Interleukin 21 in cancer and immunotherapy

Interleukin 21 in cancer and immunotherapy
PH.D. THESIS
Henrik Søndergaard, M.Sc.
2009
Faculty of Science
University of Copenhagen
Denmark
Novo Nordisk A/S
Denmark

Interleukin 21 in cancer and immunotherapy
Ph.D. Thesis 2009 © Henrik Søndergaard
ISBN 978-87-7611-388-4
Printed by SL grafik, Frederiksberg C, Denmark (www.slgrafik.dk)

Contents
Contents ..................................................................................................................... 1
Preface and acknowledgements ................................................................................. 3
Abbreviations ............................................................................................................. 4
Summary .................................................................................................................... 5
Sammendrag (Danish summary) ................................................................................ 7
Chapter 1 – Introduction and objectives .................................................................... 9
Specific objectives in part 1 ................................................................................... 10
Specific objectives in part 2 ................................................................................... 10
Chapter 2 – Background ........................................................................................... 11
Cancer and immunotherapy ................................................................................... 11
The mouse as experimental system ....................................................................... 14
Chapter 3 – Manuscripts ........................................................................................... 17
Paper I: ................................................................................................................. 19
Interleukin 21: roles in immunopathology and cancer therapy (Review)
Paper II: ................................................................................................................ 35
Interleukin 21 therapy increases the density of tumor infiltrating CD8 + T cells and inhibits
syngeneic tumor growth
Paper III: .............................................................................................................. 49
Intratumoral interleukin 21 increases anti-tumor immunity, tumor-infiltrating CD8 + T cell
density and activity, and enlarges draining lymph nodes
Paper IV: ............................................................................................................... 77
Endogenous interleukin 21 restricts CD8 + T cell expansion and is not required for tumor
immunity
Chapter 4 – Discussion ............................................................................................. 91
Chapter 5 – Conclusion ............................................................................................. 99
Chapter 6 – Future perspectives ............................................................................. 101
References ............................................................................................................. 103
Front page illustration depicts IL-21 protein structure and was provided by Kent Bondensgaard, Department of
Protein Structure and Biophysics, Novo Nordisk A/S, Denmark.
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Preface and acknowledgements
The work in this thesis has been funded by an Industrial Ph.D. fellowship from the Danish
Ministry of Science, Technology and Innovation in collaboration with Novo Nordisk A/S (NN).
The experimental work was performed at NN facilities in Måløv, Denmark, in departments of
Cancer Pharmacology, Histology, and Immunopharmacology from September 2006 to
September 2009. During this period, 6 month research was performed in the department of
Cellular Immunology at the Peter MacCallum Cancer Centre, Melbourne, Australia.
I am deeply indebted to my former and present supervisors Michael Kragh and Kresten Skak
who made this project possible and guided me into the world of science; Michael for his
invaluable help to kick-start this project with tremendous dedication, and Kresten for happily
taking over as main supervisor with his endless source of positive energy and exceptional
support throughout this entire project, I particularly enjoyed our many runs full of discussion
until our breaths ran out. I am very thankful to Niels Ødum for accepting the task as university
supervisor, safely guiding me through the university maze, and to Per Thor Straten for his
commitment as co-supervisor and for our many fruitful discussions.
I sincerely acknowledge the collaboration with my great colleagues at NN, especially, Elisabeth
Douglas Galsgaard and Birte Jørgensen for introducing me to immunohistochemistry with their
catching enthusiasm, Klaus Steensgaard Frederiksen for sharing his quantitative PCR
expertise, Peter Thygesen for his great help with pharmacokinetics, Heidi Winther and Ken
Heding who were always ready with helping hands, and to everyone in departments 903 and
479 for creating a great, fun and inspiring work atmosphere.
A deep gratitude goes to Mark J. Smyth for giving me the opportunity to visit his laboratory at
the Peter MacCallum Cancer Centre. I sincerely wish to thank Adam Uldrich for sharing his
brilliant mind and for teaching me all I know about NKT cells and fishing in Port Philip Bay. A
special thanks to Nicole Mclaughlin for her tireless help with experiments, delivered with the
same enthusiasm as when we were mountain biking. John Stagg, thank you for your great
friendship and for teaching me that surfing joyfully substitutes for science. And, thanks heaps
to all the other amazing people in the cancer immunology program who made my stay down
under unforgettable.
Finally, I wish to thank my family and friends for their great mental support and for always
reminding me of what is important, especially Kristina, for your love and support, and for
enduring our long periods of separation.
