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Dendritic Cells and Cytokine Immune Responses to Hematopoietic Stem Cell Growth Factors

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Dendritic Cells and Cytokine Immune Responses to Hematopoietic Stem Cell Growth Factors
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EKSIOGLU, ERIKA ADRIANA ( Author, Primary )
Copyright Date:
2008

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Antigens ( jstor )
Blood ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Cytokines ( jstor )
Dendritic cells ( jstor )
Immunology ( jstor )
Leukocytes ( jstor )
Tissue grafting ( jstor )
Tumors ( jstor )

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University of Florida
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University of Florida
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Copyright Erika Adriana Eksioglu. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2007

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DENDRITIC CELLS AND CYTOKI NE IMMUNE RESPONSES TO HEMATOPOIETIC STEM C ELL GROWTH FACTORS By ERIKA ADRIANA EKSIOGLU A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Erika Adriana Eksioglu

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This document is dedicated to my fam ily who are the pilla rs of my career.

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iv ACKNOWLEDGMENTS I would like to thank all th e people that made it possible for me to work on this project. First, I remain deeply indebted to my mentor Dr. Vijay Reddy for introducing me to the field of immunology of dendritic cell s. His invaluable guidance and continual support towards the completion of this project he lped me attain this degree. I would also like to thank my committee members: Dr. Sa lly Litherland and Dr. Henry Baker. Dr. Sally Litherland has not only been part of my committee but has offered advice and answered many questions. Dr. Henry Baker ha s made time out of his busy schedule to offer valuable input. From the master in biotechnology program I would like to thank Dr. Donna Duckworth and specially Joyce Conners who made sure we met all the school deadlines and keep the program running. I would also like to than k all the other people on the Dr.Reddy’s lab (Kayode Garraway, Sheridan Watkins, Andrew Winer, Shahid Mahmood, Jeff Levine, Megan Crosmer, Matt Buzzeo and Kori Watts) that in one way or another have helped me carry through this project. Their help was invaluable and their presence made work a pleasant place. I thank all the people of the stem cell la boratory at Shands for giving me technical advice on many occasions and for their help in maintain the lab. I thank the division of hematology/oncology personnel who made sure th at all the non-technical part of my work got done properly as well as being good friends. I thank Jose Iturraspe from hematopathology associates for continued advi ce. Special thanks go to Eugenia Martinez

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v not only for proofreading many of my documents and helping me in many ways but for extending her friendship to me during this time. Last, but not least I would lik e to thank my family: my parents Andres and Mariela Varela who taught me the importance of fighti ng for your goals and who helped me in so many ways: my siblings Irene, Andres and Sa ra who listened to my venting whenever things did not go as planned; and last, but not least, my husband Oguz Eksioglu who made many sacrifices so that I could improve my career and th at has been there for me in tough moments to help me achieve those goals and my son Osman Aydan who endured my comings and goings and th at has brought me so much joy. I love them all.

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vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv TABLE.......................................................................................................................... ...viii LIST OF FIGURES...........................................................................................................ix ABSTRACT....................................................................................................................... xi CHAPTER 1 GENERAL BACKGROUND......................................................................................1 1.1 Hematopoiesis.........................................................................................................1 1.2 Dendritic Cells........................................................................................................2 1.2.1 Definition of DCs.........................................................................................2 1.2.2 Maturation Stages.........................................................................................4 1.2.3 Generation of DCs for Research...................................................................6 1.2.4 Dendritic Cell Subpopulations.....................................................................8 1.3 Type 1 vs. Type 2 Immune Response...................................................................12 1.4 Cytokines..............................................................................................................16 1.4.1 Importance of The Study of Cytokines......................................................16 1.4.2 Interleukin-12.............................................................................................18 1.4.3 Granulocyte Macrophage-C olony Stimulating Factor...............................19 1.4.4 Granulocyte-Colony Stimulating Factor....................................................19 1.5 Bone Marrow and Stem Cell Transplantation......................................................20 1.5.1 Acute Graft vs Host Disease (aGVHD)......................................................21 1.5.1.1 Phase 1: Host Conditioning..............................................................22 1.5.1.2 Phase 2: Donor T Cell Activation....................................................23 1.5.1.3 PHASE 3: Inflammatory Effector Mechanisms...............................23 1.5.2 Chronic Graft vs Host Disease (cGVHD)..................................................23 2 OBJECTIVES AND SPECIFIC BACKGROUND TO PROJECT............................25 3 MATERIALS AND METHODS...............................................................................35 3.1 Culture, Medium and Cytokines...........................................................................35 3.2 Isolation and Preparation of PBMC......................................................................36 3.3 Isolation of T Cells and Monocytes from PBMC.................................................36

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vii 3.4 Enzyme-linked Immunosorbent Assay (ELISA)..................................................37 3.5 Proliferation Assay...............................................................................................37 3.6 Flow Cytometry....................................................................................................37 4 RESULTS AND DISCUSSION.................................................................................39 4.1 Effects of GM-CSF and G-CSF on The Cytokine Milieu of Ficoll Separated White Blood Cells..................................................................................................39 4.2 Effects of GM-CSF and G-CSF on the Cytokine Milieu of Ficoll Separated White Blood Cells, T Cells and T ce ll Depleted White Blood Cells.....................46 4.3 Generation and Change of the DC1 vs DC2 Ratio as Measured by Flow Cytometry..............................................................................................................54 4.4 Effects of GM-CSF and G-CSF on the Pr oliferation of T Cells Stimulated by Cultured Monocytes with G-CSF, GM-CSF or both.............................................60 5 CONCLUSIONS AND FU TURE PROSPECTS.......................................................65 LIST OF REFERENCES...................................................................................................67 BIOGRAPHICAL SKETCH.............................................................................................78

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viii TABLE Table page 4-1. Flow Cytometry Statistics of Three Separate Analyses.............................................57

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ix LIST OF FIGURES Figure page 1-1. Morphology of Dendritic Cells.....................................................................................3 1-2. Invitro Pathways of Differen tiation of Culture Dendritic Cells...................................7 1-3. Pathways of Dendritic Cell Development..................................................................11 2-1. Effects of G-CSF on T Cells.......................................................................................27 2-2. Type 1 Cytokine Profile Af ter Mixed Lymphocyte Reaction....................................28 2-3.Protecting Effects of GM-CSF in Cancer....................................................................30 2-4.GM-CSF/G-CSF Effects in Stem Cell Transplantation..............................................31 2-5.Effects of G-CSF/GM-CSF Treatment on Stem Cells, T Cells and DCs....................32 2-6.Correlation of High DC Counts Versus Survival........................................................33 2-7.Correlation of High IL-12 Versus Relapse-Free Survival...........................................33 4-1.Results of IL-12 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation...............................................................................................................41 4-2. Results of IL-10 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation...............................................................................................................42 4-3. Results of IFN Cytokine ELISA of Leukocyte Cu ltures at Different Times of Stimulation...............................................................................................................43 4-4. Results of TNF Cytokine ELISA of Leukocyte Cu ltures at Different Times of Stimulation...............................................................................................................44 4-5. Results of IL-4 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation...............................................................................................................45 4-6. Results of IL-12 Cytokine ELISA on the White Blood Cell Population With or Without T Cells........................................................................................................48

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x 4-7. Results of IL-10 Cytokine ELISA on the White Blood Cell Population With or Without T Cells........................................................................................................49 4-8. Results of IFN Cytokine ELISA on the White Blood Cell Population With or Without T Cells........................................................................................................50 4-9. Results of TNF Cytokine ELISA on the White Blood Cell Population With or Without T Cells........................................................................................................51 4-10. Results of IL-4 Cytokine ELISA on the White Blood Cell Population With or Without T Cells........................................................................................................52 4-11. Flow Cytometry Analysis of Cu ltured Monocytes Staining for DC........................56 4-12. Graphical Representation of Percentage of HLA-DR+ Lineage Cells From the Total Cells Assayed by Flow Cytometry.................................................................58 4-13. Graphical Representation of Percentage of HLA-DR+ Lineage Cells that are CD11c+ from the Total Cells Assayed by Flow Cytometry.....................................58 4-14. Graphical Representation of Percentage of HLA-DR+ Lineage Cells that are CD11c+ from the Total Cells Assayed by Flow Cytometry....................................59 4-15. Graphical Representation of Percentage of HLA-DR+ Lineage Cells that are CD11c+ from the Total Cells Assayed by Flow Cytometry....................................59 4-16. Allogeneic M ixed Lymphocyte React ions on Monocytes (experiment 1)...............62 4-17. Allogeneic M ixed Lymphocyte React ions on Monocytes (experiment 2)...............63 4-18. Allogeneic M ixed Lymphocyte React ions on Monocytes (experiment 3)...............64

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science DENDRITIC CELLS AND CYTOKI NE IMMUNE RESPONSES TO HEMATOPOIETIC CELL GROWTH FACTORS By Erika Adriana Eksioglu May 2006 Chair: Vijay Reddy Major Department: Molecular Genetics and Microbiology Dendritic cells (DCs) are key cellula r components of innate and adaptive immunity. Given their crucial role in controlling immunity, th e therapeutic role of DCs has been proposed for many diseases that involve immune activation. They can modulate the adaptive immune response between two sepa rate fates by changing the behavior of T cells: TH1 versus TH2. TH1/TH2 cells can be differentiated based on their cytokine expression: the first produces high levels of IL-12, TNF and IFN while the latter produces IL-10, IL-4 and IL-2. Previous studies have shown that monocytes can differentiate into immunostimulatory DCs when cultured with granulocyte-macrophagecolony stimulating factor (GM-C SF) and interleukin-4 (IL-4). Separate studies show that granulocyte-colony stimulating factor (G-C SF) mobilization as used in stem cell transplantation alters DC cytokine pr oduction and tilts the balance towards a TH2 response. Given the central role of DC as immune stimulators, GM-CSF may be utilized to increase DC numbers and to promote anti-t umor effects after hematopoietic stem cell

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xii transplantation. Our study examines how gr owth factors such as GM-CSF can affect cytokine expression. We hypothesize that GM-CSF will enhance immune stimulatory cytokine production by DCs and sw ay the balance towards a DC1, TH1 response. Also, administration of GM-CSF in transplants has been shown to increase the numbers of DCs in general following autologous stem cell grafts . We have shown that higher levels of DCs after allogeneic stem cell transplant correlate with longer survival of the recipients. We measured the levels of five cytokines IL-12, TNF , IFN , IL-10 and IL-4 in human PBMCs cultured with GM-CSF to test our hypothesis that GM-CSF induces a type 1 versus a type 2 cytokine profile. We then tested whether this profile seen on PBMCs was the product of T cells (TH1 or TH2) or if they were coming from the T cell depleted population. To further test our hypothesis we separated adherent monocytes from PBMCs. For this we assayed for the i nduction of an increase of DC1, DC2 or both from the monocyte population as measured by flow cytometry and if those cells were able to induce an increased al logeneic T cell prolif erative response. Our results show that there is an induction of TH1 cytokines, especially IL-12 af ter the addition of GM-CSF to the culture and that this induction comes fr om both T cell and non T cell populations. We did not observe a change in proliferati on and the DC counts as measured by flow cytometry. Flow cytometric analysis suggest s that GM-CSF does not induce an increase in the DC populations and it does not induce a ch ange in the ratio of DC1 to DC2 cells in a six day monocyte culture. Our results suggest that instead of DC s shifting the balance towards a type 1 response when stimulated with GM-CSF, as or iginally suggested, it is the T cells that have the strongest cytokine re sponse to this growth factor.

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1 CHAPTER 1 GENERAL BACKGROUND Research on the immune system continuous ly reveals new roads to novel therapies for many diseases including infections, alle rgies, cancer, and graft rejection. Among the many cells involved in this intricate sy stem are dendritic cells (DCs) whose immunobiology has already proven to be central to the function of the immune system. Understanding how DC behaves in the body may help us manipulate the immune system to possibly not only cure but prev ent a vast array of diseases. Because of their central role in the ad aptive immune response DCs have become a favorite target for research in many clinical diseases in cluding allergy, transplantation, autoimmune disease, resistance to infection, resistance to tumors and immunodeficiency. 1.1 Hematopoiesis Hematopoiesis (Greek for blood-forming) is a complex scheme of multi-lineage proliferation and differentiation th at gives rise to all the cells found in blood. It is a highly regulated system in which a large number of hematopoietic growth factors control both the production and functional activ ity of blood cells. The earliest of these growth factors to be discovered were the colony stimulating factors (CSF). These factors have been well characterized because of their obvious effects on mature cell production and/or activation. Although hematopoiesis occurs main ly in the bone marrow of adult mammals, totipotent stem cells first arise in the yol k sac during embryonic development, are later found in fetal liver, and at birth are found in high concentrations in cord blood. In adults, stem cells are found in peripheral blood at very low concentrations. The concentration

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2 increases dramatically after stem cell mob ilization into peripheral blood, a phenomenon which occurs in response to chemothe rapy or growth factor administration in vivo (for instance administra tion of G-CSF). 1.2 Dendritic Cells Our understanding of dendritic cells has boom ed in the past ten years. We are barely beginning to grasp how they cont rol the immune system and their importance during an immune response. In a short time all this information has already led to DC based cancer immunotherapy protoc ols (Ardavin et al. 2004). 1.2.1 Definition of DCs DCs are a sparsely distributed, mi gratory group of bone-marrow-derived leukocytes that are specialized for the uptake, transport, processing and presentation of antigens to T cells. At an “immature” stage of development, DCs act as sentinels in peripheral tissues, continuously sampling the antigenic environment. Any encounter with microbial products or tissue damage initiate s the migration of the DCs to lymph nodes. Mature DCs have a distinct morphology char acterized by the presence of numerous membrane processes that can extend for up to hundreds of micrometers. These processes can take the form of dendrites, pseudopods, or veils. Additional mo rphologic features of DC include high concentrations of intracellular structures related to antigen processing such as endosomes, lysosomes, and the Birbeck granules of Langerhans cells (LC) of the epidermis. DCs are highly specialized antigen presen ting cells (APCs) of the immune system. Sensitizing the immune system to specific an tigens is certainly the most pertinent function for DCs. Immature DC, located at site s of antigen entry such as the gut mucosa, is specialized for antigen capture but lack th e ability to activate T cells (Nestle et al.

