in the presence of MDSC co cultured with unloaded DC and by T cells in the presence MDSC co cultured with OVA loaded DC, at bo th 1:1 (p=0.0073) and 1:3 (p=0.0121). As negative controls T cells were added to each well at 100,000 per well alone or with 30,000 B16F10 cells per well with or without 100,000 or 33,333 splenic MDSC per well (black and white). These yielded negligible IFN release compared to T cells mixed with B16/Kb OVA cells (p < 0.004), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

PAGE 62

! &$ The above data were compiled from four replicate experiments that yielded very similar results. One ELISPO T, however, was an exception: both MDSC subsets exhibited strong and significant suppression at 1:1, MDSC: T cells (see figure 11 and Discussion ). Although at 1:1 MDSC co cultured with OVA loaded DC exhibited an average suppression more than twice as stro ng as the average suppression by MDSC co cultured with unloaded DC, the overall difference in suppression between the two MDSC subsets was not statistically significant due to the large variation in the number of spots per well among re p licates for each o f these subsets (see figure 11). However, at 1:3 the difference in suppression was statistically significant (see figure 11). In fact, MDSC co cultured with unloaded DC yielded a negligible change in IFN release, while MDSC co cultured with OVA loaded DC yielded a small but significant suppression of IFN release. Overall, splenic MDSC co cultured with OVA loaded DC exhibited consistent and significant suppression of IFN release that was significantly greater than the (mostly) negligible suppressio n exhibited by splenic MDSC co cultured with unloaded DC. This confirms that splenic MDSC mediate antigen specific suppression and, most importantly, this demonstrates that DC can transfer antigen to splenic MDSC, enabling these MDSC to suppress antigen s pecific T cell responses. This indicates that the mechanism by which peripheral lymphoid organ MDSC obtain tumor antigens in mice and humans could be through DC.

PAGE 63

! &% Figure 11 IFN release by OT I T cells in response to B16/Kb OVA cells with or without splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC. T cell release of IFN was quantified as the number of spots per well. One experiment is reported. Each condition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asterisks denote p<0.00 1. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 30,000 per well, y ielding a robust response of approximately 300 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well, at 100,000 per well (1:1 MDSC: T cells) or at 33,333 per well (1:3 MDSC: T cells ). MDSC co cultured with unloaded DC yielded clear and significant suppression of IFN release at 1:1 (dark green, p =0.0005). MDSC co cultured with OVA loaded DC yielded even higher and significant suppression of IFN release at 1:1 (dark orange, p < 0.0001), although the difference in suppression between the two MDSC subsets was not signi ficant at 1:1 (p=0.0548). At 1:3, MDSC co cultured with unloaded DC yielded an insignificant increase in IFN release (light green, p =0.04844), while MDSC co cultured with OVA loaded DC yielded a significant decrease in IFN release at 1:3 (light orange p =0.0008). The difference in suppression between the two MDSC subsets was significant at 1:3 (p=0.0077). As negative controls T cells were added to each well at 100,000 per well with 30,000 B16F10 cells per well with or without 100,000 or 33,333 spleni c MDSC per well (black and white). These yielded negligible IFN release compared to T cells mixed with B16/Kb OVA cells (p < 0.0001), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

PAGE 64

! && Tumor Derived Factors Convert Splenic MDSC from Antigen Specific Suppressors to Nonspecific Suppressors in a Dose Dependent Manner. Although our initial experiments demonstrated, as proof of principle, that DC can transfer antigens to splenic MDSC in vitro and enable them to suppress antigen specific T cell responses, our system was free of the influence of a ny tumor derived factors, to which DC and MDSC would be exposed in vivo In order to validate our findings using a system better mimicking the conditions under which DC and peripheral lymphoid organ MDSC would exist in tumor bearing organisms, we introduc ed tumor derived factors into the system by adding TES (tumor explant supernatant) to the DC culture and to the overnight DC and MDSC co culture. TES is commonly used as a source of tumor derived factors, and at high concentrations it is used to mimic the immunosuppressive tumor microenvironment (Gabrilovich & Nagaraj 2009). DC would be exposed to tumor derived factors both in the periphery and at the tumor site, so we generated DC in 20% TES starting one day after the isolation of bone marrow cells until the isolation of CD11c+ differentiated DC (day 1 to day 6). Although the amount of tumor derived factors in the periphery is low, particularly compared to the tumor site, the exact amount is unknown, so we initially performed the DC and MDSC co culture i n 20% TES. Interestingly, the antigen specific suppression that we had clearly seen in our previous experiments disappeared. We performed two experiments (partial replicates) using 20% TES in the overnight DC and MDSC co culture, and found that under the se conditions both MDSC co cultured with OVA loaded DC and MDSC co cultured with unloaded DC showed strong and significant suppression at 1:1, with no significant difference in suppression between the two MDSC subsets (see figures 12 and 13 ). At

PAGE 65

! &' 1:3, in c ontrast to the previous results in the absence of TES, MDSC co cultured with unloaded DC in 20% TES did not stimulate IFN release (see figure 12). Furthermore, at 1:3 there was no significant difference in IFN release in the presence of MDSC co cultured with unloaded DC compared to IFN release in the presence of MDSC co cultured with OVA loaded DC (see figure 12). Thi s indicates that when exposed to the concentration of tumor derived factors corresponding to 20% TES, splenic MDSC no longer required antigen obtained from DC in order to suppress antigen activated T cells: tumor derived factors induced strong nonspecific suppression by MDSC. This is consistent with findings that tumor site MDSC strongly suppress in a nonspecific manner, and that splenic MDSC transferred to the tumor site become nonspecific suppressors (Corzo et al. 2010). This also indicates that the con centration of tumor derived factors at the periphery is much lower than we initially tested. If the periphery did contain the equivalent of 20% TES, then the concentration of tumor derived factors would be high enough for splenic MDSC to nonspecifically s uppress in vitro and in vivo and tumor bearing organisms would likely suffer from overall immune suppression. Since none of these is generally the case, the concentration of tumor derived factors present at the periphery is very low (Nagaraj & Gabrilovic h 2008, Gabrilovich & Nagaraj 2009). In order to determine the amount of tumor derived factors to best mimic the conditions in the periphery (i.e., the percent TES corresponding to splenic MDSC requiring exposure to OVA through DC in order to suppress IFN release, since peripheral lymphoid organ MDSC mediate antigen specific suppression), we performed a titration by adding TES to the overnight DC and MDSC co culture from 2.5 % TES to 20 % TES.

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! &( Figure 12. Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 20% TES, experiment 1. T cell release of IFN was quantified as the number of spots per well. One experiment is reported. Each cond ition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asterisks denote p<0.001. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 15,000 per well, y ielding a robust response of approximately 350 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well, at 100,000 per well (1:1 MDSC: T cells) or at 33,333 per well (1:3 MDSC: T cells ). MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:1 (dark green, p =0.0015; dark orange, p=0.0014, respectively), with no significant difference in suppression between the two MDSC subsets at 1:1 (p=0.7923). MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded a n insignificant decrease in IFN release at 1:3 (light green, p =0.0543; light orange, p=0.1246, respectively), with no significant difference between the two MDSC subsets at 1:3 (p=0.7180). As a negative control T cells alone were added to each well at 10 0,000 per well (black and white). This yielded negligible IFN release compared to T cells mixed with B16/Kb OVA cells (p < 0.0001), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

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! &) Figure 13. Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 20% TES, experiment 2. T cell release of IFN was quantified as the number of spots per well. One experime nt is reported. Each condition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asteri sks denote p<0.001. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000 per well, y ielding a robust response of approximately 90 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well at 100,000 per well (1:1 MDSC: T cells only not enough MDSC were collected to al so use 1:3 MDSC: T cells ) MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:1 (dark green, p =0.0034; dark orange, p=0.0015, respectively), with no significant difference in suppression between the two MDSC subsets (p=0.7291). As a negative control T cells alone were added to each well at 100,000 per well (black and white). This yielded negligible IFN release compared to T cells m ixed with B16/Kb OVA cells (p= 0.0003), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

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! &* MDSC co cultured with DC in 10% TES again demonstrated very strong and significant suppression regardless of DC exposure to OVA, even at a 1:2 ratio MDSC: T cells (see figure 14). In contrast, when 5% TES was used for the co culture, at 1:2 the suppression by either MDSC subset was negligible (see figure 15). With 5% T ES there also appeared to be, at 1:1, a slight difference in suppression between MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC, although the statistical significance could not be determined ( see fi gure 15). When the amount of TE S in the co culture was then decreased to 2.5%, both MDSC subsets exhibited significant suppression at 1:1, but it was clearly and significantly weaker than the suppression by either MDSC subset at 1:2 when co cultured in 10% TES (see figures 14 and 16). At 2.5% TES, similarly to 5% TES, there was a slight difference in suppression between the two MDSC subsets, but this was not statistically significant (see figure 16). For 2.5% TES, at 1:2 MDSC co cultured with unloaded DC exhibited negligible suppressio n, while MDSC co cultured with OVA loaded DC exhibited significant suppression (see figure 16). However, the difference in suppression at 1:2 was insignificant, similarly to 1:1. Although this did not yield a condition to mimic the periphery in tumor bear ing organisms, since at 2.5% TES antigen specific suppression was not restored, overall this indicates that tumor derived soluble factors convert splenic MDSC from antigen specific to nonspecific suppressors in a dose dependent manner.

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! '+ Figure 14. S uppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 10% TES. T cell release of IFN was quantified as the number of spots per well. O ne experiment is reported. Each condition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and t hree asterisks denote p<0.001. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000 per well, y ielding a robust response of approximately 250 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well at 50,000 per well (1:2 MDSC: T cells this was chosen as the highest ratio poss ible to use in triplicate since there were not enough MDSC collected to use 1:1 MDSC: T cells). MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:2 (dark green, p =0.0035; dark orange, p=0.0139, respectively), with no significant difference in suppression between the two MDSC subsets (p=0.2564). As a negative control T cells alone were added to each well at 100,000 per well (black an d white). This yielded negligible IFN release compared to T cells m ixed with B16/Kb OVA cells (p= 0.0025), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

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! '" Figure 15. Suppression of OT I T cell IFN release in resp onse to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the presence of 5% TES. T cell release of IFN was quantified as the number of spots per well. One experiment is reported. Each condition was p erformed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asterisks denote p<0.001. Black lines brid ge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000 per well, y ielding a robust response of approximately 350 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well at 100,0 00 per well (1:1 MDSC: T cells) or at 50,000 per well (1:2 MDSC: T cells this ratio was chosen to be used again since there were enough MDSC collected to do so in triplicate ). Although at 1:1 MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC both appeared to yield a decrease in IFN release, with a slight difference in suppression between the two MDSC subsets, for each subset one of the replicate samples had to be discarded (due to leakage of the ELISPOT well s during the cell incubation period ) so there were not enough replicate sa mples to determine the statistical significance MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded an insignificant decrease in IFN release at 1:2 (dark green, p =0.4860; dark orange, p=0.0879, respectively), with no signi ficant difference between the two MDSC subsets (p=0.1173). As a negative control T cells alone were added to each well at 100,000 per well (black and white). This yielded negligible IFN release compared to T cells m ixed with B16/Kb OVA cells (p< 0.0001 ), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells.

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! '# Figure 16. Suppression of OT I T cell IFN release in response to B16/Kb OVA cells by splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC in the pre sence of 2.5% TES. T cell release of IFN was quantified as the number of spots per well. One experiment is reported. Each condition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 r eached statistical significance. One asterisk denotes p<0.05, two asterisks denote p<0.01, and three asterisks denote p<0.001. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corr esponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000 per well, yielding a robust respons e of approximately 350 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well, at 100,000 per well (1:1 MDSC: T cells) or at 50,000 per well (1:2 MDSC: T cell s this ratio was chosen to be used again since there were enough MDSC collected to do so in triplicate ). MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:1 (dark green, p =0.0010; dark orang e, p < 0.0001, respectively), with a slight but insignificant difference in suppression between the two MDSC subsets at 1:1 (p=0.2037). MDSC co cultured with unloaded DC yielded an insignificant decrease in IFN release at 1:2 (light green, p =0.1483), whil e MDSC co cultured with OVA loaded DC yielded significant suppression of IFN release at 1:2 (light orange, p=0.0019). There was no significant difference in suppression between the two MDSC subsets at 1:2, however (p=0.0809). As a negative control T ce lls alone were added to each well at 100,000 per well (black and white). This yielded negligible IFN release compared to T cells mixed with B16/Kb OVA cells (p < 0.0001), indicating the response of OT 1 T cells was specific for B16/Kb OVA cells. (For co mparison to 10% TES, the suppression at 1:1 by either MDSC subset co cultured in 2.5% TES was significantly weaker than the suppression at 1:2 by either MDSC subset co cultured in 10% TES (p=0.0025, p=0.0079, p=0.0012, p=0.0076).)

