Transcript
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7th AnnualUpstate New York

Immunology Conference

Sponsored by:

Taconic

BD Biosciences

Invitrogen

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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TABLE OF CONTENTS

Schedule of Events 7

Supporting Institutions 13

Acknowledgements 15

NYIC GrantsNIAID and Pfizer, Inc.

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

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Corporate Supporters:VWR, BioLegend, and Fisher Scientific

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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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MajorCorporateSponsors

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CorporateSupport

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CorporateContributors

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Financial Support Provided by:

Cell Signaling

eBiosciences

Guava Technologies

Hyclone

Jackson Immuno Research

Kendro

Mettler-Toledo

New England BioLabs

Ohaus Corporation

Roche

Stratagene

Thermo Electron Corporation

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PresentationAbstracts

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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

“NO ABSTRACT SUBMITTED”

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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

“NO ABSTRACT SUBMITTED”

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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 Bangia1

1Department of Immunology, Roswell Park Cancer Institute Buffalo, NY 142632Howard 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

“NO ABSTRACT PROVIDED”

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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, 2

1James 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 Hesse1

1 College of Veterinary Medicine, Cornell University, Ithaca NY2 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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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”

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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. Denkers1

1Cornell University College of Veterinary Medicine, Ithaca, NY2St. 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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PosterAbstracts

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#1

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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#2

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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#3

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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#4

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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#5

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. pneumoniae pathogen by inducing IFN-? production, which in turn en-hances TNF-a expression. The precise role of TNF-a in bacterial protection is currently being in-vestigated using TNF receptor deficient mice.

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#6

Itk Controls a Novel Checkpoint in Th2 Differentiation

Byron Au-Yeung and Dr. Deborah FowellDavid H. Smith Center for Vaccine Biology and Immunology and

Department of Microbiology and Immunology, University of Rochester, Rochester, New York

Itk is a member of the Tec family of tyrosine kinases and its best-characterized role is in controlling the calcium flux via phosphorylation of Phospholipase C ? amma. Previous studies revealed that the absence of Itk affects T helper cell responses, as IL-4 production from Itk-deficient CD4+ cells primed under Th2 skewing conditions is severely impaired while conversely, Th1-primed Itk-/- cells are able to produce IFN gamma. Th differentiation is multi-step process that takes place over re-peated rounds of exposure to antigen. We asked where the block in IL-4 production occurs in the absence of Itk? Using a bicistronic IL-4/GFP reporter mouse (4-get), we have the unique ability to track IL-4/GFP induction in single Itk-/- CD4+ cells. We found that during the first 5 days of prim-ing under Th2 conditions, Itk-/- CD4+ cells differentiate similarly to wild type (WT) cells as defined by the upregulation of IL-4 and GATA3 transcripts. However, unlike WT cells, Itk-/- cells (that had upregulated IL-4 mRNA during priming) failed to further upregulate IL-4 mRNA following res-timulation. This inability to ‘super- induce’ IL-4 transcription in the absence of Itk resulted in a fail-ure to produce IL-4 protein. These results suggest that Itk expression is not required for the initia-tion of Th2 differentiation but is essential for IL-4 production by Th2 primed effector cells. Thus, we believe Itk-deficiency reveals an important checkpoint in Th2 development at the stage of effec-tor cell activation. The studies suggest that the signals for initial Th differentiation are distinct from those needed for the release of effector function.

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#7

Role of T cells in Pneumonic Tularemia

Sofía Olmos and Dennis MetzgerCenter for Immunology & Microbial Disease, Albany Medical College

Francisella tularensis is a highly infectious facultative intracellular bacterium that causes the dis-ease tularemia. Inoculation or inhalation of as few as 10 F. tularensis organisms may cause disease in humans, and because of its high virulence, the bacterium is considered a possible bioterrorism agent. The pneumonic form of tularemia is the deadliest form of disease, even though the majority of research against this organism has focused on systemic infection. The present study was aimed at 1) examining the role of T cell subsets in defense against respiratory F. tularensis infection, as well as 2) determining the protective properties of IL-12 in pneumonic tularemia. C57BL/6 mice specifi-cally lacking αβ or γδ T cells and CD4 or CD8 cells were inoculated intranasally (i.n) with 0.5 µgIL-12 or vehicle 24 hrs before a lethal i.n challenge with 10,000 CFU of Francisella tularensis orsublethal i.n challenge with 1,000 CFU. TCRγδ KO, TCRαβ KO and CD8 KO mice were found to uniformly succumb to i.n infection with 10,000 CFU by approximately Day 13 post infection, in a manner similar to wild type mice. While treatment with IL-12 protected TCRγδ KO mice, no pro-tection was observed in CD8 KO or TCRαβ KO animals. Surprisingly, when CD4 KO mice were infected in the presence or absence of IL-12, they all survived a lethal dose of F. tularensis (100% survival compared to 0% in wild type mice) and were protected against a second challenge with even higher doses of bacteria (106/mouse). With regard to sublethal challenge doses, all mice sur-vived except the CD8 KO mice, which succumbed when infected i.n with 1,000 bacteria. Treatment of CD8 KO mice with IL-12 only partially delayed time to death. These results suggest that CD8 T cells are required to survive an intranasal challenge with F. tularensis LVS and that CD8 TCRαβ T cells are necessary for IL-12 to exert its protective role. The fact that CD4 KO mice survive lethal doses of bacteria even in the absence of IL-12 treatment was unexpected and suggests a detrimental effect of these cells in protection against pneumonic tularemia. Current studies are designed to elu-cidate the mechanisms for this observation. (Supported by NIH grant PO1 AI056320)

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#8

Age-related Defects in CD4 Helper Activity Can be Overcome by in Vitro Effector Generation

Sheri M Eaton, Eve M Burns, Troy D Randall, and Laura HaynesTrudeau Institute, Saranac Lake, NY

The decreased efficacy of vaccinations seen in elderly populations, marked by an inability to produce high titers of antibodies in response to immunization, has been well documented. This age-related defect is thought to occur at the level of the helper CD4 T cell. Our recent work using a TCR transgenic (Tg) adoptive transfer model where naïve young or aged CD4 T cells are transferred into immunized CD4-deficient hosts supports this theory. We have seen significantly reduced ex-pansion and germinal center differentiation of the Ag-specific B cell population following adoptive transfer of naïve aged donor CD4 T cells when compared to young CD4. In addition, we see re-duced antigen-specific IgG antibody titers in the serum from animals receiving aged CD4. Previous studies have shown that naïve aged CD4 T cells undergo reduced proliferation and activation, and have lower IL2 production compared to young CD4 both in vitro and in vivo. The goal of this study was to determine if the cognate helper function of aged CD4 T cells could be improved by stimulat-ing the cells in vitro, under optimum conditions, prior to adoptive transfer to immunized hosts. To do this, in vitro generated TH1 and TH2 effector populations from young and aged naïve CD4 T cells were used as donor cells. These effector populations show no age-related differences in prolif-eration or cytokine production. Upon adoptive transfer of effector populations, levels of expansion and germinal center differentiation of the Ag-specific B cell population were similar for both young and aged donors. Our results show that prior in vitro activation of aged CD4 T cells restores their ability to provide cognate helper activity. This study was supported by NIH grant AG21054 to LH.

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#9

Dendritic Cells Present Influenza Antigens Long After Clearance of Live Virus

Dawn M. Jelley-Gibbs, John P. Dibble, and Susan L. SwainTrudeau Institute, 154 Algonquin Ave, Saranac Lake, NY, 12983

The efficiency of antigen presentation to T cells can dramatically impact the quality of an immune response to pathogens. Antigen dose, type and maturation of antigen presenting cells, and duration of antigen presentation can all contribute to the efficiency of antigen presentation. We have estab-lished that influenza derived antigen is retained and presented to influenza-specific CD4 T cells for at least 1 month following resolution of infection. We have isolated B cells, monocytes/macrophages, and dendritic cells from draining lymph nodes, lungs, and spleens of mice sub- lethallyinfected 1-28 days previously with influenza virus, and co-cultured them with CFSE labeled, flu-specific CD4 T cells in vitro to determine which APC are presenting infection-derived antigens. We determined that B cells, monocytes/macrophages, and dendritic cells were all presenting flu-derivedantigen during the early stages of infection when live virus is expanding. Following resolution of in-fection, flu-derived antigen presentation became limited to dendritic cells. As expected, the fre-quency of antigen presenting cells capable of stimulating T cell division decreases with time follow-ing clearance of live virus. We plan to determine the mechanism by which antigen presenting den-dritic cells retain flu-derived antigens for presentation to CD4 T cells.

