8th Annual Upstate New York

Immunology Conference

Major Funding Provided by:

Taconic

BD Biosciences

Invitrogen

National Institutes of Allergy and Infectious Diseases

October 9-12, 2005 The Sagamore

Bolton Landing, New York

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Table of Contents Letter of Welcome ...............................................................4 Schedule of Events ..............................................................5 Institutional Support ........................................................11 Grant Support ...................................................................12 Special Thanks ..................................................................13 Major Corporate Benefactors ............................................15 Major Corporate Sponsors .................................................19 Corporate Supporters ........................................................25 Corporate Contributors .....................................................33 Sunday Keynote Speaker ..................................................35 Presentation Abstracts (Sessions I-III) ............................37 Monday Keynote Speaker ..................................................53 Presentation Abstracts (Sessions IV-VIII) ........................55 Poster Abstracts ................................................................83 Contact Information .......................................................121 The Sagamore .................................................................127 Conference Survey ..........................................................129

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8th Annual Upstate New York Immunology Conference

“Welcome Participants”

I t is with great pleasure that we welcome you to the 8th Annual Upstate New York Immunol-ogy Conference (NYIC). We hope you have all had a chance to view the NYIC website

(www.amc.edu/NYIC) which features an improved design and new information. As a result of last year’s survey, we have extended the meeting by a full day in the hopes of al-lowing for more postdoctoral and graduate student presentations and more informal gatherings among participants. We have also taken the opportunity to invite other institutions to participate in presenting and exhibiting posters. The Conference kicks off Sunday with dinner and a Keynote Speaker. Following the Keynote, there will be a cocktail reception held on the Mountain View Terrace, just outside of the Confer-ence Center. Monday afternoon from 3-6 p.m. there will be a Poster Session/Vendor Fair. Odd number posters will be viewed from 3:00-4:30 p.m. and even number posters will be viewed from 4:30-6:00 p.m. On Tuesday afternoon there will be a break from formal presentation sessions. Don’t forget you must be pre-registered for the cruise. Please check with the Conference Coordinator, Dawn Bellville, to see if there is still space available. (See the Schedule of Events for more informa-tion.) Attendees may choose to schedule activities (tennis, golf, spa, etc.) that are available at The Sa-gamore. These activities are not part of your conference package. You will be charged by The Sagamore according to their normal rates. If you are interested in golfing or tennis, it is advis-able to pre-book directly with The Sagamore. Sessions resume on Tuesday evening following dinner. We hope that you enjoy your stay at The Sagamore. But even more importantly, we hope that you find the scientific content of this year’s meeting interesting and informative and that you have ample opportunities to collaborate with fellow scientists. It is the desire of the Organizing Committee to make this meeting one of the very best. Your in-put is important to us. Please be sure to complete your conference survey and hand them in to Dawn Bellville during the Wednesday morning break. Sincerely,

Dennis W. Metzger, Ph.D. Chair, NYIC Scientific Board

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2005 Upstate New York Immunology Conference Schedule of Events

Sunday, October 9th 4:00-5:30 p.m. Hotel Registration (Registration Building) 4:00-5:30 p.m. Conference Registration (Conference Center Foyer) 5:30-7:00 p.m. Dinner (Sagamore Ballroom) 7:00-8:00 p.m. Keynote Speaker

Introduction by: Dr. James Drake (Albany Medical College)

Jeffrey Frelinger, Ph.D. Kenan Professor and Chair

Department of Microbiology and Immunology University of North Carolina

T-cell Activation is Regulated by Affinity and not Kinetics of

TRC/pMHC Interaction 8:00-9:30 p.m. Welcome Cocktail Reception (Mountain View Terrace)

Sponsored by Taconic Monday, October 10th 7:00-8:00 a.m. Breakfast (Sagamore Dining Room) 8:00-8:30 a.m. Poster Set-up (Nirvana/Wapanak) 8:30-10:15 a.m. Session I: Immune Mechanisms (Bellvue) Session Chair: Yasmin Thanavala, Ph.D. (Roswell Park Cancer Institute)

8:30-8:50 Sharon S. Evans, Ph.D. (Roswell Park Cancer Institute) Central Role of IL-6 in Directing Lymphocyte Trafficking During Fever-Range Thermal Stress

8:50-9:10 James D. Gorham, M.D., Ph.D. (Dartmouth Medical School)

TGF-beta 1 Inhibits IFN-gamma in T Helper Cells Through Novel Pathways 9:10-9:30 Beena John, Ph.D. (University of Rochester)

The Liver Modulates Systemic CD8+ T cell Memory Through a TLR-4 Dependent Mechanism

9:30-9:50 Brahm H. Segal, M.D. (Roswell Park Cancer Institute) Aspergillius fumigatus Extract Differentially Regulates Antigen Specific CD4+ and CD8+ T cell Responses to Promote Tumor Immunity

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Monday, October 10th (cont’d) 9:50-10:10 TACONIC PRESENTATION “TBA” 10:15-10:45 a.m. Break (Conference Center Foyer) 10:45-12:30 p.m. Session II: Cellular & Molecular Immunology I. (Bellvue) Session Chair: Rosemary Rochford, Ph.D. (SUNY Upstate) 10:45-11:05 Susan Swain, Ph.D. (Trudeau Institute)

Ag Presentation and Regulation of CD4 Memory Generation to Influenza

11:05-11:25 Richard Konz, Ph.D. (University of Massachusetts) High Content Polychromatic Flow Cytometry and its Impact on Immunology Research

11:25-11:45 Adriana Caballero, M.S. (Albany Medical College)

Functional Requirements for BCR-mediated Ligand Internalization and Signaling

11:45-12:05 James L. Clements, Ph.D. (Roswell Park Cancer Institute)

The SLP-76 Adaptor Protein Links Integrin Ligation with p44/42 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells

12:05-12:25 Susan Reynolds (BD Biosciences) Pharmaceutical & Technical Applications Specialist

Cellular Systems Biology: Applications for Analysis of Protein Phosphorylation and Cellular Signaling Events

12:30-1:30 p.m. Lunch (Sagamore Dining Room) 1:30-3:00 p.m. Session III: Emerging & Re-Emerging Diseases (Bellvue) Session Chair: Jeffrey Frelinger, Ph.D. (University of North Carolina) 1:30-1:50 Gary Winslow, Ph.D. (Wadsworth Center)

Chemokine-dependent Memory CD8 T cell-Mediated Pathology During Ehrlichia Infection

1:50-2:10 Michelle A. Parent, Ph.D. (Trudeau Institute) Cell-mediated Protection Against Pulmonary Yersinia pestis Infection

2:10-2:30 Timothy Sellati, Ph.D. (Albany Medical College)

Pattern Recognition and the Fate of Francisella tularensis in a Mouse Model of Tularemia

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Monday, October 10th (cont’d) 2:30-2:50 Kelly Lundsten (Invitrogen) Molecular Probes Technology Specialist Indicators for Apoptosis 3:00-6:00 p.m. Poster Session/Vendor Fair/Mixer (Nirvana/Wapanak) (Viewing of Odd Number Posters, 3:00-4:30 p.m.; Even Number Posters, 4:30-6:00 p.m.) 6:00-7:30 p.m. Dinner (Sagamore Dining Room) 7:30-8:30 p.m. Keynote Speaker

Introduction by: Dr. Edmund Gosselin (Albany Medical College)

Dennis L. Kasper, M.D. William Ellery Channing Professor of Medicine

Professor of Microbiology and Molecular Genetics Director, Channing Laboratory

Harvard Medical School

A Glimpse Into the Molecular Basis for Symbiosis Between the Intestinal Microflora and the Mammalian Immune System

Tuesday, October 11th 7:00-8:30 a.m. Breakfast (Sagamore Dining Room) 8:30-10:15 a.m. Session IV: Mucosal Immunity (Bellvue) Session Chair: Dennis W. Metzger, Ph.D. (Albany Medical College) 8:30-8:50 David L. Woodland, Ph.D. (Trudeau Institute) Memory CD8+ T cell Responses in the Lung

8:50-9:10 Hesham Nawar, M.S. (SUNY at Buffalo)

Mutants of Enterotoxins LT-IIa and LT-IIb with Altered Ganglioside-binding Activities and Diminished Toxicity are Potent Mucosal Adjuvants

9:10-9:30 Aracelis D. Fernandez, M.D. (Albany Medical College) Effect of Azithromycin on a Murine Model of Allergic Lung Inflammation 9:30-9:50 Seana Thrasher, M.S. (Cornell University)

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

9:50-10:10 Cris Kamperschroer, Ph.D. (Trudeau Institute)

SAP is Required for Initial Expansion of B Cells and Plasma Cells and for Control of Influenza Virus Upon High-dose Rechallenge

10:15-10:45 a.m. Break

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Tuesday, October 11th (cont’d) 10:45-12:30 p.m. Session V: Immune Regulation (Bellvue) Session Chair: David Woodland, Ph.D. (Trudeau Institute) 10:45-11:05 David A. Lawrence, Ph.D. (Wadsworth Center) Antibody Induction of Lupus- like Neuropsychiatric Manifestations 11:05-11:25 Kristin Zaffuto, M.S. (University of Connecticut)

The Effect of Metallothionein Gene Dose on Oxidative Stress and Signal Transduction

11:25-11:45 Matthew M. Seavey, M.Sc. (University of Rochester)

Presence of Cells Presenting Paternal Antigen in the Uterus After Mating Does Not Result in the Stimulation of Anti-paternal CD8+ T cells in vivo

11:45-12:05 Monisha Sundarrajan, Ph.D. (La Jolla Institute) IL-12 and IL-18 are Key Cytokines Involved in Differentiation of CD8+ T cells 12:05-12:25 Michael R. Nazareth, B.S. (University at Buffalo) Characterization of Fibroblasts in Human Lung Tumor Microenviron- ments: Cytokine Production, Co-regulatory Molecule Expression, and Role in the IL-12 Induced, IFN-gamma Mediated Anti-tumor Response 12:30-1:00 p.m. Lunch (Conference Center Foyer) (Pick up box lunches; Reservations for recreational activities are recommended and are at the discretion of participants – activity fees will be applied to your credit card.) 12:30-6:00 p.m. Free time 3:00-5:00 p.m. Boat Cruise on Lake George aboard The Morgan

Refreshments provided by Invitrogen (Pre-registered participants only; please be at the dock by 2:45 p.m.)

6:00-7:00 p.m. Dinner (Sagamore Dining Room) 7:00-9:00 p.m. Session VI: Cellular & Molecular Immunology II. Session Chair: David Topham, Ph.D. (University of Rochester) 7:00-7:20 David Holowka, Ph.D. (Cornell University)

New Insights into Signaling via IgE Receptors 7:20-7:40 Sergio Arce, M.D., Ph.D. (University at Buffalo)

Differential Binding of Escherichia coli Exterotoxins LT-Iia and LT-Iib and of Cholera Toxin Elicits Differences in Apoptosis, Proliferation, and Activation of Lymphoid Cells

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Tuesday, October 11th (cont’d) 7:40-8:00 Loren D. Erickson, Ph.D. (University of Virginia)

BAFF: A Dr. Jekyll & Mr. Hyde in Controlling Plasma Cell Fate

8:00-8:20 Jeff Ward, B.S. (SUNY Upstate) Regulation of CD4 Recycling by the Ras-GTPase Rab4a

8:20-8:40 Chad A. Hudson, B.S. (SUNY Upstate) The Regulation of Th2 Cytokine Production by SHP-1 8:40-9:00 Darryn Unfricht, M.S. (University of Connecticut) Grating-Coupled Surface Plasmon Resonance: A Cell and Protein Microarray Platform

Wednesday, October 12th 7:00-8:00 a.m. Breakfast (Sagamore Dining Room) 8:00-10:00 a.m. Session VII: Vaccines & Adaptive Immunity (Bellvue) Session Chair: Michael Russell, Ph.D. (University at Buffalo) 8:00-8:20 Rachel Gerstein, Ph.D. (University of M assachusetts)

Birth of a B cell: Molecular Circuits in Early B cell Development 8:20-8:50 Jeffrey H. Mills, B.S. (SUNY Upstate) Estrogen, Estrogen Receptors, and Thymocyte Development 8:50-9:10 Andras Perl, Ph.D. (SUNY Upstate)

Mitochondrial Hyperpolarization: a Checkpoint of T cell Life, Death and Autoimmunity

9:10-9:30 Adrienne Kisailus, B.S. (Roswell Park Cancer Institute) Thermal Regulation of Antigen Presenting Cells: Use of Heat as an Adjuvant for Cancer Vaccines 9:30-9:50 Amit Lugade, B.S. (University of Rochester)

Localized Tumor Irradiation Enhances Anti-tumor Immunity

10:00-10:45 a.m. Break (Conference Center Foyer) and hotel checkout

10:45-12:15 p.m. Session VIII: Microbial Pathogenesis (Bellvue) Session Chair: Gary Winslow, Ph.D. (Wadsworth Center) 10:45-11:05 Barbara A. Butcher, Ph.D. (Cornell University)

Manipulation of Macrophage Signaling During Intracellular Infection with the Protozoan Pathogen Toxoplasma gondii

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Wednesday, October 12th (cont’d) 11:05-11:25 Justin E. Wilson, B.S. (Albany Medical College) Effect of F. tularensis Invasion on the Cell Biology of Antigen Processing and Presentation by Macrophages 11:25-11:45 Romana Hochreiter, Ph.D. (SUNY Upstate)

Role of Dendritic Cells in Early Events of Gamma-herpesvirus Pathogenesis

11:45-12:05 Byron Au-Yeung, M.S. (University of Rochester) Distinct Itk-dependent Signals Required for the Release, But Not Gain, of Th2 Effector Function 12:05-12:25 Eric Yager, Ph.D. (Trudeau Institute) Effects of Aging on the CD8 T cell Response to Primary Influenza Infection 12:30-1:30 p.m. Lunch ( Sagamore Dining Room)

Depart from Conference

Thank you for participating in this year’s NYIC meeting!

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Financial Support Provided by the Following Institutions

Albany Medical College Alumni Association

Cornell University and Ve terinary College

Department of Microbiology & Immunology

Rowell Park Cancer Institute Department of Immunology

SUNY Upstate Medical University

Microbiology & Immunology Program

University at Buffalo Department of Immunology & Microbiology

University of Rochester Medical Center

Department of Microbiology & Immunology

Trudeau Institute

Wadsworth Center

Thank you for your continued support!

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Thank you to the

National Institute of Allergy and Infectious Diseases For continuing grant support to

Graduate Students and

Postdoctoral Fellows

1R13AI051522-01A1

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8th Annual Upstate New York Immunology Conference would like to take this opportunity to give

“special thanks” to:

Dr. Jim Drake and Dr. Ed Gosselin for serving as Conference Co-organizers. Dr. Dennis W. Metzger for obtaining continued financial support from NIAID and Corporate Sponsors, which help keep the meeting affordable for all. Dawn Bellville for managing all of the innumerable and multi-faceted components necessary to produce a well-organized meeting. Pat Jarrett and Lori Rehm of The Sagamore for all of their assistance, input and cooperation in accommodating the needs of the conference and its participants. Institutional Representatives for obtaining presenters from their institution, requesting finan-cial support and for promoting attendance: James Drake, Ph.D. & Ed Gosselin, Ph.D. Albany Medical College Laura Haynes, Ph.D. Trudeau Institute Matthias Hesse, Ph.D. & Jerrie Gavalchin, Ph.D. Cornell University Edith Lord, Ph.D. University of Rochester Michael Russell, Ph.D. University at Buffalo Allen Silverstone, Ph.D. & Rosemary Rochford, Ph.D. SUNY Upstate Yasmin Thanavala, Ph.D. Roswell Park Cancer Institute Gary Winslow, Ph.D. Wadsworth Center Finally, to all present and past participants, too many to name here, whose attendance, feed-back and ideas are vital to the success and growth of this annual Conference.

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Keynote Speaker

Sunday, October 9, 2005

Introduction by: Dr. James Drake

Jeffrey Frelinger, Ph.D. Kenan Professor and Chair

Department of Microbiology and Immunology University of North Carolina

“T-cell Activation is Regulated by Affinity and not Kinetics of

TRC/pMHC Interaction”

Specific recognition of antigenic pMHC complexes by TCR initiates T cell activation. In spite of long interest in the relationship between the biochemistry and subsequent immune responses, the issue of what among the physical parameters of molecular association controls T cell activity remains unresolved. Three models have been proposed to explain the relationship of TCR engagement with T cell activation. First is affinity threshold, when the affinity and hence occupancy reaches a critical amount the T cell responds. The second is posits an optimum affinity, too high and less signally occurs (or tolerance) too low and not enough occurs. The third requires that the off rate is the critical variable as the time of occupancy for each receptor is the critical variable in whether activation occurs. Each of the three major models of T cell activation make different predictions about the shape of the affinity (or off-rate) versus activity curve. The kinetic model predicts a predictable relationship with off-rate. There slow off rates should result in positive signaling, while a fast off-rate should result in negative signaling. We attempted to correlate off rates with activity; we found no significant correlation with either linear or non- linear curve fits. Indeed the peptide (K1SC9M) with the slowest off-rate, had the lowest activity, the opposite of the model’s prediction. The optimal engagement model predicts a Gaussian distribution of affinity versus activity. We cannot fit a Gaussian distribution to the data. Our data correlates best with a Boltzmann sigmoidal dose-response relation (R2 = 0.95), not a linear relationship or Gaussian distribution. In contrast, no correlation was observed between cytotoxicity and any kinetic parameter of TCR-pMHC interaction. Forcing the koff data to fit a linear regression resulted in an R2 of only .28. We were unable to get any solution to converge in the best- fit program for non-linear regressions. Our data strongly argues for an affinity threshold model of T cell activation.

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Presentation Abstracts

(Sessions I-III)

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Session I.

Central Role of IL-6 in Directing Lymphocyte Trafficking During Fever-Range Thermal Stress

Qing Chen, Kristen Clancy, Wan-Chao Wang, and Sharon S. Evans

Department of Immunology, Roswell Park Cancer Institute, Buffalo NY 14263

A longstanding question in immunology resolves around the physiological benefit of the evolutionarily conserved fever response. Fever has been correlated with improved survival during infection in endothermic and ectothermic vertebrate species despite the fact that this physiologic response occurs at great metabolic cost. Here, we report evidence for a proactive role of fever-range thermal stress in regulating multiple adhesive mechanisms that culminate in enhanced lymphocyte extravasation across specialized high endothelial venules (HEV) of lymphoid organs. Thermal stress acts directly on lymphocytes to stimulate the binding activity of two lymphocyte homing receptors, L-selectin and a4ß7 integrin. These homing molecules are responsible for mediating the initial tethering and rolling of lymphocytes along HEV in lymph nodes and Peyer’s patches. Thermal stress also induces the vascular display of endothelial adhesion molecules (ICAM-1) and chemokines (CCL21) that trigger firm arrest and transendothelial migration in HEV. Notably, thermal activation of endothelial adhesion is tightly regulated with respect to the type of vessels involved. Sus tained exposure to fever-range thermal stress selectively targets adhesion in HEV while squamous, non-activated endothelial cells of extralymphoid organs are non-responsive. A central role for the proinflammatory cytokine, IL-6, in controlling trafficking across HEV was revealed by findings that thermal stimulation of both L-selectin and ICAM-1-dependent trafficking involves a trans-signaling mechanism whereby the gp130 signal transduction molecule is engaged by a complex of IL-6 and a soluble form of the IL-6 receptor a subunit. These observations support the concept that HEV act as sentinels during febrile inflammatory responses by heightening IL-6-dependent delivery of naïve and central memory lymphocytes to secondary lymphoid organs. Supported by NIH grants CA79765, CA094045, and DOD grant W81XWH-04-1-0354.

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Session I.

