15th annual upstate new york immunology conference · search environment. last, ... mock study...
TRANSCRIPT
Major
Corporate Sponsor:
BD Biosciences
Grant
Funding:
NIH
National Institute
of Allergy and
Infectious Diseases
Trainee Awards:
American
Association of
Immunologists
15th Annual
Upstate
New York
Immunology
Conference
2012
October 21-24, 2012
The Sagamore Resort and Conference Center Bolton Landing, NY
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ANNUAL UPSTATE NE W YORK IMMUNOLO GY CONFE RENC E (NYIC) We’ve come a long way from Garnet Hill! This meeting started in 1997 as a small retreat to facilitate interactions among young scientists, institutions, and renowned experts in the field of Immunology. In just a few short years, the number of attendees grew and a larger venue was needed to meet the future needs of the Conference. This year we have a few exciting elements for attendees. As we did last year, there will be an opportunity to win one of two iPads. The raffle is open to all trainees. You must be present at the drawing to win! We are happy to announce the American Association of Immunologists (AAI) is providing 20 Young Investigator Awards. Awards will be given to those chosen for Poster Oral Presentations. There will be two Workshops given by representatives from NIH/NIAID on grants, an informative and important resource for everyone involved in today’s re-search environment. Last, but certainly not least, there will be a cruise on Lake George and an anniversary dance celebrating NYIC’s 15th year! While all these elements lend to the atmosphere, one simple principle of this Conference remains: To provide an opportunity for young and senior scientists to gather in a setting that is diverse enough to meet the needs of all attendees while remaining small enough to allow for personal interactions. While always challenging, it is the goal of the NYIC Scientific Advisory Board and the NYIC Conference Organizers to give graduate students and postdoctoral fellows the opportunity to present their research and engage in conversations that will stimulate fur-ther discussions, collaborations and interest in pursuing a new or different way of looking at their research. We hope you share our enthusiasm and enjoy your time with us!
THE SAGA MORE R ESO RT The Sagamore Resort and Conference Center celebrated it’s 125 year anniversary in 2008. If this is your first visit to the Resort, take some time to enjoy the beauty that surrounds you. Although we may have missed the grandeur of the Fall colors, there are still breath-taking views to be seen. The staff are friendly, courteous, and hard-working. If you require any information or have a special need, please see either Dawn Bellville, Administrative Conference Coordinator, or any of the Resort personnel. Many thanks to Lori Rehm (Assistant Director of Group Sales), Der-rick Hammond (Conference Services Manager), Don Vilmar (Banquet Manager), Jerid McKin-ney (Banquet Captain), Joel Clark (Function Set-up Manager) and his amazing crew, Dave Rey-nolds (CMI Communications-Audio/Visual Manager) and his crew, and all of the associates who attend to our needs. Thank you!
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Conference and Venue ................................................................................. 4 Schedule of Events ..................................................................................... 5 Platinum Corporate Sponsor BD Biosciences ...................................................................................... 14 Gold Association Sponsor American Association of Immunologists ........................................................ 16 Silver Corporate Sponsors BioLegend, Inc. ..................................................................................... 19 Cell Signaling Technologies ....................................................................... 20 DartMouse .......................................................................................... 21 eBioscience .......................................................................................... 22 Krackeler Scientific ................................................................................. 23 Life Technologies ................................................................................... 24 Bronze Corporate Sponsors Enzo Life Sciences .................................................................................. 25 FiberCell Systems, Inc. ............................................................................ 25 Worthington Biochemicals ........................................................................ 25 NYIC Scientific Advisory Board ..................................................................... 26 Institutional Financial Supporters ................................................................... 27 Grant Support .......................................................................................... 28 Keynote Speaker Gail A. Bishop, Ph.D. .............................................................................. 29 Symposium I Tumor Immunity .................................................................................... 30 Symposium II Resources for Today’s Scientific World .......................................................... 34
Table of Contents
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Workshop - Part I: NIH Presentation Types of Grants and Grant Proposals ............................................................ 37 Poster Talks Cancer Immunobiology ........................................................................... 38 Regulation of Cellular Immunology ............................................................. 44 Immunity to Human Pathogens .................................................................. 50 Immune Regulation ................................................................................ 56 Platinum Sponsor Presentation Erik Puffer, Ph.D. - BD Biosciences ............................................................. 62 Poster Abstracts ......................................................................................... 63 Symposium III Mucosal Immunity ................................................................................. 123 Symposium IV Inflammation ........................................................................................ 127 Workshop - Part II: NIH Presentation Mock Study Section ................................................................................ 131 Keynote Speaker Ralph C. Budd, M.D., Ph.D. ..................................................................... 132 Symposium V Host-Pathogen Interactions ....................................................................... 133 Symposium VI Regulation of Immune Responses ............................................................... 136 Attendee Contact Information ....................................................................... 141 Author Index ............................................................................................ 149
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15th Upstate New York Immunology Conference 2012 Schedule of Events
Sunday, October 21st 3:00-5:30 p.m. Hotel Check-In (Main Hotel Lobby)
4:30-6:00 p.m. Conference Registration (Conference Center Lobby) 6:00-6:30 p.m. Free Time 6:30-7:00 p.m. Dinner (Bellvue)
7:00-8:00 p.m. Keynote Speaker
(Bellvue) Introduction by: Jonathan A. Harton, Ph.D.
Gail A. Bishop, Ph.D. Carver College of Medicine Distinguished Professor of Microbiology & Internal Medicine Director, Interdisciplinary Graduate Immunology Program Holden Chair of Cancer Biology The University of Iowa and Iowa City VAMC
“Multiple Personalities for TRAF Molecules in Immune Regulation”
8:00-8:30 p.m. AAI Young Investigator Awards Presentations (Bellvue)
8:30-9:30 p.m. Welcome Reception (Conference Center Lobby) (Cash Bar Available)
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Monday, October 22nd 7:00-8:45 a.m. Breakfast at Leisure (La Bella Vita, Main Hotel)
9:00-10:30 a.m. Symposium I (Nirvana) Tumor Immunity Chair: Edith Lord, Ph.D. 9:00-9:30 David A. Mullins, Ph.D. (Dartmouth College) “Making Cancer Vaccines Work: Coordinating T cell Infiltration of Metastatic Melanoma” 9:30-10:00 Sandra Fernandes, Ph.D. (SUNY Upstate) “SHIP and Cancer: Novel Therapeutic Approaches via Targeted SHIP1/2 Inhibition” 10:00-10:30 Jessica L. Zourelias, B.S. (University at Buffalo) “The Aryl Hydrocarbon Receptor Plays a Critical Role
in the Prolonged Expression of IFN-γ-induced Indoleamine 2,3-dioxygenase in Dendritic Cells” 10:30-10:45 a.m. Beverage Break
10:45-11:45 a.m. Symposium II
(Nirvana) Resources for Today’s Scientific World Chair: Brent Berwin, Ph.D. 10:45-11:15 Jeffrey S. Kennedy, M.D. (Albany Medical Center) “Albany Medical Center Plan for Integrated Upstate Translational Research Program”
11:15-11:45 Michael A. Lynes, Ph.D. (University of Connecticut) “A High Content Approach to a High Content World: SPR Microarrays in Immune Analysis” 11:45-12:15 p.m. Lunch (Wapanak)
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12:15-1:45p.m. Workshop - Part I (Wapanak) NIH Presentation
Alison Deukhut Augustine, Ph.D. (NIAID) B. Duane Price, Ph.D. (NIAID) Overview of Grants Grant Applications and Process Questions and Discussion
1:45-2:15 p.m. Display Posters (Bellvue)
2:15-3:30 p.m. Poster Oral Presentations* (Albenia) Cancer Immunobiology Chair: James R. Drake, Ph.D. 2:15-2:30 Abigail L. Sedlacek, M.S. (University of Rochester) “Generation of a Dual Functioning Anti-Tumor Immune Response in the Peritoneal Cavity” (#13)
2:30-2:45 Megan Murray, B.S. (Roswell Park Cancer Institute) “CD28-mediated Pro-survival Signaling in Multiple Myeloma” (#58) 2:45-3:00 Allen Y. Chung, M.S. (University at Buffalo) “Orally-Bioavailable IL-10 Nanoparticles Reduce Intestinal Polyposis and Alleviate Inflammation-Induced Constitutional Symptoms in APC-min Mice” (#1)
3:00-3:15 Nicholas Leigh, B.S. (Roswell Park Cancer Institute) “A Novel TLR5 Agonist, CBLB502, Promotes Cytotoxic Lymphocyte Mediated Tumor Immunity” (#37) 3:15-3:30 Jason Muhitch, M.S. (Roswell Park Cancer Institute) “Overcoming Obstacles to Naïve T Lymphocyte Trafficking in Tumor-draining Lymph Nodes” (#49) *Poster abstracts selected for oral poster presentations will receive AAI Young Investigator Awards.
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2:15-3:30 p.m. Poster Oral Presentations* (Evelley) Regulation of Cellular Immunology Chair: Cynthia Leifer, Ph.D. 2:15-2:30 Weishan Huang, M.S. (Pennsylvania State University) “Functional Development of Antigen Specific Innate Memory CD8+ T cells via Selection by Hematopoietic MHC Class I” (#2) 2:30-2:45 Julie S. Lefebvre, Ph.D. (Trudeau Institute) “Impaired Function, but Not Generation, of Follicular Helper cells in Aged Mice” (#4) 2:45-3:00 Arun K. Kannan, M.S. (Cornell University) “Tec Kinase Itk Modulates Allergic Airway Inflammation by
Suppressing IFNγ(gamma) Expression in Naïve CD4+ T cells” (#36) 3:00-3:15 Daniel T. Fisher, Ph.D. (Roswell Park Cancer Institute) “Dendritic Cells Act Through IL-6 to Enhance Naïve T cell Trafficking Across High Endothelial Venules During Adaptive Immunity” (#24) 3:15-3:30 Maryann Mikucki, B.S. (Roswell Park Cancer Institute) “The Non-Redundant Role for Chemokine Receptor Cxcr3 in T cell Trafficking Into Tumor Tissue” (#15) 3:30-3:45 p.m. Beverage Break
3:45-5:00 p.m. Poster Oral Presentations* (Albenia) Immunity to Human Pathogens Chair: Kate MacNamara, Ph.D. 3:45-4:00 Donald Steiner, B.S. (Albany Medical College) “The Role of Macrophages and Neutrophils in Pulmonary Francisella tularensis Infection” (#5) *Poster abstracts selected for oral poster presentations will receive AAI Young Investigator Awards.
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4:00-4:15 Anthony J. Hickey, Ph.D. (Trudeau Institute) “Prophylactic Treatment with CpG Oligodeoxynucleotides Confers Protection Against Pulmonary and Systemic Yersinia pestis Infection” (#14) 4:15-4:30 Amanda McCabe, B.A. (Albany Medical College) “Macrophage-dependent Activation of Hematopoietic Stem Cells during Intracellular Bacterial Infection” (#54) 4:30-4:45 Egidio Torrado, Ph.D. (Trudeau Institute) “IL-27 Sustains T-bet Expression and Promotes the Development of Terminally Differentiated CD4 T cells During Tuberculosis” (#53) 4:45-5:00 William W. Reiley, Ph.D. (Trudeau Institute) “Maintenance of Peripheral T cell Responses During Mycobacterium tuberculosis Infection” (#57)
3:45-5:00 p.m. Poster Oral Presentations* (Evelley) Immune Regulation Chair: Michael Princiotta, Ph.D. 3:45-4:00 Catherine G. Burke, B.S. (University of Rochester) “Human Placental Trophoblast Cells are Hyporesponsive to Type I and Type III Interferons: Potential Role in Susceptibility of Pregnant Women to Viral Infection” (#39) 4:00-4:15 Meghan E. Bushway, M.S. (University of Rochester) “Assessment of Cytokine Responses in Human Placenta Using a Novel Whole Mount Immunofluorescence Technique” (#40) 4:15-4:30 Elise Macho Fernandez, Ph.D. (Trudeau Institute) “Lymphotoxin Controls the IL-22 Pathway to Inhibit Intestinal Inflammation” (#33) 4:30-4:45 Sesquile Ramon, Ph.D. (University of Rochester) “Specialized Proresolution Mediators Enhance Human B cell Differentiation to Antibody Secreting Cells” (#26) *Poster abstracts selected for oral poster presentations will receive AAI Young Investigator Awards.
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4:45-5:00 Jennifer Yates, B.S. (SUNY Albany School of Public Health) “IgM Memory B cells Generated During Bacterial Infection are Required for Secondary IgG Responses to Antigenic Challenge” (#47) 5:15-5:45 p.m. Dinner (Wapanak)
5:45-6:15 p.m. Platinum Sponsor Presentation
Introduction by: James R. Drake, Ph.D. Erik Puffer, Ph.D. Technical Applications Specialist BD Biosciences “Incorporating New, Bright Fluorochromes into Multicolor Panel Design” 6:15-9:30 p.m. Vendor Fair/Poster Presentation Mixer** (Bellvue)
6:45-7:45 p.m. Poster Group A (Odd Numbers) 7:45-8:45 p.m. Poster Group B (Even Numbers)
**iPad drawing during this event. Only trainees are eligible. You must be present to win!
Tuesday, October 23rd 7:00-8:15 a.m. Breakfast at Leisure (La Bella Vita, Main Hotel)
8:30-10:00 a.m. Symposium III (Nirvana) Mucosal Immunity Chair: Michael Russell, Ph.D. 8:30-9:00 Thomas H. Thatcher, Ph.D. (University of Rochester) “The Role of Pro-resolving Lipid Mediators in Attenuating Lung Inflammation”
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9:00-9:30 Alexei V. Tumanov, Ph.D. (Trudeau Institute) “Lymphotoxin-dependent Control of Inflammatory Responses in the Gut” 9:30-10:00 William O’Connor ,Jr., Ph.D. (Albany Medical College) “TH17 Cells in Health and Disease” 10:00-10:15 a.m. Beverage Break (Conference Center Foyer)
10:15-11:45 a.m. Symposium IV (Nirvana) Inflammation Chair: Jonathan A. Harton, Ph.D. 10:15-10:45 David A. Lawrence, Ph.D. (Wadsworth Center) “The Maternal Autoimmune Environment Affects Long-term Behavior in Offspring” 10:45-11:15 Cynthia Leifer, Ph.D. (Cornell University) “Nucleic Acid-Sensing Toll-like Receptor Regulation and Disease” 11:15-11:45 Lei Jin, Ph.D. (Albany Medical College) “In vivo Functions of STING in Host Defense and Vaccine Adjuvant Development” 11:45-12:00 p.m. Pick-up Box Lunch (Wapanak)
12:00-1:30p.m. Workshop - Part II (Wapanak) NIH Presentation
Alison Deukhut Augustine, Ph.D. (NIAID) B. Duane Price , Ph.D. (NIAID) Mock Study Section Panel Members: Dennis Metzger Margaret Bynoe Sharon Evans Chris Norbury
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1:30-5:00 p.m. Collaboration and Informal Discussions Leisure Activities 1:45 Meet at Dock for Cruise aboard The Morgan (Only the first 80 respondents requesting this activity may board. If you would like to be placed on the list, please see Dawn Bellville) 2:00-3:30 Scenic Cruise of Lake George Cash Bar available. Snacks provided.
5:00-6:00 p.m. Keynote Speaker
(Nirvana) Introduction by: Dennis W. Metzger, Ph.D.
Ralph C. Budd, M.D., Ph.D. Professor of Medicine Director, Vermont Center for Immunology & Infectious Disease The University of Vermont Medical Center
“Caspase Regulation of the Immune Response: the FLIP Side” 6:00-7:00 p.m. Dinner (Wapanak)
7:00-10:00 p.m. Anniversary Celebration of the (Bellvue) 15th Upstate New York Immunology Conference
Wednesday, October 24th
7:00-8:45 a.m. Breakfast at Leisure (La Bella Vita, Main Hotel)
9:00-10:30 a.m. Symposium V
(Nirvana) Host-Pathogen Interactions Chair: Terry Connell, Ph.D.
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9:00-9:30 Andrea Cooper, Ph.D. (Trudeau Institute) “Immunity to Tuberculosis – Do We Know What We Don’t Know?” 9:30-10:00 Yingru Liu, Ph.D. (University at Buffalo) “Enhancement of Adaptive Immunity to Neisseria gonnorhoeae by Inhibition of the Pathogen-induced Immunnosuppressive Mechanisms” 10:00-10:30 a.m. Break and Hotel Check out (Conference Center Foyer)
10:30-12:00 p.m. Symposium VI
(Nirvana) Regulation of Immune Responses Chair: Margaret A. Bynoe, Ph.D. 10:30-11:00 Surojit Sarkar, Ph.D. (Pennsylvania State University) “Regulation of CD8 T cell Effector and Memory Programs” 11:00-11:30 Kelvin P. Lee, M.D. (Roswell Park Cancer Institute) “Memories from the Time of Cholera” 11:30-12:00 Renee M. Laird, Ph.D.* (SUNY Upstate) “CD28 Costimulation is Dispensable for the Development and
Differentiation of γδ T Cell Effectors”
12:00-12:15 p.m. Closing Remarks and iPad Drawing**
By: James Drake, Ph.D. 12:30-2:00 p.m. Lunch & Scientific Advisory Board Meeting (La Bella Vita, Main Hotel)
Depart from Conference
**Only Trainees are eligible. Must be present to win!
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NYIC Scientific Advisory Board Institutional Representatives
Albany Medical College Jim Drake and Kate MacNamara
(NYIC Conference Organizers)
Cornell University Margaret Bynoe
Dartmouth College
Brent Berwin
Pennsylvania State University Surojit Sarkar
Roswell Park Cancer Institute
Yasmin Thanavala
SUNY Upstate Medical University Michael Princiotta
Trudeau Institute
Laura Haynes
University at Buffalo Nejat Egilmez
University of Rochester Medical Center
Edith Lord
Wadsworth Center Nicholas Mantis and William Lee
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Institutional Financial Supporters
Albany Medical College Alumni Association
Cornell University
Department of Microbiology & Immunology
Dartmouth College Department of Microbiology & Immunology
Pennsylvania State University
The Huck Institutes of the Life Sciences
Roswell Park Cancer Institute Department of Immunology
SUNY Upstate Medical University
Microbiology & Immunology Program
Trudeau Institute
University at Buffalo Department of Microbiology & Immunology
University of Rochester Medical Center
Department of Microbiology & Immunology
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Grant support provided to
Graduate Students and
Postdoctoral Fellows by the
National Institutes of Health
National Institute of Allergy and Infectious Diseases
R13AI051522
“Thank You”
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Keynote Speaker
Gail A. Bishop, Ph.D.
Carver College of Medicine
Distinguished Professor of Microbiology & Internal Medicine
Director, Interdisciplinary Graduate Immunology Program
Holden Chair of Cancer Biology
The University of Iowa and the Iowa City VAMC
Iowa City, IA 52242
“Multiple Personalities for TRAF Molecules in Immune Regulation”
The family of cytoplasmic adapter molecules known as TNF receptor associated factors
(TRAFs) were originally identified by their association with receptors of the TNFR superfamily,
for which they provide important signaling functions. It is now evident that these signaling mole-
cules serve a much wider and more diverse set of functions as participants in both membrane-
associated and cytoplasmic signaling complexes. TRAFs are now appreciated to also serve innate
immune receptors, cytokine receptors, and the TCR complex. We have found through the study of
B cell lines engineered to specifically and completely lack individual or multiple TRAFs that each
TRAF can serve distinct functions for different receptors expressed by the same cell. By producing
and studying conditional knockout mice that lack TRAF3 in specific immune cell types, we have
also learned that TRAF3 plays strikingly cell-type specific roles in immune regulation. In B lym-
phocytes, TRAF3 can either promote or constrain signaling by specific receptors, and also plays a
major role in maintenance of the balance between survival and apoptosis. In T lymphocytes, in
sharp contrast, TRAF3 plays an important positive role in TCR-mediated signaling, and shows dis-
tinct regulation of different T cell subsets. In dendritic cells, TRAF3 displays both positive and
negative signaling properties. Data illustrating each of these findings will be presented and dis-
cussed.
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Making Cancer Vaccines Work: Coordinating T-cell Infiltration of Metastatic Melanoma
David W. Mullins, Eleanor Clancy-Thompson, Matthew P. Alexander
Department of Microbiology and Immunology and Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH 03756 USA
Effective immune therapy of cancer requires infiltration of the tumor microenviron-
ment (TME) by tumor antigen (TA)-specific CD8+ T cells (TCD8), a concept demonstrated
in murine models and underscored by the prognostic significance of infiltrated TCD8 in hu-
man tumors. Infiltration of TCD8 into the TME is regulated, in part, by chemokines, yet the
spatial and temporal regulation of chemokine production in the TME remains poorly char-
acterized. We reported that the presence of circulating TA-specific TCD8 expressing the
chemokine receptor CXCR3 correlated with a survival advantage in patients with resected
stage III metastatic melanoma; further, we have observed that TCD8 cells infiltrating human
metastatic melanoma are predominantly CXCR3+. However, the induction of CXCR3+ TA-
specific TCD8 has no prognostic significance in patients with established disease. These data
suggest that CXCR3 may facilitate TCD8-mediated immune surveillance and eradication of
early metastatic lesions, but that CXCR3 is insufficient to mediate infiltration of late-stage
tumors. We hypothesized that the differential capacity of CXCR3+ TCD8 to mediate anti-
tumor efficacy may reflect the chemokine status of the TME. Therefore, we characterized
the spatial and temporal regulation of CXCR3-cognate chemokines in the metastatic mela-
noma microenvironment. In a murine model of metastatic melanoma growing in the lungs,
production of CXCR3-cognate chemokines (CXCL9, CXCL10, and CXCL11) was induced
in vascular endothelium adjacent to tumor deposits from day 6 to day 13 of tumor growth.
Chemokine production was interferon-gamma (IFN-γ)- and natural killer (NK) cell-
dependent, and the presence of chemokine correlated with the capacity of TCR transgenic
TA-specific TCD8 to infiltrate the tumor-bearing tissues in a CXCR3-dependent manner. In
late tumors (> day 13), endogenous IFN-γ and CXCR3-cognate chemokines were not de-
tected, and tumors were refractory to infiltration by TA-specific TCD8, regardless of CXCR3
expression. Exogenous IFN-γ induced CXCL9, CXCL10, and CXCL11 production in the
late stage TME and restored CXCR3-dependent TCD8 infiltration. Dysregulation of CXCR3
-cognate chemokine production in the late-stage tumor was mediated by adenosine; specific
blockade of adenosine signaling restored IFN-γ and chemokine production and infiltration
of CXCR3+ TCD8 in the TME. Thus, we conclude that early-stage and late-stage tumors are
differentially susceptible to immune-mediated infiltration and elimination by effector TCD8
as a consequence of the temporal dysregulation of IFN-γ and IFN-γ-induced chemokine
production. Based on these findings, we initiated a Phase I trial of intralesional IFN-g in
patients with dermal metastases of melanoma; our preliminary findings demonstrate that
introduction of IFN-g into the TME enhances the level of CXCL10 in tumor but not serum,
suggesting the induction of a tumor site-specific chemotactic gradient. Thus, therapies
based on active vaccination or adoptive transfer may be compromised by a paucity of
CXCR3-cognate chemotactic mediators in the microenvironment of late-stage tumor, and
restoration or induction of chemokine production in late-stage tumor may restore TCD8 infil-
tration and immune-mediated tumor control.
32
SHIP and Cancer: Novel Therapeutic Approaches via Targeted SHIP1/2
Inhibition
Sandra Fernandes,1 Gwenny M. Fuhler,1,2 Robert Brooks,1 Sonia Iyer,1
Matthew Gumbleton,1 Amanda N. Balch, 3 John D Chisholm,3 and William G Kerr 1 1Department of Microbiology and Immunology, SUNY Upstate Medical University, Syra-
cuse, NY; 2Department of Gastroenterology and Hepatology, University Medical Center
Rotterdam, Rotterdam, the Netherlands; 3Department of Chemistry, Syracuse University,
Syracuse,
New York, United States of America
We have recently demonstrated that selective inhibition of SH2 domain-containing
inositol 5‟-phosphatase 1 (SHIP1) by a small molecule 3alpha-aminocholestane (3AC), is
cytotoxic for both multiple myeloma (MM) and acute myelogenous leukemia cells in vitro.
Further biochemical analysis revealed cell cycle arrest and apoptosis were triggered in MM
cells following 3AC treatment, as determined by BrdU and AnnexinV+ staining respec-
tively. Other hallmarks of apoptosis, such as caspase and PARP cleavage, were also ob-
served. Most importantly, 3AC treatment was shown to significantly reduce tumor burden
and enhance survival in a lethal xenograft model of MM in NSG mice, while showing no
signs of morbidity or pathology associated with germline loss of SHIP1, namely eosino-
philic crystalline pneumonia and severe Crohn‟s Disease-like ileitis. As expected, the cyto-
toxity of 3AC treatment was virtually absent in cells that do not express SHIP1, such as the
AML cell line K562 or breast cancer lines MCF-7 and MDA-MB-231. Treatment of these
cells with panSHIP1/2 inhibitors, 1PIE, 2PIQ or 6PTQ, was however found to be cytotoxic.
The lead panSHIP1/2 inhibitors were shown to specifically target both the SHIP1 and
SHIP2 proteins, as they failed to inhibit OCRL, another mammalian inositol 5‟-phosphatase
in our in vitro enzymatic assays. Moreover, these panSHIP1/2 inhibitors have been found to
efficiently kill many different cancer cell lines including pediatric acute myeloid and acute
lymphoid leukemia cell lines. Finally, development of enhanced derivatives of these small
molecules with better pharmacodynamics properties has shown some promise in our enzy-
matic and in vitro cell killing assays.
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The Aryl Hydrocarbon Receptor Plays a Critical Role in the Prolonged
Expression of IFNγ-induced Indoleamine 2,3-dioxygenase in Dendritic Cells
Jessica L. Zoureliasa, Jamie L. Hardena, B. Paige Lawrenceb, & Nejat K. Egilmeza
aUniversity at Buffalo School of Medicine & Biomedical Sciences, Buffalo, NY bUniversity of Rochester School of Medicine & Dentistry, Rochester, NY
Interferon gamma (IFNγ) is the primary downstream effector cytokine induced by IL
-12, with critical roles in both innate and adaptive immunity. Along with potentiating a
strong pro-inflammatory profile, it is also a potent inducer of the immunosuppressive en-
zyme, indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DC). Studies conducted in our
laboratory have shown that the extent of the anti-tumor response elicited by a single intratu-
moral injection of IL-12/GM-CSF encapsulated microspheres is limited by IFNγ (gamma)-
driven IDO activity. Although initially induced by IFNγ (gamma), transcription of IDO in
post-therapy DC far outlasts that of IFNγ (gamma) signaling, suggesting an IFNγ (gamma)-
independent mechanism of prolonged IDO expression. To investigate the molecular
mechanisms underlying the biphasic nature of IDO expression in IFNγ (gamma)-activated
DC, our lab turned focus to the aryl hydrocarbon receptor (AhR). AhR is a ligand-activated
cytosolic transcription factor, which can induce IDO expression in tolerogenic DC. Impor-
tantly, it was recently demonstrated that the downstream metabolites of IDO activity, i.e
kynurenines, are endogenous ligands for AhR. Based on this information, we hypothesized
that IDO, once induced by IFNγ (gamma), could maintain its own expression via the
kynurenine-AhR-IDO axis. To this end, studies conducted in our laboratory show that, in
addition to IDO, IFNγ (gamma) exposure also results in the induction of AhR expression
both in splenic DC in vitro and in DC harvested from post-therapy TDLN in vivo. Further-
more, whereas the initial IFNγ (gamma)-mediated induction of IDO in DC did not require
AhR, long-term IDO expression was dependent on AhR. The results from these studies
identify an endogenous role for AhR in the IFNγ (gamma)-independent transcription of
IDO, likely through a positive feedback mechanism involving IDO-dependent production of
kynurenines.
35
Albany Medical Center
Plan for Integrated Upstate Translational Research Program
Jeffrey S. Kennedy and Vincent Verdile
Albany Medical Center, Albany, NY
In 2001, the Institute of Medicine outlined key health objectives for the 21st century.
IOM encouraged the scientific community to design new environments that enable the crea-
tion of interdisciplinary teams essential to find solutions to challenging questions necessary
to improve health outcomes. Recent data from US Medical Schools demonstrates a declin-
ing trend in the training of new clinical investigators, particularly with educational back-
grounds encompassing innovative hypothesis building, study design, data analysis, and pro-
ficiency to integrate creative interdisciplinary solutions1.
Albany Medical Center recognizes that the capital region scientific community can
be supported by enhancing the Medical College‟s effort in Clinical and Translational re-
search. We have developed a program using Design thinking2 to create a clinical research
unit that can evolve with the needs of scientists. Implementing the process will involve
creation of an incubator program, along the design of similar programs within the NIH
CTSI Institutions3, 4. The purpose is to provide a clinical resource environment at AMC that
brings together institutional knowledge in order to facilitate discovery-clinical projects.
The Clinical Center will use a human-centered approach that focuses on continuing design
experimentation that enables the Center to evolve over time. By utilizing a menu of re-
sources in 7 areas; The Conference Room; integrative hypothesis generation and Clinical
Panel; study design expertise, Grant Review; implementation, Operations Team, Data
analysis, Manuscript Review, and Translation. We expect to target and enhance specific
research teams in order to build project driven collaborative research.
1. Howard BD, David K, Steven GG. Promoting Translational and Clinical Science:
The Critical Role of Medical Schools and Teaching Hospitals. PLoS Medicine 3(9):
1492-1495.
2. Brown, T. Design Thinking. Harvard Bus Rev June 2008, p. 1-9
3. Bryne, DW et al. Clinical and Translational Research Studios: A multidisciplinary
Internal Support Program. Academic Medicine 87(8):1052-1059, 2012.
4. University of Rochester CTSI, Incubator Program; http://www.urmc.rochester.edu/
ctsi/funding/incubator-program.cfm
36
A High Content Approach to a High Content World: SPR Microarrays in
Immune Analysis
Michael A. Lynes, Ph.D.