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! '$ Tumor Derived Factors Induce Upregulation of iNOS and Arg I in Splenic MDSC in a Dose Dependent Manner. Given that nonspecific suppression by tumor site MDSC is associated with high levels of arg I and iNOS in MDSC, we evaluated the levels of arg I and iNOS expression and acti vity by splenic MDSC cultured in TES to confirm the effect of tumor derived factors on splenic MDSC that we had seen by ELISPOT (Corzo et al. 2010). Since strong nonspecific suppression was evident at 10% and 20% TES and was reduced in 5% and 2.5% TES, we cultured splenic MDSC in 10% TES, 5% TES, 2.5% TES, 1% TES, and no TES overnight. Although NO production under all conditions was undetectable by the assay used we found that iNOS expression was steadily upregulated as the amount of tumor derived factor s in the cult ure was increased (see figure 17 ). iNOS expression by MDSC cultured in 10% TES reach ed approximately five times that in the absence of TES (see figure 17). Arg I activity and expression followed a similar trend as the amount of tumor derived factors in the MDSC cult ure increased (see figures 18 and 19 ). Arg I expression in 10% TES reached approximately three and a half times that in the absence of TES (see figure 18). Arg I activity increased slightly and insignificantly from no TES to 1% T ES. Arg I activity in 10% TES was significantly increased compared to both 1% TES and no TES, and it reached approximately twice the arg I activity in no TES (see figure 19).

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! '% Figure 17. Induction of iNOS expression in splenic MDSC by tumor derived fac tors. Splenic MDSC were cultured overnight at 500,000 per well without TES (0% TES), in 1% TES, 2.5% TES, 5% TES, or 10% TES, similarly to the overnight DC and MDSC co cultures previously performed. The next day total RNA was extracted from the cells and reverse transcribed into cDNA. Real time PCR was then performed with probes for Nos2 (iNOS) and $ actin (endogenous control). The change in iNOS expression, normalized to iNOS expression by MDSC cultured without TES, was determined in duplicate for each experiment The results of two experiments are reported. Error bars show SEM (standard error of the mean). There is a steady trend of increasing iNOS expression by splenic MDSC as the amount of tumor derived factors in the overnight culture increase d, al though the limited data prevented determination of statistical significance.

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! '& Figure 18. Induction of arg I expression in splenic MDSC by tumor derived factors. Splenic MDSC were cultured overnight at 500,000 per well without TES (0% TES), in 1% TES, 2.5% TES, 5% TES, or 10% TES, similarly to the overnight DC and MDSC co cultures previously performed. The next day total RNA was extracted from the cells and reverse transcribed into cDNA. Real time PCR was then performe d with probes for ArgI (arg I) and $ actin (endogenous control). The change in arg I expression, normalized to arg I expression by MDSC cultured without TES, was determined in duplicate for each experiment The results of two experiments are reported Error bars show SEM (standard error of the mean). There is a ste ady trend of increasing arg I expression by splenic MDSC as the amount of tumor derived factors in the overnight culture increased, al though the limited data prevented determination of statistical significance.

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! '' Figure 19. Induction of arg I activity in splenic MDSC by tumor derived factors. Splenic MDSC were cultured overnight at 2 million per well without TES (0% TES), in 1% TES, 2.5% TES, 5% TES, or 10% TES, similarly to the overnight DC and MDSC co c ultures previously p erformed. The next day cell lysate was collected and arg I activity was determined by the production of urea, normalized to the total amount of protein. The results of three experiments are reported. Error bars show SEM (standard err or of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05 and two asterisks denote p<0.01 Black lines bridge two conditions that were compared for a statistically significant difference in urea production, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). There is a cl ear trend of increasing arg I activity by splenic MDSC as the amount of tumor derived factors in the overnight culture increased. There is a slight but insignificant incre ase in arg I activity from 0% TES to 1% TES (p=0.1969). There is a clear and significant increase in arg I activity in 10% TES compared to 0% TES (p=0.0064) and to 1% TES (p=0.0182).

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! '( Potential Mechanisms by whi ch DC Transfer Antigen to Splenic MDSC We have found that DC loaded with OVA can transfer OVA antigen to splenic MDSC and enable MDSC suppression of OT I T cell responses. However, it is unclear how and in what form DC transfer OVA to MDSC. DC are known to transfer MHC class I containing exosomes vesicles of endocytic origin, among themselves to activate na•ve DC, and exosomes pulsed with tumor antigens yield MHC class I/tumor antigen complexes that enable stimulation of tumor specific cytotoxic T cells (AndrŽ et al. 2004). This indicates that DC could degrade OVA into SIINFEKL, load it onto MHC class I, and transfer the MHC class I/SIINFEKL complex to MDSC via exosomes. In order to test this we performed the original experiment, but introduced a 0.4 m transwell between the OVA loaded DC and splenic MDSC during the overnight co culture. This prevented close cell contact between DC and MDSC, but would allow exosomes, which are 60 90 nm, to pass through from DC to MDSC (AndrŽ et al. 2004). This would also allow the transfer of free SIINFEKL, partial OVA protein, or MHC class I/SIINFEKL complex shedded from the DC membrane ( Knight et al. 1998, AndrŽ et al. 2004 ). In addition, this would potentially allow the transfer of SIINFEKL or MHC class I/SIINFEKL complex via tunneling nanotubes (TNT), which are 25 200 nm in diameter, though the physiological relevance of TNT in antigen transfer has not yet been demonstrated (…nfelt et al. 2005, Watkins & Salter 2005, Marzo et al. 2012). Therefore the results of this experiment would not define the role of exosomes specifically, but would indicate whether exosomes are a possible mechanism of antigen transfer from DC to MDSC, or whether close cell contact is needed.

PAGE 77

! ') We have performed this experiment once so far. We found that although MDSC co cultured directly with OVA loaded DC showed significant suppression that was visibly stronger than the suppression by MDSC co cultured with OVA loaded DC in the presence of a transwell, MDSC co cultured with OVA loaded DC in the presence of a transwell did also exhibit significant suppression, and the difference in suppression between these two MDSC subsets was not statistically significant (see figure 20). In addition, although MDSC co cultured with unloaded DC exhibited sl ight and insignificant suppression, the difference in suppression between MDSC co cultured with unloaded DC and MDSC co cultured with OVA loaded DC (with or without a transwell) was not statistically significant (see figure 20). Overall, therefore, we cann ot make any conclusions. This one experiment indicates that it may be possible that splenic MDSC require close contact with DC in order to obtain antigen and exhibit strong suppression of antigen activated T cells, which would indicate that DC may not tra nsfer antigens to MDSC via exosomes. However, the limited data and lack of statistical significance prevent a definitive answer. We plan to perform this experiment again to obtain reproducible data and ultimately determine the mechanism of antigen transf er from DC to MDSC.

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! '* Figure 20. IFN release by OT I T cells in response to B16/Kb OVA cells with or without splenic MDSC previously co cultured with unloaded DC or with OVA loaded DC +/ transwell. T cell release of IFN was quantified as the number of spots per well. One experiment is reported. Each condition was performed in at least triplicate. Error bars show SEM (standard error of the mean). A p value less than 0.05 reached statistical significance. One asterisk denotes p<0.05, t wo asterisks denote p<0.01, and three asterisks denote p<0.001. Black lines bridge two conditions that were compared for a statistically significant difference in IFN release, with the corresponding p value shown above one of the conditions (ns denotes no significant difference). OT I CD8+ T cells were added to each well at 100,000 per well and irradiated B16/Kb OVA cells were added to each well at 12,000 per well, y ielding a robust response of approximately 175 spots per well (blue). Splenic MDSC that had been co cultured with either unloaded DC or OVA loaded DC were added to each well at 100,000 per well (1:1 MDSC: T cells). MDSC co cultured with unloaded DC yield ed an insignificant decrease in IFN release at 1:1 (dark green, p =0.1479). MDSC co cultured with OVA loaded DC yielded clear and significant suppression of IFN release at 1:1 (dark orange, p =0.0028), though the difference in suppression between the two MDSC subsets was not significant (p=0.0524). MDSC co cultured with OVA loaded DC in the presence of a transwell yielded significant suppression of IFN release at 1:1 (dark red, p =0.0387), with a visible but insignificant difference in suppression compared to MDSC co cultured with OVA l oaded DC (p=0.0613). As a negative control T cells alone were added to each well at 100,000 per well (black and white). This yielded negligible IFN release compared to T cells m ixed with B16/Kb OVA cells (p= 0.0004), indicating the response of OT 1 T c ells was specific for B16/Kb OVA cells.

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! (+ Discussion In this study we evaluated the ability of splenic MDSC to obtain antigen from DC and subsequently suppress antigen specific T cell activation. It was previously thought that peripheral lymphoid org an MDSC obtained tumor antigens by migrating to the tumor site and returning to the periphery (namely traveling to lymph nodes), ultimately presenting tumor antigens to tumor specific T cells while releasing suppressive effectors (i.e. ROS, PNT) to induce T cell anergy (Kusmartsev et al. 2005). This was brought into question by recent findings that murine splenic MDSC, when introduced into the tumor site, remained there, acquired nonspecific suppressive abilities, and differentiated into TAM. Conversely, splenic MDSC re introduced into the spleen remained in the periphery as antigen specific suppressors (Corzo et al. 2010). These findings indicate that peripheral lymphoid organ MDSC acquire tumor antigens through an intermediary cell type capable of obtai ning tumor antigens, either by traveling between the tumor site and the periphery or by taking up dead tumor cell material in the periphery, and transferring them to MDSC. DC seemed the most likely candidate, so we evaluated the transfer of antigen from a ntigen loaded DC to splenic MDSC during overnight co culture by the ability of MDSC re isolated from the co culture to suppress IFN release by antigen activated T cells. We found that at 1:1 MDSC: T cells, MDSC co cultured with OVA loaded DC consistently exhibited strong and significant suppression of activated OT I (OVA specific) T cells, while MDSC co cultured with unloaded DC exhi bited slight and insignificant suppression of activated OT I T cells with the exception of one experiment (see figures 10 and 11). It is unclear why this experiment yielded different results from

PAGE 80

! (" the others. OT I T cell IFN release was higher in the p resence of B16/Kb OVA cells in this experiment compared to the others, indicating that the T cells were highly activated (see figure 11) One group has hypothesized that MDSC are induced to become potent suppressors either when they travel to the tumor si te or when they encounter strongly activated T cells (i.e. by antigen s ) but above a threshold level of T cell activation (i.e. when T cells are nonspecifically and very strongly stimulated by anti CD3 and anti CD28 antibodies) MDSC suppressive abilities are overwhelmed (Solito et al. 2011). This could explain why in the presence of highly antigen activated T cells MDSC co cultured with unloaded DC exhibited significant suppression (see figure 11). However, this idea implies that exposure to the tumor microenvironment is equivalent to exposure to highly activated T cells, such that the latter would bypass the need for splenic MDSC to obtain antigen in order to be suppressive i.e. induce splenic MDSC to suppress nonspecifically. This is in conflict w ith the results from the other experiments, where lower but still robust T cell activation occurred without significant suppression by MDSC co cultured with unloaded DC. This idea also would not explain why in this experiment the suppression by MDSC co cu ltured with OVA loaded DC was more than twice as strong as the suppression by MDSC co cultured with unloaded DC, though the difference was not statistically significant (see figure 11). In addition, this idea conflicts with the findings that tumor site MD SC but not splenic MDSC can suppress T cells highly activated by anti CD3 and anti CD28 antibodies, and that activated helper T cell mediated induction of splenic MDSC nonspecific suppression depends on MDSC MHC class II expression, not on helper T cell IF N release (Corzo et al. 2010, Nagaraj et al. 2012). It is possible that stressful conditions during cell sorting