TGF-beta 1 Inhibits IFN-gamma in T Helper Cells Through Novel Pathways

James D. Gorham, M.D., Ph.D. Dartmouth Medical School

TGF-β1 prevents the development of autoimmune disease by restraining the development of autoreactive Th1 cells.1;2 We have recently shown that TGF-β1 inhibits Th1 development in part by suppressing the expression of T-bet, an IFN-γ-induced transcription factor that promotes Th1 differentiation.3 To gain further insight into this regulatory pathway, we examined the interaction of IFN-γ and TGF-β1 in the regulation of T-bet and of IRF-1, two transcription factors required for Th1 development. In murine CD4+ T cells, IFN-γ rapidly induced expression of both T-bet and IRF-1. TGF-β1 antagonized the effects of IFN-γ, inhibiting IFN-γ’s induction of both Th1 transcription factors. These effects of TGF-β1 required new protein synthesis; accordingly, we looked at known inhibitors of the IFN-γ-Jak-Stat pathway for candidates induced by TGF-β1 that could mediate effects of TGF-β1. We found that, in the presence of IFN-γ, TGF-β1 rapidly induced in T helper cells the synthesis of the protein tyrosine phosphatase (PTP) Shp-1, but did not induce the related family member Shp-2, or or several members of the SOCS family of Jak-Stat inhibitors. Using T cells from the Shp-1-deficient mev/mev mouse strain, we demonstrated that Shp-1 is required for TGF-β1’s suppressive effects: TGF-β1’s suppression of T-bet and IRF-1 was completely abrogated in mev/mev CD4+ T cells. In addition, receptor-proximal responses to IFN-γ, such as the induction of Jak-Stat phosphorylation, were inhibited by TGF-β1 in wild-type T cells, but not in mev/mev T cells. Consistent with a direct role for Shp-1, TGF-β1’s inhibition of IFN-γ-induced Stat1 phosphorylation was sensitive to the pharmacologic PTP inhibitor pervanadate. Together, these data show that TGF-β1 suppresses IFN-γ signaling and transcriptional responses in CD4+ T cells through the PTP Shp-1. This work defines a novel pathway of cytokine cross regulation in CD4+ T cells, and identifies Shp-1 as a key player in the antagonistic interactions between TGF-β1 and IFN-γ. 1. Gorham JD, Lin JT, Sung JL, Rudner, LA, French MA: Genetic regulation of autoimmune

disease: BALB/c background TGF-β1-deficient mice develop necroinflammatory IFN-γ-dependent hepatitis. J Immunol 2001; 166:6413-6422.

2. Rudner LA/Lin JT*, Park I-K, Cates JMM, Dyer D, Franz DM, French MA, Duncan EM, White HD, Gorham JD: Necroinflammatory liver disease in BALB/c-background TGF-β1 deficient mice requires CD4+ T cells. J Immunol 2003; 170:4785-4792. (*Shared first authorship).

3. Lin JT, Martin SL, Xia L, Gorham JD: TGF-β1 uses distinct mechanisms to inhibit IFN-γ expression in CD4+ T cells at priming and at recall: differential involvement of Stat4 and T-bet. J Immunol 2005; 174:5950-5958.

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Session I.

The Liver Modulates Systemic CD8+ T cell Memory Through a TLR-4 Dependent Mechanism

Beena John, Ingo Klein and I. Nicholas Crispe.

The David H Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester NY 14642, USA

The liver is a site for accumulation and destruction of activated CD8+ T cells, and we have shown that TLR-4 regulates the trapping of activated CD8+ T cells in the liver. As a consequence of such reduced intrahepatic CD8+ T cell trapping, there was an increase in the circulating primed T cell pool and in the subsequent CD8+ T cell memory response. The frequency of memory CD8+ T cell precursors in the bone marrow, spleen, lymph nodes and liver was higher in the TLR-4 deficient mice compared to the wt mice 6 weeks after primary immunization. Upon restimulation the recall responses in the TLR-4 deficient mice were higher than that seen in the wt mice. There was however no qualitative difference in the memory cells generated in the wt or TLR-4 deficient mice. The expression of activation markers, the ability to produce effector cytokines such as IFN-ß in vitro, and the ability to lyse specific target cells in vivo, was comparable between the two groups. The memory cells generated in the wt and the TLR-4 deficient mice, when retransferred in equal numbers into wt hosts expand similarly. Thus the primary effect of the TLR-4 deficiency is in reducing the trapping and destruction of activated CD8+ T cells in the liver, which in turn contributes to an increase in the percentage of the circulating memory precursors. We also know that the primary site of the TLR-4 effect is the liver, since mice that are transplanted with TLR-4 deficient livers show the same phenotype as that seen in intact TLR-4 deficient mice.

These results reveal a new function of TLR-4, and show that the liver has an important role in regulating the magnitude of T cell responses and in the establishment of T cell memory.

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Session I.

Aspergillus fumigatus Extract Differentially Regulates Antigen Specific CD4+ and CD8+ T cell Responses to Promote Tumor Immunity

Jianming Tao1, Cheryl Eppolito1, Carly G. Dennis2 , Richard Youn2,

Brahm H. Segal1,2, and Protul A. Shrikant 1 1Department of Immunology and 2Medicine, Roswell Park Cancer Institute, Buffalo, NY

Aspergillus species are filamentous fungi (moulds) that are ubiquitous in the environment. Aspergillus infection is a major cause of morbidity and mortality in highly immunocompromised patients. Aspergillus species produce a variety of proteases, reactive oxygen scavengers, phospholipases, and hemolysins that mediate penetration of the lung epithelial barrier, nutrient acquisition, and evasion of host defenses. Gliotoxin, a metabolite produced by A. fumigatus and other fungi, has broad immunosuppressive properties against innate and antigen-specific immunity that may facilitate evasion of host defense. A. fumigatus may also evade immune recognition during germination through loss of toll- like receptor-4-mediated signal transduction. These studies suggest that A. fumigatus produces immunosuppressive products and that sensitization with an A. fumigatus (Af) extract may suppress antigen specific cellular immunity. Most of the major Aspergillus antigens are constituents of the extract as determined by western blotting using sera from patients with allergic bronchopulmonary aspergillosis. One limitation in characterizing Aspergillus antigen responses in vivo at the single cell level relates to the low number of antigen specific responder T-cells. To overcome this limitation and better understand the impact of Aspergillus associated virulence factors on host adaptive immune responses, we have used the adoptive transfer approach in which OVA peptide specific T cell receptor (TCR) transgenic OVAp-specific class-I-restricted CD8 + T cells (OT-1) and OVAp-specific class II-restricted CD4+ T cells (OT-II) are monitored during a progressive T cell response to antigen sensitization in vivo. We predicted that sensitization with a nominal OVA antigen with Af extract would suppress OVA-specific CD4+ and CD8+ T cell responses. Instead, Af extract was a potent inducer of OVA antigen specific T cell activation, proliferation and differentiation and augmented cytotoxic T lymphocyte activity in vivo. Sensitization with Af extract plus OVA enhanced CD4+ and CD8+ T cell early activation and subsequent clonal expansion primarily due to increased proliferation. The T cell expansion produced by Af extract was accompanied by differentiation into interferon (IFN)-g producing cytotoxic OT-I T cells, but no effector differentiation of the OT-II T cells occurred. The induced T cell responses were transient, as by day 10, both the OT-I and OT-II T cell numbers underwent considerable attrition. Consistent with these observations, adoptive transfer of OT-I T cells followed by sensitization with Af plus OVA was more effective than adoptive transfer and sensitization with OVA alone in controlling growth of an OVA-expressing thymoma (E.G7). Sensitization with Af extract and OVA twice led to greater control of tumor than a single sensitization. However, the Af extract only promoted type 1 effector differentiation of CD8+ T cells and the induced T cell responses were not durable. Our findings demonstrate the regulatory properties exerted by A. fumigatus associated factors on host T cell responses and have important implications in vaccine development for aspergillosis as well as harnessing the Af extract as an immunological adjuvant against tumors.

43

Session I.

Taconic Presentation

“TBA”

44

Session II.

Ag Presentation and Regulation of CD4 Memory Generation to Influenza

Susan Swain, Ph.D. Trudeau Institute, Saranac Lake, NY

“NO ABSTRACT PROVIDED”

45

Session II.

High Content Polychromatic Flow Cytometry and its Impact on Immunology Research

Richard Konz, Ph.D. University of Massachusetts

“NO ABSTRACT PROVIDED”

46

Session II.

Functional Requirements for BCR-mediated Ligand Internalization and Signaling

Adriana Caballero, Xiao-Yun Wen, Toufic O. Nashar and James R. Drake Center for Immunology and Microbial Disease Albany Medical College, Albany, NY 12208

Translocation of the B cell receptor (BCR) to membrane microdomains called lipid rafts is widely regarded as a decisive step in BCR signaling. While ligand binding is known to result in the partitioning of the BCR into lipid rafts, the exact level of raft partitioning and the effect of the ligand on this phenomenon are poorly understood. Equally conflicting reports have arisen when trying to address the functional consequences of raft manipulation or disruption on the internalization of different BCR-ligand complexes. We have addressed these questions by comparing two well-characterized BCR ligands, haptenated antigen (Ag; PC-BSA) and polyclonal anti-BCR antibody (Ab; anti-IgM). The results show that the nature of the ligand determines the level of association of ligand-BCR complexes with lipid rafts, such that Ab promotes high association of BCR with rafts compared to the Ag. Partitioning of BCR into lipid rafts is directly correlated with the signaling capacity of the ligand and inversely correlated with the ability of the ligand-BCR complexes to be internalized; suggesting that an increased association of BCR with lipid rafts favors signaling and slows the overall kinetics of BCR internalization. In agreement with this hypothesis, the internalization of Anti-IgM-BCR complexes is highly sensitive to different raft-modifying agents (i.e. MbCD and cholera toxin B subunit) and cytoskeleton disrupting drugs (i.e. cytochalsin D) when compared to the internalization of Ag-BCR complexes. Overall, these results indicate that the nature of the BCR ligand plays a significant role in modulating the amount of BCR associated with rafts and determines the relative contribution of rafts and other types of endocytic machinery to the internalization of BCR-ligand complexes.

47

Session II.

The SLP-76 Adaptor Protein Links Integrin Ligation with p44/42 MAPK Phosphorylation

and Podosome Distribution in Murine Dendritic Cells

Nancy Luckashenak, Rebecca Ryszkiewicz, Kimberley Ramsey, and James L. Clements Departments of Cell Stress Biology and Immunology,

Roswell Park Cancer Institute, Buffalo, NY The SH2-domain containing leukocyte protein of 76 kD (SLP-76) is an important molecular intermediate in multiple signaling pathways governing immune cell function. Identified originally as a component of the signaling pathways triggered by T cell receptor ligation, SLP-76 has recently been implicated as an important molecular intermediate regulating integrin signaling. Here we report that SLP-76 is expressed in CD11c+B220- dendritic cells (DCs) isolated from murine thymus or spleen, and that SLP-76 is rapidly phosphorylated on tyrosine residues upon plating of bone marrow-derived DCs (BMDCs) on integrin agonists. SLP-76 is not required for the in vitro or in vivo generation of DCs, but SLP-76 deficient BMDCs manifest multiple signaling defects following integrin ligation, including the inducible phosphoryla tion of PLCg2 and the p44/42 MAP kinases (Erk1/2). Defective integrin signaling in the absence of SLP-76 correlates with reduced adhesion in vitro and an elevated frequency of cutaneous DCs exiting ear tissue in a tissue explant model of migration. In addition, the pattern and distribution of actin-based podosome formation is markedly altered in BMDCs lacking SLP-76 following integrin engagement. These data implicate SLP-76 as an important molecular intermediate in the signaling pathways regulating multiple integrin-dependent dendritic cell functions, and add to the growing body of evidence that hematopoietic cells may employ unique molecular intermediates and mechanisms for regulating integrin signaling.

48

Session II.

Cellular Systems Biology: Applications for Analysis of Protein Phosphorylation and Cellular Signaling Events

Susan Reynolds

Pharmaceutical & 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. The use of phospho-specific antibodies in combination with flow cytometry to monitor phosphorylation events at unique residues within proteins at the single cell level is a very powerful technology. Phospho-specific antibodies tagged with fluorochromes, in combination with cell surface markers, permits the simultaneous visualization of multiple phosphorylations in distinct cell sub-populations. This presentation highlights the utility of flow cytometry and immunoassay applications for measuring major phosphorylation events during cellular activation in various cell models and whole blood systems.

49

Session III.

Chemokine-dependent Memory CD8 T cell-Mediated Pathology During Ehrlichia Infection

Gary Winslow and Constantine Bitsaktsis

Wadsworth Center, Albany, NY, USA, 12208

To address the role of cellular immunity during ehrlichia infection, we have utilized a model of monocytic ehrlichiosis that results from infection of mice by an ehrlichia isolated from an Ixodes ovatus tick (Ixodes ovatus ehrlichia; IOE). We have demonstrated that host defense requires classical immune mechanisms involving CD4 T cell-mediated, TNFa-, IL-12-, and IFN?-dependent, macrophage activation. Although these CD4 T cell responses were sufficient to protect mice from low dose IOE infection, C57BL/6 mice were not protected from a subsequent high dose (fatal) rechallenge, suggesting that protective memory T cell responses were poorly induced during primary IOE infection. When an additional low dose challenge infection was performed in an effort to boost cellular immunity, the mice succumbed to this challenge infection, even though naive mice were not susceptible to the same inoculum. The fatal recall responses were associated with high serum levels of TNFa and CCL2, and low IL-10, suggesting that cytokine-driven immunopathology was responsible. As TNFa-producing CD8 T cells have been associated with fatal high dose IOE infection, we performed low dose secondary challenge infections in CD8-deficient mice. These mice were resistant to secondary low dose challenge infection, as were mice that were administered anti-TNFa antibodies, suggesting that TNFa production initiated by CD8 T cells was responsible for fatal immunopathology. Accordingly, adoptive transfer of purified IOE memory CD8 T cells to naive mice induced fatal recall responses following low dose challenge infection. Fatal recall responses also required CCL2, which is likely responsible for monocyte recruitment to infected tissues that may also contribute to pathology. Our data indicate that IOE infection can generate a pool of CD8 memory cells that can act deleteriously upon rechallenge, and suggest that live vaccines can under some conditions induce undesirable consequences.

50

Session III.

Cell-mediated Protection Against Pulmonary Yersinia pestis Infection

Michelle A. Parent*, Kiera N. Berggren, Lawrence W. Kummer, Lindsey B. Wilhelm, Frank M. Szaba, Isis K. Mullarky, and Stephen T. Smiley

Trudeau Institute, Saranac Lake, NY

Pulmonary infection with the bacterium Yersinia pestis causes pneumonic plague, an often-fatal disease for which no vaccine is presently available. Antibody-mediated humoral immunity can protect mice against pulmonary Y. pestis infection, in an experimental model of pneumonic plague. Little is known about the protective efficacy of cellular immunity. We investigated the cellular immune response to Y. pestis in B cell-deficient µMT mice, which lack the capacity to generate antibody responses. To effectively prime pulmonary cellular immunity, we intranasally vaccinated µMT mice with live replicating Y. pestis. Vaccination dramatically increased survival of µMT mice challenged intranasally with a lethal Y. pestis dose and significantly reduced bacterial growth in pulmonary, splenic, and hepatic tissues. Vaccination also increased numbers of pulmonary T cells, and administration of T cell-depleting monoclonal antibodies at the time of challenge abrogated vaccine- induced survival. Moreover, the transfer of Y. pestis-primed T cells to naïve µMT mice protected against lethal intranasal challenge. These findings establish that vaccine-primed cellular immunity can protect against pulmonary Y. pestis infection and suggest that vaccines promoting both humoral and cellular immunity will most effectively combat pneumonic plague.

*Selected Poster Presentation

51

Session III.

Pattern Recognition and the Fate of Francisella tularensis in a Mouse Model of Tularemia

Timothy J. Sellati Center for Immunology & Microbial Disease, Albany Medical College

Albany, NY Francisella tularensis, the causative agent of pneumonic tularemia, is a bacterial threat

agent by virtue of its extreme infectivity, ease of dissemination, and substantial capacity to cause illness and death. Molecular mechanisms underlying tularemia pathogenesis remain ill defined; though, several reports have implicated Toll- like receptor 4 (TLR4) in modulation of host immunity to F. tularensis. Morbidity and mortality are increased in C3H/HeJ (TLR4-/-) mice, however, the immunological basis for this phenotype remains unclear.

Herein we report that F. tularensis induces greater matrix metalloproteinase 9 (MMP-9) activities in TLR4-/ - mice than in C3H/HeN mice. Greater MMP-9 activity was associated with increased susceptibility to infection and accelerated mortality. This association was confirmed insofar as MMP-9-/- mice survived a lethal challenge with F. tularensis. Infected TLR4 -/- mice also presented with markedly higher pulmonary bacterial burdens and more severe histopathological changes. To determine whether other elements of innate and acquired immunity were altered by TLR4 deficiency, immunomodulator levels were measured. Since pro- and anti- inflammatory cytokines play a critical role in modulating the intensity of an inflammatory response it was quite surprising to find that their levels did not differ significantly between C3H/HeN and TLR4-/ - mice. Although complex and varied, the pattern of humoral immunity also did not correlate with disease severity with TLR4 -/- mice having higher titers of protective anti-F. tularensis antibodies.

Collectively, these results suggest that TLR4-regulated MMP-9 activity plays a central role in modulating the clinical course and severity of pneumonic tularemia and identify MMPs as a novel target for therapeutic intervention.

52

Session III.

Indicators for Apoptosis

Kelly Lundsten Molecular Probes Technology Specialist

Invitrogen Apoptosis can be detected and measured by a number of cellular indications. A combination of membrane integrity, membrane structure, proteolysis, cellular metabolism, mitochondrial membrane function, nuclear condensation and fragmentation can together indicate that a cell has progressed into apoptosis versus necrosis. We will discuss a cross-section of some of the probes that can be used for this application in flow cytometry.

53

Keynote Speaker Monday, October 10, 2005

Introduction by: Dr. Edmund Gosselin

Dennis L. Kasper, M.D. William Ellery Channing Professor of Medicine

Professor of Microbiology and Molecular Genetics Director, Channing Laboratory

Harvard Medical School

“A Glimpse into the Molecular Basis for Symbiosis Between the Intestinal Microflora and the Mammalian Immune System”

We have described the novel internalization and processing of a zwitterionic polysaccharide, PSA from Bacteroides fragilis, within endosomes of antigen-presenting cells (APCs) (Cobb et al., Cell; 2004). Subsequent presentation of processed polysaccharide by major histocompatibility complex class II (MHC II) molecules activates CD4+ T cells and represents a previously undescribed pathway of antigen presentation. Through the present study, we have begun to understand the symbiotic role played by B. fragilis and PSA in their normal ecologic niche - the gastrointestinal tract. The mammalian gastrointestinal tract harbors a complex ecosystem consisting of more than one thousand species at a density of >1012 organisms per gram of cecal contents; these organisms live in homeostasis with the host immune system. Shaped by evolution, this partnership has the potential to confer symbiotic benefits. However, despite published information on the important role played by the entire intestinal flora in shaping the development of the host immune system, the identities of bacterial molecules mediating symbiosis remain undefined.

Here we show that during colonization of animals with the ubiquitous gut microorganism B. fragilis, PSA directs the cellular and physical maturation of the developing immune system. Comparison of wild-type with germ-free animals reveals that the immunomodulatory activities of PSA during B. fragilis colonization include correcting systemic T cell deficiencies and TH1/TH2 imbalances and directing lymphoid organogenesis. A specific knockout mutant of B. fragilis lacking PSA does not restore these immunologic functions. PSA presented by intestinal dendritic cells activates CD4+ T cells and elicits cytokine production consistent with the observed shifts in CD4+ subsets. PSA corrects the systemic TH2 bias found in the absence of bacterial colonization through specific induction of TH1 cytokines such as interferon-gamma and IL-12. These findings provide a molecular basis for host-bacterial symbiosis and reveal the archetypal molecule of commensal bacteria that mediates development of the host immune system.

54

55

Presentation Abstracts

(Sessions IV-VIII)

56

57

Session IV.

