EXHIBIT D1
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`EXHIBIT D1
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`VOLUME 153 I N0 1 [JULY 1, 1999
`
`' TABLE OF CONTENTS
`
`
`iillllllil Elliii
`
`1
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`6
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`11
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`15
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`21
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`Cutting Edge: Combined Treatment of TNF-a and IFN-y Causes Redistribution of
`Junctions] Adhesion Molecule in Human Endothelial Cells
`Harnnoba Ozalci', Kenji Isa-ii, Hisanori Hormone, Hidenora‘, Ara-i, Taken-ire Kama-mom,
`Kat'saya Ottawa, Akfltéfi} Iwamatsa, and Tom Kits
`
`Cutting Edge: Trimolecular Interaction of TCR with MHC Class 11 and Bacterial
`Superantigen Shows a Similar Affinity to MHC:Peptide Ligands
`Stella Redpatli, S. Man-irAimn, Cli-i'istiinaM. Lia, Anne M. O’Roarire, and Nicholas R. J. Crisco-algae
`Cutting Edge: Proteasorue involvement in the Degradation of Unassembled lg Light Chains
`Thomas O’Hare, Gregory D. Wiens,Elaiea-beth11. Wham-nib, Caroline A, Enns,
`'
`and Marin-n B. Rittenberg
`
`Cutting Edge: A Short Polypeptide Domain of HIV-l—Tat Protein Mediates Pathogenesis
`RobertA. Boykins, Renaud Mali-tenet, Unto. T. Shanka-uaram, Yong Song Giro, Sherwin F. Lee,
`Indira K. Hewlett, LarryM. Wald, Hyatt-3.1K. Kle-i-nma-n. JohnN. Brady, KennethM Yamada,
`and Salinas}: Dhaeoan
`
`and IE5
`
`Cutting Edge: Expression of the NF of Activated T Cells in Eosinophils: Regulation by IL—4
`Tan Jinqaa-n, Sha- Qna-n, Henrik H. Jacobi, Clans M, Reta-newt, Anders Mitiner, Jens B. Hansen.
`Charlotte Wiygesen, Lars P. Ryder, Hans O. Malaise-n, Hans-Jorgenill’alling, and Lars K. Poaisen
`
`CELLULAR IMMUNOLOGY AND IMMUNE REGULATION
`
`25
`
`The CD36 Subunit of the TCR Contains Endocytosis Signals
`Aldo Bom'oto, Juan Lama, FlorenceN-iedergang, Alice Danny-Vassar, Balbt'no Maroon,
`and. Andviés Aimee?“
`
`
`
`
`
`
`
`
`
`On the cover: linniunostajnjng of rat lungs for rat TlMP—2 (A and B) and rat SLPI (G andD) at 0 h (A and C) and
`4 h (B and B) after hitmpulmonaiy deposition of IgG immune complexes. In B, the arrow indicates stained
`vascular endothelial cells, while arrowheads indicate posuively staining alveolar epithelial cells. In D, solid
`
`K. J, Johnson, and P. A. Ward. 1999. Regulatory efi’ects of endogeneous protease inhibitors in acute lung in
`flammatory injury. J.
`
`
`
`
`
`The Journal ofIm-manology (ISSN 0022-1767) is published twice each month by The American Association of Im—
`munologists, 9050 Rockville Pike, Bethesda, MD 20814. Subscription rates: USA. AND POSSESSIONS: personal,
`$270.00; institutional, $470.00; single copy, $20.00. FOREIGN (incl. Canada): personal, $410.00; institutional, $605.00;
`single copy, $25.00. Air Printed Matter ra'es are available on request from Customer Service, The Journal of Immu—
`nology, Room L—2310, 9650 Rockville Pike, Bethesda, MD 20814—3998. Indexed by Current Contents and Index Medi—
`rus. Periodicals postage paid at Bethesda, MD 20814 and at additional mailing ofiices. Country of origin USA. Printed
`on acid—free recyclable paper. Postmaster: Send address changes to The Journal of Immunology, Room L—2310, 9650
`Rockville Pike, Bethesda, MD 208149998. Copyright © 1999 by The American Association of himiunologists
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`32
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`4O
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`Presentation of the Self Antigen Myelin Basic Protein by Dendritic Cells Leads to
`Experimental Autoimmune Encephalomyelitis
`Bonnie N. Dittet, Irene Visintin, Raina M. Merchant, and Cherries A. Janeway, Jr.
`
`MyeIin Oligodendrocyte Glycoprotein Induces Experimental Autoimmune
`Encephalomyelitis in the “Resistant” Brown Norway Rat: Disease Susceptibility Is
`Determined by MHC and MEG—Linked Eifects on the B Cell ReSponse
`Andreas Stej‘fiert, Usclii Bream, Marta Starch, Doris Lainbracltt—Washington, Carole Boarqnin,
`Kan-“t Wonigeit, Hans Lassmann, and Christopher Liningtmi.
