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7th Annual Upstate New York Immunology Conference Sponsored by: Taconic BD Biosciences Invitrogen National Institute of Allergy and Infectious Diseases Pfizer, Inc. November 14-16, 2004 The Sagamore Bolton Landing, New York

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  • 7th AnnualUpstate New York

    Immunology Conference

    Sponsored by:


    BD Biosciences


    National Institute of Allergyand Infectious Diseases

    Pfizer, Inc.

    November 14-16, 2004The Sagamore

    Bolton Landing, New York

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    Francisella tularensis LVS bacteria infecting murine peritoneal macro-phages. Red bacteria are internal, yellow and green are external.Cell nuclei are stained blue with DAPI.

    Submitted by: Christopher CollinsCenter for Immunology and Microbial DiseaseAlbany Medical College

    Upstate New York Immunology ConferenceCenter for Immunology and Microbial Disease

    Albany Medical College, MC-15147 New Scotland Ave.

    Albany NY 12208Phone: (518) 262-5365 Fax: (518) [email protected] www.amc.edu/NYIC

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    Schedule of Events 7

    Supporting Institutions 13

    Acknowledgements 15

    NYIC GrantsNIAID and Pfizer, Inc.


    Major Corporate Sponsors:Taconic Farms, BD Biosciences, and Invitrogen


    Corporate Supporters:VWR, BioLegend, and Fisher Scientific


    Corporate Contributors 27

    Presentation Abstracts 29

    Poster Abstracts 67

    Contact Information 115

    The Sagamore 120

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    Sunday, November 14th

    4:00-6:00 p.m. Hotel Registration (Registration Building)

    4:00-5:00 p.m. Conference Registration (Sagamore Parlor)

    5:00-6:30 p.m. Dinner (Sagamore Dining Room)

    6:30-7:30 p.m. Keynote Speaker- Introduction by: Dr. Yasmin Thanavala

    Leo Lefrancois, Ph.D.Chief, Division of ImmunologyProfessor of MedicineUniversity of Connecticut Health Center

    "The Curious Case of IL-15: CytokineTranspresentation Drives T Cell Homeostasis andDevelopment"

    8:00-9:30 p.m. Session I: Advances in Biotechnology (Bellvue) Session Chair: Dr. James R. Drake

    Gerald Bothe, Ph.D. (Taconic)“Taconic Animal Model Phenotyping”

    Susan Reynolds (BD Biosciences)Technical Applications Specialist“Analysis of Protein Phophorylation and Cellular Signaling Events by Flow Cytometry”

    Michael Grossman (Invitrogen Corporation)Biodefense Manager“Capabilities in Biodefense and Immunology”

    9:30-10:30 p.m. Cocktail Reception

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    Monday, November 15th

    7:00-8:00 a.m. Breakfast (Sagamore Dining Room)

    8:00-10:00 a.m. Session II: Current Topics in Immunology(Bellvue) Session Chair: Dr. Frances Lund

    Naveen Bangia, Ph.D. (Roswell Park)“Functions of Tapasin in Antigen Presentation by MHC Class I Molecules”

    Erica McGovern, B.S. (Albany Medical College)“Proteosome Inhibition Selectively Inhibits BCR-mediated Antigen Processing and Presentation”

    Alexandra Livingstone, Ph.D. (University of Rochester)“Role of CD40-CD40L Interactions in the Generation of Helper-dependent CD8+ T Cell Responses”

    Rebecca Emeny, Ph.D. (Wadsworth Center)“The Neuroimmune Network and Host Defense Mechanisms”

    Karen Clise-Dwyer, Ph.D. (Trudeau Institute)“Functional Defects in Recent Thymic Emigrants From Aged Mice”

    Lisa P. Daley, M.S. (Cornell)“Characterization of Llama IgG Isotypes Induced During Parelaphostrongylus Tenuis Infection”

    10:00-10:30 a.m. Break (Conference Center Foyer)

    10:30-12:30 p.m. Session III: Infection, Immunity and Vaccines(Bellvue) Session Chair: Dr. Gary Winslow

    Kenneth Ely, Ph.D. (Trudeau Institute)“Effector-memory T Cell Populations in the Lung Airways are Maintained by a Process of Continual Recruitment”

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    Monday, November 15th (cont’d)

    Noelle Polakos, M.S. (University of Rochester) “Influenza-associate Hepatitis is Induced by Intra-hepatic Accumulation of Virus-specific CD8+T cells”

    Danuta Kozbor, Ph.D. (Roswell Park)“Strategies to Enhance Cellular and Antibody Responses to the HIV Envelope Glycoprotein”

    Rhonda Kines, B.S. (Roswell Park)“An Analysis of the Immune Response to HPV-16 E7 in HLA-DR Transgenic Mice”

    Rosemary Rochford, Ph.D. (SUNY Upstate)“Immunoevasion: Lessons From a Gammaherpesvirus”

    Matthais Hesse, Ph.D. (Cornell University)“Regulating the Regulators or How to Kill PathogensWithout Killing Yourself”

    12:30-1:30 p.m. Lunch (Nirvana)

    1:30-3:30 p.m. Session IV: Mucosal Immunity(Bellvue) Session Chair: Dr. Leo Lefrancois

    Dennis W. Metzger, Ph.D. (Albany Medical College)“Mucosal Immunity to Francisella tularensis LVSafter Pneumonic Infection”

    Shabaana A. Khader, Ph.D. (Trudeau Institute)“IL-23 is Essential for the Induction of IL-17Producing Antigen-specific T cells During Tuberculosis”

    Deborah M. Brown, Ph.D. (Trudeau Institute)“Separate and Distinct Mechanisms of CD4-mediatedProtection to Lethal Influenza Infection”

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    Monday, November 15th (cont’d)

    Albert Sabirov, M.D., Ph.D. (Albany Medical College)“Intranasal Vaccination Against Pneumococcal Otitis Media in Mice”

    Nick Mantis, Ph.D. (Wadsworth Center)“Secretory IgA Functions in Innate and Adaptive Immunity to Ricin”

    Michael Russell, Ph.D. (University at Buffalo)“Immunomodulation by Enterotoxin-basedAdjuvants and Vaccine Delivery Systems”

    3:30-4:00 p.m. Break (Conference Center Foyer)

    4:30-5:30 p.m. Poster Set-Up

    4:30-7:30 p.m. Vendor Set-Up

    5:30-6:30 p.m. Dinner (Sagamore Dining Room)

    6:30-7:30 p.m. Keynote Speaker- Introduction by: Dr. Dennis W. Metzger

    Christine A. Biron, Ph.D.Esther Elizabeth Brintzenhoff Professor of Medical ScienceChairperson, Department of Molecular Biology

    and ImmunologyBrown University

    "Induction and Function of InnateImmune Responses to Viral Infection"

    7:30-10:30 p.m. Poster Session, Vendor Fair and Mixer(Wapanak and Nirvana)

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    Tuesday, November 16th

    7:00-8:00 a.m. Breakfast (Sagamore Dining Room)

    8:00-10:00 a.m. Session V: Innate Immunity(Bellvue) Session Chair: Dr. Christine Biron

    Steven Taffet, Ph.D. (SUNY Upstate)“Gap Junctions and Macrophages”

    Barbara Butcher, Ph.D. (Cornell University)“Suppression of Macrophage Proinflammatory Signaling Pathways by Toxoplasma Gondii”

    Stephen Smiley, Ph.D. (Trudeau Institute)“Infection-stimulated Fibrin Deposition Controls Hemorrhage and Limits Bacterial Growth During Literiosis”

    Edmund J. Gosselin, Ph.D. (Albany Medical College)“Human Immune Responses to Francisella tularensis”

    Meenakshi Malik, Ph.D. (Albany Medical College)“Toll-like Receptor Signaling Regulates Replicationof Francisella tularensis in a Mouse Model of Pneumonic Tularemia”

    Frances Lund, Ph.D. (Trudeau Institute)“Role of the Ectoenzyme CD38 in Regulating Chemotaxis and Adaptive Immune Responses”

    10:00-10:30 a.m. Break (Conference Center Foyer)

    10:30-12:00 p.m. Session VI: Tumor Immunity(Bellvue) Session Chair: Dr. Edith Lord

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    Tuesday, November 16tthh (cont’d)

    Chien-Chung Chang, M.S. (Roswell Park)“Structural Basis of Human High Molecular Weight Melanoma-associated Antigen Mimicry by Mouse Anti-idiotypic Monoclonal Antibody MK2-23”

    Lori Broderick, B.S. (University at Buffalo)“Evaluation of Human CD4+ Effector Memory T Cells Persisting in the Tumor Microenvironment of Non-small Cell Lung Cancer”

    Edith Kabingu,, B.S. (Roswell Park)“CD8 Dependent Control of Malignancy by Photodynamic Therapy”

    Edith Lord, Ph.D. (University of Rochester)“Immunity and the Tumor Microenvironment”

    12:00-1:00 p.m. Lunch (Trillium)

    Depart from Conference

    We would like to thank attendees for giving presentations,submitting posters and chairing sessions.

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    Financial Support Provided by the Following Institutions:

    Alumni Association of Albany Medical College

    Department of Microbiology & Immunology

    Cornell University & Veterinary College

    Department of ImmunologyRoswell Park Cancer Institute

    Microbiology & Immunology ProgramSUNY Upstate Medical University

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    Department of Immunology & Microbiology

    University at Buffalo

    NIAID Pre and Postdoctoral Training ProgramDepartment of

    Microbiology & ImmunologyUniversity of Rochester Medical Center

    Trudeau Institute

    Wadsworth Center

    Thank you for your continued support!

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    7th Annual Upstate New York Immunology Conference would like to take this opportunity to give

    “special thanks” to:

    Jim Drake, Ph.D., and Ed Gosselin, Ph.D., for serving as the Conference Co-organizers.

    Yasmin Thanavala, Ph.D., and Gary Winslow, Ph.D., for coordinating this year’s sci-entific program.

    Dawn Bellville for managing all of the innumerable and multi-faceted components necessary to produce a well-organized meeting.

