Immunology
Nascent Drosophila Toll-8 crystals. Toll receptors
are critical signaling receptors of development
and of innate immunity conserved in evolution
from flies to humans. Recombinant forms of
these receptors are used to grow crystals and
determine structures in order to establish a
structure-function relationship. Work done in
the laboratory of Luc Teyton, M.D., Ph.D.
He Zhou, M.D., Ph.D., Research Associate
Ralph A. Reisfeld, Ph.D., Professor
Yunping Luo, M.D., Ph.D., Research Associate
Department of Immunology
IMMUNOLOGY
DEPAR TMENT OF
IMMUNOLOGY
S TA F F
Richard J. Ulevitch, Ph.D.
Professor and Chairman
Bruce A. Beutler, M.D.
Professor
Roberto Baccala, Ph.D.
Assistant Professor
Gary M. Bokoch, Ph.D. *
Professor
Dennis R. Burton, Ph.D. **
Professor
Tsung-Hsien Chuang, Ph.D.
Assistant Professor
Charles G. Cochrane, M.D.
Professor Emeritus
Neil R. Cooper, M.D.
Professor Emeritus
Linda K. Curtiss, Ph.D.
Professor
Edward A. Dennis, Ph.D.
Adjunct Professor
Henrik Ditzel, M.D., Ph.D.
Adjunct Professor
Frank J. Dixon, M.D.
Professor Emeritus
Director Emeritus, Scripps
Research
Thomas S. Edgington, M.D.
Professor
2006
THE SCRIPPS RESEARCH INSTITUTE
99
Peter Ghazal, Ph.D.
Adjunct Associate Professor
David Nemazee, Ph.D.
Professor
R. Anthony Williamson, Ph.D.
Associate Professor
Jiahuai Han, Ph.D.
Professor
Glen R. Nemerow, Ph.D.
Professor
Curtis B. Wilson, M.D.
Professor Emeritus
Wendy L. Havran, Ph.D.
Professor
Per A. Peterson, M.D., Ph.D.
Adjunct Professor
Rong Xiang, M.D., Ph.D.
Assistant Professor
Shuang Huang, Ph.D.
Assistant Professor
Pascal Poignard, M.D.
Adjunct Assistant Professor
Michael Zwick, Ph.D.
Assistant Professor
Julie Jameson, Ph.D.
Assistant Professor
Ralph A. Reisfeld, Ph.D.
Professor
S TA F F S C I E N T I S T S
Jonathan G. Kaye, Ph.D.
Associate Professor
Matthias Riewald, M.D.
Assistant Professor
Richard Klemke, Ph.D.***
Associate Professor
Moores Cancer Center
San Diego, California
Hugh Rosen, M.D., Ph.D.
Professor
Norman R. Klinman, M.D.,
Ph.D.
Professor
Ulla Gissi Knaus, Ph.D.
Associate Professor
Dwight Kono, M.D.
Associate Professor
Jiing-Dwan Lee, Ph.D.
Associate Professor
Erguang Li, Ph.D.
Assistant Professor
Cheng Liu, M.D., Ph.D.
Assistant Professor
Nigel Mackman, Ph.D.
Associate Professor
Steven Brown, Ph.D.
Udayan Chatterji, Ph.D.
Wolfram Ruf, M.D.
Professor
Xin Du, Ph.D.
Colleen Fearns, Ph.D.
Vladimir Kravchenko, Ph.D.
Daniel R. Salomon, M.D.
Adjunct Associate Professor
Erica Ollmann Saphire, Ph.D.
Assistant Professor
John Mathison, Ph.D.
Anil Munshi, Ph.D.
Rafal Pawlinski, Ph.D.
Nora Sarvetnick, Ph.D.
Professor
David Schlaepfer, Ph.D.
Associate Professor
Ramona Petrovan, Ph.D.
M. Germana Sanna, Ph.D.
Laura Solforosi, Ph.D.
Linda A. Sherman, Ph.D.
Professor
Jonathan Sprent, M.D., Ph.D.
Adjunct Professor
Deborah Witherden, Ph.D.
SENIOR RESEARCH
A S S O C I AT E S
Charles D. Surh, Ph.D.
Associate Professor
Ann J. Feeney, Ph.D.
Associate Professor
Michael McHeyzer-Williams,
Ph.D.
Associate Professor
Luc Teyton, M.D., Ph.D.
Associate Professor
Philippe Gallay, Ph.D.
Associate Professor
Dianne McKay, M.D.
Assistant Professor
Argyrios N. Theofilopoulos,
M.D.
Professor
Nicholas R.J. Gascoigne,
Ph.D.
Professor
Donald E. Mosier, M.D.,
Ph.D.
Professor
Peter S. Tobias, Ph.D.
Associate Professor
Amanda Gavin, Ph.D.
Assistant Professor
Kerri A. Mowen, Ph.D.
Assistant Professor
Susan R. Webb, Ph.D.
Associate Professor
Jasimuddin Ahamed, Ph.D.
Gourab Bhattarcharjee, Ph.D.
Joao da Silva Correia, Ph.D.
Amr Abdelhamid El Sheikh,
Ph.D
Kasper Hoebe, Ph.D.
Eujing Jo, Ph.D.
Hyun-ku Lee, Ph.D.
100 IMMUNOLOGY
Rui Lin, Ph.D.
Qilin Pan, Ph.D.
Andrew Saphire, Ph.D.
2006
Eleuterio De La Camara,
Ph.D. ***
Centro Nacional de
Investigaciones
Cardiovasculares
Madrid, Spain
Yan Wu, Ph.D.****
THE SCRIPPS RESEARCH INSTITUTE
Emma Hamilton-Williams,
Ph.D.
Lars Hangartner, Ph.D.
Carsten Kreig, Ph.D.
Joerge Krueger, M.D.***
Charité Children’s Hospital
Berlin, Germany
Masaaki Hayashi, M.D.,
Ph.D.
Toru Kurokawa, Ph.D.
Natasha Hill, Ph.D.
Yumi Kurokawa, Ph.D.
Neil John Hime, Ph.D.
Young Back Kwon, Ph.D.****
Shihe Hou, Ph.D.***
Abraxis BioScience Inc.
Los Angeles, California
Cheng Yu Lai, Ph.D.
Hong Hua, Ph.D.
Elise Landais, Ph.D.
Timothy Huang, Ph.D.
Mansun Law, Ph.D.
Christoph Huber, Ph.D.
Jeff Lee, Ph.D.
Milena Iacobelli, Ph.D.
Sang-Un Lee, Ph.D.***
Cummings School of
Veterinary Medicine at
Tufts University
North Grafton,
Massachusetts
Violane Delorme, Ph.D.
R E S E A R C H A S S O C I AT E S
Djemel Ait-Azzouzene, Ph.D.
Christopher Alfonso, Ph.D.***
BD PharMingen
San Diego, California
Sandrine Arnaud-Dabernat,
Ph.D.
Celine Der Mardirossian,
Ph.D.
Benoit Besnues, Ph.D.
Anthony Don, Ph.D.
Helen Donners, Ph.D.***
Institute of Tropical Medicine
Antwerp, Belgium
Caroline Aylott, Ph.D.****
Celine Eidenschenk, Ph.D.
Ann Bellon, Ph.D.
Celia Espinoza, Ph.D.****
Michael Berger, Ph.D.
Nicolas Fazilleau, Ph.D.
Dafang Bian, Ph.D.****
Clemens Feistritzer, M.D.
Joerge Birkenfeld, Ph.D.
Christofer Flood, Ph.D.
Onur Boyman, Ph.D.***
Centre Hospitalier
Universitaire Vaudois
Immunology and Allergology
Lausanne, Switzerland
Linda Frederick, Ph.D.***
Favrille, Inc.
San Diego, California
Carlos Cantu, Ph.D.***
Digital Gene Technologies
La Jolla, California
Guo Fu, Ph.D.
Stefan Freigang, Ph.D.
Hassan Issafras, Ph.D.
Zhengfan Jiang, Ph.D.
Jennifer Lamoureux, Ph.D.
Wong Soon Justin, Ph.D.
Young Jun Kang, Ph.D.
Sung-Hyung Lee, Ph.D.***
Baylor College of Medicine
Houston, Texas
Yu-Ya Kao, Ph.D.
Cheng Li, Ph.D.****
Charles Kaplan, Ph.D.***
Genentech Corporation
San Francisco, California
Jiali Li, Ph.D.
Xiang Li, Ph.D.
Linda Kidd, Ph.D.
Michelle Fung, Ph.D.
Yang Mi Li, Ph.D.
Chang-Hoon Kim, Ph.D.
Marie Cherrier, Ph.D.
Philippe Georgel, Ph.D.***
Laboratoire
d’Immunogénétique
Moléculaire Humaine
Strasbourg, France
Roshni Chintalapati, Ph.D.
Davide Gianni, Ph.D.
Jae Ho Cho, Ph.D.****
Cristina Gil-Lamaignere,
Ph.D.***
Department of Cell Biology
Scripps Research
John B. Carey, Ph.D.
Jianming Chen, Ph.D.
Hee Ok Kim, Ph.D.****
Jun-Sub Kim, Ph.D.
Sungwoo Kim, Ph.D.***
Assistant Professor
University of Calgary
Calgary, Alberta, Canada
Yilei Li, Ph.D.***
South China Medical
University
Guangzhou, China
Ssang-Taek Lim, Ph.D.
Yang Mi Lim, Ph.D.
Edguardo Kolkowski, Ph. D.
Ting-Kun Lin, M.D., Ph.D.***
Kaiser Permanente Medical
Center
San Francisco, California
Aimee de Catherlineu, Ph.D.
Pedro Gonzalez-Cabrera,
Ph.D.
Marek Kovar, Ph.D.****
Guoxun Liu, Ph.D.****
Shrimati Datta, Ph.D.
Fang Guo, Ph.D.
Jirina Kovarova, Ph.D.****
Yuan Liu, Ph.D.
David Chodniewicz, Ph.D.****
Ben Croker, Ph.D.
Rachel Kohler, Ph.D.
IMMUNOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
101
Carina Lotz, Ph.D.***
Netherlands Cancer Institute
Amsterdam, the Netherlands
Jared Purton, Ph.D.
Joie Trifilo, Ph.D.****
Sun-Hee Yoon, Ph.D.****
Christopher Ramsey, Ph.D.
Marina Tsatmali, Ph.D.
Kenji Yoshida, Ph.D.
Christine Louis dit Sully,
Ph.D.
M. Rachel Richards, Ph.D.
David Valenta, Ph.D.
Jiangiang Yu, Ph.D.
Elise Romeo, Ph.D.
Sebastian Vallee, Ph.D.
Hui Zhang, Ph.D.
Mark Rubenstein, Ph.D.***
University of California
San Diego, California
Ester Van Leeuwen, Ph.D.
You Qing Zhang, Ph.D.
Tieming Zhao, Ph.D.
Monica Ruse, Ph.D.
Laurent Verkoczy, Ph.D.***
Duke University
Durham, North Carolina
Sophie Rutschmann, Ph.D.
Hendrik Versteeg, Ph.D.
Janelle Salkowitz-Bokal,
Ph.D.
Nicole Von Allmen-Zurcher,
Ph.D.
Huamin Zhou, Ph.D.***
Xiamen University
Xiamen, China
Prabhakar Salunkhe,
Ph.D.***
Burnham Institute
La Jolla, California
Katharina Von Lohneysen,
Ph.D.
Jorge Luis Schettini, Ph.D.
Yingchun Wang, Ph.D.***
Moores Cancer Center
San Diego, California
Yunping Luo, M.D., Ph.D.
James P. Luyendyk, Ph.D.
Michael Lyman, Ph.D.
Chitladda Mahanivong, Ph.D.
Laurent Malherbe, Ph.D.
Maria Manukyan, Ph.D.
He Zhou, Ph.D.
Annette Marleau, Ph.D.
Beatriz Maroto, Ph.D.***
Centro Nacional de
Investigaciones Oncológicas
Madrid, Spain
David Marsolais, Ph.D.
Nicolas Schrantz, Ph.D.
A S S O C I AT E S
Meng Wang, M.D.
Javier Martinez, Ph.D.
Terrence Meehan, Ph.D.
Satyajit Mitra, Ph.D.
Johann Mols, Ph.D.
Adam Mullick, Ph.D.
Perihan Nalbant, Ph.D.
Reto Andreas Schupbach,
Ph.D.
Zhao Wang, Ph.D.
Megumi Watanabe, Ph.D.
Alim Seit-Nebi, Ph.D. ***
Department of Molecular
Biology
Scripps Research
Suganya Selvarajah, Ph.D.
Chenghong Wei, Ph.D.
Christopher Wiethoff,
Ph.D.***
Loyola University
Chicago, Illinois
Ron Nepomuceno, Ph.D.
Shigeki Shimada, Ph.D.
Frank Karl Niessen, Ph.D.
Jason Smith, Ph.D.
Miyo Ota, Ph.D.
Michelle Solomon, Ph.D.****
Chia Cheng Wu, Ph.D.
Takayuki Ota, Ph.D.
Gabriel Sternik, Ph.D.
Wenyuan Wu, Ph.D.
Motoyuki Otsuka, Ph.D.
Konstantin Stoletov, Ph.D.***
Moores Cancer Center
San Diego, California
Chengran Xu, Ph.D.
Joyce Tan, Ph.D.***
Anadys Pharmceuticals
San Diego, California
Pia Yachi, Ph.D.
Rachel Tilley, Ph.D.
Michael Ye, Ph.D.
Antoine Toulon, Ph.D.
Sook Wah Yee, Ph.D.
Sandrine Pacquelet, Ph.D.
Nadige Pelletier, Ph.D.
Olivier Pertz, Ph.D.***
University of California
San Diego, California
Helle Petersen, Ph.D.
SCIENTIFIC
Justin Soon Boon Wong,
Ph.D.
Rosana Gonzales-Quintal,
Ph.D.
Marcie Rose Kritzik, Ph.D.
Nora Leaf
Ralph Pantophlet, Ph.D.
Dongyuan Xia, Ph.D.
* Joint appointment in the
Department of Cell Biology
** Joint appointment in the
Department of Molecular Biology
*** Appointment completed, new
location shown
**** Appointment complete
Yue Xu, Ph.D.
Deepak Yadav, Ph.D.
102 IMMUNOLOGY
2006
Richard Ulevitch, Ph.D.
Chairman’s Overview
rogress in biomedical research demands innovation
and depends on our ability to identify the leading
edges of science. The approach to science that
looks backward instead of forward stifles originality and
perpetuates conventional thinking. I am always impressed
by the ability of our department members to develop new
paradigms to advance scientific knowledge. The environment at Scripps Research encourages our scientists to
apply the very latest approaches to solving core problems
of immunology. This often involves taking risks, especially
in today’s world of peer-reviewed funding where out-ofthe-box thinking is not always rewarded. However, I am
pleased to report that our investigators are continuing to
take appropriate risks with their science and, as a result,
are being widely recognized for their accomplishments.
This is evidenced by the numerous scientific papers published by our department members and by the prestigious
awards and other forms of recognition bestowed upon
them. Some of these are highlighted below.
But first I would like to acknowledge some key transitions within our faculty. During the past year, Norman
Klinman retired after an incredibly productive career of
more than 30 years in the field of immunology research.
P
THE SCRIPPS RESEARCH INSTITUTE
In 2006, he received the Excellence in Mentoring Award
from the American Association of Immunologists for
“exemplary career contributions to a future generation
of scientists.” Many of Dr. Klinman’s more than 50 former trainees attended the award ceremony, which was
held in Boston in May. We wish Dr. Klinman all the best
in his future activities. We will always welcome his presence at Scripps Research.
The end of 2005 also marked the departure of
Jonathan Sprent, who returned to his native Australia
after 30 years in the United States—first at the University
of Pennsylvania and then at Scripps Research. Dr. Sprent
is currently professor at the Garvan Institute of Medical
Research in Sydney, where he will continue his seminal
work on T-cell function combining his unique knowledge
of the immune response in whole animals with molecular
models using cell-based systems. We look forward to
reading about Dr. Sprent’s new findings as they impact
immunologic memory and tolerance, transplantation
immunity, and cancer immunotherapy. His longstanding
collaborations with Charlie Surh at Scripps Research will
undoubtedly continue, and we will welcome his visits
to our campus.
Coinciding with these departures is the recent recruitment of Karsten Sauer, who joined us after a number of
years at the Genomics Institute of the Novartis Research
Foundation. Dr. Sauer specializes in studies of lymphocyte signal transduction and will bring additional strength
to our department in this important field. Defects in lymphocyte development or function underlie various immune
disorders, including immunodeficiencies, inflammatory
and autoimmune diseases, and allergy. Dr. Sauer’s studies promise to significantly expand our understanding of
these key processes in health and disease. Specifically,
his laboratory will combine state-of-the-art genomic
and proteomic profiling techniques, functional genomics,
high-throughput screening, and imaging technologies as
well as classical biochemistry, molecular biology, and
cell biology to explore the functions of novel signaling
modules. This effort will result in a new understanding
of how antigen-receptor signaling directs diverse cellular
responses and how its malfunction leads to immunologic
disease. We look forward to Dr. Sauer ’s contributions
as an active faculty member in our department.
Highlights of scientific publications provide clear
evidence of the important place our department holds
within the national and international community of scientists interested in immunology. Bruce Beutler has provided
Scripps Research with a unique resource in the form of
IMMUNOLOGY
2006
a program of random germline mutagenesis. During the
past year, he and his colleagues have provided remarkable new insights into mechanisms of innate and adaptive immunity. Key findings include those of Koichi Tabeta
and his colleagues, published in Nature Immunology, in
which an unanticipated function has been assigned to
the Unc93b1 gene product. These investigators showed
that a mutation in Unc93b1, known as the 3d mutation, disrupts signaling via Toll-like receptors 3,7, and 9,
and also alters antigen presentation. Building on these
data are findings from Kasper Hoebe and colleagues,
published this year in Immunity, have identified a novel
pathway of T-cell activation that appears to be independent of Toll-like receptor signaling. Taken together, these
two publications promise to shape our thinking about
mechanisms involved in promoting adaptive immunity
through the innate immune networks.
In addition to uncovering new biology, the germline
mutagenesis approach provides new insights into protein
structure as revealed in Proceedings of the National
Academy of Sciences by Zhengfan Jiang and his colleagues, who describe molecular details of interactions
between MyD88 and Toll-like receptors. We also look
forward to continued productivity and seminal advances
from Bruce Beutler and his group during the coming year.
Adding to the department’s strengths in innate immunity is the work done in the laboratory of Jiahuai Han.
DaSilva Correia and his group provided an unexpected
insight into a mechanism whereby Nod1 regulates the
function of the estrogen receptor. Nod1 provides a brake
on the estrogen receptor, and the absence of Nod1 results
in enhanced sensitivity to estrogen and promotes tumor
growth in a xenograft model. This work was published
in Proceedings of the National Academy of Sciences.
Members of Luc Teyton’s laboratory combine structure-function studies with both cell-based and animal
models to probe the function of the natural killer T-cell
receptor and its ligand. This work, which was published
in Nature and Nature Immunology, includes a longstanding
collaboration with Albert Bendelac at the University of
Chicago. Following on studies of immune regulatory pathways is work done by Charlie Surh and his colleagues,
which was published in Science. This report documents
an unexpected finding showing how an immune complex
of anti-IL2 and IL2 act to stimulate T-cell subsets. These
data not only provide new insights into uses of therapeutic antibodies but also alert us to concerns about unanticipated side effects. Another advance in understanding
adaptive immunity comes from Michael McHeyzer-
THE SCRIPPS RESEARCH INSTITUTE
103
Williams and members of his laboratory, whose work
on B-cell function was recently described in an article
published in Immunity.
In both the United States and worldwide, HIV and
hepatitis C infection remain significant problems for which
there are still no appropriate therapies. Dennis Burton
and his group continue their important research in these
two areas. This past year, they published articles in
Science, Nature Immunology, and Proceedings of the
National Academy of Sciences that provided insights
into key issues for advancement of new treatments. These
recent publications represent the important collaborations established by Dennis Burton and his colleagues
both inside and outside of Scripps Research.
Work from the laboratory of Hugh Rosen combines
chemical and genetic approaches to proof-of-concept
studies that will allow a better understanding of human
disease mechanisms. This work paves the way for the
development of new small-molecule therapeutics for use
in immunologic and inflammatory diseases in humans.
Specifically, publications in Nature Immunology, Nature
Chemical Biology, and other top journals illustrate the
importance of the approaches implemented by Hugh
Rosen and his group. Further illustration of the power
of genetics is found in work published in Nature Medicine by members of the J-D Lee’s laboratory. Here they
used conditional knock-out of the HSP40 gene to prove
a role for this protein in a serious human syndrome,
cardiomyopathy. This work may well lead to the development of drugs that can be used to selectively intervene in this serious medical problem.
These examples illustrate the productivity of the members of our department. More details and a complete list
of accomplishments can found in the individual reports.
I apologize in advance for not including comments about
each publication from the 2005-2006 period and urge
readers to examine the reports for more details. As always,
it is a great pleasure for me to read the material from
each of our faculty members and to reflect on the progress of the past year.
104 IMMUNOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
INVESTIGATORS’ R EPORTS
Analyzing Host Resistance:
Phenomenon to Phenotype
to Gene
M. Barnes, K. Benson, M. Berger, B. Croker, K. Crozat,
X. Du, C. Eidenschenk, K. Hoebe, Z. Jiang, B. Layton,
N. Nelson, B. Ortiz, T. Palmer, S. Rutschmann, S. Sovath,
H. Uy, K. Whitley, Y. Xia, B. Beutler
ecause microbes are an enormous threat to most
vertebrate species, mammalian evolution has
been shaped by the microbial environment, and
many examples of protein modification were driven by
the need to evade infection. On an evolutionary timescale, a fairly large fraction of the genome in mammals
has been appropriated chiefly to create resistance to
infection. Those proteins with nonredundant function
in resistance to a particular organism are components
of the host “resistome.”
To identify key resistance proteins, we use random
germ-line mutagenesis. In this process, a chemical mutagen, N-ethyl-N-nitrosourea, is used to induce a distinctive
phenotype (e.g., inadequate defense against infection).
The phenotype is then genetically mapped, and the
causal mutation is found by using DNA sequencing. Once
cause and effect have been established, we can determine precisely how a given protein contributes to defense
against infection. To date, we have created scores of
mutations that impair resistance to various pathogens;
we have mapped a total of 32 of these mutations to
chromosomal intervals. Most of the mapped mutations
have been solved at a molecular level (Fig. 1).
Many of the proteins identified in this way are previously unknown components of the resistome. Some of
the proteins serve signaling by the Toll-like receptors,
which we previously showed are key sensors by which
mammals detect infection. Other identified proteins are
important components of the cytokine response apparatus, required for the production or activity of TNF or
type I interferons. Remarkably, many of the proteins
do not yet fit into a cohesive picture. Rather, they are
puzzles waiting to be solved. Germ-line mutations can
disclose the function of proteins where no function was
previously assigned. The mutations can also reveal new
functions of a protein, if some functions were previously
known. And the mutations can shed light on the func-
B
F i g . 1 . Map locations of mutations with immunologic effects
produced to date. Shaded ovals indicate that the mutation has
been identified by using positional cloning.
tion of proteins, which in the final analysis often act
as minute “machines.” We have seen each of these
outcomes in the course of our research.
A phenotype called 3d (to connote a “triple defect” of
nucleic acid sensing) was tracked to a multispanning
membrane protein with no previously known function.
This protein, known as UNC-93B, is required for the
integrity of a specific class of endosomes, small bodies
within cells within which sensing of nucleic acids and
processing of antigens for activation of the immune system occur. The 3d mutation causes susceptibility to a
broad range of infections because microbial nucleic
acids are an important clue to the presence of infection,
and when the host remains unaware of these nucleic
acids, infections may grow out of control. In addition,
the 3d mutation prevents antigen-presenting cells from
delivering foreign proteins to T lymphocytes, particularly CD8+ cells, which are required to directly attack
and eliminate infection within tissues of the host.
Some mutations create subtle changes within proteins, revealing functions that were previously hidden
because total disruption of the protein is lethal. One
such mutation, called woodrat, causes graying of the
fur and a moderate, generalized immunodeficiency
(e.g., failure to contain infection caused by mouse
cytomegalovirus). This mutation affects an enzyme
that is of central importance in cholesterol metabolism.
Known as the site 1 protease, the enzyme cleaves a
latent transcription factor required for activation of
genes that encode other enzymes required to synthesize cholesterol. Although mice lacking the site 1 protease cannot survive to term, woodrat mutants are
fully viable, have exceptionally low blood levels of cho-
IMMUNOLOGY
2006
lesterol, and have a diminished ability to cope with
infection. Hence, a new function of the site 1 protease
has come to light.
The syndrome of shock that occurs during serious
infections of all kinds is mediated by signaling via Tolllike receptors, and for this reason, the exact structure
of the transduction apparatus that alerts the host to
the presence of lipopolysaccharide, bacterial lipopeptides, unmethylated DNA, and other signature molecules
has been the object of intense interest. The Toll-like
receptors recruit specific adapter proteins, which in
turn activate protein kinases that ultimately promote
the transcription of hundreds of genes that shape the
inflammatory response. A mutation in one such adapter
protein, MyD88, has helped us understand precisely
how the initial signal is transmitted. Called Pococurante,
this mutation has revealed the location of the receptor-adapter interface, and molecular docking studies,
performed in collaboration with I.A. Wilson and A.J.
Olson, Department of Molecular Biology, have suggested exactly how the receptor and adapter subunits
fit together (Fig. 2).
THE SCRIPPS RESEARCH INSTITUTE
105
Beutler, B. Microbial pathogenesis and the discovery of Toll-like receptor function.
In: Vaccine Adjuvants: Immunological and Clinical Principles. Hackett, C.J., Harn,
D.A., Jr. (Eds.). Humana Press, Totowa, NJ, 2005, p. 1.
Beutler, B. The Toll-like receptors. In: Genetic Susceptibility to Infection. Kaslow,
R.L., McNicholl, J., Hill, A.V.S. (Eds.). Oxford University Press, New York, in press.
Beutler, B. The Toll-like receptors: analysis by forward genetic methods. Immunogenetics 57:385, 2005.
Beutler, B., Casanova, J.-L. New frontiers in immunology. EMBO Rep. 6:620, 2005.
Beutler, B., Georgel, P., Rutschmann, S., Jiang, Z., Croker, B., Crozat, K. Genetic
analysis of innate resistance to mouse cytomegalovirus (MCMV). Brief. Funct.
Genomic Proteomic 4:203, 2005.
Beutler, B., Hoebe, K., Georgel, P., Tabeta, K., Du, X. Genetic analysis of innate
immunity: identification and function of the TIR adapter proteins. Adv. Exp. Med.
Biol. 560:29, 2005.
Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X.,
Hoebe, K. Genetic analysis of host resistance: Toll-like receptor signaling and
immunity at large. Annu. Rev. Immunol. 24:353, 2006.
Crozat, K., Georgel, P., Rutschmann, S., Mann, N., Du, X., Hoebe, K., Beutler, B.
Analysis of the MCMV resistome by ENU mutagenesis. Mamm. Genome 17:398,
2006.
Du, X., Tabeta, K., Mann, N., Crozat, K., Mudd, S., Beutler, B. An essential role
for Rxrα in development of Th2 responses. Eur. J. Immunol. 35:3414, 2005.
Fischer, H., Yamamoto, M., Akira, S., Beutler, B., Svanborg, C. Mechanism of
pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors
and adaptor protein selection. Eur. J. Immunol. 36:267, 2006.
Hoebe, K., Beutler, B. TLRs as bacterial sensors. In: Toll-like Receptors in Inflammation. O’Neil, L.A.J., Brint, E. (Eds.). Birkhauser, New York, 2006, p. 000. Progress in Inflammation Research, Parnham, M.J. (Series Ed.).
Hoebe, K., Beutler, B. TRAF3: a new component of the TLR-signaling apparatus.
Trends Mol. Med. 12:187, 2006.
Hoebe, K., Jiang, Z., Georgel, P., Tabeta, K., Janssen, E., Du, X., Beutler, B. TLR
signaling pathways: opportunities for activation and blockade in pursuit of therapy.
Curr. Pharm. Des., in press.
Hoebe, K., Jiang, Z., Tabeta, K., Du, X., Georgel, P., Crozat, K., Beutler, B.
Genetic analysis of innate immunity. Adv. Immunol. 91:175, 2006.
Huber, M., Kalis, C., Keck, S., Jiang, Z., Georgel, P., Du, X., Shamel, L., Sovath,
S., Mudd, S., Beutler, B., Galanos, C., Freudenberg, M.A. R-form LPS, the master
key to the activation of TLR4/MD2 positive cells. Eur. J. Immunol. 36:701, 2006.
F i g . 2 . The junction between a Toll-like receptor and its cytoplasmic adapter protein. Only the so-called TIR domains, which are
involved in binding, are shown.
These few examples illustrate the power of the
classical genetic approach—which proceeds from phenomenon to phenotype to gene—in analyzing immunity. However, the same approach also sheds light on
many other aspects of biology. Some of the mutations
that we have produced answer questions in the realm
of behavioral neuroscience, metabolism, and development and raise new questions in turn.
PUBLICATIONS
Beutler, B. Innate Immunity. In: Williams Hematology, 7th ed. Lichtman, M.A., et
al. (Eds.). McGraw-Hill, New York, 2005, p. 231.
Janssen, E., Tabeta, K., Barnes, M.J., Rutschmann, S., McBride, S., Bahjat,
K.S., Schoenberger, S.P., Theofilopoulos, A.N., Beutler, B., Hoebe, K. Efficient T
cell activation via a Toll-Interleukin 1 receptor-independent pathway. Immunity.
24:787, 2006.
Jiang, Z., Georgel, P., Li, C., Choe, J., Crozat, K., Rutschmann, S., Du, X., Bigby, T.,
Mudd, S., Sovath, S., Wilson, I.A., Olson, A., Beutler, B. Details of Toll-like receptor:adapter interaction revealed by germ-line mutagenesis. Proc. Natl. Acad. Sci.
U. S. A. 103:10961, 2006.
Kim, K.I., Malakhova, O., Hoebe, K., Yan, M., Beutler, B., Zhang, D.-E. Enhanced
antibacterial potential in UBP43-deficient mice against Salmonella typhimurium
infection by up-regulating type I IFN signaling. J. Immunol. 175:847, 2005.
Nemazee, D., Gavin, A.L., Hoebe, K., Beutler, B. Immunology: Toll-like receptors
and antibody responses. Nature 441:E4, 2006.
Rutschmann, S., Hoebe, K., Zalevsky, J., Du, X., Mann, N., Dahiyat, B.I., Steed, P.,
Beutler, B. PanR1, a dominant negative missense allele of the gene encoding TNFα (Tnf), does not impair lymphoid development. J. Immunol. 176:7525, 2006.
