15Th Annual Upstate New York Immunology Conference 2012

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Major Corporate Sponsor: BD Biosciences 15th Annual Grant Upstate Funding: New York NIH National Institute Immunology of Allergy and Conference Infectious Diseases

2012 Trainee Awards: American Association of Immunologists

October 21-24, 2012

The Sagamore Resort and Conference Center Bolton Landing, NY ANNUAL UPSTATE NEW YORK IMMUNOLOGY CONFERENCE (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 research 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 further 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 SAGAMORE RESORT 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! 2 Table of Contents 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

3 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

4 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)

5 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)

6 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.

7 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.

8 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.

9 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”

10 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

11 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.

12 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!

13 Platinum Corporate Sponsor

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25 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

26 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

Grant support provided to

Graduate Students and Postdoctoral Fellows by the

National Institutes of Health

National Institute of Allergy and Infectious Diseases

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“Thank You”

28 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 molecules 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 lymphocytes, 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 distinct 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 discussed.

Symposium I

Tumor Immunity

30 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 human 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 characterized. 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 melanoma 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 detected, 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 infiltration and immune-mediated tumor control. 31 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 respectively. Other hallmarks of apoptosis, such as caspase and PARP cleavage, were also observed. 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 enzymatic and in vitro cell killing assays.

32 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 enzyme, 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 intratumoral 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.

Symposium II

Resources for Today’s Scientific World

34 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 creation 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 research. 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 resources 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

35 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 macromolecules. Most commonly, these measurements are made using the Kretschmann configuration, 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 measurements 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 dynamic range. This talk will describe the technology, and will provide examples of its application to the measurement of macromolecules, bacteria, viruses, mammalian cells, and the functional characterization of mammalian cells in a high content environment.

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.

Poster Oral Presentations* (Albenia)

Cancer Immunobiology

*AAI Young Investigator Award Recipients

38 #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 release 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 response 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 omentum. 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 surprisingly 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 specific nor non-specific tumor growth was observed suggesting a long term anti-tumor response. 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 challenge. 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 involving 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. results, 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

39 #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 molecular 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 receptor. 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 interactions, 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 significantly increase MM cell survival. However, this protection is entirely abrogated by either 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 dependent fashion. To confirm that survival is also Akt-dependent, we repeated the experiments 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 expression, 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.

40 #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 constitutional symptoms and slowed the development of fatal anemia in our experimental animals. 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.

41 #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 malignant self due to the lack of a danger signal. This signal can be provided by a TLR agonist, 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 examined 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 utilized ImageStream flow cytometry to monitor NFκ(kappa)B nuclear translocation, a measure of TLR5 activation. ImageStream flow cytometry reveals that CD11b+ and CD11c+ cells, but neither NK nor T cells, directly respond to CBLB502. These findings demonstrate that the novel TLR5 agonist, CBLB502, can stimulate a robust antitumor immune response by indirectly activating cytotoxic lymphocytes through TLR5-expressing accessory immune cells.

42 #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 during 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 initial 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 therapeutically. 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 proximal 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 involved decreased expression of L-selectin on naïve lymphocytes resulting in reduced quality 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 improve 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 levels 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 improved entry of naïve T cells in TdLN may be a novel strategy to overcome immune evasion. Supported by grants from the NIH (CA79765 and AI082039).

Poster Oral Presentations* (Evelley)

Regulation of Cellular Immunology

*AAI Young Investigator Award Recipients

44 #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 significantly increased in the absence of non receptor tyrosine kinase Itk. However, their developmental path and function are not clear, particularly whether they can contribute to antigen 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 specific 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 significantly enhanced early response to antigen without prior primary stimulation. Our findings suggest a method to generate antigen specific CD8+ IMP T cells, and demonstrate that these cells promptly respond in an antigen specific fashion.

45 #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 individuals. 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 results from their incapacity to become Tfh cells or from functional defects of those Tfh remains 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 draining 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 germinal center response and consequently to the impaired humoral response generated following 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.

