Innate Immune Regulation and Cancer Immunotherapy pot

Innate Immune Regulation and Cancer
Immunotherapy

Rong-Fu Wang
Editor
Innate Immune Regulation
and Cancer Immunotherapy
Editor
Rong-Fu Wang
Baylor College of Medicine
Houston, Texas 77030, USA

ISBN 978-1-4419-9913-9 e-ISBN 978-1-4419-9914-6
DOI 10.1007/978-1-4419-9914-6
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011939215
© Springer Science+Business Media, LLC 2012
All rights reserved. This work may not be translated or copied in whole or in part without the written
permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York,
NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in
connection with any form of information storage and retrieval, electronic adaptation, computer software,
or by similar or dissimilar methodology now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they
are not identifi ed as such, is not to be taken as an expression of opinion as to whether or not they are
subject to proprietary rights.
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)
v
Contents
1 Introduction 1

Rong-Fu Wang
2 The Role of NKT Cells in the Immune Regulation
of Neoplastic Disease 7
Jessica J. O’Konek, Masaki Terabe, and Jay A. Berzofsky
3 g d T Cells in Cancer 23
Lawrence S. Lamb, Jr.
4 Toll-Like Receptors and Their Regulatory Mechanisms 39
Shin-Ichiroh Saitoh
5 Cytoplasmic Sensing of Viral Double-Stranded RNA
and Activation of Innate Immunity by RIG-I-Like Receptors 51
Mitsutoshi Yoneyama and Takashi Fujita
6 Innate Immune Signaling and Negative Regulators in Cancer 61
Helen Y. Wang and Rong-Fu Wang
7 Dendritic Cell Subsets and Immune Regulation 89
Meredith O’Keeffe, Mireille H. Lahoud, Irina Caminschi,
and Li Wu
8 Human Dendritic Cells in Cancer 121
Gregory Lizée and Michel Gilliet
9 Regulatory T Cells in Cancer 147
Tyler J. Curiel
10 Relationship Between Th17 and Regulatory T Cells
in the Tumor Environment 175
Ilona Kryczek, Ke Wu, Ende Zhao, Guobin Wang,
and Weiping Zou
vi
Contents
11 Mechanisms and Control of Regulatory T Cells in Cancer 195
Bin Li and Rong-Fu Wang
12 Myeloid-Derived Suppressor Cells in Cancer 217
Wiaam Badn and Vincenzo Bronte

13 Myeloid-Derived Suppressive Cells and Their Regulatory
Mechanisms in Cancer 231
Ge Ma, Ping-Ying Pan, and Shu-Hsia Chen
14 Cell Surface Co-signaling Molecules in the Control
of Innate and Adaptive Cancer Immunity 251
Stasya Zarling and Lieping Chen
15 Negative Regulators of NF-kB Activation and Type I
Interferon Pathways 267
Caroline Murphy and Luke A.J. O’Neill
16 Role of TGF-b in Immune Suppression and Infl ammation 289
Joanne E. Konkel and WanJun Chen
17 Indoleamine 2,3-Dioxygenase and Tumor-Induced
Immune Suppression 303
David H. Munn
18 Myeloid-Derived Suppressor Cells in Cancer:
Mechanisms and Therapeutic Perspectives 319
Paulo C. Rodríguez and Augusto C. Ochoa
19 Human Tumor Antigens Recognized by T Cells
and Their Implications for Cancer Immunotherapy 335
Ryo Ueda, Tomonori Yaguchi, and Yutaka Kawakami
20 Cancer/Testis Antigens: Potential Targets
for Immunotherapy 347
Otavia L. Caballero and Yao-Tseng Chen
21 Tumor Antigens and Immune Regulation
in Cancer Immunotherapy 371
Rong-Fu Wang and Helen Y. Wang
22 Immunotherapy of Cancer 391
Michael Dougan and Glenn Dranoff
23 Current Progress in Adoptive T-Cell Therapy of Lymphoma 415
Kenneth P. Micklethwaite, Helen E. Heslop, and Malcolm K. Brenner

24 Adoptive Immunotherapy of Melanoma 439
Seth M. Pollack and Cassian Yee
Index 467
vii
Contributors
Jay A. Berzofsky Vaccine Branch, National Cancer Institute, National
Institutes of Health , Bethesda , MD 20892 , USA
Wiaam Badn Istituto Oncologico Veneto , Via Gattamelata 64,
35128 Padova , Italy
Malcolm K. Brenner Center for Cell and Gene Therapy , Baylor College
of Medicine, The Methodist Hospital and Texas Children’s Hospital ,
Houston , TX , USA
Vincenzo Bronte Istituto Oncologico Veneto , Via Gattamelata 64 ,
35128 Padova , Italy
Otavia L. Caballero Ludwig Institute for Cancer Research, New York
Branch at Memorial Sloan-Kettering Cancer Center , New York , NY , USA
Irina Caminschi The Walter and Eliza Hall Institute , 1G Royal Parade,
Parkville , VIC 3052 , Australia
Lieping Chen Department of Oncology and the Sidney Kimmel
Comprehensive Cancer Center , Johns Hopkins University School of Medicine ,
Baltimore , MD , USA
Shu-Hsia Chen Department of Gene and Cell Medicine ,
Mount Sinai School of Medicine , 1425 Madison Avenue, Room 13-02,
New York , NY 10029-6574 , USA
Department of Surgery, Mount Sinai School of Medicine, 1425 Madison
Avenue, Room 13-02, New York, NY 10029-6574, USA
WanJun Chen Mucosal Immunology Section, Oral Infection and Immunity
Branch, National Institute of Dental and Craniofacial Research,
National Institutes of Health , Bethesda , MD 20892 , USA
Yao-Tseng Chen