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3 2001). As they mature, DCs migrate to periphe ral lymphoid organs where they lose the ability to capture antigen but now acquire MHC at the su rface and thus acquire the capacity to activate nave T cells carrying re ceptors for that antigen. DCs pick up and process antigen with high efficiency in pe ripheral tissues and c ontinuously migrate to lymphoid organs to present these antigens to specific T cells. T cells and dendritic cells (DCs) must interact to initi ate immune responses against invading pathogens (Hilkens et al. 1997, Fong et al. 2000, Banchereau et al 1998). They regulate both immunity and tolerance while other cells like B cells, T cells and NK cells are the effectors. All of these characteristics and more make these cells unique since they have the essential features for the initiation of immunity. Figure 1-1. Morphology of Dendritic Cells: Dendri tic cells purified from peripheral blood demonstrate characteristic dendrite s after several days of culture in vitro (Fong et al. 2000). One of the characteristics of DCs is a cons titutive high expression of co-stimulatory molecules such as CD80 (B7.1) and CD86 (B7.2), and molecules regulating costimulation such as CD40 are also expr essed on mature myeloid DCs. Also the expression of MHC class II gets upregulated and there is absence of lineage markers, including, CD14 (monocyte), CD3 (T cell), CD19, CD20, CD24 (B cell), CD56 (natural

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4 killer cell), and CD66b (granulocyte). Not surprisingly, in light of their antigen presenting functions, DC also express variou s adhesion and co-stimulatory molecules like CD11a (LFA-1), CD11c, CD50 (ICAM-2), CD54 (ICAM-1), CD58 (LFA-3), and CD102 (ICAM-3). However, all of these mark ers can also be found on monocytes and macrophages, but vary with differen tiation stage and activation status. 1.2.2 Maturation Stages After antigen capture in the presence of maturation signals associated with inflammation or infection, immature DCs ar e activated by toll-like receptors (TLRs), interferons (IFNs), or members of TNF-R family and undergo a complex maturation process. In vivo this process is paralleled by migra tion of DCs to T-cell rich areas of lymphoid organs, where they pr esent antigen-derived peptides to antigen-specific T cells and direct their differentiation into T effector or memory ce lls. They can also induce NK cell activation and B cell differentiation into antibody forming cells. In contrast, antigen capture in the absence of co-stimulation can lead to tolerance (Ardavin et al. 2004). DC progenitors in the bone marrow become highl y phagocytic DC precu rsors (immature DC) in peripheral tissues but when they mature present antigen and stop phagocytosing. Once mature they are able to induce clonal expa nsion and are endowed with receptors to recognize antigens: lectins, Fc receptors, toll-like receptors. They also change their chemokine receptor CCR6 to MIP3a which ma kes it home to lymph node after antigen encounter. Human DC precursor s circulating in the blood initially can express CD2, CD4, CD13, CD16, CD32, and CD33, but they gr adually lose their expression of these antigens with maturation. In contrast, adhesi on molecules, co-stimulatory molecules, and MHC antigens increase with maturation. CD80 and 86 are upregulated with activation,

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5 particularly with CD40 ligation. CD86 tends to appear earlier in maturation, while CD80, which is almost unmeasurable in blood precursor s, appears later. Freshly isolated DCs are active at pinocytosis and possess nonspecific antigen uptake receptors though at lower levels. Some express FcR (CD16, CD32) and complement receptors (CD11b, CD11c, CD35). CD11c may also act as a receptor for L PS as DC lack the classical LPS receptor, CD14, and yet respond to this stimulus. A seri es of microbial produc ts (for example CPG motifs in bacterial DNA, double-stranded viral RNA, lipopolisacharide (LPS) and necrotic cell products) activate DCs. In a ddition, DCs distinguish between tissue cells that die by the normal process of apoptosis and those that die by externally generated necrosis. The receptors that recognize thes e diverse stimuli vary from lectin-domain scavenger receptors that are similar to those on phagocytes to the TLRs, which are related to proteins from the defense systems of pl ants and insects. (Koller et al. 2002). The stimulatory milieu produced by activated DC, co mbined with the presentation of epitopes in MHC class I and class II and the expressi on of co-stimulatory molecules, contributes to the generation of potent anti gen-specific immune responses. Another characterisitic of DCs is their high stimulatory capacity. They are potent activators of nave T cells ( 10-100 times more potent at activating nave and memory T cells than other professional antigen presenting cells) and therefore important initiators of primary specific immune responses. Research groups regularly rely on the ability of DC to induce proliferation in an allogeneic mixed leukocyte reaction (MLR). Ussing this assay system, they can stimulate autologous T cells by presenting either self or exogenous antigens.

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6 1.2.3 Generation of DCs for Research There are relatively few studies on mature human DCs freshly isolated from tissue. Blood is the only readily available sour ce. Although some human blood DCs are sufficiently mature to give a proliferative response in mixe d leukocyte cultures, blood is more a source of immature DCs and progeni tor DCs. Obtaining mature DCs has proved to be a challenge not only because of the lack of markers but also because of the scarcity of this type of cells in the blood since they represent around 1% of peripheral blood mononuclear cells (PBMC). Human DC can be en riched as circulating precursors from the blood by density-based pur ification techniques. For in stance, after a period of in vitro culture and maturation, DC precursors become la rger and less dense. Gradient solutions lacking potentially immunogeni c proteins such as BSA have been employed for this separation. These different fluids are osmoti cally active to varying degrees and have additional stimulatory properties as well. As a result of their scarceness, leuka pheresis must be performed to generate sufficient numbers of DC (on average 5 x 106) for things like therapeutic vaccination in humans. Because of this problem researchers have had to depend on culture methods. Three different precursor-cell starting points have been used to generate human DCs in vitro . One approach for generating human DC -like cells from bone marrow precursors utilizes CD34+ cells (the earliest precursors) cult ured in the presence of exogenous GMCSF, usually in combination with IL-4 and/or TNF . Later these cells are selected for CD1a or CD14 and restimulated with GM-CSF. Sources for human CD34+ precursors include bone marrow, cord blood, and G-CSF-mobilized peripheral blood. As well as containing haematopoietic stem cells and proge nitor cells, this fraction contains some

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7 committed DC precursors capable of forming pure DC colonies in semisolid media. Also, stem cell factor (SCF) and/or Flt3-ligand (FL) are often adde d to increase DC yields by inducing the proliferation of DC progenitors. An alternativ e approach is to expand DC in vivo (Pulendran et al. 2001, Koller et al. 2002, Koller et al. 2002). Figure 1-2. Invitro Pathways of Differentiation of Culture Dendritic Cells (Santini et al. 2003). Recent studies suggest that blood monocytes are an immune reservoir of cells with dual potential that can be recruited to the tissues and differentiate into macrophages or DC depending on the tissue microenvironmen t. Enriched peripheral blood monocytes cultured in vitro with media supplemented with GM -CSF and IL-4 for 1 to 2 weeks differentiate into cells with immatu re DC morphology (monocytes are CD14+ and CD11c+; their precursor in blood is about 20% of blood cells). The monocytes give rise to large numbers of cells that are morphol ogically and phenotypical ly similar to the ‘‘classical’’ density purified DC. These m onocyte-derived dendri tic cells (MDCs) express MHC class II as well as low levels of co-stimulatory molecules CD80 and CD86.

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8 They also express CD1a (a tissue marker), lack CD14 (a monocyte/macrophage surface receptor) and are highly efficient in antigen capture but are poor stimulators of T cells. These cytokine-generated DC require additional maturation in vitro in order to fully stimulate in an allogeneic mixed lymphocyte reaction (MLR) or prime antigen-specific T cell responses in vitro and in vivo . After the addition of a ma turation stimulus such as TNF , lipopolisacharide (LPS) or soluble tr imeric CD40L (sCD40L) MDCs upregulate MHC II, CD80, CD86, and induce expression of CD83 (a DC specific cell surface marker). Mature DC also decrease mechan isms of antigen capture and become highly immunostimulatory just like an in vivo matured DC. Without this additional maturation step, the DC phenotype can revert to that of a monocyte (Fong et al. 2000, Koller et al. 2002). 1.2.4 Dendritic Cell Subpopulations Although there is general agreement that DC s are derived from hematopoietic stem cells, studies indicate that they can arise fr om at least two distin ct lineages. The several and often opposing roles now ascribed to them cannot all be carried out at once by the same cell at the same time, so it is theorized that there should be different sets of DCs that perform different functions. Such speci alized subtypes might represent different activation states of a single lineage, the f unctional differences depending entirely on local environmental signals (the functional plastici ty model). Alternatively, the specialized DC subtypes could be the products of entirely se parate developmental lineages. The signals that determine lineage segregation would then act earlier and the im mediate precursors of the DCs would already be separate and func tionally committed (the specialized lineage model). The reality is a confusing mixture of these two models, and a large degree of

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9 functional plasticity seems to be a gene ral feature of both DC s and progenitor DCs (Koller et al., 2002). The precise relationship between these lin eages remains controversial, partly because no specific markers for DC precursor s in humans have yet been identified. However, the heterogeneity of DC lineage a nd the possibility that the resulting DC subpopulations differ in their functions have significant implicati ons when considering DC-based strategies. What is known about th e human blood DC markers is that there is heterogeneity in their marker expression, but many of these reflect differences in the maturation or activation states of DCs rather than separate sublineages. In a few cases, human DCs have been isolated from lymphoi d tissues without any incubation steps to promote differentiation, and the mature DC s analyzed. Most human thymic DCs are CD11c+CD11b-CD45ROlo and lack myeloid markers. A minority of human thymic DCs is CD11chi CD11b+ CD45ROhi and expresses many myeloid ma rkers (Nestle et al., 2001; Koller et al., 2002). DCs in blood are generated from either myeloid or lymphoid bone marrow progenitors through intermediate DC precursor s (iDCs) that home to sites of potential antigen entry where they differentiate locally into mature DCs. Two subsets of DCs have been phenotypically described, a myeloid deri ved DC that captur es antigen in the periphery and migrated to the draining lym ph node and a lymphoid DC that resides in the lymph node. The CD11c+ resident cells in vivo (intrathymic) in the human postnatal thymus are truly myeloid DCs and have upregulated GM-CSF receptor expression. Also CD16+ cells were more effective than CD16monocytes in reverse transmigration and differentiation into DCs. CD16+ also have higher expression of co-stimulatory molecules

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10 than other monocytes. Therefor e it is suggested that CD16+ human monocytes readily develop into DCs via CCR-8 mediated signals. (Ardavin et al. 2004). In humans, the more classical myeloid DCs are derived either from a committed DC precursor or from a granulocyte/monocyte precursor. Conver sely, data derived mostly from in vivo DC reconstitution assays in th e mouse shows that the same DC subpopulations (including conventional DCs and progenitor DCs) can be generated from either myeloid or lymphoid progenitors. Myeloid DCs can also be derived from several cell types previously thought to be terminally committe d. For example, monocytes and granulocyte precursors can differentiate into DC when exposed, in vitro , to appropriate combinations of cytokines including GM-CSF or TNF with or without IL-4. Shortman and colleagues also described a population of DC derived fr om lymphoid progenitors in mice (Koller et al 2002). This other cell type appears to arise from CD4+CD8+ lymphoid precursors and can be induced to differentiate, in vitro , without GM-CSF. Knockout mouse models have also implicated a lymphoid DC lineage. In hum ans there are reports of a distinct human DC subpopulation, which would be the lymphoi d DC just described in mice, which express high levels of CD123 (IL-3 recepto r) and CD4 and lack the CD11c myeloid DC marker. Identified in blood and tonsils, these CD123+ DC precursors require IL-3 for survival and an activation signal, such as CD40L, for maturation (Nestle et al. 2001; Pulendran et al. 2001). They appear to bias CD4+ T cell priming to a TH2 response, in contrast to myeloid CD11c+ DC, which preferentially induce a TH1 response. This CD123hi DC also appear to be a major source of type I interferon; and may therefore, possess effector imm une function as well. All of these experiments do not support the existence of independent myeloid and ly mphoid DC subpopulations as previously