Memory CD8+ T cell Responses in the Lung

David L. Woodland, Kenneth H. Ely, Jacob E. Kohlmeier and Hirokazu Hikono Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983, USA

Respiratory virus infections elicit memory CD8+ T cell populations that persist for the life of the individual and are able to mediate accelerated clearance of secondary virus infections. Given the protective capacity of these cells, there has been substantial interest in developing vaccines that promote cellular immunity in the lung. It has recently emerged that memory T cells are extremely heterogeneous in terms of their anatomical distribution, phenotype, function, and longevity. However, we have little understanding of the relevance of this heterogeneity to antiviral immunity in the lung. To begin to address these issues, we have analyzed the role of different memory T cell subpopulations in mediating recall responses to secondary influenza and parainfluenza virus challenge. The data indicate that each subset of memory T cells makes a distinct contribution to pulmonary recall responses. Memory CD8+ T cells present in the lung airways and parenchyma play a key role by engaging the virus at the site of infection when viral loads are low. Memory CD8+ T cells in secondary lymphoid organs, such as the spleen and lymph nodes, are subsequently recruited to the lung, either directly after activation, or following a period of rapid proliferation. Current studies are directed at identifying subpopulations of memory T cells that are specifically recruited to the lungs, determining what signals are required for their recruitment, and characterizing the functional attributes of these cells. An understanding of the mechanics of CD8+ T cell recall responses in the lung is important for the development of vaccines that promote cellular immunity to respiratory pathogens. This work is supported by the National Institutes of Health, USA, the National Institute of Animal Health, Japan, and the Trudeau Institute, USA.

58

Session IV.

Mutants of Enterotoxins LT-IIa and LT-IIb with Altered Ganglioside -binding Activities and Diminished Toxicity are Potent Mucosal Adjuvants

Hesham Nawar1, Sergio Arce1, Kate Getman1, Michael W. Russell1,2

and Terry D. Connell 1,2 1Department of Microbiology and Immunology

2The Witebsky Center for Microbial Pathogenesis and Immunology University at Buffalo, Buffalo, NY, United States, 14214

BACKGROUND: Administration of antigens (Ags) to the mucosa often fails to elicit strong protective responses. Thus, there is a need to develop new adjuvants for potentiating immune responses to mucosally administered Ags. LT-IIa and LT-IIb, the Type II heat-labile enterotoxins of Escherichia coli, are structurally related to cholera toxin and LT (LT-I), yet have distinctive receptor-binding affinities and adjuvant properties. CT and LT-I bind to ganglioside GM1; LT-IIa binds avid ly to GD1b, less well to GD1a, and with lower affinity to GM1; LT-IIb binds with high affinity only to GD1a. Binding of LT-IIa and LT-IIb to their respective ganglioside receptors is reduced to undetectable levels in vitro when threonine at amino acids 13, 14 or 34 in the B subunit of the enterotoxins is substituted with an alternative amino acid. While the distinctive adjuvant properties of LT-IIa and LT-IIb have been reported, the role of ganglioside-binding in their adjuvant activity has not been fully investigated.

METHODS: Non-toxic mutant LT-IIa and LT-IIb enterotoxins with no detectable binding for GD1a, GD1b, or GM1, or with different binding affinity to GM1 were used to investigate the dependence of adjuvant activity upon ganglioside binding. Mice were immunized intranasally with a bacterial protein alone or in combination with wild type or mutant enterotoxin.

RESULTS: LT-IIa(T34I), which has no binding activity to GD1b, GD1a or GM1, exhibited no propensity to elicit early systemic or mucosal adjuvant activity, but primed mice for potent Ag-specific systemic memory responses. The adjuvant activities of LT-IIa(T14S), LT-IIa(T14I), and LT-IIa(T14D) were directly correlated with the differing binding affinities for GM1. Mutant LT-IIb enterotoxins possessed adjuvant activity which was equivalent to that exhibited by the wt GD1a-binding LT-IIb. Flow cytometry showed that LT-IIa(T34I) had no affinity for splenic lymphocytes. LT-Iib(T13I) retained binding activity for T cells, B cells, and macrophages.

CONCLUSIONS: The results of these experiments indicate that binding of LT-IIa to one of its receptors is essential for its immediate adjuvant properties, but not for its ability to establish memory. Binding of LT-IIb to one or more of its known ganglioside receptor(s) is not essential for its immunomodulatory properties.

59

Session IV.

Effects of Azithromycin on a Murine Model of Allergic Lung Inflammation

Aracelis D. Fernandez and Dennis W. Metzger Center for Immunology and Microbial Diseases, 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 antimicrobial properties but rather to immune modulation. In order to better characterize the mechanisms responsible for the beneficial effects of azithromycin, a murine model of allergic lung inflammation with ovalbumin (OVA) was used. Adult BALB/c mice were sensitized by intraperitoneal (i.p.) injection of OVA in alum and then challenged 14 days later with five daily intranasal doses of OVA. An i.p. injection of azithromycin or saline alone was given at the time of sensitization and/or at the time of challenge. Allergen specific antibody levels in serum and in bronchoalveolar lavage (BAL) fluid were determined by ELISA. Lungs were obtained for H & E staining to evaluate histology. Homogenates from lung tissue were examined for cytokine profile by the cytometric bead array assay. Evaluation of airway reactivity was done by plethysmography. Inflammatory cell infiltration in BAL fluid was determined by Wright-Giemsa staining. OVA specific total and IgG1 antibodies levels in serum and in BAL were reduced in the mice treated with azithromycin when compared to the saline-treated group. IL-4 and IL-5 levels were reduced in lung homogenates 24 h and 96 h after challenge in the mice that received azithromycin compared to the saline-treated group. Histology of the lungs of saline-treated mice revealed marked inflammation with prominent vasculitis after OVA sensitization and challenge; these features were virtually absent in mice treated with azithromycin. Mice that received azithromyc in showed significantly less airway reactivity upon stimulation with methacholine when compared to the saline-treated group. Also, inflammatory cell infiltration in BAL was less pronounced in the mice that received azithromycin. These findings suggest that azithromycin decreases inflammatory responses to OVA in this model of murine allergic lung inflammation. This decrease in inflammation is likely to be one of the mechanisms responsible for the clinical benefit obtained by patients with chronic pulmonary diseases who receive azithromycin. Further studies are needed to better characterize this beneficial effect.

60

Session IV.

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 Institute 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 intestinal 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 expulsion. 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 medium. 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. spiralis.

61

Session IV.

SAP is Required for Initial Expansion of B cells and Plasma Cells and for Control of Influenza Virus Upon High-Dose Rechallenge

Cris Kamperschroer1, John P. Dibble1, Dana L. Meents1, Pamela Schwartzberg2

and Susan L. Swain1 1Trudeau Institute, Saranac Lake, NY and 2National Institutes of Health, Bethesda, MD

Antibody is a crucial component of long-term immunity against infection with pathogens, but when a protein called signaling lymphocytic activation molecule (SLAM)-associated protein (abbreviated SAP) is not functional, antibody responses do not progress normally. To provide an explanation for this antibody defect, we studied the immune response to influenza virus infection and to model antigens in SAP- mice. In doing so, we observed a profound decrease in peak IgG antibody titers and in the initial expansion of antigen-specific plasma cells and B cells in SAP- mice after antigen challenge. We show that the generation of normal numbers of plasma cells requires SAP within CD4 T cells, suggesting a defect in B cell help delivered by SAP- CD4 T cells. However, the humoral immune defects in SAP - mice cannot be explained simply by a loss of helper T cells, since CD4 T cells were present throughout the immune response in SAP - mice. The reduction of antigen-specific antibody in SAP- mice was functionally significant, since control of influenza virus upon reinfection required SAP. Our data therefore indicate that the reduced antibody observed after infection of SAP- mice is at least in part due to reduced helper T cell dependent expansion of B cells and plasma cells, which results in an increased susceptibility of SAP- mice to reinfection with influenza virus.

62

Session V.

Antibody Induction of Lupus-like Neuropsychiatric Manifestations

David A. Lawrence, Valerie J. Bolivar, Chad Hudson, Tapan Mondal, Nina Pabello Wadsworth Center, Albany, NY 12201-0509

Lupus-prone male NZM88 mice, which were derived from NZB/NZW F1 mice, develop early neuropsychiatric manifestations without any signs of nephritis. In addition to autoantibodies to dsDNA and renal antigens, mice of this inbred strain express autoantibodies to numerous brain antigens. Here, we show that autoantibodies to brain antigens, assessed by Western analysis, are as individually varied as are the diverse neuropsychiatric manifestations observed in SLE patients. Additionally, a monoclonal antibody derived from the spleen of an untreated NZM88 male when injected into healthy BALB/cByJ, but not C57BL/6J, mice induced behaviors similar to those of lupus-prone NZM88 mice. This monoclonal antibody, which is specific to dynamin-1 (a protein responsible for endocytosis of neuronal vesicles) reacts preferentially with BALB/cByJ cortex. SB31 also enhances cytokine expression in the hypothalamus. Thus, even a single antibody specificity can induce multiple behavioral changes, and multiple autoantibodies to different brain antigens exist in lupus-prone mice; however, susceptibility is dependent on host genetics.

63

Session V.

The Effect of Metallothionein Gene Dose on Oxidative Stress and Signal Transduction

Kristin M. Zaffuto, Darryn W. Unfricht, and Dr. Michael Lynes University of Connecticut, Storrs, CT

Metallothionein (MT) is a small molecular weight, thiol-rich protein. This protein’s cysteine-associated thiols confer to MT its capacity to protect against heavy metal toxicity and oxidative stress. Oxidative and heavy metal challenges to the cell can induce metallothionein to release metal ions, increasing their availability to specific target proteins and amplifying signals. This also suggests that metallothionein may act early in a biological signaling cascade to regulate metal-dependent biochemical and cellular responses. Additionally, uncontrolled release of metals by excessive oxidative stress may contribute to metal toxicity. To better understand the role of metallothionein in oxidative regulation, signal transduction, and cellular activation, we used three congenic mouse strains: C57BL6/J (denoted WT), C57BL/6J-TgN(Mt1)174Bri (denoted TgN), and C57BL/6J -Mt1tm1BriMt2tm1Bri (denoted KO). The TgN has 112 extra copies of the Mt1 gene, while the KO lacks intact Mt1 and Mt2 genes. We treated cells in vitro with both hydrogen peroxide and cadmium chloride and measured lipid peroxidation as well as the activities of the antioxidant enzymes catalase and superoxide dismutase to explore differences in oxidant management between the three strains. Preliminary results show that MT gene dose does not affect lipid peroxidation levels, but does have an effect on both antioxidant enzymes. This suggests that there are compensatory actions fo r disruption or over-expression of MT. We have also identified differences in splenocyte phosphorylation patterns that develop following mitogen stimulation in the presence of absence of exogenous with heavy metals and mitogens such as LPS and Concanavalin A. Preliminary results suggest that the MT gene dose is associated with differences in the tyrosine phosphorylation patterns. These studies suggest that MT gene dose and MT protein levels play a complex but important role in management of immune and inflammatory processes, both in the presence of environmental toxins, and in the normal immune system.

Supported by NIEHS ES07408 and NIEHS ES25490.

64

Session V.

Presence of Cells Presenting Paternal Antigen in the Uterus After Mating Does Not Result in the Stimulation of Anti-paternal CD8+ T-cells in vivo

Matthew MacDonald Seavey, Tim R Mosmann. Department of Microbiology and Immunology

and the David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, 601 Elmwood Ave, Rochester, New York, 14642

Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T-cells are suppressed during pregnancy in several mouse models, and since semen has been shown to mediate immune modulation we sought to determine if exposure to paternal antigen during insemination activated or tolerized anti-paternal CD8+ T-cells. We found that father derived MHC I and II positive cells could be found in the uteri of 3 hours post-coitus (hpc) mated female mice. Flushed cells from these uteri were able to stimulate IFN-? cytokine responses from effector, but not naïve, anti-paternal CD8+ T-cells ex vivo. Also, similar MHC I and II positive cell types could be found in various male reproductive tissues involved in ejaculation. Between 3hpc and 12-16hpc maternal derived MHC I and II positive cells dramatically increased in total percentage in the uterine lumen. Flushed cells from these uteri could efficiently cross-present male H-Y antigen to effector CD8+ T-cells ex vivo, and induce IFN-? cytokine responses. Neither unprimed nor primed anti-paternal CD8+ T-cells were activated post-insemination even if the male alloantigen was cross-presented by the mother. Three days after insemination and exogenous immunization with paternal antigen, anti-paternal CD8+ T-cells could proliferate in vivo and synthesize cytokines ex vivo, suggesting that tolerance was not induced. Antigen could be transferred across the female reproductive tract as T-cells responded to intravaginal instillation of peptide antigen. However, direct allorecognition of intact cells expressing paternal MHC I failed, as highly potent dendritic cells were unable to stimulate anti-paternal CD8+ T-cell responses after intravaginal instillation. These results suggest that paternal antigens and antigen-presenting cells are introduced during coitus, but that anti-paternal CD8+ T-cell responses are blocked but not suppressed post-insemination. Supported by NIH AI51869 and the Multiple Sclerosis Society.

65

Session V.

IL-12 and IL-18 are Key Cytokines Involved in Differentiation of CD8+ T cells

*Monisha Sundarrajan, Loui MadakamutilOlga Turovskaya, Christopher Lena, Yiran Wang-Zu and Hilde Cheroutre

Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology 10355 Science Center Dr, San Diego, CA 92121

CD8 molecule on T cells are of two types, CD8ab heterodimer and the CD8aa homodimer. We have shown that CD8aa induction seem to be an absolute requirement for survival of primary effectors and for differentiation into long- lived conventional memory CD8 T cells. However, the signals involved in induction of CD8aa on activated T cells remains to be elucidated. In vitro stimulation of total splenocytes by anti-CD3 mAb together with addition of various cytokines showed that IL-2, IFN-g (initial signals) or IL- 7 (survival cytokine) did not promote the expression of CD8aa on activated T cells, whereas IL-15 and the pro- inflammatory cytokines IL-18, IL-12, IL-23, were able to expand the CD8aa+ T cells. Amongst these IL-12 and IL-18 were observed to be most efficient and these results were confirmed using specific antigen stimulated OT-1 TCR transgenic T cells as well. Consistent with an important role for these cytokines for the differentiation of memory precursor cells, IL-12 and IL-18 knockout mice are impaired in generating activation induced CD8aa primary effector cells and they lack all CD4 and CD8ab mucosal memory T cells. IL-12R and IL-18R both mediate STAT-4 dependent signaling, suggesting that STAT-4 signaling might play a critical role to promote CD8aa induction and differentiation along the memory pathway. Further studies aim at elucidating the possible mechanism or mechanism(s) that are decisive for the survival and differentiation of activated T cells into long- lived memory cells.

*Selected Poster Presentation

66

Session V.

Characterization of Fibroblasts in Human Lung Tumor Microenvironments: Cytokine Production, Co-regulatory Molecule Expression, and Role in the IL-12 Induced,

IFN-gamma Mediated Anti-tumor Response

Michael R. Nazareth, Lori Broderick, Raymond J. Kelleher, and Richard B. Bankert University at Buffalo

The local and sustained release of IL-12 from biodegradable microspheres into the microenvironment of human non-small cell lung tumor xenografts induces a cascade of events that results in the eradication of tumor cells. To determine how fibroblasts may contribute to the IL-12 induced anti- tumor response, primary cultures of tumor-associated fibroblasts were established and characterized phenotypically for cell surface markers, for changes in gene expression patterns, and for their capacity to produce and release biologically active molecules. Tumor-associated stromal fibroblasts are shown here to produce IFN-g, IFN-b, RANTES, and IP-10, which have been found to be coincident with and/or contributing to the IL-12 induced tumor arrest. Changes in gene expression patterns in fibroblasts obtained from IL-12 treated human xenografts have been observed that may also contribute, either directly or indirectly, to the tumor eradication. A portion of the human fibroblasts derived from human primary lung tumor microenvironments are shown here to constitutively express two cell membrane-associated B7 family co-regulatory molecules (B7H1/PD-L1 and B7DC/PD-L2) that are upregulated in response to IFN-g. These cell surface-associated molecules and fibroblast-synthesized biologically active factors have the potential capacity to modulate the activation, survival, and homing of tumor-associated memory T cells into the tumor microenvironment, and thereby contribute to the IL-12 induced tumor eradication.

67

Session VI.

New Insights into Signaling via IgE Receptors

David Holowka Dept. of Chemistry and Chemical Biology

Cornell University

Recent work in our laboratory to characterize the role of lipid segregation in IgE receptor

signaling in mast cells has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment to initiate the signaling cascade that leads to mast cell degranulation. Previous work showed that polymerization of F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work in our laboratory implicates cytoskeletal interactions with ordered lipid rafts in this negative regulation. Other studies reveal a novel membrane pool that participates in receptor-stimulated Ca2+ mobilization. ?These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.

68

Session VI.

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

Activation of Lymphoid Cells

Sergio Arce1,2,*, Hesham F. Nawar1,2, Michael W. Russell1,2,3, and Terry D. Connell1,2

1The Witebsky Center for Microbial Pathogenesis and Immunology, 2The Department of Microbiology and Immunology, 3The Department of Oral Biology, The

University of Buffalo, State University of New York at Buffalo, Buffalo, NY

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 enterotoxins 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. Binding 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 mutant 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 apoptosis, 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, but not LT-IIa nor LT-IIb, enhanced expression 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 likely dependent upon their capacities to recognize different ganglioside receptors on lymphocytes.

69

Session VI.

BAFF: A Dr. Jekyll & Mr. Hyde in Controlling Plasma Cell Fate

Loren D. Erickson University of Virginia Charlottesville, VA

The enduring nature of protective humoral immunity is largely dependent on the longevity of the plasma cell (PC). Conversely, it is this long-lived capacity that is a significant hindrance in antibody-mediated autoimmune diseases, like systemic lupus erythematosus. Thus, understanding the factors that determine PC development and survival takes on considerable importance in terms of both biology and therapeutics. The inextricable link between the bone marrow microenvironment and the persistence of long- lived PCs has drawn our attention to a newly- identified member of the TNF receptor family, B cell maturation antigen (BCMA), that we have shown to be critical for the survival of normal long-lived BM PCs. This function of BCMA is highly selective, as genetic deletion of BCMA or blocking BCMA appears to only influence the survival of terminally-differentiated B cells and not naïve B cells. The coordinated migration of antigen-reactive B cells to precise anatomical locations within secondary lymphoid organs and between lymphoid compartments is critical for the development of long-term humoral immunity and self-tolerance. Contrary to the behavior of normal long- lived PCs, we have found evidence that PCs from lupus-prone mice accumulate in the spleen but are virtually absent in the BM. A loss in responsiveness to CXCL12 appears to be responsible for this deficiency, and is selective to B lymphocytes committed to a PC fate. This defect in BM PC homing appears to be manifested by constitutively high levels of BAFF expression as in vivo blockade of BAFF results in the migration of PCs to BM. Importantly, PCs found in the BM of lupus mice treated with BAFF antagonist were not specific to autoantigens. Thus, while BAFF is critical for the survival of BM PCs excessive BAFF levels can prevent the migration of long-lived PCs to BM, where we propose further checkpoints of self- tolerance occurs.

70

Session VI.

