Journal of clinical investigation.
`v. 120,
`no.
`1 (Jan. 2010)
`Collection
`
`W1 JOS
`2010-01-25 06:14:5
`
`fournal of
`The
`"Clinical Investigation
`
`
`
`[LIBRARYOFfips]§=—MEDICINE y
`
`I
`[NATIONAL]
`
`PROPERTY OF THE
`NATIONAL
`LIBRARY OF
`
`UNIV. CHICAGO EX. 2017
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`S
`*
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`.)
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`. ‘ Sng UU
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`Genome & Co. v. Univ. of Chicago
`PGR2019-00002
`
`|
`
`

`

`NI
`
`CI Volume 120
`
`Number 1
`
`January 2010
`The publication of the American Society for Clinical Investigation
`
`Editor Laurence A. Turka
`
`Deputy Editors Morris Birnbaum, Jonathan A. Epstein, Gary Koretzky
`:
`h
`.
`5
`i
`aren Honey
`Executive Editor UshmaS. Neill Science Editor Brooke Grindlinger News and Reviews Editor Ka
`Senior Editors
`Craig B Thompson
`Stephen G. Emerson
`William N. Kelley
`
`Senior European Editor
`
`Marc Feldmann
`
`Associate Editors
`Nancy Bonini
`Michael F. Beers
`Yongwon Choi
`Ronald G, Coliman
`
`Consulting Editors
`E. Dale Abel
`Domenico Accili
`Rexford S. Ahima
`Sunil K, Ahuja
`Qais Al-Awqati
`Kari Alitalo
`Masayuki Amagai
`Moshe Arditi
`M. Amin Armaout
`David Artis
`Alan Attie
`Jane Aubin
`Amer Beg
`lvor Benjamin
`Jean Bennett
`Joel S. Bennett
`Bradford Berk
`Nina Bhardwaj
`Mina Bissell
`Joseph V, Bonventre
`Karin E, Bornfeldt
`Brendan Boyce
`Thomas Braciale
`David Allen Brenner
`Jonathan Bromberg
`Vincenzo Bronte
`Michael A, Brownlee
`Hal E. Broxmeyer
`Peter Carmetiat
`Marlin Carroll
`Arturo Casadevall
`Andrew Chan
`Kyong-Mi Chang
`Harold Chapman
`\srael Charo
`Youhai Chen
`David Cheresh
`Alan D. Cherrington
`Leonard Chess
`Angela Christiano
`Marco Colonna
`George Cotsarelis
`Riccardo Dalla-Favera
`Alan D'Andrea
`Chi Van Dang
`Alan Daugherty
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`Wafik S. El-Deiry
`Alan M. Gewirtz
`Stanley Goldfarb
`Klaus Kaestner
`Mark L. Kahn
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`Sudhansu Dey
`Betty A, Diamond
`Anna Mae Diehl
`Harry C. Dietz II
`Stefanie Dimmeler
`Gerald Dorn
`Michael Dustin
`Connie Eaves
`Elazer R. Edelman
`Albert O. Edwards
`George S. Eisenbarth
`Leif Ellisen
`Joel Elmaquist
`Charis Eng
`Joel D, Ernst
`Eric RA. Fearon
`Harold |, Feldman
`Dean Felsher
`Anthony W. Ferrante
`Napoleon Ferrara
`Edward A, Fisher
`Susan Fisher
`Garret FitzGerald
`Paul Frenette
`Alan Bo Frey
`Joshua Friedman
`Philippe Froquel
`Sam Gandy
`Donald E. Ganenm
`Jack Gauldie
`Alfred L, George Jr
`Andrew T Gewirlz
`Eli Gilboa
`Henry Ginsberg
`Laune Glimeher
`Todd Golde
`Gregory J. Gores
`Steven Grant
`Linda Greenbaum
`Mark |. Greene
`David Haller
`Katherine A. Hajjar
`Goran Hansson
`Robert Alexander Hegele
`Jay Heinecke
`Meenhard Herlyn
`
`Biostatistician
`
`Peter Klein
`William MLF, Lee
`Edward E. Morrisey
`Ellen Pure
`Daniel J. Rader
`
`Phyllis Gimnotty
`
`Steven L. Reiner
`Amita Sehgal
`John Stanley
`Doris Stoffers
`Jeffrey Weiser
`
`Katherine A, High
`Joy Hirsch
`Helen H. Hobbs
`Steven M. Holland
`Michael Holtzman
`Lawrence Holzman
`day Horton
`Gokhan 5, Holamisligil
`Steven Houser
`Mukesh Jain
`Carl H. June
`Takashi Kadowaki
`Raghu Kallurh
`Gerard Karsenty
`Rober 5S, Kass
`Masalo Kasuga
`Daniel P Kelly
`Eli Keshet
`Sundeep Khosla
`Richard N. Kitsis
`Thomas RA. Kleyman
`Mark A. Knepper
