18Th Annual Upstate New York Immunology Conference

20152015 18th Annual Upstate New York Immunology Conference

Major Corporate Sponsors: BD Biosciences BioLegend, Inc. Taconic Bioscience

Keynote Speakers: Supported by Yasmine Belkaid, Ph.D. NIH/NIAID Kristin A. Hogquist, Ph.D. Grant Funding

Workshop Presentation by: Yasmine Belkaid, Ph.D. Jacob Schumacher, AAI

AAI Young Investigator Awards eBioscience/affymetrix Trainee Travel Awards

Welcome to The Sagamore Resort and Conference Center Bolton Landing, NY

October 25-28, 2015

UPSTATE NEW YORK IMM UNOLOGY 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. We are happy to announce the American Association of Immu- nologists (AAI) is once again providing 10 Young Investigator Awards. eBioscience is also proving 10 Trainee Travel Awards. All award winners will give Oral Poster Presentations. There will also be two Workshops. Keynote speakers are Dr. Yasmine Belkaid (NIH/NIAID) and Dr. Kristin A. Hogquist (University of Minnesota). As part of our leisure activities, there will be a cruise on Lake George, as well as a recreational night including miniature golf, Wii, X-box, whiffle ball, and movies! Mr. Brown’s Pub will be open Tuesday night for informal discussions. Trainees will also have an opportunity to win an iPad during one of two drawings. You must be present at the drawing to win! While all these elements lend to the atmosphere, one simple principle goal 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 Confer- ence 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 Cen- ter celebrated it’s 125 year anniversary in 2008. Since that time, the resort has gone through some remarkable renovations. If this is your first visit to the Resort, take some time to enjoy the beauty that surrounds you. There are many 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 Coordinator for NYIC, or any of the Resort person- nel. Many thanks to Lori Rehm (Director of Sales), Eric Rottingen (National Sales Manager), Derrick Hammond (Conference Services Manager), Don Vilmar (Banquet Manager), Jerid McKinney (Banquet Captain), Joel Clark (Function Set-up Manager) and his amazing crew, CMI Communications-Audio/Visual Manager crew, Glen and Al, along with all of the associates who attend to our many needs. Thank you!

Table of Contents

Conference and Venue ...... 3 Schedule of Events ...... 6 Platinum Corporate Sponsors BD Biosciences ...... 16 BioLegend, Inc...... 18 Taconic Bioscience ...... 20 Silver Plus Corporate Sponsors ...... 22 eBioscience/affymetrix ...... 23 EMD Millipore ...... 24 Krackeler Scientific ...... 25 Silver Corporate Sponsors ...... 26 Dartmouth ...... 27 Lonza ...... 28 Bronze Corporate Sponsors ...... 29 American Association of Immunologists ...... 30 NYIC Scientific Advisory Board ...... 31 Institutional Financial Supporters ...... 32 Grant Support ...... 33 Keynote Speaker Kristin A. Hogquist, Ph.D...... 34 Symposium I Regulatory T-cells ...... 35 Symposium II T-cell Biology ...... 38

4. Poster Talks Session A: Adaptive Immunity ...... 41 Session B: Innate Immunity ...... 47 Corporate Presentation: BioLegend, Inc...... 53 Workshop I - Dr. Belkaid “TBA” ...... 54 Poster Talks Session C: Inflammation ...... 55 Session D: Tumor Cancer ...... 61 Corporate Presentation: BD Biosciences ...... 67 Symposium III Inflammation ...... 68 Symposium IV Immune Regulation ...... 72 Corporate Presentation: Taconic Biosciences ...... 76 Workshop II - AAI “The Urgent Importance of Advocating for Biomedical Research” ...... 77 Symposium V Antigen Presenting Cell Biology ...... 78 Keynote Speaker Yasmine Belkaid, Ph.D...... 82 Poster Listing ...... 83 Poster Abstracts ...... 84 Attendee Contact Information ...... 130 Author Index ...... 134

5. Upstate New York Immunology Conference Schedule of Events

Sunday, October 25th

3:00-5:00 p.m. Hotel Check-in (Main Hotel Lobby)

3:00-5:00 p.m. Conference Registration (Conference Center Lobby)

5:00-6:00 p.m. Free Time

6:00-6:30 p.m. Dinner (Bellvue)

6:30 p.m. Welcome and Introductions

Keynote Presentation Sponsored by BD Biosciences

Introduction: Dr. Dennis W. Metzger

Kristin A. Hogquist, Ph.D. Professor Department of Laboratory Medicine & Pathology University of Minnesota

“T Cell Adolescence”

7:45 p.m. American Association of Immunologists Young Investigator Awards and eBioscience Travel Awards (Award Presentations & Photos)

Immediately Following Awards Presentation—Meet and Greet Activities (Recreation Center)

Monday, October 26th

7:00-8:15 a.m. Breakfast at Leisure (Use Voucher Dated 10/26/15) (La Bella Vita)

8:25-8:30 a.m. Morning Announcements (Nirvana)

8:30-9:30 a.m. Symposium I: Regulatory T-Cells (Nirvana) Chair: Dr. Yasmine Belkaid

8:30-9:00 Tamer B. Chabanet, Ph.D. (Dartmouth Medical College) “Elucidating Oncogene-driven Regulatory T-Cell Responses during Melanoma Tumorigenesis”

9:00-9:30 Elizabeth A. Wohlfert, Ph.D. (University at Buffalo) “Dynamics of Tregs during Infection with T. gondii”

9:30-10:30 a.m. Symposium II: T-Cell Biology (Nirvana) Chair: Dr. Kristin Hogquist

9:30-10:00 Yina H. Huang, Ph.D. (Dartmouth Medical College) “Visualizing and Manipulating T-Cell Responses in vivo”

10:00-10:30 Scott H. Olejniczak, Ph.D. (Roswell Park Cancer Institute) “T-Cell Activation Improves MicroRNA Function”

10:30-10:50 a.m. Break (Conference Center Foyer)

10:50-12:05 p.m. Poster Talks: Session A—Adaptive Immunity (Albenia) Chairs: Dr. Drake and Dr. Wohlfert

10:50-11:05 Lisbeth A. Boule (University of Rochester) “Activation of the Aryl Hydrocarbon Receptor by Categorically Different Ligands Alters the Immune Response to Primary Influenza A Virus Infection” #4

11:05-11:20 Kevin Kenderes (SUNY Upstate Medical University)

“IgM Memory B-cells Differentiate via Multiple Pathways Following Challenge Infection” #13

11:20-11:35 Rebecca Crepeau (Dartmouth College) “miR-155 Plays a Critical Role in the Establishment and Maintenance of Latency in Murine Gammaherpesvirus Infection” #9

11:35-11:50 Jocelyn Wang (Cornel University) “Fetal Hematopoietic Stem Cells Give Rise to a Unique Population of CD8+ T-cells” #33

11:50-12:05 Adam Utley** (Roswell Park Cancer Institute) “CD28 Regulates Metabolic Fitness for ROS Dependent Long Lived Plasma Cell Survival” #41

10:50-12:05 p.m. Poster Talks: Session B—Myeloid Cell Function (Evelley) Chairs: Dr. Eyal and Dr. Hankey

10:50-11:05 Jessica L. Meyers** (University of Rochester) “Persistent Changes in Dendritic Cell Function Following Developmental Activation of the Aryl Hydrocarbon Receptor” #3

11:05-11:20 Michelle N. Messmer** (Roswell Park Cancer Institute) “Regulation of Transcription Factor IRF8 in Myeloid Progenitors is a Critical Checkpoint for Formation of Defective Myeloid Cells in Cancer” #8

11:20-11:35 Phyu Thwe** (University of Vermont) “Glycogen Metabolism Supports Effector Function and Energy Homeostasis of Dendritic Cells” #29

11:35-11:50 Kelli A. Connolly** (University of Rochester) “Inflammatory Monocytes in a Changing Tumor Microenvironment Following Radiotherapy: Implications for Immunotherapy” #37

11:50-12:05 Erika Gruber(Cornell University) “Macrophage Lipid Accumulation is Regulated by Substrate Stiffness” #42

12:05-12:15 p.m. Put Up Posters (Bellvue)

12:15-12:45 p.m. Lunch Buffet (Conference Center Foyer) (Wapanak)

12:45-1:15 p.m. Platinum Corporate Sponsor—BioLegend, Inc. (Wapanak) Ashley Cornett, Ph.D. Technical Application Scientist “Enhancing Downstream Applications through Magnetic Cell Separation”

1:15-3:00 p.m. Workshop I — Sponsored by BioLegend, Inc. (Wapanak) Yasmine Belkaid, Ph.D. “TBA”

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

3:30-4:45 p.m. Poster Talks: Session C—Inflammation (Albenia) Chairs: Dr. MacNamara and Dr. Yarovinsky

3:30-3:45 Megan Peppenelli (SUNY Upstate Medical University)

9. “A Unique Signalisome Induced by Human Cytomegalovirus

during Entry Targets The Translation of Caspase 3 Regulatory Proteins Ensuring the Survival of Infected Monocytes” #1

3:45-4:00 Julianne N.P. Smith** (Albany Medical College) “Type I IFNs Directly Promote Hematopoietic Progenitor Cell Death and Lead to Bone Marrow Failure in Severe Bacterial Infection” #25

4:00-4:15 Kerry R. Belton (Pennsylvania State University) “The Role of the Aryl Hydrocarbon Receptor and the Gut Microbiome in the Modulation of Intestinal Inflammation” #26

4:15-4:30 Elise Burger** (University of Rochester) “Loss of Intestinal Epithelial Autophagy Leads to Catastrophic Susceptibility to Acute T. gondii-mediated Inflammation” #11

4:30-4:45 Jessica L. Rastad** (Dartmouth College) “Myeloid-derived Suppressor Cells in Murine AIDS Suppress B-cell Responses in a Reactive-Oxygen- and Nitrogen- Species Dependent Manner” #7

3:30-4:45 p.m. Poster Talks: Session D—Tumor Cancer (Evelley) Chairs: Dr. Lord and Dr. Olejniczak

3:30-3:45 Kiah L. Sanders** (Dartmouth College) “Expression of Host CXCR3 Controls Early Establishment of Metastatic Melanoma in the Lung” #2

3:45-4:00 Lauren Burkard-Mandel (Roswell Park Cancer Institute) “Tumor-Derived TSLP Enhances Macrophage-mediated Metastasis” #31

10. 4:00-4:15 Danielle Twum (Roswell Park Cancer Institute) “IRF8 Transcriptionally Regulated the Macrophage Response during Tumor Immunosurveillance” #32

4:15-4:30 Adaobi Amobi** (Roswell Park Cancer Institute) “Indoleamine 2,3- Dioxygenase Regulates Density of Tumor Infiltrating CD8+ T-cells in a Murine Model of Ovarian Cancer” #40

4:30-4:45 Aditi Murthy (University of Rochester) “Impact of Hypoxia on IFNγ(gamma)-dependent Responses: Implications for Radiotherapy and Anti-tumor Immunity” #46

**AAI Young Investigator Award and Oral Poster Presentation eBioscience Trainee Travel Award and Oral Poster Presentation

5:00-6:30 p.m. Vendor/Poster Mixer Poster Viewing and Questions (Odd Numbers)

6:30-7:00 p.m. Dinner (Wapanak)

7:00-7:30 p.m. Platinum Corporate Sponsor-BD Biosciences

Andrew D. Bantly, B.S., CCy Technical Application Specialist “Using the Resolution Impact Table to Design Optimal Multicolor Flow Cytometry Panels”

7:30-9:00 p.m. Vendor/Poster Mixer (Bellvue) Poster Viewing and Questions (Even Numbers) iPad drawing during this event. Must be present to win.

9:00-9:15 p.m. Remove Posters (Posters left behind will be discarded)

11. Tuesday, October 27st

7:00-8:15 a.m. Breakfast at Leisure (Use Voucher Dated 10/27/15) (La Bella Vita)

8:25-8:30 a.m. Morning Announcements (Nirvana)

8:30-10:00 a.m. Symposium III: Inflammation (Nirvana) Chair: Dr. Brent Berwin

8:30-9:00 Elia Tait Wojno, Ph.D. (Cornell University) “The Prostaglandin D2 Receptor CRTH2 Regulates Innate Immune Responses during Type 2 Inflammation”

9:00-9:30 Felix Yarovinsky, Ph.D. (University of Rochester) “Type I Immune Response in Host Defense and Inflammation”

9:30-10:00 Margaret Bynoe, Ph.D. (Cornell University) “Purinergic Signaling at the Blood Barrier Regulates CNS Autoimmunity”

10:00-10:15 a.m. Beverage Break (Conference Center Foyer)

10:15-11:45 a.m. Symposium IV: Immune Regulation (Nirvana) Chair: Dr. Nicholas Mantis

10:15-10:45 Michael D. Robek, Ph.D. (Albany Medical College) “Virus-based Therapeutic Vaccine Vectors to Cure Chronic HBV Infection”

10:45-11:15 Raki Sudan, Ph.D. (SUNY Upstate Medical University) “LRBA is Essential for Allogeneic Responses in Bone Marrow Transplantation”

11:15-11:45 Steven M. Szczepanek, Ph.D. (University of Connecticut) “Immunologic Dysregulation in Sickle Cell Disease”

12.

12:00-12:30 p.m. Lunch Buffet (Wapanak Foyer)

12:30-1:00 p.m. Platinum Corporate Sponsor —Taconic Biosciences

Michael Seiler, Ph.D. Senior Product Manager, Humanized Models Program “Humanized Immune System Mouse Models: Recent Advances in Immuno-Oncology Applications”

1:00-2:30 p.m. Workshop II—Sponsored by Silver Corporate Sponsors Introduction by: Dr. Jonathan Harton

Jacob Schumacher Science Policy and Legislative Affairs Specialist American Association of Immunologists “The Urgent Importance of Advocating for Biomedical Research”

2:30-2:45 p.m. Meet at Dock—Prepare to board The Morgan (Please have your boarding pass and drink ticket with you.)

2:45-4:15 p.m. Cruise sponsored by Krackeler Scientific, Inc. (Refreshments provided)

4:15-6:30 p.m. Free Time

4:30-5:30 p.m. Scientific Advisory Board Meeting (Empire Room)

6:30-7:30 p.m. Dinner Buffet (Wapanak)

8:00-11:00 p.m. Mr. Brown’s Pub and Caldwell’s —Informal Discussions

13. Wednesday, October 28th

7:00-8:15 a.m. Breakfast at Leisure (Use Voucher Dated 10/28/15) (La Bella Vita)

8:25-8:30 a.m. Morning Announcements (Nirvana)

8:30-10:00 a.m. Symposium V: Antigen Presenting Cell Biology (Nirvana) Chair: Dr. Paige Lawrence

8:30-9:00 Eyal Amiel, Ph.D. (University of Vermont) “Dectin-1-dependent Changes in Dendritic Cells Metabolism Regulate NLRP3 Inflammasome Activation”

9:00-9:30 Magdia De Jesus, Ph.D. (Wadsworth Center/SUNY Albany) “The Role of Langerin+ DCs in Mucosal Immunity”

9:30-10:00 James R. Drake, Ph.D. (Albany Medical College) “MHC Class II Subsets in B-Cell Biology”

10:00-11:15 a.m. Beverage Break and Check-out (Conference Center Foyer and Hotel Lobby)

10:150-11:15 a.m. Keynote Presentation (Nirvana) Sponsored by Taconic Biosciences

Introduction: Dr. Beth Wohlfert

Yasmine Belkaid, Ph.D. Chief Mucosal Immunology Section Laboratory of Parasitic Diseases National Institutes of Health/NIAID

“Regulation of Tissue Immunity and the Microbiota”

12:15-11:30 a.m. Closing Remarks (Nirvana)  iPad drawing during this event. Must be present to win.

14.

NYIC 2014

15.

Platinum Corporate Sponsor

BD Biosciences

16.

17.

Platinum Corporate Sponsor

BioLegend, Inc.

18.

19.

Platinum Corporate Sponsor

Taconic Biosciences

20.

21.

Silver-Plus Corporate Sponsor

22. eBioscience

23.

24.

25.

Silver Corporate Sponsors

Dartmouse, Inc.

Lonza

26.

27.

28.

Bronze Corporate Sponsors

BrandTech® Scientific, Inc.

Cloud-Clone Corp.

Garland Science

Seahorse Bioscience

29.

In recognition of the significance of this meeting and work being done by Graduate Students and Postdoctoral Fellows, the American Association Of Immunologists has provided Ten(10) Young Investigator Awards.

Each will receive a monetary award, as well as the opportunity to present their research both in poster format and brief talks.

30. 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

Roswell Park Cancer Institute Yasmin Thanavala

SUNY Upstate Medical University Gary Winslow

University at Buffalo Beth Wohlfert

University of Rochester Medical Center Edith Lord

University of Vermont Eyal Amiel

Wadsworth Center/SUNY Albany Nicholas Mantis

31. Institutional Financial Supporters

Albany Medical College Alumni Association

Cornell University Microbiology & Immunology

Dartmouth College Department of Microbiology & Immunology

Roswell Park Cancer Institute Department of Immunology

SUNY Albany

SUNY Upstate Medical University Microbiology & Immunology Program

Trudeau Institute

University at Buffalo Buffalo School of Medicine Department of Microbiology & Immunology

University of Rochester Medical Center Department of Microbiology & Immunology

University of Vermont

Wadsworth Center

32.

Grant support provided to

Graduate Students and Postdoctoral Fellows by the

National Institutes of Health National Institute of Allergy and Infectious Diseases

R13AI051522

“Thank You”

33.

Keynote Speaker

Kristin A. Hogquist, Ph.D. Professor Department of Laboratory Medicine and Pathology University of Minnesota

“T-cell Adolescence”

34.

Symposium I Regulatory T-cells

Chair : Dr. Yasmine Belkaid

35.

Characterizing Natural and Induced Regulatory T cell Response During Autochthonous Melanoma Tumorigenesis

Tamer B. Chabanet, Shannon M. Steinberg, Andrea Boni, Peisheng Zhang, Brian T. Malik, Mary Jo Turk Geisel School of Medicine at Dartmouth. Lebanon, NH 03766

Regulatory T cells (Treg) are critical mediators of tumor immune suppression. While Tregs are found in established tumors, little is known about the kinetics and dynamics of the Treg accumulation and the factors that promote it during oncogene-driven tumorigenesis. In addition, the relative contribution of natural and induced Tregs to antigen-specific Treg responses during tumorigenesis is unknown. The present studies characterize Treg response kinetics and Treg conversion dynamics during early tumor development in a model of autochthonous, tamoxifen-inducible BRafV600E Pten-/- melanoma. While microscopic skin dysplasia appeared 16 days following tumor induction, FoxP3+ Treg frequency and abso- lute numbers did not significantly increase until day 26, coinciding with the development of locally invasive neoplasms. Following adoptive transfer of CD4+ T cells specific to the melanoma antigen TRP-1, antigen-specific FoxP3+ Tregs preferentially accumulated in tumor-induced skin and draining lymph nodes (dLNs), as compared to tumor-free and antigen-deficient counterparts. In contrast, depleting Tregs prior to transfer abrogated the TRP-1-specific Treg response, suggesting a predominant role for natural Tregs during melanoma tumorigenesis. Furthermore, we observed an increase in chemokine (C-C motif) ligand 17 (CCL17) prior to Treg accumulation in tumor-induced skin. CCL17 was directly regulated by BRAF and appeared to play a role in the migration of Tregs in vitro. This suggests a potential role for CCL17 in the recruitment of Tregs to sites of BRAF-driven tumorigenesis. The present study provides a critical insight into the kinetics and dynamics of the immune- suppressive response during melanoma tumorigenesis, and underscores targeting nTreg trafficking as a means of vitiating immune suppression in nascent melanoma tumors.

Funding: NCI 2R01 CA12077-06; 5T32AI007363-23; ACS RSG LIB-121864

36.

Dynamics of Tregs during Infection with T. gondii

Elizabeth Wohlfert University at Buffalo

Regulatory T cells (Tregs) are central to maintaining immunologic homeostasis and balancing immune responses to invading pathogens and preventing autoimmune responses. Underscoring the importance of their functional presence, the depletion of Tregs in adult hosts leads to lethal autoimmunity, thus demonstrating that a constant active suppression by these cells is required. The protozoan parasite Toxoplasma gondii has a complex lifecycle that inhabits multiple host tissues in and is a natural infection in mice. During acute infection with T. gondii there is a dramatic collapse of Tregs that contributes to severe immunopathology. Furthermore, T. gondii manipulates the immune regulatory compartment by se- verely disrupting mechanisms for the generation of new Tregs as evidence by thymic atrophy and the shut- down of peripherally generated Tregs to oral antigens. We are interested in understanding how this critical regulatory population is restored after this severe insult from infection. Tregs can be derived from multiple sources including thymic generation and peripheral conversion from naïve T cells. In our preliminary studies, we find that both the peripheral Treg and the thymic Treg populations collapse during peak infection. While we find peripheral Tregs are resilient and recover by three weeks post-infection, Tregs in the thymus are impaired significantly in their recovery of total numbers. This is not due to a total collapse of thymic function as the mature CD4+ and CD8+ αβT cells have begun to return to pre-infection levels. Further evidence suggesting an altered thymic environment is the observation that IFN-gamma and TNF-alpha are elevated in thymocytes three weeks post-infection compared with pre-infection levels. This lends support to the idea that the pro-inflammatory microenvironment induced by infection has not fully subsided. Nota- bly, previous studies have implicated RTE as the preferred precursor to peripheral Tregs. We find that an impairment of in vitro conversion in naïve T cells isolated from day 30 T. gondii infected mice when exposed to TGF-β. Current studies are exploring the RTE population and its ability to rebound from thymic atrophy and contribute to the newly generated Treg pool in the periphery. Future studies are needed to determine if the impairment of de novo Tregs from naïve T cells in infected mice is due to a reduction of RTE numbers or cell intrinsic alterations in the ability of the naïve T cell pool to develop into Tregs. We hypothesize the sustained drop in Tregs in the periphery has a long-term impact on host immune response through changes in thymic microenvironment and capabilities of the Treg population. Future studies will explore both the function of Tregs post-infection in addition to their diversity.

37.

Symposium II T-cell Biology

Chair : Dr. Kristin Hogquist

38. Visualizing and Manipulating T Cell Responses in vivo

Walburga Croteau, Yunfeng Feng, Jennifer Vella, Alex Bishop and Yina H. Huang Departments of Pathology and Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH

Immune-mediated control of tumor growth requires the generation and maintenance of productive CD8 T cell responses. Using in vivo imaging to guide our studies, we are investigating methods to overcome challenges that restrict tumoricidal T cell activity in the context of adoptive and CAR T cell immunotherapy. We found that altering T cell activation strength and cytokine stimulation can increase T cell infiltration into solid tumors by differentially regulating the expression of chemokine receptors and adhesion molecules. Additionally, while adoptively transferred CD8 effector T cells continue to expand within lymphoid organs in an antigen-dependent manner, they quickly disappear from skin-resident tumors and lymphoid organs within 2 weeks of transfer. In contrast, surgical resection of tumors prior to T cell contraction leads to the establishment of long-lived skin resident T cells that may provide enhanced protection against tumor rechallenge.