Regulation of CD4 Recycling by the Ras-GTPase Rab4a

*Jeff Ward, and Andras Perl

Departments of Medicine and Microbiology and Immunology SUNY Upstate Medical University, Syracuse, New York

Rab proteins are members of the Ras-GTPase family that are involved in membrane trafficking processes including vesicle formation, docking, and fusion. Rab4a has been shown to be localized to sorting endosomes and the endocytic recycling compartment and play an integral role in the recycling of the transferrin receptor (TfR) as well as regulation of the glucose transporter GLUT4. CD4 is a 55 kDa surface receptor that is constitutively endocytosed and recycled back to the cell surface at a slow rate. In order to study the function of Rab4a in lymphocytes bicistronic doxycycline- inducible vectors expressing wild type Rab4a or a Rab4a mutant locked in the GDP bound state along with GFP were stably transfected into the Jurkat E6.1 cell line. Overexpression of Rab4a resulted in a decrease of cell surface CD4 of roughly 4-fold, while expression of the dominant negative mutant resulted in a 2-fold increase as detected by mean fluorescence intensity. Quantitative Western blotting showed that the overall protein level of CD4 was also decreased in Rab4a overexpressing cells and increased in cells transfected with the dominant negative construct. Treatment of cells overexpressing Rab4a with the lysosomal inhibitor chloroquine was able to increase the total cellular level of CD4 to near that of GFP-only expressing cells, suggesting that Rab4 may regulate a critical sorting step during the intracellular trafficking of CD4. Recycling experiments using phorbol esters to internalize surface receptors have verified that Rab4a overexpressing cells are defective in their ability to return endocytosed CD4 to the cell surface while they retain the ability to recycle the CD3 receptor. This data suggests that CD4 recycles through a Rab4a dependent pathway in lymphocytes.

*Selected Poster Presentation

71

Session VI.

The Regulation of Th2 Cytokine Production by SHP-1

*Chad A. Hudson and Paul T. Massa SUNY Upstate Medical University, Syracuse, New York, USA

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling. Mice homozygous for a null allele at the SHP-1 locus (me/me or motheaten mice) have a phenotype of both severe inflammation and decreased innate immunity to viruses. We have found that this increased susceptibility to viruses may be the consequence of increased arginase I activity and decreased nitric oxide production in the central nervous system, a situation typical of Th2-skewed immune responses. Mice heterozygous at the SHP-1 locus (me/+) have no overt phenotypic differences from wild type animals (+/+), yet have a 50% reduction in SHP-1 protein expression. To determine whether me/+ mice, like motheaten mice, have a propensity for Th2-like cytokine responses, either 50 micrograms of LPS (Salmonella minnesota re595) or saline was injected intraperitoneally into me/+ and +/+ mice twice a week for 4 weeks. Serum and organs were harvested either 24 hours or 5 weeks after the first injection. At 24 hours, LPS-treated me/+ mice had significantly higher levels of serum IL-10, serum IL-13 and splenic arginase activity, while only serum IL-10 was increased in LPS-treated wild type animals. After 5 weeks, me/+ mice had significantly higher LPS-stimulated increases in Th2 cytokines including IL-13 (brain, spleen, kidney), IL-10 (brain, spleen, kidney, serum) and IL-4 (spleen, kidney), and in arginase activity (brain, spleen, kidney) than +/+ mice. No differences in the Th1 cytokine, IFN-gamma, were found. These data indicate that me/+ mice have an increased Th2- like response to LPS, suggesting that after microbial infection, me/+ mice would have an increased susceptibility to intracellular pathogens typically eradicated by Th1 responses.

*Selected Poster Presentation

72

Session VI. Grating-Coupled Surface Plasmon Resonance: A Cell and Protein Microarray Platform

Darryn W. Unfricht1, Guang-Bi Jin1, Salvador M. Fernandez2, Michael A. Lynes1

1 The Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 2 Ciencia, Inc., East Hartford, CT 06108

Grating-coupled surface plasmon resonance (GCSPR) is a method for the accurate assessment of both cell phenotype and function. In GCSPR, cells and/or proteins of interest are flowed across antibodies immobilized on a sensor chip. The chip’s surface is illuminated with monochromatic light that couples to the gold surface’s electrons to form surface plasmon. At a specific angle of incidence, the GCSPR angle, the maximum amount of coupling occurs. Shifts in the GCSPR angle can be correlated with refractive index changes following cell or analyte capture by the immobilized antibodies. In addition, GCSPR can image the cells as they are being captured. GCSPR’s multiplexed format allows for the independent assessment of up to 400 individual antibody regions. In this paper, we demonstrate GCSPR’s ability to identify cells and proteins of interest and compare results to a traditional flow cytometry system. This technology represents a fast and powerful method for the simultaneous assessment of cell phenotype and function.

This work was supported in part by grants to S.M.F. and M.A.L. from NIEHS (N44-ES35510) and NASA (NNJ04JA14C).

73

Session VII.

Birth of a B cell: Molecular Circuits in Early B cell Development

Rachel Gerstein, Ph.D. University of Massachusetts

“NO ABSTRACT PROVIDED”

74

Session VII.

Estrogen, Estrogen Receptors, and Thymocyte Development

Jeffrey H. Mills, Zhi-wei Lai and Allen E. Silverstone Microbiology & Immunology, SUNY Upstate Medical University, Syracuse, NY

Estradiol (E2) is a hormone that is known to function through estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta). E2 is also well known to cause thymic atrophy in all viviparous animals, but the mechanism of this estrogenic affect is in dispute. We had previously shown, utilizing male ERalpha knock-out (AERKO-1) mice from K. Korach (NIEHS) that AERKO-1 mice have developmentally smaller thymii compared to wild type (WT) mice but that both AERKO-1 mice and radiation chimeras with an AERKO-1 hematopoietic system still undergo E2-mediated thymic atrophy, although this atrophy is less than in WT treated mice. Our data also suggests that the target for this atrophy is in the thymocyte stem cell compartment, and that the effect is not mediated by apoptosis, but by some form of proliferation/cell cycle arrest. To further elucidate the mechanism(s) for E2 induced thymic atrophy, we compared the effects of E2 on male and female mice treated at 5 or 7 week of age, while utilizing a second ERalpha-KO (AERKO-2) strain from P. Chambon (ICS/MCI), an ERbeta-KO (BERKO) strain from D. Lubahn (U of Mo) and K. Korach, and an ERalpha/beta-?double-KO (DERKO-2) strain, using the Chambon AERKO-2 allele. Strikingly, AERKO-2 mice had full sized thymii as compared to age/sex matched WT mice, unlike AERKO-1 mice. AERKO-2 mice also appeared to be resistant to E2 induced thymic atrophy. Secondly, BERKO mice had full sized thymii as compared to age/sex matched WT mice, but were as sensitive to E2 treatment as WT mice. Thirdly, DERKO-2 mice were totally resistant to E2 induced atrophy. Fourthly, radiation hematopoietic chimeras made with DERKO-2 stem cells in WT recipients were still sensitive to E2 induced thymic atrophy, although less sensitive than WT or BERKO hematopoietic chimeras. This suggests that an E2 induced hormone from some non-hematopoietic source can mediate estrogenic thymic atrophy.

75

Session VII. Mitochondrial Hyper-polarization: a Checkpoint of T-cell Life, Death, and Autoimmunity

Andras Perl

Division of Rheumatology, Departments of Medicine, Microbiology and Immunology State University of New York Upstate Medical University

College of Medicine, 750 East Adams Street, Syracuse, NY 13210, USA

Activation, proliferation, and cell death pathway selection of T lymphocytes depend on reactive oxygen intermediates (ROI) production and ATP synthesis which are tightly regulated via the mitochondrial transmembrane potential. Mitochondrial hyperpolarization (MHP) and ATP depletion represent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of systemic lupus erythematosus (SLE) patients exhibit persistent MHP, cytoplasmic alkalinization, increased ROI production, and ATP depletion that mediate enhanced spontaneous and diminished activation- induced apoptosis and sensitize lupus T cells to necrosis. Necrotic, but not apoptotic, cell lysates activate dendritic cells and may account for increased interferon- production and inflammation in lupus patients. MHP is proposed as a key mechanism of pathogenesis and target for pharmacological intervention in SLE. Persistent MHP was associated with increased mitochondrial mass and increased mitochondrial and cytoplasmic Ca2+ content in T cells and enhanced NO production by monocytes of lupus patients. Activation of T cells through the T cell receptor initiates a biphasic elevation in cytosolic free Ca2+ concentration, a rapid initial peak observed within minutes and a plateau phase lasting up to 48 h. In response to CD3/CD28 costimulation, rapid Ca2+ fluxing was enhanced while the plateau phase was diminished in lupus T cells. NO-induced mitochondrial biogenesis in normal T cells enhanced the rapid phase and reduced the plateau of Ca2+ influx upon CD3/CD28 costimulation, thus mimicking the Ca2+ signaling profile of lupus T cells. Mitochondria constitute major Ca2+ stores and persistent MHP and NO-dependent mitochondrial biogenesis may account for altered Ca2+ handling by lupus T cells. Coordinated changes in expression of genes encoding members of the electron transport chain (ETC) and anti-oxidant defenses underlie mitochondrial dysfunction and dominate the altered gene expression profile of lupus lymphocytes. Members of the ETC may serve as novel target for pharmacological intervention in SLE.

76

Session VII.

Thermal Regulation of Antigen Presenting Cells: Use of Heat as an Adjuvant for Cancer Vaccines

*Adrienne Kisailus, Michele Pritchard, Ying Li, Elizabeth A. Repasky, Julie Ostberg

Roswell Park Cancer Institute Upon infection, antigen exposure induces antigen presenting cells, such as macrophages and dendritic cells (DCs) to take up antigen which they can use to activate T-cells. Because a local or systemic temperature increase is a cardinal feature of infection, we have hypothesized that mild thermal stress associated with fever response can help regulate antigen presenting cell activity. Fever-range whole body hyperthermia (FR-WBH, 39.5-40°C for 6 hrs) was observed to significantly enhance peritoneal macrophage phagocytosis of latex beads. Hyperthermic stimulation of macrophages was also found to increase NO production induced by LPS ± IFN-g. With regards to DCs, incubation of in vitro ear skin cultures at fever range temperatures for the first 6 hours enhanced migration of DCs out of the ear skin into culture wells. Furthermore, these emigrated dendritic cells from heated cultures displayed enhanced MHC II and CD86+ expression and were more immunostimulatory in an allo-MLR when compared to that from control cultures. Interestingly, thermally stimulated macrophages and DCs both displayed increased levels of heat shock protein 70. These data support a role for thermal stimuli in enhancing antigen presenting cell function and may be implicated for usage as a vaccine adjuvant. Indeed, when FR-WBH was used in concert with a subcutaneous tumor antigen vaccination protocol, increased survival upon tumor challenge was observed.

*Selected Poster Presentation

77

Session VII.

Localized Tumor Irradiation Enhances Anti-tumor Immunity

Amit Lugade, James Moran, Scott Gerber, John Frelinger, Edith Lord Dept of Microbiology and Immunology, University of Rochester

Intrinsic immune responses to tumors may be limited in the host for multiple reasons. One deficiency may be the lack of tumor antigen available to stimulate a response. Conventional radiation therapy may augment the immune response by generating antigen from the dying and dead tumor cells. This possibility was evaluated by assessing the effector phases of anti-tumor immune responses in mice after localized single dose tumor irradiation. Tumors were established in C57BL/6 mice using B16F0 melanoma cells expressing ovalbumin (OVA) as a model tumor antigen. Seven days after tumor cell injection, mice were treated with a single radiation dose of 15 Gy. Immune responses aga inst OVA were assessed in mice 14 days post tumor injection. Lymph nodes (TDLN) draining irradiated tumors contained increased numbers of antigen presenting cells (APC) that stimulated OVA-specific T cell hybrids in vitro and T cells that secreted gamma-interferon (IFN-γ) in response to tumor-derived antigens. Immune activation in the TDLN correlated with an increase in the number of infiltrating, CD45+ immune cells in the irradiated tumors as compared to non- irradiated tumors. These results suggest that cell death resulting from local tumor irradiation provides tumor antigen that can be processed by APC and carried to TDLN to stimulate tumor-specific T cells. Adoptive transfer studies confirmed that the increased numbers of tumor-antigen specific immune cells within irradiated tumors were due to enhanced trafficking of these cells to the tumor site. Vessels within irradiated tumors were found to have upregulated VCAM-1, an adhesion molecule on activated vasculature, which may contribute to the increased homing and infiltration of effector T cells to the tumor. Irradiation of the tumor also resulted in upregulation of MHC class I expression on the tumors cells compared to non- irradiated tumors. Upregulation of VCAM-1 and MHC class I molecules was found to be IFN-γ dependent, suggesting a role for immune activation in altering the tumor microenvironment following localized irradiation.

78

Session VIII. Manipulation of Macrophage Signaling During Intracellular Infection with the Protozoan

Pathogen Toxoplasma gondii

Barbara A. Butcher and Eric Y. Denkers Cornell University

The intracellular protozoan Toxoplasma gondii actively invades macrophages (MØ) and other cell types, and resides sequestered within a specialized parasitophorous vacuole created during cell invasion. Our recent data show that the parasite exerts major effects on proinflammatory signaling pathways in infected MØ, despite the secluded location of the Toxoplasma intracellular niche. Infected MØ fail to produce the inflammatory cytokines IL-12 and TNF-a in response to TLR ligands such as bacterial LPS. Heat-killed tachyzoites and soluble antigen extracts fail to block LPS-induced responses, demonstrating that Toxoplasma-induced inhibition requires live parasites. The inhibitory effects of T. gondii on MØ resembled anti- inflammatory effects of IL-10. Therefore, we examined activation of signal transducer and activator of transcription (STAT)3, a major signaling intermediate in the IL-10 signaling pathway, during MØ infection. Live, but not heat–killed, tachyzoites induced STAT3 phosphorylation within 2 min of infection, followed by nuclear translocation of this signaling molecule. Importantly, Toxoplasma was capable of activating STAT3 in IL-10 gene-deleted MØ. This finding rules out parasite-induced MØ IL-10 as the stimulus for STAT3 phosphorylation, instead arguing for direct activation by T. gondii. Using MØ from STAT3 gene-deleted mice, we found that the parasite could no longer suppress LPS-induced TNF-a or IL-12 production in the absence of this intracellular signaling intermediate. Our results reveal that Toxoplasma hijacks components of the IL-10 signal transduction pathway to subvert proinflammatory responses in infected MØ.

79

Session VIII.

Effect of F. tularensis Invasion on the Cell Biology of Antigen Processing and Presentation by Macrophages

*Justin E. Wilson and James R. Drake

Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

The intracellular bacterium Francisella tularensis avoids degradation and survives and

replicates within host macrophages, ultimately killing the host cell. 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 the cell biology underlying F. tularensis processing and presentation, we have established a model to study the processing of soluble and particulate antigen by alveolar and other macrophages.

To understand the biology of antigen processing we first determined the degradation kinetics of different forms of an exogenous antigen in uninfected murine macrophages via immunofluorescence microscopy. Unlike soluble antigen internalized via fluid-phase endocytosis, bead-associated antigen targeted to Fc receptors by antibody opsonization persisted for a prolonged period of time within intracellular compartments. However, when the processing and presentation of antigen internalized via different mechanisms was examined, it was determined that the Fc receptor-mediated processing and presentation of bead-associated antigen was less efficient than soluble immune complexes. These results suggest that the processing and presentation of particulate antigens, such as F. tularensis, may be significantly different than the processing of small soluble antigens.

In order to determine if F. tularensis alters global antigen processing and presentation, the effect of macrophage infection by F. tularensis on the processing and presentation of a model antigen was determined. The results demonstrate that exposure of macrophages to F. tularensis resulted in a shift in the profile of T cell-derived cytokines elicited upon antigen presentation. Further ana lysis revealed that this shift in T cell cytokine production is due to soluble factors produced upon infection of macrophage by F. tularensis. Using a well-defined model system, we have identified a potential mechanism for the inhibition of the adaptive immune response by F. tularensis that the organism may use to avoid regulation by the adaptive immune system.

This work is supported by the NIH grant AI-056320.

*Selected Poster Presentation

80

Session VIII.

Role of Dendritic Cells in Early Events of Gamma-herpesvirus Pathogenesis

Romana Hochreiter, Catherine Ptaschinski, Nancy Fiore, and Rosemary Rochford 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 are associated with several malignant human diseases, including Burkitt’s lymphoma, nasopharyngeal carcinoma, and Kaposi’s sarcoma. Because EBV and HHV-8 can replicate only in cells of human origin the closely related murine gammaherpesvirus 68 (MHV-68) is used as an experimental mouse model to more fully elucidate the mechanisms of gammaherpesvirus pathogenesis. The gammaherpesviruses gain entrance through the mucosal tissue and need to establish latency in the lymphoid compartment. The gamma-herpesvirus infections are especially interesting since the virus establishes a balance between life long latency and an immune control of the infection.

We are using infection of mice with MHV-68 to model the early events in infection. Intranasal infection of mice with MHV-68 results in replication in the lungs followed by lifelong latency in B cells, macrophages and dendritic cells (DCs). In this study we focused on the impact of MHV-68 infected DCs on the initiation of antiviral responses. We observed that DCs are productively infected with MHV-68 ex vivo and MHV-68 infected DCs are found in the lungs of mice during acute infection. Interestingly, MHV-68 infected only immature DCs and inhibited the upregulation of MHC II, CD86 and CD80, molecules essential for initiating an effective CD4+ T cell response. However, infection did not alter MHC class I levels on DCs even though the viral K3 protein, which was shown to down regulate MHC class I in fibroblasts, was expressed. In addition to reduced levels of costimulatory molecule expression we also found reduced levels of IL-12 production in infected DCs compared to cells treated with UV inactivated virus.

Taken together, MHV-68 is capable of interfering with DC maturation and/or function. Thus targeting dendritic cells could be a general mechanism for gamma-herpesviruses to allow the establishment of a persistent infection in the host. Further studies are underway to determine how infection of DCs modulates CD4+ T cell responses. In general, this data also suggests that viral infection of DCs at mucosal surfaces could affect the development of an effective anti-viral immune response.

81

Session VIII.

Distinct Itk-dependent Signals Required for the Release, But Not Gain,

of Th2 Effector Function

*Byron Au-Yeung1, Shoshana Katzman1, Katja Mohrs2, Markus Mohrs2, and Deborah Fowell1

1Department of Microbiology and Immunology and David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, Rochester, New York

2Trudeau Institute, Saranac Lake, New York Much interest has focused on the T cell receptor (TCR) and cytokine signals required for differentiation of naïve CD4+ T cells to T helper 1 (Th1) and Th2 subsets. Less well understood are the signals required for the release of effector function. Previous studies revealed an essential role for Itk in Th2, but not Th1 responses. However, it is unclear when Itk is required for Th2 development. Using an IL-4/GFP reporter (4get), we followed the fate of Itk-deficient cells during Th2 development. Surprisingly, itk-/- 4get CD4+ cells upregulated IL-4/GFP with similar kinetics in a similar proportion of cells. Further Real-Time PCR analysis revealed that other markers of Th2 differentiation (GATA-3, IL-5, IL-10, IL-13) were also induced to WT levels in the absence of Itk. However, upon restimulation, GFP+ itk-/- 4get cells failed to exert effector function, marked by defective transcriptional upregulation of IL-4 and GATA-3, which could only be restored by supplementing TCR signals with ionomycin.. The phenotype was observed in Itk-deficient cells primed in vivo. Both polyclonal re-stimulation of CD4+ CD44high CD62Llow cells and restimulation of Leishmania major-specific effectors resulted in impaired transcriptional upregulation of IL-4. Itk-dependency for IL-4 production was restricted to T cells, as itk-/- mast cells secreted WT levels of IL-4. These studies reveal the requirement for a calcium sensitive transcriptional enhancement event for the release, but not gain, of Th2 effector function. Our data suggest that the liberation of CD4 T cell effector function is under stringent control; enabling additional, post-differentiation regulation of cytokine responses.

*Selected Poster Presentation

82

Session VIII.

