Www.bloodjournal.org
`
`NATIONAL
`LIBRARY OF
`MEDICINE
`=
`
`Tolerogenic human DCs
`via intracellular CTLA4
`
`Ap1 and hematopoietic cells
`
`Seer
`and ae
`
`Mac-1 and macrophage
`efflux to lymphatics
`
`Priapism, hemolysis, and
`sickle cell disease
`
`Cover:
`
`_In situ Langerhans
`cell maturation
`
`JOURNAL OF
`
`THE AMERICAN
`
`SOCIETY OF
`
`HEMATOLOGY
`
`VOLUME 106
`
`NUMBER 9
`
`Cm Teh) ara
`
`PROPERTY OF THE
`
`A
`
`‘
`
`=
`
`Miltenyi Ex. 1016 Page 1
`
`

`

`blood
`
`JOURNAL OF
`
`THE AMERICAN
`
`SOCIETY OF
`HEMATOLOGY
`
`1 NOVEMBER 2005 - VOLUME 106, NUMBER 9
`
`CONTENTS
`
`COVER FIGURE
`
`In situ maturation of Langerhanscells induced by local injection of colchicine into
`ear skin. MHC IIis green and CD86is red. See the related article by Mizumoto et
`al, beginning on page 3082.
`
`INSIDE BLOOD
`
`
`
`2925
`
`Unraveling the hemolytic subphenotypeofsickle cell disease
`M. T. Gladwin
`
`2926
`
`Costimulatory blockade: act I
`D. H. Munn
`
`2927 Mac-1 mediates migration to lymph nodes
`J. P. Mizgerd
`
`2928
`
`RGSI16 “tightens the reins” on CXCR4
`M. Z. Ratajezak
`
`2929
`
`The SLAMfamilyof receptors: new players in platelet aggregation
`JA. Lépez
`
`2930
`
`Interferon-a: still useful after all these years
`E. J. Feldman
`
`2931
`
`Unfractionated LMWHandtherisk of HIT: are medical patients different?
`T. E. Warkentin and A. Greinacher
`
`2932
`
`‘To T-cell deplete or not
`H. E. Heslop
`
`2933
`
`Fibrin A-knob caughtin the act
`D. K. Galanakis
`
`BLOOD WORK
`
`
`
`2934 Homozygous hemoglobin C disease
`J. Lazarchick
`
`EDITORIAL
`
`
`
`2935
`
`Springtime for stem cells
`S.J. Shattil
`
`PLENARY PAPER
`
`2936 Creation of tolerogenic human dendritic cells via intracellular CTLA4:
`a novel strategy with potential in clinical immunosuppression
`P. H. Tan, J. B. Yates, S.-A. Xue, C. Chan, W. J. Jordan, J. E. Harper, M. P. Watson, R. Dong,
`M.A. Ritter, R. I. Lechler, G. Lombardi, and A. J. T. George
`
`2944
`
`Polymerization offibrin: specificity, strength, and stability of knob-hole interactions
`studied at the single-molecule level
`R. L. Litvinov, O. V. Gorkun, S. F. Owen, H. Shuman, and J. W. Weisel
`
`vii
`
`BLOOD, 1 NOVEMBER2005 - VOLUME 106, NUMBER 9
`
`CONTINUEDONviii
`
`Miltenyi Ex. 1016 Page 2
`
`Miltenyi Ex. 1016 Page 2
`
`

`

`CHEMOKINES,
`CYTOKINES, AND
`INTERLEUKINS
`
`CLINICAL TRIALS AND
`OBSERVATIONS
`
`REVIEW ARTICLE
`
`2952 Multiple roles of Rapl in hematopoietic cells: complementary versus
`antagonistic functions
`P.J. 8. Stork and T. J. Dillon
`
`
`
`ane
`
`RGS16 is a negative regulator of SDF-1-CXCR4signaling in megakaryocytes
`M. Berthebaud,C. Riviére, P. Jarrier, A. Foudi, Y. Zhang, D. Compagno, A. Galy,
`W. Vainchenker, and F. Louache
`
`
`
`2969 Outcomesof reduced-intensity transplantation for chronic myeloid leukemia:
`an analysis of prognostic factors from the Chronic Leukemia Working Party
`of the EBMT
`
`C. Crawley, R. Szydlo, M. Lalancette, A. Bacigalupo, A. Lange, M. Brune, G. Juliusson,
`A. Nagler, A. Gratwohl, J. Passweg, M. Komarnicki, A. Vitek, J. Mayer, A. Zander, J. Sierra,
`A, Rambaldi, O. Ringden, D. Niederwieser, and J. F. Apperley, for the Chronic Leukemia
`Working Party of the EBMT
`
`2977~—‘Clinical factors predictive of outcome with bortezomib in patients with relapsed,
`
`refractory multiple myeloma
`P. GG. G. Richardson, B. Barlogie, J. Berenson, S. Singhal, S. Jagannath, D. Irwin,
`S. V. Rajkumar, T. Hideshima, H. Xiao, D. Esseltine, D. Schenkein, and K. C. Anderson,
`for the SUMMITInvestigators
`
`2982
`
`Evidence for reduced B-cell progenitors in early (low-risk)
`myelodysplastic syndrome
`A. Sternberg,S. Killick, T. Litthkewood, C. Hatton, A. Peniket, T. Seidl, S. Soneji, J. Leach,
`D. Bowen, C. Chapman, G. Standen, E. Massey, L. Robinson, B. Vadher, R. Kaczmarski,
`R. Janmohammed, K. Clipsham, A. Carr, and P. Vyas
`
`2992
`
`+Brief report Successful treatment of Erdheim-Chester disease,
`a non—Langerhans-cell histiocytosis, with interferon-a
`F. Braiteh, C. Boxrud, B. Esmaeli, and R. Kurzrock
`