Department of Pathology and Laboratory Medicine ,
Weill Cornell Medical College , New York , NY , USA
viii
Contributors
Tyler J. Curiel Cancer Therapy and Research Center, University of Texas Health
Science Center , San Antonio , TX 78229 , USA
Glenn Dranoff Department of Medical Oncology and Cancer Vaccine Center,
Dana-Farber Cancer Institute and Department of Medicine , Brigham and Women’s
Hospital and Harvard Medical School , Boston , MA 02115 , USA
Michael Dougan Department of Medical Oncology and Cancer Vaccine Center,
Dana-Farber Cancer Institute and Department of Medicine , Brigham and Women’s
Hospital and Harvard Medical School , Boston , MA 02115 , USA
Takashi Fujita Laboratory of Molecular Genetics, Institute for Virus Research,
and Laboratory of Molecular Cell Biology , Graduate School of Biostudies,
Kyoto University , Kyoto , Japan
Michel Gilliet Department of Dermatology , University Hospital CHUV ,
CH-1011, Lausanne , Switzerland
Helen E. Heslop Center for Cell and Gene Therapy , Baylor College of Medicine ,
The Methodist Hospital and Texas Children’s Hospital, Houston , TX , USA
Yutaka Kawakami Division of Cellular Signaling , Institute for Advanced
Medical Research, Keio University School of Medicine , 35 Shinanomachi
Shinjuku-ku , Tokyo 160-8582 , Japan
Joanne E. Konkel Mucosal Immunology Section , Oral Infection and
Immunity Branch, National Institute of Dental and Craniofacial Research,
National Institutes of Health , Bethesda , MD 20892 , USA
Ilona Kryczek Department of Surgery , University of Michigan , Ann Arbor ,
MI 48109 , USA
Mireille H. Lahoud The Walter and Eliza Hall Institute ,
1G Royal Parade, Parkville , VIC 3052 , Australia
Lawrence S. Lamb, Jr. Department of Medicine, Division of Hematology

and Oncology , University of Alabama Birmingham , Birmingham , AL , USA
Bin Li Key Laboratory of Molecular Virology and Immunology ,
Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences , Shanghai 200025 , P.R. China
Gregory Lizée Department of Melanoma Medical Oncology ,
The University of Texas M. D. Anderson Cancer Center , Houston , TX , USA
Department of Immunology, The University of Texas M.D. Anderson Cancer
Center, Houston, TX, USA
G e M a Department of Gene and Cell Medicine , Mount Sinai School of Medicine ,
1425 Madison Avenue, Room 13-02, New York , NY 10029-6574 , USA
ix
Contributors
Kenneth P. Micklethwaite Center for Cell and Gene Therapy ,
Baylor College of Medicine , The Methodist Hospital and Texas Children’s
Hospital, Houston , TX , USA
David H. Munn Cancer Immunotherapy Program , Room CN-4141,
Augusta , GA 30912 , USA
Caroline Murphy School of Biochemistry and Immunology , Trinity College
Dublin , Dublin , Ireland
Augusto C. Ochoa Stanley S. Scott Cancer Center , Louisiana State University
Health Sciences Center , New Orleans , LA , USA
Department of Pediatrics, Louisiana State University Health Sciences Center,
New Orleans, LA, USA
Meredith O’Keeffe Centre for Immunology, Burnet Institute ,
85 Commercial Road, Melbourne , VIC 3004 , Australia
Jessica J. O’Konek Vaccine Branch, National Cancer Institute ,
National Institutes of Health , Bethesda , MD 20892 , USA
Luke A.J. O’Neill School of Biochemistry and Immunology , Trinity College
Dublin , Dublin , Ireland
Ping-Ying Pan Department of Gene and Cell Medicine , Mount Sinai School

of Medicine , 1425 Madison Avenue, Room 13-02 , New York,
NY 10029-6574 , USA
Seth M. Pollack Fred Hutchinson Cancer Research Center ,
University of Washington , 825 Eastlake Avenue East, G3630, Seattle ,
WA 98109-1023 , USA
Paulo C. Rodríguez Department of Microbiology, Immunology and Parasitology ,
Louisiana State University Health Sciences Center , New Orleans , LA , USA
Stanley S. Scott Cancer Center, Louisiana State University Health Sciences
Center, New Orleans, LA, USA
Shin-ichiroh Saitoh Division of Infectious Genetics , The Institute of Medical
Science, The University of Tokyo , Shirokanedai , Tokyo 108-8639 , Japan
Masaki Terabe Vaccine Branch, National Cancer Institute ,
National Institutes of Health , Bethesda , MD 20892 , USA
Ryo Ueda Division of Cellular Signaling , Institute for Advanced Medical
Research, Keio University School of Medicine , 35 Shinanomachi Shinjuku-ku ,
Tokyo 160-8582 , Japan
Guobin Wang Department of Surgery , University of Michigan , Ann Arbor ,
MI , USA
Helen Y. Wang
Department of Pathology and Immunology and Center for Cell
and Gene Theraphy, Baylor College of Medicine , Houston , TX 77030, USA
x
Contributors
Rong-Fu Wang Department of Pathology and immunology, The Center
for Cell and Gene Therapy , Baylor College of Medicine , Houston ,
TX 77030 , USA
K e W u Department of Surgery, Union Hospital, Tongji Medical College ,
Huazhong University of Science and Technology , Wuhan 430022 , China
L i W u The Walter and Eliza Hall Institute , 1G Royal Parade, Parkville ,
VIC 3052 , Australia

Tomonori Yaguchi Division of Cellular Signaling , Institute for Advanced
Medical Research, Keio University School of Medicine, 35 Shinanomachi
Shinjuku-ku , Tokyo 160-8582 , Japan
Cassian Yee Fred Hutchinson Cancer Research Center , University of
Washington , 825 Eastlake Avenue East, G3630, Seattle , WA 98109-1023 , USA
Mitsutoshi Yoneyama Laboratory of Molecular Genetics,
Institute for Virus Research, and Laboratory of Molecular Cell Biology ,
Graduate School of Biostudies, Kyoto University , Kyoto , Japan
PRESTO, Japan Science and Technology Agency , Saitama , Japan
Stasya Zarling Department of Oncology and the Sidney Kimmel
Comprehensive Cancer Center , Johns Hopkins University School of Medicine ,
Baltimore , MD , USA
Ende Zhao Department of Surgery , University of Michigan , Ann Arbor ,
MI 48109 , USA
Department of Surgery, Union Hospital, Tongji Medical College , Huazhong
University of Science and Technology , Wuhan 430022 , China
Weiping Zou Department of Surgery , University of Michigan , Ann Arbor ,
MI 48109 , USA
1R F. Wang (ed.), Innate Immune Regulation and Cancer Immunotherapy,
DOI 10.1007/978-1-4419-9914-6_1, © Springer Science+Business Media, LLC 2012
1 Brief Historical Background and Recent Progresses
Immune system is composed of innate and adaptive responses and plays critical
roles in cancer development and destruction. A century ago, Paul Ehrlich postulated
that cancer would be quite common in long-lived organisms if not for the protective
effects of immunity. About 50 years later, Burnet and Thomas proposed the concept
of cancer immunosurveillance based on the experimental evidence of immune rec-
ognition of tumor antigens expressed on tumor cells (Dunn et al. 2004 ) . In 1971, the
US Congress created a National Cancer Act – a War on Cancer. Among many tough
questions asked were whether the immune system can be manipulated so that it
recognizes tumor cells as foreign invaders that must be eliminated from the body