Department of Molecular and Cell Biology
University of Connecticut
Storrs, CT 06269 USA
email: [email protected]
http://web.uconn.edu/lyneslab/
Surface Plasmon Resonance (SPR) is a physical phenomenon that occurs when light
with specific characteristics interacts with a metal:dielectric interface. The incident light
excites the oscillation of free electrons at the surface of the metal (known as plasmons), and
the transfer of energy related to this excitation reduces the intensity of light reflected from
the surface. This energy transfer is sensitive to the incident illumination angle, and occurs
at a different angle when the local refractive index at the interface is altered by increases in
local mass (e.g. capture of antigen by antibody). Measurements of changes to SPR have
been used extensively as a means to assess the binding interactions between macromole-
cules. Most commonly, these measurements are made using the Kretschmann configura-
tion, in which a prism is used to establish the appropriate illumination conditions. We have
been involved studies to reconfigure SPR instrumentation, substituting a holographic dif-
fraction grating embossed on the surface of the sensor chip for the prism. This redesign has
the benefit of simplifying the manufacture and experimental configuration of the sensor
chip, and enabling a dramatic increase in the number of interactions that can be measured
from a single sample on an individual chip. We can measure more than 103 independent
interactions on a 1cm2 surface, and have reached limits of detection for cytokine measure-
ments in the 500 fg/ml range. Moreover, this configuration is compatible with sequential
grating coupled surface plasmon resonance (GCSPR) and grating coupled surface plasmon
coupled emissions (GCSPCE) measurements. This combination enables a 5 to 6 log dy-
namic range. This talk will describe the technology, and will provide examples of its appli-
cation to the measurement of macromolecules, bacteria, viruses, mammalian cells, and the
functional characterization of mammalian cells in a high content environment.
37
Workshop - Part I
NIH Presentation
Alison Deukhut Augustine, Ph.D. & B. Duane Price, Ph.D.
NIAID
Overview of Grants
Grant Applications and Process
Questions and Discussion
Handouts will be provided.
38
Poster Oral Presentations* (Albenia)
Cancer Immunobiology
*AAI Young Investigator Award Recipients
39
#13
Generation of a Dual Functioning Anti-Tumor Immune Response
in the Peritoneal Cavity
Abigail L. Sedlacek, Scott A. Gerber, and Edith M. Lord
University of Rochester, Rochester NY
Tumor cell metastasis to the peritoneal cavity (PC) is clinically observed in patients
with primary tumors of peritoneal organs, particularly colon and ovarian tumors. Upon re-
lease into the PC, tumor cells initially attach to the omentum, a tissue consisting of highly
organized immune aggregates (IA) imbedded in adipose tissue. Despite their proximity to
potential immune effector cells, tumor cells bind and thrive on these IA, which have been
shown to be inherently immunosuppressive. We hypothesized that if we could override the
suppressive function of these IA, we could generate a productive anti-tumor immune re-
sponse within the PC. To investigate anti-tumor immune responses in the PC we chose a
mouse model of colon adenocarcinoma, which has been shown to metastasize to the omen-
tum. Thus, we immunized mice i.p. with a lethally irradiated colon adenocarcinoma cell
line: C38. Immunization resulted in temporary enlargement of IA and expansion/
recruitment of multiple immune cell populations. Following specific (C38) or non-specific
(E0771 and B16) live tumor challenge, we failed to detect growth of both specific and sur-
prisingly non-specific tumors on the omentum by microscopy, flow cytometry, or colony
forming assay. Even when immunized mice were allowed to rest for 60 days, neither spe-
cific nor non-specific tumor growth was observed suggesting a long term anti-tumor re-
sponse. Depletion of T cells, B cells, or macrophages did not alter the anti-tumor response
and still resulted in a lack of tumor growth regardless of the type of tumor used to chal-
lenge. Interestingly, depletion of NK cells resulted in only non-specific tumor formation on
the omentum of immunized mice. This data suggests that NK cells are responsible for the
non-specific tumor immunity following immunization and that a redundant mechanism in-
volving both specific and non-specific immunity prevents the growth of C38. Additionally,
this phenomenon of non-specific immunity was not observed systemically. When mice
were immunized i.m. with C38, and challenged i.m. in the opposite flank with either C38 or
E0771, only C38 tumors exhibited delayed tumor growth in direct contrast to our i.p. re-
sults, thus highlighting the uniqueness of the peritoneal cavity in observing these non-
specific tumor responses. Collectively, these data demonstrate that the peritoneal cavity
represents a unique environment that under particular stimuli is capable of eliciting a long
term anti-tumor immune response able to prevent the growth of tumor which involves both
specific and non-specific mechanisms.
Supported by NIH Grants: R01: CA28332 and T32: AI007285
40
#58
CD28-mediated Pro-survival Signaling in Multiple Myeloma
Megan Murray, Jayakumar Nair, and Kelvin Lee
Roswell Park Cancer Institute, Buffalo NY
Multiple myeloma (MM) is an incurable plasma cell neoplasm which requires mo-
lecular bridges to the bone marrow microenvironment for survival. Our lab has previously
shown that one such bridge is CD28, the molecule best known as the prototypic T cell re-
ceptor. However, CD28 is also expressed on MM cells and is correlated with progressive
disease and worse outcomes, pointing to CD28 as a pro-survival signaling molecule.
To determine whether CD28 is important in the context of complex cell-cell interac-
tions, we co-cultured MM cells with dendritic cells (DC, which express the CD28 ligands
CD80 and CD86) in melphalan, a clinically relevant chemotherapy. DC were able to sig-
nificantly increase MM cell survival. However, this protection is entirely abrogated by ei-
ther a CD28 blocking antibody (CD28.6) or by CTLA4-Ig, which binds to the ligands CD80
and CD86. These experiments show that even in complex cell-cell interactions, CD28 is the
critical pro-survival molecule.
Since CD28 in T cells signals via a PI3K-Akt axis, we interrogated whether or not
this pathway is crucial for CD28-mediated MM cell survival. We observed that blockade of
PI3K signaling using chemical inhibitors abrogates CD28-mediated survival in a dose de-
pendent fashion. To confirm that survival is also Akt-dependent, we repeated the experi-
ments with an Akt inhibitor and again observed an abrogation of CD28-mediated survival
of MM cells.
It has been well established in the myeloma literature that the balance of the pro-
apoptotic molecule Bim determines apoptosis in MM cells. Importantly, Bim is transcrip-
tionally regulated by the PI3K-Akt-FoxO3a axis. When FoxO3a is phosphorylated by Akt,
it is excluded from the nucleus and is unable to upregulate Bim transcripts. Via western
blot, we confirmed that CD28 activation increases levels of phospho-FoxO3a. We next
wanted to test whether or not CD28 activation had any effect on Bim transcript levels. By
both western blot and RT-PCR, we confirmed that CD28 activation suppresses Bim expres-
sion, and CD28 blockade increases it. To confirm that this plays a functional role in MM
biology, we knocked down Bim using siRNA and cultured the cells without serum +
CTLA4-Ig. Consistent with our expectations, when we culture control cells in the absence
serum plus CD28 blockade, they die. However, in the Bim knock-down cells, we see that
they are resistant to CD28 blockade-mediated cell death. These data suggest that CD28 is
able to regulate Bim via the PI3K-Akt-FoxO3a axis, and that Bim is critical for MM cell
death or survival.
41
#1
Orally-Bioavailable IL-10 Nanoparticles Reduce Intestinal Polyposis and
Alleviate Inflammation-Induce Constitutional Symptoms in APC-min Mice
Allen Y. Chung1,2, Sarah J. Blair1, Charles M. Levea3, Thomas F. Conway1,
Jessica L. Zourelias1, Jamie L. Harden1, Nicholas G. Battaglia1, Lauren P. Virtuoso1,
M. Okyay Kilinc1, Qingseng Li1, and Nejat K. Egilmez1 1Department of Microbiology and Immunology
2Medical Scientist Training Program, University at Buffalo, Buffalo, NY 3Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY
The immunomodulatory cytokine interleukin-10 (IL-10) is essential for maintaining
immune homeostasis and resolving excessive inflammation at mucosal surfaces. Here we
utilize the inflammation-driven APC-min mouse model of gastrointestinal polyposis to
show that orally-administered IL-10 cytokine-nanoadjuvant therapy broadly reconditions
local intestinal immune environments and ameliorates intestinal disease. APC-min mice,
when orally gavaged with IL-10-encapsulated nanoparticles, manifest reduced polyp burden
and less aggressive intestinal pathology. The efficacy of therapy was partially dependent on
the conditioning effect of IL-10 on FoxP3+ T cells, as intestinal FoxP3+ T cell suppressive
capacity was enhanced in IL-10-treated mice. Orally-bioavailable IL-10 suppressed consti-
tutional symptoms and slowed the development of fatal anemia in our experimental ani-
mals. Collectively, these studies identify promising new applications for orally-bioavailable
sustained-release cytokine nanoparticles in the treatment of intestinal diseases and implicate
IL-10 as a sustainable therapeutic modality to address the inflammatory sequelae associated
with mucosal premalignancy.
42
#37
A Novel TLR5 Agonist, CBLB502, Promotes Cytotoxic Lymphocyte Mediated
Tumor Immunity
Nicholas Leigh1, Guanglin Bian1, Xilai Ding1, Hong Liu2, Lyudmila G. Burdelya3,
Andrei V. Gudkov3, and Xuefang Cao1
Author affiliations: 1Department of Immunology, 2Department of Medicine Blood and
Marrow Transplant Program, and 3Department of Cell Stress Biology, Roswell Park Cancer
Institute, Buffalo, NY 14263, USA
Toll-like receptor (TLR) mediated recognition of pathogen associated molecular pat-
terns allows the immune system to rapidly respond to pathogenic challenge. This makes
TLR agonists an attractive product that can be used to stimulate an immune response. In
particular, tumor immunity is often hindered by the inability to recognize and destroy ma-
lignant self due to the lack of a danger signal. This signal can be provided by a TLR ago-
nist, ideally providing the essential danger context to the immune system and allowing anti-
tumor immunity to ensue. An attractive TLR agonist is CBLB502, a pharmacologically op-
timized TLR5 agonist derived from Salmonella enterica flagellin. In this study, we exam-
ined the effect of CBLB502 on antitumor immunity using two lymphoma models, both of
which do not express TLR5, and thus do not directly respond to CBLB502. Using the T
cell lymphoma RMAS, which is dependent on natural killer (NK) cell mediated killing,
CBLB502 treatment after tumor inoculation provides significant protection to C57BL/6
mice from death caused by tumor growth. The protection elicited by CBLB502 is both NK
cell and perforin dependent. In addition, CBLB502 stimulates immunity against the B cell
lymphoma A20 in BALB/c mice, with clearance being dependent on CD8+ T cells. To re-
capitulate an antitumor response in vitro, we co-cultured total splenocytes with tumor cells
and treated with CBLB502. CBLB502 significantly enhances the ability of splenocytes to
control tumor growth. To determine which cell types mediate the CBLB502 effect, we util-
ized ImageStream flow cytometry to monitor NFκ(kappa)B nuclear translocation, a meas-
ure of TLR5 activation. ImageStream flow cytometry reveals that CD11b+ and CD11c+
cells, but neither NK nor T cells, directly respond to CBLB502. These findings demon-
strate that the novel TLR5 agonist, CBLB502, can stimulate a robust antitumor immune re-
sponse by indirectly activating cytotoxic lymphocytes through TLR5-expressing accessory
immune cells.
43
#49
Overcoming Obstacles to Naïve T Lymphocyte Trafficking in
Tumor-draining Lymph Nodes
Jason Muhitch, Michael Diehl, Fumito Ito, Minhyung Kim, Jeremy Waight,
Joseph Skitzki, Scott Abrams, and Sharon Evans
Department of Immunology,
Roswell Park Cancer Institute, Carlton & Elm Streets, Buffalo, NY 14263
Tumor-draining lymph nodes (TdLN) are central orchestrators for the generation of
anti-tumor immunity. These organs harbor dendritic cells that display tumor antigen and
activate rare tumor-specific naïve lymphocytes to generate an effector T cell pool. More-
over, TdLN have emerged as an important site to sustain long-term immune protection dur-
ing immunotherapy of cancer. An often-overlooked requirement for efficient dendritic cell-
T cell interactions is the entry of lymphocytes across specialized vessels in lymph nodes
termed high endothelial venules (HEVs). Recent reports have suggested that T cell entry
into lymph nodes of tumor-bearing mice may be impaired due to diminished trafficking
molecule expression on both TdLN HEVs and lymphocyes. Here we sought to confirm ini-
tial observations that HEVs within TdLN support diminished naïve T cell entry and extend
these studies to uncover if impediments to lymphocyte trafficking could be overcome thera-
peutically. Intravital microscopy studies determined that TdLN HEVs displayed an ~50%
reduction in their ability to support firm arrest of T cells, which is the penultimate step pre-
ceding extravasation. We further identified the defect in TdLN HEVs as reduced expression
of a vital homeostatic chemokine CCL21 for the transition from rolling to firm arrest. Di-
minished CCL21 expression on HEVs was restricted to the lymph node immediately proxi-
mal to the tumor and was not observed further downstream in the lymphatic network. How-
ever, in mice with large tumor burdens, we also observed defects in trafficking that in-
volved decreased expression of L-selectin on naïve lymphocytes resulting in reduced qual-
ity of rolling interactions and diminished T cell trafficking to lymph nodes throughout the
body. We next sought to determine if TdLN were responsive to inflammatory cues to im-
prove T cell entry. Immunodepletion protocols using cyclophosphamide as well as systemic
thermal therapy (STT, 39.5 ± 0.5°C, 6 h) increased lymphocyte trafficking in TdLNs to lev-
els above those observed in lymph nodes of unchallenged mice. Increased expression of
CCL21 on HEVs of TdLN following STT was found to be NF-κB–dependent. Mice treated
with cyclophosphamide or STT exhibited an increased frequency of lymphocytes undergo-
ing firm arrest in HEVs. These findings suggest that therapeutic targeting of HEVs for im-
proved entry of naïve T cells in TdLN may be a novel strategy to overcome immune eva-
sion. Supported by grants from the NIH (CA79765 and AI082039).
44
Poster Oral Presentations* (Evelley)
Regulation of Cellular Immunology
*AAI Young Investigator Award Recipients
45
#2
Functional Development of Antigen Specific Innate Memory CD8+ T cells via
Selection by Hematopoietic MHC Class I
Weishan Huang1,2, Jianfang Hu2, and Avery August1,2 1Department of Microbiology & Immunology, Cornell University, Ithaca, NY 14853
2The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park,
PA 16802
Innate memory phenotype (IMP) CD8+ T cells (CD44hiCD122+) are non-
conventional (alpha beta) T cells exhibiting features of innate immune cells, and are sig-
nificantly increased in the absence of non receptor tyrosine kinase Itk. However, their de-
velopmental path and function are not clear, particularly whether they can contribute to an-
tigen specific responses. Here we show the hematopoietic MHCI dependent generation of
antigen specific IMP CD8+ T cells using a murine bone marrow chimeric model. Transplant
of MHCI+/+ bone marrow into irradiated MHCI-/- recipients gives rise to predominantly IMP
CD8+ T cells, resembling the IMP CD8+ T cell observed in Itk-/- mice, but distinct from
CD8+ T cells derived from homeostatic expansion. This hematopoietic MHCI dependent
development of IMP CD8+ T cells is independent of the thymus and homeostatic expansion
of T cells from donor bone marrow. In contrast, transplanting MHCI-/- or MHCI-/-OTI bone
marrow into irradiated WT mice gives rise to predominantly naïve CD8+ T cells. IMP CD8+
T cells that develop via this process can respond to infection with Listeria monocytogenes
expressing ovalbumin by rapidly up-regulating IFN- (gamma) secretion upon antigen spe-
cific restimulation in vitro, compared to analogously derived naïve CD8+ T cells alone or
WT controls. More importantly, ovalbumin specific OTI IMP CD8+ T cells exhibited sig-
nificantly enhanced early response to antigen without prior primary stimulation. Our find-
ings suggest a method to generate antigen specific CD8+ IMP T cells, and demonstrate that
these cells promptly respond in an antigen specific fashion.
46
#4
Impaired Function, but Not Generation, of Follicular Helper T cells in
Aged Mice
Julie S. Lefebvre, Dawn Jelley-Gibbs, Sheri Eaton and Laura Haynes
Trudeau Institute, Saranac Lake, NY
Aging negatively impacts most aspect of the immune system. A good example of this is
the poor humoral response generated following influenza immunization in older individu-
als. To generate a strong and specific humoral response, B cells need to receive help from
CD4 T cells, particularly from the follicular helper T cell (Tfh) subset. Previous studies
have shown that intrinsic and extrinsic defects accumulate with aging that impair the helper
functions of aged CD4 T cells. Whether the inadequate help from the aged CD4 T cells re-
sults from their incapacity to become Tfh cells or from functional defects of those Tfh re-
mains to be elucidated.
Using a model of influenza vaccination in young and aged mice, we show that a higher
percentage of CD4 T cells acquires the typical Tfh markers CXCR5 and PD1 in the drain-
ing lymph nodes of aged mice compared to young mice. However, a lower proportion of B
cells acquires a germinal center phenotype (PNAhi CD38lo) suggesting that the aged Tfh
cells are functionally impaired. To identify the mechanism(s) preventing the aged Tfh cells
to support an efficient germinal center reaction, we performed real-time PCR analyses on
the RNA of purified young and aged Tfh cells. While the level of mRNA for IL-2, IL-4 and
IL-21 were similar in young and aged Tfh cells, the aged Tfh cells expressed more IL-10
and IFNgamma than young Tfh cells. Restimulation of purified Tfh cells from the spleen of
young and aged mice infected with PR8 confirmed that a greater proportion of aged Tfh
cells produced cytokines (IL-10, IL-21 and IFNgamma) than young Tfh cells.
The dysregulated signals sent by the aged Tfh cells likely contribute to the reduced ger-
minal center response and consequently to the impaired humoral response generated follow-
ing influenza immunization in aged individuals. Further experiments aimed at blocking IL-
10 and/or IFNgamma will be undertaken to confirm the putative deleterious role of these
cytokines in the germinal center reaction.
47
#36
Tec Kinase Itk Modulates Allergic Airway Inflammation by Suppressing IFNγ
(gamma) Expression in Naïve CD4+ T cells
Arun K Kannana, Nisebita Sahub,c, and Avery Augusta aDepartment of Microbiology & Immunology, Cornell University, Ithaca, NY, 14853.
bDepartment of Veterinary & Biomedical Sciences, The Pennsylvania State University,
University Park PA. cDiscovery Oncology, Genentech, Inc, South San Francisxo, CA 94080
T helper responses are critical for a productive immune response but an inappropri-
ate T helper response results in inflammatory and autoimmune disorders. Although we
know basic details of the signaling events that regulate lineage choices of CD4+ T cells,
there are still considerable gaps in the knowledge of pathways that regulate T cell differen-
tiation to T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells. Allergic asthma
is a Th2 cell dominated inflammatory disease characterized by airway inflammation and
airways hyper responsiveness. IL-2 Inducible T cell kinase (Itk) is primarily expressed in T
cells and is critical for their development, activation and function. Itk-/- mice have a defec-
tive Th2 response and are not susceptible to allergic asthma. Here we show that peripheral
Itk deficient naïve CD4+ T cells have substantially increased transcript levels of prototypic
Th1 genes, including Eomesodermin, IFNγ(gamma), T-bet and IL12Rβ(beta)1. More im-
portantly, removal of IFNγ(gamma) on the Itk-/- background rescues expression of Th2 re-
lated genes in Th cells, and House Dust Mite-driven allergic asthma in Itk-/- mice. In addi-
tion, we show that this Itk mediated signal represses IFNγ(gamma) independent of T-bet
and maintains naïve T cells in an unbiased state, until it comes in contact with cognate anti-
gen. In the absence of Itk, the genomic locus of IFNγ(gamma) is open with the promoter
and a number of conserved nucleotide regions showing enrichment of permissive H3K4me2
and loss of repressive H3K27me3 histone marks. Furthermore, these signaling pathways are
conserved in humans, as loss of Itk in human PBMC derived T cells and Jurkat cells results
in a substantial reduction in the secretion of a number of effector cytokines, at least partly
due to a failure to activate pERK. Our results indicate that Itk signals suppresses the expres-
sion of IFNγ(gamma) in CD4+ T cells, which in a positive feed forward loop, regulates the
expression of Th1 factors such as T-bet and Eomesodermin, and suppress development of
Th2 cells and Th2 responses. This work has implications for understanding T helper differ-
entiation programs, and Itk as a therapeutic target for Th2 mediated inflammatory diseases.
48
#24
Dendritic Cells Act Through IL-6 to Enhance Naïve T cell Trafficking Across
High Endothelial Venules During Adaptive Immunity
Daniel T. Fisher, Jason B. Muhitch, Trupti D. Vardam and Sharon S. Evans
Department of Immunology, Roswell Park Cancer Institute, Carlton & Elm Streets, Buffalo,
New York 14263, USA
Dendritic cells (DC) are as master regulators of the adaptive immune response, pro-
viding both cognate antigen and costimulation required for T cell activation as well as cyto-
kines (e.g., IL-12, TNF, and IL-6) that drive T cell expansion and differentiation. Addition-
ally, DCs are used therapeutically as vaccines in cancer immunotherapy. While the role of
DCs in T cell activation is well known, here we show that DCs play an unexpected role in
shaping the T cell repertoire within inflamed lymph nodes (LN) by promoting trafficking
across vascular gateways termed high endothelial venules (HEV). Immunization of mice
with bone marrow-derived DCs matured ex vivo by TLR4 and 9 agonists (LPS and ODN-
CpG, respectively), but not a TLR2 agonist (Pam3CSK4), increased the intravascular density
of two hallmark adhesion molecules, peripheral lymph node addressin (PNAd) and intercel-
lular adhesion molecule-1 (ICAM-1), selectively in HEVs of inflamed LNs. Increased
PNAd and ICAM-1 display occurred rapidly after immunization (within 12 h) and coin-
cided with improved trafficking of naïve and central memory T cells as well as increased
LN size. PNAd and ICAM-1 expression reverted to homeostatic levels 24-48 h post immu-
nization. Furthermore, preventing migration of DCs into the inflamed LN through global
blockade of chemokine receptors using the irreversible inhibitor of G-protein signaling per-
tussis toxin or using DCs deficient for the chemokine receptor CCR7 prevented induction of
PNAd and ICAM-1 required immunization with DCs with functional CCR7 chemokine re-
ceptors. Remarkably, increased adhesion molecule expression was abrogated by loss of a
single cytokine, IL-6, from immunizing DCs. These findings suggest a model whereby IL-6
–producing DC play an active role promoting the intrinsic trafficking capacity of HEVs,
thereby increasing the probability that naïve and central memory T cells come into contact
with cognate antigen-loaded DCs in inflamed LN during the early phase of the adaptive im-
mune response.
Supported by the NIH (CA79765 and AI082039), the Roswell Park Alliance Foun-
dation, and the Jeniffer Linscott Tietgen Family Foundation.
49
#15
The Non-Redundant Role For Chemokine Receptor Cxcr3 in
T Cell Trafficking Into Tumor Tissue
Maryann Mikucki1, Joseph Skitzki2, Daniel Fisher1,2, Andrew D. Luster3,
and Sharon S. Evans1
1Deptartment of Immunology and 2Department of Surgical Oncology,
Roswell Park Cancer Institute, Buffalo, NY USA; 3Center for Immunology & Inflammatory Diseases, Division of Rheumatology, Allergy &
Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown,
MA USA
Several immunotherapeutic regimens are currently under investigation for the treat-
ment of metastatic melanoma and other aggressive cancers in order to exploit the unique
capacity for T cells to mediate tumor-specific cytotoxicity and long-lasting protection
against recurrence. These strategies seek to enhance either the activity of cytotoxic tumor-
specific T cells (e.g., CTLA-4 blockade) or their frequency within the blood (e.g., adoptive
cell transfer therapy and therapeutic vaccination). Common to these treatments is the re-
quirement for effector T cells to cross from the circulation into tumor tissue via a
chemokine-dependent process, but the precise chemokine/chemokine receptor involved is
unknown. Here, we used a murine model to examine the chemokine requirements for T cell
trafficking in the tumor microenvironment. Tumor-reactive cytotoxic T cells were shown
to express three distinct chemokine receptors that are functional in vitro: CCR2, CCR5, and
CXCR3. Cognate chemokines for each of these receptors (e.g., CCL2, CCL5 and CXCL10,
respectively) were also detected in the tumor microenvironment of murine melanoma.
However, loss of a single chemokine receptor, CXCR3, severely compromised the ability of
adoptively transferred effector T cells to traffic to tumors as measured by intravital micros-
copy and short-term homing studies. CXCR3-dependent trafficking was further linked to
tumor cell apoptosis and tumor growth delay. Taken together, these studies establish the
obligate role for CXCR3 in the migration of blood-borne effector T cells into tumor tissue
and support the development of chemokine-targeted strategies to improve T cell entry dur-
ing cancer immunotherapies. Supported by the NIH (CA79765, CA085183), the Jennifer
Linscott Tietgen Family Foundation, and the Joanna M. Nicolay Melanoma Foundation.
50
Poster Oral Presentations* (Albenia)
Immunity to Human Pathogens
*AAI Young Investigator Award Recipients
51
#5
The Role of Macrophages and Neutrophils in Pulmonary Francisella tularensis
Infection
Donald Steiner and Dennis W. Metzger
Albany Medical College, Albany, NY
Francisella tularensis is the causative agent of tularemia. Pulmonary infection with
F. tularensis is highly lethal due to the profound inflammation induced in the airways.
Though F. tularensis is believed to be an intracellular pathogen that requires replication
within macrophages during respiratory infection, we have found that depletion of alveolar
macrophages by intranasal treatment with liposomal Clodronate reduced mean time to death
and total survival rates of infected mice. Furthermore, mice with a macrophage-specific
insensitivity to interferon-γ (gamma) (MIIG mice) similarly showed heightened sensitivity
to pulmonary Francisella infection. Such mice exhibited reduced lung-tissue expression of
the neutrophil chemoattractant tumor necrosis factor-α (alpha) and reduced neutrophil re-
cruitment to the lungs post-infection. Systemic neutrophil depletion reduced mean time to
death and total survival rates, with the most pronounced effect in mice depleted three days
post-infection. In mice infected with a lethal dose of bacteria, exogenous interleukin-12
promoted survival and bacterial clearance in mice, but this effect was lost upon neutrophil
depletion and in IFN-γ (gamma) knockout mice. The results suggest that rather than being
a required reservoir for bacterial replication, alveolar macrophages are necessary for de-
fense against F. tularensis infection primarily by recruiting neutrophils, which are essential
for clearing the bacterium. (Supported by NIH grant PO1 AI 056320)
52
#14
Prophylactic Treatment with CpG Oligodeoxynucleotides Confers Protection
Against Pulmonary and Systemic Yersinia pestis Infection
Anthony, J. Hickey, Lawrence W. Kummer, Frank M. Szaba, Michelle A. Parent, Lawrence
L. Johnson, Jr-Shiuan Lin, Stephen T. Smiley
Trudeau Institute, Saranac Lake, NY USA 12983
Immunomodulatory agents have the potential to serve as a new class of broad-
spectrum antimicrobials against infectious diseases for which there currently are limited
safe and effective countermeasures. An example of such a disease is plague, caused by the
gram-negative pathogen Yersinia pestis, which has resulted in three major pandemics
throughout history and has been responsible for the deaths of hundreds of millions of peo-
ple. Despite substantial efforts there currently is no safe and effective vaccine available
against plague, and the window of opportunity afforded to infected individuals for treatment
with antibiotics is small. New treatment regimens, therefore, need to be sought. We demon-
strate here that prophylactic treatment with immunomodulatory CpG oligodeoxynucleotides
(ODN) provides protection against Yersinia pestis. Specifically, intranasal administration of
CpG ODN one day prior to lethal pulmonary exposure to Y. pestis strain KIM D27 signifi-
cantly improves survival and reduces hepatic bacterial burden in mouse models. Interest-
ingly, despite conferring protection, treatment with CpG ODN increases the bacterial bur-
den of the lung. Prolonged survival and decreased hepatic bacterial burden are observed in
both B cell-deficient mMT and lymphocyte-deficient RAG2-deficient mice after pretreat-
ment with CpG ODN, signifying that adaptive immunity is not required for the treatment to
exert a protective effect. None of the above impacts, including increased pulmonary bacte-
rial burden, however, are observed in TLR9-deficient mice, indicating that they are all de-
pendent on TLR9 signaling. Finally, we demonstrate that the same treatment regimen with
CpG ODN improves the survival and reduces the hepatic bacterial burden of mice receiving
an intraperitoneal challenge with Y. pestis. This demonstrates that intranasal pretreatment
with CpG ODN can protect against both pulmonary and systemic Y. pestis infection and in-
creases the potential for its use as an immunomodulatory agent against the pathogen.
53
#54
Macrophage-dependent Activation of Hematopoietic Stem Cells during
Intracellular Bacterial Infection
Amanda McCabe, Lu Zhang, Maura Jones, and Katherine MacNamara
Albany Medical College, Albany NY
Hematopoietic stem cells (HSCs) maintain production of all cells of the blood. An
emerging idea is that during infection, HSC activation is associated with the production of
cell lineages needed for an appropriate immune response to pathogens. In a murine model
of human monocytic ehrlichiosis (HME), a tick-borne intracellular bacterial infection, we
have observed an IFNγ-dependent increase in active HSCs and reduced quiescent HSCs,
which correlated with increased monocytes. We hypothesize that the IFNγ-dependent HSC
activation and loss in quiescent HSCs is dependent upon IFNγ signaling in macrophages.