`GENE THERAPY
`
`
`
`2995 Adoptive transfer of gene-engineered CD4* helper T cells induces potent primary
`and secondary tumorrejection
`M. Moeller, N. M. Haynes, M. H. Kershaw,J. T. Jackson, M. W. L. Teng, S. E. Street, L. Cerutti,
`S. M. Jane, J. A. Trapani, M. J. Smyth, and P. K. Darcy
`
`HEMATOPOIESIS
`
`3004
`
`Three pathways to mature macrophagesin the early mouse yolk sac
`J. Y. Bertrand, A.Jalil, M. Klaine, S. Jung, A. Cumano,and I, Godin
`
`3012
`
`Soluble factor cross-talk between human bone marrow-derived hematopoietic
`and mesenchymalcells enhances in vitro CFU-F and CFU-O growth andreveals
`heterogeneity in the mesenchymal progenitor cell compartment
`D, Baksh, J. E. Davies, and P, W. Zandstra
`
`3020 G-CSF potently inhibits osteoblast activity and CXCLJ2 mRNAexpression
`in the bone marrow
`
`C. L. Semerad, M. J. Christopher, F. Liu, B. Short, P. J. Simmons, I. Winkler, J.-P. Levesque,
`J. Chappel, F. P. Ross, and D. C. Link
`
`
`
`HEMOSTASIS,
`THROMBOSIS, AND
`3028~—~Platelet aggregation induces platelet aggregate stability via SLAM family
`VASCULAR BIOLOGY
`receptor signaling
`N. Nanda, P. Andre, M. Bao, K. Clauser, F. Deguzman, D. Howie, P. B. Conley. C. Terhorst,
`and D. R. Phillips
`
`viii
`
`BLOOD, 1 NOVEMBER 2005 - VOLUME 106, NUMBER 9
`
`CONTINUED ON x
`Miltenyi Ex. 1016 Page 3
`
`Miltenyi Ex. 1016 Page 3
`
`

`

`IMMUNOBIOLOGY
`
`3035
`
`3043
`
`3049
`
`3055
`
`3058
`
`3062
`
`3068
`
`3074
`
`3082
`
`3090
`
`3097
`
`3105
`
`3114
`
`3123
`
`A novel nanobodythat detects the gain-of-function phenotype of yon Willebrand
`factor in ADAMTS13 deficiency and yon Willebrand disease type 2B
`J. J. J. Hulstein, P. G. de Groot, K. Silence, A. Veyradier, R. Fijnheer, and P. J. Lenting
`
`Aminoglycoside suppression of nonsense mutations in severe hemophilia
`P. D. James, S. Raut, G. E. Rivard, M.-C. Poon, M. Warner, S. McKenna,J. Leggo,
`and D.Lillicrap
`
`Theincidence of heparin-induced thrombocytopenia in medical patients treated
`with low-molecular-weight heparin: a prospective cohort study
`P. Prandoni, S. Siragusa, B. Girolami, and F, Fabris, for the BELZONI Investigators Group
`
`Brief report The effects of intrinsic pathway protease deficiencies
`on plasminogen-deficient mice
`Q, Cheng, Y. Zhao, W. E. Lawson, V. V. Polosukhin, J. E. Johnson, T. S. Blackwell,
`and D. Gailani
`
`Brief report Optimal biologic dose of metronomic chemotherapy regimens
`is associated with maximum antiangiogenic activity
`Y. Shaked, U. Emmenegger, S. Man, D. Cervi, F. Bertolini, Y. Ben-David, and R. S. Kerbel
`
`Elimination of human leukemia cells in NOD/SCID mice by WTI-TCR
`gene-transduced humanT cells
`S.-A. Xue, L. Gao, D. Hart, R. Gillmore, W. Qasim, A. Thrasher, J. Apperley, B. Engels.
`W. Uckert, E. Morris, and H. Stauss
`
`CD4+*+CD25* regulatory T-cell lines from human cord blood have functional
`and molecular properties of T-cell anergy
`L. Li, W. R. Godfrey, S. B. Porter, Y. Ge, C. H. June, B. R. Blazar, and V. A. Boussiotis
`
`MHEclass I1/CD38/CD9: a lipid-raft-dependentsignaling complex
`in human monocytes
`M.-T. Zilber, N. Setterblad, T. Vasselon, C. Doliger, D. Charron, N. Mooney, and C. Gelin
`
`Discovery of novel immunostimulants by dendritic-cell—based functional screening
`N. Mizumoto, J. Gao, H. Matsushima, Y. Ogawa, H. Tanaka, and A. Takashima
`
`Epstein-Barr virus LMP1 inhibits the expression of SAP gene and upregulates Th1
`cytokines in the pathogenesis of hemophagocytic syndrome
`H.-C. Chuang, J.-D. Lay, W.-C. Hsieh, H.-C. Wang, Y, Chang, S.-E. Chuang, and L.-J. Su
`
`Migration matters: regulatory ‘T-cell compartmentalization determines suppressive
`activity in vivo
`K. Siegmund, M. Feuerer, C. Siewert, S. Ghani, U. Haubold, A. Dankof, V. Krenn, M. P. Schon,
`A. Scheffold, J. B. Lowe, A. Hamann, U. Syrbe, and J. Huehn
`
`Identification of an epitope derived from the cancertestis antigen
`HOM-TES-14/SCP1 and presented by dendritic cells to circulating CD4* T cells
`F. Neumann, C. Wagner, K.-D. Preuss, B. Kubuschok, C. Schormann, S. Stevanovic.
