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`fy 17, 2005|vol. 102 | no. 20 | pp. 7051-7402
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`macular degeneration
`
` q Mcerell fabrication of branched nanotubes
`
`)) 8D structures of protein reaction PETUNIA
`
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`
`Miltenyi Ex. 1031 Page 1
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`Miltenyi Ex. 1031 Page 1
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`Miltenyi Ex. 1031 Page 2
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`

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`
`
`UreseeKOA LSDUTTOOHUPACreINACMICUec KOAed OLNRSiCALCGKLiedULcntet
`
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`

`May17, 2005 | vol. 102 | no.20 | 7051-7402
`
`
`
`
`Proceedings ofthe National AcademyofSciences ofthe United States ofAmerica
`
`www.pnas.org
`
`
`
`Cover image: Laser scanning confocalimage ofan ocular druse, the hallmark lesion associated
`with age-related macular degeneration. The complementsystem protein Sb-9 is shown in orange
`andred, and factor H, which inhibits the complement pathway,is shown in green. Theretinal
`pigment epithelium is shown in purple. Genetic variation in the factor H gene is a major
`contributor to age-related macular degeneration. See the article by Hagemanet al. on pages
`7227-7232. Imagecourtesy of Patrick Johnson (Centerfor the Study of Macular Degeneration,
`University of California, Santa Barbara).
`
`From the Cover
`
`7227 Complement system and macular degeneration
`7074 Controlled fabrication of branched nanotubes
`7145 3Dstructures of protein reaction pathway
`7198 Assessing success of vertebrate invaders
`7332 Gene flow and mutation accumulation
`
`Co n te n ts
`
`THIS WEEK IN PNAS
`
`7051 In This Issue
`
`COMMENTARIES
`
`7053 Evidence for an inflammatory processin age-related
`macular degeneration gains new support
`Dean Bok
`> See companionarticle on page 7227
`7055 The success of animal invaders
`M.Jake Vander Zanden
`=> See companion article on page 7198
`
`PHYSICALSCIENCES
`
`APPLIED MATHEMATICS
`7057 A network analysis of committees in the U.S. House
`of Representatives
`MasonA.Porter, Peter J. Mucha, M.E. J. Newman,
`and Cascy M. Warmbrand
`
`ME Freely available online through the PNAS openaccess option.
`
`:
`
`|
`
`APPLIED PHYSICAL SCIENCES
`7063 The useofoscillatory signals in the study
`of genetic networks
`Ovidiu Lipan and Wing H. Wong
`
`CHEMISTRY
`
`7069 Four-dimensionalultrafast electron microscopy
`Vladimir A. Lobastov, Ramesh Srinivasan,
`and Ahmed H.Zewail
`
`7315 Patellamide A and C biosynthesis by a microcin-like
`QW pathwayin Prochloron didemni, the cyanobacterial
`symbiontof Lissoclinum patella
`Eric W. Schmidt, James T. Nelson, David A. Rasko,
`Sebastian Sudek, Jonathan A. Eisen, Margo G. Haygood,
`and Jacques Ravel
`
`ENGINEERING
`7074 Controlled fabrication of hierarchically branched
`nanopores, nanotubes, and nanowires
`Guowen Meng, Yung Joon Jung, Anyuan Cao, Robert Vajtai,
`and Pulickel M. Ajayan
`
`STATISTICS
`7079 De novocis-regulatory moduleelicitation
`for eukaryotic genomes
`Mayetri Gupta and Jun S. Liu
`
`MiltenyiEx. T03T’Page'4! °° |"
`
`Miltenyi Ex. 1031 Page 4
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`

`

`BIOLOGICALSCIENCES
`
`ANTHROPOLOGY
`7085 A late Neandertal femur from Les Rochers-de-
`Villeneuve, France
`Cédric Beauval, Bruno Maureille, Francois Lacrampe-
`Cuyaubére, David Serre, David Peressinotto, Jean-Guillaume
`Bordes, David Cochard, Isabelle Couchoud, David
`Dubrasquet, Véronique Laroulandie, Arnaud Lenoble,
