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Staphylococcal Enterotoxin Enhancement of Inflammatory and Regulatory Cytokine Production and Humoral Responses

University of Florida Institutional Repository

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STAPHYLOCOCCAL ENTEROTOXIN ENH ANCEMENT OF INFLAMMATORY AND REGULATORY CYTOKINE PROD UCTION AND HUMORAL RESPONSES By AMY KRISTIN ANDERSON 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 2003

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I dedicate this work to my parents and my brother Tim, for always being there and supporting me in whatever I do; and to Glenn, for always be ing there for me for anything.

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ACKNOWLEDGMENTS I would like to thank Dr. Howard M. Johnson for giving me the opportunity to do research in his laboratory; and for providing ideas and insight on all the projects I have worked on. I would also like to thank my other committee members, Dr. Ed Hoffman and Dr. Peter Kima for their assistance with my research. Another thank-you goes to Dr. Mustafa Mujtaba in Dr. Johnsons lab who has assisted me with many things in the lab and my research. I would also like to acknowledge all of the other lab members as well as my fellow graduate students, Margi and Lawrence, for always helping out in any way possible. I would like to especially thank my family, who has always supported me in whatever I have chosen to do. A last special thank-you goes to my husband, Glenn, who is always there for me for anything I need. He was a large part of the motivation I needed to go to graduate school. iii

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS .................................................................................................iii LIST OF FIGURES ...........................................................................................................vi LIST OF ABBREVIATIONS ..........................................................................................vii ABSTRACT .....................................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 General Superantigen Information...............................................................................1 Interactions of Superantigens with Immune Cells and Peptide Processing..................1 Activation of Cells by Superantigens...........................................................................2 Superantigens in Disease..............................................................................................4 Uses of Superantigens in Vaccines and in the Treatment of Disease...........................5 2 MATERIALS AND METHODS.................................................................................8 Human Peripheral Blood Mononuclear cells (HPBMC) Isolation and Cell Culture....8 Human CD4+ T Cell Isolation......................................................................................8 Staphylococcal Enterotoxin Treatment of HPBMC and CD4+ T-cells........................9 Total RNA Isolation.....................................................................................................9 Microarray Procedure.................................................................................................10 Analysis of Microarray Data......................................................................................10 Western Blot for TGF3.............................................................................................10 ELISA for Human Cytokines.....................................................................................11 Mouse Studies and Detection of Specific Antibodies in Mouse Sera........................12 Proliferation Assay.....................................................................................................13 3 RESULTS...................................................................................................................14 Superantigens Enhance Specific Antibody Production to Antigens in Mice.............14 Superantigens Enhance Cytokine RNA Production in HPBMC................................15 SEA Induces Cytokine Gene Expression in HPBMC.........................................15 SEB Induces Cytokine Gene Expression in HPBMC.........................................16 SEB Induces Cytokine Gene Expression in CD4+ T cells.........................................16 Superantigens Enhance Cytokine Protein Production in HPBMC.............................24 TGF Suppresses IL-2 Induced Growth in NK92 Cells in vitro................................29 iv

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4 DISCUSSION.............................................................................................................30 APPENDIX A MICROARRAY PROTOCOL...................................................................................34 B MICROARRAY GENE LIST....................................................................................35 LIST OF REFERENCES...................................................................................................35 BIOGRAPHICAL SKETCH.............................................................................................39 v

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LIST OF FIGURES Figure page 1-1 Typical Peptide Antigen and Superantigen Interactions with T-cells and APC .......2 3-1 Antigen and isotype specificity of superantigen enhancement of antibody to BSA17 3-2 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase expression of various human cytokine genes.............................................................................19 3-3 Staphylococcal enterotoxin A induces increased expression of various Th1, Th2, and T regulatory cytokine genes..............................................................................20 3-4 Staphylococcal enterotoxin B induces increased expression of various Th1, Th2, and T regulatory Cytokine Genes.............................................................................21 3-5 Staphylococcal enterotoxin B induces increased expression of various Th1, Th2, and T regulatory cytokine genes..............................................................................22 3-6 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-2 production in human PBMC....................................................................................25 3-7 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IFNproduction in human PBMC....................................................................................26 3-8 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-10 Production in Human PBMC...................................................................................27 3-9 Staphylococcal enterotoxin A and staphylococcal enterotoxin B do not increase TGF production in human PBMC..........................................................................28 3-10 Transforming growth factor beta suppresses IL-2 induced proliferation of NK-92 cells...........................................................................................................................29 vi

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vii LIST OF ABBREVIATIONS Ab: Antibody APC: Antigen presenting cell BCA: Bicinchoninic Acid BSA: Bovine serum albumin cDNA: Complementary deoxyribonucleic acid ELISA: Enzyme linked immunosorbent assay FBS: Fetal bovine serum HPBMC: Human periphera l blood mononuclear cells HRP: Horseradish peroxidase IFN: Interferon IL: Interleukin NK: Natural killer cells PBMC: Peripheral blood mononuclear cells PBS: Phosphate buffered saline RNA: Ribonucleic acid SEA: Staphylococcal enterotoxin A SEB: Staphylococcal enterotoxin B TGF: Transforming growth factor TNF: Tumor necrosis factor

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STAPHYLOCOCCAL ENTEROTOXIN ENHANCEMENT OF INFLAMMATORY AND REGULATORY CYTOKINE PRODUCTION AND HUMORAL RESPONSES By Amy Kristin Anderson August 2003 Chair: Howard M. Johnson Cochair: Edward Hoffman Major Department: Microbiology and Cell Science Superantigens are microbial proteins that are the causative agents of food poisoning and toxic shock syndrome. Furthermore, superantigens can cause deregulation of the immune system and can exacerbate autoimmune diseases such as multiple sclerosis. Superantigens induce a burst of T-cell proliferation and activation. We analyzed in vivo and in vitro, superantigen activation of the cells of the immune system in the context of expression of genes associated with various T-cell subsets. Human peripheral blood mononuclear cells (HPBMC) treated with staphylococcal enterotoxin A (SEA) showed increased expression of the cytokines IFN-, IL-2, IL-10, TNF-, TNF-, TGF-, and IL-6 as deduced by microarray analysis of cell mRNA. A similar pattern of genes was upregulated in HPBMC treated with staphylococcal enterotoxin B (SEB). Th1, Th2 and T regulatory cells produce these cytokines. Expression for most of these cytokines is viii

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maximal 24 to 48 hours post superantigen treatment. Enzyme Linked Immuno Sorbent Assay (ELISA) tests for IFN-, IL-10 and IL-2, showed increased mRNA levels as seen on the microarray correlate with translation, with the exception of TGF, which was not detectable by ELISA or Western Blot. In addition, purified CD4+ T-cells treated with SEB show a similar pattern of upregulated cytokines as shown for superantigen treated HPBMC. In vivo experiments showed that SEA and SEB increase specific IgG (but not IgM) antibody production in mice immunized against bovine serum albumin (BSA). Thus, superantigens increase both the specific humoral and cellular immune responses against antigens. Based on the cytokine profile elicited by superantigens in vitro and the specific antibody production in vivo, superantigens enhance Th1, Th2 and T regulatory cytokine production. This property of superantigens makes them an ideal candidate to boost specific immunity; and may have prophylactic applications in the prevention of cancer and other immunemediated diseases. ix

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CHAPTER 1 INTRODUCTION General Superantigen Information Superantigens are microbial proteins produced by various bacteria and viruses including (Staphylococcus species, Streptococcus species, HIV and rabies viruses) and are powerful activators of CD4+ T-cells. (7,10) Common superantigens include the staphylococcal enterotoxins (A-E) and toxic shock syndrome toxin 1 (TSST-1) which are produced by Staphylococcus aureus (32). The enterotoxins are common causative agents of toxin-mediated food poisoning and TSSTs are implicated in toxic shock syndrome (32). Superantigens are also suspected to play a role in exacerbation of autoimmune diseases such as multiple sclerosis as well as the immunodeficiency associated with HIV infection (8,10,27,36,33). These acute and chronic disease states associated with superantigen are caused by the massive proliferation and activation of T-cells that is induced by superantigens (2). Interactions of Superantigens with Immune Cells and Peptide Processing The potency of the superantigens in relation to T-cell activation and proliferation is in part explained by its unique interaction with the T-Cell Receptor (TCR) on T cells and the major histocompatibility complex II (MHC II) on antigen presenting cells (APC) (30). As shown in Figure 1-1, superantigens bind directly to MHC II (16) and this complex interacts with a specific portion of the V region of the TCR. Each superantigen activates different V regions. TSST-1 activates T-cells with V2 while SEB activates cells with V3, V 12, V 14, V 15, V 17, and V 20 (2,15). 1

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2 The interaction with the TCR is antigen-independent and requires no further processing as seen with a typical peptide antigen. Typical peptide antigens are processed internally and presented by APC on either MHC I or MHC II molecules on their cell surface. By acting in this manner, superantigens can induce polyclonal activation of up to 20% of the total T-cell population at one time, as compared to typical peptide antigens that only activate up to 0.01 % of the total T-cell population at one time. Figure 1-1: Typical Peptide Antigen and Superantigen Interactions with T-cells and APC. (Torres, B.A., S.L. Kominsky, G.Q. Perrin, A.C. Hobeika, and H.M. Johnson. 2001. Superantigens: The good, the bad and the ugly. Exp. Med. Biol. 226:164). Activation of Cells by Superantigens Superantigens activate CD4+ T-cells. These T-cells are preferentially activated because of their increased binding affinity for MHC II (12). The CD4+ T cells can be divided into three groups: T helper 1 (Th1), T helper 2 (Th2) and T regulatory cells. Th1 cells are one T-cell group activated by superantigen. Th1 cells are associated with inflammatory responses and produce such cytokines as IL-2, IFNand tumor necrosis factor (21). IL-2 drives further T-cell proliferation and activation. One of the main functions of IFNis macrophage activation through upregulation of MHC I and MHC II. It also has powerful antiviral effects. It enhances CTL, natural killer and macrophage

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3 cell tumoricidal activity (32). Tumor necrosis factor (TNF) induces macrophages to produce NO, as well as activating the vascular endothelium. The Th2 cells are also activated by superantigens. Th2 cells are associated with humoral responses and anti-inflammatory responses. This group of cells is thought to be the classic adversary to the actions of Th1 cells (3). Th2 cells produce such cytokines as IL-4 and IL-6. Interleukin 4 is an anti-inflammatory cytokine produced by Th2 cells. Interleukin-6 is a cytokine with both anti-inflammatory and inflammatory properties. It can induce acute phase response in the liver and drives B-cell differentiation (3). A third group of cells called T-regulatory cells also are activated by superantigens, especially cells undergoing repeated treatments with superantigen (17). T regulatory cells are CD 4+ and express CD25. (18) They play important roles in the maintenance of peripheral tolerance. Typical cytokines produced by T-regulatory cytokines include TGFand IL-10 and IFNwere detected (6,17). Interleukin 10 downregulates MHC II expression and costimulatory molecules like CD 80/86, inducing anergy in CD4+ T cells (22). On the contrary, IL-10 aids in the survival of B-cells and plays a part in their differentiation. It has also been shown in mice knockouts for CD25, that stimulation with superantigens results in uncontrolled release of pro inflammatory cytokines, but that injection with CD4+ CD25 T-cells control this process (20). This indicates that regulatory cells play a role in controlling superantigen activation of T-cells. TGFis another type of regulatory cytokine. TGFinhibits IL-2 production, and has anti-proliferative effects on T-cells. TGFalso blocks the differentiation of Th1 and Th2 cells through blocking of transcription factors.

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4 Superantigens in Disease Superantigens are involved in both shortand longterm acute and chronic disease states. Staphylococcal enterotoxins A (SEA) are responsible for the most food poisoning caused by superantigens (9,32). The acute gastrointestinal illness resulting from ingestion of the toxin is short lived. Toxic shock syndrome (TSS) caused by TSST-1 is also an acute disease caused by superantigens (1,24,32). TSS became well studied in the 1980s when it became associated with tampon use in women during menstruation. Clinical manifestations of TSS include rashes, fever, severe hypotension, and possible fatal shock. While the acute effects of superantigens are somewhat severe, the involvement of superantigens in exacerbation of autoimmune diseases persists over long periods of time. The involvement of superantigens in autoimmune disease is demonstrated in a murine model of multiple sclerosis (MS), called experimental allergic encephalomyelitis (EAE), which is induced in mice by injection of myelin basic protein (MBP). The T-cell population in mice responsible for EAE has a V8+ specificity (38). Pretreatment of PL/J mice with SEB prevents induction of EAE by MBP (27). This is most likely due to anergy and deletion of VB8+ T-cells by SEB (10). However, SEB has also been shown to reactivate EAE in mice that have been immunized with MBP and recovered from an initial episode of the disease (9). This phenomenon is not limited to SEB, since SEA has also been shown to reactivate EAE (9,26) showing that different V specificities may also be involved in autoimmune disease. Superantigens are also thought to be involved in some immunodefiency diseases, such as HIV. A regulatory gene product encoded in the 3 LTR of the HIV genome