    Dennis W. Metzger, Ph.D., for efforts in obtaining continued financial support from NIAID and Corporate Sponsors, which help keep the meeting affordable for all.

    Pat Jarrett and Tim McCullough of the Sagamore for all of their assistance, input and cooperation in accommodating the needs of conference and its participants.

    Institutional Representatives for obtaining financial support and promoting atten-dance:

    Laura Haynes (Trudeau Institute)Matthias Hesse & Jerrie Gavalchin (Cornell University)Edmund Gosselin (Albany Medical College)Edith Lord (University of Rochester)Michael Russell (SUNY at Buffalo)Allen Silverstone (SUNY Upstate)Yasmin Thanavala (Roswell Park Cancer Institute)Gary Winslow (Wadsworth Center)

    Finally, all of the individuals, too many to name here, whose feedback and ideas are vital to the success and growth of this annual Conference.

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    Pfizer, Inc.is proud to provide

    an EducationalGrant

    For the7th Annual

    Upstate New YorkImmunologyConference

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    Thank you to the

    National Institute of Allergy

    and Infectious Diseasesfor continuing grant

    support forGraduate Students

    andPostdoctoral Fellows

    R13 AI51522

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    Financial Support Provided by:

    Cell Signaling


    Guava Technologies


    Jackson Immuno Research



    New England BioLabs

    Ohaus Corporation



    Thermo Electron Corporation

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    Leo Lefrancois, Ph.D.Chief, Division of Immunology

    Professor of MedicineUniversity of Connecticut Health Center

    “The Curious Case of IL-15:Cytokine Transportation Drives

    T Cell Homeostasis and Development”

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    Keynote SpeakerSunday, November 14, 2004

    The Curious Case of IL-15: Cytokine Transpresentation Drives T Cell Homeostasis and Development

    Leo LefrancoisUniversity of Connecticut Health Center, Farmington, CT

    A variety of immunological processes are dependent on the IL-15/IL-15R system. These include the maintenance of memory CD8 T cells and the development of NK/NKT cells and the CD8aa subsetsof intestinal intraepithelial T lymphocytes (IEL). In the case of memory T cell homeostasis prolif-erative signals are provided by IL-15 and survival signals are mediated by IL-7. IL-15 acts through a trimolecular receptor composed of the IL-15Ra, the IL-2/IL-15Rß and the common ? chain, ?C.However, recent findings indicate that IL-15Ra can bind IL-15 in the absence of the other receptor subunits and transpresent the cytokine to responding cells lacking IL-15Ra? but expressing IL-15Rßand ? chains. Thus, IL-15Ra-/- memory CD8 T cells undergo normal proliferation when transferred to normal but not to IL-15Ra-/- hosts, and this proliferation required bone-marrow derived cells to express IL-15Ra. In contrast, IL-15-dependent IEL subsets developed only when host parenchymal tissues expressed IL-15Ra.? Furthermore, IL-15 and IL-15Ra operated most effectively when ex-pressed by the same cell type, suggesting that IL-15 may associate with IL-15Ra intracellularly. Since the requirement for IL-15 in these processes is constitutive, this unique mechanism of action may serve to increase the effective half- life of IL-15 and allow tightly regulated directional signals to be delivered. This work was supported by the NIH.

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    Session I.

    Taconic Animal Model Phenotyping

    Gerald Bothe, Ph.D.Taconic, Inc

    Germantown, NY


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    Session I.

    Analysis of Protein Phosphorylation and Cellular Signaling Events in Flow Cytometry

    Susan Reynolds Technical Applications Specialist

    BD Biosciences

    Reversible protein phosphorylation controls the activity of numerous biological functions and its dysregulation has been implicated in numerous disease states. BDTM PhosFlow uses phospho-specific antibodies, in combination with flow cytometry, to monitor phosphorylation events at unique residues within proteins-at the single cell level. Phospho-specific antibodies tagged with unique fluorochromes, in combination with cell surface markers, permit the simultaneous visualiza-tion of multiple phosphorylations in distinct cell sub-populations. In addition, beads with distinctive fluorescent intensities have been used in combination of pairs of phospho-specific antibodies and those recognizing the total version of the protein, for detecting phosphorylation in total cell lysates.Samples are read in a flow cytometer against a phospho-protein standard. CBA significantly re-duces the time needed to monitor multiple phosphorylation events in total lysates, and adds the con-venience of an easy and more quantitative readout than most existing methodologies. This presenta-tion highlights the utility of flow cytometry and immunoassay applications for measuring major phosphorylation events during cellular activation in various cell model and whole blood systems.

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    Session I.

    Capabilities in Biodefense and Immunology

    Michael GrossmanBiodefense Manager

    Invitrogen Corporation


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    Session II.

    Functions of Tapasin in Antigen Presentation by MHC Class I Molecules

    Sarah McEvoy1, Jaana Karttunen2, Elisa Boden2, Peter Cresswell2 and Naveen Bangia11Department of Immunology, Roswell Park Cancer Institute Buffalo, NY 14263

    2Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine New Haven, CT 06510

    Tapasin is a chaperone of the endoplasmic reticulum (ER) that facilitates assembly of MHC class I molecules with peptides for transport to the cell surface. Only MHC class I molecules with bound peptide are transported to the cell surface for CTL recognition. Tapasin facilitates peptide loading by linking MHC class I to TAP, which is the source of peptides in the ER. In the absence of tapasin, TAP and MHC class I interaction is decreased and leads to an overall decrease in the total amount of MHC class I at the cell surface. The transmembrane domain (TMD) of tapasin has a positively charged lysine residue (K408) in an otherwise hydrophobic region. In other TMD con-taining proteins, it has been shown that a TMD containing a charged residue decreases protein sta-bility. These charged residues in the TMD are important for protein – protein interactions. Since tapasin TMD is required for TAP interaction, TAP is the most likely candidate to stabilize tapasin.Therefore we hypothesize that the lysine in the TMD of tapasin is destabilizing and requires interac-tion with another protein such as TAP for maximal stability. We find that mutation of K408 to a non-charged alanine (A), results in a more stable tapasin molecule. In cells expressing K408A tapasin, TAP interaction was maintained but MHC class I transport was delayed. The delay in MHC class I transport did not affect MHC class I stability as measured by resistance to thermal denatura-tion. Since K408A tapasin interacts with TAP, we propose that another molecule with a negatively charged residue in its TMD must interact with the tapasin TMD to maintain maximal stability.

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    Session II.

    Proteosome Inhibition Selectively Inhibits BCR-Mediated Antigen Processing and Presentation

    Erica McGovern and James DrakeCenter for Immunology and Microbial Disease, Albany Medical College

    B-lymphocytes internalize antigen via the B cell receptor (BCR), which then traffics to LAMP+ late endocytic compartments. Subsequently antigen is released, processed into peptides, and loaded onto MHC class II molecules. As previously reported, internalized antigen-BCR complexes selectively persist for a prolonged period in LAMP+ late endosomes, and these complexes are likely to serve as the source of antigen-derived peptides to support the observed expression of the prolonged peptide-class II complexes. This observation demonstrates that there is control over the intracellular traffick-ing of antigen-BCR complexes within multi-vesicular late endosomes. Moreover, recent studies on the epidermal growth factor receptor have demonstrated a requirement for mono-ubiquitination of the receptor for proper targeting to late multi-vesicular endocytic compartments. Therefore an es-tablished assay to follow antigen-BCR persistence was employed to examine the effect of pro-teosome inhibition, and hence ubiquitin depletion, on antigen processing and presentation. The re-sults of this analysis demonstrate that proteosome inhibition does not alter BCR mediated antigen endocytosis, or the trafficking of fluid phase markers through the endocytic pathway. However, ubiquitin depletion was found to selectively alter antigen-BCR complex trafficking within the endo-cytic pathway, as well as the subsequent processing and presentation of the BCR- internalized anti-gen. Therefore, the results suggest that ubiquitination of the BCR or a BCR associated protein may be necessary for proper antigen-BCR sorting, processing and presentation.

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    Session II.

    Role of CD40-CD40L Interactions in the Generation of Helper-dependentCD8+ T Cell Responses

    Alexandra LivingstoneUniversity of Rochester Medical Center


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    Session II.

    The Neuroimmune Network and Host Defense Mechanisms

    R.T. Emeny, J. Hornickel, T. Mondal, D. Gao and D.A. LawrenceWadsworth Center, NYSDOH

    The generation of an immune response against both infectious and immunizing agents may be sub-stantially modified by a disruption of homeostasis under stressful circumstances. Our previous stud-ies have demonstrated that one-hour cold/restraint (CR), a psychological and physical stressor, pre-ceding bacterial challenge with Listeria monocytogenes (LM) suppresses host resistance. The stress-related factors involved in this immunomodulation are derived from activation of the sympa-thetic nervous system since chemical sympathectomy (6-OHDA treatment) can abrogate the stress-induced immunosuppression. Moreover, the specific involvement of beta1 and beta2 adrenergic re-ceptors (ARs) in modifying host resistance to Listeria monocytogenes (LM) has been demonstrated by pharmacologic administration of beta blockers (atenolol and propranolol) prior to the stress treat-ment. Current in vivo studies are underway to assess the immunomodulatory effects of CR on be-ta1AR and beta2AR deficient mice. Differences were observed between experimental groups sub-jected to a sub-lethal LM infection with or without the CR treatment preceding the infection. Simi-lar to the pharmacological studies, CR impaired host resistance in wild type (FVB/NJ) and beta2AR deficient mice but not in beta 1AR deficient mice. CR-induced immunosuppression was most ap-parent in the liver as compared to the spleen. While an immunologic role for beta2AR is described in CD4+ Th1 cell differentiation, the role of beta1AR in immune regulation is uncertain. The func-tional consequences of stress on lymphocyte activity are being examined with regard to bactericidal killing activity, antigen specific CD8 T cell expansion and surface thiol expression. We have ob-served that CR reduces the amount of surface sulfhydryls on peripheral blood NK cells, a subpopu-lation necessary for LM clearance. The redox state of cells is known to affect cell trafficking as well as cytolytic and proliferative activity. Deleterious effects of stress may compromise the deve l-opment of an appropriate vaccine response and/or anti-pathogenic response. Results of these studies may further our understanding of stress-mediated immune activities and reveal target mechanisms for prophylactic treatment to alleviate stress-altered immune dysfunction.