Tabeta, K., Hoebe, K., Janssen, E.M., Du, X., Georgel, P., Crozat, K., Mudd, S.,
Mann, N., Sovath, S., Goode, J., Shamel, L., Herskovits, A.A., Portnoy, D.A.,
Cooke, M., Tarantino, L.M., Wiltshire, T., Steinberg, B.E., Grinstein, S., Beutler,
B. The Unc93b1 mutation 3d disrupts exogenous antigen presentation signaling
via Toll-like receptors 3, 7 and 9. Nat. Immunol. 7:156, 2006.
106 IMMUNOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
Wieland, C.W., Florquin, S., Maris, N.A., Hoebe, K., Beutler, B., Takeda, K.,
Akira, S., van der Poll, T. The MyD88-dependent, but not the MyD88-independent, pathway of TLR4 signaling is important in clearing nontypeable Haemophilus
influenzae from the mouse lung. J. Immunol. 175:6042, 2005.
Xia, C.H., Liu, H., Cheung, D., Cheng, C., Wang, E., Du, X., Beutler, B., Lo, W.K.
Gong, X. Diverse gap junctions modulate distinct mechanisms for fiber cell formation
during lens development and cataractogenesis. Development 133:2033, 2006.
Regulation of Cell Function by
Rho GTPases
G.M. Bokoch, J. Birkenfeld, V. Delorme, C. DerMardirossian,
A.M. DeCathelineau, D. Gianni, T. Huang, Y.-Y. Kao, J.-S. Kim,
P. Nalbant, K. Pestonjamasp, S.-H. Yoon, H. Zhang, T. Zhao,
B.P. Bohl, M. Crawford, B. Fowler, J.-Y. Seo, Z.-F. Chang*
F i g . 1 . Two-step activation mechanism for Rac GTPase–mediated
regulation of oxidant formation by the phagocyte NADPH oxidase.
* National Taiwan University, Taipei, Taiwan
ho GTPases control the assembly of the actin
and microtubule cytoskeletons, the production
of reactive oxygen species (ROS), and the activity of kinase cascades that mediate cell growth, death,
and motility. This spectrum of activities makes Rho
GTPases key components of such physiologic and pathologic processes as tumor growth and metastasis, wound
healing, neuronal connectivity, inflammatory responses,
and development. We use cellular, molecular, biophysical, and biochemical approaches to understand how
the activities of Rho GTPases are regulated, to identify
the proteins they interact with to control cell function,
and to ascertain how these regulatory processes are
abnormal in various disease states.
R
RHO GTPases AND HUMAN LEUKOCYTES
We previously established that the GTPase Rac2
regulates the formation of ROS that are used by human
phagocytic leukocytes for microbial killing and that result
in inflammatory responses. Our discovery of a functional interaction between Rac2 and cytochrome b, a
component of the membrane-bound NADPH oxidase,
independent of p67 phox , led us to propose a 2-step
mechanism for regulation of electron transfer to form
superoxide (Fig. 1). We are mapping the binding site
for Rac2 on cytochrome b to investigate the molecular
basis for regulation of ROS production by Rac2. In addition to their role in innate immunity, NADPH oxidases
participate in intracellular signaling. Regulation of nonphagocytic NADPH oxidases is largely not understood,
but we are investigating their modulation by kinase
pathways that phosphorylate regulatory components of
the oxidases.
We are using live-cell imaging in combination with
fluorescent methods to determine the spatial and tem-
poral localization of Rho GTPase activation. We are
beginning to determine the molecular signals that govern the chemotactic responses of human leukocytes.
Recently, we described the ability of Rac1 signaling in
neutrophils to stimulate RhoA activation at the rear of
cells. Such Rho GTPase cross talk promotes the development of the stable cell polarity necessary to maintain directionality of chemotaxis during inflammatory
responses. Studies of the dynamics of Cdc42 activation
during neutrophil chemotaxis are ongoing.
R E G U L AT I O N O F I N N AT E I M M U N I T Y B Y A N T H R A X
TOXINS
Bacillis anthracis inhibits the function of immune
cells by generating lethal toxin and edema toxin. As
part of a program grant funded by the Centers for Disease Control and Prevention, we are investigating the
molecular basis for the suppressive effects of the anthrax
toxins on the function of human leukocytes. We have
established that anthrax edema toxin and lethal toxin
effectively block the ability of chemoattractant receptors to stimulate the production of ROS by human neutrophils. The molecular basis for such inhibition is
currently under investigation. A requirement for Rho
GTPases in the uptake and action of anthrax toxins in
macrophages is also under study (Fig. 2).
C Y T O S K E L E TA L R E G U L AT I O N B Y R H O G T P a s e s
The p21-activated kinases (PAKs) are Rac and
Cdc42 effectors that serve as important mediators of
chemotaxis, wound healing, tumor metastasis, neurite
outgrowth, antigen presentation, and other processes
dependent on cytoskeletal polarization. In collaborative studies with G. Danuser and C. Waterman-Storer,
Department of Cell Biology, we are using quantitative
fluorescent speckle microscopy to investigate the regu-
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2006
F i g . 2 . Rho GTPase regulation of the action of anthrax lethal
toxin. Anthrax toxin is a ternary complex consisting of 1 binding
subunit, protective antigen (PA), and 2 enzymatic subunits, edema
factor (EF) or lethal factor (LF). Full-length PA (PA83) binds to
receptors on the cell surface and is cleaved by a furinlike protease
to its active form (PA63). Active PA oligimerizes, driving receptor
aggregation and internalization by endocytosis. During normal maturation and acidification of the endosomes, PA forms a channel
through which EF and LF are transported from the endosomal compartment and into the cytoplasm to act on their respective effectors. Rho GTPases may act to regulate endocytosis, endosomal
maturation, and toxin escape or activity. Figure courtesy of Aimee
DeCathelineau.
lation of leading-edge actin dynamics by PAK1 downstream of Rac GTPase. We found that PAK1 plays an
important role in coupling cell-edge protrusion mechanics to upstream signaling events and downstream motility.
The phosphorylation of cofilin, which depolymerizes
and severs actin, by PAK1 acting through LIM kinase
is an important regulatory point in cell motility. Using
a biochemical screen, we identified a unique cofilin
phosphatase, termed chronophin, that regulates stimulus-dependent activation of cofilin. Using small interfering RNA to reduce the expression of chronophin, we
discovered that this phosphatase is involved in the control of cytokinesis during cell division. Chronophin is
implicated in the formation of aneuploid cancers; it is
overexpressed in such tumors and is an autoantigen in
patients with cancer. Our recent data indicate that this
unique regulatory phosphatase orchestrates actin dynamics at the leading edge by modulating cofilin activity,
thereby increasing cancer cell motility stimulated by
epidermal growth factor. We have also linked chronophin
to cytoskeletal changes initiated during cellular energy
(ATP) depletion induced by processes such as ischemia.
THE SCRIPPS RESEARCH INSTITUTE
107
GDP dissociation inhibitors are critical regulators
of Rho GTPase function. They have been linked to kidney
disease and to the ability of cancer cells to metastasize.
We found that the interaction of GDP dissociation inhibitors with Rho GTPases is regulated by phosphorylations
initiated through various signaling pathways. Indeed,
tyrosine phosphorylation may disrupt the regulatory
capability of the inhibitors to promote cell transformation and metastasis.
Cell division also requires highly regulated actinmyosin-microtubule dynamics. We established that cross
talk between the actin and microtubule cytoskeletons
involving Rho regulation occurs via physical sequestration of the Rho guanine nucleotide exchange factor H1
(GEF-H1) by microtubules. GEF-H1 serves as a link
between mitotic spindle microtubules and the initiation
of Rho-dependent formation of cleavage furrows in dividing cells (Fig. 3). GEF-H1 activity is also controlled by
F i g . 3 . Immunofluorescent images show colocalization of endog-
enous GEF-H1 with microtubules (tubulin) in the mitotic spindle.
cell cycle–dependent kinases. Detailed analysis of the
function of GEF-H1 in cell division and motility is under
way. Of interest, GEF-H1 is abundant in blood cells
and is downregulated by recently developed drugs that
inhibit chronic leukemias.
PUBLICATIONS
Belvindrah, R., Nalbant, P., Ding, S., Wu, C., Bokoch, G.M., Müller, U. Integrinlinked kinase regulates Bergmann glial differentiation during cerebellar development. Mol. Cell. Neurosci. 33:109, 2006.
Birukova, A.A., Adyshev, D., Gorshkov, B., Bokoch, G.M., Birukov, K.G., Verin,
A.D. GEF-H1 is involved in agonist-induced human pulmonary endothelial barrier
dysfunction. Am. J. Physiol. Lung Cell Mol. Physiol. 290:L540, 2006.
Bokoch, G.M., Zhao, T. Regulation of the phagocyte NADPH oxidase by Rac
GTPase. Antioxid. Redox. Signal. 8:1533, 2006.
Chang, Y.-C., Lee, H.-H., Chen, Y.-J., Bokoch, G.M., Chang, Z.-F. Contribution of
guanine exchange factor H1 in phorbol ester-induced apoptosis. Cell Death Differ.,
in press.
Crawford, M., Aylott, C., Bourdeau, R.W., Bokoch, G.M. Bacillus anthracis toxins
inhibit human neutrophil NADPH oxidase activity. J. Immunol. 176:7557, 2006.
DeCathelineau, A.M., Bokoch, G.M. Peptide inhibitors MAP the way towards fighting anthrax. Biochem. J. 395:e1, 2006.
DerMardirossian, C., Bokoch, G.M. Phosphorylation of RhoGDI by p21-activated
kinase 1. Methods Enzymol. 406:80, 2006.
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2006
DerMardirossian, C., Rocklin, G., Seo, J.Y., Bokoch, G.M. Phosphorylation of
RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling. Mol.
Biol. Cell, in press.
Dong, X., Mo, Z., Bokoch, G.M., Guo, C., Li, Z., Wu, D. P-Rex1 is a primary Rac2
guanine nucleotide exchange factor in mouse neutrophils. Curr. Biol. 15:1874,
2005.
Huang, T.Y., DerMardirossian, C., Bokoch, G.M. Cofilin phosphatases and regulation of actin dynamics. Curr. Opin. Cell Biol. 18:26, 2006.
Pestonjamasp, K.N., Forster, C., Sun, C., Gardiner, E.M., Bohl, B., Weiner, O.,
Bokoch, G.M., Glogauer, M. Rac1 links leading edge and uropod events through
Rho and myosin activation during chemotaxis. Blood. 108:2814, 2006.
Stofega, M., DerMardirossian, C., Bokoch, G.M. Affinity-based assay of Rho
GTPase activation. Methods Mol. Biol. 332:269, 2006.
Human Antibodies and Design
of a Vaccine to HIV Type 1
M.B. Zwick, R.A. Pantophlet, R.O. Aguilar-Sino,
R.D. Astronomo, D.R. Bowley, H. Donners, A.K. Gakhal,
E. Giang, L. Hangartner, A.J. Hessell, R.C. Jensen, M. Law,
J.D. Nelson, S. Pollock, E.M. Scherer, M. Wang, R.A. Dwek,*
D. Calarese, R.M. Cardoso, R.L. Stanfield, I.A. Wilson,
D.R. Burton
* Oxford Glycobiology Institute, Oxford, England
IV type 1 (HIV-1) is a scourge on humanity.
Nearly 40 million persons are infected with the
virus, and about 20 million have died of AIDS.
It is widely recognized that a vaccine most likely is the
best way to control HIV infection worldwide. All current antiviral vaccines elicit antibody responses that
are thought to be crucial to the efficacy of the vaccines. We wish to understand antibody responses to
HIV in humans and to design vaccines that will elicit
protective responses to the virus.
We used phage display technology to generate panels of human monoclonal antibodies to HIV. We are
examining human antibody responses to the virus and
the antiviral activities of these antibodies. In particular, we generated a human monoclonal antibody, b12,
that neutralizes a broad array of different strains of HIV.
The existence of this antibody indicates that some features of HIV are conserved and are attractive targets
for vaccines. Further, b12 and a few monoclonal antibodies with similar qualities are powerful tools for
exploring antibody activity against HIV-1.
Among the first questions we have tackled were the
following: Can antibodies protect against HIV-1 infection, and, if so, under what conditions? On the basis
H
THE SCRIPPS RESEARCH INSTITUTE
of passive transfer studies in a number of animal model
systems, the answer is clearly yes. Complete protection is possible at serum titers of neutralizing antibody
greater than about 1:100, although lower titers can
provide benefit in terms of lowered or delayed viremia.
We also showed that topically applied antibody can
protect monkeys against vaginal challenge with virus.
In addition, passive transfer studies with engineered
antibodies in macaques suggest that antibody effector
functions, as well as classical neutralization, may be
important in protection against HIV.
Another major issue is the best method for eliciting protective neutralizing antibodies. Accumulated
evidence suggests that protective neutralizing antibodies are those antibodies that bind avidly to the envelope trimer on the surface of HIV-1 virions. However,
such antibodies, particularly those to conserved regions
of the envelope that are most important for vaccines,
are difficult to elicit. Apparently the envelope trimer,
which is composed of 2 glycoproteins, gp120 and
gp41, has low antigenicity and immunogenicity. Several strategies to circumvent these problems are being
investigated. One strategy is to study the interaction of
the neutralizing antibodies with envelope glycoprotein
at the molecular level and then use the knowledge
gained to design antigens capable of eliciting the relevant antibodies. In these studies, we are collaborating
with I.A. Wilson, Department of Molecular Biology. We
are also working closely with P. Dawson, Department
of Cell Biology, to design peptide immunogens and with
C.-H. Wong, Department of Chemistry, and R. Dwek,
Oxford Glycobiology Institute, to design and select carbohydrate immunogens.
Finally, we are exploring the specificities of antibodies from those rare humans who make antibodies
that neutralize a broad array of different strains of HIV.
We have evidence that a number of specificities are
involved, and we are attempting to describe these. We
are generating human monoclonal antibodies by using
not only phage display but also yeast display and the
rescue of memory B cells.
PUBLICATIONS
Braciale, T.J., Hahn, Y.S., Burton, D.R. Adaptive immune responses to viral infection. In: Fields Virology, 5th ed. Knipe, D.M., et al. (Eds.). Lippincott Williams &
Wilkins, Philadelphia, in press.
Brunel, F.M., Zwick, M.B., Cardoso, R.M.F., Nelson, J.D., Wilson, I.A., Burton, D.R.,
Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680, 2006.
Burton, D.R., Stanfield, R.L.. Wilson, I.A. Antibody versus HIV in a clash of evolutionary titans. Proc. Natl. Acad. Sci. U. S. A. 102:14943, 2005.
IMMUNOLOGY
2006
Calarese, D.A., Lee, H.-K., Huang, C.-Y, Best, M.D., Astronomo, R.D., Stanfield,
R.L., Katinger, H., Burton, D.R., Wong, C.-H, Wilson, I.A. Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12. Proc.
Natl. Acad. Sci. U. S. A. 102:13372, 2005.
Calarese, D., Scanlan, C., Lee, H.-K., Rudd, P., Wong, C.-H., Dwek, R.A., Burton, D.R., Wilson, I.A. Towards a carbohydrate-based HIV-1 vaccine. In: Carbohydrate Drug Design. Klyosov, A.A., Witczak, Z.J., Platt, D. (Eds.). Oxford University
Press, New York, 2006, p. 161. American Chemical Society Symposium Series.
Delves, P.J., Martin, S.J., Burton, D.R., Roitt, I.M. Roitt’s Essential Immunology,
11th ed. Blackwell Publishing, Oxford, England, in press.
Koff, W.C., Johnson, P.R., Watkins, D.I., Burton, D.R., Lifson, J.D., Hasenkrug,
K.J., McDermott, A.B., Schultz, A., Zamb, T.J., Boyle, R., Desrosiers, R.C. HIV
vaccine design: insights from live attenuated SIV vaccines. Nat. Immunol. 7:19,
2006.
Law, M., Sanna, P.P., Burton, D.R. Viral subversion of humoral immune responses.
In: Microbial Subversion of Immunity: Current Topics. Lachmann, P.J., Oldstone,
M.B.A. (Eds.). Caister Academic Press, Norfolk, England, 2006, p. 177.
Lindenbach, B.D., Evans, M.J., Syder, A.J., Wolk, B., Tellinghuisen, T.L., Liu,
C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., Rice, C.M.
Complete replication of hepatitis C virus in cell culture. Science 309:623, 2005.
Moore, P.L., Crooks, E.T., Porter, L., Zhu, P., Cayanan, C.S., Grise, H., Corcoran,
P., Zwick, M.B., Franti, M., Morris, L., Roux, K.H., Burton, D.R., Binley, J.M.
Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1. J. Virol. 80:2515, 2006.
O’Connor, D.H., Burton, D.R. Immune responses and HIV: a little order from the
chaos. J. Exp. Med. 203:501, 2006.
THE SCRIPPS RESEARCH INSTITUTE
109
deadliest human pathogens. Results indicate strong
cross-reactivity and immunogenicity of the different
glycoproteins. This finding supports the hypothesis
that some of the soluble forms of the glycoproteins act
as decoys.
We have also been investigating the effects of a
human monoclonal antibody that neutralizes Ebola
virus; the antibody was isolated from a patient who
was infected with the virus in the Democratic Republic of Congo and who recovered. Previously, we showed
that the antibody protects guinea pigs against challenge with Ebola virus. However, studies done in collaboration with scientists at the National Institute of
Allergy and Infectious Diseases, indicate that the antibody does not protect monkeys and indeed appears to
offer little benefit even when given at high doses. Surprisingly, viral replication apparently can proceed unhindered in the tissues of the monkeys even in the presence
of high serum concentrations of antibody. We are
attempting to understand this phenomenon and reconcile it with the ability of certain vaccines to protect
against challenge with Ebola virus.
Pantophlet, R., Burton, D.R. GP120: target for neutralizing HIV-1 antibodies.
Annu. Rev. Immunol. 24:739, 2006.
Selvarajah, S., Puffer, B., Pantophlet, R., Law, M., Doms, R.W., Burton, D.R.
Comparing antigenicity and immunogenicity of engineered gp120. J. Virol.
79:12148, 2005.
Venturini, S., Allicotti, G., Zhao, Y., Simon, R., Burton, D.R., Pinilla. C., Poignard, P.
Identification of peptides from human pathogens able to cross-activate an HIV-1gag-specific CD4+ T cell clone. Eur. J. Immunol. 36:27, 2006.
Yuste, E., Sanford, H.B., Carmondy, J., Bixby, J., Little, S., Zwick, M.B., Greenough, T., Burton, D.R., Richman, D.D., Desrosiers, R.C., Johnson, W.E. Simian
immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive
plasma. J. Virol. 80:3030, 2006.
Zhang, M.X., Bohlman, M.C., Itatani, C., Burton, D.R., Parren, P.W.H.I., St. Jeor,
S.C., Kozel, T.R. Human recombinant antimannan immunoglobulin G1 antibody
confers resistance to hematogenously disseminated candidiasis in mice. Infect.
Immun. 74:362, 2006.
Zhong, J., Gastaminza, P., Cheng, G., Kapadia, S., Kato, T., Burton, D.R.,
Wieland, S.F., Uprichard, S.L., Wakita, T., Chisari, F.V. Robust hepatitis C virus
infection in vitro. Proc. Natl. Acad. Sci. U. S. A. 102:9294, 2005.
Antibodies and Emerging Viruses
W.B. Oswald, E.O. Saphire, N.L. Sullivan,* P.B. Jahrling,*
P.W.H.I. Parren,** D.R. Burton
* National Institute of Allergy and Infectious Diseases, Bethesda, Maryland
** Genmab, B. V., Utrecht, the Netherlands
W
e are interested in determining the immunogenicity of soluble vs surface glycoproteins
of Ebola virus, a filovirus that is one of the
Toll-like Receptors and
Inflammation in Atherosclerosis
A.E. Mullick, C. Flood, N.J. Hime, M.R. Richards,
D.T. Valenta, R.J. Petrovan, P.S. Tobias, L.K. Curtiss
he ability of Toll-like receptors (TLRs) to detect a
spectrum of pathogen-derived molecules defines
the importance of the receptors in innate immunity and provides a mechanistic link between infection
and disease. Atherosclerosis is a chronic inflammatory
disease in which immune and metabolic factors interact
to initiate and propagate arterial lesions. An understanding of TLRs in atherosclerosis could clarify the etiology
of this complex process. Furthermore, the existence of
host-derived endogenous ligands for TLRs may implicate
involvement of the receptors in disease mechanisms
beyond innate immunity, such as homeostatic mechanisms to resolve injury. Our studies of atherosclerosissusceptible mouse models highlight TLR involvement
in the process of this vascular disease.
Distinguishing between local and systemic factors
that contribute to the development of atherosclerotic
lesions via activation of TLRs induced by endogenous
TLR ligands is formidable. However, bone marrow trans-
T
110 IMMUNOLOGY
2006
plantation allows alteration of gene expression for specific types of cells found in atherosclerotic lesions. Such
chimeric mice can be produced to express or lack a single gene in key effector cells, including macrophagederived foam cells, involved in the development of
atherosclerosis. Furthermore, by using reverse bone
marrow transplantation, chimeric mice can be produced
that express the gene solely in bone marrow–derived
cells. These procedures enable us to study the effects
of gene expression specific to bone marrow cells during disease progression.
Using bone marrow transplantation, we generated
atherosclerosis-susceptible chimeric mice with bone
marrow cells that lacked the gene for either TLR2 or
TLR4. Lesions containing macrophage-derived foam
cells lacking TLR2 or TLR4 developed in these animals.
Surprisingly, when chimeric mice that lacked TLR2 or
TLR4 solely in bone marrow–derived cells and that also
lacked receptors for low-density lipoprotein (LDLr–/–)
were fed an atherogenic diet for 4 months, no changes
occurred in the size of lesions in either the aortic sinus
or the aorta.
To rule out the possibility of a role for TLR2 expression by bone marrow–derived cells, we used reverse
bone marrow transplantation. Chimeras with TLR2
expression confined to bone marrow–derived cells
were produced by using female LDLr–/–TLR2–/– mice
as recipients and wild-type or TLR2–/– mice as donors.
Again, no differences in the severity of atherosclerotic
lesions were observed. All LDLr–/–TLR2–/– mice that
received bone marrow transplants had smaller lesions
than did LDLr–/– recipient mice, regardless of the donor
TLR2 genotype. Thus, a TLR2 deficiency in cells not
derived from bone marrow led to less disease. Collectively, these data suggest that endogenous ligand signaling via TLR2 and/or TLR4 affected disease outcome
and that TLR2 and/or TLR4 expression on cells not
derived from bone marrow mediated the effects of TLR2
and/or TLR4 activated by endogenous TLR ligands.
To determine the atherosclerotic effect of systemic
activation of TLR2, we administered the exogenous
TLR2 ligand synthetic N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3), a lipopeptide
that induces signaling in cells of the immune system
through TLR2, to hypercholesterolemic LDLr –/– mice.
After 10 weeks of high-fat feeding and weekly intraperitoneal injections of Pam3, the mice had a striking
dose-dependent increase in lesion severity. Compared
with lesions in control mice not given Pam3, the areas
THE SCRIPPS RESEARCH INSTITUTE
of aortic sinus and en face aortic lesions were increased
82% and more than 400%, respectively. In similarly
treated LDL–/– mice that also lacked the gene for TLR2,
no increase in lesion severity occurred.
To dissect the proatherosclerotic mechanism of
TLR2 activation, we used bone marrow transplantation to produce TLR2-deficient chimeras. LDLr–/– mice
were reconstituted with bone marrow cells from wildtype or TLR2-deficient mice. Using the same method
of TLR2 activation as before, we found a proatherosclerotic effect of Pam3 only in mice reconstituted with
wild-type bone marrow. These mice had increases in
lesion severity of 89% and 240% in the aortic sinus
and the aorta, respectively.
Hence, although both groups of mice had intact
TLR2 signaling in cells not derived from bone marrow,
only those mice that expressed TLR2 in bone marrow–
derived cells, such as monocyte-derived macrophages,
had increased atherosclerosis when activated with the
exogenous agonist. Additionally, the TLR2-induced acceleration of lesion development occurred with an unusual
pattern of atherosclerosis, with profuse abdominal
lesions. Subsequent experiments with intravenous
administration of Pam3 did not reproduce this peculiar
development of abdominal lesions, thereby implicating
a role of peritoneal inflammatory mediators in the effects
of Pam3 injected intraperitoneally.
PUBLICATIONS
Boisvert, W.A., Rose, D.M., Boullier, A., Quehenberger, Q., Sydlaske, A., Johnson, K.A., Curtiss, L.K., Terkeltaub, R. Leukocyte transglutaminase 2 expression
limits atherosclerotic lesion size. Arterioscler. Thromb. Vasc. Biol. 26:563, 2006.
Boisvert, W.A., Rose, D.M., Johnson, K.A., Fuentes, M.E., Lira, S.A., Curtiss,
L.K., Terkeltaub, R.A. Up-regulated expression of the CXCR2 ligand KC/GROα in
atherosclerotic lesions plays a central role in macrophage accumulation and lesion
progression. Am. J. Pathol. 168:1385 2006.
Curtiss, L.K. Is two out of three enough for ABCG1? Arterioscler. Thromb. Vasc.
Biol. 26:2175, 2006.
Curtiss, L.K., Valenta, D.T., Hime, N.J., Rye, K.-A. What is so special about apolipoprotein AI in reverse cholesterol transport? Arterioscler. Thromb. Vasc. Biol.
26:12, 2006.
Mullick, A.E., Tobias, P.S., Curtiss, L.K. Modulation of atherosclerosis in mice by
Toll-like receptor 2. J. Clin. Invest. 115:3149, 2005.
Mullick, A.E., Tobias, P.S., Curtiss, L.K. Toll-like receptors and atherosclerosis: key
contributors in disease and health? Immunol. Res. 34:193, 2006.
Tilley, R.E., Pedersen, B., Pawlinski, R., Sato, Y., Erlich, J.H., Shen, Y., Day, S.,
Huang, Y., Eitzman D.T., Boisvert, W.A., Curtiss, L.K., Fay, W.P., Mackman, N.
Atherosclerosis in mice is not affected by a reduction in tissue factor expression.
Arterioscler. Thromb. Vasc. Biol. 26:555, 2006.
Valenta, D.T., Bulgrien, J.J., Banka, C.L., Curtiss, L.K. Overexpression of human
apoAI transgene provides long-term atheroprotection in LDL receptor-deficient
mice. Atherosclerosis, in press.
IMMUNOLOGY
2006
Valenta, D.T., Ogier, N., Bradshaw, G., Black, A.S., Bonnet, D.J., Lagrost, L.,
Curtiss, L.K., Desrumaux, C.M. Atheroprotective potential of macrophage-derived
phospholipid transfer protein in low-density lipoprotein receptor-deficient mice is
overcome by apolipoprotein AI overexpression. Arterioscler. Thromb. Vasc. Biol.
26:1572, 2006.
Enhanced Tissue Factor
Initiation of the Coagulation
and Thrombogenic Cascades
by α2-Macroglobulin
G. Bhattacharjee, S. Arandjelovic, N. Mackman, W. Ruf,
S.L. Gonias, T.S. Edgington
he broad spectrum poteinase inhibitor α2-Macroglobulin differs from all 4 mechanistic classes of
proteinase inhibitors. It inhibits coagulation proteinases such as thrombin and factor Xa and fibrinolytic proteinases such as plasmin. a2-Macroglobulin is
also a carrier of growth factors and may regulate their
function. Receptors for a2-macroglobulin include the
low-density lipoprotein receptor-related protein (LRP)
and 78-kD glucose-regulated protein (Grp78), a highaffinity receptor. In its native conformation, a2-macroglobulin is present in plasma at high concentrations.
Upon binding to serum proteinases or small primary
amines, a2-macroglobulin is converted to the receptorrecognized form, which binds to LRP. Complexes composed of LRP, a2-macroglobulin, and proteinases are
internalized by cells.
Previously, we found that Grp78 binds tissue factor and inhibits its function. Recently, we discovered
that the receptor-recognized form of α2-macroglobulin
enhances initiation of the coagulation and thrombotic
cascades by tissue factor, generation of factor Xa, and
expression of the gene for tissue factor. This enhancement depends on binding of the receptor-recognized
form of α2-macroglobulin to LRP, but not to Grp78,
because receptor-associated protein blocks the
response. The effects of α2-macroglobulin on tissue
factor–dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but the
effects also occur in THP-1 monocytic cells. A mutant
of the receptor-recognized form of α2-macroglobulin
that does not bind LRP has no effect on tissue factor
activity or gene expression.
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THE SCRIPPS RESEARCH INSTITUTE
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Infarctive Eradication of Tumors
by Selective Tumor Vascular
Thrombosis
A. EL-Sheikh, P. Borgstrom, G. Bhattacharjee, M. Belting,
T.S. Edgington
hen fused to the extracellular domain of tissue factor (TFt), the exon-encoded polypeptide of the heparin-binding domain (HBDt)
of vascular endothelial cell growth factor localizes selectively to endothelial surfaces of intratumoral microvasculature. Tissue factor is the initiating receptor and
requisite cofactor for initiation of the thrombogenic
cascade. In tumor-bearing mice, intravenous infusion
of the fused domains, HBDt-TFt, results in selective
rapid occlusive thrombosis of tumor microvasculature.
We found that infusion of an optimal combination of
HBDt-TFt and its ligand factor VIIa in tumor-bearing
animals results in infarctive eradication of tumors and
often is curative.
Binding studies and confocal microscopy indicated
that the target for HBDt-TFt is a trimolecular complex
of chondroitin C sulfate proteoglycan, neuropilin-1, and
vascular endothelial cell growth factor receptor 2, which
is overexpressed in highly angiogenic sites of the tumor
microenvironment. HBDt-TFt also colocalized with the
trimolecular receptor complex in endothelial sprouts
from tumor tissues, and binding inhibited growth of
sprouts. In vitro, HBDt had the highest affinity for
chondroitin 6 sulfate. We are evaluating the potential
of HBDt-TFt as a therapeutic agent.
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Legumain Expression and
Cell-Surface Translocation
as a Catalytic Target for
Prodrug Delivery and Therapy
W. Wu, Y. Luo, C. Sun, Y. Liu, P. Kuo, J. Varga, R. Xiang,
R.A. Reisfeld, K.D. Janda, T.S. Edgington, C. Liu
egumain, a novel and highly specific asparaginyl
endopeptidase of the cysteine protease family, is
conserved as the only proteinase of this specificity
from plant to humans. It is highly expressed by endo-
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112 IMMUNOLOGY
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thelial, neoplastic, and stromal cells in most tumors, in
humans and animals. We developed a legumain-activated,
cell-impermeable preprodrug that incorporates doxorubicin as the cytotoxic element, although a wide variety
of cytotoxic molecules can be incorporated to make the
molecules nontoxic. Designated LEG3, the preprodrug
targets extracellular legumain on tumor microvasculature, tumor stroma, and neoplastic cells. Upon binding, it is catalytically converted to a cell-permeable
prodrug, which is then converted to doxorubicin inside
the cells, where it results in tumor-specific killing.
