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`Journal of immunology
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`

`Immunology
`
`Table of Contents
`
`MicaLeyesst:) 0;
`
`VOL. 194 | NO. 3 | February 1, 2015 | Pages 845-1386
`
`IN THIS ISSUE
`845 Driving CARs over the Threshold See article p. 911
`Finding Friends for AIRE See article p. 921
`Thymocyte Peer Pressure See article p. 1057
`One More Reason To Take Your Vitamins See article p. 1090
`The Skin-ny on Secondary Lymphoid Organs in Transplantation See article p. 1364
`
`PILLARS OF IMMUNOLOGY
`847 Laying Bare the Nude Mouse Gene
`Graham Anderson and Nicholas I. MeCarthy
`
`849
`
`Pillars Article: New Member of the Winged-Helix Protein Family Disrupted In Mouse And Rat Nude
`Mutations. Nature. 1994. 372: 103-107
`Michael Nehls, Dietmar Pfeifer, Michael Schorpp, Hans Hedrich, and Thomas Boehm
`
`BRIEF REVIEWS
`855 The Acute Respiratory Distress Syndrome: From Mechanism to Translation
`SeungHye Han and RamaK. Matlampallt
`
`CUTTING EDGE
`863 Cutting Edge: Maresin-1 Engages Regulatory ‘TCells To Limit Type 2 Innate Lymphoid Cell Activation
`and Promote Resolution of Lung Inflammation
`Nandini Krishnamoorthy, Patrick R. Burkett, Jesmond Dalli, Raja-Elie E. Abdulnour, Romain Colas, Sesquile Ramon
`Richard P. Phipps, Nicos A. Petasis, Vijay K. Kuchroo, Charles N. Serhan, and Bruce D. Levy
`
`868 Cutting Edge: Critical Role for C5aRs in the Development of Septic Lymphopenia in Mice
`Jamison J. Grailer, Fatemeh Fattahi, Rachel S. Dick, Firas S. Zetoune, and Peter A, Ware
`in DNase I—Deficient Mice
`oantibody Production
`873 Cutting Edge: AIM2 and Endosomal TLRs Differentially Regulate Arthritis and Autoantib
`A
`;
`Rebecca Baum, Shruti Sharma, Susan Carpenter, Quan-Zhen Li, Patricia Busto, Katherine A, Fitzgerald,
`Ann Marshak-Rothstein, and Ellen M. Crravallese
`*
`
`Onthe cover: Motility of the infective larval stage of the intestinal helminth /eligmnosomoides polyeyrus bakeri sh
`in temporal color code (120 frames, 60 seconds). Esser-von Bieren, J., B. Volpe, M. Kulagin, D.B. Surhesanid 2G ie
`A. Seitz, B. J. Marsland, J. S. Verbeek, and N. L. Harris. 2015. Antibody-mediated trapping of helminth lates es vor
`
`CD11b and Fey receptorI. /- Immunol. 194: 1154-1163. arvae requires
`
`
`i
`of
`The Journal ofImmunology (ISSN (022-1767) is published twice each month by The American Association
`Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994, Phone: 301-634-7197, Fax: 301-634-7829 Seeniee
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`Copyright © 2015 by The American Association of Immunologists, Inc.
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`

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`878
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`883
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`887
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`898
`
`911
`
`921
`
`929
`
`940
`
`950
`
`960
`
`973
`
`Cutting Edge: Epigenetic Regulation of Foxp3 Defines a Stable Population of CD4" Regulatory T Cells
`in ‘Tumors from Mice and Humans
`Jeremy D. Waight, Shinji Takai, Bo Marelli, Guozhong Qin, Kenneth W. Hance, Dong Zhang, Robert Tighe, Yan Lan,
`Kin-Ming Lo, Helen Sabzevari, Robert Hofmeister, and Nicholas S. Wilson
`
`Cutting Edge: Progesterone Directly Upregulates Vitamin D Receptor Gene Expression for Efficient
`Regulation of T Cells by Calcitriol
`Shankar Thangamani, Myughoo Kim, Youngmin Son, Xinxin Huang, Heejoo Kim, Jee H. Lee, Jungyoon Cho,
`Benjamin Ulrich, Hal E. Broxmeyer, and Chang H. Kim
`
`ALLERGY AND OTHER HYPERSENSITIVITIES
`
`IL-10 and Regulatory T Cells Cooperate in Allergen-Specific Immunotherapy To Ameliorate
`Allergic Asthma
`Livia Bolyn, Joachim Maxeiner, Helen Meyer-Martin, Sebastian Reuter, Susetta Finotto, Matthias Klein,
`Hansjorg Schild, Edgar Schmitt, Tobias Bopp, and Christian Taube
`
`ANTIGEN RECOGNITION AND RESPONSES
`
`Fixed Expression of Single Influenza Virus-Specific TCR Chains Demonstrates the Capacityfor
`TCR a and B—Chain Diversity in the Face of Peptide-MHC Class | Specificity
`E. Bridie Clemens, Peter C. Doherty, Nicole L. La Gruta, and Stephen J. Turner
`