39. T-cell Activation Improves microRNA Function

Scott H. Olejniczak1,2, Gaspare La Rocca1, Megan Radler1 and Craig B. Thompson1 1Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065 2Current address: Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263

Activation-induced growth and proliferation of T cells requires coordinated activation of many cellular processes including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation and set thresholds for expression of target proteins. Emerging data suggest that, despite decreased expression of Argonaute proteins, microRNA function is enhanced by T cell activation due in part to induction of the RNA-induced silencing complex scaffold protein GW182. Increased expression of GW182 in activated T cells results from effects of PI3K-Akt-mTOR and Jak-Stat-Pim signaling on the translation of GW182 mRNA. Both signaling pathways enhanced eIF4E binding to the 5’-7mG cap of GW182 mRNA, while Jak-Stat-Pim signaling also promoted eIF4A-dependent unwinding of G-quadraplexes in its 5’ untranslated region. Consistent with this, GW182 expression and microRNA function were reduced by inhibition of mTOR or Pim kinases, translation initiation complex assembly, or eIF4A function. Taken together, these data provide a mechanistic link between microRNA function and cap-dependent translation, which allows activated T cells to maintain microRNA-mediated repression of targets despite enhanced rates of protein synthesis.

40.

Poster Talks Session A Adaptive Immunity

Chairs: Dr. Mantis and Dr. Wohlfert

41. #4 Activation of the Aryl hydrocarbon Receptor by Categorically Different Ligands Alters the Immune Response to Primary Influenza A Virus Infection

Lisbeth A. Boule1,2, Guang-Bi Jin1, and B. Paige Lawrence1,2 Departments of 1Environmental Medicine and 2Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY

The aryl hydrocarbon receptor (AHR) is an environment sensing transcription factor that offers a novel target for potential new therapeutic strategies to treat immune-mediated diseases. Yet, our ability to harness the AHR to improve health and treat disease is hampered by limited knowledge of how the diverse spectrum of AHR ligands affects immune responses in vivo. For instance, exposure to certain AHR ligands alters the progression of mouse models of autoimmune or infectious diseases; however, few studies have compared the same ligands within the same disease model. Moreover, it is unknown if the immunological consequences of exposure to particular ligands are solely AHR-mediated. In this study, we directly compared the effects of the following four AHR ligands on acute primary infection with influenza A virus (IAV) in wild-type and Ahr-/- mice: 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD), 3,3’,4,4’,5-pentachlorobiphenyl-126 (PCB126), 2-(1H-Indol-3-ylcarbonyl)-4- thiazolecarboxylic acid methyl ester (ITE), and 6-formylindolo(3,2-b)carbazole (FICZ). TCDD is the prototype and best characterized AHR ligand. TCDD and PCB126 are environmental contaminants with documented human exposure. ITE is being developed as a potential pharmaceutical, whereas FICZ is a UV photodegradation product of tryptophan and may represent a naturally occurring ligand. We show here that TCDD, PCB126 and ITE, but not FICZ, reduced virus-specific antibody levels and virus-specific CD8+ T cell responses to IAV. CD4+ T cell responses to infection were altered by all four ligands; however, the direction in which the change occurred was ligand and CD4-subset specific. Compared to control treated IAV infected mice, TCDD, ITE, and PCB126 significantly reduced the number Th1 cells, whereas FICZ increased the frequency of this CD4+ T cell subset. Similar to Th1 cells, FICZ treatment during IAV infection increased the proportion of Tfh cells, while TCDD, ITE, and PCB126 reduced the number of Tfh cells generated. Th17 cells were not changed after AHR activation by any of the 4 ligands used. Infected mice treated with TCDD, PCB126, and ITE all had a greater proportion Tregs (CD25+Foxp3+CD4+ cells) compared to controls, yet FICZ did not alter the frequency of Tregs. We examined the specificity of each ligand for the AHR by using Ahr-/- mice, and determined that the majority of the changes in the adaptive immune response to IAV infection after ligand exposure require expression of the AHR. This study demonstrates that investigating AHR ligands as therapeutics will require finding a balance between the suppression of immune-mediated pathologies, while maintaining intact and sufficient host defense mechanisms.

42. #13 IgM Memory B-cells Differentiate via Multiple Pathways following Challenge Infection

Kevin Kenderes, Amber Papillion, Lisa Dishaw, and Gary Winslow SUNY Upstate Medical University

IgM memory B cells are now recognized as an important component of immunological memory, yet they are relatively understudied. It has been demonstrated IgM memory cells act as a reservoir of broadly-reactive B cells that differentiate, in germinal centers, into high affinity class-switched effector B cells following antigen encounter. However, we propose that germinal center cell differentiation and class switching are only two of a number of fates of IgM memory cells. Our experimental model uses Ehrlichia muris, an intracellular tick-borne bacterium that generates a robust IgM memory B cell population. In our model, previous studies of IgM memory B cell responses to secondary infection had not been possible, likely due to the existence of pre-existing antibodies in previously infected mice. To avoid this problem, we monitored spleen IgM memory cells following their transfer to naïve recipient mice. This approach also transferred bacteria, which initiated a primary infection in the recipient mouse, allowing us to observe the secondary response of the donor IgM memory cells. The donor B cells differentiated into IgM-producing plasmablasts early following infection, which resulted in a 4-fold increase in IgM production, relative to control mice. Other donor B cells entered germinal centers, induced AID, and underwent class switching and generated switched memory B cells. Finally, some donor B cells were apparently maintained as IgM memory cells. These data demonstrate that IgM memory cells may have the capability to maintain IgM memory, but can also differentiate into both IgM-producing plasmablasts, switched germinal center cells, and switched memory cells. We propose that this process is inhibited in part by pre-existing antibodies, which act to prevent re-infection, but may also regulate IgM memory cell differentiation, via inhibitory Fc receptors. Our findings also suggest that IgM memory cells serve as self-renewing memory stem cells capable of renewing the entire spectrum of effector and memory B cells during secondary challenge infections.

43. #9 miR-155 Plays a Critical Role in the Establishment and Maintenance of Latency in Murine Gammaherpesvirus Infection

Rebecca Crepeau, Peisheng Zhang and Edward Usherwood Geisel School of Medicine at Dartmouth

Gammaherpesvirus infection in immunosuppressed populations is associated with severe disease. MicroRNA-155 (miR-155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). We sought to determine the contribution of miR-155 in the establishment and maintenance of latency in murine gammaherpesvirus infection. Mice deficient in miR-155 had a 100-fold decrease in latent viral load, measured by qPCR, and exhibited decreases in both T follicular helper cells and germinal center B cells. Mixed bone marrow chimeric mice, however, showed a similar latent viral burden in both WT and miR-155 deficient B cells. This phenotype could be abolished with anti-viral treatment, indicating that the replication cycle during spontaneous reactivation is necessary to maintain the latent pool. When tested, miR-155 deficient animals were found to have significantly fewer reactivation competent cells, indicating that miR-155 may also play a role in the maintenance of latency. Further elucidating the relative role of miR-155 in this system will provide key insights into the development of latency in this model system.

44. #33 Fetal Hematopoietic Stem Cells Give Rise to a Unique Population of CD8+ T-cells

Jocelyn Wang1, Erin Wissink2, Norah L. Smith3, Andrew Grimson2, and Brian D. Rudd3 Field of Pharmacology1 Department of Microbiology and Immunology2, Biological and Biomedical Sciences Graduate Program. Department of Molecular Biology and Genetics3, Cornell University, Ithaca, NY 14853

Neonatal infection is a major cause of morbidity and mortality worldwide. While adults generate robust immunity to most intracellular pathogens, neonates have impaired ability to generate long-lasting immunity. We recently showed that neonatal CD8+ T cells fail to become memory cells because of an inherent propensity to rapidly proliferate and become terminally differentiated. This work has clearly demonstrated that neonatal CD8+ T cell are intrinsically different than their adult counterparts, but the underlying basis for these age- related differences has remained an open question. From a reductionist perspective, it is apparent that a number of basic models might explain why neonatal CD8+ T cell adopt different fates compared to adults during infection. First, neonatal CD8+ T cells may be less likely to develop into memory CD8+ T cells because the starting population has undergone more extensive homeostatic proliferation and may be significantly more differentiated than adults prior to infection (‘proliferation model’). Another possibility relates to the fact that neonatal and adult CD8+ T cells are derived from distinct hematopoietic stem cell populations that are metabolically and genetically very different (‘origin model’). To discriminate between these models, we directly compared the genetic profiles and behavior of neonatal and adult CD8+ T cells that had undergone similar amounts of homeostatic proliferation in the periphery or were at the same stage of development in the thymus. Interest- ingly, different aged CD8+ T cells that had undergone similar amounts of HP still expressed different gene profiles, which suggest that HP cannot fully explain the differences between adult and neonatal CD8+ T cells. Additionally, equivalent phenotypic subsets of peripheral CD8+ T cells expressed different phenotypic markers, responded differently to pro-inflammatory cytokines in vitro as well as infection in vivo. Next, we asked whether neonatal CD8+ T cells behave differently because of their different origin. To answer this question, we compared the gene expression profiles of single positive CD8+ T cells in thymus form neonatal and adult mice and found that they possessed distinct gene expression profiles. Given that neonatal CD8+ T cells exhibit a unique gene expression profile at the time they are initially created, we believe this data lends support for the origin model and suggests that neonatal CD8+ T cells respond differently to infection because they are sculpted from a different progenitor cell. In ongoing studies, we are co-transferring fetal and adult progenitor cells into adult thymii to confirm that neonatal CD8+ T cells do in fact represent a distinct lineage of CD8+ T cells. This data will also be presented and discussed.

45. #41 CD28 Regulates Metabolic Fitness for ROS Dependent Long Lived Plasma Cell Survival

Adam Utley, Daniela Ventro, Louise Carlson, and Kelvin Lee Roswell Park Cancer Institute

Sustained humoral immunity is dependent upon the production of neutralizing antigen-specific antibodies by plasma cells. Upon antigenic exposure, activated B cells differentiate into short-lived plasma cells (SLPCs) which home to secondary lymphoid organs such as the spleen where they live for days to weeks before dying by apoptosis. However, there are examples in the human population in which neutralizing antibody titers persist despite a lack of continual antigen exposure. Therefore an expanded model has been proposed wherein activated B cells differentiate into long-lived plasma cells (LLPCs) that home to the bone marrow and survive indefinitely. They are not intrinsically long-lived, but rather depend upon extrinsic survival signals within specialized niches for their persistence and long-term antibody production. Furthermore, many of these survival signals are shared with the malignant counterpart in Multiple Myeloma (MM). Our work centers on the cellular and molecular mechanisms by which MM and LLPCs survive in the bone marrow microenvironment. CD28 is best characterized as the canonical co-stimulatory molecule in T cells and is required for T cell metabolic fitness. Plasma cells and MM are highly biosynthetic and require metabolic fidelity. However, the molecular and metabolic pathways that provide this CD28-mediated survival advantage in LLPCs/MM are not well understood. Here we demonstrate that CD28 induces mitochondrial respiration in both LLPCs and MM, but not in SLPCs. Furthermore, CD28 induces mitochondrial biogenesis in LLPC and MM. By using oxygen consumption as a direct readout of mitochondrial respiration, we show that CD28 activation increases oxidative phosphorylation. A major byproduct of respiration is the production of reactive oxygen species. As expected, CD28 activation induces ROS in LLPCs specifically. ROS is a well-characterized cell-damaging agent. In order to elucidate the effects of ROS on LLPCs, we specifically inhibited ROS produced by the mitochondria with MnTBAP. Paradoxically, inhibiting ROS prevents CD28-mediated survival. Taken together the data suggest that CD28 increases mitochondrial respiration for ROS-dependent survival. By analyzing open-source RNAseq data acquired from purified populations of bone marrow LLPCs and splenic SLPCs, the LLPC population exhibits higher transcript levels of genes associated with mitochondrial respiration, oxidative phosphorylation, and the TCA cycle. However, the signals that drive this metabolic program are not characterized. Taken together the data suggests that CD28 may regulate LLPC survival and function through Slp- 76 mediated metabolic reprogramming. This makes CD28 an attractive target in vaccine design and the treatment of both autoimmune disorders and multiple myeloma.

46.

Poster Talks Session B Myeloid Cell Function

Chairs: Dr. Hankey and Dr. Lawrence

47. #3 Persistent Changes in Dendritic Cell Function following Developmental Activation of the Aryl Hy-

drocarbon Receptor

Jessica L. Meyers1, Bethany Winans1, Lisbeth Boule11,2, and B. Paige Lawrence1,2 Department of Environmental Medicine, Environmental Health Sciences Center1 and Department of Mi- crobiology and Immunology2 University of Rochester, Rochester, NY USA

The developing immune system is susceptible to environmental insults, which can lead to altered immune function later in life. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays a role in immune system development and function. The AHR binds many exogenous chemi- cals to which humans are regularly exposed, including dioxins and polychlorinated biphenyls (PCBs). Hu- man and animal data demonstrate that early life exposure to dioxins and PCBs correlates with persistent alterations in immune function. For example, developmental exposure to the prototype AHR ligand, 2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD), deregulates T cell responses to challenges such as influenza A virus (IAV) infection later in life. During acute primary infection, naïve T cells are activated by dendritic cells (DCs); however, the impact of developmental exposure on DCs is poorly understood. We therefore investigated whether developmental activation of AHR alters DC function or distribution later in life. Develop- mental exposure reduced DC function, as evidenced by a two-fold poorer capacity to stimulate the proliferation and differentiation of naïve virus-specific T cells. We next determined whether developmental exposure to TCDD changed the frequency or distribution of DC subsets in the lung and mediastinal lymph nodes (MLN). Developmental exposure did not alter the number of conventional or plasmacytoid DCs in the lung prior to or after infection. However, there were two-fold fewer DCs in the MLN after infection. A key regulator of DC trafficking from the infected lung to the MLN is chemokine receptor 7 (CCR7). The number of CCR7+ DCs and level of CCR7 expression on distinct DC subsets in the lungs were equivalent in adult offspring of TCDD and control dams. In contrast, in the MLN there were fewer CCR7+ DCs and the overall level of CCR7 expression on DCs was reduced in infected offspring of TCDD exposed dams, compared to offspring from control dams. Thus, developmental exposure to an AHR ligand diminishes the number of DCs in the MLN following IAV infection, reduces CCR7, and impairs their ability to activate naïve T lymphocytes. Given that DCs regulate T cell responses to many antigens, the discovery that maternal exposure to an AHR ligand perturbs DC function has broad implications for understanding how the early life environment shapes the integrated function of the immune system.

Funding: R01-ES017250, R01-ES023260, T32-ES07026, T32-HL066988, P30-ES01247

48. #8 Regulation of Transcription Factor IRF8 in Myeloid Progenitors is a Critical Checkpoint for For-

mation of Defective Myeloid Cells in Cancer

Michelle N. Messmer, Colleen Netherby, Scott I. Abrams Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Defective myeloid cells are commonly observed across a large range of solid cancers. These defects result in immune suppression and promote tumor progression to metastatic disease. The molecular processes underlying development of these pathological myeloid-derived suppressor cells remains unclear. Previous studies focused on characterizing these myeloid populations in peripheral tissues and the tumor microenvironment, despite the fact that the major site for normal myeloid production is the bone marrow. Since the bone marrow is sensitive to signals from the periphery, we hypothesized that tumor induced perturbations in myelopoiesis occur in the bone marrow and are the likely origin of suppressive myeloid cells. During myelopoiesis, transcription factor IRF8 within granulocyte/monocyte progenitors (GMP) determines differentiation such that high IRF8 expression favors monocytes while low IRF8 expression favors granulocytes. Our recent work using orthotopic mammary tumor models revealed that tumor-derived factors directly suppress IRF8 expression leading to accumulation of suppressive, immature myeloid cells. While these data support IRF8 acting as a negative regulator of defective myeloid accumulation, exactly when and where IRF8 is compromised during myelopoiesis remained unclear. To further elucidate how tumor growth impairs IRF8 expression, we utilized a novel mouse model expressing an IRF8-EGFP fusion protein. This allows us to investigate changes in IRF8 during both orthotopic and spontaneous mammary tumor progression. Our results show that: 1) GMP expand with increasing tumor size and increasing amounts of G-CSF in the serum of tumor-bearing hosts, and 2) GMP can be divided into IRF8hi and IRF8lo expressing populations similar to IRF8lo granulocytic and IRF8hi monocytic myeloid cells observed in the blood. Expression of IRF8 in other progenitor populations was not affected, suggesting IRF8lo GMP may be the source of expanded myeloid cells in cancer. Altogether, these data identify modulation of IRF8 in myeloid cells and their progenitors as an early consequence of tumor development and a potential target for therapeutic intervention.

Supported by: NIH RO1 CA140622

49. #29 Glycogen Metabolism Supports Effector Function and Energy Homeostasis of Dendritic Cells

Phyu Thwe, Saritha Beauchamp, and Eyal Amiel University of Vermont, Burlington, VT 05405

Dendritic cells (DCs), professional antigen presenting cells of the immune system, serve as a bridge between the innate and adaptive immune responses. Activation of DCs by a stimulus through toll-like receptors (TLRs) is coupled with an increase in energy demand, which is fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and the energy associated with DC effector function. Inhibition of glycolysis impairs the survival and effector function of activated DCs. In the prevailing model in the field of immuno-metabolism, TLR-driven glycolysis in DCs is thought to be sustained primarily by an increase in glucose uptake. While non-immune cells, such as hepatocytes and muscle cells, store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen metabolism in DCs has not been published. Our data indicate that glycogen metabolism regulates and supports the effector function of DCs. We show that DCs express the enzymes essential for glycogen metabolism and that glycogen metabolism is regulated upon TLR stimulation. We also show that the inhibition of glycogen metabolism impairs activation of these cells. These data indicate that glycogen metabolism in DCs plays an important role in energy homeostasis of these cells to support the effector function. Understanding the role and regulatory mechanisms of glycogen metabolism in DCs has important implications for the development of enhanced cellular based immunotherapy in the future.

50. #37 Inflammatory Monocytes in a Changing Tumor Microenvironment Following Radiotherapy: Implications for Immunotherapy

Kelli A. Connolly1,2, Aditi Murthy1, Brian Belt2, Edith M. Lord1, David C. Linehan2, and Scott A. Gerber1,2 Departments of Microbiology and Immunology1, and Surgery2, University of Rochester, Rochester, NY

Colorectal cancer (CRC) is responsible for approximately 500,000 deaths each year worldwide. Rapid progression of the primary tumor often leads to escape and distal metastases. Accordingly, radiotherapy (RT) is often required as a means to control the growth of the primary tumor and to reduce local recurrence. Unfortunately, therapies such as RT are often ineffective on patients with advanced stage disease creating a critical need for more effective treatments to improve tumor control and survival rates for CRC. We have established a mouse model of CRC in which Colon38, a colon adenocarcinoma cell line, is injected into syngeneic C57BL/6 mice and treated with local RT. Our data have demonstrated that the immune system mediates many of the anti-tumor effects of RT. However, the immunostimulation provided by RT appears to be a double-edged sword as we observe a concurrent increase in myeloid cells that have been associated with immunosuppression and poor prognosis. One population of myeloid cells, inflammatory monocytes (CD45+, CD11b+, Ly6chi, Ly6g-), has been shown to mediate immunosuppression in multiple tumor models. The chemokine CCL2 and its cognate receptor CCR2 on these monocytes facilitate the infiltration of these cells into tumors. Consequently, therapy that inhibits entry of these cells into tumors has resulted in remarkable clinical outcomes. However, the role of inflammatory monocytes in the context of RT and CRC is unknown. Here we have shown that RT upregulates the expression of intratumoral CCL2, and subsequently induces the infiltration of CCR2+ monocytes. Using a genetic approach, the absence of CCR2+ monocytes resulted in reduced tumor growth in CCR2KO mice when compared to WT. However, pharmacologic inhibition of CCR2 administered immediately following RT unexpectedly diminished the effectiveness of therapy resulting in increased tumor growth when compared to RT-treated control mice. Based on these data, we hypothesize that the role of inflammatory monocytes in disease progression is dependent on the inflammatory state of the tumor microenvironment to which they migrate. In support of our hypothesis, RNA-seq data from ex vivo sorted inflammatory monocytes demonstrated drastically changed genetic profiles of these phenotypically identical cells at different time points after radiotherapy. Therefore, a better understanding of the biology behind the plasticity of these inflammatory monocytes throughout treatment is important to inform future clinical trials involving monocyte-targeted immunotherapies. Future studies, using a small molecule inhibitor of CCR2, will explore the efficacy of this immunotherapy and identify an optimal schedule of administration. Further, we plan to elucidate the role of the microenvironment in promoting the potential anti-tumor function of these cells by administering agents that induce a proinflammatory tumor microenvironment, such as IL-12.

51. #42 Macrophage Lipid Accumulation is Regulated by Substrate Stiffness

Erika Gruber, Sandra Stelzer, Emma Ehrlich, Siddhartha Sinha, Cynthia Leifer Department of Microbiology & Immunology, Cornell University, Ithaca NY

Atherosclerosis is a chronic inflammatory process characterized by the accumulation of lipid-laden macrophage foam cells and other inflammatory cells within the arterial vessel wall, formation of an athero- sclerotic plaque, and tissue remodeling that results in increased arterial wall stiffness. The accumulation of lipid within macrophages is central to the progression of atherosclerosis, yet the mechanisms governing foam cell formation are not well understood. Cells probe the physical properties of their surroundings and generate chemical signals through a process known as mechanotransduction. Mechanotransduction regulates differentiation and function of numerous cell types, including mesenchymal stem cells and endotheli- al cells, yet until recently, its role in immune cell function has been largely unexplored. Our lab and others have found that substrate stiffness regulates murine macrophage cytokine secretion and phagocytic activity. Because increased arterial wall stiffness precedes formation of the lipid-rich plaque in atherosclerosis, we hypothesized that increased substrate stiffness would enhance lipid accumulation in macrophages. To investigate this, we cultured murine macrophages on functionalized tunable polyacrylamide gels that model physiologically relevant stiffnesses. We then used a combination of flow cytometry and immunofluores- cence microscopy to show that substrate stiffness regulates accumulation of the model lipid, oleic acid, as well as acetylated low density lipoprotein (LDL) in primary bone marrow-derived macrophages and RAW264.7 macrophages. Using RNAseq transcriptomic analysis, we identified sets of genes that are up- or down-regulated in RAW264.7 macrophages in response to increased substrate stiffness, including multiple genes implicated in atherosclerosis and lipid metabolism. Together, these data demonstrate that lipid accumulation by macrophages is regulated in part by substrate stiffness and mechanotransduction signaling. To our knowledge, this is the first study to investigate the potential link between two key features of atherosclerosis: increased substrate stiffness and macrophage lipid accumulation.