46 #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 inappropriate 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 differentiation 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 addition, 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 antigen. 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 expression 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 differentiation programs, and Itk as a therapeutic target for Th2 mediated inflammatory diseases.

47 #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, providing both cognate antigen and costimulation required for T cell activation as well as cytokines (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 immunization. Furthermore, preventing migration of DCs into the inflamed LN through global blockade of chemokine receptors using the irreversible inhibitor of G-protein signaling pertussis 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 receptors. 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 immune response. Supported by the NIH (CA79765 and AI082039), the Roswell Park Alliance Foun- dation, and the Jeniffer Linscott Tietgen Family Foundation.

48 #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 treatment 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 requirement 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 microscopy 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 during cancer immunotherapies. Supported by the NIH (CA79765, CA085183), the Jennifer Linscott Tietgen Family Foundation, and the Joanna M. Nicolay Melanoma Foundation.

Poster Oral Presentations* (Albenia)

Immunity to Human Pathogens

*AAI Young Investigator Award Recipients

50 #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 recruitment 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 defense against F. tularensis infection primarily by recruiting neutrophils, which are essential for clearing the bacterium. (Supported by NIH grant PO1 AI 056320)

51 #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 people. 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 demonstrate 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 significantly improves survival and reduces hepatic bacterial burden in mouse models. Interest- ingly, despite conferring protection, treatment with CpG ODN increases the bacterial burden 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 pretreatment 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 bacterial burden, however, are observed in TLR9-deficient mice, indicating that they are all dependent 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 increases the potential for its use as an immunomodulatory agent against the pathogen.

52 #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 infection resulted in proliferation and activation of BM-resident macrophages, which resulted in increased numbers of active HSCs. Ehrlichia muris infected C57BL/6 mice exhibited increased 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 reside.

53 #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 signaling 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 infection, in the absence of IL-27 activity, CD4 T cells express lower levels of the transcription 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 signaling 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.

54 #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 persistent 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 emigrants 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 peripheral 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 infection, the contribution of recent thymic emigrants declines during chronic infection. The decline 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 expansion 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 population 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 understanding of how T cell responses are maintained during persistent bacterial infections.

Poster Oral Presentations* (Evelley)

Immune Regulation

*AAI Young Investigator Award Recipients

56 #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 morbidity. 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 hyporesponsive 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 infection. 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 compromised in TBCs relative to epithelial cells. In addition, the levels of IFN-responsive gene expression 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 results 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 subsequently 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.

57 #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 interferon-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 develop 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 distinct 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)+ syncytiotrophoblast 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 cytotrophoblast cells. These data suggest that syncytiotrophoblast, which are in direct contact with maternal 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 normal versus pathological pregnancies, and for studying the effects of environmental factors on placental function and morphometry.

58 #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 severe 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 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 controls 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 intestinal 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 colitis 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.

59 #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 maintaining homeostasis. Newly identified lipid-derived mediators are key players during the process 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, including inhibition of neutrophil infiltration, reduction of T cell cytokine production and stimulation of macrophage phagocytic activity. However, their effects on B lymphocytes are unknown. 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 human 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 transcription 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

60 #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 maintaining 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 population 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 secondary 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 required 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, demonstrating 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.

61 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 parameters simultaneously. This seminar will present the basic principles for optimization of multicolor 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 previously available reagents, will be presented.