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11 proposed but instead point to a DC differen tiation model relying on contributions from both myeloid and lymphoid differentiation path ways. There is a possi bility that all DC subsets might derive from a single DC comm on precursor (Koller et al. 2002, Ardavin et al. 2004). A lymphomyeloid precu rsor population is thought to be seeding the postnatal thymus in humans (Ardavin et al. 2004). Most of the insights into human DC subsets and their development origins have come not from direct isolation of the mature DCs from tissues but from indirect studies of their development in culture from iDCs or pDCs. These studies have led to the concept of distinct pathways of human DC development, although the correspondence between the DCs generated in culture and naturally occurring DCs subtypes in human or mous e is not clear (Koller et al., 2002). Figure 1-3. Pathways of Dendritic Cell Develo pment: Representation of the interactions of cytokines in the ontogeny and function of type1 (CD11c positive) dendritic cells (pDC1) and type 2 (CD123 positive) DCs (Waller et al., 2003) Blood monocytes, termed precursor DC1, ar e the most commonly used precursor cells for generating human DCs in culture. In the presence of M-CSF they will generate macrophages but, in the prescence of GM-CSF and IL-4, DCs termed DC1 are produced after six days and little or no proliferativ e expansion is involved. Final maturation to

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12 CD14-CD38+CD86+ surface MHC IIhi DCs is achieved by stimulating with proinflamatory cytokines such as TNF or microbial products such as LPS. Helper CD4+ T cells, signaling by means of CD154, can also mature and activate these DCs (Pulendran et al. 2001, Koller et al., 2002). The precurs or for the final pathway of human DC development in culture, term ed pDC2, is the interferon producing plasmacytoid cell. They have a plasma-cell-like morp hology and a unique surface phenotype (CD4+CD123+CD11cand negative for most lineage mark ers). It has been suggested that at least some of these plasmacytoid cells are of lymphoid origin: they express many lymphoid markers, lack surface myeloid ma rkers and produce mRNA for germ-line IgK and for pre-T-cell receptor . The plasmacytoid cells respond to viral and microbial stimuli by producing class I interferons. They express intracellular but not surface MHC II, similar to iDCs. When cu ltured with IL-3 and CD154, or with microbial stimuli such as bacterial CpG or human herpes simplex virus, they become mature DCs without apparent proliferative expans ion. The progeny DCs, termed DC2, express low levels of CD11c and lack typical myeloid markers, like their precursors, but otherwise display the characteristics of mature DCs. (Pul endran et al. 2001, Koller et al., 2002) 1.3 Type 1 vs. Type 2 Immune Response A critical aspect of the immune defense ag ainst infection by pat hogens is mediated by helper function of newly activated CD4+ T helper cells. The experimental outcome of a DC-T cell interaction is often simply T cel l proliferation. Although triggering of T cells into cell cycle progression is a central function of DCs, it is now clear that DCs can also influence, and perhaps dictate, the subseque nt development of these dividing T cells. T cell activation and proliferation might lead to immunity or tolerance, to the

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13 generation/activation of effector T cells, and to T cells that secrete different patterns of cytokines, including the extreme cy tokine-polarized T helper 1 (TH1) and T helper 2 (TH2) responses (Koller et al. 2002). TH1 and TH2 cells differ in the cytokines they secrete and the type of respons e they elicit in ta rget cells expressi ng cytokine-specific receptors. The activation of the appropriate T cell subset is critical for providing protective immunity against a variety of pathogens: TH1 immunity protects against intracellular parasites such as Leishmania via macrophage activation and secrete the cytokines IL-2, IFN and TNF In contrast, TH2 immunity protects against extra-cellular pathogens, such as helminthes, and to persiste nt antigens, such as allergens, by effecting humoral immunity and secrete the cytokines IL -4, IL5, IL-10, and IL13. These same TH2 cytokines can also contribute to tumor rejection by boosting eosinophil function and increasing antibody concentrations (Dranoff 2004). One theory to explain the selectivity of T cell responses postulates that cytokine s secreted by neighboring cells drive resting nave T cells down a particular differentiati on pathway. However, Rissoan and colleagues challenged aspects of this model by sugges ting that DCs not only provide a common set of signals to initiate clonal expansion of T cells but also provide T cells with selective signals leading to either TH1 or TH2 immunity (Bottomly 1999, Rissoan et al. 1999). Two general mechanisms have been pr oposed by which DCs might maintain peripheral tolerance. The first is that a subtype of specializ ed regulatory DCs is involved; there is some evidence for such DCs, but no consensus. The second is that all DCs have a capacity for initiating tolerance or immun ity depending on the maturation or activation state of the DC (K oller et al. 2002). The ability of DC to induce immunity or tolerance is dependent on the microenvironment during antigen capture and the antigen itself.

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14 Antigens that fail to induce an inflammatory stimulus are considered safe and induce tolerance, while antigens that are accompanied by an inflammatory signal elicit an immune response directed at antigen elimination (Koller et al. 2002). Al so the differential development of nave T helper cells into functionally distinct memory TH cells, such as TH1 and TH2 cells, depends upon microenvironmental fa ctors that are present at the time of T cell activation (Hilkens et al. 1997). Recen t data indicate that the microenvironment during antigen capture polarize DC so that it pr ovides a signal in the form of release of polarizing cytokines that directs the bias of TH cells towards TH1 or TH2 cytokine production. A crucial factor in this polarization is the presence of IL-12 or IL-4 respectively during T cell rece ptor engagement (Koller et al. 2002). The concept that polarization of TH responses relies on sp ecialized DC subsets was challenged however since it was reported that different DC subpopulations either differentiated in vitro or isolated from the spleen can induce TH1 or TH2-effector cells in vitro depending on the antigen dose and the ac tivation stimulus. High an tigen doses induced a TH1 response, while low doses induced a TH2 cytokine file. On the othe r hand, the capacity of myeloid DCs versus plasmacytoid DCs to induce TH1 responses was shown to be dependent on activation with LPS and CpG, respectively, co rrelating with the hi gh expression of TLR4 and TLR9 by myeloid DCs and plasmacytoid DCs (Nestle et al. 2001; Ardavin et al. 2004). Until recently, myeloid DCs were thought to only stimulate TH1 cells by virtue of their ability to secrete IL -12 (Whittaker 2000; Whittaker et al. 2000). Recent studies in vitro and in vivo suggest that myeloid DC can induce TH1 or TH2 cytokine production in nave T cells. The determini ng factor in skewing the TH response by the DC is the

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15 secretion of IL-12 and the am ount of IL-12 secreted by the DC depends on several factors present during antigen capture as well as the anti gen itself including microbial products, the CD40 ligand (CD154), stimulation from activated T cells and the appropriate cytokine milieu (Koller et al. 2002). DC induce the development of nave TH0 cells populations into TH1 cell populations, producing both IFN and IL-4, because interaction between DC and nave TH cells does not faci litate the induction of IL-12 production. Therefore, the induction of TH1 development through DC derived IL-12 requires the action of an exogenous IL-12 inducing fact or (Hilkens et al. 1997). The functional plasticity of a given human DC subtype when exposed to different cytokines or pathogens makes it difficult to ascribe a fixe d function to a particular DC lineage. For example, the ability of huma n monocyte-derived DC1 cells to produce IL-12 and so to direct a TH1 rather than a TH2 response varies with the c onditions of DC generation and stimulation. Despite this plasticity, each human DC subtype does seem to have a different functional bias. The evidence for this come s mainly from studies on the DC1 and DC2 populations generated from precurs ors in culture. It is clear that we must move from considering DC subtypes as being static elem ents in healthy indivi duals to considering the dynamic behavior of the entire DC system in response to infections or pathological states (Koller et al., 2002). The two helper subsets also cross-regulate each other, so the balance between TH1 and TH2 cytokines can determine whether the im mune response is appropriate or will terminate in detrimental immunopa thologies. Overproduction of TH1 cytokines has been implicated in delayed-type hypersensitivity reactions and autoimmune diseases. TH2 cytokines recruit eosinophils a nd activate mast cells thus dys regulation of these cytokines

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16 can lead to allergenic and inflammatory conditions. The polarized subsets TH1 and TH2 both develop from the same TH0 precursor. The dose of an tigen, strength of signal through the T-cell receptor (TCR) and co-sti mulation all influence the initiation of TH differentiation. An important in sight was obtained from the obs ervations that the antigenactivated nave TH0 cell can be induced to differentiate into the TH1or TH2 lineage in vitro by the addition of exogenous cy tokines. Overall, the mutua lly antagonistic effects of IL-4 and IFN regulate TH1/TH2 balance and subsequent pola rization (Rengarajan et al. 2000). 1.4 Cytokines Cytokines are secreted or membranebound proteins that regulate the growth, differentiation and activation of immune cells. The cellular changes in local cytokine expression can lead to perturbations of the microenvironment in which they are secreted. These perturbations stimulate immune-cell infiltrates which in turn release additional cytokines that act in an autocrine or paracr ine fashion creating a cascade effect. Efforts to understand cytokine function during tumor de velopment and progression are complicated by the pleiotropy and apparent redundancy of cytokine action and by the ways in which the overall cytokine environment shapes the effects of individual cytokines. 1.4.1 Importance of The Study of Cytokines Cytokines are released in re sponse to a diverse range of cellular stresses, including carcinogen-induced injury, inf ection and inflammation. In these settings, cytokines function to stimulate a host re sponse that is aimed at cont rolling the cellular stress and minimizing cellular damage. Conversely, wherea s effective containment of the insult promotes tissue repair, the failure to resolve the injury can lead to persistent cytokine production and to an exacerba tion of tissue destruction.

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17 Pathogens can primarily serve as stimu lants for ongoing cytokine production and inflammation resulting in marked tissue damage . Imbalances of thes e cytokines can then promote the progression from chronic infecti on to cancer by promoting sequestration of the infectious agent by surrounding cells and promoting the growth of cells around this area. As the mixture of cytokines that is present in the tumor microenvironment shapes host immunity, therapeutic manipulation of the cytokine environment constitutes one strategy to stimulate pr otective responses. Indeed, William Coley’s pioneering work at the end of the nineteenth century, in which bacterial extracts were administered as cancer immunotherapy (Cooley’s toxins), resulted in marked alterations in cytokine levels and tumor clearance in some of the treated patients. Although th e toxicities of this approach ultimately proved limiting, probably reflecting the unintended consequences of an elicited cytokine storm that resembled toxic shock, it is noteworthy that some patients with disseminated disease achieved durable clinical bene fits (Dranoff et al., 2004). Recent investigations of the host anti-tum or response have revealed a previously unappreciated complexity of cancer-cell/immune-cell cross– talk. Studies of cytokinedeficient mice have revealed dual roles fo r the immune system in suppressing and promoting cancer formation. In these syst ems the interplay of chronic infection, inflammation and cancer immunity helps dete rmine the outcome of the host response. Future work should aim at characterizing cyto kine pathways in patients with cancer to delineate whether comparable alterations of cytokine function contribute to tumor formation in humans. If such associations could be established, it might become possible to prospectively identify indi viduals at high risk for can cer (Dranoff et al., 2004).

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18 In some cases, immune cells constitute an additional prominent component of the host response to cancer but their partic ipation in tumor pathogenesis remains incompletely understood. The presence of dens e intratumoral lymphocyte infiltrates in early-stage neoplasms are strongly correlated with reduced frequencie s of metastasis and improved patient survival in several cancer types. Compelling epidemiological data indicate that diverse forms of chronic in flammation markedly increase the risk of malignant transformation, and therefore, th at unresolved host immune reactivity can promote tumor development. One variable that might prove decisive in molding the host reaction is the mixture of cytokine that is produced in the tumor microenvironment that may be promoting or regressive of the tumo r. Therefore, manipulation of the cytokine balance may be exploited for potential can cer therapy. Therapeutic administration of cytokines may prove beneficial in over coming some defects like a tumor growing undetected, immunosuppression, inflammation in excess. 1.4.2 Interleukin-12 A powerful factor in the differential de velopment of nave T cells towards a TH1 vs. a TH2 type is IL-12. It strongly augments IFN production by activated nave T cells and, therefore, skews their development toward the memory TH1 phenotype. DCs produce physiologically significant IL-12 levels only when TH1 cell-mediated responses are beneficial (Whittaker 2000; Whittaker et al. 2000, Reddy et al 2004). LPS is also a potent inducer of production in PB MC indicating that cells stimulated by LPS are the ones secreting this cytokine. Nave T cells are poor inducers of IL -12 production in contrast to memory T cells (Hilkens et al . 1997). Mice that la ck the p40 subunit of IL-12 and IL-23 (two cytokines that stimulate IFN production) and mice that lacked NKT cells (a key

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19 source of IFN ) also developed more tumors in response to chemical carcinogens than normal mice. Systemic IL-12 elicits striking anti-tumor effects in mouse models but clinical testing was abruptly cu rtailed because of unexpected se vere toxicities. This is one of the effects obtained observed with Cooley ’s toxin which was beneficial yet proved to be deadly. IL-12 augmented tumor rejection by promoting TH1 responses, increasing lymphocyte cytotoxicity and inhibiting angiogenesis (Dranoff 2004). 1.4.3 Granulocyte Macrophage-C olony Stimulating Factor GM-CSF is a member of a large family of gl ycoprotein growth fa ctors that regulate the growth and differentiation of hematopoietic progenitor cells. It has few side effects in patients yet it has a systemic and immunologi c effect that was obs erved after repetitive daily doses of the human recombinant form of GM-CSF over extended periods of time. It is also known to act at several levels in the generation and propagation of immune responses and has several functions in the immune system includi ng the induction of the differentiation and promotes the survival of peripheral blood DCs. It also increases the activation of macrophages, granulocytes a nd NKT cells, thereby improving tumor antigen presentation (McNeel et al . 1999, Pulendran et al. 2001). 1.4.4 Granulocyte-Colony Stimulating Factor G-CSF is used to expand leukocytes in peripheral blood to mobilize of stem cells from the bone marrow into the bloodstream of patients or normal donors. It does not target tumors directly but is widely used to ameliorate the hematological toxicities of progressive cancer and cytotoxic treatments. It is also commonly used to accelerate neutrophil recovery following a high dos e of chemotherapy for various malignant diseases (Welte et al. 1996, Bella dona et al. 2002, Sloand et al. 2001).