52. Ashley Cornett, Ph.D. Technical Application Scientist, BioLegend

Enhancing Downstream Applications Through Magnetic Cell Separation

Cell sorting is a powerful application that enables researchers to separate cell subsets based on one or more distinct surface characteristics. Magnetic cell separation allows cells to be sorted using magnetic nanoparticles bound by antibodies specific for a particular surface antigen. BioLegend has developed a new magnetic cell separation system, MojoSort™, for the isolation and purification of cells from heteroge- neous populations. The MojoSort™ offers two cell separation protocols one for positive selection and the alternative for depletion. Positive selection implicates separation relies on the antibodies directly targeting the cells of interest while negative selection or depletion target unwanted cells. In either case, both sepa- rated fractions of cells can be used for downstream applications. Our versatile MojoSort™ nanoparticles deliver excellent purity and yield at an unmatched, affordable price. Further advantages include small sample volume, customizable kits with BioLegend unique content and it is compatible with multiple separation systems. The MojoSort™ Cell Separation system offers useful tools for biomedical research and drug discovery.

53.

Dr. Belkaid Workshop

54.

Poster Talks Session C Inflammation

Chairs: Dr. MacNamara and Dr. Yarovinsky

55. #1 A Unique Signalisome Induced By Human Cytomegalovirus During Entry Targets The Translation Of Caspase 3 Regulatory Proteins Ensuring The Survival Of Infected Monocytes.

Megan Peppenelli (1), Kyle Arend (2), Olesea Cojohari (1), Nathaniel Moorman (2) and Gary Chan (1). (1): SUNY Upstate Medical University, Syracuse, New York, United States (2): University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States

HCMV infection of immunocompromised individuals often leads to multi-system organ failure. The development of multi-system organ failure is dependent on the ability of HCMV to spread to peripheral organs, which is mediated by blood monocytes. In order for monocytes to mediate spread, we have previously shown HCMV to extend the short 48hr lifespan of monocytes. Mechanistically, HCMV upregulated cellular myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of active caspase 3. We now show that, compared to other myeloid survival factors, HCMV infection more efficiently upregulate Mcl-1 and the only survival factor to rapidly induce HSP27. These observations, suggest a unique virus specific mechanism of induction; thus, we examined receptor: ligand signaling events, since viral anti-apoptotic proteins are not expressed until differentiation into macrophages is complete. We determined that HCMV-induced EGFR/PI3K/AKT signaling increased the transcription of both Mcl-1 and HSP27. Yet, protein expression of Mcl-1 and HSP27 is increased via the gH/V3/src and the gB/EGFR pathways, respectively. To address this dis- crepancy between transcripts and protein we evaluated the effects of HCMV on the translation of Mcl-1 and HSP27. Indeed, we found, in contrast to myeloid survival factors, HCMV stimulated the translation of Mcl-1 and HSP27. Overall, these data indicate that inhibition of apoptosis by HCMV is through the unique signaling regulation of Mcl-1 and HSP27; ensuring short-lived monocyte survival past the normal 48hr viability checkpoint, a key event necessary for viral dissemination.

Contact: Gary Chan, [email protected]

56.

#25 Type I IFNs Directly Promote Hematopoietic Progenitor Cell Death and Lead to Bone Marrow Failure in Severe Bacterial Infection

Julianne N.P. Smith, Amanda McCabe, Katherine C. MacNamara Albany Medical College, Albany NY

Life-long blood cell production and immune function requires an appropriate balance between hematopoietic stem and progenitor cell (HSC/HSPC) fate choices. Although HSCs exist as rare, quiescent cells within the bone marrow (BM) at steady-state, they retain the potential to rapidly proliferate and differentiate in response to hematopoietic demand, such as occurs in acute infection. HSPC proliferation increases immune output but can impair stem cell function. Type I interferons (IFNα/β) modulate immune responses to most pathogens and can regulate HSPC proliferation and death in vitro and upon non- infectious immune-stimulation in vivo, however the impact of type I IFNs on HSPC response to infection remains unclear. To determine how IFNα/β impacts HSPC regulation during infection, we utilized a shock -like infection model in mice challenged with the tick-borne Ixodes ovatus ehrlichia (IOE) bacteria. We recently reported that disease severity was dependent upon the robust production of IFNα/β. At the peak of IFNα/β induction, myeloid progenitors and HSCs were depleted in wild-type (WT) BM, but were significantly protected and even expanded in mice lacking the IFNα/β receptor (IFNaR1). IFNα/β also limited infection-induced extramedullary hematopoiesis, as measured by overall splenic cellularity and myeloid progenitors. To determine whether IFNα/β depletes HSPCs via a reduction in cellular proliferation and/or an increase in cell death, we examined the cell cycle status and viability of WT and Ifnar1-/- BM cells during IOE infection. HSPC cycling was increased by infection, though significantly more cells cycled in the absence of IFNaR1. HSPCs in Ifnar1-/- mice were also less prone to IOE-induced death. Alt- hough IFNα/β did not directly deplete quiescent HSCs, nor did it impair cell cycle entry, IFNα/β directly promoted necrotic death of progenitor cells. In contrast, progenitors that lacked IFNaR1 were largely protected and preferentially underwent apoptosis during IOE infection. To determine the long-term impact of infection on HSPC function, we competitively transplanted sorted HSPCs from infected WT and Ifnar1-/- mice and measured hematopoietic repopulating activity in myeloablated recipients. WT HSPCs displayed a progressive, multilineage repopulating advantage over the Ifnar1-/- HSPC pool, which was initially superior but became exhausted throughout transplantation, consistent with more abundant hematopoietic progenitors that lack long-term activity. These data reveal an important dichotomy in the impact of IFNα/β during severe bacterial infection. We have shown that IFNα/β directly sensitizes hematopoietic progenitors to necrotic cell death, but does not directly target long-term HSCs, during IOE infection. Indeed, our data demonstrate that IFNα/β plays a critical role in reestablishing hematopoietic homeostasis in settings where the infectious challenge is resolved.

57. #26 The Role of the Aryl Hydrocarbon Receptor and the Gut Microbiome in the Modulation of

Intestinal Inflammation

Kerry R. Belton1, Limin Zhang1, Christopher Chiaro1, Gary H. Perdew1, Andrew D. Patterson1* 1Center for Molecular Toxicology and Carcinogenesis, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, Pennsylvania, 16802

Excessive dysregulation of the commensal gut microbiota is known to cause an excessive immune response leading to chronic intestinal inflammation ultimately causing generation of lesions in the mucosa (e.g., inflammatory bowel disease). Recent data has shown that the aryl hydrocarbon receptor (AHR) could be an untapped target for modulating the commensal gut microbiota as well as intestinal inflammation. To dissect this issue, male C57BL/6J mice were fed control (devoid of known AHR ligands), indole-3- carbinol (I3C), and beta-naphthoflavone (BNF) containing diets for three weeks. Mice were placed in metabowls for fecal sample collection and later euthanized to harvest tissues and biofluids. 16S rRNA gene QPCR of cecal matter revealed changes in total bacterial composition, in I3C treated mice. Liquid chromatography coupled with mass spectrometry-based metabolomics of fecal matter revealed significant excretion of bile acids. 1H nuclear magnetic resonance (NMR)-based metabolomics analysis of feces revealed profound changes in bile acids as well as glucose metabolism and bacterial fermentation products. AHR ligand containing diets caused profound changes in the expression of immunologic genes (e.g., Il-6, Il-10). Exposure to these diets displayed protective effects in intestinal inflammation (e.g., intestinal injury models) and weight loss. Overall, our data suggests that AHR ligands in the diet are capable of modulating the commensal gut microbiota as well as their metabolic function. Furthermore, our data suggests that the effects are ligand dependent. Most interestingly, the metabolomics experiments reveal changes in the commensal gut microbiota energy metabolism which could be due to a decrease in the gut microbe populations. Finally, work of this type could lead to the development of safe, low-cost, and natural dietary interventions to treat chronic gut inflammation.

58. #11 Loss of Intestinal Epithelial Autophagy leads to Catastrophic Susceptibility to Acute T. gondii-mediated Inflammation

Elise Burger, Américo López-Yglesias, Felix Yarovinsky Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY

The protozoan parasite Toxoplasma gondii triggers severe small intestinal immunopathology characterized by intestinal inflammation, Paneth cell loss and transformation of the microbiota composition. Paneth cells are the predominant intestinal secretory epithelial cells which reside at the base of the crypt and release antimicrobial peptides to regulate the intestinal microbiota. T. gondii-triggered IFN-gamma is a major inducer of Paneth cell death. However, mechanisms of T. gondii-induced IFN-gamma-dependent Paneth cell death are currently unknown. We recently observed that under steady state conditions Paneth cells undergo active autophagy, which is both microbiota- and IFN-gamma-dependent. These findings directed us to investigate a role for autophagy in T. gondii –mediated Paneth cell loss. Mice lacking the critical autophagy gene Atg5 in the intestinal epithelium (E-ATG5KO) exhibited a catastrophic increase in susceptibility to intestinal inflammation, which was characterized by complete destruction of the intestinal crypts. To further investigate these findings we generated intestinal crypt organoids to study the mechanism underlying increased sensitivity to the cytokine mediated Paneth cell death in the absence of functional autophagy. Autophagy deficient organoids exhibited significantly increased sensitivity to the TNF-induced cell death. Organoid death was further exacerbated when TNF stimulation occurred in combination with IFN-gamma, indicating syn- ergistic signaling from this inflammatory cytokine combination. Armed with this information, we returned to our in vivo model, which revealed higher expression levels of TNF receptor 2 (TNFR2) in E-ATG5KO mice than wild-type controls. This increase was further magnified during T. gondii infection in ATG5KO intestines. This indicates that E-ATG5KO mice are incapable of suppressing TNFR2 levels both at steady state and more critically during infection, leading to exaggerated sensitivity to cytokine-mediated immunopathology in the small intestine. Our results reveal that Paneth cell autophagy plays a highly protective role in the regulation of intestinal inflammation during acute T. gondii infection.

59. #7 Myeloid-Derived Suppressor Cells in Murine AIDS Suppress B-Cell Responses in a

Reactive-Oxygen- and Nitrogen-Species Dependent Manner

Jessica L. Rastad1 and William R. Green1,2. 1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH. 2Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are implicated in a variety of murine and human pathologies, including cancer and viral, bacterial, and parasitic infections. MDSCs are broadly classified into two subsets based on extensive studies of suppression of T-cells in tumor models. Granulocytic MDSCs (G-MDSCs) are polymorphonuclear and tend to utilize reactive oxygen species (ROS) and arginase-1 for their immunosuppressive function. Monocytic MDSCs (M-MDSCs), are mononuclear and primarily utilize inducible nitric-oxide synthase (iNOS) to suppress. LP-BM5 is a murine retroviral isolate that causes murine AIDS (MAIDS), which has many similarities to HIV/AIDS, including the development of profound immunodeficiency. Our lab identified that M- MDSCs increase during MAIDS pathogenesis. Increased MDSCs have subsequently been found in HIV- infected patients. Interestingly, M-MDSCs from LP-BM5-infected mice suppress not only T-cell responses, but also B-cell responses, indicating that they may be a potential therapeutic target. Suppression of T- cells is almost entirely iNOS-dependent, with suppression of B-cells showing only partial iNOS- dependence. Additionally, suppression of B-cells by M-MDSCs from LP-BM5 infected mice is independent of arginase-1, PD-1/PD-L1 interactions, IL-10 production, and indoleamine 2,3-dioxygenase activity. Our lab is the first, to our knowledge, to publish evidence of MDSC-mediated suppression of B-cells in a viral system. MDSC-mediated suppression of B-cells has since also been recently shown in a collagen- induced arthritis model. In this study we utilized a transwell system and a conditioned media assay and determined that suppression of B-cells by M-MDSCs from LP-BM5 infected mice is mostly cell-contact independent and is dependent in part on soluble mediators. Detection of NO (nitric oxide, a product of the iNOS reaction) in the transwell system indicated that iNOS is functional in the absence of cell-contact. Addition of iNOS in- hibitors to the conditioned media assay showed that iNOS was responsible for only about half of the suppression attributed to soluble-mediators. Using antioxidants, the ROS superoxide and peroxynitrite were shown to be other soluble mediators significantly contributing to MDSC suppression of B-cells. In contrast, cysteine add-back experiments indicated no evidence for involvement of cysteine-depletion as a suppressive mechanism. This study demonstrates M-MDSC-mediated suppression of B-cells by ROS, a mechanism more commonly associated with G-MDSC-mediated suppression of T-cells. These results highlight the utilization of multiple mechanisms by MDSCs to broadly inhibit B-cell function, and provide potential mechanistic targets for inhibiting or enhancing B-cell suppression by MDSCs.

60.

Poster Talks Session D Tumor Cancer

Chairs: Dr. Lord and Dr. Olejniczak

61. #2 Expression of host CXCR3 controls early establishment of metastatic melanoma in the lung

Kiah L. Sanders, Eleanor Clancy-Thompson, Thomas Perekslis, David W. Mullins Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH

Metastatic melanoma remains a difficult to treat disease with a 5-year survival rate of ~16%. Due to the aggressive nature of the disease, understanding the establishment mechanisms of metastases is crucial to the development of preventative and intervening therapies. Previous work in models of melanoma and breast cancer has demonstrated that tumor expression of the chemokine receptor CXCR3 is required for metastatic engraftment in lung, and systemic inhibition of CXCR3 dramatically reduces lung metastasis. Recent work demonstrates that macrophages may play a role in tumor progression and metastasis. In the present study, we sought to understand the role of host immune cells in mediating metastasis; specifically, we hypothesized that CXCR3-expressing macrophages mediate the establishment of melanoma metastases in the lungs. To assess the role of host CXCR3 in metastasis, we intravenously injected B16 melanoma into CXCR3WT and CXCR3-/- mice and performed quantitative PCR (qPCR) for the melanoma-specific gene tyrosinase, 24 hours and 12 days later. At both time points, CXCR3WT mice express significantly higher levels of tyrosinase. At 12 days post-injection, there are significantly more visible lung metastases in CXCR3WT compared to CXCR3-/- hosts. Given that the tumor was CXCR3-sufficient in both groups of mice, we assessed whether CXCR3 expression was also required during early engraftment on the melanoma cells. Using a small molecule inhibitor (AMG487), we found blocking CXCR3 on melanoma cells significantly decreased tumor metastasis in the lung of CXCR3wt hosts, but blockade increased metastasis to the liver, suggesting organ specific requirements for tumor-expressed chemokine receptors. To explore whether early engraftment in lung is controlled by host immune cells, we depleted CD8+ T cells or monocytes in CXCR3WT and CXCR3-/- mice. While depletion of CD8+ T cells had no effect, depletion of macrophages and Ly6Chi monocytes in circulation (leaving interstitial macrophages intact) decreased melanoma engraftment in CXCR3WT mice. This suggested the requirement for a circulating population of CXCR3-expressing macrophages to enable engraftment in the lung. To assess differences in circulating monocyte populations following B16 i.v. injection, we examined differences in F4/80+CD11b+ macrophages and CD11b+Ly6C+ monocytes in the spleen and lung. While both populations of cells increased in the lungs of CXCR3WT mice, in CXCR3-/- mice there was no significant difference following tumor injection, suggesting CXCR3-/- monocytes are defective in trafficking to the lung. Our findings suggest host expression of CXCR3 on monocytes directly impacts the ability of circulating metastatic melanoma cells to engraft in the lungs.

62. #31 Tumor-derived TSLP Enhances Macrophage-mediated Metastasis

Burkard-Mandel, Lauren & Abrams, Scott Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263

Despite advances in our understanding of solid tumor biology, breast cancer remains the second most common cause of cancer deaths in women in the United States. Recent strategies to improve therapy of breast and other solid malignancies involve targeting the patient’s immune system to bolster anti-tumor immunity. However, these strategies have largely overlooked targeting of macrophages, a major immune cell type to infiltrate breast tumors. Ordinarily, macrophages play a critical role in host defense against disease, including neoplasia. Within a tumor, though, macrophages are ‘plastic’ and can transition between distinct functional states largely influenced by the inflammatory microenvironment. These macrophages can adopt an ‘M1’ phenotype, which is thought to be effective in anti-tumor immunity. However, recent work has shown that tumor-infiltrating macrophages often adopt an ‘M2’ phenotype which can support metastatic progression of tumors. While cytokines IL-4 and IL-13 are classic drivers of M2 macrophages, additional factors can contribute to the complexity and diversity of macrophage functionality. Recent work has shown that thymic stromal lymphopoietin (TSLP), has been found to directly impact macrophage function and intensify their M2-like activities in allergy models. Interestingly, the 4T1 mammary tumor cell line was previously reported to secrete high levels of TSLP in vitro and known to generate profound myeloproliferative responses in vivo. Therefore, we hypothesize that tumor-derived TSLP augments spontaneous metastasis by enhancing the pro-tumor activity of M2 macrophages. We demonstrate that: 1) 4T1 tumor cells produce high levels of TSLP in vivo, 2) knockdown of TSLP expression in 4T1 cells leads to significant decreases in tumor growth and metastasis, 3) TSLP increases the expression of the angiogenic factor VEGF-A, as well as the chemotactic cytokine CCL17 from macrophages in vitro which has been shown to facilitate metastasis via CCR4-dependent pathways, and 4) the isogenic variants of 4T1, 67NR and 168FARN, express low levels of TSLP and CCR4, consistent with their non-metastatic phenotype. Altogether, these data suggest tumor-derived TSLP plays previously unrecognized roles in tumor metastasis acting, in part, through macrophage-dependent mechanisms.

63. #32 IRF8 Transcriptionally Regulates the Macrophage Response during tumor Immunoserveillance

Danielle Twum & Scott I. Abrams Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263

The macrophage response to pathological insults, especially in neoplasia, is regulated by transcriptional players that are activated after exposure to soluble factors present in their microenvironment. Conse- quently, macrophages are able to differentiate into two distinct phenotypes, M1 and M2. M1 macrophages are defined as ‘immune stimulatory’ cells and are associated with iNOS and IL-12 production, while M2 macrophages can be defined as ‘suppressive’ cells associated with Arg1, a T cell suppressor, and IL-10, an anti-inflammatory cytokine. In cancer, notably breast carcinoma, a high infiltration of macrophages is associated with reduced overall survival as well as progression to metastasis. Recent evidence suggests that as tumor burden increases, the M1 to M2 ratio skews significantly towards an M2-bias, co-opting their anti -inflammatory and wound healing abilities to favor tumor growth. Although several transcriptional regula- tors that drive the M2 phenotype in the tumor microenvironment have been identified, transcription factors that drive or enforce the M1 phenotype are not well defined. IRF8 (Interferon Regulatory Factor 8) is a transcriptional regulator of myeloid differentiation and is critical for the expression of genes such as iNOS and IL-12p40, hallmark markers of the M1 phenotype. We hypothesize that IRF8 is important in determining the macrophage M1 vs M2 response in mammary cancer. To test this hypothesis, we made use of an IRF8 knockout mouse model, whereby an IRF8flox/flox strain was crossed with the LysM/Cre strain, to generate progeny that specifically lacked IRF8 expression in the macrophages. As a result, we observed an increased rate of tumor growth in IRF8-/- mice compared to the wild-type controls using an orthotopic im- plantable model of mouse mammary carcinoma. Moreover, crossing the IRF8-/- mice with MMTV-PyMT mouse model of autochthonous mammary tumor development, we observed that deletion of IRF8 in macrophages enhanced spontaneous pulmonary metastasis. Altogether, these data indicate that IRF8 expression in macrophages can delay tumor onset and/or reduce tumor progression to metastasis, likely through altering the tumor-suppressing (M1) to tumor-promoting (M2) phenotypes. Thus, IRF8 may represent a poten- tially novel therapeutic target to modulate the macrophage phenotype in neoplastic diseases, such as mammary cancer, whereby this myeloid response is a critical determinant of outcome.

64. #40 Indoleamine 2,3- Dioxygenase Regulates Density of Tumor Infiltrating CD8+ T cells in a Murine Model of Ovarian Cancer

Adaobi Amobi1,4, Feng Qian4, Junko Matsuzaki3,4 and Kunle Odunsi1,2,3,4 Departments of Immuology1, and Gynecologic Oncology2, Immune Analysis Facility3 and Center for Immunotherapy4, Roswell Park Cancer Institute, Buffalo, NY, USA

Amino-acid withdrawal is an important, molecular mechanism regulating anti-tumor immune responses. The catabolism of the essential amino acid tryptophan (TRP) by indoleamine 2,3-dioxygenase (IDO1) is a central pathway that contributes to the immunosuppressive microenvironment in many types of cancer. Tumor cells and myeloid cells within the tumor microenvironment express increased levels of IDO1, which is the enzyme that initiates the first and rate-limiting step of TRP breakdown via the kynurenine pathway. IDO1 enzymatic activity results in the depletion of TRP from the local environment and, the generation of immune suppressive metabolites, such kynurenine, which subsequently inhibits T cell responses. Our lab has previously shown that ovarian cancer patients with an increase in frequency of these cells demonstrate improved survival. In addition, our lab has shown that IDO1 expression in human ovarian tumor correlates with poor prognosis and poor tumor infiltration by CD8+ T cells. Thus, IDO1 inhibition represents an attractive target for cancer immunotherapy. To establish the mechanism by which IDO1 inhibition augments immune responses in a therapeutic murine model of metastatic ovarian cancer, we utilized a murine ovarian surface epithelial cancer cell line, ID8. We generated a stable IDO1- overexpressing cell line (ID8-IDO) by transfecting murine IDO cDNA into the parental ID8 cell line and confirmed increased IDO enzyme activity. C57BL/6 mice were challenged intraperitoneally with either the parental ID8 or the ID8-IDO tumor cells. Syngeneic immunocompetent mice inoculated with the ID8-IDO displayed decreased overall survival compared with ID8 challenged mice. Additionally, the ID8-IDO tumor-bearing mice demonstrate earlier onset of tumor burden, as measured by the generation of ascites in the peritoneal cavity. To delineate the role of IDO1 derived from the tumor cells and the host cells on immune cell infiltration to the tumor site, we utilized the IDO1 genetic knockout mouse model. IDO1 knockout mice and C57BL/6 mice were challenged intraperitoneally with either the parental ID8 or the ID8-IDO tumor cells. Both C57BL/6 and IDO1 knockout mice challenged with ID8-IDO demonstrate reduced CD8+ T cell infiltration within the tumor. Interestingly, IDO knockout mice challenged with parental ID8 tumor cells that do not overexpress IDO1, demonstrate increased tumor infiltration by CD8+ T cells compared to C57BL/6 ID8 tumor-bearing mice. From these results, we conclude that the regulation of tryptophan metabolism will promote anti-tumor immune responses and permit persistence and sustained activity of effector T cells in tumor tissues. Future studies are in progress to characterize the mechanism by which IDO1 inhibition may augment vaccine induced immune responses in a murine model of ovarian cancer.