BM-resident macrophages are extremely sensitive to IFNγ and they have recently been
shown to play a role in maintaining the BM HSC niche, but how they might contribute to
HSC activation during infection has not been addressed. Here we show that bacterial infec-
tion resulted in proliferation and activation of BM-resident macrophages, which resulted in
increased numbers of active HSCs. Ehrlichia muris infected C57BL/6 mice exhibited in-
creased numbers of macrophages that expressed F4/80, Ly6G, and CD169, characteristic of
a BM-resident macrophage. Macrophages exhibited increased expression of MHCII and
CD80 during infection indicating they were activated. Furthermore, mice lacking IFNγ sig-
naling only in macrophages exhibited increased frequencies of dormant HSCs in the BM
after infection, relative to wild type infected mice. These data suggest that IFNγ signaling
in macrophages is, in part, responsible for mediating HSC activation. These results define a
novel role for macrophages in infection-induced alterations in hematopoiesis. Our current
experiments are aimed at addressing how macrophage activation during infection impacts
HSC function, and whether this interaction is direct or through the niche in which HSCs re-
side.
54
#53
IL-27 Sustains T-bet Expression and Promotes the Development of Terminally
Differentiated CD4 T cells during Tuberculosis
Egídio Torrado, Jeffrey J. Fountain, John Pearl, and Andrea M. Cooper
Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY, 12983
IL-27 is a member of the IL-12 family of cytokines that was originally described to
induce the expression of the transcription factors STAT1 and T-bet, thus promoting Th1
differentiation. Despite this apparent Th1 inducing profile, mice deficient in IL-27R signal-
ing are more resistant to Mtb infection, even though CD4 T cells in the lungs of these mice
produce lower amounts of IFN-gamma on a per cell basis.
Here we show that during M. tuberculosis infection, IL-27 activity is not required for
inducing IFN-gamma-producing CD4 T cells. However, during the chronic stage of the in-
fection, in the absence of IL-27 activity, CD4 T cells express lower levels of the transcrip-
tion factor T-bet, corresponding with a reduced accumulation of terminally differentiated
CD4 T cells in the lungs of these mice. Mechanistic analysis of the impact of IL-27 signal-
ing in Ag-specific T cells suggest improved competitive fitness of the CD4 T cells that are
deficient in IL-27 signaling, when compared to WT CD4 T cells. Subsequent studies
showed that the improved fitness of IL-27R-/- CD4 T cells is not dependent on proliferation
but likely dependent on improved survival of these cells in the M. tuberculosis generated
inflammatory environment. Finally, the transcription profile of CD4 T cells from the lungs
of Mtb-infected IL-27R-/- deficient mice suggests altered fatty acid metabolism, suggesting
an important role of IL-27 in the metabolic activity of CD4 T cells.
These data suggest that IL-27 has an important impact in the maintenance of effector
CD4 T cell responses during tuberculosis.
This work was supported by grants from the NIH (AI069121 and AI46530) and the
Trudeau Institute.
55
#57
Maintenance of Peripheral T cell Responses during Mycobacterium tuberculosis
Infection
William W. Reiley1, Susan T. Wittmer1, Lynn M. Ryan1, Sheri M. Eaton1, Laura Haynes1,
Gary M. Winslow1,2, and David L. Woodland1,3 1Trudeau Institute, Saranac Lake, NY, 2Wadsworth Center, Albany, NY, and 3Keystone
Symposia on Molecular & Cellular Biology, Silverthorne, CO
Mycobacterium tuberculosis (Mtb) establishes a latent infection that can persist for
years. The development of adaptive immunity is critical for the maintenance of infection in
the latent state and the prevention of clinical tuberculosis. While much has been learned
about Mtb over the past few decades, we still do not fully understand how acute and persis-
tent T cell responses are induced and maintained after infection. We hypothesized that long-
term T cell responses would be maintained during chronic infections by new thymic emi-
grants contributing to, and refreshing, the peripheral T cell response. Therefore, we have
dissected the contribution of new thymus-derived T cells to the maintenance of the periph-
eral CD4 and CD8 T cell response, during both acute and chronic infection. Although
newly-generated T cells contribute to the peripheral response, especially during acute infec-
tion, the contribution of recent thymic emigrants declines during chronic infection. The de-
cline is correlated with an apparent decrease in antigen presentation, or in the capability of
antigen-presenting cells to drive the activation of naive T cells, because the priming and ex-
pansion of newly-generated T cells is less efficient during chronic infection. These findings
demonstrate that although naive thymus-derived T cells can contribute to the maintenance
of immunity, this process is not the primary mechanism. Instead, other processes are likely
responsible, such as antigen-driven T cell proliferation, perhaps originating from a popula-
tion of yet undefined T cells capable of self-renewal. Our findings also reveal that antigen is
not a limiting factor for driving the activation of naive T cells during chronic infection. We
propose instead that the context of antigen presentation (i.e., the type of APC, or the quality
of co-stimulatory signals) changes throughout infection. These studies extend our under-
standing of how T cell responses are maintained during persistent bacterial infections.
57
#39
Human Placental Trophoblast Cells are Hyporesponsive to Type I and Type III
Interferons: Potential Role in Susceptibility of Pregnant Women to Viral
Infection
Catherine G. Burke*, Katherine E. Herman#, Benson Y.H. Cheng#, Luis Martinez-Sobrido #
and Shawn P. Murphy*#
Department of Obstetrics and Gynecology*, Microbiology and Immunology#, University of
Rochester School of Medicine, Rochester, NY
Pregnant women are preferentially susceptible to infection by intracellular pathogens
such as viruses. These infections are associated with severe complications of pregnancy that
include miscarriage, in utero growth restriction, preterm birth and fetal and maternal mor-
bidity. However, the factors accounting for the increased susceptibility of pregnant women
to infection are not well understood. Trophoblast cells (TBCs) form the fetal component of
the placenta, and are the only cells derived from the fertilized egg that are in direct contact
with maternal blood and tissues. We previously demonstrated that TBCs are hypo-
responsive to the pro-inflammatory cytokine interferon- gamma) [IFN- gamma)], which
is essential for immunity against tumors and certain pathogens. However, there is a gap in
our knowledge regarding the ability of TBCs to respond to type I or type III IFNs, which
induce an antiviral state and thus serve as an important first line of defense against viral in-
fection. In this study, we demonstrate that human trophoblast-derived choriocarcinoma cells
are hypo-responsive to both IFN- alpha)2 and IFN- lambda)1, despite the fact that these
cells express all of the components of the JAK-STAT pathway necessary for productive
IFN signaling. Dose response studies and kinetic analyses demonstrate that activation of the
transcription factors STAT-1 and STAT-2 in response to these IFNs is severely compro-
mised in TBCs relative to epithelial cells. In addition, the levels of IFN-responsive gene ex-
pression are significantly lower in TBCs versus epithelial cells. Furthermore, cotreatment of
TBCs with IFN- alpha)2 and the protein tyrosine phosphatase (PTP) inhibitor pervanadate
results in partial rescue of STAT activation and IFN-responsive gene expression. These re-
sults suggest that IFN responses are inhibited in TBCs by PTP(s). Since IFNs provide the
first line of defense against viral infections, we investigated the physiological relevance of
trophoblastic hypo-responsiveness to IFNs in cells pretreated with IFN- (alpha)2 and sub-
sequently infected with the IFN-sensitive Vesicular Stomatitis Virus (VSV). Pretreatment
with IFN- alpha)2 protected epithelial cells but not TBCs from subsequent infection with
VSV. Taken together, these results suggest that the inability of TBCs to respond to IFNs
may play an important role in susceptibility of pregnant women to viruses that infect the
placenta.
58
#40
Assessment of Cytokine Responses in Human Placenta Using a Novel Whole
Mount Immunofluorescence Technique
Meghan E. Bushway, Scott A. Gerber, Richard K. Miller, Edith M. Lord,
Shawn P. Murphy
University of Rochester, Rochester, NY 14642
The human placenta plays multiple critical roles in successful pregnancy, including fetal gas
and nutrient exchange and protection of the genetically distinct fetus from maternal immune-
mediated destruction. Trophoblast cells are the only cells derived from the fertilized egg that are in
direct contact with maternal blood and tissue and thus serve an important function in protection of
the fetus throughout the course of gestation. Interestingly, the pro-inflammatory cytokine inter-
feron-gamma (IFN-γ), which is critical for activating immunity against non-self, has been shown to
be present in the pregnant uterus during normal pregnancy. We hypothesized that placental cells,
especially trophoblasts, have developed unique mechanisms to remain hyporesponsive to IFN-γ
(gamma) and thus evade maternal immune recognition. To date, analysis of human placental cell
responses to cytokines or growth factors has been restricted to isolated cell populations cultured in
vitro. Furthermore, examination of the microenvironment within human placental villous structures
has traditionally been performed using classical immunohistochemistry (IHC). We sought to de-
velop a technique that would enable us to study multiple cell types within morphologically intact
villous tissue. Thus, placental whole mount immunofluorescence (WMIF) was developed in con-
junction with flow cytometry to visualize and quantify surface and intracellular molecules in dis-
tinct cell populations. Moreover, this method was utilized to examine responses to IFN-γ(gamma)
within human placental cell types, including trophoblast, resident immune cells and endothelial
cells.
Striking morphological images of developing CD31+ blood vessels were obtained using
WMIF that were not observed with classical IHC. In addition, CD45+/CD68+ fetal macrophages,
cytokeratin (CK)-7+ cytotrophoblast cells, and human chorionic gonadotropin (hCG)+ syncytiotro-
phoblast were detected within intact villous structures. Furthermore, the levels of the activated (i.e.
phosphorylated) form of the transcription factor STAT1 (pSTAT1) were examined following IFN-γ
(gamma) treatment. In placental explants treated with IFN-γ(gamma), fetal macrophages strongly
induced pSTAT1. In contrast, pSTAT1 was not detected in the syncytiotrophoblast layer or cyto-
trophoblast cells. These data suggest that syncytiotrophoblast, which are in direct contact with ma-
ternal blood, and the underlying cytotrophoblast cells are hyporesponsive to IFN-γ(gamma). This
phenomenon may represent an important mechanism of fetal evasion from maternal immunity.
WMIF combined with flow cytometry allowed for examination of multiple cell types within
intact villous structures while preserving the placental architecture. It is anticipated that this meth-
odology will provide a powerful approach for detailed analyses of the placental architecture in nor-
mal versus pathological pregnancies, and for studying the effects of environmental factors on pla-
cental function and morphometry.
59
#33
Lymphotoxin Controls the IL-22 Pathway to Inhibit Intestinal Inflammation
Elise Macho Fernandez, Ekaterina P. Koroleva, Luke Neill, and Alexei V. Tumanov
Trudeau Institute, Saranac Lake, NY 12983
As many as 2.2 million people in Europe and 1.4 million people in the United States
suffer from inflammatory bowel disease (IBD). Immune-based therapeutic approaches for
IBD, including anti-TNF therapy, have shown significant success during the last decade.
Nevertheless, many patients are still not responsive to these therapies and suffer from se-
vere side effects.
Innate lymphoid cells (ILCs) have recently emerged as key players in regulating the
balance between protective immunity and immunopathology at mucosal surfaces. However,
the cellular and molecular pathways that control crosstalk between ILCs and intestinal
epithelial cells during inflammation remain poorly understood.
IL-22 is produced by ILCs in the gut and plays a protective role in inflammatory
bowel disease by enhancing barrier integrity and epithelial innate immunity. Our recent
study revealed that lymphotoxin (LT), a member of TNF superfamily of cytokines, is criti-
cal for IL-22 production by RORgt+ ILCs in the gut during mucosal bacterial infection. By
using a conditional gene targeting approach, we found that a LT-driven positive feedback
loop controls IL-22 production by RORgt+ ILCs via lymphotoxin beta receptor (LTbR) sig-
naling on DCs.
In the present study, we aimed to determine how LT controls ILCs to inhibit intesti-
nal inflammation using a mouse model of IBD, induced by dextran sodium sulfate (DSS).
Our data show that LTbR signaling is essential for protection and recovery from acute DSS-
induced colitis. We found that the sensitivity of LTbR-deficient mice to DSS-induced coli-
tis was associated with an impaired production of IL-22 by ILCs populations in the colon.
To determine which LTbR-expressing cells were involved in protection against DSS-
induced inflammation, we generated bone marrow chimeric mice and showed that LTbR
expression in both bone marrow-derived and radioresistant stromal cells are required for the
control of intestinal inflammation. Further analysis of mice with a specific inactivation of
LTbR in distinct cell populations revealed that LTbR signaling in both intestinal epithelial
cells and dendritic cells protects from DSS-induced inflammation.
In conclusion, our data indicate that LT signaling controls intestinal inflammation
via the IL-22 protective pathway.
60
#26
Specialized Proresolution Mediators Enhance Human B cell Differentiation to
Antibody Secreting Cells
Sesquile Ramon 1, Charles N. Serhan 2, and Richard P. Phipps 1
1) Department of Microbiology and Immunology, University of Rochester, Rochester, NY;
2) Department of Anesthesiology, Brigham and Women‟s hospital, Harvard Medical
School, Boston, MA
The resolution of inflammation is an active and dynamic process critical in maintain-
ing homeostasis. Newly identified lipid-derived mediators are key players during the proc-
ess of inflammation resolution. These specialized proresolution mediators (SPM) constitute
separate classes of compounds, which include lipoxins, resolvins, protectins and maresins,
all derived from essential fatty acids. SPM regulate aspects of the immune response, includ-
ing inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimula-
tion of macrophage phagocytic activity. However, their effects on B lymphocytes are un-
known. Our study shows that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA),
resolvin D1 (RvD1) but not protectin D1 (PD1), strongly increase the ability of normal hu-
man B cells to produce IgM and IgG. The increased antibody levels are due to an increased
number of antibody-secreting cells. Furthermore, SPM regulate the expression of the tran-
scription factors Blimp-1, Xbp-1 and Pax-5, and promote B cell differentiation towards a
CD27+CD38+ antibody secreting cell phenotype. None of the SPM affect proliferation and
are non-toxic. The increase in plasma cell differentiation and antibody production coincides
with the known involvement of SPM during the late stages of inflammation and pathogen
clearance. These new findings highlight the potential applications of SPM as endogenous
and non-toxic adjuvants and anti-inflammatory therapeutic molecules.
This research is supported by DE011390 and NIH T32 DE007202
61
#47
IgM Memory B Cells Generated During Bacterial Infection are Required for
Secondary IgG Responses to Antigenic Challenge
Jennifer Yates1, Rachael Racine1, Kevin McBride3, and Gary Winslow1,2
SUNY Albany School of Public Health, Albany NY1
Wadsworth Center, Albany NY2
University of Texas MD Anderson Cancer Center, Smithville TX3
Immunological memory is a fundamental concept that is key to generating and main-
taining immunity to pathogens, and for mediating the protection afforded by vaccines. De-
spite the focus on class-switched memory B cells, several studies have validated the exis-
tence of IgM memory B cells, and have demonstrated distinct functions of IgM and IgG
memory B cell subsets. Based on the expression of CD11c, we have identified a large popu-
lation of IgM memory B cells using a natural model of infection by the bacterium Ehrlichia
muris. These CD11c+ IgM memory cells exhibit phenotypic characteristics of memory B
cells, including expression of CD73, and PD-L2. In addition, the CD11c+ IgM memory cells
lack expression of CD138, are largely quiescent, and have accumulated somatic mutations.
Although these cells did not proliferate or secrete antibody ex vivo, they produced antigen-
specific IgM upon in vitro stimulation with mitogens. The CD11c+ IgM memory B cells
were located in the splenic marginal zone, and were not detected in the blood or other sec-
ondary lymphoid organs. Their generation required CD4 T cell help, and both CD40L and
IL-21R signals. Subsequent IgG recall responses to specific antigenic challenge also re-
quired CD4 T cell help, and both CD40L and IL-21R signals. In vivo depletion of the IgM
memory cells abrogated the IgG recall response to specific antigenic challenge, demonstrat-
ing that these cells are responsible for the humoral recall response during E. muris infection.
These data confirm previous reports that IgM memory B cells can undergo class switch re-
combination and affinity maturation, following re-encounter with cognate antigen. Our
study demonstrates that IgM memory B cells maintain long-term immunity during a natural
model of infection, and may act to provide the host with greater flexibility during infection
with antigenically-variant pathogens.
62
Platinum Sponsor Presentation
Incorporating New, Bright Fluorochromes into
Multicolor Panel Design
Erik Puffer, Ph.D.
Technical Applications Specialist
BD Biosciences
Multicolor flow cytometry is a powerful tool for analyzing multiple cellular parame-
ters simultaneously. This seminar will present the basic principles for optimization of multi-
color flow cytometry panels which include considerations around fluorochrome brightness,
antigen level of expression, spillover, proper use of tandem dyes, and appropriate use of
controls. Special considerations for the detection of intracellular targets such as cytokines
and transcription factors will also be discussed. Moreover, information about the impact on
multicolor flow cytometry of the new fluorochromes, BD Horizon™ PE-CF594 and BD
Brilliant Violet™ 421, which have significantly improved brightness compared to previ-
ously available reagents, will be presented.
64
#1 Allen Y. Chung* #2 Weishan Huang*
#3 Aurelie Ray
#4 Julie S. Lefebvre* #5 Donald Steiner*
#6 Nicole Johnson
#7 Bethany Winans #8 Lisbeth A. Boule
#9 Yoichi Furuya
#10 Christopher J. Greene #11 Katrina Simmons
#12 Do-Geun Kim
#13 Abigail L. Sedlacek* #14 Anthony J. Hickey*
#15 Maryann Mikucki*
#16 Magdia De Jesus #17 David A. Hoekstra, II
#18 Fei Huang
#19 Sonia Mohinta #20 Sivakumar Periasamy
#21 E. H. Hannah Fong
#22 Renee M. Laird* #23 Giang H. Pham
#24 Daniel T. Fisher*
#25 Anastasiya Yermakova #26 Sesquile Ramon*
#27 David J. Vance
#28 David E. Place #29 Brian Franz
#30 Laura L. Goodfield
#31 Jayaleka J. Amarasinghe
#32 Benson Yee Hin Cheng
#33 Elise Macho Fernandez* #34 Sarah J. Muse
#35 Haley Spangler
#36 Arun K. Kannan* #37 Nicholas Leigh*
#38 S.M. Eaton
#39 Catherine G. Burke* #40 Meghan E. Bushway*
#41 Kathy A. Green
#42 Kathryn M. Pietrosimone #43 Joanne Y.H. Lim
#44 Anju Singh
#45 Ellen B. Duffy #46 Lu Huang
#47 Jennifer Yates*
#48 Marguerite Joly #49 Jason Muhitch*
#50 Alan M. Sanfilippo
#51 Quang-Tam Nguyen #52 Princess Rodriguez
#53 Egidio Torrado*
#54 Amanda McCabe* #55 Michael L. Davies
#56 Yubin Zhang #57 William W. Reiley*
#58 Megan Murray*
Poster Abstract Listing
*AAI Young Investigator Award Recipient
65
#1
Orally-Bioavailable IL-10 Nanoparticles Reduce Intestinal Polyposis and
Alleviate Inflammation-Induce Constitutional Symptoms in APC-min Mice
Allen Y. Chung1,2, Sarah J. Blair1, Charles M. Levea3, Thomas F. Conway1, Jessica L.
Zourelias1, Jamie L. Harden1, Nicholas G. Battaglia1, Lauren P. Virtuoso1,
M. Okyay Kilinc1, Qingseng Li1, and Nejat K. Egilmez1 1Department of Microbiology and Immunology
2Medical Scientist Training Program, University at Buffalo, Buffalo, NY 3Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY
The immunomodulatory cytokine interleukin-10 (IL-10) is essential for maintaining
immune homeostasis and resolving excessive inflammation at mucosal surfaces. Here we
utilize the inflammation-driven APC-min mouse model of gastrointestinal polyposis to
show that orally-administered IL-10 cytokine-nanoadjuvant therapy broadly reconditions
local intestinal immune environments and ameliorates intestinal disease. APC-min mice,
when orally gavaged with IL-10-encapsulated nanoparticles, manifest reduced polyp burden
and less aggressive intestinal pathology. The efficacy of therapy was partially dependent on
the conditioning effect of IL-10 on FoxP3+ T cells, as intestinal FoxP3+ T cell suppressive
capacity was enhanced in IL-10-treated mice. Orally-bioavailable IL-10 suppressed consti-
tutional symptoms and slowed the development of fatal anemia in our experimental ani-
mals. Collectively, these studies identify promising new applications for orally-bioavailable
sustained-release cytokine nanoparticles in the treatment of intestinal diseases and implicate
IL-10 as a sustainable therapeutic modality to address the inflammatory sequelae associated
with mucosal premalignancy.
66
#2
Functional Development of Antigen Specific Innate Memory CD8+ T cells via
Selection by Hematopoietic MHC Class I
Weishan Huang1,2, Jianfang Hu2, and Avery August1,2 1Department of Microbiology & Immunology, Cornell University, Ithaca, NY 14853
2The Huck Institutes of Life Sciences, The Pennsylvania State University, University Park,
PA 16802
Innate memory phenotype (IMP) CD8+ T cells (CD44hiCD122+) are non-
conventional (alpha beta) T cells exhibiting features of innate immune cells, and are sig-
nificantly increased in the absence of non receptor tyrosine kinase Itk. However, their de-
velopmental path and function are not clear, particularly whether they can contribute to an-
tigen specific responses. Here we show the hematopoietic MHCI dependent generation of
antigen specific IMP CD8+ T cells using a murine bone marrow chimeric model. Transplant
of MHCI+/+ bone marrow into irradiated MHCI-/- recipients gives rise to predominantly IMP
CD8+ T cells, resembling the IMP CD8+ T cell observed in Itk-/- mice, but distinct from
CD8+ T cells derived from homeostatic expansion. This hematopoietic MHCI dependent
development of IMP CD8+ T cells is independent of the thymus and homeostatic expansion
of T cells from donor bone marrow. In contrast, transplanting MHCI-/- or MHCI-/-OTI bone
marrow into irradiated WT mice gives rise to predominantly naïve CD8+ T cells. IMP CD8+
T cells that develop via this process can respond to infection with Listeria monocytogenes
expressing ovalbumin by rapidly up-regulating IFN- (gamma) secretion upon antigen spe-
cific restimulation in vitro, compared to analogously derived naïve CD8+ T cells alone or
WT controls. More importantly, ovalbumin specific OTI IMP CD8+ T cells exhibited sig-
nificantly enhanced early response to antigen without prior primary stimulation. Our find-
ings suggest a method to generate antigen specific CD8+ IMP T cells, and demonstrate that
these cells promptly respond in an antigen specific fashion.
67
#3
Tyk2 Signaling Pathway and Mycobacterium tuberculosis (Mtb) Infection
Aurelie Ray, Jeffrey Fountain and Andrea Cooper
Trudeau Institute, Inc. Saranac Lake, New York
The control of Mycobacterium tuberculosis (Mtb) occurs through an acquired anti-
gen-specific CD4+ T cell response and the IL-12 family of cytokines along with their re-
spective receptors are essential to this response. Not only does IL-12 play a role in modulat-
ing IFN- -mediated T-cell response, which is required for control of Mtb growth, IL-12
(p40)2 is also required for DC to migrate to the draining lymph node following Mtb activa-
tion.
The receptor for IL-12p40, IL-12R 1, is associated with the Janus kinase (JAK)
family member Tyrosine kinase 2 (Tyk2) whereas the receptor required for IL-12p35 bind-
ing, IL-12R 2, is associated with another member of the JAK family, JAK2. Activation of
IL-12R by IL-12p70 leads to phosphorylation of STAT4 and STAT3, which subsequently
induce transcription of target genes. Several studies have shown that Tyk2 is required for
IFN production in response to IL-12p70 by different cell types.
We wanted to define the role of Tyk2 in IL-12R signaling during control of Mtb in-
fection. In order to do this, we infected Tyk2 deficient mice via the aerosol route with Mtb
and followed progression of the disease.
We found that absence of Tyk2 resulted in a modest increase in bacterial burden as-
sociated with slightly reduced cytokine secretion (IL-17, IFNg) and T cell maturation.
These data suggest Tyk2 plays a minor role in the development of the protective response
during Mtb infection. It is likely therefore that other components of IL-12R signaling
(JAK2) are the principal mediators of IL-12 activity in this model.
68
#4
Impaired Function, but Not Generation, of Follicular Helper T cells in
Aged Mice
Julie S. Lefebvre, Dawn Jelley-Gibbs, Sheri Eaton and Laura Haynes
Trudeau Institute, Saranac Lake, NY
Aging negatively impacts most aspect of the immune system. A good example of this is
the poor humoral response generated following influenza immunization in older individu-
als. To generate a strong and specific humoral response, B cells need to receive help from
CD4 T cells, particularly from the follicular helper T cell (Tfh) subset. Previous studies
have shown that intrinsic and extrinsic defects accumulate with aging that impair the helper
functions of aged CD4 T cells. Whether the inadequate help from the aged CD4 T cells re-
sults from their incapacity to become Tfh cells or from functional defects of those Tfh re-
mains to be elucidated.
Using a model of influenza vaccination in young and aged mice, we show that a higher
percentage of CD4 T cells acquires the typical Tfh markers CXCR5 and PD1 in the drain-
ing lymph nodes of aged mice compared to young mice. However, a lower proportion of B
cells acquires a germinal center phenotype (PNAhi CD38lo) suggesting that the aged Tfh
cells are functionally impaired. To identify the mechanism(s) preventing the aged Tfh cells
to support an efficient germinal center reaction, we performed real-time PCR analyses on
the RNA of purified young and aged Tfh cells. While the level of mRNA for IL-2, IL-4 and
IL-21 were similar in young and aged Tfh cells, the aged Tfh cells expressed more IL-10
and IFNgamma than young Tfh cells. Restimulation of purified Tfh cells from the spleen of
young and aged mice infected with PR8 confirmed that a greater proportion of aged Tfh
cells produced cytokines (IL-10, IL-21 and IFNgamma) than young Tfh cells.
The dysregulated signals sent by the aged Tfh cells likely contribute to the reduced ger-
minal center response and consequently to the impaired humoral response generated follow-
ing influenza immunization in aged individuals. Further experiments aimed at blocking IL-
10 and/or IFNgamma will be undertaken to confirm the putative deleterious role of these
cytokines in the germinal center reaction.
69
#5
The Role of Macrophages and Neutrophils in Pulmonary Francisella tularensis
Infection
Donald Steiner and Dennis W. Metzger
Albany Medical College, Albany, NY
Francisella tularensis is the causative agent of tularemia. Pulmonary infection with
F. tularensis is highly lethal due to the profound inflammation induced in the airways.
Though F. tularensis is believed to be an intracellular pathogen that requires replication
within macrophages during respiratory infection, we have found that depletion of alveolar
macrophages by intranasal treatment with liposomal Clodronate reduced mean time to death
and total survival rates of infected mice. Furthermore, mice with a macrophage-specific
insensitivity to interferon-γ (gamma) (MIIG mice) similarly showed heightened sensitivity
to pulmonary Francisella infection. Such mice exhibited reduced lung-tissue expression of
the neutrophil chemoattractant tumor necrosis factor-α (alpha) and reduced neutrophil re-
cruitment to the lungs post-infection. Systemic neutrophil depletion reduced mean time to
death and total survival rates, with the most pronounced effect in mice depleted three days
post-infection. In mice infected with a lethal dose of bacteria, exogenous interleukin-12
promoted survival and bacterial clearance in mice, but this effect was lost upon neutrophil
depletion and in IFN-γ (gamma) knockout mice. The results suggest that rather than being
a required reservoir for bacterial replication, alveolar macrophages are necessary for de-
fense against F. tularensis infection primarily by recruiting neutrophils, which are essential
for clearing the bacterium. (Supported by NIH grant PO1 AI 056320)
70
#6
The Impact of p38 and cAMP on Cytokine Production by Human Monocytes
Infected with Borrelia burgdorferi, the Causative Agent of Lyme Disease
Nicole Johnson, Kathleen Bashant, Rebeca Patsey, Bikash Sahay, Timothy Sellati
Albany Medical College Albany, NY
Lyme disease is caused by the bacterial spirochete Borrelia burgdorferi. The disease
is easily treated in its early stage with antibiotics, but if diagnosed late, Lyme disease pa-
tients can experience severe symptoms such as arthritis, carditis, and neurological compli-
cations. It has previously been shown in mouse models that both p38-MAP kinase and
cAMP are important for tempering the inflammatory response to B. burgdorferi, which
drives the development of severe symptoms. In this study, we try to extend our current
knowledge in mouse models to in vitro human systems - the myelomonocytic THP-1 cell
line and peripheral blood monocytes (PBMCs). THP-1 cells were treated with PMA for
three days (to differentiate them into monocytes) and then exposed to a p38 inhibitor,
SB202190, or exogenous cAMP at the time of B. burgdorferi infection. The p38 inhibition
increased TNF production and lowered production of IL-10 whereas cAMP had the oppo-
site effect. This suggests that the THP-1 cells act in a similar manner to the previously
studied mouse bone marrow-derived monocytes. Human PBMCs, however reacted differ-
ently than predicted. Inhibition of p38 causes a decrease in production of both TNF and IL-
10, suggesting p38 regulation in the immortalized cell line differs from that of the primary
cells. Further studies are needed to determine the value of THP-1 cells as a surrogate for
human PBMCs. Also, to date, no cAMP studies have been conducted in human PBMCs.
71
#7
Activation of the Aryl Hydrocarbon Receptor during Development Alters the
Functional Capacity of Dendritic Cells
Bethany Winans, Guang-Bi Jin and B. Paige Lawrence
Department of Environmental Medicine, University of Rochester Medical Center,
Rochester NY
The developing immune system is susceptible to environmental insults, potentially
leading to altered immune function later in life. The aryl hydrocarbon receptor (AhR) is a
ligand-activated transcription factor that plays a role in the development and function of the
immune system. The AhR acts as an environmental sensor, binding many dioxins and poly-
chlorinated biphenyls (PCBs), pollutants to which humans are constantly exposed. Human
and animal data demonstrate that early life exposure to AhR-binding pollutants leads to per-
sistent alterations in immune function, supporting the idea that inappropriate AhR activation
influences the developing immune system. Our laboratory has shown that mice exposed to a
potent AhR agonist during development have persistent alterations in their immune re-
sponse to influenza virus challenge at maturity, including a reduced expansion and activa-
tion of CD8+ T cells. However, it is unknown whether developmental AhR signaling di-
rectly impacts CD8+ T cells, thereby altering their function, or if AhR activation leads to
functional alterations in the antigen presenting cells (APCs) required for the CD8+ T cell
response. We therefore determined if developmental activation of AhR altered dendritic cell
(DC) frequency or functional capacity. Developmentally exposed mice have fewer DCs in
their mediastinal lymph nodes following infection with influenza virus. Moreover, DC
function is altered following early life AhR activation, as isolated DCs from developmen-
tally exposed mice have a reduced capacity to stimulate naïve CD8+ T cells ex vivo. There-
fore, developmental activation of AhR leads to a reduction in both the number and function
of DCs during an influenza virus infection. These data suggest that a heretofore unexplored
effect of maternal exposure to AhR ligands is the alteration in the DC compartment, which
may contribute to the deregulated CD8+ T cell response following influenza virus infection.