Poster Abstracts

63 Poster Abstract Listing

#1 Allen Y. Chung* #31 Jayaleka J. Amarasinghe #2 Weishan Huang* #32 Benson Yee Hin Cheng #3 Aurelie Ray #33 Elise Macho Fernandez* #4 Julie S. Lefebvre* #34 Sarah J. Muse #5 Donald Steiner* #35 Haley Spangler #6 Nicole Johnson #36 Arun K. Kannan* #7 Bethany Winans #37 Nicholas Leigh* #8 Lisbeth A. Boule #38 S.M. Eaton #9 Yoichi Furuya #39 Catherine G. Burke* #10 Christopher J. Greene #40 Meghan E. Bushway* #11 Katrina Simmons #41 Kathy A. Green #12 Do-Geun Kim #42 Kathryn M. Pietrosimone #13 Abigail L. Sedlacek* #43 Joanne Y.H. Lim #14 Anthony J. Hickey* #44 Anju Singh #15 Maryann Mikucki* #45 Ellen B. Duffy #16 Magdia De Jesus #46 Lu Huang #17 David A. Hoekstra, II #47 Jennifer Yates* #18 Fei Huang #48 Marguerite Joly #19 Sonia Mohinta #49 Jason Muhitch* #20 Sivakumar Periasamy #50 Alan M. Sanfilippo #21 E. H. Hannah Fong #51 Quang-Tam Nguyen #22 Renee M. Laird* #52 Princess Rodriguez #23 Giang H. Pham #53 Egidio Torrado* #24 Daniel T. Fisher* #54 Amanda McCabe* #25 Anastasiya Yermakova #55 Michael L. Davies #26 Sesquile Ramon* #56 Yubin Zhang #27 David J. Vance #57 William W. Reiley* #28 David E. Place #58 Megan Murray* #29 Brian Franz #30 Laura L. Goodfield

*AAI Young Investigator Award Recipient 64 #1

Orally-Bioavailable IL-10 Nanoparticles Reduce Intestinal Polyposis and Alleviate Inflammation-Induce Constitutional Symptoms in APC-min Mice

65 #2

Functional Development of Antigen Specific Innate Memory CD8+ T cells via Selection by Hematopoietic MHC Class I

66 #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 antigen-specific CD4+ T cell response and the IL-12 family of cytokines along with their respective receptors are essential to this response. Not only does IL-12 play a role in modulating 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 activation. 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 binding, 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 infection. 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 associated 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.

67 #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

68 #5

The Role of Macrophages and Neutrophils in Pulmonary Francisella tularensis Infection

Donald Steiner and Dennis W. Metzger Albany Medical College, Albany, NY

69 #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 patients can experience severe symptoms such as arthritis, carditis, and neurological complications. 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 opposite 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.

70 #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 persistent 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 response to influenza virus challenge at maturity, including a reduced expansion and activation of CD8+ T cells. However, it is unknown whether developmental AhR signaling directly 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 developmentally 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.

71 #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 lasting immunological changes that persist into adulthood. These changes include greater susceptibility 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 function 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 offspring. Recently we have examined the CD4+ T cell response to influenza virus in developmentally 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 exposure 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 developmentally exposed mice after influenza virus infection.

72 #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 respiratory viral pathogens. Using a mouse model of asthma, we investigated the susceptibility of asthmatics to influenza A virus challenge. Unexpectedly, asthmatic mice were highly resistant to primary infection with the H1N1 2009 pandemic strain. However, although viral replication was initially delayed in asthmatic mice, viral lung titers ultimately reached comparable 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.

73 #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) expressed by Escherichia coli, exhibits strong mucosal adjuvant properties. Recently, we described LT-IIb(T13I), a non-toxic mutant of LT-IIb that has altered ganglioside receptor binding 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 potential of LT-IIb(T13I) as an effective second-generation adjuvant for use in biodefense vaccines.

74 #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 immune 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 component 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 conditioned 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 expression 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 incubated 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 effects on TLR4 activation in HEK Blue cells or TNFα secretion but did promoted the secretion 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 secretion 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.

75 #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 characterized 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 demonstrating that blockade of adenosine receptor signaling with broad spectrum adenosine receptor antagonists or specific antagonists that block the A2A adenosine receptor inhibits lymphocyte 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 patients with severe combined immune deficiency or SCID due to a mutation in their adenosine deaminase (ADA) gene. ADA rapidly converts adenosine to inosine resulting in adenosine‟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 significantly less severe EAE and have less immune cell infiltration in their brain. Because a common 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.