Supported by: NCI SPORE P50 CA159981

65. #46 Impact of Hypoxia on IFNγ(gamma)-Dependent Responses:

Implications for Radiotherapy and Anti-Tumor Immunity

Aditi Murthy, Scott A. Gerber, Edith M. Lord University of Rochester Medical Center, Rochester, NY

Tumor hypoxia occurs due to the increase in demand for oxygen by the rapidly growing tumor cells together with reduction in the supply of oxygen due to malformed and non-functional tumor vasculature. Tumor hypoxia offers resistance to radiotherapy (RT) and chemotherapy. Interestingly, a new paradigm has emerged suggesting that hypoxia may also suppress immunotherapy, however the mechanisms behind this observation remain undetermined. Our laboratory and others have demonstrated that IFN, an important immunotherapeutic mediator, conditions the tumor microenvironment and is important for the efficacy of radiotherapy. As a result, we hypothesized that hypoxia could inhibit the anti-tumor responses mediated by IFN resulting in a decrease of radiotherapy efficacy. Hypoxia mediated inhibition could occur in two ways. Hypoxia could be modulating the production of intratumoral IFN protein, and/or reducing the ability of cells to respond to IFN. To test the first possibility we utilized Colon-38, a murine colon adenocarcinoma tumor model, and measured intratumoral hypoxia by both flow cytometry and fluorescence microscopy using a monoclonal antibody that detects hypoxia-induced 2-nitroimidazole adducts from the drug EF5. We used this drug to demonstrate a time dependent increase of hypoxia within untreated Colon- 38 tumors. Unexpectedly, irradiation resulted in a decrease in total tumor hypoxia. We also detected various sub-populations of hypoxic immune cells in both the untreated and irradiated groups, with macrophages being the majority population. Importantly, CD8+ T cells, which are an important source of IFN and mediate effector anti-tumor responses, were present in these tumors but were not co-localized with hypoxic regions. These data suggest that CD8+ T cells are absent in hypoxic areas of the tumor presenting a mechanism of tumor escape. To address the second possibility of hypoxia induced inhibition we demonstrated in vitro that hypoxia inhibited the induction of IFN-stimulated genes in multiple human tumor cell lines and peripheral blood mononuclear cells suggesting hypoxia could inhibit the responsiveness to IFN. To mimic a clinically relevant model we have also examined the impact of an anti-tumor immunotherapeutic IL-12 on tumor hypoxia. We have previously shown that IL-12 can normalize tumor vasculature. Therefore by using a combination of RT and IL-12 immunotherapy we can generate a multi-pronged approach targeting tumor hypoxia and enhancing anti-tumor effector responses mediated by IFN. We propose that tumors that are less hypoxic (e.g. after RT and immunotherapy) are more conducive to IFN and T cell responses, resulting in enhanced tumor control.

66. Andrew D. Bantly, BS, CCy Technical Application Specialist BD Biosciences

Using the Resolution Impact Table to Design Optimal Multicolor Flow Cytometry Panels

With the ever growing list of reagents to choose from when creating multicolor flow cytometry panels, new challenges in resolving co-expressed populations may arise. This talk will discuss the Resolu- tion Impact Matrix, a visual tool that will provide help in assessing potential problems with population resolution when using two fluorochromes for co-expressed markers on populations of cells.

67.

Symposium III Inflammation

Chair : Dr. Brent Berwin

68.

The Prostaglandin D2 Receptor CRTH2 Regulates Innate Immune Responses During Type 2 Inflammation

Elia D. Tait Wojno Baker Institute for Animal Health and Department of Microbiology and Immunology, College of Veteri- nary Medicine, Cornell University, Ithaca, NY

Type 2 inflammation at mucosal surfaces such as the lung and intestine mediates protective immunity to helminth parasites and contributes to allergic disease. Type 2 inflammation is characterized by production of bioactive lipids such as prostaglandin D2 (PGD2) and epithelial cell-derived cytokines such as interleukin (IL)-33 that activate innate immune cells and CD4+ T helper type 2 (Th2) cells to produce type 2 cytokines, including IL-4, IL-5, and IL-13. These cytokines act in turn on epithelial cells, promoting mucus production, changes in epithelial cell turnover, and alterations in epithelial barrier permeability. Group 2 innate lymphoid cells (ILC2s) have recently been highlighted as key innate immune cells associated with type 2 immunity and inflammation, but the lipid and cytokine pathways that control ILC2 responses in inflamed tissues are not completely understood. Here, we show that the bioactive lipid PGD2 receptor chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) regulates ILC2 accumulation in the lung in vivo. The frequency of ILC2s that expressed CRTH2 was significantly higher in healthy human and murine peripheral blood than in the lung, suggesting that regulation of CRTH2 expression might be associated with ILC2 accumulation in the lung. Consistent with this, CRTH2-expressing murine ILC2s accumulated in the lung in response to PGD2. Further, CRTH2-deficient mice exhibited reduced ILC2 accumulation and helminth-induced type 2 inflammation in the lung compared to wild-type mice. Critically, adoptive transfer of CRTH2-sufficient ILC2s restored helminth-induced pulmonary inflammation in

CRTH2-deficient mice. Together, these data suggest that the PGD2-CRTH2 pathway regulates ILC2 accumulation and type 2 inflammation in the lung in vivo.

69. Type I Immune Response in Host Defense and Inflammation

Felix Yarovinsky Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center

Autophagy is a common cellular process required for the recycling of intracellular components. The functional autophagic machinery depends on multiple proteins which generate the double membrane bound structures in the cytosol that are ultimately delivered to lysosome for recycling. The recent experiments from our and other laboratories have demonstrated that epithelial cell exposure to high levels of IFN-gamma results in severe intestinal inflammation. Combined with the observation of IFN-gamma-dependent autophagy induction in epithelial cells, we examined if the autophagic response triggered by IFN-gammais a mechanism that contributes to intestinal inflammation. The use of autophagy reporter and epithelial cell intrinsic autophagy deficient mice allowed us to identify that under steady state condition, intestinal microbiota driven IFN-gamma is a central inducer of Paneth cell autophagy. Autophagy plays a protective role in epithelial homeostasis that was revealed during acute mucosal response to a protozoan parasite Toxoplasma gondii in vivo and in intestinal organoids in vitro. Epithelial cell restricted ATG5 deficiency results in catastrophic susceptibility to T. gondii associated with severe immunopathologiocal changes in small intestine of infected mice. Intestinal organoids further proven an essential role for autophagy for resistance to cytokine mediated epithelia cell death

70. Purinergic Signaling at the Blood Brain Barrier regulates CNS Autoimmunity

Margaret S. Bynoe Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, N.Y. 14853

Multiple sclerosis (MS) is characterized by inflammatory immune cell infiltration of the central nervous system (CNS) that attacks myelin resulting in demyelination and neuronal damage and loss. A major pathogenic characteristic amongst most MS patient cohorts is immune cell invasion of the CNS and the damage they cause. We have recently shown that the purine nucleoside, extracellular adenosine, signals the migration of immune cells into the CNS. For adenosine to be bioactive, adenosine- generating enzymes and receptors must be present on the same cell or adjacent cells due to its short half-life. We showed that mice lacking CD73 (CD73-/-), the enzyme that produces extracellular adenosine are protected from experimental autoimmune encephalomyelitis (EAE), the animal model for MS. We observed that CD73-/- mice were resistant to EAE and analysis of CNS tissue demonstrated that they lacked immune cells in their brains. However, we could induce severe EAE when CD4+ T cells from CD73-/- mice were adoptively transferred into C57BL/6-TCRα-/- mice that lacked endogenous T cells. Although CD4+ T cells from CD73-/- mice secrete more proinflammatory cytokines (IL- 17, IL-1beta) than wild type mice, they were unable to gain entry into the CNS. These results suggest that adenosine signals the entry of immune into the CNS and that its role in immune cell migration into the CNS is independent of its mechanism in immune suppression. We recently determined that adenosine signaling regulates immune cell infiltration at CNS barriers.

71.

Symposium IV Immune Regulation

Chair : Dr. Nicholas Mantis

72. Virus-based Therapeutic Vaccine Vectors to Cure Chronic HBV Infection

Tracy D. Reynolds1, John K. Rose1, and Michael D. Robek2 1Department of Pathology, Yale School of Medicine, New Haven, CT 2Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

Despite the availability of a preventative vaccine, chronic hepatitis B virus (HBV) infection affects more than 240 million people worldwide and substantially increases the risk for developing serious liver diseases such as cirrhosis and hepatocellular carcinoma. Individuals who become acutely infected with HBV generate relatively strong multi-specific T cell responses that control the virus. However, in those people that become chronically infected, the T cell response is weaker and is directed at fewer antigenic epitopes. Because the human immune system can control HBV but often fails to do so, immunotherapies including therapeutic vaccination represent a potential new approach to cure chronic hepatitis B. However, therapeutic vaccine platforms tested to date have lacked sufficient immunogenicity to overcome immune tolerance and eliminate HBV from the liver. Therefore, new safe vaccine systems that can generate an effective therapeutic immune response to HBV are needed. Compared to other vaccine platforms, viral vectors have the advantage that they efficiently elicit broad CD8 T cell and antibody responses, but concerns about toxicity and systemic spread limit their widespread clinical use. In previous studies, we found that recombinant wild type vesicular stomatitis virus (VSV) expressing the HBV middle surface envelope glycoprotein (MHBs) induces effective immune responses in mouse models of acute and chronic HBV infection. Recently, an evolved, high-titer vaccine platform consisting of Semliki Forest virus RNA replicons that express the VSV glycoprotein (VSV G) has been described. This platform generates “virus-like vesi- cles” (VLVs) that contain VSV G but no other viral structural proteins. We found that the evolved VLV vector engineered to additionally express MHBs induces functional CD8 T cell responses in mice. These responses were greater in magnitude and broader in specificity than those obtained with other immunization strategies, including recombinant protein and DNA. Additionally, a single immunization with VLV- MHBs protected mice from HBV hydrodynamic challenge, and this protection correlated with the elicita- tion of a CD8 T cell recall response. In contrast to MHBs, a VLV expressing HBV Core protein neither induced a CD8 T cell response in mice, nor protected against challenge. Finally, combining DNA and VLV -MHBs immunization led to induction of HBV-specific CD8 T cell responses in a transgenic mouse model of chronic HBV infection. The ability of VLV-MHBs to induce a multispecific T cell response capable of controlling HBV replication, and to generate immune responses in a tolerized model of chronic infection, indicates that VLV vaccine platforms may offer a unique strategy for HBV therapeutic vaccination.

73. LRBA is Essential for Allogeneic Responses in Bone Marrow Transplantation

Sudan, R1., Park, M.Y1., Srivastava, N1., Sudha, N1., Christie, Y1., Wang, J.W2., Engelman, R.W3 and Kerr, W.G1,4,5,6 1Department of Microbiology & Immunology, 4Biochemistry & Molecular Biology, 5Pediatrics, SUNY Upstate Medical University, 2 Department of Internal Medicine, University of South Florida, 3H. Lee Mof- fitt Cancer Center & Research institute, Tampa, FL, 6Chemistry Department, Syracuse University

Members of the PH-BEACH-WD40 repeat gene family (PBW) play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive Beige-like Anchor (LRBA) is a PBW protein whose immune function has not been defined. Here we show that LRBA-/- mice are viable with normal and healthful life span, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone marrow grafts. Further we demonstrate that LRBA-/- Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of γ-IFN by co-culture with IL12 and IL18 remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where Graft versus Host Disease (GvHD) is primed or initiated, and consistent with this LRBA-/- mice demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host lon- gevity while being essential for both host and donor-mediated immune responses in allogeneic transplantation and thus represent a unique and novel molecular target in transplant immunology.

74. Immunologic Dysregulation in Sickle Cell Disease

Steven M. Szczepanek, Ph.D. Center of Excellence for Vaccine Research, Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT, 06269

Sickle Cell Disease (SCD) is a well-known genetic disorder that causes red blood cells to become misshaped and rigid, leading to occlusion of blood vessels and consequent pathology in all tissues of the body. Individuals with SCD often die during childhood due to their inability to fight off common pathogens such as Streptococcus pneumoniae and Influenza virus, yet little is known about the immune dysfunctions that result in their exquisite sensitivity to these insults. Furthermore, frequent vaccination is a common clinical tool used by physicians to try and prevent life-threatening infections, yet our research has shown that common vaccines do not work well in SCD patients, or in the transgenic mouse model of SCD. Our work has made it apparent that one of the primary causes of immunologic insufficiency in SCD is due to dysfunctional B-cells, and more specifically an inability of these cells to transition into a memory phenotype. The B-1 subset of B-cells has arisen as the target of insufficient immune protection in SCD mice from S. pneumoniae infection after Prevnar-13 vaccination, while exhaustion appears to prevent functional B-cell responses to influenza vaccination in SCD patients. Increased life expectancy for patients with SCD will require a better basic science understanding of the dysfunctions of their B-cells so that translational advancements can be made in this field.

75. Michael Seiler, Ph.D. Associate Director, Product Management

Taconic Biosciences

Taconic Biosciences’ Human Immune System Engrafted Models Program: Applications in Immuno-Oncology Research

Mouse models are widely used in oncology drug discovery research but species differences can limit efficacy predictions for clinical translation. Our human immune system engraftment program is designed to accelerate drug discovery platforms for human-specific biological targets. Engrafted mice recapitulate many of the functional attributes of working immunity, and can be used to test new therapeutic targets to activate or inhibit human immune cell reactivity in vivo. An overview of our engrafted models program will include the utility of such mice in combined tumor and immune system engraftment applications for immuno-oncology drug development, and recent advances developing the next iterations of immune system reconstituted mice.

76. Jacob Schumacher Science Policy and Legislative Affairs Specialist American Association of Immunologists

The Urgent Importance of Advocating for Biomedical Research

The budget for the National Institutes of Health (NIH) more than doubled between fiscal year (FY) 1998 and FY 2003 as Congress made a bipartisan commitment to prioritize biomedical research. Today, the NIH budget is actually 22 percent below where it was in FY 2003 in inflation-adjusted dollars. There are encouraging signs that lawmakers once again recognize the urgent need to invest in NIH, due at least in part to the collective persistence of the biomedical research community’s advocacy for sustained and pre- dictable funding for the agency. It’s more important than ever that scientists continue to make their voices heard on Capitol Hill to ensure that these potential funding increases are actually realized in both the short and long term. It’s also imperative that scientists make their priorities known at the agency level, as fewer dollars require NIH and other science agencies to make difficult decisions on how funds are allocat- ed. This talk will focus on some of the key policy issues of concern to biomedical researchers and describe ways to get involved.

77.

Symposium V Antigen Presenting Cell Biology

Chair : Dr. Paige Lawrence

78. Dectin-1 –dependent Changes in Dendritic cell Metabolism Regulate NLRP3 Inflammasome Activation

Fritz, D.1, Adamik, B. 1, Galasso, N. 1, Thwe, P. 1,2, Amiel, E. 1,2 1Department of Medical Laboratory and Radiation Sciences, University of Vermont, Burlington, VT 2Vermont Center for Immunology and Infectious Disease, Immunobiology COBRE, University of Vermont, Burlington, VT

Recent advances have described an intrinsic requirement for the induction of aerobic glycolysis metabolism to support the early immune function of Toll-like Receptor (TLR) –stimulated dendritic cells (DCs) in response to bacterial ligands. Specifically, glucose-dependent fatty acid synthesis supports the high synthetic output associated with the immune effector function of DCs, particularly the processes of translation and secretion of early response cytokines. Dectin-1 is a member of the C-type Lectin Receptor (CLR) family that is expressed by DCs and primarily recognizes β-glucan molecules, a major molecular component of fungal cell walls. We show that Dectin-1 –mediated, TLR-independent, signaling in DCs mediates a rapid induction in glycolysis. This increased glucose metabolism supports early-phase inflammasome activation and IL-1β secretion, processes that play a vital role in anti-fungal defense. Ongoing work supports a model whereby glycolysis-mediated regulation of inflammasome activation is likely controlled by glucose-dependent generation of reactive oxygen species in DCs.

79. The Role of Langerin+ DCs in Mucosal Immunity

Magdia De Jesus1,2 and Nicholas J. Mantis1,2 1University at Albany School of Public Health, Biomedical Sciences, Albany, NY 2Wadsworth Center, New York State Department of Health, Albany, NY

The gastrointestinal tract is exposed to a daily onslaught of foreign antigens and microbes. Interest- ingly, it remains unclear how some individuals tolerate this constant exposure whereas others develop severe inflammatory bowel disease or food allergies. It is thought the balance between tolerance and inflammation is due in large part to dendritic cells (DCs) located in the small intestine as these are at the interface between the innate and adaptive mucosal immune system. Our goal is to understand how DCs initially sample and ultimately “decide” whether to promote tolerogenic or inflammatory responses upon antigen and microbial uptake. We have recently, identified a population of DCs within intestinal Peyer’s patches that expresses the C-type lectin receptor (CLR) Langerin. Langerin is of particular interest in the context of the gut as it binds to a variety of glycan ligands such as β-glucans, mannans, fucose and N- acetylglucosamines (GlcNAc) that are known to be present on the surface of a wide range of antigens and microbes. We have found that these Langerin+ DCs and Microfold (M-cells) can efficiently sample an array of different types of antigens; including microparticles used as delivery vehicles, opportunistic fungal pathogens such Candida albicans, allergic peanut antigens and even algae. Based on these observations, we hypothesize that Langerin+ DCs play a central role in the sampling and mediating tolerogenic and inflammatory mucosal immune responses. Elucidating these mechanisms will have important implications for mucosal therapies and vaccines in patients who suffer from recurrent inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, as well as food allergies.

80. MHC Class II Subsets in B-Cell Biology

Jim Drake Albany Medical College, Center for Immunology and Microbial Disease

MHC class II-restricted antigen processing and presentation by antigen-specific B cells results in interactions between B cells and CD4 helper T cells, to drive immunoglobulin class switching and affinity maturation. Antigen-specific B cells are unique among antigen presenting cells (APC) in that they express an antigen-specific receptor, the B cell receptor (BCR), which mediates initial B cell activation as well as antigen processing and presentation at immunologically-relevant concentrations of antigen. We have established that B cells and other APC express two distinct MHC class II conformers, formation of which is based on differential pairing of transmembane domain GxxxG dimerization motfs (M1 vs. M2 paired MHC class II), and shown that these two conformers differ both in their ability to drive T cell activation as well as B cell signaling. We have recently determined that internalized antigen-BCR (Ag-BCR) complexes physically associate with intracellular MHC class II molecules in a putative MHC class II peptide loading complex. Interestingly, it is the M1 paired MHC class II conformer that selectively associates with intracellular Ag-BCR complexes. Moreover, the M1 paired class II conformer is selectively loaded with peptide derived from the processing of BCR-bound antigen and associates with the CD79 signaling subunit of the BCR. These results reveal that the function of the BCR is not simply to facilitate antigen uptake at low concentrations, but rather to guide loading of peptides derived from the processing of cognate antigen onto the subset of M1 paired MHC class II molecules, which have unique B cell and T cell activation properties.

81. Keynote Speaker

Yasmine Belkaid, Ph.D. Chief Mucosal Immunology Section Laboratory of Parasitic Diseases National Institutes of Health/NIAID

“Regulation of Tissue Immunity and the Microbiota”

82. 2015 NYIC Poster Numbers

Poster No: Poster No: 1 Megan Peppenelli 25 Julianne N.P. Smith** 2 Kiah L. Sanders** 26 Kerry R. Belton 3 Jessica L. Meyers** 27 Amber Papillion 4 Lisbeth Boule 28 Karin M. Schneider 5 Todd A. Jusko 29 Phyu Thwe** 6 Eyal Amiel 30 Sadikshya Bhandari 7 Jessica L. Rastad** 31 Lauren Burkard-Mandel 8 Michelle N. Messmer** 32 Danielle Twum 9 Rebecca Crepeau 33 Jocelyn Wang 10 Mohammad Haque 34 Tamer B. Chabanet 11 Elise Burger** 35 Elia D. Tait Wojno 12 Americo Lopez-Yglesias 36 Margaret L. Barlow 13 Kevin Kenderes 37 Kelli A. Connolly** 14 Pamela Shen 38 Samira Mansouri 15 Anthony M. Franchini 39 Seema Patel 16 Scott Minchenberg 40 Adaobi Amobi** 17 Richard M. Jin 41 Adam Utley** 18 Elizabeth Wohlfert 42 Erika Gruber 19 Kristel Yee Mon 43 David Williamson 20 Sudie Ann Robinson 44 Danielle Califano 21 Alicia Soucy 45 Colin Chavel 22 Yoichi Furuya 46 Aditi Murthy 23 Mital Pandya 24 Nicole Nelson

**AAI Young Investigator Award and Oral Poster Presentation  eBioscience Travel Award and Oral Poster Presentation

83. #1 A Unique Signalisome Induced By Human Cytomegalovirus During Entry Targets the Translation of Caspase 3 Regulatory Proteins Ensuring The Survival Of Infected Monocytes.

HCMV infection of immunocompromised individuals often leads to multi-system organ failure. The development of multi-system organ failure is dependent on the ability of HCMV to spread to peripheral organs, which is mediated by blood monocytes. In order for monocytes to mediate spread, we have previously shown HCMV to extend the short 48hr lifespan of monocytes. Mechanistically, HCMV upregulated cellular myeloid leukemia sequence 1 (Mcl-1) and heat shock protein 27 (HSP27), to block the two proteolytic cleavages necessary for the formation of active caspase 3. We now show that, compared to other myeloid survival factors, HCMV infection more efficiently upregulate Mcl-1 and the only survival factor to rapidly induce HSP27. These observations, suggest a unique virus specific mechanism of induction; thus, we examined receptor: ligand signaling events, since viral anti-apoptotic proteins are not expressed until differentiation into macrophages is complete. We determined that HCMV-induced EGFR/PI3K/AKT signaling increased the transcription of both Mcl-1 and HSP27. Yet, protein expression of Mcl-1 and HSP27 is increased via the gH/aVb3/src and the gB/EGFR pathways, respectively. To address this discrep- ancy between transcripts and protein we evaluated the effects of HCMV on the translation of Mcl-1 and HSP27. Indeed, we found, in contrast to myeloid survival factors, HCMV stimulated the translation of Mcl-1 and HSP27. Overall, these data indicate that inhibition of apoptosis by HCMV is through the unique signaling regulation of Mcl-1 and HSP27; ensuring short-lived monocyte survival past the normal 48hr viability checkpoint, a key event necessary for viral dissemination.

