Immunology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Kerri A. Mowen, Ph.D., Assistant Professor, Department of Immunology
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
IMMUNOLOGY
2005 97
Ann J. Feeney, Ph.D.
Associate Professor
Jiing-Dwan Lee, Ph.D.
Associate Professor
Wolfram Ruf, M.D.
Associate Professor
Philippe Gallay, Ph.D.
Associate Professor
Erguang Li, Ph.D.
Assistant Professor
Daniel R. Salomon, M.D.
Adjunct Associate Professor
Richard J. Ulevitch, Ph.D.
Professor and Chairman
Nicholas R.J. Gascoigne, Ph.D.
Professor
Cheng Liu, M.D., Ph.D.
Assistant Professor
Erica Ollmann Saphire, Ph.D.
Assistant Professor
Roberto Baccala, Ph.D.
Assistant Professor
Peter Ghazal, Ph.D.
Adjunct Associate Professor
Nigel Mackman, Ph.D.
Associate Professor
Nora Sarvetnick, Ph.D.
Professor
Bruce A. Beutler, M.D.
Professor
Howard Gray, Ph.D.
Adjunct Professor
Michael McHeyzer-Williams,
Ph.D.
Associate Professor
David Schlaepfer, Ph.D.
Associate Professor
Gary M. Bokoch, Ph.D.*
Professor
Jiahuai Han, Ph.D.
Professor
Dianne McKay, M.D.
Assistant Professor
Allesandro Sette, Ph.D.
Adjunct Assistant Professor
Dennis R. Burton, Ph.D.**
Professor
Wendy L. Havran, Ph.D.
Associate Professor
Donald E. Mosier, M.D., Ph.D.
Professor
Linda A. Sherman, Ph.D.
Professor
David A. Cheresh, Ph.D.***
Professor
Moores Cancer Center
La Jolla, California
Shuang Huang, Ph.D.
Assistant Professor
Kerri A. Mowen, Ph.D.
Assistant Professor
Jonathan Sprent, M.D., Ph.D.
Professor
David Nemazee, Ph.D.
Professor
Charles D. Surh, Ph.D.
Associate Professor
Glen R. Nemerow, Ph.D.
Associate Professor
Luc Teyton, M.D., Ph.D.
Associate Professor
Per A. Peterson, M.D., Ph.D.
Adjunct Professor
Argyrios N. Theofilopoulos,
M.D.
Professor
DEPAR TMENT OF
IMMUNOLOGY
S TA F F
Robert W. Chesnut, Ph.D.
Adjunct Associate 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
Jonathan G. Kaye, Ph.D.
Associate Professor
Hidehiro Kishimoto, M.D.,
Ph.D.***
Associate Professor
Tokyo University of Science
Tokyo, Japan
Richard Klemke, Ph.D.
Associate Professor
Mary Laurie Phillips, Ph.D.
Adjunct Assistant Professor
Peter S. Tobias, Ph.D.
Associate Professor
Norman R. Klinman, M.D.,
Ph.D.
Professor
Pascal Poignard, M.D.
Adjunct Assistant Professor
Ulla Gissi Knaus, Ph.D.
Associate Professor
Ralph A. Reisfeld, Ph.D.
Professor
Daniel Von Seggern,
Ph.D.****
Assistant Professor
Dwight Kono, M.D.
Associate Professor
Matthias Riewald, M.D.
Assistant Professor
Susan R. Webb, Ph.D.
Associate Professor
Vladimir V. Kravchenko, Ph.D.
Assistant Professor
Hugh Rosen, M.D., Ph.D.
Professor
R. Anthony Williamson, Ph.D.
Associate Professor
S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F I M M U N O L O G Y : X-ray diffraction
Thomas S. Edgington, M.D.
Professor
Colleen Fearns, Ph.D.
Assistant Professor
analyses of intact adenovirus. Top right panel, Adenovirus crystals. Top left panel, Schematic diagram
of adenovirus particles and the location of protein subunits. Middle panel, Diffraction pattern obtained
at a synchrotron beamline. Bottom left panel, Unit cell dimensions and space group of adenovirus particles in the crystals. The work represents a collaboration between the laboratories of Glen R. Nemerow,
Ph.D., Department of Immunology, and Vijay Reddy, Ph.D., Department of Molecular Biology.
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98 IMMUNOLOGY
2005
Curtis B. Wilson, M.D.
Professor Emeritus
Rong Xiang, M.D., Ph.D.
Assistant Professor
Kasper Hoebe, Ph.D.
Christopher Alfonso, Ph.D.
Roshni Chintalapati, Ph.D.
Julie Jameson, Ph.D.
Sandrine Arnaud-Dabernat,
Ph.D.
Jae Ho Cho, Ph.D.
Elisabeth Jeanclos, Ph.D.****
David Chodniewicz, Ph.D.
Caroline Aylott, Ph.D.
Michael Zwick, Ph.D.
Assistant Professor
Cecile King, Ph.D.****
Liguo New, Ph.D.****
S TA F F S C I E N T I S T S
Ben Croker, Ph.D.
Ann Bellon, Ph.D.
Qilin Pan, Ph.D.
Gourab Bhattarcharjee, Ph.D.
Dafang Bian, Ph.D.
Jianhua Cui, Ph.D.***
Moores Cancer Center
La Jolla, California
Udayan Chatterji, Ph.D.
Christine Pastore, Ph.D.****
Xin Du, Ph.D.
Ramona Petrovan, Ph.D.
Onur Boyman, Ph.D.
Eleuterio De La Camara, Ph.D.
John Mathison, Ph.D.
Andrew Saphire, Ph.D.
Carlos Cantu, Ph.D.
Violane Delorme, Ph.D.
Anil Munshi, Ph.D.
Dwayne Stupack, Ph.D.***
Moores Cancer Center
La Jolla, California
Sophie Chabot, Ph.D.****
Celine Der Mardirossian, Ph.D.
Jianming Chen, Ph.D.
Sara Maree Weis, Ph.D.***
Moores Cancer Center
La Jolla, California
Zhong Chen, Ph.D ****
Becky Diebold, Ph.D.***
Emory University
Atlanta, Georgia
Marie Cherrier, Ph.D.
Anthony Don, Ph.D.
Rafal Pawlinski, Ph.D.
M. Germana Sanna, Ph.D.
Deborah Witherden, Ph.D.
SENIOR RESEARCH
A S S O C I AT E S
Jasimuddin Ahamed, Ph.D.
Joao da Silva Correia, Ph.D.
Joerge Birkenfeld, Ph.D.
Aimee de Catherlineu, Ph.D.
Yan Wu, Ph.D.
Tomasz Zal, Ph.D.***
M.D. Anderson Cancer Center
University of Texas
Houston, Texas
Amr Abdelhamid El Sheikh,
Ph.D.
R E S E A R C H A S S O C I AT E S
Amanda Gavin, Ph.D.
Djemel Ait-Azzouzene, Ph.D.
Peter Goebel, Ph.D.***
IgE Therapeutics, Inc.
San Diego, California
Seyed Alavizadeh, Ph.D.***
Moores Cancer Center
La Jolla, California
A D M I N I S T R AT I V E A S S I S TA N T S , D E PA R T M E N T O F
A D M I N I S T R AT I V E A S S I S TA N T S , D E PA R T M E N T O F
I M M U N O L O G Y : Left to right: Amanda Moore, Min Lim, Joan
Gausepohl, Gloria Jones, Lois Yamada, and Theresa Villalpando.
A D M I N I S T R AT I V E A S S I S TA N T S , D E PA R T M E N T O F
I M M U N O L O G Y : Left to right: Lindsay Smith, Katrina Schreiber,
I M M U N O L O G Y : Left to right: Patty Rutledge, Jill Crowe, Sharon
Tami Koth, Rachel Hanley, and Nancy Humphries.
Weston, Susan Ramey, and Dian Caudebec, Administrative Manager.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
IMMUNOLOGY
2005 99
Helen Donners, Ph.D.
Neil John Hime, Ph.D.
Hee Ok Kim, Ph.D.
Jeff Lee, Ph.D.
Stefan Dunzendorfer,
Ph.D.****
Shihe Hou, Ph.D.
Sungwoo Kim, Ph.D.
Sang-Un Lee, Ph.D.
Hong Hua, Ph.D.
Rachel Kohler, Ph.D.
Sung-Hyung Lee, Ph.D.
Timothy Huang, Ph.D.
Elizabeth Kompfner, Ph.D.****
Young Kyung Lee, Ph.D.****
Christoph Huber, Ph.D.
Veera Reddy Konda, Ph.D.***
Metagenics Inc.
Gig Harbor, Washington
Cheng Li, Ph.D.
Mike Eisenbraun, Ph.D.****
Celia Espinoza, Ph.D.
Nicolas Fazilleau, Ph.D.
Naaozumi Ishimaru, Ph.D.***
University of Tokushima
Tokushima, Japan
Marek Kovar, Ph.D.
Hongbin Li, Ph.D.***
Harvard Medical School
Boston, Massachusetts
Jirina Kovarova, Ph.D.
Jiali Li, Ph.D.
Hassan Issafras, Ph.D.
Carsten Kreig, Ph.D.
Xiang Li, Ph.D.
Zhengfan Jiang, Ph.D.
Joerge Krueger, M.D.
Yilei Li, Ph.D.
Hyun-Bae Jie, Ph.D.***
Harvard University
Cambridge Massachusetts
Toru Kurakawa, Ph.D.
Ssang-Taek Lim, Ph.D.
Yumi Kurokawa, Ph.D.
Yang Mi Lim, Ph.D.
Euijung Jo, Ph.D.
Rui Lin, Ph.D.
Valeria Judkowski, Ph.D.****
Sabine Kurz, Ph.D.***
Invitrogen Co.
Carlsbad, California
Wong Soon Justin, Ph.D.
Young Back Kwon, Ph.D.
Milena Iacobelli, Ph.D.
Clemens Feistritzer, M.D.
Christofer Flood, Ph.D.
Linda Frederick, Ph.D.
Stefan Freigang, Ph.D.
Guo Fu, Ph.D.
Philippe Georgel, Ph.D.
Cristina Gil-Lamaignere, Ph.D.
Jonathan Gitlin, Ph.D.***
University of Kentucky
Lexington, Kentucky
Ting-Kun Lin, M.D., Ph.D.
Young Jun Kang, Ph.D.
Cheng Yu Lai, Ph.D.
Jeffrey Lindquist, Ph.D.***
Moores Cancer Center
La Jolla, California
Emma Hamilton-Williams,
Ph.D.
Yu-Ya Kao, Ph.D.
Jennifer Lamoureux, Ph.D.
Guoxun Liu, Ph.D.
Masaaki Hayashi, M.D., Ph.D.
Charles Kaplan, Ph.D.
Mansun Law, Ph.D.
Yuan Liu, Ph.D.
Natasha Hill, Ph.D.
Ayse Kayali, Ph.D.
Hyun-Ku Lee, Ph.D.
Jeng-Fen Lo, Ph.D.****
Antje Gohla, Ph.D.****
A D M I N I S T R AT I V E A S S I S TA N T S , D E PA R T M E N T O F
A D M I N I S T R AT I V E A S S I S TA N T S , D E PA R T M E N T O F
I M M U N O L O G Y : Left to right: front row: Kathy Cairns and Bonnie
I M M U N O L O G Y : Left to right: front row: Karen Cerveny and
Towle; back row: Carol Wood, Anna Meyers, Barbara Marchand, and
Betsy Layton; back row: Ellen Klahn, Shelly Gassert, and
Kat Occhipinti.
Theresa McCarthy.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
100 IMMUNOLOGY
2005
Markus Loeffler, M.D.****
Jared Purton, Ph.D.
Konstantin Stoletov, Ph.D.
Kenji Yoshida, Ph.D.
Carina Lotz, Ph.D.
Christopher Ramsey, Ph.D.
Shibing Su, Ph.D.****
Jiangiang Yu, Ph.D.
Christine Louis dit Sully, Ph.D.
Koichi Tabeta, Ph.D.****
Hui Zhang, Ph.D.
Yunping Luo, Ph.D.
Jill Ricono, Ph.D.***
Moores Cancer Center
La Jolla, California
Joyce Tan, Ph.D.
You Qing Zhang, Ph.D.
Michael Lyman, Ph.D.
Mark Rubenstein, Ph.D.
Rachel Tilley, Ph.D.
Tieming Zhao, Ph.D.
Chitladda Mahanivong, Ph.D.
Monica Ruse, Ph.D.
Joie Trifilo, Ph.D.
He Zhou, Ph.D.
Presanta Maiti, Ph.D.****
Sophie Rutschmann, Ph.D.
David Valenta, Ph.D.
Huamin Zhou, Ph.D.
Laurent Malherbe, Ph.D.
Prabhakar Salunkhe, Ph.D.
Sebastian Vallee, Ph.D.
Zuping Zhou, Ph.D.****
Annette Marleau, Ph.D.
Yuichiro Sato, Ph.D.***
University of California San
Diego Medical Center
San Diego, California
Laurent Verkoczy, Ph.D.
Beatriz Maroto, Ph.D.
Annica Martensson, Ph.D.****
Gernot Schabbauer, Ph.D.****
Javier Martinez, Ph.D.
Terrence Meehan, Ph.D.
Monica Schaller, Ph.D.***
Basel Hospital
Basel, Switzerland
Satyajit Mitra, Ph.D.
Jorge Luis Schettini, Ph.D.
Johann Mols, Ph.D.
Nicolas Schrantz, Ph.D.
Adam Mullick, Ph.D.
Alim Seit-Nebi, Ph.D.
Eric Murphy, Ph.D.***
Moores Cancer Center
La Jolla, California
Suganya Selvarajah, Ph.D.
Perihan Nalbant, Ph.D.
Doinita Serban, Ph.D.***
Moores Cancer Center
La Jolla, California
Bishnu Nayak, Ph.D.****
S C I E N T I F I C A S S O C I AT E S
Hendrik Versteeg, Ph.D.
Katharina Von Lohneysen,
Ph.D.
Meng Wang, M.D.
Yingchun Wang, Ph.D.
Zhao Wang, Ph.D.
Chenghong Wei, Ph.D.
Christopher Wiethoff, Ph.D.
Justin Soon Boon Wong,
Ph.D.
Chia Cheng Wu, Ph.D.
Linda Sharp, Ph.D.****
Dong Wu, Ph.D.****
Xuifei Shen, Ph.D.****
Wenyuan Wu, Ph.D.
Fu Dong Shi, Ph.D.****
Yue Xu, Ph.D.
Pia Yachi, Ph.D.
Motoyuki Otsuka, Ph.D.
David Shields, Ph.D.***
Moores Cancer Center
La Jolla, California
Sandrine Pacquelet, Ph.D.
Shigeki Shimada, Ph.D.
Oliver Pertz, Ph.D.
Jason Smith, Ph.D.
Jian Ming Yang, Ph.D.***
Department of Cell Biology,
Scripps Research
Helle Petersen, Ph.D.
Laura Solforosi, Ph.D.
Michael Ye, Ph.D.
Matthew Potter, Ph.D.***
Moores Cancer Center
La Jolla, California
Michelle Solomon, Ph.D.
Jinseong Yi, Ph.D.****
Gabriel Sternik, Ph.D.
Sun-Hee Yoon, Ph.D.
Ron Nepomuceno, Ph.D.
Nathalie Niederberger,
Ph.D.****
Frank Karl Niessen, Ph.D.
Published by TSRI Press®. © Copyright 2005,
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Deepak Yadav, Ph.D.
Rosana Gonzales-Quintal,
Ph.D.
Marcie Rose Kritzik, Ph.D.
Nora Leaf
Ralph Pantophlet, Ph.D.
Susan D. Revak****
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 completed
IMMUNOLOGY
Richard Ulevitch, Ph.D.
Chairman’s Overview
he faculty of the Department of Immunology continues its long-standing commitments to the highest level of scientific achievement, the training
of graduate and postgraduate students, and participation in numerous activities outside Scripps Research. But
first an update on the demographics of the department.
The department consists of 46 full-time faculty members; 37 of the members are less than 60 years old,
and the majority are 50 years old or less. This age
distribution helps ensure the long-time vitality and
success of the department and is important to the overall
excellence of Scripps Research. The scientific successes
of our younger faculty members are well recognized as
indicated by the receipt of highly competitive career
development awards such as the Burroughs Wellcome
Career Award in Biomedical Science received by Erica
Ollmann Saphire and the highly coveted Established
Investigator Award awarded to David Schlaepfer by
the American Heart Association. Although a number of
faculty members have retired, most recently Charles
Cochrane, and more are planning to retire soon, an
active recruitment program since 1996 has added 9
new faculty members.
T
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2005
101
Another interesting statistic is that 18% of the faculty are women, all of whom are laboratory heads and
most of whom are tenured. In a recent letter in Nature
Immunology, the importance of Immunology as a career
for young female scientists was highlighted. Clearly,
the Department of Immunology far exceeds all averages
quoted in the letter. We are proud of our record.
Outside honors and appointments to review committees and top editorial boards accrue to all of the faculty.
One important activity is serving on National Institutes
of Health (NIH) review committees, and members of
our department are well represented. Serving as a
permanent member on an NIH study section is a major
time commitment; currently 5 of our faculty serve in
this capacity: Drs. Bokoch, Curtiss, Gascoigne, Kono,
and Nemazee. Their service is critical to maintaining
the integrity of the NIH review process, and their willingness to do so is appreciated by all of the members
of the department.
Large-scale research grants have become a major
focus at NIH. Consequently, it is critical that scientists
in the department be able to respond to these new funding mechanisms. Today the department has several
such grants. The most recent is a multimillion dollar
grant to establish the Scripps Research Institute Molecular Screening Center. The goal of this pilot program is
to discover small-molecule tools for translating basic
biomedical discoveries more quickly into medically relevant applications. This grant also marks a key collaboration between Scripps Research in La Jolla and Scripps
Florida. This funding is part of the NIH strategic funding plan, the Roadmap Initiative. Hugh Rosen is the
principal investigator on the grant. His intention is to
conduct high-throughput screens against various biological targets to uncover “proof of concept” molecules
useful in studying human health and in developing
new treatments for human diseases. As Dr. Rosen
remarked, “With this grant, the NIH has recognized
the unique capabilities of our established researchers
in La Jolla with our newest investigators and equipment in Palm Beach County.”
During the past year, the output of seminal advances
was high. Members of the department were authors of
more than 30 articles that appeared in high-profile journals, including Science, Nature, Cell, Nature Immunology, Nature Cell Biology, and Nature Medicine.
Most of the research described in these articles is
given in detail in the reports of the individual laboratories, but the following are a few of the highlights.
102 IMMUNOLOGY 2005
This department, long recognized for its continued
contributions to studies of the adaptive immune system,
is now recognized as one of the leaders in advancing
our understanding of innate immunity. One reason for
this recognition is the continued productivity of Bruce
Beutler and his group. During the past year, they made
several important advances. In an article published in
Nature, they describe a key role for the transmembrane
protein known as CD36 in sensing diacyglycerides. The
researchers showed that a nonsense mutation of Cd36
causes a recessive immunodeficiency in which macrophages are insensitive to the R-enantiomer of MALP-2
(a diacylated bacterial lipopeptide) and to lipoteichoic
acid. Mice homozygous for the mutation are hypersusceptible to Staphylococcus aureus infection. Studies
on macrophages from mice with the mutation revealed
that some, but not all, ligands for Toll-like receptor
(TLR) 2 are CD36 dependent. Already known as a
receptor for endogenous molecules, CD36 is also a
selective and nonredundant sensor of microbial diacylglycerides that signal via the TLR2/6 heterodimer. This
work provides new data on the role of innate immunity
during infection and during chronic inflammatory diseases such as atherosclerosis in which endogenous
diacyglycerides may be generated and trigger TLR2.
A second report by Dr. Beutler and colleagues published in Nature Immunology provides new information
on CD14 and its role in TLR4 signaling. This research is
particularly interesting because it was nearly 15 years
ago that the studies of other members of the department published in Science provided the first molecular
insights into the function of CD14 as a sensor for bacterial lipopolysaccharides.
Gary Bokoch and the members of his laboratory continue to provide new insights into the molecular physiology of phagocytic cells. During the past year, in an article
in Nature Cell Biology, they reported the biochemical isolation of chronophin, a unique cofilin-activating
phosphatase of the haloacid dehalogenase superfamily.
Chronophin directly dephosphorylates cofilin with high
specificity and colocalizes with cofilin in motile and
dividing cells. Loss of chronophin activity blocks phosphocycling of cofilin, stabilizes F-actin structures, and
causes massive defects in cell division. These findings
identify a physiologic phosphoserine protein substrate
for a mammalian haloacid dehalogenase–type phosphatase and indicate that chronophin is an important
novel regulator of cofilin-mediated actin reorganization.
Researchers in Jiahuai Han’s group recently made
a major contribution to understanding the mechanisms
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involved in stability of mRNA species that encode key
molecules in innate immunity and inflammation. Adenosine-uridine–rich elements (AREs) in the 3′ untranslated
region of unstable mRNAs dictate degradation of the
mRNAs. An RNA interference–based screen in Drosophila
S2 cells revealed that in TNF-α, components involved
in microRNA processing and function are required for
the rapid decay of mRNA that contains AREs. The
requirement for the component Dicer in the instability
of mRNA with AREs was confirmed in HeLa cells. The
researchers further observed that miR16, a human
microRNA containing a sequence complementary to
the ARE sequence is required for the turnover of RNA
that contains AREs. The role of miR16 in the decay of
mRNA that contains AREs is sequence specific and
requires the ARE-binding protein tristetraprolin. Tristetraprolin does not directly bind to miR16; rather it
interacts through association with other components
involved in microRNA processing to form a complex
with miR16 and assists in the targeting of ARE. The
targeting of ARE by microRNA therefore appears to be
an essential step in ARE-mediated mRNA degradation.
These findings provide an entirely new understanding
of the function of microRNA and a new model for studies
of AREs during a variety of host responses.
Other publications by members of the department
include reports of the pioneering research of Luc Teyton
and his group, in collaboration with A. Bendelac, University of Chicago, on natural killer T cells and the cells’ key
membrane sensor CD1. Specifically, in articles published in Science, Nature, and Nature Immunology, the
following was noted. Natural killer T cells are a distinct
lineage of T cells that coexpress a conserved αβ T-cell
receptor (TCR) and natural killer receptors. Although
the TCR of natural killer T cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified.
Dr. Teyton and his group showed that isoglobotrihexosylceramide (iGb3), a lysosomal glycosphingolipid of
previously unknown function, is recognized by both
mouse and human natural killer T cells. Impaired generation of lysosomal iGb3 in mice lacking β-hexosaminidase B results in a severe deficiency in natural killer
T cells, suggesting that this lipid also mediates development of these T cells in mice. The findings suggest that
expression of iGb3 in peripheral tissues may be involved
in controlling the responses of natural killer cells to infections and malignant neoplasms and in autoimmunity.
Further, the scientists have shown microbial, antigen-specific activation of natural killer T cells against
IMMUNOLOGY
gram-negative, lipopolysaccharide-negative α-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata.
Glycosylceramides from the cell wall of Sphingomonas
act as direct targets for mouse and human natural killer
T cells, controlling both septic shock and bacterial clearance in infected mice. In contrast, gram-negative, lipopolysaccharide-positive Salmonella typhimurium activates
natural killer T cells through recognition of iGb3 presented by lipopolysaccharide-activated dendritic cells.
These findings identify 2 novel antigenic targets of natural killer T cells in antimicrobial defense and show that
glycosylceramides are an alternative to lipopolysaccharide for innate recognition of the gram-negative, lipopolysaccharide-negative bacterial cell wall.
Finally, in collaborative studies with Ian Wilson and
his group, members of the Teyton group documented the
crystal structure of CD1d in complex with a short-chain
synthetic variant of α-galactosylceramide at a resolution
of 2.2 Å. This structure indicates the basis for the high
specificity of these microbial ligands and explains the
restriction of the α-linkage as a unique pathogen-specific
pattern recognition motif. Comparison of the binding of
altered lipid ligands to CD1d and TCRs suggested that
the differential helper T cell–like properties of natural
killer T cells may originate largely from differences in
the “loading” of the ligands in different cell types and
hence in the tissue distribution of the ligands in vivo.
Overall, this research provides a remarkably broad
set of advances in understanding the functions of natural
killer T cells and the structure of one of the key sensor
molecules. In addition, it further illustrates the ability of
members of the department to collaborate with other leaders in this field inside and outside of Scripps Research.
Members of the Department of Immunology have a
long-standing interest in the functions of T cells in various
biological models. Studies from Wendy Havran and her
group are an example of this interest. A fine balance
between rates of proliferation and apoptosis in the skin
provides a defensive barrier and a mechanism for tissue
repair after damage. Vγ3+ dendritic epidermal T cells are
primary modulators of skin immune responses. Dr. Havran
and her group showed that these cells both produce and
respond to insulin-like growth factor 1 (IGF-1) after TCR
stimulation. Mice deficient in the cells had a notable
increase in epidermal apoptosis that was abrogated by
the addition of Vγ3+ dendritic epidermal T cells or IGF-1.
Furthermore, mice deficient in the cells had reduced
activation of IGF-1 receptors at wound sites. These findings indicate critical functions for Vγ3 + dendritic epiPublished by TSRI Press®. © Copyright 2005,
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2005
103
dermal T cell–mediated IGF-1 production in regulating
skin homeostasis and repair.
Linda Sherman and members of her laboratory also
provided important new insights into the role of T cells
in tumor immunity. Studies published in Immunity, done
in collaboration with M. Theobald, Johannes Gutenberg
University, Mainz, Germany, revealed that efficient immune
attack on malignant disease requires the concerted action
of both CD8+ cytotoxic T lymphocytes (CTLs) and CD4+
T helper cells. The researchers used HLA-A*0201 (A2.1)
transgenic mice, in which the mouse CD8 molecule
cannot efficiently interact with the α3 domain of A2.1,
to generate a high-affinity, CD8-independent TCR specific for a commonly expressed, tumor-associated CTL
epitope derived from p53, a human tumor suppressor
protein. Introduction of this TCR into human T cells
resulted in CD8+ T lymphocytes with broad tumor-specific cytotoxic activity and CD4 + T cells with potent
tumor-reactive, p53A2.1-specific helper activity. Both
T-cell subsets interacted synergistically with dendritic
cell intermediates and tumor targets. The intentional
redirection of both CD4 + helper T cells and CD8+ CTLs
by the same high-affinity, CD8-independent, tumor-specific TCRs could provide the basis for novel broad-spectrum cancer immunotherapeutic agents.
Other contributions of note during the past year
include studies done by David Nemazee and members
of his laboratory on B-cell development. In developing
B cells, expression of immunoglobulin on the cell surface is an important signal to terminate expression of
recombinase activator gene (RAG) and V(D)J recombination. However, autoreactive antigen receptors promote
continued gene rearrangement and receptor editing.