Måløv, Denmark, September 2009
Henrik Søndergaard
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Abbreviations
αGC Alpha-galactosylceramide
ACT Adoptive cell transfer
Ag Antigen
ADCC Antibody-dependent ceullular
cytotoxicity
AOI Area of interest
BCR B cell receptor
CCL C-C motif ligand
CD Crohn’s disease
CIA Collagen-induced arthritis
CTL CD8 + cytotoxic T cells
CTLA-4 Cytotoxic T lymphocyte antigen 4
CXCL Chemokine CXC motif ligand
DC Dendritic cell
EAE Experimental autoimmune
encephalomyelitis
FasL Fas ligand
γ c
GC
IBD
IBF
iDC
IDO
IFN
IL
Common IL-2 receptor γ chain
Germinal center
Inflammatory Bowel disease
IRF-4-binding protein
Immature dendritic cells
Indoleamine 2,3-dioxygenase
Interferon
Interleukin
IL-21 Interleukin 21
IL-21R Interleukin 21 receptor
I.p. Intraperitoneal
I.t. Intratumoral
LN Lymph node
LPM Lamina propria monocytes
MDSC Myeloid-derived suppressor cells
mAb Monoclonal antibody
mIL-21 Murine IL-21
MCA 3’-methylcholanthrene
MM Metastatic melanoma
MMP Matrix metalloproteinase
MOG Myelin oligodendrocyte
glycoprotein
MS Multiple sclerosis
NK Natural killer cell
NKT Natural killer T cell
NOD Non-obese diabetic
OVA Ovalbumin
PBL Peripheral blood lymphocytes
PBMCs Peripheral blood mononuclear
cells
PD-L1 Programmed death receptor
ligand-1
PLP Proteolipid protein
PTI Post tumour injection
RA Rheumatoid arthritis
RCC Renal cell carcinoma
rDC Regulatory dendritic cells
RIP Rat insulin promoter
S.c. Subcutaneous
SLE Systemic lupus erythematosus
T1D Type 1 diabetes
TCR T cell receptor
TGFβ Transforming growth factor β.
T fh
T h
T h 17
TILs
TRAIL
T regs
UC
WT
Follicular helper T cells
Helper T cells
IL-17-producing helper T cells
Tumor infiltrating lymphocytes
Tumour necrosis factor–related
apoptosis-inducing ligand
Regulatory T cells
Ulcerative colitis
Wild type
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Summary
Interleukin (IL)-21 is a recently discovered cytokine with pleiotropic immunomodulatory effects
and putative anti-tumor activity. This Ph.D. thesis examines the functions of IL-21 protein as
cancer immunotherapy and the role of endogenous IL-21 in tumor immunity in preclinical
mouse models. In Chapter 1, the specific objectives for the experimental work are introduced.
Chapter 2 presents the theoretical background to cancer immunology and immunotherapy,
including specific barriers to immunotherapy and current therapeutic advances. This is followed
by a brief review of the mouse as a model organism for drug testing in cancer and immunology
with specific considerations for IL-21. Chapter 3 contains four original manuscripts; a review
manuscript introduces IL-21 and IL-21 receptor (IL-21R) immunobiology and reviews the
current knowledge concerning IL-21 in cancer therapy and immunopathology, and the
following 3 manuscripts present the experimental work of this thesis:
Paper I:
Søndergaard H. and Skak K. Interleukin 21: roles in immunopathology and
cancer therapy. Tissue Antigens. 2009 Oct. 21, (Epub ahead of print)
Paper II: Søndergaard H., Frederiksen K.S., Thygesen P.,Galsgaard E.D., Skak K.
Kristjansen P.E.G. and Kragh M. Interleukin 21 therapy increases the density of
tumor infiltrating CD8 + T cells and inhibits syngeneic tumor growth.
Cancer Immunol. Immunother. 2007, Sep;56(9):1417-28. Epub. 2007 Feb. 7
Paper III: Søndergaard H., Galsgaard E.D., Bartholomæussen M., Ødum N. and Skak K.
Intratumoral interleukin 21 increases anti-tumor immunity, tumor-infiltrating
CD8 + T cell density and activity, and enlarges draining lymph nodes.
J. Immunother. in press
Paper IV:
Søndergaard H., Coquet J.M., Uldrich A.P., McLaughlin N, Godfrey D.I.,
Sivakumar P.V. Skak K. and Smyth M.J. Endogenous interleukin 21 restricts
CD8 + T cell expansion and is not required for tumor immunity.
J. Immunol. 2009 Dec 1;183(11):7326-36. Epub 2009 Nov 13
Paper II and III focus on the anti-tumor effect of IL-21 protein therapy following
intraperitoneal, subcutaneous and intratumoral administration in two preclinical mouse cancer
models - B16 melanoma and RenCa renal cell carcinoma. Herein, the responsible effector cells
for IL-21 anti-tumor activity are determined and the effects of IL-21 are evaluated on the
density and activity of tumor infiltrating T cells and on tumor draining lymph nodes. Paper IV
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investigates the role of endogenous IL-21 in immunosurveillance, and primary and secondary
tumor immunity using various experimental tumor models in IL-21- and IL-21R-deficient mice
with focus on NK, NKT and CD8 + T cell responses.