Contact: Gary Chan, [email protected]

84. #2 Expression of Host CXCR3 Controls Early Establishment of Metastatic Melanoma in the Lung

Kiah L. Sanders, Eleanor Clancy-Thompson, Thomas Perekslis, David W. Mullins Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH

85. #3 Persistent Changes in Dendritic Cell Function following Developmental Activation of the

Aryl Hydrocarbon Receptor

Jessica L. Meyers1, Bethany Winans1, Lisbeth Boule1,2, and B. Paige Lawrence1,2 Department of Environmental Medicine, Environmental Health Sciences Center1 and Department of Microbiology and Immunology2 University of Rochester, Rochester, NY USA

Funding: R01-ES017250, R01-ES023260, T32-ES07026, T32-HL066988, P30-ES01247

86. #4 Activation of the Aryl Hydrocarbon Receptor by Categorically Different Ligands Alters the Immune Response to Primary Influenza A Virus Infection

Lisbeth A. Boule1,2, Guang-Bi Jin1, and B. Paige Lawrence1,2 Departments of 1Environmental Medicine and 2Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY

The aryl hydrocarbon receptor (AHR) is an environment sensing transcription factor that offers a novel target for potential new therapeutic strategies to treat immune-mediated diseases. Yet, our ability to harness the AHR to improve health and treat disease is hampered by limited knowledge of how the diverse spectrum of AHR ligands affects immune responses in vivo. For instance, exposure to certain AHR ligands alters the progression of mouse models of autoimmune or infectious diseases; however, few studies have compared the same ligands within the same disease model. Moreover, it is unknown if the immunological consequences of exposure to particular ligands are solely AHR-mediated. In this study, we directly compared the effects of the following four AHR ligands on acute primary infection with influenza A virus (IAV) in wild-type and Ahr-/- mice: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3’,4,4’,5- pentachlorobiphenyl-126 (PCB126), 2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester (ITE), and 6-formylindolo(3,2-b)carbazole (FICZ). TCDD is the prototype and best characterized AHR ligand. TCDD and PCB126 are environmental contaminants with documented human exposure. ITE is being developed as a potential pharmaceutical, whereas FICZ is a UV photodegradation product of tryptophan and may represent a naturally occurring ligand. We show here that TCDD, PCB126 and ITE, but not FICZ, reduced virus-specific antibody levels and virus-specific CD8+ T cell responses to IAV. CD4+ T cell responses to infection were altered by all four ligands; however, the direction in which the change occurred was ligand and CD4-subset specific. Compared to control treated IAV infected mice, TCDD, ITE, and PCB126 significantly reduced the number Th1 cells, whereas FICZ increased the frequency of this CD4+ T cell subset. Similar to Th1 cells, FICZ treatment during IAV infection increased the proportion of Tfh cells, while TCDD, ITE, and PCB126 reduced the number of Tfh cells generated. Th17 cells were not changed after AHR activation by any of the 4 ligands used. Infected mice treated with TCDD, PCB126, and ITE all had a greater proportion Tregs (CD25+Foxp3+CD4+ cells) compared to controls, yet FICZ did not alter the frequency of Tregs. We examined the specificity of each ligand for the AHR by using Ahr-/- mice, and determined that the majority of the changes in the adaptive immune response to IAV infection after ligand exposure require expression of the AHR. This study demonstrates that investigating AHR ligands as therapeutics will require finding a balance between the suppression of immune-mediated pathologies, while maintaining intact and sufficient host defense mechanisms.

87. #5 Early Life Persistent Organic Pollutant Exposure and Reduced Response to Infant

Tuberculosis Vaccination

Todd A. Jusko,1,2 Anneclaire J. De Roos,3 Sue Y. Lee,2 Kelly Thevenet-Morrison,1 Stephen M. Schwartz,4 Marc-André Verner,5 Lubica Palkovicova Murinova,6 Beata Drobná,7 Anton Kočan,8 Anna Fabišiková,9 Kamil Čonka,7 Tomas Trnovec,6 Irva Hertz-Picciotto,10 B. Paige Lawrence2 1Department of Public Health Sciences, University of Rochester School of Medicine and Dentistry, Roch- ester, New York, USA; 2Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA; 3Department of Environmental and Occupational Health, Drexel University School of Public Health, Philadelphia, Pennsylvania, USA; 4Program in Epi- demiology, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA; 5Department of Occupational and Environmental Health, School of Public Health and Université de Montréal Public Health Research Institute (IRSPUM), Université de Montréal, Mon- treal, Quebec, Canada; 6 Department of Environmental Medicine, Slovak Medical University, Brati- slava, Slovak Republic; 7Department of Toxic Organic Pollutants, Slovak Medical University, Bratisla- va, Slovak Republic; 8 Research Centre for Toxic Compounds in the Environment, Masaryk Univer- sity, Brno, Czech Republic; 9Department of Analytical Chemistry, University of Vienna, Vienna, Aus- tria; 10Division of Environmental and Occupational Health, Department of Public Health Sciences, University of California, Davis, California, USA

Background – Reasons for the highly variable and often poor protection conferred by the Mycobacte- rium bovis bacille Calmette-Guerin (BCG) vaccine are multifaceted and poorly understood. Objectives – To determine whether early life PCB (polychlorinated biphenyls) and DDE (1,1-dichloro- 2,2-bis(p-chlorophenyl)ethylene) exposure reduces 6-month infant BCG vaccine response. Methods – Data came from families participating in a prospective birth cohort in eastern Slovakia. At birth, maternal and cord blood were collected for chemical analyses, and infants were immunized with BCG. Blood was collected from infants for chemical analyses and to determine 6-month BCG-specific immunoglobulin (Ig)G and IgA levels. Multivariable linear regression models were fit to examine chemical— BCG associations among 516 mother-infant pairs, with adjustment for confounders. Results – The median 6-month infant concentration of the prevalent congener PCB-153 was 113 ng/g lipid (IQR: 37, 248), and 388 ng/g lipid (IQR: 115, 847) for DDE. Higher 6-month infant concentrations of PCB-153 and DDE were strongly associated with lower 6-month BCG-specific antibody levels. For instance, BCG-specific IgG levels were 37% lower for infants with PCB-153 concentrations at the 75th percentile compared to the 25th percentile (95% CI: -42, -32; p<0.001). Results were similar in magnitude and precision for DDE. There was also evidence that exposure to both compounds reduced BCG levels more than exposure to either compound alone. Conclusions – The associations observed in this study indicate that environmental exposures may be overlooked contributors to poorer responses to BCG vaccine. The overall association between these exposures and tuberculosis incidence is unknown.

88. #6 Dectin-1 –dependent Changes in Dendritic Cell Metabolism Regulate NLRP3 Inflammasome Activation

89. #7 Myeloid-Derived Suppressor Cells in Murine AIDS Suppress B-Cell Responses in a

Reactive-Oxygen- and Nitrogen-Species Dependent Manner

Jessica L. Rastad1 and William R. Green1,2. 1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH 2Norris Cotton Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are implicated in a variety of murine and human pathologies, including cancer and viral, bacterial, and parasitic infections. MDSCs are broadly classified into two subsets based on extensive studies of suppression of T-cells in tumor models. Granulocytic MDSCs (G-MDSCs) are polymorphonuclear and tend to utilize reactive oxygen species (ROS) and arginase-1 for their immunosuppressive function. Monocytic MDSCs (M-MDSCs), are mononuclear and primarily utilize inducible nitric-oxide synthase (iNOS) to suppress. LP-BM5 is a murine retroviral isolate that causes murine AIDS (MAIDS), which has many similarities to HIV/AIDS, including the development of profound immunodeficiency. Our lab identified that M- MDSCs increase during MAIDS pathogenesis. Increased MDSCs have subsequently been found in HIV- infected patients. Interestingly, M-MDSCs from LP-BM5-infected mice suppress not only T-cell responses, but also B-cell responses, indicating that they may be a potential therapeutic target. Suppression of T- cells is almost entirely iNOS-dependent, with suppression of B-cells showing only partial iNOS- dependence. Additionally, suppression of B-cells by M-MDSCs from LP-BM5 infected mice is independent of arginase-1, PD-1/PD-L1 interactions, IL-10 production, and indoleamine 2,3-dioxygenase activity. Our lab is the first, to our knowledge, to publish evidence of MDSC-mediated suppression of B-cells in a viral system. MDSC-mediated suppression of B-cells has since also been recently shown in a collagen- induced arthritis model. In this study we utilized a transwell system and a conditioned media assay and determined that suppression of B-cells by M-MDSCs from LP-BM5 infected mice is mostly cell-contact independent and is dependent in part on soluble mediators. Detection of NO (nitric oxide, a product of the iNOS reaction) in the transwell system indicated that iNOS is functional in the absence of cell-contact. Addition of iNOS in- hibitors to the conditioned media assay showed that iNOS was responsible for only about half of the suppression attributed to soluble-mediators. Using antioxidants, the ROS superoxide and peroxynitrite were shown to be other soluble mediators significantly contributing to MDSC suppression of B-cells. In contrast, cysteine add-back experiments indicated no evidence for involvement of cysteine-depletion as a suppressive mechanism. This study demonstrates M-MDSC-mediated suppression of B-cells by ROS, a mechanism more commonly associated with G-MDSC-mediated suppression of T-cells. These results highlight the utilization of multiple mechanisms by MDSCs to broadly inhibit B-cell function, and provide potential mechanistic targets for inhibiting or enhancing B-cell suppression by MDSCs.

90. #8 Regulation of Transcription Factor IRF8 in Myeloid Progenitors is a Critical Checkpoint for Formation of Defective Myeloid Cells in Cancer

Michelle N. Messmer, Colleen Netherby, Scott I. Abrams Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY

Defective myeloid cells are commonly observed across a large range of solid cancers. These defects result in immune suppression and promote tumor progression to metastatic disease. The molecular processes underlying development of these pathological myeloid-derived suppressor cells remains unclear. Previ- ous studies focused on characterizing these myeloid populations in peripheral tissues and the tumor microenvironment, despite the fact that the major site for normal myeloid production is the bone marrow. Since the bone marrow is sensitive to signals from the periphery, we hypothesized that tumor induced perturbations in myelopoiesis occur in the bone marrow and are the likely origin of suppressive myeloid cells. Dur- ing myelopoiesis, transcription factor IRF8 within granulocyte/monocyte progenitors (GMP) determines differentiation such that high IRF8 expression favors monocytes while low IRF8 expression favors granulocytes. Our recent work using orthotopic mammary tumor models revealed that tumor-derived factors directly suppress IRF8 expression leading to accumulation of suppressive, immature myeloid cells. While these data support IRF8 acting as a negative regulator of defective myeloid accumulation, exactly when and where IRF8 is compromised during myelopoiesis remained unclear. To further elucidate how tumor growth impairs IRF8 expression, we utilized a novel mouse model expressing an IRF8-EGFP fusion protein. This allows us to investigate changes in IRF8 during both orthotopic and spontaneous mammary tumor progression. Our results show that: 1) GMP expand with increasing tumor size and increasing amounts of G-CSF in the serum of tumor-bearing hosts, and 2) GMP can be divided into IRF8hi and IRF8lo expressing populations similar to IRF8lo granulocytic and IRF8hi monocytic myeloid cells observed in the blood. Expression of IRF8 in other progenitor populations was not affected, suggesting IRF8lo GMP may be the source of expanded myeloid cells in cancer. Altogether, these data identify modulation of IRF8 in myeloid cells and their progenitors as an early consequence of tumor development and a potential target for therapeutic intervention.

Supported by: NIH RO1 CA140622

91. #9 miR-155 Plays a Critical Role in the Establishment and Maintenance of Latency in Murine

Gammaherpesvirus Infection

Rebecca Crepeau, Peisheng Zhang and Edward Usherwood Geisel School of Medicine at Dartmouth

92. #10 Stem Cell-derived Tissue-associated Regulatory T-cells Suppress the Development of Autoimmune Diabetes

Mohammad Haque, Praneet Sandhu, and Jianxun Song Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine, Hershey, PA 17033

Regulatory T cells (Tregs) are essential for normal immune surveillance systems, and their dysfunction leads to the development of diseases, such as autoimmune disorders. Under the right circumstance, pluripotent stem cells (PSCs) can produce almost all of the cells in the body, including Tregs. PSCs provide a chance to obtain a renewable source of healthy Tregs to treat a wide array of autoimmune disorders. How- ever, the right circumstance for the development of antigen (Ag)-specific Tregs from PSCs (i.e., PSC-Tregs) has not been well defined. Ag-specific PSC-Tregs can be tissue/organ-associated and migrate into local inflamed tissues/organs to suppress the autoimmune response after adoptive transfer, thereby avoiding potential overall immunosuppression from non-specific Tregs. In this study, we developed a new approach to generate functional Ag-specific Tregs from induced PSC (iPSCs), i.e., iPSC-Tregs, which have the ability to suppress autoimmunity in a murine model of type 1 diabetes (T1D) in Ag-associated fashion. We retrovirally transduced murine iPSC with a DsRed reporter construct containing genes of major histocompatibility complex (MHC) II (I-Ab)-restricted ovalbumin (OVA)-specific T cell receptor (TCR) and the transcrip- + tional factor FoxP3. We in vitro differentiated the DsRed iPSC into OVA-specific iPSC-Tregs with an OP9 stromal cell line expressing Notch ligands DL1, DL4 and I-Ab in the presence of recombinant cytokines of rIL-7 and rFlt3L. We visualized the expression of CD3, TCR, CD4, CD25, and CTLA4 on OVA-specific iPSC-Tregs. We also in vivo differentiated the TCR and FoxP3 gene-transduced iPSC into functional OVA- specific Tregs, which had the ability to produce the suppressive cytokines of IL-10 and TGF-b, following

OVA stimulation. Moreover, adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established OVA-induced T1D model (RIP-OVA x OT-I double transgenic mice), including the inflammation and prevents the insulin secreting pancreatic beta cells from destruction. Of note, we demonstrated that the adoptive transfer significantly reduced the higher ratio of CD8+ to CD4+ T cells in diabetic mice. Our results indicate that PSCs can be used to develop Ag-specific Tregs, which have a therapeutic potential for Treg-based therapies of T1D.

93. #11 Loss of Intestinal Epithelial Autophagy leads to Catastrophic Susceptibility to Acute

T. gondii-mediated Inflammation

Elise Burger, Américo López-Yglesias, Felix Yarovinsky Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY

94. #12 T-bet-independent Th1 Response is Sufficient for Toxoplasma gondii-mediated Intestinal Immunopathology

Américo López-Yglesias, Elise Burger, Carolyn Sturge, and Felix Yarovinsky Center for Vaccine Biology and Immunology, University of Rochester, Rochester, NY

Toxoplasma gondii is a protozoan parasite recognized by innate receptors TLR11 and TLR12, leading to MyD88-dependent IL-12 production. Subsequently, IL-12 production leads to a robust CD4+ Th1 IFN-γ response which is essential for host resistance to the parasite. We have previously revealed an im- munopathological role for CD4+ T cell IFN-γ responses, characterized by: intestinal inflammation, death of Paneth cells, loss of antimicrobial peptides, and significant expansion of endogenous Escherichia coli in the small intestine. The transcription factor T-bet, encoded by Tbx21, is well described as being essential for CD4+ T cell Th1 development and IFN-γ responses. T-bet is also indispensable for the development of type I innate lymphoid cells (ILC1) which in addition to CD4+ T cells, play a role in IFN-γ-mediated host defense. Therefore, we sought to determine T-bet’s role in the regulation of IFN-g production by CD4+ T cells and ILC1s during T. gondii infection. Unexpectedly, we found that T-bet-/- mice orally infected with T. gondii exhibited strong CD4+ T cell IFN-γ responses in both the draining lymph node and spleen. Furthermore, similar to WT mice, T. gondii-infected T-bet-/- animals lost Paneth cells and developed severe intestinal inflammation. These results suggest that CD4+ T cells, but not ILC1s are responsible for IFN-γ –mediated intestinal damage, and that T-bet is dispensable for IFN-γ production. Adoptive transfers of T-bet-deficient CD4+ T cells into Rag1-/- mice revealed a T-bet-independent IFN-g and Th1 response during parasite infection. Overall, we established that the transcription factor T-bet, while required for ILC1 development, is dispensable for parasite-induced Th1 effector choice and IFN-γ-dependent intestinal immunopathology.

95. #13 IgM Memory B-cells Differentiate via Multiple Pathways following Challenge Infection

Kevin Kenderes, Amber Papillion, Lisa Dishaw, and Gary Winslow SUNY Upstate Medical University

96. #14 Cigarette Smoke Attenuates the Nasal Host Response to Streptococcus pneumoniae and Predisposes to Invasive Pneumococcal Disease in Mice

Pamela Shen1, Mathieu C. Morissette2, Dawn M.E. Bowdish2, and Martin R. Stampfli2,3 1Medical Sciences Graduate Program, McMaster University, Hamilton, Ontario, Canada. 2Department of Pathology and Molecular Medicine, McMaster Immunology Research Centre, McMaster University, Ham- ilton, Ontario, Canada. 3Department of Medicine, Firestone Institute for Respiratory Health at St. Joseph's Healthcare, McMaster University, Hamilton, Ontario, Canada

Introduction: Streptococcus pneumoniae (the pneumococcus) is a leading cause of invasive infections including bacteraemia and meningitis, with nasal pneumococcal colonization considered the first step for invasive pneumococcal disease (IPD). Cigarette smoking is well documented to be a strong risk factor for IPD. Although there is increasing awareness that cigarette smoke skews immune inflammatory processes in the lower respiratory tract, it remains unclear whether cigarette smoke compromises the upper respiratory tract (URT) bacterial host defense and if this is related to the increased risk for IPD. This is partly due to a lack of clinically relevant animal models investigating nasal pneumococcal colonization in the context of cigarette smoke exposure. Further understanding is critical as over one billion people continue to smoke worldwide. Results: We show that cigarette smoke exposure during nasal pneumococcal colonization predis- posed to IPD and mortality in mice. Cigarette smoke increased the risk of acquiring pneumococcal bacteraemia and meningitis without a requirement for prior pneumonia. In addition, we observed pneumococci in the brain without presence in either the spleen or bronchoalveolar lavage (BAL). Mechanistically, cigarette smoke attenuated the nasal expression of cytokines and chemokines, including TNF-α (alpha), IL- 12p40, CXCL-1, and CXCL-2. Smoking cessation following nasal pneumococcal colonization completely rescued mice from IPD and mortality. We found no differences in epithelial permeability or nasal clearance rates between room air and cigarette smoke-exposed mice following nasal delivery of 99mTC-DTPA. Finally, as cigarette smoke may increase bacterial adherence to host cells via upregulation of the platelet activating factor receptor (PAFR) in an IL-1α (alpha) dependent manner, we investigated the incidence of IPD using PAFR knockout (KO) and IL-1α (alpha) KO mice. However, these mice were not rescued from IPD and subsequent mortality following cigarette smoke exposure. Conclusions: Our data suggest smokers may be at increased risk of occult bacteraemia and non- hematogenous meningitis. We propose that cigarette smoke increases the incidence of IPD by compromis- ing the immune response at the mucosal surface of the URT. We found the effect of cigarette smoke exposure on IPD to be reversible upon smoking cessation, emphasizing the need for supportive smoking cessation and smoking prevention programs. However, further research should aim to restore the nasal bacterial host defense given that smoking prevalence remains high worldwide. The current study has increased our understanding of cigarette smoke's effects on the nasal bacterial host response and may contribute to the future development of effective intervention strategies.

97. #15 Aryl Hydrocarbon Receptor Activation Alters Mouse and Human Dendritic Cell Gene Expression

Profiles during Influenza A Virus Infection

Anthony M. Franchini, Guang-Bi Jin, B. Paige Lawrence University of Rochester, Department of Environmental Medicine

Activation of the aryl hydrocarbon receptor (AHR) substantially dampens host resistance to infection with influenza A viruses. We previously reported that AHR activation negatively regulates the expansion and differentiation of CD8+ cytotoxic T lymphocytes (CTL), and reduces the ability of dendritic cells (DC) to prime naïve CD8 T cells. However, the mechanism by which AHR activation diminishes DC function in vivo is unknown. Given that AHR is a ligand inducible transcription factor, we used unbiased gene expression profiling to identify gene and signaling pathways in DCs that are modulated by in vivo AHR activation. Lung DCs from infected mice that were exposed to the prototypical AHR ligand 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) or control were sorted and used for transcriptome analysis by RNA- seq. This approach revealed 473 differentially expressed genes; many of which are involved in DC func- tions. Notably, indoleamine-2,3-dioxygenase (Ido1) and programmed death ligand 1 (pdl1) were induced four-fold and nearly two fold, respectively, while transcripts for a DC-SIGN homolog cd209a and the chemokine ccl17 were decreased three-fold and five-fold over vehicle controls, respectively. AHR activation similarly elevated in Ido1 levels in DCs isolated from the mediastinal lymph nodes (MLN), the primary site of naïve T cell priming during the early phase of influenza virus infection. Accompanying increased Ido1 expression in the lung and MLN, AHR activation also significantly increased in the proportion of plasmacytoid DCs (pDCs) and regulatory T cells in the MLN of infected mice. Expanding these findings into an in vitro human system, monocyte-derived DCs (hmoDCs) from healthy human donors were exposed to TCDD or control and then influenza A virus. Targeted transcriptional analysis by qRT- PCR revealed similar changes in gene expression in the hmoDCs, including elevated ido1, and decreased cd209a transcript levels. Collectively, this provides evidence that AHR activation in DCs alters gene expression, which could induce an immunoregulatory phenotype and dampen the ability of DCs to activate naïve T cells. Given that the same molecules and pathways influence DC function in disease states beyond fighting influenza viruses, these findings have much broader implications for how the AHR regulates the function of DCs.

98. #16 Induction of Arginase-1 by TNF-α in Oligodendrocytes is Controlled by SHP-1

Scott Minchenberg, Daria LaRocca, and Paul Massa SUNY Upstate Medical University, Syracuse NY

Multiple Sclerosis (MS) is a debilitating neurological disease characterized by inflammatory demyelination in the central nervous system (CNS), and tumor necrosis factor alpha (TNF-α) has been implicated in its pathogenesis. TNF-α(alpha) is toxic to the myelin forming oligodendrocytes and their connected myelin sheaths, and its expression is elevated in MS lesions. However, the precise mechanism for TNF-α (alpha)-mediated toxicity to oligodendrocytes remains unknown. Moreover, the role of regulatory molecules, including SHP-1, in controlling both TNF-α(alpha) signaling and susceptibility to TNF-α(alpha)- mediated demyelinating diseases in the CNS is unclear. Here, we show that compared to normal littermate controls, oligodendrocytes of SHP-1-deficient mice (motheaten), which are highly susceptible to inflammatory demyelination, display a significant increase in inflammatory gene expression in response to TNF- α(alpha). Unexpectedly, the most highly induced of these inflammatory genes is arginase-1, an enzyme that converts arginine to L-ornithine with major effects on allergic-type immune responses. To elucidate the mechanism underlying this induction, we are determining how TNF-α(alpha) induces transcriptional activation of the arginase-1 promoter in an SHP-1-dependent manner. Further, we are investigating if ab- normal expression of arginase-1 in oligodendrocytes is responsible for the oligodendrocyte dysfunction and increased demyelination observed in SHP-1-deficient mice.