76 #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

Supported by NIH Grants: R01: CA28332 and T32: AI007285

77 #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 people. 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 demonstrate 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 significantly improves survival and reduces hepatic bacterial burden in mouse models. Interest- ingly, despite conferring protection, treatment with CpG ODN increases the bacterial burden 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 pretreatment 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 bacterial burden, however, are observed in TLR9-deficient mice, indicating that they are all dependent 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 increases the potential for its use as an immunomodulatory agent against the pathogen.

78 #15

The Non-Redundant Role For Chemokine Receptor Cxcr3 in T cell Trafficking into Tumor Tissue

79 #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 associated 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 capture 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, suggesting 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 associated 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 potentially use them for the development of better therapeutics and oral vaccines.

80 #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 characterized 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 translocates to the plasma membrane. Substitution of an alanine at position 25 in the pseudosubstrate 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 activation. 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 results 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 measured for apoptosis. We found that A25Y-PKCβ(beta)II induced a maximum of a 4-fold increase 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 membrane, and induces apoptosis.

81 #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 development and differentiation. CD4+ and CD8+ T cells in Itk KO mice display innate memory 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 preferentially 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 recipient 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 differentiating 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 supporting the antigen specific CD8+ T cell memory differentiation.

82 #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 example 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 suggested to contribute to the development of this disease. Activated alveolar macrophage present processed antigen to the CD8+ T cells which in turn release mediators leading to inflammation 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 development of HP, CD8+ T cells may act as negative regulators directly or indirectly, influencing the severity of the disease by affecting IL-17A expression.

83 #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 signaling, 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.

84 #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 establishment 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, progression 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 granulomas 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 oxygen species which are necessary for bacterial killing. Lungs of C56BL/6 female mice infected intranasally with Mtb express arginase. Factors released by the TDM granuloma induce 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 downregulation.

85 #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 dissemination. Similar to the previous study utilizing a non-murinized Listeria strain, bacterial burden is reduced in BatF3–/– mice compared to WT mice following infection with the murinized Listeria strain via a systemic route of infection. By contrast, we observe no difference 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.

86 #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 studies have demonstrated that targeting immunogens to Fcγ receptors (FcγR) on antigen presenting 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 activation 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

87 #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

88 #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 mechanisms 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 attachment 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 neutralizing 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 neutralize 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 antibodies against A-B toxins can neutralize by both intracellular and extracellular mechanisms.

89 #26

Specialized Proresolution Mediators Enhance Human B cell Differentiation to Antibody Secreting Cells

This research is supported by DE011390 and NIH T32 DE007202

90 #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 resolution 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 cytotoxicity 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.

91 #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 precursor 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 protein 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 number 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 mucosal 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.

92 #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 inactivated 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 inhibitory 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 immunized 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 levels 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.)

93 #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 respiratory 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 induces tolerance to these bacteria in the nasal cavity. We predict that this mechanism involves interleukin-10, which is known to suppress interferon-γ (gamma). Mice deficient in this cytokine more efficiently control B. bronchiseptica indicating that interleukin-10 contributes 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 identified secretion system during chronic B. bronchiseptica infections.

94 #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 protecting mice against enteric infection. The exposure of S. Typhimurium to Sal4 causes bacterial 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 phosphodiesterase (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 indicated 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.

95 #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 arenavirus 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, demonstrating 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 ability 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.

96 #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 severe 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 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 controls 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 intestinal 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 colitis 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.

97 #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 eukaryotic cells. The purpose of this project is to study the molecular mechanisms underlying the interaction 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 component 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 inoculated 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 inflammasome 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 inoculation. 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.

98 #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 importance 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 differentiation, 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 dependent 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 differentiation between PKCβ(beta)II and either IRF8 or IRF4 which can be tumor targets.