`
`Degradation of ZAP-70 Following Antigenic Stimulation in Human T Lymphocytes: Role of
`Calpain Proteolytic Pathway
`Doris Penna, SabinaMtttter. Fabio Martinon, Stephane Demete. Manic Itcasltima,
`and Salvatore Vatitatti
`
`Polyriboinosinic Polyribocytidylic Acid (PonCI:C)) Induces Stable Maturation of
`Functionally Active Human Dendritic Cells
`Rob M. Verdijtc, Tana—Mutts, Ben Esendam, Janine Kamp, Gees J. M. Met-tar”, Annelte Brand,
`and Etc Gummy
`
`Expression of a Variant of CD28 on a Subpopulation of Human NK Cells: Implications for
`B7—Mediated Stimulation of NK Cells
`Joanna Gatea—Lanii, David Baiting, San—Uta Gan, Leonid Krieocatcnapco, MarcetKniper,
`Jeep Gaken, Bernard Saucers-italic, and Farzin Fareaneh.
`
`The Regulatory Role of CD45 on Rat NK Cells in Target Cell Lysis
`Katintta M. Giezeman—Smits. Arno Gorter, Ronald L. P. van Vt'terbe'tghe, Jaap D. H. o. Eendenbnm,
`AtmanderM M. Eggermont, Gert Jan Ftearen, and PeterJ. K. Knppen
`
`Impaired Ability of MHC- Class H"— Dendritic Cells to Provide Tumor Protection Is
`Rescued by CD40 Ligation
`[an F. Her-mans, David S. Ritchie. Angeta Daistt, Jianping Yetta, Marilyn R. Kenny,
`and Franca Ronckese
`
`Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic
`Cell-Like Characteristics Through Discrete Signaling Pathways
`Gary K. Kcski, Gretchen N. Schwartz, David E. Weng, Brian J. Czemiectci, Citaiies Canter,
`Ronald E. Cress. and PeterA. Cohen
`
`A Novel Function of VaI4+CD4+NKT Cells: Stimulation of ID12 Production by APC in the
`innate Immune System
`Mickie Tamara. Wen—Gong Ya, Hyan-JongAhn, Motozo Yamashita, Yi-F-a Yang, Shire Ono,
`Toslrtyaki Hantaotca, TetsaKawa-no, Maseru Tanigaclii, Yasaltiko Koezntca. and Hiromi Fajiwara
`
`Matthew J. Deaths, Ross M. Kedl, and Matthew F. Mescnezr'
`
`Induction of Cyclooxygenase2 on Activated T Lymphocytes: Regulation of T Cell
`Activation by Cyclooxygenase-Z Inhibitors
`Mignet A. Inigacz, Carmen Panzan, and Manuel Fresno
`
`CD28—Mediated Regulation of mRNA Stability Requires Sequences Within the Coding
`Region of the IL-2 mRNA
`JackA. Ragheb, Mary Dean, and RonaldH. Schwartz
`
`Immrmogenicity of Apoptotic Cells In Vivo: Role of Antigen Load, APC and Cytokines
`AnnaRoncketti, Patrizia Revere, Gianticntenicalezzi. Giacomo Galati. SiteiaHettai,
`Marta Pia Piotr-t, Mfl'fia Paola Garanctni, A-ngeto 11- Manfredi, Claudio Regent, and Matteo Beltane
`ALVOGEN, EXh. 1055, p. 0167
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`62
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`71
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`77
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`82
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`93
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`102
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`111
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`120
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`130
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`137
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`143
`
`148
`
`155
`
`165
`
`174
`
`The Guanine—Nucleotide Exchange Factor Vav Is a Crucial Regulator of B Cell Receptor
`Activation and B Cell Responses to Nonrepetitive Antigens
`Martin R Bachmann, Lars Nitselzae, Connie Kraioozylt, Kerry Tedfovd, Pamela S. Oliaslii,
`Klaus l). Fischer; and. JosefM. Peaninger
`
`Bioactive lL-18 Expression Is Up—Regulated in Crohn’s Disease
`Giovanni Monteieone, Francesco Ti'apasso, TieianaPaivelle, Livia Bia-ncone, Antonella Stella,
`Rodoifo Indiana, Francesco Lneea, Alfredo FELSCO, and .F‘i-‘ancesoo Palione
`
`lFN—y— and Cell-to-Cell Contact—Dependent Cytotoxicity of Allegraft-Induced Macrophages
`Agamst Syngeneic Tumor Cells and Cell Lines: An Application of Allografting to Cancer
`Treatment
`Ryotaro Yosaida, Ynlrio Yoneda, Manabn Kniiyaina, and Takahiro Knboto
`
`Anergic CD8+ T Cells Can Persist and Function In Vivo
`Catherine A. Blisli, Stacey R. Dillon, Andrew G. Fan; and Pamela- J. Pink
`
`Diiferential Expression and Cross-Regulatory Flmction of RANTES During Mycobacterial
`{Type 1) and Schistosomal (_Type 2) Antigen-Elicited Granulomatous Inflammation
`Stephen W. Cliensne, Kelly S. Wamnington, Eric J. Allenspach. Bao Lu, Graig Gerard,
`Steven L. Kan-lash and Nicholas W. Lnkacs
`
`Transfection of Immature Murine Bone MarroweDerived Dendritic Cells with the
`Granulocyte—Macrophage Colony~Stimulating Factor Gene Potently Enhances Their In Vivo
`Antigen~Presenting Capacity
`Clara Cnriel—Leioanamniski, Kaisten Mah'flke, Mai-ta Labenr, Benthold Raters, Waiter Schmidt,
`Richard D. Granstein, Thomas A. Lager, Thomas Software, and Stephan Grabbe
`
`184
`
`A Model for CDB+ CTL Tumor lmrnunosurveillance and Regulation of Tumor Escape by
`CD4 T Cells Through an Effect on Quality of CTL
`.S'o Matsni, Jefirey I). Ahlei's, Alert 0. Vomit-eye); Masalri Tamas, Talcn illsnlcni, DaviaP. Carbone,
`Lance A. Liotta, and Jay A. Bei‘zofislcy
`
`194
`
`CD30 Overexpression Enhances Negative Selection in the Thymus and Mediates
`
`206
`
`212
`
`21'?