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20 1.5 Bone Marrow and Stem Cell Transplantation Bone marrow transplantation (BMT) is curr ently indicated in the treatment of a number of malignant and non-malignant dis eases including acute a nd chronic leukemias, myelomas, lymphomas, aplastic anemia, solid tumors, and severe immunodeficiencies. Over the past decade, stem cell transpla ntation (SCT), which includes the use of peripheral blood and cord blood in addition to stem cells separated from bone marrow, has become an established therapy for many diseases. In 1996 over 40000 SCT were performed, primarily in the US and Western Europe, for more than a dozen different clinical indications (Hor owitz MM and Rowlings PA 1997). The number of BMT and SCT performed annually is increasing at a rate of 20% to 30% per year, which is expected to continue into the foreseeable future. BMT and SCT are indicated as a treatment in a number of clinical settings because the highly prolific cells of the hematopoietic system are sensitive to many of the agents used to treat cancer patients. Chemotherapy a nd radiation therapy us ually target rapidly cycling cells, so hematopoietic cells are ablated along with the cancer cells. Consequently, patients undergoing these th erapies experience neutropenia (low neutrophil numbers, <500 per mm3), throm bocytopenia (low platelets numbers, <20000 per mm3) and anemia (low red blood cell numbers) rendering them susceptible to infections and bleeding. BMT and SCT drama tically shorten the period of neutropenia and thrombocytopenia but the patient may requi re repeated blood component transfusions for as long as six months. The time period during which the patient is neutropenic represents the greatest risk associated with these therapies and of ten requires parenteral antibiotic administrati on. In addition, some patients neve r achieve engraftment (when cell levels rise to safe levels).

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21 SCT may be performed with patient cells (autologous) that have been removed and cryopreserved prior to administ ration of chemotherapy, or w ith donor cells (allogeneic). Autologous transplants outnumber allogeneic transplants 3:2, and th e use of autologous transplants is growing more rapidly. Nevert heless, there are significant advantages and disadvantages with both tec hniques. A major concern with autologous BMT and SCT is the possibility of reintroducing tumor cells al ong with the transplant. A major obstacle in allogeneic transplantation is the high incidence of graft versus host disease (GVHD), in which donor T cells recognize th e recipient as foreign, resulting in a strong immune response against many of the recipient’s tissues. 1.5.1 Acute Graft vs Host Disease (aGVHD) The development of acute and chronic GVHD and the immunosuppression used for GVHD prophylaxis represent signi ficant risk factors for bact erial, fungal, and viral infections. Studies of experimental and c linical allogeneic BM T and SCT have shown that immunologically competent T cells must be present in the transplanted graft for GVHD to occur. Depletion of T cells or T-cell subsets prevents GVHD and eliminates the need for further immunosuppression but is associated with a hi gher rate of graft failure and increased incidence of leukemic rela pse, resulting in equi valent disease-free survival for patients who receive conventi onal prophylaxis. GVHD thus remains a major barrier to allogeneic BMT or SCT for a variety of diseases and further elucidation of the pathophysiological mechanisms involved is cr itical to its wider application. Besides inflammation or infection, BMT can also tri gger a danger signal, cau sed by secretion of a storm of proinflammatory cy tokines during the conditioning regimen. If major or minor histocompatibility antigen mismatches are present among the donor-recipient pair this storm of cytokines would then favor recogni tion of host antigen-pre senting cells (APC by

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22 donor-specific T lymphocytes). This would re sult in the secretion of a new storm of cytokines that activate other e ffectors of the immune response and lead to th e initiation of graft-versus-host disease (GVHD) (Kre nger et al. 1997). Host APC and, more particularly, host DC play a crucial role in the initiation of GVHD. Supporting these results, researchers have show n that after irradiation host DC are activated and migrate into the secondary lymph nodes we re they prime allogeneic CD8+ T cells before dying as a result of apoptosis. In agreem ent with these studies, it has been shown that treatment of BMT recipients with Flt3 ligand, a hematopoiet ic cytokine that faci litates expansion of DC, accelerates GVHD lethality. Thus , together these reports i ndicate that DCs initiate the allogeneic response. However, the impact of the allogeneic response on DC activation has never been investigat ed. (Laurin et al. 2004) Advances in basic immunology during the last decade have demonstrated how interactions between immunol ogically competent cells are governed by cytokines. Research has focused on the roles of these me diators in the pathogenesis of acute GVHD. Current evidence indicates that dysregul ated cytokine production occurs during sequential monocyte and T-cell activation. Cy tokine dysregulation after bone marrow transplantation can be conceptualized as th ree sequential parts or phases (Krenger et al. 1997). 1.5.1.1 Phase 1: Host Conditioning Agents used for BMT conditioning, whic h often includes total body irradiation and/or chemotherapy, are impor tant variables in the etio logy of acute GVHD and other transplant-related complications. Ionizing ra diation activates host cells to secrete increased levels of inflammatory cytokines. The increased systemic levels of these cytokines then can lead to endothelial cel l damage which contri butes to increased

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23 activation of donor T cells present in the donor marrow inoculum. Facilitation of T-cell activation by inflammatory cytokines may o ccur via their direct stimulatory action on T cells, or indirectly through enhanced antigen presentation or enhanced intercellular adhesion. In addition, injury to the mucosal surf ace of the gastrointestin al tract due to the conditioning regime may enable bacterial breakdown products, such as endotoxin or LPS, to enter the circulation. LPS may subsequently stimulate pr e-activated gut-associated macrophages. 1.5.1.2 Phase 2: Donor T Cell Activation Activation of alloreactive donor T cells occurs during phase 2 of GVHD. The nature of host alloantigens determines wh ich subset of donor T cells proliferates and differentiates. Activated donor T cells secrete several cytokines, predominantly IL-2 and Type 1 IFNs, which can be produced by CD4+ as well as CD8+ T cells (“TH1” and “T CTL 1” cells), inducing cytotoxic T lym phocyte (CTL) and NK cell responses, and priming monocytes to produce th e proinflammatory cytokines. 1.5.1.3 PHASE 3: Inflammatory Effector Mechanisms In aGVHD, a third set of components incl ude several inflammatory cytokines, specific anti-host cytotoxicity mediated by CTL via Fas and perforin pathways, large granular lymphocytes or NK cells, and nitric oxide (Kre nger et al. 1997). 1.5.2 Chronic Graft vs Host Disease (cGVHD) Chronic GVHD remains the most common late complication occurring following allogeneic transplantation, occurring in 25-80% of transplant recipients depending on the degree of HLA-mismatch with the donor a nd the source of stem cells. Treatment of transplant recipients introduced over two decades ago remains inadequate, although is still the ‘standard’. For patients who are refract ory to primary treatment, that is, ‘steroid-

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24 refractory’ patients, no alternative exists . Chronic GVHD is arbitrarily defined as rejection or disease occurring 100 days or more after allogeneic transplantation. While the median time to onset is 201 days following HLA-matched siblings and 133 days following unrelated donor transp lants cGVHD may occur as early as 70 days, or as late as 15 months, post-transplant. Although organ dysfunction in cGVHD is a cause of severe morbidity, the main cause of death is inf ection due to immune dysfunction associated with cGHVD and accompanying immunosuppressi ve therapy. The prior diagnosis of acute GVHD is the most important predictor for the development of chronic GVHD (Farag 2000

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25 CHAPTER 2 OBJECTIVES AND SPECIFIC BACKGROUND TO PROJECT The objective of our study was to dete rmine whether GM-CSF would skew the cytokine milieu and lead to proliferation of lymphocytes towards a type 1 phenotype. We hypothesized that GM-CSF exerted its primary effect on DCs from among all peripheral blood mononuclear cells (PBMCs). DCs then activ ate T cells that would be transferred to an allogeneic bone marrow or stem cell transpla nt recipient. These T cells in turn would induce the GVHD as well as the graft versus leukemia (GVL). The GVL effect would protect the recipient agains t tumor relapse with a t ype 1 cytokine response. As described in Chapter 1, a type 1 response produces IFN and IL-12 (which has been shown to exert a powerf ul anti-tumor effect), activ ates macrophages, NK cells and CTL cells and induces an inflammato ry cytokine production. Conversely a TH2 profile (IL-4 and IL-10) may have the opposite eff ect such as down regulating innate and acquired anti-tumor immunity. Type 1 and T ype 2 cytokines responses in T cells are cross-regulatory. Differential activation of T-cell subsets evokes immunopathogenesis of various diseases including autoimmunity, in fections, immunodefi ciencies, and GVHD. Therefore, we hypothesize a poten tial relevance in th e shift in the ratio of the type of response induced (type1 vs. type 2) in a GVHD and cGVHD. In the case of aGVHD, type 1 responses are thought responsible for in itiating acute GVHD. Type 2 responses are associated with cGVHD. There is now consider able evidence that a type 1 to type 2 immune deviations after alloge neic transplantation are associ ated respectively with either decreased aGVHD or with the development of a cGVHD syndrome that is characterized

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26 by decreased lethality and autoantibody fo rmation. In the treatment of aGVHD and cGVHD the ultimate goal would be to use agents to manipulate that ratio to prevent the induction of the inflammatory effector pha se of GVHD processes without deleting the beneficial effects that are usually mediated by alloreactive donor T cel ls also called graft versus tumor (GVT) or more specifilly graf t-versus-leukemia (GVL) effect (Krenger et al. 1997; Belladona et al. 2002). Currently the most used growth factor fo r mobilization of hematopoietic stem cells is G-CSF although GM-CSF has also been used in conjunction with it. This growth factor has been shown to increase the incidence of cGVHD although the incidence of aGVHD is not higher than that in bone marrow transp lantation. Allografts mobilized with this growth factor contain one log more CD3+CD8-CD4+ T cells than bone marrow grafts. These cells decrease the severity of acute GVHD in a murine model and are associated with decreased cytotoxic lymphocyte activity in vitro (Pan et al. 1999). The aGVHD is not increased with increased T cell mobiliz ation, possibly due to G-CSF skewing the type1/type2 ratio in favor of the more regulatory type 2 response. Sloand et al. demonstrated that the pretreatment of T ce lls with G-CSF results in diminished IFN and increased IL4 production when these cells we re subsequently subj ected to polyclonal stimulation with IL2, PHA or CD3 monoclonal antibody in vitro . The effects of G-CSF on CD4+ T cells were direct and granulocytes were not required for G-CSF mediated modulation. Even though IFN exposure results in monocyt e apoptosis, survival of monocytes in their cultures was improved by their co-culture with G-CSF treated CD4 cells, giving evidence suggesting these cel ls having the tolero genic effect of TH2. The overall effects were not due to selected deletion of the TH1 population but rather by

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27 incrementing the TH2 population. Other investigators ha ve demonstrated that G-CSF mobilized monocytes exert inhi bitory effects on T cell proliferation and responsiveness. G-CSF mobilized CD14+ cells express significantly lower levels of the co-stimulatory molecules B7-2 implying that low levels of e xpression of this molecule may contribute to the decreasing T cell responsiveness seen during transplant by providing suboptimal amounts of stimulatory signals. Similarly, monocytes obtained from G-CSF mobilized donors suppress cytotoxic responses and T-cell pr oliferation when they are co-cultivated with allogeneic T cells (Arpinat i et al. 2000, Sloand et al. 2000). Figure 2-1. Effects of G-CSF on T Cells: This figure from Sloand et al. 2000 shows that G-CSF exerts an effect on IFN and IL-4 produced by TH cells (by intracellular staining). It also show s that treated macrophages excert no change in this cytokine profile like th at seen in un-stimulated Th lymphocytes. Reddy et al. 2000 also showed that this e ffect comes only from the donor cells and not from the recipient, Furthermore the effects are mediated by TNF reduction. The reduction of IL-12 secretion shown by Reddy et al. may be one mechanism by which nave T cells are polarized toward a Th2 phenotype.