99 #36

Tec Kinase Itk Modulates Allergic Airway Inflammation by Suppressing IFNγ(gamma) Expression in Naïve CD4+ T cells

T helper responses are critical for a productive immune response but an inappropriate 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 differentiation 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 addition, 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 antigen. 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 expression 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 differentiation programs, and Itk as a therapeutic target for Th2 mediated inflammatory diseases.

100 #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

101 #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 complications 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 neutrophils are critical for efficient viral clearance. Reduced lung neutrophil numbers and reduced 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 chemokines, 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 alterations in the lung environment. This work was supported by NIH funding to L. Haynes and Trudeau Institute.

102 #39

Human Placental Trophoblast Cells are Hyporesponsive to Type I and Type III Interferons: Potential Role in Susceptibility of Pregnant Women to Viral Infection

103 #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

104 #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 disease processes, in particular against retroviruses that induce immunodeficiency. Here, we demonstrate the development of a highly immunosuppressive MDSC population 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 independent 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 target 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 assays 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 pathogenic effector role for these retrovirus-induced MDSCs.

105 #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 reactive 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 leukocyte 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 response 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 inflammatory 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 response 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.)

106 #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 demonstrated 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 increased 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 increase 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 suggests that IFN-α/β, but not IFN-γ, may be contributing to the initial chemoattraction of lymphocytes into radiation-treated tumors. Using flow cytometry analysis of immune cell populations 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 currently 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 responses.

Support: NIH R01 grant CA28332

107 #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, bacteria subvert host cell death programs to facilitate their unfettered replication within macrophages and other cell types. Release of bacteria from infected cells is coincident with severe 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 cellular 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 mitochondrial 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 infection, but present during late-phase septic disease (≥ 72 h).

108 #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 respiratory 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 strategies 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 stimulated 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.

109 #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 eosinophils protect, rather than attack Trichinella spiralis larvae in the skeletal muscle during primary infection. Accumulation of Th2 cells at sites of infection is also impaired in the absence 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 presentation 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 response is first engaged. Transfer of MHCII-/- eosinophils was effective, showing that antigen 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 decreased 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.

110 #47

IgM Memory B cells Generated During Bacterial Infection are Required for Secondary IgG Responses to Antigenic Challenge

111 #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 challenge 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 induce anaphylaxis, serum sickness or even immune complex disease. For chronically administered biologics the long-term consequences of ADA can potentially contribute to treatment failure and discontinuation of the therapeutic. In an attempt to control ADA, we identified a unique and benign induction regimen of low dose methotrexate that successfully induces 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 regulatory 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 following data suggest a unique tolerance induction regimen by low-dose, induction treatment with methotrexate.

112 #49

Overcoming Obstacles to Naïve T Lymphocyte Trafficking in Tumor-Draining Lymph Nodes

113 #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 inflammation (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 response 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.)

114 #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 induced 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. pneumoniae 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 administration of prevnar 13 plus interleukin-12 is able to enhance systemic immune responses to pneumococci and efficiently protect against bacterial carriage.

115 #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 infections, 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 responses 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 identified 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.

116 #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

117 #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 infection resulted in proliferation and activation of BM-resident macrophages, which resulted in increased numbers of active HSCs. Ehrlichia muris infected C57BL/6 mice exhibited increased 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 reside.

118 #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 immunology. Procedures for depleting leukocyte subsets range from monoclonal antibodies against surface markers to methods exploiting genes that are upregulated upon differentiation (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 methods 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 expressed from the Csf1r promoter, which is activated in macrophages and immature neutrophils. Both systems allow us to use a drug to deplete cells that have upregulated the promoter in question. We looked at depletion during infection with vaccinia virus, using an intradermal infection 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 macrophages” and Ly6G+ myeloid cells being depleted at the site of infection, and CD11b+ subsets 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.

119 #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 signals during infection is not well understood, but it is thought that activation of HSCPs during 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 hematologic abnormalities, to examine the mechanisms by which HSPC function is regulated during infection. HSPCs can respond to cytokines including interferons (IFNs), and pathogen-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 intrinsic 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.