`Jay K. Kalls
`Isser Komura
`Murray Kare
`Paul Kubes
`Boh Kulkarni
`Rajiv Kumar
`Thomas Kupper
`Antonio La Cava
`Mitchell A. Lazar
`Rudolph L. Leibet
`Klaus Ley
`James Liao
`Peter Libby
`David Lilllerap
`Richard M. Locksley
`Joseph A, Lorenzo
`Andrew R, Marks
`Jack Martin
`Kouji Matsushima
`Michael A. Matthay
`Elizabeth McNally
`Shlomo Melmed
`Michael E, Mendelsohn
`Thomas Michel
`
`Frank Miedema
`Jetlery Molkentin
`Anthony J. Muslin
`Martin G. Myers
`Enc G. Neilson
`Harry T. Orr
`Gary Owens
`Michael Parmacek
`Cam Patlerson
`Warren S, Pear
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`Pare Puigserver
`Bali Pulendran
`Ellen Pure
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`Vincent Racaniello
`Shahin Rafi
`Elaine Raines
`Gwendalyn Randolph
`Mariusz Ratajczak
`Barbara Rehermann
`Muredach P, Reilly
`Giuseppe Remuzzi
`Nicholas Restilo
`Barrett Rollins
`Mare E. Rothenberg
`Paul B. Rothman
`Anil K. Rustgy
`dunicht Sadoshuna
`J. Evan Sadler
`Robert | Satirstein
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`Mark Shlomechik
`Gerald Shulman
`Roy L. Silverstein
`M. Celeste Simon
`Lois Smith
`Weihong Song
`Robert M, Streter
`Warren Strober
`Terry Strom
`Megan Sykes
`Ira A. Tabas
`Alan A, Tall
`Sakae Tanaka
`Andrei Thomas: Tikhonenko
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`Stephen do Weiss
`David Wilkes
`Kevin don Williarns
`Joseph bo Witztum
`Kathryn Wood
`Gary Wu
`hai Wucherptennig
`Stephen Young
`Soatt Zanvwil
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`The American Soerely tor Clinical Investigation holds the nghls to and publishes the Journal ol Glinigal Investiqation The aprons eeprsoe Here pire solely
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`

`
`
`Contents
`
`Cover: Colored scanning electron
`micrograph of an adipocyte. White adipose
`tissue plays an important role in regulating
`metabolism, and its inflammationis
`linked to insulin resistance. Wueest and
`colleagues report that expression of the
`death receptor Fasis elevated in adipose
`tissue in genetic and nutritional models of
`obesity in mice andin the adiposetissue of
`patients with obesity and type 2 diabetes,
`and show that Fas is a regulator of obesity-
`associated adiposetissue inflammation,
`hepatic steatosis, and insulin resistance
`(page 191).
`
`Photo credit: Photo Researchers Inc.
`
`Cl
`
`The Journalof Clinical Investigation
`January 2010
`
`Volume 120
`
`Number1
`
`»S7
`
`|
`= ©
`|
`TRIUMPH __|
`
`Rn!
`| Editorial
`H EART |
`2
`Let’s keepthis brief
`
`In this issue
`4
`
`U.S. Nett
`
`
`
`THE STORY OF STATING
`
`| News
`
`Book reviews
`
`A
`
`=
`
`Every patienttells a story: medical
`mysteries and the art of diagnosis
`D.W. Foster
`
`Triumphof the heart:
`the story of statins
`P. Lippy
`
`|
`
`3
`
`Australian scientists say
`“G'day USA”
`K. HONEY
`
`
`
`Stem cell
`therapyfor
`neurodegenerative
`disease
`
`Review series Stem cells
`
`1
`
`20
`
`29
`
`41
`
`Si
`
`60
`
`wa
`
`Stem cells: roadmap
`to the clinic
`G.Q. DALey
`
`Repairing skeletal muscle:
`regenerative potential of
`skeletal muscle stem cells
`FS. Tepesco, A. DELLAVALLE,
`J. Diaz-Manera, G. Messina, np G. Cossu
`Pregenerative medicine:
`developmental paradigmsin
`the biology of cardiovascular
`regeneration
`B.A. Yi, 0. Werner, AND K.R. CHIEN
`Stem cells in human
`neurodegenerative
`disorders — timefor clinical
`translation?