`Jean-Baptiste Mallye, Sylvain Pasty, Jérome Primault,
`Nadin Rohland, Svante Paabo, and Erik Trinkaus
`
`BIOCHEMISTRY
`7091 Isolation and characterization of a retinal pigment
`epithelial cell fluorophore: An all-trans-retinal
`dimer conjugate
`Nathan E.Fishkin, Janet R. Sparrow, Rando Allikmets,
`and Koji Nakanishi
`
`7097 Identification and analysis of vnd/NK-2 homeodomain
`binding sites in genomic DNA
`Lan-Hsiang Wang, Rebecca Chmelik, Derek Tang,
`and Marshall Nirenberg
`
`7103 Dissecting human cytomegalovirus gene function
`sv and capsid maturation by ribozyme targeting
`and electron cryomicroscopy
`Xuekui Yu, Phong Trang, Sanket Shah, Ivo Atanasov,
`Yong-Hwan Kim, YongBai, Z. Hong Zhou, and Fenyong Liu
`7109 Humanbiliverdin reductase: A memberofthe insulin
`ly receptor substrate family with serine/threonine/tyrosine
`kinase activity
`Nicole Lerner-Marmarosh, Jenny Shen, Michael D. Torno,
`Anatoliy Kravets, Zhenbo Hu, and Mahin D. Maines
`
`7115 Structural properties of Af protofibrils stabilized
`Me by a small molecule
`Angela D. Williams, Matt Sega, Maolian Chen,
`Indu Kheterpal, MeravGeva, Valerie Berthelier,
`David T. Kaleta, Kelsey D. Cook, and Ronald Wetzel
`
`7121 Studies of yeast oligosaccharyl transferase subunits
`using the split-ubiquitin system: Topological features
`andin vivointeractions
`Aixin Yan, Elain Wu, and William J. Lennarz
`
`7127 Specific correlation between the wobble modification
`deficiency in mutant tRNAs andtheclinical features
`of a human mitochondrial disease
`Yohei Kirino, Yu-ichi Goto, Yolanda Campos,
`Joaquin Arenas, and Tsutomu Suzuki
`
`7133 Perinatal w-3 polyunsaturated fatty acid supply modifies
`brain zinc homeostasis during adulthood
`AnuraP. Jayasooriya, M. Leigh Ackland, MichaelL.
`Mathai, AndrewJ. Sinclair, Harrison S. Weisinger,
`Richard S. Weisinger, John E.Halver, Klara Kitajka,
`and Laszlé G. Puskas
`
`7139 The domainsof a cholesterol-dependentcytolysin
`undergo a major FRET-detected rearrangement
`during pore formation
`Rajesh Ramachandran, Rodney K. Tweten,
`and Arthur E. Johnson
`
`7145 Visualizing reaction pathways in photoactive yellow
`Ww protein from nanoseconds to seconds
`Hyotcherl Ihee, Sudarshan Rajagopal, Vukica Srajer,
`Reinhard Pahl, Spencer Anderson, Marius Schmidt,
`Friedrich Schotte, Philip A. Anfinrud, Michael Wulff,
`and Keith Moffat
`
`BIOPHYSICS
`7069 Four-dimensionalultrafast electron microscopy
`Vladimir A. Lobastov, Ramesh Srinivasan,
`and AhmedH.Zewail
`
`7151 Detecting remotely related proteins by their interactions
`and sequencesimilarity
`Jordi Espadaler, Ram6nAragiiés, Narayanan Eswar, Marc A.
`Marti-Renom, Enrique Querol, FrancescX. Avilés,
`Andrej Sali, and Baldomero Oliva
`
`7157 How sequence defines structure: A crystallographic
`mapof DNAstructure and conformation
`Franklin A. Hays, Amy Teegarden, ZebulonJ. R. Jones,
`Michacl Harms, Dustin Raup, Jeffrey Watson,
`Emily Cavaliere, and P. Shing Ho
`7163 Structural and functionalsimilarities between the
`capsid proteins of bacteriophages T4 and HK97 point
`to a commonancestry
`Andrei Fokine, Petr G. Leiman, Mikhail M. Shneider,
`Bijan Ahvazi, Karen M. Boeshans, Alasdair C. Steven,
`Lindsay W. Black, Vadim V. Mesyanzhinov,
`and Michael G. Rossmann
`
`7169 Investigating local conformations of double-stranded
`DNAbylow-energycircular dichroism
`of pyrrolo-cytosine
`Neil P. Johnson, Walter A. Baase, and Peter H. von Hippel
`
`CELL BIOLOGY
`7174 Point mutation in AML1 disrupts subnucleartargeting,
`prevents myeloid differentiation, and effects
`a transformation-like phenotype
`Diana Vradii, Sayyed K. Zaidi, Jane B. Lian, AndreJ.