Regulation of RAG expression and editing by B-cell
receptor signaling is poorly understood. Dr. Nemazee
and his colleagues found that in editing-competent cells,
RAG mRNA expression induced by B-cell receptor ligands
is regulated at the level of RAG transcription. In immature B cells carrying innocuous receptors, RAG expression appears to be under rapidly reversible negative
regulation. Research with transduction of a superrepressive IκBα protein indicated that NF-κB/Rel proteins
promote RAG transcription. Interestingly, cells deficient
in NF-κB overexpress RAG and undergo an exaggerated
receptor editing response. These results implicate NF-κB
transcription factors in the regulation of RAG transcription mediated by B-cell receptors. Rapidly activated
NF-κB pathways may facilitate prompt antigen receptor–regulated changes in RAG expression important for
editing and haplotype exclusion.
104 IMMUNOLOGY
2005
In closing, I can say without reservation that writing this report was a great pleasure, because it gave
me a chance to review the accomplishments of the faculty of the Department of Immunology. These accomplishments span all aspects of 21st century science:
cutting-edge contributions to knowledge of the immune
system; education of undergraduate, graduate, and
postgraduate students; and participation in important
outside activities that include serving on NIH review
committees and journal editorial boards and organizing
and participating in important national and international scientific meetings. Each member of the faculty
contributes in all these aspects of science and makes
the Department of Immunology, to me, the model that
many others aspire to emulate.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
IMMUNOLOGY
INVESTIGATORS’ R EPORTS
The Genetic Basis of
Resistance to Infection
B. Anchonda, K. Benson, B. Croker, K. Crozat, X. Du,
P. Georgel, C. Gil-Lamaignere, K. Hoebe, Z. Jiang, N. Mann,
S. Mudd, S. Rutschmann, L. Shamel, S. Sovath, K. Tabeta,
H. Uy, Z. Zhou, B. Beutler
o “universal pathogens” exist, and even the most
virulent human pathogens (e.g., HIV and smallpox virus) are innocuous in most mammalian
species. Conversely, no human has ever died of mouse
cytomegalovirus (MCMV) infection. In general, interspecies differences in susceptibility to infectious diseases cannot be explained on the basis of differences
in adaptive immunity. The combinatorial system for
generating diversity in T- and B-cell receptors is similarly effective in all mammals. Rather, innate immunity
is the characteristic that differs remarkably among species. And even among members of a single species,
differences in innate immunity may foretell life or death
in the event of an infection. Evidently, a large number
of genes confer resistance to infection. For this reason,
in humans, susceptibility to death caused by infection
has greater heritability than susceptibility to death from
any other cause.
Our broad goal is to identify all the genes required
for a strong innate immune response. Most genes that
serve this function in mice are also required for innate
immunity in humans, and, indeed, conservation of core
innate immune processes is such that many of the same
genes are even used for innate defense by fruit flies. To
find nonredundant components of the innate immune
system, we create inherited innate immunodeficiency
states through the use of a randomly acting germ-line
mutagen, N-ethyl-N-nitrosourea. Those exceptional animals that have compromised immunity are detected by
phenotypic screening. The mutations are brought to
homozygosity and mapped by using classical genetic
methods. DNA sequencing is then applied to pinpoint
the culpable mutation and hence find the gene (and
protein) with an indispensable role in disease resistance. We use 2 general types of phenotypic screens.
N
COMPONENTS OF THE TOLL-LIKE RECEPTOR SIGNAL
T R A N S D U C T I O N PAT H WAY S
We earlier determined that the mammalian Tolllike receptors (TLRs) are responsible for perceiving
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
105
diverse infections, ranging from those caused by bacteria and fungi to those caused by protozoa and viruses.
TLRs alert the host to the presence of conserved molecules that are synthesized by these microbes, such
as lipopolysaccharide, DNA with unmethylated cytosine-guanine dinucleotides, double-stranded RNA, and
various diacylglycerides and triacylglycerides. We have
implemented a focused screen for defects in signaling
from TLRs to the level of TNF production.
Using this screen, we identified 11 mutations, which
affect 10 genes. Of the 10 genes, 5 encode “known”
components of the TLR signaling apparatus, and 5
encode components that were not previously known.
Among the new elements are an adapter protein for
TLR signaling (TRIF), a coreceptor for sensing microbial
diacylglycerides (CD36), a protein kinase (feckless) that
links the sensing of double-stranded RNA to the activation of NF-κB (a key inflammatory transcription factor),
and a component of the endoplasmic reticulum (3d)
that is required for signaling via 4 of the TLRs. On the
basis of the number and types of mutations found, we
calculated that about 50 proteins are required for signaling from the TLRs to the level of TNF production.
The protein 3d is remarkable because it is required
not only for TLR signaling but also for the processing
of antigens for activation of adaptive immune responses.
Thus, 3d links the innate and adaptive immune systems. Further investigation of the mechanism of action
of 3d may reveal much about how autoimmune diseases are initiated and sustained.
SUSCEPTIBILITY TO MCMV
C57BL/6 mice normally are strongly resistant to
infection by MCMV. However, we found that a substantial fraction of the mouse genome is devoted to
the creation of resistance. Mutations in approximately
300 genes can cause profound susceptibility to MCMV.
Approximately 10% of these genes have now been
altered by mutation, and several of the mutations have
been brought to homozygosity, a necessary precondition for positional cloning.
The MCMV “resistome,” those genes that serve
nonredundant functions in resistance to this pathogen,
will gradually be defined through a forward genetic
approach. Many of the mutations so far that diminish
resistance to MCMV have broad effects on resistance
to infection overall. We therefore think that the MCMV
resistome is not much smaller than the “universal resistome,” the complement of genes required for resistance
to all microbes.
106 IMMUNOLOGY 2005
N O V E L PAT H WAY S F O R T H E A C T I VAT I O N O F
ADAPTIVE IMMUNE RESPONSES
The TLRs mediate most infection-related phenomena, including “bad” effects such as fever and shock
and “good” effects such as activation of the adaptive
immune response. This last effect has been of particular interest to immunologists, because it is an important factor in the development of vaccines. However,
we discovered that TLR-independent pathways exist
for initiation of an adaptive immune response. One
pathway impels a highly efficient CD8 T-cell response
to foreign proteins expressed by cells undergoing apoptosis (programmed death). This “death-driven” adjuvant
pathway is the most efficient means of CD8 activation ever described. It presumably evolved to permit
the detection of pathogens that trigger an apoptotic
response. Our assumption is that the host has retained
pathogen-specific pathways for cell death and uses
them to encourage an adaptive immune response.
Aberrant activation of these pathways may be important in the development of dysfunctional adaptive
immune responses to host proteins.
PUBLICATIONS
Anfossi, N., Robbins, S.H., Ugolini, S., Georgel, P., Hoebe, K., Bouneaud, C.,
Ronet, C., Kaser, A., DiCioccio, C.B., Tomasello, E., Blumberg, R.S., Beutler, B.,
Reiner, S.L., Alexopoulou, L., Lantz, O., Raulet, D.H., Brossay, L., Vivier, E. Expansion and function of CD8+ T cells expressing Ly49 inhibitory receptors specific for
MHC class I molecules. J. Immunol. 173:3773, 2004.
Hoebe, K., Janssen, E., Beutler, B. The interface between innate and adaptive
Immunity. Nat. Immunol. 5:971, 2004.
Jiang, Z., Georgel, P., Du, X., Shamel, L., Sovath, S., Mudd, S., Huber, M., Kalis, C.,
Keck, S., Galanos, C., Freudenberg, M., Beutler, B. CD14 is required for MyD88independent LPS signaling. Nat. Immunol. 6:565, 2005.
Mattner, J., DeBord, K.L., Ismail, N., Goff, R.D., Cantu, C. III, Zhou, D., SaintMezard, P., Wang, V., Gao, Y., Yin, N., Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L., Savage, P.B., Bendelac, A. Exogenous and endogenous glycolipid
antigens activate NKT cells during microbial infections. Nature 434:525, 2005.
Rietschel, E.T., Rietschel, M., Beutler, B. How the mighty have fallen: fatal infectious diseases of divine composers. Infect. Dis. Clin. North Am. 18:311, 2004.
Smythe, I., Du, X., Taylor, M.S., Justice, M.J., Beutler, B., Jackson, I.J. The
extracellular matrix gene Frem1 is essential for the normal adhesion of the embryonic epidermis. Proc. Natl. Acad. Sci. U. S. A. 101:13560, 2004.
Theofilopoulos, A.N., Baccala, R., Beutler, B., Kono, D.H. Type I interferons (α/β)
in immunity and autoimmunity. Annu. Rev Immunol. 23:307, 2005.
Zhou, Z., Hoebe, K., Du, X., Jiang, A., Shamel, L., Beutler, B. Antagonism
between MyD88- and TRIF-dependent signals in B7RP-1 up-regulation. Eur. J.
Immunol. 35:1918, 2005.
Regulation of Cell Function by
Rho GTPases
G.M. Bokoch, C. Aylott, J. Birkenfeld, M. Crawford,
V. Delorme, C. DerMardirossian, A.M. DeCathlineau,
B.A. Diebold, T. Huang, Y.-Y. Kao, P. Nalbant,
K. Pestonjamasp, Y. Wu, T. Zhao, B.P. Bohl, A. Fowler,
Beutler, B. SHIP, TGF-β, and endotoxin tolerance. Immunity 21:134, 2004.
J. Neuberg
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.
ho GTPases control the assembly of the actin
cytoskeleton, 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, angiogenesis, 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.
Beutler, B., Crozat, K., Koziol, J.A., Georgel, P. Genetic dissection of innate immunity
to infection: the mouse cytomegalovirus model. Curr. Opin. Immunol. 17:36, 2005.
Beutler, B., Hoebe, K., Georgel, P., Du, X. Forward genetic analysis of TLR pathways: a shared system for the detection of endotoxin and viral infection. In: Toll
and Toll-Like Receptors: An Immunologic Perspective. Rich, T. (Ed.). Kluwer Academic/Plenum, New York, 2005, p. 168. Molecular Biology Intelligence Unit.
Beutler, B., Hoebe, K., Georgel, P., Tabeta, K., Du, X. Genetic analysis of innate
immunity: TIR adapter proteins in innate and adaptive immune responses. Microbes
Infect. 6:1374, 2004.
Georgel, P., Crozat, K., Lauth, X., Makrantonaki, E., Seltmann, H., Sovath, S.,
Hoebe, K., Du, X., Rutschmann, S., Jiang, Z., Bigby, T., Nizet, V., Zouboulis, C.C.,
Beutler, B. A Toll-like receptor 2-responsive lipid effector pathway protects mammals
against skin infections with gram-positive bacteria. Infect. Immun. 73:4512, 2005.
Hawn, T.R., Verbon, A., Janer, M., Zhao, L.P., Beutler, B., Aderem, A. Toll-like
receptor 4 polymorphisms are associated with resistance to Legionnaires’ disease.
Proc. Natl. Acad. Sci. U. S. A. 102:2487, 2005.
Hoebe, K., Beutler, B. LPS, dsRNA and the interferon bridge to adaptive immune
responses: Trif, Tram, and other TIR adaptor proteins. J. Endotoxin Res. 10:130, 2004.
Hoebe, K., Beutler, B. Unraveling innate immunity using large scale N-ethyl-Nnitrosourea mutagenesis. Tissue Antigens 65:395, 2005.
Hoebe, K., Georgel, P., Rutschmann, S., Du, X., Mudd, S., Crozat, K., Sovath, S.,
Shamel, L., Hartung, T., Zähringer, U., Beutler, B. CD36 is a sensor of diacylglycerides. Nature 433:523, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
R
RHO GTPASES AND HUMAN LEUKOCYTES
We previously established that the GTPase Rac regulates the formation of ROS that are used by human
phagocytic cells for microbial killing and that result in
inflammatory responses. Using a cell-free recombinant
system, we established the molecular interaction of
Rac with cytochrome b, a component of the mem-
IMMUNOLOGY
brane-bound NADPH oxidase, independent of p67phox,
leading to a 2-step mechanism for regulation of electron transfer to form superoxide anion (Fig. 1). We
showed that the Rac-related GTPase Cdc42 acts as a
competitive inhibitor of Rac binding to cytochrome b.
This inhibition results in an antagonistic cross talk
between Rac and Cdc42 that modulates the formation
of ROS in leukocytes and, potentially, nonphagocytic
cells. This unique mechanism may coordinate formation of ROS with cytoskeletal dynamics during chemotaxis and phagocytosis.
2005
107
inhibitor and modulate its ability to bind Rac GTPase. We
discovered a potential positive-feedback Rac activation
cycle that involves phosphorylation of Rho GDP-dissociation inhibitor by p21-activated kinase 1 (PAK1), a
downstream effector of Rac and Cdc42 signaling.
We are using live-cell imaging in combination with
fluorescent methods to determine the spatial and temporal localization of Rho GTPase activation. We are
beginning to determine the molecular signals that govern
the chemotactic responses of human leukocytes and the
biochemical pathways that lead to assembly of the
cytoskeleton and motility. We established a primary
link between the actin and microtubule cytoskeletons
that involves regulation of Rho GTPase via physical
sequestration of the Rho GEF-H1 by microtubules. We
recently discovered that GEF-H1 is a signaling link
between microtubules in the mitotic spindle and the
initiation of Rho-dependent formation of cleavage furrows in dividing cells (Fig. 2).
F i g . 1 . Two-step activation mechanism for Rac GTPase–mediated
regulation of oxidant formation by phagocyte NADPH oxidase.
We found that an inhibitory cross talk between
leukocyte adhesion receptors and NADPH oxidase activation occurs via modulation of Rac2 GTPase activity.
Rac2 acts as a critical “molecular switch” that regulates formation of ROS in adherent cells. Integrin signaling inhibited activation of Vav1, the upstream guanine
nucleotide exchange factor (GEF) that regulates Rac2GTP formation and thus NADPH oxidase activity. We
have now identified a mechanism by which TNF-α and
certain other cytokines can overcome adhesion-induced
inhibition to allow rapid formation of ROS at inflammatory sites. This mechanism requires the activity of
proline-rich tyrosine kinase 2 to initiate signaling from
Vav1 to Rac2. These studies (1) address the important
physiologic question of how leukocytes migrate to
inflammatory sites without perpetuating continuous
oxidative damage to underlying tissue and (2) provide
additional mechanistic insight into the inflammatory
actions of TNF-α.
R E G U L AT I O N O F R H O G T PA S E S
The regulatory protein GDP-dissociation inhibitor is a
critical control point for Rho GTPase function. We are
investigating the action of kinases that phosphorylate this
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 2 . Immunofluorescent images show colocalization of endog-
enous GEF-H1 with microtubules (tubulin) in the mitotic spindle.
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 human neutrophil chemotaxis and formation of
ROS. A potential requirement for Rho GTPases in uptake
and action of the anthrax toxins in macrophages is also
under study.
C E L L R E G U L AT I O N B Y PA K S
PAKs are cellular effectors of Rac and Cdc42. The
C-terminal kinase domain of these enzymes phosphorylates substrates involved in regulating NADPH oxidase,
stress responses, and the cellular actin-myosin system.
PAKs are important mediators of chemotaxis, wound
healing, tumor metastasis, neurite outgrowth, antigen
presentation, and other processes that depend on cytoskeletal polarization.
108 IMMUNOLOGY 2005
The phosphorylation of cofilin, which depolymerizes
and severs actin, by PAK1–LIM kinase is an important
regulatory point in cytoskeletal dynamics. 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
found 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. We are investigating the regulation of cell motility and other cellular processes by this
unique regulatory phosphatase. We are also investigating the requirement for PAK function in retrograde actin
flow, a critical component of cell motility. These ongoing studies provide diverse avenues of investigation.
PUBLICATIONS
Birukov, K.G., Bochkov, V.N., Birukova, A.A., Kawkitinarong, K., Rios, A., Leitner, A., Verin, A.D., Bokoch, G.M., Leitinger, N., Garcia, J.G. Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via
Cdc42 and Rac. Circ. Res. 95:892, 2004.
Bokoch, G.M. Regulation of Innate Immunity by Rho GTPases. Trends Cell Biol.
15:163, 2005.
DerMardirossian, C., Bokoch, G.M. GDIs: central regulatory molecules in Rho
GTPase activation. Trends Cell Biol. 15:356, 2005.
Diebold, B.A., Bokoch, G.M. Rho GTPases and the control of the oxidative burst in
polymorphonuclear leukocytes. Curr. Top. Microbiol. Immunol. 291:91, 2005.
Gohla, A., Birkenfeld, J., Bokoch, G.M. Chronophin, a novel HAD-type serine protein
phosphatase, regulates cofilin-dependent actin dynamics. Nat. Cell Biol. 7:21, 2005.
Makino, A., Glogauer, M., Bokoch, G.M., Chien, S., Schmid-Schonbein, G.W.
Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases. Am.
J. Physiol. Cell Physiol. 288:C863, 2005.
Stofega, M., DerMardirossian, C., Bokoch, G.M. Affinity-based assay of Rho
GTPase activation. Methods Mol. Biol., in press.
Yuan, Z.Q., Kim, D., Kaneko, S., Sussman, M., Bokoch, G.M., Kruh, G.D.,
Nicosia, S.V., Testa, J.R., Cheng, J.Q. ArgBP2γ interacts with Akt and p21-activated kinase-1 and promotes cell survival. J. Biol. Chem. 280:21483, 2005.
Zhao, T., Bokoch, G.M. Critical role of proline-rich tyrosine kinase 2 in reversion of
the adhesion-mediated suppression of reactive oxygen species generation by human
neutrophils. J. Immunol. 174:8049, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Human Antibodies and Design
of a Vaccine to HIV Type 1
M.B. Zwick, R.A. Pantophlet, R.O. Aguilar-Sino,
R. Astronomo, D. Bowley, H. Donners, A. Gakhal,
A.J. Hessell, R.C. Jensen, M. Law, J.D. Nelson, E. Scherer,
S. Selvarajah, M. Wang, R.A. Dwek,* P.M. Rudd,*
D. Calarese, 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 42 million persons are infected with the
virus, and 22 million persons have died of AIDS.
It is widely recognized that a vaccine most likely is the
best way to control HIV-1 infection worldwide. We wish
to understand antibody responses to HIV-1 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-1. 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 potently neutralizes a broad array of different viruses. The existence of this antibody indicates
that some features of HIV-1 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 tackled were the following: Can antibodies protect against HIV-1 infection,
and, if so, under what conditions? On the basis of passive transfer studies in a number of animal model systems, the answer is clearly yes. Complete protection
from infection 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. Further, we showed that topically applied
antibody can protect monkeys against vaginal challenge with 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 strate-
H
IMMUNOLOGY
gies 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.
Currently, we are studying the epitopes recognized
by 5 broadly neutralizing antibodies: b12, 2G12, 4E10,
Z13, and 2F5. The antibody b12 recognizes a complex
epitope that overlaps the CD4-binding site of gp120.
We are using a hyperglycosylation-mutagenesis strategy to “immunofocus” responses to this epitope by
blocking nonneutralizing epitopes on gp120. The strategy is based on our previous determination of the structure of b12, docking of b12 and gp120, and extensive
mutagenesis studies of b12 and gp120.
The antibody 2G12 recognizes a cluster of sugar
residues on gp120. Solution of the structure of 2G12
in complex with sugars enabled us, in collaboration
with C.-H. Wong, Department of Chemistry, to design
synthetic sugars that are being used as immunogens
to elicit 2G12-like antibodies. The antibody 4E10 recognizes a linear epitope on gp41 (Fig. 1). We determined the structure of 4E10 with peptide bound, and
we are working with P. Dawson, Department of Cell
Biology, to design peptide immunogens that can elicit
4E10-like antibodies. The antibody Z13 recognizes an
epitope on gp41 that overlaps with that of 4E10. Both
4E10 and Z13 are providing molecular insights into
the structure and function of the membrane proximal
region of HIV-1 gp41.
2005
109
gating the molecular origins of this problem. Overall,
HIV vaccine development has been distilled down to
challenges in the design of protein, carbohydrate, and
peptide immunogens.
PUBLICATIONS
Binley, J.M., Wrin, T., Korber, B., Zwick, M.B., Wang, M., Chappey, C., Stiegler, G.,
Kunert, R., Zolla-Pazner, S., Katinger, H., Petropoulos, C.J., Burton, D.R. A comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency type 1 monoclonal antibodies. J. Virol. 78:13232, 2004.
Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan,
M.C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D.J., Skehel, J.J.,
van Die, I., Burton, D.R., Wilson, I.A., Cummings, R., Bovin, N., Wong, C.-H.,
Paulson, J.C. Printed covalent glycan array for ligand profiling of diverse glycan
binding proteins. Proc. Natl. Acad. Sci. U. S. A. 101:17033, 2004.
Bryan, M.C., Fazio, F., Lee, H.-K., Huang, C.-Y, Chang, A., Best, M.D., Calarese,
D.A., Blixt, O., Paulson, J.C., Burton, D., Wilson, I.A., Wong, C.-H. Covalent display
of oligosaccharide arrays in microtiter plates. J. Am. Chem. Soc. 126:8640, 2004.
Cardoso, R.M.F., Zwick, M.B., Stanfield, R.L., Kunert, R., Binley, J.M., Katinger, H.,
Burton, D.R., Wilson, I.A. Broadly neutralizing anti-HIV antibody 4E10 recognizes
a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22:163, 2005.
Ho, J., Uger, R.A., Zwick, M.B., Luscher, M.A., Barber, B.H., MacDonald, K.S. Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in
increased antigenicity but not neutralizing response. Vaccine 23:1559, 2005.
Koefoed, K., Farnaes, L., Wang, M., Svejgaard, A., Burton, D.R., Ditzel, H.J. Molecular characterization of the circulating anti-HIV-1 gp120-specific B cell repertoire
using antibody phage display libraries generated from pre-selected HIV-1 gp120
binding PBLs. J. Immunol. Methods 297:187, 2005.
O’Connor, D.H., McDermott, A.B., Krebs, K.C., Dodds, E.J., Miller, J.E., Gonzalez, E.J., Jacoby, T.J., Yant, L., Piontkivska, H., Pantophlet, R., Burton, D.R.,
Rehrauer, W.M., Wilson, N., Hughes, A.L., Watkins, D.I. A dominant role for
CD8+-T-lymphocyte selection in simian immunodeficiency virus sequence variation.
J. Virol. 78:14012, 2004.
Pantophlet, R., Wilson, I.A., Burton, D.R. Improved design of an antigen with
enhanced specificity for the broadly HIV-neutralizing antibody b12. Protein Eng.
Des. Sel. 17:749, 2004.
Zhang, M.Y., Shu, Y., Rudolph, D., Prabakaran, P., Labrijn, A.F., Zwick, M.B.,
Lal, R.B., Dimitrov, D.S. Improved breadth and potency of an HIV-1-neutralizing
human single-chain antibody by random mutagenesis and sequential antigen panning. J. Mol. Biol. 335:209, 2004.
Zwick, M.B., Jensen, R., Church, S., Wang, M., Stiegler, G., Kunert, R., Katinger,
H., Burton, D.R. Anti-human immunodeficiency virus type 1 (HIV-1) antibodies
2F5 and 4E10 require surprisingly few crucial residues in the membrane-proximal
external region of glycoprotein gp41 to neutralize HIV-1. J. Virol. 79:1252, 2005.
Antibodies and
Emerging Viruses
S.K. Kurz-Camacho, W. Oswald, E.O. Saphire,
P.B. Jahrling,* P.M. Rudd,** R.A. Dwek,** H. Feldman,***
D.R. Burton
F i g 1 . Model of 2F5 and 4E10, broadly neutralizing antibodies to
HIV-1, bound to the membrane proximal external region of gp41.
The fifth antibody, 2F5, recognizes another linear
epitope on gp41 (Fig. 1). Previous attempts to present
this epitope in a wide variety of immunogens did not
elicit 2F5-like neutralizing antibodies. We are investiPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
* U.S. Army Medical Research Institute of Infection Diseases, Frederick, Maryland
** Oxford Glycobiology Institute, Oxford, England
*** Bureau of Microbiology, Health Canada, Winnipeg, Manitoba
W
e are interested in determining the immunogenicity of soluble vs surface glycoproteins
of Ebola virus, a filovirus that is one of the
110 IMMUNOLOGY 2005
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.
Another focus of our Ebola virus research is the
early molecular events that occur during infection with
the virus. We found that changes in cellular gene expression occurred as early as 1 hour after Ebola virus infection of mononuclear phagocytes, the major target cell
of the virus. On the basis of our microarray data, we
hypothesized that binding and entry of the Ebola virus
glycoprotein cause the early changes in expression levels. To test this hypothesis, we produced soluble forms
of the glycoprotein and Ebola viruslike particles that
present the glycoprotein on the surface. Incubation with
the viruslike particles but not with the soluble glycoproteins caused the same changes in cellular gene and
protein expression in Ebola virus target cells as did
infection with live Ebola virus. We detected changes in
gene expression for inflammatory cytokines, chemokines,
molecules involved in the development of hemorrhage,
and potential receptors for the virus. Using the viruslike
particles, we are testing whether the indicated receptors
are involved in Ebola virus binding and signaling.
PUBLICATIONS
Wahl-Jensen, V., Kurz, S.K., Hazelton, P.R., Schnittler, H.-J., Ströher, U., Burton,
D.R., Feldmann, H. The role of Ebola virus secreted glycoproteins and virus-like
particles in activation of human macrophages. J. Virol. 79:2413, 2005.
Reverse Cholesterol Transport
and High-Density Lipoproteins
L.K. Curtiss, C. Flood, N.J. Hime, A.E. Mullick,
R.J. Petrovan, D.T. Valenta
n order to be removed from the body, cholesterol
must be dissolved into or converted to bile acids in
the liver. This biliary excretion pathway is fed by
the transport of cholesterol from peripheral tissues and
is referred to as reverse cholesterol transport. An early
step in reverse cholesterol transport is the transfer of
peripheral cell-free cholesterol to plasma high-density
lipoproteins (HDLs). The HDLs serve as transport vehicles for excess cellular cholesterol through the plasma
compartment to the liver. The major protein in HDL is
apolipoprotein AI. Importantly, the transfer of cellular
cholesterol to HDL does not occur in plasma. It occurs
in extracellular spaces such as the subendothelial space
or intima of a vessel within an atherosclerotic lesion.