`
`224
`
`232
`
`242
`
`and Giorgio Inga-immi-
`
`Development of T Lymphocytes at Extrathymic Sites
`MaiiastefaniaAntica and Roland Scollay
`
`Diversity of NK Cell Receptor Repertoire in Adult and Neonatal Mice
`AltimKnbota, Satoko Knboia, Stefan Lok-wasser, Danie L. Mayer, and Furnio Takei
`
`Humoral Response Suppression Observed with CD23 Transgenics
`Margaret E. Payet, Elaine C. Woodward, and Daniel H. Con-rad
`
`Bacterial DNA or Oligonucleotides Containing Unmethylated CpG Motifs Can Minimize
`LipoponsaccharideJnduced Inflammation in the Lower Respiratory Tract Through an IL—
`12—Dependent Pathway
`DavidA. Schwartz, Christine L. Woklfoi‘d-Lenane, fiiinothyi Quinn, and AiihnrM Kiieg
`BALB/c Invariant Chain Mutant Mice Display Relatively Eflicient Maturation of CD4+ T
`Challenge
`George Ken-Ly and Eiizaoeth K. Bikofi‘
`
`Depletion of IL—10— and TGF-JG—Producing Regulatory 316 T Cells by Administering a
`Daunomycin-Conjugated Specific Monoclonal Antibody in Early Tumor Lesions Augments
`the Activity of CTLs and NK Cells
`Naohiin Seo, Yosailci Tokni‘a, Masai-him Takiga-ioa, and Koliji Egawa.
`
`
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`MOLECULAR AND STRUCTURAL IMMUNOLOGY
`
`250
`
`259
`
`269
`
`2?8
`
`288
`
`295
`
`301
`
`A Single—Chain lL-l2 IgG3 Antibody Fusion Protein Retains Antibody Specificity and IL—12
`Bioactivity and Demonstrates Antitumor Activity
`Lisan S. Peag, Manuel L. Penichet, and Sheiie L. Morrison
`
`Predicting Regional Mutability in Antibody V Genes Based Solely on Di— and Trinucieotide
`Sequence Composition
`Gang; S. Shapiro, KayaA-oiszas, Dania! Iltle, and Lawrence J. Wysoclci
`
`Constitutive Nuclear Translocation of NF-KB in B Cells in the Absence of IKB Degradation
`Stefan Dome and Ronald B. Corley
`
`Genes Encoding Three New Members of the Leukocyte Antigen 6 Superfamiiy and a Novel
`Member of Ig Superfamily, Together with Genes Encoding the Regulatory Nuclear Chloride
`
`The Specificity of a Weak 3x8 TOR Interaction Can Be Modulated by the Glycosyiation of
`the Ligand
`Johannes Hanipl, Hansjory Schiltl, Christa Litzenberger, Miriam Baron, MickaelP. Crowley,
`and Yuck—Mira Chime
`'
`
`Enhancer—Blocking Activity Within the DNase I Hypersensitive Site 2 to 6 Region Between
`the TCR a and Dadl Genes
`Xian-Ping Zhong and MichaelS. Krangel
`
`Invariant or Highly Conserved TCR a Are Expressed on Double-Negative
`(CD3+CD4‘CD8‘) and CD8+ T Cells
`Mei Han, Lisa Hamisoii, Patricia Kean, Kimberly Steormson, Jefiirey Carrier,
`and Mam Aim Robinson
`
`HOST DEFENSE
`
`312
`
`322
`
`330
`
`337
`
`Surfactant Protein D Binds to Mycooacteiiiim tabeitvilosis Bacilli and Lipoarabinomannan
`Via Carbohydrate-Lectin Interactions Resulting in Reduced Phagocytosis of the Bacteria by
`Macrophages
`J. ScottFerg-ason, Dennis R. Voellcer, ancisX. McComnack, and Lamp 5'. Schlesinger
`
`Genetic Immunization of Mice Against Listeria monocytogenes Using Plasmid DNA
`Encoding Listeriolysin O
`KennethA. Comell, H. G. Archie Boa-wen David J. Hinrichs, and Ronald A. Bavaria;
`
`Protective Immunity from Naive CD8+ T Cells Activated In Vitro with MHC Class I Binding
`Immunogenic Peptides and IL-2 in the Absence of Specialized APCs
`Conrad Hansen Frank Zippiich, Isabelle Lento-ad, Susanne Wirlli, and Ambros W. Hilgin
`
`Schistosomelnfected 1L4 Receptor Knockout (KO) Mice, in Contrast to 1L4 KO Mice, Fail
`to Develop Granulomatous Pathology While Maintaining the Same Lyinphokine Expression
`
`Draga’aa Jankooic, Marika C. Kiilloe’rg, Nancy NooeiiATrauih, Patricia CaSpar, Jem-olclM Ward,
`Allen W. Cheeoer, William E. Paul, and Alan Sher
`
`IN FLAMMATION
`
`343
`
`Characterization of Eosinophil Adhesion to TNF—a~Activated Endotheiium Under Flow
`Conditions: as, integrins Mediate Initial Attachment, and ESelectin Mediates Rolling