The results obtained in Paper II-IV are discussed in chapter 4. Chapter 5 summarizes the main
conclusions obtained in this thesis and chapter 6 outlines the perspectives for future research
concerning IL-21 in cancer immunotherapy. A list of references is given at the end of the
thesis (excluding those in Paper I-IV).
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Sammendrag (Danish summary)
Interleukin (IL)-21 er et fornyeligt opdaget cytokin med omfattende immunmodulerende
effekter og med formodet anti-tumor aktivitet. Denne Ph.d. afhandling undersøger funktionen
af IL-21 protein som cancer immunterapi og rollen af endogent IL-21 i tumorimmunitet ved
hjælp af prækliniske musemodeller. I kapitel 1 introduceres de specifikke mål for det
eksperimentelle arbejde. I kapitel 2 gives en introduktion til cancerimmunologi og immunterapi
med fokus på specifikke barrierer overfor immunterapi og nuværende terapeutiske fremskridt.
Dette efterfølges af et kort overblik over musen som modelorganisme for afprøvning af
lægemidler indenfor cancer og immunologi med særlige betragtninger for IL-21. Kapitel 3
indeholder fire originale manuskripter; en oversigtsartikel der introducerer immunbiologien bag
IL-21 og IL-21 receptoren (IL-21R) samt giver et overblik over den aktuelle viden indenfor IL-
21 som cancerterapi og i immunpatologi, og de efterfølgende 3 manuskripter præsenterer det
eksperimentelle arbejde i denne afhandling:
Paper I:
Søndergaard H. and Skak K. Interleukin 21: roles in immunopathology and
cancer therapy. Tissue Antigens. 2009 Oct. 21, (Epub ahead of print)
Paper II: Søndergaard H., Frederiksen K.S., Thygesen P.,Galsgaard E.D., Skak K.
Kristjansen P.E.G. and Kragh M. Interleukin 21 therapy increases the density of
tumor infiltrating CD8 + T cells and inhibits syngeneic tumor growth.
Cancer Immunol. Immunother. 2007, Sep;56(9):1417-28. Epub. 2007 Feb. 7
Paper III: Søndergaard H., Galsgaard E.D., Bartholomæussen M., Ødum N. and Skak K.
Intratumoral interleukin 21 increases anti-tumor immunity, tumor-infiltrating
CD8 + T cell density and activity, and enlarges draining lymph nodes.
J. Immunother. in press
Paper IV:
Søndergaard H., Coquet J.M., Uldrich A.P., McLaughlin N, Godfrey D.I.,
Sivakumar P.V. Skak K. and Smyth M.J. Endogenous interleukin 21 restricts
CD8 + T cell expansion and is not required for tumor immunity.
J. Immunol. 2009 Dec 1;183(11):7326-36. Epub 2009 Nov 13
Paper II og III fokuserer på anti-tumor effekten af IL-21 proteinterapi givet som
intraperitoneal, subkutan og intratumoral administration i to prækliniske musemodeller for
cancer - B16 melanom and RenCa renalcelle carcinom. Heri bestemmes de celler der er
nødvendige for anti-tumor aktiviteten af IL-21 og effekten af IL-21 evalueres på densiteten og
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aktiviteten af tumor infiltrerende T celler samt på tumor drænende lymfeknuder. Paper IV
undersøger rollen af endogent IL-21 i immunovervågning, samt primær og sekundær
tumorimmunitet, ved hjælp af forskellige eksperimentelle tumormodeller i IL-21- og IL-21Rdeficiente
mus med fokus på NK, NKT and CD8 + T celle responser.
De opnåede resultater i Paper II-IV diskuteres i kapitel 4. Kapitel 5 opsummerer
hovedkonklusionerne der er opnået i denne afhandling og kapitel 6 udstikker perspektiver for
fremtidig forskning omkring IL-21 i cancerimmunterapi. Til slut er givet en liste over referencer
(eksklusive referencerne i Paper I-IV).
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Chapter 1 – Introduction and objectives
Conventional therapy is rarely curative against disseminated cancers, manifesting the need to
explore new treatment strategies. Recognition of the immune system as an intricate player in
cancer development has prompted the concept of immunotherapy, where modulation of the
immune system is proposed as a novel approach to fight cancer. Cytokines are signaling
molecules secreted by the immune system and represent one way to systemically modulate
immune responses with clinical proof of concept for the treatment of cancer (Rosenberg, 2001;
Smyth et al., 2004).