This is the first report demonstrating that early life AhR signaling perturbs DC function.
72
#8
Activation of the Aryl Hydrocarbon Receptor During Development Leads to an
Altered CD4+ T cell Response to Influenza Virus
Lisbeth A. Boule1, Bethany Winans2 and B. Paige Lawrence1,2 1Department of Immunology, Microbiology, and Virology, 2Department of Environmental
Medicine, University of Rochester Medical Center, Rochester, NY
Recent studies show that developmental exposures to certain pollutants lead to last-
ing immunological changes that persist into adulthood. These changes include greater sus-
ceptibility to pathogens and lower vaccine efficacy later in life. An intracellular receptor
activated by a variety of chemicals is the transcription factor aryl hydrocarbon receptor
(AhR). It is expressed by many cell types, including immune cells, and can alter their func-
tion upon activation. Previously, we have shown that developmental triggering of the AhR
by one of its most potent ligands, the pollutant 2,3,7,8-tetrochlorodibenzo-p-dioxin
(TCDD), results in a blunted CD8+ T cell response, characterized by a reduced ability to
proliferate and produce effector cytokines compared to cells from vehicle exposed off-
spring. Recently we have examined the CD4+ T cell response to influenza virus in develop-
mentally exposed mice. We find that there are fewer activated and virus-specific CD4+ T
cells in the draining lymph nodes (the mediastinal lymph nodes - MLN), of infected adult
mice that were developmentally exposed to TCDD. In addition, there are fewer Th1 cells in
the MLN of these mice, defined by TBet expression and IFNγ production. While, we do not
observe a change in the number of CD4+CD25+Foxp3+ regulatory T cells (Tregs), and the
percentage of Tregs is increased the MLN of infected offspring of TCDD-exposed dams
compared to vehicle exposed controls. Interestingly, instead of a decrease in the number of
activated, virus-specific CD4+ T cells in the lungs of mice developmentally exposed to
TCDD, similar to what is observed in the MLN, we show that the frequency of activated
virus-specific CD4+ T cells is increased in mice developmentally exposed to TCDD. Fur-
thermore, we have observed an increase in the frequency of Th1 cells and Tregs in the lungs
of developmentally exposed mice. Together, these data suggest that while maternal expo-
sure to TCDD leads to a suppressed CD4+ T cell response in the MLN after influenza virus
infection, the CD4+ T cell response in the lungs of these mice is enhanced. Given that the
offspring of TCDD-exposed dams only experience AhR activation during development, we
plan to further examine the epigenetic mechanisms by which developmental AhR activation
may influence the ability of CD4+ T cells to become activated and differentiate in develop-
mentally exposed mice after influenza virus infection.
73
#9
Effect of Asthma on Susceptibility to Influenza A Virus Infection
Yoichi Furuya1, Sean Roberts1, and Dennis Metzger1
1Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY 12208
Viral infections are responsible for approximately 85% of asthmatic exacerbations,
and currently, there are no effective prophylactic therapies. Despite this, little remains
known about how the asthmatic phenotype could influence immune responses against respi-
ratory viral pathogens. Using a mouse model of asthma, we investigated the susceptibility
of asthmatics to influenza A virus challenge. Unexpectedly, asthmatic mice were highly re-
sistant to primary infection with the H1N1 2009 pandemic strain. However, although viral
replication was initially delayed in asthmatic mice, viral lung titers ultimately reached com-
parable levels relative to non-asthmatic mice. Asthmatic mice also had increased numbers
of immune cells in their lungs before and after influenza virus challenge. Nevertheless, the
lungs of asthmatic mice were found to have decreased levels of various cytokines including
IFN-gamma, TNF, IL-12, and IL-5. Thus, an increased survival rate did not correlate with
viral titer; instead, increased resistance was associated with reduced pro-inflammatory lung
cytokine levels and increased numbers of immune cells at the site of infection. Currently,
we are elucidating the mechanisms responsible for enhanced resistance of asthmatic mice to
influenza virus infection.
74
#10
Enhanced Immunogenicity of a Recombinant Ricin A Subunit Vaccine When
Co-administered Intradermally with a Detoxified Type II Heat-Labile
Enterotoxin
Christopher J. Greene,1 Lorrie M. Mandell1, Chrystal Chadwick2, Joanne M. O‟Hara2,
Nicholas J. Mantis2, Terry D. Connell1 1 The Witebsky Center for Microbial Pathogenesis and Immunology, The Department of
Microbiology and Immunology, University at Buffalo, Buffalo, NY 14214 2 Division of Infectious Disease, Wadsworth Center, New York State Department of Health,
120 New Scotland Avenue, Albany, NY 12208
LT-IIb, a member of the type-II subfamily of heat labile enterotoxins (HLT) ex-
pressed by Escherichia coli, exhibits strong mucosal adjuvant properties. Recently, we de-
scribed LT-IIb(T13I), a non-toxic mutant of LT-IIb that has altered ganglioside receptor bind-
ing activity yet retains the adjuvant properties of native LT-IIb. In this study, we evaluated
the capacity of LT-IIb(T13I) to enhance the antigenic properties of RiVax, a recombinant
ricin A (RTA) subunit vaccine that has proven to be safe but not particularly immunogenic
in Phase I clinical trials. Intradermal (i.d.) co-administration of RiVax and LT-IIb(T13I) in a
mouse immunization model significantly enhanced RiVax-specific serum IgG antibody
titers and ricin toxin neutralizing activities, as compared to mice immunized with RiVax
alone. Local inflammation elicited by LT-IIb(T13I) following i.d. delivery was roughly
equivalent to that elicited by alum, and significantly less than that elicited by native LT-IIb.
RiVax-LT-IIb(T13I) immunized mice were significantly more protected in a ricin challenge
model than were mice immunized with RiVax alone. Additionally, LT-IIb(T13I) was highly
effective at stimulating RiVax-specific serum IgG and mucosal IgA antibodies when co-
administered with RiVax by the mucosal route. Collectively, these data highlight the poten-
tial of LT-IIb(T13I) as an effective second-generation adjuvant for use in biodefense vac-
cines.
75
#11
The Anti-inflammatory Effects of Vitamin D-Induced CD14 from Mammary
Epithelial Cells
Katrina Simmons1, Timothy Sellati2, and JoEllen Welsh1
1 Department of Biomedical Sciences, School of Public Health, University at Albany, Rens-
selaer, NY 2 Center for Immunology and Microbial Disease, Albany Medical College, Al-
bany, NY
Vitamin D decreases the risk for infectious diseases by regulating immune responses
at the molecular level. Very few studies have investigated how vitamin D alters the im-
mune environment of epithelial cells in barrier tissues, such as the mammary gland, that are
exposed to infectious agents. Through microarray screening of human mammary epithelial
(HME) cells treated for 24 hours with 1α(alpha),25-dihydroxyvitamin D (1α(alpha),25D)
(the active form of vitamin D), we found CD14 was elevated more than 60-fold. CD14 is a
pattern recognition receptor that binds lipopolysaccharide (LPS), a microbial cell wall com-
ponent of gram-negative bacteria. By quantitative PCR, we confirmed that CD14 mRNA
was significantly increased in HME cells treated with either 1α(alpha),25D or its precursor
metabolite 25-hydroxyvitamin D (25D), which reflects overall vitamin D status. Based on
these findings, we examined the effects of both vitamin D metabolites on the expression of
membrane CD14 (mCD14) and soluble CD14 (sCD14) by microscopy and ELISA in HME
cells. Both 1α(alpha),25D and 25D increased the accumulation of sCD14 in the condi-
tioned media of HME cells in a dose- and time- dependent manner but did not alter mCD14.
Surprisingly purified E.coli LPS, a potent inducer of CD14 in mammary gland and other
epithelial tissues as well as immune cells, did not increase the expression or secretion of
CD14 in HME cells. We also found that 1α(alpha),25D and 25D reduced the gene expres-
sion of pro-inflammatory cytokines IL-6, TNFα (alpha), and IL-1β (beta) in HME cells
stimulated with purified E.coli LPS. To determine the effects of vitamin D-induced sCD14
on TLR4 activation and cytokine secretion from monocyte-derived macrophages, we incu-
bated HEK-Blue TLR4 reporter cells (HEK-Blue) and differentiated THP-1 macrophages
with conditioned media from vitamin D-treated HME cells ± (plus or minus) purified E.coli
LPS. We found that conditioned media from vitamin D-treated cells had no significant ef-
fects on TLR4 activation in HEK Blue cells or TNFα secretion but did promoted the secre-
tion of IL-10 from differentiated THP-1 macrophages in the presence of E.coli LPS. In
conclusion, this data is the first to demonstrate effects of vitamin D on expression and se-
cretion of CD14 in mammary epithelial cells and support the central hypothesis that vitamin
D promotes an anti-inflammatory environment via secretion of sCD14 from mammary
epithelial cells.
76
#12
Depletion of Extracellular Adenosine Inhibits Inflammatory Cell Migration into
the Brains of Mice
Do-Geun Kim, Antje Krenz and Margaret S. Bynoe
Department of Microbiology and Immunology, College of Veterinary Medicine,
Cornell University, Ithaca, NY 14853 U.S.A.
Multiple sclerosis (MS) is a debilitating neuroinflammatory disease that is character-
ized by infiltration of inflammatory immune cells into the central nervous system (CNS).
Currently, there is no cure for MS and the few available treatments only lessen symptoms in
some patients, and can have debilitating side effects. Our lab has pioneered studies demon-
strating that blockade of adenosine receptor signaling with broad spectrum adenosine recep-
tor antagonists or specific antagonists that block the A2A adenosine receptor inhibits lym-
phocyte infiltration into the CNS and protects mice from a multiple sclerosis-like disease
called experimental autoimmune encephalomyelitis (EAE). In this study, we used an FDA-
approved drug called PEG-ADA (pegylated adenosine deaminase) that is used to treat pa-
tients with severe combined immune deficiency or SCID due to a mutation in their adeno-
sine deaminase (ADA) gene. ADA rapidly converts adenosine to inosine resulting in adeno-
sine‟s very short half life (about 6 seconds) in the circulation. We hypothesized that similar
to adenosine receptor blockade, depletion of extracellular adenosine by PEG-ADA may
lessen the influx of inflammatory immune cells into the brain and protect mice from EAE.
Our preliminary results show, that indeed mice treated with PEG-ADA developed signifi-
cantly less severe EAE and have less immune cell infiltration in their brain. Because a com-
mon characteristic of MS in general is that harmful immune cells invade the CNS and cause
damage, this study has the potential to benefit MS patients.
77
#13
Generation of a Dual Functioning Anti-Tumor Immune Response
in the Peritoneal Cavity
Abigail L. Sedlacek, Scott A. Gerber, and Edith M. Lord
University of Rochester, Rochester NY
Tumor cell metastasis to the peritoneal cavity (PC) is clinically observed in patients
with primary tumors of peritoneal organs, particularly colon and ovarian tumors. Upon re-
lease into the PC, tumor cells initially attach to the omentum, a tissue consisting of highly
organized immune aggregates (IA) imbedded in adipose tissue. Despite their proximity to
potential immune effector cells, tumor cells bind and thrive on these IA, which have been
shown to be inherently immunosuppressive. We hypothesized that if we could override the
suppressive function of these IA, we could generate a productive anti-tumor immune re-
sponse within the PC. To investigate anti-tumor immune responses in the PC we chose a
mouse model of colon adenocarcinoma, which has been shown to metastasize to the omen-
tum. Thus, we immunized mice i.p. with a lethally irradiated colon adenocarcinoma cell
line: C38. Immunization resulted in temporary enlargement of IA and expansion/
recruitment of multiple immune cell populations. Following specific (C38) or non-specific
(E0771 and B16) live tumor challenge, we failed to detect growth of both specific and sur-
prisingly non-specific tumors on the omentum by microscopy, flow cytometry, or colony
forming assay. Even when immunized mice were allowed to rest for 60 days, neither spe-
cific nor non-specific tumor growth was observed suggesting a long term anti-tumor re-
sponse. Depletion of T cells, B cells, or macrophages did not alter the anti-tumor response
and still resulted in a lack of tumor growth regardless of the type of tumor used to chal-
lenge. Interestingly, depletion of NK cells resulted in only non-specific tumor formation on
the omentum of immunized mice. This data suggests that NK cells are responsible for the
non-specific tumor immunity following immunization and that a redundant mechanism in-
volving both specific and non-specific immunity prevents the growth of C38. Additionally,
this phenomenon of non-specific immunity was not observed systemically. When mice
were immunized i.m. with C38, and challenged i.m. in the opposite flank with either C38 or
E0771, only C38 tumors exhibited delayed tumor growth in direct contrast to our i.p. re-
sults, thus highlighting the uniqueness of the peritoneal cavity in observing these non-
specific tumor responses. Collectively, these data demonstrate that the peritoneal cavity
represents a unique environment that under particular stimuli is capable of eliciting a long
term anti-tumor immune response able to prevent the growth of tumor which involves both
specific and non-specific mechanisms.
Supported by NIH Grants: R01: CA28332 and T32: AI007285
78
#14
Prophylactic Treatment with CpG Oligodeoxynucleotides Confers Protection
Against Pulmonary and Systemic Yersinia pestis Infection
Anthony, J. Hickey, Lawrence W. Kummer, Frank M. Szaba, Michelle A. Parent, Lawrence
L. Johnson, Jr-Shiuan Lin, Stephen T. Smiley
Trudeau Institute, Saranac Lake, NY USA 12983
Immunomodulatory agents have the potential to serve as a new class of broad-
spectrum antimicrobials against infectious diseases for which there currently are limited
safe and effective countermeasures. An example of such a disease is plague, caused by the
gram-negative pathogen Yersinia pestis, which has resulted in three major pandemics
throughout history and has been responsible for the deaths of hundreds of millions of peo-
ple. Despite substantial efforts there currently is no safe and effective vaccine available
against plague, and the window of opportunity afforded to infected individuals for treatment
with antibiotics is small. New treatment regimens, therefore, need to be sought. We demon-
strate here that prophylactic treatment with immunomodulatory CpG oligodeoxynucleotides
(ODN) provides protection against Yersinia pestis. Specifically, intranasal administration of
CpG ODN one day prior to lethal pulmonary exposure to Y. pestis strain KIM D27 signifi-
cantly improves survival and reduces hepatic bacterial burden in mouse models. Interest-
ingly, despite conferring protection, treatment with CpG ODN increases the bacterial bur-
den of the lung. Prolonged survival and decreased hepatic bacterial burden are observed in
both B cell-deficient MT and lymphocyte-deficient RAG2-deficient mice after pretreat-
ment with CpG ODN, signifying that adaptive immunity is not required for the treatment to
exert a protective effect. None of the above impacts, including increased pulmonary bacte-
rial burden, however, are observed in TLR9-deficient mice, indicating that they are all de-
pendent on TLR9 signaling. Finally, we demonstrate that the same treatment regimen with
CpG ODN improves the survival and reduces the hepatic bacterial burden of mice receiving
an intraperitoneal challenge with Y. pestis. This demonstrates that intranasal pretreatment
with CpG ODN can protect against both pulmonary and systemic Y. pestis infection and in-
creases the potential for its use as an immunomodulatory agent against the pathogen.
79
#15
The Non-Redundant Role For Chemokine Receptor Cxcr3 in
T cell Trafficking into Tumor Tissue
Maryann Mikucki1, Joseph Skitzki2, Daniel Fisher1,2, Andrew D. Luster3,
and Sharon S. Evans1
1Deptartment of Immunology and 2Department of Surgical Oncology, Roswell Park Cancer
Institute, Buffalo, NY USA; 3Center for Immunology & Inflammatory Diseases, Division of
Rheumatology, Allergy & Immunology, Massachusetts General Hospital, Harvard Medical
School, Charlestown, MA USA
Several immunotherapeutic regimens are currently under investigation for the treat-
ment of metastatic melanoma and other aggressive cancers in order to exploit the unique
capacity for T cells to mediate tumor-specific cytotoxicity and long-lasting protection
against recurrence. These strategies seek to enhance either the activity of cytotoxic tumor-
specific T cells (e.g., CTLA-4 blockade) or their frequency within the blood (e.g., adoptive
cell transfer therapy and therapeutic vaccination). Common to these treatments is the re-
quirement for effector T cells to cross from the circulation into tumor tissue via a
chemokine-dependent process, but the precise chemokine/chemokine receptor involved is
unknown. Here, we used a murine model to examine the chemokine requirements for T cell
trafficking in the tumor microenvironment. Tumor-reactive cytotoxic T cells were shown
to express three distinct chemokine receptors that are functional in vitro: CCR2, CCR5, and
CXCR3. Cognate chemokines for each of these receptors (e.g., CCL2, CCL5 and CXCL10,
respectively) were also detected in the tumor microenvironment of murine melanoma.
However, loss of a single chemokine receptor, CXCR3, severely compromised the ability of
adoptively transferred effector T cells to traffic to tumors as measured by intravital micros-
copy and short-term homing studies. CXCR3-dependent trafficking was further linked to
tumor cell apoptosis and tumor growth delay. Taken together, these studies establish the
obligate role for CXCR3 in the migration of blood-borne effector T cells into tumor tissue
and support the development of chemokine-targeted strategies to improve T cell entry dur-
ing cancer immunotherapies. Supported by the NIH (CA79765, CA085183), the Jennifer
Linscott Tietgen Family Foundation, and the Joanna M. Nicolay Melanoma Foundation.
80
#16
Mechanisms of Antigen Capture and Sampling by Peyer’s Patch
Dendritic Cells: Implications for Oral Vaccine Delivery
Magdia De Jesus1, Gary Ostroff2, and Nicholas J. Mantis1 1Wadsworth Center, New York State Department of Health, Albany, NY
2University of Massachusetts Medical School, Worcester, MA
The gut-associated lymphoid tissues (GALT), including Peyer‟s patches (PPs), are
sites of continuous antigen sampling in the intestinal mucosa. Sampling by the GALT is
highly selective, as only certain antigens are transported across the so-called follicle associ-
ated epithelium (FAE) and captured by the underlying network of dendritic cells (DCs). In
this study, we are using 2-4 µm in diameter yeast-derived β(beta)-glucan particles (GPs) as
traceable delivery vehicles to begin to define the mechanisms by which mucosal DCs cap-
ture antigens and vaccines.
Preliminary studies using the GPs reveal that sampling occurs within 2.5 hours of
oral delivery, as particles are readily visible in proximal and distal PPs. The GPs over-
whelmingly accumulate in CD11c+ DCs that reside immediately adjacent the FAE, suggest-
ing that the particles were captured by DCs following M cell trans-epithelial transport. To
begin to examine DC-GP interactions in more detail, total PP cells were incubated with
FITC-labeled GPs ex vivo for 1 h at 4oC and then subjected to flow cytometry. The results
revealed that 8% of total PP cells are CD11c+ DCs. Of this population of DCs 7% were as-
sociated with GPs. We performed parallel studies with the viable fungal pathogens Crypto-
coccus neoformans and Candida albicans. The results reveal that C.neoformans, which is
10-20 µm in diameter was too large to cross the FAE. C.albicans, on the other hand, was
sampled by PP DCs, but only when uptake was facilitated by temporarily disrupting the
mucus layer with a bolus of N-acetyl cysteine.
To begin to understand which receptors are responsible for DC-mediated sampling
of GPs and C.albicans, we have initiated a survey using immunostaining and RT-PCR of C-
type lectins receptors (CLRs) in mouse PPs. The results reveal that DC-SIGN, SIGN-R1,
SIGN-R3, Dectin-1, Dectin-2, and Langerin are expressed in murine (Balb/c) PPs under
normal conditions without exposure to exogenous antigens or adjuvant. Preliminary studies
suggest that Langerin and possibly Dectin-1 are expressed by DCs in the sub-epithelial
dome region.
As we begin to understand the receptors types and antigens that underlie selective
antigen sampling by PP DCs, we can engineer modified GPs to enhance uptake and poten-
tially use them for the development of better therapeutics and oral vaccines.
81
#17
Rewiring of the Protein Kinase C Beta 2 (PKC β(beta)II) and Bcr/Abl Signal
Transduction Pathways
David A Hoekstra II, Louise Carlson, Kelvin P. Lee
Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY, 14263
Chronic myeloid leukemia (CML) accounts for 20% of adult leukemias, and is char-
acterized by the presence of the bcr-abl fusion gene. The presence of constitutively active
Abl is essential for CML blast survival even in advanced disease, and underlies the success
that the Bcr/Abl kinase inhibitor imatinib has had. However, resistance to imatinib occurs in
a significant number of blast crisis patients and is a substantial clinical obstacle. We have
previously shown that direct activation of PKCβ(beta)II by Phorbol 12-myristate 13-acetate
(PMA) induces apoptosis and growth arrest in leukemic cells. PKCβII is kept in an inactive
state in the cytosol by interactions between its pseudosubstrate and kinase domains; upon
activation, the pseudosubstrate domain releases the kinase domain, and PKCβ(beta)II trans-
locates to the plasma membrane. Substitution of an alanine at position 25 in the pseudosub-
strate domain for a phosphomimetic glutamic acid leads to the constitutive activation of
PKCβ(beta)II. Similarly, we hypothesized that substituting the alanine at position 25 for a
phosphorylatable tyrosine (A25Y) would allow Bcr/Abl to phosphorylate the introduced
tyrosine and activate PKCβ(beta)II. Bcr/Abl mediated activation of PKCβ(beta)II would
then lead to the death of Bcr/Abl+ leukemic cells, while leaving Bcr/Abl- cells unaffected.
Using confocal microscopy, we show that following transfection WT-PKCβ(beta)II
is cytoplasmically located in media alone in Bcr/Abl+ K562 cells, and in Bcr/Abl- KG1a
cells. Addition of PMA leads to translocation to the plasma membrane, indicating activa-
tion. However when A25Y-PKCβ(beta)II constructs are transfected in, A25Y-PKCβ(beta)II
is found at the plasma membrane in K562, but not in KG1a cells in media alone. These re-
sults indicate that A25Y-PKCβ(beta)II is being activated in a Bcr/Abl dependent manner.
These observations were then quantified using ImageStream, wherein we show that A25Y-
PKCβ(beta)II is activated in media alone in K562 cells, and only upon addition of PMA in
KG1a cells. Additionally, when Bcr/Abl was stably transfected in KG1a cells, A25Y-PKCβ
(beta)II translocates to the plasma membrane in media alone, indicating activation by Bcr/
Abl. To test whether activation of A25Y-PKCβ(beta)II leads to apoptosis and growth arrest,
K562, and KG1a cells were transfected with either WT or A25Y-PKCβ(beta)II and meas-
ured for apoptosis. We found that A25Y-PKCβ(beta)II induced a maximum of a 4-fold in-
crease of apoptosis when compared to WT PKCβ(beta)II in K562 cells; however there was
no increase observed in KG1a cells. This work demonstrates that rewiring PKCβ(beta)II to
be inducible by Bcr/Abl is feasible, induces characteristic translocation to the plasma mem-
brane, and induces apoptosis.
82
#18
Itk and Antigen Specific CD8+ T cell Memory Differentiation
Fei Huang, Weishan Huang, Yuting Bai, Jessica Briggs, Tina Chew, and Avery August
Microbiology and Immunology, College of Veterinary Medicine, Cornell University
Ithaca, NY 14853
It has been shown that Itk, a Tec kinase family member, is important for T cell de-
velopment and differentiation. CD4+ and CD8+ T cells in Itk KO mice display innate mem-
ory phenotype. Since Itk KO CD8+ T cells preferentially develop into innate memory T
cells in thymus, we aimed to determine whether naïve Itk KO CD8+ T cells also preferen-
tially differentiate into memory T cells after primary response to foreign antigens. To study
the intrinsic and extrinsic effects of Itk in naïve CD8+ T cell differentiation into memory T
cells, we used an OVA-Listeria monocytogenes infection model in which naïve OTI Rag1
KO T cells or OTI Itk Rag1 KO T cells were adoptively transferred into Itk KO or WT re-
cipient mice. We found that naïve OTI ITK Rag1 KO T cells are comparable to OTI WT
Rag1 KO T cells in quantity but not in quality of memory T cell differentiation after a 5-
week infection in WT hosts. In contrast, OTI WT Rag1 KO T cells are deficient in differen-
tiating into memory T cells when placed in Itk KO hosts. Although Itk affects the CD8+ T
cell development in the thymus, its absence does not affect the number of antigen specific
memory T cell differentiation. However, Itk KO mice have an impaired environment for
naïve CD8 T cells differentiation into memory T cells during L. monocytogenes infection.
These results suggest that Itk expression in cells other than CD8+ T cells is critical for sup-
porting the antigen specific CD8+ T cell memory differentiation.
83
#19
CD8+ T cells Modulate the Progression of Hypersensitivity Pneumonitis by
Regulating IL-17A Expression in the Lungs
Sonia Mohintaa and Avery Augusta aDepartment of Microbiology & Immunology, Cornell University, Ithaca, NY, 14853
Hypersensitivity pneumonitis (HP) is an environmental lung disease characterized by
inflammation of the alveoli within the lung. HP is caused by repeated exposure to aerosol-
ized antigens into the peripheral airways leading to pulmonary fibrosis. In humans an exam-
ple of HP, also called „Farmer‟s lung,‟ is caused by a thermophilicbacteria called Saccharo-
polyspora rectivirgula (SR) due to repeated exposure to moldy hay. In mice exposed to SR,
the disease is characterized by lung fibrosis and mononuclear infiltrates mostly composed
of macrophages and T cells. IL-17A plays an important role in this disease, and Th17 cells
producing IL-17A are the major mediators of HP. In addition, CD8+ T cells have been sug-
gested to contribute to the development of this disease. Activated alveolar macrophage pre-
sent processed antigen to the CD8+ T cells which in turn release mediators leading to in-
flammation in lungs. To study the role of CD8+ T cells in HP we used a murine model of
hypersensitivity pneumonitis mediated by Saccharopolyspora rectivirgula (SR). Wild-type
mice were exposed to SR for 3 weeks to develop HP, following which CD8+ T cells were
depleted, along with continued SR exposure. Contrary to our expectation, mice which were
depleted of CD8+ T cells had a significant higher percentage and number of CD4+ T cells
producing IL-17A in the lungs. Analysis of the cytokine transcripts from the SR exposed
lungs revealed that IL-17A expression was significantly higher in the CD8 depleted mice
compared to control non-depleted SR exposed mice. These data suggests that after develop-
ment of HP, CD8+ T cells may act as negative regulators directly or indirectly, influencing
the severity of the disease by affecting IL-17A expression.
84
#20
Development of Tolerogenic Dendritic and Regulatory T cells and Recruitment
of Immature/Myeloid-derived Suppressor Cells Favors Exponential Bacterial
Growth and Survival in Respiratory Tularemia
Sivakumar Periasamy, Tabassum Rahman, and Timothy J. Sellati
Center for Immunology and Microbial Diseases
Albany Medical College, Albany NY 12208
In a mouse model of pneumonic tularemia, respiratory infection with the virulence-
attenuated Type B (live vaccine strain, LVS) or highly virulent Type A (SchuS4) strain en-
genders massive necrotizing inflammation in the lungs. Paradoxically, despite an intense
neutrophilic infiltrate and high bacterial burden, Th1-type pro-inflammatory cytokines (e.g.,
TNF, IL-1β, IL-6, and IL-12) are absent within the first ~72 h of pulmonary infection. It
has been suggested that the bacterium has the capacity to actively suppress or block NF-κB
signaling thus causing an initial delay in up-regulation of inflammatory mediators. How-
ever, our previously published findings and those presented herein contradict this paradigm
and instead strongly supports an alternative hypothesis. Rather than blocking NF-κB sig-
naling, F. tularensis infection results in the development of pulmonary tolerogenic dendritic
and regulatory T cells and recruits large numbers of immature/myeloid-derived suppressor
cells into the lungs. Also F. tularensis activate the release of anti-inflammatory cytokines
(e.g., IL-10 and TGF-β) in the lungs. As such, the highly regulated and overall anti-
inflammatory milieu established in the lung is permissive for unfettered growth and survival
of F. tularensis.
85
#21
Mycobacterium tuberculosis Infection Skews Host Immune Response Towards
Alternative Macrophage Activation
E.H. Hannah Fong, Shumin Tan, and David G. Russell
Cornell University, Ithaca NY
In immunocompetent individuals, exposure to Mycobacterium tuberculosis (Mtb)
causes chronic latent infection but not active disease. This state is brought about by estab-
lishment of lung granulomas, which comprise a core of infected macrophages enclosed by a
solid fibrous cover. Contained within the granuloma, the Mtb bacteria survive but do not
replicate or spread. Progression to active disease is accompanied by disintegration of the
granulomas, releasing infectious bacteria into the airway. In an infected individual, progres-
sion towards disintegration occurs in each granuloma independently (all granulomas do not
progress at the same rate or the same time). The establishment, maintenance, and break-
down of the granuloma involve processes that still largely remain to be elucidated.
Our laboratory developed an in vivo model of the TB granuloma where we inject
mice subcutaneously with polystyrene beads coated with Mtb cell wall lipid trehalose dimy-
colate (TDM) in a basement membrane matrix preparation (Matrigel). This generates granu-
lomas highly similar to TB granulomas.