`Target Antigen Density Governs the Efficacy of Anti-CD20-CD28-CD3 ¢€ Chimeric Antigen
`Receptor—Modified Effector CD8" T Cells
`Keisuke Watanabe, Seitaro Terakura, Anton C. Martens, Tom van Meerten, Susumu Uchiyama, Misa Imai,
`Reona Sakemura, Tatsunort Goto, Ryo Hanajiri, Nobuhiko Imahashi, Kazuyuki Shimada, Akihiro Tomtta,
`Hitoshi Kiyoi, Tetsuya Nishida, Tomoki Naoe, and Makoto Murata
`
`AUTOIMMUNITY
`
`Homeodomain-Interacting Protein Kinase 2, a Novel Autoimmune Regulator Interaction Partner,
`Modulates Promiscuous Gene Expression in Medullary Thymic Epithelial Cells
`Kristin Rattay, Janine Claude, Esmail Rezavandy, Sonja Matt, Thomas G. Hofinann, Bruno Kyewski,
`and Jens Derbinski
`
`CLINICAL AND HUMAN IMMUNOLOGY
`
`The V Gene Repertoires of Classical and Atypical Memory B Cells in Malaria-Susceptible West
`African Children
`Severin Zindcker, Christine E. Schindler, JeffSkinner, Tobias Rogosch, Michael Waisberg, Jean-Nicolas Schickel,
`Evie Meffre, Kassoum Kayentao, Aissata (Ingoiba, Boubacar Traore, and Susan K. Pierce
`
`Differential Expression ofthe ‘Transcription Factor ARID3a in Lupus Patient Hematopoietic
`Progenitor Cells
`Michelle L. Ratliff. Julie M. Ward, Joan T. Merrill, Judith A. James, and Carel F. Webb
`
`Combination Therapy with Ani-CTLA-4 and Anti-PD-1 Leads to Distinct Immunologic
`Changes In Vivo
`Rituparna Das, Rakesh Verma, Mario Sznol, Chandra Sekhar Boddupalli, Scott N. Gettinger,
`Harriet Kluger, Margaret Callahan, Jedd D. Wolchok, Ruth Halaban, Madhav V. Dhodapkar,
`and Kavita M. Dhodapkar
`
`CD26-Mediated Induction of EGR2 and IL-10 as Potential Regulatory Mechanism for CD26
`Costimulatory Pathway
`Ryo Hatano, Kei Ohbnuma, Haruna Otsuka, Eriko Kamiya, lzumi Taki, Satoshi Iwata, Nam H. Dang,
`Ko Okumura. and Chikao Morimoto
`
`Sterile DJ}; Rearrangements Reveal that Distance Between Gene Segments on the Human Ig H Chain
`Locus Influences Their Ability To Rearrange
`Tina Ostergaard Hansen, Anders Blaabjere Lange, and Torben Barington
`
`

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`983
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`990
`
`999
`
`101]
`
`1021
`
`1031
`
`1039
`
`1047
`
`IMMUNE REGULATION
`
`Immune Complexes Suppress IFN-y—Induced Responses in Monocytes by Activating Discrete Members
`of the SRC Kinase Family
`Gunther H. Boekhoudt, Anna G. McGrath, Jennifer F. A, Swisher, and Gerald M. Feldman
`
`Modulating DNA Methylation in Activated CD8* T Cells Inhibits Regulatory T Cell-Induced Binding
`of Foxp3 to the CD8" T Cell IL-2 Promoter
`Michelle M. Miller, Nuenna Akaronu, Elizabeth M. Thompson, Sylvia F. Hood, and Jonathan E. Fogle
`
`Effector T Cells Boost Regulatory T Cell Expansion by IL-2, TNF, OX40, and Plasmacytoid Dendritic Cells
`Depending on the Immune Context
`Audrey Baeyens, David Saadoun, Fabienne Billiard, Angéline Rouers, Sylvie Gregoire, Bruno Zaragoza,
`Yenkel Grinberg-Bleyer, Gilles Marodon, Eliane Piaggio, and Benoit L. Salomon
`
`Intestinal Helminths Regulate Lethal Acute Graft-versus-Host Disease and Preserve the Graft-versus- Tumor
`Effect in Mice
`Yue Li, Hung-Lin Chen, Nadine Bannick, Michael Henry, Adrian N. Holm, Abmed Metwali, Joseph F. Urban, Jr.,
`Paul B. Rothman, George J. Weiner, Bruce R. Blazar, David E. Elliott, and M. Nedim Ince
`
`Chronic Morphine-Induced MicroRNA-124 Promotes Microglial Immunosuppression by Modulating
`P65 and TRAF6
`Shuwei Qiu, Yimin Feng, Gene LeSage, Ying Zhang, Charles Stuart, Lei He, Yi Li, Yi Caudle, Ying Peng, and Deling Yin
`
`Survival of Human Circulating Antigen-Induced Plasma Cells Is Supported by Plasma Cell—Niche
`Cytokines and T Follicular Helper Lymphocytes
`Ana Ramos-Amaya, Beatriz Rodriguez-Bayona, Rubén Lopez-Blanco, Eloisa Andujar, Monica Peérez-Alegre,
`Antonio Campos-Caro, and José A. Brieva
`
`Temporal Expression of Growth Factors Triggered by Epiregulin Regulates Inflammation Development
`Masaya Harada, Daisuke Kamimura, Yasunobu Arima, Hitoshi Kobsaka, Yuji Nakatsuji, Makoto Nishida, Toru Atsumi,
`Jie Meng, Hidenori Bando, Rajeev Singh, Lavannya Sabharwal, Jing-Jing Jiang, Noriko Kumai, Nobuyuki Miyasaka,
`Saburo Sakoda, Keiko Yamauchi-Takihara, Hideki Ogura, Toshio Hirano, and Masaaki Murakami
`*
`.