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    Session II.

    Functional Defects in Recent Thymic Emigrants from Aged Mice

    Karen Clise-Dwyer, Dana Meents, Debbie Duso and Susan L. SwainTrudeau Institute, Inc., Saranac Lake, NY, USA

    It is now recognized that reduced early IL-2 synthesis by naïve CD4 T cells is in part respon-sible for the weak immune responses observed in the elderly. Reported anomalies in aged CD4 cells which may contribute to this failure to up-regulate IL-2 appear to be multifaceted and to be intrinsic to aged naïve CD4 cells. Total CD4 numbers in both mice and men remain fairly constant through-out life despite thymic involution, naïve CD4 T cell production declines dramatically as early as young adulthood. To pinpoint when the defects in naïve CD4 T cells develop, we compared recent thymic emigrants (RTE) from young and aged mice examining both monoclonal TcR transgenic CD4 T cells and those from polyclonal mice. We fluorescently labeled thymocytes in situ. Total thy-mocytes labeled exceeded 50% in both the young (6-10wk) and aged (20-28mo) groups. Dye label-ing was heterogeneous in both groups. Transgene+ or CD44lo RTE retaining the dye were isolated from secondary lymphoid tissues at 7-10 days post dye labeling. When sorted RTE were cultured either with cognate peptide and antigen presenting cells, or anti-CD3 and anti-CD28, RTE from aged mice expanded less than either RTE from young mice or naïve CD4+ cells from both young and aged mice. However, similar amounts of IL-2 were detected in each age group. The results indi-cate that defects observed in aged CD4 cells arise at least in part due to defects in thymopoeisis in-herent in the aged thymus.

    This research was supported by the NIA Grant AGA01743.

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    Session II.

    Characterization of Llama IgG Isotypes Induced During Parelaphostrongylus tenuis Infection

    Lisa P. Daley1, Lucille F. Gagliardo1, Michael S. Duffy1, and Judith A. Appleton1, 21James A. Baker Institute for Animal Health, 2Department of Microbiology and Immunology,

    College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA

    Parelaphostrongylus tenuis is a parasitic nematode that infects the central nervous system of white-tailed deer. Although infection is asymptomatic in white-tailed deer, this parasite induces an incapacitating neurologic disease in atypical hosts such as llamas. An IgG response is induced in animals infected with P. tenuis. Of the three IgG isotypes produced by llamas, IgG2 and IgG3 are unique in that they do not associate with light chains. These isotypes are called heavy-chain antibod-ies (HCAbs) and constitute approximately 50% of llama serum IgG. The IgG1 isotype is a conven-tional antibody.

    We set out to elucidate a role for HCAbs in a parasitic nematode infection. Currently, analy-sis of llama immune responses requires assays of chromatographically separated immunoglobulins. We have generated and characterized isotype-specific monoclonal antibodies in order to define the specificities of the different IgGs induced during P. tenuis infection. Of the seventeen stable hybri-domas that were cloned, detailed characterization was conducted on three hybridomas that produced monoclonal antibodies (mAbs) specific for epitopes on the γ chains of llama IgG1, IgG2 and IgG3.

    In ELISA, the mAbs detected isotypes of llama serum IgGs induced during P. tenuis infec-tion that were specific for a recombinant form of an excretory/secretory product of adult worms. Diseased animals did not produce antigen-specific HCAbs; rather, they produced the conventional isotype, IgG1, exclusively. Four of seven uninfected llamas assessed had cross-reactive IgG1; how-ever, equivalent levels of one or both HCAb isotypes were also detected. Our data suggest that these mAbs will be useful in characterizing the IgG responses to infection, developing non-invasive diag-nostic assays, as well as in describing the IgG response elicited by vaccination.

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    Session III.

    Effector-memory T Cell Populations in the Lung Airways are Maintained by a Process of Continual Recruitment

    Kenneth H. Ely, Tres Cookenham, Alan D. Roberts, and David L. WoodlandTrudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983

    Antigen-specific effector memory T cells persist in the lung airways after clearance of a respiratory virus infection. However, it is unclear how this population of memory T cells is maintained. Using Sendai virus infection of mice as a model system, we show that LFA-1 expression is selectively down-regulated on memory T cells after recruitment into the lung airways. LFA-1 analysis allowed us to identify recently recruited cells and demonstrate that memory cells are constantly replaced in the lung airways for at least a year post infection and most likely for the life of the animal. The rate of recruitment slowly declines over time, resulting in a steady decline in the absolute numbers of memory T cells present. Together, these data indicate that periphe ral memory cell sub-populationsare dynamic and depend on a systemic source of memory T cells

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    Session III.

    Influenza-associated Hepatitis is Induced by Intrahepatic Accumulation of Virus-specific CD8+ T cells

    Noelle Polakos, Judith Cornejo, John Treanor, I. Nicholas Crispe, Robert Pierce, and David TophamUniversity of Rochester School of Medicine and Dentistry, Rochester NY USA

    Rationale: The clearance of obsolete T cells by the liver during resolution of an immune re-sponse is well-described. A common feature of many acute viral infections including influenza is a transient mild hepatitis. However, the etiology and impact are yet undefined. During murine influ-enza virus infection, replication is restricted to the lung epithelium; therefore we can study interac-tions between the liver and CD8+ T lymphocytes responding to an extra-hepatic infection.

    Methods: C57BL/6 mice were infected intranasally with influenza A/HKx31 (H3N2), then challenged intranasally one month after recovery with serologically distinct influenza A/PR/8(H1N1). Influenza-specific immune responses were followed by flow cytometric tetramer staining; hepatic damage was assessed by histology and elevation of serum transaminases.

    Results: The appearance and severity of hepatitis in a murine model of influenza infection was independent both of the presence of virus in the liver and of the kinetics of viral infection in the lung. Inflammatory foci containing T cells, Kupffer cells, and apoptotic hepatocytes appeared ear-lier and more extensively in secondary as compared to primary infection, reflecting the number of activated virus-specific CD8 T cells in the system. Furthermore, virus-specific T cells accumulated in the liver up to two days prior to becoming detectable in the lung, and the intrahepatic numbers eventually rivaled those in the spleen. Deliberate infection of human subjects with influenza A re-sulted in clinically relevant increases in serum transaminases in 23% of subjects.

    Conclusions: We describe a novel pathogenic mechanism involving the accumulation of ac-tivated influenza-specific CD8 T cells in the liver and development of hepatitis. Such hepatic collat-eral damage may be a general characteristic of expanding CD8 T cell populations in response to many viral infections, and has important implications for liver pathobiology and immune homeosta-sis.

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    Session III.

    Strategies to Enhance Cellular and Antibody Responses to HIV Envelope Glycoprotein

    Danuta KozborDepartment of Immunology, Roswell Park Cancer Institute,

    Elm and Carlton Streets, Buffalo, NY 14263

    The current challenge for the design of an effective HIV vaccine is to develop immunization strategies to elicit broader immunity against diverse viral species that is both stronger and durable.We have tested interleukin 21 (IL-21) as an adjuvant for augmenting both the level and longevity of protective immune responses against HIV Envelope glycoprotein (Env, gp160) induced by DNA vaccine in mouse studies. This newly described cytokine, which is produced primarily by activated CD4+ T cells, belongs to the IL-2 family of cytokines that utilizes the common receptor γ-chain for regulating CD8+ T cell and NK cell activities as well as antibody responses. Our results demon-strated that IL-21, delivered together with the Env vaccine, had a greater ability than IL-2 and com-parable to that of IL-15 to induce protective immunity against challenge with recombinant vaccinia virus expressing homologous gp160, though complete eradication of the virus was achieved only in mice treated with both IL-21 and IL-15 at the time of immunization. The protection was associated with elevated levels of Env-specific CTL and NK cell activities in IL-21/IL-15-treated mice, which surpassed those induced by each cytokine alone. The isotype profile of Env-specific antibody re-sponses demonstrated an increased level of IgG1 than IgG2 antibody in mice treated with IL-21 at the time of vaccination, however, in mice treated simultaneously with IL-21 and IL-15, IgG1 and IgG2 antibodies were balanced and higher than those elicited by the Env vaccine only. The analysis of kinetics of IL-21-induced protection revealed further increases in the vaccine efficacy when IL-21 plasmid was delivered five days after immunization, consistent with the notion that IL-21 expo-sure to an immune system that has been primed with an antigen may lead to the optimal augmenta-tion of specific immune responses. The results from our studies provide insights into approaches for boosting the magnitude and longevity of Env-specific immune responses.

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    Session III.

    An Analysis of the Immune Response to HPV-16 E7 in HLA-DR Transgenic Mice

    Rhonda Kines*, Galina Elkin*, Shashikant Lele#, Kunle Odunsi#, T.C. Wu§, Yasmin Thanavala** Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

    # Department of Gynecologic Oncology, Roswell Park Cancer Institute, Buffalo, NY§ Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, MD

    It has been established that infection with Human Papillomavirus (HPV) is the leading cause of cervical cancer in women. Research in the past decade has followed two lines of thought: 1) What other biomarkers in conjunction with a high-risk HPV infection potentiate progression of dis-ease and 2) What is the best approach towards designing a preventative/therapeutic vaccine?

    A previous study indicated that HLA-DRB1*0401 patients may be at an increased risk of de-veloping cervical cancer following an HPV infection. Our lab hypothesized that HLA-DRB1*0401patients may not be able to mount an effective immune response to key viral antigens, thus permit-ting the virus to persist in the host and increase the relative hazard to develop cancer. Our approach is two fold: (a.) using an HLA-DRB1*0401 (DR4) transgenic mouse model to study the immune re-sponse to a viral oncoprotein, E7 and (b.) to determine the efficacy of targeting E7 DNA into the class II processing pathway (Sig-E7-LAMP-1) as a plausible approach to potentiating the immune response.