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5 (called Nef) has been shown to block binding of SEA to Raji cells; and to induce T-cell proliferation, IL-2 production, and IFNproduction (32,33). Other evidence of superantigen characteristics include selective expansion of particular V sets (V3, 5.3 and 18) (29,34) and need for APC for Nef induced activation of T-cells (37). Another study has shown that blocking Nef with an anti-Nef antibody does not support HIV replication (34) suggesting that Nef is necessary for HIV to maintain itself in a host. HIV requires activated CD4+ T cells for replication (36); and a superantigen-like molecule is one way for the virus to achieve this. These studies together show that Nef has typical characteristics of a superantigen and its action induces a pool of cells for viral replication, which aids in the spread of the virus in a host. Uses of Superantigens in Vaccines and in the Treatment of Disease Superantigen effects are generally considered to be negative, especially in the context of food poisoning, TSS, and their involvement in chronic disease states. However, the very characteristics and interactions of superantigens also make them ideal candidates for use in vaccines, prophylactically and as treatment of diseases. In order to achieve this, the proliferation and cytokine production must be controlled and biased to a certain direction. In this way, the effects of superantigens would be used to improve and bolster immune responses as opposed to deregulating immune function. A mouse model of melanoma has been used to show how superantigens can be prophylactically effective as vaccine against melanoma (14). C57BL/6 mice were injected with irradiated B16F10 melanoma cells, followed by SEA and SEB 6 and 10 days later. Three days after SEA and SEB treatment, mice were challenged with live B16F10 cells. Median survival time was >150 days for those mice injected with

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6 superantigens, as opposed to 14 to 23 days in mice with no treatment or treatment with cells only or superantigen only (14). In addition, surviving mice were rechallenged with live tumor and 75 percent survived. (14) The staphylococcal entertoxins (SE) are helping to boost anti-tumor activity through activation of a large number of T-cells already primed with the tumor antigen. Another study takes a different approach to the use of staphylococcal enterotoxins in the treatment of melanoma (23). Mice were injected with c215 transfected B16 melanoma cells followed by injection of SEA fused to a C215 tumor reactive antibody, along with IL-2 fused to the same antibody. Prolonged survival was seen in mice receiving both treatments, as opposed to each treatment alone (23). Repeated cycles of treatments also increased survival time (23). This method directs SEA directly to the tumor, with the use of the antibody. The use of IL-2 as well, adds to the proliferation induced alone by SEA. A third approach uses plasmid encoded SEB and either IL-2 or GMCSF (5). Dogs with melanoma tumors received intratumor injections of lipid complexes with plasmid DNA encoding SEB and either IL-2 or GMCSF. Partial tumor regression, and in some cases, complete regression was seen in dogs receiving this treatment (5). Little toxicity was seen in the dogs given these treatments. The treatment is directed to the tumor through intratumoral injection and SEB is produced in the animal. IL-2 acts as an additional proliferation agent, as in the previous experiment. All of the above studies show in different ways how superantigens may be used as prophylaxis for cancer as well as ways to treat cancer once tumors have already

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7 established in the body. This is only one approach to the use of superantigens in the treatment and prevention of disease. In this study, through a combination of molecular biology techniques such as cDNA microarray analysis and traditional immunological procedures, such as ELISA and western blot techniques, specific cytokine production during superantigen stimulation of HPBMC will be assessed in the context of activation of distinct groups of T cells. The studies are designed to gain insight into how superantigens modulate lymphocyte function in disease and immune therapy.

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9 CHAPTER 2 MATERIALS AND METHODS Human Peripheral Blood Mononuclear cells (HPBMC) Isolation and Cell Culture Human donor leukocyte packs were obtained from Civitan Regional Blood Center (Gainesville, FL). HPBMC were isolated using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation as per the manufacturers instructions. After the removal of the cells from the gradient and 2 washes with RPMI 1640 culture media supplemented with 10% heat-inactivated fetal bovine serum (FBS), 200 U/mL penicillin, and 200 mg Streptomycin, the cells were resuspended in fresh supplemented culture media and counted using a light microscope. 2 x 10^ 6 cells/well were plated in 24 well plates and incubated at 37C in a 5% CO2 atmosphere and used immediately for further experimentation Human CD4+ T Cell Isolation Human donor leukocyte packs were obtained from Civitan Regional Blood Center (Gainesville, FL). An enrichment cocktail (Rosette Sep)for human CD4+ T cells (Stem Cell Technologies, Vancouver, BC) was used to enrich for CD4+ T cells, followed by isolation of the cells using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation as per the manufacturers instructions. After removing cells from the gradient and 2 washes with RPMI 1640 culture media, supplemented with 10% heat inactivated fetal bovine serum (FBS), 200 U/ml penicillin, and 200 mg Streptomycin, the cells were resuspended in fresh supplemented culture media and counted using a light microscope. 8

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9 2 x 10 6 cells/well were plated in 24 well plates and incubated at 37 C in a 5% CO2 atmosphere and used immediately for further experimentation. Staphylococcal Enterotoxin Treatm ent of HPBMC and CD4+ T-cells Staphylococcal enteroto xin A (SEA) (Toxin Technology, Sarasota, FL) and B (SEB) (Toxin Technology, Sarasota, FL) were cultured with 2 x 10 6 HPBMC per well in a 24 well plate, both at a concentration of 100 ng/ml. CD4+ T-cells (2 x 10 6 cells per well) were treated with 100 ng/ml of SEB. SEB does not requir e APC for processing (4,30). Media treated cells served as a control. Six wells were used for each treatment at each time point. Cells were incubated as described above and were harvested from the cultures at time points of 8h, 16h, 24h, and 48h. Following centrifugation for 10 minutes at room temperature, cell pellets and supernatant. were separated and used for RNA isolation and ELISA/Western Blot experiments respectively. Total RNA Isolation RNA from SEA and SEB or media trea ted HPBMC as described above was obtained using the following RNA isolation kits : Absolutely RNA (Stratagene, LaJolla, CA), RNAqueous (Ambion, Austin, TX), a nd TRIZOL Reagent (GibcoBrl, Carlsbad, CA). The protocols were followed as desc ribed by the manufacturer. The RNA samples were quantitated by reading absorban ce at 260nm on a Gilford Instrument Spectrophotometer 260 (Nova Biotech, El Caj on, CA). The RNA samples were diluted 1:100 in 10mM Tris or DNAse and RNAse free water. Th e following calculation was used to determine the concentrations of RNA in g/ l: [A 260 x 100 x .040 ug/ul]. Following quantitation, RNA samples were stored at -80 C for future use in microarray experiments described below.

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10 Microarray Procedure Microarray procedures were performe d as described by the manufacturer (SuperArray, Frederick, MD). Briefly, tota l RNA was used to prepare cDNA probes. During this process the cDNA was labeled with biotinylated dUTPs. The probes were denatured and allowed to hybridize overnight at 60C to the microarray membrane. The nylon microarray membrane is spotted w ith cDNA from 96 common human cytokines purchased from SuperArray Inc (Frederick, MD). Following a series of washes and further blocking, the membrane was incubated with alkaline phosphatase-streptavidin. CDP Star substrate was then added to the membrane. The gene microarray membranes were exposed to film and developed. Th is procedure was follo wed as recommended by the manufacturer (Super Array, Frederick, MD). A schematic of the entire procedure can be found in Appendix B. Analysis of Microarray Data The film images of the media and s uperantigen treated microarray membranes were scanned and converted to a TIFF format. The images were then analyzed using the Image J 1.29 software (NIH, Bethesda, MD) and Scion Image software, to determine the pixel density value for the spot of a desired gene. Each intensity value was normalized to the positive control on its re spective membrane, then divi ded by the corresponding media control values. This value is a fold increase ratio of superantigen treated cells to media treated cells. Western Blot for TGF 3 HPBMC were cultured with or without superantigens described above and the resulting supernatants were saved at -80 C and used for western blot analysis of TGF3. The supernatants were concentrated using Amicon centriprep YM-10 filters (Millipore,

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11 Billerica, MA). A Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL) was performed on the supernatants from control (media) or supera ntigen treated cells. Equal amounts of each sample (26 g/lane) were loaded on a 15 % reducing SDS-PAGE ready gel (BioRad, Hercules, CA) and run at 100V. Overnight transfer onto nitrocellulose membrane was carried out, after which the me mbrane was block with 5% non-fat instant milk in Tris-buffered saline (pH 7.5) and .01% Tween-20 for 1 hr. The Immunoblot was incubated with a 1:1000 dilution of rabbit anti-human TGF 3 (Santa Cruz Biotechnology, Santa Cruz, CA). After wash ing, a conjugated s econdary antibody, goatanti-rabbit conjugated to horseradish peroxidase (H RP), (Santa Cruz Biotechnology, Santa Cruz, CA) was added at a 1:12,000 dilu tion and incubated for 1 h. Blots were washed and analyzed through film development. ELISA for Human Cytokines HPBMC were cultured with or without superantigens described above and the resulting supernatants were saved at -80 C a nd used for ELISA assays. The supernatants were concentrated using Amicon centriprep YM10 filters (Millipore, Billerica, MA). A BCA protein assay (Pierce, Rockford, IL) was performed on the supernatants from control (media) or superantigen treated cells. Equal amounts of each sample were used in the ELISA assays. HPBMC were treated with media or superantigen and supernatants were collected at various time points as described above. IL-2, IL-10, and IFNlevels were determined using ELISA kits. The supernatants were tested for IFNusing the CytoScreen Immunoassay kit for IFN(Biosource International, Camarillo, CA), IL-2 using the BD-Opt-EIA kit for IL-2 (BD Bi osciences), and IL-10 using the BD-OptEIA kit for IL-10 (BD Biosciences). Color development was monitored at 490nm in an ELISA plate reader (Biorad, Richmond, CA) af ter substrate solution from each respective

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12 cytokine kit was added and reaction stopped w ith the stopping solution provided in each kit. Mouse Studies and Detection of Specific Antibodies in Mouse Sera We used 6 to 8 week old female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) in these studies. Mice were bl ed before injections with the BSA. We injected 50 g of BSA intraper itoneally (i.p.) into the mice. One week later, mice were injected i.p. with PBS or a combination (25 g each) of SEA and SEB (Toxin Technology, Sarasota, FL). Mice were bled fr om the tailvein once a week for one month; and sera were stored at -20 C for further experiments. Sera from the mice were tested for BS A specific IgG and IgM antibodies using a standard ELISA protocol. Briefly, 50 l of BSA (25 ng/well) in binding buffer (0.1 M carbonate/bicarbonate, pH 9.6) were placed in wells of 96 well plates and allowed to adhere overnight at room temperature. Plates were washed in wash buffer (150 mM NaCl, 0.05% Tween 20) and free reactive site s were blocked for 2 h with 200 l/well blocking buffer (PBS (pH 7.2) containing 5% nonf at instant milk). After washing plates, sera were diluted and 50 ul were placed in the wells for 1.5 h. Plates were again washed and alkaline phosphataseconjugated anti-mous e IgG whole molecule or anti-mouse IgM (50 ul ; Sigma Aldrich, St.Loui s, MO) was added to wells. After 45 minutes, plates were washed and 200 ul of substrate (1mg/ml p-nitrophenyl phosphate in binding buffer) was added to the plates. Color was allowed to develop for 30-60 minutes, after which 50 ul of stop solution (2 M H 2 SO 4 ) was added. Absorbance was read at 405 nm using a Model 450 Bio-Rad Microplate reader (BioRad, Hercules, CA).

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13 Proliferation Assay Human NK-92 cells (ATCC, Mannassas, VA), a natural killer cell line requiring IL-2 for growth, were cultured in alpha minimum essential medium without ribonucleosides and deoxyribonucleosides with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum and 12.5% fetal bovine serum at 37 C. The cells were plated at 4 x 10 4 cells per well in a 96 well plate and treated with either media, IL-2 (Biosource International, Camarillo, CA) (30 U/ml), or IL-2 and TGF (25 ng/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) for 48 hours. Cells were then pulsed with 1 Ci per well of 3 H-thymidine for 6 hours, after which cells were harvested an cell associated radioactivity was quantified using a -scintillation counter and activity reported as mean CPM +/SD. All tests were run in replicates of six.

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14 CHAPTER 3 RESULTS Superantigens Enhance Specific Antibody Production to Antigens in Mice It has been shown previously that superantigens enhance the immune cellular response against melanoma cells in vivo in mice (15). Here a determination was made of the ability of SEA and SEB to enhance the humoral antibody response of mice primed with the T dependent antigen bovine serum albumin (BSA). Mice were first injected with BSA alone, BSA followed seven days later by SEA/SEB, SEA/SEB alone, or PBS alone. As can be seen in the ELISA measurements in Figure 3-1A, BSA alone induced an antibody response that was enhanced greater than 2 fold By SEA/SEB at a 1:100 dilution of sera. There was no antibody response to the control antigen gp120 in the same sera. This was evidenced by the low similar ELISA profiles for BSA alone, BSA followed by SEA/SEB, SEA/SEB alone, or PBS alone. Thus, the enhancement of SEA/SEB was specific for the primary antigen BSA. Furthermore, the antibody response to BSA was IgG specific, but not IgM specific. At 14 days following BSA injection there was no evidence of specific IgM antibodies to BSA in sera of mice, as per Figure 3-1B, where the BSA response was compared with that of PBS. Importantly, a comparison of mice injected with BSA and BSA followed by SEA/SEB showed the same profile of IgM response. Thus, SEA/SEB did not enhance the IgM response under the same conditions under which it enhanced the IgG response. Since specific IgG levels were increased against BSA, total IgG levels were compared among the different groups. Mice treated with BSA alone, BSA followed by SEA/SEB, SEA/SEB alone or PBS alone, did not 14