PAGE 81

! (# for this experiment could have activate d MDSC to become highly suppressive, though it would be expected that apoptosis would have occu r r ed ins tead Regardless, for all experiments at 1:3 MDSC: T cells there was a significant difference between T cell IFN release in the presence of MDSC co cultured with unloaded DC and T cell IFN release in the presence of MDSC co cultured with OVA loaded DC (see figures 10 and 11). At 1:3, MDSC co cultured with unloaded DC stimulated (or in one experiment did not affect) T cell IFN release, while MDSC co cultured with OVA loaded DC slightly and insignificantly (or in one experiment significantly) suppress ed T cell IFN release (see figures 10 and 11). IMC (immature myeloid cells) from na•ve mice, when added to the ELISPOT, increased T cell IFN release, most likely by providing co stimulation in the absence of any suppressive abilities (data not shown). MDSC, as myeloid cells, also express co stimulatory molecules It seems that in MDSC there could be a balance between co stimulation and suppression: at high enough numbers relative to T cells, the potent suppressive abilities of MDSC could outweigh any nonspecific co stimulation they might provide, and IFN release is inhibited. However, when the number of MDSC and their suppressive effectors decrease, their suppressive abilities could be diluted too much compared to highly antigen activated T cells that are provided co stimulation, to inhibit IFN releas e. This could explain the insignificant decrease in IFN release by OT I T cells when MDSC co cultured with OVA loaded DC are added at 1:3, as compared to IFN release by OT I T cells in the presence of B16/Kb OVA cells without MDSC. Taking this into a ccount, at 1:3 the significantly lower amount of IFN release in the presence of MDSC co cultured with OVA loaded DC compared to the amount of

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! ($ IFN release in the presence of MDSC co cultured with unloaded DC indicates that the former MDSC subset is supp ressive compared to the latter MDSC subset, which provided nonspecific co stimulation in the absence of significant suppression and induced overall stimulation of IFN release. This is consistent with the significant difference in suppression between MDS C subsets also observed at 1:1, where MDSC co cultured with OVA loaded DC did exhibit significant suppression. The results at 1:1 versus 1:3 are consistent with the large expansion of MDSC that occurs in the peripheral lymphoid organs of tumor bearing org anisms: since large numbers of MDSC are required to mediate robust suppression in vitro it is logical that the tumor would induce a high degree of MDSC proliferation in vivo to maximize the suppression of tumor specific T cell responses. Overall, therefo re, our results indicate that in vitro, DC can transfer antigen to splenic MDSC and enable them to suppress antigen activated T cells. This is the first time that DC have been shown to transfer antigen to MDSC providing a novel potential mechanism of tum or antig en acquisition by peripheral lymphoid organ MDSC. These findings were confirmed by our experiments introducing tumor derived factors into the DC and MDSC co culture. It has been shown in vitro that tumor site MDSC, in contrast to splenic MDSC, s uppress nonspecifically (Corzo et al. 2010). The introduction of increasing concentrations of tumor derived factors induced strong and significant suppression by splenic MDSC un exposed to antigen, which was equivalent to the suppression by splenic MDSC e xposed to antigen through DC (see figures 12 16). This mimics the conversion of splenic MDSC from antigen specific to nonspecific suppressors when they are introduced into the tumor site (Corzo et al. 2010). This indicates that in the absence of tumor de rived factors, splenic MDSC acquired antigen

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! (% from DC and needed antigen acquisition for strong suppression of antigen activated T cells, while introduction of high concentrations of tumor derived factors negated the need for splenic MDSC to obtain antigen from DC and promoted potent nonspecific suppression. Recently findings have shown that hypoxia in the tumor microenvironment and induction of expression of the transcription factor HIF 1 # mediate nonspecific suppression by MDSC residing at or introduced to the tumor site (Corzo et al. 2010 ). However, this is the first direct demonstration that tumor derived soluble factors convert splenic MDSC from antigen specific suppressors to nonspec ific suppressors in a dose dependent manner. This indicates that, in addition to hypoxia, soluble factors present in the tumor microenvironment (i.e., cytokines and growth factors) mediate nonspecific suppression by tumor site MDSC. Our findings are supp orted by the steady upregulation of iNOS expression and arg I expression and activity in splenic MDSC cultured in increasing concentrations of TES (see figures 17 19). The large and significant increase in arg I activity in MDSC cultured in 10% TES compar ed to no TES is consistent with the potent nonspecific suppression exhibited by MDSC co cultured with DC in 10% TES (see figures 14 and 19). Overall, this indicates that the induction of MDSC nonspecific suppression is likely mediated, at least in part, b y upregulation of arg I and iNOS. COX 2 is another effector that has been implicated in MDSC nonspecific suppression, and its indirect product, PGE 2 is known to be heavily present in the tumor microenvironment (Obermajer et al. 2011a, Nagaraj et al. 2012) MDSC COX 2 expression was recently found to be crucial for helper T cell mediated induction of

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! (& nonspecific suppression by MDSC (Nagaraj et al. 2012). We plan to evaluate COX 2 expression in splenic MDSC cultured overnight in 0% 10% TES and predict that it would f ollow a similar trend as arg I and iNOS expression. Conversely, since NADPH oxidase and ROS are associated with antigen specific suppression and peripheral lymphoid organ MDSC, we also plan to evaluate the expression of gp91 phox and p47 phox su bunits of NADPH oxidase, under the same conditions (Corzo et al. 2010). We predict that there would be a dose dependent downregulation of gp91 phox and p47 phox expression in splenic MDSC as the amount of tumor derived factors increases, consistent with the abrogation of antigen specific suppression and subsequent induction of nonspecific suppression. It would be interesting to determine which cytokines and growth factors present in TES mediate the conversion of splenic MDSC from antigen specific to nonspeci fic suppressors. Tumors secrete many factors that play a role in MDSC accumulation and activation, and there are likely multiple effectors involved that interact in a complex manner (Ostrand Rosenberg & Sinha 2009, Condamine & Gabrilovich 2010, Marigo et al. 2010). Generation of nonspecifically suppressing MDSC from healthy human bone marrow progenitors using a simple combination of cytokines like IL 6 and GM CSF indicates that there may be a few specific cytokines that are crucial (Marigo et al. 2010). However, this is currently beyond the scope of our study. Our original goal with the introduction of tumor derived factors into the DC and MDSC co culture was to find a condition that best mimicked the periphery in tumor bearing organisms, i.e., a conditio n where splenic MDSC are exposed to tumor derived factors at a low enough concentration during the co culture to maintain the antigen specific suppression typically exhibited by splenic MDSC in vitro and in vivo However,

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! (' since we did not achieve this at 2.5% TES, the concentration of tumor derived factors in the periphery is most likely lower than this. We plan to next evaluate suppression using 1% TES in the overnight co culture and to find an in vitro system that reproduces the periphery. Although DC a nd MDSC crosstalk has been previously demonstrated, earlier studies focused on the effect of MDSC on DC function, rather than the reverse (Ostrand Rosenberg et al. 2012). To our knowledge this is the first time that DC have been shown to influence splenic MDSC by providing antigens. It possible that DC could also affect MDSC by cytokine secretion. Uptake and processing of pathogens or pathogen derived material activate DC to undergo maturation, a process that affects cytokine secretion and that could hav e potentially occurred with DC that ingested OVA (Murphy 2012 ). However, to undergo robust activation DC usually must receive additional signaling, most commonly through recognition of molecular patterns associated with pathogens (Murphy 2012). Basal s ec retion of cytokines by DC could also influence MDSC, but this seems unlikely since MDSC co cultured with unloaded DC overall exhibited insignificant suppression (see figure 10). In addition, any weak suppression by MDSC co cultured with unloaded DC would most likely be due to arg I and iNOS expression. Although their expression by splenic MDSC is very low compared to tumor site MDSC, it is still upregulated compared to IMC, so this could account for a low level of nonspecific suppression (Corzo et al. 201 0). For one ELISPOT, splenic MDSC cultured alone (without DC) were used in addition to splenic MDSC co cultured with unloaded DC, and the former exhibited insignificant suppression equivalent to the latter. In addition, splenic MDSC cultured

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! (( alone with OV A were used in addition to splenic MDSC co cultured with OVA loaded DC, and these also exhibited significant and equivalent suppression (data not shown). (The OVA loaded DC were washed extensively before co culture with MDSC to prevent contamination with soluble OVA (see Material and Methods p. 31 ) Therefore, the equivalence of the suppression exhibited by MDSC co cultured with OVA loaded DC and the suppression exhibited by MDSC cultured alone with OVA is not due to the carry over of soluble OVA to the DC and MDSC co culture ) Although these conditions could only be performed once due to limited cell numbers, this result supports the idea that antigen transfer is the primary mechanism by which DC influence splenic MDSC suppression, and that cytokine sec retion plays a trivial role. The strong suppression of activated OT I T cells by splenic MDSC co cultured with OVA loaded DC indicates that MDSC acquired surface expression of MHC class I bound to SIINFEKL, enabling them to interact with and damage the TC R of OT I T cells and induce T cell anergy (Nagaraj et al. 2007). However, it is currently unclear how and in what form MDSC obtained OVA antigen from DC In general, foreign proteins obtained by DC undergo proteasomal degradation to form small peptides, which are loaded onto MHC class I molecules and transported to the cell surface to be presented to T cells (Murphy 2012). SIINFEKL is known to be processed from OVA and complexed with MHC class I, so it would be possible that either free SIINFEKL or the MHC class I/SIINFEKL complex would be transferred from DC to MDSC ( Murphy 2012 ). In the former case SIINFEKL would be loaded onto MHC class I endogenous to MDSC, and in the latter case the entire MHC class I/SIINFEKL complex would be derived from DC, with likely little or no processing by MDSC.

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! () T ransfer of MHC class I/antigen complex by exosomes is a possibility, since our preliminary transwell experiment did not yield conclusive results (see figure 20). Shedding from the DC membrane of MHC class I/SIINF EKL complex and secretion of SIINFEKL peptide or partial OVA protein are other possibilities that also would not require close cell contact ( Knight et al. 1998, AndrŽ et al. 2004 ) We plan to evaluate these by repeating the transwell experiments. Some re searchers have also suggested that transfer of antigen from DC could theoretically occur via TNT (Watkins & Salter 2005). TNT are long intercellular membrane bridges associated with long range signaling that have been shown to occur between cells in vitro and in vivo (Marzo et al. 2012). TNT have been shown to occur between a variety of immune cells; for example, calcium fluxes can be propagated between dendritic cells by TNT, and pathogens can use TNT to travel from macrophages to other immune cells (Wat kins & Salter 2005, Eugenin et al. 2009). However, transfer of antigen by TNT has not been demonstrated either in vitro or in vivo (Watkins & Salter 2005, Marzo et al. 2012). It is also possible that close cell contact may be involved in antigen transfer between DC and MDSC One potential cell contact dependent mechanism is the transfer of SIINFEKL by gap junction. Linear peptides of up to 16 amino acids can be transferred by gap junction, and SIINFEKL, at 8 amino acids, is a viable candidate (Saccheri et al. 2010). It was recently shown that a B16F10 cell line expressing OVA proteasomally processed OVA into SIINFEKL and transferred the peptide via gap junction to DC, which loaded it onto MHC class I molecules then transported to the cell surface (Sacch eri et al. 2010). It was also shown that DC could transfer peptides via gap junction among themselves (Saccheri et al. 2010). Furthermore, the injection of bacteria

PAGE 88

! (* treated tumor cells into mice induced expression of the gap junction protein connexin 43 by DC in tumor draining lymph nodes, and tumor cells with silenced connexin 43 expression failed to induce an anti tumor T cell response in vivo (Saccheri et al. 2010). This demonstrates that DC acquisition and transfer of antigenic peptides by gap juncti on occurs in tumor bearing organisms, indicating that MDSC could obtain SIINFEKL from DC via gap junction and load SIINFEKL onto endogenous MHC class I molecules transported to the cell surface. To investigate this possibility, we plan to evaluate the tr ansfer of calcein from DC to splenic MDSC. Calcein is a fluorescent green dye known to be transferred between cells by gap junction (Saccheri et al. 2010). DC would be pulsed with calcein and co cultured with MDSC pulsed with DDAO, a fluorescent red dye that can be used to label specific cell subsets because it undergoes covalent modification once it enters the cytosol, rendering it unable to exit back through the cell membrane (Saccheri et al. 2010). After a 4 hour co culture, calcein transfer from DC t o MDSC by gap junction, if it occurs, should be visible by fluorescence microscopy, and red labeled MDSC would be stained with green. In order to evaluate if SIINFEKL is actually transferred via gap junctio n we would perform the original experiment with t he overnight co culture between unloaded or OVA loaded DC and splenic MDSC and next day ELISPOT, but with the gap junction inhibitor heptanol added to the co culture (Saccheri et al. 2010). If the suppression by MDSC co cultured with OVA loaded DC is abro gated in the presence of heptanol, this would indicate that DC do transfer antigens to splenic MDSC via gap junction. There are, however, other cell contact dependent possibilities to evaluate. One study found that in a cell contact dependent mechanism, D C could obtain plasma