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28 Figure 2-2. Type 1 Cytokine Profile Afte r Mixed Lymphocyte Reaction: (A)The DC generated were potent antigen-presenti ng cells in a primary mixed lymphocyte reaction (B) 5 DC/well in triplicates of a 96 -well flat bottom Falcon tissue culture plate were stimulated with 100 l of 1 g/ml of LPS. Supernatants were collected 4 hr and 24 hr later, and TNF (B) and IL-12 (C) were measured by ELISA. Solid bar: Contro l; Stippled bar: G-CSF; Open bar: peritoneal macrophages (Reddy et al. 2000). We hypothesized that GM-CSF elicits a chan ge in the ratio of DC1/DC2 favoring DC1 cells. Distinct DC subsets can regulate immune responses differently and regulatory function can be altered by the microenvironmen t or by pathogens. G-CSF is required for human DC2 development from hematopoietic progenitor cells (HPCs), whereas GM-CSF is one of the growth factors required for the development of human DC1 cells from HPCs (Rissoan et al. 1999, Arpinati et al. 2000, Waller et al. 2003). In one study, rat respiratory tract DCs, were ovalbumin-pulsing and used in adoptive transfer. These cells stimulated TH2 cytokines and isotype antibodies. However, pre-treatment of these cells with GMCSF induced production of both TH 1 and TH 2 responses (Pulendran et al. 2001). G-CSF treatment of normal stem cell donors selectively increases the number of DC2, but not DC1, in the blood and periphe ral blood stem cells (PBSC) used for allogeneic transplantation. G-CSF may increas e the number of DC2 in the circulation by either stimulating their production in th e marrow, increasing survival, inducing mobilization, or decreasing migration out of the vascular space and recruitment into lymphoid organs. Yet DC2 isolated from th e blood of normal or G-CSF treated donors

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29 are unable to activate proliferation of nave allogeneic T cells until after ex vivo activation by CD40L or TNF . In contrast, DC1s freshly isol ated from human blood can activate nave alloegenic T cells. Expr ession of co-stimulatory mol ecules is relatively low or absent in circulating DC1 and DC2. Therefore, there must be other di fferences in the state of differentiation or activation of DC1 and DC 2 that account for the difference in their allostimulatory activity. Donor DCs functionally engraft in recipients of allogeneic transplants. Their persistence has been a ssociated with prolonged organ transplant survival. The variation in the DC1/DC2 ratio in the graft could have an important consequence on sensitation of the host and on engraftment outcome (Arpinati et al. 2000). However, G-CSF usage as a mobilizing agent in bone marrow and stem cell transplantations has limita tions. Allogeneic bone marrow transplant recipients who received grafts containing more donor DC2 cells mobilized with G-CSF had higher rates of relapse, and had poorer even t-free survival compared w ith recipients who received fewer donor DC2 cells (Waller et al. 2003) . Outside the hematopoietic stem cell transplant arena, GM-CSF has been shown to increase protec tive immunity to melanomas when they transduced with a gene secret ing this cytokine (S chneeberger et al. 2003, Dranoff et al. 2003, Banchereau et al. 2005). In these systems, GM-CSF stimulates an intense local immune response consisting of recruitment of DCs, macrophages and granulocytes. These findings suggest that one function of GM-CSF is to the augment antigen presentation. GM-CSF also provokes a marked expansion of DCs locally and systemically that stimulates high levels of protec tive immunity against certain cancers like melanoma. In general, GM-CSF is known to elicit the DC1 subset which has superior

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30 phagocytosis of particulate material, incl uding apoptotic tumor cells. These GM-CSF promoted DCs have higher levels of co-sti mulatory molecules which is indicative of greater functional maturation. This enhanced activity results in mo re efficient T-cell stimulation. CD4+ T cells contribute to the producti on of antitumor antibodies, which similarly play a pivotal role in GM-CSF stim ulated immunity. The delineation of specific properties of DCs that elicit high levels of tumor immunity provides important guidelines for optimizing the therapeutic use of these cells for cancer immunotherapy (Dranoff 2003) (Figure 2-3). Figure 2-3.Protecting Effects of GM-CSF in Cancer: In the model system of gene transduction with cytokines GM-CSF fared the best in protecting mice against melanoma. G-CSF conferred some prot ection but not as high as GM-CSF. Keeping the ability of the immune system to fight the tumor is something that ideally should be left in the transpla nt patient receiving stem cells (Dranoff 2003). Waller et al. has demonstrated that signifi cant increase on the content of DCs in the hematopoietic precursor graft can be achieved by using combinations of growth factors. Specifically, they compared G-CSF combin ed with GM-CSF on the content of DC subsets and T cell phenotype in cells mobili zed for transplantation. DC2 content of the patients that received both GCSF and GM-CSF was significan tly lower than that from

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31 the patients that received G-CSF alone with no significant change in the percent of DC1 cells. There was an increase in the percentage of CD4+ cells and a trend toward a higher percentage of CD34+ cells in the grafts coll ected from the patients that received G-CSF and GM-CSF together when compared to GCSF alone. The net resu lt of these studies was a shift in the DC1/DC2 ratio favoring DC 1 in the groups receiving both G-CSF and GM-CSF (see figure 2-4). Figure 2-4.GM-CSF/G-CSF Effects in Stem Cell Transplantation: Also from Waller et al. 2003 it shows that DC2 is reduced in the groups receiving GM-CSF (G-CSF alone (Cohort A), G-CSF and GM-CSF at the same time (Cohort B) and patients treated with G-CSF followed 7 days later by GM-CSF (Cohort C)). From these studies, it has been theorized that a TH1 profile is protective against leukemia relapse and therefore beneficial because of its GVL effect, while a TH2 profile may be deleterious because it may not only promot e relapse but also tumor growth and dissemination. Type 2 induced T cell hypo-re sponsiveness strategies reduce the DC2 content of the transplant graft and indu ces instead a type 1 response may enhance autologous anti-tumor immunity after au tologous hematopoietic progenitor cell transplantation. Alternatively, such cell may e nhance the graft versus tumor effect in the context of allogeneic hematopoietic progenito r cell transplantation (Sivakumaran 2001).

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32 Figure 2-5.Effects of G-CSF/GM-CSF Treatment on Stem Cells, T Cells and DCs: Stem cell content, T cell content and DC1 to DC2 ratios of patients treated with GCSF alone (Cohort A), G-CSF and GM-C SF at the same time (Cohort B) and patients treated with G-CSF followed 7 days later by GM-CSF (Cohort C). This shows the advantages of adding GM-CSF to the treatment of donor cells (from Waller et al. 2003). Two strategies exist by which to modul ate the immunotherapeu tic potential of transplantation. One can use different cyt okines to mobilize different DC and T cell phenotypes in the graft, or alternatively one can administer post-tran splantation cytokines to alter the character of the DC and T cell phenotype in the donor cells. However, T cell activation profile of lymphocyt es from patients who received post-transplantation G-CSF was skewed toward a TH2 phenotype (high IL-4, low IL12) which led to a slower immune recovery. Our recent data indicate th at patients who have a higher DC count as well as a higher IL-12 (as in a type 1 respons e) have a much better outcome (see figure 26 and 2-7).

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33 Figure 2-6.Correlation of High DC Counts Ve rsus Survival: Higher DC counts have a higher long term survival rate as comp ared to low DC count at engraftment: Kaplan-Meier plot of patient survival by DC group (low DC vs High DC) for 500 days after transplanta tion (Reddy et al. 2004). Figure 2-7.Correlation of High IL-12 Versus Re lapse-Free Survival: Recipients of grafts with higher IL-12 levels in the blood have a higher long term relapse-free survival rate as compared to low IL-12: Kaplan-Meier plot of patient relapsefree survival by IL-12 group (low IL12, medium IL-12 and high IL-12) for 500 days after transplantati on (Reddy et al. in press). The study of cytokines and/or growth factor s that can lead to a type 1 cytokine response in either DCs or T cells may help lowe r the relapse of the recipients of stem cell transplants by promoting anti-tumor effects. Of the two types of cells mentioned, DCs have the potential to lead responses toward s a more beneficial type 1 response in the

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34 donor cells which can lead to le ss relapse in the recipient. It is unknown if the interaction of GM-CSF with DCs prior to transfer into the recipien t can modulate the type of response that can lead to a bett er therapeutic outcome in the r ecipients of stem cell grafts. GM-CSF is already used regular ly in clinical settings as a growth factor to boost neutrophil counts and our research may lead to different uses for this cytokine, in the enhancement of immune responses against cancer. In summary, current therapies in stem and bone marrow transplantation can benefit from an induction of type 1 cytokine secreti on induced by the cells in the graft cells. GMCSF may have the potential to produce this desirable chan ge in cytokine secretion balance. That is the reason we are studying the effects of GM-CSF as a growth factor on normal PBMCs. Based on the above rationale ou r specific aims are (see figure 2-8 for a chart of the project): 1. To evaluate the effects of GM-CSF dire ctly on the human leukocyte population at different times by testing type 1 and type 2 cytokine secretion into the media after addition of G-CSF, GM-CSF or both. 2. To assess the subset of cells secreting cytokines by depl eting populations of T cells. 3. To study the effect of GM-CSF on cultured monocytes (progenitors of DCs) and measured by flow cytometry the capacity of this cytokine to induce a shift in the DC1/DC2 ratio. 4. To investigate wether DCs amplify the ch ange to a type 1 cytokine secretion response we tested the pro liferative capacity of T cells stimulated by monocytes exposed to G-CSF, GM-CSF or both.

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35 CHAPTER 3 MATERIALS AND METHODS 3.1 Culture, Medium and Cytokines As standard medium RPMI (Sigma, Missouri, USA) supplemented with Lglutamine, 5000U/ml penicillin (Sigma, Missour i, USA), 5000 U/ml streptomycin sulfate (Sigma, Missouri, USA) , and 10% v/v fetal bovine serum (Gibco, California, USA) was used for our cultures. Growth factors used for our standard cultur ing procedures were 2.8x106 U/ml of recombinant human GM-CSF (L eukine, Berlex, Seat tle, Washington, USA), 1x108 U/ml of recombinant human G-CSF (Neupogen, Amgen, Thousand Oaks, California, USA), 1000 U/ml recombinant hum an IL-4 (Sigma, St. Louis, Missouri, USA), 1000 U/ml recombinant human IL-12 (S igma, St. Louis, Missouri, USA), 1ug/ml LPS (Sigma, Missouri, USA) and 5 g/ml PHA (Sigma, St. Louis, Missouri, USA). PBMC were either cultured as they were isolated at a concentration of a million cells/ ml or as monocytes and T cells isolat ed using MACS magneti c bead pullout (see below) and cultured separately at 37oC/5%CO2. The PBMC (labeled as White Blood Cells WBC in the plots) were cultured in RPMI alone (control), with G-CSF, GM-CSF and G-CSF/GM-CSF in the above concentrations for either 24 hours or 48 hours. After that time either LPS or PHA was added to de ndritic cells to mature them for another 24 or 48 hours. After culture, cells were frozen to be used in subsequent projects and supernatants collected separately and frozen for later analysis by cytokine ELISAs. Same protocol was used to culture separated T cells.

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36 Separated monocytes were cultured for 5 to 6 days with no growth factors, GCSF, GM-CSF, G-CSF/GM-CSF, IL-12 and GM-CSF/IL-4 in the concentrations described above. The cells were cultured with LPS or PHA for 24 or 48 hours. At day 7 a million cells from each test were collected fo r flow cytometry. Then, the remainder of the cells were co-cultured with allogeneic T cel ls in a BrdU based a proliferation assay. 3.2 Isolation and Preparation of PBMC Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood leukocytes (leukopac, PBL) obtained fr om Lifesouth Community Blood Center (Gainesville, Florida) by discontinuous de nsity gradient separation using lymphoprep (Axis-Shield, Norway). Th e separation yields the low density (<1.077 g/cm3) mononuclear cell population at the interface wh ile the more dense erythrocytes and granulocytes will pellet at the bottom. The contents of the leukopac were diluted three times its volume in sterile 1X PBS pH 7.4 (Gibco, California, USA). Fifteen milliliters of the dilution were layered onto 10 ml of lymphoprep. The sample was then centrifuged for 25 minutes at C and 1200 rpm. The PBMC were colle cted at the interface and washed twice with PBS by centrifugation at 1 200 rpm each time for 10 minutes at 4 C. The cells were counted at the last wash in a sta ndard hematocytometer using trypan blue. 3.3 Isolation of T Cells and Monocytes from PBMC We separated T cells (CD3 positive cells) and monocytes at different times for our experiments. We used the MACS system (Militenyi Biotech, California USA) for Pan T cell isolation kit II (negative selecti on) and monocyte separation kit (negative selection). We followed the manufacturer’s prot ocol for the separation, counting cells in a hematocytometer after separation.

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37 3.4 Enzyme-linked Immunosorbent Assay (ELISA) We analyzed cytokines by ELISA: IL-12, IL-10, TNF , IFN and IL-4 (PierceEndogen). We followed Pierce-Endogen procedur e and reagents. Briefly, cytokines were captured by the specific primary monoclonal antibody and detected by biotin-labeled antiIFN, anti-TNF , anti-IL-4, anti-IL-10, or anti-IL-12 followed by strepavidinhorseradish peroxidase. The color reaction was developed by TM B microwell peroxidase substrate and stopped by the addition of an equal volume of 0.18M H2SO4. The absorbance of the assay plate was read at 450 nm using a microplate reader. Recombinant human TNF(hTNF), hIFN, hIL-4, hIL-10, and hIL-12 p40 were used as standards for ELISAs. 3.5 Proliferation Assay Proliferation of growth f actor-treated monocytes wa s measured with a BrdU ELISA (Roche Diagnostics, Manheim Germany) . Following the manufacturer’s protocol, we took the treated monocytes and plated them in a 96 well plates with different concentrations of allogeneic T cells (CD3 positive cells) for 48 hours. After the first 24 hours BrdU was added to the wells for incorp oration into prolifer ating cells. 24 hours later the plates were taken out of the in cubator and centrifuged for 10 minutes at 1200 rpm in 4C. Afterwards, they were dried with a hairdryer for 15 minutes or until completely dry. Afterwards, the ELISA pro cedure was performed and the colorimetric change measured in an ELISA plate reader. 3.6 Flow Cytometry Briefly, cultured monocytes were incubated with a lineage (lin ) cocktail (anti-CD3, CD4, CD16, CD19, CD20, CD56) conjugated w ith FITC, APC-conjugated anti-CD11c,

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38 PE-conjugated anti-CD123 and PerCP-c onjugated anti-HLA-DR for 30 min, then washed with staining buffer (PBS + 2% BS A + 0.1% Na azide) for less than 10 min, and then fixed with 2% paraformaldehyde for 20 min at 4 C. Cells were analyzed on a FacScalibur machine (BD biosciences) a nd the data was analyzed on CellQuest.