`and M. Pfreundschuh
`
`LPS induces CD40 gene expression through the activation of NF-«B and STAT-1a
`in macrophages and microglia
`H. Qin, C. A. Wilson, S. J. Lee, X. Zhao, and E. N. Benveniste
`
`CD molecules 2005: humancell differentiation molecules
`H. Zola, B. Swart, I. Nicholson, B. Aasted, A. Bensussan, L. Boumsell, C. Buckley, G. Clark,
`K. Drbal, P. Engel, D. Hart, V. Horejsi, C. Isacke, P. Macardle, F. Malavasi, D. Mason, D. Olive,
`A. Saalmueller, S. F Schlossman, R. Schwartz-Albiez, P. Simmons, T. F. Tedder, M. Uguccioni,
`and H. Warren
`
`x
`
`BLOOD, 1 NOVEMBER 2005 « VOLUME 106, NUMBER 9
`
`CONTINUEDONxii
`Miltenyi Ex. 1016 Page 4
`
`Miltenyi Ex. 1016 Page 4
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`

`

`NEOPLASIA
`
`3127
`
`3134
`
`3142
`
`3150
`
`3160
`
`3166
`
`3175
`
`3183
`
`3191
`
`3200
`
`3206
`
`Human CTLA4 knock-in mice unravel the quantitative link between tumor
`immunity and autoimmunity induced by anti-CTLA-4 antibodies
`K. D. Lute, K. F. MayJr, P. Lu, H. Zhang, E. Kocak, B, Mosinger, C. Wolford, G. Phillips,
`M.A. Caligiuri, P. Zheng, and Y. Liu
`
`
`
`RAS-blocking bisphosphonate zoledronic acid inhibits the abnormalproliferation
`and differentiation of juvenile myelomonocytic leukemia cells in vitro
`Y. Ohtsuka, A. Manabe, H. Kawasaki, D. Hasegawa, Y. Zaike, S. Watanabe, T. Tanizawa,
`T. Nakahata, and K. Tsuji
`
`Overexpression of Shp2 tyrosine phosphatase is implicated in leakemogenesis
`in adult human leukemia
`R. Xu, Y. Yu, 8. Zheng, X. Zhao, Q. Dong, Z. He, Y. Liang, Q. Lu, Y. Fang, X. Gan, X. Xu,
`S. Zhang, Q. Dong, X. Zhang, and G.-S. Feng
`
`MDM2antagonists induce p53-dependentapoptosis in AML: implications
`for leukemia therapy
`K. Kojima, M. Konopleva, I. J. Samudio, M. Shikami, M. Cabreira-Hansen, T. McQueen,
`V. Ruvolo,T. Tsao, Z. Zeng, L. T. Vassilev, and M. Andreeff
`
`Myelomacells suppress bone formation by secreting a soluble Wntinhibitor, sFRP-2
`T. Oshima, M. Abe. J. Asano, T. Hara, K. Kitazoe, E, Sekimoto, Y. Tanaka, H. Shibata,
`T. Hashimoto, S. Ozaki, S. Kido, D. Inoue, and T. Matsumoto
`
`Loss of EBNA1-specific memory CD4* and CD8* T cells in HIV-infected patients
`progressing to AIDS-related non-Hodgkin lymphoma
`E. Piriou, K. van Dort, N. M. Nanlohy, M. H. J. van Oers, F. Miedema, and D. van Baarle
`
`Mutations in the ATM genelead to impaired overall and treatment-free survival
`that is independent of JGVH mutation status in patients with B-CLL
`B. Austen,J. E. Powell, A. Alvi, I. Edwards, L. Hooper, J. Starezynski, A. M. R. Taylor,
`C. Fegan, P. Moss, and T. Stankovic
`
`Diffuse large B-cell lymphoma subgroups havedistinct genetic profiles that
`influence tumorbiology and improve gene-expression—based survival prediction
`S. Bea, A. Zettl, G. Wright, I. Salaverria, P. Jehn, V. Moreno, C. Burek, G, Ott, X. Puig, L. Yang,
`A. Lopez-Guillermo, W. C. Chan, T. C. Greiner, D. D. Weisenburger, J. O. Armitage,
`R. D. Gascoyne, J. M. Connors, T. M. Grogan, R. Braziel, R. I. Fisher, E. B. Smeland,
`S. Kvaloy, H. Holte, J. Delabie, R. Simon, J. Powell, W. H. Wilson, E. 8. Jaffe,
`E. Montserrat, H.-K. Muller-Hermelink, L. M. Staudt, E. Campo, and A. Rosenwald,
`for the Lymphoma/Leukemia Molecular Profiling Project
`
`Tachpyridine, a metal chelator, induces G;cell-cycle arrest, activates checkpoint
`kinases, and sensitizes cells to ionizing radiation
`J. Turner, C. Koumenis, T. E. Kute, R. P. Planalp, M. W. Brechbiel, D. Beardsley, B. Cody,
`K. D. Brown, FE. M.Torti, and S. V. Torti
`
`Tumornecrosis factor-c inhibits hTERT gene expression in human myeloid normal
`and leukemiccells
`
`O. Beyne-Rauzy, N. Prade-Houdellier, C. Demur, C. Recher, J. Ayel, G. Laurent,
`and V. Mansat-De Mas
`