PAGE 89

! )+ membrane from other APC and lymphocytes and could also transfer plasma membrane to other DC (Harshyne et al. 2001). Plasma membrane was found to be physically pulled from one DC to another and contained in endocytic vesicles. Furthe rmore, DC expressing a melanoma antigen could transfer it to other DC, enabling them to activate melanoma specific T cells (Harshyne et al. 2001). This indicates that DC could transfer plasma membrane containing the MHC class I/SIINFEKL complex directly t o splenic MDSC. In addition, it is necessary to discriminate between transfer of free peptide or partial protein, and transfer of MHC class I/SIINFEKL complex. To do this we would co culture splenic MDSC with OVA loaded DC generated from the bone marrow of $ 2 microglobulin knockout mice, and evaluate MDSC suppression of activated OT I T cells. MHC class I molecules comprise two polypeptide chains, the # chain and $ 2 microglobulin so $ 2 microglobulin knockout mice contain unformed and unstable MHC cla ss I molecules incapable of binding to antigen (Murphy 2012, JAX Mice Database: B6;129P2 B2m tm1Unc /J [date unknown] ). If MDSC co cultured with OVA loaded DC generated from these mice exhibit insignificant suppression compared to MDSC co cultured with OVA loaded DC generated from B6 mice, this would indicate that splenic MDSC acquisition of antigen from DC depends on functional MHC class I expression by DC, signifying that DC transfer antigen complexed with MHC class I to MDSC. We also need to evaluate the ability of other APC, namely macrophages, to transfer antigen to splenic MDSC, since they too could be a source of tumor antigens for peripheral lymphoid organ MDSC. Macrophages, like DC are APC known to take up

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! )" extracellular antigens, particularly apop totic material (Harshyne et al. 2001, Murphy 2012). Both DC and macrophages can take up material fro m dead tumor cells, particularly in tumor draining lymph nodes (lymph node resident DC) and in peripheral blood and tissues (migratory DC), but a recent st udy found that only CD169 expressing macrophages were essential for the cross presentation of tumor antigens from dead tumor cells (Asano et al. 2011). Dead tumor cells were transported via the lymphatic system to draining lymph nodes, where resident CD16 9+ macrophages ingested them and cross presented tumor antigens to tumor specific T cells. R esident and migratory DC exhibited poor tumor antigen uptake and depletion of CD169+ macrophages, not CD11c+ DC, abrogated tumor antigen cross presentation to T c ells (Asano et al. 2011). This indicates that macrophages are the APC that primarily take up tumor antigens, implying that they could be responsible for transferring tumor antigens to peripheral lymphoid organ MDSC. However, the possibility that migrato ry DC obtained some tumor antigens and contributed to cross presentation could not be ruled out (Asano et al. 2011) Interestingly, the study that showed DC could obtain and transfer antigen containing plasma membrane also found that DC could obtain and t ransfer plasma membrane from macrophages, but macrophages lacked the ability to do so (Harshyne et al. 2001). This indicates that even if macrophages are primarily responsible for obtaining antigens from dead tumor cells, it seems unlikely that they could transfer them to peripheral lymphoid organ MDSC. It does seem likely that DC, even if they do not acquire antigens from dead tumor cells directly, could acquire tumor antigens from macrophages and then transfer them to peripheral lymphoid organ MDSC. In addition, it may even be possible

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! )# that migratory DC traveling to the tumor site could obtain plasma membrane from live tumor cells (as well as antigens from dead tumor cells) and then migrate to the lymph nodes. There either they could transfer tumor ant igens to resident lymph node DC, which would then transfer tumor antigens to peripheral lymphoid organ MDSC, or they could transfer tumor antigens directly to peripheral lymphoid organ MDSC. Above all, we need to explore the phenomenon of tumor antigen t ransfer to peripheral lymphoid organ MDSC in vivo to determine if DC provide tumor antigens to peripheral lymphoid organ MDSC if macrophages provide tumor antigens to peripheral lymphoid organ MDSC, if macrophages provide tumor antigens to DC that then tr ansfer the tumor antigens to peripheral lymphoid organ MDSC, or if a different mechanism is involved E lucidation of the mechanism of peripheral lymphoid organ MDSC tumor antigen acquisition would lead to better characterization of the factors that enable and influence MDSC immunosuppression, with po tential implications for future treatments that could target these factors.

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! )$ Conclusion In conclusion, we have demonstrated that DC are capable of transferring antigens to splenic MDSC, thereby enabling sp lenic MDSC to suppress antigen activated T cells. We have also demonstrated that soluble factors derived from solid tumors convert splenic MDSC from antigen specific suppressors to nonspecific suppressors in a dose dependent manner that is accompanied by upregulation of iNOS and arg I. We plan to next elucidate the particular mechanism of antigen transfer from DC to MDSC. Ultimately we plan to validate our findings in tumor bearing organisms to determine the mechanism by which peripheral lymphoid organ M DSC obtain tumor antigens. It is necessary to elucidate the mechanism of tumor antigen acquisition by peripheral lymphoid organ MDSC, as well as better understand the factors that mediate antigen specific and nonspecific suppression by MDSC in different compartments. This would aid in the development of immunothe rapies that activate anti tumor immune r esponses, eliminate the complex immunosuppressive network recruited by the tumor and mediated by MDSC, and ultimately promote progression free survival (Ga brilovich et al. 2012). Efforts to target MDSC have so far yielded significant progress: for example IL 12 treatment was found to reprogram murine MDSC to stimulate T cells, resulting in tumor regression (Mauti et al. 2011). In addition, the use of all trans retinoic acid to induce the differentiation of patient MDSC has resulted in activation of anti tumor responses (Mirza et al. 2006). However, much progress remains to be made to characterize and effectively target MDSC (Nagaraj et al. 2012).

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! )% Append ix I: List of Abbreviations APC antigen presenting cells Arg I arginase I ArgI gene encoding arginase I B6 C57BL/6 or black 6; B6 mice are a co mmonly used inbred mouse strain B16F10 mouse melanoma cell line B16/Kb OVA cells B16F10 cells transfe cted to express a fusion MHC class I/SIINFEKL peptide complex CD3 co receptor that forms part of the T cell receptor complex CD3 zeta/ chain ; main component of the TCR complex initiating the signal transduction cascade leading to T cell activation a fter antigen binding CD4 co receptor expressed by helper T cells CD8 co receptor expressed by cytotoxic T cells CD11b myeloid cell surface marker CD11c cell surface marker expressed by differentiated dendritic cells CD28 co stimulatory receptor e xpressed by T cells CDK4 cyclin dependent kinase 4 COX 2 cyclooxygenase 2 DC dendritic cells EL 4 mouse lymphoma cell line ELISPOT enzyme linked immunosorbent spot assay EP E prostanoid receptor FACS fluorescence activated cell sorting

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! )& G CSF granulocyte colony stimulating factor GM CSF granulocyte/macrophage colony stimulating factor G MDSC granulocytic myeloid derived suppressor cells Gr 1 myeloid cell surface marker; co expression of Gr 1 with CD11b defines murine myeloid derived sup pressor cells HIF 1 # hypoxia inducible factor 1alpha HRP horseradish peroxidase IFN interferon gamma IFN R interferon gamma receptor IL interleukin IL 2R interleukin 2 receptor IMC immature myeloid cell iNOS inducible nitric oxide synthase JAK Janus kinase M CSF macrophage colony stimulating factor MDSC myeloid derived suppressor cell MHC m ajor histocompatibility complex M MDSC monocytic myeloid derived suppressor cells NO nitric oxide Nos2 gene encoding inducible nitric oxide synthase OT I OVA TCR I or OVA specific, class I restricted TCR; cytotoxic T cells from OT I mice (OT I T c ells) specifically recognize t he MHC class I/SIINFEKL complex OVA ovalbumin

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! )' PAMPs pathogen associated molecular patterns PHA phytohemagglutinin PNT peroxynitrite ROS reactive oxygen species SIINFEKL 8 amino acid peptide derived from ovalbumin that binds to MHC class I STAT signal transducer and activator of transcription TAM tumor associated macrophages TCR T cell receptor TES tumor explant supernatant TNT tunneling nanotubes VEGF vascular endothelial growth factor

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! )( Append ix II: Abbreviated Version of Material and Methods Cell Culture. Generation of DC. Whole bone marrow was extracted from B6 GFP mouse femur and tibia and cells were cultured in complete RPMI 1640 (supplemented with 10% FBS (fetal bovine serum) and 1% antibi otics) with 20 ng/mL IL 4 and 10 ng/mL GM CSF from day 0 to day 6 to induce differentiation into DC. On day 3 the media was replenished. On day 5 OVA (100 g/mL) was added to half the culture overnight. On day 6 CD11c+ DC were isolated. DC and MDSC co culture. On day 6, after CD11c+ DC were isolated, they were co cultured overnight in complete RPMI 1640 supplemented with 10 ng/mL GM CSF with Gr 1+ MDSC freshly isolated from an EL 4 tumor bearing mouse spleen. DC were co cultured with MDSC 1:5 1:2, DC: MDSC. For comparison, Gr 1+ MDSC were cultured overnight in complete RPMI 1640 supplemented with 10 ng/mL GM CSF without DC and with and without OVA (100 g/mL) On day 7 all cells were collected. DC and MDSC co culture with transwell. The overnight co culture was performed as previously stated, but one condition was added: OVA loaded DC were co cultured with MDSC separated by a 0.4 m transwell. Generati on of DC in TES. DC were generated as previously stated, but on day 1 of the culture TES from an EL 4 tumor bearing mouse was added to the culture at 20% TES (see attached TES generation protocol). DC and MDSC co culture in TES. The overnight co cultur e was performed as previously stated, but TES from an EL 4 tumor bearing mouse was added to the co culture at 2.5%, 5%, 10%, or 20% TES.

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! )) MDSC culture in TES. Gr 1+ MDSC freshly isolated from an EL 4 tumor bearing spleen were cultured overnight at 2x10 6 MD SC in 200 L (for arg I activity and NO production assays) or at 500,000 MDSC in 200 L (for iNOS and arg I expression assays) in complete RPMI 1640 supplemented with 10 ng/mL GM CSF without TES or with TES from an EL 4 tumor bearing mouse added at 1%, 2.5 %, 5%, or 10%. B16/Kb OVA cells. B16/Kb OVA cells were cultured in complete DMEM. Cells were split every 2 3 days after becoming confluent. The selection antibiotic G418 was added once a week (10 L per mL of media). B16F10 cells. B16F10 cells were cul tured in complete DMEM. Cells were split every 2 3 days after becoming confluent. Cell Isolation. T cells. Splenocytes from an OT I mouse spleen were collected and CD8+ T cells were isolated by biotinylated anti CD8 ab (antibody) and streptavidin microbe ad labeling and magnetic column separation (see attached cell isolation by MACS protocol). DC. CD11c+ DC were isolated by biotinylated anti CD11c ab and streptavidin microbead labeling and mag netic column separation. MDSC. Splenocytes from an EL 4 tumor b earing mouse spleen were collected and Gr 1+ MDSC were isolated by biotinylated anti Gr 1 ab and streptavidin microbead labeling and magnetic column separation. After overnight co culture with DC, GFP CD11c CD11b+Gr 1+ MDSC were separated from GFP+CD11c+ DC and collected by flow cytometry cell sorting using the FACSAria (see attached MDSC sort protocol). Cells were labeled with APC conjugated anti CD11c, PE Cy 7 conjugated anti CD11b, and

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! )* PE conjugated anti Gr 1 abs DAPI was used to evaluate cell viabil ity and exclude dead cells. Evaluation of T C ell IFN Secretion by ELISPOT. MDSC mediated suppression of IFN production by OT I CD8+ T cells in response to irradiat ed (60 Gy) B16/Kb OVA cells was evaluated by ELISPOT (see attached ELISPOT protocol). 1 00,000 CD8+ T cells per well were used with 30,000, 15,000, or 12,000 B16/Kb OVA cells per well. MDSC co cultured with unloaded DC +/ TES, MDSC co cultured with OVA loaded DC +/ TES, MDSC co cultured with OVA loa ded DC separated by transwell, M DSC cultur ed alone, or MDSC cultured alone with OVA (100 g/mL) were added to the ELISPOT at 1:1, 1:2, and/or 1:3 ratios (MDSC: T cells) depending on the total number of MDSC collected T cells with B16/Kb OVA cells (without MDSC) were used as a positive control. T cells with irradiated B16F10 cells with or w ithout MDSC were used in place of B16 Kb OVA cells, as a negative control. T cells alone were used as an additional negative control. The cells were incubated at 37 ¡ C for 40 46 hours. The number of spots per well was counted in triplicate for each cond ition (or duplicate if restricted by cell numbers) and calculated by an automatic ELISPOT counter. Evaluation of NO Production. After overnight MDSC culture with or without TES, cell supernatant for each sample was collected, and the amount of nitrite pre sent due to production of NO by iNOS was measured by spectrophotometry (see attached NO production protocol).