and whether viruses play a role in human cancer. In 1980s, Steven Rosenberg and
his colleagues developed adoptive cell therapy (ACT) for the treatment of mela-
noma cancer patients using lymphocyte activated killed (LAK) cells, providing the
fi rst direct evidence that the immune system can be manipulated to achieve thera-
peutic effi cacy of cancer treatment (Rosenberg, 2011 ). In 1990s, many human tumor
antigens such as MAGE and NY-ESO-1 have been identifi ed from melanoma and
many other types of cancer using tumor-reactive T cells, thus setting the stage for
the development of cancer vaccines in the twenty-fi rst century (Wang and Rosenberg,
1999 ). Indeed, the fi rst dendritic cell (DC)-based vaccine was approved in 2010 by
the U.S. Food and Drug Administration (FDA) for the treatment of patients with
prostate cancer.
R F. Wang (*)
Center for Cell and Gene Therapy , Baylor College of Medicine , Houston , TX 77030 , USA
Department of Pathology and Immunology , Baylor College of Medicine ,
Houston , TX 77030 , USA
e-mail:
Chapter 1
Introduction
Rong-Fu Wang
2 R F. Wang
In the last 10 years, a large body of evidence not only supports the concept of
cancer immunosurveillance, but also proposes cancer immunoediting – a dynamic
interaction between the host immune system and cancer cells. Innate immunity is
the fi rst line of host defense against pathogens and transformed tumor cells. Innate
immune cells including NK, NKT, and dgT cells have been shown to play a critical
role in protecting the host against cancer (Dunn et al. 2004 ; Smyth et al. 2001 ) .
Both macrophages and DCs function as major sensors of invading pathogens and
transformed cells via a limited number of germline-encoded pattern recognition
receptors (PRRs), and play an important role in modulating infl ammation and
immune responses (Akira et al. 2006 ) . Adaptive immunity is involved in the elimi-

nation of pathogens and transformed tumor cells in the late phase of host defense
and generates more specifi c immunity and immunological memory. This notion is
further supported by direct experimental evidence, showing that the immune sys-
tem can restrain cancer growth in an equilibrium phase (i.e., expansion of trans-
formed cells is held in check by immunity) (Koebel et al. 2007 ) . Thus, both innate
and adaptive immune response play a key role in eliminating and controlling tumor
growth. However, there is a continuous dynamic battle between immune cells and
tumor cells, which, in some circumstances, favors the growth of the latter. The
failure of the initial immune responses to control infections/tissue injury leads to
chronic infl ammation, which in turn modulates tumor growth. The concept that
links chronic infl ammation and cancer development was proposed long ago. In
1863, Rudolf Virchow fi rst proposed that cancer originates at sites of chronic
infl ammation. Although the mechanisms by which chronic infl ammation directly
contributes to cancer are poorly understood, activation of the innate immune
response (in particular the NF- k B pathway), through Toll-like receptor (TLR)-
mediated recognition of invading pathogens or damaged tissues, serves as a link
between chronic infl ammation and cancer (Clevers 2004 ; Condeelis and Pollard
2006 ; Greten et al. 2004 ; Karin and Greten 2005 ) . Whether infl ammation pro-
motes or suppresses tumor development depends upon many factors, including the
cytokine milieu and the presence or absence of other immune cells (Greten et al.
2004 ; Karin and Greten 2005 ) . For example, IL-1, IL-6, IL-8, and TGF- b released
by immune cells and tumor cells promote angiogenesis, tumor growth, and dif-
ferentiation of Th1, Th17, and Treg cells. Recent studies show that TLR and IRAK
sequence polymorphism is an important risk factor for prostate cancer (Sun et al.
2005 ; Xu et al. 2005 ) . Chronic infl ammation induced by Helicobacter pylori
infection is the leading cause of stomach cancer, while infl ammatory bowel dis-
eases including ulcerative colitis and Crohn’s disease are closely associated with
colon cancer (Coussens and Werb 2002 ; Karin et al. 2006 ) . Similarly, hepatitis B
and C viral infections are the leading factor contributing to liver cancer (Coussens
and Werb 2002 ; Karin et al. 2006 ) . Thus, these studies clearly demonstrate that

pathogens including bacteria and viruses can trigger innate immune responses,
which in turn affect tumor development, through various innate PRRs. Besides
TLRs, NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), AIM2-like
receptors, and C-type lectin-like receptors have been identifi ed and characterized
as major innate immune receptors or sensors for detecting structure-conserved
3
1 Introduction
molecules, so-called pathogen-associated molecular patterns (PAMPs), as well as
endogenous ligands released from damaged cells, termed damage-associated
molecular patterns (DAMPs). Recognition of PAMPs or DAMPs by PRRs triggers
the activation of several key signaling pathways, including NF- k B, type I inter-
feron (IFN), and infl ammasome, leading to the production of infl ammatory cytok-
ines. Because of the importance of these key signaling pathways, their tight
regulation is essential for both innate and adaptive immunities; otherwise, aber-
rant immune responses may occur, leading to severe or even fatal consequences
such as bacterial sepsis, autoimmune, and chronic infl ammatory diseases. In the
last few years, we have witnessed signifi cant and rapid progress being made in the
areas of innate immune signaling and infl ammation-associated cancer develop-
ment. Thus, infl ammation mediated by innate immune cells that are designed to
fi ght pathogen infections and tissue damages and heal wounds can result in their
inadvertent support of multiple hallmark capabilities associated with cancer,
thereby manifesting the now widely appreciated tumor-promoting consequences
of infl ammatory responses.
Although the fi rst DC-based cancer vaccine (Provenge) has been approved by
FDA as a treatment option for prostate cancer patients, it is very expensive and its
clinical effi cacy remains to be improved (extending only 4 months of patient sur-
vival). There are many reasons that could account for relatively ineffectiveness of
current cancer vaccines in general. Among them include immunological escape,
negative regulations or immune suppression in tumor microenvironment, because
all solid tumors are embedded in a stromal environment consisting of immune

cells, such as macrophages and lymphocytes, and non-immune cells such as
endothelium and fi broblasts. Recent studies suggest that CD4
+
regulatory T (Treg)
cells and myeloid-derived suppressor cells at tumor sites potently suppress the
CD4
+
and CD8
+
T-cell responses elicited by vaccination, thus promoting tumor
growth (Wang and Wang, 2007 ). In addition, there are many suppressive cytok-
ines and negative signaling molecules in signaling pathways that dampen strong
immune responses. Blocking negative signaling molecules on immune cell sur-
face by specifi c antibody could be one way to enhance antitumor immune
response. Recent approval of anti-CTLA-4 antibody by FDA as a treatment for
melanoma patient this year is another milestone for T-cell-based immunotherapy
of cancer.
2 Parts of the Book
Part I of the book discusses innate immune signaling and infl ammatory responses to
pathogens and cancer. Chapters 2 and 3 introduce innate lymphocytes, including
NKT and gamma–delta T cells and their roles in innate immune response to cancer.
Chapters 4 and 5 offer an overview of TLR- and RLR-mediated innate immune
signaling and their role in detecting invading pathogens and initiating infl ammatory
response to pathogen infection. Chapter 6 discusses the innate immune signaling
4
R F. Wang
pathways induced by TLRs, RLRs, and NLRs and the control of their immune
responses by negative regulators. Activation of these innate immune receptors
expressed on many cell types by invading pathogens triggers NF- k B, type I inter-
feron, and infl ammasome pathways, leading to production of proinfl ammatory