    The data indicates that DR4 Tg mice, when immunized with E7 protein antigen, make equivalent immune responses, measured by both in vitro T cell proliferation and cytokine secretion, when compared to other strains (HLA-DRB1*03 and BALB/c). When the mice were immunized with DNA encoding E7 alone, T cells obtained from DR4 mice proliferated significantly less, as well as secreted less IFN-? and more IL-10, when compared to BALB/c mice. In contrast, T cells from DR4 mice immunized with DNA encoding Sig-E7-LAMP-1 showed a significantly increased proliferative response.

    HLA transgenic mice provide us a unique opportunity to understand the immune response to HPV-16 protein E7 and to identify potentially immunogenic peptides for future vaccine studies.

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    Session III.

    Immunoevasion: Lessons from a Gammaherpesvirus

    Romana Hochreiter and Rosemary RochfordDept. of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY

    The human pathogens, Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8), are members of the Gammaherpesvirinae which also includes the murine gammaherpesvirus-68 (MHV-68). Because EBV and HHV-8 can replicate only in cells of human origin, studies on the patho-genesis of these viruses are limited and highlight the need for a small animal model system such as MHV-68 infection of mice. Our laboratory is focused on the early stages of viral infection to iden-tify how the virus is able to escape the host immune response and establish latency. Following in-tranasal (i.n.) inoculation, MHV-68 establishes an acute infection in the lungs. Concurrent with this acute infection, we found expression of viral latent genes in the mediastinal lymph nodes (MLN) as early as 2 dpi. Like other members of the gammaherpesvirus family, MHV-68 encodes several pro-teins that either have been demonstrated to or are postulated to subvert the host immune response.These include M3, a highly secreted chemokine binding protein; M11, a bcl-2 homologue; ORF4, a complement-regulatory protein; ORF74, a G-protein coupled receptor, and K3, a protein that pro-motes degradation of MHC class I expression on the surface of mouse fibroblasts. All are expressed during the viral lytic cycle suggesting a role in the acute phase of replication. However, many of these proteins have now been found to not be necessary for lytic replication in the lungs nor do they affect host immunity during the acute phase of infection. For example, we determined that mice in-fected with a virus deleted of the M3 coding sequence had no effect on host chemokine or cellular inflammatory response in the lungs. Our more recent studies have focused on the role of dendritic cells (DC) in MHV-68 infection. We found that immature DCs infected ex vivo can be productively infected with MHV-68 and respiratory DC are infected with MHV-68 in vivo suggesting that DCscan serve as reservoirs for viral dissemination. Interestingly, MHV-68 infection of DCs resulted in the loss of CD86, an important co-stimulatory molecule required for initiation of T cell responses.However, infection did not result in changes in MHC-Class I levels even though the viral K3 protein was expressed. This suggests that K3 mediated modulation of Class I and co-stimulatory molecules is cell type dependent. Together, these studies illustrate the complexities of studying viral immune evasion proteins and the importance of well-described animal model systems for the analysis of their function.

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    Session III.

    Regulating the Regulators or How to Kill Pathogens Without Killing Yourself

    Ian A. White1, Martin Baumgart1, Charles A. Scanga2, Alan Sher2 and Matthias Hesse11 College of Veterinary Medicine, Cornell University, Ithaca NY

    2 Laboratory of Parasitic Diseases, NIH, Bethesda MD

    Inducing an immune response against invading pathogenic organisms confronts the immune system with a serious dilemma. The immune response has to be potent enough to stop the invasion and eradicate or at least diminish the amount of pathogen. However, many effector mechanisms of the immune system kill pathogenic organisms effectively but they can also inflict severe harm on the host. The immune system employs a variety of mechanisms to control potentially host-destructive immunity while mounting an attack against pathogenic invaders. However, these im-mune-suppressive mechanisms can compromise an effective anti-pathogen response. There is accu-mulating evidence that certain pathogens exploit these mechanisms, which allows them to establish persistent infections. Therefore it is necessary to regulate the regulators. During the last decade spe-cialized T cells (Treg) with immune-suppressive capacities have been convincingly demonstrated. Interestingly, natural-occurring CD4+CD25+ Treg constitutively express the cell surface receptor glucocorticoid- induced TNF receptor family-related gene (GITR). Ligation of GITR abrogates Treg-mediated immune suppression in vitro and in vivo and acts also as costimulatory signal for ac-tivation of effector T cells. The ligand for GITR (GITRL) was recently identified and is expressed on most antigen-presenting cells (APC). We analyzed expression of GITRL by macrophages, which were stimulated either classically or alternatively. Our results reveal that only classical activated macrophages upregulate GITRL expression. Expression of GITRL requires a Toll- like-receptor me-diated signal. In contrast, alternative activation of macrophages fails to induce GITRL expression. We conclude that recognition of potentially highly dangerous pathogens attenuates the functional activity of Treg and thereby promotes the development of a powerful Th-1-immune response. On the contrary, Th-2–inducing pathogens, such as schistosomes, do not cause a control of Treg activ-ity, which could help to establish a polarized Th-2 response.

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    Session IV.

    Mucosal Immunity to Francisella tularensis LVS after Pneumonic Infection

    Dennis W. Metzger, Nathalie S. Duckett, and María C. LópezCenter for Immunology & Microbial Disease, Albany Medical College,

    Albany, NY 12208

    Little is known about the role of immune cell-derived cytokines in protection from respiratory infec-tion with F. tularensis. Intracellular cytokine staining followed by FACS analysis was used to in-vestigate lung cytokine expression after intranasal inoculation of BALB/c and C57BL/6 mice with sublethal (1,000 CFU) or lethal (10,000 CFU) doses of LVS. IFN-? KO (GKO) mice and exoge-nous IL-12 treatment were employed to determine the importance of these cytokines for protection.There were found to be significant increases in the percentages of lung immune cells producing IFN-? after intranasal LVS infection. For example, 72 hr after infection with 1000 CFU, C57BL/6 mice were found to contain 25% ± 2% IFN-γ+ cells in the lung compared to 5% ± 2% IFN-γ+ cells in uninfected mice. Infected BALB/c contained 7% ± 1% IFN-γ+ lung cells vs. 3% ± 0.1% in unin-fected mice. There were no significant changes in expression of IL-4, TNF-α or IL-2-secreting cells after infection. Detailed phenotypic analyses showed that the main source of IFN-?-secreting lung cells during F. tularensis LVS infection were CD11b+ DX5+ αβ TCR- natural killer cells. GKO mice all failed to survive intranasal challenge with a dose that was sublethal for wild-type mice, demonstrating the pivotal role of IFN-? in protection against respiratory LVS. Similarly, IL-12p35and IL-12p40 KO mice all succumbed to normally sublethal amounts of intranasal F. tularensisLVS. IL-12 treatment before infection with 10,000 CFU of LVS induced complete protection in wild-type BALB/c or C57BL/6 mice; such protection was not observed in GKO mice. The results indicate that IFN-? produced by NK cells is a key cytokine involved in defense against respiratory infection with F. tularensis LVS. Furthermore, exogenous IL-12 treatment can prevent infection through a mechanism that is at least partially dependent upon IFN-? expression. (Supported by NIH grant PO1 AI056320)

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    Session IV.

    IL-23 is Essential for the Induction of IL-17 Producing Antigen-specific T cells During Tuberculosis.

    Shabaana A. Khader, John E. Pearl, Kaori Sakamoto*, Leigh Gilmartin, Dawn M. Jelly-Gibbs,Nico Ghilardi§,

    Fred deSauvage§ and Andrea M. Cooper1

    Trudeau Institute, Inc., Saranac Lake, New York 12983; §Genentech Inc., South San Francisco, California 94080

    Tuberculosis, caused by Mycobacterium tuberculosis kills more than 3 million people per year. IL-12p70, a heterodimeric cytokine made up of two disulphide linked subunits p35 and p40, promotes IFN-γ production by antigen-specific T cells and is believed to be the keystone of the protective cell-mediated immune response to this disease. Absence of the p40 subunit is more detrimental than the absence of p35 subunit to the generation of protective immune responses in M.tuberculosis mur-ine infection. The fact that both the p35 and p40 mice lack IL-12p70, yet the p40 deficient mice are more susceptible, implicated a role for other p40 dependent molecules such as IL-23. IL-23 is an IL-12p40-dependent cytokine composed of the IL-12p40 subunit covalently bound to a p19 subunit and has been implicated in the induction of IFN-γ and IL-17 production by CD4 T cells. Here we show that in the absence of the IL-23 p19 subunit, mycobacterial growth is controlled and that there is neither diminution of IFN-γ-producing antigen-specific CD4 T cells nor local IFN-γ mRNA ex-pression. Conversely, there is an almost total loss of both IL-17-producing antigen-specific CD4 T cells and local production of IL-17 mRNA within the lungs of Mycobacterium tuberculosis-infectedIL-23p19-deficient mice. We also show that IL-23 is required for the induction of an IL-17 produc-ing antigen-specific phenotype in naïve CD4 T cells in vitro and that absence of IL-12p70 actually promotes an increase in the number of IL-17-producing antigen-specific CD4 T cells. The absence of IL-17 does not alter expression of the anti-mycobacterial genes, iNOS and LRG-47; it does how-ever result in increased expression of the gene for TNF-α. The absence of IL-23 or IL-17, both of which are implicated in mediating inflammation, surprisingly fails to substantially affect the granu-lomatous response to tuberculosis in the lung possibly as a result of the increased TNF-α induction.

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    Session IV.