Pharmacokinetics and tissue distribution of LEG3
compared with doxorubicin alone confirmed selective
local prodrug activation leading to 30- to 100-fold
increases of doxorubicin in tumor cell nuclei but with
100-fold reductions of doxorubicin in normal tissues.
Importantly, this prodrug completely inhibited growth
of a variety of multidrug-resistant human tumors in
mice, produced tumor eradication, and significantly
extended survival without any evidence of myelosuppression or toxic cardiac effects.
THE SCRIPPS RESEARCH INSTITUTE
PUBLICATIONS
Bhattacharjee, G., Ahamed, J., Pedersen, B., EL-Sheikh, A., Mackman, N., Ruf,
W., Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the
coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol.
25:1737, 2005.
EL-Sheikh, A., Borgstrom, P., Bhattacharjee, G., Belting, M., Edgington, T.S. A
selective tumor microvasculature thrombogen that targets a novel receptor complex
in the tumor angiogenic microenvironment. Cancer Res. 65:11109, 2005.
Lin, R., Maeda, S., Liu, C., Karin, M., Edgington, T.S. A large noncoding RNA is a
marker for murine hepatocellular carcinoma and a spectrum of human carcinomas.
Oncogene, in press.
Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R.,
Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation
to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer
Res. 66:970, 2005.
Control of V(D)J Recombination
and Formation of the Antibody
Repertoire in Normal and
Autoimmune Mice
A.J. Feeney, C.R. Espinoza, J. Lamoureux, M. Cherrier,
C.R. Xu, J. Carey, S. Salerno, L. Watson
Association of a Large
Noncoding RNA Riboregulator
With Neoplasia
R. Lin, S. Maeda, C. Liu, M. Karin, T.S. Edgington
arge noncoding RNAs account for an unexpectedly
large proportion of the transcribed genome but
have been sparsely analyzed. We found a novel
murine gene encoding a 7000-base mRNA-like transcript. This gene and mammalian counterparts lack
credible or conserved open reading frames, characteristics of large noncoding RNAs.
In murine hepatocellular carcinomas induced with
a procarcinogen and in all samples of human hepatocellular carcinomas analyzed, expression of this gene
was enhanced. Compared with normal liver, hepatocellular carcinomas had a 6- to 7-fold increase of this
noncoding RNA. Thus, this large noncoding RNA is a new
marker for hepatocellular carcinoma. The RNA was also
significantly overexpressed in all 5 nonhepatocellular
human carcinomas analyzed. Expression was enhanced
in cells enriched at the mitotic stage, advancing this large
noncoding RNA as a generic marker for carcinomas
and suggesting a role for it as a riboregulator (regulatory RNA) in the molecular cell biology of neoplasia.
L
main focus of our laboratory is the molecular
analysis of factors that influence the composition of the antibody repertoire and elucidation
of the mechanisms that control the V(D)J rearrangement process. In each precursor B lymphocyte, a different set of V, D, and J genes recombine to form exons
for the light and heavy chains of the antibody molecule. Each locus has many V, D, and J genes, but the
gene segments are not used equally. One of our goals
is to understand the basis of this nonrandom use of
gene segments.
We previously showed that much of this bias occurs
because V genes undergo recombination with different
intrinsic frequencies due to differences in the recombinase signal sequence, the binding site for the recombinase, flanking each gene segment. The recombinase
signal sequence is composed of a relatively conserved
heptamer and nonamer flanking a “spacer” of conserved
length but only modestly conserved sequence. Few genes
have consensus sequences, however, and changes in this
natural variation in the recombinase signal sequence
can greatly affect recombination frequency in vitro and
in vivo.
In addition, other factors clearly influence recombination frequencies; currently we are focusing on the
role of transcription factors and chromatin modifications
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IMMUNOLOGY
2006
in controlling accessibility to V(D)J recombination and
recombination frequency. Genes in loci that are undergoing V(D)J recombination are often associated with
histones that are acetylated. We hypothesized that the
extent of histone modification affects the frequency
of recombination of individual genes, and indeed we
observed a positive correlation between the relative
rearrangement frequency of several individual genes
in vivo and the extent of acetylation of histones H3
and H4 associated with those genes, as assessed by
chromatin immunoprecipitation. Other modifications
associated with repressed or activated genes are now
being investigated.
We have also uncovered a novel role for the transcription factor Pax5 in promoting V(D)J rearrangement.
Pax5 is essential for B-cell development. In the absence
of Pax5, VH to DJH rearrangement is severely impaired.
We found Pax5 binding sites in the coding regions of
many V H genes. Furthermore in collaboration with
Z. Zhang and M. Cooper, University of Alabama, Birmingham, we showed that Pax5 binds to the recombinase proteins RAG1 and RAG2. Hence, we propose
that Pax5 may recruit RAG1 or RAG2 to the recombinase signal sequence or may stabilize the interaction
of the RAG complex with its binding site.
In other studies, we are examining the breakdown
of B-cell tolerance in autoimmunity. When precursor
B cells successfully recombine both heavy- and lightchain gene segments, they express a B-cell receptor for
the first time. If the receptor is autoreactive, then the
immature B cell normally continues to undergo lightchain V-J rearrangement until an innocuous receptor is
made. This process is termed receptor editing and is
an important checkpoint in B-cell tolerance. We have
evidence that this process is not functioning as efficiently
in lupus-prone mice as in nonautoimmune mice, and
we are investigating why this difference occurs. Such
misregulation of this key checkpoint could lead to the
release of autoreactive B cells into the periphery, where
they can become activated to secrete autoantibodies
and cause autoimmune disease.
PUBLICATIONS
Espinoza, C.R., Feeney, A.J. The extent of histone acetylation correlates with differential rearrangement frequency of individual VH genes in pro-B cells. J.
Immunol. 175:6668, 2005.
Espinoza, C.R., Feeney, A.J. Quantifying chromatin accessibility of individual gene
family members by combining ligation-mediated PCR with real-time PCR. Biotechniques 41:404, 2006.
Watson, L.C., Moffatt-Blue, C.S., MacDonald, R.Z., Kompfner, E., Aït-Azzouzene,
D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeney, A.J. Paucity of V-DD-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT–/– and TdT+/+ mice. J. Immunol. 172:1120, 2006.
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Zhang, Z., Espinoza, C.R., Yu, Z., Stephan, R., He, T., Williams, G.S., Burrows,
P.D., Hagman, J., Feeney, A.J., Cooper, M.D. Transcription factor Pax5 (BSAP)
transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin
genes. Nat. Immunol. 7:616, 2006.
Syndecans and HIV Type 1
Pathogenesis
M. Bobardt, U. Chatterji, S. Selvarajah, A. de Parseval,*
J.H. Elder,* P.A. Gallay
* Department of Molecular Biology, Scripps Research
he syndecans belong to the heparan sulfate family
of proteoglycans. In syndecans, the sulfation
pattern of the heparan sulfate chains dictates
the ligand specificity. HIV type 1 (HIV-1) has maximized
its use of syndecans. It uses them as receptors to facilitate infection of macrophages. It uses them as receptors on the endothelium to enhance endurance of the
virus in the hostile environment and to trans infect
circulating T cells. HIV-1 also exploits syndecans to
facilitate transport of the virus through the blood-brain
barrier and the genital epithelium. Thus, the interplay
between HIV-1 and syndecans may profoundly affect
HIV-1 pathogenesis.
Sexual transmission is the most common mode of
infection in the global HIV-1 epidemic. In the absence
of an effective vaccine, additional strategies to prevent
new HIV-1 infections are urgently needed. Mucosal
dendritic and Langerhans cells are the first cells that
HIV-1 encounters. These cells may thus play a crucial
role in HIV-1 transmission. Interactions between HIV-1
and dendritic or Langerhans cells are poorly understood.
Although researchers initially proposed that C-type
lectin receptors such as DC-SIGN, the mannose receptor, and langerin mediated contact between HIV-1 and
dendritic or Langerhans cells, blocking of CD4, CCR5,
and C-type lectin receptors only partly prevents HIV-1
capture by dendritic cells. This finding led researchers
to postulate the existence of unidentified HIV-1 receptors on dendritic cells.
We found that syndecan-3, which is normally poorly
expressed or even absent on many cell types and tissues, is highly expressed on both dendritic cells and
Langerhans cells. More importantly, we obtained evidence that syndecan-3 plays a key role in HIV-1 capture and trans infection of T cells by dendritic cells.
Because dendritic and Langerhans cells may be the
first Trojan horses that HIV-1 exploits to colonize
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114 IMMUNOLOGY
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humans, understanding the contribution of syndecan-3
in the capture and transfer of HIV-1 by dendritic cells
and Langerhans cells to T cells is imperative to develop
new therapies to prevent HIV-1 infections.
PUBLICATIONS
Binley, J.M., Ngo-Abdalla, S., Moore, P., Bobardt, M., Chatterji, U., Gallay, P.,
Burton, D.R., Wilson, I.A., Elder, J.H., de Parseval, A. Inhibition of HIV Env binding to cellular receptors by monoclonal antibody 2G12 as probed by Fc-tagged
gp120. Retrovirology. 3:39, 2006.
Bobardt, M.D., Chatterji, U., Selvarajah, S., Van der Schueren, B., David, G.,
Kahn, B., Gallay, P.A. Cell-free HIV-1 transcytosis through primary genital epithelial cells. J. Virol., in press.
de Parseval, A., Bobardt, M.D., Chatterji, A., Chatterji, U., Elder, J.H., David, G.,
Zolla-Pazner, S., Farzan, M., Lee, T.H., Gallay, P.A. A highly conserved arginine in
gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs. J.
Biol. Chem. 280:39493, 2005.
Saphire, A.C., Gallay, P.A., Bark, S.J. Proteomic analysis of human immunodeficiency virus using liquid chromatography/tandem mass spectrometry effectively distinguishes specific incorporated host proteins. J. Proteome Res. 5:530, 2006.
Innate Intracellular Immunity
and Infection With HIV Type 1
U. Chatterji, M. Bobardt, S. Selvarajah, P.A. Gallay
onhuman cells contain intracellular innate factors
that inhibit infection by HIV type 1 (HIV-1) by
targeting the incoming viral capsid core, which
makes up the shell that surrounds the viral genome. The
first intracellular primate restriction factor identified,
TRIM5α, is a member of the tripartite motif (TRIM)
family of proteins. However, the mechanism by which
TRIM5α blocks HIV-1 infection is unknown.
We asked if TRIM5α blocks HIV-1 by diverting the
incoming capsid core into an abortive degradation pathway. We compared the degradation status of incoming
cores in restrictive and nonrestrictive cells. We found
that monkey, but not human, TRIM5α provokes destruction of capsids immediately after HIV-1 entry. The
capsid degradation is specific because the integrity of
other abundant viral proteins is preserved. TRIM5α
directs capsids to a nonproteasome degradation pathway. Capsids delivered into the vesicular compartment
undergo similar degradation in restrictive and nonrestrictive cells. In contrast, capsids delivered into the
cytosol of nonrestrictive cells remain intact, whereas
capsids delivered into the cytosol of restrictive cells
are destroyed.
Our finding that TRIM5α specifically degrades
cytosolic capsids suggests that cytosolic capsids represent authentic infectious events, whereas vesicular
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THE SCRIPPS RESEARCH INSTITUTE
capsids represent abortive infectious events. Further
work is required to determine whether TRIM5α-mediated capsid degradation and capsid core uncoating
represent linked or distinct events.
An understanding of the nature of restrictions to
HIV-1 infection after the virus enters cells is critical
for several reasons. First, information on the viral and
cellular factors that modulate these processes will shed
light on the poorly understood series of events that
govern the fate of capsids after entry. Second, speciesspecific barriers to HIV-1 infection present obstacles
to the development of animal models for the study of
HIV-1 pathogenesis and treatment. Finally, an understanding of this critical part of the HIV-1 life cycle
may suggest approaches to intervene in transmission
of the virus or its spread within the host.
PUBLICATIONS
Barry, S.M., Melar, M., Gallay, P., Hope, T.J. Review of the twelfth West Coast
Retrovirus Meeting. Retrovirology 2:72, 2005.
Chatterji, U., Bobardt, M.D., Gaskill, P., Sheeter, D., Fox, H., Gallay, P.A. TRIM5α
accelerates degradation of cytosolic capsid associated with productive HIV-1 entry.
J. Biol. Chem., in press.
Chatterji, U., Bobardt, M.D., Stanfield, R., Ptak, R.G., Pallansch, L.A., Ward,
P.A., Jones, M.J., Stoddart, C.A., Scalfaro, P., Dumont, J.M., Besseghir, K.,
Rosenwirth, B., Gallay, P.A. Naturally occurring capsid substitutions render HIV-1
cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl
monkey cells. J. Biol. Chem. 280:40293, 2005.
Imaging Molecular Interactions
in T-Cell Development and
Activation
N.R.J. Gascoigne, J. Ampudia, G. Fu, C. Goergen,
K. Holmberg, H.-C. Hung, H.-O. Kim, C. Lotz, A. Munshi,
G. Sternik, S. Vallee, P. Yachi, M.A. Zal. T. Zal, N. Bosco,*
R. Ceredig,* M. Daniels,** E. Palmer**
* U548 INSERM, Grenoble, France
** University Hospital, Basel, Switzerland
IMAGING OF INTERACTIONS BETWEEN T-CELL
RECEPTORS AND CORECEPTORS IN T-CELL
A C T I VAT I O N
luorescence resonance energy transfer (FRET) is
a physical phenomenon that occurs at distances
less than 10 nm. Thus, FRET between fluorescent proteins, for example, between cyan and yellow
fluorescent proteins, attached to proteins of interest
can be used to investigate interactions between proteins in living cells.
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IMMUNOLOGY
2006
Using FRET, we showed that the coreceptor CD8
and the T-cell receptor (TCR) signal-transducing protein CD3ζ are recruited to the “immunologic synapse,”
where they interact when antigenic MHC-peptide complexes are presented to T cells. No FRET occurs when
weaker (e.g., TCR antagonist) ligands are used. We
showed previously that endogenous MHC-peptides aid
in recognizing antigenic MHC-peptide complexes. The
interaction between CD8 and endogenous MHC-peptides improves TCR recognition of antigenic MHC-peptides, including the ability to associate with CD8. This
surprising finding suggests how T cells can respond to
small amounts of antigens in a “sea” of nonstimulatory MHC-peptides.
We are now testing the relative importance of TCR
and CD8 interactions with the endogenous MHC-peptides in helping stimulation by antigenic MHC-peptide
complexes. We also compared the formation of immunologic synapses and the TCR-CD8 interaction in a
system in which the affinity of the interaction between
TCRs and MHC-peptides is known. The strength of weak
agonists is more closely related to the speed at which
they recruit TCRs to the synapse and start to induce
FRET than it is to the affinity of the interaction between
TCRs and MHC-peptides.
In collaboration with E. Palmer, University Hospital, Basel, Switzerland, we investigated new signaling
induced through the TCR that differs between positive
and negative selection. The induction of FRET appears
to explain why some agonists are stronger or weaker
than would be predicted on the basis of their affinities, and the kinetics of FRET differ between positiveand negative-selecting stimuli. We are directly testing
the potentially different roles of CD8αα and CD8αβ in
formation of the immunologic synapse and the dynamics of association of the kinase Lck with CD8 before
and during antigenic stimulation.
ROLE OF THE PROTEIN KINASE C η ISOFORM IN
THE IMMUNOLOGIC SYNAPSE
We previously showed that the η isoform of protein
kinase C (PKC) is upregulated during positive selection
of developing thymocytes. Of the PKC isoforms, only
PKCθ is known to have a special role in T cells, where
it is recruited to the immunologic synapse during antigen recognition. The finding that mice deficient in PKCθ
have normal thymic selection suggested that PKCη could
be replacing PKCθ in the developing thymocytes.
We found that PKCη is also naturally recruited to
the synapse in mature thymocytes and T cells. In the
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115
absence of PKCθ, PKCη is expressed at an earlier stage
of thymocyte development, where it functions in place of
PKCθ. Inhibition of PKCη expression during thymocyte
development by short hairpin RNAs results in inhibition
of T-cell development. We are now using short hairpin
RNA and mice lacking the gene for PKCη to determine
the roles of PKCη in T-cell activation and development.
I D E N T I F I C AT I O N O F A N O V E L P R O T E I N I M P O R TA N T I N T - C E L L D I F F E R E N T I AT I O N
We identified a novel protein that is expressed primarily during thymocyte differentiation. It is expressed
during the stages at which TCR genes undergo rearrangement, and it interacts with the cell-cycle and DNA
damage-repair enzyme ATM. Transgenic expression of
the gene that encodes the protein provides some protection against DNA double-strand breaks induced by
ionizing radiation, suggesting that the novel protein
acts in synergy with ATM. The protein also interacts
with phospholipase C γ1, important in T-cell signaling.
We have produced a strain of mice that lack the gene
for this novel protein. Mice that lack the gene have
defects in thymic positive selection and reduced phosphorylation of phospholipase C γ1 in response to TCR
stimulation. We intend to identify the role of this protein in T-cell signaling and development.
TCR ENDOCYTOSIS, RECYCLING, AND
U B I Q U I T I N AT I O N
Because allelic exclusion of the TCR α-chain is
maintained after translation, many mature T cells
express 2 α-chain proteins. However, expression of
2 α-chains on the cell surface is rare. We previously
showed that functional allelic exclusion is attained in
the thymus through TCR signaling involving the kinase
Lck and the ubiquitin ligase Cbl, which controls degradation of endocytosed TCRs. We are developing a
transgenic minigene system to analyze the effects of
expression of 2 α-chains on the cell surface. We are
using FRET between ubiquitin monomers and TCR
subunits labeled with fluorescent proteins to analyze
ubiquitination of TCRs after endocytosis. In collaboration with R. Ceredig, INSERM, Grenoble, France, we
examined the TCR α-chain repertoire of specialized
CD25+CD4+ regulatory T cells. We found that the repertoire is as diverse as that of mainstream CD4+ T cells.
We discovered a new property of the dye FM4-64 that
enables us to selectively image the nuclear membrane
in living cells. This discovery should be useful to cell
biologists studying nuclear trafficking of proteins and
diseases of the nuclear envelope.
116 IMMUNOLOGY
2006
PUBLICATIONS
Bosco, N., Hung, H.-C., Pasqual, N., Jouvin-Marche, E., Marche, P.N., Gascoigne,
N.R.J., Ceredig, R. Role of the T cell receptor α-chain in the development and phenotype of naturally arising CD4+CD25+ T cells. Mol. Immunol. 43:246, 2006.
Daniels, M.A., Teixeiro, E., Gill, J., Hausmann, B., Roubaty, D., Holmberg, K.,
Werlen, G., Holländer, G., Gascoigne, N.R.J., Palmer, E. Thymic selection threshold defined by compartmentalization of Ras/MAK signalling. Nature, in press.
Yachi, P.P., Ampudia, J., Zal, T., Gascoigne, N.R. Altered peptide ligands induce
delayed CD8-T cell receptor interaction: a role for CD8 in distinguishing antigen
quality. Immunity. 25:203, 2006.
Zal, T., Zal, M.A., Lotz, C., Goergen, C.J., Gascoigne, N.R. Spectral shift of fluorescent dye FM4-64 reveals distinct microenvironment of nuclear envelope in living
cells. Traffic, in press.
Zambricki, E., Zal, T., Yachi, P., Shigeoka, A., Sprent, J., Gascoigne, N., McKay,
D. In vivo anergized T cells form altered immunological synapses in vitro. Am. J.
Transplant. 6:2572. 2006.
Signaling Pathways in the
Innate Immune System
J. Chen, R.T. Cook, Y. Kang, S. Lee, J. Mols, M. Otsuka,
S. Shimada, C.C. Wu, C. Xie, Y. Xu, J. Han
he p38 MAP kinase pathway plays a crucial function in the cellular response after infection by
pathogens or inflammatory stimulation. Our interests are the functions of p38 in innate immunity in fruit
flies, in Toll-like receptor (TLR) signaling, and in control
of the cell cycle. We are also interested in the involvement of microRNAs in the innate immune system.
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A N E W PAT H WAY I N T H E I M M U N E R E S P O N S E T O
BACTERIAL INFECTION IN DROSOPHILA
Using the P-element insertion-excision technique,
we generated mutant fruit flies that lacked individual
p38 isoforms and a double mutant that lacked both
p38 isoforms. As widely described previously, the Toll
and Imd pathways are essential in Drosophila for protection against microbial infection. In contrast, we
showed that the p38 pathway mediates another defense
mechanism independent of those 2 pathways and that
the deficiency of either Toll or Imd does not impair p38
activation after infection.
In the p38 double mutants, melanization of the
hindgut, which is related to bacterial infection, develops during the larval stage. Furthermore, p38 regulates some of the functions of heat-shock factor, and
expression of heat-shock proteins mediated by heatshock factor is, at least in part, responsible for the
p38 anti-infectious functions, revealing a new innate
immune system in Drosophila.
THE SCRIPPS RESEARCH INSTITUTE
M E D I AT I O N O F T L R 4 S I G N A L I N G B Y T H E 4 - 1 B B
LIGAND
TLRs are an important first contact between host
and microorganisms after infection. Once activated, the
receptors generate downstream signaling pathways
leading to gene expression. The 4-1BB ligand is highly
induced and expressed at the cell surface of macrophages after inflammatory stimulation. We showed that
once expressed, the ligand can interact with TLRs at
the cell surface of macrophages, but mostly activation
of the TLR4 signaling pathway leads to p38 activation. These results revealed a new mechanism in which
macrophages undergo a 2-step activation process,
involving expression and translocation of 4-1BB ligand
to the cell surface and afterward interaction with TLRs,
sustaining the inflammatory responses.
A N T I V I R A L R E S P O N S E S M E D I AT E D B Y M I C R O R N A
We found that some microRNAs interfere with propagation of vesicular stomatitis virus (VSV) and VSVinduced cell death. MicroRNAs are single-stranded
RNA molecules of 19–23 nucleotides that originate
from longer and imperfectly matching hairpin precursors processed by several ribonucleoprotein complexes.
The cytosolic enzyme Dicer converts hairpin precursors into 19- to 23-nucleotide RNA duplexes and
therefore is of key importance for the maturation of
microRNAs.
We generated Dicer-deficient mice and investigated
macrophage sensitivity to VSV infection. Macrophages
from Dicer-deficient mice had greater VSV propagation
and VSV-induced cell death than did control macrophages from wild-type mice. Furthermore, we found
that the anti-VSV functions of Dicer are at least partly
mediated by endogenous microRNAs that target viral
RNA genes encoding the RNA-directed RNA polymerase complex. Therefore, microRNAs may be a broadly
important element of the innate immune response to
viral infection in mammals.
R E G U L AT I O N O F R N A I N T E R F E R E N C E B Y T N F
S T I M U L AT I O N
Regulation of the mRNA stability of cytokines by
inflammatory stimuli is widely documented and is
linked to p38 activity. Previously, we showed that adenine-uridine–rich elements (AREs) in the 3′ untranslated
region of cytokine mRNAs dictate mRNA degradation
in a microRNA-dependent manner. Recently, we found
that exogenously added short interfering RNAs and the
endogenous ARE-microRNA systems act as regulators
of mRNA stability and that both systems are regulated
IMMUNOLOGY
2006
by inflammatory stimulation. Indeed, TNF-α stimulation
blocks both short interfering RNA– and ARE-dependent
mRNA degradation. The mechanism of action involves
lengthening of the poly (A) tail and a partial decrease
of the activity of the RNA-induced silencing complex
that does not impair the synthesis and maturation of
short interfering RNAs and microRNAs. We conclude
that RNA interference is based on mechanisms regulated
by inflammatory stimulation, linking the ARE-dependant mRNA stabilization to the function of short interfering RNAs.
PUBLICATIONS
Al Sarraj, J., Vinson, C., Han, J., Thiel, C. Regulation of GTP cyclohydrolase I
gene transcription by basic region leucine zipper transcription factors. J. Cell.
Biochem. 96:1003, 2005.
da Silva Correia, J., Miranda, Y., Austin-Brown, N., Hsu, J., Mathison, J., Xiang,
R., Zhou, H., Li, Q., Han, J., Ulevitch, R.J. Nod1-dependent control of tumor
growth. Proc. Natl. Acad. Sci. U. S. A. 103:1840, 2006.
Fu, J., Yang, Z., Wei, J., Gu, J. Nuclear protein NP60 regulates p38 MAPK activity.
J. Cell Sci. 119(Pt. 1):115, 2006
Han, J. MyD88 beyond Toll. Nat. Immunol. 7:370, 2006.
Han, J., Ulevitch, R.J. Limiting inflammatory responses during activation of innate
immunity. Nat. Immunol. 6:1198, 2005.
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Zhuang, S., Yan, Y., Han, J., Schnellmann, R.G. p38 Kinase-mediated transactivation of the epidermal growth factor receptor is required for dedifferentiation of renal
epithelial cells after oxidant injury. J. Biol. Chem. 280:21036, 2005.
Specificity and Function of
Intraepithelial γδ T Cells
W.L. Havran, M. Haynes, J.M. Jameson, C.H. Kim,
E. Kolkowski, H.K. Komori, T. Meehan, R. Mills, A. Toulon,
M. Watanabe, J. Whitelock, D. Witherden
e have a long-term interest in interactions
between intraepithelial γδ T cells and their
neighboring epithelial cells. We focus on
interactions in the thymus, skin, and intestine. We
are investigating the development, specificity, and
function of these γδ T cells. Our results have defined
unique properties of these cells and support a specialized role for intraepithelial γδ T cells in immune
surveillance, wound repair, inflammation, and protection from malignant tumors.
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MOLECULES REQUIRED FOR γδ T CELL
A C T I VAT I O N
Liu, W., Rui, H., Wang, J., Lin, S., He, Y., Chen, M., Li, Q., Ye, Z., Zhang, S.,
Chan, S.C., Chen, Y.G., Han, J., Lin, S.C. Axin is a scaffold protein in TGF-β signaling that promotes degradation of Smad7 by Arkadia. EMBO J. 25:1646, 2006.
Lu, G., Kang, Y.K., Han, J., Herschman, H.R., Stefani, E., Wang, Y. TAB-1 modulates intracellular localization of p38 MAP kinase and downstream signaling. J.
Biol. Chem. 281:6087, 2006.
Meng, F., Yamagiwa, Y., Taffetani, S., Han, J., Patel, T. IL-6 activates serum and
glucocorticoid kinase via a p38α mitogen-activated protein kinase pathway. Am. J.
Physiol. Cell. Physiol. 289:C971, 2005.
Shi, G.X., Han, J., Andres, D.A. Rin GTPase couples nerve growth factor signaling
to p38 and b-Raf/ERK pathways to promote neuronal differentiation. J. Biol. Chem.
280:37599, 2005.
Tang, J., Qi, X., Mercola, D., Han, J., Chen, G. Essential role of p38β in K-Ras
transformation independent of phosphorylation. J. Biol. Chem. 280:23910, 2005.
Xie, C., Zhang, N., Zhou, H., Li, J., Li, Q., Zarubin, T., Lin, S., Han, J. Distinct
roles of basal steady-state and induced H-ferritin in tumor necrosis factor-induced
death in L929 cells. Mol. Cell. Biol. 25:6673, 2005.
Xu, Y., Huang, S., Liu, Z.G., Han, J. Poly(ADP-ribose) polymerase-1 signaling to
mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation. J. Biol. Chem. 281:8788, 2006.
Zarubin, T., Han, J. Activation and signaling of the p38 MAP kinase pathway. Cell
Res. 15:11, 2005.
Zhou, H., Zarubin, T., Ji, Z., Min, Z., Zhu, W., Downey, J.S., Lin, S., Han, J. Frequency and distribution of AP-1 sites in the human genome. DNA Res. 12:139,
2005.
Zhou H., Zheng, M., Chen, J., Xie, C., Kolatkar, A.R., Zarubin, T., Ye, Z., Akella,
R., Lin, S., Goldsmith, E.J., Han, J. Determinants that control the specific interactions between TAB1 and p38β. Mol. Cell. Biol. 26:3824, 2006.
Zhu, X., Mei, M., Lee, H.G., Wang, Y., Han, J., Perry, G., Smith, M.A. p38 Activation mediates amyloid-β cytotoxicity. Neurochem. Res. 30:791, 2005.
In murine skin, γδ T cells express an invariant
γδ T cell receptor that recognizes an unknown antigen
expressed by damaged or malignant neighboring keratinocytes. We have now produced soluble skin γδ T cell
receptor molecules for use as a tool to detect expression and facilitate isolation and characterization of this
unidentified antigen. Future structural studies will determine how these T-cell receptors interact with antigen.
We propose that in addition to antigen, damaged
keratinocytes express molecules that participate in
activation of skin γδ T cells by binding to coreceptors
or costimulatory molecules on the T-cell surface. Skin
γδ T cells do not express classical molecules, including CD4, CD8, and CD28, known to affect activation
of αβ T cells.
We recently identified several molecules expressed
by the skin γδ T cells and keratinocytes that provide
important costimulatory signals for activation of γδ
T cells. One such molecule, JAML, is uniquely costimulatory for intraepithelial γδ T cells. We have identified
another JAM family member, the coxsackievirus-adenovirus receptor, as a ligand for JAML that is expressed on
epithelial cells in the skin and intestine. Interactions
between JAML and the coxsackievirus-adenovirus receptor may play important roles in γδ T cell responses during wound repair and other epithelial challenges.
118 IMMUNOLOGY
2006
We also found that the semaphorin Sema4D (CD100)
is expressed by skin and intestinal γδ T cells upon
activation. CD100 binds to a new member of the
plexin superfamily of semaphorin receptors, plexin-B2,
expressed on epithelial cells. We found that interactions between CD100 and plexin-B2 deliver signals
to both the intraepithelial γδ T cells and epithelial
cells in the skin and intestine.
THE SCRIPPS RESEARCH INSTITUTE
inflammatory disorders and may be useful in designing
or testing new therapies.
PUBLICATIONS
Havran, W.L., Jameson, J.M., Witherden, D.A. Epithelial cells and their neighbors, III: interactions between intraepithelial lymphocytes and neighboring epithelial cells. Am. J. Physiol. Gastrointest. Liver Physiol. 289:G627, 2005.
Komori, H.K., Meehan, T.F., Havran, W.L. Epithelial and mucosal γδ T cells. Curr.