I
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We are investigating how apolipoprotein AI promotes the efficient transfer of excess cholesterol from
peripheral cells (i.e., intimal macrophage foam cells)
to HDL. We have published convincing in vivo data
that apolipoprotein E and apolipoprotein AI participate
in the efflux of free cholesterol from atherosclerotic
lesions. We found that both plasma-derived apolipoprotein AI and macrophage-produced apolipoprotein E
participate in the efficient efflux of free cholesterol
from aortas containing atherosclerotic lesions in atherosclerosis-prone mice. The in vivo specificity for apolipoprotein AI in efflux of cholesterol from macrophages
in atherosclerotic lesions is a direct function of the
ability of the apolipoprotein to dissociate from spherical HDL and to form transiently stable, lipid-poor apolipoprotein AI, which can accept macrophage ATP
binding cassette A1–transported free cholesterol and
phospholipid. We are studying this phenomenon in
mice that lack receptors for low-density lipoprotein.
Mice deficient in receptors for low-density lipoprotein are severely hyperlipidemic, and detectable atherosclerotic lesions develop in the aorta and aortic sinus
within weeks if the animals are fed a high-fat diet. We
are focusing on the role of multiple macrophage nuclear
liver X receptor–inducible genes in reverse cholesterol
transport. We are determining the role played by
phospholipid transfer protein (PLTP) in the generation
of lipid-poor or lipid-free apolipoprotein AI in vivo and
in vitro. To examine the role of macrophage-derived
PLTP in cholesterol metabolism and atherosclerosis,
we performed bone marrow transplantations in mice
deficient in receptors for low-density lipoprotein; the
mice were lethally irradiated and were reconstituted
with either wild-type or PLTP-deficient bone marrow
cells. The transplanted animals were fed a high-fat
diet for 16 weeks to induce atherosclerosis. We found
that macrophage PLTP deficiency led to increases in
the total plasma levels of cholesterol and in the extent
of atherosclerotic lesions, suggesting an atheroprotective role of macrophage-derived PLTP in the intima.
We are also determining the role of cholesteryl ester
transfer protein (CETP) in this same process in vivo and
in vitro. Both PLTP and CETP are expressed by macrophages, can generate lipid-poor apolipoprotein AI from
spherical HDL, are present in atherosclerotic lesions,
and are induced by ligation of liver X receptors.
Finally, we are studying the role played by the lipoprotein triglyceride hydrolases lipoprotein lipase and
hepatic lipase in the generation of lipid-poor apolipo-
IMMUNOLOGY
protein AI in vitro. Both of these lipases also are
expressed by macrophages, are present in atherosclerotic lesions, and are upregulated by ligation of nuclear
liver X receptors. Macrophage-derived lipoprotein lipase
and hepatic lipase promote the formation of lipid-poor
apolipoprotein AI from mature HDL. This remodeling
of HDL facilitates a reduction in the size of HDL particles to provide free apolipoprotein AI substrate for PLTP
and CETP lipid transfer. Both lipid transfer proteins and
neutral triglyceride lipases participate in HDL remodeling and in macrophage-mediated efflux of cholesterol
from atherosclerotic lesions. Therefore a number of
nuclear liver X receptor–inducible gene products are
expressed by macrophages in response to the accumulation of cholesteryl ester and participate in reverse cholesterol transport to prevent the formation of foam cells.
PUBLICATIONS
Bradshaw, G., Gutierrez, A., Miyake, J.H., Davis, K.R., Li, A.C., Glass, C.K., Curtiss, L.K., Davis, R.A. Facilitated replacement of Kupffer cells expressing a paraoxonase-1 transgene is essential for ameliorating atherosclerosis in mice. Proc. Natl.
Acad. Sci. U. S. A. 102:11029, 2005.
Jahangiri, A., Rader, D.J., Marchadier, D., Curtiss, L.K., Bonnet, D.J., Rye, K.-A.
Evidence that endothelial lipase remodels high density lipoproteins without mediating the dissociation of apolipoprotein A-I. J. Lipid Res. 25:896, 2005.
Mullick, A.E., Tobias, P.S., Curtiss, L.K. Modulation of atherosclerosis in mice by
Toll-like receptor 2. J. Clin. Invest., in press.
Schneider, M., Witztum, J.L., Young, S.G., Ludwig, E.H., Miller, E., Tsimikas, S.,
Curtiss, L.K., Marcovina, S.M., Taylor, J.M., Lawn, R.W., Innerarity, T.L., Pitas,
R.E. High level lipoprotein (a) expression in transgenic mice: evidence for oxidized
phospholipid in lipoprotein (a) but not in low density lipoprotein. J. Lipid Res.
46:769, 2005.
Tobias, P., Curtiss, L.K. Paying the price for pathogen protection: Toll receptors in
atherogenesis. J. Lipid Res. 46:404, 2005.
Wiedmer, T., Zhao, J., Li, L., Zhou, Q., Hevener, A., Olefsky, J.M., Curtiss, L.K.,
Corr, M., Witztum, J.L. Adiposity, dyslipidemia and insulin resistance in mice with
targeted deletion of phospholipid scramblase 3 (PLSCR3). Proc. Natl. Acad. Sci.
U. S. A. 101:13296, 2004.
Expression of a Novel Large
Noncoding RNA in Neoplasia,
Proliferation, and Differentiation
R. Lin, C. Liu, T.S. Edgington, S. Maeda,* M. Karin*
* University of California, San Diego, California
ecent genomic and transcriptomic studies have
revealed the existence of a substantial number
of large noncoding RNAs. Only a few of these
RNAs have been characterized, and little is known of
their functional roles. Using differential display analysis of CD31-enriched tumor microvascular endothelial
R
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2005
111
cells from a syngeneic murine colon carcinoma, we
discovered a novel large noncoding RNA of approximately
360 base pairs. The full-length gene is a 7000-base
mRNA-like transcript but lacks extensive or conserved
open reading frames.
Bioinformatics analysis suggested that the full-length
gene is highly conserved among vertebrates, implying
functional significance. More important, Northern blot
analysis and in situ hybridization indicated that expression of the full-length transcript is highly upregulated
in carcinogen-induced primary hepatomas and is a
novel marker for hepatomas and for other neoplasms
so far analyzed.
We designated this noncoding RNA hepatomin.
We found that hepatomin transcription is induced in
liver undergoing regeneration, skin undergoing wound
healing, hair follicles during growth of hair, and cultured
cells synchronized at the mitosis stage.
These results suggest that hepatomin plays roles in
proliferation and differentiation. We conclude that hepatomin is a member of the new class of large noncoding
RNAs and a new marker for hepatomas. This discovery
can provide unique murine models for analysis of the
role of this noncoding RNA in neoplasia, proliferation, and
differentiation as an apparent regulator of the changes
in gene expression associated with these processes.
Novel Redirected Molecular
Expression, Architecture,
and Properties in the
Microvasculature of Tumors
A. El-Sheikh, G. Bhattacharjee, Z. Ruggeri,* T.S. Edgington,
P. Borgstrom**
* Department of Molecular and Experimental Medicine, Scripps Research
** Sidney Kimmel Cancer Center, La Jolla, California
eoplastic cells induce remarkable changes in
their new angiogenic microvasculature as they
seek nutrients, oxygen, and the freedom to
invade and metastasize. The loss of normal vascular
development and architecture results in a bizarre, functionally incompetent maze of capillary-like vessels lined
by endothelium with strikingly divergent gene transcription and aberrant display of cell-surface proteins. Understanding the aberrations may broaden our knowledge
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112 IMMUNOLOGY 2005
of the molecular biology of these complex angiogenic
and postangiogenic vascular mazes, and some of the
molecules involved may provide novel targets for therapeutic intervention.
We discovered an endothelial receptor complex that
appears unique for the tumor microvascular environment.
Vascular endothelial growth factor receptor 2 and its
coreceptor neuropilin-1 can be found elsewhere within
the vascular tree, but the association of an oligosaccharide (chondroitin C sulfate) to create a ternary complex is unique to the tumor microvasculature. This
complex is a novel target for therapeutic strategies.
We developed a thrombogen specific for tumor vasculature that recognizes this ternary complex selectively on tumor neoangiogenic vessels. This soluble
hybrid protein, HBDt-TFt, incorporates the modified
exon 7–encoded heparin-binding domain of the gene
for vascular endothelial growth factor at its N terminus
with the extracellular domain of tissue factor at the
C terminus. The hybrid can selectively dock on the surface of tumor endothelium at this trimolecular complex
and initiate the coagulation protease cascade. Subsequently, the tumor microvasculature is thrombosed,
resulting in local infarctive eradication of tumor. Using
intravital microscopy, we optimized the dose and rate
of infusion of HBDt-TFt and monitored microcirculation
within the tumor as well as thrombus formation. In
addition, using fluorescein-labeled platelets, we showed
a central participation of platelets in tumor-specific
microvascular thrombosis.
Regulation of Tissue
Factor–Mediated Initiation
of the Coagulation Cascade
by Grp78
G. Bhattacharjee, J. Ahamed, B. Pedersen, A. El-Sheikh,
N. Mackman, W. Ruf, C. Liu, T.S. Edgington
e used biopanning with phage-displayed peptidyl libraries to discover peptide probes that
bind selectively to the surface of the endothelium in atherosclerotic plaques. EKO130, the peptide with the highest affinity among those tested, binds
the 78-kD glucose-regulated protein (Grp78). This protein participates in many pathologic processes, including
regulation of the coagulation cascade. To characterize
W
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how Grp78 regulates coagulation, we analyzed the
effect of the protein on tissue factor–mediated procoagulant activity in mouse brain endothelial cells and
in macrophage-like cells.
We found that Grp78 is present on the endothelium
and on monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 increased procoagulant activity. We also found that Grp78 negatively
regulates procoagulant activity by interacting directly
with the extracellular domain of tissue factor on the
cell surface.
These findings indicate that Grp78 negatively regulates the functional activity of tissue factor by binding
directly to tissue factor and subsequently inhibiting
it. Determining how Grp78 regulates the function of
tissue factor may provide insight into the pathobiology
of atherosclerosis and associated arterial thrombosis.
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,
L. Watson, P. Lao, R.Z. MacDonald
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. In
each precursor B lymphocyte, a different set of V, D,
and J genes recombines 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 heptamers and nonamers, however, and changes in this natural variation in the recombinase signal sequence can greatly affect recombination
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IMMUNOLOGY
frequency in vitro and in vivo. Surprisingly, even differences in the sequence of the spacer can greatly influence recombination frequency, and these differences
also contribute to nonrandom use of genes in vivo.
However, other factors clearly influence recombination frequencies, and currently, we are focusing on
the role of transcription factors and chromatin modifications in controlling accessibility to V(D)J recombination and recombination frequency. Genes in loci that
are undergoing 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 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.
We also use a novel system in which certain
immunoglobulin gene rearrangements can be induced
in a nonlymphoid cell line after the transient transfection by vectors expressing a B cell–specific transcription
factor, E2A or EBF, and the recombinases. We showed
that E2A induces rearrangement of VκI genes. However,
the VκII and VκIII genes, which are interspersed with
the VκI genes within the IgVκ locus, seldom rearrange
after E2A transfection. EBF induces VλJλ rearrangement, but mainly of only a single Vλ gene.
Thus, this induction of accessibility of genes is not
uniform across the locus. Neighboring genes can be
differentially induced to rearrange, suggesting localized
control of accessibility for rearrangement. Current studies
are aimed at elucidating the mechanism of this localized gene-specific control. Transfection with E2A does
not induce much acetylation of the histones associated
with VκI genes, but the rearranged genes are associated with acetylated histones, suggesting that once a
V gene becomes accessible, it efficiently rearranges.
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 the same
in lupus-prone mice as in nonautoimmune mice, and
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2005
113
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
Li, L., Salido, E., Zhou, Y., Battacharyya, S., Yannone, S.M., Dunn, E., Meneses, J.,
Feeney, A.J., Cowan, M.J. Targeted disruption of the Artemis murine counterpart
results in SCID and defective V(D)J recombination that is partially corrected with
bone marrow transplantation. J. Immunol. 174: 2420, 2005.
Syndecans and HIV Type 1
Pathogenesis
M. Bobardt, U. Chatterji, A. de Parseval,* J. Elder,* P. Gallay
* Department of Molecular Biology, Scripps Research
yndecans are transmembrane receptors highly
expressed on adherent cells (e.g., macrophages,
epithelial or endothelial cells) but poorly expressed
on suspension cells (e.g., T lymphocytes). The syndecan family has 4 members, syndecan-1 through syndecan-4. The ectodomain in each syndecan has linear
heparan sulfate chains, which are composed of a repetition of a sulfated disaccharide motif. The sulfation
pattern of the heparan sulfates dictates the ligand specificity of the syndecan.
Syndecans function as receptors for HIV type 1
(HIV-1). Pretreatment of target cells with heparinase
that removes heparan sulfates from syndecans dramatically reduces HIV-1 infectivity. We showed that syndecans also serve as in trans (i.e., on the surface of cells
opposite each other) receptors for HIV-1. Specifically,
HIV-1 binds syndecans richly expressed on the endothelium. HIV-1 bound to syndecans remains infectious
for a week, whereas cell-free virus loses its infectivity
after a single day. Most importantly, HIV-1 attached to
the endothelium via syndecans is an in trans source of
infection for circulating T cells. These findings suggest
that syndecan-rich endothelium can provide a microenvironment that amplifies HIV-1 replication in T cells.
Last, we showed that syndecans on brain microvascular
endothelial cells play a significant role in HIV-1 transmigration through the blood-brain barrier. Altogether these
observations suggest that syndecans, by acting as in
cis (i.e., on the surface of the same cell) and in trans
receptors, may profoundly affect HIV-1 pathogenesis.
We recently found that a single conserved arginine
at position 298 in the HIV envelope glycoprotein gp120
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114 IMMUNOLOGY 2005
governs HIV-1 binding to syndecans. An amine group
on the side chain of this residue is necessary for syndecan utilization by HIV-1. We showed that HIV-1
binds syndecans via a 6-O sulfation, indicating that
this binding is not the result of random interactions
between basic residues and negative charges but the
result of specific contacts between gp120 and a welldefined sulfation in syndecans. Surprisingly, arginine
at position 298 that mediates HIV-1 binding to syndecans also mediates HIV-1 binding to the major HIV-1
entry receptor CCR5.
We postulated that HIV-1 recognizes similar motifs
on syndecans and CCR5. In support of this hypothesis,
we found that the 6-O sulfation recognized by HIV-1
on syndecans mimics the sulfated tyrosines recognized
by HIV-1 in the N terminus of CCR5. Our finding that
CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which
chemokines use these 2 disparate receptors and suggests that the gp120-chemokine mimicry may represent a common strategy in microbial pathogenesis.
PUBLICATIONS
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., in press.
Gallay, P. Syndecans and HIV-1 pathogenesis. Microbes Infect. 6:617, 2004.
Innate Intracellular Immunity
and Infection With HIV Type 1
not have any idea of how this restriction factor blocks
HIV-1 infection.
An understanding of the nature of restrictions to
HIV-1 infection after cellular entry of the virus in primates 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, species-specific barriers to HIV-1 infection present obstacles to the development of animal
models for the study of HIV-1 pathogenesis, treatment, and prophylaxis. Finally, an understanding of
this critical part of the HIV-1 life cycle may suggest
approaches to intervene in HIV-1 transmission to or
spread within the host.
PUBLICATIONS
Chatterji, U., Bobardt, M.D., Stanfield, R., Ptak, R.C., Pallansch, L.A., Ward,
P.A., Jones, M.J., Stoddart, C.A., Scalfaro, P., Dumont, J.-M., Besseghir, K.,
Rosenwirth, B., Gallay, P.A. A naturally occurring capsid motif renders HIV-1
cyclophilin A independent in human cells and TRIM-cyclophilin resistant in owl
monkey cells. J. Biol. Chem., in press.
Galigniana, M.D., Morishima, Y., Gallay, P.A., Pratt, W.B. Cyclophilin-A is bound
through its peptidylprolyl isomerase domain to the cytoplasmic dynein motor protein complex. J. Biol. Chem. 279:55754, 2004.
Molecular Interactions in T-Cell
Development and Activation
N.R.J. Gascoigne, J. Ampudia, G. Fu, K. Holmberg,
H.-C. Hung, H.-O. Kim, C. Lotz, A. Munshi, N. Niederberger,
G. Sternik, S. Vallee, P. Yachi, M.A. Zal, T. Zal, M. Gronski,*
P. Ohashi,* M.Y. Lin,** S.M. Hedrick,** N. Bosco,***
R. Ceredig,*** M. Cahalan****
U. Chatterji, M. Bobardt, P. Gallay
* Ontario Cancer Institute, Toronto, Ontario
onhuman primate 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. These restriction factors block HIV-1 replication
at steps before integration but subsequent to entry. The
first intracellular primate restriction factor identified,
TRIM5α, is a member of the tripartite motif (TRIM)
family of proteins. The introduction of TRIM5α derived
from species that highly restrict HIV-1 (e.g., Rhesus
macaque, African green or owl monkeys) into cells that
are normally permissive (e.g., human cells) makes the
cells nonpermissive to HIV-1. However, although
TRIM5α was identified more than 2 years ago and
several laboratories are actively studying it, we still do
** University of California, San Diego, California
N
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*** U548 INSERM, Grenoble, France
**** University of California, Irvine, California
IMAGING MOLECULAR INTERACTIONS IN LIVING
C E L L S I N T - C E L L A C T I VAT I O N
e used live-cell fluorescence deconvolution
microscopy and fluorescence resonance
e nergy transfer (FRET) microscopy to investigate molecular movement and intermolecular interactions during T-cell activation. FRET between cyan and
yellow fluorescent proteins is effective at ranges of
less than 10 nm and is therefore ideal to investigate
interactions between proteins in living cells.
Using FRET, we showed that the coreceptor CD8
and the T-cell receptor (TCR) signal-transducing protein
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IMMUNOLOGY
CD3ζ are recruited to the “immunologic synapse,” where
they interact when antigen is presented to the T cell.
No FRET occurs when weaker (e.g., TCR antagonist)
ligands are used. We compared formation of the immunologic synapse and the TCR-coreceptor interaction in
a system in which the affinity of the interaction between
TCRs and MHC-peptides is known. We found that 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 interactions between TCRs and MHC-peptides.
The induction of FRET appears to explain why some
agonists are stronger or weaker than would be predicted
on the basis of their affinities. By controlling the level
of antigenic MHC-peptide complexes presented to T cells,
in the presence or absence of natural endogenous nonstimulatory MHC-peptides, we found that the endogenous complexes aided in the recognition of the antigenic
complexes. The interaction between CD8 and the endogenous MHC-peptide improves the TCR recognition of
the antigenic MHC-peptide, 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.
In a series of experiments in collaboration with
P. Ohashi, Ontario Cancer Institute, Toronto, Ontario, we
found that the strength of TCR interaction with MHC
peptide ligands alters the ability of an autoantigen to
induce diabetes, so that a reduced affinity results in a
reduced incidence of disease. We also used fluorescent
tetrameric class I MHC molecules to measure binding
of TCR-MHC-peptide complexes on T cells to compare
binding of negative- and positive-selecting ligands.
Although crystallographic studies indicate that the
coreceptor CD4 exists as a dimer, biochemical or biological evidence for this finding has been weak. We
made CD4 chimeras with cyan and yellow fluorescent
proteins to probe dimerization on the cell surface. We
found a weak constitutive association, which was
greatly increased at the immunologic synapse during
antigen recognition.
TWO-PHOTON MICROSCOPY OF TCR AND
CORECEPTOR MOVEMENT IN LIVING TISSUES
Two-photon microscopy allows visualization of cells
deep in tissues and thus allows observation of T cells
interacting with antigen-presenting cells during an
immune response or of thymocytes interacting with
thymic stromal cells during development. We produced
transgenic mice that express the fluorescent chimeric
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2005
115
CD8 and CD3ζ molecules, as well as transgenic TCRs,
and are collaborating with M. Cahalan, University of
California, Irvine, to use 2-photon microscopy to investigate movement and interaction of TCRs and coreceptors. We found that the cell-surface molecules form
synapses within tissues, and we are investigating how
the synapses form during the initiation of an immune
response, during responses to a solid tumor, and during thymocyte development.
ROLE OF THE PROTEIN KINASE η ISOFORM IN THE
IMMUNOLOGIC SYNAPSE
We found that the η isoform of protein kinase C
(PKCη) is upregulated after TCR ligation in developing
thymocytes and in natural positive selection. 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 absence of PKCθ, PKCη is expressed
at an earlier stage of thymocyte development, where it
functions in place of PKCθ.
G E N E E X P R E S S I O N I N E A R LY T - C E L L
D I F F E R E N T I AT I O N
We identified a novel protein with strongly regulated
expression during thymocyte differentiation. The protein
is expressed during the stages of TCR gene rearrangement. It interacts with the cell-cycle and DNA damage–
repair enzyme ATM and with phospholipase Cγ1, which
is important in T-cell signaling. We are now using small
interfering RNA, gene ablation, and transgenic techniques to investigate the possible role of the protein in
development and T-cell signaling.
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
poor, many mature T cells express 2 α-chain proteins.
However, expression of 2 α-chains on the cell surface
is quite rare. We previously showed that functional
allelic exclusion is attained in the thymus at the start
of positive selection and that this exclusion is posttranslationally regulated. We found that the positively
selected αβ combination remains on the surface when
the TCR is stimulated, whereas the other αβ combination is endocytosed. Endocytosis and allelic exclusion
are controlled by TCR signaling involving the kinase
Lck and the ubiquitin ligase Cbl, which controls degra-
116 IMMUNOLOGY 2005
dation of endocytosed TCRs. To analyze ubiquitination
of TCRs after endocytosis, we are using FRET between
ubiquitin monomers and TCR subunits labeled with
fluorescent proteins. 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.
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 CD25+CD4+ T cells. Mol. Immunol., in press.
Gronski, M.A., Boulter, J.M., Moskophidis, D., Nguyen, L.T., Holmberg, K.,
Elford, A.R., Deenick, E.K., Kim, H.O., Penninger, J.M., Odermatt, B., Gallimore, A.,
Gascoigne, N.R.J., Ohashi, P.S. TCR affinity and negative regulation limit autoimmunity. Nat. Med. 10:1234, 2004.
Lin, M.Y., Zal, T., Ch’en, I.L., Gascoigne, N.R.J., Hedrick, S.M. A pivotal role for
the multifunctional calcium/calmodulin-dependent protein kinase II in T cells: from
activation to unresponsiveness. J. Immunol. 174:5583, 2005.
Niederberger, N., Buehler, L.K., Ampudia, J., Gascoigne, N.R.J. Thymocyte stimulation by anti-TCRβ, but not by anti-TCRα, leads to induction of developmental
transcription program. J. Leukoc. Biol. 77:830, 2005.
Yachi, P.P., Ampudia, J., Gascoigne, N.R.J., Zal, T. Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological
synapse. Nat. Immunol. 6:785, 2005.
ligand influences ligand-induced MAP kinase activation but has no effect on NF-κB activation. Studies are
under way to define the mechanisms that selectively
activate MAP kinase and NF-κB.
INTRACELLULAR SIGNALING
The p38 MAP kinase pathway is important in TLRinduced cellular changes. One of our interests is to
determine how the signal is transduced from TLRs to the
p38 pathway. We evaluated the contribution of MAP
kinase kinase (MKK)–dependent and MKK-independent
p38 activation. We found that both MKK-dependent and
MKK-independent pathways contribute to p38 activation; the MKK-dependent pathway is the primary one in
most situations. We also found that MKK-independent
p38 activation can occur with or without dependence on
TAB1, the activating protein for transforming growth factor-β–activated kinase 1. MyD88, TRIF, and TRAF6 are
clearly the signaling molecules between the TLRs and
p38 activation. However, which molecules link these
adaptors and the p38 pathway is still not clear; we are
identifying these proteins. We think that information on
the proteins will bridge the gap in our knowledge of
how the p38 pathway is activated by TLRs.
R E G U L AT I O N O F G E N E E X P R E S S I O N
Cellular Activation Mechanisms
in the Innate Immune System
J. Chen, M. Cheung, Y. Kang, S. Lee, Y. Li, J. Mols,
A. Seit-Nebi, M. Otsuka, C.-C. Wu, Y. Xu, T. Zarubin,
H. Zhou, J. Han
acterial pathogens and inflammatory cytokines
are important players in the innate immune
response. Our interests are the signal transduction pathways activated by pathogens and cytokines.
We study the signaling events at different cellular levels.
B
S I G N A L I N G AT T H E C E L L S U R FA C E
Toll-like receptors (TLRs) are an important point of
first contact between host and microbe. Once activated,
the receptors generate signals that culminate in the
induction of genes important for host defense. TLRs often
need to cooperate with other proteins on the cell surface
to function. For example, CD14 and MD2 are involved in
TLR4-mediated activation by lipopolysaccharide.
We found that 4-1BB (CD137) ligand is another
protein that interacts with TLRs on the cell surface.
Unlike CD14 and MD2, 4-1BB ligand affects intracellular signaling rather than ligand recognition. 4-1BB
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
The p38 pathway plays a key role in posttranscriptional regulation of cytokine gene expression. We used
Drosophila melanogaster S2 cells to screen a number of
genes for their requirement in mRNA decay mediated by
adenine-uridine–rich elements (AREs). We found that
proteins in the Dicer and Ago families are required for
ARE-mediated mRNA decay. We further confirmed the
requirement of Dicer in mammalian cells. Because
Dicer and Ago family members are required for processing microRNA, we evaluated whether any microRNA
is required for ARE-mediated mRNA degradation.
Sequence analysis revealed that miR16, a human
microRNA containing a sequence complementary to the
ARE sequence, has complementary sequence to miR16,
and we confirmed that miR16 is required for the quick
ARE-mediated decay of mRNA. Because miR16 and
ARE can only form a maximum of 8 pairs, most likely
miR16 alone is not sufficient to guide the targeting of
AREs by the RNA-induced gene-silencing complex (RISC).
We hypothesized that ARE-binding proteins may
have a role and further determined that the ARE-binding protein tristetraprolin is required for miR16 to target ARE-containing mRNA. Tristetraprolin does not
directly interact with miR16, but via association with
an Ago family member forms a complex with miR16.
IMMUNOLOGY
Thus, the initiation of ARE-induced mRNA decay requires
miR16-containing RISC and tristetraprolin. Currently,
we are focusing on the signaling mechanism between
the p38 pathway and the involvement of RISC and
tristetraprolin in ARE-mediated mRNA decay.
M A C R O P H A G E D E AT H
The activation and life span of macrophages are
important in innate immunity. Although macrophage
death in vivo has not been well evaluated, it can be
induced by different stimuli, such as a combination of
cytokines and lipopolysaccharide or anthrax lethal
toxin. We used 2 systems, macrophage death induced
by lipopolysaccharide plus the caspase inhibitor vZAD
and macrophage death induced by anthrax lethal toxin,
to study the death process of macrophages.
Previously, we showed that induction of the orphan
nuclear receptor Nur77 plays a role in macrophage death
induced by lipopolysaccharide plus vZAD. We recently
found that macrophage death induced by this combination had a phenotype of autophagy and depends on
reactive oxygen species and poly(ADP-ribose) polymerase. We also found that macrophage death induced by
lipopolysaccharide plus vZAD can be effectively blocked
by serine protease inhibitors. Thus, multiple cellular
events may be involved in this type of macrophage
death. To study macrophage death induced by anthrax
lethal toxin, we used random mutagenesis to identify
genes that are required for lethal toxin–induced death.