99. #17 Getting the Balance Right: Immunoregulation of Host-defense and Tissue Repair in Skeletal Muscle

during T. gondii Infection

Richard M. Jin, Sarah J. Blair, Elizabeth M. Schiavoni, Ira J. Blader, and Elizabeth A. Wohlfert Department of Microbiology and Immunology, School of Medicine and Biomedical Sciences, University at Buffalo

Proper maintenance and repair of skeletal muscle tissue in response to insult is heavily reliant on innate immune populations, namely macrophages. The temporally distinct transition from proinflammatory (M1) to pro-regenerative (M2) phenotypes is crucial for the initiation of an appropriate regenerative response and if disrupted this transition results in debilitating chronic injury and fibrosis. Recent studies have described a role for regulatory T cells (Tregs) in promoting the transition from M1 to M2- mediated phases of repair. Toxoplasma gondii infection results in a non-resolving persistent infection with a preference for tissue encystment in the central nervous system and skeletal muscle. Importantly, infection greatly disrupts peripheral Tregs during peak infection. Here, we use infection with T. gondii as a model to investigate how the inflammatory landscape established during infection in skeletal muscle influences resident Treg physiology and its consequences on the coordination of skeletal muscle repair. Characterization of the resident inflammatory environment reveals a highly Th1-polarized setting in which IFNγ (gamma) is produced by both Τh1 and CD8+ T cells. Early during infection, resident skeletal muscle Treg frequencies dramatically decrease and fail to return to naïve levels during persistent infection (up to day 100 post- infection). Further, infection significantly alters the homeostatic proportions of polarized macrophages leading to a predominance of M1:M2 macrophages that does not return to the steady state levels. Given these observations, we hypothesized that decreased Treg frequencies were responsible for the persistence of elevated M1:M2 proportions, resulting in dysregulated repair. Contrary to our hypothesis that removal of Tregs would deplete M2 macrophage populations, we found that M2 reemerged while M1 were reduced following systemic depletion of Tregs. Furthermore, we observed enhanced muscle fiber regeneration that occurred in the absence of increased inflammation suggesting increased regeneration was not confounded by immunopathology caused by Treg depletion. Ongoing studies are investigating the mechanism by which T. gondii induced resident skeletal muscle Tregs impair the tissue regeneration program.

100. #18 The Development of T Regulatory Cells during Toxoplasma gondii Infection Induced Thymic Atrophy

Elizabeth Schiavoni, Sarah Blair, Richard Jin, Joseph Iloure, and Elizabeth Wohlfert University at Buffalo School of Medicine and Biomedical Sciences, State University of New York

Most vertebrate thymi gradually atrophy with age, affecting the host’s ability to respond to infectious challenge due to a loss of new naïve T cells. A variety of infectious organisms are capable of induc- ing transient thymic atrophy. We study how infection-induced (II) thymic atrophy influences thymic Treg (tTreg) development and function; an unexplored area of broad implications to Treg biology as temporary thymic atrophy occurs in a varied settings such as extreme stress and pregnancy. Previous studies have shown that infecting mice with T. gondii induces thymic atrophy and causes the systemic collapse of Treg populations, making mouse infection with T. gondii an ideal model to study how thymic atrophy may affect tTreg development. We hypothesize the sustained drop in tTregs has a long -term impact on host immune response through changes in the thymic microenvironment and the capabilities of Treg populations. As expected we have found mature single positive CD4+ and CD8+ T cell levels decreased due to T. gondii infection induced thymic atrophy. Interestingly, while CD4+ and CD8+ T cell numbers recover following infection, tTregs have a prolonged impairment in their development suggesting a differential response within the thymus of infected hosts. Histologic analysis of the thymus shows a sustained breakdown in the distinct thymic regions of the cortex and medulla at peak infection and beyond with some structural recovery by seven weeks post-infection. We examined Treg population function by measuring TCR Vbeta diversity and observed changes in Vbeta expression patterns under toxoplasmosis conditions such as increased Vbeta 5.1/5.2 expression in Treg cells in the thymus and spleen, while Vbeta 3 and 14 decreased among Tregs in the thymus and Vbeta8.3 decreased among Tregs in the spleen. Our work contributes key information to understanding Treg development during infection and offers new insights into the recovery of this critical regulatory population from an immune response.

101. #19 Vitamin D deficiency Impairs Innate Memory CD8+ T Cell Development in Early Life

Kristel Yee Mon1, Brian D. Rudd1 1Department of Immunology & Infectious Diseases, College of Veterinary Medicine, Cornell University

Malnutrition is a serious public health issue facing women and children and is linked to increased morbidity and mortality. Many countries have reported a high prevalence of vitamin D deficiency in women of child-bearing age and nursing mothers, with adverse consequences on immune health for the fetus and growing infants. Previous studies have highlighted a role for Vitamin D in adult T cell activation and differentiation during infection, but we lack critical knowledge regarding the influence of Vitamin D on the ontogeny of the CD8+ T cell compartment. Using extensive flow cytometric phenotyping analysis of na- ïve CD8+ T cells during early stages of life, our studies showed that maternal Vitamin D deficiency impairs the development of a specialized subset of cells recently distinguished as ‘innate memory’ cells. Spe- cifically, we found neonatal mice raised on a Vitamin D deficient diet and whose maternal parent was on the same diet contained significantly fewer NKG2A+CD122+ CD8+ T cells compared to those on a control diet. Given that T-bet and Eomes are known to drive the development of this innate memory CD8+ T cell subset in humans, we examined their expression by flow cytometry and observed reduced expression levels of both transcription factors in CD8+ T cells from Vitamin D deficient mice. Lastly, a hallmark fea- ture of innate memory phenotype is their ability to secrete cytolytic molecules in response to proinflammatory cytokine stimulation. To examine whether CD8+ T cells from Vitamin D deficient mice undergo less bystander activation, we stimulated cells in vitro with IL-12 and IL-18 and found that Vitamin D deficient CD8+ T cells produced significantly less GzmA and GzmB. Collectively, this data demonstrates that Vitamin D promotes the development of innate memory CD8+ T cells in neonatal life, thereby high- lighting a potential therapeutic role of Vitamin D supplementation for mothers and neonates. Further studies will determine the relationship between Vitamin D and innate memory cells in conferring immune protection in neonates. We are also currently using RNAseq to establish the mechanistic links between maternal Vitamin D status and the development of innate memory CD8+ T cells in their offspring.

102. #20 Classically Activated Peripheral Macrophages in an Adenosine Poor Environment has a Pertinent Role in Beta Amyloid Clearance in APP Transgenic Mice.

Sudie Ann Robinson, Margaret S. Bynoe Cornell University College of Veterinary Medicine, Department of Microbiology and Immunology, Ithaca NY

Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disease that accounts for approximately 60-70% of dementia cases. Currently, there is no cure for AD and treatment options only tem- porarily alleviate some symptoms. It is becoming apparent that people that consume diets high in fats and simple carbohydrates develop a variety of metabolic diseases and this is associated with higher risk of AD. Previous studies have shown that adenosine receptors (ARs) are pertinent contributors in modulation of cognitive function in AD. Specifically, long term administration of an adenosine receptor antagonist (caffeine) reduced beta amyloid plaque deposition in the brain resulting in increased cognition in an Alz- heimer’s transgenic mouse model. As such, we set out to study the effects of chronic and acute systemic inflammation, induced by high fat diet (HFD), on beta amyloid deposition and clearance via recruited macrophages and microglia in an adenosine rich and poor environment. In this study, we aim to investigate the effects of a high fat diet (HFD) on peritoneal macrophage activation, recruitment to the central nervous system (CNS) and clearance of beta amyloid plaque deposition. We observed that peritoneal macrophages in an adenosine poor environment tend to be more activated than those of their standard diet (SD) counterparts and have increased levels of pro-inflammatory cytokines, including TNF-alpha and IL-1-beta. We hypothesize that this increased activation state has the potential to persist as macrophages are recruited to the CNS in an effort to assist with clearance of beta amyloid deposits. This could account for the decrease in plaque deposition seen in amyloid precursor protein (APP) transgenic mice lacking extracellular adenosine that were fed HFD for an extended period of time. These data suggest that chronic inflammation in the periphery is sufficient to induce neurodegeneration and that extracellular adenosine plays an important role in the removal or attempt at beta amyloid clearance by microglia and recruited macrophages.

103. #21 Immune Protection against Pulmonary F. tularensis Infection

Alicia Soucy and Dennis W. Metzger Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

F. tularensis, the causative agent of tularemia, is a CDC Tier 1 agent, and inhalation of only 10 CFU can result in fatal disease. The F. tularensis tularensis (SchuS4) strain is highly virulent when in- haled, and can cause severe disease in humans. Currently, there is no licensed vaccine against this pathogen; however, it is known that vaccinating BALB/c mice with a less virulent F. tularensis holartica strain (LVS) can protect against pulmonary challenge with SchuS4; however, vaccination does not protect C57Bl/6 mice. We hypothesize that as a result of vaccination, BALB/c mice establish a strong recall immune response, which results in the control of SchuS4 infection. To test this, we primed and boosted mice intranasally with LVS. Following vaccination, BALB/c mice had high levels of both B cells and effector memory CD4+ T cells in the lungs. Compared to C57Bl/6 mice, BALB/c mice also had higher levels of Francisella-specific IgG in the blood 21 days after LVS immunization. Following challenge with SchuS4, LVS-vaccinated BALB/c mice produced greater levels of IL-12p40, IL-17, IL-22, and the antimicrobial peptide, S100A9. In contrast, C57Bl/6 mice produced greater levels of pro-inflammatory cytokines later in infection. Histological analysis showed that BALB/c mice had less lung necrosis at day 8 following SchuS4 challenge compared to C57Bl/6 mice. BALB/c mice also had less total protein in the bronchoalveolar lavage fluid by day 10 post-infection. These findings suggest that BALB/c mice mount a stronger recall response compared to C57Bl/6 mice, which aids in controlling SchuS4 and tissue damage. Future studies are being conducted to determine the precise mechanisms responsible for protection of BALB/c mice from pulmonary SchuS4 infection. (Supported by NIH P01AI056320)

104. #22 TGF-β Production Associated with Allergic Asthma Prevents Influenza Virus Induced Immunopathology

Yoichi Furuya and Dennis Metzger Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY 12208

Asthma is believed to be a risk factor for influenza infection, however little remains known about how the asthmatic phenotype influences anti-viral immune responses. We therefore investigated the impact of asthma on susceptibility to influenza infection by using a mouse model of allergic airway inflammation. Unexpectedly, asthmatic mice were highly resistant to primary infection with the H1N1 2009 pandemic strain. Notably, the increased resistance was not attributed to enhanced viral clearance but instead was due to reduced lung inflammation. Asthmatic mice exhibited a significantly reduced cytokine storm as well as reduced total protein levels and cytotoxicity in the airways, indicators of decreased tissue injury. Further, we show that asthmatic mice have significantly increased levels of TGF-β1 and that the heightened resistance of asthmatic mice is abrogated in the absence of TGF-β receptor II. Thus, we conclude that the transient increase in TGF-β expression following an acute asthma episode is responsible for temporary protection via suppression of influenza-induced immunopathology.

105. #23 Next Generation Amplicon Sequencing Accelerates Bovine Leukocyte Antigen Class I Typing and

Provides New Insight to Expressed Gene Diversity

Mital Pandya1, Korin Eckstrom1, James Vincent2, William T. Golde3, and John Barlow1* 1University of Vermont, Burlington, Vermont; 2Vermont Genetics Network, Burlington, Vermont, 3Plum Island Animal Disease Center, Agricultural Research Service, USDA, Greenport, New York

Major histocompatibility complex (MHC) class I molecules are critical in the induction of adaptive immune responses of mammals, including cattle. Current techniques for identification of expressed alleles within a herd of cattle are time consuming and costly. Recent advances in next generation sequencing (NGS) technologies allow for the sequencing of highly polymorphic regions of the genome, such as the MHC locus. These high-throughput methodologies have been applied to several species, such as humans, nonhuman primates, and swine, but have not been widely used in cattle. Herein, we described a compre- hensive method for genotyping bovine leukocyte antigen (BoLA) from 27 Holstein cattle by using Illumina MiSeq, cloning and Sanger sequencing, and polymerase chain reaction sequence-specific primers (PCR- SSP). These methods are used to evaluate either a 375-bp complementary DNA (cDNA) PCR amplicon spanning the highly polymorphic peptide binding region or the approximately 1kb full-length BoLA class I region. We present a streamlined bioinformatics pipeline for analyzing Illumina MiSeq data. Although each genotyping method described above has potential errors, Illumina MiSeq provided the best depth of coverage for the alleles found in this herd. The present study demonstrated Illumina MiSeq can be an effi- cient genotyping platform for complex, multilocus systems such as cattle MHC. While Illumina MiSeq provides a viable alternative to known methods, quality checking with stringent requirements is needed to differentiate artifacts from true alleles. This approach improves our understanding of the genetic specificity of BoLA, lays the foundation for studying the cell-mediated immune responses, and enhances vaccine design; improving ongoing efforts to protect cattle from devastating infectious diseases.

106. #24 Development of an Adjuvant-Free Intranasal Vaccine Platform Via Targeting Fc Receptors

Nicole Nelson, Li Barie, & Edmund J. Gosselin Albany Medical College

The first line of defense against most pathogens is located at mucosal sites such as the nasal tissue and oral mucosa. Although there are multiple vaccine strategies in use today to help prevent mucosal pathogens from causing infection, very few produce optimal immune protection at mucosal sites. In addition, most vaccines require adjuvant which contains safety concerns such as toxicity. Therefore, new vaccine platforms need to be designed to generate strong mucosal immunity, peripheral immunity, and protection without adjuvant. We have previously shown, using a novel fusion protein (FP) construct, that targeting protein antigen to Fc receptors intranasally not only circumvents the need for adjuvant, but also generates mucosal immunity and protection against the extracellular mucosal pathogen Streptococcus pneumoniae. However, optimal protection with this FP design requires three separate administrations. In this study, we plan to determine the mechanism by which protection is generated after administration of the FP. The outcome of this study will therefore allow us to improve upon the original FP design and develop optimal protection using fewer administrations. To date the FP has been verified to be present in transfected cell su- pernatants and purified via nickel affinity chromatography. Next, the protein will be fluorescently labeled and followed in vivo with fluorescent microscopy and flow cytometry to determine the in vivo trafficking of the FP.

107. #25 Type I IFNs Directly Promote Hematopoietic Progenitor Cell Death and Lead to Bone Marrow

Failure in Severe Bacterial Infection

Julianne N.P. Smith, Amanda McCabe, Katherine C. MacNamara Albany Medical College, Albany NY

Life-long blood cell production and immune function requires an appropriate balance between hematopoietic stem and progenitor cell (HSC/HSPC) fate choices. Although HSCs exist as rare, quiescent cells within the bone marrow (BM) at steady-state, they retain the potential to rapidly proliferate and differentiate in response to hematopoietic demand, such as occurs in acute infection. HSPC proliferation increases immune output but can impair stem cell function. Type I interferons (IFNα/β) modulate immune responses to most pathogens and can regulate HSPC proliferation and death in vitro and upon non- infectious immune-stimulation in vivo, however the impact of type I IFNs on HSPC response to infection remains unclear. To determine how IFNα/β impacts HSPC regulation during infection, we utilized a shock- like infection model in mice challenged with the tick-borne Ixodes ovatus ehrlichia (IOE) bacteria. We recently reported that disease severity was dependent upon the robust production of IFNα/β. At the peak of IFNα/β induction, myeloid progenitors and HSCs were depleted in wild-type (WT) BM, but were significantly protected and even expanded in mice lacking the IFNα/β receptor (IFNaR1). IFNα/β also limited infection-induced extramedullary hematopoiesis, as measured by overall splenic cellularity and myeloid progenitors. To determine whether IFNα/β depletes HSPCs via a reduction in cellular proliferation and/or an increase in cell death, we examined the cell cycle status and viability of WT and Ifnar1-/- BM cells during IOE infection. HSPC cycling was increased by infection, though significantly more cells cycled in the absence of IFNaR1. HSPCs in Ifnar1-/- mice were also less prone to IOE-induced death. Although IFNα/β did not directly deplete quiescent HSCs, nor did it impair cell cycle entry, IFNα/β directly promoted necrotic death of progenitor cells. In contrast, progenitors that lacked IFNaR1 were largely protected and preferentially underwent apoptosis during IOE infection. To determine the long-term impact of infection on HSPC function, we competitively transplanted sorted HSPCs from infected WT and Ifnar1-/- mice and measured hematopoietic repopulating activity in myeloablated recipients. WT HSPCs displayed a progressive, multilineage repopulating advantage over the Ifnar1-/- HSPC pool, which was initially superior but became exhausted throughout transplantation, consistent with more abundant hematopoietic progenitors that lack long-term activity. These data reveal an important dichotomy in the impact of IFNα/β during severe bacterial infection. We have shown that IFNα/β directly sensitizes hematopoietic progenitors to necrotic cell death, but does not directly target long-term HSCs, during IOE infection. Indeed, our data demonstrate that IFNα/β plays a critical role in reestablishing hematopoietic homeostasis in settings where the infectious challenge is resolved.

108. #26 The Role of the Aryl Hydrocarbon Receptor and the Gut Microbiome in the Modulation of Intestinal Inflammation

Excessive dysregulation of the commensal gut microbiota is known to cause an excessive immune response leading to chronic intestinal inflammation ultimately causing generation of lesions in the mucosa (e.g., inflammatory bowel disease). Recent data has shown that the aryl hydrocarbon receptor (AHR) could be an untapped target for modulating the commensal gut microbiota as well as intestinal inflammation. To dissect this issue, male C57BL/6J mice were fed control (devoid of known AHR ligands), indole-3-carbinol (I3C), and beta-naphthoflavone (BNF) containing diets for three weeks. Mice were placed in metabowls for fecal sample collection and later euthanized to harvest tissues and biofluids. 16S rRNA gene QPCR of cecal matter revealed changes in total bacterial composition, in I3C treated mice. Liquid chromatography coupled with mass spectrometry-based metabolomics of fecal matter revealed significant excretion of bile acids. 1H nuclear magnetic resonance (NMR)-based metabolomics analysis of feces revealed profound changes in bile acids as well as glucose metabolism and bacterial fermentation products. AHR ligand containing diets caused profound changes in the expression of immunologic genes (e.g., Il-6, Il-10). Exposure to these diets displayed protective effects in intestinal inflammation (e.g., intestinal injury models) and weight loss. Overall, our data suggests that AHR ligands in the diet are capable of modulating the commensal gut microbiota as well as their metabolic function. Furthermore, our data suggests that the effects are ligand dependent. Most interestingly, the metabolomics experiments reveal changes in the commensal gut microbiota energy metabolism which could be due to a decrease in the gut microbe populations. Finally, work of this type could lead to the development of safe, low-cost, and natural dietary interventions to treat chronic gut inflammation.

109. #27 Regulation of IgM+ Memory B-cells by the Inhibitory Receptor FcgRIIb

Amber Papillion and Gary Winslow Upstate Medical University, Syracuse, NY

Our laboratory has described a long-term IgM+ memory population in the spleens of mice infected with Ehrlichia muris (J. Immunol. 191:1240). Among the many surface markers that distinguish the memory cells from conventional B cells is the inhibitory receptor FcgRIIb, which exhibited 4-fold higher expression, relative to conventional B cells. We hypothesized FcgRIIb negatively regulates IgM+ memory cells by binding immune complexes present during low-level chronic infection. To investigate this question, we monitored the IgM+ memory cell population in infected FcgRIIb-deficient mice. Thirty days post- infection, the IgM+ memory B cells were increased by 4-fold in frequency in FcgRIIb-deficient mice, compared to control mice. This increase in the frequency of spleen IgM+ memory cells was due to an increase in the number of IgM+ memory cells, and not simply a decrease in the number of other B cells present. FcgRIIb-deficient mice also produced IgM+ memory cells six days earlier than controls. Preliminary studies revealed the IgM+ memory population generated in FcgRIIb-deficient mice was identical in phenotype to IgM+ memory cells generated in infected wild-type mice. The increase in the IgM+ memory population was associated with an increase in antigen-specific IgG. These data indicate that FcgRIIb plays an important role in regulating the expansion and/or persistence of IgM+ memory cells. We propose the inhibitory receptor acts to limit the contribution of IgM+ memory cells to recall responses under conditions where antigen-specific IgG is sufficient to control infection.

110. #28 Theiler’s Murine Encephalomyelitis Virus Infection of Macrophages Depends on M1-Differentation

Karin M. Schneider, Neva B. Watson and Paul T. Massa SUNY Upstate Medical University, Syracuse NY

Macrophages are common targets for infection by many pathogenic viruses including Theiler’s Mu- rine Encephalomyelitis Virus (TMEV). Recent studies in our laboratory suggest that granulocyte- macrophage colony-stimulating factor (gm-CSF) positively influences viral infection of macrophages compared to macrophages differentiated under the influence of macrophage colony-stimulating factor (m-CSF). GM-CSF is an inflammatory cytokine shown to prime monocytes towards inflammatory M1-like macrophages to a greater degree than that seen with macrophage colony-stimulating factor (m-CSF). However, exactly how these phenotypic properties including secretion of proinflammatory cytokines and increased phagocytic activity may increase macrophage susceptibility to virus infection is not well understood. To study this further, C3H bone-marrow cells were differentiated in culture with either the standard protocol using recombinant m-CSF or gm-CSF for 6 days, and then inoculated with TMEV (BeAn) and further incubated for 0, 12, 24 or 48hrs. RNA or protein analysis, virus titers, flow cytometery and virus binding assays were performed to characterize the virological parameters between the different macrophage cultures. Our analysis of gm-CSF-differentiated, TMEV-infected macrophages showed higher levels of viral RNA and proinflammatory cytokines and molecules compared to infected m-CSF and mock treated cells. Based on our results, the higher susceptibility of GM-CSF-differentiated macrophages to TMEV-infection is due to an increase in virus uptake rather than receptor binding. Thus, TMEV appears to prefer an M1-skewed phenotype for replication that is stimulated by gm-CSF compared to m-CSF differentiation profile. In conclu- sion, we provide compelling evidence implicating a role for gm-CSF in increasing macrophage susceptibility to TMEV infection most likely by increasing virus uptake rates that may be applicable to other macrophage-tropic virus infections.