`
`CD40L Induces Functional Tunneling Nanotube Networks Exclusively in Dendritic Cells Programmed
`by Mediators of Type 1 Immunity
`Colleen R. Zaccard, Simon C. Watkins, Pawel Kalinski, Ronald J, Fecek, Aartka L. Yates, Russell D. Satter,
`Velpandi Ayyavoo, Charles R. Rinaldo, and Robbie B. Mailliarad
`
`IMMUNE SYSTEM DEVELOPMENT
`
`1057
`
`Stable Interactions and Sustained TCR Signaling Characterize Thymocyte—Thymocyte Interactions that
`Support Negative Selection
`Heather J. Melichar, Jenny O. Ross, Kayleigh T. Taylor, and Ellen A. Robey
`
`1062
`
`1069
`
`LO80
`
`IMMUNOGENETICS
`
`The HumanIL-23 Receptor rs1 1209026 A Allele Promotes the Expression of a Soluble 1L-23R—Encoding
`mRNA Species
`Raymond Y. Yu, Jonathan Brazaitis, and Grant Gallagher
`
`IMMUNOTHERAPY AND VACCINES
`
`Induction of Potent CD8 T Cell Cytotoxicity by Specific Targeting of Antigen to Cross-Presenting
`Dendritic Cells In Vivo via Murine or Human XCRI
`Evelyn Hartung, Martina Becker, Annabell Bachem, Nele Reeg, Anika Jikel, Andreas Hutloff, Harald Weber,
`Christoph Weise, Claudia Giesecke, Volker Henn, Stephanie Gurka, Konstantinos Anastassiadis, Hans W. Mages,
`and Richard A, Kroczek
`
`CD8 T Cell Tolerance to a ‘Tumor-Associated Self-Antugen Is Reversed by CD4 T Cells Engineered
`To Express the Same T’ Cell Receptor
`Sara Ghorashian, Pedro Veliga, Ignatius Chua, Anne-Marie McNicol, Ben Carpenter, Angelika Holler, Emma Nicholson,
`Maryam Almadi, Mathias Zech, Shao-Au Xue, Wolfgang Uckert, Emma Morris, Ronjou Chakraverty, and Hans], Stauss
`
`

`

`1090
`
`1100
`
`1112
`
`1122
`
`1131
`
`1141
`
`1154
`
`1164
`
`1169
`
`1178
`
`1190
`
`1199
`
`INFECTIOUS DISEASE AND HOST RESPONSE
`
`The a-Tocopherol Form of Vitamin E Reverses Age-Associated Susceptibility to Streptococcus pneumoniae
`Lung Infection by Modulating Pulmonary Neutrophil Recruitment
`Ela N. Bou Ghanem, Stacie Clark, Xiaogang Du, Dayong Wu, Andrew Camilli, John M. Leong, and Simin N. Meydani
`
`A Major Role for Myeloid-Derived Suppressor Cells and a Minor Role for Regulatory T’ Cells in
`Immunosuppression during Staphylococcus aureus Infection
`Christina Tebartz, Sarah Anita Horst, Tim Sparwasser, Jochen Huebn, Andreas Beineke, Georg Peters, and Eva Medina
`
`Atg7 Enhances Host Defense against Infection via Downregulation of Superoxide but Upregulation of
`Nitric Oxide
`Xuefeng Li, Yan Ye, Xikun Zhou, Canhua Huang, and Min Wu
`
`IRAK4 as a Molecular Target in the Amelioration of Innate Immunity—Related Endotoxic Shock and
`Acute Liver Injury by Chlorogenic Acid
`Sun Hong Park, Seung-Il Baek, Jieun Yun, Seungmin Lee, Da Young Yoon, Jae-Kyung Jung, Sang-Hun Jung,
`Bang Yeon Hwang, Jin Tae Hong, Sang-Bae Han, and Youngsoo Kim
`
`Diverse Roles for T-bet in the Effector Responses Required for Resistance to Infection
`Gretchen Harms Pritchard, Aisling O'Hara Hall, David A, Christian, Sagie Wagage, Qun Fang, Gaia Muallem,
`Beena John, Arielle Glatman Zaretsky, William G. Dunn, Jacqueline Perrigoue, Steven L. Reiner,
`and Christopher A. Hunter
`
`CD8"* T Cells Complement Antibodies in Protecting against Yellow Fever Virus
`Maria BR. Bassi, Michael Konesgaard, Maria A. Steffensen, Christina Fenger, Michael Rasmussen, Karsten Skjodt,