`Lit-mien. H. [i‘lfina-n, Philip H. M. Kaijper, Jan A. M. can tier Linden, Jan-Willemcl Lammers,
`Jaap Jan Zioaginga, and Leo Koeiideimcn
`ALVOGEN, Exh. 1055‘ p_ 0169
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`351
`
`359
`
`Involvement of Thioredoxin in Rheumatoid Artlnitis: Its Costirnuiatory Roles in the TNF—a-
`Induced Production of llr6 and 1&8 from Cultured Synovial Fibroblasts
`Shin-icni Yoshiaa, Tetsnji Karon, Tosh-ifumi Tetsnka, Kamrico Uno, Nobao Matsai,
`and Takashi Okamoio
`
`Deficiency of 5-Iipoxygenase Abolishes Sex-Related Survival Differences in MRL—ipiflpr
`Mice
`Jennifer-L. Goatet, Robert C. Grifiiths, Phillip Ruiz, Robert F. Spaniey, David S. Pisetslcy,
`Severity H. Kolier, and Thomas M, Cofinian
`
`367
`
`P~Se1ectin Binding Promotes the Adhesion of Monocytes to VCAM—l Under Fiovv
`Conditions
`
`Tadayiii’ci Yago, Maniom Tsnknda, and Mntsakiko Minami
`
`374
`
`380
`
`38'?
`
`396
`
`403
`
`412
`
`420
`
`427
`
`434
`
`Estrogen Protects Against Cellular Infiltration by Reducing the Expressions of E-Selectin
`and IL~6 in Endotoxin-Induced Uveitis
`Noriko Miyamoio, Miokiiio Mancini, Ian-mi Sneama, Kiyoshi Saanma, Kaori Kohayaslii,
`and Yoshiliito Honda
`
`Differential Cytokine Modulation and T Cell Activation by Two Distinct Classes of
`Thalidomide Analogues That Are Potent Inhibitors of TNF—oz
`Lawn. G. Corral, Patrick A. J. Hastett, George W. Mailer, Roger Chen, Lit—Min Wong,
`ChristopiierJ. Ooampo, Rebecca 1". Patterson, David I. Stiiiing, and Gina Kapian
`
`Pulmonary Surfactant Protein A Modulates the Cellular Response to Smooth and Rough
`Lipopolysaccharides by Interaction with CD14
`Hitonii Sano, Himshi Sohina, Tatsnski Maia, Shin—ism Noninra, Dennis R. Voeiker,
`and Yoshio Knroki
`
`Decreased Expression of Th2 Type Cytokine mRNA Contributes to the Lack of Allergic
`Bronchial Inflammation in Aged Rats
`Kyoiaro Ida, Hiroshi Hayakawa, Taliesin Yogi, Atsahiko Sara, Ynlrio Koide, Amish-i Yoshida,
`Masato Uchijiina, Takofnmi Sada, Kingo Chida, and HiroioskiNakamara
`
`Mouse Monocyte—Derived Chemokine Is Involved in Airway Hyperreactivity and Lung
`Inflammation
`Jose-Angel Goneaio, Yang Pan, Clare M. Lloyd, Gui—Qnan Jia, Gary Ya, Barry Dussaalt,
`Christine A. Powers, Amanda E. I. B‘o-adfoot, Anthony J. Coyie, David Gearing,
`and Jose-Carlos Gutierrez—Ramos
`
`The Murine Antiapoptotic Protein A1 Is Induced in lnflan'lmatory Macrophages and
`Constitutiveiy Expressed in Neutrophils
`Amos Oriofsky, Robert D. Soniogyi, Lonis M Weiss, and Michael B. Prysioiosky
`
`Induction of Phosphorylation and Intracellular Association of CC Chemokine Receptor 5
`and Focal Adhesion Kinase in Primary Human CD4+ T Cells by Macrophage-Tropic HIV
`Envelope
`(Tia-adia- Cioaica James Aiihos, Margaret Rails, Mauro Vaccarezza, Andrea Rubbers, Agoslino Rina,
`Kathryn Wildt, Oren Cohen, and Anthony S. Fanci
`
`NF-KB Regulation by IKB Kinase in Primary Fibroblast—Like Synoviocytes
`Karifi‘ied R. Aapperle, Brydon L. Bennett, David L. Boyle, Pant—Peter Tak, Anthony M. Manning,
`and Gary S. Fi‘restein
`
`Osteoclast Difi‘erentiation Factor Acts as a Multifunctional Regulator in Murine Osteoclast
`Difl‘erentiation and Function
`Eijiro Jimi, Shaiclii Akiyaina, Taro Ilsa-Militia“, Nobno Okahashi, Kanichiro Kobayaslii,
`Nob-aynki Udagaioa, Tatsnji Nishihara, Naoynki Takahashi, and Tatsao Sada
`ALVOGEN, Exh. 1055. p. 0170
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`ALVOGEN, Exh. 1055, p. 0170
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`CLINICAL IMM u NOLOGY
`
`443
`
`448
`
`456
`
`466
`
`4-76
`
`483
`
`491
`
`507
`
`525
`
`533
`
`535
`
`537
`
`In Situ T Cell Responses Against Melanoma Comprise High Numbers of Locally Expanded
`T Cell Clonotypes
`Per inorSt-raten. Per Giudoerg. Kirsten: Gipnbcek, Mia RiiseHansen. AleareiE Kirkin, Tina Sereniet.