`van Wijnen, JanetL. Stein, and GaryS. Stein
`
`7180 Sca-1 expression identifies stem cells
`in the proximal region of prostatic ducts with high
`capacity to reconstitute prostatic tissue
`Patricia E. Burger, Xiaozhong Xiong, Sandra Coetzee,
`Sarah N. Salm, David Moscatelli, Ken Goto,
`and E. Lynette Wilson
`
`7186 Balance of actively generated contractile and resistive
`forces controls cytokinesis dynamics
`Wendy Zhang and Douglas N. Robinson
`
`DEVELOPMENTAL BIOLOGY
`7192 Late-emigrating neuralcrest cells in the roof plate
`Wy arerestricted to a sensory fate by GDF7
`Liching Lo, Emma L. Dormand,and David J. Anderson
`
`ECOLOGY
`7198 Invasion success of vertebrates in Europe
`and North America
`Jonathan M.Jeschke and David L. Strayer
`= See Commentary on page 7055
`
`| May 17,2005 | vol.102 | no.20 | v
`PNAS
`Miltenyi Ex. 1031 Page 5
`
`Miltenyi Ex. 1031 Page 5
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`

`

`EVOLUTION
`7203 Conservation and evolvability in regulatory networks:
`WY Theevolution of ribosomalregulation in yeast
`AmosTanay, Aviv Regev, and Ron Shamir
`7209 Clathrin heavy andlight chain isoforms originated by
`SW independent mechanismsof gene duplication during
`chordate evolution
`Diane E. Wakeham, Laurent Abi-Rached, Mhairi C. Towler,
`Jeremy D. Wilbur, Peter Parham, and Frances M. Brodsky
`GENETICS
`7215 Insights into TOR function and rapamycin response:
`Me Chemical genomicprofiling by using a high-density
`cell array method
`Michael W.Xie, Fulai Jin, Heejun Hwang, Seungmin Hwang,
`Vikram Anand, Mara C. Duncan,and Jing Huang
`7221 Genomewideproduction of multipurposealleles
`for the functional analysis of the mouse genome
`Frank Schniitgen, Silke De-Zolt, Petra Van Sloun,
`Melanie Hollatz, ThomasFloss, Jens Hansen, Joachim
`Altschmied, Claudia Seisenberger, Norbert B. Ghyselinck,
`Patricia Ruiz, Pierre Chambon, Wolfgang Wurst,
`and Harald von Melchner
`
`7227 A commonhaplotype in the complementregulatory
`SM genefactor H (HF1/CFH) predisposes individuals
`to age-related macular degeneration
`Gregory S. Hageman, Don H. Anderson, Lincoln V. Johnson,
`Lisa S. Hancox, Andrew J. Taiber, Lisa I. Hardisty,
`Jill L. Hageman, HeatherA. Stockman, James D. Borchardt,
`Karen M. Gehrs, Richard J. H. Smith, GiulianaSilvestri,
`Stephen R. Russell, Caroline C. W. Klaver, Irene Barbazetto,
`Stanley Chang, Lawrence A. Yannuzzi, GaetanoR.Barile,
`John C. Merriam, R. Theodore Smith, Adam K.Olsh,
`Julie Bergeron, Jana Zernant, JoannaE. Merriam,Bert Gold,
`Michael Dean, and Rando Allikmets
`> See Commentary on page 7053
`
`IMMUNOLOGY
`7233 Chromosomalclustering of genes controlled
`by the aire transcription factor
`Jonathan B. Johnnidis, Emily S. Venanzi, Debra J. Taxman,
`Jenny P.-Y. Ting, Christophe O. Benoist, and Diane J. Mathis
`7239 HIV-1-specific IFN-y/IL-2-secreting CD8 T cells support
`We CD4-independentproliferation of HIV-1-specific
`CD8T cells
`Simone C. Zimmerli, Alexandre Harari, Cristina Cellerai,
`Florence Vallelian, Pierre-Alexandre Bart,
`and GiuseppePantaleo
`7245 Laser-capture microdissection of plasmacells from
`subacute sclerosing panencephalitis brain reveals
`intrathecal disease-relevant antibodies
`Mark P. Burgoon, Kathryne M. Keays, Gregory P. Owens,