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! *+ Evaluation of Arg I Activity. After overnight MDSC culture with or without TES, cell lysate for each sample was collected, and the amount of ur ea produced by arginine hydrolysis was measured by spectrophotometry (see attached arg I activity protocol). The total amount of protein present in the lysate was quantifie d by Bio Rad protein assay kit Arg I activity was determined as the concentration of urea present in each sample normalized to the concentration of total protein present in each sample. Evaluation of Arg I and iNOS E xpression. After overnight MDSC culture with or without TES, total RNA was extracted by E.Z.N.A. Total RNA Kit I and rev erse transcribed into cDNA, with water used as a blank (see attached RNA extraction protocol). Expression of arg I and iNOS was deter mined by real time PCR performed on the cDNA samples using Taqman probes directed against A rgI (arg I) and Nos2 (iNOS), wi th $ actin used as an endogenous control (see attached reverse transcription protocol). Bio Rad software was used to analyze the real time PCR results and determine Ct values. The fold change in arg I and iNOS expression compared to MDSC cultured overnig ht in 0% TES was determined for each sample from the Ct values and normalized to $ actin expression (see attached real time PCR protocol). Evaluation of SIINFEKL/MHC Class I Expression by B16/Kb OVA C ells. We evaluated and verified the maintenance of SII NFEKL/MHC class I expression by B16/Kb OVA cells using flow cytometry with an LSRII cytometer (see attached B16/Kb OVA staining protocol). Cells were labeled with APC conjugated anti pMHC CI OVA ( SIINFEKL/MHC class I ) ab. DAPI was used to evaluate cell v iability and exclude dead cells.

PAGE 100

! *" Statistical Analysis. Two tailed student's t test with Welch's correction for significantly different variances was performed fo r all data using Prism software, with p<0.05 reaching statistical significance.

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! *# Appendix III: Step by Step Protocols B16F10 Cell and B16/Kb OVA Cell Culture 1. Seed approximately 1 million adherent cells in 10 12 mL complete DMEM (10% FBS, 1% antibiotics) in a 50 mL tissue culture flask. Incubate at 37 ¡C. Split every 2 3 days. When splitting cells keep 1 million in culture and freeze the rest if not needed for assays. Once a week add selection antibiotic G418 (10 L per mL of media) to transfected B16/Kb OVA cell culture. Generation of DC from GFP B6 Mouse Bone Marrow 1. Take hind legs from 2 GFP B6 mice. Clean off all skin, fat, and muscles. Discard fibula and separate tibia from femur. Place clean tibia and femur in 5 10 mL PBS or complete media in a 15 mL centrifuge tube 2. Cut each bone in half. Place bones in 5 10 mL complete RPMI or PBS in a 6 well plate. Fill a syringe with PBS and plunge all marrow out of each bone. Collect marrow in PBS and wash once, 1800 rpm, 5 minutes, 5/5 brake/acceleration. Resuspend in 5 8 mL PBS. 3. Filter marrow cells through 70 m cell strainer. Collect cells and wash with PBS. Resuspend in 3 mL ACK to lyse red blood cells. 4. Incubate at room temperature for 2 minutes. Wash once with PBS and once with complete media. Count cells. 5. Resuspend cells in complete RPMI at 1 2 million c ells per mL. Supplement media with 10 ng/mL GM CSF and 20 ng/ml IL 4. Add cells to 24 well flat bottom plates at 1 2 millions cells per well. Incubate at 37 ¡C for 6 days. Change

PAGE 102

! *$ media on day 3. On day 5 add 100 g/mL OVA protein to half of the cells so that there are two separate subsets: OVA loaded and unloaded. 6. On day 6 collect cells and isolate CD11c+ DC from each subset (OVA loaded and unloaded). Generation of DC from GFP B6 Mouse Bone Marrow with TES. 1. Follow above protocol but add EL 4 TES on day 1 to DC culture so that the media comprises 20% TES throughout the rest of the DC culture. DC and MDSC Co Culture 1. On day 6, a fter isolation of CD11c+ DC (OVA loaded and unloaded) from GFP B6 bone marrow culture and isolation of Gr 1 + MDSC from a tumor bea ring EL 4 mouse spleen co culture each DC subset with MDSC at a ratio of 1:5 DC: MDSC to 1:2 DC: MDSC For comparison, c ulture any leftover MDSC alone (without DC), with and without OVA (100 g/mL). Culture cells in a 6 well flat bottom plate at 500,000 1.5 million cells per mL per well in complete media supplemented with 10 ng/mL GM CSF. Incubate cells at 37 ¡C overnight. 2. Collect cells day 7 for cell sorting. DC and MDSC Co Culture with Transwell 1. Follow the above protocol but add an extra condition : MDSC co cultured with OVA loaded DC, separated by a 0.4 m transwell insert. For ease of next day collection for sort for each transwell condition, suspend MDSC in 3 mL and add directly to well. Suspend DC in 2 mL and add into insert

PAGE 103

! *% DC and MDSC Co Cu lture with TES 1. Follow DC and MDSC Co Culture protocol bu t add TES to the overnight co cult ure so that the media comprises 2.5%, 5%, 10%, or 20% TES. MD SC Culture with and without TES 1. A fter isolation of Gr 1 + MDSC from a tumor bearing EL 4 mouse spleen culture MDSC in a 96 well U bottom plate at 2 million cells per 200 L per well (for arg I activity and NO production assays) or at 500,000 cells per 200 L per well (for arg I and iNOS expression assays) in complete media supplemented with 10 ng/mL GM CSF. Add TES to the culture with as many of the following conditions a s cell numbers permit: 0% TES, 1% TES, 2.5% 5% TES, and 10% TES. Incubate cells at 37 ¡C overnight. Generation of EL 4 TES 1. Isolate primary tumor (and secondary tumor(s)) from EL 4 tumor bearing mouse. 2. Separate tumor in half and place each half in 20 mL co mplete RPMI in a tissue culture dish. 3. For each tumor half, cut evenly into small pieces, approximately 10 mm 2 each. 4. For each tumor half, collect all media and tumor pieces and place into a 50 mL tissue culture flask. Incubate at 37 ¡C overnight. 5. The next day collect all media and tumor pieces into 50 mL centrifuge tube. Spin at 1800 rpm, 10 min, 5/5 acceleration/brake. 6. Collect supernatant and aliquot into 1.5 2 mL microfuge tubes. Spin at 12,000 rpm, 10 min, 5/5 acceleration/brake.

PAGE 104

! *& 7. Collect supernatant a nd and aliquot into 1.5 2 mL microfuge tubes. Freeze at 80¡C. When needed thaw to room temperature and add directly to media. B16F10 Cell and B16 Kb OVA Cell Collection and Irradiation 1. Remove media and rinse flask with PBS. Add 4 mL 0.25% trypsin and incubate cells at room temperature for 3 4 minutes. Tap flask to remove cells. Add 25 mL complete DMEM. Wash cells twice with complete DMEM. Resuspend cells in complete DMEM and count. Collect at this step for pMHC CI OVA staining. 2. For ELISPOT, take a liquot of cells (2 3 million B16F10 cells and 2 3 million B16/Kb OVA cells) in about 5 mL complete DMEM and irradiate with X rays at 60 Gy. 3. Wash cells twice, once with complete DMEM and once with complete RPMI. Count cells. Resuspend in complete RPMI for ELISPOT. Preparation of Splenocytes from OT I Transgenic Mouse Spleen and from EL 4 Tumor Bearing Mouse Spleen 1. Extract whole spleen from mouse, place in 5 8 mL PBS or complete RPMI 2. Filter spleen through 70 m cell strainer Collect cells and wash once with PBS, 1800 rpm, 5 minutes, 5/5 brake/acceleration. Resuspend cells in 3 mL ACK to lyse red blood cells. 3. Incubate at room temperature for 2 minutes. Wash twice with PBS. Count. Resuspend in MACS buffer for isolation of CD8+ OT I T cells or EL 4 Gr 1 + MDSC.

PAGE 105

! *' CD11c+ DC, CD8+ T Cell, and Gr 1+ MDSC Isolation by Positive Selection using Biotinylated Primary Antibody, Streptavidin Microbeads, MACS Column, and Magnetic Separator (Adapted from Miltenyi Biotec protocol.) 1. Collect bone marrow cells (OVA loaded DC and unloaded DC cultures) or splenocytes (from OT 1 mouse spleen or EL 4 tumor bearing mouse spleen) and count. Wash in MACS buffer and resuspend in MACS buffer at 50 million cells per mL (500 L total for fewer than 25 million cells). 2. Add biotinyla ted primary antibody (biotin anti CD11c antibody, biotin anti CD8 antibody, or biotin anti Gr 1 antibody) at 1 L per one million cells. Incubate for 30 minutes at 4 ¡C. 3. Wash cells once in MACS buffer. Resuspend in 1 mL of MACS buffer for 50 150 million cells, or 500 L for 20 50 million cells in a 15 mL centrifuge tube. 4. Add streptavidin microbeads at 1 L per one million cells. Incubate for 20 minutes at 4 ¡C. 5. Wash cells once in MACS buffer. Resuspend 10 50 million cells in 500 L MACS buffer; resuspend 50 150 million cells in 1 mL; scale up for cell numbers greater than 150 million. 6. Place appropriate column in the field of a MACS separator and prepare by rinsing with MACs buffer. Use LS column for loading of 50 million or more cells. Use MS column for loading of fewer than 50 million cells if number of expected target cells does not exceed 10 million. If e xpected number of target cells exceeds 10 million use LS column.

PAGE 106

! *( a. Rinse LS column with 3 mL MACS buffer. b. Rinse MS column with 500 L MACS buffer. 7. Apply cell suspension to column. Collect unlabeled cells that pass through. Wash column three times with MACS buffer. Perform each wash only after column reservoir has emptied each time. a. Wash LS column with 3 mL MACS buffer each time. b. Was h MS column with 500 L MACS buffer each time. 8. Remove column from magnetic separator and place in new collection tube. 9. Pipette MACS buffer onto column and immediately flush out magnetically labeled target cells by firmly pushing plunger into the column. a. Pi pette 5 mL MACS buffer onto LS column. b. Pipette 1 mL MACS buffer onto MS column. 10. Wash target cells with complete media. Count. Wash again with complete media and resuspend in complete media for co culture overnight (CD11c+ DC and Gr 1+ MDSC) or for ELISPO T (OT I T cells). MDSC Sort Protocol (S terile) Flow Cytometry 1. Collect all cell subsets after overnight culture un loaded DC co cultured with MDSC overnight, OVA loaded DC co cultured with MDSC overnight MDSC cultured overnight with OVA loaded DC separa ted by transwell (collect only MDSC directly from the well), MDSC cultured alone overnight, and MDSC cultured alone with OVA overnight Collect adherent and non adherent cells. 2. Wash all cell subsets once with media or PBS. Count cells. Separate cells in to 5 mL sterile sort tubes.