`Jasper Zenilien, and Jiirge-n C. Becker
`
`Repeated Administration of Adenovirai Vectors in Lungs of Human CD4 Transgenic Mice
`Treated with a Nondepleiing CD4 Antibody
`Narenara Chirmule, Almsegea Maren, Sarah, Enlen Haecker, John Taeelaar, Guano-ping Gao,
`Steven E. Roper, Joseph V. Hughes, and James M. Wilson
`
`1'L-5 Increases Expression of 5-Lipoxygenase—Activating Protein and Translocates 5-
`Lipoxygenase to the Nucleus in Human Blood Eosinophils
`Andrew S. Cowbmn, Stephen T. Holgale, and Anthony P. Sampson
`
`Skin Homing (_Cutaneous LymphocyteAssociated Antigen—Positive) CD8+ T Cells Respond
`to Superantigen and Contribute to EosinOphilia and IgE Production in Atopic Dermatitis
`Mabeccel Ainslie, Hans—Hive Simon, Lorenz Weigl, OlioerKreyclen, KnrtBlaser, and Ce’emi A. Akdis
`
`A Complementary Peptide Vaccine That Induces T Cell Anergy and Prevents Experimental
`Allergic Neuritis in Lewis Rats
`Shigern Aroga, Masaniro Kishimoto, Salolco Doi, and Kenji Nakasnima
`
`C‘niistopheRonsin, Veronique Changé‘cott, isaoelle Pcullion, Nicolas Almonclze, Catnenlne (lanolin,
`and Fredémc Trieoel
`
`Altered Memory T Cell Difierentiation in Patients with Early Rheumatoid Arthritis
`Alla Skapenko, Jory Wenaler. Peter-E. Lipslcy, Joachim R. Kalaen, and HendrikSchulze—Koops
`
`Endogenous CD8+ T Cell Expansion During Regression of Monoclonal EBV—Associated
`Posttransplant Lymphoproliferative Disorder
`Vijay P. Khaki, Robert A. Baiooclzi, Rnooi Pang, Adam R. Oberkircher. Jean M. Dolee, Pamela M.
`Ward, Geoffrey P. Herein, and Michael A. Caliginri
`
`Eflicient Transfer of a Tumor Antigen-Reactive TCR to Human Peripheral Blood
`Lymphocytes Confers Anti-Tumor Reactivity
`Timothy M. Clay, Mary C. Caster, Jessica Sachs, Pal-rick. Hana, StevenA. Rosenberg,
`and Michael I. Nisliimnra
`
`Induced Expression of B7-1 on Myelorna Cells Following Retroviral Gene Transfer Results
`in Tumor—Specific Recognition by Cytotoxic T Cells
`Karin Torte, Xne-Gnang Zliang, Eric Legonfe, Catherine Heniog, Majiarla’ehtali,
`Jean-Francois Rossi, anal Bemard Klein
`
`Early Autoantibody Responses in Prediabetes Are IgGl Dominated and Suggest Antigen-
`Speciflc Regulation
`Ezio Bonifacio, Miriam Sciipoli, Katharina Kindel, Martin Filclitenonscn, and Anette—G. Ziegler
`
`Increased Apoptosis in Patients with Major Depression: A Preliminary Study
`EranEilat, ShlomoMendlooic, AdielDoron, Vern Zakntli, anal ZoiSpirer
`
`Author Index
`
`Information for Authors
`
`ALVOGEN’ EXh' 1055‘ p' 0171
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`
`
`ALVOGEN, Exh. 1055, p. 0171
`
`

`

`Editorial Policies and Practices
`
`Submission Form: Full Length Articles
`
`AAI Membership Application
`
`Submission Form: Cutting Edge
`
`AAI Trainee Membership Application
`
`ALVOGEN, Exh. 1055, p. 0172
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`ALVOGEN, Exh. 1055, p. 0172
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`

`

`This material may be protected by Copyright law (Title 17 US. Code)
`
`i
`
`Difl‘erential Cytokine Modulation and T Cell Activation by
`Two Distinct Classes of Thalidomide Analogues That Are
`Potent Inhibitors of TNF-o:I
`
`Laura G. Corral,” Patrick A. J. Haslettf' George W. Muller,* Roger Chenfi‘ Lu-Min Wongfi‘
`Christopher J. Ocampo,T Rebecca T. Patterson,* David I. Stirlingfi‘ and Gilla KaplanT
`
`TNF-a mediates both protective and detrimental manifestations of the host immune response. Our previous worlc has shown
`thalidomide to be a relatively selective inhibitor of TNF-o: production in vivo and in vitro. Additionally, we have recently reported
`that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF—o:
`activity and reduced or absent toxicities, novel TNF-a inhibitors were designed and synthesized. When a selected group of these
`compounds was examined for their immunomodulatory activities, dilferent patterns of cytokine modulation were revealed. The
`tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors,
`inhibited TNF—o: production, increased IL-10 production by LPS-induced PBMC, and had little elt‘ect on T cell activation; the
`other class of compounds, similar to thalidomide, were not pbosphodiesterase 4 inhibitors and markedly stimulated T cell pro-
`liferation and [L-2 and IFN-‘y production. These compounds inhibited TN'F-oc, lL-lB, and 11-6 and greatly increased IL-fil}
`production by LPS-induced PBMC. Similar to thalidomide, the elfect of these agents on 1L-12 production was dichotomous; IL-12
`was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The
`latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes
`of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of djfl'erent immunopathological
`disorders. The Journal of Immunology, 1999, 163: 380—386.