`0. LINDVALL AND Z. KOKAIA
`
`The therapeutic promiseof
`the cancer stem cell concept
`N.Y. Frank, T. SCHATTON,
`AND MH, FRANK
`
`Progress towardtheclinical
`application of patient-specific
`pluripotent stem cells
`E. KISKINIS AND K. EGAN
`
`Enabling stem cell therapies
`through synthetic stem cell-
`niche engineering
`R. PEERANI AND P.W. ZANDSTRA
`
`The bioethics of stem cell
`research and therapy
`|, Hyun
`
`TheJournal of Clinical Investigation
`
`hup://wewjcLorg
`
`Volume 120
`
`Number|
`
`January 2010
`
`i
`
`

`

`contents
`
`103
`Oncogenic mTOR
`signaling
`
`127
`Cellular adaptation
`to hypoxia
`
`
`
`
`
`
`142
`
`Targeting fatty acid
`oxidation in leukemia
`
`254
`Cardiac fibroblasts
`and pressure overload
`
`Commentaries
`
`Putting the brakes on BTLAin T cell-mediated cancer immunotherapy
`C.M. Pautos Anp C,H. JUNE
`Relatedarticle, page 157
`Sodium channels gone wild: resurgent current from neuronal and muscle
`channelopathies
`S.C. Cannon ano B.P. BEAN
`Related article, page 369
`
`Newroles for Notch in tuberous sclerosis
`W.S. Pear
`Related articles, pages 93 and 103
`
`Oxidant stress derails the cardiac connexon connection
`G.F. TOMASELLI
`Relatedarticle, page 266
`Ramping up RANTESin the acute responseto arterial injury
`T. Hua ano MLL. Oo
`Related article, page 303
`
`Research articles
`
`93
`
`157
`
`168
`
`179
`
`The evolutionarily conserved TSC/Rheb pathwayactivates Notch in tuberous
`sclerosis complex and Drosophila external sensory organ development
`M. Karsowniczex, D. Zitserman, D. KHABIBULLIN, T. HARTMAN, J. Yu, T. Morrison, E. NicoLas, R. SQuiLtace,
`F. Roeciers, AnD E.P. HENSKE
`
`Related Commentary, page 84
`Mammaliantarget of rapamycin regulates murine and humancell differentiation
`through STAT3/p63/Jagged/Notch cascade
`J. Ma. Y. Meno, DJ. Kwiatkowski, X. Cen, H. Pene, Q. Sun, X. ZHa, F. Wane, Y. Wane,Y. JING, S. ZHANG,
`R. Cen, L. Wane, E. Wu, G. Cal, 1. Mainowska-Kotoozieu, Q. Liao, ¥. Liu, ¥. Zuao, Q. Sun, K. Xu, J. Dal,
`J. Han, L. Wu, B.C. Zhao, H. SHEN, AND H. ZHANG
`
`Related Commentary, page 84
`Ablation of C/EBPf alleviates ER stress and pancreatic B cell failure through
`the GRP78 chaperonein mice
`T. Matsupa, ¥. Kino, S. Asanara, T. Kaisuo, T, TANAKA, N. HaSHiMoTO, Y. SHIGEYAMA, A. TAKEDA, T. INOUE,
`¥ Sipurant, M. Kovanaci, T. Hosooka, M. Matsumoto, H. Inoue, T. Uchipa, M. Koike, ¥. Uchiyama,
`S. Anima, AnD M. KasuGa
`
`The unfolded protein response protects human tumorcells during hypoxia
`through regulation of the autophagy genes MAP1LC3B and ATG5
`K.M.A. Rouscuop, T. van pen Beucken, L. Dupois, H. Niessen, J. Bussink, K. SAVELKOULS, T. KEULERS,
`H. Muucic, W. Lanouyt, JW. Voncken, P. LamBiN, A.J. VAN DER Kocet, M. Koritzinsky, ano B.G. Wouters
`
`Pharmacologicinhibition of fatty acid oxidation sensitizes human leukemia
`cells to apoptosis induction
`|. Samunio, R. Hanmancey, M. Fiest, H. Kantaruian, M. Konopveva, B. KorcHin, K. KALUARACHGHI,
`W. Bornmann, S. Duvvurt, H. TAEGTMEYER, AND M. ANDREEFE
`
`BTLA mediatesinhibition of human tumor-specific CD8* T cells that
`can bepartially reversed by vaccination
`L. Derreé, J.-P. Rivas, C. Janpus, S. Pastor, D. Rimoto, P Romero, O. Micietin,