120 #57

Maintenance of Peripheral T cell Responses during Mycobacterium tuberculosis Infection

121 #58

CD28-mediated Pro-survival Signaling in Multiple Myeloma

Megan Murray, Jayakumar Nair, and Kelvin Lee Roswell Park Cancer Institute, Buffalo NY

122

Symposium III

Mucosal Immunity

123 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 contributes 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 failure 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 resolution 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 monocytes 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 human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL- 10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil 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.

124 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, collectively known as inflammatory bowel disease (IBD). The pathogenesis of IBD is associated with a dysregulated mucosal immune response. Innate lymphoid cells (ILCs) have recently emerged as important regulators of the balance between protective immunity and immunopathology 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 understood. We found that lymphotoxin (LT), a member of TNF family of cytokines, produced 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 represents 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 microenvironment 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. rodentium infection. Our data suggest that alternative, but not canonical NF-kB signaling pathway 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.

125 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 variety 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 inflammatory 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 research. Recent studies from our group and others highlighting novel mechanisms controlling TH17 cells, and newly appreciated roles of TH17-associated cytokines, will be discussed.

126

Symposium IV

Inflammation

127 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 offspring 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 relatively 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 influences affect the social behavior of offspring.

128 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 selective 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 cytoplasmic motif that regulates access to different endosomal compartments. This motif is selectively required for the production of proinflammatory cytokines, but not type I interferon. 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 inflammatory diseases.

129 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 essential for cytosolic DNA induced type I IFN production. Studies done in STING-/- mice (Tmem173) have demonstrated that STING is critical for host defense against DNA virus infection. However, increasing evidence suggests that STING may also be important 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 significance and mechanism by which STING functions in host defense against these non-viral infections have not been demonstrated. Using a conditional STING-/- mice (Tmem173), we show here that STING mediates host defense against L.M. infection 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 effective mucosal vaccine adjuvant.

130

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.

131 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 receptors, 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 activate caspase-8 during cell growth situations. Thus, c-FLIPL acts as a molecular switch between 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 another 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.

132

Symposium V

Host-Pathogen Interactions

133 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 tuberculosis (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 manipulation 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.

134 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 characterized 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 regulatory T cells (Tr1). Blockade of TGF-β or IL-10 by neutralizing antibodies significantly enhanced 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 promote 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 microspheres 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 involving immunoinhibitory cytokines (TGF-β and IL-10) and regulatory T cells, and that this effect can be manipulated to enhance specific protective immune responses.

135

Symposium VI

Regulation of Immune Responses

136 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 immunity 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 inflammation. 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 infection, 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 suppression of the effector program, Tregs aided in the acquisition of hallmark memory T cell properties such as polyfunctionality, antigen-independent longevity and secondary expansion. 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 quiescent state of the memory CD8 T cells from effector cells during an adaptive immune response.

137 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 remains 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 demonstrated 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 chemotherapy-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 support 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.

138 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 effectors. 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 effector 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 effector development and differentiation but also highlight differences in the requirements for the generation of αβ and γδ T cell effectors. .

139

Please join us next year for the

16th Annual Upstate New York Immunology Conference

October 27-29, 2013 The Sagamore Resort and Conference Center

140 Attendee Contact Information Amarasinghe, Jayaleka Budd, Ralph Wadsworth Center University of Vermont 518-526-2486 802-656-2286 [email protected] [email protected]

Avery, August Burke, Catherine Cornell University University of Rochester 607-253-3401 585-273-1840 [email protected] [email protected]

Augustine, Alison Deckhut Bushway, Meghan NIH/NIAID University of Rochester 301-496-7551 585-273-1840 [email protected] [email protected]

Bellville, Dawn Bynoe, Margaret Albany Medical College Cornell University 518-262-5365 607-253-1023 [email protected] [email protected]