Effects of Aging on the CD8 T cell Response to Primary Influenza Infection

*Eric Yager, Kathleen Lanzer and Marcy Blackman Trudeau Institute, 154 Aligonquin Avenue, Saranac Lake, NY 12983

Aged mice are compromised in their ability to respond to primary viral infections. It has been speculated that age-related reductions in the size of the naïve T cell pool, owing to decreased thymic output and increased proportions of activated memory-phenotype CD44hi CD8 T cells, may lead to contraction of the T cell repertoire and reduced responsiveness to new infections. Spectratype analyses of purified naïve CD8 T cells from aged mice suggested that perturbations might exist in the naïve CD8 T cell repertoire of aged mice. To examine the consequences of a perturbed naïve repertoire on primary immune responses, the CD8 T cell response to influenza infection was characterized in aged mice. It was found that the percentage of CD8 T cells elicited in the lung airways of infected mice specific for the immunodominant influenza epitope NP366-374 was greatly reduced in aged mice compared to young mice. In contrast, the CD8 T cell response generated against the immunodominant influenza epitope PA224-233 appeared to be largely unchanged with increased age. Consistent with reports in the literature, aged mice infected with influenza exhibited delayed viral clearance and viral persistence in lungs of aged mice was correlated with reductions in the diversity of the responding CD8 T cell repertoire. These studies suggest that alterations in the naïve CD8 T cell repertoire of aged mice may lead to the generation of a less diverse CD8 T cell response against primary influenza infection thus compromising the efficacy of viral clearance.

*Selected Poster Presentation

83

Poster Abstracts

84

85

Poster Abstracts Found in Presentation Abstract Section

Poster No. Title Page # 2 Characterization of Fibroblasts in Human Lung Tumor 66 Microenvironments: Cytokine Production, Co-regulatory Molecule Expression, and Role in the IL-12 Induced, IFN-g Mediated Anti-tumor Response

# 3 SAP is Required for Initial Expansion of B cells and Plasma Cells 61 and for Control of Influenza Virus Upon High Dose Rechallenge # 7 Role of Dendritic Cells in Early Events of Gamma-herpesvirus 80 Pathogenesis # 15 Functional Comparison of Rat Bone Marrow-derived Mucosal 60 Mast Cells with the Mucosal Mast Cell Line RBL-2H3 # 18 Effect of F. tularensis Invasion on the Cell Biology of Antigen 79 Processing and Presentation by Macrophages # 19 Effects of Azithromycin on a Murine Model of Allergic Lung 59 Inflammation # 20 Functional Requirements for BCR-mediated Ligand Internalization 46 and Signaling # 23 The Regulation of Th2 Cytokine Production by SHP-1 71 # 24 IL-12 and IL-18 are Key Cytokines Involved in Differentiation 65 of CD8+ T cells # 25 Cell-mediated Protection Against Pulmonary Yersinia pestis Infection 50 # 29 Regulation of CD4 Recycling by the Ras-GTPase Rab4a 70 # 34 The SLP-76 Adaptor Protein Links Integrin Ligation with p44/42 47 MAPK Phosphorylation and Podosome Distribution in Murine Dendritic Cells # 38 The Liver Modulates Systemic CD8+ T cell Memory Through a 41 TLR-4 Dependent Mechanism

86

Poster Abstracts Found in Presentation Abstract Section

Poster No. Title Page # 39 Presence of Cells Presenting Paternal Antigen in the Uterus After 64 Mating Does Not Result in the Stimulation of Anti-paternal CD8+ T-cells in vivo # 40 Mutants of Enterotoxins LT-IIa and LT-IIb with Altered 58 Ganglioside-binding Activities and Diminished Toxicity are Potent Mucosal Adjuvants # 47 Thermal Regulation of Antigen Presenting Cells: Use of Heat as 76 an Adjuvant for Cancer Vaccines # 49 Effects of Aging on the CD8 T cell Response to Primary Influenza 82 Infection # 50 Distinct Itk-dependent Signals Required for the Release, 81 But Not Gain, of Th2 Effector Function # 53 Chemokine-dependent Memory CD8 T cell-Mediated Pathology 49 During Ehrlichia Infection

87

Poster # 1

Compensatory Role for IL-17 in the Absence of IFN-g During Influenza Infection

Deborah M. Brown, Allison Dilzer and Susan L. Swain. Trudeau Institute, 154 Algonquin Ave. Saranac Lake, NY 12983.

Interferon-gamma (IFN-g) is an important component of the anti-viral response; however, studies using IFN-g deficient mice demonstrate that this cytokine is not required for influenza viral clearance. Although CD8 cell function in IFN-g-/ - mice is not compromised, the CD4 cell response in the absence of IFN-g has not been fully elucidated. This study investigated the CD4 response to influenza in the presence, or absence of IFN-g. As previously demonstrated, CD8 functions appeared to be similar between WT and g-/ - mice given a sublethal (500 EIU) dose of influenza. The CD4 response to specific influenza peptides was measured in IFN-g-/- mice using an anti-IL-17 ELISpot assay. Although the frequency of IL-17 producing CD4 cells was lower in IFN-g mice, the kinetics of the response was similar between WT and IFN-g-/- mice. As an additional measure of CD4 function, proliferation assays demonstrated that CD4 cells from IFN-g-/- mice could expand better upon in vitro restimulation with influenza infected APCs. Finally, to determine if CD4 cells from IFN-g-/- mice were protective in vivo, CD4 cells were isolated from draining lymph nodes (DLN) or lung of sublethally infected mice and transferred to normal BALB/c mice that were then given a lethal dose of influenza. Surprisingly, mice given CD4 effectors from WT mice survived a high dose of influenza while mice given CD4 cells from IFN-g-/- mice succumbed to infection. It remains to be determined whether CD4 cells from IFN-g-/- mice lose their ability to produce IL-17 upon transfer, or whether this cytokine is a compensatory mechanism for promoting survival in IFN-g-/- mice. Experiments are underway to determine if blocking IL-17 during influenza infection has any effects on clearance of viral burden, or survival. Taken together, these data indicate that CD4 cells may utilize many different effector mechanisms in response to viral infections.

Supported by PHS grants F32-AI056962 (DMB) and PO1-HL63925.

88

Poster # 4

Developmental Lead Exposure Modifies Early Brain Cytokine levels and Induces Long-term Changes in Behavior

Nina G. Pabello, Jane Kasten-Jolly, Valerie J. Bolivar, David A.Lawrence

Wadsworth Center, Albany, NY 12208 Lead (Pb) is one of the oldest-established poisons known and Pb- exposure continues to be a major public health problem, particularly to persons in areas of low socioeconomic status. The developing nervous system is particularly sensitive to the effects of Pb, and exposure during development induces a wide array of physiological, biochemical, and behavioral dysfunctions. In addition to the effects on the CNS, Pb also has effects on the immune system. Pb decreases host resistance to a variety of infectious agents and influences sickness behavior. While the effects of Pb on the central nervous system (CNS) and the immune system have been studied separately, it is now becoming clear that these two systems interact and share signaling molecules. Cytokines are constitutively expressed in the CNS and have been implicated in normal CNS development and neuromodulation. Cytokines can also mediate sickness behavior and are involved in several neurodegenerative and neurobehavioral processes. In this study the effects of developmental Pb exposure on brain cytokine expression was examined. We also performed a battery of behavioral tasks on adult offspring developmentally exposed to Pd in order to determine whether the effects of Pb are persistent. BALB/c female mice were bred, and on gestational day 8 (E8) pregnant dams were exposed to Pb (0.1mM) or control drinking water from E8 to postnatal day 21 (PND21). On PND21 pups were perfused, and brains were processed for RT-PCR or ELISA. Some pups were weaned until 5-8 months of age for behavioral testing. We found developmental Pb exposure altered whole brain gene expression of several cytokines in the brain in a gender-dependent manner measured by quantitative RT-PCR. Interleukin-18 (IL-18), one of the most highly expressed of the cytokines examined, was increased by Pb exposure especially in females. IL-18 protein was differentially affected in several brain regions. The substantia nigra and hippocampus were two areas containing higher levels of IL-18 and these levels were increased by Pb exposure. Several neurobehavioral deficits (activity, rearing, turning, morris-water maze) were evident in adult offspring exposed to Pb, particularly in females. However, aggressive behaviors in males, as assessed by a resident-intruder paradigm, is reduced in adult mice developmentally exposed to Pb. These results suggest that exposure to Pb during early development affects regional brain cytokine levels which have been implicated in proper CNS development. Furthermore, low level developmental Pb exposure results in long- lasting neurobehavioral alterations.

89

Poster # 5

The Regulation of Arginine Metabolism and Innate Immunity by SHP-1 in the Central Nervous System

Paul T. Massa, Kathryn L. Bonaparte, Chad A. Hudson and George P. Christophi

SUNY Upstate Medical University, Syracuse, New York We have previously shown that the SH2 domain-containing protein tyrosine phosphatase SHP-1 plays a critical role in controlling Theiler’s murine encephalomyelitis virus(TMEV) replication in CNS glia. Increased virus replication in SHP-1-deficient glia in vitro may be related to presently unknown roles for SHP-1 in innate anti-viral state. In the present study, we observed a profound decrease in expression of inducible nitric oxide synthase (iNOS/NOS2) in response to the TLR3 ligand dsRNA, despite high levels of iNOS mRNA, in SHP-1-deficient compared to wild type littermate glia. Nitric oxide (NO) production induced by dsRNA was also significantly less in SHP-1-deficient glia compared to normal littermate cells. Because both iNOS expression and NO production are controlled by polyamine concentrations within cells, it was important that we observed abnormally high constitutive expression and activity of arginase I, in cultured glia of SHP-1-deficient compared to wild type mice. Arginase I converts arginine to ornithine thus constituting an important biosynthetic step in the production of polyamines within cells. As expected, increased exogenous polyamines decreased NO production and increased TMEV replication in CNS glia. Accordingly, IL-4 and IL-13, two cytokines known to induce arginase I, also increased TMEV replication. These findings provide the first evidence of regulation of arginase I and NO production by SHP-1 in CNS cells via pathways that may involve polyamine synthesis. Thus, we propose that SHP-1 is a critical regulator of polyamine synthesis and iNOS expression important in innate immune responses to pathogen infections in CNS cells. This work was supported by a grant from the NIH(NINDS).

90

Poster # 6

B cell Activation Through TLR9 Signaling Potentiates Gammaherpesvirus Infection

Catherine Ptaschinski, Romana Hochreiter, Nancy Fiore and Rosemary Rochford Department of Microbiology and Immunology

SUNY Upstate Medical University, Syracuse, NY

Epstein-Barr Virus (EBV) and Kaposi’s Sarcoma Associated Herpes Virus (KSHV) are members of the gammaherpesvirus family. Because EBV and KSHV can replicate only in cells of human origin, experimental animal models are needed to understand the pathogenesis of these viruses. We have been using the well-characterized animal model, murine gammaherpesvirus-68 (MHV-68) infection of mice to study gammaherpesvirus pathogenesis. Memory B cells are the primary site of gammaherpesvirus latency. However, the mechanism by which these cells become infected in vivo is unclear. MHV-68 is present in splenic B cells within six days post-infection in vivo, but does not readily infect these cells ex vivo. We therefore wished to determine what factors are involved in the infection of B cells. Murine B cells express both TLR4 and TLR9, whose ligands are bacterial lipopolysaccharide (LPS) and unmethylated CpG DNA, respectively. Purified mouse B cells were treated with either 1 µM CpG DNA or 25 µg/ml LPS, or were left untreated. After 24 hours, these cells were infected with a recombinant MHV-68 that expresses GFP. Cells were then visualized by microscopy and flow cytometry to determine the frequency of GFP expressing cells. We found that only those cells stimulated with CpG or LPS became infected with the virus. In a second experiment, purified B cells were treated with CpG and LPS concurrent with viral infection. No infection was seen as evidenced by lack of GFP expression. These data suggest that activation of the B cells is required for MHV-68 infection. Studies are underway to further identify the factors necessary for efficient infection of B cells.

91

Poster # 8

CD4+CD25+ Treg Cells Partially Contribute to IL-10-mediated Leishmania major Susceptibility, but Only in the Absence of IL-4 Receptor Signaling

Hisashi Nagase and Nancy Noben-Trauth

The George Washington University Medical Center, Washington, DC We have demonstrated that IL-4 receptor alpha (IL-4Ra)-deficient BALB/c mice remain highly susceptible to infection with Leishmania major substrain LV39 due to residual levels of IL-10. Therefore, we addressed whether IL-10-secreting CD4+CD25+ regulatory T cells (Treg) mediate susceptibility of IL-4Ra-/- mice to L. major LV39. The role of Treg cells in L. major infection has not been fully resolved, and their activation may depend on the site of infection or L. major substrain used. BALB/c IL-4Ra mice were infected in the footpad with 105 L. major LV39 parasites and injected with 1 mg anti-CD25 (clone PC61.5) weekly. After 6 weeks infection, anti-CD25 treated IL-4Ra mice had a 200-fold decrease in the number of L. major parasites per mg of infected tissue and 50-fold decrease of parasite number in the draining lymph node. In the same experiment, anti-CD25 treatment of wild-type BALB/c mice with did not show any appreciable decrease in parasite number, even though the percentage of CD4+CD25+ T cells were comparably reduced in the draining lymph node. In dramatic contrast to anti-CD25 treatment, IL-4Ra mice treated with anti- IL-10R mAb had no detectable parasites in the footpad, confirming our previous results and emphasizing the importance of IL-10 in mediating L. major susceptibility. To investigate the immune responses at the site of infection, we used an ear dermis infection model established by Yasmine Belkaid. In the infected IL-4Ra ear dermis, we found a two-fold increase in the percent of CD4+CD25+ T cells compared to BALB/c mice. Even more striking, we found a significant percentage of Class II+ cells secreting IL-12p40 in L. major-infected IL-4Ra ear dermis. Despite the apparent production of IL-12p40, along with decreased Th2 responses, IFNg levels remained at basal levels in the draining lymph node of IL-4Ra mice. Taken together, these results imply that in the absence of IL-4Ra, susceptibility may be mediated, in part, by CD4+CD25+ T cells. In intact BALB/c mice, the mechanisms of susceptibility most likely involve both IL-4/IL-13 and IL-10, which may be secreted by Treg cells, other CD4+ T cell subsets, as well as non-T cell populations.

92

Poster # 9

Effects of Physiological Thermal Stress on Tumor Vascular Function

Daphne A. Scott*, John T. Schueckler*, Bonnie L. Hylander, William Kraybill, Elizabeth A. Repasky

Departments of Immunology and Surgery, Roswell Park Cancer Institute, Buffalo, NY 14263

Tumor vasculature is structurally and functionally abnormal in several different respects, which can create a barrier to efficient delivery of therapeutic agents to the interior of the tumor. For more effective delivery to occur, a therapeutic drug must be able to access a functional tumor microvessel and must be able to pass unidirectionally from the vessel to the tumor bed and remain there long enough to cause tumor regression. However, the high interstitial pressure in tumors results in many vessels being compressed and non-functional; moreover, abnormal intratumoral pressure and vessel leakiness prevents a sufficient build-up of drug in the tumor stroma. Strategies for facilitating vascular extravasation and therapeutic delivery include increasing vascular pressure to reperfuse tumor vessels and increasing overall release of drug from blood vessels to the tumor microenvironment. Our laboratory has shown that a fever-range whole body hyperthermia (FR-WBH) exposure exerts an anti- tumor effect and that this is in part due to enhanced delivery of immune effector cells to the tumor bed. In this context, an increase in tumor vessel diameters has also been seen. We hypothesize that thermal sensitivity of blood vessels (due to a natural response to either conserve or lose heat) is responsible for these alterations in blood flow. Previous work in our lab supports this hypothesis, and suggests that FR-WBH can also be used to overcome barriers to drug delivery. We have demonstrated that FR-WBH enhances the anti-tumor efficacy of liposome encapsulated doxorubicin (DOXIL) in both the syngeneic CT26 mouse tumor model as well as in human tumor xenograft/ SCID mouse models and suggests that this effect is mediated by changes in tumor vasculature. Dye perfusion studies show that treatment with FR-WBH increased the percentage of perfused vessels in the tumor bed when tumors are grown in a subcutaneous location. We have now extended and confirmed these initial studies and have examined the effect of WBH on vascular perfusion in the 4T1 orthotopic breast tumor model as well as determining the effect of WBH on tumor hypoxia. Initial data reveals a 47 percent increase in perfused vessels per field in subcutaneous tumors treated with FR-WBH and a 53 percent increase in perfused vessels per field in orthotopic tumors treated with FR-WBH. These data are now being correlated with areas of tumor hypoxia. This work was supported by NIH grants CA94045 and CA71599.

*These authors contributed equal amounts of work to this project.

93

Poster # 10

Immunomodulatory Activities of LT-IIa and LT-IIb Enterotoxin Adjuvants Depend Upon Binding to Surface Receptors Other Than Ganglioside GM1

Hesham Nawar, Sergio Arce, Michael W. Russell, and Terry D. Connell

Department of Microbiology and Immunology The Witebsky Center for Microbial Pathogenesis and Immunology

University at Buffalo, Buffalo, NY, United States, 14214 BACKGROUND: Administration of antigens (Ags) to the mucosa often fails to elicit strong protective responses. Thus, there is a need to develop new adjuvants for potentiating immune responses to mucosally administered Ags. LT-IIa and LT-IIb, the Type II heat-labile enterotoxins of Escherichia coli, are structurally related to cholera toxin and LT, yet have distinctive receptor-binding affinities and adjuvant properties. CT and LT-I bind to ganglioside GM1; LT-IIa binds avidly to GD1b, less well to GD1a, and with low affinity to GM1; LT-IIb binds essentially only to GD1a. While the distinctive adjuvant properties of LT-IIa and LT-IIa have been reported, the role of ganglioside-binding in their adjuvant activity has not been investigated. METHODS: LT-IIa(T34I) and LT-IIb(T13I), two non-toxic mutant enterotoxins with no detectable binding for GD1a, GD1b, or GM1 were used to investigate the dependence of adjuvant activity upon ganglioside binding. Mice were immunized intranasally with a bacterial protein alone or in combination with LT-IIa, LT-IIa(T34I), LT-IIb or LT-IIb(T13I). RESULTS: LT-IIa(T34I) exhibited no propensity to elicit early systemic or mucosal adjuvant activity, but primed mice for potent Ag-specific systemic memory responses. LT-IIb(T13I) possessed adjuvant activity which was equivalent to that exhibited by the wt GD1a-binding LT-IIb. Flow cytometry showed that LT-IIa(T34I) had no affinity for splenic lymphocytes. LT-IIb(T13I) retained binding activity for T cells, B cells, dendritic cells, and macrophages. Binding of LT-IIb(T13I) was diminished on macrophages pre-treated with periodate or neuraminidase or cultured in the presence of PDMP, a reagent which inhibits ganglioside synthesis. CONCLUSIONS: The receptor required for adjuvant activity by LT-IIb(T13I), and likely by wt LT-IIb, is an unidentified glycosphingolipid. These results suggest that LT-IIa and LT-IIb have potential use as safe mucosal adjuvants for human application.

94

Poster # 11

Immunomodulatory Effect of CpG ODNs on a Live Vaccine Against Cutaneous Leishmaniasis

Susan Mendez1, Wenhui Wu1 and Yasmine Belkaid2

1Dept. of Microbiology, Immunology and Tropical Medicine; The George Washington University, Washington DC

2Laboratory of Parasitic Diseases; NIAID, NIH, Bethesda MD Leishmania major (Lm) is the causative agent of zoonotic cutaneous leishmaniasis, the most widely distributed form of cutaneous leishmaniasis (CL) in the Old World. Despite numerous attempts to develop a vaccine against CL, the inoculation of live, non-attenuated Lm to produce a lesion that heals, referred to as “leishmanization” is the only vaccine against ZCL that provides life long protection to date. We have recently developed a live vaccine that contains live Lm parasites and immunostimulatory oligodeoxynucleotides (CpG ODNs). The addition of CPG ODNs to the vaccine reduced or completely eliminated the lesion due to the vaccination without compromising long term protection. In order to determine the mechanism, we vaccinated C57Bl/6 healing mice and extracted DCs and Treg at different time points to evaluate their phenotype and function. Our data revealed that the addition of CpG ODNs to the live vaccine induced an increased influx of effector cells to the vaccination site during the first two weeks after vaccination. We also detected a transient downregulation of Treg function, since although they maintained their proliferative capacity, were unable to produce IL-10. Moreover, we proved that DC activation was enhanced by the presence of CpG ODNs in the vaccine cocktail. Suppression of Treg function seems to be at least partially mediated by an enhanced IL-6 production by the DCs. Neutralization of IL-6 partially restored Treg function in vivo and in vitro. Understanding the mode of action of CpG ODNs in live vaccination against Leishmania and the effect on Treg may be useful to establish the functional basis to design other vaccines containing CpG ODNs, such as recombinant or DNA vaccines.