    Separate and Distinct Mechanisms of CD4 Mediated Protection to Lethal Influenza Infection

    Deborah M. Brown, Dana Meents and Susan L. SwainTrudeau Institute, 154 Algonquin Ave. Saranac Lake, NY 12983

    Interferon-gamma (IFN-γ) is an important component of the anti-viral response; however, the contri-bution of CD4 derived IFN-γ in the response to influenza remains poorly characterized. To further investigate the role of IFN-γ in protection against lethal influenza infection, TCR transgenic mice, recognizing the peptide HA126-138 from influenza hemagglutinin, were used. CD4 T cell effectors from TCR transgenic wildtype (WT) or TCR Tg IFN-γ-/- mice were generated in vitro in the pres-ence of Th1 polarizing conditions. Surprisingly, both in vitro generated WT and IFN γ-/- effectors could confer protection against lethal infection. The ability of in vitro generated effectors to pro-mote survival correlated with perforin mediated in vitro cytolytic activity. Both WT and IFN-γ-/-CD4 cells localize to the lung upon i.v. transfer and increase recruitment of host CD8 cells; how-ever, protection mediated by in vitro generated effectors did not require host T cells or IFN-γ. In ad-dition, influenza viral titers were decreased in the lungs of mice given in vitro generated effectors in the first 6 days compared to control mice. To determine if in vivo generated effectors could also pro-mote survival, CD4 cells were isolated from draining lymph nodes (DLN) or lung of sublethally in-fected BALB/c or IFN-γ-/- mice and transferred to normal BALB/c mice that were subsequently in-fected with a lethal dose of influenza. Surprisingly, mice given CD4 effectors from WT mice sur-vived a high dose of influenza while mice given CD4 cells from IFN-γ-/- mice succumbed to infec-tion. CD4 effectors generated in vivo could proliferate upon restimulation, but did not demonstrate cytolytic activity directly ex vivo, suggesting that these effectors utilize IFN-γ as a major mecha-nism to provide protection. Experiments are underway to determine whether in vivo generated effec-tors can promote survival via an indirect pathway that may require other host cells types. Thus, CD4 effectors may utilize both IFN-γ dependent and IFN-γ independent mechanisms to control lethal in-fluenza infection. Supported by PHS grants F32-AI056962 (DMB) and PO1-HL63925.

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    Session IV.

    Intranasal Vaccination Against Pneumococcal Otitis Media in Mice

    Albert Sabirov and Dennis W. MetzgerCenter for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208

    Streptococcus pneumoniae is the leading bacterial cause of acute otitis media (OM) in in-fants and young children. The recently introduced pneumococcal conjugate vaccine, which is ad-ministered via the intramuscular route, is poorly protective against development of OM. In the pre-sent study, we tested the efficacy of intranasal (i.n.) vaccination with conjugate vaccine and inter-leukin-12 (IL-12) as a mucosal adjuvant against development of OM in neonatal mice. We em-ployed two models of streptococcal OM: after direct middle ear challenge, and after intranasal cha l-lenge, which leads to nasopharyngeal colonization (NC). Neonatal wild-type (WT), IFN-γ knockout(IFN-γ−/−), and IgA knockout (IgA-/ -) mice were immunized i.n. with type 14 pneumococcal conju-gate vaccine on days 7 and 14 after birth and treated with IL-12 (vaccine+IL-12 group) or PBS vehi-cle (vaccine only group) on days 7-10 and 14. I.n. vaccination of WT mice in the presence of IL-12was found to significantly enhance the levels of specific antibodies (IgA, IgG1, IgG2a and total) in ME washes, nasal washes, and serum, and the levels of IFN-γ in the middle ear (ME). However, IL-12-mediated enhancement of mucosal and serum antibody responses did not occur in IFN-γ −/−mice.Increased numbers of specific IgA antibody-producing cells as well as IgA- and IgG-positive cells were detected in the ME and nasal mucosa after i.n. vaccination of WT mice in the presence of IL-12. Vaccine+IL-12 treated WT mice showed enhanced bacterial clearance from the ME, decreased NC and reduced incidence of OM. In addition, direct middle ear challenge of vaccine+IL-12 treated WT mice with a lethal dose of bacteria resulted in 78% survival. However, examination of nasal washes showed that IgA-/- mice failed to clear bacteria as efficiently as WT mice. There was no dif-ference in the incidence of OM and NC between immunized and unimmunized IgA-/ - mice. This suggests the importance of pneumococcus-specific IgA in protection against OM and NC. These re-sults indicate that i.n. vaccination of neonatal mice in the presence of IL-12 is able to enhance mid-dle ear mucosal and systemic immune responses to pneumococci, responses that were IgA and IFN-γ dependent, and efficiently protect against both OM and invasive infection.

    Supported by NIH grant AI41715 and a grant from Philip Morris, Inc.

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    Session IV.

    Secretory IgA in Innate and Adaptive Immunity Against Ricin

    Nicholas Mantis1,2, Oluwakemi Sonuyi2, Stephanie Farrant2, and Carolyn McGuinness1.1Division of Infectious Disease, Wadsworth Center, and 2GI Cell Biology Laboratory,

    Children’s Hospital Boston

    Secretory IgA antibodies are heavily glycosylated, protease resistant, polymeric immu-noglobulins. As the predominant antibody class in secretions of the upper respiratory tract and gas-trointestinal tract, SIgA represents a first line of defense against mucosal pathogens and toxins.Here we demonstrate that SIgA can function in both innate and acquired immunity to the shiga- liketoxin ricin. Ricin, a member of the A-B family of toxins, is comprised of an enzymatic A subunit (RTA) and a galatose-specific lectin B subunit (RTB). In solid phase binding assays, ric in bound to N- and O- linked oligosaccharide side chains on secretory component and the heavy chains of human IgA1 and IgA2, independent of the antibody variable domains. Ricin had no detectable affinity for human IgG. sIgA (but not IgG) reduced ricin attachment to the apical surfaces of polarized intesti-nal epithelial cells grown in culture and to the lumenal surfaces of human duodenum in tissue sec-tion overlay assays. These data indicate that oligosaccharide side chains on sIgA may serve as ‘decoy’ receptors for ricin, thereby reducing (but not completely eliminating) the effective dose of toxin that gains access to the intestinal epithelium. Furthermore, these results demonstrate that ac-quired immunity may be necessary to completely safeguard against toxin exposure in vivo. To test the latter possibility we have produced a panel of monoclonal, polymeric IgA antibodies directed against RTA and RTB. Neutralizing antibodies against both sub units were identified. Antibodies against RTB prevent toxin attachment to epithelial cell surfaces, whereas antibodies against RTAappear to function intracellularly. We are currently testing whether these antibodies are protective in an animal model of gastrointestinal ricin poisoning. This work was supported by the National In-stitutes of Health (USA).

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    Session IV.

    Immunomodulation by Enterotoxin-based Adjuvants and Vaccine Delivery Systems

    Michael W. Russell*, Terry D. Connell*, Hesham F. Nawar*, Sergio Arce*, Christine M. Gockel*, and George Hajishengallis†.

    *Witebsky Center for Microbial Pathogenesis and Immunology, Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY

    †Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Science Center, New Orleans, LA

    Heat- labile enterotoxins (cholera toxin, CT, and the E. coli labile toxins, LT-I, LT-IIa, and LT-IIb) have been extensively used as potent adjuvants and coupled delivery systems for enhancing responses to mucosally delivered vaccines. The mechanisms of action are incompletely understood, but binding of the B subunits to cell-surface ganglioside receptors has been thought to be critical.The role of toxic enzyme activity within the A subunits is controversial, as some reports describe adjuvant activity by active-site mutants of CT and LT-I that lack detectable enzymic activity, or by recombinant B subunits, whereas others show that enhanced immune responses depend on the pres-ence of A subunits having at least residual toxic activity that synergizes with the binding activity of the B subunits, or that antigens coupled to recombinant B subunits alone are immunogenic, or con-versely, tolerogenic. Several factors contribute to these diverse findings, including the particular toxin used, whether intact holotoxin or pentameric B subunit, and the nature of the coupling be-tween antigen and B subunit.

    In contrast to CT, LT-IIa and LT-IIb were ineffective in inducing cytokine release from hu-man monocytic THP-1 cells, and they differentially regulated the induction of cytokine release stimulated by bacterial LPS. The pentameric B subunits of LT-IIa and LT-IIb, however, stimulated cytokine release which, in the case of LT-IIb.B5, was antagonized by the holotoxin. Moreover, a mutant of LT-IIb (T13I) that has no detectable binding to gangliosides possessed in vivo adjuvant activity (by the intranasal route) that was equivalent to that of the wild-type GD1a-binding LT-IIb.However, a non-binding mutant of LT-IIa (T34I) did not retain adjuvant activity as revealed by mu-cosal antibody responses, although it enhanced priming for anamnestic recall of antibody responses by the antigen alone. Both LT-IIa.B5 and LT-IIb.B5 were found to induce cytokine responses in monocytic cells that were dependent upon interaction with TLR2. Different patterns of binding of enterotoxins to lymphoid cells were observed, and both LT-IIa and CT selectively induced apoptosisin CD8+ T cells. These findings show that the immunoenhancing activities of different enterotoxins do not necessarily depend on either ganglioside binding or toxic enzyme activity, and reveal novel mechanisms of immunomodulation by the type II enterotoxins.

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  • 55

    Christine A. Biron, Ph.D.Esther Elizabeth Brintzenhoff Professor of Medical Science

    Chairperson,Department of Molecular Biology and Immunology

    Brown University

    “Induction and Function of Innate Immune Responses to Viral Infection”

  • 56

    Keynote SpeakerMonday, November 15, 2004

    Innate Responses to Viral Infections - Controlling Accessibility of STAT Signaling Pathways to Modify Interferon Effects

    Christine A. BironDepartment of Molecular Microbiology and Immunology, Division of Biology and Medicine,

    Brown University, Providence, RI, USA 02912

    The mechanisms, by which particular innate cytokine responses are induced and function in the con-text viral infections, are being characterized in our laboratory. The oldest known innate cytokines are the type 1 interferons (IFNs). These factors were originally characterized by their ability acti-vate antiviral functions, and the signal transducers and activators of transcription (STATs) were first defined by their role in delivering the signal from type 1 IFNs to induce the expression of antiviral gene products. The type 1 IFNs and STAT molecules are now known to regulate a broad range ofbiological responses to cytokines. We have been evaluating the expression and func tion of the STATs as they relate to shaping innate and adaptive immune responses to lymphocytic choriomen-ingitis virus (LCMV) and murine cytomegalovirus (MCMV) infections of mice. The hypothesis be-ing tested is that STAT levels are modified during endogenous responses to focus the targets of cy-tokine exposure to defined subsets as needed. The picture emerging is that particular STATs, and normal regulation of access to individual STAT signaling pathways, modifies the consequences of exposure to cytokines to help shape the subset immune responses elicited. This concept helps re-solve many of the paradoxical reported effects of cytokines, and moves control of immune re-sponses from cytokines to intracellular signaling pathways.