Opin. Immunol. 18:534, 2006.
A ROLE FOR INTRAEPITHELIAL γδ T CELLS IN
EPITHELIAL TISSUE REPAIR
We recently showed a role for skin γδ T cells in the
reepithelialization stage of wound repair. The γδ T cells
are activated at wound sites and produce cytokines,
including the epithelial growth factors KGF-1 and KGF-2.
In the absence of skin γδ T cells, keratinocyte proliferation and tissue reepithelialization after wounding are
defective. Recent results indicated that a keratinocyteresponsive γδ T-cell receptor is necessary for activation
of the T cells by damaged keratinocytes during wound
healing and is also required for the maintenance of
T cells in the epidermis. In addition, we found that
the skin γδ T cells are necessary for the recruitment
of inflammatory cells into the wound site. In a novel
mechanism, γδ T cell–produced KGFs stimulate production of hyaluronan by epidermal cells, which then
controls migration of macrophages into wounds.
Skin γδ T cells play roles not only in the repair of
damaged tissue but also in the normal maintenance of
the epidermis. Insulin-like growth factor 1 is required
by keratinocytes in the skin for maintenance and during wound healing. We determined that after activation
skin γδ T cells produce this growth factor that affects
wound healing and apoptosis in the skin. Together
these results indicate a role for skin γδ T cells in multiple aspects of wound repair and for homeostasis of
the epithelium.
In previous studies, we showed that intestinal intraepithelial γδ T cells play a similar role in responding
to tissue damage in a model of colitis. Our recent results
indicate that in the absence of CD100-mediated signals,
increased damage and delayed repair occur, indicating
an important role for costimulation through these molecules in γδ T-cell functions in the gut. Results in both
models support our hypothesis that intraepithelial γδ T
cells respond to epithelial damage or disease and play
important roles in tissue repair and epithelial homeostasis. Future studies should provide information that
will further define the role of γδ T cells in epithelial
Mechanisms of γδ T-Cell
Dysfunction in Nonhealing
Wounds
J.M. Jameson, R. Mills, K. Taylor
kin γδ T cells are activated by stressed or damaged keratinocytes to produce molecules such
as insulin-like growth factor 1 (IGF-1) and keratinocyte growth factors that are important for the
maintenance of skin homeostasis and wound repair.
We are examining skin γδ T cells in nonhealing wounds
to investigate the mechanisms that inhibit normal
wound healing.
S
C R O S S TA L K O F γδ T C E L L S A N D K E R AT I N O C Y T E S
IN THE SKIN IN DIABETES
Treatment of nonhealing wounds is a considerable
problem for patients and healthcare services. In murine
models of diabetes, levels of IGF-1 and keratinocyte
growth factors in wounds contribute to defective wound
repair. When these growth factors are replenished, the
markedly delayed wound repair is reversed. Previously,
we showed that skin γδ T cells play roles in wound
reepithelialization via the production of growth factors.
Now we are investigating whether decreased levels of
growth factors and impaired proliferation/migration of
keratinocytes in nonhealing wounds in mice with diabetes are due to dysregulation of skin γδ T cells. Initially, we are focusing on how the cross talk between
the key growth factors produced by skin γδ T cells and
the factors’ receptors expressed on keratinocytes is
defective in mice with diabetes.
In addition, we are examining mechanisms that may
contribute to skin γδ T-cell dysfunction in diabetes,
including changes in insulin or IGF-1 receptor signaling
and inhibition by glucocorticoids. The decreased levels
of IGF-1 in the epidermal compartment may result in
reduced IGF-1 receptor signaling in skin γδ T cells, a
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2006
situation that may exacerbate dysfunction of skin γδ
T cells.
γδ T-CELL FUNCTION IN RESPONSE TO
RAPAMYCIN
Rapamycin is approved by the Food and Drug
Administration for prophylaxis of acute rejection of transplanted organs. Patients who receive rapamycin have an
increased incidence of complications of wound healing.
Because these patients have difficulties with wound
healing, rapamycin may not only be targeting allograftspecific αβ T lymphocytes but also be suppressing skin
γδ T cells. In preliminary experiments, we observed
defects in the proliferation of skin γδ T cells and the production of IGF-1 in the presence of rapamycin. We are
investigating the mechanism of this suppression.
Once we better understand the role of T cells in
tissue repair, it may be possible to design therapies
that enhance the ability of these immune cells to heal
ulcers and chronic wounds. Our objective is to determine the mechanisms by which skin γδ T cells, normally important in wound repair of healthy wild-type
mice, are not functioning properly in chronic wounds.
Regulators of T-Lymphocyte
Development and Function
J. Kaye, P. Aliahmad, M. Fung, O. Goularte, C. Krieg,
N. Sanathara
recursor cells in the thymus undergo a complex
developmental program before seeding peripheral lymphoid organs as mature T lymphocytes.
Developmental checkpoints in the thymus, termed
β-selection, positive selection, and negative selection,
narrow the repertoire of T-cell antigen specificities to
those that are not overtly autoreactive but maintain
weak reactivity against self-MHC-peptide complexes.
We are interested in the mechanisms that determine
the fate of developing T cells and the control of gene
expression during these processes. Our identification
of a cell-surface protein that is first expressed in the
T-cell lineage during positive selection led to studies
on regulation of the immune response and the potential of this protein as a novel therapeutic target.
P
R E G U L AT I O N O F T H Y M O C Y T E S E L E C T I O N B Y A
NUCLEAR ARCHITECTURAL PROTEIN
We identified thymocyte selection–associated high
mobility group (HMG) box protein (TOX) several years
THE SCRIPPS RESEARCH INSTITUTE
119
ago. Members of the HMG box protein superfamily share
one or more copies of a sequence-related and structurally
related DNA-binding domain that can recognize distorted
DNA structures and modify chromatin by bending DNA.
In general, HMG box proteins function as architectural
factors that regulate gene expression by promoting formation of transcriptional complexes or by acting as components of chromatin remodeling complexes.
We found that TOX belongs to a small subfamily of
evolutionarily conserved proteins whose members share
almost identical HMG box sequences. The HMG box
sequence in TOX can recognize distorted DNA but is a
relatively poor bender of DNA, because of the lack of
a critical internal wedge residue.
Expression of TOX in the thymus is tightly regulated.
Signaling through the serine/threonine phosphatase
calcineurin is required for positive selection of thymocytes, and the gene for TOX is a target of this signaling
pathway. TOX is expressed in early thymocyte progenitors and then is transiently upregulated during β-selection and positive selection. To analyze the function of
this nuclear factor, we produced transgenic mice that
express either wild-type or a mutant TOX and genetargeted mice that lack TOX.
Our data indicate that expression of TOX is sufficient to initiate the differentiation of immature thymocytes to the CD8+ T-cell lineage, even in the absence of
signals mediated by T-cell antigen receptors. Both the
DNA-binding domain and the N-terminal domain of TOX
are required for this in vivo activity. Although TOX is not
a T cell–specific protein, mice that lack the gene for
TOX are grossly normal but have a specific block in an
early stage of thymic positive selection. We are using
these tools to delineate the mechanism of action of this
critical regulator of the fate of T cells in the thymus.
N E G AT I V E R E G U L AT I O N O F T - C E L L R E S P O N S E S
The functional outcome of engagement of the T-cell
antigen receptor is modulated by secondary signals,
which can have costimulatory or coinhibitory functions.
We isolated a gene that encodes a cell-surface protein
of the immunoglobulin superfamily, now designated
BTLA (B- and T-lymphocyte attenuator), that is upregulated during positive selection and that is expressed
by mature lymphocytes and antigen-presenting cells.
Evidence indicates that this protein can act as a negative regulator of lymphocyte activation. We produced
mice that lack the gene for BTLA and panels of monoclonal antibodies specific for BTLA to analyze the in
vivo function of this protein.
120 IMMUNOLOGY
2006
One of our monoclonal antibodies acts as an agonist
for this inhibitory molecule, thereby inhibiting facets of
T-cell activation. In vivo studies indicated that BTLA is
a negative regulator of homeostatic expansion of T cells
and production of CD8+ memory T cells. For vaccines
against intracellular infections and tumors, development
of methods to regulate CD8+ T-cell responses and memory formation is paramount, and thus BTLA may be a
useful therapeutic target in this regard. Preliminary data
also suggest that one of our monoclonal antibodies to
BTLA prolongs allograft survival in mice, and another
antibody may ameliorate disease in a murine model of
inflammatory bowel disease.
PUBLICATIONS
Aliahmad, P., Kaye, J. Commitment issues: linking positive selection signals and
lineage diversification in the thymus. Immunol. Rev. 209:253, 2006.
Krieg, C., Boyman, O., Fu, Y.-X., Kaye, J. T cell intrinsic regulation of homeostasis
and CD8+ memory cell generation by BTLA. Nat. Immunol., in press.
Krieg, C., Han, P., Stone R., Goularte, O.D., Kaye, J. Functional analysis of B and
T lymphocyte attenuator engagement on CD4+ and CD8+ T cells. J. Immunol.
175:6420, 2005.
Regulation of the Innate
Immune Response in
Inflammation and Infection
U.G. Knaus, B. Desnues, M. Lehmann, K. von Loehneysen,
S. Luxen, M. Manukyan, S. Pacquelet, M. Ruse, M. Tsatmali,
M. Valo, M. Ye
nnate immune cells are the first line of defense in
the fight against invading pathogens. We focus primarily on understanding molecular mechanisms that
phagocytes and the pulmonary epithelium use to protect
the host from the injury and how some responses wind
up damaging the host. For example, second messengers
such as reactive oxygen species (ROS) or nitric oxide
that are produced during infection can have beneficial
as well as detrimental effects. The overall outcome
depends on precise spatial and temporal regulation of
these second messengers by the affected cell populations. The intracellular signaling pathways that control
these turn on–turn off mechanisms are an ideal target
for intervention in disease.
Almost all processes connected to pathogen uptake,
pathogen elimination, and sustained inflammation are
governed by small GTPases of the Ras superfamily. Our
research centers on the Rho GTPases Rac, Cdc42,
I
THE SCRIPPS RESEARCH INSTITUTE
and Rho, which are essential regulators for various
leukocyte functions ranging from production of ROS to
chemotaxis and phagocytosis. Generation of superoxide is accomplished by a Rac-dependent NADPH oxidase (Nox) upon stimulation with chemotactic factors
or phagocytic stimuli.
GTPases of the Rho family are also involved in signaling cascades, which originate from pathogen-activated
Toll-like receptors. Toll-like receptors 2, 3, and 4 stimulated by microbial products derived from bacteria and
viruses activate Rac1, Cdc42, and RhoA, which regulate pathways required for activation of gene transcription. We are studying different aspects of signaling by
Toll-like receptors in several primary human cell types,
including macrophages and neutrophils, and in genetically altered mouse models. We are also examining
the impact of this signaling on the functions, such as
apoptosis and upregulation of proinflammatory mediators, of innate immune cells.
Another area of research is the interaction and
communication between innate immune cells and the
pulmonary epithelium. To this end, we established an
in vitro reconstitution system for lung epithelium that
we use to examine signaling mechanisms initiated by
pathogens. The differentiated and fully functional lung
epithelium also serves as a model for studies of lung
barrier function and the influence of bacteria-derived
ligands and toxins on transmigration of neutrophils. In
addition, we are investigating processes that lead to
uptake of pathogens or environmental particles and
the impact of these pathogens or particles on airway
epithelial functions.
Recently, ROS-generating Nox proteins have been
identified in epithelial cells, and work is in progress to
study the molecular basis for ROS generation by these
novel proteins. Nox proteins may serve as compartmentalized signaling modules, thereby activating or inhibiting
signaling cascades via superoxide, or as an epithelial
host defense mechanism via hydrogen peroxide–generating Nox/Duox isoforms. Because of their tissue-specific
distribution and distinct localization patterns, Nox proteins might have highly specialized functions and undergo
isoform-dependent regulation. For example, Nox4, an
oxidase expressed in colon tissue and melanomas, is
constitutively active in certain conditions and does not
require any of the known oxidase components for superoxide generation. Elucidating physiologic stimuli and
control mechanisms for these Nox proteins combined
with structure-function studies will help define the
biological functions of Nox in health and disease.
IMMUNOLOGY
2006
PUBLICATIONS
Chan, A.Y., Coniglio, S.J., Chuang, Y.Y., Michaelson, D., Knaus, U.G., Philips,
M.R., Symons, M. Roles of the Rac1 and Rac3 GTPases in human tumor cell invasion. Oncogene 24:7821, 2005.
Martyn, K.D., Frederick, L.M., von Loehneysen, K., Dinauer, M.C., Knaus, U.G.
Functional analysis of Nox4 reveals unique characteristics compared to other
NADPH oxidases. Cell. Signal. 18:69, 2006.
Martyn, K.D., Kim, M.J., Quinn, M.T., Dinauer, M.C., Knaus, U.G. p21-Activated
kinase (Pak) regulates NADPH oxidase activation in human neutrophils. Blood
106:3962, 2005.
Ruse, M., Knaus, U.G. New players in TLR-mediated immunity: PI3K and small
Rho GTPases. Immunol. Res. 34:33, 2006.
Regulatory Mechanisms for
Tumor Carcinogenesis
M. Hayashi, J.-F. Lo, S.-W. Kim, J.-D. Lee
T H E F O U R T H M A P K I N A S E PAT H WAY
ig mitogen-activated kinase 1 (BMK1), also
called extracellular signal–regulated kinase 5, a
newer member of the mammalian MAP kinase
family, is activated by angiogenic growth factors. Using
a mouse model in which expression of the gene for
BMK1 can be deleted, we showed that the BMK1 pathway is required for tumor-associated angiogenesis and
consequent tumor growth through the BMK1/ribosomal
S6 kinase/ribosomal protein S6 pathway. To investigate
and define the function and molecular actions of the
BMK1 pathway in endothelial cells and in angiogenesis, we are investigating the signaling pathways that
interact with or are regulated by the BMK1 cascade in
endothelial cells and are exploring the mechanism of
action of the BMK1 cascade during angiogenesis. These
studies should provide new information on the regulatory mechanisms of neovascularization. We hope to
use the results to identify novel and important targets
for a more effective and specific therapeutic intervention for human cancer by inhibition of tumor-associated angiogenesis.
B
THE TUMOR SUPPRESSOR/PROTEIN CHAPERONE
TID1
Tid1 is the human counterpart of the Drosophila
tumor suppressor Tid56. Mutations that cause loss of
function of the gene for Tid56 result in tumorous imaginal discs due to continuous cell proliferation without
differentiation. Using yeast 2-hybrid screening, we
found that Tid1 interacts with the signaling domain of
the receptor protein-tyrosine kinase ErbB2/Her2. Subsequent studies indicated that increased expression of
THE SCRIPPS RESEARCH INSTITUTE
121
Tid1 in breast cancer cells overexpressing ErbB2 facilitated the ubiquitination and degradation of ErbB2
and growth inhibition of the cells. Moreover, using
RNA interference to deplete the physiologic levels of
Tid1 in breast cancer cells, we discovered that the
metastatic potential of Tid1-depleted cells was substantially enhanced. This enhancement was due to
increased production of IL-8 through upregulation of
the nuclear transcription factor NF-κB in the Tid1depleted cells.
Thus, because Tid1 attenuates signals generated
from the ErbB2 receptor and negatively regulates the
activity of NF-κB, we hypothesize that Tid1, like its
Drosophila counterpart, may be an important tumor
suppressor, especially in breast carcinogenesis and
metastasis. To evaluate this hypothesis in an animal
model, we have established a mouse model in which
the gene for Tid1 can be deleted specifically in mammary epithelial cells. We are investigating and evaluating the mechanistic role of Tid1 as a tumor suppressor
in the regulation of tumorigenesis and metastasis of
breast cancer not only at the molecular and cellular
levels but also in an organismal context.
PUBLICATIONS
Abbasi, S., Lee, J.-D., Su, B., Chang, X., Alcon, J.L., Yang, J., Kellems, R.E., Xia, Y.
Protein kinase-mediated regulation of calcineurin through the phosphorylation of
modulatory calcineurin-interacting protein 1. J. Biol. Chem. 281:7717, 2006.
Hayashi, M., Fearns, C., Eliceiri, B., Yang, Y., Lee, J.-D. Big mitogen-activated
protein kinase 1/extracellular signal-regulated kinase 5 signaling pathway is essential for tumor-associated angiogenesis. Cancer Res. 65:7699, 2005.
Hayashi, M., Imanaka-Yoshida, K., Yoshida, T., Wood, M., Fearns, C., Tatake,
R.J., Lee, J.-D. A critical role of mitochondrial Hsp40 in preventing dilated cardiomyopathy. Nat. Med. 12:128, 2006.
Kim, S.W., Hayashi, M.l., Lo, J.F., Fearns, C., Xiang, R., Lazennec, G., Yang, Y.,
Lee, J.-D. Tid1 negatively regulates the migratory potential of cancer cells by
inhibiting the production of interleukin-8. Cancer Res. 65:8784, 2005.
Zhou, H., Luo, Y., Lo, J.-F., Kaplan, C.D., Mizutani, M., Mizutani, N., Lee, J.-D.,
Primus, F.J., Becker, J.C., Xiang, R., Reisfeld, R.A. DNA-based vaccines activate
innate and adaptive antitumor immunity by engaging the NKG2D receptor. Proc.
Natl. Acad. Sci. U. S. A. 102:10846, 2005.
Host-Pathogen Interactions:
Mechanisms and Applications
E. Li, S.P. Lad, J. Li
icrobial pathogens can be classified into 2
broad categories: those that infect the host
accidentally and those that do so for growth.
The outcome of an infection by “accidental” pathogens
is commonly associated with severe host inflammatory
M
122 IMMUNOLOGY
2006
responses and is often lethal. In contrast, obligate intracellular pathogens such as chlamydiae and rickettsiae
have developed efficient yet poorly defined mechanisms
to evade host immune surveillance and secure a favorable habitat. We focus on host responses to viral and
bacterial infections. We use infections with adenovirus
and the obligate intracellular bacterial pathogen Chlamydia trachomatis as models systems.
Chlamydia trachomatis infection affects 140 million
persons worldwide. It is also the most notifiable disease
in the United States; it leads to 50,000–100,000 new
cases of pelvic inflammatory diseases and infertility
each year. Although an inflammatory cellular response
and chronic inflammation are the underlying mechanisms
of chlamydial diseases, a hallmark of a chlamydial infection is its asymptomatic nature. We found that Chlamydia can downregulate host inflammatory responses by
converting a regulatory molecule of the inflammation
pathway to a negative inhibitor of the same pathway.
The host responds to viral and bacterial infections
by using the nuclear transcription factor NF-κB to modulate genes involved in inflammation and innate immunity. NF-κB consists of a heterodimeric complex
composed of 2 subunits, commonly p50/NF-κB1 and
p65/RelA, which are sequestered in the cytoplasm and
are made inactive through their association with inhibitory molecules, including IκBα. Bacterial and viral infections, proinflammatory cytokines, and stimulation with
lipopolysaccharide all can induce rapid degradation of
IκBα, resulting in the release and nuclear translocation
of the NF-κB complex for gene regulation.
In addition to protecting IκBα from degradation
induced by TNF-α, chlamydial infections promote
p65/RelA cleavage. The N-terminal cleavage product
functions as a dominant negative inhibitor of the NF-κB
pathway and hence can block NF-κB–modulated gene
expression associated with inflammatory responses.
We are identifying and characterizing the bacterial
protease that causes the cleavage.
We are also designing and modifying adenovirus
for targeted gene delivery. Although widely used for
gene therapy studies, adenovirus-based vectors cannot
target specific tissues. We generated modified adenoviruses that are equipped with a tumor-targeting antibody, enabling the selective delivery of therapeutic genes
to tumor cells by antibody-guided “missiles.”
PUBLICATIONS
Li, J., Lad, S., Yang, G., Lou, Y., Iacobelli-Martinez, M., Primus, F.J., Reisfeld,
R.A., Li, E. The adenovirus fiber shaft contains a trimerization element supporting
peptide fusion for targeted gene delivery. J. Virol., in press.
THE SCRIPPS RESEARCH INSTITUTE
The Cysteine Protease Network
in Tumor Progression
and Therapy
C. Liu, W. Wu, Y. Liu, F. Guo, K. Shumilak, Q. Fang
eoplasms in humans arise via a multistage
process that involves the functions of a protease network. We first reported that legumain,
a lysosomal asparaginyl endopeptidase with a caspaselike catalytic site, is highly expressed in a majority of
rodent and human solid tumors. Compared with adjacent normal tissues, both tumor cells and tumor stromal cells must survive under substantially lower oxygen
tension within the tumor microenvironment. We found
that legumain expression is induced by hypoxia and
occurs early during tumor development. We showed
that legumain enhances tumor cell metastasis and
invasion and protects cells from apoptosis through a
complex and precise regulation of a cathepsin and
caspase network (Fig. 1).
N
F i g . 1 . Legumain promotes tumor cell invasion and metastasis
by binding to cell-surface integrins and activates both matrix metalloproteinase 2 (MMP2) and cathepsin L. It also protects cells from
programmed cell death by catalytically inactivating caspase 9. It
prevents Bid activation by cathepsin B by binding to and modulating the activity of the cathepsin.
IMMUNOLOGY
2006
S U P P R E S S I O N O F T U M O R I N VA S I O N , M E TA S TA S I S ,
AND ANGIOGENESIS BY INHIBITION OF LEGUMAIN
We showed that legumain is present in the tumor
microenvironment, where it binds to cell-surface integrins such as αvβ3 and α5β1. Complexes consisting of
legumain and αvβ3 are predominantly at the front of
migrating cells. Binding of legumain to αvβ3 significantly enhances the activity of legumain toward physiologic substrates such as pro–matrix metalloproteinase
2 and procathepsin L. Therefore, integrins are both
receptors and cofactors of legumain.
Inhibition of legumain activity by a high-affinity
asparaginyl endopeptidase inhibitor suppressed angiogenesis and tumor cell invasion in vitro and in vivo.
Systemic administration of the inhibitor in mice
reduced tumor growth, vascular density, and tumor
invasiveness in breast cancer models. Importantly, this
treatment inhibited both spontaneous and experimental lung metastasis. These data indicate that legumain
is a critical modulator of pericellular proteolytic cascades and an effective target for cancer therapy.
L E G U M A I N P R O T E C T I O N A G A I N S T M U LT I P L E
P R O G R A M M E D C E L L D E AT H PAT H WAY S
Caspases do not occur in plants, and legumain is
the effector protease for plant cell apoptosis. However,
we found that in mammals legumain evolved to have
an antiapoptotic activity. Overexpression of legumain
protects cells from multiple programmed cell death
pathways. We showed that during TNF-induced cell
death, lysosomal proteases diffuse into cytoplasm.
Legumain exerts its antiapoptosis activity by depleting
procaspase 9 and inactivating caspase 9 in apoptosomes. Prolegumain can be activated by caspase 3.
Therefore, the presence of legumain serves as a brake
for the caspase cascade. In addition, legumain binds
directly to cathepsin B and suppresses autoactivation
of the cathepsin and cathepsin B–mediated activation
of the proapoptotic protein Bid.
These findings indicate that legumain plays a critical role in mitochondria and in apoptosis mediated by
death receptors. Inhibition of legumain activity and
expression sensitizes tumor cells to natural death cues
and chemotherapeutic agents, providing a novel technique for cancer intervention.
PUBLICATIONS
Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf, W.,
Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. 25:1737, 2005.
THE SCRIPPS RESEARCH INSTITUTE
123
Wu, W., Luo, Y., Sun, C., Liu, Y., Kuo, P., Varga, J., Xiang, R., Reisfeld, R.,
Janda, K.D., Edgington, T.S., Liu, C. Targeting cell-impermeable prodrug activation
to tumor microenvironment eradicates multiple drug-resistant neoplasms. Cancer
Res. 66:97, 2006.
Role of the Tissue
Factor–Thrombin
Pathway in Thrombosis,
Inflammation, and Cardiac
Ischemia-Reperfusion Injury
R. Pawlinski, R.E. Tilley, J.P. Luyendyk, L. Kidd, E. Romeo,
N. Mackman
e are interested in the role of tissue factor (TF)
in hemostasis, thrombosis, inflammation,
myocardial infarction, and cardiac remodeling. TF is the primary initiator of blood coagulation and
plays an essential role in hemostasis by activating blood
coagulation after vessel injury. However, aberrant TF
expression within the vasculature induces thrombosis
in a variety of diseases, including sepsis (Fig. 1). In
W
F i g . 1 . Role of the tissue factor–thrombin pathway in thrombo-
sis and cardiac remodeling. Abbreviations: LPS, lipopolysaccharide;
I/R, ischemia-reperfusion.
addition, we have shown that TF-dependent generation
of thrombin contributes to the size of infarcts and cardiac
remodeling after cardiac ischemia-reperfusion injury.
THROMBOSIS
In sepsis, bacterial lipopolysaccharide released by
gram-negative bacteria induces TF expression by intravascular cells. This expression leads to disseminated
intravascular coagulation, tissue ischemia, and inflammation. Notably, inhibition of TF reduces both coagulation and inflammation in animal models of sepsis. We
are interested in the relative contribution of TF expression by monocytes, endothelial cells, and platelets to
124 IMMUNOLOGY
2006
THE SCRIPPS RESEARCH INSTITUTE
lipopolysaccharide-induced coagulation. In collaboration with A. Weyrich, University of Utah, Salt Lake City,
we recently found that lipopolysaccharide-stimulated
human platelets express TF.
To delete the gene for TF in the different cell types,
we generated floxed TF mice. These mice have the TF
gene flanked by specific sequences (loxP sites) that can
be used for tissue-specific expression of TF via the Cre
recombinase system. In the first experiments, floxed
TF mice were crossed with mice that express the Cre
recombinase in myeloid cells. These mice had reduced
levels of TF expression in lipopolysaccharide-stimulated
macrophages in a murine endotoxemia model, indicating
that monocytes are a major source of intravascular TF
in endotoxemia. Future studies will determine how deleting the TF gene in either endothelial cells or platelets
affects lipopolysaccharide-induced coagulation. We plan
to design novel strategies targeting intravascular TF that
will reduce lipopolysaccharide-induced coagulation without increasing the risk of bleeding.
remodeling and improved left ventricular function 2
weeks after ischemia-reperfusion injury. These results
suggest that PAR1 contributes to cardiac remodeling
after ischemia-reperfusion injury.
In parallel studies, we determined the effect of overexpressing PAR1 in cardiomyocytes on heart morphology
and function. Cardiac hypertrophy developed in a transgenic mouse model, and heart function was impaired.
These data indicate that PAR1 signaling in cardiomyocytes induces hypertrophy and cardiac remodeling.
Future studies will determine the role of TF-dependent
generation of thrombin in the activation of PAR1 in
the heart.
I N F L A M M AT I O N
Kamimura, M., Viedt, C., Dalpke, A., Rosenfeld, M.E., Mackman, N., Cohen,
D.M., Blessing, E., Preusch, M., Weber, C.M., Kreuzer, J., Katus, H.A., Bea, F.
Interleukin-10 suppresses tissue factor expression in lipopolysaccharide-stimulated
macrophages via inhibition of Egr-1 and a serum response element/MEK-ERK1/2
pathway. Circ. Res. 97:305, 2005.
We are interested in the role of the phosphatidylinositol-3′-kinase (PI3K)–Akt intracellular signaling pathway
in lipopolysaccharide-induced inflammation and coagulation. We have shown that activation of the PI3K-Akt
pathway inhibits lipopolysaccharide induction of inflammatory mediators and TF in human monocytic and
endothelial cells and in a murine endotoxemia model.
Interestingly, several compounds that improved survival
in sepsis also activated the PI3K-Akt pathway. For
example, we found that a low dose of insulin, which
did not affect blood glucose levels, reduced inflammation
and improved survival in a murine endotoxemia model
in a PI3K-dependent manner. More recently, we found
that simvastatin, a widely used cholesterol-lowering
drug, inhibits lipopolysaccharide induction of inflammation and coagulation in mice. Importantly, the protective
effect of simvastatin was abolished by inhibition of PI3K.
Future studies will determine how insulin and simvastatin reduce lipopolysaccharide-induced TF and cytokine expression in vitro and in vivo.
MYOCARDIAL INFARCTION AND CARDIAC
REMODELING
We have shown that the TF-thrombin pathway contributes to infarction after cardiac ischemia-reperfusion
injury. More recently, we found that a deficiency of
protease-activated receptor 1 (PAR1), a receptor for
thrombin, did not affect the size of infarcts. However,
PAR1 deficiency was associated with reduced cardiac
PUBLICATIONS
Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf, W.,
Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol. 25:1737, 2005.
Hayashi, M., Matsushita, T., Mackman, N., Ito, M., Adachi, T., Katsumi, A.,
Yamamoto, K., Takeshita, K., Kojima, T., Saito, H., Murohara, T., Naoe, T. Fatal
thrombosis of antithrombin-deficient mice is rescued differently in the heart and liver
by intercrossing with low tissue factor mice. J. Thromb. Haemost. 4:177, 2006.
Luyendyk, J.P., Tilley, R.E., Mackman, N. Genetic susceptibility to thrombosis.
Curr. Atheroscler. Rep. 8:193, 2006.
Mackman, N. Role of tissue factor in hemostasis and thrombosis. Blood Cells Mol.
Dis. 36:104, 2006.
Mackman, N. Tissue-specific hemostasis in mice. Arterioscler. Thromb. Vasc. Biol.
25:2273, 2005.
Motton, D.D., Mackman, N., Tilley, R.E., Rutledge, J.C. Postprandial elevation of tissue factor antigen in the blood of healthy adults. Thromb. Haemost. 94:504, 2005.
Rushworth, S.A., Chen, X.-L., Mackman, N., Ogborne, R.M., O’Connell, M.A.
Lipopolysaccharide-induced heme oxygenase-1 expression in human monocytic
cells is mediated via Nrf2 and protein kinase C. J. Immunol. 175:4408, 2005.
Schabbauer, G., Mackman, N. Tissue factor expression by the endothelium. In:
The Endothelium: A Comprehensive Reference. Aird, W. (Ed.). Cambridge University Press, New York, in press.
Tilley, R.E., Mackman, N. Tissue factor in hemostasis and thrombosis. Semin.
Thromb. Haemost. 32:5, 2006.
Tilley, R.E., Pedersen, B., Pawlinski, R., Sato, Y., Erlich, J.H., Shen, Y., Day, S.,
Huang, Y., Eitzman, D.T., Boisvert, W.A., Curtiss, L.K., Fay, W.P., Mackman, N.
Atherosclerosis in mice is not affected by a reduction in tissue factor expression.
Arterioscler. Thromb. Vasc. Biol. 26:555, 2006.