In order for Mtb to establish chronic infection and evade the host immune response,
it has to manipulate the host immune system. One mechanism is through the polarization of
macrophage response towards alternative activation. Alternatively activated macrophages
inhibit inflammation and bacterial killing. One marker of alternative activation is increase
in arginase production. Arginase competes with nitric oxide synthase for their common sub-
strate L-arginine, diverting L-arginine away from production of NO and other reactive oxy-
gen species which are necessary for bacterial killing. Lungs of C56BL/6 female mice in-
fected intranasally with Mtb express arginase. Factors released by the TDM granuloma in-
duce an increase in arginase activity in bone marrow macrophages. Macrophages isolated
from the TDM granuloma also express alternative activation markers CD36 and mannose
receptor.
In order to survive in an immunocompetent individual, Mtb needs to evade the host‟s
immune response. It does so in part by skewing the immune response away from pro-
inflammatory, bactericidal activities, and towards alternative activation and immune down-
regulation.
86
#22
Differential Requirement of CD8α(alpha)+/CD103+ Dendritic Cells for
Dissemination Versus T cell Priming Following Mucosal Infection with
Listeria monocytogenes
Renee M. Laird, Benjamin J. Wolf, Sandra M. Hayes and Michael F. Princiotta
Department of Microbiology and Immunology
SUNY Upstate Medical University, Syracuse, NY 13210
Dendritic cells (DCs) are essential in priming the cytotoxic T cell response following
infection with bacterial pathogens such as Listeria. Notably, recent studies have shown that
DCs also function in systemic dissemination of this pathogen. BatF3–/– mice, which lack
both CD8α+ and CD103+ DCs, are resistant to systemic Listeria infection, with BatF3–/–
mice exhibiting up to a 2 log lower bacterial burden compared with wild-type (WT) mice.
However, the systemic route of infection utilized in this previous study does not mimic that
in humans. Listeria infection in humans occurs via a mucosal route, which is the result of
the binding of the bacterial internalin A (InlA) protein with E-cadherin on the surface of
epithelial cells. Murine E-cadherin does not bind wild-type Listeria InlA. For this reason,
we are using a “murinized” Listeria strain engineered to express a modified form of InlA
that binds murine E-cadherin. We infected both WT and BatF3–/– mice with murinized Lis-
teria via a mucosal or systemic route to determine the role of DCs in bacterial dissemina-
tion.
Similar to the previous study utilizing a non-murinized Listeria strain, bacterial bur-
den is reduced in BatF3–/– mice compared to WT mice following infection with the mur-
inized Listeria strain via a systemic route of infection. By contrast, we observe no differ-
ence in bacterial burden in WT and BatF3–/– mice following infection with murinized Lis-
teria via a mucosal route of infection. Despite no difference in Listeria burden, we find that
BatF3–/– mice fail to mount a CD8+ T cell response to the murinized Listeria strain. These
data demonstrate that DCs are dispensable for bacterial dissemination following a mucosal
infection with Listeria, but are indispensable to prime the CD8+ T cell response.
87
#23
Use of Antigen-pulsed Dendritic Cells to Further Study
Immune Complex Mediated Protection Against Tularemia
Giang H. Pham, Bibiana V. Iglesias, Sarah Rosa and Edmund J. Gosselin
Center for Immunology and Microbial Disease
Albany Medical College, Albany, NY12208
Francisella tularensis is the causative agent of tularemia, a debilitating disease of
humans and other mammals. It is a category A biothreat agent for which, to date, there is no
approved vaccine and the correlates of protection are not well understood. Numerous stud-
ies have demonstrated that targeting immunogens to Fcγ receptors (FcγR) on antigen pre-
senting cells (APC) can enhance humoral and cellular immunity in vitro and in vivo. Impor-
tantly, recent studies from our laboratory have shown that immune complex consisting of
inactivated F. tularensis (iFt) and anti-LPS monoclonal antibody (iFt-mAb) enhances B and
T cells responses, as well as protection against infection, when administered intranasally. In
this study, we examine the impact of iFt-mAb on antigen presentation by dendritic cells
(DC), DC maturation, and cytokine secretion. We find that, iFt-mAb complex enhances ac-
tivation of iFt-specific T cells. In addition, iFt-mAb complex enhances the secretion of TNF
- , IL-6, IL-10, as well as leukocyte activation chemokine MCP-1. Taken together, our data
suggest that targeting FcγR on DC using iFt-mAb complex enhances T cells activation, the
maturation of DC, and the secretion of pro-inflammatory cytokines by DC, thus providing a
potential explanation for the immune stimulatory activity of iFt-mAb complex following
mucosal administration.
This work is supported by a grant from National Institute of Health; Grant RO1AIO76408
88
#24
Dendritic Cells Act Through IL-6 to Enhance Naïve T cell Trafficking Across
High Endothelial Venules During Adaptive Immunity
Daniel T. Fisher, Jason B. Muhitch, Trupti D. Vardam and Sharon S. Evans
Department of Immunology, Roswell Park Cancer Institute, Carlton & Elm Streets,
Buffalo, New York 14263, USA
Dendritic cells (DC) are as master regulators of the adaptive immune response, pro-
viding both cognate antigen and costimulation required for T cell activation as well as cyto-
kines (e.g., IL-12, TNF, and IL-6) that drive T cell expansion and differentiation. Addition-
ally, DCs are used therapeutically as vaccines in cancer immunotherapy. While the role of
DCs in T cell activation is well known, here we show that DCs play an unexpected role in
shaping the T cell repertoire within inflamed lymph nodes (LN) by promoting trafficking
across vascular gateways termed high endothelial venules (HEV). Immunization of mice
with bone marrow-derived DCs matured ex vivo by TLR4 and 9 agonists (LPS and ODN-
CpG, respectively), but not a TLR2 agonist (Pam3CSK4), increased the intravascular density
of two hallmark adhesion molecules, peripheral lymph node addressin (PNAd) and intercel-
lular adhesion molecule-1 (ICAM-1), selectively in HEVs of inflamed LNs. Increased
PNAd and ICAM-1 display occurred rapidly after immunization (within 12 h) and coin-
cided with improved trafficking of naïve and central memory T cells as well as increased
LN size. PNAd and ICAM-1 expression reverted to homeostatic levels 24-48 h post immu-
nization. Furthermore, preventing migration of DCs into the inflamed LN through global
blockade of chemokine receptors using the irreversible inhibitor of G-protein signaling per-
tussis toxin or using DCs deficient for the chemokine receptor CCR7 prevented induction of
PNAd and ICAM-1 required immunization with DCs with functional CCR7 chemokine re-
ceptors. Remarkably, increased adhesion molecule expression was abrogated by loss of a
single cytokine, IL-6, from immunizing DCs. These findings suggest a model whereby IL-6
–producing DC play an active role promoting the intrinsic trafficking capacity of HEVs,
thereby increasing the probability that naïve and central memory T cells come into contact
with cognate antigen-loaded DCs in inflamed LN during the early phase of the adaptive im-
mune response.
Supported by the NIH (CA79765 and AI082039), the Roswell Park Alliance Foun-
dation, and the Jeniffer Linscott Tietgen Family Foundation.
89
#25
A Neutralizing Antibody Against the Ricin Binding Subunit Interferes with
Toxin Intracellular Trafficking
Anastasiya Yermakova1, Tove Irene Klokk3, Kirsten Sandvig3, and Nicholas J. Mantis 1,2 1Department of Biomedical Sciences, University at Albany School of Public Health, and
2Division of Infectious Diseases, Wadsworth Center, New York State Department of
Health, Albany, NY 3Institute for Cancer Research, Oslo, Norway
Antibodies are remarkable in their capacity to inactivate even the most potent protein
toxins, including anthrax, botulinum, diphtheria, and ricin. However, the underlying mecha-
nisms by which this occurs are poorly understood. In this study, we have produced and
characterized a collection of monoclonal antibodies (mAbs) directed against the B subunit
(RTB) of ricin toxin. RTB plays two roles in ricin cytotoxicity; it promotes toxin attach-
ment to host cell surfaces, and it mediates the intracellular trafficking of the toxin from the
plasma membrane to the endoplasmic reticulum (ER). We screened ~2,000 RTB-specific
murine B cell hybridomas, and identified 2 mAbs (SylH3 and 24B11) with toxin neutraliz-
ing activity. Both of these mAbs, passively protected mice against a 5xLD50 dose of ricin,
however, only SylH3 prevented ricin binding to cell surfaces, indicating that 24B11 must
neutralize ricin by a mechanism other than simply interfering with receptor attachment. In a
modified Vero cell cytotoxicity assay we found that 24B11 was able to completely neutral-
ize ricin in vitro even after the toxin had bound to cell surfaces. Using confocal microscopy
we determined that 24B11 internalized with ricin and remained colocalized with it for up to
4 hours. We observed that in the absence of mAb, by 4 hours, ricin traffics to and enters the
trans-Golgi network (TGN). In contrast, the addition of 24B11, appears to re-route the toxin
away from the TGN. Furthermore, staining for various endosomal compartments revealed
that by 90 minutes of internalization, the majority of the ricin-24B11 complexes colocalize
with late endosomes, suggesting that the toxin is destined for lysosomal degradation. Bio-
chemical assays using a modified form of ricin which becomes sulfated upon reaching the
Golgi, revealed that there was significantly less ricin sulfation in the presence of 24B11,
further confirming that toxin is re-routed away from the TGN. These data provide insight
into toxin antibody interactions, and introduce a new paradigm that anti-B subunit antibod-
ies against A-B toxins can neutralize by both intracellular and extracellular mechanisms.
90
#26
Specialized Proresolution Mediators Enhance Human B cell Differentiation to
Antibody Secreting Cells
Sesquile Ramon 1, Charles N. Serhan 2, and Richard P. Phipps 1
1) Department of Microbiology and Immunology, University of Rochester, Rochester, NY;
2) Department of Anesthesiology, Brigham and Women‟s hospital, Harvard Medical
School, Boston, MA
The resolution of inflammation is an active and dynamic process critical in maintain-
ing homeostasis. Newly identified lipid-derived mediators are key players during the proc-
ess of inflammation resolution. These specialized proresolution mediators (SPM) constitute
separate classes of compounds, which include lipoxins, resolvins, protectins and maresins,
all derived from essential fatty acids. SPM regulate aspects of the immune response, includ-
ing inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimula-
tion of macrophage phagocytic activity. However, their effects on B lymphocytes are un-
known. Our study shows that the novel SPM 17-hydroxydosahexaenoic acid (17-HDHA),
resolvin D1 (RvD1) but not protectin D1 (PD1), strongly increase the ability of normal hu-
man B cells to produce IgM and IgG. The increased antibody levels are due to an increased
number of antibody-secreting cells. Furthermore, SPM regulate the expression of the tran-
scription factors Blimp-1, Xbp-1 and Pax-5, and promote B cell differentiation towards a
CD27+CD38+ antibody secreting cell phenotype. None of the SPM affect proliferation and
are non-toxic. The increase in plasma cell differentiation and antibody production coincides
with the known involvement of SPM during the late stages of inflammation and pathogen
clearance. These new findings highlight the potential applications of SPM as endogenous
and non-toxic adjuvants and anti-inflammatory therapeutic molecules.
This research is supported by DE011390 and NIH T32 DE007202
91
#27
Interactions of Murine Monoclonal and Single-Domain Camelid Antibodies with
Ricin Toxin’s A and B Subunits
David J. Vance1, Jacqueline M. Tremblay2, Charles B. Shoemaker2, and Nicholas J. Mantis1 1Division of Infectious Diseases, Wadsworth Center, Albany, NY
2Tufts Cummings School of Veterinary Medicine, North Grafton, MA
Ricin toxin is a member of the RNA N-glycosidase family of ribosome-inactivating
proteins (RIPs) that includes abrin and Shiga toxin. Ricin‟s A subunit (RTA) catalyzes the
removal of a specific adenine residue in 28S rRNA that leads to termination of protein syn-
thesis and cell death. Ricin‟s B subunit (RTB) is galactose-specific lectin that promotes
toxin attachment and entry into host cells. In previous studies we generated a collection of
RTA- and RTB-specific neutralizing (and non-neutralizing) mouse monoclonal antibodies
(mAbs). Here we used a phage-displayed peptide library approach to define at high resolu-
tion the specific residues that are recognized by a select number of high affinity mAbs. Of
particular importance is the resolution of two overlapping neutralizing B cell epitopes
within a solvent exposed, immunodominant α-helix (residues Y91-T116) on RTA that is
thought to be a critical epitope for inclusion within recombinant RTA-based vaccines. Most
recently we have produced a collection of ricin-specific camelid single domain antibodies
(VHHs) against RTA and RTB. VHHs are unique in that they tend to have longer CDR3
domains than conventional murine immunoglobulins, thereby permitting them to penetrate
antigen crevices, such as enzymatic active sites. We immunized alpacas with RTA and
RTB, and created a library of phage-displayed VHHs from peripheral blood mononuclear
cells. Clones that strongly recognized RTA or RTB were selected, expressed, purified and
characterized for their ability to bind ricin holotoxin and neutralize ricin in a Vero cell cyto-
toxicity assay. We now have a collection of RTA- and RTB-specific VHHs with a range of
toxin neutralizing activities and affinities for ricin. We have initiated characterization of the
most potent neutralizing VHHs and have begun testing them as therapeutics in a mouse
model of ricin challenge. This collection of VHHs will be invaluable in our ongoing effort
to understanding the molecular determinants of antibody-mediated toxin neutralization.
92
#28
Inflammasome Independent Interleukin-1 Contributes to Control of
Bordetella pertussis Infection
David E. Place, Sarah J. Muse, and Eric T. Harvill
The Pennsylvania State University, University Park, PA
Interleukin-1 signaling has previously been implicated in protecting murine hosts
during infection by Bordetella pertussis, but the initial steps involved in processing the pre-
cursor forms of the two IL-1 subtypes have yet to be studied in great detail. In this work we
have examined the role of inflammasome-mediated processing of IL-1β (beta) by the pro-
tein caspase1, a cysteine protease contained in the majority of inflammasome complexes.
Our results demonstrate that while the IL-1 receptor is critical for controlling bacterial num-
ber and pathology, caspase1 is dispensable during primary infection as well as vaccine-
induced protection against B. pertussis in the mouse model. These data suggest that IL-1β
(beta) produced in a caspase1-independent manner or IL-1α (alpha) or both are required for
effective pulmonary immune responses. Indeed, our preliminary data demonstrate that IL-
1β (beta) is only partially required for protection, suggesting a role for IL-1α (alpha) in mu-
cosal immunity against Bordetella. Future experiments are aimed at the study of individual
IL-1 subtypes and their role in infection and vaccination immune responses. Importantly,
this work highlights the role of non-canonical, inflammasome-independent processing of IL
-1β (beta) during infection by this pathogen and may indicate that IL-1 is processed differ-
ently during certain respiratory infections than in other tissues.
93
#29
The Absence of the Inhibitory Receptor FcγRIIB Results in Enhanced
Protection Against Francisella tularensis Challenge Following Immunization
Brian Franz, Ying Li, Constantine Bitsaktsis, Bibiana Iglesias, Giang Pham, Sarah Rosa,
and Edmund J. Gosselin
Center of Immunology and Microbial Disease, Albany Medical College, Albany,
New York, 12208
We have previously demonstrated that intranasal (i.n.) immunization with inacti-
vated Francisella tularensis LVS (iFt) targeted to Fcγ receptors can induce protection
against a lethal mucosal challenge with F. tularensis. These studies also showed that this
protection is antibody and interferon gamma (IFNγ) dependent. FcγRIIB is the only inhibi-
tory Fc receptor (FcR) in the FcγR family, and has been reported to regulate the production
of IgG. Therefore, we wished to determine the impact of FcγRIIB on iFt-induced immune
protection against F. tularensis (LVS) challenge. First, we tested the impact of FcγRIIB on
survival by challenging both FcγRIIB knockout (KO) mice and C57BL/6 (WT) mice with
varying doses of LVS. The results showed little difference in survival between KO and WT
mice. Next, we immunized KO and WT mice with iFt organisms on day 0 and day 21, and
subsequently challenged these mice with a lethal dose of LVS on day 35. The KO mice im-
munized with iFt showed significantly increased survival as compared to WT mice. Fur-
thermore, IgG and IgA production was elevated in both serum and bronchoalveolar lavage
fluid (BALF) from KO mice. In addition, cytokine production was examined, and IFNγ lev-
els were enhanced in KO mice. In summary, these data suggest that FcγRIIB influences the
production of IgG, IgA, and IFNγ, which are all factors that are thought to play a major role
in survival against a lethal LVS challenge. (Supported by NIH grant numbers P01
AI056320 and R01 AI076408.)
94
#30
The Role of Interleukin-10 in Chronic B. bronchiseptica Nasal Cavity Infection
Laura L. Goodfield, Olivier Y. Rolin, Liron Bendor, and Eric T. Harvill
The Pennsylvania State University, University Park, PA
Although animals are colonized by huge numbers of commensals, and sometimes
pathogens, for their entire lives, the mechanistic basis for the persistence of bacterial resi-
dents is poorly understood. Bordetella bronchiseptica, a respiratory pathogen that can infect
a wide variety of animals, induces a strong Th1 inflammatory response and is eventually
cleared from the lower respiratory tract. However, B. bronchiseptica generally persists in
the nasal cavity in high numbers for the life of the animal. Once cleared from the lower res-
piratory tract of wild type mice, the systemic inflammatory immune response, measured by
the production of interferon-γ (gamma), remains elevated. However, the local response
within the persistently colonized nasal cavity returns to basal levels, with interferon-γ
(gamma) levels falling below the limit of detection. These data suggest that either these
bacteria have mechanisms to suppress interferon-γ (gamma) production or that the host in-
duces tolerance to these bacteria in the nasal cavity. We predict that this mechanism in-
volves interleukin-10, which is known to suppress interferon-γ (gamma). Mice deficient in
this cytokine more efficiently control B. bronchiseptica indicating that interleukin-10 con-
tributes to the maintenance of chronic B. bronchiseptica infection in the nasal cavity. In
addition, we recently described an important virulence system in B. bronchiseptica, the type
VI secretion system, and now examine its contribution to nasal cavity persistence. Ongoing
studies will also investigate the cell types responsible for interleukin-10 production and
their activation status in the nasal cavity, as well as the contributions of this newly identi-
fied secretion system during chronic B. bronchiseptica infections.
95
#31
YhjH, a cyclic-di-GMP Phosphodiesterase Modulates Salmonella enterica
serovar Typhimurium Adaptation to IgA Antibodies at Mucosal Surfaces
Jayaleka J. Amarasinghe and Nicholas J. Mantis
Wadsworth Center, New York State Department Of Health, Albany, NY 12208
Sal4 is a monoclonal, polymeric IgA antibody directed against the O-antigen (O-Ag)
of Salmonella enterica serovar Typhimurium that has been shown to be sufficient in pro-
tecting mice against enteric infection. The exposure of S. Typhimurium to Sal4 causes bac-
terial paralysis (loss of flagella-based motility) within minutes, alterations in the outer
membrane integrity and subsequent biofilm formation. We have previously reported that
Sal4-induced biofilm formation was mainly regulated by a diguanylate cyclase, YeaJ. YeaJ
produces cyclic di-GMP (c-di-GMP) signaling molecules which function as a key regulator
of bacterial motility and biofilm formation. Here, we demonstrate that WT (pYeaJ), a c-di-
GMP overproducing strain is capable of overriding Sal4-mediated motility arrest. Coincid-
ing with these data, a deletion mutant of yhjH, a gene that encodes for a specific phosphodi-
esterase (PDE), which degrades cyclic di-GMP in S. Typhimurium is immune to Sal4-
mediated motility arrest in an agar environment. The complementation of the ΔyhjH mutant
by introducing a plasmid containing a functional copy of the yhjH gene completely restores
the Sal4-mediated motility arrest in this strain. In addition, real-time qPCR analysis indi-
cated that expression of yhjH gene is down-regulated by approximately 3 fold upon 1 h of
Sal4 exposure compared to the control. Our data indicate that the presence of enhanced c-di
-GMP levels within the cell allow S. Typhimurium to better adapt to environmental stress
signals such as IgA antibodies. Based on these data, we propose that interaction between
Sal4 and S. Typhimurium reveals a mechanism by which enteric pathogens may sense and
respond to host immunity at mucosal surfaces.
96
#32
Assessing Arenavirus Resistance to Type I Interferon
Benson Yee Hin Cheng1, Juan Carlos de la Torre3, Luis Martínez-Sobrido1,2
Department of Microbiology & Immunology1, Center Biodefense Immune Modeling2, Uni-
versity of Rochester, Rochester, New York; Department of Immunology and Microbial
Science3, The Scripps Research Institute, La Jolla, California
Several members of the Arenaviridae family cause severe hemorrhagic fever (HF)
disease in humans that is associated with high morbidity and significant mortality and thus
pose a serious public health problem in their regions of endemicity. However, increased
travel to and from these regions has resulted in importation of arenaviral HF cases into met-
ropolitan areas, increasing the global health threat of this virus family. Several arenaviruses
are included as category A agents because they could potentially be used as agents of
bioterrorism and recent evidence indicates that the globally distributed prototypic arenavi-
rus lymphocytic choriomeningitis virus (LCMV) is likely a neglected human pathogen of
clinical significance in congenital infections. Public health concerns posed by human-
pathogenic arenaviruses are escalated by the lack of FDA-licensed vaccines and that current
antiviral therapy is limited to off-label use of the partially effective nucleoside analog ri-
bavirin. All these reasons underscore the importance of developing novel antiviral strategies
and effective vaccines to combat arenavirus infections, a task that would be facilitated by a
better understanding of the molecular and cellular biology of arenaviruses.
Interferons (IFNs) are innate pro-inflammatory cytokines that provide the first line of
defense against viral infections by activating ≥300 antiviral proteins encoded by IFN-
stimulated genes (ISGs). We have recently reported that the virally-encoded nucleoprotein
(NP) is able to block the induction of type I IFN (IFN-I) during infection by binding and
inhibiting IkappaB epsilon (IKKε)-mediated phosphorylation and nuclear translocation of
the interferon regulatory factor 3 (IRF-3). Here, we provide experimental evidence that
LCMV is able to infect and replicate efficiently in cells pre-treated with IFN-I, demonstrat-
ing the ability of LCMV to resist the antiviral state induced by IFN-I. Additionally, LCMV
resistance to IFN-I is independent of counteracting the antiviral state, as it was unable to
rescue infection of the IFN-I sensitive Newcastle Disease Virus (NDV). Furthermore, qRT-
PCR analysis demonstrates that LCMV-infected cells treated with IFN-I maintain their abil-
ity to upregulate ISGs. This is, to our knowledge, the only negative-strand RNA virus that is
resistant to the antiviral state induced by IFN-I.
Understanding how arenaviruses uniquely overcome host innate immunity will guide
development of new anti-virals and possibly open new avenues in IFN-based therapies
against arenavirus infection. Studies to elucidate mechanisms of arenavirus resistance to
IFN-I will be discussed.
97
#33
Lymphotoxin Controls the IL-22 Pathway to Inhibit Intestinal Inflammation
Elise Macho Fernandez, Ekaterina P. Koroleva, Luke Neill, and Alexei V. Tumanov.
Trudeau Institute, Saranac Lake, NY 12983
As many as 2.2 million people in Europe and 1.4 million people in the United States
suffer from inflammatory bowel disease (IBD). Immune-based therapeutic approaches for
IBD, including anti-TNF therapy, have shown significant success during the last decade.
Nevertheless, many patients are still not responsive to these therapies and suffer from se-
vere side effects.
Innate lymphoid cells (ILCs) have recently emerged as key players in regulating the
balance between protective immunity and immunopathology at mucosal surfaces. However,
the cellular and molecular pathways that control crosstalk between ILCs and intestinal
epithelial cells during inflammation remain poorly understood.
IL-22 is produced by ILCs in the gut and plays a protective role in inflammatory bowel dis-
ease by enhancing barrier integrity and epithelial innate immunity. Our recent study re-
vealed that lymphotoxin (LT), a member of TNF superfamily of cytokines, is critical for IL-
22 production by RORgt+ ILCs in the gut during mucosal bacterial infection. By using a
conditional gene targeting approach, we found that a LT-driven positive feedback loop con-
trols IL-22 production by RORgt+ ILCs via lymphotoxin beta receptor (LTbR) signaling on
DCs.
In the present study, we aimed to determine how LT controls ILCs to inhibit intesti-
nal inflammation using a mouse model of IBD, induced by dextran sodium sulfate (DSS).
Our data show that LTbR signaling is essential for protection and recovery from acute DSS-
induced colitis. We found that the sensitivity of LTbR-deficient mice to DSS-induced coli-
tis was associated with an impaired production of IL-22 by ILCs populations in the colon.
To determine which LTbR-expressing cells were involved in protection against DSS-
induced inflammation, we generated bone marrow chimeric mice and showed that LTbR
expression in both bone marrow-derived and radioresistant stromal cells are required for the
control of intestinal inflammation. Further analysis of mice with a specific inactivation of
LTbR in distinct cell populations revealed that LTbR signaling in both intestinal epithelial
cells and dendritic cells protects from DSS-induced inflammation.
In conclusion, our data indicate that LT signaling controls intestinal inflammation
via the IL-22 protective pathway.
98
#34
The Effect of Caspase 1 on B. bronchiseptica Type VI Secretion System
Mediated Virulence
Sarah J. Muse, Sara E. Hester, Laura L. Goodfield, David E. Place, and Eric T. Harvill
Department of Veterinary and Biomedical Sciences, The Pennsylvania State University,
University Park, PA 16802
Bordetella bronchiseptica is a Gram-negative respiratory pathogen that has a wide
range of mammalian hosts and is closely related to the subspecies B. pertussis and B.
parapertussis, the causative agents of whooping cough. We recently discovered that the B.
bronchiseptica strain RB50 has a functional Type VI secretion system (T6SS) (Weyrich,
Rolin, unpublished), a “needle-like” apparatus with homology to a T4 phage tail that puta-
tively secretes effector proteins associated with virulence. In other bacteria, T6SS has been
linked to quorum sensing and inter-bacterial competition as well as virulence against eu-
karyotic cells.
The purpose of this project is to study the molecular mechanisms underlying the in-
teraction between the B. bronchiseptica T6SS and the innate inflammatory response in the
lungs during infection. One of the key mediators of inflammation is caspase 1, a compo-
nent of the inflammasome which is responsible for the activation of the cytokine precursors
of IL-1β and IL-18.
To investigate the effect of the T6SS on immune cells in vitro, BMDM were inocu-
lated with RB50 and RB50∆clpV (T6SS ATPase) at MOIs of 1 and 10. The presence of the
T6SS resulted in significantly decreased levels of caspase 1 protein and affected other in-
flammasome components. To determine the effects in vivo, wild type C57BL/6 and Casp1-
/- mice were inoculated with RB50 and RB50∆clpV. Flow cytometry analysis indicated
that the T6SS and caspase 1 affect leukocyte recruitment to the lungs 7 days post inocula-
tion. In addition, both the T6SS and caspase 1 are associated with increased bacterial colo-
nization of the respiratory tract after 14 days post inoculation. Overall, these results suggest
that B. bronchiseptica uses the Type VI secretion system to manipulate the inflammatory
response to respiratory infection.
99
#35
Downstream Targets of PKCβ(beta)II in Dendritic Cell Differentiation
Haley Spangler, Matt Farren, Louise Carlson, Scott Abrams, Kelvin Lee
Roswell Park Cancer Institute, Buffalo NY
Dendritic cells (DCs) are antigen presenting cells capable of inducing both innate
and adaptive immune responses. In a tumor setting their differentiation from progenitor
cells is inhibited by tumor derived factors, resulting in an accumulation of immature DCs
(or myeloid derived suppressor cells). However, many of the mechanisms by which tumors
regulate DC differentiation are currently unknown. Our lab has previously shown the im-
portance of protein kinase C βII (PKCβ(beta)II) in human DC differentiation, while others
have shown roles for interferon regulatory factor 8 and 4 (IRF8 and IRF4) in myeloid cell
differentiation. We then sought to determine whether a relationship exists between these
two IRF molecules with PKCβ(beta)II in DC differentiation.
We have found that IRF8 knockout mice have fewer conventional DCs than wild
type mice. We also found that with PKCβ(beta)II inhibition there is decreased DC differen-
tiation, as well as decreased expression of IRF8. Similarly, when PKCβ(beta)II is activated,
IRF8 expression is upregulated. However, when progenitor cells are treated with GM-CSF
(a known activator of PKCβ(beta)II) we see an upregulation in IRF4 expression and less so
in IRF8. When progenitor cells are treated with FLT3L (a known activator of IRF8 in DC
differentiation) we have found that PKCβ(beta)II is activated. The progenitor cells were
then treated with FLT3L and a PKCβ(beta)II inhibitor, and we saw a PKCβ(beta)II depend-
ent upregulation of IRF8. We have therefore demonstrated roles for both PKCβ(beta)II and
IRF8, and PKCβ(beta)II and IRF4 in myeloid DC differentiation, depending on the DC-
generation stimuli. This indicates to us that there are potential relationships in DC differen-
tiation between PKCβ(beta)II and either IRF8 or IRF4 which can be tumor targets.