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#10

Characterization of Llama IgG Isotypes Induced During Parelaphostrongylus tenuis Infection

Lisa P. Daley1, Lucille F. Gagliardo1, Michael S. Duffy1, and Judith A. Appleton1, 2

1James 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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#11

Detection of Syndecan-1 in Muscle Cells Infected with Trichinella spiralis

Daniel P. Beiting,* 1 Pyong Woo Park,2 Judith A. Appleton1

1James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA

2Department of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA

Syndecans are a family of cell surface, transmembrane proteoglycans found on all adherent cells. Members of the syndecan family are comprised of a core protein modified by numerous, highly sulfonated heparan sulfate chains that mediate interactions with extracellular matrix, growth factors, cytokines and chemokines. One member of this family, syndecan-1, is expressed on the sur-face of epithelial cells, endothelial cells, plasma cells, and immature skeletal muscle cells. In this study we show that mature muscle cells infected with Trichinella spiralis are induced to express syndecan-1. Immunohistochemical analysis of nurse cell syndecan-1 demonstrated cytoplasmic and extracellular distribution of the protein, rather than conventional, cell surface localization. To exam-ine the role of syndecan-1 in the intracellular habitat of T. spiralis, we infected wild-type and synde-can-1 deficient mice by intravenous injection of T. spiralis newborn larvae. Nurse cells developed to maturity, and the inflammatory response to muscle infection was largely unchanged in the ab-sence of syndecan-1. In addition to syndecan-1, we also detected perlecan, a related proteoglycan, and heparan sulfate associated with the nurse cells in both wild-type and syndecan-1 deficient mice.This suggests that other heparan sulfate-bearing proteoglycans may compensate for a loss of synde-can-1.

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#12

Brain-specific Autoantibodies in Lupus-prone Mice Affects Behavior

David LawrenceWadsworth Center

“NO ABSTRACT PROVIDED”

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#13

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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#14

Azithromycin Decreases Humoral Immune Responses in Mice

Aracelis D. Fernandez 1, 2 and Dennis W. Metzger 1.1Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY and

2 Department of Pediatrics, Albany Medical College, Albany, NY

Azithromycin has been used in patients with illnesses characterized by pulmonary inflammation such as cystic fibrosis and asthma, with clinical benefit. This effect may be unrelated to its antim-icrobial properties but rather to immune modulation. We have analyzed the effect of azithromycin on murine immune responses using both a pneumococcal vaccine and infection model, and an aller-gic lung inflammation model. Adult BALB/c mice were inoculated i.p. with azithromycin or PBS and immunized one hr later with a heptavalent protein-conjugated pneumococcal vaccine(Prevnar®). After immunization, the mice were bled to measure primary serum antibody responses and then challenged intranasally with S. pneumoniae type 14 to analyze resistance to bacterial colo-nization. Spleen cells from a separate group of treated and immunized mice were cultured four weeks after priming to analyze recall proliferation responses. In order to study the effect of azithro-mycin on a murine model of allergic inflammation, mice were sensitized i.p. with ovalbumin (OVA) in alum and then challenged 14 days later with five daily intranasal doses of OVA in normal saline solution. Allergen specific antibody levels in serum and in bronchoalveolar lavage fluid (BAL) were determined by ELISA. The immunized mice pretreated with azithromycin had a marked de-crease in total serum antibody responses to Prevnar® when compared to the mice pretreated with PBS. Similar results were obtained for serum IgG1 antibody levels. Spleen cell suspensions from vaccinated mice treated with azithromycin were also found to have impaired memory responses to the Prevnar® recall antigen compared to control groups and nasal washings from animals treated with azithromycin contained a higher number of colony forming units than those from immunized mice pretreated with PBS. Similarly, azithromycin treatment decreased total antibody responses in BAL fluids after OVA sensitization and challenge. Similar results were obtained for IgG1 anti-OVA levels in both BAL and serum. We conclude that the macrolide antibiotic, azithromycin, ex-erts a significant suppressive effect on the murine immune system independently from its antimicro-bial properties. (Supported by NIH grant AI41715)

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#15

Differential Binding of Escherichia coli Enterotoxins LT-IIa and LT-IIb andof Cholera Toxin Elicits Differences in Apoptosis, Proliferation, and

Activation of Lymphoid Cells

Sergio ArceUniversity at Buffalo

Cholera toxin (CT), LT-IIa, and LT-IIb are potent adjuvants which induce distinct T-helper(Th)-cell cytokine profiles and IgG subclass and IgA antibody responses. To determine if the distinct immune regulatory effects observed for LT-IIa, LT-IIb and CT are elicited by binding of the entero-toxins to their cognate ganglioside receptors, the lineages of lymphoid cells that interact with the three enterotoxins and their effects on various lymphocyte responses in vitro were evaluated. Bind-ing patterns of LT-IIa, LT-IIb, and CT to several lymphoid cell populations were distinctive for each enterotoxin. LT-IIa and CT, but not LT-IIb, induced apoptosis in CD8+ T cells. LT-IIa(T34I), a mu-tant with no detectible binding to gangliosides, did not induce apoptosis. Blockade of GM1 on the surface of CD8+ T cells by LT-IIa(T14I), a mutant that binds only to GM1 but does not induce apop-tosis, did not inhibit induction of apoptosis by LT-IIa. Mitogen-induced proliferation of CD8+ T cells was abrogated by treatment with CT, while resting CD8+ T cells which were sensitive to LT-IIa- induced apoptosis became more resistant to apoptosis after mitogen-activation. Exposure to CT, but not to LT-IIa or LT-IIb, inhibited mitogen-driven CD4+ T cell proliferation and expression of CD25 and CD69. In mitogen-stimulated B cells, CT and LT-IIa, but not LT-IIb, enhanced expres-sion levels of CD86, while only CT induced B cell differentiation into plasma cells. Thus, LT-IIa,LT-IIb and CT exhibit distinguishable immunomodulatory properties which are dependent upon their capacities to recognize different ganglioside receptors on lymphocytes.

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#16

Toxoplasma gondii-stimulated anemia results from IFNγ-driven hemorrhage

I.K. Mullarky, K. Berggren, F. Szaba, M. Parent, and S.T. SmileyTrudeau Institute, Saranac Lake NY

The ability of various infections to stimulate anemia has been well documented. However the mechanisms behind this phenomenon remain largely undefined. Utilizing the protozoan parasite, Toxoplasma gondii, we have identified an interferon gamma (IFN?)-dependent anemia during the early stages of infection. To characterize the cause of infection-stimulated anemia, erythropoiesis and survival time of circulating red blood cells were analyzed in mice following peroral infection with ME49 strain of T. gondii. Erythropoiesis was measured through a flow cytometric assay identi-fying young red blood cells by RNA/DNA content. Additionally, intravenous injection of mice with sulfo-NHS-LC-biotin allowed for quantification of red blood cells throughout infection. We demon-strated a decrease in reticulocyte production as well as an increase in red blood cell loss during in-fection. However, infection of IL-6 knockout mice restored erythropoiesis while having no effect on IFN?-dependent anemia. These findings suggest that IFN?-dependent anemia is a consequence of hemorrhage. This conclusion is further supported by both histopathology and fecal blood data.

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#17

Cell Mediated Immunity Against Yersinia Infections

M.A. Parent1, K.N. Berggren1, F.M. Szaba1, I.K. Mullarky1, J. J. Adamovicz2,and S.T. Smiley1

1Trudeau Institute, Saranac Lake, NY 129832USAMRIID Plague Vaccine Program, Fort Detrick MD 21702

Vaccination strategies directed against Yersinia pestis have focused on the development of a humoral immune response. While these regimens generate effective protection against the bubonic form of infection, they provide incomplete protection against pneumonic infection. Since humoral immunity is unable to afford full protection from infection, we have investigated the possible role of cell-mediated protection against Yersinia. Vaccination studies were conducted using the Fraction 1 (F1) and V proteins of Y. pestis, both known virulence factors that stimulate some protection in the infected host. In order to assess the presence of a cell-mediated response, V-protein specific pep-tides were generated covering the entire length of the protein and were used to screen for the pres-ence of V-protein specific CD4+ T cells in an F1-V vaccinated host. Utilization of the F1-V pro-tein subunit in complete Freunds' adjuvant (CFA) resulted in the identification of V protein specific CD4+ T cells that demonstrate a type 1 cytokine response. This cell-mediated (TH1), V-specificCD4+ T cell response was detected from several of the 63 peptides screened and was characterized by the production of interleukin-2, interferon-gamma, and by T cell proliferation. To document the role of cell-mediated immunity during Yersinia infections F1-V and V-peptide vaccinated mice were challenged with 10 LD50 of Yersinia enterocolitica O8:WA. Y. enterocolitica does not produced the F1 protein and antibodies specific for Y. pestis V protein do not cross protect against Y. entero-colitica O8:WA V protein, therefore, any resulting protection would likely be due to a cell-mediatedmechanism. F1-V vaccinated mice were protected against Yersinia infection with no organisms re-covered, while control groups demonstrated detectable colony forming units. V1, V2 and V3 pep-tide vaccinated mice were protected as demonstrated by increased survival when compared to the Vneg control group. These findings suggest that F1-V and V peptide vaccination can stimulate cell-mediated immunity against Yersinia.