`Bente Finsen, Anette Stryhn, Soren Buus, Jan P. Christensen, and Allan R. Thomsen
`
`Antibody-Mediated Trapping of Helminth Larvae Requires CD11b and Fey Receptor|
`Julia Esser-von Bieren, Beatrice Volpe, Manuel Kulagin, Duncan B. Sutherland, Romain Guiet, Arne Seitz,
`Benjamin J. Marsland, J. Sjef Verbeek, and Nicola L. Harris
`
`Complement Component C5 Recruits Neutrophils in the Absence of C3 during Respiratory Infection
`with Modified Vaccinia Virus Ankara
`;
`Philip J. R. Price, Zoltan Banki, Angelika Scheideler, Heribert Stoiber, Admar Verschoor, Gerd Sutter,
`and Michael H. Lebmann
`
`INNATE IMMUNITY AND INFLAMMATION
`
`‘TRIF-Dependent Innate ImmuneActivation Is Critical for Survival to Neonatal Gram-Negative Sepsis
`Alex G. Cuenca, Dallas N. Joiner, Lori F. Gentile, Angela L. Cuenca, James L. Wynn, Kindra M. Kelly-Scumpia,
`Philip O. Scumpia, Kevin FE. Bebrns, Philip A. Efron, Dina Nacionales, Chao Lui, Shannon M. Wallet, Westley H. Reeves,
`Clayton E. Mathews, and Lyle L. Moldawer
`
`Radiation Exposure Induces Infammasome Pathway Activation in Immune Cells
`Veit M. Stoecklein, Akinori Osuka, Shizu Ishikawa, Madeline R. Lederer, Lorenz Wanke-Jellinek, and James A, Lederer
`
`TLR2 Modulates Antibodies Required for Intestinal Ischemia/Reperfusion-Induced Damage and
`Inflammation
`Michael R. Pope and Sherry D. Fleming
`
`A Second Stimulus Required for Enhanced Antifungal Activity of Human Neutrophils in Blood Is Provided
`by Anaphylatoxin C5a
`Kerstin Hiinniger, Kristin Bieber, Ronny Martin, Teresa Lehnert, Mare Thilo Figge, Jiirgen Liffler, Ren-Feng Guo,
`Niels C. Riedemann, and Oliver Kurzai
`
`Neutrophil Priming Occurs in a Sequential Manner and CanBeVisualized in Living Animals by Monitoring
`IL-1 Promoter Activation
`Yi Yao, Hironort Matsushima, Jennifer A, Obtola, Shuo Geng, Ran Lu, and Akira Takashima
`
`Zebrafish [RFT Regulates IFN Antiviral Response through Binding to IFNeb1l and IFNd3 Promoters
`Downstream of MyD88 Signaling
`Hui Feng, Yi-Bing Zhang, Qi-Min Zhang, Zhi Li, Qi-Ya Zhang, and Jian-Fane Gui
`
`

`

`MiR-127 Modulates Macrophage Polarization and Promotes Lung InflammationandInjury by Activating
`the JNK Pathway
`Hangjie Ying, Yanhua Kang, Hang Zhang, Dongjin Zhao, Jingyan Xia, Zhe Lu, Huanhuan Wang, Feng Xn, and Liyun Shi
`
`Protective Role for Caspase-11 during Acute Experimental Murine Colitis
`Katarzyna Oficjalska, Mathilde Raverdeau, Gabriella Aviello, Siobhan C. Wade, Ana Hickey, Katherine M. Sheehan,
`Sinead C. Corr, Elaine W. Kay, Luke A. O'Neill, Kingston H. G. Mills, and Emma M. Creagh
`
`Apoptosis-Associated Speck-like Protein Containing a CARD Forms Specks but Does Not Activate
`Caspase-1 in the Absence of NLRP3 during Macrophage Swelling
`Vincent Compan, Fatima Martin-Sanchez, Alberto Baroja-Mazo, Gloria Lopez-Castejon, Ana I. Gomez,
`Alexei Verkhratsky, David Brough, and Pablo Pelegrin
`
`Endogenous Intracellular Cathelicidin Enhances TLR9 Activation in Dendritic Cells and Macrophages
`Yukinobu Nakagawa and Richard L, Gallo
`
`Complement Deficiency Promotes Cutaneous Wound Healing in Mice
`Kenurtzelis, Periklis G. Foukas, Maciej M. Markiewski, Robert A. DeAngelis, Mara Guariento,
`Stavros Rafail,
`loannis
`Daniel Ricklin, Elizabeth A, Grice, and John D. Lambris
`
`1292
`