IMMUNOLOGY
2006
Regulating Adaptive Immunity
M.G. McHeyzer-Williams, L.J. McHeyzer-Williams,
L.P. Malherbe, N.R. Fazilleau, N. Pelletier
R E G U L AT I O N O F B - C E L L I M M U N I T Y B Y H E L P E R
T CELLS
elper T cells are the master regulators of adaptive immunity that control the development of
antigen-specific B-cell immunity. We seek to
define the cellular and molecular details of the major
developmental checkpoints that regulate the fate of
these helper T cells and B cells in vivo. We recently
extended our studies to the earliest innate immune
events that initiate and shape this adaptive immune
response. If we can understand the rules that control
adaptive immunity, we can design safe and effective
protein subunit vaccines.
H
SELECTION OF ANTIGEN-SPECIFIC HELPER T CELLS
We recently described an affinity threshold model for
selecting antigen-specific helper T cells in the response
to protein antigens. The unique characteristic of this
model is how clonal diversity is achieved and maintained through the progressive loss of lower affinity cells
rather than through the preferential expansion of the
high-affinity clonotypes. Our evidences suggests that
selection is not due to interclonal competition but is a
more intrinsic threshold with a “set point” for selection.
Hence, in order to manipulate antigen-specific clonal
diversity, this affinity threshold must be altered. We
are examining multiple variables of vaccination to
define these rules, with emphasis on the impact of
immune adjuvant on the priming of adaptive immunity.
EVOLUTION OF MEMORY B CELLS
The rapid evolution of memory B cells occurs at the
cellular level and is controlled by multiple cognate
checkpoints during the development of adaptive immunity. Although clonal selection is the fundamental process that underpins adaptive immunity, surprisingly little
is understood about the mechanism of its action across
multiple junctures of development of antigen-specific
memory B cells in vivo. Our recent studies focus on the
consolidation of B-cell memory upon antigen recall in
the boost phase of vaccination with protein antigens.
Cellular and molecular analysis of the memory response
reveals the developmental staging of memory B cells
that express different antibody subtypes. These studies
highlight the importance of antibody isotype as a fundamental cellular division within antigen-specific Bcell memory.
THE SCRIPPS RESEARCH INSTITUTE
125
ANTIGEN-EXPERIENCED DENDRITIC CELLS
Dendritic cells are the most efficient innate initiators
of adaptive immunity. Immune adjuvants substantially
affect the developmental response of subsets of dendritic
cells that in turn regulate separable functions in the
helper T cell and B-cell compartments in vivo. Using
antibodies that reveal specific peptide–MHC II complexes on dendritic cells, we can directly isolate antigen-experienced dendritic cells to evaluate the effect of
immune adjuvants on priming of the cells in vivo.
PUBLICATIONS
Alfonso, C., McHeyzer-Williams, M.G., Rosen, H. CD69 down-modulation and
inhibition of thymic egress by short- and long-term selective chemical agonism of
sphingosine 1-phosphate receptors. Eur J. Immunol. 36:149, 2006.
McHeyzer-Williams, L.J., Malherbe, L.P., McHeyzer-Williams, M.G. Checkpoints
in memory B-cell evolution. Immunol. Rev. 211:255, 2006.
McHeyzer-Williams, L.J., Malherbe, L.P., McHeyzer-Williams, M.G. Helper T cell
regulated B cell immunity. Curr. Trends Microbiol. Immunol. 311:59, 2005.
McHeyzer-Williams, L.J., McHeyzer-Williams, M.G. Memory B cell development.
In: The Autoimmune Diseases, 4th ed. Rose, N.R., Mackay, I.R. (Eds.). Elsevier,
St. Louis, 2006, p. 157.
McHeyzer-Williams, M.G., McHeyzer-Williams, L.J., Malherbe, L.P. B cells discriminate the rules of engagement. Immunity 24:125, 2006.
Adaptive and Innate Responses
to Alloantigens
D.B. McKay, A. Shigeoka
urgical and medical advances have provided an
opportunity for life to patients with end-stage
organ disease. One of the most remarkable
advances has been the replacement of organs through
transplantation. Despite remarkable technological
advances, the host immune system must be suppressed
to prevent rejection of the allogeneic transplanted organ.
The suppression of host immunity requires the life-long
use of toxic nonspecific immunosuppressive medications. One experimental method has allowed survival
of transplanted organs without the use of immunosuppressive medications: intravenous exposure of the organ
recipient to donor antigens before transplantation. In
several animal models and human clinical trials, exposure to donor antigens before transplantation downregulated the T-cell responses of recipients to donor antigens.
One focus of our research is the intracellular signaling events that lead to the induction of peripheral T-cell
tolerance by exposure to donor antigens. We found that
intravenous infusion of semiallogeneic donor cells into
S
126 IMMUNOLOGY
2006
recipient mice leads to a series of events that culminate in acquired unresponsiveness to donor antigens
and tolerance to allografts. We discovered that several
proximal T-cell receptor–coupled signaling molecules are
altered in peripheral T cells of the recipient mice. We
are investigating how these proximal molecules may be
regulated in T cells from recipients that do not reject
their transplanted organs.
In addition, we are interested in the initial events
that regulate activation of recipient T cells, namely, the
events that regulate activation of the cells that present
donor antigens. Several recent studies in animals have
suggested that the family of evolutionarily conserved
cell-surface Toll-like receptors might respond to ligands
released by apoptotic and necrotic tissue and that such
ligands might provide an important molecular trigger
for adaptive responses to ischemic injury. More recently,
we have been investigating how binding of Toll-like
receptors leads to activation of responses to donor
antigens and how to target these mechanisms to prevent allograft recognition and rejection.
PUBLICATIONS
Josephson, M.A., McKay, D.B. Management of pregnancy in the transplant recipient. Adv. Chronic Kidney Dis., in press.
McKay, D.B., Adams, P., Bumgardner, G.L., Davis, C.L., Fine, R.N., Krams, S.M.,
Martinez, O.M., Murphy, B., Pavlakis, M., Tolkoff-Rubin, N., Sherman, M.S.,
Josephson, M. Reproduction and pregnancy in transplant recipients: current practices. Prog. Transplant. 16:127, 2006.
McKay, D.B., Josephson, M. Pregnancy in transplant recipients of solid organs:
effects on mother and child. N. Engl. J. Med. 354:1281, 2006.
McKay, D., Shigeoka, A., Rubinstein, M., Surh, C., Sprent, J. Simultaneous deletion of MyD88 and Trif delays major histocompatibility and minor antigen mismatch allograft rejection. Eur. J. Immunol. 36:1994, 2006.
Zambricki, E., Zal, T., Yachi, P., Shigeoka, A., Sprent, J., Gascoigne, N., McKay,
D. In vivo anergized T cells form altered immunological synapses in vitro. Am. J.
Transplant. 6:2572, 2006.
Blocking CCR5 to Inhibit HIV
Type 1 Infection
D.E. Mosier, R. Nedellec, C. Pastore, A. Ramos,
J. Salkowitz-Bokal, S. Pontow,* L. Ratner,* O. Hartley,**
R. Offord,** J. Overbaugh,*** M. Lederman****
* Washington University School of Medicine, St. Louis, Missouri
** Centre Médical Universitaire, Geneva, Switzerland
*** University of Washington, Seattle, Washington
**** Case Western Reserve University, Cleveland, Ohio
H
IV type 1 (HIV-1), the cause of the AIDS pandemic, infects human cells by sequential binding
of the viral envelope protein to the cell-surface
THE SCRIPPS RESEARCH INSTITUTE
receptors CD4 and CCR5. Because CCR5 binding occurs
after CD4 binding, CCR5 is defined as a coreceptor. The
importance of CCR5 in HIV-1 infection was first appreciated because some persons have a natural mutation
that prevents expression of CCR5. These persons are
resistant to HIV-1 infection, and they have few clinical
consequences of lacking CCR5. These observations led
to the development of CCR5-blocking agents to prevent
HIV-1 infection.
However, HIV-1 can undergo mutations that allow
a second chemokine receptor, CXCR4, to replace the
binding function of CCR5. We have studied the costs
to viral fitness of those mutations and their importance
in resistance to CCR5 inhibitors.
A N T I V I R A L C O M P O U N D S T H AT TA R G E T C C R 5
The normal function of CCR5 is to bind chemokines
and signal cell migration. RANTES (CCL5) is the CCR5binding chemokine with the most potent activity against
HIV-1, but its activity is limited. We prepared synthetic
modifications of the N-terminal domain of RANTES.
We found that the most potent of these compounds,
PSC-RANTES, is 1000 times more effective than
native RANTES at inhibiting HIV-1 infection. A single
injection of PSC-RANTES before inoculation of virus
prevented HIV-1 infection in 100% of mice with severe
combined immunodeficiency repopulated with human
peripheral blood leukocytes. Brief exposure of human
cells to PSC-RANTES leads to prolonged internalization of CCR5.
These properties led to the formulation of PSCRANTES as a topical microbicide to prevent sexual
transmission of HIV-1. Treatment with PSC-RANTES
can prevent vaginal transmission of a chimeric virus
consisting of simian immunodeficiency virus and HIV
in the rhesus macaque model. To ensure that PSCRANTES will be effective against HIV-1 variants
present in Africa, we examined the sensitivity of a
number of recently transmitted isolates from Kenya.
Fortunately, all of the isolates were more sensitive to
PSC-RANTES inhibition than were laboratory HIV-1
isolates from the United States.
M U TAT I O N A L C O S T S O F C O R E C E P T O R S W I T C H I N G
One concern about CCR5-blocking agents such as
PSC-RANTES is that they might select for resistant
viruses that can infect via other chemokine receptors,
such as CXCR4. Although previously we showed that
such “coreceptor switch” mutants can arise during treatment, a detailed analysis revealed that most mutants
have a loss of fitness during coreceptor switching that
IMMUNOLOGY
2006
coincides with a period when neither CCR5 nor CXCR4
supports efficient virus infection.
The mutations in the HIV-1 envelope that drive coreceptor switching occur mainly in the exposed variable
loops (V1/V2 and V3), and different HIV-1 isolates
require as few as 1 mutation or as many as 7 mutations to switch from use of CCR5 to use of CXCR4. Poor
replication on both CCR5- and CXCR4-expressing target cells and increased sensitivity to both CCR5 and
CXCR4 inhibitors were common features of viruses that
were switching coreceptors.
To more fully understand the cost of each mutation
associated with changing coreceptor binding from CCR5
to CXCR4, we reconstructed all possible mutational
pathways between 4 parental CCR5-using viruses and
their CXCR4-using descendants separated by 3–7 mutations. We used site-directed mutagenesis to introduce all
possible combinations of single and multiple mutations
in the HIV-1 envelope gene. These mutated envelopes
were combined with an envelope-deficient reporter
virus to make HIV-1 particles capable of only a single
cycle of infection.
Mutations in variable loops 1 and 2 of the envelope
improved the use of CCR5 but did not permit infection
via CXCR4. Mutations in variable loop 3 led to use of
CXCR4 for viral entry, but only poorly. Combinations of
mutations in all 3 variable loops improved the ability
of the virus to use CXCR4. The sequence in which
mutations were introduced was critical. The probability of coreceptor switching is thus constrained by having to make the right mutation at the right place at the
right time.
Using chimeric coreceptors with the 4 extracellular
domains derived from either CCR5 or CXCR4, we also
mapped the domains of CCR5 or CXCR4 required for
infection by each of the mutated envelopes. The initial
stage of coreceptor switching favored use of the second
extracellular domain of CXCR4, and 2 mutants required
only this domain for infection. This result allows the
mapping of mutations near the tip of variable region 3
of the envelope that must confer binding to extracellular loop 2 of CXCR4. The results from these mapping
studies of envelope interactions with coreceptors will
enable us to predict the likelihood of resistance-conferring mutations in patients during clinical trials of
CCR5 inhibitors.
PUBLICATIONS
Pastore, C., Nedellec, R., Ramos, A., Pontow, S., Ratner, L., Mosier, D.E. Human
immunodeficiency virus type 1 coreceptor switching: V1/V2 gain-of-fitness mutations compensate for V3 loss-of-fitness mutations. J. Virol. 80:750, 2006.
THE SCRIPPS RESEARCH INSTITUTE
127
Control of Cytokine Expression
by Arginine Methylation
K.A. Mowen, D.M. Hill, S. Hemmers, K. Bonham
elper T cells can be divided into 2 distinct populations on the basis of their immune specificity and cytokine profiles. Type 1 helper T cells
produce IFN-γ and are responsible for cell-mediated
immunity; type 2 helper T cells secrete IL-4 and are
associated with the humoral immune response. These
2 types of cells have been associated with susceptibility to malignant, infectious, allergic, and autoimmune
diseases. The improper development of type 2 helper
T cells can lead to allergy and asthma, and an overactive response by type 1 helper T cells can lead to autoimmune diseases such as type 1 diabetes.
Because of the opposing roles of the 2 types in
immune function, the development and migration of
helper T cells must be tightly regulated. Indeed, the
discrete subsets, type 1 and type 2, reciprocally antagonize the maturation and behavior of each other in the
immune response, resulting in a population of helper
T cells that is primarily type 1 or type 2. Thus, manipulating the ratio of type 1 to type 2 T helper cells provides an intriguing avenue of therapy, and understanding
the molecular events that control lineage-specific cytokine expression may provide useful tools for modulating the helper T cell response.
Although several lineage-specific and nonspecific
transcription factors are required for the development
and function of type 1 and type 2 helper T cells, less
is known about the events that occur after the reactivation of type 1 and type 2 effector populations and
result in the disparate cytokine profiles of the 2 types
of helper T cells. Signal transduction pathways use posttranslational modifications to translate changes in the
extracellular milieu into environment-sensitive gene
expression in a timely and efficient fashion.
Phosphorylation of serine, threonine, and tyrosine
residues and protein ubiquitination has been widely
studied. Although methylation of arginine residues was
discovered more than 30 years ago, it has only recently
aroused renewed interest. Arginine methylation of
proteins by members of the protein arginine methyltransferase (PRMT) family regulates the subcellular
localization of the methylated proteins and modulates
protein-protein interactions.
H
128 IMMUNOLOGY
2006
We discovered a unique contribution of arginine
methylation to cytokine gene expression downstream of
signaling by T-cell receptors. Our goal is to investigate
more broadly the role for arginine methylation in immune
function, including further study of helper T cells and
other immune cell types. We also plan to examine the
upstream regulation of PRMT expression and activity
and to characterize the effects of ablation or suppression of PRMT expression. Understanding the role of
posttranslational modifications, such as arginine methylation, of proteins that are key in regulating cytokine
production will give us novel targets in diseases induced
or exacerbated by the cytokine environment, such as
inflammatory arthritis.
Analysis of Immune Learning
in B Lymphocytes
D. Nemazee, A. Gavin, D. Aït-Azzouzene, C. Huber, T. Ota,
J. Vela, B. Duong, P. Skog, M. Lim
he main goal of our research is to understand
how lymphocytes distinguish between self and
nonself antigens. Because antigen receptors on
lymphocytes are assembled from component parts
through an essentially random mechanism, many lymphocytes have self-reactive receptors. Regulation of such
autoreactive specificities may be important to prevent
autoimmune disease and to ensure efficient response
to microbes.
The development of B lymphocytes is a multistep process punctuated by the somatic generation of antibody
heavy and light chain genes through DNA recombination,
which is catalyzed by the products of recombinase
activator gene 1 (RAG-1) and RAG-2. Because V(D)J
recombination is imperfect and error prone, pre-B and
B cells are endowed with sensing mechanisms to detect
protein expression of heavy chains and assembled heavy
and light chains (i.e., intact surface IgM). A major function of the expression of immunoglobulin in immature
B cells is signaling to downregulate recombinase activity and to stimulate developmental progression. Newly
formed B-cell receptors are also screened for autoreactivity. These quality control mechanisms rely on signaling by antigen receptors.
Previously, we showed that B cells with autoreactive
receptors do not downregulate recombination because
of excessive signaling through the antigen receptor,
T
THE SCRIPPS RESEARCH INSTITUTE
resulting in “receptor editing,” a process in which previously expressed genes for antibody light chains are
inactivated and replaced by secondary DNA recombination. More recent data indicated that editing can
also play an important role in inactivating and replacing receptor genes that are underexpressed at the protein level. In this situation, subnormal expression of
unligated surface immunoglobulin does not provide a
needed signal.
These recent results suggest that quality control of
newly formed B lymphocytes is surprisingly stringent
and that through recombinase regulation B cells are
often able to “repair” unacceptable light-chain genes
by replacing the unacceptable genes with new genes.
Because of the apparent efficiency of the editing process, we suspect that we have uncovered a major cellular “proofreading” pathway.
A key question of current interest is how signaling
through the antigen receptor regulates editing. A major
nuclear end point is the regulation of RAG transcription.
We are assessing the biochemical signaling pathways by
which the signal from antigen receptors regulate RAG
transcription. Recent studies suggested that NF-κB and
rel transcription factors may be involved in both positive
and negative regulation of the RAG genes. We have also
made progress in our understanding of the triggering
involved in B-cell positive selection, in which innocuous
B-cell receptors, via tonic signaling, activate a signaling
cascade that involves the activity of phosphatidylinositol3′-kinase and recruited effectors, including phospholipase C γ2 and Akt. Tonic signaling refers to the weak
signal generated by the unoccupied B-cell receptor for
antigen. This pathway appears to be inactivated in
autoreactive immature B cells, a finding that probably
explains why the time frame of editing is limited.
To assess the role of receptor editing in preventing
unwanted autoreactivity, we generated mice that have
a defect in receptor editing. These mutant mice lack a
functional recombining sequence/κ light chain–deleting
element, which is involved in destructive editing of loci
for κ light chains in cells that go on to rearrange either
a second allele for κ light chains or genes for λ light
chains. These mice are being assessed for their ability
to produce autoantibodies and to accelerate autoimmune disease when crossbred with mice that are prone
to autoimmunity.
In other studies, we focused on the cues that mature
B cells use to distinguish self from nonself. Fully mature
recirculating B cells can be rapidly inactivated and
IMMUNOLOGY
2006
induced to apoptosis when confronted with tissue antigen, whereas the same cells are able to respond to
antigens expressed by microbes. We are investigating
both the death pathway involved in self-tolerance and
the nature of the signals that prevent this pathway in
responses to nonself antigens. Recently, we found that
the ability of B cells to distinguish self from nonself in
this setting is independent of T lymphocytes and instead
most likely involves a novel pathway of self-recognition.
We are testing the hypothesis that signaling by Toll-like
receptors is not required for this discrimination. We
are also exploring the idea that immune tolerance in
mature B cells depends on specific costimulation by
self-tissue, a mode of signaling akin to missing selfrecognition by natural killer cells.
PUBLICATIONS
Aït-Azzouzene, D., Gavin, A.L., Skog, P., Duong, B., Nemazee, D. Effect of cell:cell
competition and BAFF expression on peripheral B cell tolerance and B-1 cell survival in transgenic mice expressing a low level of Igκ-reactive macroself antigen.
Eur. J. Immunol. 36:985, 2006.
Aït-Azzouzene, D., Verkoczy, L., Duong, B., Skog, P., Gavin, A.L., Nemazee, D.
Split tolerance in peripheral B cell subsets in mice expressing a low level of Igκreactive ligand. J. Immunol. 176:939, 2006.
Collins, C.E., Gavin, A.L., Migone, T.S., Hilbert, D.M., Nemazee, D., Stohl, W. B
lymphocyte stimulator (BLyS) isoforms in systemic lupus erythematosus: disease
activity correlates better with blood leukocyte BLyS mRNA levels than with plasma
BLyS protein levels. Arthritis Res. Ther. 8:R6, 2005.
Gavin, A.L., Hoebe, K., Duong, B., Ota, T., Martin, C., Beutler, B., Nemazee, D.
Minor role for toll-like receptor signaling in adjuvant-enhanced antibody responses.
Science, in press.
Nemazee, D. Receptor editing in lymphocyte development and central tolerance.
Nat. Rev. Immunol. 6:728, 2006.
Nemazee, D., Gavin, A., Hoebe, K., Beutler, B. Immunology: Toll-like receptors
and antibody responses. Nature 441:E4, 2006.
Watson, L.C., Moffatt-Blue, C.S., McDonald, R.Z., Kompfner, E., Aït-Azzouzene,
D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeney, A.J. Paucity of V-DD-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and Tdt+/+ mice. J. Immunol. 177:1120, 2006.
Toll-like Receptor 9 Signaling by
Adenoviruses That Use CD46
Receptors
M. Martinez-Iacobelli, G.R. Nemerow
uman adenoviruses are potent activators of the
innate immune response, a feature that limits
their use for in vivo gene transfer. However, the
precise mechanisms by which different adenoviruses
trigger innate immunity have not been fully elucidated.
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In recent studies, we analyzed different types of adenoviruses that use coxsackievirus-adenovirus receptors
or CD46 as their primary receptors for the ability of the
viruses to trigger innate immune responses as measured
by production of type I interferon in human peripheral
blood mononuclear cells.
We found that adenoviruses that use CD46 preferentially induced production of IFN-α and that this finding
most likely was due to activation of Toll-like receptor 9,
because an oligonucleotide antagonist of the receptor
inhibited cytokine expression. Moreover, empty/immature adenovirus particles that lacked double-stranded
DNA did not induce interferon production even though
they were fully capable of binding to and entering host
cells. In further studies, we found that epithelial cells
that supported equivalent levels of infection by adenoviruses that used coxsackievirus-adenovirus receptors
and by adenoviruses that used CD46 produced significantly higher amounts of interferon upon infection by
the viruses that used CD46.
These findings indicate that distinct receptor-mediated entry pathways may play a pivotal role in activation
of Toll-like receptor 9 by adenovirus. The findings also
have implications for the development of safer adenovirus vectors for clinical applications.
Improving Adenovirus
Transduction of Human
Myeloid Cells
R. Nepomuceno, L. Pache, G.R. Nemerow
endritic cells are ideal targets for immunomodulatory regimens to treat genetic or acquired
diseases. However the lack of coxsackievirusadenovirus receptors on dendritic cells has stymied
transfer of genes to these cell types via type 5 adenoviruses. In recent studies, we found that the fiber knob
derived from adenovirus type 37 (37FK) could significantly enhance adenovirus-mediated gene transfer to
primary human monocytes and to dendritic cells but
not to T and B lymphocytes. Fiber knobs derived from
other adenovirus strains, including type 5 and type 16,
did not have this ability. Enhancement of gene transfer
by 37FK depended on sialic acid, because removal of
sialic acid residues by treatment with neuraminidase
abrogated gene transfer. Moreover, lectins with speci-
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ficity for α2,6-linked sialic acid residues, but not lectins
with specificity for α2,3-linked sialic acid residues, could
inhibit gene transfer by 37FK. In further investigations,
we found that 37FK bound directly to adenovirus particles and thereby increased virus binding to monocytic cells.
We concluded that an electrostatic interaction
between the positively charged 37FK and the negatively charged virus capsid and cell-surface sialic acid
residues results in the formation of a ternary complex
that potentiates adenovirus infection and gene transfer. These findings may point the way for improving
gene delivery to dendritic cells.
Adenovirus-Mediated Disruption
of Endosomes and Development
of Nanoparticle Delivery Methods
J.G. Smith, C. Wiethoff,* S. Police,** C.Y. Lai,
V. Kickhoefer,*** L. Rome,*** G.R. Nemerow
* Loyola University Medical Center, Maywood, Illinois
** Cell Genesys, Inc., San Francisco, California
*** University of California, Los Angeles, California
he mechanisms by which nonenveloped viruses,
including adenovirus, penetrate the barrier of the
host cell endosomal membrane are not well understood. In previous studies, we identified an internal
capsid protein, designated protein VI, that may mediate
the disruption of the early endosome upon partial disassembly of the virion at low pH. To test this hypothesis, we recently examined the infectivity of a panel of
mutant adenoviral particles that contain single amino
acid substitutions in the putative membrane-reactive
domain of protein VI.
Using a quantitative fluorescence imaging device,
we found that several of the particles with mutant protein VI molecules had mildly reduced infectivity compared with that of wild-type virions. Further biochemical
assays revealed that the reduced infectivity was not due
to defects in virion assembly or disassembly. Currently,
we are evaluating the membrane lytic and endosomedisrupting properties of the mutant adenoviruses; we
expect that some of the mutants will have defects in
these activities. In keeping with this expectation, we
found that mutations in protein VI that affect virus
infection also reduce binding of protein VI to liposomes.
Finally, efforts are under way to determine if wild-type
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THE SCRIPPS RESEARCH INSTITUTE
protein VI molecules can be incorporated into naturally
occurring nanoparticles in an attempt to improve gene
delivery to host cells.
Structure Analyses of Adenovirus
via Electron Cryomicroscopy and
X-ray Diffraction
S. Saban,* P. Stewart,* V. Reddy, G.R. Nemerow
* Vanderbilt University School of Medicine, Nashville, Tennessee
denovirus is one of the largest macromolecular
complexes whose structure has been analyzed by
using electron cryomicroscopy or x-ray diffraction. In ongoing studies, we are using electron cryomicroscopy and images of approximately 2000 particles to
determine the adenovirus structure to 6–7 Å. At this level
of resolution, we can clearly resolve multiple α-helices
present in the penton base, protein VI, hexon, and protein IIIa. These studies have also revealed the location
and potential associations of the adenovirus proteins
located on the inner capsid surface that help stabilize
the virus before its disassembly in the early endosome.
The pseudoatomic model generated from the electron
cryomicroscopy data was used to facilitate structural
analyses of the virus by x-ray diffraction. We found that
large single crystals of adenovirus diffracted to about
5-Å resolution at different synchrotron beam lines.
Recent studies indicated that the adenovirus crystals
can be frozen, thereby allowing the collection of nearly
complete electron cryomicroscopy data sets from single
crystals. Generation of electron density maps from x-ray
diffraction data revealed distinct features of the inner
core of the virus. This new information may provide
further insights into the assembly and disassembly of
adenovirus as well as the mode of viral DNA uncoating and recognition by Toll-like receptor 9.
A
PUBLICATIONS
Maginnis, M.S., Forrest, J.C., Kopecky-Bromberg, S.A., Dickeson, S.K., Santoro,
S.A., Zutter, M.M., Nemerow, G.R., Bergelson, J.M., Dermody, T.S. β1 Integrin
mediates internalization of mammalian reovirus. J. Virol. 80:2760, 2006.
Nepomucena, R., Pache, I., Nemerow, G.R. Enhancement of gene transfer to
human myeloid cells by adenovirus-fiber complexes. Mol. Ther., in press.
Nicklin, S.A., Wu, E., Nemerow, G.R., Baker, A.H. The influence of adenovirus
fiber structure and function on vector development for gene therapy. Mol. Ther.
12:384, 2005.
Saban, S.D., Silvestry, M., Nemerow, G.R., Stewart, P.L. Visualization of α-helices in
a 6 Å resolution cryoEM structure of adenovirus allows refinement of capsid protein
assignments. J. Virol., in press.
IMMUNOLOGY
2006
Wodrich, H., Cassany, A., D‚Angelo, M.A., Guan, T., Nemerow, G., Gerace, L.
Adenovirus core protein pVII is translocated into the nucleus by multiple import
receptor pathways. J. Virol. 80:9608, 2006.
Enhancement of Lymphocyte
Cross Talk by Engagement of
the NKG2D Receptor
H. Zhou, Y. Luo, C.D. Kaplan, J.A. Krueger, S.H. Lee,
R. Xiang, R.A. Reisfeld
cells targeting tumor-associated antigens are
readily detectable in cancer patients who have
received cancer vaccines, but often the cells do
not eradicate tumors. Thus, established tumors can
apparently induce immune tolerance through as yet
poorly defined mechanisms. We hypothesized that
immunization strategies that target different areas of
the immune system could overcome this immune tolerance. We used interactions between the NKG2D
receptor and its ligand to test this hypothesis, because
the receptor occurs at the crossroad between innate
and adaptive immunity.
NKG2D, a stimulatory lectinlike receptor, is
expressed on natural killer cells, activated CD8+ T
cells, γζ T cells, and activated macrophages and
mediates costimulatory signals for CD8+ T cells and
stimulatory signals for natural killer cells and macrophages. In addition, NKG2D ligands are related to
MHC class I molecules, which in mice include products
of the H60 ligand. Importantly, in syngeneic mice,
ectopic expression of NKG2D ligands causes natural
killer cell–mediated rejection of transfected tumor cells
and primes cytotoxic T lymphocytes, which are
responsible for rejecting subsequent challenges by
tumor cells that do not express the NKG2D ligand.
In previous studies, we showed that engagement
of the NKG2D receptor markedly improved the antitumor efficacy of a DNA vaccine encoding both the
NKG2D ligand H60 and the inhibitor of apoptosis protein survivin. This combination vaccine activated both
innate and adaptive antitumor immunity and resulted
in improved protection against tumors of different origins and with different levels of expression of NKG2D.
More recently, we found that this combination vaccine
induces intense cross talk between dendritic cells,
natural killer cells, and T cells. Depletion of natural
killer or CD8 + T cells led to a decrease in activation
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131
of dendritic cells, indicating the positive helper function
of these cells in the activation of dendritic cells in vivo.
We also found that depletion of CD8+ T cells or natural killer cells during the priming, but not the effector
phase, led to reduced activity of natural killer or cytotoxic T cells, respectively. Thus, indirect cross talk exists
between these cells and is mediated through dendritic
cells or by direct interactions between natural killer
cells and CD8+ T cells.
In contrast to natural killer and CD8+ T cells, CD4+
T cells appear to negatively regulate these effector cells.
Depletion of CD4+ cells during the priming phase led to
the activation of dendritic cells, natural killer cells, and
CD8 + T cells and to enhanced activity of natural killer
cells, suggesting the existence of CD4+CD25+ T regulatory cells. Because CD4+CD25+ cells lack expression of
the NKG2D receptor, even after activation, this finding
could explain the lack of enhancement of activation and
activity of T regulatory cells despite the profound activation of dendritic cells, natural killer cells, and CD8+
T cells. Thus, by engaging the NKG2D receptor, our
vaccine preferentially activated natural killer and CD8+
T cells, a situation that might have tilted the balance
toward immune surveillance and breakage of peripheral
tolerance to tumor antigens mediated by T regulatory
cells. Our finding that depletion of CD4+ T cells during
the effector phase also reduced the activity of cytotoxic T cells suggests that CD4+ T-cell help is required
to maintain antigen-specific CD8+ T cells in vivo.