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#18

The IL-12 p40 Subunit is Required for Dendritic Cell Maturation and T cell Priming Following Mycobacterial Infection

Shabaana A Khader, Santiago Partida-Sanchez, Guy Bell, Dawn M Jelly-Gibbs,Nico Ghilardi§, Fred deSauvage §, Frances E Lund and Andrea M Cooper

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

Tuberculosis (TB), 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-g 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 detri-mental than the absence of p35 subunit in the murine model of TB. The fact that both the p35 and p40 mice lack IL-12p70, yet the p40 deficient mice are more susceptible, implicates other p40 de-pendent molecules such as IL-23 or the p40 homodimer as mediators of protection in TB. We have recently determined that the absence of the cytokine IL-23 (which is composed of a four-alpha helix molecule, p19 and forms a disulfide-bonded heterodimer with the p40 subunit) does not compromise the cell-mediated immune response and that the IL-23 p19 deficient mice are not more susceptiblethan the intact mice to TB. In light of this new finding, we have reexamined the basis for the higher susceptibility of IL-12p40 deficient mice. Two events are crucial to the induction of adaptive immu-nity. The first is the activation of tissue DCs upon encounter with the pathogen and their mobiliza-tion to the secondary lymphoid organs. The second is the priming and activation of antigen-specificT cells to a TH1 phenotype. We investigated the role of p40 and p35 in both of these events and found that IL-12p40 homodimer is required for DC activation and subsequent ability of these cells to migrate to the draining lymph node. We also found that DCs activation and migration, while not optimal, did occur in the absence of IL-12p35. This inability of IL-12p40 deficient mice to mobilize tissue DCs in response to pathogen sets them apart from the IL-12p35 deficient mice and may pro-vide the underlying mechanism for their increased susceptibility to TB.

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#19

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 re-sponsible 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 con-stant throughout 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 com-pared 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 thymocytes labeled exceeded 50% in both the young (6-10wk) and aged (20-28mo)groups. Dye labeling 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 indicate that defects observed in aged CD4 cells arise at least in part due to de-fects in thymopoeisis inherent in the aged thymus.

This research was supported by the NIA Grant AGA01743.

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#20

Effect of Mode of Presentation on Macrophage Response to Mycobacterial Trehalose 6,6’-dimycolate

Kaori Sakamoto, Rachel E. Geisel, Elizabeth R. Rhoades and David G. RussellDept. of Microbiology and Immunology

Veterinary Medical Center, Cornell UniversityIthaca, NY 14853

Trehalose 6,6’-dimycolate (TDM), or cord factor, is a major component of the thick waxy cell wall of mycobacteria and is responsible for much of the proinflammatory activity of Complete Freund’s Adjuvant (CFA); however the mechanism by which this effect occurs is still unclear. Of the peripheral lipids of Mycobacterium bovis Bacille Calmette-Guerin, trehalose mycolates induce the highest levels of proinflammatory cytokines (such as TNF-α, IL-1α, IL-6) when presented to murine bone marrow-derived macrophages (mBMDM) on the surface of 90µm polystyrene micro-spheres. These results correlate with in vivo studies using these coated microspheres when injected into mice in an artifical peritoneal granuloma model. TDM-coated microspheres stimulatemBMDM in a TLR2- and TLR4-independent, MyD88-dependent manner. Microarray results com-paring MyD88-/- and wild-type mBMDM responses to TDM-coated 90 µm microspheres show that most of the responses to TDM are MyD88-dependent and the most upregulated genes are involved in the immune response and tissue remodeling. Examination of the few MyD88-independent genes suggests the engagement of another receptor. Interestingly, when TDM is coated onto microspheres of phagocytosable sizes (10µm and less in diameter), proinflammatory cytokines are not induced.Inhibition of phagocytosis using cytochalasin D results in cytokine induction by phagocytosable TDM-coated microspheres. There is decreased cytokine production, however, in response to non-phagocytosable TDM-coated microspheres (25 and 90 µm diameter), suggesting that cytokine in-duction is proportional to the number of macrophage receptors engaged. This effect is trehalose my-colate-specific as other mycobacterial cell wall lipids do not exhibit this size dependence. Since TDM may only be present on a large surface in CFA or in the late stage caseous granuloma as part of residual lipid pellicles, these results, particularly the induction of numerous tissue remodeling en-zymes by TDM, may elucidate possible mechanisms of tissue destruction by CFA and capsule deg-radation during reactivation of tuberculosis.

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#21

Increased Expression of Arginase-I in Glia of SHP1 Deficient Mice Relates to Decreased iNOS Expression and Anti-viral State

Kathryn Beuler and Paul MassaDepartment of Microbiology and Immunology, SUNY Upstate Medical University,

750 East Adams ST, Syracuse, NY 13210

We have previously shown that the SH2 domain-containing protein tyrosine phosphatase SHP1 plays a critical role in controlling viral replication in myelin-forming oligodendrocytes and virus-induced demyelination in the central nervous system (CNS). In the present study, we have observed an abnormally high constitutive expression of arginase I in oligodendrocytes of SHP1-deficient mice that may relate to increased virus replication in these cells. Increased arginase I expression corre-lated with a profound decrease expression of inducible nitric oxide synthase (iNOS) protein in SHP1-deficient oligodendrocytes. Accordingly, nitric oxide production was also significantly less in SHP1-deficient compared to normal littermate cells. Oligodendrocytes of SHP1-deficient mice also displayed inducible expression of arginase I after co-treatment with IL4 and IL10, cytokines that act through increased STAT6 activation. We are currently examining whether amplified expression of arginase I in SHP1-deficient glia competes with iNOS for arginine availability. Additionally, the role of arginine deficiency on decreased iNOS translation in oligodendrocytes is being investigated.In sum, these studies are directed at determining the importance of SHP-1 in regulating arginine me-tabolism and antiviral activities of nitric oxide in the CNS.

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#22

Ricin Induces IL-8 Secretion in Human Monocyte/Macrophages by Activating the p38 MAP Kinase Pathway

Tessa-Vera Gonzalez1, Stephanie A. Farrant 1, Gary Edwards2, and Nicholas J. Mantis1,2

1Harvard Medical School and Children’s Hospital Boston, Boston, MA 2Division of Infectious Diseases, Wadsworth Center, Albany, NY

Background: Exposure of mucosal tissues, including the respiratory and gastrointestinaltracts, to the shiga- like toxin ricin induces pathological inflammation that includes an influx of poly-morphonuclear (PMN) cells. Macrophages are the first cells to show signs of pathological changes in response to toxin exposure, and it is hypothesized that these cells may initiate inflammatory cas-cades that exacerbate tissue damage associated with ricin. In this study we demonstrate that human monocytes/macrophages secrete IL-8 in response to sub- lethal doses of ricin.

Methods: The human monocyte/macrophage cell lines 28SC or U937 were treated with ricin (1-1000 ng/ml) and at designated time points supernatants were assayed for IL-8 by ELISA.

Results: IL-8 secretion was detected as early as six hours post-toxin exposure, and continued as late as 40 hr. 28SC cells did not secrete IL-8 when exposed to ricin toxoid, ricin B subunit, or the protein synthesis inhibitor cycloheximide. The human monocyte/macrophage cell line U937 also released IL-8 in response to toxin. As predicted from the literature, we detected an increase in p38 MAPK activity in 28SC cells as early as 3h after ricin exposure. Treatment of 28SC cells with the p38 MAPK inhibitor SB203580 before or after the addition of ricin completely suppressed IL-8 se-cretion.

Conclusion: We conclude that ricin induces IL-8 secretion in human monocyte/macrophage cell lines by activating the p38 MAPK pathway. These data raise the possibility that p38 MAPK in-hibitors may potentially serve as therapeutic agents to suppress ricin- induced mucosal inflammation.

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#23

Frank SzabaTrudeau Institute, Saranac

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#24

Effects of Ethanol on CD4 T Cell Function

Kimberly Hale and William LeeWadsworth Center, Albany NY

Excessive alcohol consumption can impair numerous physiological processes, including those of the immune system. Animal and clinical studies have shown that various immune cells, especially T lymphocytes, are negatively affected by ethanol. However, the mechanism(s) that underlie T cell dysfunction are unclear. Since CD4 T cells are central to overall immunity, it is important to under-stand how ethanol dampens T cell function and to devise methods to restore activity. We are exam-ining the effect(s) of ethanol consumption on DO11.10, CD4+T cell function, specifically prolifera-tion in response to specific antigen (OVA323-339), in an in vivo model. Host mice (BALB/c) will be fed ethanol (LED-30%), liquid control diet, or regular diet for different periods of time prior to adoptive transfer, this will allow us to determine the effect(s) of ethanol consumption for various pe-riods of time. The effect(s) of ethanol will be investigated in both male and female mice. Once the effect(s) of ethanol consumption on CD4+ T cell function have been established, we plan to ascer-tain whether these affects are direct or indirect.

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#25

A Model System to Study the Effect of F. tularensis Invasion on the Cell Biology of Antigen Processing and Presentation by Macrophages

Justin E. Wilson, Erica McGovern, and James R. DrakeCenter for Immunology and Microbial Disease, Albany Medical College, Albany, NY

In most instances, respiratory pathogens encounter macrophages upon entry into the lungs, whereupon they are phagocytosed, processed, and presented to T lymphocytes. Bacterial species such as Legionella pneumophila and Francisella tulerensis avoid degradation and survive and repli-cate within the host macrophage. Nevertheless, processing and presentation of bacterial antigens to CD4+/CD8+ T cells is critical to the establishment of an effective immune response, and clearance of infection. In order to elucidate the mechanism of MHC-restricted processing and presentation, we have established a model to study the processing of particulate antigen by alveolar and other macro-phages.