`The Endoplasmic Reticulum Adaptor Protein ERAdPInitiates NK Cell Activation via the Ubel 3-Mediated
`NF-KB Pathway
`Jun Chen, Lu Hao, Chong Li, Buging Ye, Ying Du, Honglian Zhang, Bo Long, Pingping Zhu, Benyu Lin, Liulin Yang,
`Peifeng Li, Yong Tian, and Zusen Fan
`
`1304
`
`Distinct Cellular Sources of Hepoxilin Aj and Leukotriene B, Are Used To Coordinate Bacterial-Induced
`Neutrophil ‘T'ransepithelial Migration
`Michael A. Pazos, Waheed Pirzai, Lael M. Yonker, Christophe Morissean, Karsten Gronert, and Bryan P. Hurley
`
`1316
`
`Knockdown of the Antiapoptotic Bcl-2 Family Member A1/Bfl-1 Protects Mice from Anaphylaxis
`Eleonora Ottina, Katarina Lyberg, Maja Sochalska, Andreas Villunger, and Gunnar P. Nilsson
`
`1323
`
`MOLECULAR AND STRUCTURAL IMMUNOLOGY
`Phosphatidylinositol 4—Phosphate 5—Kinase a and Vav1 Mutual Cooperationin CD28-Mediated Actin
`Remodeling and Signaling Functions
`Michela Muscolini, Cristina Camperio, Nicla Porciello, Silvana Caristi, Cristina Capuano, Antonella Viola,
`Ricciarda Galandrini, and Loretta Tuesto
`
`1334
`
`MUCOSAL IMMUNOLOGY
`IL-2RB—Dependent Signaling and CD103 Functionally Cooperate To Maintain Volerance in the Gut
`Mucosa
`Niaomei Yuan, Michael [. Dee, Norman H, Altman, and Thomas R. Malek
`
`1347
`
`1357
`
`TRANSPLANTATION
`IFN-y Production by Memory Helper T Cells Is Required for CD40-Independent Alloantibody Responses
`Victoria Gorbacheva, Ran Fan, Xi Wang, William M. Baldwin, HI, Robert L. Fairchild, and Anna Valujskikh
`
`Anti-IL-12/23 p40 Antibody Attenuates Experimental Chronic Graft-versus-Host Disease via Suppression
`of IFN-y/IL-17—Producing Cells
`Sachiyo Okamoto, Hideaki Fujiwara, Hisakazu Nishimori, Ken-ichi Matsuoka, Nobubaru Fujii, Fisei Kondo,
`Takehiro Tanaka, Akihiko Yoshimura, Mitsune Tanimoto, and Yoshinobu Maeda
`
`1364
`
`Both Rejection and Tolerance of Allografts Can Occurin the Absence of Secondary Lymphoid Tissues
`Cavit D. Kant, Yoshinobu Akiyama, Katsunori Tanaka, Susan Shea, Yohei Yamada, Sarah E. Connolly, Jose Marino,
`Georges Tocco, and Gilles Benichou
`
`NOVEL IMMUNOLOGICAL METHODS
`I'SLP Expression: Analysis with a ZsGreen ‘TSLP Reporter Mouse
`Cedric Dewas, Xi Chen, Tetsuya Honda, Ikka Junttila, Jay Linton, Mark C. Udey, Stephen F. Porcella.
`Daniel E. Sturdevant, Lionel Feigenbaum, Lily Koo, Joy Williams, and William E. Paul
`
`

`

`1381
`
`1382
`
`1383
`
`LETTERS OF RETRACTION
`Retraction: Guanosine Inhibits CD40 Receptor Expression and Function Induced by Cytokines and B
`Amyloid in Mouse Microglia Cells
`lolanda D'Alimonte, Vincenzo Flati, Elena Toniato, Stefano Martinotti, Michel P. Rathbone, Shucui Jiang,
`Patrizia Di lorio, Francesco Caciagli, and Renata Ciccarelli
`
`CORRECTIONS
`Correction: Role of Neutrophils in IL-17—Dependent Immunity to Mucosal Candidiasis
`Anna R. Huppler, Heather R. Conti, Nydiaris Hernandez-Santos, Toni Darville, Partha 8. Biswas, and Sarah L. Gaffen
`
`Correction: AID-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Populations
`Zhangguo Chen, Sheila Ranganath, Sawanee S. Viboolsittiseri, Maxwell D, Eder, Xiaomi Chen, Mihret T. Elos,
`Shunzong Yuan, Erica Hansen, and Jing H. Wang
`
`1384
`
`AUTHOR INDEX
`
`

`

`
`
` i i . Code
`
`This material may be protected by Copyright law(Title 17 U.S.