In our experimental model in which attenuated Salmonella typhimurium specifically delivers the DNA
encoding the pH60/survivin vaccine to Peyer’s patches,
these secondary lymphoid tissues most likely are the
location for T-cell priming. The ability of the vaccine
to increase homing of dendritic cells and natural killer
cells to Peyer’s patches but decrease the homing of
CD4 + T cells could presumably be due to changes in
the homing receptor profile of the cells. The upregulation of the chemokine receptor CCR7 we observed especially on CD8+ T cells could occur if a greater percentage
of naive CD8+ T cells home to Peyer’s patches, because
CCR7 is highly expressed on naive T cells and mediates
their homing to these secondary lymphoid tissues. In
this situation, these findings may indicate that the
pH60/survivin vaccine also induced changes in stromal cells in Peyer’s patches, especially because such
cells are the source of ligands of CCR7, such as chemokines CCL19 and CCL21. Because lymphocytes
activated in Peyer’s patches express homing receptors,
132 IMMUNOLOGY
2006
the cells most likely home to the periphery, where they
can combat tumor cells or become quiescent in the
absence of antigen. Currently, determining their exact
fate is difficult because such lymphocytes lack specific
and stable markers.
Taken together, we showed that by preferentially
activating and attracting positive regulators and reducing
negative regulators in Peyer’s patches, our pH60/survivin
DNA vaccine induced increased lymphocyte cross talk
and thereby established a microenvironment more suitable for activation of natural killer cells and T-cell priming. The success of this vaccine in combating tumors
of different origins and with different levels of NKG2D
expression in prophylactic and therapeutic models and
in inducing long-lived immune memory indicates that
activation of the innate and adaptive arms of the immune
system is an attractive strategy to overcome tumorinduced peripheral tolerance.
PUBLICATIONS
Abdollahi, A., Griggs, D.W., Zieher, H., Roth, A., Lipson, K.E., Saffrich, R.,
Grone, H.J., Hallahan, D.E., Reisfeld, R.A., Debus, J., Niethammer, A.G., Huber,
P.E. Inhibition of αvβ3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy. Clin. Cancer Res. 11:6270, 2005.
THE SCRIPPS RESEARCH INSTITUTE
protective effects of APC is incompletely understood.
Protein C is activated to APC by the key procoagulant
enzyme thrombin bound to thrombomodulin on the
surface of endothelial cells. APC in turn downregulates
generation of thrombin in a negative feedback loop.
Previously, we showed that APC signaling in endothelial cells requires binding to endothelial protein C
receptor and activation of protease-activated receptor 1
(PAR1), the thrombin receptor. Thrombin-PAR1 signaling has well-established proinflammatory effects, including disruption of endothelial barrier function, raising
the question of how the same receptor can also mediate protective effects of APC. Large-scale gene expression profiling indicated that APC-PAR1 downregulated
transcript levels of proapoptotic proteins and that some
of these transcripts were upregulated by thrombin-PAR1.
Furthermore, APC-PAR1 had powerful endothelial barrier protective effects through cross-activation of the
sphingosine 1-phosphate (S1P) signaling pathway
(Fig. 1). Incubation of an endothelial monolayer with
Allen, B.J., Raja, C., Rizvi, S., Li, Y., Tsui, W., Graham, P., Thompson, J.F., Reisfeld, R.A., Kearsley, J. Intralesional targeted alpha therapy for metastatic
melanoma. Cancer Biol. Ther. 4:1318, 2005.
Mahanivong, C., Krueger, J.A., Bian, D., Reisfeld, R.A., Huang, S. A simplified
closing strategy for the generation of an endothelial cell selective recombinant adenovirus vector. J. Virol Methods 135:127, 2006.
Osenga, K.L., Hank, J.A., Albertini, M.R., Gan, J., Sternberg, A.G., Eickhoff, J.,
Seeger, R.C., Matthay, K.K., Reynolds, C.P., Twist, C., Krailo, M., Adamson, P.C.,
Reisfeld, R.A., Gillies, S.D., Sondel, P.M. A phase 1 clinical trial of the hu14.18IL2 (EMD 273063) as a treatment for children with refractory or recurrent neuroblastoma and melanoma: a study of the Children’s Oncology Group. Clin. Cancer
Res. 12:1750, 2006.
Schrama, D., Reisfeld, R.A., Becker, J.C. Antibody targeted drugs as cancer therapeutics. Nat. Rev. Drug Discov. 5:147, 2006.
Schrama, D., Voigt, H., Eggert, A.O., Xiang, R., Reisfeld, R.A., Becker, J.C. Therapeutic efficacy of tumor-targeted IL2 in Ltα–/– mice depends on conditioned T
cells. Cancer Immunol. Immunother. 55:861, 2006.
Zhou, H., Luo, Y., Kaplan, C.D., Krueger, J.A., Lee, S.H., Xiang, R., Reisfeld, R.A.
A DNA-based cancer vaccine enhances lymphocyte cross talk by engaging the
NKG2D receptor. Blood 107:3251, 2006.
Protective Protease-Activated
Receptor 1 Signaling by the
Protein C Pathway
M. Riewald, C. Feistritzer, R.A. Schuepbach, R. Lenta
A
ctivated protein C (APC), an anticoagulant serine protease, has been approved for treatment
of severe sepsis, but the molecular basis for the
F i g . 1 . Inflammatory disorders such as sepsis are associated
with increased permeability of the endothelial cell monolayer at the
blood-tissue interface. APC-mediated enhancement of endothelial
barrier integrity depends on binding of APC to endothelial protein C
receptor and activation of PAR1, cellular sphingosine kinase-1 (SK1),
and S1P receptor-1 (S1P1). Thrombin can affect barrier integrity in
at least 3 ways: proinflammatory signaling by higher concentrations
can disrupt endothelial barrier integrity, incubation with low concentrations has a barrier-enhancing effect, and activation of protein
C by thrombin on the endothelial cell surface is linked to powerful
autocrine protective signaling by the generated APC.
low concentrations of thrombin had a similar barrier
protective effect.
Taken together, these results establish that PAR1
can mediate opposite effects on gene expression and
barrier integrity, and they reveal an unexpected role for
cross-communication between the prototypical barrierprotective S1P and barrier-disruptive PAR1 pathway.
IMMUNOLOGY
2006
Using the anticoagulant double mutant thrombin
W215A/E217A, we recently showed that activation of
protein C by thrombin on the endothelial cell surface
is mechanistically linked to highly efficient PAR1-dependent autocrine protective signaling by the generated
APC. These results suggest that W215A/E217A may
have powerful protective effects in systemic inflammation through signaling by the generated APC.
To dissect how signaling by the same receptor leads
to different biological outcomes, we are using a panel
of monoclonal antibodies to analyze how specific populations of PAR1 are affected by APC and thrombin on
the endothelial surface. Genetically modified mouse
strains that express in endothelial cells PAR1 variants
that are efficiently activated by APC but not by thrombin are currently used in models of systemic inflammation to dissect the in vivo role of PAR1 signaling by
exogenous and endogenously generated APC.
PUBLICATIONS
Feistritzer, C., Lenta, R., Riewald, M. Protease-activated receptors-1 and -2 can
mediate endothelial barrier protection: role in factor Xa signaling. J. Thromb.
Haemost. 3:2798, 2005.
Feistritzer, C., Mosheimer, B.A., Sturn, D.H., Riewald, M., Patsch, J.R., Wiedermann, C.J. Endothelial protein C receptor-dependent inhibition of migration of
human lymphocytes by protein C involves epidermal growth factor receptor. J.
Immunol. 176:1019, 2006.
Chemical and Genetic
Approaches to Disease
H. Rosen, G. Sanna, E. Jo, P. Gonzalez-Cabrera, A. Don,
S. Cahalan, D. Marsolais, S. Brown, M.-T. Schaeffer,
J. Chapman
ymphocytes develop in the thymus (T cells) and
bone marrow (B cells) and upon maturation egress
from their sites of development to enter the bloodstream. Because the numbers of lymphocytes with specific receptors for antigen are limited, the probability of
random productive collision of specific lymphocyte, antigen, and antigen-presenting cell in a permissive environment for an efficient immune response is low. In
the immune system, this probability is enhanced by
rapid recirculation of lymphocytes through secondary
lymphoid organs, so that each lymphocyte has many
opportunities to respond to its specific antigen. A sufficient number of blood lymphocytes are therefore essential for the development of efficient immune responses
and are maintained by the recirculation of lymphocytes
through the secondary lymphoid organs.
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133
Using small synthetic druglike organic molecules,
we elucidated specific molecular gatekeepers that control the numbers of recirculating lymphocytes. These
compounds alter lymphocyte trafficking and induce
clinically useful immunosuppression by activating a
single sphingosine 1-phosphate (S1P) receptor subtype, S1P1. Using 2-photon fluorescence and selective
agonists and antagonists of this receptor, we directly
imaged the control of lymphocyte egress from lymph
nodes in living systems.
M O L E C U L A R C O N T R O L O F LY M P H O C Y T E M I G R AT I O N
Molecular control of the migration of lymphocyte
subsets within the recirculation pathway is a fundamental issue of therapeutic importance. Although transplantation involves the sensitization of an immunologically
naive host, treatment of most autoimmune diseases
requires intervention in a sensitized host that already
has autoreactive effector T cells in the periphery. We
approached this problem by examining the role of
the S1P system in the control of lymphocyte egress
from lymph nodes and thymus, and using chemical
approaches, we revealed differences between intrinsic
lymphocyte and barrier mechanisms that alter lymphocyte migration. The rapid reversibility of agonist-mediated lymphocyte arrest coupled with its competitive
reversal by molar excess of antagonist strongly support
an endothelial barrier mechanism.
ROLE OF SIGNALING LIPIDS IN THE CONTROL OF
LUNG INTEGRITY
Pulmonary abnormalities, including acute respiratory distress syndrome, are characterized by disruption
of pulmonary integrity and edema that compromise respiratory function. S1P is a lipid mediator synthesized
and/or stored in mast cells, platelets, and epithelial cells,
and its production is upregulated by the proinflammatory cytokines IL-1 and TNF. We used agonists and
antagonists of receptors to examine this system.
S1P1, found on lung capillaries, tightens capillary
junctions and protects from leakage. Antagonists of
S1P1 therefore promote lung leakage from the vascular
side. We found that changes in signaling-lipid regulation of lung barrier function from either vascular or
endothelial interfaces induce acute pulmonary edema.
The S1P-receptor axis may therefore be an important
independent variable in the control of lung barrier function, and its activation and modulation in serious human
diseases such as acute respiratory distress syndrome
are under study.
S T R AT E G I C O U T L O O K
The S1P system thus regulates adaptive immunity
in at least 3 discrete ways: egress of naive cells from
134 IMMUNOLOGY
2006
lymph nodes, sequestration of effector T cells in lymph
nodes, and egress of mature medullary T cells from the
thymus. The system can therefore alter both the peripheral diversity of lymphocyte responses and the efficiency
of T-cell activation by misdirecting T cells to the wrong
lymph nodes and by inhibiting the egress of antigenspecific effector T cells from lymph nodes after antigen activation and clonal proliferation.
These effects can alter adaptive immune responses
and the expression of tissue damage while providing
potentially significant advantages to patients by sparing
innate host defenses to bacteria and pathogenic fungi.
The fine molecular control of this system and its effect
on immune responses as a fundamental approach to
organization of the immune system and potential therapeutic agents will remain our primary focus.
The recent discovery of a critical role for chemically
tractable S1P receptors in the innate immune system is
a new focus in molecular pathogenesis of inflammatory
lung disease that is of long-term interest to us.
THE SCRIPPS RESEARCH INSTITUTE
PUBLICATIONS
Alfonso, C., McHeyzer-Williams, M.G., Rosen, H. CD69 down-modulation and
inhibition of thymic egress by short- and long-term selective chemical agonism of
sphingosine 1-phosphate receptors. Eur. J. Immunol. 36:149, 2006.
Chun, J., Rosen, H. Lysophospholipid receptors as potential drug targets in tissue
transplantation and autoimmune diseases. Curr. Pharm. Des. 12:161, 2006.
Gong, Q., Ou, Q., Ye, S., Lee, W.P., Cornelius, J., Diehl, L., Lin, W.Y., Hu, Z., Lu,
Y., Chen, Y., Wu, Y., Meng, Y.G., Gribling, P., Lin, Z., Nguyen, K., Tran, T., Zhang,
Y., Rosen, H., Martin, F., Chan, A.C. Importance of cellular microenvironment and
circulatory dynamics in B cell immunotherapy. J. Immunol. 174:817, 2005.
Li, Z., Chen, W., Hale, J.J., Lynch, C.L., Mills, S.G., Hajdu, R., Keohane, C.A.,
Rosenbach, M.J., Milligan, J.A., Shei, G.J., Chrebet, G., Parent, S.A., Bergstrom,
J., Card, D., Forrest, M., Quackenbush, E.J., Wickham, L.A., Vargas, H., Evans,
R.M., Rosen, H., Mandala, S. Discovery of potent 3,5-diphenyl-1,2,4-oxadiazole
sphingosine-1-phosphate-1 (S1P1) receptor agonists with exceptional selectivity
against S1P2 and S1P3. J. Med. Chem. 48:6169, 2005.
Martinez, X., Kreuwel, H.T., Redmond, W.L., Trenney, R., Hunter, K., Rosen, H.,
Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in nonobese
diabetic mice is restored by insulin-dependent diabetes resistance alleles. J.
Immunol. 175:1677, 2005.
Rosen, H., Goetzl, E.J. Sphingosine 1-phosphate and its receptors: an autocrine
and paracrine network. Nat. Rev. Immunol. 5:560, 2005.
Sanna, M.G., Wang, S.K., Gonzalez-Cabrera, P.J., Don, A., Marsolais, D., Matheu,
M.P., Wei, S.H., Parker, I., Jo, E., Cheng, W.C., Cahalan, M.D., Wong, C.H.,
Rosen, H. Enhancement of capillary leakage and restoration of lymphocyte egress
by a chiral S1P1 antagonist in vivo. Nat. Chem. Biol. 2:434, 2006.
THE SCRIPPS RESEARCH INSTITUTE MOLECULAR
SCREENING CENTER
The Scripps Research Institute Molecular Screening Center is a national center for small-molecule screening and is part of the National Institutes of Health (NIH)
Molecular Libraries Screening Centers Network of the
NIH Roadmap. The Scripps center is distributed between
the La Jolla and the Florida campuses; its component
parts are assay development, chemistry, assay implementation, and pharmacokinetics. These 4 cores are unified
in a single data environment by the Informatics Core.
The mission of the center is to use the NIH library
of more than 60,000 individual compounds to screen
molecular and cell-based targets, which are accepted
through an NIH-wide peer-reviewed application process,
for proof-of-concept small-molecule probes. Researchers
at the Scripps center have successfully identified and
published proof-of-concept molecules in the center’s
first year of operations. Compounds discovered by this
process are public information that can be accessed
by all scientists through the PUBCHEM database of the
National Center for Biotechnology Information.
The Scripps center joins human excellence with
state-of-the-art robotics and informatics. The combination can provide new insights into the basic science of
small-molecule probes of physiologic and pathologic
function, move scientific fields forward, and, over time,
provide new, significant insights into therapies for
human diseases.
Wei, S.H., Rosen, H., Matheu, M.P., Sanna, M.G., Wang, S.K., Jo, E., Wong, C.-H.,
Parker, I., Cahalan, M.D. Sphingosine 1-phosphate type 1 receptor agonism
inhibits transendothelial migration of medullary T cells to lymphatic sinuses. Nat.
Immunol. 6:1228, 2005.
Protease Pathways in
Inflammation, Angiogenesis,
and Cancer
J. Ahamed, M. Kerver, T. Kurokawa, Y. Kurokawa, F. Niessen,
H. Petersen, H. Versteeg, P.J. Hogg,* M. Friedlander,
B.M. Mueller,** W. Ruf
* University of New South Wales, Sydney, Australia
** La Jolla Institute for Molecular Medicine, San Diego, California
D I S U L F I D E / T H I O L E X C H A N G E A S A R E G U L AT O R Y
SWITCH FOR RECEPTOR FUNCTION
ctivation of coagulation by the cell-surface receptor tissue factor (TF) is induced by binding of its
ligand, the serine protease factor VIIa. In addition,
the TF-VIIa complex triggers cell signaling by cleaving
and activating the G protein–coupled protease-activated
receptor 2 (PAR2), a highly relevant promigratory receptor in tumor biology. However, the physiologic significance of direct TF-VIIa signaling remained unclear,
because activation of coagulation generates a number
of proteases that may override direct TF signaling pathways. We identified a surprisingly simple mechanism
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IMMUNOLOGY
2006
by which TF-dependent coagulation is disabled while
preserving TF-VIIa signaling.
An extracellular disulfide (Cys186-Cys209) in TF fits
the criteria for an “allosteric” disulfide; these disulfides
are typically labile because of their bond geometry.
Activation of coagulation requires this disulfide, but
mutational breaking of the disulfide did not impair
TF-VIIa signaling. Protein disulfide isomerase (PDI)
targets this disulfide to disable coagulation. PDI is
an abundant intracellular chaperone and thiol/disulfide exchange catalyst required for protein folding,
but TF-VIIa is regulated by extracellular PDI. PDI suppresses TF coagulant activity by nitric oxide–dependent
redox pathways and protein S nitrosylation, indicating
an unexpected link between oxidative cardiovascular
stress and thrombosis. These data also exemplify a new
role for PDI as an extracellular switch for the functional
specificity of a receptor.
THE SCRIPPS RESEARCH INSTITUTE
Koizume, S., Jin, M.S., Miyagi, E., Hirahara, F., Nakamura, Y., Piao, J.H., Asai,
A., Yoshida, A., Tsuchiya, E., Ruf, W., Miyagi, Y. Activation of cancer cell migration and invasion by ectopic synthesis of coagulation factor VII. Cancer Res.
66:9453, 2006.
Ruf, W. Flow perturbation is linked to endothelial PAR signaling. Arterioscler.
Thromb. Vasc. Biol. 26:962, 2006.
Ruf, W. Is APC activation of endothelial cell PAR1 important in severe sepsis? Yes.
J. Thromb. Haemost. 3:1912, 2005.
Ruf, W., Mueller, B.M. Thrombin generation and the pathogenesis of cancer.
Semin. Thromb. Hemost. 32(Suppl. 1):61, 2006.
Versteeg, H.H., Ruf, W. Emerging insights in tissue factor-dependent signaling
events. Semin. Thromb. Hemost. 32:24, 2006.
Structural Analysis of the
Host-Pathogen Interface
E. Ollmann Saphire, D.M. Abelson, M.L. Fusco,
P R O T E A S E S I G N A L I N G PAT H WAY S I N A N G I O G E N E -
C.R. Kimberlin, J.E. Lee, D.R. Burton, M.K. Hart*
SIS AND CANCER
* U.S. Army Medical Research Institute for Infectious Diseases, Frederick,
We are interested in protease pathways in cancer
and angiogenesis, with a particular focus on signaling by
PARs. Previously, in ex vivo models, we showed that mice
lacking the cytoplasmic domain of TF have enhanced
angiogenesis. Recently, we analyzed the role of PARs
in angiogenesis in vivo and found that PAR2, but not the
thrombin receptor PAR1, plays a central role in angiogenesis. We further found that blockade of TF-VIIa
suppresses angiogenesis in vivo and thus established
that TF-VIIa signaling through PAR2 is the major pathway
in angiogenesis. We plan to further analyze the respective roles of PARs in tumor progression and angiogenesis.
We have also characterized species-specific antibodies that block TF-VIIa signaling through PAR2 on
tumor cells. We found that these antibodies suppress
tumor growth in xenograft models, providing evidence
that PARs not only regulate angiogenesis in host cells
but are an important determinant in tumor progression.
We will evaluate the role of PARs in transgenic mouse
models to provide additional genetic evidence for the
specific role of protease pathways in tumor progression.
PUBLICATIONS
Ahamed, J., Versteeg, H.H., Kerver, M., Chen, V.M., Mueller, B.M., Hogg, P.J.,
Ruf, W. Disulfide isomerization switches tissue factor from coagulation to cell signaling. Proc. Natl. Acad. Sci. U. S. A. 103:13932, 2006.
Bhattacharjee, G., Ahamed, J., Pedersen, B., El-Sheikh, A., Mackman, N., Ruf,
W., Liu, C., Edgington, T.S. Regulation of tissue factor-mediated initiation of the
coagulation cascade by cell surface Grp78. Arterioscler. Thromb. Vasc. Biol.
25:1737, 2005.
Chen, V.M., Ahamed, J., Versteeg, H.H., Berndt, M.C., Ruf, W., Hogg, P.J. Evidence for activation of tissue factor by an allosteric disulfide bond. Biochemistry
45:12020, 2006.
135
Maryland
e are crystallizing proteins that play key roles
in the pathogenesis and lethality of viruses
that cause hemorrhagic fever. The resulting
crystal structures will provide (1) information for the
design of vaccines and inhibitors against the viruses
as the microbes exist naturally and (2) structural templates that will enable us to anticipate and rapidly
respond to newly emerging and synthetic versions of
the viruses and viral proteins.
W
EBOLA VIRUS
At least 10 recognized outbreaks of Ebola virus in
humans have occurred; in each outbreak, 50%–90% of
those infected died. Survival depends on the ability of
the host to mount early and strong immune responses.
However, filoviruses have evolved mechanisms by which
the host immune system is suppressed. For example, the
viral nucleocapsid proteins VP35 and VP24 block interferon-mediated activation of immunomodulatory genes.
Structural analysis of these proteins, alone and in complex with the human proteins that they bind, will provide
insights into viral replication and immunosuppression
and will provide the structural basis for the design of
antiviral compounds and attenuated viral strains.
An additional, unusual feature of the Ebola viral
genome is its ability to encode 2 different glycoproteins,
sGP and GP, from the same gene. These 2 glycoproteins share 295 amino acids of N-terminal sequence,
but a transcriptional editing event causes them to have
136 IMMUNOLOGY
2006
different C-terminal sequences that result in unique
patterns of disulfide bonding, structures, and roles in
pathogenesis. Comparative structural analysis of sGP
and GP should explain how 2 structures arise from the
same sequence, provide templates for the design of
vaccines that elicit antibodies that target the virus rather
than the secreted proteins, and illustrate structural mechanisms by which the virus escapes immune surveillance.
Additional crystal structures of these proteins in
complex with rare human antibodies derived from survivors of Ebola virus infection or with immunotherapeutic agents under development by the U.S. Army
will assist in vaccine design. We recently determined
the crystal structure at 2.0-Å resolution of a potential
immunotherapeutic agent, 13F6-1-2, in complex with
its GP epitope (Fig. 1). 13F6-1-2 contains a rare Vλx
THE SCRIPPS RESEARCH INSTITUTE
determine structural features of epitopes associated
with neutralization and enhancement (Fig. 2).
F i g . 2 . Crystals of envelope protein E of dengue virus serotype 1.
F i g . 1 . Crystal structure of Fab 13F6-1-2 in complex with its
Ebola virus glycoprotein epitope. The antibody light chain is black,
the heavy chain is gray, and the GP peptide is illustrated in balland-stick form.
light chain and has several unusual structural features
in its combining site.
DENGUE VIRUS
Dengue virus is a mosquito-borne flavivirus that
causes up to 100 million infections each year. Infection with dengue virus results in either dengue fever or
the much more severe disease dengue hemorrhagic
fever. Dengue hemorrhagic fever usually occurs upon
secondary infection with a different viral subtype or in
infants born to dengue virus–immune mothers. This
potential antibody-mediated enhancement of infection
is a major concern in the testing and use of vaccines
against dengue virus because antibodies elicited by the
vaccines could trigger severe disease. To aid in vaccine design, we are determining crystal structures of
envelope proteins of contemporary field isolates of
dengue virus, alone and in complex with antibodies, to
PUBLICATIONS
Cárdenas, W.B., Loo, Y.-M., Gale, M., Jr., Hartman, A.L., Kimberlin, C.R.,
Martínez-Sobrido, L., Saphire, E.O., Nichol, S.N., Basler, C.F. Ebola virus VP35
protein binds double-stranded RNA and inhibits α/β interferon production induced
by RIG-I signaling. J. Virol. 80:5168, 2006.
Autoimmune Mechanisms and
Compensatory Responses
N. Sarvetnick, M. Cleary, S. Dabernat, S. Datta, D. Dietz,
C. Fine, N. Hill, H. Hua, M. Kritzik, A. Marleau, P. Secrest,
A. Stotland, D. Yadav, Y.Q. Zhang
ype 1 diabetes occurs when self-reactive T cells
destroy the insulin-producing beta cells in the
islets in the pancreas. The assumption has been
that the fault lies exclusively in the immune system,
but increasingly findings suggest that the targets of
autoimmunity, the islets, may also be defective. Genetic
linkage analysis of nonobese diabetic mice has led to
the identification of critical intervals that confer susceptibility to diabetes. One of these regions, Idd9, is
associated with strong protection from disease when it
T
IMMUNOLOGY
2006
is replaced with the B10 allele. Interestingly, we found
that genes at the Idd9 locus associated with susceptibility
to diabetes control islet resilience to CD8+ T cell–mediated autoimmunity.
Susceptible islets are hyperresponsive to the cytokines TNF and IFN-γ, resulting in increased expression
of the death receptor Fas. Fas upregulation in beta cells
is mediated by TNF receptor 2 (TNFR2), and in nonobese
diabetic mice, colocalization of the receptor with the
adaptor TNF receptor–associated factor 2 in beta cells
is altered. The gene for TNFR2 lies within the candidate
Idd9 interval, and the diabetes-associated variant contains
a mutation adjacent to the binding site for TNF receptor–
associated factor 2. A component of diabetes susceptibility is therefore determined by the target of the autoimmune response, and protective TNFR2 signaling in
islets may inhibit early cytokine-induced damage required
for the development of destructive autoimmunity.
Because insulin-dependent diabetes mellitus is due
to selective destruction of insulin-producing cells, strategies that promote growth of beta cells provide a means
to prevent or reverse this type of diabetes. One approach
is to replace insulin-producing cells by using genetic
engineering or by guiding stem cells (pancreas progenitors) to differentiate into beta cells. The progression of
pancreatic progenitor cells to beta cells is governed by
basic helix-loop-helix transcription factors, which are
regulated by inhibitor of differentiation proteins that
bind to and inhibit the function of the factors. Transcription of inhibitor of differentiation proteins is induced by
bone morphogenetic proteins (BMPs).
We showed that BMP signaling is necessary and
sufficient for proliferation of pancreatic progenitor cells
and that this signaling is correlated with an increase
in the expression of inhibitor of differentiation proteins.
Using a mouse model of regenerating pancreas, we
found that injection of an antibody that inhibits BMP4
significantly reduced cell proliferation and caused an
increase in NeuroD, a basic helix-loop-helix factor
required for the differentiation of pancreatic islet cells.
Therefore, our results indicate that stimulation by BMP4
blocks the differentiation of endocrine progenitor cells
and instead promotes their expansion, thereby revealing a novel model of signaling that explains the balance
between expansion and differentiation of pancreatic
duct epithelial progenitors.
PUBLICATIONS
Flodstrom-Tullberg, M., Hultcrantz, M., Stotland, A., Maday, A., Tsai, D., Fine,
C., Williams, B., Silverman, R., Sarvetnick, N. RNase L and double-stranded
RNA-dependent protein kinase exert complementary roles in islet cell defense during coxsackievirus infection. J. Immunol. 174:1171, 2005.
THE SCRIPPS RESEARCH INSTITUTE
137
Horwitz, M.S., Ilic, A., Fine, C., Sarvetnick, N. Induction of antigen specific
peripheral humoral tolerance to cardiac myosin does not prevent CB3-mediated
autoimmune myocarditis. J. Autoimmun. 25:102, 2005.
Hua, H., Zhang, Y.Q., Dabernat, S., Kritzik, M.N., Dietz, D., Sterling, L., Sarvetnick, N. BMP4 regulates pancreatic progenitor cell expansion through Id2. J. Biol.
Chem. 281:13574, 2006.
Kayali, A.G., Stotland, A., Gunst, K.V., Kritzik, M., Liu, G., Dabernat, S., Zhang,
Y.Q., Wu, W., Sarvetnick, N. Growth factor-induced signaling of the pancreatic
epithelium. J. Endocrinol. 185:45, 2005.
Kim, S.H., Gunst, K.V., Sarvetnick, N. STAT4/6-dependent differential regulation
of chemokine receptors. Clin. Immunol. 118:250, 2006.
Marleau, A.M., Sarvetnick, N. T cell homeostasis in tolerance and immunity. J.
Leukoc. Biol. 78:575, 2005.
Martinez, X., Kreuwel, H.T., Redmond, W.L., Trenney, R., Hunter, K., Rosen, H.,
Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in nonobese
diabetic mice is restored by insulin-dependent diabetes resistance alleles. J.
Immunol. 175:1677, 2005.
Solomon, M., Flodstrom-Tullberg, M., Sarvetnick, N. Differences in suppressor of
cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c
and spontaneously-diabetic NOD recipient mice. Transplantation 15:1104, 2005.
Zhang, Y.Q., Kritzik, M., Sarvetnick, N. Identification and expansion of pancreatic
stem/progenitor cells. J. Cell. Mol. Med. 9:331, 2005.
Promotion of Cell Migration
and Invasion by Tyrosine
Kinase Signaling
D.D. Schlaepfer, J.A. Bernard-Trifilo, X.L. Chen, A. Chi,
D.A. Hanson, S. Hou, S.T. Lim, Y.M. Lim, S.K. Mitra,
J.E. Molina, S. Uryu, A. Wang
e wish to understand how intracellular signaling networks promote complex biological
processes such as cell motility, cell invasion,
and tumor metastasis. We hypothesize that critical intracellular signaling proteins exist within cells that act as
signal “integrators” to process environmental stimuli that
control cell movement. These proteins should be activated by various extracellular inputs and act to regulate
multiple downstream signaling pathways. One such
integrator is focal adhesion kinase (FAK), an intracellular protein-tyrosine kinase that is associated with both
transmembrane integrin and growth factor receptors.
W
CONNECTIONS TO GROWTH FACTOR RECEPTORS
The ErbB family of protein-tyrosine kinase receptors
includes epidermal growth factor receptor (ErbB-1),
ErbB-2, ErB-3, and ErB-4. Overexpression of ErbB-2
is associated with poor prognosis and invasiveness in
cancer in humans. An important early event implicated
in controlling cell migration induced by growth factors
138 IMMUNOLOGY
2006
is FAK activation. Using cells that lacked the gene for
FAK, FAK-reconstituted fibroblasts, and human breast
carcinoma cells in which FAK expression was inhibited
by short inhibitory RNA, we dissected the function of
FAK in ErbB-2/ErbB-3 oncogenic transformation and
cell invasion. We found that these processes depend
on FAK. In many cells, FAK activation promotes binding of the protein-tyrosine kinase c-Src to FAK, thereby
generating a dual FAK-Src signaling complex. In these
studies, ErbB-2/ErbB-3–induced oncogenic transformation depended on a FAK-Src and MAP kinase activation,
whereas ErbB-2/ErbB-3–induced cell invasion was
FAK-Src dependent and independent of MAP kinase.