By monitoring the levels of immune complex (IC) binding to FcγRII, we have determined the optimal conditions for the generation of soluble ICs of hen egg lysozyme (HEL) and an anti-HELmonoclonal 2D1. Similarly, HEL-coated glass or latex beads can be oposnized with 2D1 mAb for subsequent FcγRII -mediated phagocytosis by alveolar macrophages. Immunofluorescence micros-copy-based analysis has revealed the colocalization of beads with the late endosome/lysozome marker, LAMP, demonstrating internalization and delivery to late endocytic compartments. To ana-lyze the presentation of HEL on MHC class II molecules, we are utilizing two antibodies specific for either total I-Ak class II molecule (11-5.2) or HEL46-61–I-Ak complexes (C4H3). Preliminary ex-periments demonstrate that cells pulsed overnight with HEL-2D1 ICs, generate significant levels of HEL46-61–I-Ak-complexes which can be detected via C4H3 staining. These results demonstrate the establishment of a system for the study of antigen processing in macrophages, which will be useful for the analysis of the antigen processing and presentation in Francisella tulerensis-infected macro-phages. This work is supported by the NIH grant AI-056320.

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#26

Effects of Photodynamic Therapy (PDT) Dose on the Immune System

Philaretos C. Kousis, Sally Schneider, Barbara W. Henderson and Sandra O. GollnickRoswell Park Cancer Institute

In photodynamic therapy (PDT), four main factors can be identified that influence the treat-ment outcome: the amount of photosensitizer and oxygen present in the treated site, the total amount of light energy delivered to the local site (fluence) and the rate of light delivery (fluence rate). Low fluence rate treatments conserve the oxygenation status of the treated site during treatment, regard-less of fluence. High fluence (high dose) treatments given at oxygen conserving low fluence rates result in the induction of low levels of inflammation, while low fluence treatments given at equiva-lent fluence rates, result in high levels of inflammation. Using these two treatment regimens we have examined the effects of PDT induced low and high levels of inflammation on the immune sys-tem. Experiments conducted in immunocompromized SCID mice have shown that the presence of a competent immune system is required for the full curative potential of PDT, regardless of the degree of inflammation elicited. However, the quality of the immune response is influenced by the degree of local inflammation. Preliminary experiments show that the high inflammation group has a higherfrequency of cells able to recognize and respond to tumor antigen by the production of IFN-? than the low inflammation group. Furthermore, when the high inflammation group is challenged with tu-mor cells, it seems able to control a bit better tumor growth, than the low inflammation group, as very preliminary data has shown. Overall, treatment regimens that result in high inflammation, i.e. low fluence, low fluence rate regimens, are better able to stimulate the anti-tumor immune response. The determination of the effects of fluence on the immune response can lead to the development of treatment regimens that result in robust immune responses that may be more effective at controlling tumors present outside the treatment field.

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#27

Visualization of IFN-γ-expressing Cells After Primary and Secondary Influenza Virus Infection

Katrin D. Mayer, Katja Mohrs, Sherry R. Crowe, Paul Rhyne∗, David L. Woodland and Markus Mohrs

Trudeau Institute, Saranac Lake, NY 12983; ∗Upstate USA, Inc., Lake Placid, NY 12946

IFN-γ production is a hallmark of the immune response to infection with Influenza virus. How-ever, it is not known whether T cells are homogeneous in their capacity to produce IFN-γ, whether this potential varies between lymphoid and non- lymphoid sites at the single cell level, how this is related to primary and secondary challenges, and how rapid cytokine production is accomplished.

We used bicistronic IFN-γ-eYFP reporter mice (Yeti) and MHC class I tetramers to visualize and functionally characterize IFN-γ-expressing cells directly ex vivo after both primary and secon-dary infection with Influenza virus. eYFP reporter fluorescence nine days after primary Influenza virus infection correlated directly with an increased capacity to produce IFN-γ by both CD4+ and CD8+ T cells, including antigen-specificCD8+ T cells. Despite the expression of the eYFP reporter the production of the IFN-γ protein was dependent on restimulation suggesting a mechanism of post-transcriptional regulation. Both CD4+ and CD8+ T cells were broadly heterogeneous in their capacity to produce IFN-γ based on their eYFP reporter brightness and highly fluorescent cells were only present in the infected lung. Anti-gen-specific CD8+ T cells were almost exclusively eYFP+ in all organs with eYFP fluorescence in-tensities being also highest at the site of infection. The overall eYFP fluorescence peaked on day nine post primary infection in all organs and all cell types. Antigen-specific CD8+ T cells remained eYFP+ for at least six months after primary infection. Equivalent levels of eYFP brightness were only reached seven days after a secondary challenge with a serologically distinct Influenza virus. Most interestingly, recall experiments with mutant viruses lacking certain MHC class I-restrictedepitopes revealed that the capacity of CD8+ memory cells to produce IFN-γ is largely dependent on the presence of the respective epitope.

Our results demonstrate a functional heterogeneity of IFN-γ-expressing T cells even within antigen-specific cell populations. Moreover, the functional heterogeneity in IFN-γ production of an-tigen-specific CD8 T cells after secondary infection is increased and the increase of eYFP reporter brightness is antigen-dependent. The identification of cells poised for IFN-γ production is therefore highly relevant to assess the functional potential of immunological memory.

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#28

Ex Vivo HHV-7 Enhancement of HIV-1 Replication

Karen M. Duus, Lishan Su*, and J. Webster-Cyriaque*^Albany Medical College, Albany, NY *Lineberger Comprehensive Cancer Center,

Department of Microbiology and Immunology, School of Medicine; ^Department of Dental Ecology, School of Dentistry, University of North Carolina, Chapel Hill, NC

Background: Human herpesvirus 7 (HHV-7), a T-cell –tropic herpesvirus, is one of the most be-nign (and thus successful) human pathogens. HHV-7 infects small children; 95% have seroconvert by age 5, and many shed the virus asymptomatically via saliva for the rest of their lives. The ab-sence of HHV-7-mediated pathology extends to immunosuppressed individuals, in sharp contrast to other members of the herpesvirus family. Results of one study suggested that prior HHV-7 infection inhibited HIV-1 replication in some pediatric patients. However, effects of HHV-7 upon HIV-1 rep-lication and T cell depletion are unknown. The ability of the virus to infect human cord blood cells, CD4+ T cells, and the SUPT-1 thymocyte- like cell line suggested that HHV-7 would infect the thy-mus and secondary lymphoid organs. We hypothesized that HHV-7 infection could inhibit HIV replication and T cell depletion in thymus and lymphoid explants.

Methods: Tissue explants were first infected with cell- free supernatant from mock infected or HHV-7-infected SUPT-1 cells. Cell- free HIV-1 supernatant from mock-, NL4- or HXB2-infectedPBMC was used to infect the explants 24 hours post-HHV-7 infection. Cells were harvested from the explants at various times post- infection (3-9 days). Surviving cells were counted by trypan blueexclusion and analyzed for virus replication by p24 ELISA (HIV) or PCR (HHV-7). Cells were analyzed for CD4, CD8 and CD3 expression by fluorescence activated cell sorter (FACS).

Results: Our results demonstrated that HHV-7 replicated in the thymus explants, but did not sig-nificantly deplete cells compared to the mock- infected tissue. A slight decrease in the CD4/CD8 ra-tio during the culture period suggested that CD4 expression on CD4 single-positive (SP) cells was decreased in the HHV-7- infected tissue. However, unlike HIV-1 infection, there was no significant decrease in the relative percentage of CD4+CD8+ double-positive (DP) thymocytes associated with HHV-7 infection of the thymus. Surprisingly, the results suggested that HHV-7 co- infection en-hanced HIV-1 replication. In addition, co- infection of HHV-7 enhanced the replication of HXB2, an HIV-1 clone that does not normally replicate to detectable levels in the thymus.

Conclusions: HHV-7 infection of thymus explants did not significantly deplete T cells, but co-infection with HHV-7 significantly enhanced HIV-1 replication.