cytokines. Tight control of these innate immune responses is critical to the mainte-
nance of immune homeostasis. Unchecked immune responses, otherwise, lead to
harmful, even fatal consequence to the host.
Part II of the book introduces the concept of immune regulation and cell- mediated
immune suppression. Chapters 7 and 8 provide overviews of our current under-
standing of human and mouse dendritic cell (DC) subsets and their regulatory
mechanisms, since DC functions as sensors of invading pathogens and professional
antigen-presenting cells to initiate innate and adaptive immune responses. Chapter
9 discusses regulatory T (Treg) cells in cancer and their clinical relevance to cancer
therapy. Chapter 10 offers an overview of Treg cells and IL-17-producing T (Th17)
cells in cancer, while chapter 11 discusses the regulation and function of Treg and
Th17 cells by TLR-mediated signaling. Chapters 12 and 13 introduce and discuss
myeloid-derived suppressor cells and their role in cancer.
Part III of the book introduces the concept of negative regulators and immune
suppressive molecules that may serve as therapeutic targets of cancer immunother-
apy. Chapter 14 provides an overview of co-stimulatory and co-inhibitory molecules
in T cell activation. Some of these molecules such as cytotoxic T-lymphocyte
antigen 4 (CTLA-4), also known as cluster of differentiation 152 and programmed
death receptor 1 (PD-1) have been extensively studied as therapeutic drug targets
for cancer treatment. Chapter 15 discusses the negative regulators of NF- k B and
type I IFN signaling. Chapter 16 offers an overview of TGF- b signaling pathway
and its role in the regulation of Treg and Th17 cells. Chapters 17 and 18 discuss
several key immune suppressive molecules such as the catabolic enzymes indoleam-
ine 2,3-dioxygenase (IDO) and arginase in cancer.
Part IV of the book introduces the concept of cancer antigens, cancer vaccines,
and adoptive T-cell therapy. Chapters 19 – 21 provide a broad overview of our
understanding of antibody- and T-cell-recognized cancer-associated antigens.
Identifi cation of these cancer antigens has set the stage for the development of
effective cancer vaccine therapy. Chapter 22 discusses the current strategies of can-
cer immunotherapy. Chapters 23 and 24 demonstrate the effectiveness of T-cell-

based therapy for the treatment of various types of cancer.
In summary, this book highlights emerging concept and research areas about the
underlying mechanisms of innate immunity, and signaling regulation and immune
suppression and offers novel ideas and strategies to develop therapeutic cancer
drugs by blocking these negative signaling pathways, suppressive cells and
molecules. Major progresses in the understanding of tumor antigens, immune
suppressive molecules, and suppressive cell population have made therapeutic can-
cer vaccines and drugs a reality. Recent approval of DC-based cancer vaccines and
anti-CTLA-4 antibody-based drugs by FDA are two examples in the pipeline of
therapeutic anticancer drugs or vaccines that are being developed.
5
1 Introduction
References
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124:783–801
Clevers H (2004) At the crossroads of infl ammation and cancer. Cell 118:671–674
Condeelis J, Pollard JW (2006) Macrophages: obligate partners for tumor cell migration, invasion,
and metastasis. Cell 124:263–266
Coussens LM, Werb Z (2002) Infl ammation and cancer. Nature 420:860–867
Dunn GP, Old LJ, Schreiber RD (2004) The immunobiology of cancer immunosurveillance and
immunoediting. Immunity 21:137–148
Greten FR, Eckmann L, Greten TF, Park JM, Li ZW, Egan LJ, Kagnoff MF, Karin M (2004)
IKKbeta links infl ammation and tumorigenesis in a mouse model of colitis-associated cancer.
Cell 118:285–296
Karin M, Greten FR (2005) NF-kappaB: linking infl ammation and immunity to cancer develop-
ment and progression. Nat Rev Immunol 5:749–759
Karin M, Lawrence T, Nizet V (2006) Innate immunity gone awry: linking microbial infections to
chronic infl ammation and cancer. Cell 124:823–835
Koebel CM, Vermi W, Swann JB, Zerafa N, Rodig SJ, Old LJ, Smyth MJ, Schreiber RD (2007)
Adaptive immunity maintains occult cancer in an equilibrium state. Nature 450:903–907

Rosenberg SA (2011). Cell transfer immunotherapy for metastatic solid cancer-what clinicians
need to know. Nat Rev Clin Oncol
Smyth MJ, Crowe NY, Godfrey DI (2001) NK cells and NKT cells collaborate in host protection
from methylcholanthrene-induced fi brosarcoma. Int Immunol 13:459–463
Sun J, Wiklund F, Zheng SL, Chang B, Balter K, Li L, Johansson JE, Li G, Adami HO, Liu W et al
(2005) Sequence variants in Toll-like receptor gene cluster (TLR6-TLR1-TLR10) and prostate
cancer risk. J Natl Cancer Inst 97:525–532
Wang RF, Rosenberg SA (1999) Human tumor antigens for cancer vaccine development. Immunol
Rev 170:85–100
Wang HY, Wang RF (2007) Regulatory T cells and cancer. Curr Opin Immunol 19:217–223
Xu J, Lowey J, Wiklund F, Sun J, Lindmark F, Hsu FC, Dimitrov L, Chang B, Turner AR, Liu W
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7R F. Wang (ed.), Innate Immune Regulation and Cancer Immunotherapy,
DOI 10.1007/978-1-4419-9914-6_2, © Springer Science+Business Media, LLC 2012
1 D e fi nition of NKT Cells
Natural killer T (NKT) cells are a subset of T cells that have phenotypic and
functional characteristics of both T cells and NK cells, expressing both a T cell
receptor and NK lineage markers (Godfrey et al. 2004 ) . NKT cells are defi ned by
their ability to recognize lipid antigens presented by the non-classical MHC class Ib
molecule CD1d (Godfrey et al. 2004 ; Bendelac et al. 2007 ; Tupin et al. 2007 ) .
Although NKT cells make up only a small percentage of lymphocytes (1–2% of
mouse spleen and 0.01–2% of human peripheral blood mononuclear cells), they
play very important roles in many aspects of the immune system because they can
regulate many other cell types such as macrophages, dendritic cells (DCs), CD8
+
T,
and NK cells and are uniquely equipped to link innate and adaptive immune
responses (Taniguchi et al. 2003 ; Kronenberg 2005 ; Bendelac et al. 2007 ) .