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15 show any significant differences in total IgG levels, as shown in Figure 3-1C. Thus, an enhancement effect of SEA/SEB on the total IgG levels was not observed. Furthermore, the enhancement of the an tibody response to BSA by SEA/SEB could not be attributed to non-specific enhancement of total IgG. Therefore, the SEA/SEB enhancement of the antibody response to BSA was antigen specific and of the IgG isotype. Superantigens Enhance Cytokine RNA Production in HPBMC A determination of the ability of SEA and SEB to induce increased cytokine production was first made through microarray anal ysis of RNA in cells treated with the SAgs. Human PBMC were tr eated with 100 ng/ml of SEA or SEB, or culture media alone. Cells were harvested from the culture at timepoints of 8, 16, 24, and 48 hours. Total RNA was extracted from the cells a nd was used to synthesize cDNA. The cDNA was allowed to hybridize to a nitrocellulose membrane that was spotted with various human common cytokine genes. Representative microarray images of media, SEA, and SEB treatments are shown in Figure 3-2. An alysis of the microarray membranes and supernatants of the treated cells revealed that both SEA and SEB induce increased gene expression in HPBMC as compared to those of media control cells. Appendix B contains a list of all the cytokines coded fo r the on the microarray membrane. SEA Induces Cytokine Gene Expression in HPBMC As shown in Figure 3-3, SEA induces upre gulation of several genes including IFN, IL-2, CD40L, IL-10, IL-6, TNF TGF and IL-1 with as much as 2-25 fold increase over media in HPBMC. IFN expression was similar at all four time points (5-15 fold increase over media), where as e xpression of IL-2, IL-10 and IL-1 were maximal at 16 hours after SEA incubation. TGF, IL-6 and CD40L had maximal expression at 24

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16 hours as compared to media controls. SEA induction of TNF gene expression was approximately 2 fold greater than media at 8 hours, where as it was approximately 16 fold greater than media at 16-48 hours. Thus, SEA increased cytokine gene expression in HPBMC as compared to media treated cells. SEB Induces Cytokine Gene Expression in HPBMC The cytokine gene expression induced by SEB in HPBMC was determined next. As shown in Figure 3-4, SEB increased cytokine expression of several genes as compared to media controls, although expression levels were lower than that was seen with SEA treatment. SEB induction of IFNand IL-1 was maximal at 16 hours, IL-6 and TGF at 24 hours, and IL-10, TNF and CD40L at 48 hours as compared to the media control. IL-10 and TGF are regulatory cytokines, so the relative delay in their gene expression could indicate a signal to modulate Th1 cell cy tokines. Therefore, SEB also upregulates cytokine gene expression in HPBMC. SEB Induces Cytokine Gene Expression in CD4+ T cells Previously, it has been shown that supe rantigens activate CD4+ T cells (37). Here we investigated the profile of cytokine gene expression in CD4+ T cells treated with SEB. SEB does not require antigen presenting cells for processing (4,30), thus CD4+ T cells were purified from HPBMC using th e Rosette Sep procedure as described in Materials and Methods, and then treated with 100 ng/ml of SEB for timepoints of 8, 16, 24, and 48 hours. As shown in Figure 3-5, sim ilar cytokine genes we re upregulated as those seen above for HPBMC. Expression levels for TGF CD40L, and TNF were greater than 50 times than that of media at 24 hours. IFNexpression was maximal at 24 hours, IL-2 at 48 hours and IL-6 at 8 hour s. Thus, SEB also stimulates cytokine production in CD4+ T-cells.

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17 Figure 3-1: Antigen and isotype specificity of superantigen enhancement of antibody to BSA. Mice were injected with BSA alone, BSA followed by SEA/SEB, SEA/SEB alone or PBS under the same conditions as in Materials and Methods. A) Sera were tested by ELISA for IgG Abs to BSA or gp120 B) Sera were tested by ELISA for IgM Abs to BSA. C) Total IgG levels for the sera of A are presented in C. Students t test: A)BSA vs BSA and SEA/SEB, P<0.001; BSA and SEA/SEB, BSA vs gp120, p<0.001. No comparisons were significant in B and C. Data are representative of three experiments, each performed in triplicate.

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18 0.00.20.40.60.81.01.21.41:30001:10001:3001:100DilutionAbsorbance (405 nM) BSA: PBS only BSA: BSA BSA: BSA+SAg BSA: SAg only GP120: PBS only GP120: BSA GP120: BSA+SAg GP120: SAg onlyA 1:30001:10001:3001:1000.00.20.40.60.81.01.21.4 B BSA only BSA+SAg SAg only PBS onlyAbsorbance (405 nM)Dilution 0100200300400500600700800PBSSAgBSABSA+SAgconcentration IgG (ug/ml)C C B A

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19 MEDIA SEA SEB Figure 3-2: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase expression of various human cytokine genes. Human PBMC were treated with 100 ng/ml of SEA or SEB or media alone and cultured as described in methods. Cells were harvested from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was extracted from these cells and used to make cDNA. The cDNA was allowed to hybridize overnight with a nylon membrane spotted with cDNA spotted with various common human cytokines. After washes and incubation with AP-streptavidin, and substrate, the membranes were exposed to film and developed. Images seen here represent 24 hours post treatment.

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20 051015202530 IFNg IL-2IL-10TGFb IL-6TNFb IL-1bC D40LFold Increase (SEA/Media) 8 hours 16 hours 24 hours 48 hours IFNIL-2 IL-10 TGF IL-6 TNF IL-1 CD40L Figure 3-3: Staphylococcal enterotoxin A induces increased expression of various Th1, Th2, and T regulatory cytokine genes. Human PBMC were treated with 100 ng/ml of SEA and cultured as described in methods. Cells were harvested from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was extracted from these cells and used to make cDNA. The cDNA hybridized overnight to a nylon membrane spotted with cDNA of common human cytokines. The complete list of cytokine genes on the nylon membrane is presented in Appendix 3. After washes and incubation with AP-streptavidin, and substrate, the membranes were exposed to film and developed. Pictures were scanned into TIFF format and the software program ScionImage was used to determine pixel density for each spot. Background values were subtracted from each value, followed by normalization to a positive control on each membrane. This normalized value for SEA treated cells was divided by the same value for media to obtain the fold increase in gene expression of each cytokine.

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21 051015202530 Fold Increase (SEB/Media) 8h 16h 24h 48h IFN-yIL-2IL-10IL-6TNFBIL1BTGFBCD40L IFNIL-2 IL-10 TGF IL-6 TNF IL-1 CD40L Figure 3-4: Staphylococcal enterotoxin B induces increased expression of various Th1, Th2, and T regulatory Cytokine Genes. Human PBMC were treated with 100 ng/ml of SEB and cultured as described in methods. Cells were harvested from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was extracted from these cells and used to make cDNA. The cDNA hybridized overnight to a nylon membrane spotted with cDNA of common human cytokines. The complete list of cytokine genes on the nylon membrane is presented in Appendix 3. After washes and incubation with AP-streptavidin, and substrate, the membranes were exposed to film and developed. Pictures were scanned into TIFF format and the software program ScionImage was used to determine pixel density for each spot. Background values were subtracted from each value, followed by normalization to a positive control on each membrane. This normalized value for SEB treated cells was divided by the same value for media to obtain the fold increase in gene expression of each cytokine.

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22 Figure 3-5: Staphylococcal enterotoxin B induces increased expression of various Th1, Th2, and T regulatory cytokine genes. Human CD4+ T cells were treated with 100 ng/ml of SEB and cultured as described in methods. Cells were harvested from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was extracted from these cells and used to make cDNA. The cDNA hybridized overnight to a nylon membrane spotted with cDNA of common human cytokines. The complete list of cytokine genes on the nylon membrane is presented in Appendix B. After washes and incubation with AP-streptavidin, and substrate, the membranes were exposed to film and developed. Pictures were scanned into TIFF format and the software program ScionImage was used to determine pixel density for each spot. Background values were subtracted from each value, followed by normalization to a positive control on each membrane. This normalized value for SEB treated cells was divided by the same value for media to obtain the fold increase in gene expression of each cytokine. A) Fold increase of IFN-, IL-2, IL-10 and IL-6 in CD4+ T cells treated with SEB. B) Fold increase of TGF, TNF, CD40L, and IL-1 in CD4+ T cells treated with SEB.

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23 01020 IFN-yIL-2IL-10IL -6Fold Increase (SEB/Media) 8 hours 16 hours 24 hours 48 hours A IFNTGF TNF CD40L IL-1 050100150200250TGFB3TNFBCD40LIL1BFold Increase (SEB/Media) 8 hours 16 hours 24 hours 48 hours B

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24 Superantigens Enhance Cytokine Protein Production in HPBMC Since superantigens enhance the expression of various cytokine genes, we next determined cytokine protein levels in supernatants of human PBMC treated with SEA or SEB. ELISA and Western Blots were performed on the supernatants of HPBMC treated with 100 ng/ml of either SEA, SEB or media. IL-2, IFN-, IL-10 and TGF levels were determined. As shown in Figure 3-6, IL-2 was present in cultures treated with SEA and SEB, but not in media. Concentrations of IL-2 were greater than 600 ng/ml in both SEA and SEB treated groups at all four timepoints. Media treated cells supernatants had IL-2 levels of less than 50 ng/ml at all timepoints. This was in contrast to the IL-2 mRNA levels, which decreased after 16 hours. Similarly, IFNand IL-10 protein levels were higher in supernatants from SEA and SEB treated cells than the media treated cells. IFNlevels in cell supernatants were higher at 16 hours than at 24 hours, but were maximal at 48 hours (Figure 3-7). Media treated cell supernatants had low IFNprotein (<0.05 OD). IL-10 levels were highest at 48 hours (Figure 3-8) (>1500 ng/ml). Media treated cells had no detectable IL-10 at any of the timepoints. These protein levels were fairly consistent with the mRNA levels seen on the microarray. Higher concentrations of IL-10 at the later time points may be acting to down regulate the action of IL-2 (See discussion). Although TGF gene expression was increased as shown on the microarray, there was no detectable protein in the supernatants as determined by ELISA (data not shown) and Western Blots (Figure 3-9). Although TGF may be expressed at a later timepoint than 48 hours, cells begin to die after 48 hours incubation with superantigen and generally undergo apoptosis or anergy. Therefore, cytokine measurements may be inaccurate after 48 hours. Thus, superantigens also induce cytokine protein production in conjunction with increased cytokine gene expression in HPBMC in culture.

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25 010020030040050060070080090010008162448Time (hours)IL-2 concentration (pg/ml) SEA SEB Media Figure 3-6: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-2 production in human PBMC. HPBMC were treated with 100 ng/ml of either SEA or SEB, or culture medium alone. Cells were cultured as described in methods. Supernatants were harvested from the cultures at timepoints of 8, 16, 24, and 48 hours. An ELISA was performed on the supernatant to determine levels of IL-2. Data shown here represent mean and SD of duplicate experiments.

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26 00.10.20.30.40.50.60.78162448Time (hours)Absorbance @ 490nm SEA SEB Media Figure 3-7: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IFNproduction in human PBMC. HPBMC were treated with 100 ng/ml of either SEA or SEB, or culture medium alone. Cells were cultured as described in methods. Supernatants were harvested from the cultures at timepoints of 8, 16, 24, and 48 hours. An ELISA was performed on the supernatant to determine levels of IFN-. Data shown here represent mean and SD of duplicate experiments.

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27 050010001500200025008162448Time (hours)IL-10 concentration (pg/ml) SEA SEB Media Figure 3-8: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-10 Production in Human PBMC. HPBMC were treated with 100 ng/ml of either SEA or SEB, or culture medium alone. Cells were cultured as described in methods. Supernatants were harvested from the cultures at timepoints of 8, 16, 24, and 48 hours. An ELISA was performed on the supernatant to determine levels of IL-10. Data shown here represent mean and SD of duplicate experiments.

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28 A B C D E F G H I J K Figure 3-9: Staphylococcal enterotoxin A and staphylococcal enterotoxin B do not increase TGF production in human PBMC. HPBMC were treated with 100 ng/ml of either SEA or SEB, or culture medium alone. Cells were cultured as described in methods. Supernatants were harvested from the cultures at timepoints of 8, 16, 24, and 48 hours. A Western Blot was performed on the supernatants to determine levels of TGF. Data shown here represent 24 and 48 hour timepoints. A: TGF MW marker (25 kDa), B: blank, C: blank, D: 24h SEA, E: 24h SEB, F: 24h media, G: 48h SEA, H: 48h SEB, I: 48h media, J: media control K: Molecular weight ladder

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29 TGF Suppresses IL-2 Induced Growth in NK92 Cells in vitro NK92 cells, an IL-2 dependent cell line, were treated with either media, IL-2 (30 U/ml), or IL-2 and TGF (25 ng/ml) for 48 hours. Cells were then pulsed with 3 H-Thymidine for 6 hours and cell associated radioactivity was counted on a -scintillation counter. As shown in Figure 3-10, cells cultured with IL-2 and TGF had almost 50% less proliferation than those cells cultured with IL-2 alone. Cells cultured with media alone, had low proliferation, showing that IL-2 is necessary for significant proliferation. Therefore, TGF can suppress IL-2 induced proliferation. 020004000600080001000012000MediaIL-2 IL2+TGFb(25n g/ m Treatmentcpm Figure 3-10: Transforming growth factor beta suppresses IL-2 induced proliferation of NK-92 cells. Human NK92 cells were plated at 4 x 104 cells per well in a 96 well plate and treated with either media, IL-2 (30 U/ml), or IL-2 and TGF (25 ng/ml) for 48 hours. Cells were then pulsed with 1 Ci per well of 3H-thymidine for 6 hours, after which cells were harvested and cell associated radioactivity was quantified using a -scintillation counter and activity reported in CPM. All experiments were performed in replicates of six. IL-2 and TGF ( 25n g /ml ) 29

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30 CHAPTER 4 DISCUSSION In the first part of this study the immunoenhancing effects of superantigens on the humoral arm of the immune response were studied. Immunization of mice with the prototype T-dependent Ag BSA followed by SEA/SEB resulted in increased IgG antibody response to BSA. Thus, enhancement effects of superantigens were specific for the primary antigen BSA. The results presented here, combined with those of previous findings on superantigen enhancement of tumor-specific immunity to mouse melanoma (14), are evidence that superantigens such as SEA and SEB can significantly boost Ag-specific immune responses. Microarray experiments of human PBMC treated with SEA, SEB or media alone showed increases in mRNA levels in various cytokines. These cytokines include but are not limited to IFN-, IL-2, TNF-, IL-6, IL-10 and TGF-. The presence of IL-2 mRNA is seen as early as 8 hours, peaking at 16 hours, followed by a decline after that through 48 hours (Figure 3-3). IFNmRNA expression levels peak at 24 hours, followed by a decline (Figure 3-3, Figure 3-4, Figure 3-5). TNFmRNA expression increases at 16 h and is maintained through 48 hours (Figure 3-3). These three cytokines are indicative of a typical Th1 inflammatory type response. In the case of Th2 cytokine expression, IL-6 mRNA expression levels are maximal at 24 hours followed by a rapid decline (Figure 3-3). TGFand IL-10, which are produced by T regulatory CD4+ T cells are maximal at 16 and 24 hours respectively. The studies on the cytokine profile of 30