`D. Otive, avo D.E. Speiser
`
`Related Commentary, page 76
`
`CD27 sustains survival of CTLsin virus-infected nonlymphoid tissue in mice
`by inducing autocrine IL-2 production
`V. Peperzan, Y. Xiao, E.A.M. VeRAAR, AND J. Borst
`
`Lnk regulates integrin cllbB3 outside-in signaling in mouseplatelets, leading
`to stabilization of thrombus developmentin vivo
`H. Takizawa, S. Nishimura, N. TAKAYAMA, A. Oba, H. Nishiki, ¥. Morita, S. Kakinuma, S. YaMazan,
`S. Okamura, N. Tamura, S. Goto, A. SawacucHi, |. Manase, K. TaKATSU, H. NakAUCHI, S. TAKAKI, AND K. Ero
`
`The Journal ofClinical Investigation
`
`hieep: Awww jelorg
`
`Volume 120
`
`Number 1
`
`January 2000
`
`

`

`contents
`
`191
`
`203
`
`214
`
`223
`
`242
`
`254
`
`266
`
`230
`
`290
`
`303
`
`Deletion of Fas in adipocytes relieves adipose tissue inflammation and hepatic
`manifestations of obesity in mice
`S. Wueest, R.A. Raroto, D.M. Scuumann, J.M. RytKa, A. SeHitoknecnT, 0. Nov, A.V. CHervonsky, A. RupicH,
`E.J. ScHoente, M.Y. Donat, AND D. Konrab
`Urea-induced ROS generation causesinsulin resistance in mice with chronic
`renal failure
`M. D’Apotiro, X. Du, H. Zone, A. Gatucci, L. Mauri, T. Trivisano, M. Pettoeto-Mantovani, A. CAMPANOZZ1I,
`V. Raia, JE. Pessin, M. BROWNLEE, AND |. GIARDINO
`
`CD20 deficiency in humansresults in impaired T cell-independent
`antibody responses
`T.W. Kuwpers, RJ. Benpe, PA. Baars, A. Grumeecs, |.A.M. Derks, K.M. Dotman, T. BEAUMONT,
`TF. Tepper, C.J.M. van Noeset, E. ELberine, AND R.A.W. van LIER
`
`Genetic and epigenetic silencing of SCARA5 may contribute to human
`hepatocellular carcinoma by activating FAK signaling
`J. Huane, D.-L. ZHene, F.-S. Qin, N. Cenc, H. CHEN, B.-B. Wan, Y.-P. Wane,
`H.-S. Xiao, AnD Z.-G. HAN
`
`Cardiac mastcells causeatrial fibrillation through PDGF-A—mediated fibrosis
`in pressure-overloaded mousehearts
`C. Liao, H. Akazawa, M. Tamacawa, K. Ito, N. Yasupa, Y. Kuo, R. Yamamoto, Y, Ozasa, M. Fusimoto,
`P. Wane, H. Nakaucui, H. NakaAYA, AND |. KoMURO
`
`Cardiac fibroblasts are essential for the adaptive response of the murine heart
`to pressure overload
`N. Taxepa, |. Manape, Y, UcHino, K. Ecucni, S. Matsumoto, S. Nishimura, T. SHinpo, M. Sano, K, Otsu,
`P. Sniper, S.J. Conway, anp R. Nacat
`Limited forward trafficking of connexin 43 reducescell-cell coupling in stressed
`human and mouse myocardium
`J.W. Smyth,T.-T. Hone, D. Gao, J.M. Vocan, B.C, Jensen, T.S. Fone, P.C. Simpson, D.Y.R. STAINIER,
`N.C. Gui, AnD R.M. SHaw
`
`Related Commentary, page 87
`
`Cardiac signaling genes exhibit unexpected sequencediversity in sporadic
`cardiomyopathy, revealing HSPB7 polymorphisms associated with disease
`S.J. Markovich, D.J. Van Booven, A. Hinoes, M.Y. Kane, T.E. Draucey, RL.M. Vaccania, R.D. Mitra,
`M.P. Reitty, T.P. Cappo, ano G.W. DornII
`TGF-B1-induced expression of human Mdmcorrelates with late-stage
`metastatic breast cancer
`S. Araki, JA. Erret, C.N. BATUELLO, K. BiuanGi-VISHEHSARAEI, X.-J. Xie, D. Daniecpour, K.E. PoLok,
`D.A. BooTHMaN, AND L.D. Mayo
`
`Stat3-dependentacute Rantes production in vascular smooth musclecells
`modulates inflammation following arterial injury in mice