Upon activation, NKT cells can rapidly release many cytokines, such as IFN- g ,
IL-4, IL-13, and IL-17, and also stimulate other cells to produce cytokines, such as
IL-12 from CD1d-expressing APCs (Matsuda et al. 2003 ; Stetson et al. 2003 ;
Michel et al. 2007 ; Rachitskaya et al. 2008 ) . NKT cells express IFN- g and IL-4
mRNA, even in the absence of TCR stimulation, suggesting that they are poised to
quickly respond once stimulated (Matsuda et al. 2003 ; Stetson et al. 2003 ) . The
balance of cytokines determines which downstream immune cells are activated,
and thus NKT cells help to steer the adaptive immune system in the desired
direction.
J.J. O’Konek • M. Terabe (*) • J. A. Berzofsky
Vaccine Branch, National Cancer Institute , National Institutes of Health ,
Bethesda , MD 20892 , USA
e-mail: ;
Chapter 2
The Role of NKT Cells in the Immune
Regulation of Neoplastic Disease
Jessica J. O’Konek , Masaki Terabe , and Jay A. Berzofsky
8 J.J. O’Konek et al.
1.1 Type I NKT Cells
NKT cells are a heterogeneous population which can be further subdivided into two
groups, type I and type II. Type I NKT cells express an invariant TCR receptor a
chain (V a 14J a 18 in mice and V a 24J a 18 in humans) which pairs with V b 2, 7, and
8.2 in mice and V b 11 in humans (Imai et al. 1986 ; Koseki et al. 1989 ; Porcelli et al.
1993 ; Dellabona et al. 1994 ; Lantz and Bendelac 1994 ; Makino et al. 1995 ; Godfrey
et al. 2004 ) . Type I NKT cells often express other markers such as NK1.1, CD44,
and CD69; however none of these are expressed on all type I NKT cells and cannot
be used to defi ne this population (Chiu et al. 1999 ; Kronenberg 2005 ; McNab et al.
2007 ) . Both CD4
+
and CD4

−
CD8
−
double negative (DN) populations of type I NKT
cells exist in mice, and some express CD8 a a or CD8 a b in humans (Bendelac et al.
1994 ; Gadola et al. 2002 ) . In humans, CD4
+
type I NKT cells were found to express
both Th1 and Th2 cytokines, while DN type I NKT cells mainly expressed Th1
cytokines (Gumperz et al. 2002 ; Lee et al. 2002 ) . Tissue distribution has also been
implicated in the function of type I NKT cells. NKT cells derived from the liver
could stimulate tumor rejection, while those from the thymus or spleen could not
(Crowe et al. 2005 ) . The role of type I NKT cells is often investigated in J a 18
−/−

mice which lack only type I NKT cells. CD1d
−/−
mice are also useful tools since they
lack all NKT cells. There are no known markers specifi c for type II NKT cells, and
a knockout mouse expressing only type I NKT cells and not type II does not exist.
NKT cells are often defi ned by staining with CD1d-tetramers loaded with a -galac-
tosylceramide ( a -GalCer) for type I NKT cells or sulfatide for type II cells (Benlagha
et al. 2000 ; Matsuda et al. 2000 ; Karadimitris et al. 2001 ; Jahng et al. 2004 ) .
Despite being very limited in their TCR b repertoire with an invariant TCR a
chain, type I NKT cells recognize a range of lipid antigens (Brutkiewicz 2006 ;
Behar and Porcelli 2007 ; Tupin et al. 2007 ) . Recently discovered NKT cell recogni-
tion of a variety of microbial lipids from Sphingomonas , Ehrlichia , and Borrelia
organisms suggests that type I NKT cells play a role in host defense (Kinjo et al.
2005 ; Mattner et al. 2005 ; Wu et al. 2005 ; Brutkiewicz 2006 ; Tupin et al. 2007 ) .
Only a few endogenous glycolipid antigens have been discovered to stimulate NKT

cells including phosphatidylinositol, isoglobotrihexosylceramide, and disialogan-
glioside GD3 (Gumperz et al. 2000 ; De Silva et al. 2002 ; Wu et al. 2003 ; Zhou et al.
2004 ) . The most widely investigated antigen for type I NKT cells is a -GalCer, a
glycolipid derived from a marine sponge (Kobayashi et al. 1995 ; Morita et al. 1995 ;
Kawano et al. 1997 ; Taniguchi et al. 2003 ) . Upon stimulation with a -GalCer, type I
NKT cells rapidly release large amounts of both Th1 (IFN- g ) and Th2 (IL-4, IL-13)
cytokines and promote anti-tumor immunity. The mechanism of a -GalCer-medi-
ated tumor protection was shown to require IFN- g and IL-12 (Cui et al. 1997 ; Fuji
et al. 2000 ; Chiodoni et al. 2001 ; Hayakawa et al. 2002 ; Smyth et al. 2002 ) and
involved IFN- g -activated NK cells (Hayakawa et al. 2001 ; Smyth et al. 2001 ; 2002 )
as well as activated CD4
+
and CD8
+
T cells (Nakagawa et al. 2004 ; Osada et al.
2004 ; Hong et al. 2006 ) .
Distinct from conventional T cells, for which different cytokine profi les can be
induced against the same peptide-MHC complex, the structure of antigens seems to
9
2 The Role of NKT Cells in the Immune Regulation of Neoplastic Disease
play a critical role in determining the cytokine profi le induced in type I NKT cells.
Modifi cations of the length and saturation of the lipid chains of a -GalCer can lead
to different binding affi nities to CD1d as well as altered TCR signaling (McCarthy
et al. 2007 ) . Truncation of the lipid chains has been associated with more
Th2-skewed immune responses using glycolipids such as OCH (Miyamoto et al.
2001 ; Oki et al. 2004 ) . More recently, it has been shown that glycolipids modifi ed
to include an aromatic ring in their acyl or sphingosine tail were more potent than
a -GalCer in activating and expanding human NKT cells (Fujio et al. 2006 ; Chang
et al. 2007 ) .
Although the majority of structure function studies are focusing more on altera-