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31 superantigen activated cells and the enhancement of the humoral response against BSA presented here and the studies on enhancement of cellular response against melanoma cells suggest distinct T cell populations are being activated by superantigens (14). Thus, superantigens activate Th1, Th2, and T regulatory cells in vivo and in vitro. In conjunction with the microarray studies, ELISA and Western blot studies were run to determine if the increases in mRNA levels were associated with translation of the message. ELISA (Figure 3-7) and Western Blots for IFN(data not shown) showed increases in IFNover time, with very little production of the IFN in media treated cells. Furthermore, IL-2 levels increased as early as 8 hours and remained constant through 48 hours (Figure 3-6). This is in contrast to the mRNA levels of IL-2, which started to decrease at the same time, possibly due to action of T regulatory cells. This may be due to a lag period between RNA message decline and seeing an actual decline in the protein produced. Carrying out the experiment for a much longer period of time may show the actual decrease in protein levels. However, this may prove to be difficult, as cells exposed to these high a concentrations of SEA or SEB begin to die after 48 hours. The fact that IL-10 protein expression in superantigen treated cells increased over time and is maximal at 48 hours (Figure 3-8) indicates how the T regulatory cells control the Th1 and Th2 cell types and plays an important role in helping the immune system to recover from encounters with superantigen. TGF expression increases after superantigen stimulation of cells, but is not detected at the protein level one to two days after superantigen stimulation (Figure 3-9). Others have shown that IL-10 enhances the expression of TGF (4,13,25). Thus, TGF protein production may occur beyond the 48 hour time point measured here but may be difficult to detect for the same reason 31

PAGE 41

32 discussed above for IL-2. It has been previously shown in mice lacking CD4 25+ T-cells, which is the phenotype of T regulatory cells, have sustained production of inflammatory cytokines and that this production can be corrected by injections with CD4 25+ T-cells (20). As per Figure 3-9, TGF suppresses IL-2 induced proliferation by almost 50% in NK92 cells, an IL-2 dependent cell line. This suggests how the regulatory cytokines produced at later time points during superantigen stimulation, may act to down regulate the effects of Th1 cytokines produced earlier. It also identifies a potential target to block regulatory cytokines or a particular cell type, to bias and sustain an inflammatory or humoral response for a longer period of time. Thus, similar to an increase in RNA expression, IL-10, IL-2 and IFNprotein expression increase in culture supernatants taken from superantigen treated cells. Superantigens enhance specific humoral and cellular responses against antigens, such as BSA in vivo and this immune enhancement is due to the action of superantigen on CD4+ T cells that produce inflammatory, helper and regulatory cytokines in a time dependent manner. Inflammatory cytokines produced by Th1 cells are induced initially by superantigen after which regulatory and suppressor cytokine, produced by Th2 and T regulatory cells, levels increase. There is an inherent characteristic of superantigen effects on naive vs Ag-primed T cells that is a plus for their immunoenhancing properties. Naive T cells initially undergo cell division when treated with superantigens, followed shortly by anergy and/or deletion. Ag-primed T cells also expand when treated with superantigen but, in contrast, do not undergo the anergy/deletion characteristic of naive T cells (11). Thus, the V -specific polyclonal expansion associated with superantigens is tilted toward 32

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33 primed Ag-specific T cell. This effect may be of beneficial use in vaccinations to boost immunity to a particular pathogen or disease. 33

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APPENDIX A MICROARRAY PROTOCOL Figure A-1: Microarray Protocol (GEArray Q series protocol from Superarray, Frederick, MD) 34

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APPENDIX B MICROARRAY GENE LIST Allograft inflammatory factor 1 c-fos induced growth factor Interleukin 4 Bone morphogenetic protein 1 Hepatocyte growth factor Interleukin 5 bone morphogenetic protein 2 interferon, alpha 1 Interleukin 6 Growth differentiation factor 10 Interferon, alpha 2 Interleukin 7 Bone morphogenetic protein 4 Interferon, alpha 4 Interleukin 8 Bone morphogenetic protein 6 Interferon, alpha 5 Interleukin 9 bone morphogenetic protein 8 Interferon, alpha 6 Leptin Colony stimulating factor 1 Interferon, alpha 7 Lymphotoxin-alpha Colony stimulating factor 2 Interferon, beta 1, fibroblast Lymphotoxine-beta Colony stimulating factor 3 Interferon, gamma Platelet-derived growth factor-BB Homo sapiens erythropoietin Interferon, omega 1 Platelet-derived growth factor alpha polypeptide Fibroblast growth factor 1 Insulin like growth factor IA Pleiotrophin Fibroblast growth factor 10 Insulin-like growth factor 2 Transforming growth factor, alpha Fibroblast growth factor 11 Interleukin 10 Transforming growth factor, beta 1 Fibroblast growth factor 12 Interleukin 11 Transforming growth factor, beta 2 Fibroblast growth factor 12B Interleukin 12A, p35 Transforming growth factor, beta 3 Fibroblast growth factor 14 Interleukin 12B,p40 Thrombopoietin Fibroblast growth factor 16 Interleukin 13 Tumor necrosis factor Fibroblast growth factor 17 Interleukin 14 Tumor necrosis factor (ligand) superfamily, member 10 Fibroblast growth factor 19 Interleukin 15 Tumor necrosis factor (ligand) superfamily, member 11 Fibroblast growth factor 2 Interleukin 16 Homo sapiens TNF (ligand) superfamily, member 4 Fibroblast growth factor 20 Interleukin 17 CD40 ligand Fibroblast growth factor 21 Interleukin 18 Ligand for Fas Fibroblast growth factor 23 Interleukin 19 CD27 ligand/CD70 antigen Fibroblast growth factor 3 Interleukin 1, alpha CD30 ligand Fibroblast growth factor 4 Interleukin 1, beta Tumor necrosis factor (ligand) superfamily, member 9 Fibroblast growth factor 5 Interleukin 2 Vascular endothelial growth factor Fibroblast growth factor 6 Interleukin 20 Vascular endothelial growth factor B Fibroblast growth factor 7 Interleukin 22 Vascular endothelial growth factor C Fibroblast growth factor 9 Interleukin 3 PUC18 Plasmid DNA (negative control) Ribosomal protein L13a (positive control) Glyceraldehyde-3-phosphate dehydrogenase (positive control Homosapiens peptidylprolyl isomerase A Beta Actin (positive control) Table B-1: Microarray gene list. (GEArray Q series protocol from Superarray, Frederick, MD) 35

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35 LIST OF REFERENCES 1. Altemeier, W.A., S.A. Lewis, P.M. Schievert, M.S. Bergdoll, H.J. Bjornson L. Taneck, and B.A. Crasss. 1982. Staphylococcus aureus associated toxic shock syndrome: Phage typing and toxin capability tested. Ann. Intern. Med. 96:978 2. Callahan, J.E., A. Herman, J.W. Kappler, and P. Marrack. 1990. Stimulation of B10.BR T cells with superantigenic staphylococcal toxins.J. Immunol. 144:2473. 3. Cameron, S.B., M.C. Nawijn, W.W.S. Kum, H.F.J. Savelkoul, and A.W. Chow. 2001. Regulation of helper T cell responses to staphylococcal superantigens. European Cytokine Network. 12:210 4. De Winter, H., D. Elewaut, O. Turovskaya, M. Huflejt, C. Shimeld, A. Hagenbaugh, S. Binder, I. Takahashi, and M. Kronenberg. 2002. Regulation of mucosal immune responses by recombinant interleukin 10 produced by intestinal epithelial cells in mice. Gastroenterology. 129:1829 5. Dow, S.W., R.E. Elmslie, A.P. Willson, L. Roche, C. Gorman, and T.A. Potter. In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma. J. Clin. Invest. 101:2406. 6. Florquin, S., Z. Amraoui, and M. Goldman. 1996. Persistant production of Th2 type cytokines and Polyclonal B cell activation after chronic administration of staphylococcal enterotoxin B in mice. Journal of Autoimmunity. 9:609. 7. Fraser, J., V. Arcus, P. Kong, E. Baker, and T. Proft. 2000. Superantigenspowerful modifiers of the immune system. Molecular Medicine Today. 6:125. 8. Friedman S., D. Posnett, and J. Tumang. 1991. A potential role for microbial superantigens in pathogenesis of systemic autoimmune disease. Arth. Rheum. 34:468. 9. Johnson, H.M., E.J. Butfiloski, L. Wegrzyn, and J.M Soos. 1993. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. PNAS. 90:8543. 35 35

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36 10. Johnson, H.M., B.A. Torres, and J.M. Soos. 1994. Superantigens: Structure and relevance to human disease. Proc. Soc. Exp. Biol. Med. 212:99. 11. Kawabe, Y., and A.Ochi. 1990. Selective anergy of V beta 8+ CD4+ T cells in Staphylococcus enterotoxins B primed mice. J Exp Med. 172:1065 12. Labrecque, N., J. Thibodeau, and R.P. Sekaly. 1993. Interactions between staphylococcal superantigens and MHC class II molecules. Seminars in Immunology. 5:23 13. Levings, M.K., R. Bacchetta, U. Schulz, and M.G. Roncarlo. 2002. The role of IL-10 and TGFin the differentiation and effector function of T Regulatory cells. Int. Arch. Immunol. 129:263 14. Kominsky, S.L., B.A. Torres, A.C Hobeika, F.A. Lake, and H.M. Johnson. 2001. Superantigen enhanced protection against a weak tumor specific antigen: implications for prophylactic vaccination against cancer. Int. J. Cancer. 94:834. 15. Marrack, P., and J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science. 248:705. 16. Mollick JA, R.G. Cook, R.R Rich. 1989. Class II MHC molecules are specific receptors for staphylococcal enterotoxin A. Science. 244:817. 17. Noel, C., S. Florquin, M. Goldman, and M.Y. Braun. 2001. Chronic exposure to superantigen induces regulatory CD4+ T cells with IL-10 mediated suppressive activity. International Immunology. 13:431. 18. Papiernik, M., and A. Banz. 2001. Natural regulatory CD4 T cells expressing CD 25. Microbes and Infection. 3:937. 19. Perrin, G.Q., H.M. Johnson, and P.S. Subramaniam. 1999. Mechanism of Interleukin-10 Inhibition of T helper cell activation by superantigen at the level of the cell cycle. Blood. 93:208. 20. Pontoux, C., A. Banz, and M. Papiernik. 2002. Natural CD 4 CD 25+ regulatory T cells control the burst of superantigen-induced cytokine production: the role of IL-10. International Immunology. 14:233. 21. Rink, L. J. Luhm, M. Koester, and H. Kirchner. 1996. Induction of a cytokine network by superantigens with parallel Th1 and Th2 stimulation. J Interferon Cytokine Res. 16:41. 22. Roncarolo, M.G., R. Bachetta, C. Bordignon, S. Narula, and M.K. Levings. 2001. Type I T regulatory cells. Immunological Reviews. 182:68. 36

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37 23. Rosendahl, A., K. Kristensson, M. Carlsson, N.J. Skartved, K. Riesbeck, M. Sogaard, and M. Dohlsten. 1999. Long-term survival and complete cures of B16 melanoma-carrying animals after therapy with tumor targeted IL-2 and SEA. Int. J. Cancer. 81:156. 24. Schlievert, P.M., K.N. Shands, B.B. Dan, G.P. Schmid, N.D. Nishimura. 1981. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxic shock syndrome. J. Infect. Dis. 143:509 25. Seder, R.A., T. Marth, M.C. Sieve, W. Strober. J.J. Letterio, A.B. Roberts, and B. Kelsall. 1998. Factors involved in the differentiation of TGF-beta-producing cells from nave CD4+ T cells: IL-4 and IFN-gamma have opposing effects, while TGF-beta positively regulates its own production. J. Immunol. 160:5719. 26. Soos, J.M., A.C Hobeika, E.J.Butfiloski, J. Schiffenbauer, and H.M. Johnson. 1995. Accelerated induction of experimental allergic encephalomyelitis in PL/J mice by a non-V8-specific superantigen. PNAS. 92:6082. 27. Soos, J.M., J. Schiffenbauer, and H.M. Johnson. 1993. Treatment of PL/J mice with the superantigen, staphylococcal enterotoxin B, prevents development of experimental allergic encephalomyelitis. Journal of Neuroimmunology. 43:39. 28. Soos, J.M., J. Schiffenbauer, B.A. Torres, and H.M. Johnson. 1997. Superantigen as virulence factors in autoimmunity and immunodeficiency diseases. Medical Hypotheses. 48:253 29. Tanabe, T., B.A. Torres, P.S. Subramaniam, and H.M. Johnson. 1996. V Activation by HIV nef protein: Detection by a simple amplification procedure. Biochem. Biophys. Res. Commun. 230:509. 30. Taub, D.D., and T.J. Rogers. 1992. Direcet activation of murine T-cells by staphylococcal enterotoxins. Cell. Immunol. 148:1240. 31. Torres, B.A., and H.M. Johnson. 1994. Identification of an HIV-1 nef peptide that binds to HLA class II antigens. Biochem. Biophys. Res. Commun. 200:1059. 32. Torres, B.A., S.L. Kominsky, G.Q. Perrin, A.C. Hobeika, and H.M. Johnson. 2001. Superantigens: The good, the bad and the ugly. Exp. Med. Biol. 226:164. 33. Torres, B.A., J.M. Soos, G.Q. Perrin, and H.M. Johnson. 2000. Microbial superantigens and immunological deregulation. Washington DC: ASM press 183-197. 37