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#29

SLP-76 is Expressed in Murine Dendritic Cells and Regulates Integrin Receptor-Mediated Signaling

Nancy Luckashenak , Kimberly Ramsey and James L. ClementsDepartment of Immunology • Roswell Park Cancer Institute • Buffalo, NY 14263

The dendritic cell (DC) is the most potent and dynamic antigen presenting cell within the im-mune system, capable of inducing both tolerance and/or initiation of a primary immune response. A number of DC subsets with distinct immunological functions have been described in vivo. How-ever, little is known about the intracellular signaling pathways that regulate the development and/or function of these DC subsets. In an attempt to elucidate these pathways, we have chosen to focus our experiments on the hematopoietic adaptor protein SLP-76. Preliminary data generated in our laboratory indicates that murine DCs express SLP-76. Recently, a role for SLP-76 as a regulator of cytoskeletal reorganization and the cellular processes dependent on these changes has been de-scribed in other hematopoietic cell types. As DCs are highly dependent on cytoskeletal rearrange-ment for their biological activity, we will examine DC function in the absence of SLP-76. We hy-pothesize that the SLP-76 hematopoietic adaptor protein regulates cytoskeletal reorganization in DCs and is necessary for optima l DC biological function. We are able to generate DCs from the bone marrow (BMDCs) of SLP-76 deficient mice for our studies and will assess several DC functions that rely on cytoskeletal rearrangement and the intracellular signaling pathways that may be involved. Recent data from our laboratory indicates that SLP-76-/- deficient BMDCs adhere poorly to fibronectin. In addition, we observe a greater frequency of DCs migrating from the ears of SLP-76 deficient mice. We have also identified SLP-76 phosphorylation upon ligation of β1 and β2

integrins in BMDCs. Interestingly, under these conditions we observe the constitutive association of SLP-76 with the guanine nucleotide exchange factor Vav in our DCs, in stark contrast to the phosphotyrosine inducible association previously observed in other hematopoietic cell types. To further delineate the function of SLP-76 in integrin-mediated signaling pathways and DC function, we are currently testing the ability of SLP-76 deficient BMDCs to adhere specifically via β1, β2, andβ3 integrins. Additionally, we will establish if integrin associated downstream signaling cascades (i.e. Erk and Ca2+ flux) are disrupted in the absence of SLP-76 in BMDCs.

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#30

Lack of IL-27 Signaling Reduces T Cell Loss in Disseminated Progressive Mycobacterial Disease

Alejandra Solache1, John Pearl1, Leigh Gilmartin1, Guy Bell1, Nico Ghilardi2, Fred deSauvage2 and Andrea M. Cooper1.

1Trudeau Institute, Inc., Saranac Lake, NY, 12983. 2Department of Molecular Biology, Genentech, Inc, South San Francisco, CA 94080

The protective strong Th1 immune response that controls mycobacteria can also lead to T cell-mediated immunopathology resulting in significant tissue destruction. Induction of IFN-γ pro-ducing CD4 T cells depends to a large degree on IL-12p70. Recently other IL-12 related cytokines such as IL-27 have been implicated in the induction of Th1 cytokines during T cell activation. IL-27 is thought to begin the polarization of Th1 T cells by inducing Th1 transcription factor, T-Bet,via T cell cytokine receptor (TCCR or WSX-1)-mediated activation of STAT-1 and by induc ing the expression of IL-12Rβ2 and thus responsiveness of the T cells to IL-12p70 signaling.

In this study mice lacking the TCCR molecule and therefore unable to mediate IL-27 in-duced signals were intravenously infected with Mycobacterium avium 724 and the progression of disease monitored by bacterial burden, T cell response and histopathology. Following infection with this virulent mycobacteria in intact mice, CD4 T cell numbers expand but then contract, in contrast this same population in TCCR-KO mice expands and remains high throughout. This enhanced lym-phocytic response is also seen in the granulomas of TCCR-KO mice and correlates with improved control of bacteria. The number of IFN-γ producing cells and the level of IFN-γ mRNA remain simi-lar between the intact and TCCR-KO mice. However, by directly comparing the levels of mRNA within purified CD4 T cells from infected B6 and TCCR-KO mice we found that the absence of IL-27 signaling results in reduced mRNA for T-Bet which correlates with reduced IL-12Rβ2 and re-duced induction of the IFN-γ gene.

Our data support the hypothesis that IL-27 signaling is not essential for the induction of Th1 responses but that it does serve to limit the size of the CD4 T cell population during mycobacterial disease. It also provides a model whereby IL-27 modulates the ability of TH1 T cells to be more highly polarized as a result of increased T-bet and IL-12Rβ2, thus the absence of IL-27 reduces the polarization of CD4 T cells during infection- induced activation.

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#31

Characterization of GFP-Francisella tularensis LVS After Pneumonic Infection

Shawn D. Baron, Maria C. López, and Dennis W. MetzgerCenter for Immunology and Microbial Disease

Albany Medical Center, Albany, NY

Francisella tularensis is a small, gram-negative intracellular pathogen that is the agent of the severe and often fatal disease, tularemia. It has been considered a potential biological weapon since it was shown to be virulent at low doses when inoculated intranasally (i.n.). Results of our previous intra-cellular cytokine staining experiments showed local production of IFN-? in the lungs of infected mice by CD11b+ cells after pneumonic F. tularensis infection. The goal of the present study was to histologically identify these cells in lung sections from infected animals and determine their spatial proximity to bacterial- infected cells. For this purpose, the F. tularensis LVS strain was transformed to express GFP (kindly provided by Dr. Jing-Ren Zhang) and its localization in the lungs of C57BL/6 mice after i.n. infection was visualized by fluorescent microscopy. An initial survival study showed that the virulence of the GFP-labeled bacteria was similar to non-transformed LVS bacteria. Groups of four mice were then infected i.n. with 10,000 CFUs of GFP-labeled bacteria, and sacrificed 6, 24, 48, 72, and 96 hr after infection. Cells in the lung expressing CD11b and CD11c were identified using fluorescent antibody staining. Histological analysis showed inflammation around alveolar regions of the lungs, which were not seen in sham-infected animals. The results showed double positive staining for PE-CD11b and GFP-F. tularensis 24 hr. after infection, sug-gesting that alveolar macrophages are the main cells responsible for uptake of GFP-F. tularensis 24 hr. after pneumonic infection. Therefore, we conclude that since GFP-labeled bacteria have an in-fectivity pattern similar to the original LVS strain, use of this strain will allow us to follow the route of infection through the animal via immunofluorescent staining. (Supported by NIH grant PO1 AI056320)

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#32

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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#33

Functional Comparison of Rat Bone Marrow-derived Mucosal Mast Cells with the Mucosal Mast Cell Line RBL-2H3

Seana Thrasher1, David Holowka2, and Judith Appleton1

1J.A. Baker Institiute for Animal Health, Cornell University, Ithaca, NY, 14853;2Department of Chemistry and Chemical Biology, Cornell University, Ithaca NY, 14853

Our aim is to define the role of mast cells in expulsion of Trichinella spiralis from the intes-tinal epithelium. Although correlative evidence suggests that expulsion is dependent on mast cells, the role of mucosal mast cells has not been defined. We hypothesize that specific IgG antibodies, complexed with parasite antigens, interact with Fc receptors on mast cells to promote parasite expul-sion. To address this question, we cultured mast cells from bone marrow of Lewis strain rats (BMMC) and compared their functional properties with those of the rat mucosal cell line, RBL-2H3.Large numbers of BMMC were generated within 2-3 weeks of culture in SCF/IL-3 containing me-dium. We confirmed the mucosal phenotype of BMMC by Alcian blue staining and by detection of rat mast cell protease-2 (RMCP-2). When compared with RBL-2H3 cells, BMMC were more granular and produced greater quantities of RMCP-2. Using flow cytometric analysis, we found that Fc receptors on both cell types bound immune complexes formed with IgG1, IgG2a, IgG2b but not IgG2c. Only IgG2a complexes triggered degranulation. RBL-2H3 cells degranulated in response to complexes formed with IgE, while BMMC failed to degranulate, despite showing similar binding properties. Our results indicate that rat BMMC are readily prepared, model mucosal mast cells well, and should be useful in dissecting the roles of IgG and mast cells in intestinal immunity to T. spi-ralis.

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#34

Comparision of the Immune Response Generated Following Delivery of B+T Peptide and Adjuvant by a “Needle Free” Device vs. Traditional

Needle and Syringe

Vy Thao Tran*, Richard Stout †, Eric Mishkin† and Yasmin Thanavala**Roswell Park Cancer Institute, † Bioject Inc., Bedminister, NJ.

Nontypeable Haemophilus Influenza (NTHI) is a pathogen that causes repeated infections in adults and children. P6 is a highly conserved outer membrane lipoprotein that exists amongst all strains of NTHI. The B+T peptide is a chimeric peptide that consists of the immunodominant T cell epitope of P6 and a surface expressed P6 specific B cell epitope. Thus, there is considerable interest in evaluating this peptide as a potential vaccine candidate.

The need for needle-free delivery systems for vaccination is important due to the risk that needles and syringes have on sanitation, blood-borne diseases, sharps injury and patient compliance.The Biojector® needle free device works by forcing liquid medication at high speed through a tiny orifice held against the skin. This creates an ultra- fine stream of high-pressure medication that penetrates the skin, depositing the medication in the tissue beneath.

We evaluated the magnitude of the T cell proliferative response and the anti P6 specific Ab titers when the B+T peptide was delivered via needle and syringe injection or using the needle-freeBiojector®.

P6 specific antibody responses were measured in sera of mice immunized with alum ad-sorbed B+T peptide or B+T peptide mixed with the adjuvant RIBI, using needle and syringe or the Biojector®. Data indicate that higher titers of anti-P6 antibodies were obtained in mice immunized with the Biojector®. Further, when RIBI was used as the adjuvant, mice showed a greater anti-P6Ab response compared to antibody titers in animals where alum was used as the adjuvant. T cellproliferation was also evaluated in mice immunized with B+T peptide and RIBI/alum, delivered us-ing either needle and syringe or the Biojector®. Our data indicates that mice immunized with the Biojector® elicited a greater in vitro T cell proliferative response to P6 and the peptide used for im-munization compared to mice immunized via traditional needle and syringe. Overall, more striking differences in responses were seen when RIBI was used as the adjuvant.