`
`I
`
`nmor necrosis factor a, a highly pleiotropic cytokine pro—
`
`duced primarily by monocytes and macrophages, plays a
`
`central role in the host protective immune response to
`bacterial and viral infections. For example, TNF-oe is essential for
`granulonia formation and the control of bacterial dissemination in
`experimental tuberculosis in mice (1, 2). In addition, TNF—or added
`to infected cells in vitro inhibits the replication of both DNA and
`RNA viruses (3. 4). However, the cytokine may also play a role in
`the pathogenesis of disease. Perhaps the best evidence for this is
`the dramatic reduction in disease activity observed in rheumatoid
`arthritis and inflammatory bowel disease after treaunent of patients
`with neutralizing anti-TNF—cr Abs (5, 6). Additionally, elevated
`levels of TN'F—o have been associated with the fevers. malaise, and
`weight loss that accompany chronic infections (7), and reductions
`in TNF-d levels have been linked with an amelioration of clinical
`symptoms in a number of disease states {8—11}.
`Our previous work has shown thalidomide to be a relatively
`selective inhibitor of TNF-Q‘. production by human monocytes in
`Vivo and in vitro. Leprosy patients with erythema nodosum lepro-
`sum treated with thalidomide. experience a reduction of serum
`TNF-o levels with a concomitant abrogation of clinical symptoms
`
`
`“‘Celgenc Corporation, Warren, N] 0?0591 and 'iahoratory of Cellular Physiology
`iLI'ICl immunology, The Rockefeller University. New York, NY 10021
`
`Received for publication December 13. 1998. Accepted for publication April 22. 1999.
`The costs of publication of diis article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advei'riscoicm in accordance
`with IS U.S.C. Section 1134 solely to indicate dils fact.
`
`1 This work was supponcd by Col gene CorporaLion. UK was supported by Public
`Health Service National institutes of Health Research Grant Al-ZEE-Ifi: P.A.J.H. was
`supponed by General Clinical Research Center Grant MOI-121200102.
`
`3 Address correspondence and neprinl requests to L. G. Con-a}. Laboratory of Cellular
`Physiology and Immunology. Rockefeller University, 1230 York Avenue. New York
`NY 1002!. E-mail address: corrall@rockvax.rockefeller.edu
`
`Copyright ® 1999 by The American Association of Immunologists
`
`(9), In patients with tuberculosis, with or without HIV infection,
`thalidomide lowers plasma TNF-oc protein levels and leukocyte
`TNF-cu mRNA levels in association with an accelerated weight
`gain (8). In vitro, thalidomide has been shown to selectively par-
`tially (504096) inhibit "INF—o: produced by inonocytes and mac-
`rophages stimulated with LPS (12).
`Recently, we have reported the ability of thalidomide to co-
`stimulate T cells in vitro (13). Thus, in addition to its monocytc
`cytokinc—inhibitory activity, thalidomide exerts a costimularory or
`adjuvant ellect on T cell responses that includes increased produc—
`tion of IL—2 and IFN—y. This efi'ect may contribute to the human“-
`modulating efi'ccts of the drug.
`To obtain drugs that are more etficient TNF—a inhibitors than
`thalidomide, structural analogues of the parent molecule have been
`synthesized and examined for inhibition of TNF-or productibn. Re—
`cently, some of these thalidomide analogues have been describe
`(12, 14, 15, 47). On a molar basis, these reported compounds are
`up to 50,000-fold more potent
`than thalidomide at
`inhibiting
`TNF—o: production by PBMC in vitro. In this study, we have se—
`lected six of these compounds and evaluated them for their effects
`on the production of other monocyte cytokines, as well as their
`ilnrnunomodulatory effects on T cells.