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38 34. Torres, B.A., T. Tanabe, and H.M. Johnson. 1996a. Characterization of nef induced CD4 T cell proliferation. Biochem. Biophys. Res. Commun. 225:54. 35. Torres, B.A., T. Tanabe, H.M. Johnson. 1996b. Replication of HIV-1 in human peripheral blood mononuclear cells activated by exogenous nef. AIDS. 10:1042. 36. Torres, B.A., T. Tanabe, P.S. Subramaniam, J.K. Yamamoto, and H.M. Johnson. 1998. Mechanism of HIV pathogenesis: Role of superantigens in disease. Alcohol Clin. Exp. Res. 22:188S. 37. Torres, B.A., T. Tanabe, J.K. Yamamoto, and H.M. Johnson. 1996. HIV encodes for its own CD4 T-cell superantigen mitogen. Biochem. Biophys. Res. Commun. 225:672 38. Zamvil, S.S., D.J. Mitchell, N.E. Lee, A.C. Moore, M.K. Waldor, K. Sakai, J.B. Rothbard, H.O. McDevitt, L. Steinman, and H. Acha-Orbea. 1988. Predominant expression of a T cell receptor V beta gene subfamily in auto immune encephalomyelitis. J. Exp. Med. 167:1586. 38

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BIOGRAPHICAL SKETCH Amy Kristin Anderson was born in New Jersey on May 2, 1977. Her family got larger, when her brother Tim was born in March of 1980. Her family lived in NJ until she was 10 years old, when they moved to Tinmouth, Vermont in December of 1987. Amy enjoyed living in the rural town of about 400 people, having a horse and attending smaller sized elementary and high schools. After graduation from Mill River Union High School in 1995, Amy began attending the University of New Hampshire, on a partial academic scholarship, where she pursued a Bachelor of Science in Medical Laboratory Science. Amy graduated magna cum laude from UNH in 1999 and got a job at Duke Medical Center in Durham, NC, in the immunohematology lab as a Medical Technologist. While living in NC, Amy met Glenn, who was working on his Master of Science at North Carolina State University. Glenns job search after graduation in 2000, took him all the way to Florida and Amy decided to look at graduate school there. Amy began attending the University of Florida in the fall of 2001 to pursue a Master of Science degree, studying in the department of Microbiology and Cell science. In November of 2001, Amy decided to do her masters research work in the laboratory of Dr. Howard M. Johnson. Amy and Glenn were married on June 28, 2003 in Vermont. After graduation, Amy plans to pursue a career in the biotechnology industry, hopefully doing research. 39


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Creator: Anderson, Amy Kristin
Publication Date: 2003
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STAPHYLOCOCCAL ENTEROTOXIN ENHANCEMENT OF INFLAMMATORY
AND REGULATORY CYTOKINE PRODUCTION AND HUMORAL RESPONSES
















By

AMY KRISTIN ANDERSON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2003
































I dedicate this work to my parents and my brother Tim, for always being there and
supporting me in whatever I do; and to Glenn, for always being there for me for anything.















ACKNOWLEDGMENTS

I would like to thank Dr. Howard M. Johnson for giving me the opportunity to do

research in his laboratory; and for providing ideas and insight on all the projects I have

worked on. I would also like to thank my other committee members, Dr. Ed Hoffman

and Dr. Peter Kima for their assistance with my research. Another thank-you goes to Dr.

Mustafa Mujtaba in Dr. Johnson's lab who has assisted me with many things in the lab

and my research. I would also like to acknowledge all of the other lab members as well

as my fellow graduate students, Margi and Lawrence, for always helping out in any way

possible. I would like to especially thank my family, who has always supported me in

whatever I have chosen to do. A last special thank-you goes to my husband, Glenn, who

is always there for me for anything I need. He was a large part of the motivation I needed

to go to graduate school.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ......... .................................................................................... iii

LIST O F FIG U R E S .... ...................................................... .. ....... ............... vi

LIST OF A BBREV IA TION S ................................................. ............................. vii

A B S T R A C T .......................................... .................................................. v iii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

General Superantigen Information ........................................... .. ............... 1
Interactions of Superantigens with Immune Cells and Peptide Processing..................
A ctivation of Cells by Superantigens ........................................ ........ ............... 2
Superantigens in D disease .................................................................................... ... 4
Uses of Superantigens in Vaccines and in the Treatment of Disease...........................5

2 M ATERIALS AND M ETHODS ........................................ ........................... 8

Human Peripheral Blood Mononuclear cells (HPBMC) Isolation and Cell Culture....8
Human CD4+ T Cell Isolation.......................................... .... ...................8
Staphylococcal Enterotoxin Treatment of HPBMC and CD4+ T-cells...................
T total R N A Isolation ................... .... .......................... .. ...... ........ .......... .. ....
M icroarray Procedure ................................................ ........ .. ............ 10
A analysis of M icroarray D ata ............................................................................... 10
W western Blot for TGF 3 .................................. ......................................... 10
ELISA for Human Cytokines ...................... .............. .... .. ... ...............11
Mouse Studies and Detection of Specific Antibodies in Mouse Sera.....................12
Proliferation Assay ........................................ ........ ......... .... 13

3 R E S U L T S ......... ................................................ .......................................14

Superantigens Enhance Specific Antibody Production to Antigens in Mice .............14
Superantigens Enhance Cytokine RNA Production in HPBMC ..............................15
SEA Induces Cytokine Gene Expression in HPBMC ............... .....................15
SEB Induces Cytokine Gene Expression in HPBMC .......................................16
SEB Induces Cytokine Gene Expression in CD4+ T cells......................................16
Superantigens Enhance Cytokine Protein Production in HPBMC ...........................24
TGFB Suppresses IL-2 Induced Growth in NK92 Cells in vitro ............................29











4 D ISC U SSIO N ......... .......... ......... ............................... ........................... 30

APPENDIX

A MICROARRAY PROTOCOL .......................................................................34

B MICROARRAY GENE LIST ........................................................................35

L IST O F R E F E R E N C E S ............................................................................ ............... 35

B IO G R A PH IC A L SK E T C H ...................................................................... ..................39













































v















LIST OF FIGURES


Figure p

1-1 Typical Peptide Antigen and Superantigen Interactions with T-cells and APC .......2

3-1 Antigen and isotype specificity of superantigen enhancement of antibody to BSA 17

3-2 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase expression
of various hum an cytokine genes .............. .................................... ....... ........ 19

3-3 Staphylococcal enterotoxin A induces increased expression of various Thl, Th2,
and T regulatory cytokine genes ........................................ ......................... 20

3-4 Staphylococcal enterotoxin B induces increased expression of various Thl, Th2,
and T regulatory Cytokine G enes....................................... .......................... 21

3-5 Staphylococcal enterotoxin B induces increased expression of various Thl, Th2,
and T regulatory cytokine genes.. ........................ .............................................. 22

3-6 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-2
production in hum an PB M C .. ............................. ............................................... 25

3-7 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IFN-y
production in hum an PBM C .............................................................................. 26

3-8 Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-10
Production in Hum an PBM C .............................................................................27

3-9 Staphylococcal enterotoxin A and staphylococcal enterotoxin B do not increase
TGFp production in human PBMC...........................................................28

3-10 Transforming growth factor beta suppresses IL-2 induced proliferation of NK-92
c e lls ........................................................................... 2 9















LIST OF ABBREVIATIONS


Ab: Antibody

APC: Antigen presenting cell

BCA: Bicinchoninic Acid

BSA: Bovine serum albumin

cDNA: Complementary deoxyribonucleic acid

ELISA: Enzyme linked immunosorbent assay

FBS: Fetal bovine serum

HPBMC: Human peripheral blood mononuclear cells

HRP: Horseradish peroxidase

IFN: Interferon

IL: Interleukin

NK: Natural killer cells

PBMC: Peripheral blood mononuclear cells

PBS: Phosphate buffered saline

RNA: Ribonucleic acid

SEA: Staphylococcal enterotoxin A

SEB: Staphylococcal enterotoxin B

TGF: Transforming growth factor

TNF: Tumor necrosis factor















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

STAPHYLOCOCCAL ENTEROTOXIN ENHANCEMENT OF INFLAMMATORY
AND REGULATORY CYTOKINE PRODUCTION AND HUMORAL RESPONSES


By

Amy Kristin Anderson

August 2003

Chair: Howard M. Johnson
Cochair: Edward Hoffman
Major Department: Microbiology and Cell Science

Superantigens are microbial proteins that are the causative agents of food

poisoning and toxic shock syndrome. Furthermore, superantigens can cause deregulation

of the immune system and can exacerbate autoimmune diseases such as multiple

sclerosis. Superantigens induce a burst of T-cell proliferation and activation. We

analyzed in vivo and in vitro, superantigen activation of the cells of the immune system in

the context of expression of genes associated with various T-cell subsets. Human

peripheral blood mononuclear cells (HPBMC) treated with staphylococcal enterotoxin A

(SEA) showed increased expression of the cytokines IFN-y, IL-2, IL-10, TNF-ac, TNF-P,

TGF-P, and IL-6 as deduced by microarray analysis of cell mRNA. A similar pattern of

genes was upregulated in HPBMC treated with staphylococcal enterotoxin B (SEB).

Thl, Th2 and T regulatory cells produce these cytokines. Expression for most of these

cytokines is









maximal 24 to 48 hours post superantigen treatment. Enzyme Linked Immuno Sorbent

Assay (ELISA) tests for IFN-y, IL-10 and IL-2, showed increased mRNA levels as seen

on the microarray correlate with translation, with the exception of TGF3, which was not

detectable by ELISA or Western Blot. In addition, purified CD4+ T-cells treated with

SEB show a similar pattern of upregulated cytokines as shown for superantigen treated

HPBMC. In vivo experiments showed that SEA and SEB increase specific IgG (but not

IgM) antibody production in mice immunized against bovine serum albumin (BSA).

Thus, superantigens increase both the specific humoral and cellular immune responses

against antigens. Based on the cytokine profile elicited by superantigens in vitro and the

specific antibody production in vivo, superantigens enhance Thl, Th2 and T regulatory

cytokine production. This property of superantigens makes them an ideal candidate to

boost specific immunity; and may have prophylactic applications in the prevention of

cancer and other immune- mediated diseases.














CHAPTER 1
INTRODUCTION

General Superantigen Information

Superantigens are microbial proteins produced by various bacteria and viruses

including (Staphylococcus species, Streptococcus species, HIV and rabies viruses) and

are powerful activators of CD4+ T-cells. (7,10) Common superantigens include the

staphylococcal enterotoxins (A-E) and toxic shock syndrome toxin 1 (TSST-1) which are

produced by Staphylococcus aureus (32). The enterotoxins are common causative agents

of toxin-mediated food poisoning and TSST's are implicated in toxic shock syndrome

(32). Superantigens are also suspected to play a role in exacerbation of autoimmune

diseases such as multiple sclerosis as well as the immunodeficiency associated with HIV

infection (8,10,27,36,33). These acute and chronic disease states associated with

superantigen are caused by the massive proliferation and activation of T-cells that is

induced by superantigens (2).

Interactions of Superantigens with Immune Cells and Peptide Processing

The potency of the superantigens in relation to T-cell activation and proliferation

is in part explained by its unique interaction with the T-Cell Receptor (TCR) on T cells

and the major histocompatibility complex II (MHC II) on antigen presenting cells (APC)

(30). As shown in Figure 1-1, superantigens bind directly to MHC II (16) and this

complex interacts with a specific portion of the V3 region of the TCR. Each superantigen

activates different V3 regions. TSST-1 activates T-cells with Vp2 while SEB activates

cells with Vp3, V3 12, V3 14, V3 15, V3 17, and V3 20 (2,15).






2


The interaction with the TCR is antigen-independent and requires no further processing

as seen with a typical peptide antigen. Typical peptide antigens are processed internally

and presented by APC on either MHC I or MHC II molecules on their cell surface. By

acting in this manner, superantigens can induce polyclonal activation of up to 20% of the

total T-cell population at one time, as compared to typical peptide antigens that only

activate up to 0.01 % of the total T-cell population at one time.

A. Antigen-presenting cell B. Antigen-presenting cell

MHC Peptide
class antigen
II Superantigen





Beta chain
Beta variable region
Alpha chain (Vp)
chain

T cell T cell
Figure 1-1: Typical Peptide Antigen and Superantigen Interactions with T-cells and
APC. (Torres, B.A., S.L. Kominsky, G.Q. Perrin, A.C. Hobeika, and H.M. Johnson.
2001. Superantigens: The good, the bad and the ugly. Exp. Med. Biol. 226:164).


Activation of Cells by Superantigens

Superantigens activate CD4+ T-cells. These T-cells are preferentially activated

because of their increased binding affinity for MHC II (12). The CD4+ T cells can be

divided into three groups: T helper 1 (Thl), T helper 2 (Th2) and T regulatory cells. Thl

cells are one T-cell group activated by superantigen. Thl cells are associated with

inflammatory responses and produce such cytokines as IL-2, IFN-y and tumor necrosis

factor (21). IL-2 drives further T-cell proliferation and activation. One of the main

functions of IFN- y is macrophage activation through upregulation of MHC I and MHC

II. It also has powerful antiviral effects. It enhances CTL, natural killer and macrophage









cell tumoricidal activity (32). Tumor necrosis factor (TNF) induces macrophages to

produce NO, as well as activating the vascular endothelium.