tions of the lipid portion of a -GalCer, the sugar moiety, as well as its linkage to the
ceramide tails, also infl uences the response of the NKT cells, as this is the portion
of the antigen which the TCR interacts with. For example, while a -GalCer is a
potent stimulator of cytokine production, a -glucosylceramide can also stimulate
type I NKT cells, while b -galactosylceramide has been found to downregulate TCR
expression without causing cytokine release or activation of effector cells (Ortaldo
et al. 2004 ) . A subsequent study attributed differences in the activity of theses gly-
colipids to their affi nity for theTCR (Sidobre et al. 2002 ) . A C-glycosidic analog of
a -GalCer induces more IFN- g production and is a more potent inhibitor of tumor
growth (Schmieg et al. 2003 ) . b -linked glycosylceramides have been shown not to
induce signifi cant anti-tumor immune responses, compared with a -linked glycosyl-
ceramides (Ortaldo et al. 2004 ; Parekh et al. 2004 ) . Although IFN- g has been found
to be the key mediator for type I NKT-mediate anti-tumor immunity together with
a -GalCer and other type I NKT agonists (Smyth and Godfrey 2000 ; Berzofsky and
Terabe 2008 ) , we have recently discovered that b -mannosylceramide is just as
potent in eliciting an anti-tumor immune response similar to a -GalCer, despite
failing to induce signifi cant IFN- g production (O’Konek et al. 2011 ).
Recently, nonglycosidic lipid antigens, such as threitolceramide, were found to
be type I NKT cell agonists (Silk et al. 2008 ) . While these lipid/CD1d complexes
were found to have weaker binding affi nities for the TCR compare to a -GalCer/
CD1d, they could stimulate type I NKT cells to promote DC maturation and activa-
tion of antigen-specifi c T cells. Interestingly, the decreased affi nity for TCR resulted
in less activation-induced anergy and decreased lysis of DCs presenting the antigen.
Thus despite limited TCR usage, NKT cells can discriminate a wide variety of
antigen structures to initiate the proper immune response.
As mentioned above, a -GalCer was originally discovered for its anti-tumor prop-
erties, as it promotes type I NKT-dependent tumor rejection in a wide variety of
mouse models (Kawano et al. 1998 ; Fuji et al. 2000 ; Chiodoni et al. 2001 ; Hayakawa
et al. 2001, 2002, 2003 ; Miyagi et al. 2003 ; Nakagawa et al. 2004 ; Osada et al. 2004 ;
Ambrosino et al. 2007 ) . a -GalCer can induce protection against chemical or onco-

gene-driven tumor formation (Hayakawa et al. 2003 ) . Loading tumor cells with
a -GalCer induce antitumor immunity (Chung et al. 2007 ; Shimizu et al. 2007 ) .
Because a -GalCer is such a potent stimulator, it also causes type I NKT cells to
become anergic (Wilson et al. 2003 ; Harada et al. 2004 ; Parekh et al. 2005 ) . Blockade
of the interaction between PD-1 and PD-L during a -GalCer treatment prevented this
10 J.J. O’Konek et al.
anergy, and in mice which lacked PD-1, repeated injection of a -GalCer did not
induce anergy of type I NKT cells (Parekh et al. 2009 ) . Thus PD-1/PD-L blockade
could be a potential therapeutic target to enhance the antitumor effect of a -GalCer,
allowing it to be administered repeatedly.
Even in the absence of exogenous stimulation by a -GalCer, type I NKT cells can
prevent tumor formation (Cui et al. 1997 ) . Mice lacking type I NKT cells are more
susceptible to methylcholanthrene-induced carcinogenesis (Smyth et al. 2000, 2001 ;
Crowe et al. 2002 ; Nishikawa et al. 2003 ) . Both J a 18
−/−
and CD1d
−/−
p53
+/−
mice
exhibit a faster onset of tumorigenesis and decreased survival compared to p53
+/−

mice, suggesting that type I NKT cells can suppress spontaneous tumorigenesis
(Swann et al. 2009 ) .
Although it has been reported that type I NKT cells can kill tumor cells in vitro
(Kawano et al. 1998 ) , the immune response initiated by stimulation of type I NKT
cells relies on activation of other effector mechanisms to ultimately kill tumor cells
(Smyth et al. 2000 ) . Type I NKT cells have been shown to activate NK and CD8
+

T
cells (Carnaud et al. 1999 ; Toura et al. 1999 ; Eberl and MacDonald 2000 ; Smyth
et al. 2002 ; Fujii et al. 2003b ) . NKT cells also promote maturation and production
of IL-12 by DCs through interaction of CD40L on NKT cells with CD40 on DCs,
and a -GalCer can induce DC maturation by mimicking the effect of Toll-like recep-
tor agonists (Fujii et al. 2004 ) . This NKT-mediated anti-tumor response is likely
initiated by the production of IFN- g by activated type I NKT cells, leading to the
recruitment of NK and CD8
+
T cells which directly lead to tumor cell lysis. The
sequential production of IFN- g by NKT cells followed by NK cells recruitment has
been shown to be necessary for tumor protection induced by a -GalCer (Smyth et al.
2002 ) . Additionally, type I NKT cells have been shown to activate B cells, resulting
in increased Ig secretion and generation of better antibody responses (Galli et al.
2003 ) . NKT-mediated cytotoxic activity has also been demonstrated to occur
through several mechanisms including perforin/granzyme, Fas ligand, and TNF-
related apoptosis-inducing ligand (TRAIL) (Kawano et al. 1999 ; Nieda et al. 2001 ;
Gumperz et al. 2002 ) .
A role for type I NKT cells has also been demonstrated in humans. In vitro it was
shown that stimulating human NKT cells with a -GalCer induced NK-mediated
lysis of human tumor cells (Ishihara et al. 2000 ) . Studies have reported a decreased
type I NKT cell number in the blood of patients with advanced cancer (Tahir et al.
2001 ; Giaccone et al. 2002 ; Dhodapkar et al. 2003 ) , and levels of circulating type I
NKT cells inversely correlated with survival in patients with head and neck squamous
cell carcinoma (Molling et al. 2007 ) . It was also reported that type I NKT cells from
cancer patients have decreased capacity to make IFN- g , proliferate, and respond to
a -GalCer when compared to healthy controls (Tahir et al. 2001 ; Yanagisawa et al.
2002 ; Dhodapkar et al. 2003 ; Fujii et al. 2003a ; Crough et al. 2004 ) . In colon
carcinoma, a correlation was observed between the number of type I NKT cells
infi ltrating the tumor and survival (Tachibana et al. 2005 ) . It is also important to