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    Session V.

    Gap Junctions and Macrophages

    I. Alvarez de Mora, M. Delmar and S. M. TaffetSUNY Upstate

    The expression of connexin43 (Cx43) was studied in murine bone marrow derived macrophages (BMDM) and in the J774A.1 murine macrophage- like cell line. Cx43 expression was non detectable in BMDMs untill exposure to bacterial Lipopolysaccharide (LPS). However, in the J774A.1 cells, Cx43 expression was detected at basal levels and increased after exposure to LPS. Elevated Cx43 expression was detected at protein and RNA levels by immunoblot and ribonuclease protection as-says respectively. The protein had a detectable immunoreactive band at 43 kD. The extent of stimu-lation was dependent on the concentration of LPS used, with detectable induction with 0.1 ng/ml and maximal induction with 1 µg/ml. The increase in RNA and protein expression was detected at 6 hour after LPS (1 µ g/ml) treatment and continue to increase through 24 hours. In order to deter-mine the signaling pathways that were involved in LPS-mediated induction of CX43, specific in-hibitors were used. As NF-?B is pivotal in the LPS-induced transcription of many genes, BMDM and J774A.1 cells were treated with TPCK or TLCK, agents known to block activation of NF-?B.Both agents prevented the LPS-induced increase in CX43 expression. Pre-treatment of either BMDM orJ774A.1 cells with the p38 inhibitor SB202190 (10µM) for 1 hour prior to LPS-stimulation significantly decreased both Cx43 protein and mRNA expression. In contrast, pre-treatment of J774A.1 cells with the MEK1 inhibitor, PD98059 (20 µM), had no effect on Cx43 ex-pression, and very little or not significant effect in BMDMs. The JNK inhibitor, SP600125 (10µM), on the other hand, increased Cx43 expression in both cell types. These results imply that the MAPK p38 pathway, as well as the NF-?B pathway have a role in CX43 expression in BMDMs and J774A.1 cells.

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    Session V.

    Suppression of Macrophage Proinflammatory Signaling Pathways by Toxoplasma gondii

    Barbara A. Butcher1, Leesun Kim1, Peter J. Murray2, and Eric Y. Denkers11Cornell University College of Veterinary Medicine, Ithaca, NY

    2St. Jude Children’s Research Hospital, Memphis, TN

    Intracellular infection with the protozoan Toxoplasma gondii suppresses LPS-induced macrophage IL-12 and TNF-γ production, but the underlying molecular mechanisms are enigmatic. Here, we tested the hypothesis that the parasite employs an IL-10-STAT3 anti- inflammatory signaling path-way to accomplish its down regulatory agenda. We found that live T. gondii elicited rapid, potent and sustained STAT3 phosphorylation and nuclear translocation in infected macrophages. In con-trast, neither heat killed parasites nor soluble parasite antigens induced this response. Furthermore,using gene-targeted macrophages we show that neither IL-10 nor IL-6, cytokine activators of STAT3, is responsible for Toxoplasma-triggered STAT3 phosphorylation in infected cells. Mostimportantly, using STAT3 deleted macrophages, we demonstrate that loss of this molecule elimi-nates the parasite's ability to inhibit macrophage proinflammatory response to endotoxin. Our results demonstrate that Toxoplasma subverts proinflammatory signaling pathways through direct, cytokine independent, STAT3 activation.

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    Session V.

    Infection-stimulated Fibrin Deposition Controls Hemorrhage and Limits Bacterial Growth During Listeriosis

    Stephen SmileyTrudeau Institute, Saranac Lake NY

    Septic bacterial infections are major causes of human mortality. While infection-stimulatedcoagulation clearly contributes to septic pathology, here we demonstrate that coagulation also per-forms critical host protective functions during bacterial infection. Specifically, we demonstrate that gene-targeted fibrin(ogen)-deficient mice, in comparison to genetically matched control mice, dis-play increased mortality upon infection with the Gram-positive facultative intracellular bacterium Listeria monocytogenes. To distinguish effects of fibrinogen from those of fibrin, we treat wild type mice with warfarin, an anticoagulant that suppresses fibrin formation without impacting fibrinogen levels. Warfarin-treatment exacerbates listeriosis, suggesting that fibrin is the key mediator of pro-tection. With regard to the underlying protective mechanisms, we demonstrate that fibrin sup-presses anemia, reduces hemorrhagic pathology, and limits bacterial growth during listeriosis. The precise mechanism underlying the fibrin-mediated constraint of bacterial growth remains to be fully explained, but appears to proceed independently of fibrin’s putative capacity to limit the dissemina-tion of bacteria from the peritoneal cavity. Although the pathologic potential of excessive fibrin deposition is well appreciated, our findings underscore the multiple protective capacities of infec-tion-associated coagulative responses, and help to explain why anticoagulant therapies generally fail to reduce septic pathology.

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    Session V.

    Human Immune Responses To Francisella tularensis

    Edmund J. Gosselin and Diane R. GosselinAlbany Medical College, Albany, NY

    Background: Interest in F. tularensis has grown significantly over the past couple of years, due primarily to its potential as a bioterrorism agent. Currently however, there is no acceptable vac-cine, and there is still a great deal to be learned about the human immune response to F. tularensis.

    Methods: In an effort to evaluate the human immune response to this organism, fixed, UV inactivated, and live F. tularensis organisms were incubated with human immune cells.

    Results: In every instance, a titratable proliferative response was observed in the presence of F. tularensis organisms, even in the absence of prior exposure. This was the case using multiple do-nors and multiple preparations of F. tularensis. The proliferation could not be reproduced using su-pernatants obtained from fixed bacteria incubated up to a week in PBS, suggesting soluble products are not involved. Furthermore, the proliferation could not be attributed to replication of live bacteria remaining in the fixed F. tularensis preparations. Analysis of the responding cell populations indi-cated that Natural Killer (NK) cells were the dominant cell population responding followed by cyto-toxic (CD8) T cells. Conclusions : NK and CD8 T cells represent a major portion of the initial hu-man immune response to F. tularensis organisms, with the latter population being most responsive to live organisms. Since, T cells are likely to play an important role in protection against F. tularen-sis infection following immunization, information obtained from these studies will be directly rele-vant to the development of vaccines against F. tularensis using attenuated or inactivated F. tularen-sis organisms.

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    Session V.

    Toll-like Receptor Signaling Regulates Replication of Francisella tularensis in aMouse Model of Pneumonic Tularemia

    Meenakshi Malik1, Chandra S. Bakshi1, Steven A. Lotz1, Kevin Regan1, Pauline Carrico1,Sophie C. Gangloff2, Sanna M. Goyert3, J. Andrés Melendez1, and Timothy. J. Sellati1

    1Albany Medical College, Albany NY, 2University of Reims, France,3North Shore-Long Island Jewish Research Institute/NYU School of Medicine, Manhasset, NY

    Background: Francisella tularensis, the causative agent of tularemia, is a Gram-negativebacterial threat agent due to its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death. Pneumonic tularemia is characterized by lung inflammation involving focal necrosis and infiltration of perivascular and peribronchiolar tissues by neutrophils and alveolar macrophages. The molecular mechanisms underlying tularemia pathogenesis remain ill defined.Herein, we studied the role of the pattern recognition receptors (PRRs) CD14, TLR2, TLR4 in the inflammatory response to F. tularensis and hypothesize that binding of F. tularensis to PRRs modu-lates the intensity and duration of disease.

    Methods: Wild type (WT) and PRR-/- mice were intranasally infected with the live vaccine strain (LVS) of F. tularensis and their survival was monitored. Upon sacrifice, tissues were har-vested for histological evaluation and determination of bacterial burden, and sera were collected to measure immunomodulator levels and characterize the humoral response.

    Results: Comparing survival curves, TLR4-/ - and TLR2 -/-, but not CD14-/- mice were more susceptible to infection and exhibited accelerated mortality compared with WT mice. Histologi-cally, it was observed that cellular infiltrates in all genotypes were composed primarily of neutro-phils and alveolar macrophages. However, the pulmonary and extrapulmonary tissues of TLR2-/-

    and TLR4-/ - mice were considerably more inflamed. These differences also were reflected in mark-edly higher bacterial burdens. An important correlation exists between pulmonary pathophysiology and levels of matrix metalloproteinase 9 (MMP-9), an extracellular matrix-degrading protease. Not surprisingly, TLR4 -/- mice expressed significantly higher levels of MMP-9 than did their WT coun-terparts.

    To determine whether innate and acquired immunity were altered by PRR deficiency, immu-nomodulator levels were measured. Since pro-inflammatory cytokines play a critical role in modu-lating the intensity of an inflammatory response it was quite surprising to find that their levels did not differ significantly between WT and PRR-/ - mice. Although complex and varied, the pattern of humoral immunity in WT and PRR-/ - mice did not correlate with disease severity insofar as TLR4-/-

    mice had higher titers of anti-F. tularensis antibodies compared to WT mice.Conclusion: These results show that TLR2 and TLR4, but not CD14, play a critical role in

    modulating the clinical course and severity of pneumonic tularemia and may identify novel immu-notherapeutic strategies to combat disease caused by this respiratory contagion.

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    Session V.