The Th2 cells are also activated by superantigens. Th2 cells are associated with

humoral responses and anti-inflammatory responses. This group of cells is thought to be

the classic adversary to the actions of Thl cells (3). Th2 cells produce such cytokines as

IL-4 and IL-6. Interleukin 4 is an anti-inflammatory cytokine produced by Th2 cells.

Interleukin-6 is a cytokine with both anti-inflammatory and inflammatory properties. It

can induce acute phase response in the liver and drives B-cell differentiation (3).

A third group of cells called T-regulatory cells also are activated by

superantigens, especially cells undergoing repeated treatments with superantigen (17). T

regulatory cells are CD 4+ and express CD25. (18) They play important roles in the

maintenance of peripheral tolerance. Typical cytokines produced by T-regulatory

cytokines include TGF-3 and IL-10 and IFN-y were detected (6,17). Interleukin 10

downregulates MHC II expression and costimulatory molecules like CD 80/86, inducing

energy in CD4+ T cells (22). On the contrary, IL-10 aids in the survival of B-cells and

plays a part in their differentiation. It has also been shown in mice knockouts for CD25,

that stimulation with superantigens results in uncontrolled release of pro inflammatory

cytokines, but that injection with CD4+ CD25 T-cells control this process (20). This

indicates that regulatory cells play a role in controlling superantigen activation of T-cells.

TGF-3 is another type of regulatory cytokine. TGF-3 inhibits IL-2 production, and has

anti-proliferative effects on T-cells. TGF-3 also blocks the differentiation of Thl and

Th2 cells through blocking of transcription factors.









Superantigens in Disease

Superantigens are involved in both short- and long- term acute and chronic

disease states. Staphylococcal enterotoxins A (SEA) are responsible for the most food

poisoning caused by superantigens (9,32). The acute gastrointestinal illness resulting

from ingestion of the toxin is short lived. Toxic shock syndrome (TSS) caused by

TSST-1 is also an acute disease caused by superantigens (1,24,32). TSS became well

studied in the 1980's when it became associated with tampon use in women during

menstruation. Clinical manifestations of TSS include rashes, fever, severe hypotension,

and possible fatal shock.

While the acute effects of superantigens are somewhat severe, the involvement of

superantigens in exacerbation of autoimmune diseases persists over long periods of time.

The involvement of superantigens in autoimmune disease is demonstrated in a

murine model of multiple sclerosis (MS), called experimental allergic encephalomyelitis

(EAE), which is induced in mice by injection of myelin basic protein (MBP). The T-cell

population in mice responsible for EAE has a V38+ specificity (38). Pretreatment of

PL/J mice with SEB prevents induction of EAE by MBP (27). This is most likely due to

energy and deletion of VB8+ T-cells by SEB (10). However, SEB has also been shown

to reactivate EAE in mice that have been immunized with MBP and recovered from an

initial episode of the disease (9). This phenomenon is not limited to SEB, since SEA has

also been shown to reactivate EAE (9,26) showing that different V3 specificities may

also be involved in autoimmune disease.

Superantigens are also thought to be involved in some immunodefiency diseases,

such as HIV. A regulatory gene product encoded in the 3' LTR of the HIV genome









(called Nef) has been shown to block binding of SEA to Raji cells; and to induce T-cell

proliferation, IL-2 production, and IFN-y production (32,33). Other evidence of

superantigen characteristics include selective expansion of particular V3 sets (V33, 5.3

and 18) (29,34) and need for APC for Nef induced activation of T-cells (37). Another

study has shown that blocking Nef with an anti-Nef antibody does not support HIV

replication (34) suggesting that Nef is necessary for HIV to maintain itself in a host. HIV

requires activated CD4+ T cells for replication (36); and a superantigen-like molecule is

one way for the virus to achieve this. These studies together show that Nef has typical

characteristics of a superantigen and its action induces a pool of cells for viral replication,

which aids in the spread of the virus in a host.

Uses of Superantigens in Vaccines and in the Treatment of Disease

Superantigen effects are generally considered to be negative, especially in the

context of food poisoning, TSS, and their involvement in chronic disease states.

However, the very characteristics and interactions of superantigens also make them ideal

candidates for use in vaccines, prophylactically and as treatment of diseases. In order to

achieve this, the proliferation and cytokine production must be controlled and biased to a

certain direction. In this way, the effects of superantigens would be used to improve and

bolster immune responses as opposed to deregulating immune function.

A mouse model of melanoma has been used to show how superantigens can be

prophylactically effective as vaccine against melanoma (14). C57BL/6 mice were

injected with irradiated B16F10 melanoma cells, followed by SEA and SEB 6 and 10

days later. Three days after SEA and SEB treatment, mice were challenged with live

B16F10 cells. Median survival time was >150 days for those mice injected with









superantigens, as opposed to 14 to 23 days in mice with no treatment or treatment with

cells only or superantigen only (14). In addition, surviving mice were rechallenged with

live tumor and 75 percent survived. (14) The staphylococcal entertoxins (SE) are helping

to boost anti-tumor activity through activation of a large number of T-cells already

primed with the tumor antigen.

Another study takes a different approach to the use of staphylococcal enterotoxins

in the treatment of melanoma (23). Mice were injected with c215 transfected B16

melanoma cells followed by injection of SEA fused to a C215 tumor reactive antibody,

along with IL-2 fused to the same antibody. Prolonged survival was seen in mice

receiving both treatments, as opposed to each treatment alone (23). Repeated cycles of

treatments also increased survival time (23). This method directs SEA directly to the

tumor, with the use of the antibody. The use of IL-2 as well, adds to the proliferation

induced alone by SEA.

A third approach uses plasmid encoded SEB and either IL-2 or GMCSF (5).

Dogs with melanoma tumors received intratumor injections of lipid complexes with

plasmid DNA encoding SEB and either IL-2 or GMCSF. Partial tumor regression, and in

some cases, complete regression was seen in dogs receiving this treatment (5). Little

toxicity was seen in the dogs given these treatments. The treatment is directed to the

tumor through intratumoral injection and SEB is produced in the animal. IL-2 acts as an

additional proliferation agent, as in the previous experiment.

All of the above studies show in different ways how superantigens may be used as

prophylaxis for cancer as well as ways to treat cancer once tumors have already









established in the body. This is only one approach to the use of superantigens in the

treatment and prevention of disease.

In this study, through a combination of molecular biology techniques such as

cDNA microarray analysis and traditional immunological procedures, such as ELISA and

western blot techniques, specific cytokine production during superantigen stimulation of

HPBMC will be assessed in the context of activation of distinct groups of T cells. The

studies are designed to gain insight into how superantigens modulate lymphocyte

function in disease and immune therapy.














CHAPTER 2
MATERIALS AND METHODS

Human Peripheral Blood Mononuclear cells (HPBMC) Isolation and Cell Culture

Human donor leukocyte packs were obtained from Civitan Regional Blood Center

(Gainesville, FL). HPBMC were isolated using Histopaque 1077 (Sigma-Aldrich, St.

Louis, MO) density gradient centrifugation as per the manufacturers instructions. After

the removal of the cells from the gradient and 2 washes with RPMI 1640 culture media

supplemented with 10% heat-inactivated fetal bovine serum (FBS), 200 U/mL penicillin,

and 200 mg Streptomycin, the cells were resuspended in fresh supplemented culture

media and counted using a light microscope. 2 x 10A6 cells/well were plated in 24 well

plates and incubated at 370C in a 5% CO2 atmosphere and used immediately for further

experimentation

Human CD4+ T Cell Isolation

Human donor leukocyte packs were obtained from Civitan Regional Blood Center

(Gainesville, FL). An enrichment cocktail (Rosette Sep)for human CD4+ T cells (Stem

Cell Technologies, Vancouver, BC) was used to enrich for CD4+ T cells, followed by

isolation of the cells using Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) density

gradient centrifugation as per the manufacturers instructions. After removing cells from

the gradient and 2 washes with RPMI 1640 culture media, supplemented with 10% heat

inactivated fetal bovine serum (FBS), 200 U/ml penicillin, and 200 mg Streptomycin, the

cells were resuspended in fresh supplemented culture media and counted using a light

microscope.









2 x 106 cells/well were plated in 24 well plates and incubated at 370C in a 5% C02

atmosphere and used immediately for further experimentation.

Staphylococcal Enterotoxin Treatment of HPBMC and CD4+ T-cells

Staphylococcal enterotoxin A (SEA) (Toxin Technology, Sarasota, FL) and B

(SEB) (Toxin Technology, Sarasota, FL) were cultured with 2 x 106 HPBMC per well in

a 24 well plate, both at a concentration of 100 ng/ml. CD4+ T-cells (2 x 106 cells per

well) were treated with 100 ng/ml of SEB. SEB does not require APC for processing

(4,30). Media treated cells served as a control. Six wells were used for each treatment at

each time point. Cells were incubated as described above and were harvested from the

cultures at time points of 8h, 16h, 24h, and 48h. Following centrifugation for 10 minutes

at room temperature, cell pellets and supernatant. were separated and used for RNA

isolation and ELISA/Western Blot experiments respectively.

Total RNA Isolation

RNA from SEA and SEB or media treated HPBMC as described above was

obtained using the following RNA isolation kits: Absolutely RNA (Stratagene, LaJolla,

CA), RNAqueous (Ambion, Austin, TX), and TRIZOL Reagent (GibcoBrl, Carlsbad,

CA). The protocols were followed as described by the manufacturer. The RNA samples

were quantitated by reading absorbance at 260nm on a Gilford Instrument

Spectrophotometer 260 (Nova Biotech, El Cajon, CA). The RNA samples were diluted

1:100 in 10mM Tris or DNAse and RNAse free water. The following calculation was

used to determine the concentrations of RNA in [tg/[tl: [A260 x 100 x .040 ug/ul].

Following quantitation, RNA samples were stored at -800 C for future use in microarray

experiments described below.









Microarray Procedure

Microarray procedures were performed as described by the manufacturer

(SuperArray, Frederick, MD). Briefly, total RNA was used to prepare cDNA probes.

During this process the cDNA was labeled with biotinylated dUTP's. The probes were

denatured and allowed to hybridize overnight at 600C to the microarray membrane. The

nylon microarray membrane is spotted with cDNA from 96 common human cytokines

purchased from SuperArray Inc (Frederick, MD). Following a series of washes and

further blocking, the membrane was incubated with alkaline phosphatase-streptavidin.

CDP Star substrate was then added to the membrane. The gene microarray membranes

were exposed to film and developed. This procedure was followed as recommended by

the manufacturer (Super Array, Frederick, MD). A schematic of the entire procedure can

be found in Appendix B.

Analysis of Microarray Data

The film images of the media and superantigen treated microarray membranes

were scanned and converted to a TIFF format. The images were then analyzed using the

Image J 1.29 software (NIH, Bethesda, MD) and Scion Image software, to determine the

pixel density value for the spot of a desired gene. Each intensity value was normalized to

the positive control on its respective membrane, then divided by the corresponding media

control values. This value is a fold increase ratio of superantigen treated cells to media

treated cells.

Western Blot for TGFP3

HPBMC were cultured with or without superantigens described above and the

resulting supernatants were saved at -80 C and used for western blot analysis of TGF-33.

The supernatants were concentrated using Amicon centriprep YM-10 filters (Millipore,









Billerica, MA). A Bicinchoninic Acid (BCA) protein assay (Pierce, Rockford, IL) was

performed on the supernatants from control (media) or superantigen treated cells. Equal

amounts of each sample (26 [tg/lane) were loaded on a 15 % reducing SDS-PAGE ready

gel (BioRad, Hercules, CA) and run at 100V. Overnight transfer onto nitrocellulose

membrane was carried out, after which the membrane was block with 5% non-fat instant

milk in Tris-buffered saline (pH 7.5) and .01% Tween-20 for 1 hr. The Immunoblot was

incubated with a 1:1000 dilution of rabbit anti-human TGFi3 (Santa Cruz

Biotechnology, Santa Cruz, CA). After washing, a conjugated secondary antibody, goat-

anti-rabbit conjugated to horseradish peroxidase (HRP), (Santa Cruz Biotechnology,

Santa Cruz, CA) was added at a 1:12,000 dilution and incubated for 1 h. Blots were

washed and analyzed through film development.

ELISA for Human Cytokines

HPBMC were cultured with or without superantigens described above and the

resulting supernatants were saved at -80 C and used for ELISA assays. The supernatants

were concentrated using Amicon centriprep YM-10 filters (Millipore, Billerica, MA). A

BCA protein assay (Pierce, Rockford, IL) was performed on the supernatants from

control (media) or superantigen treated cells. Equal amounts of each sample were used in

the ELISA assays. HPBMC were treated with media or superantigen and supernatants

were collected at various time points as described above. IL-2, IL-10, and IFN-y levels

were determined using ELISA kits. The supernatants were tested for IFN-y using the

CytoScreen Immunoassay kit for IFN-y (Biosource International, Camarillo, CA), IL-2

using the BD-Opt-EIA kit for IL-2 (BD Biosciences), and IL-10 using the BD-OptEIA

kit for IL-10 (BD Biosciences). Color development was monitored at 490nm in an

ELISA plate reader (Biorad, Richmond, CA) after substrate solution from each respective









cytokine kit was added and reaction stopped with the stopping solution provided in each

kit.

Mouse Studies and Detection of Specific Antibodies in Mouse Sera

We used 6 to 8 week old female C57BL/6 mice (The Jackson Laboratory, Bar

Harbor, ME) in these studies. Mice were bled before injections with the BSA. We

injected 50 [tg of BSA intraperitoneally (i.p.) into the mice. One week later, mice were

injected i.p. with PBS or a combination (25 |tg each) of SEA and SEB (Toxin

Technology, Sarasota, FL). Mice were bled from the tailvein once a week for one month;

and sera were stored at -200C for further experiments.