`Alanna M.Ritchie, Pradeep R. Rai, Carlyne D. Cool,
`and Donald H.Gilden
`7251 Interleukin 10 attenuates neointimalproliferation
`~~ andinflammationin aortic allografts by a heme
`oxygenase-dependent pathway
`Sifeng Chen, Matthias H. Kapturczak, Clive Wasserfall,
`Olena Y. Glushakova, Martha Campbell-Thampson,
`Jessy S. Deshane, Reny Joseph, Pedro E. Cruz,
`William W. Hauswirth, Kirsten M. Madsen, Byron P. Croker,
`KennethI. Berns, Mark A. Atkinson, Terence R.Flatte,
`C. Craig Tisher, and Anupam Agarwal
`
`7257 The Toll pathwayis importantfor an antiviral response
`in Drosophila
`Robert A. Zambon, Madhumitha Nandakumar,
`Vikram N. Vakharia, and Louisa P. Wu
`7263 Coevolution of TCR-MHCinteractions: Conserved MHC
`Wy tertiary structure is not sufficient for interactions
`with the TCR
`Hye-Jung Kim, Donglin Guo, and Derek B.Sant’Angelo
`7268 Serum IgG mediates mucosal immunity against
`rotavirus infection
`Larry E. Westerman, Harold M. McClure, Baoming Jiang,
`Jeffrey W. Almond, and RogerI. Glass
`
`MEDICALSCIENCES
`7274 Micelacking multidrug resistance protein 3 show altered
`i morphine pharmacokinetics and morphine-6-
`glucuronide antinociception
`Noam Zelcer, Koen van de Wetering, Michel Hillebrand,
`Elise Sarton, Annemieke Kuil, Peter R. Wielinga,
`ThomasTephly, Albert Dahan, Jos H.Beijnen, and Piet Borst
`7280 Utility of siRNA against Keap1as a strategy to stimulate
`ue a cancer chemopreventive phenotype
`Tim W.P. Devling, Christopher D. Lindsay,
`Lesley I]. McLellan, Michael McMahon, and John D. Hayes
`7286 c-Myc regulates cell size and ploidy but is not essential
`for postnatal proliferation in liver
`Esther Baena, Alberto Gandarillas, Mireia Vallespinds,
`Jennifer Zanet, Oriol Bachs, Clara Redondo,Isabel Fabregat,
`Carlos Martinez-A., and Ignacio Moreno de Alboran
`7292 Recombinant granulocyte colony-stimulating
`factor-transferrin fusion protein as an oral
`myelopoietic agent
`Yun Bai, David K. Ann, and Wei-Chiang Shen
`7297 The Nkx6.1 homeodomaintranscription factor
`SW suppresses glucagon expression and regulates
`glucose-stimulated insulin secretion in islet beta cells
`Jonathan C.Schisler, Per Bo Jensen, David G. Taylor,
`ThomasC.Becker, Filip Krag Knop, Shiro Takekawa,
`Michael German, Gordon C. Weir, Danhong Lu,
`Raghavendra G. Mirmira, and Christopher B. Newgard
`MICROBIOLOGY
`7303 Predicted highly expressed genes in archaeal genomes
`Samuel Karlin, Jan Mrazek, Jiong Ma, and Luciano Brocchieri
`7309 Genomic and proteomic comparisons betweenbacterial
`and archaeal genomesand related comparisons with
`the yeast and fly genomes
`SamuelKarlin, Luciano Brocchieri, Allan Campbell,
`MarthaCyert, and Jan Mrazek
`7315 Patellamide A and C biosynthesis by a microcin-like
`SW pathwayin Prochloron didemni, the cyanobacterial
`symbiontof Lissoclinum patella
`Eric W. Schmidt, James T. Nelson, David A. Rasko,
`Sebastian Sudek, Jonathan A. Eisen, Margo G. Haygood,
`and Jacques Ravel
`7321 Cellulose utilization by Clostridium thermocellum:
`Bioenergetics and hydrolysis product assimilation
`Yi-Heng Percival Zhang and Lee R. Lynd
`7326 Yeast genome-widescreenreveals dissimilar sets of host
`genesaffecting replication of RNA viruses
`Tadas Panavas, Elena Serviene, Jeremy Brasher,
`and Peter D. Nagy
`
`PNAS | May 17,2005 |