`The small molecule tyrosine kinase inhibitor AMN107 inhibits TEL-PDGFRB
`and FIP1L1-PDGFRain vitro and in vivo
`E. H.Stover, J. Chen, B. H. Lee, J. Cools, E. McDowell, J. Adelsperger, D. Cullen, A. Coburn,
`S. A. Moore, R. Okabe, D. Fabbro, P. W. Manley, J. D. Griffin, and D. G. Gilliland
`
`xii
`
`BLOOD, 1 NOVEMBER2005 « VOLUME 106, NUMBER 9
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`CONTINUEDONxvi
`Miltenyi Ex. 1016 Page 5
`
`Miltenyi Ex. 1016 Page 5
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`

`

`3214
`
`3223
`
`3227
`
`3234
`
`Characterization of 8p21.3 chromosomaldeletions in B-cell lymphoma:
`TRAIL-RI and TRAIL-R2 as candidate dosage-dependent tumor suppressor genes
`F. Rubio-Moscardo, D. Blesa, C. Mestre, R. Siebert, T. Balasas. A. Benito, A. Rosenwald,
`J. Climent, J. I. Martinez, M. Schilhabel, E. L. Karran, $. Gesk, M.Esteller, R. deLeeuw,
`L. M. Staudt, J. L. Fernandez-Luna, D. Pinkel, M. J. S. Dyer, and J. A. Martinez-Climent
`
`Brief report Transduction of CLL cells by CD40 ligand enhances
`an antigen-specific immune recognition by autologous T cells
`C. Mayr, D. M. Kofler, H. Biining, D. Bund, M. Hallek, and C.-M. Wendtner
`
`Jak3 negatively regulates dendritic-cell cytokine production and survival
`K. Yamaoka, B. Min, Y.-J. Zhou, W. E. Paul, and J. J. O'Shea
`
`A specific role of integrin Mac-1 in accelerated macrophageefflux to the lymphatics
`C. Cao, D. A. Lawrence, D. K. Strickland, and L. Zhang
`
`PHAGOCYTES
`
`RED CELLS
`
`
`
`STEM CELLSIN
`HEMATOLOGY
`
`TRANSPLANTATION
`
`3242
`
`3251
`
`3256
`
`3264
`
`3268
`
`3271
`
`3285
`
`3293
`
`3300
`
`Intracellular labile iron pools as direct targets of iron chelators:
`a fluorescence study of chelator action in living cells
`H.Glickstein, R. B. El, M. Shvartsman, and Z. 1. Cabantchik
`
`A novel molecularbasis for § thalassemia intermedia poses newquestions
`aboutits pathophysiology
`A. Premawardhena, C. A. Fisher, N. F. Olivieri, S. de Silva, J. Sloane-Stanley, W. G. Wood,
`and D. J. Weatherall
`
`The hydroxyurea-induced small GTP-binding protein SAR modulates y-globin gene
`expression in human erythroid cells
`D.C. Tang, J. Zhu, W. Liu, K. Chin, J. Sun, L. Chen, J. A. Hanover, and G. P. Rodgers
`
`Hemolysis-associated priapism in sickle cell disease
`V. G. Nolan, D. F. Wyszynski, L. A. Farrer, and M. H. Steinberg
`
`Brief report Novel urine hepcidin assay by mass spectrometry
`E. Kemna, H. Tjalsma, C. Laarakkers, E. Nemeth, H. Willems, and D, Swinkels
`
`
`
`Mitochondrial DNAspectra of single human CD34*cells, T cells, B cells,
`and granulocytes
`Y. Ogasawara, K. Nakayama, M. Tarnowka,J. P. McCoyJr, S. Kajigaya, B. C. Levin,
`and N.S. Young
`
`
`
`Absenceof inducible costimulator on alloreactive T cells reduces graft versus host
`disease and induces Th2 deviation
`V. M. Hubbard, J. M. Eng, T. Ramirez-Montagut, K. H. Tjoe, S. J. Muriglan, A. A. Kochman,
`T. H.Terwey, L. M. Willis, R. Schiro, G. Heller, G. F. Murphy, C. Liu, O. Alpdogan,
`and M. R. M. van den Brink
`
`Differential effects of proteasomeinhibition by bortezomib on murine acute
`graft-versus-host disease (GVHD): delayed administration of bortezomib results
`in increased GVHD-dependentgastrointestinal toxicity
`K. Sun, D. E. C. Wilkins, M. R. Anver, T. J. Sayers, A. Panoskaltsis-Mortari, B. R. Blazar,
`L. A. Welniak, and W. J. Murphy
`
`Critical role for CCR5in the function of donor CD4*CD25* regulatory T cells
`during acute graft-versus-host disease
`C. A. Wysocki, Q. Jiang, A. Panoskaltsis-Mortari, P. A. Taylor, K. P. McKinnon,L. Su,
`B. R. Blazar, and J. S. Serody
`
`xvi
`
`BLOOD, 1 NOVEMBER 2005 + VOLUME 106, NUMBER 9
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`Miltenyi Ex. 1016 Page 6