95

Poster # 12

NK and CD8 T Cells Dominate the Response by Human Peripheral Blood Mononuclear Cells to Inactivated Francisella tularensis Live Vaccine Strain (LVS)

Edmund J. Gosselin, Diane R. Gosselin, and Steven A. Lotz

Center for Immunology and Microbial Disease, MC-151, Albany Medical College, Albany, NY 12208

Francisella tularensis is a category A bio-threat agent, and thus has recently generated significant research interest. F. tularensis LVS is an attenuated form of the virulent F. tularensis organism, and has previously been utilized as a vaccine. However, due to safety concerns, it is no longer approved for this purpose. Thus, the use of inactivated organisms is preferable for vaccine purposes. While, numerous studies have been done examining human PBMC, and in particular CD4 T cell, responses to inactivated F. tularensis, there has been no study identifying the individual human cell populations within a mixed PBMC population, which respond to this organism. Thus, we sought to address this deficit. In contrast to previous studies, our results indicate that NK and CD8 T cells comprise the majority of cells responding to F. tularensis LVS. In addition, data suggest CD8 T cell responses are maximal, when utilizing antibiotic-treated organisms, and minimal when using formaldehyde-fixed organisms. Given the belief that CD8 T cells can play an important role in protection against F. tularensis infection, these studies have direct relevance to the development of F. tularensis vaccines using inactiva ted organisms. In addition, significant new knowledge is added to our understanding of the human immune response to F. tularensis LVS.

This work was supported by the National Institutes of Health, Grant number P01 AI056320.

96

Poster # 13 Active Immunization with SdrG Reduces Staphylococcus epidermidis Infection in a Murine

Challenge Model

Adrienne Scott, Bret Sellman, Yekaterina Timofeyeva, Jasdeep Nanra & Steve Baker Wyeth Vaccine Research, Pearl River, NY 10965, USA

Staphylococcus epidermidis is a major nosocomial pathogen and is commonly associated with indwelling medical devices infections. A foreign object implanted into the body is quickly coated with various plasma proteins, including fibronectin and fibrinogen.

S. epidermidis possesses a number of proteins that contribute to specific adhesion to these molecules. One such protein is SdrG (Fbe), a 119 kDa fibrinogen binding protein localized on the surface of S. epidermidis and is present in most strains of S. epidermidis. Using a mouse model of infection, the immunogenicity and functionality of antibodies to either the N1N2N3 (amino acids 50-597) or N2N3 (amino acids 273-597) domains of the N-terminal A region of rSdrG were investigated. Immunizing mice with either form of rSdrG produced an antibody response that crossreacted with both; however, only mice vaccinated with SdrG(N2N3) exhibited a significant reduction (p<0.01) in the number of S. epidermidis recovered from the spleen. Our data suggest that SdrG shows potential as a vaccine antigen to protect against infections caused by S. epidermidis.

97

Poster # 14

Recombinant VSV Vectors Expressing the HSV-2 gD Antigen are Immunogenic and Protective in Mice

Min Guo, David Cooper, Priscilla C. Calderon, Kevin J. Wright, Farooq Nasar, David Clarke,

Aaron Abramovitz, Narendar Kalyan, Jacek Kowalski, Michael Hendry, John Eldridge, Stephen Udem, and Robert J. Natuk

Wyeth, Pearl River NY

Currently, HSV-2 vaccine design has focused around the premise that cellular immune responses directed against HSV-2 antigen are fundamental for vaccine efficacy. A variety of experimental vectored vaccine approaches have been investigated. Here we describe the use of the recombinant vesicular stomatitis virus (rVSV) vector platform. This approach offers an attractive mechanism for the induction of robust cellular and humoral immune responses directed against selected human pathogen target antigens. Two rVSV vectors were constructed by cloning the HSV-2 gD gene into the VSVIndiana genome (rVSVIndiana-gD) and into a VSVIndiana genome that had the Indiana G gene replaced with the G gene from the related Chandipura virus (rVSVChandipura-gD). The creation of rVSV vectors with two different serotypes permits the boosting vector to circumvent the neutralizing antibodies generated against the priming vector. BALB/c mice were primed with rVSVChandipura-gD and boosted with rVSVIndiana-gD to evaluate their ability to induce anti-HSV-2 immune responses and to protect recipient mice from a lethal vaginal HSV-2 challenge. Mice immunized with rVSV-gD responded with a Th1 polarized anti-gD cellular response (as measured by HSV-2 specific CTL lysis and IFN-? expression) and a Th1 polarized anti-gD humoral immune response (as measured by strong anti-gD IgG2a titers and strong anti-HSV-2 neutralization titers). Intranasal administration of rVSV-gD induced stronger cellular and humoral immune responses than those responses induced by intramuscular administration. Furthermore, a single intranasal administration of rVSV-gDChandipura induced protective immunity against a lethal HSV-2 vaginal challenge, whereas mice immunized intramuscularly required both priming and booster immunizations for protection. These prototypic VSV vectors, without further attenuation, have proven to exhibit some morbidity and mortality in mice when inoculated intracranially. Efforts are underway to generate further attenuated rVSV vectors that are less virulent but still retain their immunogenicity. In summary, rVSV-gD vectors were employed to elicit anti-HSV-2 Th1-like cellular and humoral immune responses that were protective in a mouse challenge model.

98

Poster # 16 Atypical expression of JNK/SAPK in mouse neutrophils reveals a selective requirement for

JNK2 in Toxoplasma gondii-induced IL-12 release

Woraporn Sukhumavasi and Eric Denkers College of Veterinary Medicine, Cornell University NY

The mitogen-activated protein kinase (MAPK) family member c-Jun N-terminal kinase (JNK)/stress-activated MAP kinase (SAPK) is involved in cellular stress and defense responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are thought to be ubiquitously expressed, but JNK3 is largely restricted to tissues of the brain, testis and heart. Here, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been little studied. We employed anti-MAPK Western blot ana lysis to examine expression patterns of JNK/SAPK in wild-type and JNK2-/- polymorphonuclear leukocytes (PMN). Surprisingly, neut rophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed equally high levels of both JNK1 and JNK2 MAPK. Lack of JNK1 was found in thioglycollate-elicited neutrophils purified from the mouse peritoneal cavity, and the same expression pattern was apparent in PMN isolated from the bone marrow of normal mice. We used PMN infection with the protozoan Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced PMN JNK2 phosphorylation within 10 min of infection, a response that was sustained for up to 60 min. Transwell experiments suggested that JNK2 activation was a response to secreted tachyzoite factors rather than to infection itself. Furthermore, chemical blockade of JNK signaling, as well as use of JNK2-/- neutrophils, revealed that this MAPK family member was required for optimal PMN IL-12 production. These findings are important because 1, they reveal a previously unrecognized nontypical expression pattern of JNK in mouse neutrophils, and 2, they demonstrate a requirement for JNK2 in PMN IL-12 production in response to a microbial pathogen.

99

Poster # 17

Superoxide Dismutase-B (sodB) Deficient Mutants of Francisella tularensis are Hypersensitive to Oxidative Stress and Defective in Macrophage Colonization

Chandra S Bakshi, Meenakshi Malik, Kevin Regan, Vitaly Pavlov*,

J. Andres Melendez and Timothy J. Sellati Center for Immunology and Microbial Disease Albany Medical College, Albany NY 12208

*State Research Center for Applied Microbiology, Obolensk, Russia

Francisella tularensis (F. t.), the etiologic agent of tularemia is a small gram-negative

coccobacillus. Based on its high infectivity and the ease of intentional aerosol dissemination F. t. is designated as a category A biological agent. Its ability to invade and multiply in a wide range of eukaryotic cells suggests that F. t. harbor a powerful genetic machinery to adapt to the hostile intracellular environment. The intracellular survival of F. t. is dictated by its ability to produce a battery of antioxidants such as superoxide dismutases (SODs), catalases and peroxidases. SODs play a major role in protection against oxidative stress by catalyzing the dismutation of superoxide (O2

-) to hydrogen peroxide (H2O2) that is generated during aerobic and intracellular growth. Two types of SODs have been identified in F. t., a constitutively expressed cytoplasmic FeSOD encoded by the sodB gene and a periplasmic CuZnSOD encoded by the sodC gene. The FeSOD protects organisms from endogenously generated O2

- while the CuZnSOD protects them from exogenous O2

-. In other bacterial pathogens SODs are putative virulence factors, however, in F. t. a similar role has not been confirmed.

To characterize the role of F. t. SODs in virulence we constructed a mutant strain with altered sodB gene (F. t. ~sodB) activity by allelic replacement. The F. t. ~sodB mutant was characterized with respect to its ability to survive under various acellular growth conditions and oxidative stresses and its ability to invade MH-S cells, a murine alveolar macrophage cell line. In contrast to the wild type F. t. strain, F. t. ~sodB mutants exhibited significantly reduced FeSOD activity and increased sensitivity to paraquat, H2O2 and peroxynitrite. After four hours of co-incubation with MH-S cells one log fewer F. t. ~sodB mutants were recovered than wild type F. t. To conclude, the present study demonstrates that FeSOD in F. t. is essential for aerobic growth, resistance to redox cycling compounds such as paraquat, H2O2, preoxynitrite and for the invasion of and/or survival within macrophages. The potential of F. t. ~sodB mutant as a vaccine candidate is under investigation in a mouse model of pneumonic tularemia.

100

Poster # 21

Virion-Associated HLA-DR Enhancement of HIV-DR Pathogenesis

Cherie Fontaine and Karen Duus Center for Immunology & Microbial Disease, Albany Medical College

HIV/AIDS has become a pandemic of unimagined proportions, infecting 14,000 people/day. In order to develop effective antiviral therapies virus-host cell interactions must be understood in depth. Many enveloped viruses incorporate host cell proteins into their envelope when budding from infected cell membranes. In the last decade it has become evident that incorporation of host cell proteins into HIV-1 may confer a selective advantage to the virus for survival in the host. The host cell MHC class II molecule, specifically HLA-DR (DR), is ubiquitously incorporated into the HIV-1 envelope. MHC class II on the virion envelop has been found to cause an increase in viral replication and cell death in PBMCs. Our hypothesis is that incorporation of DR enhances HIV-1 pathogenesis in the thymus by increasing viral replication and enhancing apoptosis in uninfected bystander cells. The levels of class II molecules on the three different cell lines used in this study were assessed by immunofluoresence microscopy and flow cytometry. Producing various HIV-1 virus strains in cells containing or lacking class II molecules allowed the assessment of the pathogenicity conferred by different levels of these molecules on the virion envelope. Pathogenicity was quantified by detecting apoptotic cells and also by trypan blue staining. It was determined that infection of the cell lines DR+ CEM-T4 and DR- SUPT-1 with MHC class II+ virions caused higher rates of death via apoptosis and necrosis and also produce higher infectious titers. Clones of SUPT-1 and A293T cells stably transfected with the MHC II transactivator, and expressing different levels of MHC class II will also be used to test whether the levels of the molecule on the virion’s envelope will have an effect on the pathogenicity caused by it.

Thymocytes do not normally express high levels of HLA-DR. To determine the consequences of virion-associated DR on the initial infection of DR- cells, a marker-virus engineered to express the murine heat-stable antigen (CD24), which is incorporated into the infected cell membrane, was generated in DR+ and DR- cells, and used to infect DR- SUPT-1 cells. The results suggested that expression of DR on the initial infecting virion strongly influenced subsequent pathogenicity. Also, we were able to detect CD24 transferred to SUPT-1 cells when infecting virions fused with the target cells. Our results to date suggest that DR in the virion’s envelope confers a selective advantage ove r DR- virions. DR incorporation into the HIV-1 envelope may have the same effect in human thymus tissue cultured ex vivo, and will be investigated in future experiments.

101

Poster # 22

Chronic Foot-shock Induced Changes in Cytokine Production: Possible Neuroimmune Connections to Schizophrenia

Ling Cao and Jan A. Moynihan

University of Rochester, Rochester, New York IL-2, a critical cytokine in T lymphocyte activation and function, has been suggested to play an important role in the pathophysiology of the psychotic disorder schizophrenia. Both increased serum IL-2 levels and decreased mitogen-stimulated IL-2 production from peripheral blood mononuclear cells (PBMC) have been repeatedly reported in schizophrenia patients. Furthermore, a shift towards a Th1 cytokine profile in schizophrenic patients has also been found in terms of the IFN-gamma/IL-4 ratio. Previously, we have observed hyper- locomotor activity in chronically foot-shocked (CFS) adult male BALB/c mice. Since hyper- locomotor activity in rodents is often thought to be a behavioral manifestation of psychotic and schizophrenia- like disorders, we investigated the possible changes of IL-2 production in the CFS model both in vivo and in vitro. Adult male BALB/c mice subjected to CFS were singly housed in shock boxes, while control mice were housed in their home cage (HC). Foot-shock was delivered randomly 6 to 14 times per day (random period of time between 5 to 30 sec at 0.5 mA) for 5 weeks. Both spleen and whole brain IL-2 levels were significantly elevated at the early stage of CFS treatment (peaked at day 2 in the spleen and week 1 in the brain) compared to HC control animals. However, IL-2 was not detectable in serum in both HC and CFS mice. Additionally, splenocytes from HC and CFS mice at weeks 1, 2, 4 and 6 were isolated and stimulated with Conconavalin A (Con A) in vitro. IL-2 production was significantly decreased from cells in CFS mice compared to those in HC control mice at weeks 1, 2 and 4. Significantly decreased IL-4 and IL-10 levels in CFS mice were also detected in vitro at weeks 1 and 2, while decreased IL-6 levels were found at week 1. No significant differences in IFN-gamma production were found. Altogether, CFS induces changes in cytokine and behavior similar to the clinical profile observed in schizophrenia patients. Thus, CFS may be used to further investigate the underlying neuroimmunological mechanisms in schizophrenia.

102

Poster # 26

Alterations in CD8+ T cell Priming via the Direct and Cross Presentation Pathways

Amanda M. Schell*, Frank Koczot† and Christopher C. Norbury* * Dept. of Microbiology and Immunology, MC H107,

Penn State M.S. Hershey College of Medicine, Hershey, PA 17033. † Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National

Institutes of Health, Bethesda MD, 20892-0440, USA CD8+ T cells (TCD8+) play a crucial role in immunity to viruses. Antiviral TCD8+ are initially activated by professional APCs (pAPC) that are directly infected by viruses (direct priming) or following uptake of exogenous antigen transferred from virus infected cells (cross priming). To date, potential differences in the efficiency of TCD8+ priming via each of these pathways, along with variation in the phenotype of the resulting effector and memory TCD8+, have not been thoroughly examined. In this study we examine the induction of antigen-specific TCD8+ generated following immunization with recombinant adenoviruses that express influenza A virus nucleoprotein under the control of tissue-specific promoters. We show that the induction of TCD8+ in response to tissue-targeted antigen is less efficient than the response to ubiquitously-expressed antigen, and that the resulting primary and memory TCD8+ home primarily to the draining lymph node. Reduced duration of presentation via the cross priming pathway correlates with reduced differentiation of CD8+ T cells into memory cells. These results have significant implications for the design of vaccine vectors intended to induce protective TCD8+.

103

Poster # 27 IL-12 Conditions Antigen Induced CD8+ T cell Responses for Homeostatic Expansion in a

STAT4 Dependent Manner

Qingsheng Li and Protul A. Shrikant Department of Immunology, Roswell Park Cancer Institute

Elm and Carlton Streets, Buffalo, NY 14263 Host deterrence of intracellular infectious challenge requires differentiation of antigen specific CD8+ T cells into effector and memory cells. Memory cells are thought to be selected from the pool of antigen induced effector cells with increased capacity for survival and sensitivity to cytokines like IL-7 and IL-15 that augment Lymphopenia-Induced Proliferation (LIP). Evidently, IL-12 programmed effector differentiation of antigen activated CD8+ T cells requires STAT4 and our observations confirm this understanding. However, the impact of IL-12 conditioning for survival and homeostatic expansion remains relatively uncharacterized. Our results demonstrate that IL-12 render “fitness” to antigen activated CD8+ T cells for survival via increased Bcl3 and Bcl2 expression and sensitize them to IL-7 and IL-15 by enhancing the expression of IL-7Ra, IL-15Ra, CD122 and CD132. The observed increase in IL-12 induced CD8+ T cell “fitness” stringently requires STAT4. As predicted by these observations, IL-12 conditioned CD8+ T cells have heightened capacity for homeostatic expansion in a STAT4 dependent manner. These results demonstrate that the ability of IL-12 to enhance “fitness” for homeostatic expansion is STAT4 dependent and suggests that the presence of IL-12 during antigen presentation may achieve greater CD8+ T cell memory.

104

Poster # 28

The Role of STAT4 in IL-21 Mediated Antigen Specific CD8+ T cell Responses

Cheryl Eppolito and Protul A. Shrikant Roswell Park Cancer Institute, Department of Immunology

Buffalo, NY 14263 The recently identified cytokine gc cytokine IL-21 produces durable tumor immunity when administered in vivo. To better understand the molecular mechanisms that regulate IL-21 induced CD8+ T cell responses, we tested the impact of IL-21 on naïve TCR transgenic CD8+ T cells activated with latex microspheres bearing MHC/peptide and chimeric B7.1. Our results demonstrate that IL-21 addition rapidly activates STAT4, but unlike IL-12 cannot maintain the enhanced phosphorylation state of STAT4 for more than 2 hours. Like IL-12, IL-21 did not promote antigen dependent proliferation but could augment IFNg production and CTL maturation of CD8+ T cells. The increased effector differentiation was accompanied with reduction of CD44 whereas increased IL-7Ra expression. However, the IFNg production achieved with IL-21 was not as robust as that observed with IL-12. Surprisingly, loss of STAT4 abrogated the IL-12 but not the IL-21 mediated effector differentiation into effector cells. However, the IL-21 induced IL-7Ra and CD44 expression was reduced on antigen activated STAT4 deficient CD8+ T cells. In addition, IL-21 promoted survival of antigen activated CD8+ T cells by enhancing the expression of Bcl3 and survivin mRNA, the loss of STAT4 did not affect the CD8+ T cell survival. These observations indicate that the activation of STAT4 by IL-21 does not directly regulate antigen specific CD8+ T cell responses but may be involved in combination with other STAT’s like STAT3 by forming heterodimeric complexes.

105

Poster # 30

IL-21 Regulates CD8+ T cell Responses in an Antigen Dependent Manner

Protul A. Shrikant, Cheryl Eppolito, and Qingsheng Li Roswell Park Cancer Institute Department of Immunology

Buffalo, NY 14263 The family of cytokines that utilize the common gamma chain for signaling T cell responses includes the recently identified cytokine IL-21. Our recent publication indicates the superior capacity of IL-21 than IL-2 or IL-15 to achieve durable tumor immunity in vivo. To better understand the mechanisms by which IL-21 regulates CD8+ T cell responses we have used a reductionist approach in which naïve TCR transgenic CD8+ T cells are activated with inert antigen presenting constructs in the absence or presence of various gamma chain cytokines. The results obtained demonstrate that in comparison to IL-2 and IL-15, IL-21 robustly activates STAT 1, 3, 4 and weakly the STAT 5 pathways, both in the presence or absence of antigen stimulation. The dose dependant IL-21 signaling in the absence of antigen stimulation does not promote homeostatic proliferation but significantly reduces IL-7Ra and CD44 expression without altering CD62L expression, whereas in the presence of antigen stimulation it increases IL-7Ra, CD44 and CD62L expression. Moreover, IL-21 treatment of naïve CD8+ T cells in the absence of antigen induces IFN? production although no CTL functions were noted. In contrast, IL-21 was potent at promoting CTL maturation, but only high dose of IL-21 could support the transient IFN? production achieved by antigen stimulation. The continuing investigations are designed to demonstrate the molecular mechanisms underpinning these observations and the biological implications of our observations.