Berwin, Brent Cheng, Benson Dartmouth Medical School University of Rochester 603-650-6899 585-276-4847 [email protected] [email protected]

Bishop, Gail Chung, Allen University of Iowa University at Buffalo 319-335-7945 716-829-8765 [email protected] [email protected]

Blaauboer, Steven Connell, Terry Albany Medical College University at Buffalo 518-252-0052 716-829-3364 [email protected] [email protected]

Boule, Lisbeth Cooper, Andrea University of Rochester Trudeau Institute 585-275-2013 518-891-3080 [email protected] [email protected]

141 Davies, Michael Evans, Sharon Pennsylvania State University Roswell Park Cancer Institute 717-531-0624 716-845-3421 [email protected] [email protected]

De Jesus, Magdia Fernandes, Sandra Wadsworth Center SUNY Upstate Medical University 518-402-4081 315-440-2822 [email protected] [email protected]

Dewan, Kalyan Fernandez, Elise Macho Pennsylvania State University Trudeau Institute 814-470-5281 518-891-3080 [email protected] [email protected]

D’Hondt, Rebecca Fisher, Daniel Wadsworth Center Roswell Park Cancer Institute 518-402-4081 716-845-3157 [email protected] [email protected]

Drake, James Fong, Ern Hwei Hannah Albany Medical College Cornell University 518-262-9337 646-306-3373 [email protected] [email protected]

Duus, Karen Franz, Brian Albany Medical College Albany Medical College 518-262-1176 518-262-6972 [email protected] [email protected]

Duffy, Ellen Furuya, Yoichi Albany Medical College Albany Medical College 518-262-4447 518-262-6220 [email protected] [email protected]

Eaton, Sheri Gerber, Scott Trudeau Institute University of Rochester 518-891-3080 585-275-6747 [email protected] [email protected]

142 Glass, Aaron Herman, Katherine SUNY Upstate Medical University University of Rochester 315-525-1043 513-225-5766 [email protected] [email protected]

Goodfield, Laura Hickey, Anthony Pennsylvania State University Trudeau Institute 814-865-9134 518-891-3080 [email protected] [email protected]

Gorham, James Hu, John Dartmouth College University at Buffalo 603-650-8373 585-991-8047 [email protected] [email protected]

Green, Kathy Huang, Fei Dartmouth College Cornell University 603-724-3963 530-304-2651 [email protected] [email protected]

Green, William Huang, Lu Dartmouth College Cornell University 603-724-3964 607-592-9218 [email protected] [email protected]

Greene, Christopher Huang, Weishan University at Buffalo Cornell University 716-829-5426 814-321-7414 [email protected] [email protected]

Harton, Jonathan Jelley-Gibbs, Dawn Albany Medical College Trudeau Institute 518-262-4445 518-524-5972 [email protected] [email protected]

Hayes, Sandy Jin, Lei SUNY Upstate Medical University Albany Medical College 315-464-7692 518-262-0053 [email protected] [email protected]

143 Johnson, Nicole Ku, Amy Albany Medical College Roswell Park Cancer Institute 518-262-8141 661-618-0250 [email protected] [email protected]

Joly, Marguerite Laird, Renee Genzyme/Sanofi SUNY Upstate Medical University 508-270-2498 315-464-7690 [email protected] [email protected]

Kalia, Vandana Lawrence, David Pennsylvania State University Wadsworth Center 814-863-8533 518-474-8285 [email protected] [email protected]

Kannan, Arun Lefebvre, Julie Cornell University Trudeau Institute 814-321-2867 518-891-3080 [email protected] [email protected]

Kennedy, Jeff Lee, Kelvin Albany Medical College Roswell Park Cancer Institute 781-249-6128 716-845-4106 [email protected] [email protected]

Kim, Dogeun Lee, William Cornell University Wadsworth Center 607-379-3774 518-474-3543 [email protected] [email protected]

Kirimanjeswara, Girish Leifer, Cynthia Pennsylvania State University Cornell University 814-863-5250 607-253-4258 [email protected] [email protected]