note that in humans expression of V a 24 alone cannot be used to defi ne type I NKT
cells as a population of V a 24-negative cells was found to bind and respond to
a -GalCer, possibly suggesting a new subset of NKT cells (Gadola et al. 2002 ) . Both
in humans and mice, type I NKT cells are being defi ned more commonly by staining
11
2 The Role of NKT Cells in the Immune Regulation of Neoplastic Disease
with CD1d-tetramers loaded with a -GalCer or an analog (Benlagha et al. 2000 ;
Matsuda et al. 2000 ; Karadimitris et al. 2001 ) .
1.2 Type II NKT Cells
In contrast to type I NKT cells, type II NKT cells express a diverse TCR repertoire
(Cardell et al. 1995 ; Godfrey et al. 2004 ) . Type II NKT cells also are CD1d restricted;
however these cells are much less characterized than type I NKT cells, and there are
no good markers to defi ne these cells. Type II NKT cells recognize a distinct set of
lipid antigens from type I NKT cells. While sulfatide is the prototypical antigen for
type II NKT cells (Jahng et al. 2004 ; Zajonc et al. 2005 ) , some type II NKT cell
hybridomas have been reported not to recognize sulfatide (Park et al. 2001 ; Jahng
et al. 2004 ) , suggesting that type II NKT cells may be further subdivided on the
basis of antigen recognition. Recently, it was reported that alterations of the fatty
acid chain of sulfatide alter the degree to which it can stimulate a type II NKT cell
hybridoma (Roy et al. 2008 ; Blomqvist et al. 2009 ) . This suggests that alternating
the structure of sulfatide may infl uence its action, as has been observed with type I
NKT stimulation and modifi cations of the structure of a -GalCer.
Unlike type I NKT cells, which can be characterized by specifi c markers
(V a 14J a 18 TCR, recognition of a -GalCer), type II NKT cells are far less defi ned.
It has been reported that a subset of type II NKT cells may be stained using CD1d-
sulfatide tetramers (Jahng et al. 2004 ) ; however, this has not yet seen as widespread
use as CD1d- a -GalCer tetramers. Further characterization of type II NKT cells will
depend upon future discovery of markers specifi cally expressed on these cells.
Type II NKT cells have been implicated in suppressing immune responses which
result in the development of autoimmune diseases. For example, in the NOD mouse

model of type I diabetes, overexpression of type II NKT cells prevented disease onset
(Duarte et al. 2004 ) , and similarly type II NKT cells have been found to suppress
experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis
(Jahng et al. 2004 ) and concanavalin A-induced hepatitis (Halder et al. 2007 ) . In con-
trast to the protective role of type I NKT cells in tumor models, type II NKT cells have
been shown to suppress anti-tumor immunity and enhance tumor growth (Moodycliffe
et al. 2000 ; Terabe et al. 2000, 2003a ) . For example, it has been demonstrated that
15-12RM fi brosarcoma, 4T1 mammary tumors, CT26-L5 subcutaneous tumors, and
CT26 lung metastases, which grow well in wild-type and J a 18KO mice, are rejected
in CD1d
−/−
mice (Terabe et al. 2005 ) . Blockade of CD1d with monoclonal antibodies
inhibited tumor growth, presumably by inhibiting type II NKT cells (Terabe et al.
2005 ; Teng et al. 2009a, b ) . Stimulation of type II NKT cells with the glycolipid sul-
fatide suppressed immunosurveillance (Ambrosino et al. 2007 ) . In humans, a subset
of type II NKT cells which recognize lysophosphatidylcholine has been identifi ed in
the blood of patients with multiple myeloma (Chang et al. 2008 ) . In human bone mar-
row, type II NKT cells have been shown to suppress autoimmune T cell responses by
releasing Th2 cytokines (Exley et al. 2001 ) . In a similar manner, type II NKT cells
may also suppress anti-tumor immune responses in humans.
12 J.J. O’Konek et al.
1.3 Interaction Between Type I and Type II NKT Cells
A new immunoregulatory axis was discovered in tumor immunity where type I and
type II NKT cells not only have opposite roles but also counterregulate each other
(Ambrosino et al. 2007 ; Terabe and Berzofsky 2007, 2008 ; Ambrosino et al. 2008 ;
Berzofsky and Terabe 2008 ) . When type II NKT cells are activated in vitro with
sulfatide, they are able to suppress the proliferation of activated type I NKT cells
(Ambrosino et al. 2007 ) . This result was verifi ed in vivo, as sulfatide suppressed
a -GalCer induced protection against 15-12RM subcutaneous tumors and CT26
lung metastases (Ambrosino et al. 2007 ) . From these studies, a new immunoregula-

tory axis was defi ned in which the interaction between type I and type II NKT cells
may be analogous to that of Th1 and Th2 cells. Understanding of the interaction
between type I and type II NKT cells is critical, as the success of immunotherapies
may depend on which way the balance of this axis is shifted. One goal of future
anti-tumor therapies should be to enhance the activity of type I NKT cells while
simultaneously blocking type II NKT cells.
1.4 Interaction Between NKT Cells and Other Cell Types
NKT cells have been shown to interact with other immune components (Terabe and
Berzofsky 2008 ) . As described above, type I NKT cells can also induce maturation
of DCs and activation of NK cells.
CD4
+
CD25
+
T regulatory (Treg) cells have been well characterized for their
ability to suppress other cells of the immune system (Sakaguchi 2004 ) . The interac-
tion between NKT cells and Tregs has not been well-characterized; however,
evidence suggests that such crosstalk does exist. In a mouse lung metastasis model,
it was reported that Tregs reduced the number of type I NKT cells in tumor-bearing
mice, resulting in increased tumor burden (Nishikawa et al. 2003 ) . Human Tregs
can suppress proliferation and function of type I NKT cells activated by a -GalCer-
loaded DCs (Azuma et al. 2003 ) . Increased number of Tregs and decreased type I
NKT cells in cancer correlate with worse prognosis or more advanced cancer (Tahir
et al. 2001 ; Dhodapkar et al. 2003 ; Curiel et al. 2004 ; Tachibana et al. 2005 ; Molling
et al. 2007 ) . Interestingly, in the setting of autoimmune disease, type I NKT cells
and Tregs cooperate with one another (Roelofs-Haarhuis et al. 2003 ) . Also in auto-
immune disease, type I NKT cells appeared to increase Treg cell numbers through
IL-2 production (Liu et al. 2005 ; La Cava et al. 2006 ) . Further characterization of
how type I NKT cells as well as type II NKT cells interact with Tregs is needed.
Myeloid-derived suppressor cells (MDSC), which are defi ned as CD11b