PAGE 107

! *) 3. Wash again with PBS and resuspend in 100 L per tube MACS buffer. Use 100,000 cells for each compensation control tube. 4. Stain cells in the dark for sort. Incubate in the dark for 20 minutes at room temperature. a. S tain cells with APC conjugated anti CD11c antibody (2 g/mL), PE Cy7 conjugated anti CD11b antibody (2 g/mL), and PE conjugated anti Gr 1 antibody (2 g/mL). Keep unstained cells and cells suspended in DAPI buffer as controls. Keep unstained GFP+ cells as a control. Keep cells stained only with APC conjugated anti CD11c antibody (2 g /mL) ( unloaded DC + MDSC ), only with PE Cy7 conjugated anti CD11b antibody (2 g/mL) (MDSC cultured alone +/ OVA ), and only with PE conjugated anti Gr 1 antibody ( 2 g/mL) ( MDSC cultured alone +/ OVA ) for single stain compensation controls. 5. Wash cells once with PBS and resuspend in 100 L per tube DAPI buffer, or 100 L per tube MACS buffer for unstained and compensation controls. Make collection tubes with FBS for MDSC co cultured with unloaded DC, MDSC co cultured with OVA loaded DC, MDSC co cultured with OVA loaded DC separated by transwell, MDSC cultured alone, and MDSC cultured alone with OVA 6. Sort MDSC subsets on FACSAria cell sorter ideally collect 400,000 500,000 MDSC (see figure 21 for collection parameters). 7. Collect sorted MDSC subsets and w ash once with complete RPMI Resuspend in complete RPMI

PAGE 108

! ** Figure 21. Parameters for collecting MDSC from the DC and MDSC co culture by FACS. Figure provided by Dr. Thomas Condamine. Splenic MDSC were separated from DC after the overnight co culture us ing morphology, lack of expression of GFP and CD11c, and clear co expression of Gr 1 and CD11b. Live cells that did not express DAPI were selected. The MDSC population was very distinct from the DC population, so contamination of the collected MDSC by DC was negligible.

PAGE 109

! "++ ELISPOT Protocol ( A dapted from Mabtech protocol. ) Day 1 (sterile conditions) 1. Pre wet each well of MultiScreen IP plate with 75 L per well of 70% ethanol for 2 minute s Decant and wash with 200 L per well sterile PBS 6 times Once the membrane is pre wet with alcohol, do not allow membrane to dry for the duration of the assay. 2. Coat each well with 100 L anti IFN antibody (10 g/mL) diluted in sterile PBS. Incubate covered plate overnight at 4¡C. Day 2 (sterile conditions) 1. Decant primary antibody solution and wash off unbound antibody with 200 L per well sterile PBS 6 times. 2. Block membrane with 20 0 L per well cell medium (co mplete RPMI) for at least 30 minutes at room temperature. 3. When cells are ready, decant medium and plate out 200 300 L cells (OT I CD8+ T cells, irradiated B16F10 cells, irradiated B16/Kb OVA cells, and sorted MDSC subsets) per well and incubate 40 46 hour s at 37 ¡C. Do three wells per condition, or at least two if low cell numbers prevent use of triplicates. Day 4 1. Decant cells and wash with 2 00 L per well PBS 6 times. 2. Coat plate with 100 L secondary biotinylated anti IFN antibody (2 g/mL)

PAGE 110

! "+" diluted in PBS. Incubate at room temperature for 2 hours. 3. Decant secondary antibody solution and wash with 2 00 L per well PBS 6 times. 4. Coat plate with Streptavidin HRP solution diluted 1000 times in PBS Incubate at room temperature for 1 hour. 5. Decant Streptavidin HRP solution and wash with 2 00 L per well PBS 6 times. 6. Add 100 L per well TMB substrate developing solution. Leave for approximately 5 minutes. In order to avoid over development, do not leave for more than 10 minutes. Stop when spots are clearly vis ible but are not so numerous that they seem to merge. 7. Wash plate extensively in DI water to stop spot development. Blot on paper towel and gently dry underside of membranes to ensure all substrate is removed. Let plate dry overnight in the dark. Store i n the dark. Day 5 1. Analyze plate and count number of spots per well using automatic ELISPOT counter from Cellular Technology. Each spot corresponds to IFN release by one OT I CD8+ T cell. Measurement of NO Production 1. After culturing MDSC overnight in 0% 10% TES in a 96 well plate, spin plate for 3 minutes at 2500 rpm, 5/5 brake/acceleration to pellet cells. 2. Remove supernatant. For each well take 100 L of supernatant for NO production assay. Freeze remaining 100 L at 80 ¡C to use for later assays if necessary. Save cell pellet for arginase I activity assay.

PAGE 111

! "+# 3. Add 100 L of supernatant to 96 well flat bottom plate. 4. Make sodium nitrite standards by ser ial dilution of 0.25 M sodium nitrite solution (1 M sodium nitrite = 69 mg in 1 mL DI water). Add 100 L of each standard to the 96 well flat bottom plate. 5. Add 100 L of Greiss reagent (100 mg sulfanilamide + 10 mg N (1 napthyl)ethyl enediamine + 0.5 H 3 P O 4 in 10 mL DI water) to each sample and each standard and incubate at room temperature for 10 minutes. 6. Measure absorbance of samples at 550 nm with spectrophotometer and determine sodium nitrite concentration in each by using standard curve generated from absorbance values of serially diluted sodium nitrite standards. Use concentration of sodium nitrite in each sample to determine relative production of NO and activity of iNOS. Mea surement of Arg I Activity 1. After pelleting of cells and removal of supernat ant for NO production assay, add 120 L of 0.1% Triton X 100 (50 uL Triton X 100 in 50 mL DI water) per well. Mix well and transfer each sample to a 1.5 mL eppendorf tube. Incubate for 30 min at room temperature to lyse cells. 2. Spin cells in microfuge for 30 min at 14,000 rpm, 5/5 brake/acceleration. Collect supernatant. 3. During centrifugation make up Bio Rad Protein Assay standards. Add 10 L of each standard to 190 L of Bio Rad Protein Assay Buffer, pre diluted in DI water

PAGE 112

! "+$ 1:5 (1 mL 1X Bio Rad Protein Assay Buffer + 4 mL DI water) in 96 well flat bottom plate. Add 190 L of pre diluted Bio Rad Protein Assay Buffer to each well for each sample to be analyzed. 4. Once samples are done spinning, take 10 L lysate and add each to the 190 L of pre diluted Bio Rad Protein Assay Buffer. Take 100 L of remaining lysate and add to new 1.5 mL eppendorf tube. Keep plate for analysis to be done concurrently with urea production/arg I activity (see step 10 below). 5. For each sample of 100 L lysate, add 100 L Tris HC l (157.6 mg in 40 mL water ) and 10 L MnCl 2 Heat in 56 ¡C water bath for 10 minutes to activate arg I. 6. Add 100 L 0.5 M L arginine (3.5 g in 40 mL DI water, pH 9.7) to each sample. Incubate for 1 hour in 37 ¡C water bath. 7. During the 1 hour incubation, p repare urea standards by serial dilution of 100 mM urea (60 mg in 10 mL water). Also prepare solution of: H 2 SO 4 (96%) + H 3 PO 4 (85%) + DI water, 1:3:7 (2 mL H 2 SO 4 (96%) + 6 mL H 3 PO 4 (85%) + 14 mL DI water). Also prepare solution of 9% # isonitrosopropioph enone, dissolved in 100% EtOH (90 mg # isonitrosopropiophenone in 1 mL 100% EtOH). 8. Stop reaction after the 1 hour incubation by adding to each sample 900 L of H 2 SO 4 / H 3 PO 4 /DI water solution. Also add to each urea standard. 9. To each sample and each urea standard add 40 L of 9% # isonitrosopropiophenone solution. Incubate for 30 minutes at 95 ¡C on heat

PAGE 113

! "+% block. 10. After the 30 minute incubation add 100 L of ea ch sample and each urea standard to 96 well flat bottom plate and measure absorbance at 540 nm with spectrophotometer. Determine urea concentration in each sample by using standard curve generated from absorbance values of serially diluted urea standards. Measure absorbance at 595 nm of protein assay standards and samples and determine protein concentration in each sample by using standard curve generated from absorbance values of serially diluted protein assay standards. Determine concentration of urea in each sample relative to the concentration of total protein in each sample to determine relative activity of arg I. Extraction of total RNA from MDSC ( Adapted from E.Z.N.A. Total RNA Kit I Handbook) 1. Collect cells for each TES condition from 96 well plat e. Centrifuge cells at 5 min at 500g in a 1.5 mL eppendorf microfuge tube and remove supernatant. 2. For each condition add 350 L TRK lysis buffer. Vortex thoroughly to homogenize 3. Add 350 L of 70% ethanol to homogenized lysate and mix well by pipetting up and down 3 5 times. 4. Transfer sample to HiBind RNA column column placed in supplied 2 mL collection tube. Centrifuge for 60 s ec at 10,000g at room temperature. Discard flow through and reuse collection tube for next step. 5. Add 500 L of RNA Wash buffer I by pipetting directly onto column. Centrifuge

PAGE 114

! "+& for 60 seconds at 10,000g at room temperature. Discard flow through and reuse c ollection tube for next step. 6. Add 500 L of RNA Wash buffer II (diluted with absolute ethanol) by pipetting directly onto column. Centrifuge for 60 seconds at 10,000g at room temperature. Discard flow through and reuse collection tube for next step. 7. Add 5 00 L of RNA Wash buffer II by pipetting directly onto column. Centrifuge for 60 seconds at 10,000g at room temperature. Discard flow through and place column in new supplied 2 mL collection tube. 8. Centrifuge column for 2 minutes at maximum speed to comple tely dry the HiBind matrix. 9. Transfer column into clean 1.5 mL Eppendorf centrifuge tube and elute RNA by adding 30 mL of DEPC treated water directly onto the center of the column matrix. Centrifuge at maximum speed for 60 seconds at room temparature. Col lect RNA eluted in water in the 1.5 mL Eppendorf tube. Discard column. 10. Once RNA is eluted keep on ice it is ready to be reverse transcribed into total cDNA. Reverse trans cription of total RNA into cDNA 1. Keep all samples and reagents on ice. For each sa mple, add all of total RNA eluted to a labeled PCR tube. For blank add 30 L of DEPC treated water to labeled PCR tube. 2. For each sample add random primers (4 L), dNTPs (1.6 L), RT buffer (4 L), RNase inhibitor (1 L), and RT enzyme MMLV (1 L) and mix by pipetting. 3. Add samples to thermocycler and run program for reverse transcription of total

PAGE 115

! "+' RNA: 10 min at 25 ¡C, 2 h at 37 ¡C, 5 min at 75 ¡C, and finally hold indefinitely at 4 ¡C. 4. Once program is finished, freeze cDNA samples at 80 ¡C until ready to p erform real time PCR for arg I and iNOS expression. Determination of MDSC arg I and iNOS expression by real time PCR 1. Keep all samples and reagents on ice in dark when not in use. Perform addition of samples and reagents in designated hood in the dark. For each gene add 10.5 L of the corresponding 20X probe and primer mix (Taqman probes for argI (arg I), Nos2 (iNOS), and $ actin (endogenous control)) to a labeled 1.5 mL Eppendorf tube. Add 105 L of 2X master mix (containing AmpliTaq Gold DNA polymerase), and 178.5 L DI water. Mix thoroughly by pipetting. 2. For each gene, add 18 L of the above mix to each well of 96 well white PCR plate. Use two wells for each gene for each condition (duplicate for each gene). For each condition add 2 L of sample to ea ch well. Mix by pipetting. Cover plate in clear plastic film and keep covered in tin foil. 3. Add plate to real time PCR thermocycler and run real time PCR program for Taqman probes: 10 min at 95 ¡C, and 40 cycles of: 15 sec at 95 ¡C, 5 min at 75 ¡C. Analy ze results using Bio Rad qPCR software. Determine Ct values for each sample. Report gene expression as fold change in expression relative to baseline expression (arg I and iNOS expression by MDSC cultured in 0% TES) normalized to the endogenous control e xpression ( $ actin expression) in each sample. Use the formula: fold change in expression = 2 '' Ct

PAGE 116

! "+( B16/Kb OVA Staining Protocol Flow Cytometry 4. C ollect B16/Kb OVA cells and B16F10 cells. Resuspend cells in complete DMEM and count. 5. Aliquot cells in 5 m L flow tubes 100,000 200,000 cells per tube, and wash with PBS. Resuspend in 100 L per tube MACS buffer. 6. Stain B16/Kb OVA cells and B16F10 cells in the dark with APC conjugated anti pMHC CI OVA (SIINFEKL/MHC class I) antibody (2 g/mL). Incubate in the dark for 20 minutes at room temperature. Keep unstained cells, cells stained only with APC conjugated anti pMHC CI OVA antibody (2 g/mL), and cells suspended only in DAPI buffer for compensation controls. 7. Wash cells once with PBS and resuspend in 100 L per tube DAPI buffer, or 100 L per tube MACS buffer for unstained and compensation controls. 8. Analyze cells on LSRII flow cytometer. 9. Analyze pMHC CI OVA expression using FlowJo software. Verify that B16/Kb OVA cells maintain pMHC CI OVA expression throug h multiple passages.