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#35

The Role of Antigen in Recruitment and Proliferation ofCD4 T cells During Influenza Infection

Timothy J Chapman1, Maria R Castrucci2, Linda M Bradley3, David J Topham1

1University of Rochester School of Medicine and Dentistry, Rochester, NY2Istituto Superiore di Sanita, Rome, Italy

3Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121

CD4+ T cells significantly contribute to both primary and secondary immune protection against viruses. However, few effective model systems exist for tracking epitope-specific CD4+ T cells at the single-cell level during the infection. To address this, we used reverse genetics to engi-neer the OVA323-339 peptide into the neuraminidase stalk of influenza/A/WSN. The recombinant vi-rus, WSN-OVAII, elicits an immune response comparable to related parental WSN strains in both viral clearance and kinetics of the cellular response. An OVA323-339-specific immune response could be measured in the lung and draining lymph node of C57BL/6 mice throughout the course of infec-tion. When OT-II or DO11.10 TcR transgenic cells were injected prior to infection, these cells could be found in the BAL and lung tissue during the acute infection with either WSN-OVAII or a control influenza A virus. However, transgenic cells only proliferated when OVA323-339 was present in the virus. These data suggest that antigen is required for CD4+ T cell acquisition of proliferativepotential at the site of infection. The experiments also suggest that non-antigen-specific “bystander” CD4+ T cells are present in the infected lung with limited impact on their activation.

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#36

Connexin43 Expression in Cultured Cells of the Monocyte/Macrophage Lineage

Isabel Alvarez de Mora,1 M. Delmar,2 S. M. Taffet11 Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY

2 Pharmacology, SUNY Upstate Medical University, Syracuse, NY

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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#37

Time-dependent Distribution of Naturally Occurring Regulatory T cells in a Pul-monary Granuloma Model of Schistosomiasis

Martin Baumgart, Fae Tompkins and Matthias HesseCornell University College of Veterinary Medicine

Recent data indicate an important immunoprotective role for CD4+CD25+ regulatory T cells (Treg) in a murine model of schistosomiasis. Adoptive transfer experiments revealed that absence of natu-ral occurring Treg results in enhanced inflammatory responses to schistosome infections. However, the heterogeneity of the CD4+CD25+ T cell population found at the site of inflammation and in various lymphoid organs during the infection is not well defined. We developed an adoptive transfer model in immuno-deficient RAG mice, which allowed the identification of different donor CD4+ T cell populations in schistosome-egg immunized mice. Our data demonstrate for the first time the accumulation of natural occurring Treg in granulomatous tissue and draining lymph nodes in schis-tosomiasis. Furthermore, the CD4+CD25+ T cell population comprises a mixture of T cells from ef-fector T cell and Treg origin in all examined tissues and at each time point. Strikingly, our data indi-cate a shift from high effector and low Treg frequencies in early acute inflammation to a reverse situation at the late response. It is conceivable that natural occurring Treg contribute to the resolu-tion of the inflammatory response after the parasite eggs were neutralized by the host immune sys-tem. Further studies will be conducted to investigate the functional profile of individual, well-defined Treg populations in our model.

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#38

Gestational Exposure to Mercury in DBF1 Mice

Karsten Pilones and Jerrie GavalchinSUNY Upstate Medical University

The immunomodulatory effects of metal xenobiotics such as mercury have been well de-scribed. In particular, its generalized lymphoproliferative effects and propensity to skew towards a Th2-type, MHC Class II-dependent cytokine response has been thought to influence appearance of autoimmune disease in certain susceptible murine animal models.

We sought to determine how prenatal exposure to mercury in a non-autoimmune mouse model might affect lymphocytic development. To this end, after overnight breeding of Balb/c dams and DBA/1 mice the pregnant dams were exposed to mercuric chloride-treated drinking water at 10 ppm. The DBF1 pups were sacrificed at Day 18 of gestation and the fetal thymus and liver harvested for immunophenotyping. No observable differences were seen in pup weight although average litter size in the control group was almost twice that of the exposed group (8.5 vs 4.7); moreover, mer-cury-treated dams had fetal resorptions while none were seen in unexposed mice. Thymus and liver cellularity between treatment groups was not found to be significant. Gender differences in pheno-types were also examined; specifically, we saw increases in SP (CD4+) and DN (CD4-/CD8-)populations in the thymus that was significant for male pups. We likewise saw an expansion of B220+ cells in the liver for both male (p<0.03) and female (p<0.001) pups. Previous studies from our lab have shown that dysregulation of the idiotypic network, particularly the expansion of T-andB-lymphocytes reactive against the idiotypic determinant IdLNF1, is key in the development of lu-pus nephritis in the autoimmune SNF1 mouse. When hepatic lymphocytes from DBF1 mice exposed to mercury in utero were stained for B-cells expressing IdLNF1 , we observed a significant increase in these populations in both male (p<0.01) and female (p<0.01) pups.

These preliminary studies show that prenatal exposure to mercury at 10ppm induces gender-specific phenotypic changes in the developing thymus and liver in a non-susceptible mouse.Whether these changes persist postnatally and into adulthood is the subject of continuing studies. More importantly, we shall conduct similar studies in an autoimmune mouse model and look at how mercury, in conjunction with other factors such as gender, time of exposure and genetic back-ground, might alter lymphocytic development and cytokine expression that predisposes towards ap-pearance of autoimmune disease.

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#39

The Infected Tissue Microenvironment Further Modifies CD4+ T Cell Effector Function

Shoshana Katzman and Dr. Deborah FowellDavid H. Smith Center for Vaccine Biology and Immunology

Department of Microbiology and ImmunologyUniversity of Rochester, Rochester, NY, United States

Effector function of activated CD4+ T cells is an integral component in determining the course of disease. Differentiation is thought to occur in the lymph node with subsequent departure of effector cells to find their cognate antigen. What happens at the tissue site of infection, where the pathogen resides, is less clear.

We use the murine model of Leishmania major infection to study T helper cell differentia-tion. To follow cytokine production at the single cell level, we use T cell receptor transgenic CD4+

T cells specific for an immunodominant epitope of the Leishmania major protein, LACK, in combi-nation with IL-4 reporter mice to be able to track antigen specific effector cytokine production. Sur-prisingly, within the draining lymph nodes (DLN) of susceptible Balb/c mice, single cell assays re-veal a heterogeneous population containing both IL-4, and, potentially anti- leishmaniacidal, IFNγproducing cells. Preliminary data indicate a narrowing of the cytokine repertoire in favor of non-pathogen clearing, IL-4 producing cells, at the tissue site of infection in comparison to the heteroge-neous DLN. These results suggest that the infected tissue modifies the type of effector response, ei-ther by selective homing of effector cells, or alternatively, further modification of effector function.Thus, the tissue site of infection may make an important contribution to disease susceptibility.

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#40

Wiskott-Aldrich Syndrome Protein (WASp) Controls Two Distinct Stages in CD4+ T Helper Cell Differentiation

Vanessa Morales-Tirado, Sara Johansson and Deborah FowellDavid H. Smith Center for Vaccine Biology and Immunology,

Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14624

Signals through the TCR can lead to the activation and differentiation of CD4+ T helper cells (Th) from an uncommitted CD4+ T cells to Th1 and Th2 effector cells. Each subtype has discrete cytokine profiles. The expression of distinct cytokines in turn determines the ability of the immune response to help in the clearance of pathogens and antibody production, respectively. Remodeling of the actin cytoskeleton has been suggested as an integral part for the sustaining of TCR signaling events after the formation of the immunological synapse. The Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of actin cytoskeleton, interacting with actin monomers and the Arp2/3 complex. We investigated the role of this protein during the delivery of signals from the TCR for T helper cell fate decisions by using WASp-deficient mice.

We have identified two distinct WASp-dependent control points: regulation of cytokine gene transcription and the control of cytokine secretion. The final secretion of both Th1 and Th2 cyto-kines is severely impaired in the absence of WASp. However, the two lineages require WASp at dif-ferent stages of differentiation. For Th2 development, we observe a transcriptional block in the ab-sence of WASp, with defects in signals for sustained IL-4 cytokine gene activation. In contrast, IFNg gene expression and protein synthesis proceeds normally in the absence of WASp. We previ-ously reported a striking defect in IFNg cytokine secretion. WASp-deficient cells contain similar levels of intracellular IFNg protein when compared to wild type cells but are unable to polarize cy-tokines to the immunological synapse for directed secretion. These results suggest that signals that determine Th differentiation are distinct from those, as yet poorly defined, signals for the induction of effector T cell function. Here, we analyze the Th2 signature cytokine IL-4. We observed a tran-scriptional defect for IL-4 by defects in signals for IL-4 cytokine gene activation when Real Time-PCR was performed. As a result, IL-4 cytokine was synthesized at a reduced frequency when com-pared to wild type C57Bl/6 mice, in addition to defects in cytokine secretion. These observations suggest different roles for WASp during different stages of CD4+ T cell development.