111. #29 Glycogen Metabolism Supports Effector Function and Energy Homeostasis of Dendritic Cells

Phyu Thwe, Saritha Beauchamp, and Eyal Amiel University of Vermont, Burlington, VT 05405

Dendritic cells (DCs), professional antigen presenting cells of the immune system, serve as a bridge between the innate and adaptive immune responses. Activation of DCs by a stimulus through toll-like receptors (TLRs) is coupled with an increase in energy demand, which is fulfilled by a TLR-driven burst in glycolytic metabolism. Up-regulation of glycolysis in activated DCs provides these cells with molecular building blocks and the energy associated with DC effector function. Inhibition of glycolysis impairs the survival and effector function of activated DCs. In the prevailing model in the field of immuno-metabolism, TLR-driven glycolysis in DCs is thought to be sustained primarily by an increase in glucose uptake. While non-immune cells, such as hepatocytes and muscle cells, store glucose in the form of glycogen as an intracellular energy reserve, the role of glycogen metabolism in DCs has not been published. Our data indicate that glycogen metabolism regulates and supports the effector function of DCs. We show that DCs express the enzymes essential for glycogen metabolism and that glycogen metabolism is regulated upon TLR stimulation. We also show that the inhibition of glycogen metabolism impairs activation of these cells. These data indicate that glycogen metabolism in DCs plays an important role in energy homeostasis of these cells to support the effector function. Understanding the role and regulatory mechanisms of glycogen metabolism in DCs has important implications for the development of enhanced cellular based immunotherapy in the future.

112. #30 Stress Proteins in the Extracellular Environment: a Novel Class of Immunomodulator

Sadikshya Bhandari, Xiuyun Yin, Michael A. Lynes, University of Connecticut, Storrs CT

Metallothioneins (MTs) are small, cysteine rich stress proteins that play a central role as a reservoir of essential divalent heavy metal cations such as zinc and copper, and also can diminish the effects of toxic heavy metals such as mercury and cadmium. Historically, MT has been considered to be an intracellular protein with roles to play in the management of heavy metals, as a regulator of cellular redox potential, and as a buffer of free radicals. The presence of MT in a variety of fluids and extracellular spaces (e.g. serum, urine, milk, prostatic fluids, and bile, and in liver sinusoids, bronchoalveoli, and pancreatic acini) has suggested an additional extracellular role for this protein. We have previously shown that MT can bind to the surface of lymphocytes, is a potent chemoattractant for Jurkat T cells (ATCC TIB-152), and that this effect can be blocked with antagonists of G-protein coupled receptors. Intriguingly, when Jurkat T cells are pre- incubated with MT, there is a decline in the subsequent chemotactic response to SDF 1- A similar capacity to block SDF-1 effects was observed with the breast cancer cell line, MDA-MB 231 (ATCC HTB- 26). These influences of MT on chemotaxis suggest that MT released under cellular stress can play an im- munomodulatory role in inflammatory disease, and that is may also influence the progression of metastatic cell movement. If so, manipulations of these phenomena may represent a pathway for therapeutic intervention.

113. #31 Tumor-derived TSLP Enhances Macrohage-mediated Metastasis

Burkard-Mandel, Lauren & Abrams, Scott Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263

114. #32 IRF8 Transcriptionally Regulates the Macrophage Response during Tumor Immunosurveillance

Twum, Danielle & Abrams, Scott I. Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263

The macrophage response to pathological insults, especially in neoplasia, is regulated by transcriptional players that are activated after exposure to soluble factors present in their microenvironment. Conse- quently, macrophages are able to differentiate into two distinct phenotypes, M1 and M2. M1 macrophages are defined as ‘immune stimulatory’ cells and are associated with iNOS and IL-12 production, while M2 macrophages can be defined as ‘suppressive’ cells associated with Arg1, a T cell suppressor, and IL-10, an anti-inflammatory cytokine. In cancer, notably breast carcinoma, a high infiltration of macrophages is associated with reduced overall survival as well as progression to metastasis. Recent evidence suggests that as tumor burden increases, the M1 to M2 ratio skews significantly towards an M2-bias, co-opting their anti- inflammatory and wound healing abilities to favor tumor growth. Although several transcriptional regula- tors that drive the M2 phenotype in the tumor microenvironment have been identified, transcription factors that drive or enforce the M1 phenotype are not well defined. IRF8 (Interferon Regulatory Factor 8) is a transcriptional regulator of myeloid differentiation and is critical for the expression of genes such as iNOS and IL-12p40, hallmark markers of the M1 phenotype. We hypothesize that IRF8 is important in determining the macrophage M1 vs M2 response in mammary cancer. To test this hypothesis, we made use of an IRF8 knockout mouse model, whereby an IRF8flox/flox strain was crossed with the LysM/Cre strain, to generate progeny that specifically lacked IRF8 expression in the macrophages. As a result, we observed an increased rate of tumor growth in IRF8-/- mice compared to the wild-type controls using an orthotopic im- plantable model of mouse mammary carcinoma. Moreover, crossing the IRF8-/- mice with MMTV-PyMT mouse model of autochthonous mammary tumor development, we observed that deletion of IRF8 in macrophages enhanced spontaneous pulmonary metastasis. Altogether, these data indicate that IRF8 expression in macrophages can delay tumor onset and/or reduce tumor progression to metastasis, likely through altering the tumor-suppressing (M1) to tumor-promoting (M2) phenotypes. Thus, IRF8 may represent a poten- tially novel therapeutic target to modulate the macrophage phenotype in neoplastic diseases, such as mammary cancer, whereby this myeloid response is a critical determinant of outcome.

115. #33 Fetal Hematopoietic Stem Cells Give Rise to a Unique Population of CD8+ T-cells

Jocelyn Wang1, Erin Wissink2, Norah L. Smith3, Andrew Grimson2, and Brian D. Rudd3 Field of Pharmacology1 Department of Microbiology and Immunology2, Biological and Biomedical Sci- ences Graduate Program. Department of Molecular Biology and Genetics3, Cornell University, Ithaca, NY 14853

Neonatal infection is a major cause of morbidity and mortality worldwide. While adults generate robust immunity to most intracellular pathogens, neonates have impaired ability to generate long-lasting immunity. We recently showed that neonatal CD8+ T cells fail to become memory cells because of an inherent propensity to rapidly proliferate and become terminally differentiated. This work has clearly demonstrated that neonatal CD8+ T cell are intrinsically different than their adult counterparts, but the underlying basis for these age-related differences has remained an open question. From a reductionist perspective, it is apparent that a number of basic models might explain why neonatal CD8+ T cell adopt different fates compared to adults during infection. First, neonatal CD8+ T cells may be less likely to develop into memory CD8+ T cells because the starting population has undergone more extensive homeostatic proliferation and may be significantly more differentiated than adults prior to infection (‘proliferation model’). Another possibility relates to the fact that neonatal and adult CD8+ T cells are derived from distinct hematopoietic stem cell populations that are metabolically and genetically very different (‘origin model’). To discriminate between these models, we directly compared the genetic profiles and behavior of neonatal and adult CD8+ T cells that had undergone similar amounts of homeostatic proliferation in the periphery or were at the same stage of development in the thymus. Interestingly, different aged CD8+ T cells that had undergone similar amounts of HP still expressed different gene profiles, which suggest that HP cannot fully explain the differences between adult and neonatal CD8+ T cells. Additionally, equivalent phenotypic subsets of peripheral CD8+ T cells expressed different phenotypic markers, responded differently to pro-inflammatory cytokines in vitro as well as infection in vivo. Next, we asked whether neonatal CD8+ T cells behave differently because of their different origin. To answer this question, we compared the gene expression profiles of single positive CD8+ T cells in thymus form neonatal and adult mice and found that they possessed distinct gene expression profiles. Given that neonatal CD8+ T cells exhibit a unique gene expression profile at the time they are initially created, we believe this data lends support for the origin model and suggests that neonatal CD8+ T cells respond differently to infection because they are sculpted from a different progenitor cell. In ongoing studies, we are co-transferring fetal and adult progenitor cells into adult thymii to confirm that neonatal CD8+ T cells do in fact represent a distinct lineage of CD8+ T cells. This data will also be presented and discussed.

116. #34 Characterizing Natural and Induced Regulatory T cell Response During Autochthonous Melanoma Tumorigenesis

Tamer B. Chabanet, Shannon M. Steinberg, Andrea Boni, Peisheng Zhang, Brian T. Malik, Mary Jo Turk Geisel School of Medicine at Dartmouth. Lebanon, NH 03766

Funding: NCI 2R01 CA12077-06; 5T32AI007363-23; ACS RSG LIB-121864

117. #35

The Prostaglandin D2 Receptor CRTH2 Regulates Innate Immune Responses during

Type 2 Inflammation

Elia D. Tait Wojno Baker Institute for Animal Health and Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, NY

Type 2 inflammation at mucosal surfaces such as the lung and intestine mediates protective immunity to helminth parasites and contributes to allergic disease. Type 2 inflammation is characterized by production of bioactive lipids such as prostaglandin D2 (PGD2) and epithelial cell-derived cytokines such as interleukin (IL)-33 that activate innate immune cells and CD4+ T helper type 2 (Th2) cells to produce type 2 cytokines, including IL-4, IL-5, and IL-13. These cytokines act in turn on epithelial cells, promoting mucus production, changes in epithelial cell turnover, and alterations in epithelial barrier permeability. Group 2 innate lymphoid cells (ILC2s) have recently been highlighted as key innate immune cells associated with type 2 immunity and inflammation, but the lipid and cytokine pathways that control ILC2 responses in inflamed tissues are not completely understood. Here, we show that the bioactive lipid PGD2 receptor chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2) regulates ILC2 accumulation in the lung in vivo. The frequency of ILC2s that expressed CRTH2 was significantly higher in healthy human and murine peripheral blood than in the lung, suggesting that regulation of CRTH2 expression might be associated with ILC2 accumulation in the lung. Consistent with this, CRTH2- expressing murine ILC2s accumulated in the lung in response to PGD2. Further, CRTH2-deficient mice exhibited reduced ILC2 accumulation and helminth-induced type 2 inflammation in the lung compared to wild-type mice. Critically, adoptive transfer of CRTH2-sufficient ILC2s restored helminth-induced pulmonary inflammation in CRTH2-deficient mice. Together, these data suggest that the PGD2-CRTH2 pathway regulates ILC2 accumulation and type 2 inflammation in the lung in vivo.

118. #36 Total Body Irradiation Weakens the Cutaneous Immune Response to Candida ablicans Infections

Margaret L. Barlow, Constantine G. Haidaris, Julie L. Ryan, Scott A. Gerber, and Edith M. Lord University of Rochester Medical Center, Rochester NY

Radiation exposure results in several health complications, including a weakening of the immune system due to loss of precursor cells in the bone marrow and altered function of immune cells resident in other tissues, including the skin. Several immune cell populations reside in the skin, which serve an important immunological barrier function. In a previous study, we observed that a sub-lethal radiation dose resulted in a reduced concentration of cutaneous dendritic cells (cDCs) that persisted for several weeks. As these are potent antigen-presenting cells, we hypothesized this could leave the skin more vulnerable to infection following radiation. In immunocompromised situations such as this, commensal organisms often cause opportunistic infections. One such skin-resident organism is Candida albicans. To test this hypothesis, we infected hairless mice with C. albicans following a sub-lethal 6 Gy dose of total-body irradiation (TBI). Mice were infected intradermally in the ear with C. albicans at various time points following irradiation. Afterward we assessed the severity of infection at the local skin site and in the kidneys as a re- flection of systemic dissemination. When the mice were infected seven days post radiation, at a time when the cDCs were maximally depleted, we observed local control at the skin site, but greater dissemination to the kidneys in irradiated mice. Interestingly, if challenged at later time points, there was no increase in dissemination, indicating that the radiation effect is acute. As expected, we also observed a reduction of circulating leukocytes following radiation, at the acute infection end point. Upon infection, we assumed these populations would increase in response to the organism. However, this reduction persisted in irradiated mice, which could reflect a loss of precursor cells due to radiation-induced bone marrow damage. To further characterize the local response, we examined the infected ear tissue by measuring the populations of immune cells present. We observed a reduction of CD4+ and CD8+ T cells, with an increase in granulocytes, in irradiated mice after infection. To examine the possible mechanisms responsible for the reduction of T cells, we also measured the expression of various cytokines and chemokines in infected ears. Several molecules involved in T cell infiltration and function, including interferon gamma (IFNγ), CXCL9, and IL-9, were decreased. These changes suggest that a sub-lethal dose of radiation is able to alter the immune environment of the skin, leaving an individual at a greater risk of opportunistic fungal infection.

Supported by NIH Grants: R01 CA28332, U19AI091036, and T32AI07285

119. #37 Inflammatory Monocytes in a Changing Tumor Microenvironment Following Radiotherapy:

Implications for Immunotherapy

Colorectal cancer (CRC) is responsible for approximately 500,000 deaths each year worldwide. Rapid progression of the primary tumor often leads to escape and distal metastases. Accordingly, radiotherapy (RT) is often required as a means to control the growth of the primary tumor and to reduce local recurrence. Unfortunately, therapies such as RT are often ineffective on patients with advanced stage disease creating a critical need for more effective treatments to improve tumor control and survival rates for CRC. We have established a mouse model of CRC in which Colon38, a colon adenocarcinoma cell line, is injected into syngeneic C57BL/6 mice and treated with local RT. Our data have demonstrated that the immune system mediates many of the anti-tumor effects of RT. However, the immunostimulation provided by RT appears to be a double-edged sword as we observe a concurrent increase in myeloid cells that have been associated with immunosuppression and poor prognosis. One population of myeloid cells, inflammatory monocytes (CD45+, CD11b+, Ly6chi, Ly6g-), has been shown to mediate immunosuppression in multiple tumor models. The chemokine CCL2 and its cognate receptor CCR2 on these monocytes facilitate the infiltration of these cells into tumors. Consequently, therapy that inhibits entry of these cells into tumors has resulted in remarkable clinical outcomes. However, the role of inflammatory monocytes in the context of RT and CRC is unknown. Here we have shown that RT upregulates the expression of intratumoral CCL2, and subsequently induces the infiltration of CCR2+ monocytes. Using a genetic approach, the absence of CCR2+ monocytes resulted in reduced tumor growth in CCR2KO mice when compared to WT. However, pharmacologic inhibition of CCR2 administered immediately following RT unexpectedly diminished the effectiveness of therapy resulting in increased tumor growth when compared to RT-treated control mice. Based on these data, we hypothesize that the role of inflammatory monocytes in disease progression is dependent on the inflammatory state of the tumor microenvironment to which they migrate. In support of our hypothesis, RNA-seq data from ex vivo sorted inflammatory monocytes demonstrated drastically changed genetic profiles of these phenotypically identical cells at different time points after radiotherapy. Therefore, a better understanding of the biology behind the plasticity of these inflammatory monocytes throughout treatment is important to inform future clinical trials involving monocyte-targeted immunotherapies. Future studies, using a small molecule inhibitor of CCR2, will explore the efficacy of this immunotherapy and identify an optimal schedule of administration. Further, we plan to elucidate the role of the microenvironment in promoting the potential anti-tumor function of these cells by administering agents that induce a proinflammatory tumor microenvironment, such as IL-12.

120. #38 Mucosal Vaccine Adjuvant Cyclic di-GMP Induces STING-Dependent Pulmonary Dendritic Cell Activation

Samira Mansouri, Steven M. Blaauboer, and Lei Jin Albany Medical College, Albany NY

Vaccination is the best tool to prevent infectious diseases. A majority of infectious microorganisms either colonize or cross mucosal surfaces to enter the host. Mucosal vaccinations prevent the onset of disease and block early colonization, and reduce the risk of horizontal transmission, thus, are superior to the current systemic vaccinations. The challenge for mucosal vaccinations, however, is to develop safe and effective mucosal adjuvants. Cyclic di-GMP (CDG) is a promising mucosal vaccine adjuvant candidate that elicits protective immunity against Steptococcus pneumonia and Staphylococcus aureus. Our lab showed that the mucosal adjuvant activity of CDG is mediated by STING-induced TNF signaling in vivo. STING (stimulator of interferon genes) is a receptor for CDG. We investigated the role of STING in mediating the mucosal adjuvant activity of CDG in vivo and our results demonstrate that CDG enhances antigen uptake, processing, and activation in pulmonary dendritic cells, leading to the production of TH polarizing cytokines. Cyclic di-GMP induced antigen uptake and activation was dependent on STING expression in CD11c+ cells, indicating that this STING dependence is intrinsic to dendritic cells. Our results uncover a promising new mucosal vaccine strategy that optimizes antigen uptake and processing by pulmonary dendritic cells to elicit T cell responses.

121. #39 STING Mediated TI-2 Antigen-induced Antibody Response Depends on the Route of Administration

Seema Patel, Jesse Rabinowitz, and Lei Jin Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY

Antibody responses can be classified into two distinct categories: T cell-dependent (TD), whereas B cell receives help from a T cell during stimulation, and T cell-independent (TI), whereas B cell is stimulated without T cell help. A TI type 1 (TI-1) response is elicited by microbial ligands that involve signaling through the B cell receptor (BCR) and Toll-like receptors (TLRs). A T cell-independent type 2 (TI-2) responses is typically elicited by multivalent, highly repetitive motifs, such as bacterial capsular polysaccha- rides and viral capsids. TI-2 antigens bind to BCR with extensive crosslinking, resulting in prolonged and persistent signaling and ultimately B cell activation [1]. Multiple polysaccharide vaccines have been licensed in U.S. They include pneumococcal vaccine Pneumovax23, meningococcal vaccine Menomune, and Salmonella typhi vaccine Typhim Vi. Recent research suggests that 4-hydroxy-3- nitrophenylacetyl Ficoll (NP-Ficoll), a model TI-2 antigen, up-regulate endogenous retrovirus (ERV) RNAs and activates nucleic acid sensors like cGMP-AMP Synthase (cGAS) upon intra-peritoneal (i.p) administration. cGAS is known to activate the Stimulator of Interferon Gene (STING/MPYS) pathway [2]. In this study we sought to find out if STING is activated against NP-Ficoll, a TI-2 antigen, when administered via the intramuscular route, which is clinically more relevant. Our results show antibody response to NP-Ficoll is STING dependent upon intra peritoneal and STING independent upon intramuscular administration of antigen. We concluded that the STING-mediated TI-2 response depends on the immunization route.

122. #40 Indoleamine 2,3- Dioxygenase Regulates Density of Tumor Infiltrating CD8+ T cells in a Murine Model of Ovarian Cancer

123. #41 CD28 Regulates Metabolic Fitness for ROS Dependent Long Lived Plasma Cell Survival

Adam Utley, Daniela Ventro, Louise Carlson, and Kelvin Lee Roswell Park Cancer Institute

Sustained humoral immunity is dependent upon the production of neutralizing antigen-specific antibodies by plasma cells. Upon antigenic exposure, activated B cells differentiate into short-lived plasma cells (SLPCs) which home to secondary lymphoid organs such as the spleen where they live for days to weeks before dying by apoptosis. However, there are examples in the human population in which neutralizing antibody titers persist despite a lack of continual antigen exposure. Therefore an expanded model has been proposed wherein activated B cells differentiate into long-lived plasma cells (LLPCs) that home to the bone marrow and survive indefinitely. They are not intrinsically long-lived, but rather depend upon extrinsic survival signals within specialized niches for their persistence and long-term antibody production. Furthermore, many of these survival signals are shared with the malignant counterpart in Multiple Myelo- ma (MM). Our work centers on the cellular and molecular mechanisms by which MM and LLPCs survive in the bone marrow microenvironment. CD28 is best characterized as the canonical co-stimulatory molecule in T cells and is required for T cell metabolic fitness. Plasma cells and MM are highly biosynthetic and require metabolic fidelity. Howev- er, the molecular and metabolic pathways that provide this CD28-mediated survival advantage in LLPCs/ MM are not well understood. Here we demonstrate that CD28 induces mitochondrial respiration in both LLPCs and MM, but not in SLPCs. Furthermore, CD28 induces mitochondrial biogenesis in LLPC and MM. By using oxygen consumption as a direct readout of mitochondrial respiration, we show that CD28 activation increases oxidative phosphorylation. A major byproduct of respiration is the production of reactive oxygen species. As expected, CD28 activation induces ROS in LLPCs specifically. ROS is a well-characterized cell-damaging agent. In order to elucidate the effects of ROS on LLPCs, we specifically inhibited ROS produced by the mitochondria with MnTBAP. Paradoxically, inhibiting ROS prevents CD28-mediated survival. Taken together the data suggest that CD28 increases mitochondrial respiration for ROS-dependent survival. By analyzing open-source RNAseq data acquired from purified populations of bone marrow LLPCs and splenic SLPCs, the LLPC population exhibits higher transcript levels of genes associated with mitochondrial respiration, oxidative phosphorylation, and the TCA cycle. However, the signals that drive this metabolic program are not characterized. Taken together the data suggests that CD28 may regulate LLPC survival and function through Slp- 76 mediated metabolic reprogramming. This makes CD28 an attractive target in vaccine design and the treatment of both autoimmune disorders and multiple myeloma.

124. #42 Macrophage Lipid Accumulation is Regulated by Substrate Stiffness

Erika Gruber, Sandra Stelzer, Emma Ehrlich, Siddhartha Sinha, Cynthia Leifer Department of Microbiology & Immunology, Cornell University, Ithaca NY

125. #43 A Novel Francisella tularensis Live Vaccine Strain Expressing Ovalbumin Reveals Memory and

Effector CD8 T Cell Responses during Vaccination and Re-challenge

David Williamson1,2, David Place1,2, Yevgeniy Yuzefpolskiy3, Kalyan Dewan1, Rachel Markley1,2, Bhuvana Katkere1, Surojit Sarkar 3, Vandana Kalia3, Girish Kirimanjeswara1. 1The Dept. of Veterinary and Biomedical Science, 2Immunology and Infectious Disease Graduate Pro- gram, The Pennsylvania State University, University Park, PA. 3Department of Immunology, University of Washington, Seattle, WA.