`J.C. Kovacic, R. Gupta, A.C. Lee, M. Ma, F. Fane, C.N. Tovsert, A.D. Watts, LE. BeLtran, H. San,
`G. CHEN, C. ST. HILAIRE, AND M. BoeHm
`
`A
`315
`
`331
`
`343
`
`357
`
`Related Commentary, page 90
`The Rho/Rac exchangefactor Vav2 controls nitric oxide-dependent responses
`in mouse vascular smooth musclecells
`V. Sauzeau, M.A. Sevitta, M.J. Montero, ano X.R. Busteo
`IL-17 produced by neutrophils regulates IFN-y—mediated neutrophil migration
`in mouse kidney ischemia-reperfusion injury
`L. Li, L. Huane, A.L. Verais, H. Ye, A. Baswa, V. Narayan, R.M. Stricter, D.L. Rosin, ano M.D. Okusa
`
`The cytolytic molecules Fas ligand and TRAILare required for murine thymic
`graft-versus-host disease
`|.-K. Na, S.X. Lu, NLL. Yim, G.L. Govopers, J. Tsai, U. Rao, O.M. Smith, C.G. Kine, D. Sux,
`D. HirnscHHorn-Cymerman, L. PaLomaa, 0. Penack, A.M. HoLLAnp, R.R. Jena, A. GHosh, H. TRAN,
`T. Mercuous, C. Liu, G.D. Sempowski, M. Ventevocet, N. BEAUCHEMIN, AND M.R.M. VAN DEN BRINK
`
`FoxO1 expression in osteoblasts regulates glucose homeostasis through
`regulation of osteocalcin in mice
`M.-T. Racheo, A. Kove, B.C. Sitva, D.Y. June, S. Gray, H. Onc, J.-H. Paik, R.A. DePinno, U.K. Kim,
`G. Karsenry, anp S. KousTeNt
`
`
`
`The Journal ofClinical Investigation—hietp://wwwjciorg Volume 120° Number tb January 2010
`
`
`
`
`
`331
`Neutrophil migration
`in kidney injury
`
`266
`Cardiac connexon
`connections
`
`Metastatic breast
`cancer
`
`315
`Regulating arterial
`contractility
`
`
`
`
`
`
`

`

`369
`
`Humanvoltage-gated sodium channel mutations that cause inherited neuronal
`and muscle channelopathies increase resurgent sodium currents
`B.W.Jarecki, A.D. Piekarz, J.0. Jackson II, ano T.R. Cummins
`Related Commentary, page 80
`
`Technical advance
`
`372
`
`Amelioration of emphysemain mice throughlentiviral transduction of long-
`lived pulmonary alveolar macrophages
`A.A. Witson, G.J. Murery, H. Hamakawa, L.W. Kwok, S. Srinivasan, A.-H. Hovav, R.C. MULLIGAN,
`S. Amar, B. Suki, ano D.N. Korron
`
`357
`Bone control of
`glucose homeostasis
`390=2009 American Society for Clinical Investigation Presidential Address
`Can wekeep the “academic” in academic medicine?
`N.C. ANDREWS
`
`Supplement
`
`Article amendments
`
`394
`
`contents
`
`
`
`
`379
`Genetherapyfor
`emphysema
`
`
`
`TheJournal ofClinical Investigation—hutp://wwwjccorg Volume 12000 Number) January 2000
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`
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`

`

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`‘This material may be protected by Copyright law(Title17 US. Code)
`
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`
`Related Commentary, page 76 Fa Wwe
`
`
`BTLA mediates inhibition of human
`tumor-specific CD8* T cells that can
`be partially reversed by vaccination
`
`Laurent Derre,' Jean-Paul Rivals,2 Camilla Jandus,' Sonia Pastor,3? Donata Rimoldi,'
`Pedro Romero,! Olivier Michielin,'’4* Daniel Olive,? and Daniel E. Speiser'
`
`‘Ludwig Institute for Gancer Research, Lausanne, Switzerland. “University Hospital Center and University af Lausanne (CHUV),
`Lausanne, Switzerland. INSERM UMR891, Institut Paoli Calmettes, Marseille, France.