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`Miltenyi Ex. 1016 Page 6
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`

`

`3308
`
`3314
`
`3322
`
`Influence of T-cell depletion on chronic graft-versus-host disease: results of a
`multicenter randomizedtrial in unrelated marrow donortransplantation
`S. Z. Pavletic, S. L. Carter, N. A. Kernan, J. Henslee-Downey, A. M. Mendizabal,
`E. Papadopoulos, R. Gingrich, J. Casper. S. Yanovich, and D, Weisdorf,
`for the membersof the National Heart, Lung. and BloodInstitute Unrelated Donor Marrow
`Transplantation Trial
`
`Conditioning with 8-Gy total body irradiation and fludarabine forallogeneic
`hematopoietic stem cell transplantation in acute myeloid leukemia
`M.Stelljes. M. Bornhauser, M. Kroger. J. Beyer, M. C. Sauerland, A. Heinecke, B. Berning,
`C. Scheffold, G. Silling, T. Buchner, A. Neubauer, A. A. Fauser, G. Ehninger, W. E. Berdel,
`and J. Kienast, for the Cooperative German Transplant Study Group
`
`CCR2 is required for CD8-induced graft-versus-host disease
`T. H. Terwey, T. D. Kim, A. A. Kochman, V. M. Hubbard,S. Lu, J. L. Zakrzewski,
`T. Ramirez-Montagut, J. M. Eng,S. J. Muriglan, G. Heller, G. F. Murphy, C. Liu,
`T. Budak-Alpdogan, O. Alpdogan, and M..R. M. van den Brink
`
`OTHER DEPARTMENTS
`
`xxv Author guide
`
`xxxii
`
`Future articles
`
`ib
`
`Classified advertising
`
`SUBMISSION INSTRUCTIONS
`
`
`
`All manuscripts, including figures, should be submitted electronically at
`http://blood.manuscriptcentral.com to Editor-in-Chief Sanford J. Shattil, MD. Before submitting
`your paper, review Blood’s Author Guide at http://www.bloodjournal.org. If you need help during
`the submission process, contact the Editorial Office by phone at 202-776-0548orvia e-mail at
`editorial @hematology.org.
`
`XX
`
`BLOOD, 1 NOVEMBER 2005 - VOLUME 106, NUMBER 9
`
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`Miltenyi Ex. 1016 Page 7
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`

`

`
`This material may be protected byCopyrightlaw (Title 17 U.S. Code)
`
`
`
`GENE THERAPY
`
`Adoptive transfer of gene-engineered CD4*~helperT cells induces potent primary
`and secondary tumorrejection
`Maria Moeller, Nicole M. Haynes, Michael H. Kershaw, Jacob T. Jackson, Michele W.L. Teng, Shayna E. Street, Loretta Cerutti,
`Stephen M.Jane, JosephA. Trapani, Mark J. Smyth, and Phillip K. Darcy
`
`Because CD4*Tcells play a keyrole in
`cytokines, proliferate, and lyse erbB2+
`mor responses in these mice correlated
`aiding cellular immune responses, we
`tumor targets following antigen ligation
`with localization and persistence of gene-
`wanted to assess whether increasing
`in vitro. In adoptive transfer experiments
`engineeredT cells at the tumorsite. Strik-
`numbers of gene-engineered antigen-
`using severe combined immunodeficient
`ingly, mice that survived primary tumor
`restricted CD4* T cells could enhance an
`(scid) mice, we demonstrated that injec-
`challenge could reject a subsequentre-
`antitumor response mediatedby similarly
`tion of equivalent numbers of antigen-
`challenge. Overall, this study has high-
`gene-engineered CD8* T cells.
`In this
`specific engineered CD8*+ and CD4+ T
`lighted the therapeutic potential of using
`study, we have used retroviral transduc-
`cells led to significant improvement in
`combined transfer of antigen-specific
`tion to generate erbB2-reactive mouse
`gene-modified CD8* and CD4*Tcells to
`survival of mice bearing established lung
`T-cell populations composed of various
`metastases compared with transfer of
`significantly enhance T-cell adoptive
`proportions of CD4* and CD8* cells and
`unfractionated (largely CD8*) engineered
`transfer strategies for cancer therapy.