`
`Materials and Methods
`Preparation of cells
`
`PBMC were isolated hum the blood of healthy volunteers by Ficoll—
`Hypaque [Pharmacia Piscataway. NJ) density cenuifugation as previously
`described (12). T lymphocytes were purified from PBMC by resetting Willi
`neuraminidase-treated sheep erythrocytes and subsequent incubatioz'. OT
`eiythrocyte-rosetting cells on a nylon wool column. Nonadhercnt cells
`eluted from the column were >93% C03 Ag positive by flow cytotr-sr-‘J‘l‘
`lFACStar, Becton Dickinson. San Jose, CA). Leukocytes were cultured in
`RPMI medium (Life Technologies. Grand Island, NY) supplemented with
`
`oozz—iiswswssino
`
`ALVOGEN, Exh. 1055. P- 0173
`
`ALVOGEN, Exh. 1055, p. 0173
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`

`

`The Journal of Immunology
`
`(use ABT human serum. 2 mM L—glutamine, 1.00 Ll/ml penicillin, and 100
`itgr'l’l'll streptomycin (Life Technologies).
`
`Thalidomide and analogues
`
`Thalidomide and analogues (Celgene, Warren, NJ) were dissolved in
`PMSO (Sigma, St. Louis, MO); further dilutions were made in culture
`medium immediately before use. The final DMSO concentration in all
`,fngE‘lYS was 0.25%. The following structural analogues were used: ClI—A is
`compound 3a (l4) and CC—1069 (12): CH-B is compound (EC—3052 (16)
`with the carboxyrnethyl group replaced by an amide moiety; CH—C is an
`amino-substituted analogue of compound 4b (14); compounds Cl-A, Cl-B,
`and Cl—C are amino-substituted analogues of thalidomide. Cl-A is 53, CI—B
`is its and CI-C is 14 (47).
`
`t-"BMC stir-nutrition by LPS
`
`3- Elli/[C (E X 105 cells) incubated in 96-well flat-bottom polystyrene Costar
`USSUC culture plates (Corning, Corning, NY) were stimulated by l ugtml
`1.?5 from Salmonella minnesota R595 (List Biological Labs, Campbell.
`CA) for the induction of TN'F—o: , 11,-1.8, 11-6, 11-8, lL~10, and 11-12 (12).
`if'ells were incubated with or without thalidomide or analogues for 20 h,
`and supernatants were collected for the determination of cytokine levels by
`ELISA.
`
`PBMC stimulation by anti-CD3 Ab
`
`PBMC (l X 10” cells) were stimulated by cross-linking of the TCR by
`immobilized monoclonal mouse anti-human CD3 (Orthoclorre OKT3, a
`kind gift of Dr. R. Zivin, Ordrobiotech, Raritan, NJ) as previously de~
`scribed (13). The anti—CD3 Ab was diluted to 10 ugi’ml in 100 pl PBS and
`coated onto 48—well flat‘bottorn polystyrene Falcon tissue culture plates
`(Elector: Dickinson, Franklyn Lakes, NJ) by overnight incubation at 4°C.
`Appropriate dilutions of thalidomide and analogues were added at the start
`or' the cell cultures. Supematants were collected at 24, 48, and 72 h and
`assayed for 11-10, lL-IZ, and TNF~a levels. Cells were collected at 48 h
`for evaluation of CD40 ligand (CD40L)3 and CD3 surface expression by
`two—color flow cytometry (anti-CD40L. PharMingen. San Diego, CA; anti—
`iT."'D3. Becton Dickinson, San Jose, Ca).
`
`1?" cell stimulation and proffer-anon assays
`
`Purified T cells (2 X It)5 cellslwell) in 96—well Costar tissue culture plates
`tCol'ning) previously coated with anti-CD3 mAb (as above) were treated
`daily with thalidomide or analogues for up to 120 Ir. Supernatants were
`=oar-vested for lFN—y assay at 7‘2 h. T cell—proliferative responses were as-
`sayed by measuring [3H}thymidine (NEN Products, Boston, MA) incorpo-
`ration during the last 18 h of l20-h cultures. DNA was harvested onto fiber
`mats with an automatic cell harvester (Skatron, Stirling, VA), and [JHJthy—
`r'nicline incorporation was measru'ed with a LKB 1205 Betaplate liquid
`scintillation counter (Wallac, Gaithersburg, MD)
`
`Phospltodt'estemse 4 (P1384) inhibition assay
`P054 inhibition was evaluated in purified extracts of promonocytic U937
`cells using a modified method of Hill and Mitchell (17) as previously
`described (14). Cells (1 x 109) were washed in PBS and lysed in cold
`homogenization buffer (20 mM Tris~'HCI, pH 7.1; 3 mM 2~ME;
`1 mM
`MgCl: 0.] mM EGTA, 1 uM PMSF,
`1 ugimi leupeptin). After homoge—
`nization with a Dounce homogenizer, the supernatant was collected by
`centrifugation and loaded onto a Sephacryl 8—200 column equilibrated widr
`l'-i.tmogenization butler. PDE4 was eluted in homogenization butter, and
`enzyme activity was determined in 50 mM Tris—HCl, pH 7.5, 5 mM MgClQ,
`and 1 MM cAMP (of which 1% was [3H]cAMP) as described in detail by
`Thompson et al. (18). Reactions were performed at 30°C for 30 min and
`terminated by boiling for 2 min. Briefly, cyclic 3’,S’-[3H]AMP was con—
`verted to 5'—[’H]AMP by phosphcdiesterase, The separation of 5’—
`lelAMP from 3’.fi'-l3H]AM'P was achieved by enzymatically converting
`5"-[5HlAMP to [flHladcnosine with nucleotidase present in snake venom
`l-Sigma, V-0376), l mglrnl at 30°C for is min, Adenosine was separated
`from the unreacted cyclic substrate by addition of 200 Jrtl of AGl—XS resin
`lBio-Rad, Hercules. CA) that absorbs cyclic 3’,S'—[3H1AMP, Samples were
`then spun at 3000 rpm for 5 min, and 50 al of the aqueous phase were
`taken for counting of aderrosine radioactivity by liquid scintillation tech-
`niques. Enzyme activity was determined in the presence of varying con-
`rTontrations of compounds.