`‘Multidisciplinary Oncology Center, Lausanne University Hospital (CHUV), Lausanne, Switzerland
`
`The function ofantigen-specific CD8' T cells, which may protect against both infectious and malignantdiseases,
`can be impairedbyligationoftheir inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and
`programmedcell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA)was identified as a novel inhib-
`itory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLAtriggering leads to decreased
`antimicrobial and autoimmuneT cell responses in mice, but its functions in humansare largely unknown. Here
`we have demonstrated that as humanviral antigen-specific CD8*T cells differentiated from naive to effector
`cells, their surface expression of BTLA was gradually downregulated. In marked contrast, haman melanoma
`tumor antigen-specific effector CD8* T cells persistently expressed highlevels of BTLA in vivo and remained
`susceptible to functionalinhibitionbyits ligand herpes virus entry mediator (HVEM). Such persistence ofBTLA
`expression wasalso found in tumorantigen-specific CD8* T cells from melanomapatients with spontaneous
`antitumor immuneresponses and after conventional peptide vaccination. Remarkably, addition of CpG oligo-
`deoxynucleotidesto the vaccine formulationled to progressive downregulation ofBTLAinvivo and consequent
`resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8'
`cancer-specific T cells, and appropriate immunotherapymaypartially overcomethis inhibition,
`
`Introduction
`Src homology2 (SH2) domain containing phosphatase | (SHIP. 1)/
`SHP-2 association, resulting in decreased T cell proliferanion and
`Activation of lymphocytes is controlled by 2 classes of signals:
`cytokine production (12-14). In Bells, BTLA regulates B cell recep-
`firsc, by those criggered through the Tcell receptor upon interac:
`ror signaling by reducing the phosphorylation of syk, B cell linker
`tion with antigenic peptide bound to MHC molecules; and second,
`protein (BLNK), and phospholipase Cy2 (PLCy2) (15),
`by signals delivered by binding of coreceptors to their ligands on
`Band T cell development is normal in BYLA-defierent mice,
`antigen-presenting cells (1). Coreceptors consist of costimulate
`Maturelymphocytes, however, are functionally altered and show
`ry and coinhibirory receptors (2-7). Preclinical and clinical dara
`enhanced generation of memory Tcells and memory responses
`indicate that the co-inhibirory receptors CTL-associared protein 4
`(16). BTLA defiereney was found to enhance protection from
`(CTLA-4) and programmedcell death [ (PID-1) are co-responsible
`murine malaria (17) and to aggravate experimental autoimmune
`for the suppression of humaneffector Tcell responses to intec-
`encephalomyelitis (12) and allergic airway inflammacon (18) and
`tious diseases and cancer (5, 6); the therapeutic blockade of these
`was associated with spontaneous development of an autonmmune
`2 parhways isin promising clinical development. Lymphocytes can
`heparitis-like disease (19), More recently, BTLA has been shown to
`express additional inhibitory receptors, such as killer-inhibitory
`be involvedin peripheral T cell rolerance induction (20) and in early
`
`receptors and C-leetin- type receptors (8)—both oftheseclasses,
`control of tissue damageand of antibacterial immunity(21).
`however, are expressed by only small subsets of T cells (8, 9).