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#41

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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#42

Intrahepatic Presentation of Antigen Causes Proliferation of Functional Effector CD8 T-cells

Ingo Klein and I. Nicholas CrispeUniversity of Rochester School of Medicine and Dentistry, Rochester NY USA

Introduction: Several phenomena in liver immunobiology seem to favor tolerance induction over immunity and it has been hypothesized that this is mediated by antigen presentation in the liver. To investigate the effect of intrahepatic antigen presentation on activation and proliferation of CD8 T-cells, we used orthotopic mouse liver transplantation in order to restrict antigen presentationto the liver.

Methods: C57BL/6 livers were orthotopically transplanted into bm8 mice, that are unable to present SIINFEKL peptide on MHC Class I. OT-I cells were then adoptively transferred into trans-plant recipients that could only present the relevant antigen within the transplanted livers following SIINFEKL injection.

Results: OT-I cells encountering antigen only in the transplanted liver were activated, un-derwent extensive proliferation, and developed effector functions based on IFN? production. These results were confirmed by using [bm8->B6] bone marrow chimeras as liver donors, to eliminate the effect of passenger leukocytes from the graft.

Conclusions: The restricted presentation of antigen in the liver has no immunosuppressive effect on the activation of CD8 T-cells.

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#43

CD28 Costimulation tips the immuno-regulatory balance

Angela Hughson, Teresa L. Sukiennicki, Dorothy K. Sojka and Deborah J. FowellDavid H. Smith Center for Vaccine Biology and Immunology,

Department of Microbiology and Immunology, University of Rochester,Rochester, NY 14624

CD4+ CD25+ regulatory T cells (T regs) are emerging as key regulators of immune re-sponse. Although first described to regulate peripheral self-tolerance and to protect against autoim-munity, the CD25+ T regs have also been implicated in suppressing a number of other immune re-sponses including those to infectious agents, tumors and transplants. Given their broad suppressive action, it is unclear how the activity of T regs is regulated to ensure beneficial immune responses to infectious stimuli proceed unabated while other responses are suppressed. The up-regulation of B7 expression (CD80 and CD86) by antigen presenting cells (APC) represents a central event in the ac-tivation of naïve T cells to infectious agents and may serve as a mechanism to escape regulatory T cell tolerance.

Provision of CD28 costimulatory signals (anti-CD28 mAbs) abrogates suppressive activity in vitro. However it is not known if CD28 signaling mediates its anti-suppressive effects by directly abrogating T reg activity or by rendering effector T cells unresponsive to suppression. Here we re-strict CD28 signaling to either the T regs or responders and use a FACS-based suppression assay to follow proliferation of both CD4+CD25- responders and CD25+ T regs in co-culture. CD28 signals targeted to CD4+CD25+ T cells did not abrogate their suppressive activity. Rather, CD28 signaling targeted to the responders rendered them refractory to T reg suppression. Thus the regula tion of CD28 costimulation may play a key role in determining the ability of anti-pathogenic, and perhaps auto-reactive, T cells to evade T reg mediated suppression.

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#44

Regulation of Recall Responses by CD8+ Cells During an Intracellular Bacterial Infection

Constantine BitsaktsisWadsworth Center, Albany NY

Ixodus ovatus Ehrlichiae (IOE) has been shown to cause fatal monocytic ehrlichiosis in immuno-competent mouse strains. A Type 1 response is essential in resolving a low-dose infection. The in-volvement of CD4+ (but not CD8+) cells as well as antibodies is equally important. Immunization of C57Bl/6 mice with a sublethal dose of IOE failed to protect against a lethal IOE challenge while protection was achieved by using a heterologous bacterial strain (E. muris). Although primary re-sponses were sufficient to clear the sublethal (low-dose) IOE infection, secondary challenge with a low-dose inoculum proved to be fatal for immunocompetent mice. Our preliminary data have sug-gested that the latter effect was mediated by CD8+ cells, as CD8-deficient mice did not succumb to a secondary low-dose IOE challenge. Adoptive transfer of total CD8+ cells from sublethally IOE infected mice into naïve hosts, rendered the latter susceptible to a low dose IOE infection. We hy-pothesize that CD8+ T cells act directly or indirectly to induce fatal immunopathology in response to secondary IOE infection, or that the CD8+ cells interfere or block CD4-mediated secondary re-sponses. Our findings are suggesting a potentially novel mechanism regarding the regulation and outcome of an intracellular bacterial infection at the cellular level.

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#45

HSV-1 Amplicons as Immunization Vectors: Mechanism of Action andImplications for Tumor Immunotherapy

David Simon, Scott Gerber, Rick Willis, William Bowers Ph.D., Howard Federoff M.D. Ph.D., Edith Lord Ph.D., and John Frelinger Ph.D.

University of Rochester

Prostate cancer is a leading form of cancer among men, and while it has been shown that sur-gery and hormone treatment can increase survival in early stages, unfortunately, there is no effective treatment for metastatic disease. Specific immunotherapy is an attractive strategy for the treatment of metastatic disease, since immune cells are able to disseminate throughout the host and have the ability to directly lyse target cells. A number of viral vectors are currently being evaluated as a means to deliver tumor antigens and other immunomodulatory proteins, with the long-range goal of engendering effective anti-tumor responses.

HSV-1 amplicons, plasmid based viral vectors based on the HSV-1 virus but encoding no viral gene products, are attractive potential immunization vectors. Their broad host range specificity, large coding capacity, high expression levels of encoded transgene, and lack of expression of any viral protein, are attractive features for their use in immunization studies. The current work will fo-cus on the fundamental biology and use of amplicons as immunization vectors. These studies will yield insights into the mechanism by which amplicons engender immune responses and provide crit ical information as to how they might be enhanced, which will be essential for developing effec-tive tumor immunotherapy.

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#46

Chronic Irregular Foot-shock Induces Decreases in the Antibody Response to KLH and Depressive Changes in Behavior

Ling Cao and Jan A. MoynihanUniversity of Rochester, Rochester, NY

Both stress and depression have been associated with similar changes in immune responses and stress has been recognized as an important modulator in the development of depression. In this study, chronic irregular inescapable foot-shock (FS) induced antibody responses to KLH and behav-ioral changes were evaluated using adult male BALB/c mice. Mice subjected to FS were single housed in shock boxes. Foot-shock was delivered randomly 6 to 14 times per day (any period of time between 5 to 30 sec. at 0.5 mA) for 5 weeks. FS induced a significant loss of body weight within the first week of FS followed by a slow weight gain. FS mice showed significantly lower body weights than the home cage control mice (HC) throughout the experimental period (5 weeks).Basal serum corticosterone, IL-6 and TNFα were found to be significantly higher in the FS mice at week 3 compared to home cage control mice (HC), indicating chronic stress induced activation of the hypothalamus-pituitary-adrenal (HPA) axis and a pro-inflammatory response. When mice were immunized with KLH at week 3, significant decreases in antibody responses was detected 7 and 14 days later, including total serum IgM, IgG, IgG1 and IgG2a. However, in vitro KLH stimulation of splenocytes from HC or FS mice did not indicate reduced production of cytokines, including no changes in IFNγ, IL-2, IL-4 and IL-10 levels. Flow cytometric analysis of splenic leukocytes showed a slightly increased T cell population but significantly decreased B cell population in FS group 3 days after KLH injection, consistent with reduced antibody production. Furthermore, al-though a FS- induced decrease in chocolate milk consumption (a measure of anhedonia) was not found, significantly reduced marble burying behavior was observed in FS group. This suggests FS induced defensive behavior, a distinct type of anxiety, or provoked new object- induced anxiety. Al-together, chronic FS induced HPA axis activation, pro- inflammatory responses and certain behav-ioral changes in BALB/c mice that are associated with antibody responses to KLH.

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#47

SHP-1 Haploinsufficiency Mediated by LPS

Chad A. Hudson and Paul T. MassaSUNY Upstate Medical University, Syracuse, NY

The protein tyrosine phosphatase SHP-1 is known to play an important role in the regulation of im-munity and the loss of SHP-1 activity has numerous deleterious effects on innate immunity and host resistance in both the periphery and the central nervous system. Mice heterozygous for a null allele (me) at the SHP-1 locus (C3HFeJ me/+) have been shown to have reduced expression of SHP-1compared to wild type animals. To investigate if this reduction of SHP-1 yields a susceptibility to immune dysregulation, LPS from the rough strain Salmonella minnesota Re595 was employed as an immune stimulus both in vivo and in vitro. SHP null (me/me) mice are thought to have altered argi-nase/iNOS balance and here we show me/me mice have higher constituitive levels of arginase activ-ity in the brain and spleen. To measure the effects of heterozygosity in vivo, 200 µl of either saline or 250 µg/ml LPS was injected intraperitonealy into C3HFeJ me/+ and C3HFeJ +/+ mice twice a week for four weeks and then organs were harvested and measured for arginase activity. Both Me/+ and wild type LPS-treated mice had significantly higher arginase activity in the spleen than salinecontrols, but the LPS-treated me/+ were increased to a level significantly higher than LPS-treatedwild types. In the brain, LPS-treated me/+ showed significantly increased arginase activity not seen in wild type animals. The increase in me/+ brain arginase was mirrored by behavioral changes. Us-ing the light-dark box paradigm of anxiety/fear, it was found that LPS-treated me/+ mice showed significantly greater anxiety than both saline controls and wild type animals. Acute responsiveness to LPS was assessed via IL-1β production of me/+ and wild type splenocytes incubated for 24 hours with 0, 1, 5, 10 and 20 µg/ml of LPS. Two-way ANOVA showed heterozygote splenocytes had sig-nificantly higher levels of IL-1β production than wild type and at 5 µg/ml, p=0.001. In all, these findings show that there is a haploinsufficiency of SHP-1 in C3HFeJ me/+ that is produced via im-munostimulation and indicate that SHP-1 is a critical regulator of innate immunity and accompany-ing behavioral responses.