The identified genes are being characterized.
PUBLICATIONS
Cao, J., Semenova, M.M., Solovyan, V.T., Han, J., Coffey, E.T., Courtney, M.J.
Distinct requirements for p38α and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death. J. Biol. Chem.
279:35903, 2004.
Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F.,
Lin, S.-C., Gram, H., Han, J. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120:623, 2005.
Li, J., Li, Q., Xie, C., Zhou, H., Wang, Y., Zhang, N., Shao, H., Chan, S.C., Peng, X.,
Lin, S.-C., Han, J. β-Actin is required for mitochondria clustering and ROS generation in TNF-induced caspase-independent cell death. J. Cell Sci. 117(Pt.
20):4673, 2004.
Qi, X., Tang, J., Pramanik, R., Schultz, R.M., Shirasawa, S., Sasazuki, T., Han, J.,
Chen, G. p38 MAPK activation selectively induces cell death in K-ras-mutated
human colon cancer cells through regulation of vitamin D receptor. J. Biol. Chem.
279:22138, 2004.
Rui, Y., Xu, Z., Lin, S., Li, Q., Rui, H., Luo, W., Zhou, H.M., Cheung, P.Y., Wu, Z.,
Ye, Z., Li, P., Han, J., Lin, S.C. Axin stimulates p53 functions by activation of
HIPK2 kinase through multimeric complex formation. EMBO J. 23:4583, 2004.
2005
117
Shen, G., Hebbar, V., Nair, S., Xu, C., Li, W., Lin, W., Keum, Y.S., Han, J., Gallo,
M.A., Kong, A.N. Regulation of Nrf2 transactivation domain activity: the differential
effects of mitogen-activated protein kinase cascades and synergistic stimulatory
effect of Raf and CREB-binding protein. J. Biol. Chem. 279:23052, 2004.
Specificity and Function of
Intraepithelial γδ T Cells
W.L. Havran, G. Cauvi, M. Haynes, J. Jameson,
H.K. Komori, T. Meehan, R. Mills, L. Sharp, 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 epithelial γδ T cells in immune surveillance, wound repair,
inflammation, and protection from malignant tumors.
W
M O L E C U L E S R E Q U I R E D F O R γδ T - C E L L A C T I V A T I O N
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 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
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, AMICA/JAML, is uniquely
costimulatory for γδ T cells. We also found that the
semaphorin Sema4D (CD100) is expressed by skin γδ
T cells upon activation and binds to a new member of
the plexin superfamily of semaphorin receptors, plexinB2, expressed on keratinocytes. These novel receptors
and receptor-ligand pair most likely play key roles in
the interactions between skin γδ T cells and keratinocytes
during homeostasis and in skin disorders. We will also
examine the role of these molecules in interactions
between intestinal intraepithelial γδ T cells and epithelial cells during colitis.
A R O L E F O R I N T R A E P I T H E L I A L γδ T C E L L S I N T H E
REPAIR OF EPITHELIAL TISSUE
Sakai, A., Han, J., Cato, A.C., Akira, S., Li, J.D. Glucocorticoids synergize with
IL-1β to induce TLR2 expression via MAP kinase phosphatase-1-dependent dual
Inhibition of MAPK JNK and p38 in epithelial cells. BMC Mol. Biol. 5:2, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
We recently showed a role for skin γδ T cells in the
reepithelialization stage of wound repair. The γδ T cells
118 IMMUNOLOGY 2005
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. 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 inflammatory disorders and
may be useful in designing or testing new therapies.
PUBLICATIONS
Baccala, R., Witherden, D., Gonzalez-Quintial, R., Dummer, W., Surh, C.D.,
Havran, W.L., Theofilopoulos, A.N. γδ T cell homeostasis is controlled by IL-7
and IL-15 together with subset-specific factors. J. Immunol. 174:4606, 2005.
Jameson, J.M., Cauvi, G., Sharp, L.L., Witherden, D.A., Havran, W.L. γδ T cellinduced hyaluronan production by epithelial cells regulates inflammation. J. Exp.
Med. 201:1269, 2005.
Sharp, L.L., Jameson, J.M., Cauvi, G., Havran, W.L. Dendritic epidermal T cells
regulate skin homeostasis through local production of insulin-like growth factor 1.
Nat. Immunol. 6:73, 2005.
Sharp, L.L., Jameson, J.M., Witherden, D.A., Komori, H.K., Havran, W.L. Dendritic epidermal T-cell activation. Crit. Rev. Immunol. 25:1, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Lysophosphatidic Acid–Induced
Expression of Urokinase in
Ovarian Cancer Cells
H. Li, C. Mahanivong, D. Bian, S. Huang
scites fluid and plasma of patients with ovarian
cancer have high concentrations of lysophosphatidic acid (LPA). In experimental models,
LPA promotes invasion and metastasis of ovarian cancer cells by enhancing cell migration, upregulating
expression of angiogenic factors, and promoting cell
survival and proliferation. Recently, we focused on
how LPA affects the expression of urokinase, an invasion-associated protease, in ovarian cancer cells.
We found that LPA can upregulate expression of
urokinase in the majority of ovarian cancer cell lines
but not in any normal ovary surface epithelial cells.
Using an invasive ovarian cancer cell line as a model
system, we explored the signaling molecules and pathways essential for LPA-induced upregulation of urokinase. Using specific inhibitors and dominant-negative
forms of signaling molecules, we found that the pathway associated with the GTP-binding protein Gi, but
not the pathways associated with the GTP-binding proteins G12/13 and Gq, mediates this LPA-induced event.
Moreover, both constitutively active H-Ras (V12) and
Raf-1 (BXB) enhanced urokinase expression, whereas
dominant-negative H-Ras (N17) and Raf-1 (301A)
blocked LPA-induced urokinase upregulation, suggesting that the Ras-Raf pathway works downstream of Gi
to mediate this LPA-induced process. Surprisingly, dominant-negative MAP kinase kinase (MEK) 1 or extracellular
signal–regulated kinase (ERK) 2 had only a marginal
inhibitory effect on LPA-induced urokinase upregulation, suggesting that a signaling pathway distinct from
Raf-MEK1/2-ERK is the prominent pathway responsible for this process.
In other studies, we showed that LPA activates the
transcription factor NF-κB in a Ras-Raf–dependent manner and that blocking NF-κB activation with either nonphophorylable IκB or dominant-negative IKKβ abolished
LPA-induced upregulation of urokinase and activation
of the urokinase promoter. Furthermore, introducing
mutations to delete the NF-κB binding site of the
urokinase promoter resulted in more than an 80%
reduction in LPA-induced activation of the promoter.
In contrast, LPA-induced activation of the promoter
A
IMMUNOLOGY
was only moderately affected by introducing mutations
in the AP1 binding sites of the promoter. These results
suggest that the Gi–Ras–Raf–NF-κB signaling cascade
is responsible for LPA-induced upregulation of urokinase
in ovarian cancer cells.
PUBLICATIONS
Chen, L.-Y., Doerner, A., Lehmann, P.F., Huang, S., Zhong, G., Pan, Z.K. A novel
protein kinase C (PKCε) is required for fMet-Leu-Phe-induced activation of NF-κB
in human peripheral blood monocytes. J. Biol. Chem. 280:22497, 2005.
Jing, Q., Huang, S., Guth, S., Zarubin, T., Motoyama, A., Chen, J., Di Padova, F.,
Lin, S.-C., Gram, H., Han, J. Involvement of microRNA in AU-rich element-mediated mRNA instability. Cell 120:623, 2005.
Li, H., Ye, X., Mahanivong, C., Bian, D., Chun, J., Huang, S. Signaling mechanisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells. J .Biol. Chem. 280:10564, 2005.
Yu, J., Bian, D., Mahanivong, C., Chang, R.K., Zhou, W., Huang, S. p38 Mitogen-activated protein kinase regulation of endothelial cell migration depends on
urokinase plasminogen activator expression. J. Biol. Chem. 279:50446, 2004.
Regulators of T-Cell Development
and Lymphocyte Function
J. Kaye, P. Aliahmad, O. Garijo, P. Han, C. Krieg
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 developmental processes. Our identification of a
cell-surface protein that is upregulated on developing
thymocytes also led to studies on regulation of the
immune response and the potential of this protein as
a novel therapeutic target.
P
A N U C L E A R P R O T E I N I N V O LV E D I N R E G U L AT I O N
OF THYMOCYTE SELECTION
We identified thymocyte selection–associated high
mobility group (HMG) box protein (TOX) several years
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 forPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
119
mation 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.
The protein is expressed in early thymocyte progenitors and then transiently upregulated during β-selection and positive selection. Using transgenic animals
that express wild-type TOX or mutants of the protein,
we investigated the role of this nuclear factor in positive selection and the associated commitment to the
CD4 + or CD8 + T-cell lineages. 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. Interestingly, expression of TOX coupled
with IL-7–mediated signals appears to be sufficient to
induce both the development and parital maturation of
CD8 + lineage T cells.
Signaling through the serine/threonine phosphatase
calcineurin is required for positive selection of thymocytes. We found that expression of the gene for TOX is
regulated by this signaling pathway. These studies led
to a model in which upregulation of TOX is a critical
component of cell-lineage decisions during positive
selection. We are producing mice that lack the gene
for TOX to shed further light on the role of TOX in vivo.
A C E L L - S U R FA C E LY M P H O C Y T E A N D A N T I G E N P R E S E N T I N G C E L L P R O T E I N W I T H R E G U L AT O R Y
FUNCTION
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. One of our monoclonal
120 IMMUNOLOGY 2005
antibodies acts as an agonist for this coinhibitory molecule, thereby inhibiting facets of T-cell activation,
including T-cell proliferation and IL-2 secretion. We are
testing the ability of this antibody to modulate immune
responses in several model systems.
PUBLICATIONS
Kreig, C., Han, P., Stone, R., Goularte, O.D., Kaye, J. Functional analysis of BTLA
engagement of CD4+ and CD8+ T cells. J. Immunol., in press.
Parinaz, A., Kaye, J. Commitment issues: linking positive selection signals and lineage diversification in the thymus. Immunol. Rev., in press.
Mechanisms of Cell Migration
and Cancer Progression
R.L. Klemke, Y. Wang, K. Stoletov, O. Pertz, W. Wang,
C. Green, M. Holcomb, R. Hanley
ormal cell migration plays an essential role in
embryonic development, wound repair, and
immune function. However, aberrant cell motility
contributes to cancer progression, inflammation, and
developmental defects. Cell movement is a dynamic process that involves the coordinated protrusion of a leading
pseudopodium (lamellipodium) followed by retraction of
the rear compartment of the cell through actin-myosin–
mediated events. Directed cell migration, or chemotaxis,
occurs as a response to a chemokine gradient and is
thought to be one of the key mechanisms by which cancer cells metastasize (spread) to distinct tissues in the
body. We are investigating how complex signaling networks temporally and spatially regulate cell migration
and cancer metastasis in vitro and in vivo.
For our studies, we developed a method to isolate
the leading pseudopodium from the rear compartment
of a migrating cell. In collaboration with J. Yates, Department of Cell biology, we used multidimensional protein
identification technology and large-scale proteomics to
identify several hundred proteins that localized to this
structure, including Lasp-1, which is an actin-binding
scaffolding protein that is overexpressed in 8%–12%
of metastatic breast cancers. We discovered that Lasp-1
translocates to newly forming focal adhesions and membrane ruffles in response to chemokines and that c-Abl
tyrosine kinase regulates this process. Using small interfering RNA techniques, we showed that Lasp-1 is
required for proper cell migration and survival.
We are also investigating the role of the small
GTPase RhoC in mediating pseudopodial dynamics
N
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
and cancer cell invasion. Our findings indicate that
overexpression of RhoC, but not its related isoform
RhoA, leads to pseudopodia with a distinct morphology and increased dynamics. Using fluorescence resonance energy transfer microscopy and large-scale
proteomics, we are investigating the signaling events
that lead to this altered phenotype and how the phenotype is related to metastasis. Our goals are to continue characterizing RhoC and Lasp-1 and to identify
and characterize novel phosphotyrosine signaling proteins that regulate pseudopodial formation during
chemotaxis and cancer invasion.
We are also investigating how c-Abl tyrosine kinase
regulates cell migration and survival properties in normal and metastatic cells. We discovered that c-Abl
negatively regulates the CAS/Crk signaling module,
which is necessary for normal cell migration and survival. Interestingly, inhibition of the 26S proteasome
promotes c-Abl activation. Increased c-Abl activation
facilitates the phosphorylation of tyrosine 221 of Crk,
leading to the disassembly of CAS/Crk complexes, the
destabilization of the cytoskeleton, and the induction
of apoptosis.
Angiogenesis and vascular remodeling facilitate
cancer progression by providing a nutrient supply to
the tumor and potential pathways for tumor cell metastasis. However, studying these processes in live animals has been difficult. Therefore, we developed a
novel xenograft model of human tumor formation and
angiogenesis that allows high-resolution and real-time
imaging of these dynamic processes in living zebra
fish. Transgenic zebra fish engineered to express green
fluorescent protein in all blood vessels were injected
with human tumor cells expressing red fluorescent
protein. Using intravital confocal microscopy, we found
that the transplanted cancer cells rapidly homed to
existing blood vessels and formed solid tumors. The
invasive tumor cells then permeabilized the vessel
wall, creating looplike structures that subdivided into
channels and formed new functional vessels. These
distinct structures form in proximity to tumor cells, are
dynamic, and undergo continual remodeling during
tumor progression.
Our findings are the first high-resolution observations of tumor formation and tumor-induced vascular
remodeling in a living organism. This model is also
readily amendable to testing of pharmacologic agents
and genetic manipulation of conserved vertebrate angiogenic processes mediated by vascular endothelial cell
IMMUNOLOGY
growth factor, ephrin-B2, and basic fibroblast growth
factor. Our results will provide a better understanding
of cancer metastasis and provide diagnostic markers
of cancer progression.
PUBLICATIONS
Chodniewicz, D., Klemke, R.L. Guiding cell migration through directed extension
and stabilization of pseudopodia. Exp. Cell Res. 301:31, 2004.
Emami, S., Klemke, R.L. Regulation of cell motility by Abl family kinases. In: Abl
Family Kinases in Development and Disease. Koleske, T. (Ed.). Landes Bioscience,
Georgetown, TX, 2005. Available at: http://www.eurekah.com/abstract.php?chapid
=2426&bookid=185&catid=56.
Wozniak, M.A., Kwong, L., Chodniewicz, D., Klemke R.L., Keely, P.J. R-Ras controls membrane protrusion and cell migration through the spatial regulation of Rho
and Rac. Mol. Biol. Cell 16:84, 2005.
Regulation of the Innate
Immune Response in
Inflammation and Infection
U.G. Knaus, A. Bamberg, M. Lehmann, K. von Loehneysen,
S. Luxen, S. Pacquelet, M. Ruse, 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 of the 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, and Rho, which are essential regulators for
various leukocyte functions ranging from production of
ROS to chemotaxis and phagocytosis. Generation of
superoxide anion is accomplished by a Rac-dependent
NADPH oxidase (Nox) upon stimulation with chemotactic factors or phagocytic stimuli. We have identified
several Rac effector protein kinases, p21-activated
kinases (PAKs), in leukocytes, and we are investigat-
I
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The Scripps Research Institute. All rights reserved.
2005
121
ing the role of PAKs in the generation of superoxide
anion. Additionally, we identified and are characterizing downstream targets of PAKs, which are relevant
for innate immune cell functions.
GTPases of the Rho family are also involved in signaling cascades, which originate from pathogen-activated Toll-like receptors. Toll-like receptors 2 and 4,
stimulated by microbial products derived from gram-positive and gram-negative bacteria, activate Rac1 and
RhoA, which initiate 2 independent pathways required
for RelA transactivation and subsequent NF-κB–dependent
gene transcription. We are studying different aspects of
signaling by Toll-like receptors in several primary human
cell types, including monocytes and neutrophils, and
genetically altered mouse models and the impact of
this signaling on innate immune cell functions such as
apoptosis and upregulation of proinflammatory mediators.
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 (Fig. 1). The differentiated and fully functional
lung epithelium also serves as a model for studies of
F i g . 1 . Transmission electron micrograph of a 3-dimensional culture of human airway epithelium grown in air-liquid interface culture for 33 days (3900X).
122 IMMUNOLOGY 2005
lung barrier function and the influence of bacteria-derived
ligands and toxins on transmigration of neutrophils. In
addition, we will investigate processes leading to uptake
of pathogens or environmental particles and the impact
of these pathogens 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.
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, in press.
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., in press.
Martyn, K.D., Kim, M.J., Quinn, M.T., Dinauer, M.C., Knaus, U.G. p-21 Activated
kinase (Pak) regulates NADPH oxidase activation in human neutrophils. Blood, in press.
Yamauchi, A., Marchal, C.C., Molitoris, J., Pech, N., Knaus, U., Towe, J., Atkinson, S.J., Dinauer, M.C. Rac GTPase isoform-specific regulation of NADPH oxidase
and chemotaxis in murine neutrophils in vivo: role of the C-terminal polybasic
domain. J. Biol. Chem. 280:953, 2005.
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
B
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
can be deleted, we showed that the BMK1 pathway
delivers an antiapoptotic signal in endothelial cells
and plays a critical role in neovascularization during
development and in response to stimulation by angiogenic growth factors. Our recent data suggest that
BMK1 regulates the function of endothelial cells not
only at the transcriptional level but also at the translational level. Because endothelial cells are a key component in the vasculature and are important for the
formation of new blood vessels, most likely the BMK1
pathway regulates angiogenesis through its function in
endothelial cells.
Moreover, because angiogenesis contributes to tumor
growth, we hypothesize that the BMK1 pathway is
involved in tumor-induced neovascularization, which is
vital for sustaining tumor growth. To shed light on the
role of the BMK1 pathway in various aspects of cancer
development, we use multidisciplinary approaches,
including molecular, cellular, genetic, and pathologic
methods, to elucidate the molecular mechanisms of
BMK1 in angiogenesis and oncogene-dependent tumorigenesis. The results will provide insights into new strategies for therapeutic interventions of carcinogenesis.
THE TUMOR SUPPRESSOR 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. To date, the mechanism of tumor suppression of Tid56 in Drosophila and the cellular function
of Tid1 in human tumorigenesis are poorly understood.
We discovered that the signaling domain of the receptor protein-tyrosine kinase ErbB2 interacts with Tid1
protein. We also found that increased expression of Tid1
in breast cancer cells overexpressing ErbB2 promotes
ubiquitinization and proteosomal degradation of ErbB2,
resulting in potent inhibition of ErbB2-dependent intracellular signaling and proliferation/survival of the cells.
To evaluate and characterize the role of Tid1 in
breast tumorigenesis in adult animals, we have established a mouse model in which the gene for Tid1 can
be deleted specifically in mammary epithelial cells.
These animals can be used to closely mimic the effect
in humans of Tid1 removal on the onset and progression of breast cancer associated with Tid1 dysfunction. The mice can also be used to screen and test
agents used for treatment of breast tumors involving a
Tid1 defect.
IMMUNOLOGY
PUBLICATIONS
Hayashi, M.. Lee, J.-D. Role of the BMK1/ERK5 signaling pathway: lessons from
knockout mice. J. Mol. Med. 82:800, 2004.
Kim, S.-W, Chao, T.H., Xiang, R., Lo, J.F., Campbell, M.J., Fearns, C., Lee, J.-D.
Tid1, the human homologue of a Drosophila tumor suppressor, reduces the malignant activity of ErbB-2 in carcinoma cells. Cancer Res. 24:7732, 2004.
Lo, J.F., Zhou, H., Fearns, C., Reisfeld, R.A., Yang, Y., Lee, J.-D. Tid1 is required
for T cell transition from double-negative 3 to double-positive stages. J. Immunol.
174:6105, 2005.
Host-Pathogen Interactions:
Mechanisms and Applications
E. Li, S.P. Lad, E.Y. Fukuda, J. Li
ur research activities focus on host responses
to viral and bacterial infections. We use infections with adenovirus and the obligate intracellular bacterial pathogen Chlamydia trachomatis as
model systems.
Chlamydia trachomatis is the most common cause
of sexually transmitted infection that can lead to pelvic
inflammatory disease and infertility in females. Chlamydial infection of nonimmune cells produces inflammatory factors, including IL-8 and interferons, that play a
critical role in the disease process of Chlamydia infection. IL-8 induces inflammatory responses and promotes
angiogenesis, resulting in tissue damage and scarring
that are characteristic of chlamydial disease. On the
other hand, interferon treatment was reported to inhibit
growth of Chlamydia. Unlike the inflammatory responses
to most invasive bacterial pathogens that induce rapid
cytokine production with endotoxins or peptidoglycans,
IL-8 production during Chlamydia infection is delayed
and relies on bacterial replication. Growth of Chlamydia induces host lipid remodeling for bacterial lipid
uptake. We found that this process causes production
of inflammatory factors.
Chlamydia infection of cervical epithelial cells, the
primary target of chlamydial infection, activates cytosolic
phospholipase A 2 for release of arachidonic acid and
upregulates cyclooxygenase 2 for conversion of arachidonic acid to prostaglandins, including prostaglandin E2.
Both Chlamydia and prostaglandin E 2 induce IL-8
release through the extracellular signal–regulated kinase/
MAP kinase pathway, and inhibition of chlamydial growth
or lipid remodeling reduces IL-8 release.
We also examined the host antimicrobial response
to Chlamydia infection. We found that cervical epithelial cells produce IFN-β in response to Chlamydia
O
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The Scripps Research Institute. All rights reserved.
2005
123
infection. Production of IFN-β induces signal transducer
and activator of transcription 1 (STAT1), STAT2, and
interferon-stimulated gene factor 3γ, components of the
Janus kinase–STAT signal transduction pathway. The
upregulation and activation of this pathway are critical
for host clearance of chlamydial infection because
Chlamydia growth is inhibited in cells with upregulated
STAT1 expression; STAT1-deficient cells support Chlamydia propagation more efficiently. Using DNA and protein expression profiles and small interfering RNA, we
also showed that STAT1 upregulation is critical for
antimicrobial gene expression. These studies provide
insights into host innate immune responses to Chlamydia
infection. They also highlight the importance of maintaining a balanced habitat for parasitic pathogens as
obligate intracellular organisms.
We are also working on the design and modification of adenovirus for targeting gene delivery. Although
widely used for gene therapy studies, adenovirus-based
vectors cannot target specific tissues. We generated
modified adenoviruses that selectively infect cancer cells
with upregulated growth factor receptors. Our ultimate
goal is to generate modified viruses by fusing tissuespecific antibody to these viruses for selectively targeting tumor cells.
PUBLICATIONS
Fukuda, E.Y., Lad, S.P., Mikolon, D.P., Iacobelli-Martinez, M., Li, E. Activation of
lipid metabolism contributes to interleukin-8 production during Chlamydia trachomatis infection of cervical epithelial cells. Infect. Immun. 73:4017, 2005.
Lad, S.P., Fukuda, E.Y., Li, J., de la Maza, L.M., Li, E. Up-regulation of the
JAK/STAT1 signal pathway during Chlamydia trachomatis infection. J. Immunol.
174:7186, 2005.
The Microenvironment of Tumors
and Protease-Activated Prodrugs
W. Wu, P. Kuo, L. Wong, C. Liu
nvasion and metastasis are critical features that
define a malignant tumor. The ability of cancer
cells to break through tissue boundaries and penetrate into surrounding normal tissues involves the actions
of diverse extracellular proteases from multiple enzymatic classes. This enrichment of proteolytic activity
is one of the distinctive features of the tumor microenvironment, in contrast to the microenvironment of normal tissues. These cellular proteases also participate
in a wide range of biological and pathologic processes,
I
124 IMMUNOLOGY 2005
such as the formation of new blood vessels, signal transduction, and cell survival.
According to recent reports, humans have 553 genes
that encode proteases. We use an integrated approach
to characterize the repertoire of extracellular proteases
operating in the tumor microenvironment (i.e., the
“cancer degradome”) and the functional modulators,
target substrates, and specificity of the enzymes. We
call this approach cancer degradomics. Currently, we
are focusing on a subset of cell-surface anchored or
associated proteases, such as legumain, type II membrane serine protease 4, prostate-specific membrane
antigen, and a number of novel protease-encoding
genes, that are drastically overexpressed in a high percentage of human cancers. Certain protease inhibitors
(survivin) and cofactor molecules (tissue factor) are also
upregulated in tumors. We showed that legumain activates both cathepsin cysteine proteases and matrix
metalloprotease 2 and that overexpression of legumain
in tumors increases tumor invasion and metastasis. Similarly, type II membrane serine protease 4 activates
matrix metalloprotease 9 and promotes cancer cell
migration and metastasis. In contrast, expression of
prostate-specific membrane antigen in prostate cancers
reportedly reduces invasive potentials. These findings
indicate a high level of complexity involving the interactive protease network in cancers.
The cellular proteases and the inhibitors that constitute the cancer degradome are valuable prognostic
and diagnostic markers as well as attractive targets for
cancer imaging and therapy. The proteolytic specificities of peptide substrates provide modular chemical
tools for the rational design of protease-activated prodrugs. For example, a novel legumain-activated, cellimpermeable doxorubicin prodrug, LEG-3 (Fig. 1), is
activated exclusively in the tumor microenvironment.
After administration of LEG-3, a profound increase
occurs in the end product doxorubicin in the nuclei of
cells in tumors, but little increase in other tissues.
This protease-activated prodrug completely arrested
growth of a variety of neoplasms, including multidrugresistant tumors, in vivo and significantly extended
survival without evidence of myelosuppression or cardiac
toxic effects. The design of prodrugs activated by proteases in the tumor microenvironment can be extended
to other proteases and chemotherapeutic compounds
and therefore provides new potential for the rational
development of more effectively targeted cancer therapeutic agents.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Chemical structure (A) and optimized structural model
(B) of prodrug LEG-3.
PUBLICATIONS
Luo, Y., Zhou, H., Mizutani, M., Mizutani, N., Liu, C., Xiang, R., Reisfeld, R. A
DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 induces long-lived
T-cell memory against tumor recurrence. Cancer Res. 65:3419, 2005.
Tissue Factor, Coagulation
Proteases, and Protease-Activated
Receptors in Hemostasis,
Thrombosis, and Inflammation
R. Pawlinski, G. Schabbauer, M.T. Lin, R.E. Tilley,
J. Luyendyk, J. Nieva,* N. Mackman
* Scripps Cancer Center, La Jolla, California
issue factor (TF), the initiator of blood coagulation, is expressed by cells that surround blood
vessels. After vascular injury, TF is exposed to
clotting factors in the blood, and coagulation is initiated. The formation of a clot stops further blood loss
from the vasculature. Aberrant expression of TF also
contributes to thrombosis and inflammation in a variety
of disease states, such as sepsis, cancer, and atherosclerosis. We are interested in the role of TF in hemostasis, thrombosis, and inflammation.