CONNECTIONS TO INTEGRINS
Overexpression of FRNK, the FAK C-terminal domain,
can inhibit FAK activity, in part by disrupting FAK association with integrins. Analyses of breast tumor samples
revealed that elevated FAK expression occurs with the
development of benign ductal hyperplasia into invasive
carcinomas. We inhibited FAK activity or FAK expression in murine 4T1 breast carcinoma cells via transient
expression of FRNK or stable expression of anti-FAK
short hairpin RNA, respectively.
Expression of anti-FAK short hairpin RNA resulted
in the inhibition of 4T1 cell invasion in vitro and spontaneous 4T1 metastasis after implantation of the cells in
Balb/c mice.* Transient reexpression of wild-type but
not kinase-inactive FAK in 4T1 cells with the anti-FAK
short hairpin RNA promoted in vivo lung metastasis,
and this finding was associated with increased expression of urokinase plasminogen activator. Because the
inhibition of FAK within 4T1 cells was also associated
with increased host survival after tumor cell implantation, our results support the pharmacologic targeting
of FAK activity as a means to inhibit tumor spread.
TA R G E T S O F FA K A C T I V I T Y P R O M O T I N G T U M O R
PROGRESSION
Using stable FRNK overexpression to inhibit FAK
in 4T1 breast carcinoma cells, we found that FRNK
overexpression was not associated with alterations in
cell proliferation or anchorage-independent cell survival
in vitro. Instead, FRNK-expressing 4T1 cells secreted
less vascular endothelial cell growth factor (VEGF) and
formed small avascular tumors, findings associated with
the inhibition of a signaling linkage involving FAK, phosphorylation of FAK at tyrosine 925, and MAP kinase that
regulates expression of VEGF. The biological importance
of this FAK signaling pathway was confirmed through
reconstitution experiments with Src transformation of
THE SCRIPPS RESEARCH INSTITUTE
fibroblasts lacking the gene for FAK in which point
mutations affecting FAK catalytic activity or phosphorylation disrupted the ability of FAK at tyrosine 925 to
promote tumor growth–associated MAP kinase and
VEGF expression. Inhibition of FAK in breast, prostate,
and neuroblastoma cells also resulted in reduced VEGF
expression. These studies provide the first biological
support for Y925 FAK phosphorylation and define a novel
role for FAK activity in promoting a MAP kinase–associated angiogenic switch during tumor progression.
INTEGRIN SIGNALING INDEPENDENT OF FAK
In cell culture, fibroblasts that lack the gene for FAK
have defects in motility but not in proliferation. The
fibronectin-binding integrins α5β1 and α4β1 generate
signals pivotal for cell migration through distinct yet
undefined mechanisms. For α5β1, β1-mediated activation of FAK promotes c-Src recruitment to FAK and the
formation of a FAK-Src signaling complex that promotes
motility. Interestingly, expression of human α4 integrin
in fibroblasts that lack the gene for FAK forms a functional α4β1 receptor that promotes motility of the cells,
equal to that of wild-type FAK-containing fibroblasts
stimulated with α5β1. This α4β1-stimulated signaling
connection was initiated by the cytoplasmic domain of
α4 integrin and involved the activation of Src-family
protein-tyrosine kinases in the absence of FAK. Currently,
we are elucidating the molecular linkage of α4 integrin to
Src and how this signaling pathway promotes neuroblastoma motility, invasion, and tumor progression.
PUBLICATIONS
Benlimame, N., He, Q., Jie, S., Xiao, D., Xu, Y.J., Loignon, M., Schlaepfer, D.D.,
Alaoui-Jamali, M.A. FAK signaling is critical for ErbB-2/ErbB-3 receptor cooperation for oncogenic transformation and invasion. J. Cell Biol. 171:505, 2005.
Bernard-Trifilo, J.A., Lim, S.T., Hou, S., Schlaepfer, D.D., Ilic, D. Analyzing FAK
and Pyk2 in early integrin signaling events. In: Current Protocols in Cell Biology.
Bonifacino, J.S., et al. (Eds.). Wiley, New York, 2006, Chap. 14.7.1.
Hsia, D.A., Lim, S.T., Bernard-Trifilo, J.A., Mitra, S.K., Tanaka, S., den Hertog,
J., Streblow, D.N., Ilic, D., Ginsberg, M.H., Schlaepfer, D.D. Integrin α4β1 promotes focal adhesion kinase-independent cell motility via α4 cytoplasmic domainspecific activation of c-Src. Mol. Cell. Biol. 25:9700, 2005.
Hu, B., Jarzynka, M.J., Guo, P., Imanishi, Y., Schlaepfer, D.D., Cheng, S.Y.
Angiopoietin 2 induces glioma cell invasion by stimulating matrix metalloprotease 2
expression through the αvβ1 integrin and focal adhesion kinase signaling pathway.
Cancer Res. 66:775, 2006.
Mitra, S.K., Lim, S.T., Chi, A., Schlaepfer, D.D. Intrinsic focal adhesion kinase
activity controls orthotopic breast carcinoma metastasis via the regulation of urokinase expression in a syngeneic tumor model system. Oncogene 25:4429, 2006.
Mitra, S.K., Mikolon, D., Molina, J., Hsia, D.A., Hanson, D.A., Chi, A., Lim, S.T.,
Bernard-Trifilo, J.T., Ilic, D., Stupack, D.G., Cheresh, D.A., Schlaepfer, D.D.
Intrinsic FAK activity and Y925 phosphorylation facilitate an angiogenic switch in
tumors. Oncogene 25:5969, 2006.
Urbinati, C., Bugatti, A., Giacca, M., Schlaepfer, D., Presta, M., Rusnati, M. αvβ3
integrin-dependent activation of focal adhesion kinase mediates NF-κB activation
and motogenic activity by HIV-1 Tat in endothelial cells. J. Cell Sci. 118(Pt.
17):3949, 2005.
IMMUNOLOGY
2006
The Consequences of T-Cell
Recognition of Self-Antigens and
Tumor Antigens
L.A. Sherman, X. Martinez, C.-H. Wei, J. Wong,
E. Hamilton-Williams, J.A. Biggs, K.L. Marquardt, R.L. Trenney
he consequence of antigen recognition by naive
CD8+ T cells can be either tolerance or immunity,
depending on the activation status of the antigenpresenting dendritic cells. If a CD8+ T cell recognizes
antigen on a quiescent dendritic cell that has relatively
low levels of expression of costimulatory molecules, then
activation of the T cell results in deletion and tolerance.
Inflammatory signals, such as those due to the presence
of foreign pathogens and activated lymphocytes, activate
dendritic cells to express cell-surface costimulatory
molecules and cytokines. If CD8 + T cells recognize
antigen on activated dendritic cells, the costimulatory
molecules and cytokines prevent deletion and promote
the clonal expansion of the T cells and the development
of effector functions.
Understanding the signals that result in either T-cell
deletion or immunity is of importance in preventing autoimmunity, which represents a failure to control selfdestructive T lymphocytes. This understanding is also
important in promoting tumor immunity, in which the
goal is to promote the autoimmune destruction of tumor
cells. We are comparing the consequence of the interaction of naive CD8+ T lymphocytes with a transgenic
self-antigen (the influenza virus hemagglutinin) expressed
by the insulin-producing beta cells in the pancreatic
islets in 3 different types of mice: normal mice, diabetesprone nonobese diabetic (NOD) mice, and mice in which
the beta cells express an oncogene that promotes spontaneous transformation and production of tumors.
In all 3 types of mice, the interaction between antigen and naive CD8+ T lymphocytes specific for hemagglutinin first occurs in the pancreatic lymph nodes. There
antigen is recognized on dendritic cells that obtain it in
from beta cells in the islets and cross-present it to naive
T cells in the lymph nodes. In normal mice, this interaction results in an abortive activation of the T cells and
subsequent deletion of the potentially autoreactive T cells
specific for hemagglutinin.
T
TOLERANCE OF TISSUE-RESIDENT MEMORY CD8+
T CELLS
Memory CD8 + T cells are compartmentalized on
the basis of their ability to circulate through either
THE SCRIPPS RESEARCH INSTITUTE
139
lymphoid tissue (central memory cells) or blood and
parenchymal tissue (tissue-resident memory cells).
Compartmentalization is based on cell-surface expression of CD62L, an adhesion molecule required for migration of T cells across high endothelial venules into
secondary lymphoid tissue. Tissue-resident memory
cells express low levels of CD62L and are therefore
excluded from secondary lymphoid tissue.
Previously, we showed that central memory CD8 +
T cells are efficiently tolerized by either soluble peptide
or tissue-derived antigen that is cross-presented in
draining lymph nodes. More recently, we determined
whether tissue-resident memory cells are also tolerized by these 2 forms of antigen.
Soluble peptide was highly efficient at tolerizing
tissue-resident memory cells in all tissues tested except
the brain. This finding may be due to the inability of
soluble peptide to cross the blood-brain barrier. Crosspresented antigen was able to tolerize central memory
cells but not tissue-resident memory cells. This difference in tolerance may occur because the cells are
excluded from entry into secondary lymphoid tissue,
the site at which dendritic cells cross-present antigen
derived from tissue. Our results are also consistent
with the possibility that some tissue-resident memory
cells may not circulate out of tissue.
MECHANISMS OF PROTECTION FROM TYPE 1
D I A B E T E S B Y G E N E T I C P O LY M O R P H I S M S
The spontaneous diabetes that develops in NOD
mice is similar to type 1 diabetes in humans. The disease process involves destruction of the insulin-producing beta cells in the pancreas by CD8+ T lymphocytes.
In humans and mice, genetic regions have been identified in which allelic polymorphism predisposes individuals to type 1 diabetes. We are studying the effects of
such allelic polymorphism, designated insulin-dependent
diabetes (Idd) loci, on the establishment of CD8 +
T-cell tolerance.
Congenic mice that express protective alleles at
Idd3/5 have normal abortive activation of islet antigenspecific CD8+ T cells in the pancreas, suggesting that
tolerance is restored at the earliest time when naive
CD8 + T cells first encounter antigen. In contrast, in
NOD mice, such CD8+ T cells accumulate in the pancreatic lymph nodes and then enter the islets. This
difference in the accumulation of CD8+ T cells in the
pancreatic lymph nodes occurs in the absence of all
CD4 + T cells. We are testing the hypothesis that this
difference may be intrinsic to the interaction between
140 IMMUNOLOGY
2006
T cells and cross-presenting dendritic cells in NOD and
Idd3/5 mice.
ROLE OF CD4+ HELPER T CELLS IN PROMOTING
TUMOR CELL DESTRUCTION BY CD8+ T CELLS
CD4 + helper T cells can enhance the performance
of CD8 + T cells in different ways, including enhanced
clonal expansion during activation of CD8 + T cells,
enhanced tissue infiltration by the activated effector
CD8 + T cells, and enhanced survival of the effector
CD8 + T cells. We are assessing the effects of CD4 +
helper T cells at various times after activation of CD8+
T cells to evaluate the ability of the helper cells to promote destruction of tumor cells by CD8+ T cells.
PUBLICATIONS
Cohen, C.J., Zheng, Z., Bray, R., Zhao, Y., Sherman, L.A., Rosenberg, S.A., Morgan, R.A. Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53
TCR [published correction appears in J. Immunol. 177:5746, 2006]. J. Immunol.
175:5799, 2005.
Martinez, X., Kreuwel, H.T.C., Redmond, W.L., Trenney, R., Hunter, K., Rosen,
H., Sarvetnick, N., Wicker, L.S., Sherman, L.A. CD8+ T cell tolerance in
nonobese diabetic mice is restored by insulin-dependent diabetes resistence alleles.
J. Immunol. 175:1677, 2005.
Wei, C.-H., Trenney, R., Sanchez-Alavez, M., Marquardt, K., Woodland, D.L.,
Henriksen, S.J., Sherman, L.A. Tissue resident memory CD8+ T cells can be
deleted by soluble, but not cross-presented antigen. J. Immunol. 175:6615, 2005.
Regulation of Homeostasis of
Mature T Cells
C.D. Surh, C. Ramsey, J. Purton, E.M.M. van Leeuwen,
J.Y. Lee, D. Kim, O. Boyman,* C. Ahn,** J. Sprent***
* Universitaire Vaudois, Lausanne, Switzerland
** Seoul National University Hospital, Seoul, Korea
*** Garvan Institute of Medical Research, Darlinghurst, Australia
he homeostasis of mature T cells is largely governed by 2 related cytokines, IL-7 and IL-15,
which bind to the receptors belonging to the common γ-chain (CD132) receptor family. Other members
of the CD132 family include receptors for IL-2, IL-4,
IL-9, and IL-21. In conjunction with signals from contact with self-peptide–MHC ligands, IL-7 controls survival of naive T cells. Memory T cells, which are at a
higher state of activation than naive T cells, depend
on both IL-7 and IL-15 for survival and for intermittent
cell division. Memory CD4+ T cells are generally more
dependent on IL-7 than on IL-15 for their homeostasis,
whereas memory CD8 + T cells rely primarily on IL-7
for survival and on IL-15 for periodic cell division. Indica-
T
THE SCRIPPS RESEARCH INSTITUTE
tive of the cells’ cytokine requirements, the dimeric
receptor for IL-7 (CD127-CD132) is expressed at high
levels on naive and memory T cells, whereas the dimeric
receptor for IL-15 (CD122-CD132) is expressed at negligible levels on naive T cells, low levels on memory
CD4+ T cells, and high levels on memory CD8+ T cells.
Neither IL-7 nor IL-15 is produced by T cells; both are
produced by epithelial, stromal, and antigen-presenting cells.
IL-2, which is produced by T cells for autocrine purposes, is closely related to IL-15. Recognition of IL-2 by
T cells is mediated by the high-affinity trimeric receptor
CD25-CD122-CD132; the dimeric form of the recepter
(CD122-CD132) recognizes IL-15. Indeed the IL-15
receptor can also recognize IL-2 at a lower affinity.
Interestingly, injecting a monoclonal antibody to IL-2,
a technique that is thought to deplete the cytokine,
increases the background turnover rate of memory
CD8 + T cells. This finding was interpreted to indicate
that IL-2, unlike IL-15, dampens the homeostasis of
memory CD8+ T cells. Recent work, however, indicates
that this interpretation is untrue.
Instead of depleting IL-2, the monoclonal antibody
actually boosts the biological activity of the interleukin.
Hence, the ability of the monoclonal antibody to IL-2
to elevate turnover of memory CD8 + T cells does not
occur in the absence of IL-2, and, more important,
injecting the monoclonal antibody complexed with IL-2
dramatically induces rapid proliferation of memory
CD8+ T cells. Indeed, administration of the antibody–IL-2
complex induced 100- to 200-fold greater expansion
of memory CD8+ T cells than did administration of IL-2
alone. The complex stimulated CD8+ T cells through
the dimeric IL-15 receptor rather than through the
trimeric IL-2 receptor, because stimulatory activity of
the complex was also evident on CD25−CD8 + T cells.
Exactly how the bound monoclonal antibody
enhances the activity of IL-2 is unknown. Nonetheless,
it is clear that monoclonal antibodies augment the cytokine activity in vivo but not under in vitro conditions, and
that the Fc part of the antibody is required for its intensifying role. These findings suggest that the monoclonal
antibody boosts the cytokine activity by concentrating
the cytokine onto the cell surface of antigen-presenting cells and/or by prolonging the half-life of the cytokine. The specificity of the monoclonal antibody also
determines its ability to enhance the activity of IL-2 to
IL-2 receptors. Thus, one particular monoclonal antibody to IL-2 could stimulate T cells expressing the
IMMUNOLOGY
2006
trimeric IL-2 receptor, such as regulatory T cells, but
not memory CD8 + T cells that express the dimeric
IL-15/IL-2 receptor.
The ability of the monoclonal antibody to boost the
activity of the bound cytokine appears to be generally
applicable because the biological activity of other cytokines, such as IL-4 and IL-7, can also be enhanced by
binding to specific monoclonal antibodies. The ability
to induce expansion and activation of specific subsets
of T cells by administering complexes composed of
cytokine plus monoclonal antibody may offer a new
way to modulate the immune response for therapeutic
purposes. The immune response can be boosted against
tumors and infectious agents by activating naive and
memory T cells; alternatively, allergens and tissue grafts
can be suppressed by inducing expansion of regulatory
T cells and the immune responses against self.
PUBLICATIONS
Boyman, O., Kovar, M., Rubinstein, M.P., Surh, C.D., Sprent, J. Selective stimulation of T cell subsets with antibody-cytokine immune complexes. Science
311:1924, 2006.
Davey, G.M., Starr, R., Cornish, A.L., Burghardt, T., Alexander, W.S., Carbone,
F.R., Surh, C.D., Heath, W.R. SOCS-1 regulates IL-15-driven homeostatic proliferation of antigen-naive CD8 T cells, limiting their autoimmune potential. J. Exp.
Med. 202:1099, 2005.
Gattinoni, L., Finkelstein, S.E., Klebanoff, C.A, Antony, P.A., Palmer, D.C.,
Spiess, P.J., Hwang, L.N., Yu, Z., Wrzesinski, C., Heimann, D.M., Surh, C.D.,
Rosenberg, S.A., Restifo, N.P. Removal of homeostatic cytokine links by lymhodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells.
J. Exp. Med. 202:907, 2005.
Lee, S.K., Surh, C.D. Role of interleukin-7 in bone and T-cell homeostasis.
Immunol. Rev. 208:169, 2005.
Ramsey, C., Hässler, S., Marits, P., Kämpe, O., Surh, C.D., Peltonen, L., Winqvist, O. Increased antigen presenting cell-mediated T cell activation in mice and
patients without the autoimmune regulator. Eur. J. Immunol. 36:305, 2006.
Surh, C.D., Boyman, O., Purton, J.F., Sprent, J. Homeostasis of memory T cells.
Immunol. Rev. 211:154, 2006.
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141
on artificial bilayers with recombinant forms of TCRαβ,
CD3δε, CD3γε, and CD8αβ is in progress. We use a
combination of single-molecule, multicolor imaging by
total internal reflection fluorescence microscopy, in collaboration with K. Fish, University of Pittsburgh, Pittsburgh, Pennsylvania, and electron microscopy to examine
the dynamics and membrane relationships of each subunit within the complex. Similar observations are carried
out in the presence of MHC ligands displayed in solution or at the surface of polystyrene beads and liposomes.
Interactions of MHC and TCR molecules with their
respective membrane could provide simple switches
essential to T-cell activation. This hypothesis is supported by our determination, in collaboration with A.K.
Mitra, University of Auckland, Auckland, New Zealand,
of the structure of an MHC molecule attached to a phospholipid bilayer that shows parallel orientation of the
long axis of the molecule with the lipid leaflet. In collaboration with I.A. Wilson, Department of Molecular
Biology, we are determining 3-dimensional structures
of CD3, TCR complexes, and CD8αβ.
AUTOIMMUNE DIABETES
We are using MHC multimers to detect antigen-specific T-cell populations in diabetes-prone nonobese
diabetic mice. Pathogenic T cells are characterized by
analyzing secretion of cytokines and use of TCRs by
single cells. We are also trying to treat insulin-dependent diabetes by depleting antigen-specific T cells in
vivo during the preclinical phase of the disease. For
this therapy, we are using MHC molecules to deliver
doxorubicin liposomes to autoreactive T cells. The
specificity of the intervention will limit side effects
and complications of general immunosuppression.
L I N K S B E T W E E N I N N AT E A N D A D A P T I V E I M M U N I T Y
Surh, C.D., Sprent, J. On the TRAIL of homeostatic memory T cells [published
correction appears in Nat. Immunol. 7:672, 2006]. Nat. Immunol. 7:439, 2006.
Tan, J.T., Surh, C.D. T cell memory. Curr. Top. Microbiol. Immunol., in press.
Structure-Function Studies of
Innate and Adaptive Immunity
L. Teyton, B. Atteberry, K. Bennett, C. Cantu, S.Y. Chang,
L. Develioglu, S. Freigang, H. Issafras, M. Holt, E. Landais,
M. Ota, N. Schrantz, J. Sim, R. Stefanko,
N. Von Allmen-Zurcher, C. Wang, K. Yoshida
A C T I VAT I O N O F T - C E L L R E C E P T O R S
O
ur goal is to understand the molecular switches
that lead to activation of T cells. Assembly of
functional complexes of T-cell receptors (TCRs)
We are studying lipid binding to CD1 to determine
the factors that govern the presentation of the lipids to
T cells. A family of lipid transfer proteins known as
saposins, which are involved in the catabolism of lipids,
are critical for the loading of natural glycolipids onto
CD1 and the selection of natural killer T cells. Other
lipid transfer proteins most likely account for the loading of other endogenous and exogenous ligands. In
collaboration with A. Bendelac, University of Chicago,
and P.B. Savage, Brigham Young University, Provo,
Utah, we are using RNA interference, genetic techniques,
and recombinant biochemistry to study CD1 within
the context of lipid metabolism. At a structural level,
we are examining recognition of dissimilar ceramides
such as α-galactosyl ceramide (Fig. 1) and isoglobotrihexosyl ceramide (β-linked) by a TCR bearing a unique
142 IMMUNOLOGY
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THE SCRIPPS RESEARCH INSTITUTE
Genetics of Systemic
Autoimmunity and T-Cell
Homeostasis in Autoimmunity,
Aging, and Cancer
A.N. Theofilopoulos, D.H. Kono, R. Baccala,
R. Gonzalez-Quintial, M.K. Haraldsson, D. Aït-Azzouzene,
J. Schettini, R.M. Chintalapati, C.A. Louis-Dit-Sully
e have continued our research on predisposing and effector genes in murine models of
systemic lupus erythematosus (SLE), homeostatic T-cell disturbances in systemic autoimmunity
and aging, and the potential use of homeostatic T-cell
proliferation for inducing efficient antitumor responses.
W
F i g . 1 . Side view of α-galactosyl ceramide bound to murine
CD1d. The galactose is accessible for TCR recognition.
α-chain (Vα14 in mice, Vα24 in humans) and a limited set of Vβ partners.
I N N AT E I M M U N E R E C E P T O R S
Recognition of unique features of the prokaryotic
world is embedded in a series of receptors of the innate
immune system called pattern recognition molecules.
Each of these receptors can sense the presence of a
family of unique prokaryotic compounds such as glycolipids, proteoglycans, DNA, or RNA and allow activation of macrophages, dendritic cells, and neutrophils.
We are collaborating with R. Ulevitch and P. Tobias,
Department of Immunology, to decipher the structural
basis of this mode of recognition. We expressed recombinant forms of receptor family members from Drosophila,
mice, and humans to compare the biophysical and
structural characteristics of the receptors and to delineate new activation pathways.
PUBLICATIONS
Benlagha, K., Wei, D.G., Veiga, J., Teyton, L., Bendelac, A. Characterization of
the early stages of thymic NKT cell development. J. Exp. Med. 202:485, 2005.
Shore, D.A., Teyton, L., Dwek, R.A., Rudd, P.M., Wilson, I.A. Crystal structure of
the TCR coreceptor CD8αα in complex with monoclonal antibody YTS 105.18 Fab
fragment at 2.88 Å resolution. J. Mol. Biol. 358:347, 2006.
Wei, D.G., Lee, H., Park, S.H., Beaudoin, L., Teyton, L., Lehuen, A., Bendelac,
A. Expansion and long-range differentiation of the NKT cell lineage in mice expressing CD1d exclusively on cortical thymocytes. J. Exp. Med. 202:239, 2005.
Zajonc, D.M., Cantu, C. III, Mattner, J., Zhou, D., Savage, P.B., Bendelac, A.,
Wilson, I.A., Teyton, L. Structure and function of a potent agonist for the semiinvariant natural killer T cell receptor. Nat. Immunol. 8:810, 2005.
GENETIC BASIS OF SYSTEMIC AUTOIMMUNITY
Susceptibility to SLE is inherited as a multifactorial trait, and genetic predisposition is a major if not an
essential factor in the disease. We are defining the role
and identity of susceptibility genes that promote SLE
in both spontaneous and induced mouse models of the
disease. Previously, we identified loci that predispose
mice to SLE in NZB, NZW, BXSB, and MRL strains,
which are SLE prone, and in C57BL/6 mice, which are
not predisposed to autoimmune disease. We also identified a disease-resistance locus in DBA/2 mice. We
subsequently analyzed the contribution of several of
these loci to autoimmunity in interval congenic lines. The
NZB x NZW F2-related Lbw2, Lbw5, and Lbw7 (chromosomes 4, 7, and 1, respectively); the MRL x B6-Fas lpr
F2-related Lmb1–Lmb4 (chromosomes 4, 5, 7, and 10),
and the NZB x DBA/2 and SJL x DBA/2 F 2 -related
Hmr1 (chromosome 1) were among those analyzed.
On the basis of these studies. we concluded that
lupus-prone strains have substantial genetic heterogeneity, strains not predisposed to autoimmune disease
can harbor susceptibility genes, a single locus often
promotes multiple traits, and specific combinations of
loci determine clinical manifestations.* Moreover, phenotypes do not always correlate with the initial mapping
results, and the genetic background plays a major role
in shaping locus-induced phenotypes. Further fine-mapping studies indicated that identifying the underlying
genes by screening for alterations in gene expression
still requires the evaluation of subcongenics.
Substantial progress was also made in defining
Lmb3 as a nonsense mutation of the gene that encodes
IMMUNOLOGY
2006
coronin-1A, and actin-binding protein that plays a role
in the formation of filaments. Surprisingly, this mutation
was present in the C57BL/6-Faslpr/Scr substrain, which
is not predisposed to autoimmunity, and not in the MRL
strain, indicating that Lmb3 most likely is a diseasemodifying or resistance allele. Currently, we are determining the role of coronin-1 in normal immune responses
and autoimmunity. We are also more precisely mapping
and identifying the genes for other loci.
TYPE I INTERFERONS IN SLE
Type I interferons (IFN-α/β) have important effects
in the innate and adaptive immune systems and may
play a central role in the pathogenesis of autoimmune
diseases, including SLE. We used several approaches
to define the mechanisms of these interferons and to
curtail their adverse effects.
We previously found that deletion of the common
receptor for type I interferons resulted in significant disease reduction in lupus-predisposed NZB mice. Although
this finding provides strong support for the involvement
of IFN-α/β in SLE, it has yet to be shown that blockade
of IFN-α/β activity can inhibit active disease. We previously showed the efficacy of intramuscular injections
of nonviral vectors encoding the fusion protein consisting of the IFN-γ receptor and the Fc fragment of IgG1
in ameliorating SLE in MRL-Faslpr lupus-prone mice. A
similar strategy to block type I interferons is being used
to treat the prototypic NZB/W strain of lupus-prone
mice. Early assessments indicate that blockade of
IFN-α/β can indeed cause regression of active disease.
In collaborative studies with R. Schreiber and his
colleagues, Washington University of Medicine, St. Louis,
Missouri, we will ascertain the efficacy of a recently
developed mouse monoclonal antibody to mouse IFN-α
receptor 1. Overall, our previous and current studies
have provided the impetus for the potential use of
interferon blockers as therapies in human SLE and
other autoimmune diseases.
A major issue in the role of type I interferons in SLE
is the stimuli that induce and sustain the production
of these cytokines. Although exogenous stimuli, such as
infectious agents, may induce IFN-α/β through engagement of Toll-like receptors (TLRs) and precipitate disease,
the primary involvement of the endogenous stimuli
remains less defined. Therefore, a major focus has been
on identify the endogenous (self) stimuli acting under
“sterile” conditions.
Studies have shown that products of apoptotic/
necrotic cells or nucleic acids, when combined with
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143
autoantibodies from the sera of patients with SLE,
induce plasmacytoid dendritic cells to produce large
quantities of IFNα/β. The requirement of forming a complex with autoantibodies is consistent with the idea that
mammalian nucleic acids themselves are compartmentalized away from the endosomes where IFN-α/β–inducing TLR3, TLR7, TLR8, and TLR9 reside. Although
this mechanism is important for disease perpetuation,
primary inducers not predicated on preexisting autoantibodies need to be identified. A likely candidate might
be apoptotic material that under certain conditions
could constitute a “danger signal.” Recent studies, in
collaboration with K. Hoebe and B. Beutler, Department
of Immunology, indicated that early apoptotic cells can
indeed act as efficient inducers of type I interferons and
that the responding cells are not plasmacytoid dendritic
cells, but precursors of B220 −CD8 + lymphoid-type
dendritic cells. Importantly, induction was mediated
by a TLR-independent pathway.
On the basis of these findings, we propose that
induction of IFN-α/β in SLE encompasses 2 types of
apoptosis-generated stimuli that act sequentially in the
disease process. The early-phase stimulus does not
require autoantibodies or TLR engagement and is generated by stressed cells with propensity to apoptosis;
this material is taken up by lymphoid-type dendritic
cells and initiates production of IFN-α/β, leading to
activation of antigen-presenting cells, priming of previously quiescent nontolerant T and B cells, and production of autoantibodies. IFN-α/β may also promote
maturation and survival of T and B cells directly as
well as by inducing production of B-cell trophic factors from activated dendritic cells.
The late-phase stimulus consists of apoptosis/necrosis materials and associated nucleic acids complexed
with autoantibodies. These complexes are directed into
endosomal compartments of plasmacytoid or conventional dendritic cells and B cells, through receptors for
IgG or autoreactive B-cell receptors, where they engage
TLRs and amplify production of interferons and responses
of autoimmune T and B cells.* We are conducting experiments to define the responsiveness of plasmacytoid
and conventional dendritic cells to apoptotic materials
in SLE and to assess the 2-step hypothesis in disease
pathogenesis.
CYCLIN KINASE INHIBITORS IN SYSTEMIC
AUTOIMMUNITY
We previously found that the cell-cycle inhibitor
p21 is a nonredundant effector molecule for SLE in
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autoimmune-prone BXSB mice; lack of p21 resulted in
enhanced apoptosis of T and B lymphocytes, decreased
numbers of activated/memory CD4+ T cells, and disease reduction. These findings support our earlier
hypothesis that increased numbers of activated/memory CD4 + T cells in SLE could be caused by repeated
stimulation by self-antigens leading to accumulation of
cell cyclin kinase inhibitors, such as p21 and p27, and
a replicative senescence-like state. Upon stimulation,
these senescence-like T cells would be resistant to apoptosis and proliferation because of their inability to cycle
but could produce autoimmunity-promoting proinflammatory factors.