PAGE 117

! "+) References Almand B, Clark JI, Nikitina E, van Beynen J, English NR, Knight SC, Carbone DP, Gabrilovich DI. 2001. Increased Production of Immature Myeloid Cells in Cancer Patients: A Mechanism of Immunosuppression in Cancer. J I mmunol 166(1):678 689. Alvarez DA. Interferon gamma Receptor Deficiency [internet]. [updated 2005]. Davidson (NC): Davidson College Department of Biology; [cited 2012 April 12] Available from http://www.bio.davidson.edu/courses/immunology/Students/spring2006/V_Alvare z/IFNgRdeficiency.html. AndrŽ F, Chaput N, Schartz NE, Fl ament C, Aubert N, Bernard J, Lemonnier F, Raposo G, Escudier B, Hsu DH, Tursz T, Amigorena S, Angevin E, Zitvogel L. 2004. Exosomes as potent cell free peptide based vaccine. I. Dendritic cell derived exosomes transfer functional MHC class I/peptide compl exes to dendritic cells. J Immunol 172(4):2126 2136. Asano K, Nabeyama A, Miyake Y, Qiu CH, Kurita A, Tomura M, Kanagawa O, Fujii S, Tanaka M. 2011. CD169 positive macrophages dominate antitumor immunity by crosspresenting dead cell associated antigens. Im munity 34(1):85 95. Bingisser RM, Tilbrook PA, Holt PG, Kees UR 1998. Macrophage derived nitric oxide Regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway. J Immunol 160(12):5729 5734. BioAssay Systems QuantiChrom Ure a Assay Kit [internet]. [Date unknown]. Hayward (CA): BioAssay Systems; [cited 2012 April 12] Available from

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! "+* http://www.bioassaysys.com/products.php?q=Urea BioRad Protein Assay Kit I [internet]. [Date unknown]. Hercules (CA): BioRad Laboratories, Inc.; [ cited 2012 April 12] Available from http://www.bio rad.com/prd/en/US/LSR/PDP/d4d4169a 12e8 4819 8b3e ccab019c6e13/Bio Rad_Protein_Assay Brito C, Naviliat M, Tiscornia AC, Vuillier F, Gualco G, Dighiero G, Radi R, Cayota AM. 1999. Peroxynitrite inhibits T lymphocyte activation and proliferation by promoting impairment of tyrosine phosphorylation and peroxynitrite driven apoptotic death. J Immunol 162(6):3356 3566. Bronte V, Serafini P, De Santo C, Marigo I, Tosello V, Mazzoni A, Segal DM, Staib C, Lowel M, Sutter G, Colombo MP, Zanovello P. 2003. IL 4 induced arginase 1 suppresses alloreactive T cells in tumor bearing mice. J Immunol 170( 1 ):270 278. Chow A, Brown BD, Merad M. 2011. Studying the mononuclear phagocyte system in the molecular age. Nat Rev Immu nol 11(11):788 798. Clarke SR, Barnden M, Kurts C, Carbone FR, Miller JF, Heath WR. 2000. Characterization of the ovalbumin specific TCR transgenic line OT I: MHC elements for positive and negative selection. Immunol Cell Biol 78(2):110 117. Condamine T, G abrilovich DI. 2011. Molecular mechanisms regulating myeloid derived suppressor cell differentiation and function. Trends Immunol 32(1):19 25. Coqueret O. 2002. Linking cyclins to transcriptional control. Gene 299(1 2):35 55 Corzo CA, Condamine T, Lu L, C otter MJ, Youn JI, Cheng P, Cho HI, Celis E, Quiceno DG, Padhya T, McCaffrey TV, McCaffrey JC, Gabrilovich DI. 2010. HIF 1 regulates function and differentiation of myeloid derived suppressor cells in the

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! ""+ tumor microenvironment. J Exp Med 207(11):2439 2453. Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD 2002. Cancer immunoediting: from immunosu rveillance to tumor escape. Nat Immunol 3(11):991 998 eMICE: Mouse Cancer Models [internet]. [Date unknown]. Bethesda (MD): National Cancer Institute ; [cited 2012 April 12]. Available from http://emice.nci.nih.gov/aam/mouse. Eugenin EA, Gaskill PJ, Bermanab JW. 2009. Tunneling nanot ubes (TNT) are induced by HIV infection of macrophages : A potential mechanism for intercellular HIV trafficking Cell Immunol 254(2): 142 148. Filipazzi P, Valenti R, Huber V, Pilla L, Canese P, Iero M, Castelli C, Mariani L, Parmiani G, Rivoltini L. 2007. Identification of a new subset of myeloid suppressor cells in peripheral blood of melanoma patients with modulation by a granulocyte macrophage colony stimulation factor based antitumor vaccine. J Clin Oncol 25(18):2546 2553. Frese KK, Tuveson DA. 2007. Ma ximizing mouse cancer models. Nat Rev Cancer 7(9):645 658. Fricke I, Mirza N, Dupont J, Lockhart C, Jackson A, Lee JH, Sosman JA, Gabrilovich DI. 2007. Vascular Endothelial Growth Factor Trap Overcomes Defects in Dendritic Cell Differentiation but Does Not Improve Antigen Specific Immune Responses. Clin Cancer Res 13(16):4840 4848. Gabrilovich DI, Velders MP, Sotomayor EM, Kast WM. 2001. Mechanism of Immune Dysfunction in Cancer Mediated by Immature Gr 1 + Myeloid Cells. J Immunol

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! """ 166(9):5398 5406. Gabrilov ich DI & Nagaraj S. 2009. Myeloid derived suppressor cells as regulators o f the immune system. Nature Rev Immun ol 9 (3):162 174 Gabrilovich DI, Ostrand Rosenberg S, Bronte V. 2012. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol 12(4):2 53 268. Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19(1):71 82. Griffiths AJF, Wessler SR, Lewontin RC, Carroll SB 2008. I ntroduction to Genetic Analysis, 9 th ed. New Yor k (NY): W.H. Freeman and Co. Harari O & Liao JK. 2004. Inhibition of MHC II gene transcription by nitric oxide and antioxidants. Curr Pharm Des 10(8):893 898. Harshyne LA, Watkins SC, Gambotto A, Barratt Boyes SM. 2001. Dendritic cells acquire antigens fro m live cells for cross presentation to CTL. J Immunol 166(6):3717 3723. Hoechst B, Ormandy LA, Ballmaier M, Lehner F, KrŸger C, Manns MP, Greten TF, Korangy F. 2008. A New Population of Myeloid Derived Suppressor Cells in Hepatocellular Carcinoma Patients Induces CD4 + CD25 + Foxp3 + T Cells. Gastroenterology 135(1):234 243. Huang B, Pan PY, Li Q, Sato AI, Levy DE, Bromberg J, Divino CM, Chen SH 2006. Gr 1+CD115+ immature myeloid suppressor cells mediate the development of tumor induced T regulatory cells and T cell anergy in tumor bearing host. Cancer Res 66(2):1123 1131.

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! ""# Invitrogen's Flow Cytometry Resource Center [internet]. [Date unknown]. Grand Island (NY): Invitrogen Life Technologies; [cited 2012 April 12] Available from http://www.invitrogen.com/site/u s/en/home/Products and Services/Applications/Cell Analysis/Flow Cytometry/Flow Cytometry Technical Resources.html. Invitrogen's Griess Reagent Kit, for nitrite quantification [internet]. [Date unknown]. Grand Island (NY): Invitrogen Life Technologies; [cit ed 2012 April 12] Available from http://products.invitrogen.com/ivgn/product/G7921. Invitrogen's High Capacity cDNA Reverse Transcription Kit [internet]. [Date unknown]. Grand Island (NY): Invitrogen Life Technologies; [cited 2012 April 12] Available fro m http://products.invitrogen.com/ivgn/product/4368813?ICID==%3Dcvc rt rt pcr c1t1. I nvitrogen's Real Time PCR Learning Area [internet]. [Date unknown]. Grand Island (NY): Invitrogen Life Technologies; [cited 2012 April 12] Available from http://www.applie dbiosystems.com/absite/us/en/home/applications technologies/real time pcr/rtpcr learn.html JAX Mice Database: C57BL/6 Tg( TcraTcrb)1100Mjb/J [internet] [Date unknown]. Bar Harbor (ME): The Jackson Laboratory; [cited 2012 April 12]. Available from http://j axmice.jax.org/strain/003831.html. JAX Mice Database: C57BL/6J [internet]. [Date unknown]. Bar Harbor (ME): The Jackson Laboratory; [cited 2012 April 12]. Available from http://jaxmice.jax.org/strain/000664.html.

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! ""$ JAX Mice Database: B6;129P2 B2m tm1Unc /J [in ternet]. [Date unknown]. Bar Harbor (ME): The Jackson Laboratory; [cited 2012 April 12]. Available from http://jaxmice.jax.org/strain/002070.html Kerkar SP, Goldszmid RS, Muranski P, Chinnasamy D, Yu Z, Reger RN, Leonardi AJ, Morgan RA, Wang E, Marincola FM, Trinchieri G, Rosenberg SA, Restifo NP 2011. IL 12 triggers a programmatic change in dysfunctional myeloid derive d cells within mouse tumors. J Clin Invest 121(12):4746 4757. Knight SC, Iqball S, Roberts MS, Macatonia S, Bedford PA. 1998. Transfer of antigen between dendritic cells in the stimulation of primary T cell proliferation. Eur J Immunol 28(5):1636 1644. Ko JS, Rayman P, Ireland J, Swaidani S, Li G, Bunting KD, Rini B, Finke JH, Cohen PA. 2010. Direct and Differential Suppression of Myeloid De rived Suppressor Cell Subsets by Sunitinib Is Compartmentally Constrained. Cancer Res 70(9):3526 3536. Kusmartsev SA, Li Y, Chen SH. 2000. Gr 1+ Myeloid Cells Derived from Tumor Bearing Mice Inhibit Primary T Cell Activation Induced Through CD3/CD28 Costim ulation. J Immunol 165(2):779 785. Kusmartsev S & Gabrilovich DI. 2003. Inhibition of myeloid cell differentiation in cancer: the role of reactive oxygen species. J Leuk Biol 74(2):186 196. Kusmartsev S, Nefedova Y, Yoder D, Gabrilovich DI 2004. Antigen s pecific inhibition of CD8+ T cell response by im mature myeloid cells in cancer i s mediated by reactive oxygen species. J Immunol 172(2):989 999. Kusmartsev S, Gabrilovich DI. 2005. STAT1 Signaling Regulates Tumor Associated

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! ""% Macrophage Mediated T Cell Delet ion. J Immunol 174(8):4880 4891. Kusmartsev S, Nagaraj S, Gabrilovich DI. 2005. Tumor Associated CD8+ T Cell Tolerance Induced by Bone Marrow Derived Immature Myeloid Cells. J Immunol 175(7):4583 4592. Lee JM, Seo JH, Kim YS, Ko HJ, Kang CY. 2011. The rest oration of myeloid derived suppressor cells as functional antigen presenting cells by NKT cell help and all trans retinoic acid treatment. Int J Cancer [Epub ahead of print] doi: 10.1002/ijc.26411. Lu T, Ramakrishnan R, Altiok S, Youn JI, Cheng P, Celis E Pisarev V, Sherman S, Sporn MB, Gabrilovich D 2011. Tumor infiltrating myeloid cells induce tumor cell resistance to cy totoxic T cells in mice. J Clin Invest 121(10):4015 4029. Mabtech ELISpot technique [internet]. [Date unknown]. Cincinnati (OH): Mabte ch, Inc.; [cited 2012 April 12]. Available from http://www.mabtech.com/main/Page.asp?PageId=16&PageName=About+ELISpo t. Marigo I, Bosio E, Solito S, Mesa C, Fernandez A, Dolcetti L, Ugel S, Sonda N, Bicciato S, Falisi E, Calabrese F, Basso G, Zanovello P, C ozzi E, Mandruzzato S, Bronte V. 2010. Tumor induced tolerance and immune suppression depend on the C/EBPbeta transcription factor. Immunity 32(6):790 802. Marzo L, Gousset K, Zurzolo C. 2012. Multifaceted roles of tunneling nanotubes in intercellular comm unication. Front Physiol 3(72):1 14. Mauti LA, Le Bitoux MA, Baumer K, Stehle JC, Golshayan D, Provero P, Stamenkovic I 2011. Myeloid derived suppressor cells are implicated in regulating