T
H E M O S TA S I S
TF is essential for hemostasis because complete
deficiency of TF is not compatible with life. TF-depen-
IMMUNOLOGY
dent clotting is counterbalanced by 3 anticoagulant
molecules/pathways: the complex formed by TF and
coagulation factor VIIa is inhibited by TF pathway inhibitor; the protein C pathway inactivates the clotting cofactors coagulation factors Va and VIIIa; and antithrombin
inhibits all coagulation proteases, including thrombin.
A complete deficiency in any of these 3 pathways leads
to embryonic lethality due to thrombosis. We hypothesized that reducing the levels of TF would rescue embryos
deficient in these anticoagulants.
Crossbreeding of mice with low levels of TF rescued
embryos deficient in TF pathway inhibitor or the protein C pathway but not those deficient in antithrombin.
In adult mice, the relative levels of the 3 anticoagulant
pathways were different in different tissues, indicating
that the different anticoagulants acted in a tissue-specific manner.
THROMBOSIS
Recently, researchers have proposed a role for bloodborne TF in the propagation of a thrombus. The pool
of blood-borne TF is incorporated into growing thrombi.
However, different injury models have resulted in conflicting data. Using a murine laser-induced microvascular thrombosis model, we found that blood-borne TF
contributes to the growth of the thrombus. In contrast,
in a photochemical injury model of the common carotid
artery, thrombus formation was driven primarily by
TF derived from the blood vessel wall.
Currently, we are collaborating with J. Nieva, Scripps
Cancer Center, to investigate the hypothesis that cancer
patients have elevated levels of blood-borne TF and that
this elevation contributes to the increased incidence of
deep-vein thrombosis. We developed a new functional
assay that measures TF activity in microparticles and
platelets from the blood. We compared levels of functional blood-borne TF from healthy volunteers and from
cancer patients. We found low levels of TF in healthy
volunteers and elevated levels in the blood of cancer
patients. Preliminary data revealed that a patient with
a high level of blood-borne TF also had a deep-vein
thrombosis. Our discovery that elevated levels of
blood-borne TF are found in cancer patients and may
correlate with the occurrence of deep-vein thrombosis suggests that these patients may benefit from antiTF therapy.
I N F L A M M AT I O N
Another disease in which TF expression is induced
and contributes to intravascular coagulation is sepsis.
In sepsis, lipopolysaccharide released by gram-negative
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2005
125
bacteria activates the innate immune system, a situation
that leads to inflammation and disseminated intravascular coagulation. The phosphatidylinositol-3′-kinase
(PI3K) signaling pathway is activated by numerous
stimuli and affects many cellular processes. Previously, we showed in vitro that the PI3K pathway suppresses lipopolysaccharide induction of TF and TNF-α
in human monocytic cells. More recently, we showed
that the PI3K pathway inhibits lipopolysaccharideinduced expression of inflammatory mediators and
coagulation in a murine endotoxemia model. Insulin is
a potent activator of the PI3K pathway and is used in
the treatment of sepsis in humans. We found that low
doses of insulin, lower than the level that affects glucose metabolism, reduce inflammation in an endotoxemia model in a PI3K-dependent manner. Further
studies will determine the mechanism by which the
PI3K pathway inhibits lipopolysaccharide signaling.
PUBLICATIONS
Chou, J., Mackman, N., Merrill-Skoloff, G., Pedersen, B., Furie, B.C., Furie, B.
Hematopoietic cell-derived microparticle tissue factor contributes to fibrin formation
during thrombus propagation. Blood 104:3190, 2004.
Day, S.M., Reeve, J., Pedersen, B., Farris, D.M., Myers, D.D., Im, M., Wakefield,
T.W., Mackman, N., Fay, W.P. Macrovascular thrombosis is driven by tissue factor
derived primarily from the blood vessel wall. Blood 105:192, 2005.
Frank, R.D., Schabbauer, G., Holscher, T., Sato, Y., Tencati, M., Pawlinski, R.,
Mackman, N. The synthetic pentasaccharide fondaparinux reduces coagulation,
inflammation and neutrophil accumulation in kidney ischemia-reperfusion injury.
J. Thromb. Haemost. 3:531, 2005.
Mackman, N. Mouse models in haemostasis and thrombosis. Thromb. Haemost.
92:440, 2004.
Morrow, D.A., Murphy, S.A., McCabe, C.H., Mackman, N., Wong, H.C., Antman,
E.M. Potent inhibition of thrombin with a monoclonal antibody against tissue factor
(Sunol-cH36): results of the PROXIMATE-TIMI 27 trial. Eur. Heart J. 26:682, 2005.
Pawlinski, R., Mackman, N. Tissue factor, coagulation proteases, and protease-activated receptors in endotoxemia and sepsis. Crit. Care Med. 32(5 Suppl.):S293, 2004.
Pawlinski, R., Pedersen, B., Erlich, J., Mackman, N. Role of tissue factor in
hemostasis, thrombosis, angiogenesis and inflammation: lessons from low tissue
factor mice. Thromb. Haemost. 92:444, 2004.
Pedersen, B., Holscher, T., Sato, Y., Pawlinski, R., Mackman, N. A balance
between tissue factor and tissue factor pathway inhibitor is required for embryonic
development and hemostasis in adult mice. Blood 105:2777, 2005.
Pyo, R.T., Sato, Y., Mackman, N., Taubman, M.B. Mice deficient in tissue factor
demonstrate attenuated intimal hyperplasia in response to vascular injury and
decreased smooth muscle cell migration. Thromb. Haemost. 92:451, 2004.
Schabbauer, G., Tencati, M., Pedersen, B., Pawlinski, R., Mackman, N. PI3K-Akt
pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler. Thromb. Vasc. Biol. 24:1963, 2004.
Yu, J.L., May, L., Lhotak, V., Shahrzad, S., Shirasawa, S., Weitz, J.I., Coomber,
B.L., Mackman, N., Rak, J.W. Oncogenic events regulate tissue factor expression
in colorectal cancer cells; implications for tumor progression and angiogenesis.
Blood 105:1734, 2005.
126 IMMUNOLOGY 2005
Helper T Cell–Regulated
B-Cell Immunity
tion mechanism establishes and maintains functional
heterogeneity among antigen-experienced helper T cells
in vivo.
DEVELOPMENT OF ANTIGEN-SPECIFIC B CELLS
M.G. McHeyzer-Williams, L.J. McHeyzer-Williams,
L.P. Malherbe, A.P. O’Connor
roduction of high-affinity antibodies is considered the most effective long-term protection
against reinfection by pathogens. Hence, vaccines need to promote strong, long-lasting memory in
the antigen-specific B-cell compartment to ensure persistent adaptive immunity in vivo. We seek to understand the rules that govern the helper T cell–regulated
development of the antigen-specific B-cell memory. We
focus our efforts on well-characterized murine models
of immunity to protein antigens.
P
ANTIGEN-EXPERIENCED DENDRITIC CELLS
Dendritic cells initiate most aspects of adaptive
immunity. Primarily, their capacity for antigen uptake,
antigen processing, and presentation of foreign peptides
recruits antigen-specific helper T cells. Using antibodies to complexes consisting of specific peptides and
MHC class II molecules, we can identify antigen-experienced dendritic cells in the secondary lymphoid organs
draining sites of protein vaccination. The extent and
dynamics of this cellular response vary with adjuvant
and dose of antigen, providing an important quantitative index of immunogenicity in vivo. We propose that
these differences critically affect the fate and the function of naive antigen-specific helper T cells recruited
into the adaptive phase of the immune response. This
approach provides new and objective ways to evaluate
the impact of vaccination regimens on the quality of
adaptive immunity in vivo.
DEVELOPMENT OF ANTIGEN-SPECIFIC HELPER
T CELLS
The specificity of clones that respond to foreign
antigens is the earliest defining attribute of adaptive
immunity. The rules that underpin this selection process for helper T cells have been difficult to assess
experimentally. Using an adoptive transfer model and
binding of T cells to complexes consisting of peptide
and MHC class II molecules, we detected and quantified
the selective loss of antigen-specific clonotypes that
express lower affinity antigen receptors. This affinitythreshold selection is followed by the unbiased propagation of preferred clonotypes regardless of binding
half-lives or affinity. We propose that this unique selecPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
The cellular organization of B-cell memory affects
the quality and quantity of the immune response to
antigen rechallenge. We are studying the complex development and cellular organization of antigen-specific
memory B cells. Using antigen binding, cell-surface
phenotype, single-cell analysis of the diversity of the
antigen receptor repertoire, and gene expression analysis
directly ex vivo, we are determining a comprehensive
and unique view of subsets of memory B cells after
initial antigen priming and after challenge with the
priming antigen later on in vivo. We propose a linear
progression of development from typical memory phenotype B cells that exit the germinal center reaction to
a compartment of distinct and persistent preplasma
memory B cells that appear to be the immediate cellular precursors of high-affinity plasma cells.
PUBLICATIONS
Malherbe, L., Hausl, C., Teyton, L., McHeyzer-Williams, M.G. Clonal selection of
helper T cells is determined by an affinity threshold with no further skewing of TCR
binding properties. Immunity 21:669, 2004.
McHeyzer-Williams, L.J., Malherbe, L.P., McHeyzer-Williams, M.G. Helper T cell
regulated B cell immunity. Curr. Top. Microbiol. Immunol., in press.
McHeyzer-Williams, L.J., McHeyzer-Williams, M.G. Analysis of antigen-specific
B cell memory directly ex vivo. Methods Mol. Biol. 271:173, 2004.
McHeyzer-Williams, L.J., McHeyzer-Williams, M.G. Antigen-specific memory B cell
development. Annu. Rev. Immunol. 23:487, 2005.
McHeyzer-Williams, M.G. Memory B cell development. In: The Autoimmune Diseases, 4th ed. Rose, N.R., Mackay, I.R. (Eds.). Elsevier, St. Louis, in press.
Adaptive and Innate Responses
to Alloantigens
D.B. McKay, A. Shigeoka, E. Zambricki
urgical and medical advances have provided an
opportunity for life to patients who would otherwise succumb to end-stage organ disease. Despite
remarkable technological advances, most efforts to prevent rejection of transplanted tissues and organs have
relied on treatment with nonspecific immunosuppressive medications that are toxic and require life-long use.
One experimental method has allowed survival of transplanted organs without the use of immunosuppressive
medications: intravenous exposure of the organ recipi-
S
IMMUNOLOGY
ent 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 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 the initial activation of the cells that
present donor antigens.
In other research, we are investigating the mechanisms that mediate the expansion of activated T cells
in response to transplantation antigens. The long-range
goal of this project is to develop effective ways to block
proliferative signals and thereby prevent allograft rejection. Transplantation of allogeneic tissues induces vigorous proliferation of host T cells specific for donor
alloantigens. Activation and cell division of recipient
T cells are differentially regulated by intracellular signals evoked through the ligation of cell-surface receptors. A classic example is the binding of IL-2 to its
receptor, which culminates in 3 distinct intracellular
signaling pathways, including 2 that are important for
T cell proliferation: the Janus kinase–signal transducer
and activator of transcription pathway and the Ras–MAP
kinase pathway. A third pathway, the phosphatidylinositol-3′-kinase–Atk kinase pathway, induces both
proapoptotic and antiapoptotic signals, explaining the
dual role of IL-2.
Antibodies that specifically block binding of IL-2 to
its receptor have been used in clinical transplantation.
We are evaluating the intracellular signaling mechanisms
that lead to effective blockade of the ligation of the
receptors of growth factors. In the microenvironment of
an allogeneic organ, several growth factors may influence the proliferation of potentially alloreactive T cells,
and understanding these factors may lead to better use
of therapeutic blockade of growth factors.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
127
PUBLICATIONS
McKay, D.B., Josephson, M.A., Armenti, V.T., August, P., Coscia, L.A., Davis,
C.L., Davison, J.M., Easterling, T., Friedman, J.E., Hou, S., Karlix, J., Lake, K.D.,
Lindheimer, M., Matas, A.J., Moritz, M.J., Riely, C.A., Ross, L.F., Scott, J.R.,
Wagoner, L.E., Wrenshall, L., Adams, P.L., Bumgardner, G.L., Fine, R.N., Goral,
S., Krams, S.M., Martinez, O.M., Tolkoff-Rubin, N., Pavlakis, M., Scantlebury, V.
Women's Health Committee of the American Society of Transplantation. Reproduction and transplantation: report on the AST Consensus Conference on Reproductive
Issues and Transplantation. Am. J. Transplant. 5:1592, 2005.
Zambricki, E., Shigeoka, A., Kishimoto, H., Sprent, J., Burakoff, S., Carpenter,
C., Milford, E., McKay, D. Signaling T-cell survival and death by IL-2 and IL-15.
Am. J. Transplant. 5:2623, 2005.
CCR5 and CXCR4, Receptors
for HIV Type 1
D.E. Mosier, C. Pastore, A. Ramos, R. Nedellec, O. Hartley,*
R. Offord,* M. Lederman**
* Centre Médical Universitaire, Geneva, Switzerland
** Case Western Reserve University, Cleveland, Ohio
IV type 1 (HIV-1) is the cause of the AIDS
pandemic. The first step in HIV-1 infection is
sequential binding of the virus to the cell-surface 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 naturally resistant to HIV-1 infection, and they have no apparent clinical consequences
of lacking CCR5. These observations led to research
programs to develop CCR5-blocking agents to prevent
HIV-1 infection.
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.
H
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 is the CCR5-binding
chemokine with the most potent activity against HIV-1,
but it is poor at inhibiting the infection of macrophages.
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
and that it completely blocks infection of macrophages. A
single injection of PSC-RANTES before inoculation of
virus prevents HIV-1 infection of 100% of mice with
severe combined immunodeficiency repopulated with
128 IMMUNOLOGY 2005
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 PSC RANTES as a topical microbicide to prevent sexual
transmission of HIV-1. Recently, treatment with PSCRANTES prevented vaginal transmission of a chimeric
virus consisting of simian immunodeficiency virus and
HIV in the rhesus macaque model. Preclinical development of this compound is progressing toward the
first trials in humans.
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 recent detailed analysis revealed that
most mutants have a loss of fitness during coreceptor
switching that 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 in 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 a parental CCR5-using virus
and a CXCR4-using descendent virus separated from
the parent virus by 5 mutations. We used site-directed
mutagenesis to introduce all 32 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.
We found that 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. About
30% of possible mutations were noninfectious.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Maximum likelihood analysis indicated 1 favored
mutational pathway in 120 sequential possibilities. The
probability of coreceptor switching is thus constrained
by having to make the right mutation at the right place
at the right time. In the lottery of ongoing viral mutation, a coreceptor switch event is a rare winner.
PUBLICATIONS
Hartley, O., Gaertner, H., Wilken, J., Thompson, D., Fish, R., Ramos, A., Pastore, C.,
Dufour, B., Cerini, F., Melotti, A., Heveker, N., Picard, L., Alizon, M., Mosier, D.,
Kent, S., Offord, R. Medicinal chemistry applied to a synthetic protein: development of
highly potent HIV entry inhibitors. Proc. Natl. Acad. Sci. U. S. A. 101:16460, 2004.
Lederman, M.M., Veazey, R.S., Offord, R., Mosier, D.E., Dufour, J., Mefford, M.,
Piatak, M., Jr., Lifson, J.D., Salkowitz, J.R., Rodriguez, B., Blauvelt, A., Hartley, O.
Prevention of vaginal SHIV transmission in rhesus macaques through inhibition of
CCR5. Science 306:485, 2004.
Mosier, D.E. HIV-1 envelope evolution and vaccine efficacy. Curr. Drug Targets
Infect. Disord. 5:171, 2005.
Control of Cytokine Expression
by Arginine Methylation
K.A. Mowen, J.W. Fathman
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 cellmediated 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 helper T cells provides
an intriguing avenue of therapy, and understanding the
molecular events that control lineage-specific cytokine
expression may provide useful tools to modulate 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
H
IMMUNOLOGY
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 have 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.
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 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,
L. Verkoczy, 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 genes
T
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The Scripps Research Institute. All rights reserved.
2005
129
for antibody heavy and light chains 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, 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 regulates
RAG transcription. Recent results suggested that NF-κB
and rel transcription factors may be involved in both
positive and negative regulation of the RAG genes. In
addition, we are using DNA array analysis and other
screening methods to look more closely at changes in
gene expression during and subsequent to the receptor
editing response. We found that a relatively small fraction of genes is differentially expressed, including a
handful of previously uncharacterized mRNAs, which
we are analyzing further.
In other studies, we focused on the cues that
mature B cells use to distinguish self from nonself.
130 IMMUNOLOGY 2005
Fully mature recirculating B cells can be rapidly inactivated and 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 selftolerance 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 exploring the idea that immune
tolerance in mature B cells depends on specific costimulation by self-tissue.
PUBLICATIONS
Aït-Azzouzene, D., Verkoczy, L., Peters, J., Gavin, A., Skog, P., Vela, J.L., Nemazee,
D. An immunoglobulin Cκ-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system. J. Exp.
Med. 201:817, 2005.
Gavin, A., Aït-Azzouzene, D., Mårtensson, A., Duong, B., Verkoczy, L., Skog, J.L.,
Skog, P., Nemazee, D. Peripheral B lymphocyte tolerance. Keio J. Med. 53:151, 2004.
Gavin, A.L., Duong, B., Skog, P., Aït-Azzouzene, D., Greaves, D.R., Scott, M.L.,
Nemazee, D. ∆BAFF, a splice isoform of BAFF, opposes full-length BAFF activity in
vivo in transgenic mouse models. J. Immunol. 175:319, 2005.
Peters, B., Sidney, J., Bourne, P., Bui, H.H., Buus, S., Doh, G., Fleri, W., Kronenberg, M., Kubo, R., Lund, O., Nemazee, D., Ponomarenko, J.V., Sathiamurthy, M., Schoenberger, S.P., Stewart, S., Surko, P., Way, S., Wilson, S., Sette,
A. The immune epitope database and analysis resource: from vision to blueprint.
PLoS Biol. 3:e91, 2005.
Verkoczy, L., Aït-Azzouzene, D., Skog, P., Mårtensson, A., Lang, J., Duong B.,
Nemazee, D. A role for nuclear factor κB/rel transcription factors in the regulation
of the recombinase activator genes. Immunity 22:519, 2005.
Verkoczy, L.K., Mårtensson, A.S., Nemazee, D. The scope of receptor editing and
its association with autoimmunity. Curr. Opin. Immunol. 16:808, 2004.
improved gene delivery to human hematopoietic cells
that express this receptor.
Important new advances in data acquisition and
image processing resulted in major improvements in
resolution compared with the resolution in earlier studies. For example, electron cryomicroscopy density was
observed for hexon residues missing from the crystal
structure that included hypervariable regions and the
epitope of a neutralizing antibody. On the inner capsid
surface, density was revealed at the base of the hexons
and below the penton base that most likely correspond
to minor adenovirus proteins, including protein VI.
On the basis of the new structural information, we
proposed a new model for Ad35F. In particular, the
model presents 2 possible orientations for protein IX,
either binding on the capsid surface or extending away
from the capsid, consistent with the use of the C terminus of protein IX for the insertion of exogenous ligands
to redirect adenovirus vectors to alternative receptors.
These studies increase our knowledge of adenovirus
capsid assembly and antibody neutralization and thus
may promote further improvements in gene delivery to
hematopoietic cell types.
Targeting a General Biochemical
Pathway in Viral Infections Via
Cyclic D,L-α-Peptides
W.S. Horne, C.M. Wiethoff, C. Cui, K.M. Wilcoxen,
Structural Analyses of an
Adenoviral Vector Targeted to
Hematopoietic Cells
S. Saban,* R.R. Nepomuceno, G.R. Nemerow, P.L. Stewart*
* Vanderbilt University Medical Center, Nashville, Tennessee
espite recent advances in the uses of adenovirus vectors for vaccines and gene delivery, we
still lack basic knowledge of the structure of
intact adenovirus particles. In recent studies, we used
electron cryomicroscopy and image reconstruction to
determine the 3-dimensional structure of an adenovirus vector, Ad35F, at 9-Å resolution. This viral vector
recognizes the receptor CD46, a member of the complement regulatory protein family, thereby allowing
D
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
M. Amorin, M.R. Ghadiri, G.R. Nemerow
iverse human viruses have coevolved to exploit
the acidification of endosomal compartments
to gain entry into host cells. Recently, we used
a supramolecular approach to selectively target and
inhibit viral infections through this central pathway.
We used a high-throughput screen with an adenovirus
vector encoding green fluorescent protein to select an
8-residue cyclic D ,L-α-peptide from a directed combinatorial library that specifically inhibited the development of low pH inside endocytic vesicles, thereby
arresting escape of virus from these compartments.
The peptide had no adverse effect on cell viability
and was only able to exert its inhibitory activity when
added to cells in the presence of the virus. Confocal
fluorescence microscopic studies with labeled adenovirus particles indicated that the peptide did not hinder
D
IMMUNOLOGY
viral attachment or entry but rather extinguished the
pH gradient inside cell endosomes. Influenza virus
that uses a mode of entry similar to that of adenovirus
was also inhibited by the peptide. Our results suggest
that self-assembling cyclic peptides may provide a
broad-spectrum and alternative approach to the design
of antiviral drugs.
Inhibiting Expression of
Proinflammatory Cytokines
With Adenoviruses
M. Iacobelli-Martinez, R.R. Nepomuceno, J. Connolly,*
G.R. Nemerow
* Isis Pharmaceuticals, Inc., Carlsbad, California
ost adenovirus serotypes bind to host cells
via the coxsackievirus-adenovirus receptor,
but subgroup B and subgroup D (adenovirus
37) viruses recognize the receptor CD46. Interestingly,
diverse microbial pathogens that use CD46 for infection downregulate the expression of IL-12, a cytokine
involved in both the innate and the adaptive immune
responses. We determined whether adenovirus serotypes
that use CD46 alter the expression of proinflammatory
cytokines.
We found that subgroup B adenoviruses type 16
and 35 and subgroup D adenovirus type 37, but not
subgroup C adenoviruses type 2 or 5, significantly
reduced expression of IL-12 by peripheral blood mononuclear cells stimulated by IFN-γ and lipopolysaccharide.
IL-12 mRNA, as well as mRNA encoding other mediators, such as IL-1α, IL-β, the receptor for IL-1α, and
IL-6, were also downregulated upon interaction with
adenoviruses that use CD46. Analysis of transcription
factor activity required for cytokine expression indicated that adenoviruses that used CD46 preferentially
inhibited the DNA-binding activity of the transcription
factor CCAAT/enhancer-binding protein β (C/EBP-β).
Expression of C/EBP-β protein induced by IFN-γ was
also impaired by adenoviruses that use CD46, consistent with the reduced DNA-binding activity of C/EBP-β.
Interference with IFN-γ signaling events by adenoviruses
that use CD46, but not adenoviruses that use the coxsackievirus-adenovirus receptor, revealed a potentially
critical difference in the host immune response against
adenovirus vectors, a situation that has implications
for gene delivery and vaccine development.
M
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
131
PUBLICATIONS
Horne, W.S., Wiethoff, C.M., Cui, C., Wilcoxen, K.M., Amorin, M., Ghadiri, M.R.,
Nemerow, G.R. Antiviral cyclic D,L-α-peptides: targeting a general biochemical
pathway in virus infections. Bioorg. Med. Chem. 13:5145, 2005.
Hsu, C., Boysen, M., Gritton, L.D., Frosst, P.D., Nemerow, G.R., Von Seggern,
D.J. In vitro dendritic cell infection by pseudotyped adenoviral vectors does not correlate with their in vivo immunogenicity. Virology 332:1, 2005.
Iacobelli-Martinez, M., Nepomuceno, R.R., Connolly, J., Nemerow, G.R. CD46utilizing adenoviruses inhibit C/EBPβ-dependent expression of proinflammatory
cytokines. J. Virol. 79:11259, 2005.
Saban, S.D., Nepomuceno, R.R., Gritton, L.D., Nemerow, G.R., Stewart, P.L.
CryoEM structure at 9Å resolution of an adenovirus vector targeted to hematopoietic cells. J. Mol. Biol. 349:526, 2005.
Wiethoff, C.M., Wodrich, H., Gerace, L., Nemerow, G.R. Adenovirus protein VI
mediates membrane disruption following capsid disassembly. J. Virol. 79:1992,
2005.
DNA Vaccine–Induced
Long-Lived T-Cell Memory
Against Tumor Recurrence
Y. Luo, H. Zhou, J. Krueger, C. Kaplan, S.H. Lee, R. Xiang,
R.A. Reisfeld
ong-lived T-cell memory is a major objective of
vaccination against tumors because it provides
continuous protection against tumor dissemination and recurrence. Key requirements for successful
protection against tumors include an increased number
of tumor antigen–specific CD8+ T cells in an immune
host and the capability of CD8+ T memory cells to
proliferate, secrete inflammatory antitumor cytokines,
and repeatedly kill recurring tumor cells more effectively than naive CD8+ T cells do.
We achieved these objectives with a DNA vaccine
that encodes Fos-related antigen 1 (Fra-1), a murine
transcription factor. The antigen was fused to polyubiquitin, modified by cotransformation with a gene encoding secretory murine IL-18, and carried to secondary
lymphoid organs (i.e., Peyer’s patches) by attenuated
Salmonella typhimurium. This DNA vaccine was effective in 2 different breast carcinoma models and in a
non–small lung carcinoma model. In syngeneic mice, it
induced a strong antitumor immunity that could be maintained as a long-lived specific immune response and
resulted in eradication of tumor metastases. In addition, IL-18 enhanced this immune response by activating
both T cells and natural killer cells while upregulating
expression of MHC class I antigens and promoting the
differentiation of CD4+ T cells to type 1 helper T cells.
L
132 IMMUNOLOGY 2005
The vaccine also was effective in a therapeutic
setting. It markedly suppressed growth and dissemination of established pulmonary metastases of both D2F2
breast and D121 non–small cell lung carcinomas. The
primary mediators of this protective immunity were
CD8+ T cells, which secreted IFN-γ, a proinflammatory
cytokine associated with type 1 helper T cells, and
were activated in both lymphoid and nonlymphoid tissues, especially in tumor tissues of lung and liver.
Furthermore, our finding that a single vaccination can
upregulate CD4+ T cells releasing IL-2 in Peyer’s patches
supports the contention that the mechanism of tumor
protection depends on CD4 + T cells and is mediated
by effector functions of helper T cells.
We also showed that the vaccine induced the establishment and long-term maintenance of immunologic
memory CD8 + T cells in successfully vaccinated mice.