`In humans, BELA expression maybe alrered by specific imimu-
`Amore recently described co-inhibitory receptoris Band T lym-
`nocherapywith allergens, as shown for allergic rhinitis (22). How-
`phoeyte attenuator (BTLA; Cl 1272), an unmunoglobulin-like mol-
`ever, only little is Known about the role and function of BTLA in
`ecule belonging to che CD28:B7 family, which ts expressed bythe
`humans, and there are no data vet available on antigen-specific T
`majority of lymphocytes (6, 10-12), Interestingly, rts ligand, herpes
`cells. In chis study, we showfor che first time to our knowledge
`virus enery mediator (HIVEM), 15 a memberof the TNF receptor
`thar BTLAis downregulated during human CD8° Tcell differen-
`(TNER) supertamaly (10, 11). This receptor system also includes lym-
`tiation to effector cells. This was, however, not the case for tumor
`photosxin-a, LIGHT (CD258), and CD160, which are present at the
`antigen-specific (Melan-AM""l-specific) Tcells, whichpersistent
`cytoplasmic membraneofcells of different histological origin. They
`ly expressed BTLA despice effector cell differentiation in unvac-
`may compete for their ligand HVEM, whichts present on T, B, and
`cinated melanoma patents, In contrast, when CpGs were used as
`NK cells, OC's, and myeloid cells, and also a variery of tumorcells (6,
`10-12). The hgation of BTLA by HVEM leads to phosphorylation
`adjuvant for vaccination, Melan-AM"! !-speeihe T cells downregu-
`lated BTLA, developedstrony effector functions, and becameinde-
`of mmmunoreceptor tyrosine-based inhibirion morits ({TTMs) and
`pendent of BTLA-mediated inhibition, Finally, the BTLA ligand
`HVEMwasfound to be expressed by melanoma tumors in situ and
`mediated functional inhibition of BTLA' T cells.
`
`Conflict ofinterest: The wichors buve dechited That no contlice or iyrerest exists
`Citation for this avticle: | (fin fiver 12015% 167 (2000), dew WALZICEIOOTO
`
`The Journal of Clinical Investigation
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`Figure 1
`BTLA (CD272) is downregulated with progressive CDB'Tcell differentiation, except in tumor antigen-specilic CD&: T cells, Direct ex vivo
`analysis of CD8* T cells from PBMCsbyflow cytometry. (A) Representative examples of BTLA expression by Melan-AM4"" '— and virus-specitic
`(EBV, CMV, and influenza virus [FLU]) CD8' T cells from healthy donors (HD) and melanoma patients. BTLA' T cells were distinguished from
`BTLA’ T cells using a threshold established according to the autologous naive T cell subset, whichis always BTLA positive, Numbers indicate
`percentageofpositive or negative cells. (B) Comparison of BTLA expressionon total CD8' and virus- and Melan-A-specific T cells, from healthy
`individuals and unvaccinated melanomapatients, “*P < 10°for each of the 2 populations of Melan-A-specific T cells comparedwithall 5 other
`populations. (C-E) BTLA expression in subsets of CD8" T cells of healthy individuals (n = 58), upon definition of early andlate differentiation
`stages by CD45RA and CCR7 expression(i.e. naive [N, CD45RA'CCR7'], central memory [CM, CD45RA CCR7’], effector memory [EM,
`CD45RACCR7’], and effector memory RA’ [EMRA, CD45RA‘CCR7| cells), gated as shown in C; a representative example demonstrates
`the narrow positive peak observed in phenotypic naive cells. (D) Statistical assessment of BTLA* CD8* T cell subsets. (E) GMFI data, showing
`results compatible with those presented in D. Overall GMFI was determined by including the whole range from BTLA-negative to-positive cells,
`as shown in C. GMFI data were normalized to values determined in autologous naive CDB' T cells.
`
`Volum120
`Nuuwnlaer
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`Janay 20
`158 The Journal of Clinical Investigation—hetpy/wwwyenorg
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`research article
`
`Results
`Virws- but not Lonorsspecific effector CD8° Tells downregulate BILA. Vo
`investigate the expression oF HELA by Caumorspecific (Melan-AMoe! 1)
`and virus-specific (EBV, CMV, and influenza virus) human CD8'
`T cells, we performedex vivo multicolor flow cyrometry analysis
`on PBMCs fromhealthy donors and untreated melanoma patients
`(Figure TA). In healthy mdividuals, Melan-AM!)! specific CD8"
`T cells were predominantly BTLA’ (Figure 1B), with significantly
`higher BLA expression than on virus-spectfie T cells or on total
`CD8’ T cells. This could be explained by their differentiationstag-
`es, since Melan-AM™! | specific T cells are almost always naive
`in healthy donors (Supplemental Figure 1; supplemental mate-
`rial available online with chis article; doi: 10.1172/JCI40070DS8 1),
`in contrast to virus-specilic CI8* T cells (23). Correspondingly,
`naive T cells are BELA positive in the murine system (11, 24, 25).
`Therefore, we studied BTLA expression on coral CD8* 'T cells, in
`relarion to their differentiation stage as defined by expression of
`CCR7 and CD4SRA(26) in 58 healthyindividuals (Figure 1,C and
`1). Almoseall phenotypic naive T cells (N) and the majority of
`cenrral-memory T cells (CM) expressed BTLA, In contrast, effec-
`ror memory‘T’ cells (EM) and effector memory RA' Tcells (EMRA)
`were frequently BTLA negative, demonstrating decreased BTLA
`expression correlating with CD8* T cell differentiation.