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! ""& permissiveness for tumor meta stasis during mouse gestation. J Clin Invest 121 (7):2794 2807 Mellman I, Coukos G, Dranoff G. 2011. Cancer immu notherapy comes of age. Nature 480(7378):480 489 Millipore MultiScreen HTS Filter Plates for Elispot [internet]. [Date unknown]. Billerica (MA): EMD Millipore; [cited 2012 April 12] Available from http://www.millipore.com/catalogue/module/c9039 Miltenyi Biotec MACS Technology [internet]. [Date unknown]. Auburn (CA): Miltenyi Biotec Inc; [cited 2012 April 12] Available from http://www.miltenyibiotec.com/en/NN_1036_MACS_Technology.as px. Mirza N, Fishman M, Fricke I, Dunn M, Neuger AM, Frost TJ, Lush RM, Antonia S, Gabrilovich DI. 2006. All trans retinoic acid improves differentiation of myeloid cells and immune response in cancer patients. Cancer Res 66(18):9299 9307. Molon B, Ugel S Del Pozzo F, Soldani C, Zilio S, Avella D, De Palma A, Mauri P, Monegal A, Rescigno M, Savino B, Colombo P, Jonjic N, Pecanic S, Lazzarato L, Fruttero R, Gasco A, Bronte V, Viola A. 2011. Chemokine nitration prevents intramural infiltration of antigen sp ecific T cells. J Exp Med 208(10):1949 1962. Movahedi K, Guilliams M, Van den Bossche J, Van den Bergh R, Gysemans C, Beschin A, De Baetselier P, Van Ginderachter JA. 2008. Identification of discrete tumor induced myeloid derived suppressor cell subpopulat ions with distinct T cell suppressive activity. Blood 111(8):4233 4244. Mundy Bosse BL, Lesinski GB, Jaime Ramirez AC, Benninger K, Khan M, Kuppusamy P, Guenterberg K, Kondadasula SV, Chaudhury AR, La Perle KM, Kreiner M,

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! ""' Young G, Guttridge DC, Carson WE 3rd. 2011. Myeloid derived suppressor cell inhibition of the IFN response in tumor bearing mice. Cancer Res 71(15):5101 51 10. Murphy K. 2012. Janeway's Immunobiology, 8 th ed. New York (NY) : Garland Science Publishing Nagaraj S, Gupta K, Pisarev V, Kinarsk y L, Sherman S, Kang L, Herber DL, Schneck J, Gabrilovich DI. 2007. Altered recognition of antigen is a mechanism of CD8+ T cell tolerance in cancer. Nat Med 13(7):828 85. Nagaraj S & Gabrilovich DI. 2008. Tumor escape mechanism governed by myeloid derived suppressor cells. Cancer Res 68(8):2561 2563. Nagaraj S, Youn JI, Weber H, Iclozan C, Lu L, Cotter MJ, Meyer C, Becerra CR, Fishman M, Antonia S, Sporn MB, Liby KT, Rawal B, Lee JH, Gabrilovich DI. 2010. Anti inflammatory triterpenoid blocks immune suppre ssive function of MDSCs and improves immune response in cancer. Clin Cancer Res 16(6):1812 1823. Nagaraj S, Nelson A, Youn JI, Cheng P, Quiceno D, Gabrilovich DI. 2012. Antigen specific CD4+ T cells regulate function of myeloid derived suppressor cells in cancer via retrograde MHC class II signaling. Cancer Res 72(4):928 938. Nausch N, Galani IE, Schlecker E, Cerwenka A. 2008. Mononuclear myeloid derived "suppressor" cells express RAE 1 and activate natural killer cells. Blood 112(10):4080 4089. Obermajer N Muthuswamy R, Lesnock J, Edwards RP, Kalinski P. 2011. Positive feedback between PGE2 and COX2 redirects the differentiation of human

PAGE 126

! ""( dendritic cells toward stable myeloid derived suppressor cells. Blood 118(20):5498 505. Obermajer N, Muthuswamy R, Oduns i K, Edwards RP, Kalinski P. 2011. PGE2 Induced CXCL12 Production and CXCR4 Expression Controls the Accumulation of Human MDSCs in Ovarian Cancer Environment. Cancer Res 71(24):7463 7470. Ochoa AC, Zea AH, Hernandez C, Rodriguez PC. 2007. Arginase, Prostag landins, and Myeloid Derived Suppressor Cells in Renal Cell Carcinoma. Clin Cancer Res 13(2):721s 726s. …nfelt B, Purbhoo MA, Nedvetzki S, Sowinski S, Davis DM. 2005. Long distance calls between cells connected by tunneling nanotubes. Sci STKE 2005(313):pe 55 pe57. Orange DE, Jegathesan M, Blachre NE, Frank MO, Scher HI, Albert ML, Darnell RB. 2004. Effective antigen cross presentation by prostate cancer patients' dendritic cells: implications for prostate cancer immunotherapy. Prostate Cancer Prostatic Dis 7(1):63 72. Ostrand Rosenberg S & Sinha P. 2009. Myeloid Derived Suppressor Cells: Linking Inflammation and Cancer. J Immunol 182(8):4499 4506. Ostrand Rosenberg S, Sinha P, Beury DW, Clements VK. 2012. Cross talk between myeloid derived suppressor cells (MDSC), macrophages, and dendritic cells enhances tumor induced immune suppression. Semin Cancer Biol [Epub ahead of print] http://dx.doi.org/1 0.1016/j.semcancer.2012.01.011. Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello P, Mandruzzato S, Bronte V. 2010. Myeloid derived suppressor cell heterogeneity and subset definition. Curr Opin

PAGE 127

! "") Immunol. 22(2):238 244. Priceman SJ, Sung JL, Shaposhnik Z, Burton JB, Torres Collado AX, Moughon DL, Johnson M, Lusis AJ, Cohen DA, Iruela Arispe ML, Wu L. 2010. Targeti ng distinct tumor infiltrating myeloid cells by inhibiting CSF 1 receptor: combating tumor evasion of antiangiogenic therapy. Blood 115(7):1461 1471. Ribechini E, Greifenberg V, Sandwick S, Lutz MB. 2010. Subsets, expansion and activation of myeloid derive d suppressor cells. Med Microbiol Immunol 199(3):273 281. Rodriguez PC, Zea AH, Culotta KS, Zabaleta J, Ochoa JB, Ochoa AC 2002. Re gulation of T cell receptor CD3 zeta chain expression by L arginine. J Biol Chem 277(24):21123 21129 Rodriguez PC, Hernande z CP, Quiceno D, Dubinett SM, Zabaleta J, Ochoa JB, Gilbert J, Ochoa AC. 2005. Arginase I in myeloid suppressor cells is induced by COX 2 in lung carcinoma. J Exp Med 202(7):931 939. Rodriguez PC, Quiceno DG, Ochoa AC 2007. L arginine availability regulat es T lym phocyte cell cycle progression. Blood 109(4): 1568 1573. Rodriguez PC, Ernstoff MS, Hernandez C, Atkins M, Zabaleta J, Sierra R, Ochoa AC. 2009. Arginase I Producing Myeloid Derived Suppressor Cells in Renal Cell Carcinoma Are a Subpopulation of Activated Granulocytes. Cancer Res 69(4):1553 1560. Saccheri F, Pozzi C, Avogadri F, Barozzi S, Faretta M, Fusi P, Rescigno M. 2010. Bacteria induced gap junctions i n tumors favor antigen cross presentation and

PAGE 128

! ""* antitumor immunity. Sci Transl Med 2(44):44ra57. Schroder K, Hertzog PJ, Ravasi T, Hume DA 2004. Interferon g amma : an overview of signals, mec hanisms, and functions. J Leuk Biol 75(2):163 189. SchŸler T, Blank enstein T. 2003. Cutting Edge: CD8+ Effector T Cells Reject Tumors by Direct Antigen Recognition but Indirect Action on Host Cells. J Immunol 170(9):4427 4431. Scotton CJ, Wilson JL, Scott K, Stamp G, Wilbanks GD, Fricker S, Bridger G, Balkwill FR. 2002. M ultiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res 62(20):5930 5938. Serafini P, Meckel K, Kelso M, Noonan K, Califano J, Koch W, Dolcetti L, Bronte V, Borrello I. 2006. Phosphodiesterase 5 inhibition augm ents endogenous antitumor immunity by reducing myeloid derived suppressor cell function. J Exp Med 203(12):2691 2702. Serafini P, Mgebroff S, Noonan K, Borrello I. 2008. Myeloid derived suppressor cells promote cross tolerance in B cell lymphoma by expandi ng regulatory T cells. Cancer Res 68(13):5439 5449. Sinha P, Clements VK, Ostrand Rosenberg S. 2005. Reduction of Myeloid Derived Suppressor Cells and Induction of M1 Macrophages Facilitate the Rejection of Established Metastatic Disease. J Immunol 174(2): 636 645. Sinha P, Clements VK, Fulton AM, Ostrand Rosenberg S. 2007. Prostaglandin E2 Promotes Tumor Progression by Inducing Myeloid Derived Suppressor Cells. Cancer Res 67(9):4507 4513.

PAGE 129

! "#+ Solito S, Bronte V, Mandruzzato S. 2011. Antigen specificity of immun e suppression by myeloid derived suppressor cells. J Leuk Biol 90(1):31 36. Sompayrac, L. 2003. How the Immune System Works, 2 nd ed. Malden (MA): Blackwell Publishing. Srivastava MK, Bosch JJ, Thompson JA, Ksander BR, Edelman MJ, Ostrand Rosenberg S. 2008. Lung cancer patients' CD4(+) T cells are activated in vitro by MHC II cell based vaccines despite the presence of myeloid derived suppressor cells. Cancer Immunol Immunother 57(10):1493 1504. Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM 2005. Gem citabine selectively eliminates splenic Gr 1 + /CD11b+ myeloid suppressor cells in tumor bearing animals and enhances antitumor immune activity. Clin Cancer Res 11(18):6713 6721 Talmadge JE. 2007. Pathways mediating the expansion and immunosuppressive acti vity of myeloid derived suppressor cells and thei r relevance to cancer therapy. Clin Cancer Res 13(18):5243 5248. Umemura N, Saio M, Suwa T, Kitoh Y, Bai J, Nonaka K, Ouyang GF, Okada M, Balazs M, Adany R, Shibata T, Takami T. 2008. Tumor infiltrating myel oid derived suppressor cells are pleiotropic inflamed monocytes/macrophages that bear M1 and M2 type characteristics. J Leuk Biol 83(5):1136 44. Vuk Pavlovi S, Bulur PA, Lin Y, Qin R, Szumlanski CL, Zhao X, Dietz AB. 2010. Immunosuppressive CD14+HLA DRlo w/ monocytes in prostate cancer. Prostate 70(4):443 455. Watkins SC, Salter RD. 2005. Functional connectivity between immune cells mediated

PAGE 130

! "#" by tunneling nanotubules. Immunity 23(3):309 318. Watanabe S, Deguchi K, Zheng R, Tamai H, Wang LX, Cohen PA, Shu S 2008. Tumor Induced CD11b+ Gr 1+ Myeloid Cells Suppress T Cell Sensitization in Tumor Draining Lymph Nodes. J Immunol 181(5):3291 3300. Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. 2008. Subsets of myeloid derived suppressor cells in tumor bearing mic e. J Immunol 181(8):5791 5802. Youn JI & Gabrilovich DI. 2010. The biology of myeloid derived suppressor cells: the blessing and the curse of morphological and functional heterogeneity. Eur J Immunol 40(11): 2969 2975. Zea AH, Rodriguez PC, Atkins MB, Hern andez C, Signoretti S, Zabaleta J, McDermott D, Quiceno D, Youmans A, O'Neill A, Mier J, Ochoa AC. 2005. Arginase producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion. Cancer Res 65(8):3044 3048. Zhang H, Nguyen Jackson H, Panopoulos AD, Li HS, Murray PJ, Watowich SS. 2010. STAT3 controls myeloid progenitor growth during emergency granulopoiesis. Blood 116(14):2462 2471.


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