`Code)
`
`
`
`
`The Journal of Immunology
`
`Combination Therapy with Anti-CTLA-4 and Anti-PD-1
`Leadsto Distinct Immunologic Changes In Vivo
`Rituparna Das,*’ Rakesh Verma,*** Mario Sznol,**" Chandra Sekhar Boddupalli,*"
`Scott N. Gettinger,**" Harriet Kluger,*"’ Margaret Callahan," Jedd D. Wolchok,*
`Ruth Halaban,’ Madhav V. Dhodapkar,**" and Kavita M. Dhodapkar**"4
`Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to re
`though both PD-1 and CTLA-4 dampenthe T cell activation,the
`markable antitumoreffects. Al-
`in vivoeffects of these drugs in humansremaintobe clearly defined-
`Tobetterunderstandbiologiceffectsoftherapy,weanalyzedblood/tumortiss
`sit
`immunecheckpoint blockade, We show that blockade of CTLA-4, PD-1, or combination ofthe two leadstodistinct genomic
`functionalsignaturesinvivoinpurified humanTcellsandmonocytes.Therapy-inducedchangesarem
`in monocytes and involve largely nonoverlappingchanges in coding genes, including alternatively
`ore prominentin T cells that
`RNAs.Pathway analysis revealed that CTLA-4blockadeinduce
`rain
`Spliced transcripts and noncoding
`Sa proliferative signature
`memory Tcells, whereas PD-1 blockade instead leads to changes in genes implicated j
`predominantlyin a subset of transitional
`blockadeleads to nonoverlapping changesin gene expression, including proliferation-ass¢
`n cytolysis and NKcell function, Combination
`apies also have differential effects on plasmalevels of CXCLI10, soluble IL-2R, and IL
`ciated and chemokine genes. These ther
`following anti-PD-1 therapy may be incomplete in the tumor T cells even in the
`-la. Importantly, PD-1 receptor occupancy
`circulating T cells. These data demonstrate that, despite shared property of check
`setting of complete receptor occupancy 2
`alone, or in combination haye distinct immunologic effects in vivo. Improved unders
`Point blockade, Abs against PD-1, CTLA-
`agents in patients will support rational development ofimmune-based combinations
`tanding of pharmacodynamiceffects ofthese
`against cancer, The Journal ofImmunology:
`
`2015, 194: 950-959,
`
`ntigen-specific Tcell activationis regulated by a balance
`ofcostimulatory and coinhibitory Signals, such as those
`mediated by inhibitory receptors CTLA-4 and PD-I (1).
`Absthat block these inhibitory receptors or their ligands such as
`PDL-1 have led to impressive antitumor effects in several cancers
`(2, 3). CTLA-4 blockade with Ipilimumab (Yervoy; Bristol-Myers
`Squibb, Princeton, NJ) was the first
`treatment demonstrated to
`improve survival of patients with stage IV melanoma in ran-
`domized trials (4). Clinical
`trials with anti-PD-| Ab (such as
`Nivolumab) have demonstrated promising clinical activity in di-
`verse tumor types, including melanoma, renal cell carcinoma, and
`lung cancer (5-8),
`In preclinical models, combined blockade of
`both PD-1 and CTLA-4 ledto greaterantitumoreffects than either
`therapy alone, and,
`in a recent clinical trial,
`the combination of
`Nivolumab and Ipilimumab led to a distinet pattern of antitumor
`
`on Wheels (to K.M.D.),
`
`*Depurtment of Medicine, Yale University School of Medicine, New Haven, CT
`06520;
`'Smil
`i
`icine, New Haven,
`
`CT 06520;
`udwig Center for Cancer Immunotherapy, Memorial Sloan-Kettering
`Cancer Center, New York, NY 10065; “Department ofDermatology, Yale University
`School of Medicine, New Haven, CT 06520): and "Department of Pediatrics, Yale
`University School of Medicine, New Haven, CT 06520
`Received for publication July 9, 2014, Acceptedfor publication November 18, 2014,
`This work was Supportedin part by National Institutes of Health Grants ROI-A10792299
`(t0 K.M.D.), CA106802 (to M.V.D.), CAI35110(to M.V.D.), K24CA 172123 (to H.R),
`and P50-CA121974 (to RLH. and K.M.D.) and the Dana Foundation and Hyundai Hope
`Address correspondence and reprint requests to Dr Kavita M. Dhodapkar, Yale
`University School of Medicine, 333 Cedar Street, New Haven, CT 06520. E-mail
`address: kavita.dhodapkar@yale.edu
`Theonline version of this article contains supplemental material.
`Abbreviations used in this article: Combo, combination; Seq, sequential,
`Copyright ©2015 by The American Association ofImmunologists, Ine. 0022- I767/15/$25,00
`www.jimmunolorg/cgi/doi/10.4049/jimmunol, 1401686
`
`-Prarences
`i
`raples have clear differen¢
`
`activity, with rapid and deep tumor regressions in a substantial
`Proportion of melanoma Patients (9, 10),
`Clinical studies of PD-1 and CTLA-4 blockade in cancer
`patients have shownthat the twothe
`in the frequency and pattern of immune-related adverse events
`(3, 11). Whereas the Preclinical models of PD-1 or CTLA4
`blockade have to date been Poorly predictive of the pattern ah
`immune-related adverse events observed in the clinic. genetic
`deletion of PD-| or CTLA-4 lead
`lo very different effects
`S$
`mice. CTLA-4 knockout mice suf
`fer from alethal lymphopro-
`liferative disease, whereas deficie
`ney of PD-1 leads to less sever®
`phenotype with strain-specific
`autoimmunity (12-16). Although
`both PD-1 and CTLA-4
`act
`to dampen Tcell activation Via
`shared Signaling pathw
`ays, differences in sites of action have
`been proposed to help understand the differences in patterns of
`autoimmunity as well as antitumoreffects with PD-1 and CTLA-
`4 blockade. For example, the effects of CTLA-4 may be mostly
`in lymphoid tissue, whereas PD-|
`interactions may primarily
`occur in the periphery, Inhibitory Signaling via both PD-1 and
`CTLA-4 in human T cells in culture was shown to converge 0
`certain nodes such as inhibition of Akt phosphorylation, al-
`though the proximate events may differ, such as the involvement
`of phosphatase PP2A with CTLA-4, but not PD-1 (17-19), Im-
`proved understanding ofthe changesin gene expression in vivO
`in humans using genome-wide approaches in specific immune
`cells in response to checkpoint blockade therapy may provide
`new insights into the mechanisms of antitumor and autoimmune
`effects with these agents.