100
#36
Tec Kinase Itk Modulates Allergic Airway Inflammation by Suppressing
IFNγ(gamma) Expression in Naïve CD4+ T cells
Arun K Kannana, Nisebita Sahub,c, and Avery Augusta aDepartment of Microbiology & Immunology, Cornell University, Ithaca, NY, 14853.
bDepartment of Veterinary & Biomedical Sciences, The Pennsylvania State University,
University Park PA. cDiscovery Oncology, Genentech, Inc, South San Francisxo, CA 94080
T helper responses are critical for a productive immune response but an inappropri-
ate T helper response results in inflammatory and autoimmune disorders. Although we
know basic details of the signaling events that regulate lineage choices of CD4+ T cells,
there are still considerable gaps in the knowledge of pathways that regulate T cell differen-
tiation to T helper 1 (Th1), T helper 2 (Th2) and T helper 17 (Th17) cells. Allergic asthma
is a Th2 cell dominated inflammatory disease characterized by airway inflammation and
airways hyper responsiveness. IL-2 Inducible T cell kinase (Itk) is primarily expressed in T
cells and is critical for their development, activation and function. Itk-/- mice have a defec-
tive Th2 response and are not susceptible to allergic asthma. Here we show that peripheral
Itk deficient naïve CD4+ T cells have substantially increased transcript levels of prototypic
Th1 genes, including Eomesodermin, IFNγ(gamma), T-bet and IL12Rβ(beta)1. More
importantly, removal of IFNγ(gamma) on the Itk-/- background rescues expression of Th2
related genes in Th cells, and House Dust Mite-driven allergic asthma in Itk-/- mice. In addi-
tion, we show that this Itk mediated signal represses IFNγ(gamma) independent of T-bet
and maintains naïve T cells in an unbiased state, until it comes in contact with cognate anti-
gen. In the absence of Itk, the genomic locus of IFNγ(gamma) is open with the promoter
and a number of conserved nucleotide regions showing enrichment of permissive H3K4me2
and loss of repressive H3K27me3 histone marks. Furthermore, these signaling pathways are
conserved in humans, as loss of Itk in human PBMC derived T cells and Jurkat cells results
in a substantial reduction in the secretion of a number of effector cytokines, at least partly
due to a failure to activate pERK. Our results indicate that Itk signals suppresses the expres-
sion of IFNγ(gamma) in CD4+ T cells, which in a positive feed forward loop, regulates the
expression of Th1 factors such as T-bet and Eomesodermin, and suppress development of
Th2 cells and Th2 responses. This work has implications for understanding T helper differ-
entiation programs, and Itk as a therapeutic target for Th2 mediated inflammatory diseases.
101
#37
A Novel TLR5 Agonist, CBLB502, Promotes Cytotoxic Lymphocyte Mediated
Tumor Immunity
Nicholas Leigh1, Guanglin Bian1, Xilai Ding1, Hong Liu2, Lyudmila G. Burdelya3,
Andrei V. Gudkov3, and Xuefang Cao1
Author affiliations: 1Department of Immunology, 2Department of Medicine Blood and Mar-
row Transplant Program, and 3Department of Cell Stress Biology, Roswell Park Cancer In-
stitute, Buffalo, NY 14263, USA
Toll-like receptor (TLR) mediated recognition of pathogen associated molecular pat-
terns allows the immune system to rapidly respond to pathogenic challenge. This makes
TLR agonists an attractive product that can be used to stimulate an immune response. In
particular, tumor immunity is often hindered by the inability to recognize and destroy ma-
lignant self due to the lack of a danger signal. This signal can be provided by a TLR ago-
nist, ideally providing the essential danger context to the immune system and allowing anti-
tumor immunity to ensue. An attractive TLR agonist is CBLB502, a pharmacologically op-
timized TLR5 agonist derived from Salmonella enterica flagellin. In this study, we exam-
ined the effect of CBLB502 on antitumor immunity using two lymphoma models, both of
which do not express TLR5, and thus do not directly respond to CBLB502. Using the T
cell lymphoma RMAS, which is dependent on natural killer (NK) cell mediated killing,
CBLB502 treatment after tumor inoculation provides significant protection to C57BL/6
mice from death caused by tumor growth. The protection elicited by CBLB502 is both NK
cell and perforin dependent. In addition, CBLB502 stimulates immunity against the B cell
lymphoma A20 in BALB/c mice, with clearance being dependent on CD8+ T cells. To re-
capitulate an antitumor response in vitro, we co-cultured total splenocytes with tumor cells
and treated with CBLB502. CBLB502 significantly enhances the ability of splenocytes to
control tumor growth. To determine which cell types mediate the CBLB502 effect, we util-
ized ImageStream flow cytometry to monitor NFκ(kappa)B nuclear translocation, a meas-
ure of TLR5 activation. ImageStream flow cytometry reveals that CD11b+ and CD11c+
cells, but neither NK nor T cells, directly respond to CBLB502. These findings demon-
strate that the novel TLR5 agonist, CBLB502, can stimulate a robust antitumor immune re-
sponse by indirectly activating cytotoxic lymphocytes through TLR5-expressing accessory
immune cells.
102
#38
Impaired Recruitment of Inflammatory Cells to Aged Lungs Following
Influenza Infection Contributes to Delayed Virus Clearance
S. M. Eaton, P. A. Lanthier, M. P. Tighe, D. M. Jelley-Gibbs, L. Haynes
Trudeau Institute, Saranac Lake, NY
Elderly people are very susceptible to influenza virus and often exhibit complica-
tions following infection including secondary bacterial infections and exacerbation of lung
diseases. Using mouse models, it has been well established that, much like humans, aged
animals exhibit a delay in clearing sublethal influenza virus infection compared to young.
However, the mechanisms responsible for this delay are not well understood. In this study,
we have determined that following influenza infection, the levels of early IFN production
(days 3-6) are significantly lower in aged lungs. Reduced IFN levels lead to the reduced
production of important IFN -dependent chemokines by innate lung cells, including CCL3,
CCL4 and CXCL10, which are necessary for the proper recruitment of inflammatory cells
such as neutrophils. This observation is important since it is now appreciated that neutro-
phils are critical for efficient viral clearance. Reduced lung neutrophil numbers and re-
duced lung immunopathology were observed in aged flu-infected animals, indicating an
overall less vigorous immune response. This diminished initial response plays an important
role in the delay in virus clearance. In addition, it also subsequently contributes to lingering
inflammation in aged lungs, including high levels of inflammatory cytokines and chemoki-
nes, which can contribute to exacerbating pre-existing lung disease. It is likely that these
early impairments also impact responses to secondary infections in the aged due to altera-
tions in the lung environment. This work was supported by NIH funding to L. Haynes and
Trudeau Institute.
103
#39
Human Placental Trophoblast Cells are Hyporesponsive to Type I and Type III
Interferons: Potential Role in Susceptibility of Pregnant Women to
Viral Infection
Catherine G. Burke*, Katherine E. Herman#, Benson Y.H. Cheng#, Luis Martinez-Sobrido #
and Shawn P. Murphy*#
Department of Obstetrics and Gynecology*, Microbiology and Immunology#,
University of Rochester School of Medicine, Rochester, NY
Pregnant women are preferentially susceptible to infection by intracellular pathogens
such as viruses. These infections are associated with severe complications of pregnancy that
include miscarriage, in utero growth restriction, preterm birth and fetal and maternal mor-
bidity. However, the factors accounting for the increased susceptibility of pregnant women
to infection are not well understood. Trophoblast cells (TBCs) form the fetal component of
the placenta, and are the only cells derived from the fertilized egg that are in direct contact
with maternal blood and tissues. We previously demonstrated that TBCs are hypo-
responsive to the pro-inflammatory cytokine interferon- gamma) [IFN- gamma)], which
is essential for immunity against tumors and certain pathogens. However, there is a gap in
our knowledge regarding the ability of TBCs to respond to type I or type III IFNs, which
induce an antiviral state and thus serve as an important first line of defense against viral in-
fection. In this study, we demonstrate that human trophoblast-derived choriocarcinoma cells
are hypo-responsive to both IFN- alpha)2 and IFN- lambda)1, despite the fact that these
cells express all of the components of the JAK-STAT pathway necessary for productive
IFN signaling. Dose response studies and kinetic analyses demonstrate that activation of the
transcription factors STAT-1 and STAT-2 in response to these IFNs is severely compro-
mised in TBCs relative to epithelial cells. In addition, the levels of IFN-responsive gene ex-
pression are significantly lower in TBCs versus epithelial cells. Furthermore, cotreatment of
TBCs with IFN- alpha)2 and the protein tyrosine phosphatase (PTP) inhibitor pervanadate
results in partial rescue of STAT activation and IFN-responsive gene expression. These re-
sults suggest that IFN responses are inhibited in TBCs by PTP(s). Since IFNs provide the
first line of defense against viral infections, we investigated the physiological relevance of
trophoblastic hypo-responsiveness to IFNs in cells pretreated with IFN- (alpha)2 and sub-
sequently infected with the IFN-sensitive Vesicular Stomatitis Virus (VSV). Pretreatment
with IFN- alpha)2 protected epithelial cells but not TBCs from subsequent infection with
VSV. Taken together, these results suggest that the inability of TBCs to respond to IFNs
may play an important role in susceptibility of pregnant women to viruses that infect the
placenta.
104
#40
Assessment of Cytokine Responses in Human Placenta Using a Novel Whole
Mount Immunofluorescence Technique
Meghan E. Bushway, Scott A. Gerber, Richard K. Miller, Edith M. Lord,
Shawn P. Murphy
University of Rochester, Rochester, NY 14642
The human placenta plays multiple critical roles in successful pregnancy, including fetal gas
and nutrient exchange and protection of the genetically distinct fetus from maternal immune-
mediated destruction. Trophoblast cells are the only cells derived from the fertilized egg that are in
direct contact with maternal blood and tissue and thus serve an important function in protection of
the fetus throughout the course of gestation. Interestingly, the pro-inflammatory cytokine inter-
feron-gamma (IFN-γ), which is critical for activating immunity against non-self, has been shown to
be present in the pregnant uterus during normal pregnancy. We hypothesized that placental cells,
especially trophoblasts, have developed unique mechanisms to remain hyporesponsive to IFN-γ
(gamma) and thus evade maternal immune recognition. To date, analysis of human placental cell
responses to cytokines or growth factors has been restricted to isolated cell populations cultured in
vitro. Furthermore, examination of the microenvironment within human placental villous structures
has traditionally been performed using classical immunohistochemistry (IHC). We sought to de-
velop a technique that would enable us to study multiple cell types within morphologically intact
villous tissue. Thus, placental whole mount immunofluorescence (WMIF) was developed in con-
junction with flow cytometry to visualize and quantify surface and intracellular molecules in dis-
tinct cell populations. Moreover, this method was utilized to examine responses to IFN-γ(gamma)
within human placental cell types, including trophoblast, resident immune cells and endothelial
cells.
Striking morphological images of developing CD31+ blood vessels were obtained using
WMIF that were not observed with classical IHC. In addition, CD45+/CD68+ fetal macrophages,
cytokeratin (CK)-7+ cytotrophoblast cells, and human chorionic gonadotropin (hCG)+ syncytiotro-
phoblast were detected within intact villous structures. Furthermore, the levels of the activated (i.e.
phosphorylated) form of the transcription factor STAT1 (pSTAT1) were examined following IFN-γ
(gamma) treatment. In placental explants treated with IFN-γ(gamma), fetal macrophages strongly
induced pSTAT1. In contrast, pSTAT1 was not detected in the syncytiotrophoblast layer or cyto-
trophoblast cells. These data suggest that syncytiotrophoblast, which are in direct contact with ma-
ternal blood, and the underlying cytotrophoblast cells are hyporesponsive to IFN-γ(gamma). This
phenomenon may represent an important mechanism of fetal evasion from maternal immunity.
WMIF combined with flow cytometry allowed for examination of multiple cell types within
intact villous structures while preserving the placental architecture. It is anticipated that this meth-
odology will provide a powerful approach for detailed analyses of the placental architecture in nor-
mal versus pathological pregnancies, and for studying the effects of environmental factors on pla-
cental function and morphometry.
105
#41
Myeloid-Derived Suppressor Cells in Murine Retrovirus-induced AIDS:
Inhibition of the T-cell and B-Cell Responsiveness that Define the
Immunodeficiency
Kathy A. Green, W. James Cook, William R. Green
Geisel School of Medicine at Dartmouth, Lebanon, NH
Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease
settings, especially in many tumor systems. Compared to their involvement in tumor micro-
environments, however, MDSCs have been less well studied in response to infectious dis-
ease processes, in particular against retroviruses that induce immunodeficiency.
Here, we demonstrate the development of a highly immunosuppressive MDSC popula-
tion that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired
immunodeficiency. These MDSCs express a cell-surface marker signature (CD11b+, Gr-1+,
Ly6C+) characteristic of monocytic type MDSCs; exhibit profound inhibition of immune
responsiveness by a cell-dose and substantially iNOS-dependent mechanism that is inde-
pendent of arginase activity, PD-1/PD-L1 expression, and IL-10 production; and display
levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-
infected w.t. vs. knock-out mouse strains, differentially susceptible to pathogenesis. These
MDSC suppressed not only T-cell, but also B-cell, responses, the latter an understudied tar-
get for MDSC inhibition. The direct MDSC immunosuppression of B-cell responses was
confirmed by use of: purified B responder cells, multiple B-cell stimuli, and independent
assays measuring B-cell expansion. Retroviral load measurements and infectious center as-
says indicated that the suppressive Ly6Glow/+ /Ly6C+/CD11b+-enriched MDSC subset is
positive for LP-BM5-RNA, albeit at a significantly lower level than that of non-fractionated
splenocytes from LP-BM5-infected mice.
These results, including the strong, direct MDSC inhibition of B-cell responsiveness, are
novel for murine retrovirus-induced immunosuppression, and, as this broadly suppressive
function mirrors that of the LP-BM5-induced disease syndrome, support a possible patho-
genic effector role for these retrovirus-induced MDSCs.
106
#42
A Bacterial Stress Protein with Chemotactic Properties
Kathryn M. Pietrosimone1, Stephanie R. Davis1, Debbie Laukens2, Michael A. Lynes1
1University of Connecticut 2University of Ghent
Metallothionein (MT) is a small molecular weight protein that is rich in cysteine
(cys) residues. In mammalian cells, MT expression increases under stressful conditions
such as exposure to divalent heavy metal cations, reactive oxygen species (ROS), or reac-
tive nitrogen species (RNS). The cys residues are essential to detoxifying ROS and RNS,
and sequestering heavy metals. MT also shares sequence similarities with chemokines, and
has been shown to act as a chemoattractant for human Jurkat T cells and some primary leu-
kocyte cell populations. A bacterial MT, SmtA, found in certain bacterial species such as
Synechococcus and Pseudomonas, shares some molecular features with eukaryotic MT
based on relative size, sequence comparisons and cys motifs. The expression of SmtA by
the pathogen Pseudomonas aeruginosa suggests that SmtA could influence an immune re-
sponse to bacterial infection by detoxifying ROS produced by inflammatory cells and by
influencing immune cell trafficking. To test this hypothesis, pGEX-6p-smtA plasmid was
transfected into Escherchia coli MC1061 and exposed to various oxidative and inflamma-
tory conditions. SmtA-expressing E.coli was protected from the oxidative stress resulting
from hydrogen peroxide exposure, but not from other oxidants. SmtA purified from this
plasmid was used to measure the protein‟s influence on Jurkat T cell trafficking. Chemo-
taxis was measured using standard Micro-Boyden chambers, and the automated real-time
ECIS/Taxis assays. SmtA induced a chemotactic response by Jurkat T cells, but this re-
sponse was not as robust as the response to SDF-1 . Prior exposure of Jurkat T cells to
SmtA suppresses the subsequent response to SDF-1 . SmtA‟s ability to detoxify ROS and
influence immune cell movement may represent important avenues by which P. aeruginosa
survives the innate immune response, and may be a novel target for therapeutic interven-
tions during infection.
(Presented in Kathryn‟s absence due to inability to attend.)
107
#43
Distinct Roles of Type I and Type II Interferons in Radiation Therapy for B16
Melanoma
Joanne Y.H. Lim, Scott A. Gerber, Edith M. Lord
University of Rochester Medical Center, Rochester, NY
Endogenous type I interferons (IFN-α/β) and interferon gamma (IFN-γ) have both
been implicated in immune responses against cancer. Interestingly, studies done using viral
infection models have demonstrated that IFN-α/β and IFN-γ play non-redundant roles in the
immune system and they act synergistically, enhancing responses that are induced by the
two different signaling pathways. However, this phenomenon, known as priming, has not
been well-characterized in the context of anti-tumor immunity. Our lab previously demon-
strated using a B16 murine melanoma model that local high dose (15Gy) radiation therapy
induced high intratumoral levels of IFN-γ, which had several downstream effects like in-
creased MHC class I expression on tumor cells that in turn enhanced recognition by CD8+ T
cells. Overall, these IFN-γ-dependent effects led to increased tumor control, evident by
drastically decreased rate of tumor growth following treatment.
In the current study, we hypothesize that local radiation therapy (RT) increases the
levels of IFN-α/β within the tumor, and this is required to enhance IFN-γ responses so that
maximal therapeutic potential can be achieved. Indeed, local RT resulted in an early, tran-
sient spike in intratumoral IFN-α levels about two days after treatment. Interestingly, an in-
crease in IFN-γ levels was observed four days post-treatment and remained elevated for at
least five days. When B16 tumors were grown in mice that lack functional IFN-I receptors
(IFNABR KO), the delayed increase in IFN-γ following RT was completely abrogated. To
investigate the mechanism involved, we examined the transcription of interferon-inducible
genes, including the CXCR3 chemokines CXCL9 (MIG) and CXCL10 (IP-10). Following
RT, intratumoral transcript levels of MIG and IP-10 were increased to similar extents in
both WT and IFNγKO mice, but remained at basal levels in IFNABR KO mice. This sug-
gests that IFN-α/β, but not IFN-γ, may be contributing to the initial chemoattraction of lym-
phocytes into radiation-treated tumors. Using flow cytometry analysis of immune cell popu-
lations within the tumors, we confirmed that the levels of intratumoral CXCR3 chemokines
correlate strongly with tumor infiltration by T lymphocytes. Our data suggest that IFN-I
play a critical role in the increase of CXCR3 chemokine levels within the tumor after RT.
These chemokines are important for the recruitment of activated T cells into the tumor,
which then produce IFN-γ, increasing IFNγ-induced immune responses locally. We are cur-
rently studying the effects of combining RT and IFN-α therapy, with the goal of further im-
proving T cell recruitment and activation, hence IFN-γ-associated anti-tumor immune re-
sponses.
Support: NIH R01 grant CA28332
108
#44
Francisella tularensis Triggers Lipoxin A4-induced Programmed Necrosis
and Bax-dependent Mitochondrial Apoptosis/secondary Necrosis
Anju Singh, Sivakumar Periasamy, Tabassum Rahman, Bikash Sahay, Florin M. Musteata
Meenakshi Malik, Chandra Shekhar Bakshi, Jeffrey G. Ault, Carmen A. Mannella, and
Timothy J. Sellati
Center for Immunology and Microbial Disease, Albany Medical College, Albany NY,
12208
Infection with Francisella tularensis SchuS4 causes severe pulmonary disease in
mice, non-human primates, and tularemia patients. The high infectivity and virulence of
Francisella tularensis (Ft) subspecies tularensis coupled with its ease of aerosolization
make it an ideal bioweapon. During respiratory tularemia, caused by inhalation of Ft, bac-
teria subvert host cell death programs to facilitate their unfettered replication within macro-
phages and other cell types. Release of bacteria from infected cells is coincident with se-
vere tissue histopathology and a sepsis-like syndrome that ultimately results in mortality
rates of between 30% - 60%. A common feature of early respiratory infection (≤ 72 h) is
robust induction of host cell death. However, the mechanism(s) by which this complex cel-
lular process is initiated remains incompletely-defined as does the impact of F. tularensis
on mitochondria, the host cell‟s organellar „canary in a coal mine‟. We report that Ft facili-
tates its survival and exponential replication by triggering programmed necrotic cell death
through release of lipoxin A4 followed by Bax-dependent mitochondrial apoptosis/
secondary necrosis marked with an increase in prostaglandin E2 (PGE2) levels. These
events encompass: i) decreased mitochondrial membrane potential and consequent mito-
chondrial oncosis, ii) increased mitochondrial superoxide generation, and iii) release of
damaged mitochondria into the pulmonary extracellular milieu. Notably, host recognition
of and response to accumulating necrotic cell debris and not the bacterium itself elicits the
TNF, IL-1β, IL-6, IL-12p70, IFN-γ and elevated PGE2 which are absent during early infec-
tion, but present during late-phase septic disease (≥ 72 h).
109
#45
IL-1β Production by Inactivated Francisella tularensis LVS Complexed with
Anti-Ft Monoclonal Antibody is Mediated by Fc-gammaR Signaling and the
NLRP3 Inflammasome
Ellen B. Duffy and Jonathan A. Harton
Albany Medical College, Albany, NY
Francisella tularensis (Ft), an intracellular bacterium, causes the often fatal respira-
tory disease tularemia at a low infectious dose, and has been listed as a category A biothreat
agent. A vaccination strategy using formaldehyde-inactivated Ft LVS (iFt) complexed with
a monoclonal antibody against Ft LVS LPS (mAb) provided complete protection against
lethal LVS intranasal infection and improved protection against lethal intranasal SchuS4
infection in mice. Our hypothesis was that this protection was mediated by pro-
inflammatory responses to the vaccine components. Determination of a vaccination strate-
gies ability to elicit a pro-inflammatory response, especially one that produces IL-1β, may
be useful as a screening mechanism for potential efficacy of the strategy. We examined the
innate immune response elicited by this vaccination strategy in a human monocyte cell line.
PMA-treated THP-1 cells were used as a model human macrophage-like cell and treated
with iFt-mAb (allowed to complex overnight at 4 degrees C) for 24 h after which super-
natants were analyzed for IL-1β (beta) by ELISA. Antibodies against Fc-gamma receptors
and TLR2 were used to block signaling from these surface receptors. Chemical inhibitors
of Src family kinases (PP1 or PP2), Syk (piceatannol), and cellular ROS (APDC and NAC)
were used to probe the signaling pathways downstream of surface receptors. While iFt
alone produced slightly more IL-1β than untreated controls, the complexed iFt with mAb
treatment produced significantly more IL-1β. The production of IL-1β was dependent on
FcgammaRI and TLR2 as blocking antibodies reduced IL-1β. Specifically, FcgammaRI is
responsible for transmitting signal upon binding of mAb, while TLR2 signaling is stimu-
lated by the iFt. In addition, there appears to be some cooperation between the signaling
through FcgammaRI and the TLR2. IL-1β production was also dependent on Syk and Src-
family kinases as chemical inhibitors reduced IL-1β. THP-1 cells deficient in NLRP3 or
Nlrp3-deficient mouse bone marrow-derived macrophages showed little to no IL-1β, and
chemical inhibitors of ROS reduced IL-1β indicating that the NLRP3/Nlrp3 inflammasome
and ROS are involved.
110
#46
Eosinophils Promote Immune Responses to Preserve Parasitic Nematode Larvae
Lu Huang1, Nebiat G. Gebreselassie1, Lucille F. Gagliardo1, Nancy A. Lee2, James J. Lee3,
and Judith A. Appleton1. 1Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University,
Ithaca, NY 14853 USA. 2Department of Hematology and Oncology and 3Biochemistry and
Molecular Biology, Mayo Clinic Arizona, Scottsdale, AZ 85259 USA.
By using two different eosinophil-ablated mouse strains, we have shown that eosino-
phils protect, rather than attack Trichinella spiralis larvae in the skeletal muscle during pri-
mary infection. Accumulation of Th2 cells at sites of infection is also impaired in the ab-
sence of eosinophils. To further elucidate the mechanism of how eosinophils preserve T.
spiralis and regulate T cell responses in muscle, we examined the requirement for adaptive
immunity, the time when eosinophils exert their effect, the necessity for antigen presenta-
tion by eosinophils, and eosinophil-mediated effects on dendritic cells. Parasite growth was
normal in Rag1-/- mice and was coincident with enhanced expression of genes associated
with alternatively activated macrophages, suggesting that eosinophils may preserve larvae
in an innate context. Furthermore, the presence of eosinophils only during the earliest stages
of muscle infection was sufficient to support parasite growth. Thus, eosinophils are required
to be present at the time newborn larvae first arrive in the muscle and the local immune re-
sponse is first engaged. Transfer of MHCII-/- eosinophils was effective, showing that anti-
gen presentation by eosinophils is not required to preserve. Lastly, the number of myeloid
dendritic cells (DCs) (CD11c+CD4+CD8α-CD11b+) in cervical lymph nodes was de-
creased in the absence of eosinophils, suggesting that eosinophils promote accumulation of
myeloid DCs, which may be necessary for accumulation of Th2 cells. Taken together, our
findings suggest that eosinophils exert their influence at the very early stages of initiating
the immune response that preserve T. sprialis larvae.
111
#47
IgM Memory B cells Generated During Bacterial Infection are Required for
Secondary IgG Responses to Antigenic Challenge
Jennifer Yates1, Rachael Racine1, Kevin McBride3, and Gary Winslow1,2
SUNY Albany School of Public Health, Albany NY1
Wadsworth Center, Albany NY2
University of Texas MD Anderson Cancer Center, Smithville TX3
Immunological memory is a fundamental concept that is key to generating and main-
taining immunity to pathogens, and for mediating the protection afforded by vaccines. De-
spite the focus on class-switched memory B cells, several studies have validated the exis-
tence of IgM memory B cells, and have demonstrated distinct functions of IgM and IgG
memory B cell subsets. Based on the expression of CD11c, we have identified a large popu-
lation of IgM memory B cells using a natural model of infection by the bacterium Ehrlichia
muris. These CD11c+ IgM memory cells exhibit phenotypic characteristics of memory B
cells, including expression of CD73, and PD-L2. In addition, the CD11c+ IgM memory cells
lack expression of CD138, are largely quiescent, and have accumulated somatic mutations.
Although these cells did not proliferate or secrete antibody ex vivo, they produced antigen-
specific IgM upon in vitro stimulation with mitogens. The CD11c+ IgM memory B cells
were located in the splenic marginal zone, and were not detected in the blood or other sec-
ondary lymphoid organs. Their generation required CD4 T cell help, and both CD40L and
IL-21R signals. Subsequent IgG recall responses to specific antigenic challenge also re-
quired CD4 T cell help, and both CD40L and IL-21R signals. In vivo depletion of the IgM
memory cells abrogated the IgG recall response to specific antigenic challenge, demonstrat-
ing that these cells are responsible for the humoral recall response during E. muris infection.
These data confirm previous reports that IgM memory B cells can undergo class switch re-
combination and affinity maturation, following re-encounter with cognate antigen. Our
study demonstrates that IgM memory B cells maintain long-term immunity during a natural
model of infection, and may act to provide the host with greater flexibility during infection
with antigenically-variant pathogens.
112
#48
Low-Dose Induction Regimen of Methotrexate Induces Long-Lived Immune
Tolerance to Biologics Potentially Through the Induction of Regulatory B cells
Joly M., Tata P., Richards S., Joseph A.
Genzyme/Sanofi
Anti-drug antibodies (ADA) generated in response to biologics can potentially chal-
lenge the efficacy and safety of treatment. ADA can influence drug pharmacokinetics and
pharmacodynamics. In addition, ADA can impact patient safety and in the worst cases in-
duce anaphylaxis, serum sickness or even immune complex disease. For chronically admin-
istered biologics the long-term consequences of ADA can potentially contribute to treat-
ment failure and discontinuation of the therapeutic. In an attempt to control ADA, we iden-
tified a unique and benign induction regimen of low dose methotrexate that successfully in-
duces long-lived immune tolerance to a variety of biologics including Myozyme®, anti-
mouse thymocyte globulin (mATG) and Alemtuzumab. Surprisingly, the mechanism by
which methotrexate induces immune tolerance does not seem to be through cell depletion or
T regulatory cell induction. Rather, methotrexate appears to enrich IL-10-secreting regula-
tory B cells during the first week of treatment. Additionally, studies in IL-10 knock-out
mice suggest that IL-10 appears necessary for methotrexate induced tolerance. The follow-
ing data suggest a unique tolerance induction regimen by low-dose, induction treatment
with methotrexate.
113
#49
Overcoming Obstacles to Naïve T Lymphocyte Trafficking in
Tumor-Draining Lymph Nodes
Jason Muhitch, Michael Diehl, Fumito Ito, Minhyung Kim, Jeremy Waight,
Joseph Skitzki, Scott Abrams, and Sharon Evans
Department of Immunology,
Roswell Park Cancer Institute, Carlton & Elm Streets, Buffalo, NY 14263
Tumor-draining lymph nodes (TdLN) are central orchestrators for the generation of
anti-tumor immunity. These organs harbor dendritic cells that display tumor antigen and
activate rare tumor-specific naïve lymphocytes to generate an effector T cell pool. More-
over, TdLN have emerged as an important site to sustain long-term immune protection dur-
ing immunotherapy of cancer. An often-overlooked requirement for efficient dendritic cell-
T cell interactions is the entry of lymphocytes across specialized vessels in lymph nodes
termed high endothelial venules (HEVs). Recent reports have suggested that T cell entry
into lymph nodes of tumor-bearing mice may be impaired due to diminished trafficking
molecule expression on both TdLN HEVs and lymphocyes. Here we sought to confirm ini-
tial observations that HEVs within TdLN support diminished naïve T cell entry and extend
these studies to uncover if impediments to lymphocyte trafficking could be overcome thera-
peutically. Intravital microscopy studies determined that TdLN HEVs displayed an ~50%
reduction in their ability to support firm arrest of T cells, which is the penultimate step pre-
ceding extravasation. We further identified the defect in TdLN HEVs as reduced expression
of a vital homeostatic chemokine CCL21 for the transition from rolling to firm arrest. Di-
minished CCL21 expression on HEVs was restricted to the lymph node immediately proxi-
mal to the tumor and was not observed further downstream in the lymphatic network. How-
ever, in mice with large tumor burdens, we also observed defects in trafficking that in-
volved decreased expression of L-selectin on naïve lymphocytes resulting in reduced qual-
ity of rolling interactions and diminished T cell trafficking to lymph nodes throughout the
body. We next sought to determine if TdLN were responsive to inflammatory cues to im-
prove T cell entry. Immunodepletion protocols using cyclophosphamide as well as systemic
thermal therapy (STT, 39.5 ± 0.5°C, 6 h) increased lymphocyte trafficking in TdLNs to lev-
els above those observed in lymph nodes of unchallenged mice. Increased expression of
CCL21 on HEVs of TdLN following STT was found to be NF-κB–dependent. Mice treated
with cyclophosphamide or STT exhibited an increased frequency of lymphocytes undergo-
ing firm arrest in HEVs. These findings suggest that therapeutic targeting of HEVs for im-
proved entry of naïve T cells in TdLN may be a novel strategy to overcome immune eva-
sion. Supported by grants from the NIH (CA79765 and AI082039).