106

Poster # 31

The Cellular Immune Response to Human Papillomavirus E7: A Possible Predictor of Viral Persistence and Delayed Cervical Disease Regression

Rhonda Kines,1 Kunle Odunsi,2 Helen Trottier,3 Sukbong Koh,4 Janet Kornegay,5 Shashikant

Lele,2 Eduardo Franco,3 Yasmin Thanavala1 1 Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY, USA

2 Department of Gynecological Oncology, Roswell Park Cancer Institute, Buffalo, NY, USA

3 Division of Cancer Epidemiology, McGill University, Montreal, Canada 4 Catholic University of Daegu, School of Medicine,

Department of Obstetrics and Gynecology, Daegu, Korea 5 Roche Molecular Diagnostics, Inc., Alameda, CA, USA

Most women infected with human papillomavirus (HPV) clear their infection, however, a small percentage will develop chronic infection that can lead to cervical cancer. In this prospective, longitudinal study, we analyzed the time to cytological regression and time to clear HPV infection and determined if there was a correlation to the in vitro T-cell proliferative responses to HPV-16 E7 in 107 women. Women were examined every 3-6 months for a period of 32 months. At each visit, Papanicolaou cytology and HPV DNA testing were performed and blood samples were collected for antigen specific in vitro lymphocyte stimulation assays. Kaplan-Meier survival analysis was used to estimate time to lesion regression and the time to HPV clearance as determined by patient immune response to E7. Although not statistically significant, we observed a longer mean and median time to disease regression among immunological responders compared to non-responders with either ASCUS/LSIL or HSIL. We also observed a delay in time to clear viral infection for immunological responders compared to non-responders infected with an HPV-16 related type or a high-risk type other than 16 or 16 related however this was not statistically significant. Interestingly, only immunological non-responders infected with HPV-16 were able to clear their infection at a more rapid rate and in greater number when compared to immunological responders (p=0.0439). Thus, the E7 specific immune response imparts little help towards lesion regression or viral clearance. This information is of value when considering E7 as a target antigen in therapeutic vaccine design.

107

Poster # 32

Neurotransmitter down-regulates adaptive immunity to Listeria

R.T. Emeny, J. Hornickel, J. Ault, T. Mondal, D. Gao and D.A. Lawrence Wadsworth Center, NYSDOH, Albany, NY

The generation of an immune response against both infectious and immunizing agents may be substantially modified by an increase in allostatic load as can occur with chemical, physical and/or psychological stressors. Our previous studies have demonstrated that one-hour cold/restraint (CR), a psychological and physical stressor, preceding bacterial challenge with Listeria monocytogenes (LM) suppresses host resistance. The stress-related factors involved in this immunomodulation are derived from activation of the sympathetic nervous system and release of norepinephrine (NE). Beta1- and beta2-adrenergic receptor ($1AR and $2AR) deficient mice have been employed to demonstrate that the negative effects of NE on host defenses are delivered through $1AR. The mechanisms involved in the NE-$1AR-immunosuppression is posited to be related to cellular changes in reduction-oxidation (redox) potential of certain immune cells. CR reduced the amount of surface sulfhydryls on peripheral blood T cells, B cells, and NK cells. Surprisingly, CR increased the glutathione content of monocyte/macrophages. In vitro exposure of peritoneal macrophages to NE increased production of nitric oxide. NE also preferentially enhanced the IL-4 to IFN( ratio of stimulated spleen cells. We hypothesize that CR enhances early innate mechanisms, including NO release, which inhibits efficient activation of Th1 cells by shifting them into an unresponsive state as well as generating an environment more conducive for humoral immunity, as suggested by Murata and colleagues.

108

Poster # 33

Vaccination with Inactivated F. tularensis LVS Protects Against Respiratory Tularemia

Shawn D. Baron and Dennis W. Metzger Center for Immunology and Microbial Disease, Albany Medical Center, Albany, NY

Francisella tularensis (F.t.) is a small, gram-negative intracellular pathogen that causes severe and often fatal disease, tularemia. It has been considered a potential biological weapon owing to its virulence even at very low doses when inoculated intranasally (i.n.). Vaccination attempts against tularemia have been focused primarily on the use of low doses of an avirulent strain of F. t., known as live vaccine strain (LVS), which has been shown to provide protection in mice against subsequent challenge with virulent strain. In the present study, we performed experiments to determine if inactivated F.t. LVS induces protection against subsequent challenge with high doses of LVS in mice. The F.t. LVS was inactivated either by 2% paraformaldehyde fixation, heat inactivation, or UV-irradiation. Six to eight week old BALB/c and C57Bl/6 mice were immunized i.n. with 108 CFU of fixed F. t. with IL-12 as an adjuvant. A total of two boosts were given days 14 and 28 following primary immunization. Unvaccinated mice were used as controls. All of the mice were challenged i.n. with a lethal dose (104 CFU) of live F.t. LVS at day 35 of the primary immunization. Both the UV-irradiated and heat inactivated vaccines provided 90-100% protection in C57Bl/6 and BALB/c mice. However, only 30% of the BALB/c mice vaccinated with 2% paraformaldehyde fixed bacteria were protected as compared to 100% of C57Bl/6 mice. All unimmunized C57Bl/6 mice, as opposed to only 50% of the BALB/c mice succumbed to the lethal challenge. Although the inactivated vaccine could induce protection in both strains of mice, this protection was not correlated with increased systemic antibody responses. Further studie s are in progress to elucidate the mechanism of protection and to determine whether the inactivated vaccines can protect against type-A F.t. Schu S4.

109

Poster # 35

Proteomic Analysis and Molecular Characterization of in Dermatophagoides farinae Induced Allergic Inflammation and Sensitization

Chih-Lung, Chen1 and Chun-Keung Yu2.

1Division of tissue engineering, BMEC, ITRI, Hsinchu, Taiwan 310, ROC. 2Department of Microbiology and Immunology, College of Medicine, National Cheng-Kung University,

Tainan, Taiwan 310, ROC.

The dust mite (Dermatophagoides farinae; Der f) is a major source of inhaled allergens that increase the risk of developing asthma. We carried out two-dimensional electrophoresis (2DE) analysis and established BALF protein profiles of mice undergoing Der f, lipopolysaccharide (LPS), and ovalbumin (OVA) challenge. Approximately 250-300 spots were represented on the 2DE after challenge. Some spots were associated to a particular challenge. But the majority of the induced spots overlapped of all these three experimental treatments. (81.5 % of Der f, 89.6 % of LPS and 97.4 % of OVA). Repetitively Der f challenged mice revealed a Th2 pheno type that included infiltration of neutrophils, eosinophils, and periodic acid Schiff-positive goblet cells around the blood vessels, and abundant pulmonary levels of epithelial growth factor and GATA3 mRNA. Mice challenged only a single time with Der f displayed a qualitatively similar, but quantitatively lessened, response, as well as an acute inflammatory response that included clustering of several acute phase proteins, host defense proteins, antioxidant enzymes, and cytoskeletal proteins. However, fo llowing the increase of Der f-challenge time, airway had eosinophilia combined with production of acute phase proteins mediated the oxidative-antioxidant driven chronic airway inflammation. On the other hand, systemic prime Der f to mice elicited more severe airway inflammation. 2DE results of the Der f sensitized mice demonstrated unique expression patterns of complement C3, inducible cytokines, and chemokine ligand. The increased knowledge of protein expression in this asthmatic model system may help identity candidate therapeutic targets.

110

Poster # 36

Concurrent Intestinal Helminth Infection Provides Protection from Influenza Infection

Stephen C. Jones, Peter Sayles, Susan L. Swain Trudeau Institute, Saranac Lake, NY

Intestinal helminth infections such as pinworm and roundworm remain endemic in many areas of the world that are also stricken with influenza infection every year. Given that parasitic helminthes generally elicit a strong Th2 type immune response that has been shown in numerous models to nonspecifically influence bystander responses, we sought to understand how concurrent intestinal helminth infection would affect immunity to influenza. To this end, C57Bl/6 mice were orally infected with the intestinal nematode Heligmosomoides polygyrus (H. polygyrus) 8 days prior to influenza infection. H. Polygyrus is a natural mouse parasite whose life cycle is completely restricted to the intestinal submucosa and lumen, allowing us the unique opportunity to study the influence of concurrent infections in two distinct anatomical locations. Remarkably, we found that infection with H. polygyrus protected animals from a lethal dose of the WSN-OVAII (H1N1) recombinant influenza virus, and that this correlated with a significant decrease in viral RNA copies in the lungs of dual infected mice. This protection is dependent upon the host CD4+ T cell response to H. polygyrus, as protection is lost in dual infected CD4+ T cell knock out animals. To directly assess how dual infection influences the anti- influenza response, B6 mice were adoptively transferred with naïve OTII transgenic CD4+ T cells prior to H. polygyrus infection. These cells respond specifically to the WSN-OVAII virus based upon its engineered expression of their cognate OVA323-339 epitope. Although we observed similar numbers of activated OTII CD4+ T cells in the spleen, lymph nodes and lungs of animals with and without concurrent H. polygyrus infection, there was significantly less IFNg production in the spleens and lungs of dual infected animals. The finding of higher flu specific IgG1 antibody titers in these animals supported these observations. Thus, in this model, concurrent intestinal helminth infection protects mice from influenza infection, despite a trend towards a more Th2 type antigen specific influenza response.

111

Poster # 37

The role of TNF-a in IL-12-dependent IFN-? production from NK cells

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

Stimulation of splenocytes or lung lymphocytes by IL-12 together with heat-killed pneumococci (HKP) resulted in effective production of IFN-g. We also found that i.n. IL-12 treatment can only induce increased level of IFN-g in BALF when there was also pulmonary pneumococcal infection. However, simulation of splenocytes with IL-12 or HKP alone did not induce IFN-g production, nor was significant IFN-g production in BALF of BALB/c mice after treatment with IL-12 or pneumococci alone. Addition of neutralizing Ab specific for TNF-a completely inhibited the production IFN-g even in presence of IL-12 and HKP. In agreement with these findings, stimulation of spleoncytes from either TNF-a-/- or TNF-R-/- mice with IL-12 plus HKP did not result in IFN-g protection. However mixing splenocytes from TNF-a-/- and TNF-R-/- mice resulted in enhanced production of IFN-g which could be completely inhibited by adding anti-TNF-a neutralizing Ab. Using flow cytometry analysis, it was found that the majority of cells able to produce IFN-g after stimulation with IL-12 and HKP were NK cells. Splenocytes from SCID mice, which have high percentage of NK cells, produced greatly enhanced levels of IFN-g after stimulation with IL-12 and HKP compared to normal mice. As demonstrated by RT-PCR analysis, stimulation of splenocytes with IL-12 plus HKP resulted in increased transcription of IFN-g but not IL-12R compared to incubation with IL-12 or HKP alone. Taken together, these data suggest that TNF-a is a cofactor for IL-12 induced production of IFN-g by resting NK cells during pneumococcal infection.

112

Poster # 41

Novel Mechanisms of Immunomodulatory Activity by Type II Enterotoxins Depend on

Toll-like Receptor 2

Michael W. Russell, Terry D. Connell, Richard I. Tapping, and George Hajishengallis. Department of Microbiology and Immunology, University at Buffalo, Buffalo, NY

Department of Microbiology, University of Illinois, Urbana, IL Department of Microbiology, Immunology, and Parasitology, Louisiana State University,

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 is thought to be critical, and the role of toxic enzyme activity within the A1 subunits is controversial. Our recent findings that ganglioside-binding mutants of LT-IIa or LT-IIb may retain adjuvant activities, and that recombinant chimeric proteins containing antigen coupled to CTB subunit are immunogenic, not tolerogenic, suggest other mechanisms of immunomodulation besides those involving known ganglioside receptors or toxic enzyme activity due to the A1 subunits. The abilities of intact enterotoxins or B subunits to stimulate monocytic/macrophage cells in vitro were examined by release of inflammatory cytokines and by NF-kappaB activation. LT-IIa, LT-IIb, and CT regulated cytokine induction in LPS-activated cells. However, in contrast to CT, neither LT-IIa nor LT-IIb alone stimulated cytokine release in human THP-1 cells or mouse macrophages. B subunits of LT-IIa and LT-IIb, but not CTB, stimulated cytokine production that was specifically inhibited by antibody to TLR2. Transfected HEK 293 cells that expressed TLR2 (and TLR1) responded to stimulation by LT-IIa-B or LT-IIb-B with activation of NF-kappaB. Macrophages from mice genetically deficient in TLR2 could not be stimulated by LT-IIa-B or LT-IIb-B. Type II enterotoxins exert adjuvant activity by novel mechanisms that act in part through TLR2. Together with our other findings, these results reveal novel mechanisms of immunomodulation by type II enterotoxins involving pathways independent of toxicity and conventional ganglioside receptors, and offer the prospect of developing safe and effective mucosal adjuvants.

113

Poster # 42

Screening for Toxoplasma gondii Regulated Transcriptional Response in LPS-Activated Macrophages

Chiang W. Lee and Eric Y. Denkers

Cornell University, College of Veterinary Medicine, Ithaca, NY Toxoplasma gondii infected macrophages (Mf) are blocked in production of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-a) upon activation with lipopolysaccharide (LPS). Here, we used pathway-focused cDNA arrays to identify additional T. gondii regulated transcriptional responses. Parasite infection decreased 54 (inclusive of IL-12 and TNF-a) and increased expression of 7 of 77 LPS-activated cytokine and cytokine-related genes. Interestingly, we found that the LPS-induced transcriptional response of the anti- inflammatory cytokine IL-10 was synergistically increased by T. gondii, results that we validated by conventional RT-PCR and ELISA. Importantly, although the parasite exerted disparate effects in LPS-signaling leading to TNF-a versus IL-10 production, both responses required functional Toll- like receptor 4 (TLR4). We suggest that these effects may represent defense mechanisms to avoid or delay induction of antimicrobial activity and/or T cell-mediated immunity during Toxoplasma infection.

114

Poster # 43

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

Shoshana Katzman and Deborah Fowell

Department of Microbiology and Immunology David H. Smith Center for Vaccine Biology and Immunology,

Aab Institute for Biomedical Sciences University of Rochester, Rochester, New York 14642

Effector function of activated CD4+ T cells is an integral component in determining the course of disease. Differentiation of these cells 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 differentiation. Surprisingly, within the draining lymph nodes (DLN) of susceptible Balb/c mice, single cell assays reveal a heterogeneous population containing both IL-4, and, potentially anti-leishmaniacidal, IFNg producing cells. Preliminary data indicate an early 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 heterogeneous DLN. There are many possibilities to account for this restriction. One possibility is differential recruitment/exclusion of cytokine producing cells at the tissue site of infection. Preliminary studies show that IFNg-producing cells can be found in other tissues of the body at this early time-point, but not at the tissue site of infection. This suggests that CD4+ effector T cells capable of combating Leishmania major infection are generated within the lymph node and are able to traffic to the periphery, but are unable to be recruited to or retained at the site of infection. A second possibility is that there are differential signals needed for the release of effector function once at the tissue site of infection. We have a unique model to track IL-4 transcription using IL-4 reporter mice (4get) in which further up-regulation of GFP is indicative of cytokine production. At the tissue site of infection we find a further up-regulation GFP in comparison to that seen in the DLN, indicating a transcriptional control at the tissue site of infection. These data suggest differential signaling between the lymph node and the site of infection, which results in a modification of CD4+ T cell effector function.

115

Poster # 44

Fever-Range Whole Body Hyperthermia Prevents the Onset of Autoimmune (Type 1) Diabetes in Nonobese Diabetic Mice

Maegan L. Capitano, Bradley R. Ertel, Elizabeth A. Repasky, and Julie R. Ostberg

Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263

Administration of a fever-range whole body hyperthermia (WBH) has been shown to

have various immunoregulatory functions in mice, including the ability to delay tumor growth in a manner that appears to be dependent on NK cells. Interestingly, NK cells have also recently been found to play a role in the ability of complete Freund’s adjuvant (CFA) to prevent diabetes in nonobese diabetic (NOD) mice. This led to the hypothesis that, similar to CFA, fever-range WBH can prevent NOD mice from becoming diabetic in a manner dependent on NK cells. To test this theory, fever-range WBH (39.5oC, 8 hrs) was administered weekly for thirty-two weeks in combination with weekly blood glucose monitoring. It was observed that fever-range temperatures could prevent the onset of diabetes without a significant increase in diabetes incidence after the termination of treatment. However, when mild WBH was administered once a month for eight months, no preventative benefit was observed. The results indicate that, like CFA, certain WBH treatment schedules can prevent the onset of Type 1 diabetes in NOD mice. However, whether this affect is NK cell dependent or involves other regulatory mechanisms remains to be determined.

Supported by NIH PO1 CA94045 and R21 CA098852

116

Poster # 45

Metallothionein Gene Affects Cadmium-induced Decreasing T cell-dependent humoral Immune Response in Mice

Guang-Bi Jin, Xiuyun Yin, and Michael A. Lynes

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT

Cadmium is an industrial and environmental pollutant that can act as potent suppressor of immune function. One of the stress response protein synthesized by cells in response to cadmium is metallothionein (MT), a low molecular weight, cysteine-rich protein that binds to heavy-metal cations and can protect cells from the toxic effects of these metals. A number of reports indicate that cadmium toxicity is enhanced in MT gene knockout mice. We have shown that MT can significantly influence immune functions both in vivo and in vitro. Mice that have a targeted gene disruption of Mt1 and Mt2 display an augmented humoral response compared to normal wild type controls. We hypothesized that elevated MT level might also result in altered immune function. To test this hypothesis, we employed three stains of mice, C57BL/6J (denoted WT), C57BL/6J-TgN (Mt1)174Bri (denoted TgN), and C57BL/6J –Mt1tm1Bri Mt2tm1Bri (denoted KO), which share the same genetic background. These mice were exposed to 100 µM cadmium chloride or zinc chloride in the drinking water for 45 days. This dose was selected as the highest dose tested that caused no changes in the gross indications of health status of the mice. Vehicle control mice received only tap water. Three days after the start of treatment, each mouse was immunized intra-peritoneally with chicken ovalbumin (OVA) and boosted with a second injection of OVA on day 13. Serum samples were collected on day 0, 17, 24, 31,38 and 45. Cadmium treatment decreased serum anti-OVA antibody level in KO mice, while both cadmium and zinc treatments reduced serum anti-OVA antibody level in TgN mice. At the dose used, neither cadmium nor zinc affected anti-OVA response in WT mice. Treatment of the three strains mice with cadmium or zinc did not effect total serum IgG or serum IgM, nor did it effect the CD3+, Ig+, CD4+, or CD8+ subpopulations of cells. Our results suggest that KO mice experience immunosuppression by cadmium due to an absence of MT and consequent damage to the cell’s functions. TgN mice were also immunosuppressed by cadmium, which may have resulted from the release of MT to the circulation. Normal immune responses exist in the context of an optimum level of MT. When MT levels are elevated beyond this optimal range by toxicant exposure, significant declines in immune function may ensue. These observations may suggest avenues by which various stressors (e.g. metals, irradiation, infection and autoimmune disease) produce changes in immune capacity. This research was supported by the NIEHS grant #ES07408

117

Poster # 46

Defects in TCR Signals for IFNg Cytokine Secretion in the Absence of the Wiskott-Aldrich Syndrome protein (WASp) are Rescued by APC Interactions

Vanessa Morales-Tirado, Sara Johansson, Shoshana Katzman & Deborah Fowell

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14624

Uncommitted CD4+ T cells can polarize into two distinct groups, Th1 and Th2, with discrete cytokine profiles. Different components of the T helper cell differentiation process can change the strength and duration of signals. The Immunological Synapse has been suggested to play a role in the stabilization of signals upon T cell: APC conjugate formation. Synapse formation is cytoskeleton-driven, allowing the rearrangement and segregation of molecules at the T cell: APC interface. The Wiskott-Aldrich Syndrome protein (WASp) is a key regulator of the actin cytoskeleton. In the absence of WASp, actin polymerization and cytoskeletal movement, hence synapse formation is affected. We previously reported a striking defect in IFNg cytokine release by WASp -/- CD4+ T lymphocytes upon TCR-derived signals in an APC-independent system. Here, we investigated IFNg cytokine release in an APC-dependent system in the absence of WASp. Surprisingly, signals, other than the TCR, provided by Wild type (WT) APCs enable WASp-deficient CD4+ T cells to secrete IFNg cytokine. Despite in vitro restoration of IFNg secretion, Th1 responses remained defective in vivo in WAS-/- mice. Using Leishmania major infection, where protection requires a Th1 response, WASp-deficient mice were unable to clear pathogen. This was not due to enhanced Th2 responses but reflected a reduced IFNg capacity. Thus, in vivo, additional defects remain in the absence of WASp. From these experiments we concluded signals derived from TCR in vitro, in an APC-independent system, are not enough to restore cytokine secretion defect in the absence of WASp. Provision of WT APCs restores the cells ability to secrete cytokine, bypassing the necessity of WASp. Our in vivo data shows WASp deficient mice are not able to heal and clear L. major, an intracellular pathogen requiring a Th1 response, despite being able to produce the required cytokine. Our results suggest this could be due to defects in the magnitude of cytokine produced, not being enough to promote clearance of pathogens, or defects in lymphocyte trafficking. In humans, defects in WASp leads to problems with both humoral and cellular immunity. Humans cannot recover from bacterial and viral infections, suggesting defects in clearance of intracellular pathogens (defective Th1 response).