`In particular,
`it
`is important
`to in
`derstand whether combination checkpoint blockade in vivo leads
`to distinet or synergistic biologic effects compared with block-
`ade of individual checkpoints in humans,
`
`

`

`The Journal of Immunology
`
`Materials and Methods
`Patients
`
`Peripheral blood and tumortissue was obtained from patients (1 = 45)
`undergoing immune checkpoint blockadeafter obtaining informed consent
`under a separate protocol for the collection of research samples approved
`by the Yale University Institutional Review Board. This included patients
`receiving anti-PD-1 (2 = 24), anti-CTLA-4 (i = 9), or combination
`(Combo) therapy Gr = 12; 9 concurrent, 3 sequential [Seq]).
`
`Cell separation for gene expression analysts
`PBMCs were obtained by density gradient centrifugation process using
`Ficoll Paque Plus (GE Health Care Life Sciences). Monocytes were sorted
`from PBMCsusing anti-human CD14 microbeads (Miltenyi Biotec) using
`the manufacturer's protocol (20), Sample separation was performedusing
`
`
`MACS-LS columns (Miltenyi Biotec), The CD14
`fraction was further
`
`Subjected to a second round of separation for T cells
`using Human Par
`T Cell Isolation Kit
`| (Miltenyi Biotec). Purity of sorted populations was
`Monitored by flow cytometry (Supplemental Fig. 1A). Monocytes and
`l cells obtained by MACS bead separation were pelleted, suspendedin
`RLTbuffer (Qiagen), and stored at —80°C for RNA isolation.
`Geneexpression analysis ofpurified monocytes and T cells
`RNAwasextracted from purified monocytes and Tcells using the RNeasy
`Mini kit
`from Qiagen. We employed Affymetrix GeneChip Human
`lranscriptome 2.0 microarrays for gene expression profiling to allow
`analysis of coding as well as noncoding andalternatively splicedtranscripts.
`Paired pretherapy and posttherapy samples from each patient for cach cell
`'ype (monocyte or T cells) were compared directly to evaluate therapy-
`induced changes. We used Genespring GX 12.5 platform to analyze the
`changes in coding genes, exon workflowofthe Partek GS 6.6 to analyze the
`alternatively spliced exons 2.0 genechip, as described by Whistleret al. (2 l,
`22), and Partek GS 6.6 platform to analyze changes in the noncoding
`genes,
`
`Analysis ofcoding genes
`Data on coding genes were analyzed using Genespring GX 12.5 platform
`(20). Data were Imported via exon expression workflow employing
`RMAI6or PLIER16 (normalization) for analyzing the coding genes using
`the specific annotation support file for humantranscriptome array 2.0 (as
`Provided by Genespring GX). Experiment grouping for each treatment
`Cohort (anti-PD-| alone, anti-CTLA-4 alone, concurrent anti-PD-1 +
`tni-CTLA-4: Combo and PD-1 following CTLA-4; Seq) was created for
`both T cells and monocytes, and interpretation for
`the posttreatment
`“ompared with pretreatment samples was generated, Quality control
`analysis was used in principal component analysis and for probe hybrid-
`"ation intensities. For the identification of the differentially regulated
`Coding genes between post- and pretreatment samples for all
`treatment
`£roups under eachcell type, the locus filter was set on coding only genes
`(ATA gene chip 2.0 covers 44,699 genes/transeript clusters). We applied
`4 T test with a p value cutoff set at 0.05, followed bythe unsupervised
`Clustering analysis using Genespring clustering workflow (Euclidean dis-
`ance metric and Ward's linkagerule), Further statistical analysis involved
`“Pplication ofa fold-change thresholdof1.3; differentially regulated genes
`Were then manually curated to include coding genes only,
`D
`:
`Pathway analysis
`Pathway analysis ofdifferentially expressed genes was performed using the
`welicore pathway analysis platform (20). Differentially regulated gene
`lists with p< 0.05 andfold change of 1.3 were used from each treatment
`£foup as input
`for the Metacore pathway platform, and differentially
`"egulated pathways maps and gene ontology terms were generated.
`Analysis ofalternatively spliced genes
`Analysis to detect the potentially alternatively spliced genes was performed
`using the Exon workflow ofthe Partek GS 6.6 by taking into account exon
`
`Probes from Affymetrix HTA 2.0 genechip, as described by Whistler et al.