    Role of the Ectoenzyme CD38 in Regulating Chemotaxis and Adaptive Immune Responses

    Santiago Partida-Sanchez, Laura Rivero-Nava, Stephen Goodrich, Kim Kusser, Troy Randall and Frances Lund

    Trudeau Institute, Saranac Lake NY

    Mice lacking CD38, an ecto-enzyme that generates several calcium mobilizing metabolites includ-ing cyclic ADP-ribose (cADPR) and ADP-ribose, make reduced T cell dependent antibody re-sponses. We show that CD38 and cADPR regulate calcium mobilization in chemokine stimulated dendritic cells (DCs) and that this calcium signal is required for the chemotaxis of immature and mature DCs to a number of chemokines including CCL2, CCL19, CCL21 and CXCL12. In the ab-sence of CD38, dendritic cell precursors migrate poorly from the bone marrow and blood to sites of inflammation and mature DCs migrate inefficiently from sites of inflammation to secondary lym-phoid organs. The reduced trafficking of CD38 deficient DCs results in inefficient T cell priming and significantly reduced humoral immune responses. Together these data show that CD38 via its ability to catalyze the formation of calcium mobilizing metabolites can act at the intersection of in-nate and adaptive immune responses by controlling the migration of DCs and DC precursors.

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    Session VI.

    Structural Basis of Human High Molecular Weight Melanoma-AssociatedAntigen Mimicry by Mouse Anti-idiotypic Monoclonal Antibody MK2-23

    Chien-Chung Chang1, Francisco G. Hernandez-Guzman2, Wei Luo 1, Xinhui Wang1,Debashis Ghosh2, and Soldano Ferrone1

    1Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY and 2Hauptman-Woodward Medical

    Research Institute, Inc., Buffalo, NY

    Because of its expression in a high percentage of melanoma lesions with limited intra- and inter- lesional heterogeneity, its high expression on melanoma cells and its restricted distribution in normal tissues, the human high molecular weight-melanoma associated antigen (HMW-MAA), a cell membrane proteoglycan, has been used as a target to implement active specific immunotherapy in patients with melanoma. Like most human tumor associated antigens, the HMW-MAA is a self-antigen and is poorly immunogenic in hosts with constitutive expression of this antigen. To over-come unresponsiveness to self-HMW-MAA, we have implemented active specific immunotherapy in patients with melanoma utilizing a mimic of HMW-MAA as an immunogen. The mimic we have used is the mouse anti- idiotypic (anti- id) mAb MK2-23, which mimics the determinant recognized by the anti-HMW-MAA mAb 763.74. mAb MK2-23 induced HMW-MAA-specific antibodies in about 60% of the immunized patients and anti-mouse Ig antibodies in all the patients. The develop-ment of HMW-MAA-specific immunity was associated with regression of metastatic lesions in a few patients and with a statistically significant survival prolongation. These clinical findings have stimulated interest in the characterization of the molecular basis of HMW-MAA mimicry by anti- idmAb MK2-23, since this information may contribute to the optimization of the design of immuniza-tion strategies with HMW-MAA peptide mimics. To this end, we have determined the structure of the anti- id mAb MK2-23 Fab’ fragments by X-ray crystallography at 2.50 angstrom resolution. Analysis of the crystal has shown that the light chain complementarity-determining region (CDR) 1 (L1) and the heavy chain CDR 3 (H3) of anti- id mAb MK2-23 are unusually protruded and form a unique β-strand/loop structure, suggesting their involvement in the basis of HMW-MAA mimicry. In addition, the amino acid sequences of L1 and H3 show significant homology with two nearby re-gions within a predicted extracellular chondroitin sulfate proteoglycan repeat domain of the HMW-MAA. These structural findings are paralleled by the following immunological results. A peptide (PMK2-23H3) derived from the anti- id mAb MK2-23 H3 inhibits the binding of anti-HMW-MAAmAb 763.74 to HMW-MAA-bearing melanoma cells in vitro. Furthermore, peptide PMK2-23H3induces anti-HMW-MAA humoral immune responses in BALB/c mice. Taken together, these data suggest that the 11 amino acid- long motif of anti- id mAb MK2-23 H3 plays a major role in the HMW-MAA mimicry by anti- id mAb MK2-23.

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    Session VI.

    Evaluation of Human CD4+ Effector Memory T Cells Persisting in the Tumor Microenvironment of Non-small Cell Lung Cancer

    Lori Broderick, Sandra Yokota, Maurice Barcos, Carleton C. Stewart, Raymond J. Kelleher, Jr. and Richard B. Bankert

    Department of Microbiology & Immunology and the Witebsky Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY

    The chronic inflammatory state associated with non-small cell lung cancer and its functional signifi-cance and capabilities have remained somewhat of an enigma, as the T lymphocytes, despite their presence within the tumor microenvironment, appear to be quiescent and unresponsive to the pro-gressing tumor. The hypothesis is put forth that the dynamic microenvironment of most human, pri-mary non-small cell lung tumors contains quiescent memory T cells which can escape normal host immune regulatory mechanisms by the local administration of IL-12 to eradicate tumor cells in situ by indirect mechanisms that are dependent upon IFN-?. Using a human/SCID mouse chimeric model, the early cellular events that occur within the human tumor microenvironment in response to IL-12 have been studied. The majority CD3+ T cell population was found to display an activated (CD45RO+) phenotype consistent with that of a CD4+ effector memory T cell, i.e. positive for CD3, CD4, CD45RO, CXCR3+, CD28+, CD44+ and CD11a+, and IL-12 receptor (ß1 subunit) and was negative for CD27, CD45RA, and CD62L. The finding of IFN-a producing plasmacytoid den-dritic cells in the tumor microenvironment is significant as these cells may contribute to the long-term persistence of the CD4+ memory T cells which are shown to be responsible for the IL-12 in-duced tumor eradication. In spite of the persistence of tumor-associated memory T cells in the hu-man lung tumor microenvironment, they fail to adequately respond to the progressing tumor, the reason for which is widely debated. Preliminary data suggest that these cells fail to re-set their T cell receptor mediated signaling potential and/or their activation and proliferation are suppressed by co-regulatory molecules present on stromal fibroblasts and tumor cells. Hence, these cells persist in a non-responsive state until antigen is cleared from the microenvironment or until the cells are acti-vated by an alternative pathway such as that which is initiated by IL-12. Manipulation of the tumor microenvironment has important implications for immunotherapy against solid tumors in that it may be possible to reduce tumor bulk, if not completely eradicate the primary tumor, by activating spe-cific anti-tumor cells in the vicinity of the tumor. Furthermore, local death of tumor cells may lead to tumor antigen release and the development of a systemic anti-tumor response.

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    Session VI.

    CD8 Dependent Control of Malignancy by Photodynamic Therapy

    Edith Kabingu and Sandra O GollnickPDT Center, Roswell Park Cancer Institute

    Photodynamic therapy (PDT) is a treatment modality for cancer and other non-cancerousconditions. It uses photo-reactive drugs that get activated upon exposure to light of a specific wave-length. The interaction of drug and light produces reactive oxygen in tissues that results in direct cytotoxicity. PDT also shuts down surrounding vasculature and induces a strong inflammatory re-sponse. These events all contribute to direct cell death and induction of a specific host anti- tumorimmune response. Using the EMT6 murine mammary carcinoma cell line in BALB/c mice, we have shown that in situ PDT of the primary tumor of mice bearing both EMT6 subcutaneous tumors (1o) and lung tumors (2o) results in a significant reduction in the number of lung tumors present (anaverage of 6.5 ± 3.9 tumors per lung) compared to mice that get surgery of their primary tumors (an average of 41.2 ± 8.5 tumors per lung). This response is tumor specific and requires the presence of an intact immune system in the host since immune compromised SCID mice did not show the same reduction in secondary tumors after PDT of the primary tumor. These data suggests a role for the adaptive immune system in the reduction of distant tumors after local PDT. Studies where CD4+,CD8+ T cells, or both have been depleted in this experimental model have further indicated a role for CD8+ T cells that appears to be independent of CD4+ T cells. In these studies, we observed that mice depleted of CD8+ T cells were completely hampered in their ability to control both 1o and 2o

    tumors whereas the CD4+ T cell depleted mice controlled secondary tumor growth just as well as control mice that were not depleted of T cells. It is not clear how the CD8+ T cells become activated effector cells in the absence of CD4+ T cell help. We have begun to investigate the mechanism by which CD8+ T cells may become activated without CD4+ T cell help. In the same EMT6-BALB/cmodel we have observed that DCs isolated from the TDLNs of PDT treated mice are better able to stimulate T cells compared to DCs from the TDLNs of mice that get surgical removal of their pri-mary tumor. PDT may directly activate DCs thereby foregoing the need for CD4 help. This study overall suggests PDT as a potential therapy for controlling systemic metastases through induction of a specific host anti-tumor immune response mediated by CD8+ T cells.

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    Session VI.

    Immunity and the Tumor Microenvironment

    Edith M. LordDepartment of Microbiology & Immunology

    University of Rochester

    Immunotherapy is an attractive alternative therapy for cancer due to its potential specificity and limited side effects. However, due to the difficulty in generating effective responses against large tumors and in immune cells functioning within the adverse conditions of the tumor microenvi-ronment, its use as an adjuvant therapy to target primarily metastatic tumor growth has been pro-posed. Recently we have been studying the growth of tumor cells in the peritoneal cavity as a model of direct metastases of intraperitoneal tumors such as ovarian and colon. A major site of me-tastases for these tumors is the greater omentum. This is a sheath of well-vascularized adipose tis-sue embedded with clusters of immune cells, which serves to cushion and protect the peritoneal or-gans from infection. These immune cell clusters, termed milky spots, have been known for many years, but their function still remains poorly defined. Using a whole mount technique developed by my laboratory and immunofluorescence microscopy, we have found that these clusters are ex-tremely well vascularized, and they have a clear structural organization characterized by an outer rim of macrophages surrounding an area of B cells. In the center is an area, composed predomi-nately of T cells with interspersed dendritic cells. Further, confocal microscopy studies indicate that a layer of mesothelial cells overlays the immune cells and an extensive extracellular matrix, which contains both collagen I and III. We have observed that when cells from many different tumor lines are injected into the peritoneal cavity, they all localize preferentially to the milky spots and grow rapidly. Based on these results, we hypothesize that the high level of pro-angiogenic vasculature within the milky spots combined with the preferential attachment of tumor cells to the collagen ma-trix are critical for the rapid tumor growth observed. This raises the intriguing possibility that blocking collagen attachment could have a marked effect on the establishment of metastases. Alter-natively or in combination, immunotherapy with sensitized immune cells, which also localize to the milky spots, could be used to target remaining tumor cells. Thus, immunotherapy could provide an essential adjuvant to improve the treatment outcome for the large percentage of ovarian cancer pa-tients whose disease will progress using current treatments. Our studies to learn more about the mi-croenvironment both within the peritoneal cavity and within the tumors will provide information needed for the development of effective immunotherapy.Supported by RO1CA28332 and a grant from the Sally Edelman and Harry Gardner Cancer Founda-tion.