`T cells. Transferred CD4* T cells had to be
`then determined the antitumorreactivity
`(Blood. 2005;106:2995-3003)
`of these mixtures. Gene-modified CD4+
`antigen-specific (not just activated) and
`and CD8* T cells were shown to specifi-
`secrete interferon y (IFN-y) to potentiate
`cally secrete Tc1 (T cytotoxic-1) or Tc2
`the antitumor effect. Importantly, antitu-
`
`© 2005 by The American Society of Hematology
`
`Introduction
`
`eon
`
`of engineered CD8* T cells alone.?!’? However, despite these
`promising studies, the issue of whether the addition of antigen-
`specific engineered CD4* T-cell help may enhance antitumor
`immunity, long-term persistence, and secondary tumor rejection
`has not been properly tested in vivo for adoptively transferred
`T cells.
`
`Muchattention has been directed at the genetic modification of T
`cells and their therapeutic potential in the adoptive immunotherapy
`ofcancer. T cells engineered to express chimeric surface receptors
`that
`incorporate an extracellular single-chain antibody domain
`(scFv) and a transmembraneand cytoplasmic signaling domain can
`specifically direct antitumorimmuneresponses in a major histocom-
`patibility complex (MHC)-independent mannerattargets normally
`In the interest of generating an optimal antitumor response, we
`capable of evading immune recognition. These targets also often
`wanted to determine whether increasing the proportion of engi-
`lack important costimulatory ligands for maximalT-cell activation.
`neered CD4* T cells could more effectively eradicate disease. In
`The therapeutic potential of engineered T cells extends from
`this study we have used retroviral transduction for expression ofthe
`studies demonstrating specific antigen binding and target-cell lysis
`scFy-CD28-{ receptor, reactive against the erbB2 human breast
`in vitro! and in a range of different mouse tumor models**to their
`cancer—associated antigen, on the surface of both CD8* and CD4+
`successful transfer into patients with minimal side effects in phase
`splenic mouseTcells. Strikingly, we show that coinfusing equiva-
`| clinical trials.?
`lent numbers of engineered CD8* and CD4* T cells led to
`A number of studies have demonstrated the importance of
`completeeradication of established tumorin mice, whereas transfer
`CD4* T-cell help in elimination of infectious disease and for
`of mainly CD8* T cells was less effective. The enhancedefficacy
`antitumor immunity.!°-!! CD4* T cells have been demonstrated to
`was entirely dependenton antigenic specificity of the CD4* T cells
`be critical for maintenance of CD8* T-cell numbers, their recruit-
`and correlated with tumorlocalization and long-term persistence of
`ment to the tumorsite,!?""4 and induction of a memory response.!5-!6
`both CD8* and CD4* T cells. Interestingly, mice “cured” of
`In patients infected with HIV,
`the coinfusion of genetically
`ptimary tumor could subsequently reject a secondary challenge
`modified CD4* and CD8* T cells has been demonstrated to
`with erbB2* tumor. Thus, these studies show that CD4* T cells are
`overcome the lack of T-cell persistence observed with transfusion
`an important component in successful adoptive immunotherapy
`
`
`From the Cancer Immunology Program, Peter MacCallum Cancer Centre, East
`Melbourne, Victoria, Australia; Howard Hughes Medical
`Institute and
`Department of Microbiology and Immunology, University of California, San
`Francisco, CA; and Rotary Bone Marrow Research Laboratory, Royal
`Melbourne Hospital, Parkville, Victoria, Australia.
`
`Submitted December 23, 2004; accepted July 5, 2004. Prepublished online as
`Blcod First Edition Paper, July 19, 2005; DOI 10.1182/blood-2004-12-4906.
`Supported by the Susan G. Komen Breast Cancer Foundation and the Cancer
`Council of Victoria research grants. M.H.K. and P-K.D. were supported by
`National Health and Medical Research Council of Australia (NHMRC) R. D.
`
`Wright Research Fellowships. M.J.S. and J.A.T. were supported by NHMRC
`Principal Research and Senior Principal Research Fellowships, respectively.
`
`Reprints: Phillip K. Darcy, Cancer Immunology Program, Peter MacCallum
`Cancer Centre, Locked Bag 1, A’Beckett St, East Melbourne, 8006,Victoria,
`Australia; e-mail: phil.darcy @ petermac.org.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked “advertisement” in accordance with 18 U.S.C. section 1734.
`
`© 2005 by The American Society of Hematology
`
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`MOELLER etal
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`BLOOD, 1 NOVEMBER 2005 - VOLUME 106, NUMBER 9
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`capacity of transducedTcells to produce cytokines (IFN~y, IL-2, granulo-
`with retrovirus-transduced CD8* cytotoxic T lymphocytes (CTLs),
`cyte-macrophage colony-stimulating factor [GM-CSF], IL-4) after erbB2
`thus raising important implications for optimizing adoptive T-cell
`antigen ligation was determined by enzyme-linked immunosorbent assay
`therapy for cancer treatment in patients.
`(ELISA) (PharMingen) andthe proliferative capacity of transduced T cells
`
`was assessed in a
`[?H]-thymidine incorporation assay, as described
`previously.35
`
`Materials and methods
`
`Cell culture
`
`MDA-MB-435 human mammary carcinoma cells, the erbB2-expressing
`MDA-MB-435 cells, 471.2 mouse mammary carcinoma cells, and the
`erbB2-expressing 4T1.2-erbB2 cells were cultured in Dulbecco modified
`Eagle medium (DMEM) supplemented with 10% (vol/vol) fetal calf serum
`(FCS), 2 mM glutamine, 100 U/mL penicillin, and 100 .g/mL streptomycin
`(Life Technologies, Grand Island, NY). Retrovirus-producing GP+E86
`cells were cultured in DMEM containing 0.5 mg/mL G418 (Life
`Technologies).