114
#50
Resistance to Invasive Pneumococcal Infection in Mice with Acute Allergic Lung
Inflammation
Alan M. Sanfilippo and Dennis W. Metzger
Albany Medical College, Albany, NY
Streptococcus pneumoniae is the most common cause of bacterial pneumonia which
results in 175,000 hospitalizations as well as 40,000 deaths in the United States annually.
With the introduction of a conjugate vaccine, overall disease burden from S. pneumoniae
has declined. However, recent work has identified a link between asthma and invasive
pneumococcal disease (IPD) prompting the Advisory Committee on Immunization Prac-
tices to recommend that all adults with asthma receive the pneumococcal conjugate vaccine.
To study the underlying mechanism responsible for an increased risk of IPD for individuals
with asthma, we used an acute murine model of ovalbumin (OVA)-induced allergic lung
inflammation. We hypothesized that mice with acute OVA-induced allergic lung inflamma-
tion (ALI) would be more susceptible than naive mice to an S. pneumoniae infection. Fol-
lowing OVA challenge, BALB/c mice were infected intranasally (i.n.) with a lethal dose of
S. pneumoniae and morbidity and mortality were monitored. Surprisingly, mice with acute
ALI were more resistant to infection than naïve controls. This increased resistance was
found to be highest 10 days following the final OVA challenge, despite reduced cytokine
production after infection. In the absence of pneumococcal infection, histological and flow
cytometry analysis showed that following OVA challenge inflammatory cell infiltration was
most apparent between 24 and 96 hours, though not completely cleared 10 days later. Mice
with ALI also had significantly increased early pneumococcal clearance from the lungs
across a wide range of infectious challenges. We plan to further characterize the cellular re-
sponse to pulmonary S. pneumoniae infection in mice with acute ALI to determine the
mechanisms responsible for the observed increased resistance. (Supported by NIH grant
R01 AI41715.)
115
#51
Increased Protection against Pneumococcal Disease by Administration of
Prevnar 13 Plus Interleukin-12
Quang-Tam Nguyen, Sean Roberts, and Dennis W. Metzger
Center for Immunology & Microbial Disease, Albany Medical College,
Albany, New York 12208
Streptococcus pneumoniae is a major pathogen responsible for bacterial pneumonia
in adults and children. Worldwide, over one million children suffer from pneumonia in-
duced by S. pneumoniae. The pneumococcal vaccine, prevnar 13 contains thirteen serotypes
of pneumococcal polysaccharide (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) which
are conjugated to the carrier protein CRM197. The recent development of pneumococcal
conjugate vaccines has led to a dramatic improvement in protection against invasive disease
in infants and children, but these vaccines have been found to be only 50 to 60% protective
against mucosal bacterial carriage. In this study, we found that intramuscular administration
of prevnar 13 plus interleukin-12 (IL-12) enhanced protection against mucosal S. pneumo-
niae infection in mice. Immunized mice treated with IL-12 had increased IgG and IgM anti-
pneumococcal polysaccharide (PPS) antibody responses to PPS3 and PPS14. In addition,
after intranasal challenge with type 14 pneumococci, the prevnar 13 plus IL-12-immunized
mice exhibited improved bacterial clearance in the lung and blood compared with the mice
immunized with prevnar 13 alone. These results suggest that the intramuscular administra-
tion of prevnar 13 plus interleukin-12 is able to enhance systemic immune responses to
pneumococci and efficiently protect against bacterial carriage.
116
#52
Blasting with the Dog and Cat Genomes to Identify and Clone Ferret Genes
Princess Rodriguez, Heather Glenn, Eric Yager and Karen Duus
Albany Medical College, Albany NY 12208
The ferret model has been established as one of the best models for respiratory infec-
tions, including influenza A, because they are highly susceptible to this virus and emulate
symptoms (runny nose, fever, coughing) seen in humans (Belser et al., 2011). In addition,
ferrets are able to transmit the virus to other ferrets and humans which is another key aspect
of human influenza infections (Belser et al., 2011). The genome of the ferret has not been
fully sequenced and is not yet annotated. Therefore, there are no commercially available
monoclonal antibodies against ferret proteins that would enable cell-mediated immune re-
sponses against influenza A to be evaluated. This lack of resources limits the usefulness of
the ferret as a model for influenza A. We hypothesized that we can identify, PCR-amplify,
and clone activated ferret PBMCs using the published sequences of related species (eg. Fe-
lis catus and Canis lupus familiaris) that display reasonable high-scoring pair matches
against ferret gene sequences (Mustela putorius furo). To date we have tentatively identi-
fied 14 genes, amplified 8 ferret genes, and cloned 6 of them. Using this information, we
further hypothesize that we will be able to generate B-cell mediated responses in mice
against ferret proteins by DNA vaccination using the particle-mediated epithelial delivery
(PMED/gene gun) technique. Our goal is to generate mouse monoclonal antibody reagents
with which to study ferret cell-mediated immune responses in influenza vaccinated and
challenged ferrets.
117
#53
IL-27 Sustains T-bet Expression and Promotes the Development of Terminally
Differentiated CD4 T cells during Tuberculosis
Egídio Torrado, Jeffrey J. Fountain, John Pearl, and Andrea M. Cooper
Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY, 12983
IL-27 is a member of the IL-12 family of cytokines that was originally described to
induce the expression of the transcription factors STAT1 and T-bet, thus promoting Th1
differentiation. Despite this apparent Th1 inducing profile, mice deficient in IL-27R signal-
ing are more resistant to Mtb infection, even though CD4 T cells in the lungs of these mice
produce lower amounts of IFN-gamma on a per cell basis.
Here we show that during M. tuberculosis infection, IL-27 activity is not required for
inducing IFN-gamma-producing CD4 T cells. However, during the chronic stage of the in-
fection, in the absence of IL-27 activity, CD4 T cells express lower levels of the transcrip-
tion factor T-bet, corresponding with a reduced accumulation of terminally differentiated
CD4 T cells in the lungs of these mice. Mechanistic analysis of the impact of IL-27 signal-
ing in Ag-specific T cells suggest improved competitive fitness of the CD4 T cells that are
deficient in IL-27 signaling, when compared to WT CD4 T cells. Subsequent studies
showed that the improved fitness of IL-27R-/- CD4 T cells is not dependent on proliferation
but likely dependent on improved survival of these cells in the M. tuberculosis generated
inflammatory environment. Finally, the transcription profile of CD4 T cells from the lungs
of Mtb-infected IL-27R-/- deficient mice suggests altered fatty acid metabolism, suggesting
an important role of IL-27 in the metabolic activity of CD4 T cells.
These data suggest that IL-27 has an important impact in the maintenance of effector
CD4 T cell responses during tuberculosis.
This work was supported by grants from the NIH (AI069121 and AI46530) and the
Trudeau Institute.
118
#54
Macrophage-dependent Activation of Hematopoietic Stem Cells during
Intracellular Bacterial Infection
Amanda McCabe, Lu Zhang, Maura Jones, and Katherine MacNamara
Albany Medical College, Albany NY
Hematopoietic stem cells (HSCs) maintain production of all cells of the blood. An
emerging idea is that during infection, HSC activation is associated with the production of
cell lineages needed for an appropriate immune response to pathogens. In a murine model
of human monocytic ehrlichiosis (HME), a tick-borne intracellular bacterial infection, we
have observed an IFNγ-dependent increase in active HSCs and reduced quiescent HSCs,
which correlated with increased monocytes. We hypothesize that the IFNγ-dependent HSC
activation and loss in quiescent HSCs is dependent upon IFNγ signaling in macrophages.
BM-resident macrophages are extremely sensitive to IFNγ and they have recently been
shown to play a role in maintaining the BM HSC niche, but how they might contribute to
HSC activation during infection has not been addressed. Here we show that bacterial infec-
tion resulted in proliferation and activation of BM-resident macrophages, which resulted in
increased numbers of active HSCs. Ehrlichia muris infected C57BL/6 mice exhibited in-
creased numbers of macrophages that expressed F4/80, Ly6G, and CD169, characteristic of
a BM-resident macrophage. Macrophages exhibited increased expression of MHCII and
CD80 during infection indicating they were activated. Furthermore, mice lacking IFNγ
signaling only in macrophages exhibited increased frequencies of dormant HSCs in the BM
after infection, relative to wild type infected mice. These data suggest that IFNγ signaling
in macrophages is, in part, responsible for mediating HSC activation. These results define a
novel role for macrophages in infection-induced alterations in hematopoiesis. Our current
experiments are aimed at addressing how macrophage activation during infection impacts
HSC function, and whether this interaction is direct or through the niche in which HSCs re-
side.
119
#55
Viral Pathogenesis and Myeloid Cell Trafficking Under Comparative Conditions
of Macrophage Depletion
Michael L. Davies, Lauren A. Weiler and Chris C. Norbury
Milton S. Hershey College of Medicine, Penn State University, Hershey PA
Cell type-specific depletion methods are important experimental tools for in vivo immu-
nology. Procedures for depleting leukocyte subsets range from monoclonal antibodies
against surface markers to methods exploiting genes that are upregulated upon differentia-
tion (e.g. diphtheria toxin receptor [DTR] expressed from the CD11b or CD11c promoter).
However, it is difficult to use these properties to target a particular functional cell type. This
is particularly true for myeloid lineages, which are known for their plasticity and the tissue-
specific nature of their differentiation. In vivo, cells such as tissue-infiltrating monocytes are
increasingly recognized as differentiating into a continuum of mature states rather than a
few clearly defined branches. Thus it has been observed that lineage-based transgenic meth-
ods are often “leaky”, having off-target effects, in addition to incomplete effects on targeted
cells due to the promoter being activated at low levels rather than simply switched on and
off.
In this study we compared a functional depletion method, injection of clodronate-loaded
liposomes (CLL), to two transgenic depletion methods – LysM:cre x iDTR, and MaFIA. In
LysM:cre x iDTR mice, high-affinity DTR is expressed in lysozyme-producing cells, which
include granulocytes and some monocytic cells. In MaFIA mice, a “suicide gene” is ex-
pressed from the Csf1r promoter, which is activated in macrophages and immature neutro-
phils. Both systems allow us to use a drug to deplete cells that have upregulated the pro-
moter in question.
We looked at depletion during infection with vaccinia virus, using an intradermal infec-
tion model that is normally restricted to the skin. Our results suggest that CLL effects the
most comprehensive ablation of the phagocytes that prevent virus spread and replication,
and is less toxic to the animals. However, the effect of CLL is less specific to myeloid cells,
also producing a massive decline in splenic lymphocytes, particularly when given intrave-
nously. MaFIA or LysM:cre x iDTR depletion led to both “classical inflammatory macro-
phages” and Ly6G+ myeloid cells being depleted at the site of infection, and CD11b+ sub-
sets in the spleen being less well-defined. MaFIA depletion is more comprehensive for both
effects. Although these depletions can be effective in rendering mice more susceptible to
infection, it is difficult to identify which depleted cells are responsible for this effect.
120
#56
MyD88-Signaling in CD4 T Cells Promotes Hematopoietic Stem and
Progenitor Cell Expansion by Optimizing IFN Production
Yubin Zhang1, Maura Jones1, Gary M. Winslow2 and Katherine C. MacNamara1 1 Center for Immunology and Microbial Disease, Albany Medical College, NY 12208, USA 2 Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA
How hematopoietic stem and progenitor cells (HSPCs) respond to inflammatory sig-
nals during infection is not well understood, but it is thought that activation of HSCPs dur-
ing infection is important for generating cells responsible for host defense. We have used a
mouse model of human monocytic ehrclihiosis (HME), a disease that causes dramatic he-
matologic abnormalities, to examine the mechanisms by which HSPC function is regulated
during infection. HSPCs can respond to cytokines including interferons (IFNs), and patho-
gen-associated molecules via the expression of Toll like receptors (TLR). We previously
found that HSPC activation and proliferation during Ehrlichia muris (E. muris) infection
was dependent on IFN-gamma (IFN ). Here we sought to investigate the role of MyD88, an
adaptor molecule for TLR and IL-1 family cytokine signaling, in the regulation of HSPCs
during E. muris infection. We found that proliferation of Lin-negative Sca-1+ cKit+ (LSK)
cells, which contains active HSPCs, was reduced in MyD88-deficient mice. Correlating
with reduced proliferation, we observed reduced numbers and frequencies of LSK cells, and
reduced numbers of monocytes in MyD88-deficient mice. However, in radiation-induced
mixed bone marrow chimeras comprised of wild type and MyD88-deficient cells, both wild
type and MyD88-deficient cells were equally expanded, indicating that MyD88-signaling
was not intrinsically required for increased LSK cells. Reduced LSK expansion in MyD88-
deficient mice correlated with diminished IFNγ production, as compared to wild type mice.
Thus, we next tested whether MyD88 was critical for IFN production. We identified CD4
T cells as the primary cellular source of IFN within the bone marrow, and found that in-
trinsic MyD88-signaling was required for CD4 T cells to produce optimal amounts of IFNγ.
Our data demonstrate that bone marrow LSK expansion driven by E. muris infection is not
mediated by direct MyD88-signaling; rather, intrinsic MyD88-signaling in CD4 T cells may
promote LSK expansion by optimizing IFN production.
121
#57
Maintenance of Peripheral T cell Responses during Mycobacterium tuberculosis
Infection
William W. Reiley1, Susan T. Wittmer1, Lynn M. Ryan1, Sheri M. Eaton1, Laura Haynes1,
Gary M. Winslow1,2, and David L. Woodland1,3 1Trudeau Institute, Saranac Lake, NY, 2Wadsworth Center, Albany, NY, and 3Keystone
Symposia on Molecular & Cellular Biology, Silverthorne, CO
Mycobacterium tuberculosis (Mtb) establishes a latent infection that can persist for
years. The development of adaptive immunity is critical for the maintenance of infection in
the latent state and the prevention of clinical tuberculosis. While much has been learned
about Mtb over the past few decades, we still do not fully understand how acute and persis-
tent T cell responses are induced and maintained after infection. We hypothesized that long-
term T cell responses would be maintained during chronic infections by new thymic emi-
grants contributing to, and refreshing, the peripheral T cell response. Therefore, we have
dissected the contribution of new thymus-derived T cells to the maintenance of the periph-
eral CD4 and CD8 T cell response, during both acute and chronic infection. Although
newly-generated T cells contribute to the peripheral response, especially during acute infec-
tion, the contribution of recent thymic emigrants declines during chronic infection. The de-
cline is correlated with an apparent decrease in antigen presentation, or in the capability of
antigen-presenting cells to drive the activation of naive T cells, because the priming and ex-
pansion of newly-generated T cells is less efficient during chronic infection. These findings
demonstrate that although naive thymus-derived T cells can contribute to the maintenance
of immunity, this process is not the primary mechanism. Instead, other processes are likely
responsible, such as antigen-driven T cell proliferation, perhaps originating from a popula-
tion of yet undefined T cells capable of self-renewal. Our findings also reveal that antigen is
not a limiting factor for driving the activation of naive T cells during chronic infection. We
propose instead that the context of antigen presentation (i.e., the type of APC, or the quality
of co-stimulatory signals) changes throughout infection. These studies extend our under-
standing of how T cell responses are maintained during persistent bacterial infections.
122
#58
CD28-mediated Pro-survival Signaling in Multiple Myeloma
Megan Murray, Jayakumar Nair, and Kelvin Lee
Roswell Park Cancer Institute, Buffalo NY
Multiple myeloma (MM) is an incurable plasma cell neoplasm which requires mo-
lecular bridges to the bone marrow microenvironment for survival. Our lab has previously
shown that one such bridge is CD28, the molecule best known as the prototypic T cell re-
ceptor. However, CD28 is also expressed on MM cells and is correlated with progressive
disease and worse outcomes, pointing to CD28 as a pro-survival signaling molecule.
To determine whether CD28 is important in the context of complex cell-cell interac-
tions, we co-cultured MM cells with dendritic cells (DC, which express the CD28 ligands
CD80 and CD86) in melphalan, a clinically relevant chemotherapy. DC were able to sig-
nificantly increase MM cell survival. However, this protection is entirely abrogated by ei-
ther a CD28 blocking antibody (CD28.6) or by CTLA4-Ig, which binds to the ligands CD80
and CD86. These experiments show that even in complex cell-cell interactions, CD28 is the
critical pro-survival molecule.
Since CD28 in T cells signals via a PI3K-Akt axis, we interrogated whether or not
this pathway is crucial for CD28-mediated MM cell survival. We observed that blockade of
PI3K signaling using chemical inhibitors abrogates CD28-mediated survival in a dose de-
pendent fashion. To confirm that survival is also Akt-dependent, we repeated the experi-
ments with an Akt inhibitor and again observed an abrogation of CD28-mediated survival
of MM cells.
It has been well established in the myeloma literature that the balance of the pro-
apoptotic molecule Bim determines apoptosis in MM cells. Importantly, Bim is transcrip-
tionally regulated by the PI3K-Akt-FoxO3a axis. When FoxO3a is phosphorylated by Akt,
it is excluded from the nucleus and is unable to upregulate Bim transcripts. Via western
blot, we confirmed that CD28 activation increases levels of phospho-FoxO3a. We next
wanted to test whether or not CD28 activation had any effect on Bim transcript levels. By
both western blot and RT-PCR, we confirmed that CD28 activation suppresses Bim expres-
sion, and CD28 blockade increases it. To confirm that this plays a functional role in MM
biology, we knocked down Bim using siRNA and cultured the cells without serum +
CTLA4-Ig. Consistent with our expectations, when we culture control cells in the absence
serum plus CD28 blockade, they die. However, in the Bim knock-down cells, we see that
they are resistant to CD28 blockade-mediated cell death. These data suggest that CD28 is
able to regulate Bim via the PI3K-Akt-FoxO3a axis, and that Bim is critical for MM cell
death or survival
124
The Role of Pro-resolving Lipid Mediators in Attenuating Lung Inflammation
Thomas H. Thatcher, Hsi-Min Hsiao, Richard P. Phipps and Patricia J. Sime
Division of Pulmonary and Critical Care Medicine, University of Rochester
Introduction: Cigarette smoking is a profound pro-inflammatory stimulus that con-
tributes to multiple acute and chronic diseases. Many of these diseases persist or even
worsen even after smoking cessation, suggesting that part of the disease pathology is a fail-
ure to resolve inflammation. Until recently, it was believed that resolution of inflammation
occurred passively after removal of the inflammatory stimulus. It is now known that resolu-
tion of inflammation is an active process regulated by small lipid mediators derived mainly
from dietary omega-3 polyunsaturated fatty acids, including the resolvins, protections and
maresins. We hypothesized that resolvin D1 (RvD1) would have potent anti-inflammatory
and pro-resolving effects in a model of cigarette smoke-induced lung inflammation.
Methods: Primary human lung fibroblasts, small airway epithelial cells and mono-
cytes were treated with IL-1b or cigarette smoke extract and RvD1 in vitro, and production
of pro-inflammatory mediators was determined. Mice were exposed to dilute mainstream
cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking
cessation. The effect on lung inflammation and lung macrophages was assessed.
Results: RvD1 suppressed production of pro-inflammatory mediators by primary hu-
man cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with
cigarette smoke exposure significantly reduced neutrophilic lung inflammation and produc-
tion of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-
10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutro-
phil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given
after the final smoke exposure.
Conclusions: RvD1 has potent anti-inflammatory and pro-resolving effects in cells
and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic
approach to resolve lung injury in COPD and other chronic inflammatory lung diseases.
Funding Sources: This work was supported by National Institutes of Health Grants R01
HL088325, HL088325-02S1, HL66988, ES07026, 8UL1TR000042 and ES01247.
125
Lymphotoxin-dependent Control of Inflammatory Responses in the Gut
Ekaterina P. Koroleva, Elise Macho Fernandez, Alexei V. Tumanov
Trudeau Institute, Saranac Lake, NY 12983
Inflammation in the gut is a hallmark of Crohn`s disease and ulcerative colitis, col-
lectively known as inflammatory bowel disease (IBD). The pathogenesis of IBD is associ-
ated with a dysregulated mucosal immune response. Innate lymphoid cells (ILCs) have re-
cently emerged as important regulators of the balance between protective immunity and im-
munopathology at mucosal surfaces. Secretion of specific cytokines by distinct populations
of ILCs is thought to mediate protective or pathogenic immune effects in the gut. However,
the pathways that mediate crosstalk between ILCs, dendritic cells and intestinal epithelial
cells during inflammation and in response to mucosal bacterial infection remain poorly un-
derstood. We found that lymphotoxin (LT), a member of TNF family of cytokines, pro-
duced by RORgt+ ILCs is critical for protection of mice against mucosal bacterial pathogen
Citrobacter rodentium (C. rodentium). C. rodentium is a natural mouse pathogen that repre-
sents a model of human infectious colitis caused by pathogenic E. coli. The LT-dependent
protective mechanism involves early neutrophil recruitment and secretion of IL-22 by
RORgt+ lymphoid inducer tissue cells (LTi) after infection. By using a conditional gene
targeting approach, we demonstrate that a LT-driven positive feedback loop controls IL-22
production by RORgt+ LTi cells via LTβR signaling in DCs. Furthermore, expression of
LT by RORgt+ ILCs was essential for the development of isolated lymphoid follicles- terti-
ary lymphoid tissues the gut. These data suggest that LT provides a spatial microenviron-
ment to facilitate interaction among ILCs, DC, epithelial cells, and adaptive immune cells in
immune response to mucosal bacterial pathogen. Since stimulation of LTbR activates NF-
kB signaling pathway, we analyzed mice deficient in distinct NF-kB subunits in C. roden-
tium infection. Our data suggest that alternative, but not canonical NF-kB signaling path-
way is essential for control of infection. Analysis of mice with conditional inactivation of
LTbR and NF-kB components in specific cells in the gut will allow us to further investigate
the critical pathways to control inflammation and protections against mucosal pathogens. In
summary, our data suggest that LT pathway regulates ILCs and that manipulation of LTbR
signaling could be used to control inflammatory responses in the gut.
126
TH17 cells in Health and Disease
William O’Connor Jr., Ph.D.
Center for Immunology and Microbial Disease
Albany Medical College
Effector lymphocytes play an important role in ensuring host protection against a va-
riety of potentially pathogenic organisms. TH17 cells, a recently identified class of helper T
cells, are instrumental in mounting a robust response to microorganisms at the mucosa
while limiting the extent of bystander tissue damage that often accompanies inflammatory
episodes. When improperly regulated, inflammation can lead to a myriad of chronic in-
flammatory and auto-inflammatory disorders in mice and men. Indeed, TH17 cells have
been associated with Multiple Sclerosis, Inflammatory Bowel Diseases, Rheumatoid Arthri-
tis, and Psoriasis, among others.
Understanding TH17 cell function and the role of TH17-associated cytokines in the
initiating and resolving phases of inflammation is an active and exciting area of current re-
search. Recent studies from our group and others highlighting novel mechanisms control-
ling TH17 cells, and newly appreciated roles of TH17-associated cytokines, will be dis-
cussed.
128
The Maternal Autoimmune Environment Affects Long-term behavior of
Offspring
Yubin Zhang, Yong Heo, Donghong Gao, Kerri Kluetzman, Veronica Miller, and
David Lawrence
Wadsworth Center, New York state Department of Health
Autism spectrum disorders (ASD) are neurodevelopmental disorders with unknown
etiology. Here, we show that BTBR T+tf/J (BTBR) mice, a mouse strain with behaviors
that resemble autism, have elevated serum levels of IgG and anti-brain antibodies as well as
greater than normal levels of IgG and proinflammatory cytokines in the brain. The BTBR
mice also have enhanced activation of peripheral CD4+ T cells with preferential expression
of Vβ6 chains. The CD4+CD25+Vβ6+ and Vβ6
- splenic cells of BTBR mice have elevated
levels of IL-4, IFN- and IL-17, suggesting no preferential CD4+ T subset skewing/
polarization. BTBR mice appear to be in a constitutive state of inflammation, including an
elevated percentage of peripheral blood neutrophils. CD4+Vβ6+ T cells derived from
C57BL/6 (B6) mice were selectively expanded in both BTBR and B6 hosts that received an
equal number of BTBR and B6 bone marrow cells. The high level of IgG production by
BTBR B cells was dependent on T cells from BTBR mice. B6 offspring derived from B6
dams that were gestationally injected with purified IgG from sera of BTBR mice, but not
IgG of B6 mice, developed significantly impaired social behavior. Additionally, B6 off-
spring that developed in BTBR dams had impaired social behavior, while BTBR offspring
that developed in B6 dams had improved social behavior. All of the immunological and be-
havioral parameters of BTBR mice were compared with those of B6 mice, which have rela-
tively normal behaviors. Interestingly, the aberrant behavior of the BTBR mice may relate
to altered mitochondrial functions, in that BTBR mice with B6 mitochondria have improved
behavior. The results indicate maternal antibodies (Abs) and possibly other maternal influ-
ences affect the social behavior of offspring.
129
Nucleic Acid-Sensing Toll-like Receptor Regulation and Disease
Cynthia Leifer
Associate Profession of Microbiology and Immunology
Cornell University
Nucleic acid structures are highly evolutionarily conserved, and when self nucleic
acids are aberrantly detected by Toll-Like Receptors (TLRs) they contribute to autoimmune
disease. Multiple regulatory mechanisms have been identified that may prevent response to
self-nucleic acids including intracellular compartmentalization. Recognition of DNA by
TLR9 occurs specifically in the endosomal compartment, and recent studies have shown
that TLR9 is proteolytically processed to an 80 kilodalton form (p80) in endosomes. Fur-
thermore, recognition of CpG DNA in different endosomal compartments leads to the selec-
tive production of proinflammatory cytokines, or type I interferon. We have identified a
novel cleavage event that generates a soluble version of the TLR9 ectodomain (sTLR9).
Generation of sTLR9 was partially dependent on cathepsin S, a protease active at neutral
pH. Most importantly, sTLR9 inhibited TLR9-dependent signaling. We also identified a cy-
toplasmic motif that regulates access to different endosomal compartments. This motif is
selectively required for the production of proinflammatory cytokines, but not type I inter-
feron. In response to CpG DNA stimulation, the proteolytically processed 80 kDa fragment
is tyrosine phosphorylated. Although tyrosine 888 is not itself phosphorylated, the structure
of this motif is necessary for both TLR9 phosphorylation and selectively for TNF-α
production in response to CpG DNA. Together these data reveal two new facets of TLR9
regulatory biology that could be exploited for therapeutically modulating TLR9 in inflam-
matory diseases.
130
In vivo Functions of STING in Host Defense and Vaccine Adjuvant Development
Lei Jin††, Andrew Getahun*, Heather M. Knowles, Jennifer Mogan*, Linda J. Akerlund*,
Anne-Laure Perraud*, and John C. Cambier*† †Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY;
*Integrated Department of Immunology, University of Colorado Denver School of Medi-
cine and National Jewish Health, Denver, CO
STING (stimulator of type I IFN genes), a.k.a MPYS, MITA, TMEM173, is essen-
tial for cytosolic DNA induced type I IFN production. Studies done in STING-/- mice
(Tmem173<tm1Gnb>) have demonstrated that STING is critical for host defense against
DNA virus infection. However, increasing evidence suggests that STING may also be im-
portant for host defense against non-viral pathogens such as Francisella tularensis, Brucella
abortus, Streptococcus pneumoniae, Listeria monocytogenes (L.M), Chlamydia muridarum,
Mycobacterium tuberculosis and Plasmodium falciparum infections. The in vivo signifi-
cance and mechanism by which STING functions in host defense against these non-viral
infections have not been demonstrated. Using a conditional STING-/- mice
(Tmem173<tm1Camb>), we show here that STING mediates host defense against L.M. in-
fection in vivo via a mechanism distinct from its ability to activate type I IFN. STING is
also important for the adjuvant activity of DNA vaccine. In this talk, we will also be dis-
cussing the role of STING, in vivo, in the development of cyclic di-GMP as a safe and ef-
fective mucosal vaccine adjuvant.
131
Workshop - Part II
NIH Presentation
Alison Deukhut Augustine, Ph.D. & B. Duane Price, Ph.D.
NIAID
Mock Study Section
Panel Members:
Dennis Metzger
Margaret Bynoe
Sharon Evans
Chris Norbury
Questions and Discussion
Handouts will be provided.
132
Keynote Speaker
Ralph C. Budd, M.D., Ph.D.
Professor of Medicine
Director, Vermont Center for Immunology & Infectious Disease
The University of Vermont
Burlington, VT
“Caspase Regulation of the Immune Response: the FLIP Side”
Since the discovery that caspase-8 is critical not only for apoptosis by death recep-
tors, but also for cell survival and growth, an important issue has been defining how caspase
-8 activity is tightly regulated to control these seemingly opposite functions. A logical can-
didate is the catalytically-inactive caspase-8 paralogue, c-FLIP-Long (c-FLIPL). c-FLIPL is
able to inhibit caspase-8 activation following death receptor ligation, as well as partly acti-
vate caspase-8 during cell growth situations. Thus, c-FLIPL acts as a molecular switch be-
tween cell growth and death pathways. This seminar will provide a molecular model of c-
FLIP function in these two situations in T cells. It will then apply this to the study of an-
other pathway that is regulated by caspase-8 and c-FLIP, the RIG-I helicase pathway of de-
tecting viral RNA. The model provides an explanation why certain viruses usurped c-FLIP
from the mammalian genome, but only a truncated short form of c-FLIP.
134
Immunity to Tuberculosis - Do We Know What We Don't Know?
Andrea Cooper
Trudeau Institute, Saranac Lake, NY
Abstract: Immunity to tuberculosis has been key to human survival over the last 40-
70,000 years. This is a powerful statement and reflects the fact that Mycobacterium tuber-
culosis (Mtb, the causative agent of tuberculosis) has been associated with modern human
populations since before the migration out of Africa. Mtb is an exquisite manipulator of the
human imune response and utilizes this immune response to keep its host alive long enough
to transmit to many other potential hosts. My talk will focus on the induction and manipula-
tion of the antigen-specific T cell response by Mtb once it is delivered to the vertebrate lung
via an aerosol infection. The hypothesis is that Mtb induces very strong CD4 T cell immune
responses but very slowly following infection. The slowness of the response results in a
bacterially-defined primary lesion which is then able to regulate the T cell response locally
in order to generate a lesion capable of transmitting the bacterium to other hosts.