Koroleva, Ekaterina Leigh, Nicholas Trudeau Institute Roswell Park Cancer Institute 518-354-8097 716-845-3553 [email protected] [email protected]

144 Lim, Joanne Martinez-Sobrido, Luis University of Rochester University of Rochester 585-478-9325 585-276-4733 [email protected] [email protected]

Liu, Yingru McCabe, Amanda University at Buffalo Albany Medical College 716-201-9423 518-262-0922 [email protected] [email protected]

Lopez-Pinet, Odelys Metzger, Dennis University of Rochester Albany Medical College 585-275-6747 518-262-6750 [email protected] [email protected]

Lord, Edith Mikucki, Maryann University of Rochester Roswell Park Cancer Institute 585-275-5855 716-845-3157 [email protected] [email protected]

Lynes, Michael Mohinta, Sonia University of Connecticut Cornell University 860-486-4350 814-321-3029 [email protected] [email protected]

MacNamara, Kate Muhitch, Jason Albany Medical College Roswell Park Cancer Institute 518-262-0921 716-845-3157 [email protected] [email protected]

Mantis, Nicholas Mullins, David Wadsworth Center Dartmouth College 518-402-2750 603-653-9922 [email protected] [email protected]

Markley, Rachel Murphy, Shawn Pennsylvania State University University of Rochester 640-613-1406 585-273-3910 [email protected] [email protected]

145 Murray, Megan Pham, Giang Roswell Park Cancer Institute Albany Medical College 716-845-8231 518-262-6972 [email protected] [email protected]

Muse, Sarah Place, David Pennsylvania State University Pennsylvania State University 814-865-9134 585-469-5111 [email protected] [email protected]

Nguyen, Quang-Tam Price, B. Duane Albany Medical College NIH/NIAID 515-262-6220 301-451-2592 [email protected] [email protected]

Norbury, Christopher Princiotta, Michael Pennsylvania State University SUNY Upstate Medical University 717-531-7204 315-464-7683 [email protected] [email protected]

O’Connor, William Ramon, Sesquile Albany Medical College University of Rochester 518-262-6548 585-275-7836 [email protected] [email protected]

Papillion, Amber Ray, Aurelie SUNY Upstate medical University Trudeau Institute 315-464-7690 518-817-3965 [email protected] [email protected]

Pearl, John Reiley, William Trudeau Institute Trudeau Institute 518-891-3080 518-891-3080 [email protected] [email protected]

Periasamy, Sivakumar Rodriguez, Princess Albany Medical College Albany Medical College 518-496-8026 518-262-1437 [email protected] [email protected]

146 Russell, Michael Surojit Sarkar University at Buffalo Pennsylvania State University 716-829-2790 814-867-4034 [email protected] [email protected]

Sanfilippo, Alan Taffet, Steven Albany Medical College SUNY Upstate Medical University 518-462-6220 315-464-5419 [email protected] [email protected]

Sedlacek, Abigail Thatcher, Thomas University of Rochester University of Rochester 585-275-6747 585-273-5170 [email protected] [email protected]

Sellati, Timothy Torrado, Egidio Albany Medical College Trudeau Institute 518-262-8140 518-891-3080 [email protected] [email protected]

Simmons, Katrina Tumanov, Alexei Wadsworth Center Trudeau Institute 518-210-6077 518-891-3084 [email protected] [email protected]

Singh, Anju Vance, David Albany Medical College Wadsworth Center 518-262-7715 518-402-4081 [email protected] [email protected]

Steiner, Donald Winans, Bethany Albany Medical College University of Rochester 518-262-6220 585-275-2013 [email protected] [email protected]

Sun, Keer Yates, Jennifer Albany Medical College SUNY Albany School of Public Health 518-262-6220 518-473-2794 [email protected] [email protected]

147 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]

148 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

149 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

150 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

151 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

152 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

153 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

154 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

155 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

156