`{21, 22), Exons with a p value <0.05 were included for analysis. We ob-
`ined number of exon probe sets in each transcript cluster (with corre-
`‘ponding transcript cluster identification derived from the meta-probe set
`file) and discarded clusters with > 100 probe sets or <10 probe sets and
`eluded exon clusters with alt-splice p value 0.00001. The list of alter-
`
`hatively spliced genes was fillered on gene clusters with p values —0.05 and
`4 fold change of + 1.3 between pretherapy and posttherapy samples to ob-
`
`lain Splice variants that were also differentially regulated at the gene level.
`
`951
`
`Analysis of noncoding genes
`
`For analyzing the noncoding data, we used the Partek GS 6.6, postimporting
`data, and we employedthe filter on noncoding genomic loci (22.829) to
`retain only the noncoding probe sets on the HTA 2.0 genechip fromall
`samples. Statistical analysis involved one-way ANOVA, followed by ap-
`plication of a fold change threshold of1.3 to generate noncodingtranscript
`lists (unadjusted p value, 0.05), All the noncoding probe sets/transeript lists
`were further curated manually (via University of California Santa Cruz.
`Ensembl). Venn diagrams were generated between different
`treatment
`groups under each cell type.
`
`Quantitative PCR
`
`RNA fromT cells isolated frompatients pretherapy and posttherapy was
`usedto validate the microarray data forthe expression of Ki-67 and ICOS by
`quantitative PCR using the assays on demand primer probes (Applied
`Biosciences). Expression of GAPDH was monitored as a housekeeping
`gene. Reactions were sel up in triplicates using EZ PCR Core reagents’
`(Applied Biosciences), according to manufacturer's protocol, Relative
`expression of target genes was calculated using comparative threshold
`cycle method.
`
`Hmmunoassay for detecting plasma levels of various cytokines
`
`Plasma collected from patient samples before and atier therapy (ipilimumab
`alone, # = 5; nivolumabalone, 2 = 20; concurrent ipilimumab+ nivolumab
`[Combo], 1 = 6; and Seq nivolumab in patients with prior ipilimumab,
`n= 3) and stored at —20°C was thawed and used for the assay. Samples
`were used undiluted and in duplicate. Milliplex MAP Human Cytokine/
`Chemokine Magnetic Bead Panel Kit (MPXHCYTO-60K-PMX39: EMD
`Millipore) for 96-well plate assay was usedfor the simultaneous quanti-
`lication of 39 human cytokines and chemokines (epidermal growth factor,
`
`eotaxin, fibroblast growth fuctor-2, Fl-3 ligand, fractalkine, G-CSE, GM-
`CSE. growth-related oncogene,
`IFN-«2,
`IFN-y, [L-la, IL-1, L- Tree,
`IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8. 1L-9, TL-10, IL-12 [p40], IL-12
`[p70], IL-13, IL-1S, IL-17A, IFN--y-inducible protein- LO, MCP-1, MCP-3,
`macrophage-derived chemokine, MIP-la, MIP-1B, sCD4OL., soluble IL-
`2Re, TGF-a, TNF-«, TNE-B, vascular endothelial growth factor) using the
`protocol provided by the manufacturer, xPONENT software (Luminex)
`was usedto deteet, quantitate, and analyze the samples on the Luminex
`100 instrument.
`
`Detection ofcytokines secreted by tunor- infiltrating Tcells
`Tumors were processedinto single-cell suspension by manual dissociation
`and citherleft untreated or treated with anti-CD3/CD28 beads for 48 hin
`96-well round-bottom plates in RPMI with 5% pooled human serum,
`Cell supernatant was collected at 48 h and analyzed for eytokines and
`chemokines using the Luminex assay, as described above,
`
`Imimunophenotyping of PBMCs
`
`Cryopreserved patient presample and postsample PBMCs were thawed
`together and stained with dead cell exclusion dye and fuorochrome-
`conjugated anti-human Abs CD3, CD4. and CD8 (all from BD Pharmin-
`gen) and CD56(BioLegend), CD25 (clone 4E3: Miltenyi Biotec), CD45RO
`(BD Horizon), as well as PD-1 (clone J105: eBioscience). For some
`samples, cells were fixed and permeabilized, After permeabilization ofcells,
`fluorochrome-conjugated Abs against human granzyme B (BD Biosciences)
`and Ki-67 (cBioscience) were used to stain and detect the respective intra-
`cellular molecules. For detection of cytokine production, cells were rested
`overnight after thawing andthenstimulated with PMA and jonomyein, both at
`500 ng/mlinthe presence ofprotein transport inhibitor BD Golgi Stop (0.7 ul/
`mil). After 5 h ofstimulation, the cells were stained with the dead cell ex-
`clusion dye, fixed, permeabilized, and stained with fuorochrome-conjugated
`Abs against human CD3, CD4, CDS, and IFN-y (all from BD Biosciences).
`All
`live cell stains were acquired on BD-LSR Fortes
`i, and the data were
`analyze