Francisella tularensis is a gram negative bacterial pathogen capable of causing lethal disease in humans via the respiratory route. Intra-nasal vaccination with an attenuated live vaccine strain (LVS) pro- tects mice from subsequent challenge with the highly virulent Schu S4 strain, however intradermal vaccination is not protective. To-date, understanding of the immune responses associated with protective immunity has been hampered by a lack of tools to identify pathogen-specific T cells. In this work, we have developed a novel tool for studying CD8 T cell responses to the Francisella tularensis Live Vaccine Strain (LVS) using a codon-optimized fragment of ovalbumin (OVA), a model antigen, tethered to Francisella vgrG, a protein secreted by the type VI secretion system. We have shown that Francisella-specific CD8 T cells expand after the first week of infection and persist within the local lung environment and spleen for ten weeks beyond clearance of the bacterium. Kinetics of CD8 effector (Teff) and central (Tcm) memory cells were also examined in lung, draining lymph node, and spleen throughout a primary infection and rechallenge with a lethal dose of LVS-OVA. We also show that Francisella-specific CD8 T cells produce IFNγ but not IL-17A during primary and recall responses. Preliminary data indicate that a higher number of LVS-OVA specific CD8 T cells are found in the lungs following intranasal vaccination as compared to intradermal. Together, our data show that LVS-OVA can be used to study multiple aspects of Francisella- specific CD8 T cell responses, which will enable us to develop more protective vaccines for tularemia.

126. #44 IFN Restricts Group II Innate Lymphoid Cells and Causes Increased Susceptibility to

Lethal Influenza A Infection

Danielle Califano, Yoichi Furuya and Dennis W. Metzger Center for Immunology & Microbial Disease, Albany Medical College, Albany, NY 12208

Influenza A viruses are the leading cause of human respiratory infections, resulting in a significant number of hospitalizations and deaths each year. Influenza viruses elicit substantial production of IFNγ, however most studies indicate that IFNγ does not influence survival following influenza infection. Recent- ly, IFN was shown to inhibit IL-33-dependent activation of group 2 innate lymphoid cells (ILC2s), during both helminth infection and fatty acid metabolism. The role of IFN in ILC2 regulation during influenza infection is unknown. ILC2s are activated in response to IL-33 or IL-25 to promote rapid production of IL-5 and IL-13, leading to eosinophil recruitment, mucus secretion, goblet cell hyperplasia and airway hyper-reactivity (AHR). Furthermore, ILC2s secrete the epidermal growth factor amphiregulin, which promotes epithelial cell proliferation and lung regeneration following virus-induced tissue damage. ILC2s have been implicated in a wide range of biological processes including the development of allergies and asthma, protective immunity against parasitic worms, and the regulation of adipocyte development and metabolic homeostasis. ILC2s also play diverse roles during influenza, such as stimulating IL-13-induced AHR, activating tissue repair mechanisms and promoting lung homeostasis during the recovery phase of infection. Currently there is limited information about the regulation of ILC2s during influenza infection and moreover, previous studies have been conducted in Rag-deficient mice, which do not take into consideration the adaptive immune response. Our results, using immunocompetent mice, demonstrate that IFNγ restricts ILC2 activity in the lung following lethal challenge with the pandemic H1N1 A/CA/04/2009 influenza virus. IFNγ-deficiency results in enhanced ILC2 activity, characterized by increased production of IL-5 and amphiregulin, but not IL-13. Moreover, we show that IFNγ-deficient mice exhibit decreased mortality following infection, which is dependent on IL-5. These data point to ILC2-mediated protection against influenza, possibly though diminished inflammation and increased tissue regeneration. Interestingly, IFN-/- mice were not protected from lethal challenge with mouse-adapted A/PR8/34 (H1N1) virus, which elicited a predominately IL-13-producing ILC2 response. We describe for the first time the detrimental role of IFNγ in the pathogenesis of influenza, and further suggest that IL-5 is a protective factor, which promotes survival following lethal challenge. Our data also indicate that influenza A infection elicits diverse ILC2 responses dependent on viral strain. These results suggest possible therapeutic approaches against influenza infection that would involve limiting IFN production in favor of enhanced ILC2-mediated immune responses.

127. #45 PKCβ(beta)II Dependent Development of Dendritic Cells

Colin Chavel, Inna Lindner, and Kelvin Lee Department of Immunology, Roswell Park Cancer Institute, Buffalo NY 14263

Dendritic cells are professional antigen presenting cells that are important activators of adaptive immune responses. Dendritic cells have also been shown to play a role in the activation of CD8+ T-cells that target tumor cells. Our lab has previously shown that PKCβ(beta)II is essential for dendritic cell differentiation. We have also shown that tumor derived factors, particularly IL-6, inhibit dendritic cell differentiation in part by repressing the expression of PKCβ(beta)II. However, the pathways activated downstream of PKCβ(beta)II during dendritic cell differentiation have yet to be characterized. To study this, we used the K562 cell line, which has been previously shown to be able differentiate into dendritic cells and PMA, a known chemical activator of PKCβ(beta)II. We have shown previously that PMA specifically activates PKCβ(beta)II in K562 cells and not the other conventional PKC kinases. Addition of PMA to K562 cells showed the activation of both the ERK1/2 and NFκ(kappa)B pathways at different time points. Inhi- bition of ERK1/2 during dendritic cell differentiation revealed a role for ERK1/2 in the growth arrest associated with this process. Chemical inhibition of either pathway yielded dendritic cells with a reduced capacity to stimulate T-cell proliferation. RelB is a transcription factor for the NFκ(kappa)B pathway and its expression increases upon activation of the NFκ(kappa)B pathway. Inhibition of ERK1/2 also showed an increase in RelB levels during dendritic cell differentiation. Our lab has also shown that mature dendritic cells reduce Foxo3a protein levels with the stimulation of CD80/CD86 through an Akt dependent mechanism. Inhibition of either the ERK1/2 or NFκ(kappa)B pathways showed an increase in Foxo3a levels, indicating a role for the ERK1/2 and NFκ(kappa)B pathways in Foxo3a regulation during differentiation into mature dendritic cells. Taken together, these results show an integral role for the ERK and NFκ(kappa)B pathways downstream of PKCβ(beta)II activation during dendritic cell differentiation.

128. #46 Impact of Hypoxia on IFNγ(gamma)-Dependent Responses: Implications for Radiotherapy and Anti-Tumor Immunity

Aditi Murthy, Scott A. Gerber, and Edith M. Lord University of Rochester Medical Center, Rochester, NY

Tumor hypoxia occurs due to the increase in demand for oxygen by the rapidly growing tumor cells together with reduction in the supply of oxygen due to malformed and non-functional tumor vasculature. Tumor hypoxia offers resistance to radiotherapy (RT) and chemotherapy. Interestingly, a new paradigm has emerged suggesting that hypoxia may also suppress immunotherapy, however the mechanisms behind this observation remain undetermined. Our laboratory and others have demonstrated that IFNγ, an important immunotherapeutic mediator, conditions the tumor microenvironment and is important for the efficacy of radiotherapy. As a result, we hypothesized that hypoxia could inhibit the anti-tumor responses mediated by IFNγ resulting in a decrease of radiotherapy efficacy. Hypoxia mediated inhibition could occur in two ways. Hypoxia could be modulating the production of intratumoral IFNγ protein, and/or reducing the ability of cells to respond to IFNγ. To test the first possibility we utilized Colon-38, a murine colon adenocarcinoma tumor model, and measured intratumoral hypoxia by both flow cytometry and fluorescence microscopy using a monoclonal antibody that detects hypoxia-induced 2-nitroimidazole adducts from the drug EF5. We used this drug to demonstrate a time dependent increase of hypoxia within untreated Colon-38 tumors. Unexpectedly, irradiation resulted in a decrease in total tumor hypoxia. We also detected various sub-populations of hypoxic immune cells in both the untreated and irradiated groups, with macrophages being the majority population. Importantly, CD8+ T cells, which are an important source of IFNγ and mediate effector anti-tumor responses, were present in these tumors but were not co- localized with hypoxic regions. These data suggest that CD8+ T cells are absent in hypoxic areas of the tumor presenting a mechanism of tumor escape. To address the second possibility of hypoxia induced inhibition we demonstrated in vitro that hypoxia inhibited the induction of IFNγ-stimulated genes in multiple human tumor cell lines and peripheral blood mononuclear cells suggesting hypoxia could inhibit the responsiveness to IFNγ. To mimic a clinically relevant model we have also examined the impact of an anti-tumor immunotherapeutic IL-12 on tumor hypoxia. We have previously shown that IL-12 can normalize tumor vasculature. Therefore by using a combination of RT and IL-12 immunotherapy we can generate a multi-pronged approach targeting tumor hypoxia and enhancing anti-tumor effector responses mediated by IFNγ. We propose that tumors that are less hypoxic (e.g. after RT and immunotherapy) are more conducive to IFNγ and T cell responses, resulting in enhanced tumor control.

129. NYIC 2015 Participant Contact Information

Allen, Joselyn Boule, Lisbeth Pennsylvania State University University of Rochester 340-227-0155 703-937-7516 [email protected] [email protected]

Amiel, Eyal Burger, Elise University of Vermont University of Rochester 802-656-0522 585-276-7731 [email protected] [email protected]

Amobi, Adaobi Burke, Catherine Roswell Park Cancer Institute University of Rochester 716-583-0875 716-949-2802 [email protected] [email protected]

Barlow, Margaret Burkard-Mandel, Lauren University of Rochester Roswell Park Cancer Institute 216-410-5140 716-845-3352 [email protected] [email protected]

Belkaid, Yasmine Bynoe, Margaret NIAID/NIH Cornell University 301-496-4882 607-253-4023 [email protected] [email protected]

Bellville, Dawn Califano, Danielle Albany Medical College Albany Medical College 518-262-5365 518-262-0096 [email protected] [email protected]

Berwin, Brent Chabanet, Tamer B. Dartmouth College Dartmouth College 603-208-7446 330-667-0094 [email protected] [email protected]

Bhandari, Sadikshya Chavel, Colin University of Connecticut Roswell Park Cancer Institute 580-647-0228 716-845-8231 [email protected] [email protected]

130.

Connolly, Kelli Furuya, Yoichi University of Rochester Albany Medical College 716-946-5527 518-262-0096 [email protected] [email protected]

De Jesus, Magdia Gerber, Scott Wadsworth Center/SUNY Albany University of Rochester 347-224-9964 585-276-7380 [email protected] [email protected]

Dhume, Shaffina Ginsberg, Arielle Baxalta BD Biosciences 203-272-1638 908-642-2892 [email protected] [email protected]

Drake, James (Jim) Gruber, Erika Albany Medical College Cornell University 518-262-9337 607-235-1366 [email protected] [email protected]

Elliott, Michael Hankey, Pamela University of Rochester Pennsylvania State University 585-273-4793 814-863-0128 [email protected] [email protected]

Emmons, Tiffany Haque, Mohammad Roswell Park Cancer Institute Pennsylvania State University 716-845-1372 717-810-8950 [email protected] [email protected]

Feng, Mengyang Harton, Jonathan Pennsylvania State University Albany Medical College 814-777-8829 518-262-4445 [email protected] [email protected]

Franchini, Anthony Hogquist, Kristin University of Rochester University of Minnesota 518-495-9755 612-625-1616 [email protected] [email protected]

131.

Huang, Yina Lord, Edith Dartmouth Medical College University of Rochester 603-650-7545 585-749-8604 [email protected] [email protected]

Jackson, Kimberly Lynes, Michael University of Rochester University of Connecticut 585-690-1127 860-486-4350 [email protected] [email protected]

Jin, Richard MacNamara, Kate University at Buffalo Albany Medical College 503-866-5785 518-262-0921 [email protected] [email protected]

Jusko, Todd Mahapatra, Riddhi University of Rochester Roswell Park Cancer Institute 585-273-2849 716-908-8041 [email protected] [email protected]

Kenderes, Kevin Maharaj, Shivana SUNY Upstate Medical University Roswell Park Cancer Institute 315-464-7671 716-380-0223 [email protected] [email protected]

Lawrence, B. Paige Mansouri, Samira University of Rochester Albany Medical College 585-730-2605 518-262-0052 [email protected] [email protected]

Lawrence, David Mantis, Nicholas Wadsworth Center/SUNY Albany Wadsworth Center/SUNY Albany 518-486-9154 518-473-7487 [email protected] [email protected]

Lopez-Yglesias, Americo Messmer, Michelle University of Rochester Roswell Park Cancer Institute 206-852-2707 716-816-6896 [email protected] [email protected]

132.

Meyers, Jessica Patel, Seemaben University of Rochester Albany Medical College 724-944-6483 518-262-0052 [email protected] [email protected]

Minchenberg, Scott Peppenelli, Megan SUNY Upstate Medical University SUNY Upstate Medical University 516-524-5812 315-464-7682 [email protected] [email protected]

Murthy, Aditi Pujanauski, Lindsey University of Rochester Journal of Immunology/AAI 508-813-3363 703-499-0346 [email protected] [email protected]

Nelson, Nicole Qiao, Guanxi Albany Medical College Roswell Park Cancer Institute 518-262-1650 716-364-8252 [email protected] [email protected]

O’Connor, William Rastad, Jessica Albany Medical College Dartmouth Medical College 518-262-6548 603-277-1048 [email protected] [email protected]

Olejniczak, Scott Robek, Michael Roswell Park Cancer Institute Albany Medical Center 716-912-2723 518-264-2580 [email protected] [email protected]

Pandya, Mital Robinson, Sudie Ann University of Vermont Cornell University 614-439-8680 607-262-0295 [email protected] [email protected]

Papillion, Amber Sanders, Kiah SUNY Upstate Medical University Dartmouth Medical College 337-580-1924 603-359-0251 [email protected] [email protected]

133.

Schneider, Karin Szczepanek, Steven

SUNY Upstate Medical University University of Connecticut 315-559-8953 860-486-8101 [email protected] [email protected]

Schumacher, Jacob Tangeman, Matthew AAI Technician 503-516-3587 218-224-2165 [email protected] [email protected]

Shen, Pamela Thwe, Phyu McMaster University University of Vermont 289-339-3681 414-241-3367 [email protected] [email protected]

Singel, Kelly Twum, Danielle Roswell Park Cancer Institute Roswell Park Cancer Institute 716-845-3138 716-845-3352 [email protected] [email protected]

Smith, Julianne Utley, Adam Albany Medical College Roswell Park Cancer Institute 518-262-0922 336-847-4725 [email protected] [email protected]

Snyder, Lindsay Wang, Jocelyn Pennsylvania State University 607-379-3219 505-801-2980 Cornell Univesity [email protected] [email protected]

Soucy, Alicia Williamson, David Albany Medical College Pennsylvania State University 518-262-6220 814-769-9805 [email protected] [email protected]

Sudan, Raki Wojno, Elia Tait SUNY Upstate Medical University Cornell University 315-751-7254 908-672-2160 [email protected] [email protected]

134.

Wohlfert, Beth University at Buffalo 716-829-3969 [email protected]

Yarovinsky, Felix University of Rochester 240-750-0720 [email protected]

Yee Mon, Kristel Cornell University 347-462-7290 [email protected]

Yeoh, Beng San Pennsylvania State University 814-880-4388 [email protected]

135. Authors Index CS – Corporate Speaker P# - Poster Number

KS - Keynote Speaker S – Speaker O - Oral Poster Presentation WP – Workshop Presenter Page (s) ************************************************************************************* Abrams, Scott I...... 49, 63, 64, 90, 113, 114 Adamik, B...... 79, 89 Amiel, Eyal (S, P6) ...... 50, 79, 89, 112 Amobi, Adaobi (O, P40) ...... 65, 123 Arend, Kyle ...... 56, 84 Bantly, Andrew D. (CS) ...... 67 Barie, Li ...... 107 Barlow, John ...... 106 Barlow, Margaret L. (P36) ...... 119 Beauchamp, Saritha ...... 50, 112 Belkaid, Yasmine (KS, WP) ...... 54, 82 Belton, Kerry R. (O, P26) ...... 58, 109 Bett, Brian ...... 120 Bhandari, Sadikshya (P30) ...... 113 Bishop, Alex ...... 39 Blaauboer, Steven M...... 121 Blair, Sarah J...... 99, 101 Blader, Ira J...... 100 Boni, Andrea ...... 36, 117 Boule, Lisbeth A. (O, P4) ...... 42, 48, 86, 87 Bowdish, Dawn M.E...... 97 Burger, Elise (O, P11) ...... 59, 94 Burkard-Mandel, Lauren (O, P31) ...... 63, 114 Bynoe, Margaret S. (S) ...... 71, 103 Califano, Danielle (P44) ...... 127 Carlson, Louise ...... 46, 124 Chabanet, Tamer B. (S, P34) ...... 36, 117 Chan, Gary ...... 56, 84 Chavel, Colin (P45) ...... 128 Chiaro, Christopher ...... 58, 107 Christie, Y...... 74 Clancy-Thompson, Eleanor ...... 62, 85 Cojohari, Olesea ...... 56, 84 Conka, Kamil ...... 88

136. Connolly, Kelli A. (O, P37) ...... 51, 120

Cornett, Ashley (CS) ...... 53 Crepeau, Rebecca (O, P9) ...... 44, 92 Croteau, Walburga ...... 39 De Jesus, Magdia (S) ...... 80 Dewan, Kalyan ...... 126 Dishaw, Lisa ...... 43, 96 Drake, James R. (S) ...... 81 Drobna, Beata ...... 88 Eckstrom, Korin ...... 106 Ehrlich, Emma ...... 42, 125 Engelman, R.W...... 74 Fabisikova, Anna ...... 88 Feng, Yunfeng ...... 39 Franchini, Anthony M. (P15) ...... 98 Fritz, D...... 79, 89 Furuya, Yoichi (P22) ...... 105, 127 Galasso, N...... 81, 89 Gerber, Scott A...... 51, 66, 119, 120, 129 Golde, William T...... 106 Gosselin, Edmund J...... 107 Green, William R...... 60, 90 Grimson, Andrew ...... 45, 116 Gruber, Erika (O, P42) ...... 52, 125 Haidaris, Constantine G...... 119 Haque, Mohammad (P10) ...... 93 Hertz-Picciotto, Irva ...... 88 Hogquist, Kristin A. (KS) ...... 34 Huang, Yina H. (S) ...... 39 Iloure, Joseph ...... 101 Jin, Guang-Bai ...... 42, 87, 98 Jin, Lei ...... 121, 122 Jin, Richard M. (P17) ...... 100, 101 Jusko, Todd A. (P5) ...... 88 Kalia, Vandana ...... 126 Katkere, Bhuvana ...... 126 Kenderes, Kevin (O, P13) ...... 43, 96 Kerr, W.G...... 74 Kirimanjeswara, Girish ...... 126 Kocan, Anton ...... 86

137. La Rocca, Gaspare ...... 40, 99 Lawrence, B. Paige ...... 42, 48, 86, 87, 88, 98

Lee, Kelvin ...... 46, 124, 128 Lee, Sue Y...... 88 Leifer, Cynthia ...... 52, 125 Lindner, Inna ...... 128 Linehan, David C...... 51 Lopez-Yglesias, Americo (P12) ...... 59, 94, 95 Lord, Edith M...... 51, 66, 119, 121, 129 Lynes, Michael A...... 113 MacNamara, Katherine C...... 57, 108 Malik, Brian T...... 36, 117 Mansouri, Samira (P38) ...... 119 Mantis, Nicholas J...... 80 Markley, Rachel ...... 126 Massa, Paul T...... 99, 111 Matsuzaki, Junko ...... 65, 121 McCabe, Amanda ...... 57, 108 Messmer, Michelle N. (O, P8) ...... 49, 91 Metzger, Dennis W...... 104, 105. 127 Meyers, Jessica L. (O, P3) ...... 48, 86 Minchenberg, Scott (P16) ...... 99 Mon, Kristel Yee (P19) ...... 100 Moorman, Nathaniel ...... 56, 84 Morissette, Mathieu C...... 97 Mullins, David W...... 62, 85 Murinova, Lubica Palkovicova ...... 88 Murthy, Aditi (O, P46) ...... 51, 120 Nelson, Nicole (P24) ...... 107 Netherby, Colleen ...... 49, 91 Odunsi, Kunle ...... 65, 123 Olejniczak, Scott H. (S) ...... 40 Pandya, Mital (P23) ...... 106 Papillion, Amber (P27) ...... 43, 96, 110 Park, M.Y...... 76 Patel, Seema (P39) ...... 122 Patterson, Andrew D...... 58, 109 Peppenelli, Megan (O, P1) ...... 56, 84 Perdew, Gary H...... 58, 107 Perekslis, Thomas ...... 62, 85

138. Place, David ...... 126

Qian, Feng ...... 65, 123 Rabinowicz, Jesse ...... 122 Radler, Megan ...... 40 Rastad, Jessica L. (O, P7) ...... 60, 90 Reynolds, Tracy D...... 73 Robek, Michael D. (S) ...... 73 Robinson, Sudie Ann (P20) ...... 103 Rose, John K...... 73 Rudd, Brian T...... 45, 102, 116 Ryan, Julie L...... 119 Sanders, Kiah L. (O, P2) ...... 662, 85 Sandhu, Praneet ...... 93 Sarkar, Surojit ...... 126 Schiavoni, Elizabeth M...... 100, 101 Schneider, Karin M. (P28) ...... 111 Schumacher, Jacob (WP) ...... 77 Schwartz, Stephen M...... 88 Seiler, Michael (CS) ...... 76 Shen, Pamela (P14) ...... 97 Sinha, Siddhartha ...... 52, 125 Smith, Julianne N. (O, P25) ...... 57, 108 Smith, Nora L...... 45, 116 Song, Jianxun ...... 93 Soucy, Alicia (P21) ...... 104 Srivastava, N...... 74 Stampfli, Martin r...... 97 Steinberg, Shannon M...... 36, 117 Stelzer, Sandra ...... 52, 125 Sturge, Carolyn ...... 95 Sudan, R. (S) ...... 74 Sudha, N...... 74 Szczepanek, Steven M. (S) ...... 75 Thevenet- Morrison, Kelly ...... 88 Thwe, Phyu (O, P29) ...... 50, 79, 89, 112 Thompson, Craig B...... 42 Trnovec, Tomas ...... 88 Turk, Mary Jo ...... 36, 117 Twum, Danielle (O, P32) ...... 64, 115 Usherwood, Edward ...... 44, 92

139. Utley, Adam (O, P41) ...... 46, 124 Vella, Jennifer ...... 39

Ventro, Daniela ...... 46, 124 Verner, Marc-Andre ...... 88 Vincent, James ...... 106 Wang, J.W...... 74 Wang, Jocelyn (O, P33) ...... 45, 116 Watson, Neva B...... 111 Williamson, David (P43) ...... 126 Winans, Bethany ...... 48, 86 Winslow, Gary ...... 43, 96, 110 Wissink, Erin ...... 45, 116 Wohlfert, Elizabeth A. (S, P18) ...... 37, 100, 101 Wojno, Elia D. Tait (S, P35) ...... 69, 118 Yarovinsky, Felix (S) ...... 59, 70, 94, 95 Yin, Xiuyun ...... 113 Yuzefpolskiy, Yevgeniy ...... 126 Zhang, Limin ...... 58, 109 Zhang, Peisheng ...... 36, 44, 92, 117

140.