`
`Mice
`
`BALB/c and BALB/cscid/scid (scid) mice were purchased from the Walter
`and Eliza Hall
`Institute of Medical Research (Melbourne, Australia).
`BALB/c interferon yy (IFN-y)-deficient (BALB/c IFN-y~/~) mice were
`bred at the Peter MacCallum Cancer Centre. Mice 6 to 12 weeks of age
`were used in experiments that were performed in accordance with the Peter
`MacCallum Cancer Centre’s animal experimental ethics committee
`guidelines.
`
`Immunohistochemistry
`
`Hematoxylin and eosin staining and immunohistochemistry were per-
`formed on frozen sections. Antibodies used were FITC-antimouse CD4,
`biotin-antimouse CD8b.?, allophycocyanin (APC)-antimouse CD11b, FITC-
`antimouse CD11b (all from Becton Dickinson, San Jose, CA), biotin-anti-
`mye tag (Abcam, Cambridge, United Kingdom), isotype controls, and a
`streptavidin Alexa 594 secondary (Molecular Probes, Eugene, OR). The
`microscopes used were a Leica DMBRE (Leica, Wetzlar, Germany) with a
`Plan (PL) FLUOTAR, numerical aperture (NA) 0.7 objective lens and
`Bio-Rad MRC 1024 confocal (Bio-Rad, Hercules, CA) for confocal images
`and a Zeiss Axioskop 2 (Zeiss, Hertfordshire, United Kingdom) with a
`Plan-NEC FLUAR, NA 0.7/40 X objective lens and RT SE Diagnostic
`Instruments SPOT camera (Diagnostic Images, Sterling Heights, MI) for
`H&E images. Original magnification was x 400. Tissue sections were
`mounted with DakoCytomation Fluorescent Mounting Medium (DakoCyto-
`mation, Glostrup, Denmark) or stained for H&E. Image acquisition and
`processing software was LaserSharp 2000 or SPOT Basic Version 4.1 and
`Adobe Photoshop CS2, respectively.
`
`PCR
`
`Generation of scFv receptor-transduced CD8* and CD4*
`mouseT cells
`
`Adoptive transfer model
`
`The detection of gene-engineered T cells following adoptive transfer in
`mice was assessed by polymerase chain reaction (PCR) amplification of the
`neomycin phosphotransferase gene. Mice bearing 5-day MDA-MB-435
`A chimeric gene construct composed of the scFv—anti-erbB2 monoclonal
`tumor were giveninjections of scFv-CD28-¢-transduced CD8* and CD4"
`antibody (mAb), a membrane-proximal hinge region of human CD8, and
`T cells and were eye bled or had spleens removed at various time points.
`the transmembrane and cytoplasmic regions of the mouse CD28signaling
`Red blood cells were lysed using ACK lysis buffer (room-temperature
`chain fused to the cytoplasmic region of human T-cell receptor €
`(TCR-C;
`ammonium chloride potassium for 5 minutes), washed, and cells resus-
`scFv-anti-erbB2 CD28-{) was clonedinto the retroviral vector pLXSN as
`pended at 5 X 108 cells/200 L. gDNA from peripheral blood or spleno-
`previously described. A stable GP+E86 ecotrophic packaging cell line
`cytes was subsequently purified for PCR using QlAamp DNA Blood
`expressing the scFv—anti-erbB2 CD28-¢ receptor wasisolated as previously
`MiniKit per the manufacturer’s instructions (Qiagen, Clifton Hill, Austra-
`described.5 Transduction of mouse splenic T lymphocytes was performed as
`lia). Neomycin sense primer was 5'-TGGCTGCTATTGGGCGAAGT-3’;
`described previously.”!* To generate transduced CD8* and CD4*Tcells,
`antisense was 5’-TATCACGGGTAGCCAACGCT-3’, mouse f-actin sense
`each T-cell subset was initially isolated by labeling with anti-CD4 or
`primer was 5'-AGGCGGTGCTGTCCTTGTAT-3’; and antisense was 5'-
`anti-CD8 magnetic beads (Miltenyi Biotec, Auburn, CA) and passed
`GGAAGGAAGGCTGGAAGAGT-3’. The forward andreverse primers for
`through a magnetic-activated cell sorting (MACS) depletion column. The
`both neomycin and B-actin genes were designed to amplify fragments of
`efficiency of
`isolating separate T-cell
`subsets was verified by flow
`approximately 400 base pairs (bp) using Platinum Pfx DNA polymerase
`cytometry. Enriched CD8*, CD4*, or unfractionated T-cell cultures (107
`(Invitrogen Life Sciences, Carlsbad, CA).
`cells) were immediately cocultured with retrovirus-producing packaging
`cells (5 X 105) for 72 hours in DMEM supplemented with 4 g/mL
`polybrene, 5 .g/mL phytohemagglutinin (PHA; Sigma, Saint Louis, MO),
`and 100 U/mL human recombinant
`interleukin-2 (rIL-2; Chiron, Em-
`eryville, CA).