When passively transferred to mice with severe combined immunodeficiency disease, such memory cells
maintained sufficient memory in the absence of tumor
antigen to markedly suppress dissemination and growth
of a lethal challenge of D2F2 breast tumor cells. Importantly, after vaccination, CD8 + T cells in the tumor
microenvironment also migrated to nonlymphoid tissues such as lungs, where some of the T cells were
found as long-lived, dormant memory T cells ready
to respond to reencounter with the same tumor antigen.
Strikingly, such lymphocytes released more IFN-γ and
contained a higher percentage of CD8 + cytotoxic
T lymphocytes and memory CD8+ T cells than did their
splenic counterparts, suggesting that these CD8+ T cells
can play a key role in the cellular response against tumor
metastases. In fact, these results point to the existence
of a population of extralymphoid effector memory T cells
poised for an immediate antitumor immune response.
On the basis of data in the literature that T-cell proliferation induced by IL-18 depends on IFN-γ, we examined the effect of IL-18 on T-cell turnover. We found that
expression of secretory IL-18 in our vaccine induced the
rapid turnover of CD8+ T memory cells in mice within
24 hours after immunization and that such cells can be
maintained in lymphoid tissues as well as locally in lung
tumor tissues. Interestingly, CD8+ T cells obtained from
the lungs of our vaccinated mice proliferated and released
IFN-γ after exposure to antigen in vitro. Consequently, we
conclude that persistently activated T cells and memory
CD8+ T cells in the lung can play a key role in the cellular immune response against tumor metastases.
We also demonstrated in our tumor models that a
specific memory T-cell response is induced and mainPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
tained in the absence of tumor antigen. We showed that
CD8 + T cells adoptively transferred from successfully
vaccinated mice to syngeneic mice with severe combined immunodeficiency and parked there for 7 or 30
days could maintain an effective and long-lived memory
in the absence of both tumor antigen and naive T cells.
In fact, 6 of 8 of these successfully reimmunized mice
lived 3 times longer than control mice did in the absence
of any detectable tumor growth up to 56 days after
challenge with tumor cells.
Taken together, our data indicate that an oral DNA
vaccine encoding ubiquitinated Fra-1 and IL-18 can
protect mice against a lethal challenge of murine breast
cancer cells and of non–small cell lung carcinoma cells.
Moreover, this vaccine can break T-cell tolerance to the
Fra-1 self-antigen and generate a long-lived memory
T-cell immune response against recurring breast cancer,
which can be maintained constantly in both lymphoid
and nonlymphoid organs in mice with severe combined
immunodeficiency in the absence of tumor antigen.
PUBLICATIONS
King, D.M., Albertini, M.R., Schalch, H., Hank, J.A., Gan, J., Surfus, J., Mahvi, D.,
Schiller, J.H., Warner, T., Kim, K.M., Eickhoff, J., Kendra, K., Reisfeld, R.A.,
Gillies, S.D., Sondel, P. Phase I clinical trial of the immunocytokine EMD 273063
in melanoma patients. J. Clin. Oncol. 22:4463, 2004.
Lo, J.F., Zhou, H., Fearns, C., Reisfeld, R.A., Yang, Y., Lee, J.D. Tid1 is required
for T cell transition from double-negative 3 to double-positive stages. J. Immunol.
174:6105, 2005.
Loeffler, M., Kruger, J.A., Reisfeld, R.A. Immunostimulatory effects of low-dose
cyclophosphamide are controlled by inducible nitric oxide synthase. Cancer Res.
65:5027, 2005.
Luo, Y., Zhou, H., Mizutani, M., Mizutani, N., Liu, C., Xiang, R., Reisfeld, R.A. A
DNA vaccine targeting Fos-related antigen 1 enhanced by IL-18 Induces long-lived
T-cell memory against tumor recurrence. Cancer Res. 65:3419, 2005.
Mizutani, N., Luo, Y., Mizutani, M., Reisfeld, R.A., Xiang, R. DNA vaccines suppress angiogenesis and protect against growth of breast cancer metastases. Breast
Dis. 20:81, 2004.
Niethammer, A.G., Wodrich, H., Loeffler, M., Lode, H.N., Emmerich, K., Abdollahi, A., Krempien, R., Debus, J., Huber, P.E., Reisfeld, R.A. Multidrug resistance-1
(MDR-1): a new target for T cell-based immunotherapy. FASEB J. 19:158, 2005.
Xiang, R., Mizutani, N., Luo, Y., Chiodoni, C., Zhou, H., Mizutani, M., Ba, Y., Becker,
J.C., Reisfeld, R.A. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res. 65:553, 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.
Zhou, H., Luo, Y., Mizutani, M., Mizutani, N., Reisfeld, R.A., Xiang, R. T cellmediated suppression of angiogenesis results in tumor protective immunity. Blood,
in press.
IMMUNOLOGY
2005
133
Protective Protease-Activated
Receptor 1 Signaling by the
Protein C Pathway
M. Riewald, C. Feistritzer, R.A. Schuepbach, R. Lenta
hrombin and activated protein C (APC) are major
regulators of the blood coagulation system. Thrombin not only is the key procoagulant enzyme but
also activates the anticoagulant protein C pathway on
the surface of endothelial cells. The generated APC
inhibits blood coagulation by downregulating prothrombin activation in a negative feedback loop. Results from
animal models and clinical trials indicate that APC has
potent protective effects in systemic inflammation that
are independent of its anticoagulant function, and recently
recombinant APC was approved to treat patients with
severe sepsis. The molecular basis for the anti-inflammatory effects of APC is incompletely understood.
Previously, we showed that APC signaling in endothelial cells requires binding to endothelial protein C
receptor and activation of protease-activated receptor 1
(PAR-1), the thrombin receptor. Thrombin–PAR-1 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. Incubation of
an endothelial monolayer with APC and low concentrations of thrombin potently enhanced barrier integrity
through PAR-1–dependent transactivation of the barrier
protective sphingosine 1-phosphate (S1P) signaling
pathway (Fig. 1). These results revealed an unexpected
role for cross-communication between the prototypical
barrier protective S1P and barrier-disruptive PAR-1
pathways and suggest that S1P signaling may mediate
protective effects of APC in sepsis. In addition, largescale gene expression profiling indicated that thrombin
and APC can have distinct PAR-1–dependent effects in
inflammatory cytokine-perturbed endothelial cells.
APC–PAR-1, but not thrombin–PAR-1, downregulated
transcript levels of several proapoptotic proteins, including p53 and thrombospondin-1.
Taken together, these results indicate that the same
receptor, PAR-1, can mediate different biological effects
depending on the rate of receptor activation, and they
suggest that APC elicits powerful protective responses
precisely because it is a relatively poor PAR-1 activator compared with thrombin. We are elucidating how
T
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . PAR-1 can mediate opposite effects on endothelial bar-
rier integrity. Inflammatory disorders such as sepsis are associated
with increased permeability of the endothelial cell monolayer at
the blood-tissue interface. Proinflammatory signaling by thrombin
through PAR-1 can disrupt endothelial barrier integrity. APC enhanced
endothelial barrier integrity dependent on binding to endothelial
protein C receptor (EPCR) and activation of PAR-1, cellular sphingosine kinase-1 (SK1), and S1P receptor-1 (S1P1). Thus, barrier
protection by APC proceeds via cross talk between the barrier-disruptive PAR-1 and barrier-protective S1P pathways. Incubation of
cells with low concentrations (~40 pM) of thrombin had an equally
potent barrier-enhancing effect dependent on the S1P pathway.
differences in the level of receptor activation translate
into activation of different cellular signaling pathways.
To dissect the contributions of PAR-1 activation by
APC and thrombin, we designed PAR-1 variants that
are efficiently activated by APC but not by thrombin.
Transgenic mice expressing these variants in endothelial cells have been generated and will be analyzed in
sepsis models to define the in vivo roles of PAR-1 signaling in systemic inflammation.
PUBLICATIONS
Feistritzer, C., Mosheimer, B.A., Kaneider, N.C., Riewald, M., Patsch, J.R., Wiedermann, C.J. Thrombin affects eosinophil migration via protease-activated receptor-1. Int. Arch. Allergy Immunol. 135:12, 2004.
Feistritzer, C., Riewald, M. Endothelial barrier protection by activated protein C
through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation.
Blood 105:3178, 2005.
Riewald, M., Ruf, W. Protease-activated receptor-1 signaling by activated protein C
in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J. Biol.
Chem. 280:19808, 2005.
134 IMMUNOLOGY 2005
Chemical and Genetic
Approaches to Adaptive
and Innate Immunity
H. Rosen, G. Sanna, C. Alfonso, E. Jo, P. Gonzalez-Cabrera,
A. Don, M. Peterson, Y. Gon
ymphocytes develop in the thymus (T cells) and
bone marrow (B cells) and upon maturation leave
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 this number is maintained by the
recirculation of lymphocytes through the secondary
lymphoid organs.
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.
L
F i g . 1 . Postulated contributions of lymphocytes and endothelial
cells to mediation of lymphocyte trafficking by the S1P-S1P1 system. A, Stromal gate control: Lymphocytes pass through open
endothelial junctions (left). S1P or synthetic agonists ligate S1P1
receptors on the surfaces of endothelial cells, stimulating Rac
GTPase-dependent endothelial junctional tightening. Lymphocytes
cannot pass through the closed gate of sinus-lining endothelial
cells (right) and accumulate in the lymph node. B, S1P1 intrinsic
lymphocyte control: S1P normally stimulates migration of T cells
from the lymph node into the sinus with concentration-dependent
responses (left). High concentrations of S1P may downregulate
S1P1 expression on T cells, and S1P1 antagonists may block S1P
chemotactic signaling of T cells to promote the retention of T cells
in the lymph node (right). Both mechanisms could contribute
simultaneously to the control of T-cell trafficking.
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, most autoimmune diseases require
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 we have delineated 2 potentially
synergistic mechanisms that alter lymphocyte migration (Fig. 1).
CHEMICAL PROBES OF RECEPTOR INTERACTIONS,
A C T I VAT I O N , A N D FAT E
One of our goals has been to define the rules for
chemical tractability of therapeutic targets in signaling
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
lipids. Using high-throughput screening of commercial
chemical libraries, we identified potent selective agonists of the S1P1 receptor. These agonists produced
lymphopenia in blood by sequestering lymphocytes in
lymph nodes, but not in the spleen.
The minimal signals required for lymphocyte sequestration are being defined by using selective S1P1-specific agonists that generate prolonged signals upon
ligand stimulation and induce receptor internalization
but rapid recycling to the cell surface. Receptor docking
and mutagenesis studies with A. Parrill, University of
Memphis, Memphis, Tennessee, and G. Tigyi, University
of Tennessee, Memphis, Tennessee, indicated that these
ligands overlap the binding site for the natural lipid
mediation S1P, and that key requirements for headgroup
interactions for S1P (Fig. 2) can be replaced by ion-
IMMUNOLOGY
2005
135
cells, disrupts tight junctions between epithelial cells
but not capillary endothelium and results in breakdown of the lung barrier. We also have evidence for a
synergistic interaction between the S1P-S1P3 axis and
exposure to tumor necrosis factor. Whereas neither
tumor necrosis factor nor S1P induces pathologic
changes in the lungs when given alone in subthreshold doses, the 2 molecules produce severe breakdown
of lung barriers with lethal pulmonary leakage when
administered together. These data have led us to a
model in which S1P controls physiology in different
systems through the use of discrete receptor subtypes
that have different cellular and spatial distributions
and through downstream signal coupling (Fig. 3).
F i g . 2 . A space-filling model of the S1P 1 -selective ligand
SEW2871 docked into the receptor shows the critical headgroup
mimetic interactions. Image courtesy of A. Parrill, University of
Memphis, Memphis, Tennessee.
dipole interactions in this synthetic tetra-aromatic chemical series. These studies suggest that continuous agonism is a requirement for sequestration.
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 tumor necrosis
factor. We suspected that S1P could be an independent regulator of lung barrier function and therefore a
contributor to lung injury. In collaboration with J. Chun,
Department of Molecular Biology, and M. Woods and
B. Kiosses, Core Microscopy Facility, we used a combination of chemical and genetic approaches in mice
lacking genes for S1P receptor subtypes to examine
lung barrier integrity. We found that barrier integrity is
regulated through S1P3 activation, whereas lymphocyte recirculation is controlled by S1P1.
It is now apparent that different S1P receptor subtypes regulate lung barrier function in spatially distinct
and functionally opposite ways. S1P1, found on lung
capillaries, tightens capillary junctions and protects
from leakage, whereas S1P3, found on lung epithelial
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Spatially and mechanistically distinct S1P receptor subtypes have opposing effects on pulmonary epithelial and endothelial
barriers. S1P modulates epithelial and endothelial barrier function.
Fig. 3.
S1P-induced S1P 3 activation in alveolar epithelium results in
increased permeability via opening of tight junctions and loss of
zonula occludens protein, most likely through Rho activation. In
contrast, activation of S1P1 receptors on endothelial cells activates
Rac1 GTPase, inducing downstream assembly and stabilization
of cell-cell junctions with reorganization of the actin cytoskeleton
and VE-cadherin. G indicates G protein; filled circles indicate cellcell junctions.
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
lymph nodes, sequestration of effector T cells in lymph
nodes, and egress of mature medullary T cells from
the thymus. The system can therefore affect both the
peripheral diversity of lymphocytic responses and the
efficiency of T-cell activation by misdirecting T cells to
the wrong lymph nodes and by inhibiting the egress of
antigen-specific effector T cells from lymph nodes after
antigen activation and clonal proliferation.
136 IMMUNOLOGY 2005
These effects can alter adaptive immune responses
and the expression of tissue damage while providing
potentially important 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, where the
S1P system regulates lung epithelial barrier function,
is a new focus in molecular pathogenesis of inflammatory lung disease that is of long-term interest to us.
Regulation of Protease
Signaling Pathways
W. Ruf, J. Ahamed, M. Belting,* A. Dorfleutner,
E. Hintermann,** M. Kerver, B.M. Mueller,*** F. Niessen,
T. Kurokawa, Y. Kurokawa, Y. Lee, M. Majumdar,
H. Peterson, Y. Takada,**** H.H. Versteeg
* University of Lund, Lund, Sweden
** Department of Cell Biology, Scripps Research
*** La Jolla Institute for Molecular Medicine, San Diego, California
**** University of California, Davis, California
THERAPEUTIC INTERVENTION WITH TISSUE
PUBLICATIONS
Goetzl, E.J., Rosen, H. Regulation of immunity by lysosphingolipids and their
G protein-coupled receptors. J. Clin. Invest. 114:1531, 2004.
Gon, Y., Wood, M.R., Kiosses, W.B., Jo, E., Sanna, M.G., Chun, J., Rosen, H.
S1P3 receptor-induced reorganization of epithelial tight junctions compromises lung
barrier integrity and is potentiated by TNF. Proc. Natl. Acad. Sci. U. S. A.
102:9270, 2005.
Hale, J.J., Doherty, G., Toth, L., Li, Z., Mills, S.G., Hajdu, R., Keohane, C.A.,
Rosenbach, M., Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D.,
Rosen, H., Mandala, S. The discovery of 3-(N-alkyl)aminopropylphosphonic acids
as potent S1P receptor agonists. Bioorg. Med. Chem. Lett. 14:3495, 2004.
Hale, J.J., Doherty, G., Toth, L., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.,
Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Forrest, M., Sun, S.Y.,
West, S., Xie, H., Nomura, N., Rosen, H., Mandala, S. Selecting against S1P3
enhances the acute cardiovascular tolerability of 3-(N-benzyl)aminopropylphosphonic
acid S1P receptor agonists. Bioorg. Med. Chem. Lett. 14:3501, 2004.
Hale, J.J., Lynch, C.L., Neway, W., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Parent, S.A., Chrebet, G., Bergstrom, J.,
Card, D., Ferrer, M., Hodder, P., Strulovici, B., Rosen, H., Mandala, S. A rational
utilization of high-throughput screening affords selective, orally bioavailable 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonists. J. Med.
Chem. 47:6662, 2004.
Hale, J.J., Neway, W., Mills, S.G., Hajdu, R., Keohane, C.A., Rosenbach, M.,
Milligan, J., Shei, G.J., Chrebet, G., Bergstrom, J., Card, D., Koo, G.C., Koprak,
S.L., Jackson, J.J., Rosen, H., Mandala, S. Potent S1P receptor agonists replicate
the pharmacologic actions of the novel immune modulator FTY720. Bioorg. Med.
Chem. Lett. 14:3351, 2004.
Hale, J.J., Yan, L., Neway, W.E., Hajdu, R., Bergstrom, J.D., Milligan, J.A., Shei,
G.J., Chrebet, G.L., Thornton, R.A., Card, D., Rosenbach, M., Rosen, H., Mandala, S. Synthesis, stereochemical determination and biochemical characterization
of the enantiomeric phosphate esters of the novel immunosuppressive agent
FTY720. Bioorg. Med. Chem. 12:4803, 2004.
Jo, E., Sanna, M.G., Gonzalez-Cabrera, P.J., Thangada, S., Tigyi, G., Osborne,
D.A., Hla, T., Parrill, A.L., Rosen, H. S1P1-selective in vivo-active agonist from
high-throughput screening: off-the-shelf chemical probes of receptor interactions,
signaling and fate. Chem. Biol. 12:703, 2005.
Rosen, H. Chemical approaches to the lysophospholipid receptors. Prostaglandins
Other Lipid Mediat. 77:179, 2005.
Yan, L., Hale, J.J., Lynch, C.L., Budhu, R., Gentry, A., Mills, S.G., Hajdu, R.,
Keohane, C.A., Rosenbach, M.J., Milligan, J.A., Shei, G.J., Chrebet, G., Bergstrom, J.,
Card, D., Rosen, H., Mandala, S.M. Design and synthesis of conformationally constrained 3-(N-alkylamino)propylphosphonic acids as potent agonists of sphingosine-1-phosphate (S1P) receptors. Bioorg. Med. Chem. Lett. 14:4861, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
FA C T O R – S I G N A L I N G PAT H WAY S
e are interested in protease systems that
regulate inflammation, cancer, and angiogenesis. Activation of the coagulation pathway by
tissue factor (TF) triggers cell signaling events that contribute to lethality in sepsis. Therapy with activated
protein C, the natural counterbalance of TF-initiated
coagulation, markedly improves survival in severe sepsis.
We recently showed that activated protein C elicits
a unique cellular response in inflamed endothelial cells
through protease-activated receptor 1 (PAR-1) signaling.
The established gene profile is consistent with antiapoptotic and anti-inflammatory protection of the endothelium. Importantly, the pattern of gene induction is
distinct from the profile of the proinflammatory protease
thrombin, which signals through the same G protein–coupled receptor PAR-1. Thus, signaling specificity appears
to be determined by accessory protease-binding receptors, and protease-specific effects appear to be crucial
for therapeutic benefit.
How to appropriately target direct TF-initiated cell
signaling pathways remains an active area of research.
Inhibition of TF can improve survival in animal models
of lethal hemorrhagic fever, similar to the results of
earlier studies of bacterial septicemia. However, clinical
trials with a recombinant form of TF pathway inhibitor,
the physiologic inhibitor of TF, have been disappointing. We established that the recombinant inhibitor is a
poor inhibitor of signaling of the TF initiation complex
at doses that are highly effective in blocking activation
of coagulation. Because bleeding complications are
the major concern with TF-directed inhibitors, the ratio
of anticoagulant efficiency to antisignaling potency of
TF-targeted inhibitors is a consideration in using such
drugs to treat inflammatory disorders or cancer.
W
IMMUNOLOGY
We are identifying downstream signaling responses
that predict inhibitory potency of TF-directed strategies.
We are also continuing basic mechanistic studies on
the specificity of the TF signaling pathway; our goal is
to identify inhibitors with highly selective antisignaling
activities. We identified a candidate that specifically
blocks signaling by the complex consisting of TF and
coagulation factor VIIa without impairing the coagulant response. Such strategies may allow intervention
in TF signaling pathways while reducing the risk of
impairments in hemostasis.
2005
137
stream of the TF cytoplasmic domain and the cross
talk of TF with PARs and integrins.
PUBLICATIONS
Ahamed, J., Belting, M., Ruf, W. Regulation of tissue factor-induced signaling by endogenous and recombinant tissue factor pathway inhibitor 1. Blood 105:2384, 2005.
Belting, M., Ahamed, J., Ruf, W. Signaling of the tissue factor coagulation pathway in angiogenesis and cancer. Arterioscler. Thromb. Vasc. Biol. 25:1545, 2005.
Dorfleutner, A., Hintermann, E., Tarui, T., Takada, Y., Ruf, W. Cross-talk of integrin α3β1 and tissue factor in cell migration. Mol. Biol. Cell 15:4416, 2004.
Majumdar, M., Tarui, T., Shi, B., Akakura, N., Ruf, W., Takada, Y. Plasmininduced migration requires signaling through protease-activated receptor 1 and
integrin α9β1. J. Biol. Chem. 279:37528, 2004.
R E G U L AT I O N O F I N T E G R I N F U N C T I O N B Y T F
CYTOPLASMIC DOMAIN SIGNALING
We discovered an important regulatory role of the
TF cytoplasmic domain in tumor and developmental
angiogenesis. In diabetic eye diseases, we showed
that the cytoplasmic domain of TF is phosphorylated
specifically in neovasculature associated with pathologic changes but not in normal endothelial cells or in
the vessel wall. PAR-2, the target for TF-VIIa signaling, was also expressed in neovasculature. These data
suggest that the TF-VIIa complex is an important regulator of vascular cell signaling in angiogenesis. Results
of recent in vitro studies lend further support to the
concept that the TF–PAR-2 pathway regulates cell
migration in angiogenesis.
The extracellular domain of TF is involved in an
interaction with integrins, but we found no evidence
that the TF-integrin interaction results in competition
or inhibition of binding to the extracellular matrix by
these adhesive receptors. Rather, TF specifically suppressed cell migration on laminin 5 that depends on
activation of integrin α3β1. Inhibition of promigratory
α3β1 depended on the TF cytoplasmic domain. Mutagenesis indicated that integrin function is suppressed
when the TF cytoplasmic domain is not phosphorylated. However, phosphorylation of the TF cytoplasmic
domain by TF-VIIa–mediated PAR-2 signaling is sufficient to release integrin inhibition. Thus, protease-driven signaling pathways of TF regulate cell migration
by targeting the cross talk between the cytoplasmic
domain of TF and integrins.
Laminin 5 is a component of basement membranes,
including the subendothelial matrix. In addition, α3β1
is targeted by antiangiogenic molecules. We are using
genetic approaches to define components of the TF–VIIa–
PAR-2 signaling pathway and the relationship between
the pathway and integrins during angiogenesis in vivo.
In vitro, we are mapping the signaling pathways downPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Peerschke, E.I.B., Petrovan, R.J., Ghebrehiwet, B., Ruf, W. Tissue factor pathway
inhibitor-2 (TFPI-2) recognizes the complement and kininogen binding protein
gCIqR/p33 (gCIqR): implications for vascular inflammation. Thromb. Haemost.
92:811, 2004.
Riewald, M., Ruf, W. Protease-activated receptor-1 signaling by activated protein C
in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J. Biol.
Chem. 280:19808, 2005.
Ruf, W. Emerging roles of tissue factor in viral hemorrhagic fever. Trends Immunol.
25:461, 2004.
Shi, X., Gangadharan, B., Brass, L.F., Ruf, W., Mueller, B.M. Protease-activated
receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis. Mol.
Cancer Res. 2:395, 2004.
Structural Analysis of the
Host-Pathogen Interface
E. Ollmann Saphire, M.L. Havert, D.M. Abelson,
C.R. Kimberlin, J.E. Lee
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 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 virus
and viral proteins.
W
EBOLA AND MARBURG VIRUSES
At least 10 recognized outbreaks of infection with
Ebola virus in humans have occurred; in each outbreak,
50%–90% of those infected died. Six outbreaks of infection by the closely related Marburg virus have also
occurred. In outbreaks of Marburg virus, typically
25%–40% of those infected die; however, in a recent
outbreak in Angola, mortality was 90%. To date no
vaccines or treatments are available for infections caused
by either virus.
138 IMMUNOLOGY 2005
With these 2 viruses, death usually occurs 7–12
days after infection. Events early in infection and innate
immune responses are critical for survival in those
infected. However, filoviruses have evolved mechanisms
by which the host immune system is suppressed. For
example, the viral protein VP35 is a required component of the Ebola and Marburg viral capsids and transcription complexes. VP35 also blocks activation of
immunomodulatory genes by type I interferon and may
play a significant role in viral suppression of the host
immune system. Hence, structural analysis of the VP35
protein will provide insights into viral replication and
type I interferon suppression and will provide the structural basis for the design of antiviral compounds and
attenuated viral strains.
An additional, unusual feature of the genome of
Ebola virus 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
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 and should provide templates for the
design of vaccines that elicit antibodies that target the
virus rather than the secreted proteins.
In contrast, Marburg virus expresses only the membrane-embedded GP. Although Ebola and Marburg
viruses are closely related, antibodies to Ebola GP do
not cross-react with Marburg GP. Comparative structural analysis of Ebola and Marburg GP should illustrate the fusion machinery required for infection and
the structural mechanisms by which the viruses escape
from immune surveillance. Additional crystal structures
of these proteins in complex with rare human antibodies derived from survivors of infection will assist in
vaccine design.
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 vacPublished by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
cines 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 determine structural features of epitopes associated with
neutralization and enhancement.
Autoimmune Mechanisms and
Compensatory Responses
N. Sarvetnick, M. Cleary, S. Dabernat, D. Dietz, C. Fine,
N. Hill, H. Hua, A. Ilic, H.-B. Jie, V. Judkowski, A. Kayali,
C. King, M. Kritzik, X. Li, G. Liu, A. Maday, A. Marleau,
E. Rodriguez, P. Secrest, M. Solomon, L. Sterling,
A. Stotland, W. Wu, D. Yadav, Y.Q. Zhang
nfection with coxsackievirus is associated with the
development of autoimmunity in humans, and infection with the CB4 strain of this virus is strongly
linked to the development of type 1 diabetes mellitus.
However, the development of autoimmunity in general
depends on the availability of autoimmune T cells in
the periphery. Normally, autoreactive T cells are deleted
during negative selection in the thymus, and few T cells
with high affinity for self-antigens gain access to the
body. We tested the hypothesis that CB4 infection
inhibits negative selection in the thymus, allowing the
maturation of self-reactive T cells and migration of the
cells into the periphery.
We found that novel central tolerance mechanisms
are responsible for coxsackievirus-induced autoimmunity.
We have now determined the role of coxsackievirus
in the generation of an autoimmune T-cell repertoire.