`
`vol. 102 | no.20 |
`
`vii
`
`Miltenyi Ex. 1031 Page 6
`
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`

`

`7332
`
`7338
`
`7344
`
`7350
`
`7356
`
`7362
`
`7368
`
`Global divergence of microbial genome sequences
`mediated by propagating fronts
`Kalin Vetsigian and Nigel Goldenfeld
`
`NEUROSCIENCE
`Prefrontal cortex and flexible cognitive control:
`Rules without symbols
`Nicolas P. Rougier, David C. Noelle, Todd S. Braver,
`Jonathan D. Cohen, and Randall C. O’Rcilly
`Noncholinergic excitatory actions of motoneurons
`in the neonatal mammalianspinalcord
`George Z. Mentis, Francisco J. Alvarez, Agnes Bonnot,
`Dannette S. Richards, David Gonzalez-Forero,
`Ricardo Zerda, and Michael J. O’Donovan
`
`Functional organization of humanoccipital-callosal
`fiber tracts
`Robert F. Dougherty, Michal Ben-Shachar, Roland Bammer,
`Alyssa A. Brewer, and Brian A. Wandell
`Brain response to putative pheromones
`in homosexual men
`Ivanka Savic, Hans Berglund,and Per Lindstr6m
`The neurotrophin receptor p75‘™® modulates long-term
`depression and regulates the expression of AMPA
`receptor subunits in the hippocampus
`Harald Rosch, Rudiger Schweigreiter, Tobias Bonhoeffer,
`Yves-Alain Barde, and Martin Korte
`
`G protein-dependentpresynaptic inhibition mediated
`by AMPAreceptorsat the calyx of Held
`Hideki Takago, Yukihiro Nakamura,
`and Tomoyuki Takahashi
`
`7374 Ventralized dorsal telencephalic progenitors in Pax6
`mutant mice generate GABAinterneuronsofa lateral
`ganglionic eminence fate
`Todd T. Kroll and Dennis D. M. O’Leary
`PHARMACOLOGY
`7380 Selective anxiolysis produced by ocinaplon,
`a GABAa receptor modulator
`A. Lippa, P. Czobor, J. Stark, B. Beer, E. Kostakis,
`M. Gravielle, S. Bandyopadhyay,S. J. Russek, T. T. Gibbs,
`D.H.Farb,and P. Skolnick
`PHYSIOLOGY
`7386 Alternative Gnas gene products have opposite effects
`on glucose andlipid metabolism
`Min Chen, Oksana Gavrilova, Jie Liu, Tao Xie, Chuxia Deng,
`Annie T. Nguyen, Lisa M. Nackers, Javier Lorenzo,
`Laura Shen, and Lee S. Weinstein
`POPULATION BIOLOGY
`7392 Phylogeography of Barbary macaques (Macaca sylvanus)
`andtheorigin of the Gibraltar colony
`Lara Modolo, Walter Salzburger, and Robert D. Martin
`
`SOCIAL SCIENCES
`
`ECONOMICSCIENCES
`7398 Emotion expression in human punishment behavior
`Erte Xiao and Daniel Houser
`
`xi-xii Author Index
`xiii Subscription Form
`
`| May 17,2005 | vol.102 | no.20 |
`PNAS
`Miltenyi Ex. 1031 Page 7
`
`ix
`
`Miltenyi Ex. 1031 Page 7
`
`

`

`HIV-1-specific IFN-y/IL-2-secreting CD8 T cells
`support CD4-independentproliferation
`of HIV-1-specific CD8 T cells
`simone C. Zimmerli, Alexandre Harari, Cristina Cellerai, Florence Vallelian, Pierre-Alexandre Bart,
`and Giuseppe Pantaleo*
`ler Universitaire Vaudois,
`Departmentof Medicine, Division of ImmunologyandAllergy, Laboratory of AIDS Immunopathogenesis, Centre Hospital
`University of Lausanne, 1011 Lausanne, Switzerland
`Communicated by AnthonyS. Fauci, NationalInstitutes of Health, Bethesda, MD, March 23, 2005(received for review December 18, 2004)
`Functional and phenotypic characterization of virus-specific CD8 T
`helper CD4T cells in sustaining Ag-specific CD8 T cell prolifera-
`cells against cytomegalovirus, Epstein-Barr virus, influenza(flu),
`tion (13).