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39 CHAPTER 4 RESULTS AND DISCUSSION 4.1 Effects of GM-CSF and G-CSF on The Cytokine Milieu of Ficoll Separated White Blood Cells To determine the effect of GM-CSF on the overall population of human white blood cells, leukocytes were separated from a buffy coat obtained from Lifesouth by ficoll gradient centrifugation and washed twi ce with 1X PBS pH 7.4. Viable cells were counted in the last wash in a hematocy tometer by trypan blue exclusion. Then a concentration of 1x106cells/ml in sterile RPMI media (supplemented with 10% fetal bovine serum and 1000 U/ml of pennicillum-stre ptomycin) was added to each well. Final volume was 25 ml in a 50 ml sterile tissue fl ask. Growth factors were added to each flask in the following fashion: a control (no grow th factors added, labeled “C”), G-CSF, GMCSF, or both G-CSF and GM-CSF together as described in chapter 3. The cultures were incubated with these factors for either 24 or 48 hours followed by a maturation step for another 24 or 48 hours. Two different matura tion stimuli were test ed: lipopolisacharide (LPS) or lectin (PHA). LPS matures DC s and monocytes, while PHA stimulates lymphocyte maturation. The purpose of using LPS and PHA was to determine whether GM-CSF affects mature monocytes or lymphoc ytes respectively. At the end of the culture period (48 hours, 72 hours or 96 hour s, depending on the culture), cells were centrifuged at 1200 rpm for ten minutes. Then supernatant and cells were separated and cells were cryo-preserved in FBS with 10% DMSO. Culture supernatants were measured by cytokine sandwich ELISA. Cytokine s related to a type 1 (IL-12, TNF , IFN or type

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40 2 response (IL-10 and IL-4). Results of the ELISA are displayed in figures 4-1 through 4-5. All of the plots represen t three separate runs of th e experiment. Error bars are calculations of the standard error for the three separate runs. It is important to note that human blood cells obtained from leukopacs have an inherent variation since they come from a heterogeneous volunteer donor popula tion. The demographical data for each donor leukopac was not provided for our re search purposes (Koller et al. 2002). The data in figures 4-1 through 4-3 dem onstrate that type 1 cytokines IL-12, TNF and IFN are up-regulated when GM-CSF is adde d to the culture media in the first 24 hours with IL-12 and TNF having the most marked effect by GM-CSF stimulation. This supports our initial hypot hesis that GM-CSF induces a type 1 response in the unseparated white blood cell population. Our data also indicate that IL-12 levels increase after addition of GM-CSF in a t ype dependent manner whereas TNF and IFN are secreted equally among cells stimulated with LPS or PHA in the first 96 hours. Surprisingly, when we assayed the type 2 cytokines, we found no IL-4 in the LPS stimulated white blood cells, whic h probably indicates that IL-4 is not secreted by mature monocytes, dendritic cells or macrophages as indicated by Sloand et al. (2000) and by Arpinati et al. (2000). However, since we we re able to get IL-4 from PHA stimulated cells our data confirms that IL-4 is secr eted by lymphocytes. By contrast IL-10 was down-regulated by the addition of GM-CSF. We noted that there was no difference in the IL-4 production between G-CSF and GM-CSF treatment by PHA stimulated cells. In summary, we evaluated IL-4 in our cultures based on the Sloand et al. (2000) data that showed that G-CSF induced a change in the T cell profile of cancer patients

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41 IL-12 ELISA (Results reflect the means of three separate experiments)0 0.1 0.2 0.3 0.4 0.5 0.6LPS 24/24 C LPS 24/24 G LPS 24/24 GM LPS 24/24 G/GM PHA 24/24 C PHA 24/24 G PHA 24/24 GM PHA 24/24 G/GM LPS 24/48 C LPS 24/48 G LPS 24/48 GM LPS 24/48 G/GM PHA 24/48 C PHA 24/48 G PHA 24/48 GM PHA 24/48 G/GM LPS 48/24 C LPS 48/24 G LPS 48/24 GM LPS 48/24 G/GM PHA 48/24 C PHA 48/24 G PHA 48/24 GM PHA 48/24 G/GM LPS 48/48 C LPS 48/48 G LPS 48/48 GM LPS 48/48 G/GM PHA 48/48 C PHA 48/48 G PHA 48/48 GM PHA 48/48 G/GMTreatment nameConcentration of IL-12 (ug/ml) Figure 4-1. Results of IL-12 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation. Supernatants of the cultur e with untreated control (C), G-CSF (G), GM-CSF (GM) or both (G/GM) were measured for IL-12. Cultures were treated cultured for either 24 or 48 hours followed by stimulation with either LPS or PHA for another 24 to 48 hours as shown in the figure. The Results are from three separate experiments. The bars indicate the calculated standard error from triplicate experiments.

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42 IL-10 ELISA (Results represent the mean of three different experiments)0 0.5 1 1.5 2 2.5 3 3.5 4LPS 24/24 C LPS 24/24 G LPS 24/24 GM LPS 24/24 G/GM PHA 24/24 C PHA 24/24 G PHA 24/24 GM PHA 24/24 G/GM LPS 24/48 C LPS 24/48 G LPS 24/48 GM LPS 24/48 G/GM PHA 24/48 C PHA 24/48 G PHA 24/48 GM PHA 24/48 G/GM LPS 48/24 C LPS 48/24 G LPS 48/24 GM LPS 48/24 G/GM PHA 48/24 C PHA 48/24 G PHA 48/24 GM PHA 48/24 G/GM LPS 48/48 C LPS 48/48 G LPS 48/48 GM LPS 48/48 G/GM PHA 48/48 C PHA 48/48 G PHA 48/48 GM PHA 48/48 G/GMTreatment nameConcentration of IL-10 (ug/ml) Figure 4-2. Results of IL-10 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation. Supernatants of the cultur e with control media (C), GCSF (G), GM-CSF (GM) or both (G/GM) were measured for IL-10. Cultures were treated cultured for either 24 or 48 hours followed by stimulation with either LPS or PHA for another 24 to 48 hours as shown in the figure. The Results are from three separate experiments. The bars indicate the calculated standard error from triplicate experiments.

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43 IFN ELISA (results represent mean of three separate experiments)0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1LPS 24/24 C LPS 24/24 G LPS 24/24 GM LPS 24/24 G/GM PHA 24/24 C PHA 24/24 G PHA 24/24 GM PHA 24/24 G/GM LPS 24/48 C LPS 24/48 G LPS 24/48 GM LPS 24/48 G/GM PHA 24/48 C PHA 24/48 G PHA 24/48 GM PHA 24/48 G/GM LPS 48/24 C LPS 48/24 G LPS 48/24 GM LPS 48/24 G/GM PHA 48/24 C PHA 48/24 G PHA 48/24 GM PHA 48/24 G/GM LPS 48/48 C LPS 48/48 G LPS 48/48 GM LPS 48/48 G/GM PHA 48/48 C PHA 48/48 G PHA 48/48 GM PHA 48/48 G/GMTreatment nameConcentration of IFN (ug/ml) Figure 4-3. Results of IFN Cytokine ELISA of Leukocyte Culture s at Different Times of Stimulat ion. Supernatants of the culture with control media (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were measured for IFN . Cultures were treated cultured for either 24 or 48 hours followed by stimulation with either LPS or PHA for another 24 to 48 hours as shown in the figure. The Results are from three separate experiments. The bars indicate the calculated standard erro r of triplicate experiments. Values of cultures stimulated for 48 hours and mature for 48 hours with PHA (48/48 PHA) cultures were much more than 1 ug/ml of IFN . The top of the bars for those samples are omitted to illustrate the changes in the other samples.

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44 TNF ELISA (Results are the mean of 3 separate experiments)0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6LPS 24/24 C LPS 24/24 G LPS 24/24 GM LPS 24/24 G/GM PHA 24/24 C PHA 24/24 G PHA 24/24 GM PHA 24/24 G/GM LPS 24/48 C LPS 24/48 G LPS 24/48 GM LPS 24/48 G/GM PHA 24/48 C PHA 24/48 G PHA 24/48 GM PHA 24/48 G/GM LPS 48/24 C LPS 48/24 G LPS 48/24 GM LPS 48/24 G/GM PHA 48/24 C PHA 48/24 G PHA 48/24 GM PHA 48/24 G/GM LPS 48/48 C LPS 48/48 G LPS 48/48 GM LPS 48/48 G/GM PHA 48/48 C PHA 48/48 G PHA 48/48 GM PHA 48/48 G/GMTreatment nameConcentration of TNF (ug/ml) Figure 4-4. Results of TNF Cytokine ELISA of Leukocyte Culture s at Different Times of Stimulat ion. Supernatants of the culture with control media (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were measured for TNF . Cultures were treated cultured for either 24 or 48 hours followed by stimulation with either LPS or PHA for another 24 to 48 hours as shown in the figure. The Results are from three separate experiments. The bars indicate the calculated standard error from triplicate experiments

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45 IL-4 ELISA (Results represent mean of three separate experiments)0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08LPS 24/24 C LPS 24/24 G LPS 24/24 GM LPS 24/24 G/GM PHA 24/24 C PHA 24/24 G PHA 24/24 GM PHA 24/24 G/GM LPS 24/48 C LPS 24/48 G LPS 24/48 GM LPS 24/48 G/GM PHA 24/48 C PHA 24/48 G PHA 24/48 GM PHA 24/48 G/GM LPS 48/24 C LPS 48/24 G LPS 48/24 GM LPS 48/24 G/GM PHA 48/24 C PHA 48/24 G PHA 48/24 GM PHA 48/24 G/GM LPS 48/48 C LPS 48/48 G LPS 48/48 GM LPS 48/48 G/GM PHA 48/48 C PHA 48/48 G PHA 48/48 GM PHA 48/48 G/GMTreatment nameConcentration of IL-4 (ug/ml) Figure 4-5. Results of IL-4 Cytokine ELISA of Leukocyte Cultures at Different Times of Stimulation. Supernatants of the culture with control media (C), G-CSF (G), GM-CSF (GM) or both (G/GM) were measured for IL-4. Cultures were treated cultured for either 24 or 48 hours followed by stimulati on with either LPS or PHA for another 24 to 48 hours as shown in the figure. The Results are from three separate experiments. The ba rs indicate the calculated standard error from triplicate experiments.

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46 towards TH2 cells. Therefore, G-CSF was utilized in our experiments as a type 2 inducing control. Yet, our results don’t seem to show a difference between G-CSF and GM-CSF in IL-4 secretion. Still, IL-10 levels in our cultures indicated that there is a type 2 respons e induction with G-CSF. GM -CSF also induced levels higher than non-stimulated control cell a lthough less than that seen in the G-CSF stimulated cells. The lower levels of IL -10 with GM-CSF are only seen with LPS matured cells as compared to PHA matured cells. This suggests that some type 2 cytokines such as IL-10 are produced by the activa ted non-lymphocyte population while mature lymphocytes (indicated by PHA matured cells) produce others like IL-4. However, IL-10 production could be due also to DC2 cells since they are thought to be of lymphoid origin as descri bed earlier. Overall, GM-CSF increases type 1 cytokines over a two day period, wh ile G-CSF induced type 2 cytokines or at least increased the secr etion of type 2 cytokine s like IL10. When G-CSF and GM-CSF are together the cell’s cytokine secretion resembles that by cells treated with GM-CSF alone. These results probabl y indicate that the effects of GM-CSF override the effects of G-CSF or that G-CSF doesn’t have a strong inhibitory effect over GM-CSF. 4.2 Effects of GM-CSF and G-CSF on the Cytokine Milieu of Ficoll Separated White Blood Cells, T Cells and T cell Depleted White Blood Cells. To differentiate the role of T cells in cytokine production from the rest of the white blood cell population when stim ulated with either G-CSF or GM-CSF we repeated the previous experiment. Cu ltures either had whole white blood cells, T cells or non-T cells (T cell depleted white blood cells). To obtain these cells we obtained buffy coats as before and se parated by ficoll gradient centrifugation.