135
Enhancement of Adaptive Immunity to Neisseria gonorrhoeae by Inhibition of
the Pathogen-induced Immunosuppressive Mechanisms
Yingru Liu, Nejat K. Egilmez and Michael W. Russell
Department of Microbiology & Immunology, and Witebsky Center for Microbial Patho-
genesis and Immunology, University at Buffalo, Buffalo, NY
Infection with N. gonorrhoeae triggers an intense inflammatory response character-
ized by an influx of neutrophils in the genital tract, yet natural gonococcal infection does
not induce a state of specific protective immunity. Herein we show in a mouse model that
N. gonorrhoeae selectively elicits Th17-governed innate immune defenses and suppresses
Th1/Th2-dependent adaptive immune responses. Induction of TGF-β by N. gonorrhoeae is
critically involved in the differential effect on Th1, Th2 and Th17 immunity. However, N.
gonorrhoeae only slightly increases the numbers of CD4+Foxp3+ Treg cells. In contrast,
gonococcal infection induces abundant production of IL-10 and the type 1 subset of regula-
tory T cells (Tr1). Blockade of TGF-β or IL-10 by neutralizing antibodies significantly en-
hanced Th1 and Th2 reactivity of mouse CD4+ T cells to N. gonorrhoeae. While anti-IL-10
reversed N. gonorrhoeae-mediated inhibition of Th1 cell development more effectively,
anti-TGF-β reversed the inhibition of Th2 cell development to a greater extent. Blockade of
TGF-β in combination with blockade of IL-10/Tr1 activities had a synergic effect to pro-
mote host Th1 and Th2 immune responses against N. gonorrhoeae. Treatment of mice with
anti-TGF-β or/and anti-IL-10 antibodies by intraperitoneal injections during gonococcal
challenge led to faster clearance of infection and the production of circulating and vaginal
anti-gonococci antibodies, and induced protection against secondary infection. Furthermore,
treatment of mice intravaginally with anti-TGF-β or/and anti-IL-10 in slow-release micro-
spheres had similar results. Our results suggest that N. gonorrhoeae as a well-adapted
pathogen has evolved strategies to suppress Th1- and Th2-mediated adaptive immunity in-
volving immunoinhibitory cytokines (TGF-β and IL-10) and regulatory T cells, and that this
effect can be manipulated to enhance specific protective immune responses.
137
Regulation of CD8 T cell Effector and Memory Programs
Laura A. Penny, Yevgeniy Yuzefpolskiy, Vandana Kalia, Surojit Sarkar
The Huck Institutes of the Life Sciences,
The Pennsylvania State University, PA 16802, USA
Naïve CD8 T cells get rapidly activated and enter into an effector program following
encounter with a pathogen. Upon pathogen clearance, a small pool of the effector cytotoxic
T lymphocytes (memory precursors) preferentially survives and gives rise to long-lived
memory CD8 T cells. Designing effective vaccines that will induce robust protective immu-
nity is hinged heavily on our understanding of the mechanisms that regulate the generation
and maintenance of such long-lived memory CD8 T cells.
We have previously shown that CD8 T cell memory precursors are generated early
during effector differentiation and are programmed by the duration of antigen and inflam-
mation. Mechanisms governing the processes by which the memory precursors convert
from a highly activated effector state into a relatively quiescent memory state remain un-
clear. Is the effector to memory transition of CD8 T cells a default pathway occurring as a
result of abrogation of infection-related stimuli, or is there an active machinery that facili-
tates this process? Based on the role of Tregs in dampening effector programs in autoreac-
tive CD8 T cells, we hypothesized that the effector to memory transition of CD8 T cells in-
volves an active cell-extrinsic suppression through CD4+FoxP3+ regulatory T cells (Tregs).
By transiently deleting Tregs during distinct stages of CD8 T cell responses to acute viral in-
fection, our experiments revealed a novel role of Tregs in dampening the effector program of
CD8 T cells during generation and maintenance of memory cells. Through an active sup-
pression of the effector program, Tregs aided in the acquisition of hallmark memory T cell
properties such as polyfunctionality, antigen-independent longevity and secondary expan-
sion. Thus, contrary to previous notion of effector to memory differentiation being a passive
follow-up to pathogen clearance, these studies demonstrate that Tregs play an active role in
this process by curtailing T cell proliferation and downregulation of effector functions.
Our data shows that Tregs are critical for the generation and maintenance of the qui-
escent state of the memory CD8 T cells from effector cells during an adaptive immune re-
sponse.
138
Memories from the Time of Cholera
Kelvin P. Lee, M.D.
Roswell Park Cancer Institute
Buffalo, NY
Multiple myeloma (MM) is a neoplasm of transformed plasma cells (PC) that com-
prises 20% of all hematologic malignancies, and despite new chemotherapeutic agents re-
mains incurable. The normal counterpart of MM cells are non-proliferating BM-resident
long-lived PC (LLPC). LLPC are responsible for the sustain antibody titers necessary for
durable protective humoral immunity against a wide range of pathogens – in particular
agents that cause episodic epidemics. Studies in both murine models and normal human
subjects suggest that predicted natural lifespan of individual LLPC exceeds that of the host
itself, and this is of particular relevance given that “immortalization” is one of the two hits
required for malignant transformation. The longevity of the LLPC is however not PC-
intrinsic, as it is clear that LLPC are critically dependent on specific BM stromal niches for
their survival. This also is true for MM cells, and thus defining the largely uncharacterized
molecular and cellular components specifically involved in the LLPC/MM – BM may point
to fundamental immunobiology as well as novel therapeutic targets. We have recently dem-
onstrated that CD28, the prototype T cell costimulatory receptor that is also expressed on
PC and MM cells, plays an essential PC-intrinsic role in sustaining the survival of BM
LLPC. We have also found that CD28 overexpression in myeloma patient gene expression
profiles is associated with disease progression and poor prognosis patient subgroups
(especially the maf subgroup), and that activation of CD28 on MM cells induces PI3K and
NFκB signaling and protects against chemotherapy-induced death. We have subsequently
determined that specific signaling pathways downstream of CD28, PI3K and Vav, regulate
different aspects of the MM/PC to receptor activation. Of particular interest is CD28-
induced upregulation of BLIMP-1, an essential transcriptional regulator in normal PC - but
whose role in MM biology has been largely unexplored. A pro-survival role for CD28 in
MM/PC points to BM stromal cells BMSC expressing the CD28 ligands CD80 and/or
CD86 as the cellular partners in the myeloma pro-survival niche. Consistent with this, we
found that myeloid CD80/CD86+ dendritic cells (DC) confer significant resistance to che-
motherapy-induced apoptosis to MM cells. We have also found CD28 ligation of CD80/
CD86 “backsignals” to the DC, inducing it to produce the pro-survival cytokine IL-6 and
the immunosuppressive enzyme indoleamine 2, 3 dioxygenase (IDO)/CD86 – which has
been described in the context of T cell activation but not in MM. Altogether these data sup-
port a model where CD28-CD80/CD86 is a central molecular bridge between MM cells
(and normal BM PC) and supportive myeloid cells (including DC) in the BM niche, directly
transducing a prosurvival signal to the myeloma cell and inducing a stromally generated pro
-survival/immunosuppressive milieu.
139
CD28 Costimulation is Dispensable for the Development and Differentiation
of γδ T Cell Effectors
Renee M. Laird, Benjamin J. Wolf, Michael F. Princiotta, and Sandra M. Hayes
Department of Microbiology and Immunology, State University of New York Upstate
Medical University, Syracuse, NY 13210
Both antigen recognition and CD28 costimulation are required for the activation of
naïve αβ T cells and their subsequent differentiation into cytokine-producing or cytotoxic
effectors. However, because of contradictory results, it remains unresolved as to whether
CD28 costimulation is also necessary for the activation and differentiation of γδ T cell ef-
fectors. Recently, it has been shown that γδ T cells acquire effector functions in the thymus
following encounter with self-ligands. This is in contrast to αβ T cells, which acquire effec-
tor functions in secondary lymphoid tissues following encounter with foreign antigens. In
light of these newly identified requirements for γδ effector cell development, we re-
examined the role of CD28 in γδ T cell effector fate specification and function. We report
that, although IFNγ-producing (γδ-IFNγ) effectors express higher levels of CD28 than IL-
17-producing (γδ-17) effectors, CD28-deficiency has no effect on the development of either
γδ-17 or γδ-IFNγ cells. Also, following infection with Listeria monocytogenes, we found
that the activation, expansion and differentiation of γδ-17 and γδ-IFNγ cells were normal in
CD28-/- mice. Thus, these data not only indicate that CD28 is not essential for γδ T cell ef-
fector development and differentiation but also highlight differences in the requirements for
the generation of αβ and γδ T cell effectors.
.
140
Please join us next year for the
16th Annual Upstate New York
Immunology Conference
October 27-29, 2013
The Sagamore Resort and Conference Center
141
Amarasinghe, Jayaleka Wadsworth Center 518-526-2486 [email protected] Avery, August Cornell University 607-253-3401 [email protected] Augustine, Alison Deckhut NIH/NIAID 301-496-7551 [email protected] Bellville, Dawn Albany Medical College 518-262-5365 [email protected] Berwin, Brent Dartmouth Medical School 603-650-6899 [email protected] Bishop, Gail University of Iowa 319-335-7945 [email protected] Blaauboer, Steven Albany Medical College 518-252-0052 [email protected] Boule, Lisbeth University of Rochester 585-275-2013 [email protected]
Budd, Ralph University of Vermont 802-656-2286 [email protected] Burke, Catherine University of Rochester 585-273-1840 [email protected] Bushway, Meghan University of Rochester 585-273-1840 [email protected] Bynoe, Margaret Cornell University 607-253-1023 [email protected] Cheng, Benson University of Rochester 585-276-4847 [email protected] Chung, Allen University at Buffalo 716-829-8765 [email protected] Connell, Terry University at Buffalo 716-829-3364 [email protected] Cooper, Andrea Trudeau Institute 518-891-3080 [email protected]
Attendee Contact Information
142
Davies, Michael Pennsylvania State University 717-531-0624 [email protected] De Jesus, Magdia Wadsworth Center 518-402-4081 [email protected] Dewan, Kalyan Pennsylvania State University 814-470-5281 [email protected] D’Hondt, Rebecca Wadsworth Center 518-402-4081 [email protected] Drake, James Albany Medical College 518-262-9337 [email protected] Duus, Karen Albany Medical College 518-262-1176 [email protected] Duffy, Ellen Albany Medical College 518-262-4447 [email protected] Eaton, Sheri Trudeau Institute 518-891-3080 [email protected]
Evans, Sharon Roswell Park Cancer Institute 716-845-3421 [email protected] Fernandes, Sandra SUNY Upstate Medical University 315-440-2822 [email protected] Fernandez, Elise Macho Trudeau Institute 518-891-3080 [email protected] Fisher, Daniel Roswell Park Cancer Institute 716-845-3157 [email protected] Fong, Ern Hwei Hannah Cornell University 646-306-3373 [email protected] Franz, Brian Albany Medical College 518-262-6972 [email protected] Furuya, Yoichi Albany Medical College 518-262-6220 [email protected] Gerber, Scott University of Rochester 585-275-6747 [email protected]
143
Glass, Aaron SUNY Upstate Medical University 315-525-1043 [email protected] Goodfield, Laura Pennsylvania State University 814-865-9134 [email protected] Gorham, James Dartmouth College 603-650-8373 [email protected] Green, Kathy Dartmouth College 603-724-3963 [email protected] Green, William Dartmouth College 603-724-3964 [email protected] Greene, Christopher University at Buffalo 716-829-5426 [email protected] Harton, Jonathan Albany Medical College 518-262-4445 [email protected] Hayes, Sandy SUNY Upstate Medical University 315-464-7692 [email protected]
Herman, Katherine University of Rochester 513-225-5766 [email protected] Hickey, Anthony Trudeau Institute 518-891-3080 [email protected] Hu, John University at Buffalo 585-991-8047 [email protected] Huang, Fei Cornell University 530-304-2651 [email protected] Huang, Lu Cornell University 607-592-9218 [email protected] Huang, Weishan Cornell University 814-321-7414 [email protected] Jelley-Gibbs, Dawn Trudeau Institute 518-524-5972 [email protected] Jin, Lei Albany Medical College 518-262-0053 [email protected]
144
Johnson, Nicole Albany Medical College 518-262-8141 [email protected] Joly, Marguerite Genzyme/Sanofi 508-270-2498 [email protected] Kalia, Vandana Pennsylvania State University 814-863-8533 [email protected] Kannan, Arun Cornell University 814-321-2867 [email protected] Kennedy, Jeff Albany Medical College 781-249-6128 [email protected] Kim, Dogeun Cornell University 607-379-3774 [email protected] Kirimanjeswara, Girish Pennsylvania State University 814-863-5250 [email protected] Koroleva, Ekaterina Trudeau Institute 518-354-8097 [email protected]
Ku, Amy Roswell Park Cancer Institute 661-618-0250 [email protected] Laird, Renee SUNY Upstate Medical University 315-464-7690 [email protected] Lawrence, David Wadsworth Center 518-474-8285 [email protected] Lefebvre, Julie Trudeau Institute 518-891-3080 [email protected] Lee, Kelvin Roswell Park Cancer Institute 716-845-4106 [email protected] Lee, William Wadsworth Center 518-474-3543 [email protected] Leifer, Cynthia Cornell University 607-253-4258 [email protected] Leigh, Nicholas Roswell Park Cancer Institute 716-845-3553 [email protected]
145
Lim, Joanne University of Rochester 585-478-9325 [email protected] Liu, Yingru University at Buffalo 716-201-9423 [email protected] Lopez-Pinet, Odelys University of Rochester 585-275-6747 [email protected] Lord, Edith University of Rochester 585-275-5855 [email protected] Lynes, Michael University of Connecticut 860-486-4350 [email protected] MacNamara, Kate Albany Medical College 518-262-0921 [email protected] Mantis, Nicholas Wadsworth Center 518-402-2750 [email protected] Markley, Rachel Pennsylvania State University 640-613-1406 [email protected]
Martinez-Sobrido, Luis University of Rochester 585-276-4733 [email protected] McCabe, Amanda Albany Medical College 518-262-0922 [email protected] Metzger, Dennis Albany Medical College 518-262-6750 [email protected] Mikucki, Maryann Roswell Park Cancer Institute 716-845-3157 [email protected] Mohinta, Sonia Cornell University 814-321-3029 [email protected] Muhitch, Jason Roswell Park Cancer Institute 716-845-3157 [email protected] Mullins, David Dartmouth College 603-653-9922 [email protected] Murphy, Shawn University of Rochester 585-273-3910 [email protected]
146
Murray, Megan Roswell Park Cancer Institute 716-845-8231 [email protected] Muse, Sarah Pennsylvania State University 814-865-9134 [email protected] Nguyen, Quang-Tam Albany Medical College 515-262-6220 [email protected] Norbury, Christopher Pennsylvania State University 717-531-7204 [email protected] O’Connor, William Albany Medical College 518-262-6548 [email protected] Papillion, Amber SUNY Upstate medical University 315-464-7690 [email protected] Pearl, John Trudeau Institute 518-891-3080 [email protected] Periasamy, Sivakumar Albany Medical College 518-496-8026 [email protected]
Pham, Giang Albany Medical College 518-262-6972 [email protected] Place, David Pennsylvania State University 585-469-5111 [email protected] Price, B. Duane NIH/NIAID 301-451-2592 [email protected] Princiotta, Michael SUNY Upstate Medical University 315-464-7683 [email protected] Ramon, Sesquile University of Rochester 585-275-7836 [email protected] Ray, Aurelie Trudeau Institute 518-817-3965 [email protected] Reiley, William Trudeau Institute 518-891-3080 [email protected] Rodriguez, Princess Albany Medical College 518-262-1437 [email protected]
147
Russell, Michael University at Buffalo 716-829-2790 [email protected] Sanfilippo, Alan Albany Medical College 518-462-6220 [email protected] Sedlacek, Abigail University of Rochester 585-275-6747 [email protected] Sellati, Timothy Albany Medical College 518-262-8140 [email protected] Simmons, Katrina Wadsworth Center 518-210-6077 [email protected] Singh, Anju Albany Medical College 518-262-7715 [email protected] Steiner, Donald Albany Medical College 518-262-6220 [email protected] Sun, Keer Albany Medical College 518-262-6220 [email protected]
Surojit Sarkar Pennsylvania State University 814-867-4034 [email protected] Taffet, Steven SUNY Upstate Medical University 315-464-5419 [email protected] Thatcher, Thomas University of Rochester 585-273-5170 [email protected] Torrado, Egidio Trudeau Institute 518-891-3080 [email protected] Tumanov, Alexei Trudeau Institute 518-891-3084 [email protected] Vance, David Wadsworth Center 518-402-4081 [email protected] Winans, Bethany University of Rochester 585-275-2013 [email protected] Yates, Jennifer SUNY Albany School of Public Health 518-473-2794 [email protected]
148
Yermakova, Anastasiya Wadsworth Center 518-402-4081 [email protected] Zhang, Yubin Albany Medical College 518-262-0922 [email protected] Zourelias, Jessica University at Buffalo 724-831-6731 [email protected]
149
Authors Index
KS - Keynote Speaker
O - Oral Poster Presentation
P# - Poster Number
S - Speaker Page (s)
*************************************************************************************
Abrams, Scott ................................................................................. 43, 99, 113
Akerlund, Linda J. ............................................................................ 130
Alexander, Matthew P. ...................................................................... 31
Amarasinghe, Jayaleka J. (P31) ............................................................. 95
Appleton, Judith A. .......................................................................... 110
Ault, Jeffrey G. ............................................................................... 108
August, Avery ................................................................................. 45, 47, 66, 82, 83, 100
Balch, Amanda N. ............................................................................ 32
Bakshi, Chandra Shekhar .................................................................... 108
Bai, Yuting ..................................................................................... 82
Bashant, Kathleen ............................................................................ 70
Battaglia, Nicholas G. ........................................................................ 41, 65
Bendor, Liron ................................................................................. 94
Bian, Guanglin ................................................................................ 42, 101
Bishop, Gail A. (KS) ......................................................................... 29
Bitsaktsis, Constantine ....................................................................... 93
Blair, Sarah J. ................................................................................. 41, 65
Boule, Lisbeth A. (P8) ....................................................................... 72
Briggs, Jessica ................................................................................. 82
Brooks, Robert ............................................................................... 32
Budd, Ralph C. (KS) ......................................................................... 132
Burdelya, Lyudmila G. ...................................................................... 42, 101
Burke, Catherine G. (P39, O) .............................................................. 57, 103
Bushway, Meghan E. (P40, O) ............................................................. 58, 104
Bynoe, Margaret S. ........................................................................... 76
Cambier, John C. ............................................................................. 130
150
Cao, Xuefang .................................................................................. 42, 101
Carlson, Louise ............................................................................... 81, 99
Chadwick, Chrystal .......................................................................... 74
Cheng, Benson Yee Hin (P32) .............................................................. 57, 96, 103
Chew, Tina .................................................................................... 82
Chisholm, John D. ........................................................................... 32
Chung, Allen Y. (P1, O) .................................................................... 41, 65
Clancy-Thompson, Eleanor ................................................................. 31
Connell, Terry D. ............................................................................ 74
Conway, Thomas F. .......................................................................... 41, 65
Cook, W. James .............................................................................. 105
Cooper, Andrea M. (S) ...................................................................... 54, 67, 117, 134
Davies, Michael L. (P55) .................................................................... 119
Davis, Stephanie R. .......................................................................... 106
de la Torre, Juan ............................................................................. 96
De Jesus, Magdia (P16) ...................................................................... 80
Diel, Michael .................................................................................. 43, 113
Ding, Xilai ..................................................................................... 42, 101
Duffy, Ellen B. (P45) ........................................................................ 109
Duus, Karen ................................................................................... 116
Eaton, Sheri M. (P38) ....................................................................... 46, 55, 68, 102, 121
Egilmez, Nejat K. ............................................................................ 33, 41, 65, 135
Evans, Sharon S. ............................................................................. 43, 48, 49, 79, 88, 113
Farren, Matt ................................................................................... 99
Fernandes, Sandra (S) ........................................................................ 32
Fernandez, Elise Macho (P33, O) .......................................................... 59, 97, 125
Fisher, Daniel T. (P24, O) .................................................................. 48, 49, 79, 88
Fong, E.H. Hannah (P21) ................................................................... 85
Fountain, Jeffrey J. ........................................................................... 54, 67, 117
Franz, Brian (P29) ............................................................................ 93
Fuhler, Gwenny M. .......................................................................... 32
Furuya, Yoichi (P9) .......................................................................... 73
151
Gagliardo, Lucille F. ......................................................................... 110
Gao, Donghong ............................................................................... 128
Gebreselassie, Nebiat G. .................................................................... 110
Gerber, Scott A. .............................................................................. 39, 58, 77, 104
Getahun, Andrew ............................................................................ 130
Glenn, Heather ............................................................................... 116
Goodfield, Laura L. (P30) ................................................................... 94, 98
Gosselin, Edmund J. ......................................................................... 87, 93
Green, Kathy A. (P41) ...................................................................... 105
Green, William R. ........................................................................... 105
Greene, Christopher J. (P10) ............................................................... 74
Gudkov, Andrei V. ........................................................................... 42, 101
Gumbleton, Matthew ........................................................................ 32
Harden, Jamie L. ............................................................................. 33, 41, 65
Harton, Jonathan A. ......................................................................... 108
Harvill, Eric T. ............................................................................... 92, 94, 98
Hayes, Sandra M. ............................................................................. 86, 139
Haynes, Laura ................................................................................. 46, 55, 68, 102
Heo, Yong ..................................................................................... 128
Herman, Katherine E. ....................................................................... 57, 103
Hester, Sara E. ................................................................................ 98
Hickey, Anthony J. (P14, O) ............................................................... 52, 78
Hoekstra II, David A. (P17) ................................................................. 81
Hsiao, His-Min ................................................................................ 124
Hu, Jianfang ................................................................................... 45, 66
Huang, Fei (P18) ............................................................................. 82
Huang, Lu (P46) .............................................................................. 110
Huang, Weishan (P2,O) ..................................................................... 45, 66, 82
Iglesias, Bibianna V. .......................................................................... 87
Ito, Fumito .................................................................................... 43, 113
Iyer, Sonia ..................................................................................... 32
Jelley-Gibbs, Dawn M. ..................................................................... 46, 68, 102
152
Jin, Guang-Bi .................................................................................. 71
Jin, Lei (S) ..................................................................................... 130
Johnson, Lawrence P. ....................................................................... 52, 78
Johnson, Nicole (P6) ......................................................................... 70
Joly, Marguerite (P48) ...................................................................... 112
Jones, Maura .................................................................................. 53, 118, 120
Joseph, A. ..................................................................................... 112
Kalia, Vandana ................................................................................ 137
Kannan, Arun K. (P36, O) .................................................................. 47, 100
Kennedy, Jeffrey S. (S) ...................................................................... 35
Kerr, William G. ............................................................................. 32
Kilinc, M. Okyay ............................................................................. 41, 65
Kim, Do-Geun (P12) ........................................................................ 76
Kim, Minhyung ............................................................................... 43, 113
Klokk, Tove Irene ............................................................................ 89
Kluetzman, Kerri ............................................................................. 128
Knowles, Heather M. ........................................................................ 130
Koroleva, Ekatrina P. ........................................................................ 59, 97, 125
Krenz, Antje .................................................................................. 76
Kummer, Lawrence W. ..................................................................... 52, 78
Laird, Renee M. (P22, S) ................................................................... 86, 139
Lanthier, P.A. ................................................................................. 102
Laukens, Debbie .............................................................................. 106
Lawrence, B. Paige ........................................................................... 33, 71, 72
Lawrence, David A. (S) ..................................................................... 128
Lee, James J. .................................................................................. 110
Lee, Kelvin P. (S) ............................................................................ 40, 81, 99, 122, 138
Lee, Nancy A. ................................................................................. 110
Lefebvre, Julie S. (P4, O) ................................................................... 46, 68
Leifer, Cynthia (S) ............................................................................ 129
Leigh, Nicholas (P37, O) .................................................................... 42, 101
Levea, Charles M. ............................................................................ 41, 65
153
Li, Qinseng .................................................................................... 41, 65
Li, Ying ........................................................................................ 93
Lim, Joanne Y.H. (P43) ..................................................................... 107
Lin, Jr-Shiuan ................................................................................. 52, 78
Liu, Hong ...................................................................................... 42, 101
Liu, Yingru (S) ................................................................................ 135
Lord, Edith M. ................................................................................ 39, 58, 77, 107
Luster, Andrew D. ........................................................................... 49, 79
Lynes, Michael A. (S) ........................................................................ 36, 106
MacNamara, Katherine C. .................................................................. 53, 118, 120
Malik, Meenakshi ............................................................................. 108
Mandell, Lorrie ............................................................................... 74
Mannella, Carmen A. ........................................................................ 108
Mantis, Nicholas J. ........................................................................... 74, 80, 89, 91, 95
Martinez-Sobrido, Luis ...................................................................... 57, 96, 103
McBride, Kevin ............................................................................... 61, 111
McCabe, Amanda (P54, O) ................................................................. 53, 118
Metzger, Dennis W. ......................................................................... 51, 69, 73, 114, 115
Mikucki, Maryann (P15, O) ................................................................ 49, 79
Miller, Richard K. ............................................................................ 58, 104
Miller, Veronica .............................................................................. 128
Mogan, Jennifer .............................................................................. 130
Mohinta, Sonia (P19) ........................................................................ 83
Muhitch, Jason B. (P49, O) ................................................................. 43, 48, 88, 113
Mullins, David W. (S) ....................................................................... 31
Murphy, Shawn P. ............................................................................ 57, 58, 103, 104
Murray, Megan (P58, O) .................................................................... 40, 122
Muse, Sarah J. (P34) ......................................................................... 92, 98
Musteata, Florin M. .......................................................................... 108
Nair, Jayakumar .............................................................................. 40, 122
Neill, Luke .................................................................................... 59, 97
Nguyen, Quant-Tam (P51) ................................................................. 115
154
Norbury, Chris C. ............................................................................ 119
O’Connor Jr., William (S) ................................................................. 126
O’Hara, Joanne M. ........................................................................... 74
Ostroff, Gary ................................................................................. 80
Parent, Michelle A. .......................................................................... 52, 78
Patsey, Rebeca ................................................................................ 70
Pearl, John .................................................................................... 54, 117
Penny, Laura A. .............................................................................. 137
Periasamy, Sivakumar (P20) ................................................................ 84, 108
Perraud, Anne-Laure ........................................................................ 130
Pham, Giang H. (P23) ....................................................................... 87, 93
Phipps, Richard P. ............................................................................ 60, 90, 124
Pietrosimone, Kathryn M. (P42) ........................................................... 106
Place, David E. (P28) ........................................................................ 92, 98
Princiotta, Michael F. ........................................................................ 86, 139
Puffer, Erik (S) ................................................................................ 62
Racine, Rachael ............................................................................... 61, 111
Rahman, Tabassum ........................................................................... 84, 108
Ramon, Sesquile (P26, O) .................................................................. 60, 90
Ray, Aurelie (P3) ............................................................................. 67
Reiley, William W. (P57, O) .............................................................. 55, 121
Richards, S. ................................................................................... 112
Roberts, Sean ................................................................................. 73, 115
Rodriguez, Princess (P52) .................................................................. 116
Rolin, Oliver Y. .............................................................................. 94
Rosa, Sarah .................................................................................... 87, 93
Russell, David G. ............................................................................. 85
Russell, Michael W. ......................................................................... 85, 135
Ryan, Lynn M. ................................................................................ 55, 121
Sahay, Bikash .................................................................................. 70, 108
Sahu, Nisebita ................................................................................. 47, 100
Sandvig, Kirsten .............................................................................. 89
155
Sanfilippo, Alan M. (P50) ................................................................... 114
Sarkar, Surojit (S) ............................................................................ 137
Sedlacek, Abigail L. (P13, O) .............................................................. 39, 77
Sellati, Timothy J. ............................................................................ 70, 75, 84, 108
Serhan, Charles N. ........................................................................... 60, 90
Shoemaker, Charles B. ....................................................................... 91
Sime, Patricia J. ............................................................................... 124
Simmons, Katrina (P11) ..................................................................... 75
Singh, Anju (P44) ............................................................................ 108
Skitzki, Joseph ................................................................................ 43, 49, 79, 113
Smiley, Stephen T. ........................................................................... 52, 78
Spangler, Haley (P35) ....................................................................... 99
Steiner, Donald (P5, O) ..................................................................... 51, 69
Szaba, Frank M. ............................................................................... 52, 78
Tan, Shumin ................................................................................... 85
Thatcher, Thomas H. (S) .................................................................... 124
Tighe, M.P. ................................................................................... 102
Torrado, Egidio (P53, O) ................................................................... 54, 117
Trembley, Jacqueline M. .................................................................... 91
Tumanov, Alexei V. (S) ..................................................................... 59, 97, 125
Vance, David J. (P27) ....................................................................... 91
Vardam, Trupti D. ........................................................................... 48, 88
Verdile, Vincent .............................................................................. 35
Virtuoso, Lauren P. .......................................................................... 41, 65
Waight, Jeremy ............................................................................... 43, 113
Weiler, Lauren A. ............................................................................ 119
Welsh, JoEllen ................................................................................ 75
Winans, Bethany (P7) ........................................................................ 71, 72
Winslow, Gary M. ........................................................................... 55, 61, 111, 120, 121
Wittmer, Susan T. ........................................................................... 55, 121
Wolf, Benjamin J. ............................................................................ 86, 139
Woodland, David L. ......................................................................... 55, 121
156
Yager, Eric .................................................................................... 116
Yates, Jennifer (P47, O) .................................................................... 61, 111
Yermakova, Anastasiya (P25) ............................................................... 89
Yuzefpolskiy, Yevgeniy ...................................................................... 137
Zhang, Lu ...................................................................................... 53, 118
Zhang, Yubin (P56) .......................................................................... 120, 128
Zourelias, Jessica L. (S) ...................................................................... 33, 41, 65