`ed
`and HIV-1 were performedonthebasisof the ability of CD8Tcells
`Recentstudies (14-16) investigating antiviral memory CD4 T
`to secrete IFN-y andIL-2, to proliferate, and to express CD45RA and
`cell responses have shownthatthe combinedassessment of IL-2 and
`CCR7. Two functional distinct populations of CD8 T cells were
`IFN-y is instrumental to distinguish functionally distinct popula-
`identified: (i) dual IFN-y/IL-2-secreting cells and (ii) single IFN-y-
`tions of memory CD4Tcells and patternsofantiviral immune
`secreting cells. Virus-specific IFN-y/IL-2-secreting CD8 T cells were
`responsesassociated with different conditionsofvirus persistence
`CD45RA~CCR7-, whereas single IFN-y CD8 T cells were either
`and control.
`CD45RA-CCR7— or CD45RA+CCR7-. The proportion of virus-
`In the presentstudy, we have performed functional and pheno-
`specific IFN-y/IL-2-secreting CD8 T cells correlated with that of
`typic characterization of antiviral CD8 T cell responses specific for
`proliferating CD8T cells, and the loss of HIV-1-specific IL-2-secret-
`HIV-1, CMV,EBVandinfluenza(flu) on thebasis of their ability
`ing CD8T cells was associated with that of HIV-1-specific CD8Tcell
`to proliferate, to secrete IL-2 and IFN-y, and to express CD45RA
`proliferation. Substantial proliferation of virus-specific CD8Tcells
`and CCR7. Ourresults indicate:(i) a wide heterogeneity ofantiviral
`(including HIV-1-specific CD8 T cells) was also observed in CD4 T
`CD8T cell immuneresponses underdifferent conditions ofvirus
`cell-depleted populations or after stimulation with MHC class |
`persistence; (i) a combined loss of virus-specific IFN-y/IL-2-
`tetramer-peptide complexes. IL-2 was the factor responsible for
`secreting and -proliferating CD8 T cells in progressive HIV-1
`the CD4-independent CD8T cell proliferation. These results indi-
`infection;
`(iii) a typical phenotype of effector cells,
`i.e.,
`cate that IFN-y/IL-2-secreting CD8 T cells may promote antigen-
`CD45RA-CCR7-,for the IFN-y/IL-2-secreting CD8T cells; (iv)
`specific proliferation of CD8 T cells even in the absence of helper
`a correlation between the proportion of virus-specific IL-2-
`CD4T cells.
`secreting and -proliferating CD8 T cells; and (v) the occurrence of
`Ag-specific CD8 T cell proliferation also in experimental condi-
`tions, excluding the involvementof Ag-specific helper CD4Tcells.
`C D8T cells playacritical role in the controlofviral infections
`Materials and Methods
`(reviewedin ref. 1). Several studies have shown a wide heter-
`Study Groups. The 21 subjects with progressive chronic HIV-1
`ogeneity ofmemory CD8 and CD4Tcells with multiple phenotypes
`infection enrolledin this study were naive to antiviral therapy, with
`and functions in response to virus infections (2-7). Functionally
`distinct populations of CD8T cells can be defined by the expression
`CD4Tcell counts of >250 cells per microliter (mean + SE: 810 +
`of CD45RA and CCR7(8) andare able to proliferate and/or to
`39) and plasma viremia counts of =5,000 HIV-1 RNAcopies per
`secrete cytokines such as IL-2, IFN-y, and TNF-a after antigen
`ml (mean + SE: 41,854 + 12,339). Five HIV-1-infected patients
`(Ag)-specific stimulation (9-11). The determination of quantitative
`with nonprogressive disease,i.c., LTNPs,as defined by documented
`and qualitative changes of virus-specific CD8 T cells in rapidly
`HIV-1 infection for >14 years, stable CD4 T cell counts of >500
`controlled acute, more slowly controlled or uncontrolled chronic
`cells per microliter (mean + SE: 912 + 125) and plasma viremia of
`infections showed that high load of lymphocytic choriomeningitis
`<1,000 HIV-1 RNAcopies per ml (mean + SE: 97 + 38) werealso
`included. Patient 1010 has a documented HIV-1 infection since
`virus resulted in the progressive diminution ofthe ability of CD8 T
`cells to produce IL-2, TNF-a, and IFN-y (9). Ofinterest, the
`March 1999. He wastreated with antiviral therapy at the time of
`capacityto secrete cytokines could berestoredif the viral load was
`primary infection and remained onantiviral therapy for 18 months.