`
`the
`The antitumor activity of transduced T cells was assessed against
`MDA-MB-435-erbB2 tumorcell line as previously described.* Briefly. scid
`mice were given intravenous injections of 5 X 10° human MDA-MB-435-
`erbB2 breast carcinomacells to establish pulmonary metastases. Unfraction-
`ated scFv-CD28-¢-—transduced T cells (107), transduced CD8* or CD4* T
`cells alone (107), or a 1:1 combination of transduced CD8* and CD4* T
`cells (5 * 10° of each) from BALB/c donor mice were injected intrave-
`nously into groups of 5 to 10 mice at day 5 after tumor inoculation. For
`experiments evaluating the minimum number of engineered CD4 cells
`required to achieve total tumor regression, mice received different ratios of
`transduced CD8* and CD4* T cells. The adoptive transfer of T cells
`transduced with an irrelevant scFv receptor (scFv-a-CEA-y) or mock
`transduced with an empty vector pLXSN served as controls. In addition,
`adoptive transfer of scFv-transduced CD8* and CD4* T lymphocytes
`(5 X 106 of each, day 5)
`from either BALB/c-wild-type or BALB/c
`IFN-y~/~ donor mice was used to evaluate the role of IFN-y released by
`Antigen-specific cytotoxicity, cytokine secretion, and
`either T-cell subset in the antitumor effect. All mice were monitored daily
`proliferation by transducedTcells
`for tumor growth, Tumor growth was assessed as follows: (1) in survival
`experiments, mice that were morbid were killed and the day of death and
`lung weights recorded or (2) mice were killed at days 6, 12, or 16 and
`Miltenyi Ex. 1016 Page 9
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`Flow cytometry
`
`Expression of the a-erbB2-CD28-¢ chimeric receptor on the surface of
`CD8+, CD4*, or unfractionated transduced mouse T cells was determined
`by indirect immunofluorescence with a c-myc tag antibody (Ab), followed
`by staining with a phycoerythrin (PE)—labeled anti-mouse immunoglobulin
`mAb (BD Biosciences, San Jose, CA). Background fluorescence was
`assessed using the PE-labeled anti-mouse immunoglobulin mAbalone.
`Cell-surface phenotyping of transduced cells was determined by direct
`staining with fluorescein isothiocyanate (FITC)—labeled anti-CD4 (RM4-5;
`PharMingen, San Diego, CA) and PE-labeled anti-CD8 (53-6.7; PharMin-
`gen) mAbs,as previously described.45--!8
`
`Theability of transduced T cells to specifically mediate target-cell lysis was
`assessed in a 6-hour chromium-release assay, as described previously.'? The
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`ENHANCED TUMOR REJECTION BY ENGINEERED CD4TCELLS
`
`2997
`
`harvested lungs were fixed in 10% formalin, embedded, sectioned, and
`cells, CD8* and CD4* T cells were isolated by magnetic bead depletion
`stained with hematoxylin and eosin for histologic examination or frozen
`priorto transduction. Using this approach, we were able to achieve good
`sections of lungs were made for immunohistochemical analysis,
`levels of expression of the chimeric a-erbB2-CD28-¢ receptor in CD8*
`and CD4*Tcells afier staining with a c-myc anti-tag antibody (Figure
`Tumorrechallenge experiments
`1D-F) compared to mock-transduced CD8* or CD4* T cells (Figure
`1H,J), although expression was consistently found to be higher in CD8*
`T cells than CD4* T cells. This mayreflect less efficient cell cycling by
`CD4* T cells following PHA/IL-2 stimulation during the transduction
`process. Importantly, the T-cell populations consisted of more than 95%
`CD8* or CD4*T cells following transduction (Figure 1C,E,G,D. There
`wasnegligible expression ofthe chimeric receptorin either the CD8~ or
`CD4~ populations (data not shown),
`
`Mice surviving the primary MDA-MB-435 tumor long-term (> 100 days)
`were rechallenged with an intravenousinjection of 5 X 10° human MDA-
`MB-435-erbB2 tumor cells and survival monitored over 100 days.
`In
`another set of experiments, mice were rechallenged with a subcutaneous
`injection of the mouse mammary carcinoma 4T1.2-erbB2 cells or 4T1.2
`parental cells at 5 X 10* (high dose) or 5 X 10° (low dose). Survival of
`mice was monitored daily and defined as the period with noovert signs of
`distress, as assessed by 2 independent observers. Subcutaneous tumor
`growth was measured bya caliper square along the perpendicular axes of
`Transduced CD8* and CD4* mouseTcells mediate
`the tumors. Data were recorded as either percentage survival or the mean
`antigen-specific cytokine secretion, proliferation,
`tumorsize (mm?,product ofthe 2 perpendicular diameters) + SEM.
`
`and tumor-cell lysis
`
`Results
`
`Expression of the scFv-CD28-¢ receptor in transduced CD8*
`and CD4* primary mouseT cells
`
`Consistent with our previous studies, we have demonstrated that
`retroviral transduction ofunfractionated PHA/IL-2-stimulated splenic T
`cells consistently resulted in a high proportion of CD8* T cells
`(80%-85%) but
`low numbers of CD4* T cells (10%-15%; Figure
`1A-B).4°7!8 To determine whether we could achieve expression ofthe
`chime