118

Poster # 48

A Proteomics Approach to Characterizing Human Colon Tumor

Sensitivity to Killing by Apo2L/TRAIL

Marlieke de Bruijn1, Sarah Hejaily2, Dr. Latif Kazim3, and Dr. Elizabeth Repasky1

1Department of Immunology, 2Department of Molecular and Cellular Biophysics and Biochemistry, 3Department of Cell Stress Biology

Roswell Park Cancer Institute, Buffalo, NY

There is considerable interest in identifying molecules that can selectively induce death of tumor cells while sparing normal cells. Currently, there is enthusiasm for the clinical potential of the cytokine, Apo2L/TRAIL (Tumor Necrosis Factor Apoptosis-Inducing Ligand), because this molecule has been shown to induce apoptosis in a large number of human tumor cell lines, both in vitro and in vivo, while sparing normal cells. However, it is also clear that a significant number of long established malignant cell lines are resistant to Apo2L/TRAIL. This implies that the response of actual patients will be variable. Our lab has found that Apo2L/TRAIL is able to significantly suppress growth of some tumors, however, some patients’ tumors are more resistant to killing by Apo2L/TRAIL. Currently, studies that evaluate the sensitivity of a patient’s tumor to Apo2L/TRAIL involve measuring tumor size in mice over time as the treatment (or a control) is administered. Subsequent analyses to elucidate the mechanism of sensitivity/resistance rely on measuring the relative levels of individual proteins known to be present in the death receptor-activated apoptotic pathway. Instead of looking for single proteins that are known to be relevant to indicate sensitivity or resistance after treatment with Apo2L/TRAIL, we are interested in analyzing many proteins simultaneously. Our approach is to use 2-D DIGE (differential gel electrophoresis) and protein profiling of whole tumor sections with MALDI (matix-assisted laser desorption- ionization) to study protein differences between Apo2L/TRAIL-sensitive and –resistant tumor specimens. Our hypothesis is that human tumors can be characterized as Apo2L/TRAIL-sensitive or Apo2L/TRAIL-resistant based on proteomic studies with 2-D DIGE or from protein profiles of whole tissue sections acquired with MALDI. By acquiring protein profiles of tumors and identifying individual proteins of interest from these panels, we could increase our understanding of the proteins responsible for determining whether a patient’s tumor would respond to treatment. Ongoing 2-D DIGE experiments reveal that there are differences between tumors of varying sensitivity to Apo2L/TRAIL. We have begun to identify this panel of proteins and future studies will investigate the function of these proteins as how they relate to Apo2L/TRAIL sensitivity in human colon tumors.

119

Poster # 51

Pyrin-Only Protein 2 (POP2): A Novel Modulator of NF-kappaB

Jonathan A. Harton, Laurel L. Sandler, and Felipe Bedoya Department of Medical Microbiology and Immunology and the Immuology Program,

H. Lee Moffitt Cancer Center, University of South Florida, Tampa, FL 33612

Proteins containing Pyrin and Caspase recruitment (CARD) domains have been implicated in the regulation of inflammation and apoptosis through activities influencing NF-kB activation, IL-1β production, or both. Pyrin domain containing proteins include members of the CATERPILLER (CLR) family (also known as NOD, NALP, and NACHT), Apoptotic Speck Containing Protein (ASC), and the Mediterranean Fever protein Pyrin. Homotypic interactions between pyrin and CARD domains participate in the assembly of both the so-called “apoptotic speck” seen during apoptosis and the inflammasome complex which liberates the active form of IL-1β . ‘Pyrin-only’ proteins are attractive as negative regulators of pyrin domain-mediated functions and one such protein, POP1, has been reported. We have identified and initially characterized a second Pyrin-only protein (POP2), more highly similar to the pyrin domains of the CLR family than to the prototypic pyrin domains of pyrin and ASC. POP2 is a single exon gene located on human chromosome 3 (3q28). The gene is 294nt in length and codes for a 97 amino acid polypeptide. A large number of putative phosphorylation sites, suggest the potential for kinase-mediated regulation of POP2. Phylogenetic analysis confirms that POP2 is highly similar to CLR pyrin domains and unlikely to be a paralog of POP1. POP2 is expressed principally in leukocytes. Localization studies reveal that POP2 is expressed throughout the cell. In vitro, POP2 inhibits the activity of p65 NF-kB in a dose-dependent fashion. Accordingly, the activity of tumor necrosis factor alpha (TNFa; a strong NF-kB agonist) is also repressed by POP2. Together these observations suggest that POP2 acts to inhibit NF-kB and NF-kB dependent processes.

120

Poster # 52

Early Kinetic Window of Target T cell Susceptibility to CD25+ Regulatory T cell Activity

Dorothy K. Sojka, Angie Hughson, Teresa L. Sukiennicki, and Deborah J. Fowell

Department of Microbiology and Immunology David H. Smith Center for Vaccine Biology and Immunology,

Aab Instistute for Biomedical Sciences, University of Rochester, Rochester, NY

Peripheral tolerance is maintained in part by thymically-derived CD25+ CD4+ T cells (Tregs). Their mechanism of action has not been well characterized. Therefore, to get a better understanding of Treg action we investigated the kinetics of murine Treg activity in vitro. Tregs were suppressive within a surprisingly narrow kinetic window: necessary and sufficient only in the first 6-10h of culture. Visualization of this timeframe, using a sensitive single-cell assay for IL-2, revealed the early elaboration of target cell IL-2 producers in the first 6 h despite the presence of CD25+ CD4+ Tregs. However, after 6h, a rapid rise in the number of IL-2 producers in the absence of Tregs was dramatically abrogated by the presence of Tregs. Importantly, the timing of suppression was dictated by the kinetics of target T cell activation suggesting that early target T cell signals may alter susceptibility to suppression. Modulating target T cell activation signals with provision of CD28, IL-2 or high antigen dose all abrogated suppression of proliferation late in culture. However, only CD28 signals enabled target T cells to resist the early Treg- induced downregulation of IL-2. Therefore the quality of early target T cell activation signals, in particular engagement of CD28, represents an important control-point in the balance between vulnerability and resistance to Treg suppression.

121

Judith Appleton, Ph.D. Cornell University Jaa2@cornell.edu Sergio Arce, Ph.D. University at Buffalo arce@buffalo.edu Byron Au-Yeung, M.S. University of Rochester Byron_auyeung@urmc.rochester.edu Vickie Babineau, B.S. University of Connecticut Darryn.unfricht@huskymail.uconn.edu Chandra Shekhar Bakshi, D.V.M., Ph.D. Albany Medical College bakshis@mail.amc.edu Corey Balinski, M.S. University of Connecticut cbalins@yahoo.com Shawn Baron, B.S. Albany Medical College Barons@mail.amc.edu Dawn Bellville Albany Medical College bellvid@mail.amc.edu Constantine Bitsaktsis, Ph.D. Wadsworth Center cbitsakt@wadsworth.org Seth Blumerman, Ph.D. Trudeau Institute sblumerman@trudeauinstitute.org Deborah Brown, Ph.D. Trudeau Institute Dbrown@trudeauinstitute.org Barbara Butcher, Ph.D. Cornell University Bab26@cornell.edu

NYIC 2005 Participant Contact Information

Adriana Caballero, M.S. Albany Medical College caballa@mail.amc.edu Ling Cao, Ph.D. University of Rochester Ling_Cao@urmc.rochester.edu Maegan Capitano Roswell Park Cancer Institute Maegan.capitano@roswellpark.org Chih-Lung Chen, Ph.D. Industrial Technology Research Institute Ccl0805@yahoo.com.tw James Clements, Ph.D. Roswell Park Cancer Institute James.clements@roswellpark.org Terry D. Connell, Ph.D. University at Buffalo Connell@ucsu.buffalo.edu Marlieke de Bruijn, B.S. Roswell Park Cancer Institute Marlieke.debruijnL@roswellpark.org James R. Drake, Ph.D. Albany Medical College drakej@mail.amc.edu Douglas Durrant, B.S. Albany Medical College durrand@mail.amc.edu Richard Dutton, Ph.D. Trudeau Institute Dutton@northnet.org Karen Duus, Ph.D. Albany Medical College duusk@mail.amc.edu Charlotte Egan, Ph.D. Cornell University Cee22@cornell.edu

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Rebecca Emeny, Ph.D. Wadsworth Center Rte1@health.state.ny.us Cheryl Eppilito, Ph.D. Roswell Park Cancer Institute Cheryl.eppilito@roswellpark.org Loren Erickson, Ph.D. University of Virginia Lde9w@virginia.edu Sharon Evans, Ph.D. Roswell Park Cancer Institute Sharon.evans@roswellpark.org Aracelis Fernandez, M.D. Albany Medical College fernana@mail.amc.edu Nancy Fiore, B.S. SUNY Upstate fioren@upstate.edu Cherie Fontaine, B.S. Albany Medical College Fontaic@mail.amc.edu Jeffrey Frelinger, Ph.D. University of North Carolina jfrelin@med.unc.edu Rachel Gerstein, Ph.D. University of Massachusetts Rachel.gerstein@umassmed.edu Matthew Giannandrea, M.S. University of Rochester Matthew_giannandrea@urmc.rochester.edu James Gorham, Ph.D. Dartmouth Medical School James.D.Gorham@Dartmouth.edu Edmund Gosselin, Ph.D. Albany Medical College gossele@mail.amc.edu

Min Guo, M.S. Wyeth Guom1@wyeth.com Guang-ming Han, Ph.D. Wadsworth Center hanguangmingdoctor@yahoo.com.cn Sean Harrison, Ph.D. Millennium Pharmaceuticals, Inc. Harrison@mpi.com Jonathan Harton, Ph.D. University of South Florida jharton@hsc.usf.edu Sarah Hejaily, B.S. Roswell Park Cancer Institute Sarah.hejaily@roswellpark.org Romana Hochreiter, Ph.D. SUNY Upstate hochreir@upstate.edu David Holowka, Ph.D. Cornell University Dah24@cornell.edu Chad Hudson, B.S. SUNY Upstate Hudsonc@upstate.edu David Janik Wadsworth Center Djanik@wadsworth.org Guang-Bi Jin, Ph.D. University of Connecticut Guang-bi.jin@uconn.edu Beena John, Ph.D. University of Rochester Beena_john@urmc.rochester.edu Stephen Jones, Ph.D. Trudeau Institute Sjones@trudeauinstitute.org

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Cris Kamperschroer, Ph.D. Trudeau Institute ckamperschroer@trudeauinstitute.org Dennis L. Kasper, M.D. Harvard Medical School Dennis_Kasper@hms.harvard.edu Shoshana Katzman, M.S. University of Rochester Shoshana_katzman@urmc.rochester.edu Girish Kirimanjeswara, D.V.M., Ph.D. Albany Medical College kirimag@mail.amc.edu Adrienne Kisailus, B.S. Roswell Park Cancer Institute Adrienne.kisailus@roswellpark.org Richard Konz, Ph.D. University of Massachusetts Richard.Konz@umassmed.edu Ryan Larson, B.S. Trudeau Institute rlarson@trudeauinstitute.org David Lawrence, Ph.D. Wadsworth Center David.Lawrence@wadsworth.org Chiang Lee, Ph.D. Cornell University Cwl23@cornell.edu Qingsheng Li, Ph.D. Roswell Park Cancer Institute Qingsheng.li@roswellpark.org Edith Lord, Ph.D. University of Rochester Edith_Lord@urmc.rochester.edu Amit Lugade, B.S. University of Rochester Amit_lugade@urmc.rochester.edu

Eithne MacMahon, Ph.D. Kings College London Eithne.macmahon@kcl.ac.uk Helen Mahler, B.S. University at Buffalo hmmahler@buffalo.edu Paul Massa, Ph.D. SUNY Upstate massap@upstate.edu Alex Maue, Ph.D. Trudeau Institute amaue@trudeauinstitute.org Susana Mendez, Ph.D. George Washington University mtmsxm@gwumc.edu Dennis W. Metzger, Ph.D. Albany Medical College metzged@mail.amc.edu Jeff Mills, B.S. SUNY Upstate Millsjh@upstate.edu Vanessa Morales-Tirado, M.S. University of Rochester Vanessa_morales@urmc.rochester.edu Hesham Nawar, M.S. University at Buffalo hfnawar@acsu.buffalo.edu Bisweswar Nandi, Ph.D. Wadsworth Center bnandi@wadsworth.org Michael Nazareth, B.S. University at Buffalo Nazareth@buffalo.edu Thien Nguyen, B.S. SUNY Upstate nguyent@upstate.edu

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Nancy Noben-Trauth, Ph.D. George Washington University nnoben@gwu.edu Chris Nobury, Ph.D. Penn State College of Medicine Ccn1@psu.edu Sofia Olmos, Ph.D. Albany Medical College olmoss@mail.amc.edu Nina Pabello, Ph.D. Wadsworth Center npabello@aol.edu Linda Paredes, M.S. Roswell Park Cancer Institute Linda.paredes@roswellpark.org Michelle Parent, Ph.D. Trudeau Institute mparent@trudeauinstitute.org Andras Perl, Ph.D. SUNY Upstate perla@upstate.edu Karsten Pilones, B.S. SUNY Upstate pilonesk@upstate.edu Aparna Prasad Wadsworth Center Gainaparna@rediffmail.com Catherine Ptaschinski, B.S. SUNY Upstate ptaschic@upstate.edu Rachael Racine, B.S. Wadsworth Center Rachaelr23@yahoo.com Deepak Rawool, D.V.M., Ph.D. Albany Medical College rawoold@mail.amc.edu

Rosemary Rochford, Ph.D. SUNY Upstate rochforr@upstate.edu Michael Russell, Ph.D. University at Buffalo russellm@buffalo.edu Viktoriya Rybalko, B.S. University of Rochester Viktoriya_rybalko@urmc.rochester.edu Bikash Sahay, D.V.M., Ph.D. Albany Medical College Bikashs@mail.amc.edu Felix Santiago, B.S. University of Rochester Felix_santiago@urmc.rochester.edu Adrienne Scott, M.S. Wyeth Pharmaceuticals Scotta3@wyeth.com Daphne Scott Roswell Park Cancer Institute Daphne.scott@roswellpark.org Matthew Seavey, M.Sc. University of Rochester seavey@rocketmail.com Brahm Segal, M.D. Roswell Park Cancer Institute Brahm.segal@roswellpark.org Timothy J. Sellati, Ph.D. Albany Medical College sellatt@mail.amc.edu Protul Shrikant, Ph.D. Roswell Park Cancer Institute Protul.shrikant@roswellpark.org Rajendra Singh, D.V.M., Ph.D. Albany Medical College singhr@mail.amc.edu

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Dorothy Sojka University of Rochester Dorothy_sojka@urmc.rochester.edu Elizabeth Sorensen, B.S. University of Rochester Elizabeth_Sorensen@urmc.rochester.edu Woraporn Sukhamavasi Cornell University Ws66@cornell.edu Keer Sun, M.S. Albany Medical College sunk@mail.amc.edu Monisha Sundarranjan, Ph.D. La Jolla Institute Monisha@liai.org Susan Swain, Ph.D. Trudeau Institute sswain@trudeauinstitute.org Yasmin Thanavala, Ph.D. Roswell Park Cancer Institute Yasmin.thanavala@roswellpark.org Seana Thrasher, M.S. Cornell University Smt26@cornell.edu David Topham, Ph.D. University of Rochester David_topham@urmc.rochester.edu Darryn Unfricht, M.S. University of Connecticut Darryn.unfricht@huskymail.uconn.edu Jeff Ward, B.S. SUNY Upstate Wardj@upstate.edu Justin Wilson, B.A. Albany Medical Center Wilsonj@mail.amc.edu

Gary Winslow, Ph.D. Wadsworth Center Gary.winslow@wadsworth.org David Woodland, Ph.D. Trudeau Institute dwoodland@trudeauinstitute.org Eric Yager, Ph.D. Trudeau Institute Eyager@trudeauinstitute.org Xiuyun Yin, Ph.D. University of Connecticut Xiuy2002@yahoo.com Jeffrey Yu, B.S. University at Buffalo Jjyu@buffalo.edu Kristin Zaffuto, M.S. University of Connecticut Kristin.zaffuto@uconn.edu

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The Sagamore Bolton Landing, NY

Once again we would like to thank the staff of The Sagamore for their exceptional service and for hosting this event.

Pat Jarett, Conference & Catering Coordinator

Lori Rehm, National Sales Manager Don Vilmar, Banquet Manager

Joel and the Housemen Banquet Captains

Concierge Staff Housekeeping

Sagamore Dining Room Staff Wait Staff

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Conference Evaluation Form

Please rate all items between 1 and 5 as follows: 1= Greatly Satisfied, 2= Mostly Satisfied, 3= Somewhat Satisfied, 4= Disappointed, 5= Very Disappointed Pre-Conference Arrangements 1) Ease of on-line registration _____ 2) Web site information _____ 3) Questions answered by phone or email _____ Conference Content 1) Scientific Content _____ 2) Keynote Presentations _____ 3) Poster Session/Vendor Fair/Mixer _____ 4) Session Themes _____ 5) Session Format (15 min. presentation/5 min Q & A) _____ 6) Selection of Speakers (PIs, Postdocs, Students) _____ Conference Organization 1) Ease of Check-in _____ 2) Organization of Conference Program _____ 3) Comfort of room/accommodations _____ 4) Meeting room set-up & organization _____ 5) Amount of recreational/social time allotted _____ 6) Additional day/shorter days _____

Feedback For the Future Did you like having more recreational/free time? Would you like more organized activities? The conference will continue to be held in October, next year’s dates are October 15-18, 2006. Do you plan on attending?

Please complete back of page

8th Annual Upstate New York Immunology Conference Sagamore Hotel and Conference Center, Bolton Landing NY

October 9-12, 2005

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Name 2-3 Keynote Speakers (Please provide first and last names and their affiliation.) that you would be interested in hearing at future meetings? Do you know anyone who would like information on future meetings? If so, please provide contact information (email, phone, etc.). What did you like least about the meeting? What did you like best about the meeting? Comments: Name: ____________________________________ Email: _______________________________________________

Please hand survey to Dawn Bellville prior to leaving the conference or mail/fax to:

NYIC Attn: Dawn Bellville Albany Medical College, MC-151 47 New Scotland Ave. Albany NY 12208 or Fax to (518) 262-5748, attention Dawn

Thank you for your time in completing this evaluation.