`brought under control(9).
`Heinterrupted therapy spontaneously in December 2000. During
`IL-2 production from virus-specific CD8 T cells has been the
`the last 4 years, he constantly had levels of viremia of <50 HIV-1
`object of few studies in humans. Recentstudies have shownthat a
`RNAcopies per ml and CD4Tcell countin the range of1,400 cells
`variable percentage of cytomegalovirus (CMV)- and Epstein-Barr
`per microliter. In addition, blood from 28 HIV-negative subjects
`virus (EBV)-specific CD8T cells were able to secrete IL-2 (10, 11),
`was obtained from the local blood bank or from laboratory co-
`whereas IL-2 wasnot produced by melanoma-1-specific CD8T cells
`workers. The studies were approved by the Institutional Review
`obtained from patients with stage 1V melanoma(10). With regard
`Board of the Centre Hospitalier Universitaire Vaudois.
`to HIV-1 infection, no studies have investigated the ability of
`HIV-1-specific CD8 T cells to secrete IL-2. However, it has been
`shown that HIV-1-specific CD8 T cells of HIV-1-infected subjects
`Freely available online through the PNASopenaccessoption.
`with nonprogressive disease,
`i.e.,
`long-term nonprogressors
`Abbreviations: EBV, Epstein-Barrvirus; CMV, cytomegalovirus; Ag, antigen; LTNP, long-
`term nonprogressor; CFSE, carboxyfluorescein succinimidyl ester; SEB, staphylococcal en-
`(LTNPs), had greater proliferation capacity as compared with
`terotoxin B.
`HIV-1-specific CD8 T cells from progressors (12), and this finding
`*To whom correspondence should be addressedat: Laboratory of AIDS Immunopathogen-
`wasassociatedwith a better ability to control virus replication (12).
`esis, Division of ImmunologyandAllergy, Centre Hospitalier Universitaire Vaudois, Rue
`A recentstudy has shownthat the loss of HIV-1-specific CD8 T cell
`Bugnon, 1011 Lausanne, Switzerland. E-mail: giuseppe.pantaleo@hospvd.ch.
`proliferation wasassociated with the loss of HIV-1-specific helper
`© 2005 by The National AcademyofSciences of the USA
`CD4Tcells and has proposeda critical role of HIV-1-specific
`
`IMMUNOLOGY
`
`www.pnas.org/cgi/doi/10.1073/pnas.0502393102
`
`| no.20 | 7239-7244
`| May 17,2005 | vol. 102
`PNAS
`Miltenyi Ex. 1031 Page 8
`
`Miltenyi Ex. 1031 Page 8
`
`

`

`FACScalibur or an LSR II and analyzed by using CELLQUEST and
`DIVAsoftware (Becton Dickinson). The numberof nongated events
`ranged between 10° and 10° events.
`
`Ex Vivo Proliferation Assay. After an overnight rest, cells were
`washed twice, resuspended at 1 X 106 ml in PBS, and incubated for
`7 min at 37°C with 0.25 4M carboxyfluorescein succinimidyl ester
`(CFSE; Molecular Probes). The reaction was quenched with 1
`volumeof FCS, and cells were washed andcultured in the presence
`of anti-CD28 Ab(0.5 g/ml) (Becton Dickinson).Cells wereeither
`stimulated with HIV-1 peptide pools (1 g/ml of each peptide),
`individual peptides (5 ug/ml), or tetramers (0.31 wg/ml). Staphy-
`
`significant difference was found betweenthe percentage of HIV-
`1-specific IFN-y/IL-2-secretingcells in progressive HIV-1 infection
`and that found in the virus-specific IFN-y/IL-2-secreting CD8 T -
`cells (P < 0.05) of the other virus infections (Fig. 1B). Wealso
`evaluated the proportion of IL-2-secreting cells within IFN-y-
`secreting CD8T cells. Cumulative data of this analysis are shown
`in Fig. 1C. The proportion of CMV-specific (12.7 + 1.8%, n = 11)
`and EBV-specific (19.2 + 3.2%, n = 10) IL-2-secreting CD8T cells
`wassignificantly higher (P < 0.05) compared with that of HIV-1-
`specific IL-2-secreting CD8T cells (2.3 + 0.6%, n = 21) (Fig. 1C).