`lC$0 values were determined from dose—re-
`Sponses curves derived from at least three independent experiments done in
`
`
`' Abbreviations used in this paper: CD40L. CD40 ligand: PDE4. pliosphodicsterase 4.
`
`381
`
`duplicate. IC50 values were calculated by nonlinear regression analysis
`(variable slope) using Prism by GraphPad Software (San Diego, CA).
`Cytokr'rte assays
`
`Culture supernatants were harvested at indicated times and frozen imme-
`diately at —?0°C until assayed in triplicate or duplicate. TNF—n , IL—lfi,
`lL-ZZ, Ila-6, lL—S, lL-l 0. IL- I 2 (p40 and p70}, and IFN—y levels were mea-
`sured by ELISA (Endogen, Cambridge, MA)
`as described by the
`manufacturer.
`
`Statistical analysis
`
`Data were evaluated by the Friedman test, a nonparametric ANOVA. in
`view of the small sample size. The SPSS computer program was used.
`Significance was set at p < 0.05.
`
`Results
`
`Efect of thalidomide analogues on LPS—t’ndttccd cytokinc
`production
`
`A group of thalidomide analogues were selected for their capacity
`to inhibit TN'F-o: production by LPS—stimulated PBMC, Their ICSU
`values for TNF—ot (the concentration at which each compound was
`able to inhibit TNF-ot
`levels by 50%) were established when
`screening these agents (Table I). Although all compounds were
`efiicient TNF—oc inhibitors,
`their dose—response curves were not
`identical (data not shown). For some compounds, dose—response
`curves were the classical sigmoidal curves seen for pharmacolog-
`ical antagonists (class II compounds, seebelow) (12). Other com—
`pounds, however, showed a flatter, thalidomide~like dose response
`(class [ compounds, see below) (12). Subsequent experiments were
`conducted with the compounds at three concentrations: their ap—
`proximate TNF~a ICSD; 3 times the TNF-oc ICSQ; and 10 times the
`"INF-a ICSO. Rolipranr, a known TNF-or inhibitor (19), was used as
`a control. The elfect of drese drugs on other LPS-induced cyto-
`kines was also investigated. Compounds were added at the men—
`tioned concentrations to LPS-stitnulated human PBMC, and cyto—
`kine secretion into the culture supernatant was evaluated. Fig.
`1
`shows the effect of the drugs on the production of TNF-o , IL—lB,
`IL-6, IL-S, 11,-10, and IL-12. Compounds clearly segregated into
`two different classes according to their elfects on LPS—induced
`lL- 118, IL—fi, LL—IO, and IL—12 cytolrines. One class of compounds
`(class I) showed significant inhibition of llwIJB, at their TNF—or
`ICSO, and almost complete inhibition at higher concentrations,
`whereas compounds from class II had a more modest inhibitory
`effect, albeit significant at the higher concentrations (Fig. 1). Sim-
`ilarly, class 1 compounds significantly inhibited lL-fi
`levels,
`whereas class H compounds did not afect l'L-6 production (Fig. l).
`lL—S levels were not significantly affected by either class of corn-
`pounds, although class I showed a very minor trend toward inhi—
`bition of IL—8 production (Fig. 1).
`
`Table I. TNF-ot and PDE4 inhibition
`
`Icfio Values fair)“
`———____._____
`
`Compound
`TN'F-a
`PDE4
`
`Thalidomide
`194
`>500
`CI—A
`0.01
`3> l 00
`CI—B
`0, 10
`> 100
`CI—C
`0.04
`> 100
`CII-A
`12.6
`9.4
`CH-B
`20.6
`15.0
`CH-C
`0.21
`0,04
`
`0.15Rolipram 0.40
`
`“ TNF—a 1C5“ values were determined in LPS—stinrulated human PBMC from
`dose—response curves derived from four independent experiments widt did'erent do~
`nors. PDE4 ICSO values were determined in U93T—purilied enzyme from dose-response
`curves derived from three independent experiments. 1Cfin values were calculated by
`nonlinear regression analysis (variable slope) using Prism by GruphPad Software.
`ALVOGEN, Exh. 1055, p. 0174
`
`ALVOGEN, Exh. 1055, p. 0174
`
`

`

`382
`
`THALIDOMIDE ANALOGUES AS DIFFERENTIAL CYTOKINE MODULATORS
`
`—D— Elli-A
`-L'-—- Gil—B
`
`TNF—o
`
`100-
`
`—0—- ClI-C
`-—6—C|-A
`—I—CI-B
`—A— CI—C
`-—X— Rolipram
`
`
`
`hit
`
`50—
`
`25-
`
`O_——-,—_r.._._'__
`
`U-'—i—