Recently, we found that CB4 infects the thymus. We
extended this observation to show that productive infection leads to dramatic changes in the developmental
processes that occur during T-cell differentiation in the
thymus. In investigating this novel aspect of virusinduced autoimmunity, we determined that CB4 infection inhibits the negative selection of self-reactive T cells
in the thymus.
On the basis of our recent data, we selected several interesting mechanisms for further study. We are
using a diversified molecular approach to gain information that will bring a novel mechanism linking virus
infection with autoimmunity into focus, fueling new
directions in therapy for virus-induced autoimmunity.
The cytokines IFN-γ and TNF-α play major roles in
the destruction of pancreatic islets during the develop-
I
IMMUNOLOGY
ment of diabetes and in the acute rejection of islet tissue allografts. The protein termed suppressor of cytokine
signaling-1 (SOCS-1) negatively regulates interferon
signaling by inhibiting activation of the proteins Janus
kinase and signal transducer and activator of transcription. We investigated whether modulation of interferon
signaling by SOCS-1 could prevent the destruction of
pancreatic islet tissue allografts in mice.
We found that islets expressing SOCS-1 that were
transplanted beneath the kidney capsule of MHC-mismatched recipient mice had delayed allograft rejection
and reversed streptozotocin-induced diabetes for at least
2 weeks longer than did normal islets. Surprisingly,
although SOCS-1 negatively regulates interferon signaling, the islets expressing SOCS-1 responded to
stimulation with IFN-γ and upregulated class I MHC selfantigens, suggesting that this negative regulation was
not a factor in improved islet allograft survival. Islets
expressing SOCS-1 were significantly more resistant
than normal islets to cytokine-induced cell death after
treatment with TNF-α alone or with TNF-α plus IFN-γ.
Protection against cytokine-induced cytotoxic effects
correlated with degradation of the IκB inhibitor of the
upstream transcription factor NF-κB and inhibition of
the transcription factor interferon regulatory factor-1,
reflecting enhanced NF-κB–regulated cell survival signals. Our findings indicate that intragraft expression of
SOCS-1 makes islets insensitive to the deleterious
effects of cytokines and will be important in the development of therapies to prevent acute allograft rejection.
Activins regulate the growth and differentiation of
a variety of cells. During the development of islets in
the pancreas, activins are required for the specialization of pancreatic precursors from the gut endoderm
during midgestation. We probed the role of activin signaling during the development and regeneration of
pancreatic islet cells. We found that both activins and
activin receptors are upregulated in duct epithelial cells
during islet differentiation. Interestingly, the expression
of endogenous cellular inhibitors of activin signaling,
follistatin and Cripto, were also augmented. Inhibition
of activins significantly enhanced survival and expansion of pancreatic epithelial cells but decreased the
numbers of differentiated cells. Our results suggest
that the homeostasis of growth and terminal differentiation requires a precise context-dependent regulation
of activin signaling. Follistatin participates in this process by promoting proliferation of precursor cells during pancreas growth.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
139
PUBLICATIONS
Jie, H.-B., Sarvetnick, N. The role of NK cells and NK cell receptors in autoimmune disease. Autoimmunity 37:147, 2004.
Judkowski, V., Rodriguez, E., Pinilla, C., Masteller, E., Bluestone, J.A., Sarvetnick, N., Wilson, D.B. Peptide specific amelioration of T cell mediated pathogenesis in murine type 1 diabetes. Clin. Immunol. 113:29, 2004.
Judkowski, V., Allicotti, G.M., Sarvetnick, N., Pinilla, C. Peptides from common
viral and bacterial pathogens can efficiently activate diabetogenic T-cells. Diabetes
53:2301, 2004.
Yadav, D., Judkowski, V., Flodstrom-Tullberg, M., Sterling, L., Redmond, W.L.,
Sherman, L., Sarvetnick, N. B7-2 (CD86) controls the priming of autoreactive
CD4 T cell responses against pancreatic islets. J. Immunol. 173:3631, 2004.
Zhang, Y.-Q, Cleary, M.M., Si, Y., Liu, G.. Eto, Y., Kritzik, M., Dabernat, S., Kayali, A.G., Sarvetnick, N. Inhibition of activin signaling induces pancreatic epithelial
cell expansion and diminishes terminal differentiation of pancreatic beta-cells. Diabetes 53:2024, 2004.
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. Molina, S. Uryu
e wish to understand how intracellular signaling networks coordinate a complex biological response such as cell motility. In order
for a cell to correctly process different environmental
stimuli, these networks must contain critical intracellular signaling proteins that act as signal “integrators.”
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 sites of binding of integrin receptors to matrix proteins such as fibronectin. FAK catalytic
activity is enhanced by integrin binding to fibronectin,
and FAK tyrosine phosphorylation is increased in a receptor-proximal fashion by transmembrane growth factor
and G protein–linked receptors.
In many cells, increased FAK phosphorylation promotes binding of the protein tyrosine kinase c-Src to
FAK, thereby generating a dual FAK-Src protein tyrosine
kinase signaling complex. Because genetic inactivation
of FAK or Src results in the inhibition of cell migration,
our research strategy is to perform rescue and/or gainof-function assays in cells that lack FAK expression. We
are elucidating the importance of FAK catalytic activity, FAK-Src signaling connections, novel interactions
W
140 IMMUNOLOGY 2005
between FAK and its binding partners, and the molecular basis for FAK relocalization to distinct intracellular
sites after treatment with motility-promoting stimuli.
Additionally, we are investigating the effects of the upregulated expression of the FAK-related proline-rich tyrosine kinase 2 in cells that lack FAK.
During cancer progression, tumor cells can become
highly motile and invasive. These properties contribute
to tumor spread and metastasis. Elevated FAK expression has been correlated with increased tumor growth
and metastasis. However, the molecular signaling connections that link FAK to tumorigenesis remained undefined. The possibility that FAK-mediated signaling may
promote increased tumor growth has generated much
interest in determining the molecular mechanisms of FAK
function in both normal cells and tumor cells.
We found that FAK-Src signaling promotes an invasive cell phenotype in a manner that is distinct from
the signaling role of FAK in promoting cell motility. In
these studies, we are using live-cell imaging, recombinant viral vectors, RNA interference, cell culture–based
signaling, and in vivo tumor growth and experimental
metastasis assays to elucidate the molecular signaling
connections of FAK in tumorigenesis.
PUBLICATIONS
Mitra, S.K., Hanson, D.H., Schlaepfer, D.D. Focal adhesion kinase: in command
and control of cell motility. Nat. Rev. Mol. Cell. Biol. 6:56, 2005.
The Consequences of T-Cell
Recognition of Self-Antigens
and Tumor Antigens
L.A. Sherman, H.T.C. Kreuwel, W.L. Redmond, M.A. Lyman,
C.H. Wei, J.A. Biggs, K.L. Marquardt, R.L. Trenney,
J. Martinez, B. Marincek
he consequence of antigen recognition by naive
CD8 + T cells can be either tolerance or immunity, depending on the activation status of the
antigen-presenting dendritic cells. If a CD8+ cell recognizes antigen on a quiescent dendritic cell that
expresses relatively low levels 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 cellsurface costimulatory molecules and cytokines. If
T
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
CD8 + T cells recognize antigen on activated dendritic
cells, the costimulatory molecules and cytokines prevent deletion and promote the clonal expansion of the
CD8 + 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 self-destructive 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 (Ins-HA mice), diabetes-prone
nonobese diabetic mice (NOD-InsHA mice) and mice
in which the beta cells express an oncogene that promotes spontaneous transformation and production of
tumors (RIP-Tag2-InsHA mice).
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 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.
DELETION OF NAIVE CD8+ T CELLS
Using the transgenic animals that were our source
of naive CD8 + T cells specific for hemagglutinin, we
examined the requirements for peripheral deletion in
vivo. We found that independent of the amount of
antigen used for stimulation, a single dose of antigen
did not result in complete clonal deletion. Instead, further antigenic exposure was required to completely
eliminate all of the activated T cells. Consecutive stimulations with low doses of antigen were highly effective
in promoting deletion. In contrast, although stimulation
with high doses of antigen initially led to the programmed cell death of many of the activated T cells,
it induced hyporesponsiveness in part of the responding
cells, thereby sparing the cells from further activation
and deletion. These data explain why some conditions
promote tolerance through clonal deletion whereas
others promote anergy. Furthermore, the data provide
a framework for devising protocols for effective deletion of potentially autoreactive T cells.
IMMUNOLOGY
R E S T O R AT I O N O F C D 8 + T - C E L L T O L E R A N C E I N
NOD MICE
The development of autoimmune diseases such as
type 1 diabetes is mediated by multiple genetic and
environmental factors. Although genes that may control
type 1 diabetes can now be identified, defining the
resulting cellular events mediated by each locus is a
major challenge. In congenic NOD mice, the genetic
regions that control diabetes, designated as insulindependent diabetes (Idd) loci, have been replaced by
resistant alleles obtained from nondiabetic strains of
mice. We hypothesize that critical genetic susceptibility loci regulate the maintenance of self-specific CD8+
T cells.
We compared the fate of islet-reactive CD8+ and
CD4 + T cells in diabetes-susceptible NOD mice with
the fate of the same kinds of cells in diabetes-resistant
NOD congenic mice with protective alleles at Idd3,
Idd5.1, and Idd5.2 (Idd3/5 strain) or at Idd9.1, Idd9.2,
and Idd9.3 (Idd9 strain). We found that protection
from diabetes in each instance is correlated with functional tolerance of islet-specific CD8+ T cells; however,
this tolerance is achieved in different ways. In Idd3/5
mice, tolerance occurs during the initial activation of
islet-specific CD8+ and CD4+ T cells in the pancreatic
lymph nodes, where the presence of CD25 + regulatory T cells prevents accumulation of the CD8+ and
CD4 + T cells. In contrast, resistance alleles in Idd9
mice do not prevent the accumulation of islet-specific
CD8+ and CD4+ T cells in the pancreatic lymph nodes,
suggesting that tolerance occurs at a later checkpoint.
These results underscore the variety of ways that autoimmunity can be prevented and indicate the elimination
of islet-specific CD8+ T cells as a common indicator of
high-level protection.
FAT E O F L O W - A F F I N I T Y T U M O R - S P E C I F I C C D 8 +
T CELLS IN TUMOR-BEARING MICE
An important issue in tumor immunology is how to
best activate and mobilize the low-avidity self-specific
and tumor-specific T cells that remain in the T-cell repertoire after the development of central and peripheral tolerance. We generated transgenic mice that express a
low-avidity T-cell receptor (clone 1 mice) specific for
hemagglutinin, a model self-antigen. When CD8+ T cells
from clone 1 mice were transferred into InsHA mice,
little proliferation occurred in response to low amounts
of cross-presented hemagglutinin, indicating that the
low-avidity clone 1 CD8+ T cells ignore the cross-presented hemagglutinin self-antigen. In contrast, the
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
141
expression of hemagglutinin as a tumor-associated antigen on spontaneous hemagglutinin-expressing beta
cell tumors in RIP-Tag2-InsHA mice led to high levels
of cross-presented antigen that could activate the lowaffinity clone 1 T cells. However, because of the absence
of inflammatory signals, this activation resulted in deletion of the clone 1 cells. This model should be useful
in optimizing protocols for immunotherapy of solid
tumors with low-affinity tumor-specific T cells.
PUBLICATIONS
Kuball, J., Schmitz, F.W., Voss, R.H., Ferreira, E.A., Engel, R., Guillaume, P.,
Strand, S., Romero, P., Huber, C., Sherman, L.A., Theobald, M. Cooperation of
human tumor-reactive CD4+ and CD8+ T cells after redirection of their specificity
by a high-affinity p53A2.1-specific TCR. Immunity 22:117, 2005.
Lyman, M.A., Nugent, T.C., Marquardt, K.L., Biggs, J.A., Pamer, E.G., Sherman,
L.A. The fate of low affinity tumor specific CD8+ T cells in tumor-bearing mice. J.
Immunol. 174:2563, 2005.
Redmond, W.L., Marincek, B.C., Sherman, L.A. Distinct requirements for deletion versus anergy during CD8 T cell peripheral tolerance in vivo. J. Immunol.
174:2046, 2005.
Redmond, W.L., Sherman, L.A. Peripheral tolerance of CD8 T lymphocytes. Immunity 22:275, 2005.
Yadav, D., Judkowski, V., Flodstrom-Tullberg, M., Sterling, L., Redmond, W.L.,
Sherman, L., Sarvetnick, N. B7-2 (CD86) controls the priming of autoreactive
CD4 T cell response against pancreatic islets. J. Immunol. 173:3631, 2004.
T-Cell Specificity and
the Thymus
J. Sprent, O. Boyman, J.-H. Cho, N. Ishimaru, M. Kovar,
M.P. Rubinstein
ature T cells arise in the thymus through a
process of positive and negative selection
directed to MHC molecules complexed with
self-peptides. In the secondary lymphoid tissues, T cells
survive for prolonged periods through covert signaling
elicited by interaction with self-peptide–MHC complexes
and various cytokines. Contact with complexes composed of foreign peptide and MHC molecules causes
T cells to proliferate and differentiate into effector cells,
with subsequent production of small numbers of memory cells.
M
S T I M U L AT I O N O F N A I V E C D 8 + T C E L L S B Y
FRAGMENTS OF ANTIGEN-PRESENTING CELLS
Typical immune responses of naive CD8+ T cells
are stimulated by foreign peptide–MHC complexes on
viable antigen-presenting cells (APCs) such as dendritic
cells and take place in the T-dependent areas of the
lymphoid tissues. Under certain conditions, however,
142 IMMUNOLOGY 2005
CD8 + cells can respond to small membrane vesicles
(exosomes) secreted by APCs. In addition, CD8 + cells
can be stimulated by fragments of plasma membranes
derived from sonicated APCs. These membrane fragments can be directly immunogenic for purified naive
CD8 + T cells in the absence of APCs, but only when
the fragments express specific peptide-MHC complexes
and also coexpress both B7 and intracellular adhesion
molecule 1 (the ligands for CD28 and lymphocyte
function–associated antigen 1, respectively). Membrane
fragments from APCs are also immunogenic in vivo
and can be used to prime mice for tumor rejection.
CYTOKINE CONTROL OF CD8+ T MEMORY CELLS
CD8+
T memory cells are kept alive and divide intermittently through contact with cytokines, especially IL-15.
Another cytokine, IL-2, can have a negative effect on
CD8 + memory cells, as indicated by the finding that
the number of CD8+ memory cells are greatly increased
in mice that lack IL-2 or components of the receptor
for IL-2 such as CD25 (IL-2Rα) and CD122 (IL-2/IL15Rβ). Unexpectedly, we found massive proliferation
of normal CD8 + memory cells after the cells were
transferred to mice that lack the gene for IL-2 or the
gene for the IL-2 receptor. Why CD8+ memory cells
proliferate under these conditions is unclear.
I N F L U E N C E O F N F - κB O N T - C E L L P R O L I F E R AT I O N
Stimulation of T cells and other lymphoid cells is
controlled by heterodimers of NF-κB1 (p50) and NF-κB2
(p52) bound to RelA and RelB subunits, respectively.
In the case of NF-κB2, activation and nuclear translocation are under the control of NF-κB–inducing kinase,
which is mutated in aly/aly mice. Confirming the results
of others, we found that unseparated aly/aly CD4 +
T cells have poor proliferative responses, implying an
important role for NF-κB2 in proliferation. Surprisingly,
however, quite different results occur when CD4+ T cells
are separated into purified subsets of naive (CD44lo)
and memory (CD44 hi) phenotype cells. Thus, CD44lo
cells have enhanced proliferative responses and IL-2
synthesis, whereas CD44lo cells have reduced responses.
Significantly, inhibition occurs when CD44hi cells are
added to CD44 lo cells. These findings indicate that
primary responses of T cells can be negatively influenced by NF-κB2.
PUBLICATIONS
Ganesh, K.A., Thomas, S., Thompson, L., Sprent, J., Murali-Krishna, K. Type I
interferons act directly on CD8 T cells to allow clonal expansion and memory formation in response to viral infection. J. Exp. Med., in press.
Goodnow, C.C., Sprent, J., de St. Groth, B.F., Vinuesa, C.G. Cellular and genetic
mechanisms of self tolerance and autoimmunity. Nature. 435:570, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Sprent, J. Direct stimulation of naive T cells by antigen-presenting cell vesicles.
Blood Cells Mol. Dis. 35:17, 2005.
Sprent, J. Proving negative selection in the thymus. J. Immunol. 174:3841, 2005.
Sprent, J. Swapping molecules during cell-cell interactions. Sci. STKE pe8, 2005,
March 1 issue.
Surh, C.D., Sprent, J. Regulation of mature T cell homeostasis. Semin. Immunol.
17:183, 2005.
Regulation of Homeostasis of
Mature T Cells
C.D. Surh, J. Tan, C. Ramsey, J. Purton, J.Y. Lee,
W.C. Kieper, J.T. Burghardt, D. Kim, C. Ahn*
* Seoul National University Hospital, Seoul, Korea
he pool of mature T cells is constantly regulated
by homeostatic mechanisms to remain at a steady
size and maintain predictable proportions of naive
and memory T cells. Homeostatic signals are received
as a result of interactions of T cells with self-MHC molecules and the cytokines IL-7 and IL-15. Thus, contact
with self-MHC molecules and IL-7 is required for survival of naive T cells, and a combination of IL-7 and
IL-15 is required for survival of memory T cells.
A hallmark of homeostatic regulation is the ability
of both naive and memory T cells to undergo spontaneous homeostatic proliferation in response to severe
T-cell depletion. A slower rate of homeostatic proliferation is also evident for memory T cells under normal
T cell–sufficient conditions, but not for most naive T cells.
Because the requirements for homeostatic proliferation
closely mirror the requirements for cell survival, most
likely the survival signals delivered by low levels of
IL-7 and IL-15 become mitogenic signals when the
concentrations of these cytokines are elevated.
Homeostatic proliferation of naive T cells is driven
by signals induced by contact of the cells with low-affinity ligands composed of self-antigens, MHC molecules,
and peptides that have been intensified by elevated levels of IL-7. Thus, polyclonal naive T cells transferred
into syngeneic wild-type mice treated to be lymphopenic
undergo mostly homeostatic proliferation as indicated
by the cell division rate of 1 division every 24–36 hours.
In congenitally T cell–deficient syngeneic hosts, however,
a fraction of polyclonal naive T cells undergoes a prodigious rate of proliferation and outcompetes the rest of
the naive cells that are undergoing slow homeostatic
proliferation. Such rapid T-cell proliferation resembles
proliferation driven by high-affinity foreign antigens,
T
IMMUNOLOGY
because the resultant T cells have characteristics of
effector cells. The proliferation also occurs largely
independent of homeostatic factors, that is, in the
absence of IL-7 and in T cell–sufficient hosts that are
nonetheless devoid of functional T-cell immunity.
Consistent with the idea that foreign antigens induce
such a rapid form of proliferation, congenitally immunodeficient mice raised under germ-free conditions had
only slow homeostatic proliferation, not the rapid T-cell
proliferation that occurred in conventionally raised
immunodeficient mice. Thus, polyclonal proliferation
of naive T cells in T cell–deficient hosts can be driven
predominantly by either self-antigens or foreign antigens, depending on the host’s previous state of T-cell
immunocompetency. The finding in germ-free mice
also indicates that much of the homeostatic proliferation of naive T cells occurs in the absence of any foreign antigens.
Among the various subsets of T cells, memory
CD4 cells are probably the least well understood in terms
of their homeostatic requirements. Recent research indicates that survival of antigen-specific memory CD4 cells
is supported by IL-7, whereas spontaneously generated
polyclonal memory phenotype cells can also use signals
from contact with self-MHC-peptide ligands. One potential problem with studying memory phenotype cells in
normal mice is that these cells appear to be heterogeneous in terms of their homeostatic requirements. Thus,
whereas antigen-specific memory CD4 cells are acutely
dependent on IL-7 for survival, a population of polyclonal memory phenotype cells is independent of IL-7
(and IL-15) for survival and homeostatic proliferation.
The origin and the homeostatic requirements of
these IL-7/IL-15–independent memory phenotype CD4
cells are unclear, but compared with antigen-specific
memory CD4 cells, these cells appear to be at a higher
state of activation, because they undergo a faster rate
of homeostatic proliferation. The factors that support
the generation and maintenance of various populations
of memory phenotype T cells are currently unknown.
Nonetheless, these cells may play a significant role in
influencing the homeostatic behavior of other subsets
of T cells through competition for homeostatic factors.
PUBLICATIONS
Baccala, R., Witherden, D., Gonzalez-Quintial, R., Dummer, W., Surh, C.D.,
Havran, W.L., Theofilopoulos, A.N. γδ T cell homeostasis is controlled by IL-7 and
IL-15 together with subset-specific factors. J. Immunol. 174:4606, 2005.
2005
143
Surh, C.D., Sprent, J. Regulation of mature T cell homeostasis. Semin. Immunol.
17:183, 2005.
Structure-Function Studies of
Innate and Adaptive Immunity
L. Teyton, B. Atteberry, K. Bennett, H. Burt, C. Cantu,
S. Chabot, S.Y. Chang, W. Cheung, S. Freigang, H. Issafras,
C. Li, N. Schrantz, J. Sim, R. Stefanko, 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
ur goal is to understand the molecular switches
that lead to activation of T cells. Assembly of
functional complexes of T-cell receptors (TCRs) 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, Molecular and Integrative Neurosciences
Department, 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 membranes could provide simple switches
essential to T-cell activation. This hypothesis is supported
by our structure determination, in collaboration 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.
O
AUTOIMMUNE DIABETES
We are using MHC multimers to detect antigenspecific T-cell populations in 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 the side effects and complications of general immunosuppression.
L I N K 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
Kieper, W.C., Troy, A., Burghardt, J.T., Ramsey, C., Lee, J.Y., Jiang, H.-Q., Dummer, W.,
Shen, H., Cebra, J.J., Surh, C.D. Recent immune status determines the source of
antigens that drive homeostatic T cell expansion. J. Immunol. 174:3158, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
We are studying lipid binding to CD1 to determine
the factors that govern the presentation of the lipids to
144 IMMUNOLOGY 2005
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 exogenous ligands. In collaboration with A. Bendelac, University of Chicago, we are using RNA interference, genetic techniques, and recombinant biochemistry
to study CD1 within the context of lipid metabolism.
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
Goff, R.D., Gao, Y., Mattner, J., Zhou, D., Yin, N., Cantu, C. III, Teyton, L., Bendelac, A., Savage, P.B. Effects of lipid chain lengths in α-galactosylceramides on
cytokine release by natural killer T cells. J. Am. Chem. Soc. 126:13602, 2004.
Kelker, M.S., Foss, T.R., Peti, W., Teyton, L., Kelly, J.W., Wuthrich, K., Wilson,
I.A. Crystal structure of human triggering receptor expressed on myeloid cells 1
(TREM-1) at 1.47 Å. J. Mol. Biol. 342:1237, 2004.
Malherbe, L., Hausl, C., Teyton, L., McHeyzer-Williams, M.G. Clonal selection of
helper T cells is determined by an affinity threshold with no further skewing of TCR
binding properties. Immunity 21:669, 2004.
Mattner, J., Debord, K.L., Ismail, N., Goff, R.D., Cantu, C. III, Zhou, D., SaintMezard, P., Wang, V., Gao, Y., Yin, N., Hoebe, K., Schneewind, O., Walker, D., Beutler, B., Teyton, L., Savage, P.B., Bendelac, A. Exogenous and endogenous glycolipid
antigens activate NKT cells during microbial infections. Nature 434:525, 2005.
Ranheim, E.A., Tarbell, K.V., Krogsgaard, M., Mallet-Designe, V., Teyton, L.,
McDevitt, H.O., Weissman, I.L. Selection of aberrant class II restricted CD8+
T cells in NOD mice expressing a glutamic acid decarboxylase (GAD)65-specific
T cell receptor transgene. Autoimmunity 37:555, 2004.
Reiser, J.B., Teyton, L., Wilson, I.A. Crystal structure of the Drosophila peptidoglycan
recognition protein (PGRP)-SA at 1.56 Å resolution. J. Mol. Biol. 340:909, 2004.
Robey, I.F., Peterson, M., Horwitz, M.S., Kono, D.H., Stratmann, T., Theofilopoulos, A.N., Sarvetnick, N., Teyton, L., Feeney, A.J. Terminal deoxynucleotidyltransferase deficiency decreases autoimmune disease in diabetes-prone nonobese
diabetic mice and lupus-prone MRL-Faslpr mice. J. Immunol. 172:4624, 2004.
Zhou, D., Mattner, J., Cantu, C. III, Schrantz, N., Yin, N., Gao, Y., Sagiv, Y., Hudspeth, K., Wu, Y.P., Yamashita, T., Teneberg, S., Wang, D., Proia, R.L., Levery,
S.B., Savage, P.B., Teyton, L., Bendelac, A. Lysosomal glycosphingolipid recognition by NKT cells. Science 306:1786, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
Genes and Genetics of Systemic
Autoimmunity and T-Cell
Homeostasis in Autoimmunity
and Cancer
A.N. Theofilopoulos, D.H. Kono, R. Baccala, R. Chintalapati,
R. Gonzalez-Quintial, M.K. Haraldsson, C.A. Louis-Dit-Sully,
K.M. Pollard,* J. Schettini
* Department of Molecular and Experimental Medicine, Scripps Research
ur main interests are identifying predisposing
loci and genes in murine models of systemic
autoimmunity, clarifying the role of type I interferons in systemic lupus erythematosus (SLE), determining why activated/memory phenotype T cells accumulate
in SLE and why cyclin-dependent kinase inhibitors are
increased in these cells, and characterizing factors that
influence acute homeostatic proliferation of T-cell subsets and the relevance of this process in autoimmunity
and cancer.
O
GENETIC BASIS OF SYSTEMIC AUTOIMMUNITY
Susceptibility to SLE is in large part determined by
genetic predisposition. Thus, defining the specific genes
and how certain alterations lead to autoimmunity should
yield new insights into the pathogenesis of SLE and
facilitate the development of innovative approaches to
disease management. Because of the complexity of
defining susceptibility genes in humans, we use both
spontaneous and induced models of SLE in well-characterized inbred mouse strains. Previously, we identified
loci that predispose mice to spontaneous manifestations
of SLE in NZB, NZW, BXSB, MRL-Faslpr, and C57BL/6Faslpr strains and a DBA/2 locus associated with resistance to mercury-induced autoimmunity.
Currently, we are identifying the underlying genes
and their specific roles in autoimmunity for 4 loci:
Lbw2, Lbw5, Lmb3, and Hmr1. Lbw2 is a locus on
chromosome 4 in NZB mice that promotes spontaneous
activation of B cells, production of autoantibodies, glomerulonephritis, and autoimmune hemolytic anemia.