Sera from the mice were tested for BSA specific IgG and IgM antibodies using a

standard ELISA protocol. Briefly, 50 [l of BSA (25 ng/well) in binding buffer (0.1 M

carbonate/bicarbonate, pH 9.6) were placed in wells of 96 well plates and allowed to

adhere overnight at room temperature. Plates were washed in wash buffer (150 mM

NaC1, 0.05% Tween 20) and free reactive sites were blocked for 2 h with 200 rl/well

blocking buffer (PBS (pH 7.2) containing 5% nonfat instant milk). After washing plates,

sera were diluted and 50 ul were placed in the wells for 1.5 h. Plates were again washed

and alkaline phosphatase- conjugated anti-mouse IgG whole molecule or anti-mouse IgM

(50 ul; Sigma Aldrich, St.Louis, MO) was added to wells. After 45 minutes, plates were

washed and 200 ul of substrate (Img/ml p-nitrophenyl phosphate in binding buffer) was

added to the plates. Color was allowed to develop for 30-60 minutes, after which 50 ul of

stop solution (2 M H2SO4) was added. Absorbance was read at 405 nm using a Model

450 Bio-Rad Microplate reader (BioRad, Hercules, CA).









Proliferation Assay

Human NK-92 cells (ATCC, Mannassas, VA), a natural killer cell line requiring

IL-2 for growth, were cultured in alpha minimum essential medium without

ribonucleosides and deoxyribonucleosides with 2 mM L-glutamine adjusted to contain

1.5 g/L sodium bicarbonate and supplemented with 0.2 mM inositol, 0.1 mM 2-

mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum and 12.5% fetal bovine serum

at 37 C. The cells were plated at 4 x 104 cells per well in a 96 well plate and treated

with either media, IL-2 (Biosource International, Camarillo, CA) (30 U/ml), or IL-2 and

TGFp (25 ng/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) for 48 hours. Cells were

then pulsed with 1 ptCi per well of 3H-thymidine for 6 hours, after which cells were

harvested an cell associated radioactivity was quantified using a P-scintillation counter

and activity reported as mean CPM +/- SD. All tests were run in replicates of six.














CHAPTER 3
RESULTS

Superantigens Enhance Specific Antibody Production to Antigens in Mice

It has been shown previously that superantigens enhance the immune cellular response

against melanoma cells in vivo in mice (15). Here a determination was made of the

ability of SEA and SEB to enhance the humoral antibody response of mice primed with

the T dependent antigen bovine serum albumin (BSA). Mice were first injected with

BSA alone, BSA followed seven days later by SEA/SEB, SEA/SEB alone, or PBS alone.

As can be seen in the ELISA measurements in Figure 3-1A, BSA alone induced an

antibody response that was enhanced greater than 2 fold By SEA/SEB at a 1:100 dilution

of sera. There was no antibody response to the control antigen gpl20 in the same sera.

This was evidenced by the low similar ELISA profiles for BSA alone, BSA followed by

SEA/SEB, SEA/SEB alone, or PBS alone. Thus, the enhancement of SEA/SEB was

specific for the primary antigen BSA. Furthermore, the antibody response to BSA was

IgG specific, but not IgM specific. At 14 days following BSA injection there was no

evidence of specific IgM antibodies to BSA in sera of mice, as per Figure 3-1B, where

the BSA response was compared with that of PBS. Importantly, a comparison of mice

injected with BSA and BSA followed by SEA/SEB showed the same profile of IgM

response. Thus, SEA/SEB did not enhance the IgM response under the same conditions

under which it enhanced the IgG response. Since specific IgG levels were increased

against BSA, total IgG levels were compared among the different groups. Mice treated

with BSA alone, BSA followed by SEA/SEB, SEA/SEB alone or PBS alone, did not









show any significant differences in total IgG levels, as shown in Figure 3-1C.

Thus, an enhancement effect of SEA/SEB on the total IgG levels was not observed.

Furthermore, the enhancement of the antibody response to BSA by SEA/SEB could not

be attributed to non-specific enhancement of total IgG. Therefore, the SEA/SEB

enhancement of the antibody response to BSA was antigen specific and of the IgG

isotype.

Superantigens Enhance Cytokine RNA Production in HPBMC

A determination of the ability of SEA and SEB to induce increased cytokine

production was first made through microarray analysis of RNA in cells treated with the

SAg's. Human PBMC were treated with 100 ng/ml of SEA or SEB, or culture media

alone. Cells were harvested from the culture at timepoints of 8, 16, 24, and 48 hours.

Total RNA was extracted from the cells and was used to synthesize cDNA. The cDNA

was allowed to hybridize to a nitrocellulose membrane that was spotted with various

human common cytokine genes. Representative microarray images of media, SEA, and

SEB treatments are shown in Figure 3-2. Analysis of the microarray membranes and

supernatants of the treated cells revealed that both SEA and SEB induce increased gene

expression in HPBMC as compared to those of media control cells. Appendix B contains

a list of all the cytokines coded for the on the microarray membrane.

SEA Induces Cytokine Gene Expression in HPBMC

As shown in Figure 3-3, SEA induces upregulation of several genes including IFN-

y, IL-2, CD40L, IL-10, IL-6, TNFP, TGFP and IL-11, with as much as 2-25 fold increase

over media in HPBMC. IFNy expression was similar at all four time points (5-15 fold

increase over media), where as expression of IL-2, IL-10 and IL-13 were maximal at 16

hours after SEA incubation. TGF-3, IL-6 and CD40L had maximal expression at 24









hours as compared to media controls. SEA induction of TNF3 gene expression was

approximately 2 fold greater than media at 8 hours, where as it was approximately 16

fold greater than media at 16-48 hours. Thus, SEA increased cytokine gene expression in

HPBMC as compared to media treated cells.

SEB Induces Cytokine Gene Expression in HPBMC

The cytokine gene expression induced by SEB in HPBMC was determined next.

As shown in Figure 3-4, SEB increased cytokine expression of several genes as compared

to media controls, although expression levels were lower than that was seen with SEA

treatment. SEB induction of IFN-y and IL-13 was maximal at 16 hours, IL-6 and TGF3

at 24 hours, and IL-10, TNF3, and CD40L at 48 hours as compared to the media control.

IL-10 and TGFP are regulatory cytokines, so the relative delay in their gene expression

could indicate a signal to modulate Thl cell cytokines. Therefore, SEB also upregulates

cytokine gene expression in HPBMC.

SEB Induces Cytokine Gene Expression in CD4+ T cells

Previously, it has been shown that superantigens activate CD4+ T cells (37).

Here we investigated the profile of cytokine gene expression in CD4+ T cells treated with

SEB. SEB does not require antigen presenting cells for processing (4,30), thus CD4+ T

cells were purified from HPBMC using the Rosette Sep procedure as described in

Materials and Methods, and then treated with 100 ng/ml of SEB for timepoints of 8, 16,

24, and 48 hours. As shown in Figure 3-5, similar cytokine genes were upregulated as

those seen above for HPBMC. Expression levels for TGF3, CD40L, and TNF3 were

greater than 50 times than that of media at 24 hours. IFN-y expression was maximal at

24 hours, IL-2 at 48 hours and IL-6 at 8 hours. Thus, SEB also stimulates cytokine

production in CD4+ T-cells.









Figure 3-1: Antigen and isotype specificity of superantigen enhancement of antibody to
BSA. Mice were injected with BSA alone, BSA followed by SEA/SEB,
SEA/SEB alone or PBS under the same conditions as in Materials and
Methods. A) Sera were tested by ELISA for IgG Abs to BSA or gpl20 B)
Sera were tested by ELISA for IgM Abs to BSA. C) Total IgG levels for the
sera of A are presented in C. Students t test: A)BSA vs BSA and SEA/SEB,
P<0.001; BSA and SEA/SEB, BSA vs gpl20, p<0.001. No comparisons were
significant in B and C. Data are representative of three experiments, each
performed in triplicate.






















-- BSA PBS only
14
-- BSA BSA
1 2 -0- BSA BSA+SAg

1 0 BSA SAg only

08 --GP120 PBS only
---GP120 BSA
06
-e-GP120 BSA+SAg
04 --GP120 SAg only

02 -

00
1 3000 1


1000 1 300

Dilution


--BSA only

10 -*-BSA+SAg
-A-SAg only

08 -x-PBS only


06-


04-


02-

00
1 3000 1 1000 1 300 1100

Dilution


E

S500
0
. 400


300
o
200


100


0


SAg BSA BSA+SAg


1 100









MEDIA SEA SEB




















Figure 3-2: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase
expression of various human cytokine genes. Human PBMC were treated with
100 ng/ml of SEA or SEB or media alone and cultured as described in
methods. Cells were harvested from the cultures at time points of 8, 16, 24,
and 48 hours. Total RNA was extracted from these cells and used to make
cDNA. The cDNA was allowed to hybridize overnight with a nylon
membrane spotted with cDNA spotted with various common human
cytokines. After washes and incubation with AP-streptavidin, and substrate,
the membranes were exposed to film and developed. Images seen here
represent 24 hours post treatment.




















30
8 hours 0 16 hours E 24 hours E 48 hours

,25






CU

-o
20 -





015 -



IFN-y IL-2 IL-10 TGFp IL-6 TNFp IL-10 CD40L

Figure 3-3: Staphylococcal enterotoxin A induces increased expression of various Thl,
Th2, and T regulatory cytokine genes. Human PBMC were treated with 100
ng/ml of SEA and cultured as described in methods. Cells were harvested
from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was
extracted from these cells and used to make cDNA. The cDNA hybridized
overnight to a nylon membrane spotted with cDNA of common human
cytokines. The complete list of cytokine genes on the nylon membrane is
presented in Appendix 3. After washes and incubation with AP-streptavidin,
and substrate, the membranes were exposed to film and developed. Pictures
were scanned into TIFF format and the software program ScionImage was
used to determine pixel density for each spot. Background values were
subtracted from each value, followed by normalization to a positive control on
each membrane. This normalized value for SEA treated cells was divided by
the same value for media to obtain the fold increase in gene expression of
each cytokine.























30

25

20

15
Ca
S10
-0
0 5

0


IFN-y IL-2 IL-10 TGFP IL-6 TNFp IL-10 CD40L


Figure 3-4


:Staphylococcal enterotoxin B induces increased expression of various Thl,
Th2, and T regulatory Cytokine Genes. Human PBMC were treated with 100
ng/ml of SEB and cultured as described in methods. Cells were harvested
from the cultures at time points of 8, 16, 24, and 48 hours. Total RNA was
extracted from these cells and used to make cDNA. The cDNA hybridized
overnight to a nylon membrane spotted with cDNA of common human
cytokines. The complete list of cytokine genes on the nylon membrane is
presented in Appendix 3. After washes and incubation with AP-streptavidin,
and substrate, the membranes were exposed to film and developed. Pictures
were scanned into TIFF format and the software program ScionImage was
used to determine pixel density for each spot. Background values were
subtracted from each value, followed by normalization to a positive control on
each membrane. This normalized value for SEB treated cells was divided by
the same value for media to obtain the fold increase in gene expression of
each cytokine.









Figure 3-5: Staphylococcal enterotoxin B induces increased expression of various Thl,
Th2, and T regulatory cytokine genes. Human CD4+ T cells were treated
with 100 ng/ml of SEB and cultured as described in methods. Cells were
harvested from the cultures at time points of 8, 16, 24, and 48 hours. Total
RNA was extracted from these cells and used to make cDNA. The cDNA
hybridized overnight to a nylon membrane spotted with cDNA of common
human cytokines. The complete list of cytokine genes on the nylon membrane
is presented in Appendix B. After washes and incubation with AP-
streptavidin, and substrate, the membranes were exposed to film and
developed. Pictures were scanned into TIFF format and the software program
ScionImage was used to determine pixel density for each spot. Background
values were subtracted from each value, followed by normalization to a
positive control on each membrane. This normalized value for SEB treated
cells was divided by the same value for media to obtain the fold increase in
gene expression of each cytokine. A) Fold increase of IFN-y, IL-2, IL-10 and
IL-6 in CD4+ T cells treated with SEB. B) Fold increase of TGFO, TNF3,
CD40L, and IL-10 in CD4+ T cells treated with SEB.














* 8 hours O 16 hours O 24 hours 48 hours


TGFP TNFP


CD40L


* 8 hours 0 16 hours O 24 hours O 48 hours


i-ri


-li-K


TNFB CD40L


20







10

0
a

-e


-5
0


fc


250


IL-10


-200
C-

S150


1 100
0



0-
0


". -L-


"""


""


TGFB3


IL1B









Superantigens Enhance Cytokine Protein Production in HPBMC

Since superantigens enhance the expression of various cytokine genes, we next

determined cytokine protein levels in supernatants of human PBMC treated with SEA or

SEB. ELISA and Western Blots were performed on the supernatants of HPBMC treated

with 100 ng/ml of either SEA, SEB or media. IL-2, IFN-y, IL-10 and TGF3 levels were

determined. As shown in Figure 3-6, IL-2 was present in cultures treated with SEA and

SEB, but not in media. Concentrations of IL-2 were greater than 600 ng/ml in both SEA

and SEB treated groups at all four timepoints. Media treated cells supernatants had IL-2

levels of less than 50 ng/ml at all timepoints. This was in contrast to the IL-2 mRNA

levels, which decreased after 16 hours. Similarly, IFN-y and IL-10 protein levels were

higher in supernatants from SEA and SEB treated cells than the media treated cells.