A more recent T-cell transfer study confirmed that
lack of p21 in T cells is sufficient to reduce clinical
manifestations of autoimmunity. Moreover, BXSB mice
lacking the gene for p27, another cell-cycle inhibitor,
also have reduced mortality compared with littermate
BXSB mice that have the gene, further supporting our
hypothesis. We are also examining the role of p21 in
organ-specific autoimmunity, and we have generated
type 1 diabetes–prone nonobese diabetic mice that lack
the gene for p21. We have backcrossed (>10 generations) the p21 knockout gene onto the type 1 diabetes–prone nonobese diabetic strain of mice. Studies will
continue to define in more detail the role of cyclin
kinase inhibitors in autoimmunity and to determine
the potential for modulation of the cell cycle in therapy for SLE.
T - C E L L H O M E O S TA S I S A N D S L E
Systemic autoimmunity is essentially a disease that
can be defined on the basis of disturbances in homeostasis of lymphoid cells. We previously speculated that
frank lymphopenia and associated excess of T-cell trophic
cytokines (IL-7 and IL-15) leading to ”acute homeostatic T-cell proliferation” might be an inducing mechanism for SLE autoimmune processes. We found that
acute homeostatic T-cell proliferation of adoptively
transferred T cells indeed recapitulated systemic autoimmunity in lymphopenic recipient mice predisposed to
SLE. Currently, we are investigating whether a similar
phenomenon might be mediated by downregulation of
receptors for IL-7 and/or IL-15 in accumulating chronically activated autoreactive T cells, a process that might
create an excess of these cytokines and promote proliferation of newly generated autoreactive T cells. To assess
this possibility, we studied MRL-Fas lpr mice in which a
Fas mutation leads to massive expansion of double-negative (CD4−CD8 −) T cells.
THE SCRIPPS RESEARCH INSTITUTE
We found that these T cells had significantly
reduced expression of CD127 (the receptor for IL-7)
and CD122 (the receptor for IL-15). In accordance
with our hypothesis, nonlymphopenic MRL-Fas lpr
recipients supported proliferation of transferred isogenic
CD8 + T cells, whereas nonlymphopenic normal hosts
did not. Moreover, although single-positive CD4+ and
single-positive CD8+ as well as double-negative T cells
proliferated efficiently in a lymphopenic MRL-Fas lpr
recipient, survival and repopulation occurred with single-positive, but not double-negative, T cells.
These studies support the concept that excess of
IL-7 and IL-15 created by downregulation of the corresponding receptors in expanded autoreactive T cells
creates an environment that mimics the lymphopenic
condition of cytokine excess, triggering proliferation of
newly emerging autoreactive T cells. Verification of
these results may indicate the usefulness of blocking
T-cell trophic cytokines and/or the receptors of these
cytokines to interfere with autoimmune responses. We
have started experiments to address this possibility.
T - C E L L H O M E O S TA S I S A N D A G I N G
Aging has been associated with several T-cell defects,
but whether these defects are intrinsic or are imposed
by changes in the microenvironment is unclear. To
address this issue, we adoptively transferred labeled
T cells into young and old lymphopenic mice and examined degrees of “acute homeostatic expansion.” The
results indicated that aging is associated with impaired
homeostatic T-cell proliferation. The proliferation was not
due to a primary T-cell defect but rather to changes in
the microenvironment. Adoptively transferred T cells from
aged donors indeed proliferated normally in lymphopenic
young recipients, whereas T cells from young donors had
reduced proliferation in aged lymphopenic hosts.
One possibility for this aging-associated defect in the
microenvironment is reduced levels of IL-7 and IL-15,
which are necessary for homeostatic expansion. Therefore, we treated old, sublethally irradiated recipients
with recombinant mouse IL-7. We found that the defect
was largely corrected. The results suggest that T-cell
trophic cytokine reconstitution may be an effective
means to correct immunologic senescence.
T - C E L L H O M E O S TAT I C P R O L I F E R AT I O N T O B R E A K
TOLERANCE TO TUMOR ANTIGENS
We previously proposed that lymphopenia-induced
homeostatic proliferation mediated by excess of trophic
cytokines and recognition of self-peptide–MHC may be
a way to activate low-affinity T cells that recognize tumor
IMMUNOLOGY
2006
antigens. Our earlier studies with a melanoma model
indicated the validity of this approach when coupled
with tumor cell immunization. We are now assessing
the efficacy of this approach in established and
metastasizing tumors, specifically in mouse models of
breast and prostate carcinomas.
We found that although the size of subcutaneous
tumors was again reduced, metastasis was marginally
affected, probably because of the time required for
regeneration of lymphocytes and acquisition of a
diverse repertoire.
Currently, we are assessing the potential usefulness
of protocols in which homeostatic proliferation as a
priming event is combined with administration of complexes consisting of IL-7 and nonneutralizing antibodies to IL-7. This approach is based on the recent novel
finding of our collaborators O. Boyman, C.D. Surh, and
J. Sprent, Department of Immunology, that such complexes induce massive and accelerated expansion of
CD8 + (and CD4+) T cells. Preliminary results indicate
that this modified protocol significantly reduces both the
size of the primary tumor and the degree of metastasis. We think that this and other contemplated protocols to promote priming of T cells may be promising
approaches to tumor immunotherapy.
PUBLICATIONS
Homann, D., Dummer, W., Wolfe, T., Rodrigo, E., Theofilopoulos, A.N., Oldstone,
M.B., von Herrath, M.G. Lack of intrinsic CTLA-4 expression has minimal effect on
regulation of antiviral T-cell immunity. J. Virol. 80:270, 2006.
Janssen, E., Tabeta, K., Barnes, M.J., Rutschmann, S., McBride, S., Bahjat, K.S.,
Schoenberger, S.P., Theofilopoulos, A.N., Beutler, B., Hoebe, K. Efficient T cell activation via a Toll-interleukin 1 receptor-independent pathway. Immunity 24:787, 2006.
Kono, D.H., Theofilopoulos, A.N. Genetics of autoantibody production in mouse
models of lupus. In: Autoantibodies and Autoimmunity: Molecular Mechanisms in
Health and Disease. Pollard, K.M. (Ed.). Wiley-VCH, New York, in press.
Kono, D.H., Theofilopoulos, A.N. Genetics of murine models of autoimmunity. In:
Dubois’ Systemic Lupus Erythematosus, 7th ed. Wallace, D.J., Hahn, B.H. (Eds.).
Williams & Wilkins, Baltimore, in press.
Kono, D.H., Theofilopoulos, A.N. Genetics of SLE in mice. Springer Semin.
Immunopathol. 28:83, 2006.
Sfikakis, P.P., Gourgoulis, G.M., Moulopoulos, L.A., Kouvatseas, G., Theofilopoulos, A.N., Dimopoulos, M.A. Age-related thymic activity in adults following
chemotherapy-induced lymphopenia. Eur. J. Clin. Invest. 35:380, 2005.
Watson, L.C., Moffatt-Blue, C.S., McDonald, R.Z., Kompfner, E., Aït-Azzouzene,
D., Nemazee, D., Theofilopoulos, A.N., Kono, D.H., Feeny A.J. Paucity of V-D-D-J
rearrangements and VH replacement events in lupus prone and nonautoimmune
TdT–/– and TdT+/+ mice. J. Immunol. 177:1120, 2006.
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145
Initiation of Inflammation by the
Innate Immune System
P.S. Tobias, H.-K. Lee, L.K. Curtiss,* P. Dawson,**
T. Kirkland,*** D. Liebler****
* Department of Immunology, Scripps Research
** Department of Cell Biology, Scripps Research
*** University of California, San Diego, California
**** Vanderbilt University, Nashville, Tennessee
e focus on understanding the mechanisms
by which cells use the innate immune system
to initiate defensive inflammatory responses.
First, we seek to understand the structural features of
the Toll-like receptors (TLRs) and their allied proteins
lipopolysaccharide-binding protein, CD14, MD-2, and
CD36, which enable the receptors to bind their ligands.
Second, we seek to understand the structural changes
by which binding of a microbial ligand to the extracellular domain of the receptor leads to signal transduction
across the cell membrane and initiation of intracellular
signaling cascades. Third, we seek to understand the
involvement of endogenous and exogenous inflammatory stimuli in atherosclerosis.
Ten TLRs are known. For most of these, ligands
derived from microorganisms are known; binding to
the ligands initiates signaling, leading to expression of
inflammatory mediators and other defensive responses.
In addition, some TLRs that may be involved in sterile
inflammatory conditions such as arthritis or atherosclerosis may have endogenous ligands. However, these
ligands are not yet clearly identified.
To understand the structural features of ligand-receptor binding, we use using 2 approaches. In the traditional mutation approach, amino acid residues in the
proteins are mutated, and the proteins are then studied
for functional changes. In the second approach, we use
cross-linking agents to create covalent attachments
of the ligands to the proteins. The proteins are then
degraded chemically to determine the site of attachment. Currently, we are using these approaches to
study binding of endotoxin, bacterial lipopeptides, and
polyinosinic-polycytidylic acid to CD14.
Binding of ligands to TLRs starts an intracellular
signaling cascade that results in activation of a number
of cellular responses. Prominent hypothesized mechanisms by which ligand binding to TLRs incurs transmembrane signaling are (1) the ligand induces dimerization
of receptors and (2) binding of the ligand induces con-
W
146 IMMUNOLOGY
2006
formational changes in the receptor. Our studies indicate
that pairs of TLRs are associated even in the absence
of ligand and that the pairs undergo a conformational
change upon ligand binding. We are using a variety of
approaches to understand the structural basis for associations among the TLRs and their associated intracellular signaling partners.
Atherosclerosis is an inflammatory disease of the
large arteries. Evidence suggests that inflammatory
components derived from microbes can induce progression of atherosclerosis. However, most of the development of atherosclerotic lesions is due to endogenous
inflammatory factors. Because the TLR system is so
intimately involved with inflammation, we are determining whether the TLRs are involved in atherosclerosis.
Our initial data clearly indicate that TLR2, whether
activated by endogenous ligands or by exogenous ligands,
promotes progression of atherosclerosis. Unexpectedly,
we found that TLR2 expressed on non–bone marrow–
derived cells detects endogenous TLR2 ligands that
promote atherosclerosis, whereas bone marrow–derived
cells detect exogenous TLR2 ligands that promote
atherosclerosis. For these experiments, we are using
mouse models of the disease and mice deficient in
individual TLRs.
PUBLICATIONS
Lee, H.K., Dunzendorfer, S., Soldau, K., Tobias, P.S. Double-stranded RNA-mediated TLR3 activation is enhanced by CD14. Immunity 24:153, 2006.
Mullick, A.E., Tobias, P.S., Curtiss, L.K. Modulation of atherosclerosis in mice by
Toll-like receptor 2. J. Clin. Invest. 115:3149, 2005.
Viriyakosol, S., Tobias, P.S., Kirkland, T.N. Mutational analysis of membrane and
soluble forms of human MD-2. J. Biol. Chem. 281:11955, 2006.
Molecular Mechanisms of
Host-Pathogen Interactions
R.J. Ulevitch, V.V. Kravchenko, C. Fearns, T.-H. Chuang,
J.C. Mathison, Q. Pan, J. da Silva Correia, K. Iwata,
K.D. Janda, G. Kaufmann, M. Meijler
nfection by microbial pathogens often sets in motion
chains of events that cause severe injury to the host,
and nowhere is this phenomenon illustrated more
dramatically than in the response by humans to infection by gram-negative bacteria. In his book Lives of a
Cell, Lewis Thomas characterized the host response to
the endotoxin, or lipopolysaccharide, of gram-negative
bacteria as being “read by our tissues as the very worst
I
THE SCRIPPS RESEARCH INSTITUTE
of bad news. . . . There is nothing intrinsically poisonous about endotoxin, but it must look awful, or feel
awful, when sensed by cells. Cells believe that it signifies the presence of gram-negative bacteria, and they
will stop at nothing to avoid this threat.” In other words,
the innate immune response to infection has caused a
serious disease in humans.
Clearly, much human suffering could be eased if
such overzealous host responses could be tempered.
However, such responses, when not overzealous, are a
normal part of the host’s homeostatic mechanisms,
designed to respond to the threat of infection by gramnegative bacteria. Accordingly, we are attempting to
(1) define the mechanisms of innate immunity and (2)
learn how to control these responses without compromising host defenses against pathogens. Recently, we
contributed to the understanding of innate immunity
through studies of Toll-like receptors (TLRs) and of effector mechanisms that mediate host responses to infection.
It is now well appreciated that the innate immune
system is positioned at the intersection of multiple host
pathways, including those for microbial and viral recognition, enhancement of adaptive immune responses, and,
possibly, cancer immunosurveillance. Each pathway
depends on ligand recognition by specific cellular receptors that are either membrane bound (plasma membrane
as well as endosomal compartments) or cytosolic. The
most important class of membrane-bound receptors are
the TLRs. Among cytosolic receptors, an important
family known as the NLR/ Nod/Caterpillar family has
been identified. Within this family, 2 proteins, Nod1 and
Nod2, are involved in recognition of bacterial ligands
distinct from the ligands for TLRs. Activation of TLR
and Nod signaling pathways leads to production of
multiple cytokines with proinflammatory and anti-inflammatory activities. Such responses are central to host
responses to infection. However, when a breakdown
occurs in the normal regulatory mechanisms that control these pathways, disease may result.
Perhaps the most well-understood link between innate
immunity and human disease is in the host response
to infection. When dysregulation of innate immune
responses occurs, clinical syndromes such as septic
shock and acute respiratory distress syndrome ensue.
Dysregulation of innate immune responses may also
play a role in human diseases in which chronic inflammation is responsible for disease progression, including autoimmune and autoinflammatory diseases. Genetic
studies in humans have revealed strong associations
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2006
among various members of the Nod family of proteins
and human diseases.
During the past year, we made considerable progress in several different areas. We have identified several unique pathways of innate immunity. These findings
are briefly summarized here.
C A S PA S E 1 2 A S A N E G AT I V E R E G U L AT O R O F
I N N AT E I M M U N I T Y
Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in
resistance to sepsis. During the past year we described
a novel role for a caspase in dampening responses to
bacterial infection. We showed that in mice, gene-targeted deletion of caspase-12 makes animals resistant
to peritonitis and septic shock. The resulting survival
advantage was conferred by the ability of the caspase12–deficient mice to clear bacterial infection more
efficiently than did wild-type littermates. Caspase-12
dampened the production of the proinflammatory cytokines IL-1β, IL-18 (IFN-γ–inducing factor), and IFN-γ,
but not TNF-α and IL-6, in response to various bacterial components that stimulate TLR and Nod pathways.
The IFN-γ pathway was crucial in mediating survival
of caspase-12–deficient mice that had sepsis; administration of neutralizing antibodies to IFN-γ receptors
ablated the survival advantage that otherwise occurred
in these animals.
Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease
function of caspase-12 was not necessary for this effect,
because the catalytically inactive caspase-12 mutant
Cys299Ala also inhibited caspase-1 and IL-1β production to the same extent as did wild-type caspase-12.
In this regard, caspase-12 seems to be the counterpart of cFLIP, an antiapoptotic protein, for regulating
the inflammatory branch of the caspase cascade. In
mice, caspase-12 deficiency confers resistance to
sepsis, and its presence exerts a dominant-negative
suppressive effect on caspase-1, resulting in enhanced
vulnerability to bacterial infection and septic mortality.
These findings have broad implications for new therapies
for sepsis and related problems.
HOMOSERINE LACTONES AND HOST IMMUNITY
Receptors in the innate immune system function as
sensors of infection and trigger the immune responses
through ligand-specific signaling pathways. The ligands
are pathogen-associated products, such as components
of bacterial walls and viral nuclear acids. A common
response to such ligands is the activation of mitogen-
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147
activated protein kinase p38; double-stranded viral
RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α. Now we have shown
that p38 and phosphorylation of eukaryotic translation
initiation factor 2α are 2 biochemical markers of the
effects induced by N-(3-oxo-acyl)homoserine lactones,
the secreted products of a number of gram-negative
bacteria, including Pseudomonas aeruginosa, an
opportunistic pathogen in humans. Furthermore,
N-(3-oxo-dodecanoyl)homoserine lactone induces distension of mitochondria and the endoplasmic reticulum
and transcription of the gene for c-jun. These effects
occur in a wide variety of cell types, including alveolar
macrophages and bronchial epithelial cells, and require
the structural integrity of the lactone ring motif and its
natural stereochemistry.
These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the
innate immune system. However, we found that signaling mediated by N-(3-oxo-dodecanoyl)homoserine
lactone does not require the presence of the canonical
innate immune system receptors, TLRs or Nod1 and
Nod2. These data offer a new understanding of the
effects of N-(3-oxo-dodecanoyl)homoserine lactone on
host cells and its role in persistent airway infections
caused by P aeruginosa. Our results should be useful
in the development of new therapeutic interventions
for devastating diseases such as cystic fibrosis in which
P aeruginosa or other homoserine lactone–producing
organisms are important in disease progression.
ROLE OF NOD1 IN THE GROWTH OF ESTROGENSENSITIVE BREAST TUMOR CELL LINES
Nod1, a cytosolic protein that senses ligands containing meso-diaminopimelic acid derived from peptidoglycan, plays a role in host responses to invasive
bacteria. We have identified a novel function for Nod1:
control of tumor formation. We used cell lines derived
from the human breast cancer epithelial cell line MCF-7
in a xenograft model of mice with severe combined
immunodeficiency to characterize a pathway linking
Nod1 to the growth of estrogen-sensitive tumors.
In MCF-7 cells, the absence of Nod1 correlates
with tumor growth, an increased sensitivity to estrogen-induced cell proliferation, and a failure to undergo
Nod1-dependent apoptosis. Conversely, overexpression
of Nod1 in MCF-7 cells results in inhibition of estrogen-dependent tumor growth and reduction of estrogen-induced proliferative responses in vitro. We are
using a combination of genetics and protein pathway
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mapping to investigate the molecular details of this
Nod1 pathway.
PUBLICATIONS
da Silva Correia, J., Miranda, Y., Austin-Brown, N., Hsu, J., Mathison, J., Xiang, R.,
Zhou, H., Li, Q., Han, J., Ulevitch, R.J. Nod1-dependent control of tumor growth.
Proc. Natl. Acad. Sci. U. S. A. 103:1840, 2006.
Kravchenko, V.V., Kaufmann, G.F., Mathison, J.C., Scott, D.A., Katz, A.Z., Wood,
M.R., Brogan, A.P., Lehmann, M., Mee, J.M., Iwata, K., Pan, Q., Fearns, C.,
Knaus, U.G., Meijler, M.M., Janda, K.D., Ulevitch, R.J. N-(3-Oxy-acyl)homoserine
lactones signal cell activation through a mechanism distinct from the canonical
pathogen-associated molecular pattern recognition receptor pathways. J. Biol.
Chem. 281:28822, 2006.
Pan, Q., Kravchenko, V., Katz, A., Huang, S., Ii, M., Mathison, J.C., Kobayashi,
K., Flavell, R.A., Schreiber, R.D., Goeddel, D., Ulevitch, R.J. NF-κB-inducing
kinase regulates selected gene expression in the Nod2 signaling pathway. Infect.
Immun. 74:2121, 2006.
Raetz, C.R., Garrett, T.A., Reynolds, C.M., Shaw, W.A., Moore, J.D., Smith, D.C.,
Jr., Ribeiro, A.A., Murphy, R.C., Ulevitch, R.J., Fearns, C., Reichart, D., Glass,
C.K., Benner, C., Subramaniam, S., Harkewicz, R., Bowers-Gentry, R.C., Buczynski, M.W., Cooper, J.A., Deems, R.A., Dennis, E.A. Kdo2-lipid A of Escherichia
coli, a defined endotoxin that activates macrophages via TLR-4. J. Lipid Res.
47:1097, 2006.
Saleh, M., Mathison, J.C., Wolinski, M.K., Bensinger, S.J., Fitzgerald, P., Droin,
N., Ulevitch, R.J., Green, D.R., Nicholson, D.W. Enhanced bacterial clearance
and sepsis resistance in caspase-12-deficient mice. Nature 440:1064, 2006.
Wirz, S.A., Tobias, P.S., Ulevitch, R.J., Aribibe, L., Bartfai, T. TLR2 is required for
the altered transcription of p75NGF receptors in gram positive infection. Neurochem. Res. 31:297, 2006.
Prion Diseases: Insights Into the
Biology of an Infectious Protein
L. Solforosi, A. Bellon, Z. Cheng, P. Sidiropoulos, G. Abalos,
J. Cruite, R.A. Williamson
he prion diseases, or transmissible spongiform
encephalopathies, are diseases of protein conformation that cause profound neurodegeneration
and death. They include bovine spongiform encephalopathy, also known as mad cow disease; scrapie in sheep;
and chronic wasting disease, which is spreading rapidly
in deer and elk within the United States. The epidemic
of bovine spongiform encephalopathy in the United
Kingdom predated the emergence of a variant form of
Creutzfeldt-Jacob disease (vCJD) in humans, the incidence of which can be most readily explained by the
consumption of foods contaminated with the prion that
causes bovine spongiform encephalopathy. The transmission of vCJD prions via blood products obtained
from apparently healthy donors in whom vCJD later
developed has reignited concern about the widespread
dissemination of prions in humans. Alarmingly, retrospective immunocytochemical detection of prion disease
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THE SCRIPPS RESEARCH INSTITUTE
in archived tissue samples suggests that at least 3000
persons in the United Kingdom are currently incubating
vCJD asymptomatically.
Uniquely, the infectious agent in transmissible
spongiform encephalopathies, the prion, is though to
be composed largely of PrP Sc, an abnormally shaped
version of the cellular prion protein PrPC, a molecule
of unknown function found in all healthy individuals.
Once established within an infected host, prions replicate by converting the normal PrPC form of the protein
into additional molecules of the disease-associated
form. Over time, PrPSc accumulates in the CNS, and
its appearance is closely associated with profound
neuropathologic changes.
In the favored model of prion propagation, PrPSc
acts as a template, sequestering endogenous PrPC and
triggering its conformational rearrangement into nascent
PrPSc and prion infectivity. Although almost none of
the molecular details of this pivotal process are understood, the persistence of individual prion strains (each
of which is associated with a distinct disease phenotype) suggests that assembly of the prion replicative
complex is a tightly choreographed process.
Implicit in this view of prion propagation is a direct
and specific binding interaction between PrP C and
PrP Sc . To systematically map defined regions of PrP
sequence that bind tightly to PrP Sc , we generated a
large and comprehensive panel of motif-grafted recombinant antibodies containing successive and overlapping
polypeptide grafts that collectively span PrP residues
19–231. In PrPSc-binding studies conducted under
stringent conditions with these hybrid-antibody reagents,
we identified 3 distinct and independent high-affinity
PrPSc recognition motifs.
The first of these binding motifs lies at the N-terminal region of the mature PrP molecule, within residues
PrP 23–33; the second motif, within PrP residues
98–110; and the third, within PrP residues 136–158.
Mutational analyses of these PrP Sc -binding regions
revealed that reactivity of the 23–33 and 98–110 PrP
peptide segments depends largely on the presence of
multiple positively charged amino acid residues. Intriguingly, in an acidic environment akin to that found
intracellularly within the endocytic pathway in which
PrPSc is found, PrP grafts corresponding to the N-terminal region of PrP, between residues 29 and 100,
also acquire the ability to strongly recognize misfolded
PrP conformers. These studies yield new insight into
critical peptidic components composing one face of the
IMMUNOLOGY
2006
prion replicative interface and suggest the potential for
a 2-phase PrPC-PrPSc binding interaction that is pH
dependent. Importantly, elucidating how these different PrP conformers interact now enhances the prospect
of efficiently inhibiting their association and thereby
halting prion replication and disease.
Endoglin (CD105) as a Target
for a DNA Vaccine Against
Breast Cancer
S.H. Lee, N. Mizutani, M. Mizutani, Y. Luo, H. Zhou,
C.D. Kaplan, R. Xiang
ntiangiogenic therapy has become an attractive
concept for tumor therapy because the growth
of new capillary blood vessels from preexisting
vasculature is an essential feature of tumor growth and
metastasis. The goal of this approach has been to deliver
antiangiogenic agents to appropriate targets in the tumor
vasculature to eliminate or suppress the blood supply
to tumors, ablating or suppressing growth of the tumors
without seriously disturbing blood flow to normal tissues.
Endoglin (CD105) is a suitable target for such an
antiangiogenic strategy because it is a coreceptor in
the transforming growth factor β (TGF-β) receptor complex that is overexpressed on proliferating endothelial
cells in the neovasculature of breast tumors. CD105
and its ligand TGF-β are important modulators of angiogenesis, and expression of endoglin on proliferating
endothelial cells is upregulated by TGF-β and hypoxic
conditions. In solid tumors, such as breast cancer,
endoglin is almost exclusively expressed on endothelial
cells of both peritumoral and intratumoral blood vessels and on tumor stromal components.
We tested the hypothesis that antiangiogenic or
antitumor effects can be achieved in a prophylactic
setting by using an oral DNA vaccine encoding murine
endoglin carried by double-attenuated Salmonella
typhimurium to a secondary lymphoid organ, that is,
Peyer’s patches in the small intestine. We found that
this DNA-based vaccine elicited the activation of antigen-presenting dendritic cells and induced immune
responses mediated by CD8 + T cells against endoglinpositive murine endothelial cells. For these studies, we
used a syngeneic model of D2F2 murine breast carcinoma cells, which do not express endoglin.
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Expression of endoglin after oral vaccination was
verified in Peyer’s patches by using confocal microscopy, indicating that CD11c+ dendritic cells express
endoglin intracellularly. Moreover, the endoglin vaccine
markedly suppressed D2F2 breast tumor metastases to
the lung, resulting in a 60% prolongation in life span.
In vivo, suppression of pulmonary metastases was abrogated by depletion of CD8+ T cells but not by depletion
of CD4+ T cells. Antitumor activity of the vaccine correlated with T-cell activation as indicated by upregulation
of CD28 and with activation of dendritic cells as indicated by increased expression of CD80 and CD86 on
CD11c + dendritic cells. Immunization with the DNA
vaccine evoked the generation of endoglin-specific cytotoxic T lymphocytes that lysed endoglin-positive murine
endothelial cells. Importantly, tumor angiogenesis was
markedly suppressed in Matrigel assays, indicating a
significant decrease in neovascularization only in mice
immunized with the endoglin vaccine.
Taken together, our data suggest that a CD8 +
T cell–mediated immune response effectively suppressed
dissemination of pulmonary metastases of D2F2 breast
carcinoma cells by eliminating proliferating endothelial
cells, causing suppression of angiogenesis in the tumor
vasculature. We anticipate that vaccine strategies such
as this one will contribute to future therapies for
breast cancer.
PUBLICATIONS
Lee, S.H., Mizutani, N., Mizutani, M., Luo, Y., Zhou, H., Kaplan, C.D., Kim.
S.W., Xiang, R., Reisfeld, R.A. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol. Immunother. 55:1565, 2006.
Zhou, H., Luo, Y., Mizutani, M., Mizutani, N., Reisfeld, R.A., Xiang R. T cellmediated suppression of angiogenesis results in tumor protective immunity. Blood
106:2026, 2005.
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2006
The Membrane-Proximal
External Region of gp41 and
HIV Type 1 Vaccine Design
J.D. Nelson, R. Jensen, I.A. Wilson, P.E. Dawson,
D.R. Burton, M.B. Zwick
IV type 1 (HIV-1) is a major world health problem for which the preferred solution is an
effective vaccine. However, typical candidates
for HIV-1 vaccines have not come close to eliciting the
concentration of neutralizing antibodies associated with
protection. HIV-1 neutralizing antibodies target the envelope glycoproteins gp120 and gp41, which assemble as
a trimer, (gp120-gp41)3, on the surface of the virion.
We have shown that of the roughly 6 reported broadly
neutralizing monoclonal antibodies against HIV-1, 3 of
them, 2F5, 4E10, and Z13, bind to the membrane-proximal external region (MPER) of gp41. These antibodies,
particularly 4E10, can neutralize primary isolates of
HIV-1 from around the globe with remarkable breadth
and potency.
Clearly, it would be desirable to exploit the MPER,
and gp41 in general, for vaccine development, but
eliciting neutralizing antibodies against gp41 has not
been straightforward. One problem is that the structure
of gp41 in the native trimer is unknown. Another difficulty is that the envelope trimer is labile and produces
nonfunctional forms of gp41, including gp120-gp41
monomers and “stumps” of gp41 from which gp120
has been shed. Thus, the poor concentrations of neutralizing antibodies to gp41 elicited by vaccination and
during natural infection apparently are due at least in
part to a poor presentation to the immune system of
native gp41 relative to nonfunctional forms of gp41.
In collaboration with I.A. Wilson, Department of
Molecular Biology, and P.E. Dawson, Department of
Cell Biology, we identified a minimal peptide epitope
that binds with high affinity to 4E10 and adopts a
largely helical conformation in the 4E10-bound complex. To our knowledge, no structure-function analysis
has been done on an antibody whose natural epitope
is closer to the membrane than that of 4E10. Thus,
we have an excellent opportunity not only to better
understand the MPER of gp41 and its potential in
HIV-1 vaccine design but also to gain insight into the
role of lipid in antibody recognition near a membrane.
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We are particularly interested now in performing a
similarly detailed structure-function analysis of the
monoclonal antibody Z13, because its epitope on the
MPER overlaps those of 2F5 and 4E10 and therefore
is important for understanding all 3 sites on gp41. By
clarifying the effects that the membrane and constraints
imposed by the envelope trimer have on the MPER, our
findings should enable the design of HIV-1 vaccine
candidates with stable, homogeneous, and nativelike
presentations of the MPER of gp41.
PUBLICATIONS
Brunel, F.M., Zwick, M.B., Cardoso, R.M.F., Nelson, J.D., Wilson, I.A., Burton,
D.R., Dawson, P.E. Structure-function analysis of the epitope for 4E10, a broadly
neutralizing human immunodeficiency virus type 1 antibody. J. Virol. 80:1680,
2006.
Moore, P.L., Crooks, E.T., Porter, L., Zhu, P., Cayanan, C.S., Grise, H., Corcoran,
P., Zwick, M.B., Franti, M., Morris, L., Roux, K.H., Burton, D.R., Binley, J.M.
Nature of nonfunctional envelope proteins on the surface of human immunodeficiency virus type 1. J. Virol. 80:2515, 2006.
van Houten, N.E., Zwick, M.B., Menendez, A., Scott, J.K. Filamentous phage as
an immunogenic carrier to elicit focused antibody responses against a synthetic
peptide. Vaccine 24:4188, 2006.
Yuste, E., Sanford, H.B., Carmody, J., Bixby, J., Little, S., Zwick, M.B., Greenough, T., Burton, D.R., Richman, D.D., Desrosiers, R.C., Johnson, W.E. Simian
immunodeficiency virus engrafted with human immunodeficiency virus type 1 (HIV1)-specific epitopes: replication, neutralization, and survey of HIV-1-positive
plasma. J. Virol. 80:3030, 2006.
Zwick, M.B. The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring. AIDS 19:1725, 2005.