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    Phagocytosis of Francisella tularensis by macrophages

    Christopher R. Collins1, Daniel J. Loegering2, James R. Drake1, and Michelle R. Lennartz3

    Albany Medical College Centers for: 1Immunology and Microbial Disease, 2Cardiovascular Studies,3Cell Biology and Cancer Research, 47 New Scotland Avenue, Albany NY 12208

    Francisella tularensis is a human macrophage pathogen and the causative agent of the dis-ease, tularemia. F. tularensis is an intracellular pathogen which has been classified as a category A bioterrorism agent, based on its ability to be weaponized, extraordinarily low infective dose, and high morbidity/mortality rate. While Francisella infects macrophages, the mode of entry, as well as the intracellular trafficking of the internalized bacteria, is not well characterized. The live vac-cine strain (LVS) of F. tularensis, which is non-pathogenic in humans, efficiently kills mice and provides an excellent model of lethal disease. Results from a 21 day survival study demonstrate that C57Bl/6 mice are more sensitive to infection than Balb/c. Using a computerized immunofluores-cence microscopy based technique to quantify internalization of F. tularensis LVS in vitro, It wasdetermined that the increased susceptibility to infection is paralleled by higher levels of bacterial up-take by isolated peritoneal and alveolar macrophages from C57Bl/6 mice. In contrast, bone marrow derived macrophages do not show this difference, suggesting that the activation or maturation state of tissue macrophages influences their ability to ingest LVS. This disparity in overall uptake in tis-sue macrophages appears to be due to strain variation in the number of mature phagocytic cells, rather than inherent differences in the phagocytic capacity of macrophages. Phagocytosis of F. tu-larensis by alveolar macrophages was found to be inhibited by cytochalasin D, indicating an actin mediated, phagocytic mode of entry. Ongoing experiments seek to determine whether the activation state impacts uptake and proliferation of the bacteria, as well as to identify the receptors involved in phagocytosis of F. tularensis with a particular focus on testing the hypothesis that Complement Re-ceptor 3 (CR3) is involved in uptake. Supported by NIH grants # AI056320 & GM50821

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    Proteosome Inhibition Selectively Inhibits BCR-Mediated Antigen Processing and Presentation

    Erica McGovern and James DrakeCenter for Immunology and Microbial Disease, Albany Medical College

    B-lymphocytes internalize antigen via the B cell receptor (BCR), which then traffics to LAMP+ late endocytic compartments. Subsequently antigen is released, processed into peptides, and loaded onto MHC class II molecules. As previously reported, internalized antigen-BCR complexes selectively persist for a prolonged period in LAMP+ late endosomes, and these complexes are likely to serve as the source of antigen-derived peptides to support the observed expression of the prolonged peptide-class II complexes. This observation demonstrates that there is control over the intracellular traffick-ing of antigen-BCR complexes within multi-vesicular late endosomes. Moreover, recent studies on the epidermal growth factor receptor have demonstrated a requirement for mono-ubiquitination of the receptor for proper targeting to late multi-vesicular endocytic compartments. Therefore an es-tablished assay to follow antigen-BCR persistence was employed to examine the effect of pro-teosome inhibition, and hence ubiquitin depletion, on antigen processing and presentation. The re-sults of this analysis demonstrate that proteosome inhibition does not alter BCR mediated antigen endocytosis, or the trafficking of fluid phase markers through the endocytic pathway. However, ubiquitin depletion was found to selectively alter antigen-BCR complex trafficking within the endo-cytic pathway, as well as the subsequent processing and presentation of the BCR- internalized anti-gen. Therefore, the results suggest that ubiquitination of the BCR or a BCR associated protein may be necessary for proper antigen-BCR sorting, processing and presentation.

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    Enhanced Humoral and Cellular Immune Responses to Antigens Targeted to Fcgamma Receptor Type I

    Elisaveta Adamova, Mary C. Walsh, Diane R. Gosselin, Karen Hale, Mark T. Preissler, and Edmund J. GosselinCenter for Immunology and Microbial Disease, Albany Medical College, Albany, NY

    There is a continuing need for alternatives to human adjuvants such as alum. One alternative involves the targeting of antigen (Ag) to human FcgammaRI on professional APC. A number of studies have demonstrated that targeting Ag to huFcgammaRI enhances T cell activation in vitro and Ag-specific antibody (Ab) production in vivo. However, only one paper has been published, thus far, examining the effect of FcgammaRI-specific Ag targeting in vivo, using an adjuvant- free sys-tem. This study however, utilized a single, high dose of Ag (100 microg/mouse). Furthermore, none of the above studies examined the primary immune response to FcgammaRI-targeted Ag, the influ-ence of immunization route, cytokine profiles, or whether similar enhancement could be achieved in a mouse strain other than FVB/N, a strain not commonly used in immunological investigations. In this study, we utilized C57BL/6 mice to demonstrate enhanced Ab responses to low doses of FcgammaRI-targeted Ag (2.5 to 10 microg/mouse), in the absence of adjuvant. We examined both primary and secondary immune responses, the potential influence of immunization route, as well as the Ig isotypes and cytokines produced.

    These studies suggest that in the absence of adjuvant, enhancement of the immune response can be achieved in C57BL/6 mice, in primary as well as secondary immune responses. Intradermal immunization appears to be optimal, and as little as 2.5 microg of FcgammaRI-targeted Ag/mouse is required. Furthermore, while isotype analysis suggests a tendency towards IgG1, and thus a Th2-type response, cytokine profiles suggest an enhanced Th1 T cell response as well.

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    The Role of Host T cells in the Recovery from Influenza Infection of Mice Receiving Effector Tc1 and Tc2 cells

    Timothy Powell, David Dwyer and Richard DuttonTrudeau Institute, Saranac Lake NY

    Naive CD8 T cells can be activated to generate two subsets of cells: Tc1 that produce large amounts of IFN-γ and Tc2 that produce IL-4, IL-5 and less IFN-γ. When these effector cells are ad-optively transferred into influenza infected mice, the Tc1 promote a more rapid recovery from the infection than the Tc2 as measured by recovery of body weight and virus clearance compared with PBS controls. The precise mechanism of protection by Tc1 cells and the role of host T cells are not known. We wished to determine in this study how the presence or absence of host lymphocytes al-tered this Tc1 or Tc2 mediated recovery from virus infection.

    Mice were depleted of T cells by adult thymectomy, irradiation and reconstitution with T de-pleted bone marrow (ATx.BM mice) and then infected with influenza followed by adoptive transfer of Tc1, Tc2 or PBS alone. Normal littermate controls were also treated similarly. These experi-ments showed that Tc1 and Tc2 cells were able to promote recovery from influenza in the absence or presence of host T cells as measured by survival after infection with lethal doses of influenza or recovery of initial weight. However using a higher dose of virus the Tc2 cells became less able to promote recovery from infection indicating a role for the host lymphocytes in limiting virus spread or reduction of virus associated pathology. The most striking observation was that ATx.BM mice lost less weight after influenza infection than their wildtype littermate controls, indicating a T cell dependent component responsible for weight loss during infection. Reconstitution of ATx.BM in-fluenza infected mice with Tc1 cells, and to a lesser extent Tc2 cells, restored weight loss to levels similar to that seen in WT littermate controls given influenza plus PBS or Tc1 cells. Therefore we propose that host T cells have a role in weight loss and the general pathology associated with this infection. This could be a direct effect by secretion of cytokines such as IL-1, IL-6 or TNF-α or an indirect effect such that host T cells or Tc1 cells recruit other cells such as granulocytes which se-crete these cytokines. In conclusion we find that recovery from influenza using adoptive transfer of Tc1 and Tc2 cells can be independent of host T cells and that the presence of host T cells contrib-utes to virus associated immunopathology

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    The Protective Role of IL-12 in Innate Immunity against Pneumococcal Pneumonia.

    Keer Sun and Dennis W. MetzgerCenter for Immunology & Microbial Disease, Albany Medical College, Albany, NY 12208

    Innate immunityplays an important role in elimination of pneumococcus in the host before development of specific adaptive immunity. Since pro- inflammatory cytokines have been found to influence certain components of the innate antibacterial response, we wanted to determine whether IL-12 could elicit protective innate immune responses against the extracellular pathogen, S. pneumo-niae. BALB/c mice were infected intranasally with serotype 3 S. pneumoniae 24 hrs after intranasal administration of mIL-12. Control mice received vehicle (1% NMS in PBS) 24 hrs before infection.All IL-12 treated mice showed significantly more IFN-γ in broncho-alveolar lavage fluid (BALF) 12 hrs after pulmonary infection, and they also tended to have increased levels of TNF-α compared to the PBS treated group. There were also significantly more PMN in lung tissue and BALF of IL-12treated mice 48 hrs after infection. As the infection progressed, lower numbers of bacteria were found in the lungs of IL-12 treated mice compared to vehicle treated controls and most importantly, administration IL-12 significantly improved the survival rate of mice after pulmonary infection compared to the control group that received vehicle only. Using IFN-?-/- mice, it was found that IFN-? was essential for IL-12-induced resistance against pneumococcal pneumonia. IFN-?was able to promote TNF-a production by alveolar macrophages during in vitro stimulation with heat-killedpneumococci, and in vivo neutralization of IFN-? by antibody treatment reduced TNF-a expressionin the BALF of IL-12 treated mice. However, neutralization of TNF-a did not attenuate production of IFN-?. Taken together, the data suggest that IL-12 is able to improve innate defense in the lung against the extracellular S. p