`Theproportion (25.6 + 3.6%, n = 7) of flu-specific IL-2-secreting
`CD8T cells wassignificantly higher (P < 0.05) compared with that
`
`HiVv-1
`Progressor 2077
`Gated on
`CDS Tells
`
`cMV
`Subject 205
`Gated on
`CDE Tcells
`
`A
`
`9
`=
`
`EBV
`Subject 195
`Gated on
`__CD8*Tcells
`
`FLU
`Subject 35
`Gated on
`_CD8* Tcells
`oorm)S
`=e
`3
`£
`a
`a
`a
`
`
`
`
`
`
`3
`g
`&
`BR
`
`B
`4a
`Fe
`= z
`¢ i
`Be
`a2
`3s
`Sr
`3%
`382
`ee
`$8
`83
`fa
`
`026%
`IFN-y
`
`Fig. 1. Analysis of different virus-specific IFN-y- and IL-2-secreting CD8T cells after stimulation withsingle peptides. (A) Distribution of IFN-y- andIL-2-secreting
`virus-specific CD8Tcells. Cells were stimulated withsingle peptides. One representative profile is shownfor HIV-1-, CMV-, EBV-, or flu-specific CD8Tcell responses.
`Thecluster of events shownin red correspondsto the responder CD8T cells, i.¢., secreting IFN-y orIL-2, and the blue clusters correspond to the nonresponder
`cells. (B) Cumulative data on the percentage (mean = SE)of IFN-y/IL-2-secretingcells within the differentvirus-specific CD8Tcell responses. (C) Cumulative data
`on the proportion (mean + SE) ofIL-2-secreting cells within IFN-y-secreting CD8Tcells. +, P< 0.05.
`
`
`
`ea
`EBV
`n=10
`
`c
`Flu
`n=7
`
`n=21
`
`cmv
`n=11
`
`aSa8
`BiH
`£39 40
`wes=S 30
`gee22
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`7240 | www.pnas.org/cgi/doi/10.1073/pnas.0502393102
`
`Zimmerli etal.
`
`Miltenyi Ex. 1031 Page 9
`
`Miltenyi Ex. 1031 Page 9
`
`

`

`A
`
`Progressor 2113
`Gated on CD8*T cells
`
`B
`
`LTNP 2061
`LTNP 2069
`LTNP2073
`Gated on CD8*T cells
`Unstimulated Unstimulated Unstimulated
`
`of HIV-1- and CMV-specific but not with that of EBV-specific
`[L-2-secreting CD8 T cells (Fig. 1C). Finally, the proportion of
`EBV-specific IL-2-secretingcells was also significantly higher com-
`pared with that of CMV-specific IL-2-secreting CD8Tcells (P <
`0.05) (Fig. 1C). CMV-, EBV-, and flu-specific CD8T cell responses
`were also studied in HIV-1-infected individuals either by using
`peptides specific to CMV and EBV (n = 7) and flu (7 = 6) or a pool
`of 21 CMV-, EBV-, andflu-derived peptides in 30 HIV-1-infected
`subjects. The proportion of CMV-, EBV-, or flu-specific IL-2-
`secreting CD8 T cells in HIV-1-infected subjects was notsignifi-
`cantly different from that observed in HIV-negative subjects
`(P > 0.05).
`To exclude the possibility that the lack of detection of HIV-1-
`specific IFN-y/IL-2-secreting CD8 T cells was specific of the
`response to certain peptides, we performedstimulation with pep-
`tide pools spanning gag, pol, and nef proteins of HIV-1. A
`representative flow cytometry profile ofone (of 21) HIV-1-infected
`subjects with progressive disease (progressors) is shownin Fig. 24.
`Despite the presence of HIV-1-specific TIFN-y-secreting CD8 T
`cells after stimulation with different HIV-1 peptide pools, IL-2-
`secreting CD8 T cells were not detected (Fig. 24).