`In many instances, BTLA receptor levels displayed a pattern of
`continuous surface expression without a clear transition from
`negative to positive T cells. However, phenotypically naive CD8'
`T cells were consistently positive, with a distinct narrow peak, We
`therefore used rhis populationas a reference to set the threshold
`for BTLA’ cells (figure 1C). Moreover, we also evaluated the data
`in terms of geometric mean fluorescence intensity (GMFI) val-
`vies normalized to the GMEFI of the autologous naive CD8° Tcell
`subset, The resules (Figure 1E) closely resembled those based on
`the assessmentof percentage oF BTLA' cells (Figure 1D), Thus,
`using autologous naive CD8° T cells as the internal ATLArefer-
`ence allowed consistent standardization and normalization of
`both percentage and GMPdata.
`Analyzing PBMCs of melanoma patients revealed that BTLA
`expression was high among Melan-AM"Tl specific T cells,
`surprisingly even among memory and effector cells (Figure 1B
`and Figure 2, A and B). In contrase, toral CDS" Tcells showed
`reduced BTLA expression, with progressive differentiation to
`effector cells (Figure 2B),
`For comprehensive immune characterization, it is also impor-
`tant to investigate T cells from diseased organs/tissues, Therefore,
`we analyzed lymphocytes from metastatic lymph nodes direetly
`ON VIVO and assessed BTLA expression by Melan-AMART I specific T
`cells as foundin 13 of 18 normal lymph nodes andin 6 of6 tumor-
`infiltrated lymph nodes. Once again, the Melan-AMART specific
`memory andeffector T cells were largely BTLA’, and the remain-
`ing, CD8" T cells bore significantly lower levels of BTLA(Figure2,
`Cand PD). Thus, the expression ofthe inhibitory receptor BTLA
`washigh for Melan-AN“"' specific Tcells bothin circulation and
`in solid tissue, despite the fact these cells engage in considerable
`“spontaneous,” disease-driven responses in most panents with
`progressive melanoma (27),
`In sitte expression of HVEM by melanomacells and T cell mbibition
`by BULA-HVEM ligation. We next sought co determine whether
`BLA could negatively regulate T cell function via binding to
`HVEM expressed by cancercells. The characterization of cultured
`melanomacell lines (Figure 3A) revealed that 26 of 40 expressed
`
`HVEM (19 of 40 highly and 7 of40 weakly), whereas none of them
`expressed BULA or LIGHT (Figure 318), Subsequently, we assessed
`in situ HIVEMexpression by immunohistochemistry on paraffin-
`embedded metastases from patients (Figure 3C). We found chae
`50%and 25%of metastases were strongly and moderately positive
`forHVEMexpression, respectively, while the remaiming 25%were
`weakly positive or negative (Table 1),
`To test whether HVEMexpression by melanoma cells could
`inhibit the function of antigen-specific T cells, we analyzed LFN-y
`production by Melan-AM¢®™ specific CD8' Tcell clones upon
`triggering with HVEM-expressing melanomacell lines. Por these
`experiments, wefirst characterized our new mAb, BTLA-8.2, and
`foundthat it efficiently blocked the interaction berween BTLA
`and HVEM (Supplemental Figure 2A). Subsequent experiments
`showed that Tcell clones properly secreted IFPN-y in the presence
`of IgG] control antibody, as expeeted. In contrasr, IPN-y produc-
`tion was significantly increased for clones expressing BYLA upon
`addition of blocking mAb BTLA-8.2 during che stimulation, In
`contrast, no change in [FN-y secretion by a clone lacking BTLA
`expression was observed (clone 618-420, Figure 3D), These data
`demonstrate functional inhibition of tumor-specific CDS* T cells
`via BTLA-LVEMinteractions.
`Downregulation ofBTLA upon vacemation with peptide and CpG. Next,
`we addressed whether vaccination of melanonia patients altered
`BTLA expression by tumor antigen-specific CD8" Tcells, Patients
`received s.c. vaccinations with Melan-AM“"'! peptides in conven:
`tional vaccine formulations, e.g, emulsified in Incomplete Freund's
`adjuvane (IPA) (28), A second cohort of patients was