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47 Once the cells were centrifuged and counte d, half of the cells were used for the white blood cell cultures and the rest we re processed through magnetic activated cell sorting procedures by negative selection of CD3+ T cell fraction as described in chapter 3 (materials and methods). Af ter separation the T cell fraction and the non-T cell fraction (T cell depleted white blood cells) were cultured the same way as the white blood cells described in the pr evious section. Cells were cultured for 48 hours followed by 24 hours of maturation with LPS or PHA. These times were chosen because the cytokines of interest were obtained at higher levels under those culture conditions (see figures 41 through 4-5). The cytokine secretion profiles are shown in figures 4-6 through 4-10. All of the plot s represent three separate runs of the experiment. Error ba rs are calculations of the standard error for the three separate runs. The separated cells had similar cytokine secretion patterns, as compared to the whole white blood cell population, with type 1 cytokines being up regulated by GM-CSF induced cells. Of this group of cytokines, IFN was at background levels in the T cells popula tion by itself and therefore was produced mostly by the T cell depleted population. IFN was up-regulated by the presence of GM-CSF in the culture. These results suggest that al l stimulated white blood cells participate in the secretion of IFN and that the secretion of th is cytokine was up-regulated by GM-CSF and not G-CSF. In addition, G-CSF alone may down-regulate IFN . Similar results are observed with TNF which was not secreted by T cells and it was secreted instead by the T cell depleted population. TNF was also upregulated regulated in all cases when GM-CSF was present. IL-12 production was

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48 IL12 ELISA OF T versus Non T cells (three separate experiments)0 0.1 0.2 0.3 0.4 0.5 0.6 0.7Leuko 3 WBC LPS C Leuko 3 WBC LPS G Leuko 3 WBC LPS GM Leuko 3 WBC LPS G/GM Leuko 3 WBC PHA C Leuko 3 WBC PHA G Leuko 3 WBC PHA GM Leuko 3 WBC PHA G/GM Leuko 3 T Cells LPS C Leuko 3 T Cells LPS G Leuko 3 T Cells LPS GM Leuko 3 T Cells LPS G/GM Leuko 3 T Cells PHA C Leuko 3 T Cells PHA G Leuko 3 T Cells PHA GM Leuko 3 T Cells PHA G/GM Leuko 3 Non T Cells LPS C Leuko 3 Non T Cells LPS G Leuko 3 Non T Cells LPS GM Leuko 3 Non T Cells LPS G/GM Leuko 3 Non T Cells PHA C Leuko 3 Non T Cells PHA G Leuko 3 Non T Cells PHA GM Leuko 3 Non T Cells PHA G/GMTypes of cells and treatment amount (ug/ml) Figure 4-6. Results of IL-12 Cytokine ELISA on the White Blood Cell Population With or Without T Cells. Supernatants of the culture with nothing (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were m easured for this cytokine. Cultures were treated cultured for 24 with its respective growth factor follo wed by stimulation with either LPS or PHA for 48 hours as shown in the figure.

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49 IL-10 ELISA OF T versus Non T cells (three experiments)0.000 0.500 1.000 1.500 2.000 2.500 3.000 3.500 4.000 4.5001 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Types of cells and treatmentConcentration of IL-10 (ug/ml) Figure 4-7. Results of IL-10 Cytokine ELISA on the White Blood Cell Population With or Without T Cells. Supernatants of the culture with nothing (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were m easured for this cytokine. Cultures were treated cultured for 24 with its respective growth factor follo wed by stimulation with either LPS or PHA for 48 hours as shown in the figure.

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50 IFN ELISA OF SEPARATED CELLS 0 0.5 1 1.5 2 2.5 3 3.5 WBC LPS C WBC LPS G WBC LPS GM WBC LPS G/GM WBC PHA C WBC PHA G WBC PHA GM WBC PHA G/GM T Cells LPS C T Cells LPS G T Cells LPS GM T Cells LPS G/GM T Cells PHA C T Cells PHA G T Cells PHA GM T Cells PHA G/GM Non T Cells LPS C Non T Cells LPS G Non T Cells LPS GM Non T Cells LPS G/GM Non T Cells PHA C Non T Cells PHA G Non T Cells PHA GM Non T Cells PHA G/GMTypes of cell and treatmentQuantity of IFN (ng/ml) Figure 4-8. Results of IFN Cytokine ELISA on the White Blood Cell Population With or Without T Cells. Supernatants of the culture with nothing (C), G-CSF (G), GM-CSF (G M) or both (G/GM) were measured for this cytokine. Cultures were treated cultured for 24 with its respectiv e growth factor followed by s timulation with either LPS or PHA for 48 hours as shown in the figure.

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51 TNF ELISA OF T versus Non T cells (three experiments)0 0.5 1 1.5 2 2.5 WBC LPS C WBC LPS G WBC LPS GM WBC LPS G/GM WBC PHA C WBC PHA G WBC PHA GM WBC PHA G/GM T Cells LPS C T Cells LPS G T Cells LPS GM T Cells LPS G/GM T Cells PHA C T Cells PHA G T Cells PHA GM T Cells PHA G/GM Non T Cells LPS C Non T Cells LPS G Non T Cells LPS GM Non T Cells LPS G/GM Non T Cells PHA C Non T Cells PHA G Non T Cells PHA GM Non T Cells PHA G/GMTypes of Cells and treatment Amount of TNF (ug/ml) Figure 4-9. Results of TNF Cytokine ELISA on the White Blood Cell Population With or Without T Cells . Supernatants of the culture with nothing (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were m easured for this cytokine. Cultures were treated cultured for 24 with its respective growth factor follo wed by stimulation with either LPS or PHA for 48 hours as shown in the figure.

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52 IL4 ELISA OF T versus Non T Cells (three experiments)0 1 2 3 4 5 6 WBC LPS C WBC LPS G WBC LPS GM WBC LPS G/GM WBC PHA C WBC PHA G WBC PHA GM WBC PHA G/GM T Cells LPS C T Cells LPS G T Cells LPS GM T Cells LPS G/GM T Cells PHA C T Cells PHA G T Cells PHA GM T Cells PHA G/GM Non T Cells LPS C Non T Cells LPS G Non T Cells LPS GM Non T Cells LPS G/GM Non T Cells PHA C Non T Cells PHA G Non T Cells PHA GM Non T Cells PHA G/GMType of cells and treatmentQuantity of IL-4 (ug/ml) Figure 4-10. Results of IL-4 Cytokine ELISA on the White Blood Cell Population With or Without T Cells. Supernatants of the culture with nothing (C), G-C SF (G), GM-CSF (GM) or both (G/GM) were m easured for this cytokine. Cultures were treated cultured for 24 with its respective growth factor follo wed by stimulation with either LPS or PHA for 48 hours as shown in the figure.

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53 similar to that seen in the whole white blood cell population. The PHA activated T cell population pr oduced higher levels of IL-12. This could be important since mobilized blood has ten times more T cells than normal bone marrow products alone. It is thought th at mobilized blood cells have a very important role in fighting against th e tumor by producing the beneficial GVL effect. They also help during engraftmen t engraftment (discussed in chapter 1). Although DCs may be responsible for the init ial secretion of IL-12, T cells appear to be producing most of the cytokine that can shift the balance towards a type 1 immune response. Non-T cells are likely to be important in th e initiation of the selection of the type of response while th e T cells drive the beneficial response in stem cell transplantation as observed by Reddy et al. (2004, 2005). The type 2 cytokine secretion effect s of G-CSF observed were not downregulated significantly by GM-CSF. The P HA stimulated T cell fraction secreted high levels of IL-4 in all experiments while the LPS fraction had no IL-4. IL-10 was induced only in the T cell depleted fraction. PHA-stimulated cells had lower levels of IL-10; especially GM-CSF stim ulated cells which had almost no IL-10. G-CSF stimulated higher IL-10 producti on in the T cell-depleted fraction. Overall, GM-CSF induced a type 1 cyt okine response as early as one day after stimulation. Even though the literature says that GM-CSF induces a type 1 response, most of these studies were done only in combination with other cytokines like IL-4, and used monocyte/ cultured dendritic ce ll population over a period of 6 to 7 days of culture. Our e xperiments show that GM-CSF stimulation of white blood cells up-regulates a type 1 response as early as the first 24 hours of

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54 culture. It was also shown here that GM-CSF not only increases cytokine secretion in the white blood cell population, but apparently most of this secretion, especially of IL-12, comes from the T cell population. 4.3 Generation and Change of the DC1 vs DC2 Ratio as Measured by Flow Cytometry As stated in chapter 2, DC plays an integral part in survival after HSC transplantation. From the works of Arpi nati et al. 2000 we know that G-CSF acts by changing the DC2 population which induces a change towards type 2 responses in the T helper cell group. Furthermore, DCs are a very rare subpopulation in normal peripheral blood. Most studies in vitro require that DC be prepared from monocytes or CD34+ progenitor cells by culturing them in GMCSF and IL-4 for 6 to 7 days. We were in terested in the effect of GM-CSF in DC by itself, as per our hypothesis. Therefore we tested monocytes in culture with GCSF, GM-CSF or G-CSF/GM-CSF over 6 da ys for their capacity to induce the proliferation of allogeneic T cells in culture. tTested GM-CSF ‘s effects by culturing without IL-4 or TNF . Culture conditions resemb le those of the cultures from the previous sections. Monocyte cu ltures were assessed by flow cytometry for their maturation to DC1 and DC2 subt ypes contents. As a control, a set of monocytes was cultured with GM-CSF/ IL-4 known to up-regulate a DC1 phenotype. We should expect the DC2 subtype to be lower in concentration in our cultures because they are thought to aris e primarily from lymphoid progenitors. However DC2 can arise from monocyte progenitors under th e right conditions, and these monocyte-derived DC can then lead to type 2 responses in T cells.

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55 Cells in the monocyte culture were stained to identify DC subsets as described in chapter 3 with lineage (lin) cocktail (anti-CD3, CD4, CD16, CD19, CD20, CD56) conjugated with FITC, AP C-conjugated anti-CD11, PE-conjugated anti-CD123 and PerCP-conjugated anti-HLADR. Isotype controls were run with the monocyte fraction that had no growth f actor treatment (not shown). The flow cytometry was performed in the ICBR’s Fl ow Cytometry Core at the University of Florida. FacScan was utilized to ru n the stained samples and analyzed by CellQuest in their facilities. DCs are a complicated target in flow cytometry due to the complexity of markers needed for their identification since they are identified by a combination of marker s. In humans, DCs do not carry lineage specific markers, such as CD3 on T cells , are HLA-DR positive and either have CD11c expression ( DC1) or CD123 which is the IL3 receptor chain (DC2) (reviewed in chapter 1, Nunez et al. 2004, Bueno et al. 2005, BD Biosciences recommendations). The analysis started by evaluating HLA-DR versus the lineage (Lin) cocktail markers. To determine the gate s and the analysis, plots of cell count versus each marker were done. This helped differentiate the populations inside the plots where two stains were compare d. The first gate was set was for Linand HLA-DR+ cells. The next two plots were of either CD11c or CD123 with HLADR+. Next, two wide gates were drawn; one for DC1 and one for DC2. An analysis is shown on figure 4-11. All an alyses were done in the same way. To compensate for the wide gates we used the contents of those gates to draw a Forward Scatter (FSC) and Side Scatter (SSC) plots (shown in Fig 4.11).

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56 Figure 4-11. Flow Cytometry Analysis of Cultured Monocytes Staining for DC: Separated monocytes were cultured in me dia with control media (C), G-CSF (G), GM-CSF (GM), both (G/GM), Interleuki n 12 (IL-12) or GM-CSF/IL-4 as described in materials and methods for 6 days prior to the addition of LPS. At that point 1 million cells were collected and stained for DC1/DC2 with conjugated fluorescent antibodies.

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57 Table 4-1. Flow Cytometry Statistics of Three Separate Analyses: Separated monocytes were cultured in media w ith control media (C), G-CSF (G), GM-CSF (GM), both (G/GM), Interle ukin 12 (IL-12) or GM-CSF/IL-4 as described in materials and method s for 6 days prior to the addition of LPS. At that point 1 million cells were collected and stained for DC1/DC2 with conjugated fluoresce nt antibodies. Different dates show different cultures (different buffy coats). Results are percentages of gated monocytes obtained with an alysis with CellQuest as described in the text. Experiment 1 LPS added and cultured 48 hours Sample name % DR+ Lin% CD11c % CD123 % Ratio DC1/DC2 MoDC C 63.1721.0111.381.85 MoDC GCSF 63.5820.8110.761.93 MoDC GMCSF 65.5823.5912.11.95 MoDC G/GMCSF 64.8222.4313.071.72 MoDC IL12 64.522.1612.571.76 MoDC GM/IL4 65.0123.2411.761.98 Experiment 2 LPS added and cultured 48 hours Sample name DR+ LinCD11c CD123 Ratio DC1/DC2 MoDC C 83.6460.435.541.69 MoDC GCSF 76.1763.4846.241.37 MoDC GMCSF 66.557.4146.541.23 MoDC G/GMCSF 64.8753.351.921.02 MoDC IL12 76.3863.2644.281.42 MoDC GM/IL4 88.5558.2134.931.67 Experiment 3 LPS added and cultured 48 hours Sample name DR+ LinCD11c CD123 Ratio DC1/DC2 MoDC C 83.4751.919.942.6 MoDC GCSF 77.9847.4721.833.26 MoDC GMCSF 40.156.512.212.95 MoDC G/GMCSF 39.6710.055.981.68 MoDC IL12 49.211.237.221.56 MoDC GM/IL4 48.7714.962.545.89

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58 Percentage of DR+Lincells from the monocyte cultures0 10 20 30 40 50 60 70 80 90 100MoDC CMoDC GCSFMoDC GMCSF MoDC G/GMCSF MoDC IL12MoDC GM/IL4 TreatmentsPercentages Figure 4-12. Graphical Representa tion of Percentage of HLA-DR+ LineageCells From the Total Cells Assayed by Flow Cytometry. Monocytes were cultured with control media (n othing), G-CSF, GM-CSF, both (G/GM), IL-12 or GM-CSF/IL-4 as a positive control. This graph represents an average of the va lues reported on table 4-1. Bars represent standard deviations of triplicate experiments. Percentage of DR+Lincells that are CD11c+ (DC1)0 10 20 30 40 50 60 70 80 90 100MoDC CMoDC GCSFMoDC GMCSF MoDC G/GMCSF MoDC IL12MoDC GM/IL4 TreatmentPercentages Figure 4-13. Graphical Representa tion of Percentage of HLA-DR+ LineageCells that are CD11c+ from the Total Cells Assayed by Flow Cytometry. Monocytes were cultured with c ontrol media (nothing), G-CSF, GMCSF, both (G/GM), IL-12 or GM-CSF/IL-4 as a positive control. This graph represents an average of th e values reported on table 4-1. Bars represent standard deviations of triplicate experiments.