The Lbw2 locus appears to contain at least 3 subloci
that affect different component phenotypes mapped to
this interval. Lbw5 is a recessive locus on chromosome 7 in NZW mice that enhances production of IgG
autoantibodies, glomerulonephritis, and autoimmune
hemolytic anemia. Further mapping of Lbw5 suggests
that this interval contains as least 2 subloci.
IMMUNOLOGY
The dominant MRL Lmb3 is also a locus on chromosome 7, but it occurs at a different, more distal location
than does Lbw5. Lmb3 congenic MRL-Faslpr mice containing an introgressed chromosome 7 fragment of
C57BL/6 have marked reductions in lymphoproliferation, production of autoantibodies, glomerulonephritis,
and early mortality. This locus was recently mapped to
a 0.8 Mb-sized interval, and a likely candidate gene
with a functional mutation has been identified. The
Hmr1 locus on chromosome 1 does not confer resistance to deposition of glomerular immune complexes
in congenic NZB and SJL mice that have the DBA/2
Hmr1 interval, suggesting that epistatic interactions
with other DBA/2 resistance genes are required. In
support of this notion, the reciprocal congenic DBA/2
mice with the NZB Hmr1 region are susceptible to
mercury-induced autoimmunity. We are mapping the
number and location of the various subloci and are
identifying and characterizing possible genes in the
reduced intervals.
TYPE I INTERFERONS IN SLE
Type I interferons (IFN-α/β) are highly pleiotropic
cytokines that affect both innate and adaptive immune
responses. Long-standing observations indicate the
central role of these cytokines in the pathogenesis of
SLE in humans and in animal models. In our studies
of SLE-prone NZB mice that lack the common receptor
for IFN-α/β, we clearly delineated the pathogenic role
of these effector molecules. Compared with mice that
had the receptor, mice that lacked the receptor had
significant decreases in humoral, cellular, and histologic characteristics of SLE and increases in survival.
Several questions remain unanswered, however,
particularly about the mechanisms associated with
endogenous stimuli for production of IFN-α/β. We are
determining the efficacy of a nonviral vector that encodes
the IFN-α/β–binding IFNAR2 chain fused to the Fc
fragment of IgG1 in inhibiting disease when applied
either at the early (prophylactic) stage or the late
(therapeutic) stage of the disease in various models of
spontaneous SLE. The efficacy of this approach will
provide the basis for translating these results to similar
contemplated efforts for treatment of SLE in humans.
We are also interested in differentiating the effects
of IFN-α from those of IFN-β and in defining the postulated central role of plasmacytoid dendritic cells as the
major producers of IFN-α/β in this disease. Most importantly, in collaboration with B. Beutler and K. Hoebe,
Department of Immunology, we are creating congenic
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
145
SLE mice that lack adaptor molecules involved in the
production of IFN-α/β mediated by the Toll-like receptors. We are also concentrating on identifying the exact
nature of the endogenous (self) factors that stimulate
production of IFN-α/β, particularly the roles of apoptotic
materials and immune complexes composed of autoantibodies and particles containing DNA and/or RNA.
CYCLIN-DEPENDENT KINASE INHIBITORS IN
SYSTEMIC AUTOIMMUNITY
In recent studies, we focused on the role of the
cell-cycle inhibitor p21 in normal immune responses
and autoimmunity. The cell cycle, which plays a critical role in determining both the fate and the differentiation of cells, is highly regulated by complexes of
proteins, including cyclin, cyclin-dependent kinases,
and cyclin-dependent kinase inhibitors, that are themselves controlled by external stimuli via a number of
signaling pathways. Previously, in SLE-prone BXSB
mice, we found that high levels of certain cyclin-dependent kinase inhibitors, such as p21, p18, and p27,
are present in activated/memory (CD44 hi) phenotype
CD4+ T cells, a population commonly increased in SLE.
Therefore, we hypothesized that repeated stimulation
of T cells reactive to self-antigens might lead to a state
similar to “replicative senescence,” in which T cells
are no longer cycling but are resistant to apoptosis,
accumulate, and transcribe autoimmune-promoting
proinflammatory cytokines. In support of this notion,
we found that male BXSB mice lacking p21 had a
marked reduction in SLE-like disease associated with
both enhanced apoptosis of T and B lymphocytes and
significant decreases in the number of activated/memory CD4 + T cells. Recently, we created diabetes-susceptible nonobese diabetic mice that lacked the gene
for p21. In sharp contrast to the situation in SLE-prone
mice, p21 deficiency had no effect on the development
and severity of diabetes, indicating that p21 plays different roles in systemic and organ-specific diseases.
Currently, we are addressing the role of p21 in other
SLE-prone strains, immune responses to foreign antigens and viral infection, and the observed reduction in
Fas-mediated apoptosis.
H O M E O S TAT I C T - C E L L P R O L I F E R AT I O N I N
AUTOIMMUNITY AND CANCER
Homeostasis is defined as the ability of a biological system to maintain its internal equilibrium by adjusting critical physiologic properties. Recent studies have
largely defined the factors that control homeostasis of
naive and memory T cells under states in which the
146 IMMUNOLOGY 2005
number of lymphocytes is sufficient or is markedly
reduced (lymphopenia). Of particular relevance to autoimmunity is the phenomenon termed “acute homeostatic
T-cell proliferation,” which signifies proliferation of the
remaining T cells after a lymphopenia-inducing event
(e.g., treatment with cytotoxic drugs, viral infection) to
reestablish a pool with normal numbers of lymphocytes.
Efficient acute homeostatic proliferation appears to be
based on recognition of self-peptide–MHC complexes and
signaling by trophic cytokines, such as IL-7 and IL-15.
We recently hypothesized that such lymphopeniamediated T-cell proliferation may be a contributing factor
to autoimmunity, and we have discussed several examples in the literature in which lymphopenia was paradoxically associated with autoimmune phenomena. Our
recent experiments in SLE-prone mice that lack the gene
for the α-chain of the T-cell receptor and thus lack
T cells, provided evidence that homeostatic proliferation of syngeneic cells in this empty environment can
recapitulate an SLE-like disease. Others have shown that
increased proliferation but inefficient survival of T cells
can lead to lymphopenia and can be a contributing factor in the organ-specific autoimmune disease of nonobese
diabetic mice.
Thus, the perplexing association of lymphopenia
with autoimmunity might be explained on the basis of
compensatory self-mediated homeostatic proliferation
of T cells. Overall, we postulate that in normal mice,
the rare occurrence of lymphopenia and physiologic
proliferation of a polyclonal T-cell population containing few (if any) autoreactive cells will be a physiologic
process without pathologic consequences. Similarly, in
animals that have more autoreactive T cells, a rare
occurrence of homeostatic proliferation of T cells most
likely will be innocuous. In contrast, in animals predisposed to autoimmunity, lymphopenia might contribute to the initiation and/or progression of recurrent
or chronic disease (Fig. 1).
T cells that express γδ T-cell receptors constitute
a considerable fraction of lymphocytes in secondary
lymphoid organs and blood and predominate in the
mucosa and epithelia of various tissues. Considerable
evidence indicates that γδ T cells have important immunologic functions, including antitumor activities, and
may contribute to the pathogenesis of autoimmune diseases. Among subsets of T cells, γδ T cells uniquely have
a tissue distribution based on their antigen receptors, but
what defines the preferential homing and homeostasis of
these cells is unknown. To address this question, we
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
F i g . 1 . Postulated mechanisms for recurrent lymphopenia-induced
expansion of autoreactive T cells and autoimmunity. In animals with
a normal genetic background, the rare occurrence of lymphopenia
leads to homeostatic proliferation (HP) and survival of diverse
T cells (white areas, nonautoreactive; black areas, autoreactive)
without predominance of the few potentially autoreactive clones in
the periphery. Similarly, in animals with a genetic predisposition to
autoimmunity (autoimmune background), which have a higher frequency of potentially autoreactive T cells, the rare occurrence of
homeostatic proliferation might not lead to autoimmunity because
the frequency of the autoreactive cells remains low and other
requirements are absent. By contrast, in animals with a genetic predisposition to autoimmunity in which lymphopenia occurs recurrently
or chronically, proliferation and selection of autoreactive T cells
together with other factors such as adequate antigen presentation
and costimulation might lead to autoimmunity.
studied the resources that control the homeostasis of
γδ T cells in secondary lymphoid organs.
We found that γδ and αβ T cells are controlled by
partially overlapping resources, because lymphopeniainduced acute homeostatic proliferation of γδ T cells
was inhibited by an intact αβ T-cell compartment and
both γδ and αβ T cells were dependent on IL-7 and
IL-15. Significantly, acute homeostatic proliferation of
γδ T cells also required depletion of γδ cells. Thus,
homeostasis of γδ T cells is maintained by trophic cytokines commonly used by other types of lymphoid cells
and by additional, as yet unidentified, γδ-specific ligands.
Efforts to develop effective antitumor immunotherapies are hampered by the difficulty of overcoming tolerance against tumor antigens, which in most instances are
normal gene products that are overexpressed, preferentially expressed, or reexpressed in cancer cells. Because
lymphopenia-induced homeostatic proliferation of T cells
is mediated by recognition of self-peptide–MHC complexes and because the expanded cells acquire some
effector functions, we hypothesized that lymphopenia-
IMMUNOLOGY
induced homeostatic proliferation could be used to break
tolerance against tumor antigens. Our earlier studies with
W. Dummer, Genentech, Inc., South San Francisco, California, A.G. Niethammer and R. Reisfeld, Department of
Immunology, in mouse models of melanoma and colon
carcinoma indicated that availability of tumor antigens
during homeostatic proliferation of T cells indeed leads to
effective antitumor autoimmunity with specificity and
memory. We hypothesize that this effect is mediated by a
reduction in the activation threshold of low-affinity tumorspecific T cells, leading to preferential engagement and
proliferation of the cells in the presence of a high concentration of tumor antigens.
We are further defining the parameters of this
approach, particularly its efficacy in the treatment of
primary and metastatic tumors with different immunologic and histologic characteristics. Our emphasis is
on mechanistic issues, such as the exact process by
which tolerance is broken, modes of antigen presentation (direct vs indirect), the efficacy of refined T-cell
subsets, and the potentiating effects of appropriate
vaccines and cytokines. Overall, we think that because
of its simplicity, this approach will have considerable
application in the treatment of malignant neoplasms in
humans because it relies on conventional lymphopeniainducing cancer therapies, tumor-specific vaccination
at the early phases of lymphopenia, and, optimally,
infusion of autologous lymphocytes.
PUBLICATIONS
Baccala, R., Gonzalez-Quintial, R., Dummer, W., Theofilopoulos, A.N. Tumor
immunity via homeostatic T cell proliferation: mechanistic aspects and clinical perspectives. Springer Semin. Immunopathol. 27:75, 2005
Baccala, R., Kono, D.H., Theofilopoulos, A.N. Interferons as pathogenic effectors
in autoimmunity. Immunol. Rev. 204:9, 2005.
Baccala, R., Theofilopoulos, A.N. The new paradigm of T-cell homeostatic proliferation-induced autoimmunity. Trends Immunol. 26:5, 2005.
Baccala, R., Witherden, D., Gonzalez-Quintial, R., Dummer, W., Surh, C.D.,
Havran, W.L., Theofilopoulos, A.N. γδ T cell homeostasis is controlled by IL-7 and
IL-15 together with subset-specific factors. J. Immunol. 174:4606, 2005.
Haraldsson, M.K., dela Paz, N.G., Kuan, J.G., Gilkeson, G.S., Theofilopoulos,
A.N., Kono, D.H. Autoimmune alterations induced by the New Zealand Black
Lbw2 locus in BWF1 mice. J Immunol. 174:5065, 2005.
Kono, D.H., Theofilopoulos, A.N. Genetics of autoantibody production in mouse
models of lupus. In: Autoantibodies and Autoimmunity. Pollard, K.M. (Ed.). WileyVCH, New York, in press.
Pollard, K.M., Arnush, M., Hultman, P., Kono, D.H. Costimulation requirements of
induced murine systemic autoimmune disease. J. Immunol. 173:5880, 2004.
Pollard, K.M., Hultman, P., Arnush, M., Hildebrand, J.A., Kono, D.H. Immunology
and genetics of xenobiotic-induced autoimmunity. In: From Animal Models to Human
Genetics: Research on the Induction and Pathogenicity of Autoantibodies. Conrad, K.,
et al. (Eds.). Pabst Science Publishers, Lengerich, Germany, 2004, p. 130.
Theofilopoulos, A.N., Baccala, R., Beutler, B., Kono, D.H. Type I interferons (α/β)
in immunity and autoimmunity. Annu. Rev. Immunol. 23:307, 2005.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
147
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 of the 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 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 crosslinking agents to create covalent attachments of the
ligands to the proteins. The proteins are then degraded
chemically to determine the site of attachment.
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 conformational changes in the receptor. Our studies indicate
that pairs of TLRs are associated even in the absence
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148 IMMUNOLOGY 2005
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, drives progression of atherosclerosis. For these
experiments, we are using mouse models of the disease
and mice deficient in individual TLRs.
PUBLICATIONS
Dunzendorfer, S., Lee, H.-K., Soldau, K., Tobias, P.S. TLR4 is the signaling but
not the lipopolysaccharide uptake receptor. J. Immunol. 173:1166, 2004.
Dunzendorfer, S., Lee, H.-K., Tobias, P.S. Flow-dependent regulation of endothelial Tolllike receptor 2 expression through inhibition of SP1 activity. Circ. Res. 95:684, 2004.
Tobias, P., Curtiss, L.K. Paying the price for pathogen protection: Toll receptors in
atherogenesis. J. Lipid Res. 46:404, 2005.
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 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.
I
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
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. Most recently, we contributed to the understanding of innate immunity through
our 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 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 antiinflammatory 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 abnormalities such
as septic shock and acute respiratory distress syndrome
occur. 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 among various members of the Nod family of
proteins and human diseases.
During the past year, we made considerable progress in several different areas. First we established mice
that lack both copies of the gene for Triad3A, an E3
ubiquitin ligase that controls the expression of some
TLRs. We are breeding these mice to begin studies of
IMMUNOLOGY
the cellular phenotype that occurs when the innate
immune system is activated. At the same time, we are
continuing our studies of the interactions between
Triad3A and potential endogenous substrates. Although
it is too early to know the exact consequences of the
deletion of Triad3A, our initial observations suggest that
homozygous Triad 3A–/– mice have lower body weight
and size during the first 8 weeks of life than do their
wild-type littermates.
We continue to study the biological functions of
Nod1 and Nod2. We discovered a signaling pathway
in MCF-7 cells in which Nod1 negatively regulates the
response of this human breast cancer cell line to estrogen and also influences the level of expression of the
estrogen receptor. We found that MCF-7 cells containing Nod1 had greater proliferative responses in tissue
culture than did cells lacking Nod1. Most importantly,
in a xenograft model of tumor growth in mice with severe
combined immunodeficiency, growth of MCF-7 cells
containing Nod1 was greater than growth of cells lacking Nod1. We are working out the molecular details of
the pathway leading from Nod1 to the estrogen receptor.
We are also continuing our studies on the biological
function of Nod2, with a specific emphasis on its role in
the assembly of a macromolecular protein complex that
induces release of the cytokine IL-1 from cells after activation with the Nod2 ligand. Using cells genetically deficient in specific components associated with IL-1 release,
we showed that the Nod2 pathway requires the kinase
receptor interacting protein-2 and an adaptor protein
known as ASC. Currently, we are using cells deficient in
other proteins that may also be implicated in the release
of IL-1. Our long-term goal is to identify the nature of
this activating complex so that strategies can be devised
to block IL-1 release in chronic inflammatory diseases.
Finally, in collaboration with K.D. Janda, Department of Chemistry, we are studying a class of bacterial products known as quorum-sensing factors. These
factors play a crucial role in the adaptation of bacteria
to the host and serve as a means for bacteria to communicate with one another. Quorum-sensing factors
are involved in the induction of virulence factors and
the establishment of biofilms. Thus, this class of bacterial molecules clearly plays a role in pathogenesis of
infectious processes.
Now we are investigating the role in quorum sensing of members of the homoserine lactone family, which
induce inflammation and cell death via their effects on
host cells. Inflammation and cell death induced by homoserine lactones may be important processes in diseases
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
149
such as cystic fibrosis in which the local concentrations
of the quorum-sensing factor can be quite high in the
lung and in which inflammation and cell remodeling
and/or death are unchecked. By understanding how the
quorum-sensing molecules activate host cells, we may
be able to devise new therapeutic strategies for treatment of diseases in which these molecules are involved.
PUBLICATIONS
Fort, M.M., Mozaffarian, A., Stover, A.G., da Silva Correia, J., Johnson, D.A.,
Crane, R.T., Ulevitch, R.J., Persing, D.H., Bielefeldt-Ohmann, H., Probst, P., Jeffery, E., Fling, S.P., Hershberg, R.M. A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease. J. Immunol.
174:6416, 2005.
Ulevitch, R.J. Therapeutics targeting the innate immune system. Nat. Rev. Immunol.
4:512, 2004.
Ulevitch, R.J., Mathison, J.C., da Silva Correia, J. Innate immune responses during infection. Vaccine 22(Suppl. 1):S25, 2004.
Regulation of CD4+ T-Cell
Responses by Accessory Molecules
S. Webb, N.S. Kim, J. Kovarova, L.H. Lopez, J.G. Melton,
R.R. Mendoza
ctivation of CD4+ T cells leads to the development of a heterogeneous collection of functionally distinct effector cells. For several years, we
have focused on how the activities of these various effector cells are regulated. Both cytokines and interactions
between T-cell receptors and peptide-MHC molecules
make important contributions to the effector activity of
CD4+ T cells. We have shown that the particular array
of accessory molecule receptors engaged during priming
of naive CD4+ cells also critically influences the subsequent activities and survival of developing effector cells.
In previous studies with Drosophila cell lines transfected with selected murine MHC molecules and accessory molecule ligands, we discovered an important role
for interactions between lymphocyte function–associated
antigen 1 (LFA-1) and intracellular adhesion molecule
1 (ICAM-1) in the regulation of cytokine production. In
these studies, engaging LFA-1 during priming suppressed
synthesis of IL-4 and IL-10 and enhanced secretion of
IFN-γ and IL-2. Two broad possibilities might explain
how interactions between LFA-1 and ICAM-1 mediate
these effects. One possibility is that LFA-1 regulates
cytokine production by promoting cell adhesion and
thus strengthening the relative degree of signaling via
T-cell receptors and costimulatory receptors. Alternatively,
or additionally, ligation of LFA-1 triggers signaling events
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150 IMMUNOLOGY 2005
that lead to altered activation of transcription factors
and, ultimately, expression of cytokine genes.
To distinguish between these possibilities, we are
generating a panel of LFA-1 constructs with selected
mutations within the cytoplasmic domain of the LFA-1
β-chain to interfere selectively with either adhesion or
putative signal transduction pathways. These constructs
will be expressed in LFA-1–deficient CD4+ T cells during primary peptide-dependent stimulation. By monitoring the effects of these mutations on the subsequent
capacity of LFA-1 to alter cytokine production, we will
begin to define how LFA-1 alters the function of CD4+
T cells.
In other studies, we are using Drosophila cell lines
cotransfected with murine MHC molecules, inducible
costimulator (ICOS) ligand, programmed cell death 1
(PD-1) ligands, and/or B7.1 to examine the costimulatory activity of members of the CD28 family. These
Drosophila cells are used to present peptide antigen to
T-cell receptor transgenic CD4+ cells.
We tested a currently popular hypothesis that ICOS,
in contrast to CD28, preferentially costimulates secondary responses. To our surprise, cells expressing
ICOS ligand did not effectively costimulate proliferative
responses and/or cytokine production by either naive
or primed CD4+ T cells in the absence of B7 expression.
These results raise the possibility that the primary function of ICOS is to amplify CD28-initiated responses by
increasing the effective level of engagement of costimulatory receptors after initial proliferation of the T cells.
This possibility will be tested in future experiments.
PD-1, like cytotoxic T lymphocyte antigen 4, has
been implicated in the negative regulation of T-cell
responses. To study the mechanisms by which PD-1
engagement suppresses the function of CD4+ cells, we
expressed the PD-1 ligands, PD-L1 and PD-L2, in
Drosophila cells expressing class II MHC molecules
with or without B7 and/or ICAM-1. Expression of PD-1
ligands on the Drosophila cells during peptide-mediated
stimulation of naive CD4 + cells did not significantly
inhibit either proliferative responses of or cytokine production by CD4 + cells. In preliminary experiments,
Drosophila antigen-presenting cells expressing PD-1
ligands did induce higher and/or more sustained production of the inhibitory cytokine IL-10 than did antigenpresenting cells lacking PD-1 ligand expression. This
finding raises the possibility that the negative regulation
of T-cell responses by PD-1 may function indirectly via
IL-10 rather than directly via PD-1–mediated signaling.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
We are also studying the influence of selected accessory molecules in generating CD4+ effector cells that
provide help for CD8 + cells in an in vivo model for
type 1 diabetes. These experiments are a collaboration
with L.A. Sherman, Department of Immunology.
Prion Diseases: Insights Into the
Biology of an Infectious Protein
L. Solforosi, M. Schaller, A. Bellon, G. Moroncini,
E. Ollmann Saphire, G. Abalos, J. Cruite, E. Wiseman,
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.
Human consumption of foodstuffs contaminated with
the prions that cause bovine spongiform encephalopathy led to the emergence of a variant of CreutzfeldtJakob disease (vCJD). To date, more than 180 cases
of vCJD have occurred, primarily in the United Kingdom. More recently, transmission of vCJD via blood
products obtained from apparently healthy donors in
whom vCJD later developed has been documented.
These events have reignited concern about the widespread dissemination of prion diseases in humans.
Because no diagnostic test for early-stage prion infections exists, the risks to public health cannot be accurately quantified.
Uniquely, the infectious agent in transmissible
spongiform encephalopathies, the prion, is thought to
be composed largely of PrP Sc, an abnormally shaped
version of the cellular prion protein PrPC, a molecule
of unknown function that is 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 diseaseassociated form, PrP Sc , through a templating-type
mechanism that is poorly understood. Over time, PrPSc
accumulates in the CNS, and its appearance is closely
associated with profound neuropathologic changes.
We have developed antibody reagents that specifically recognize different components of either the normal PrPC or the abnormal PrPSc conformers of the prion
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IMMUNOLOGY
protein. We have used these immunologic reagents
to gain insights into several aspects of prion biology
that remain poorly understood. For example, the mechanisms through which the accumulation of PrPSc within
the CNS leads to the destruction of brain tissues are
undetermined, although the presence of PrP Sc in and
of itself appears to be insufficient to promote damage
in the absence of PrPC.
We hypothesized that PrPC may contribute directly
to the prion-induced neurodegenerative cascade, perhaps through an unknown signaling pathway. To test
this possibility experimentally, we introduced recombinant monoclonal IgG antibodies that recognize PrPC
into the brain in mice. Upon binding to and effectively
cross-linking PrP C on the surface of neuronal cells, the
antibodies rapidly triggered extensive neuronal death
by apoptosis. These findings indicate that PrPC may
be co-opted twice in prion diseases, once as a substrate for conformational conversion into nascent PrPSc
molecules and additionally as a signaling vehicle that
promotes neuronal injury and death, perhaps after
cross-linking by oligomeric forms of PrPSc.
In additional experiments, we are using PrPSc-specific antibodies to map the association between PrPC
and PrP Sc , a key event in the formation of the prion
replicative complex. Elucidating how these different
PrP conformers interact will enhance the prospect of
efficiently inhibiting their association and thereby halting prion replication and disease. Finally, our comprehension of prion disease would be greatly enhanced if
a detailed molecular structure of PrP Sc were solved.
We are using the PrPSc-reactive antibodies in an effort
to cocrystalize the disease-associated form of PrP. In
the longer term, lessons learned in the study of prion
disease most likely will increase our understanding of
other more common neurodegenerative conditions that
are intimately linked to abnormally folded proteins, such
as Alzheimer’s and Parkinson’s diseases.
PUBLICATIONS
Deleault, N.R., Geoghegan, J.C., Nishina, K., Kascsak, R., Williamson, R.A.,
Supattapone, S. Protease-resistant prion protein amplification reconstituted with
partially purified substrates and synthetic polyanions. J. Biol. Chem. 280:26873,
2005.
Yadavalli, R., Guttmann, R.P., Seward, T., Centers, A.P., Williamson, R.A., Telling,
G.C. Calpain-dependent endoproteolytic cleavage of PrPSc modulates scrapie prion
propagation. J. Biol. Chem. 279:21948, 2004.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.
2005
151
Legumain as a Target for
a Genetic Vaccine Against
Breast Cancer
R. Xiang, C. Dolman, C. Liu, D. Markowitz, Y. Luo
n our efforts to develop cancer immunotherapies,
we are using legumain as a target to induce a strong,
cell-mediated immunity against breast cancer cells.
Legumain, an asparaginyl endopeptidase, is a newly
discovered stress protein that is an almost ideal target
for our DNA-based vaccines. First, positional gene
expression profiling of tumor tissues in a search for
genes that are upregulated in tumors and tumor vasculature indicated that legumain is highly expressed in
many murine breast tumor tissues. In contrast, expression of legumain is very low or absent in all normal
tissues from which breast tumors arise. In addition, the
protein is overexpressed by endothelial cells in the breast
tumor vasculature and by tumor-associated macrophages
in the breast tumor microenvironment. It is well known
that tumor-associated macrophages are recruited by
chemokine gradients established by tumor cells into
the tumor stroma, where the macrophages mediate
immunosuppression by making T cells ineffective and
by promoting angiogenesis, leading to increased tumor
cell growth and metastases. Importantly, cell-surface
expression of legumain occurs under stress, such as
tumor growth in vivo, but not in tissue culture.
We obtained the following evidence in our initial
experiments. First, we found that legumain was overexpressed in vivo by most of the solid tumors we tested,
especially on neoplastic cells, neovasculature, and
tumor-associated macrophages, but not by the corresponding cultured tumor cell lines in tissue culture.
Most normal tissues had either no or essentially undetectable levels of legumain expression. Second, we
found that legumain is a stress-responsive protein
induced under certain conditions such as heat shock,
drug treatment, and hypoxia and is associated with
tumor invasion, dissemination of metastases, and tumor
angiogenesis. Third, in a prophylactic setting, the
legumain-based DNA vaccine induced suppression of
both growth of 4T1 primary breast tumors and dissemination of spontaneous pulmonary metastases. Finally,
this antitumor effect could be achieved by suppression
of angiogenesis in the tumor vasculature combined
with tumor-cell killing mediated by cytotoxic T lym-
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152 IMMUNOLOGY 2005
phocytes and/or antibodies. Taken together, the results
indicate that legumain can be used as an effective target for a DNA vaccine against breast cancer. This
approach may lead to the rational design of such vaccines for future clinical application.
Published by TSRI Press®. © Copyright 2005,
The Scripps Research Institute. All rights reserved.