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NYIC 2004 Participants Contact Information

Elisaveta AdamovaAlbany Medical [email protected](518) 262-6972

Diana AlbuAlbany Medical [email protected](518) 262-6220

Isabel Alvarez de MoraSUNY [email protected](315) 464-7651

Sergio Arce, Ph.D.SUNY [email protected](716) 829-2748

Sarah AustinUniversity of [email protected](585) 259-2942

Shekar Bakshi, Ph.D.Albany Medical College [email protected](518) 262-8141

Naveen Bangia, Ph.D.Roswell Park Cancer [email protected](716) 845-8540

Shawn BaronAlbany Medical [email protected](518) 262-6750

Martin Baumgart, Ph.D.Cornell [email protected](607)253-3366

Daniel BeitingCornell [email protected](607) 256-5647

Dawn K. BellvilleAlbany Medical [email protected](518) 262-5365

Kiera BerggrenTrudeau [email protected](518) 891-3080

Allison BierlyCornell [email protected](610) 805-8328

Christine Biron, Ph.D.Brown [email protected](401) 863-9585

Constantine Bitsaktsis, Ph.D.Wadsworth Center [email protected](518) 473-2794

Gerald [email protected](518) 537-5200

Lori BroderickSUNY at [email protected](716) 845-8536

Deborah Brown, Ph.D.Trudeau [email protected](518) 891-3084

Barbara Butcher, Ph.D.Cornell [email protected](607) 253-3270

Byron Au-YeungUniversity of [email protected](585) 273-2902

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Ling Cao, Ph.D.University of [email protected](518) 859-5363

Chien-Chung ChangRoswell Park Cancer [email protected](716) 845-1755

Timothy ChapmanUniversity of [email protected](585) 273-1408

Karen Clise-Dwyer, Ph.D.Trudeau [email protected](518) 891-3080

Christopher CollinsAlbany Medical [email protected](518) 262-6962

Lisa DaleyCornell [email protected](607) 256-5647

James Drake, Ph.D.Albany Medical [email protected](518) 262-9337

Karen Duus, Ph.D.Albany Medical [email protected](518) 262-1176

David DwyerTrudeau [email protected](518) 891-3080

Sheri EatonTrudeau [email protected](518) 891-3080

Gary EdwardsSUNY at [email protected](518) 402-4081

Kenneth Ely, Ph.D.Trudeau [email protected](518) 891-3080

Rebecca EmenyWadsworth Center [email protected](518) 474-6509

Aracelis Fernandez, M.D.Albany Medical [email protected](518) 262-5332

Soldano Ferrone, Ph.D.Roswell Park Cancer [email protected](716) 845-8534

Deborah Fowell, Ph.D.University of [email protected](585) 273-3600

Jerrie Gavalchin, Ph.D.SUNY [email protected](607) 279-8179

Scott GerberUniversity of [email protected](585) 275-6747

Edmund Gosselin, Ph.D.Albany Medical [email protected](518) 262-5562

Karen Hale, Ph.D.Wadsworth [email protected](518) 473-2794

Laura Haynes, Ph.D.Trudeau [email protected](518) 891-3080

Matthias Hesse, Ph.D.Cornell [email protected](607) 253-3389

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Romana Hochreiter, Ph.D.SUNY [email protected](315) 464-5466

Chad HudsonSUNY [email protected](518) 859-5364

Angie HughsonUniversity of [email protected](585) 273-3681

Dawn Jelley-Gibbs, Ph.D.Trudeau [email protected](518) 891-3080

Edith KabinguRoswell Park Cancer [email protected](716) 845-8836

Carolyn KalinkaErie County Medical Center [email protected](716) 898-4138

Cris Kamperschroer, Ph.D.Trudeau [email protected](518) 891-3080

Shoshana KatzmanUniversity of [email protected](585) 273-2902

Shabaana A. Khader, Ph.D.Trudeau [email protected](518) 891-3080

Rhonda KinesRoswell Park Cancer [email protected](716) 881-1564

Ingo Klein, Ph.D.University of [email protected](585) 507-6662

Philaretos KousisRoswell Park Cancer [email protected](716) 845-8167

Danuta Kozbor, Ph.D.Roswell Park Cancer [email protected](716) 845-8668

David Lawrence, Ph.D.Wadsworth [email protected](518) 402-5684

Leo Lefrancois, Ph.D.University of [email protected](860) 679-3242

Michelle Lennartz, Ph.D.Albany Medical [email protected](518) 262-5217

Yili LinAlbany Medical [email protected](518) 262-5174

Alexandra Livingstone, Ph.D.University of [email protected](585) 275-9407

Edith Lord, Ph.D.University of [email protected](585) 275-5855

Steve LotzAlbany Medical [email protected](518) 262-5174

Nanacy LuckashenakRoswell Park Cancer [email protected](715) 845-1384

Frances Lund, Ph.D.Trudeau [email protected](518) 891-3080

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Meenakshi Malik, Ph.D.Albany Medical [email protected](518) 262-8141

Nicholas Mantis, Ph.D.Wadsworth [email protected](518) 402-2750Paul Massa, Ph.D.SUNY [email protected](315) 464-7606

Katrin MayerTrudeau [email protected](518) 891-3080

Dana MeentsTrudeau [email protected](518) 891-3080

J. Andrés Melendez, Ph.D.Albany Medical [email protected](518) 262-8791

Dennis W. Metzger, Ph.D.Albany Medical [email protected](518) 262-6750

Katja MohrsTrudeau [email protected](518) 891-3084

Lupita Montoya, Ph.D.Rensselaer Polytechnic [email protected](518) 276-2532

Vanessa Morales-TiradoUniversity of [email protected](585) 273-2902

Tim Mosmann, Ph.D.University of [email protected](585) 275-9120

Isis Mullarky, Ph.D.Trudeau [email protected](518) 891-3080

Erica McGovernAlbany Medical [email protected](518) 262-9338Monika [email protected](908) 231-3304

Sofia Olmos, Ph.D.Albany Medical [email protected](518) 262-6220

Fernando OntiverosUniversity of [email protected](585) 273-1437

Nina Pabello, Ph.D.Wadsworth [email protected](518) 262-474-5057

Heather PageAlbany Medical [email protected](518) 262-8141

Michelle Parent, Ph.D.Trudeau [email protected](518) 891-3080

Karsten PilonesSUNY [email protected](315) 416-1209

Noelle PolakosUniversity of [email protected](585) 273-1408

Tim Powell, Ph.D.Trudeau [email protected](518) 891-3080

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Troy Randall, Ph.D.Trudeau [email protected](518) 891-3080

Rosemary Rochford, Ph.D.SUNY [email protected](315) 646-5468Michael Russell, Ph.D.SUNY at [email protected](716) 829-2790

Albert Sabirov, Ph.D.Albany Medical [email protected](518) 262-6220

Kaori SakamotoCornell [email protected](607) 253-3390

Allen Silverstone, Ph.D.SUNY [email protected](315) 464-5871

David SimonUniversity of [email protected](585) 275-6318

Steve Smiley, Ph.D.Trudeau [email protected](518) 891-3080

Abigail [email protected](888) Taconic; (888) 822-6642

Dorothy SojkaUniversity of [email protected](585) 273-2902

Alejandra Solache, Ph.D.Trudeau [email protected](518) 891-3080

Keer SunAlbany Medical [email protected](518) 262-6220

Frank SzabaTrudeau [email protected](518) 891-3080Steven Taffet, Ph.D.SUNY [email protected](315) 464-5419

Yasmin Thanavala, Ph.D.Roswell Park Cancer [email protected](716) 845-8536

Seena ThrasherCornell [email protected](607) 256-5679

David Topham, Ph.D.University of [email protected](585) 273-1403

Vy Thao TranRoswell Park Cancer [email protected](no phone number provided)

Justin WilsonAlbany Medical [email protected](518) 262-9338

Gary Winslow, Ph.D.Wadsworth [email protected](518) 482-5430

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The Sagamore Resort and Conference Center

Dinners and Keynote Speakers, as well as breakfast, will be in the Sagamore Dining Room in the main hotel.

All Sessions will be held in the Bellvue room at the Conference Center.

The Poster Session/Vendor Fair will be held in Nirvana/Wapanak in the Conference Center.

Maps are available in the main hotel.

Should you have any special needs, please see Dawn Bellville.

Special Thanks to the Sagamore for hosting this event!


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