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59 Percetage of DR+Lincells that are CD123+ (DC2)0 20 40 60 80 100 MoDC CMoDC GCSFMoDC GMCSF MoDC G/GMCSF MoDC IL12MoDC GM/IL4 TreatmentsPercentages Figure 4-14. Graphical Representation of Percentage of HLA-DR+ LineageCells that are CD11c+ from the Total Cells Assayed by Flow Cytometry. Monocytes were cultured with c ontrol media (nothing), G-CSF, GMCSF, both (G/GM), IL-12 or GM-CSF/IL-4 as a positive control. This graph represents an average of th e values reported on table 4-1. Bars represent standard deviations of triplicate experiments. Ratio of DC1/DC20.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00MoDC CMoDC GCSFMoDC GMCSF MoDC G/GMCSF MoDC IL12MoDC GM/IL4 TreatmentsRatio Figure 4-15. Graphical Representation of Percentage of HLA-DR+ LineageCells that are CD11c+ from the Total Cells Assayed by Flow Cytometry. Monocytes were cultured with c ontrol media (nothing), G-CSF, GMCSF, both (G/GM), IL-12 or GM-CSF/IL-4 as a positive control. This graph represents an average of th e values reported on table 4-1. Bars represent standard deviations of triplicate experiments.

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60 This ensures that only the cells that are in the upper region are the DCs. Finally another FSC vs SCC plot was added in multic olor to verify that the cells marked are in the right position on the overall m easured population and a gate for overall DC count added to it. This analysis was used for all the cells collected from each experiment for a total of three experime nts. Counts for DC1 and DC2 populations are tabulated in table 4-1. Graphical repr esentations of the percentages of DCs from the flow cytometry from table 41 are shown in figures 4-12 though 4-15. Even though there is certain amount of individual patient variation in the analysis, there were visible populations of each type of DC which were more determined with the cell count plots. The numbers of DCs ranged from 15% to 65% of the total cell population for DC1 and 2% to 40% for DC2 (Table 4-1). GM-CSF treatments did not yield higher DC counts in general as compared to the control culture where no growth factors we re added, to the G-CSF group or to the GM-CSF/IL-4 control. There were no si gnificant changes in the total number of HLA-DR+Lingroups or the DC1 or DC2 groups . When comparing the ratios only GM-CSF/IL-4 group is higher in DC1 which is consistent with its control purpose. (Figures 4-12 th rough 4-15, Table 4-1). 4.4 Effects of GM-CSF and G-CSF on the Proliferation of T Cells Stimulated by Cultured Monocytes with G-CSF, GM-CSF or both The immunogenicity of DCs was me asured in an allogeneic mixed lymphocyte reaction (MLR). Briefly, white blood cells were separated by ficoll and immediately allowed to adhere to a flask. Adherent cells are an enriched monocyte population and were cultured after washing and counting. Culture conditions resemble the ones mentioned for the other experiments except that the

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61 culture lasts for 6 days with maturati on on the 6 day of culture with LPS (as described in chapter 3). After the last 48 hours of incubation, cells were irradiated to arrest their proliferation and then co -cultured with allogeneic T cells (from ficoll separated leukocytes and then one round of magnetic separated cell sorting as described in chapter 3, materials a nd methods). Allogeneic reactions were chosen for the study to correlate our e xperiments with the results seen from allogeneic transplants. BrdU incorporati on was used to measure the proliferation of T cells induced by stimulated monocytes. As seen in figures 4-16 through 4-18 , monocytes treated with GM-CSF did not induce a higher T cell prol iferation as compared to the other cells. It is similar to the GM-CSF and IL-4 treated group. G-CSF also gave a similar T cell proliferation level. Our results are comp atible to Arpinati et al. 2000 in that macrophages stimulated with G-CSF do not demonstrate an increased proliferation in a mixed lymphocyte react ion. In their study, T cells treated with G-CSF and then tested for proliferation showed an inhibitory effect. Future studies are needed to test whether th e T cell phenotypic changes influenced by GM-CSF come from the T cell subtypes (helper CD4+ cells vs. cytotoxic CD8+ cells.

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62 Effects of GCSF and GMCSF on an Allogeneic Mixed Lymphocyte Reaction with Treated Monocytes0.000 0.020 0.040 0.060 0.080 0.100 0.120 0.140 0.160 0.180 0.200 Control 1:1 1:10 Dilution FactorOD Nothing GCSF GMCSF G/GMCSF IL12 GM/IL4 Figure 4-16. Allogeneic M ixed Lymphocyte R eactions on M onocytes (experiment 1): Monoc ytes were culture with control media (nothing), G-CSF, GM-CSF, both (G/GM), IL-12 and GM-CSF/IL-4 as a positive control. T cells were also run with or without PHA as a negative and positive c ontrol to test proliferati on (not shown on graph). E ach point in the series represents three wells. Error bars indicat e the standard deviat ion of three wells.

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63 Effects of GCSF and GMCSF on an Allogeneic Mixed Lymphocyte Reaction with Treated Monocytes 0.000 0.200 0.400 0.600 0.800 1.000 1.200 Control 1:1 1:10 Dilution FactorOD Nothing GCSF GMCSF G/GMCSF IL12 GM/IL4 Figure 4-17. Allogeneic M ixed Lymphocyte R eactions on M onocytes (experiment 2): Monoc ytes were culture with control media (nothing), G-CSF, GM-CSF, both (G/GM), IL-12 and GM-CSF/IL-4 as a positive control. T cells were also run with or without PHA as a negative and positive c ontrol to test proliferati on (not shown on graph). Th e three graphs are three separate experiments. Each point in the series represents three wells. Error bars indicate the standard deviation of three well.

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64 Effects of GCSF and GMCSF on an Allogeneic Mixed Lymphocyte Reaction with Treated Monocytes0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350 Control 1:1 1:10 Dilution FactorOD Nothing GCSF GMCSF G/GMCSF IL12 GM/IL4 Figure 4-18. Allogeneic M ixed Lymphocyte R eactions on M onocytes (experiment 3): Monoc ytes were culture with control media (nothing), G-CSF, GM-CSF, both (G/GM), IL-12 and GM-CSF/IL-4 as a positive control. T cells were also run with or without PHA as a negative and positive cont rol to test proliferation ( not shown on graph. Each point in the series represents three wells. Error bars indicate the standard de viation of three wells

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65 CHAPTER 5 CONCLUSIONS AND FUTURE PROSPECTS Our results demonstrate that the levels of IL-12, TNF and IFN were increased after addition of GM-CSF to a culture of PBMC, and that th e levels of IL-10 and IL-4 were decreased. These findings indicate that type 1 cytokines are higher while type 2 cytokines when GM-CSF has added to the cultu re media. We found that IL-12 levels in the whole leukocyte population increased, and that there was a highe r concentration of IL-12 produced by T cells isolated from GM-CSF stimulated cultures. Further research is indicated to determine if treating allogeneic stem cell grafts with GM-CSF can lead to improved survival of transplant recipien ts by stimulating the production of IL-12 posttransplant since high levels of IL-12 have been associated with improved clinical outcomes with less relapse and without an increase in GVHD (Reddy et al. 2004, 2005). Our type 2 cytokine results differ from those of Sloa nd et al. (2000) who showed that G-CSF exposed T cells induce an imm une response towards T h2 by eliciting levels of IL-4. We found that the leve ls of IL-10 (another type 2 cytokine) produced during the first 24 to 48 hours of culture of leukocytes is the result not of th e T cell population but of the T cell depleted population. The question of why GM-CSF decreases the levels of Il10 but not of IL-4 merits further study. One hypothesis is that IL-10 down regulation might be the result of two possible mechan isms. First, that GM-CSF regulated type 1 response and second that it decreases type 2 cytokines, further shifting the balance towards an immunogenic pro-inflammatory re sponse. However, T cell proliferation results may vary depending on the incubation period of monocytes af ter stimulation with

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66 G-CSF and GM-CSF. Additional studies are needed to confirm our findings. GM-CSF stimulation of adherent monocytes incr ease the overall DC population nor did it significantly change the DC1/DC 2 ratio therefore, this lack of GM-CSF effects on DC growth may be another explanat ion for the lack of increased T cell proliferation in the mixed lymphocyte reactions. Further work will be needed to corrobor ate the findings obtained here. Such research might include culturing T cells of 6 to 7 days with th e growth factors such as the monocyte cultures. This work might establish whether the T cells are more susceptible to the effects of GM-CSF. Our work with GM-C SF is consistent with our initial hypothesis of a type 1 response and is also consistent with the literatu re on the subject. Our results also indicate that the effect of GM-CSF on donor cells is the result no t of an altered ratio of DC1/DC2 but rather of a pr ocess in which T cells are induced to produce the majority of the cytokines that shift the balance.

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72 Moss P, Rickinson A, January 2005, “Cellula r immunotherapy for viral infection after HSC transplantation,” Nature Re views Immunology, Volume 5, pp 9-20. Murphy KM, Reiner SL, December 2002, “The Lineage decisions of helper T cells,” Nature Reviews Immunology, Volume 2, pp 933-944. Nagata S, McKenzie C, Pender SLF, Bajaj-Elliott M, Fairclough PD, Walker-Smith JA, Monteleone G, MacDonald TT, 2000, The Journal of Immunology, Volume 165, pp 5315-5321. Nestle FO, Banchereau J, Hart D, July 2001, “Dendritic cells: on the move from bench to bedside,” Nature Medicine, Volume 7, Number 7, pp 761-765. Mikolich-Zugich J, Slifka MK, Messaoudi I, February 2004, “The many important facets of T-cell repertoire diversity,” Na ture Reviews Immunology, Volume 4, pp 123132. Nouri-Shirazi M, Banchereau J, Bell D, Burkeholder S, Kraus ET, Davoust J, Palucka KA, “Dendritic cells capture killer tumor cells and present their antigens to elicit tumor specific immune responses,” The Journal of Immunology, Volume 165, pp 3797-3803. Nunez R, Garay N, Bruno A, Villafane C, Bruno E, Filgueira L, July 2004, “Functional and structural characterization of tw o populations of human monocyte-derived dendritic cells,” Experimental and Molecular Pathology, Volume 77, pp 104-115. Orange DE, Jegathesan M, Blachere NE, Fr ank MO, Scher HI, Albert ML, Darnell RB, September 2004, “Effective antigen cross-pr esentation by prostate cancer patients’ dendritic cells: implications for prosta te cancer immunotherapy,” Prostate Cancer and Prostatic Diseases, Volume 7, pp 63-72. Pan L, Teshima T, Hill GR, Bungard D, Brinson YS, Reddy VS, Cooke KR, Ferrara JLM, Hune 15th 1999, “Granulocyte colony-stimulating factor-mobilized allogeneic stem cell transplantation maintains gr aft-versus-leukemia effects through a perforin-dependent pathwa y while preventing graft-ve rsus-host-disease,” Blood, Volume 93, Number 12, pp 4071-4079. Parajuli P, Mosley RL, Pisarev V, Chavez J, Ulrich A, Varney M, Singh RK, Talmadge JE, June 2001, “Flt3 ligand and granulocyt e-macrophage colony-stimulating factor preferentially expand and stimulate di fferent dendritic and T-cell subsets,” Experimental Hematology, Volume 29, pp 1185-1193. Pasare C, Medzhitov R, November 2004, “To ll-dependent control mechanisms of CD4 T cell activation,” Immunity, Volume 21, pp 733-741.

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78 BIOGRAPHICAL SKETCH Erika Adriana Eksioglu was born and raised in Caracas, Venezuela. She started her studies in biology at the Univer sidad Simon Bolivar also in Caracas. After two years she transferred to the University of Florida and entered the College of Agriculture to pursue a major in microbiology and cell sc iences. She obtained her Bachelor of Science degree in December 1998. After college she worked at the Department of Pharmacology at the University under Dr. Phillip Scarpace until September 1999. She then moved to Atlanta, Georgia, where she worked at Yerkes Primate Research Center for 3 years at the Tetramer Core Facility under Dr. John Altman and Dr. J ohn Lippolis. In August 2003 she moved back to Gainesville to pursue her master’s degree at th e University of Florida in the College of Medicine. There she was under the mentorship of Dr. Vijay Reddy and worked on the role of growth factors in modulation of the immune response and on dendritic cell biology in the division of hema tology/oncology. After graduation she will continue on to pursue a doctorate also at the Univers ity of Florida’s Co llege of Medicine.