IFN-y levels in cell supernatants were higher at 16 hours than at 24 hours, but were

maximal at 48 hours (Figure 3-7). Media treated cell supernatants had low IFN-y protein

(<0.05 OD). IL-10 levels were highest at 48 hours (Figure 3-8) (>1500 ng/ml). Media

treated cells had no detectable IL-10 at any of the timepoints. These protein levels were

fairly consistent with the mRNA levels seen on the microarray. Higher concentrations of

IL-10 at the later time points may be acting to down regulate the action of IL-2 (See

discussion). Although TGF3 gene expression was increased as shown on the microarray,

there was no detectable protein in the supernatants as determined by ELISA (data not

shown) and Western Blots (Figure 3-9). Although TGF3 may be expressed at a later

timepoint than 48 hours, cells begin to die after 48 hours incubation with superantigen

and generally undergo apoptosis or energy. Therefore, cytokine measurements may be

inaccurate after 48 hours. Thus, superantigens also induce cytokine protein production in

conjunction with increased cytokine gene expression in HPBMC in culture.



















* SEA E SEB O Media


T T


8 16


24 48


Time (hours)



Figure 3-6: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase IL-2
production in human PBMC. HPBMC were treated with 100 ng/ml of either
SEA or SEB, or culture medium alone. Cells were cultured as described in
methods. Supernatants were harvested from the cultures at timepoints of 8,
16, 24, and 48 hours. An ELISA was performed on the supernatant to
determine levels of IL-2. Data shown here represent mean and SD of
duplicate experiments.


1000
900
800
700
600
500
400
300
200
100
0

















U SEA 1 SEB O Media


8 16


24 48


Time (hours)


Figure 3-7: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase
IFN-y production in human PBMC. HPBMC were treated with 100 ng/ml of
either SEA or SEB, or culture medium alone. Cells were cultured as described
in methods. Supernatants were harvested from the cultures at timepoints of 8,
16, 24, and 48 hours. An ELISA was performed on the supernatant to
determine levels of IFN-y. Data shown here represent mean and SD of
duplicate experiments.















2500


2000


1500


S1000
500
d 500


* SEA 1 SEB E Media


24 48


Time (hours)


Figure 3-8: Staphylococcal enterotoxin A and staphylococcal enterotoxin B increase
IL-10 Production in Human PBMC. HPBMC were treated with 100 ng/ml of
either SEA or SEB, or culture medium alone. Cells were cultured as described
in methods. Supernatants were harvested from the cultures at timepoints of 8,
16, 24, and 48 hours. An ELISA was performed on the supernatant to
determine levels of IL-10. Data shown here represent mean and SD of
duplicate experiments.


Elm




















A B C D E F G H I JK







Figure 3-9: Staphylococcal enterotoxin A and staphylococcal enterotoxin B do not
increase TGFP production in human PBMC. HPBMC were treated with 100
ng/ml of either SEA or SEB, or culture medium alone. Cells were cultured as
described in methods. Supernatants were harvested from the cultures at
timepoints of 8, 16, 24, and 48 hours. A Western Blot was performed on the
supernatants to determine levels of TGF3. Data shown here represent 24 and
48 hour timepoints. A: TGFP MW marker (25 kDa), B: blank, C: blank, D:
24h SEA, E: 24h SEB, F: 24h media, G: 48h SEA, H: 48h SEB, I: 48h media,
J: media control K: Molecular weight ladder









TGFp Suppresses IL-2 Induced Growth in NK92 Cells in vitro

NK92 cells, an IL-2 dependent cell line, were treated with either media, IL-2 (30

U/ml), or IL-2 and TGFp (25 ng/ml) for 48 hours. Cells were then pulsed with 3H-

Thymidine for 6 hours and cell associated radioactivity was counted on a P-scintillation

counter. As shown in Figure 3-10, cells cultured with IL-2 and TGFp had almost 50%

less proliferation than those cells cultured with IL-2 alone. Cells cultured with media

alone, had low proliferation, showing that IL-2 is necessary for significant proliferation.

Therefore, TGFP can suppress IL-2 induced proliferation.


12000


10000


8000


6000


4000


2000

0
Media IL-2 IL-2 and TGFp
Treatment (25ng/ml)

Figure 3-10: Transforming growth factor beta suppresses IL-2 induced proliferation of
NK-92 cells. Human NK92 cells were plated at 4 x 104 cells per well in a 96
well plate and treated with either media, IL-2 (30 U/ml), or IL-2 and TGFp
(25 ng/ml) for 48 hours. Cells were then pulsed with 1 tCi per well of 3H-
thymidine for 6 hours, after which cells were harvested and cell associated
radioactivity was quantified using a P-scintillation counter and activity
reported in CPM. All experiments were performed in replicates of six.















CHAPTER 4
DISCUSSION

In the first part of this study the immunoenhancing effects of superantigens on the

humoral arm of the immune response were studied. Immunization of mice with the

prototype T-dependent Ag BSA followed by SEA/SEB resulted in increased IgG

antibody response to BSA. Thus, enhancement effects of superantigens were specific for

the primary antigen BSA. The results presented here, combined with those of previous

findings on superantigen enhancement of tumor-specific immunity to mouse melanoma

(14), are evidence that superantigens such as SEA and SEB can significantly boost Ag-

specific immune responses.

Microarray experiments of human PBMC treated with SEA, SEB or media alone

showed increases in mRNA levels in various cytokines. These cytokines include but are

not limited to IFN-y, IL-2, TNF-P, IL-6, IL-10 and TGF-3. The presence of IL-2 mRNA

is seen as early as 8 hours, peaking at 16 hours, followed by a decline after that through

48 hours (Figure 3-3). IFN-y mRNA expression levels peak at 24 hours, followed by a

decline (Figure 3-3, Figure 3-4, Figure 3-5). TNF-3 mRNA expression increases at 16 h

and is maintained through 48 hours (Figure 3-3). These three cytokines are indicative of

a typical Thl inflammatory type response. In the case of Th2 cytokine expression, IL-6

mRNA expression levels are maximal at 24 hours followed by a rapid decline

(Figure 3-3). TGF-3 and IL-10, which are produced by T regulatory CD4+ T cells

are maximal at 16 and 24 hours respectively. The studies on the cytokine profile of









superantigen activated cells and the enhancement of the humoral response against BSA

presented here and the studies on enhancement of cellular response against melanoma

cells suggest distinct T cell populations are being activated by superantigens (14).

Thus, superantigens activate Thl, Th2, and T regulatory cells in vivo and in vitro.

In conjunction with the microarray studies, ELISA and Western blot studies were

run to determine if the increases in mRNA levels were associated with translation of the

message. ELISA (Figure 3-7) and Western Blots for IFN-y (data not shown) showed

increases in IFN-y over time, with very little production of the IFNy in media treated

cells. Furthermore, IL-2 levels increased as early as 8 hours and remained constant

through 48 hours (Figure 3-6). This is in contrast to the mRNA levels of IL-2, which

started to decrease at the same time, possibly due to action of T regulatory cells. This

may be due to a lag period between RNA message decline and seeing an actual decline in

the protein produced. Carrying out the experiment for a much longer period of time may

show the actual decrease in protein levels. However, this may prove to be difficult, as

cells exposed to these high a concentrations of SEA or SEB begin to die after 48 hours.

The fact that IL-10 protein expression in superantigen treated cells increased over time

and is maximal at 48 hours (Figure 3-8) indicates how the T regulatory cells control the

Thl and Th2 cell types and plays an important role in helping the immune system to

recover from encounters with superantigen. TGFP expression increases after

superantigen stimulation of cells, but is not detected at the protein level one to two days

after superantigen stimulation (Figure 3-9). Others have shown that IL-10 enhances the

expression of TGFP (4,13,25). Thus, TGFP protein production may occur beyond the 48

hour time point measured here but may be difficult to detect for the same reason









discussed above for IL-2. It has been previously shown in mice lacking CD4 25+ T-cells,

which is the phenotype of T regulatory cells, have sustained production of inflammatory

cytokines and that this production can be corrected by injections with CD4 25+ T-cells

(20). As per Figure 3-9, TGFP suppresses IL-2 induced proliferation by almost 50% in

NK92 cells, an IL-2 dependent cell line. This suggests how the regulatory cytokines

produced at later time points during superantigen stimulation, may act to down regulate

the effects of Thl cytokines produced earlier. It also identifies a potential target to block

regulatory cytokines or a particular cell type, to bias and sustain an inflammatory or

humoral response for a longer period of time. Thus, similar to an increase in RNA

expression, IL-10, IL-2 and IFN-y protein expression increase in culture supernatants

taken from superantigen treated cells.

Superantigens enhance specific humoral and cellular responses against antigens,

such as BSA in vivo and this immune enhancement is due to the action of superantigen

on CD4+ T cells that produce inflammatory, helper and regulatory cytokines in a time

dependent manner. Inflammatory cytokines produced by Thl cells are induced initially

by superantigen after which regulatory and suppressor cytokine, produced by Th2 and T

regulatory cells, levels increase. There is an inherent characteristic of superantigen effects

on naive vs Ag-primed T cells that is a plus for their immunoenhancing properties. Naive

T cells initially undergo cell division when treated with superantigens, followed shortly

by energy and/or deletion. Ag-primed T cells also expand when treated with superantigen

but, in contrast, do not undergo the anergy/deletion characteristic of naive T cells (11).

Thus, the VP-specific polyclonal expansion associated with superantigens is tilted toward









primed Ag-specific T cell. This effect may be of beneficial use in vaccinations to boost

immunity to a particular pathogen or disease.













APPENDIX A
MICROARRAY PROTOCOL


RNA


} Probe labeling
1-2 hour

}Hybrid~zwUewn
overnIgHt


DeteCUMln
2 3h- r


RHw image
3, D~a~a extracuan
F l) aw cdata
Dow Deanalysis with
G IArray Anrtymar

rztza tabliff


an Data
analysis


Figure A-i: Microarray Protocol (GEArray Q
MD)


series protocol from Superarray, Frederick,

















APPENDIX B
MICROARRAY GENE LIST


Allograft inflammatory factor 1
Bone morphogenetic protein 1
bone morphogenetic protein 2
Growth differentiation factor 10
Bone morphogenetic protein 4
Bone morphogenetic protein 6
bone morphogenetic protein 8
Colony stimulating factor 1
Colony stimulating factor 2

Colony stimulating factor 3
Homo sapiens erythropoietin

Fibroblast growth factor 1
Fibroblast growth factor 10
Fibroblast growth factor 11
Fibroblast growth factor 12
Fibroblast growth factor 12B
Fibroblast growth factor 14
Fibroblast growth factor 16
Fibroblast growth factor 17

Fibroblast growth factor 19

Fibroblast growth factor 2

Fibroblast growth factor 20
Fibroblast growth factor 21
Fibroblast growth factor 23
Fibroblast growth factor 3
Fibroblast growth factor 4

Fibroblast growth factor 5
Fibroblast growth factor 6
Fibroblast growth factor 7
Fibroblast growth factor 9

Glyceraldehyde-3 -phosphate
dehydrogenase (positive control


c-fos induced growth factor
Hepatocyte growth factor
interferon, alpha 1
Interferon, alpha 2
Interferon, alpha 4
Interferon, alpha 5
Interferon, alpha 6
Interferon, alpha 7
Interferon, beta 1, fibroblast

Interferon, gamma
Interferon, omega 1

Insulin like growth factor IA
Insulin-like growth factor 2
Interleukin 10
Interleukin 11
Interleukin 12A, p35
Interleukin 12B ip-)4
Interleukin 13
Interleukin 14

Interleukin 15

Interleukin 16

Interleukin 17
Interleukin 18
Interleukin 19
Interleukin 1, alpha
Interleukin 1, beta

Interleukin 2
Interleukin 20
Interleukin 22
Interleukin 3

Homosapiens peptidylprolyl
isomerase A


Interleukin 4
Interleukin 5
Interleukin 6
Interleukin 7
Interleukin 8
Interleukin 9
Leptin
Lymphotoxin-alpha
Lymphotoxine-beta

Platelet-derived growth factor-BB
Platelet-derived growth factor alpha
polypeptide
Pleiotrophin
Transforming growth factor, alpha
Transforming growth factor, beta 1
Transforming growth factor, beta 2
Transforming growth factor, beta 3
Thrombopoietin
Tumor necrosis factor
Tumor necrosis factor (ligand)
superfamily, member 10
Tumor necrosis factor (ligand)
superfamily, member 11
Homo sapiens TNF (ligand)
superfamily, member 4
CD40 ligand
Ligand for Fas
CD27 ligand/CD70 antigen
CD30 ligand
Tumor necrosis factor (ligand)
superfamily, member 9
Vascular endothelial growth factor
Vascular endothelial growth factor B
Vascular endothelial growth factor C
PUC18 Plasmid DNA (negative
control)
Ribosomal protein L13a (positive
control)
Beta Actin (positive control)


Table B-l: Microarray gene list. (GEArray Q series protocol from Superarray, Frederick,
MD)
















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BIOGRAPHICAL SKETCH

Amy Kristin Anderson was born in New Jersey on May 2, 1977. Her family

got larger, when her brother Tim was born in March of 1980. Her family lived in NJ

until she was 10 years old, when they moved to Tinmouth, Vermont in December of

1987. Amy enjoyed living in the rural town of about 400 people, having a horse and

attending smaller sized elementary and high schools. After graduation from Mill

River Union High School in 1995, Amy began attending the University of New

Hampshire, on a partial academic scholarship, where she pursued a Bachelor of

Science in Medical Laboratory Science. Amy graduated magna cum laude from

UNH in 1999 and got a job at Duke Medical Center in Durham, NC, in the

immunohematology lab as a Medical Technologist. While living in NC, Amy met

Glenn, who was working on his Master of Science at North Carolina State University.

Glenn's job search after graduation in 2000, took him all the way to Florida and Amy

decided to look at graduate school there. Amy began attending the University of

Florida in the fall of 2001 to pursue a Master of Science degree, studying in the

department of Microbiology and Cell science. In November of 2001, Amy decided to

do her master's research work in the laboratory of Dr. Howard M. Johnson. Amy and

Glenn were married on June 28, 2003 in Vermont. After graduation, Amy plans to

pursue a career in the biotechnology industry, hopefully doing research.