+
Gr-1
+

cells, are immature myeloid lineage cells capable of producing arginase, nitric
oxide, and TGF- b to suppress other immune cells (Gabrilovich 2004 ; Bronte and
Zanovello 2005 ) . Accumulation of MDSCs has been well characterized in many
mouse tumor models as well as in human cancer patients (Pak et al. 1995 ; Almand
et al. 2001 ; Schmielau and Finn 2001 ; Gabrilovich 2004 ; Bronte and Zanovello
13
2 The Role of NKT Cells in the Immune Regulation of Neoplastic Disease
2005 ) . Type II NKT cells produce IL-13 which, along with TNF- a , stimulates
MDSCs to produce TGF- b , resulting in the inhibition of CD8
+
T cell-mediated
tumor lysis in multiple mouse tumor models (Terabe et al. 2003a, b ; Fichtner-Feigl
et al. 2005, 2008 ; Renukaradhya et al. 2008 ) . IL-13, which can be made by type II
NKT cells, can also induce arginase expression in MDSCs (Gallina et al. 2006 ) .
MDSCs can in turn suppress the function of type I NKT cells. In a B16 melanoma
model where MDSCs suppress type I NKT cells, a -GalCer is a poor inducer of anti-
tumor immunity; however, if the number of MDSCs was reduced using retinoic
acid, a -GalCer was able to protect against tumor formation (Yanagisawa et al.
2006 ) . It has also been reported in a model of infl uenza that activated type I NKT
cells can reduce the suppressive activity of MDSCs in both mice and humans (De
Santo et al. 2008 ) . Recently, it was reported that type I NKT cells from human
tumors can kill tumor-associated macrophages which are considered to be a subset
of MDSCs (Song et al. 2009 ) . Conversely, IL-13 from type II NKT cells can activate
tumor-associated macrophages (Sinha et al. 2005 ) . These studies support a role for
type I NKT cells in suppressing and type II NKT cells in activating MDSCs.
2 Clinical Trials/Therapeutics

Activation of type I NKT cells using a -GalCer and other glycolipid antigens has
generated much preclinical success in mice, leading to several clinical trials in
humans (reviewed in (Motohashi and Nakayama 2009 ) ). To date, all of the trials
have used a -GalCer to manipulate NKT cells, but preclinical success achieved with
other glycolipids suggests that these may progress into the clinic trials. Phase I
clinical trials have used soluble a -GalCer (Giaccone et al. 2002 ) , a -GalCer-pulsed
autologous DCs (Chang et al. 2005 ; Ishikawa et al. 2005 ; Kunii et al. 2009 ) , or
adoptive transfer of NKT cells expanded ex vivo with a -GalCer (Motohashi et al.
2006 ) in patients with melanoma, glioma, lung, breast, colorectal, liver, kidney,
prostate, and head and neck cancers. These trials have demonstrated that a -GalCer
is well-tolerated with no dose-limiting toxicity (Giaccone et al. 2002 ; Ishikawa
et al. 2005 ) . In some patients, this treatment induced expansion of type I NKT cells
as well as an increase in IFN- g -producing PBMCs and memory CD8
+
T cells, sug-
gesting that this may have the potential to induce an anti-tumor immune response.
However, a -GalCer has had limited success so far in patients. A few possible
explanations have been suggested. The frequency of type I NKT cells is much lower
in humans than in mice (Kronenberg 2005 ) , and as noted above, cancer in advanced
stages often correlates with reduced number of type I NKT cells. Patients in these
trials also had much more advanced disease than the mice in which a -GalCer
showed signifi cant greater therapeutic effect. This therapy may also be hindered by
the anergy induced by a -GalCer, since in mice it has been demonstrated that follow-
ing injection of a -GalCer, type I NKT cells can not be restimulated for at least 1
month (Fujii et al. 2002 ) . The lack of success with a -GalCer in humans compared
with mice also may be due in part to the presence of anti- a -linked sugar antibodies
in humans which the mouse lacks (Galili et al. 1987, 1988 ; Yoshimura et al. 2001 ) .
14 J.J. O’Konek et al.
More success has been observed with the adoptive transfer of a -GalCer-pulsed
DCs. In contrast to the administration of soluble glycolipid, adoptive transfer of DCs

loaded with a -GalCer resulted in prolonged NKT activation in mice without the
induction of anergy (Fujii et al. 2002 ) . Several clinical trials have studied the effects
of injecting monocyte-derived immature DCs loaded with a -GalCer (Nieda et al.
2004 ; Chang et al. 2005 ; Ishikawa et al. 2005 ) . This therapy was also shown to be
well-tolerated and gave more promising results compared with soluble a -GalCer. A
similar trial using mature DCs showed better NKT expansion in vivo, although these
cells displayed diminished ability to secrete IFN- g (Chang et al. 2005 ) . Because many
patients with advanced cancers have defects in NKT cell number or function, an adop-
tive transfer study was carried out in which in vitro expanded NKT cells were admin-
istered to patients with lung cancer (Motohashi et al. 2006 ) . Two out of three patients
who received the higher dose of NKT cells showed increased numbers of IFN- g -
producing cells and had stable disease. A recent Phase I/II study of adoptive transfer
of whole PBMCs cultured with IL-2 and GM-CSF and subsequently pulsed with
a -GalCer for the treatment of non-small cell lung cancer reported that 10 of 17 patients
displayed an increased number of IFN- g producing cells following treatment
(Motohashi et al. 2009 ) . The patients who showed increased IFN- g producing cells
had signifi cantly longer median survival. This suggests that IFN- g production
following a -GalCer administration can be a predictive marker of success and may be
a useful screening tool for selecting patients who may benefi t from this treatment.
Manipulating NKT cells alone may not be suffi cient to eradicate tumors in patients,
and combinatorial approaches may prove to be more successful. For example, a -GalCer
can function as a vaccine adjuvant by promoting the generation of antigen-specifi c
T cells (Gonzalez-Aseguinolaza et al. 2002 ; Silk et al. 2004 ) and overcoming oral
tolerance by inducing the upregulation of costimulatory molecules on dendritic cells
(Chung et al. 2004 ) . Recently, a -GalCer has been shown to be an effective mucosal
adjuvant for inducing antigen-specifi c immune responses following administration of
HIV peptides (Courtney et al. 2009 ) and for inducing protective immunity against
sexually transmitted HSV-2 infection in mice (Lindqvist et al. 2009 ) . Because a -GalCer-
pulsed DCs can stimulate cytokine production without inducing anergy, they may also
be attractive candidates in the adjuvant setting. Combining a -GalCer with monoclo-

nal antibodies against TRAIL and 4-1BB, which induce apoptosis of tumor cells and
activation of T cells, respectively, induced regression and complete rejection of estab-
lished tumors in mice (Teng et al. 2007 ) . Taken together, data from these preclinical
mouse models suggest that clinical approaches combining multiple agents which take
advantage of the interactions of NKT cells with other immune cells may work better
than stimulating NKT cells with a -GalCer alone.
3 Conclusion
NKT cells act as pivotal regulatory as well as effector cells bridging the gap between
the innate and adaptive immune systems. The balance along the immunoregulatory
axis between type I and type II NKT cells may play a key role in many immune