Interleukin (IL)-21 is a recently discovered cytokine produced by CD4 + T cells and NKT cells
(Parrish-Novak et al., 2000; Coquet et al., 2007), and targets a broad range of immune cells
within both the lymphoid and myeloid lineage (Spolski and Leonard, 2008). IL-21 has shown
encouraging anti-tumor activity in various mouse models, but so far most studies have been
performed in models with little clinical relevance, like artificial cytokine secreting tumors,
models immunogenically enhanced with foreign antigens, or by the use of cytokine producing
plasmids (Leonard and Spolski, 2005).
However, it remains unknown if these results are reproducible using recombinant IL-21 protein
therapy in more clinically relevant models. Also, there is limited in vivo data describing the
anti-tumor mechanisms of IL-21, and it remains elusive whether the anti-tumor effect of IL-21
is caused by an increase in the number or function of effector cells, improved homing, better
survival or perhaps other secondary effects. Finally, there is a general lack of knowledge
concerning feasible routes of administration of IL-21 and potential biomarkers of IL-21 antitumor
activity. Altogether, it will be essential to clarify these issues for the understanding and
development of IL-21 as a cancer immunotherapy.
Extensive work in experimental animals have recently associated endogenous IL-21 signaling
with the development of several major autoimmune diseases including, systemic lupus
erythematosus (SLE) (Bubier et al., 2009), type 1 diabetes (T1D) (Spolski et al., 2008;
Sutherland et al., 2009), rheumatoid arthritis (RA) (Young et al., 2007), inflammatory bowel
disease (IBD) (Fina et al., 2008), and possibly multiple sclerosis (MS) (Nurieva et al., 2007).
Together, these data highlight that IL-21 is mainly a proinflammatory cytokine and for this
reason it has been suggested that neutralization of IL-21 could be of potential benefit to
patients suffering from these diseases (Ettinger et al., 2008). However, the role of endogenous
IL-21 during cancer development, growth and metastasis remains unknown and has potential
ramifications for such approaches.
9

Therefore, the objectives of this thesis is divided into two parts; part 1 is the main part (Paper
II and III) and will focus on the evaluation of IL-21 protein as a cancer immunotherapy and
part 2 (Paper IV) will focus on the role of endogenous IL-21 in tumor immunity.
Specific objectives in part 1
Here, two clinically relevant murine cancer models will be established to investigate the
therapeutic anti-tumor activity of IL-21 protein using the murine orthologue protein (mIL-21)
and the following questions will be addressed:
1. Can IL-21 protein therapy given by various routes of administration inhibit established
subcutaneous tumor-growth in the B16 melanoma and RenCa renal cell carcinoma
model?
2. How does IL-21 anti-tumor effect of local administration compare to systemic
administration?
3. What immune cells are responsible for the anti-tumor activity of IL-21?
4. How does IL-21 therapy affect the density and activity tumor infiltrating T cells and the
number and proliferation of T cells in tumor draining lymph nodes?
Specific objectives in part 2
Here, IL-21- and IL-21R-deficient mice will be subjected to various experimental tumors and
the following question will be addressed:
1. How does IL-21 signaling-deficiency affect tumor immunosurveillance, primary tumor
immunity conducted by NK, NKT and CD8 + T cells and secondary CD8 + T cell memory?
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Chapter 2 – Background
Cancer and immunotherapy
Cancer is a leading cause of death world-wide and accounted for a total of 7.4 million deaths in
2004 (~13% of all deaths); a number which is expected to rise rapidly with the increase in
global ageing 1 . Cancer is a generic term for a large group of diseases that can arise from all
nucleated cells in our body. The hallmarks of cancer is a transformation of normal cells by a
series of inherited and acquired genetic mutations, which provide growth and survival
advantages, and eventually generate malignant neoplasms able to invade adjacent tissues and
spread to distant organs (Hanahan and Weinberg, 2000). The spreading of cancer cells known
as metastasis is a defining feature of the disease and is the major cause of death from cancer.
Chemotherapy is still the first line treatment of most disseminated cancers, but despite the
arrival of more than 20 new compounds in the last decade, chemotherapy is only curative in
very few cases (Savage et al., 2009). Daily, patients and physicians are faced with the
shortcomings of these conventional treatments and clearly new approaches are needed.
Cancer immunotherapy is a novel approach aiming to harness our immune system to combat
cancer, and has the potential to specifically target cancer cells with limited systemic toxicity.
Our immune system is a tremendously potent defense system, which protects us from a large
and versatile array of microbial intruders, and the idea of using its inherent strengths to fight
cancer is appealing. In 1970, the concept of cancer immunosurveillance was conceived, which
proposed the existence of immunological mechanisms that eliminate potentially dangerous
mutant cells (Burnet, 1970). Since then, this concept has been substantiated by evidence that
both the innate and adaptive parts of the immune system indeed recognize, shape and partly
inhibit cancer development (van der et al., 1991; Shankaran et al., 2001; Smyth et al., 2001;
Dunn et al., 2002). Still, cancers clearly develop in the presence of a competent immune
system, showing that the immune system alone is not equipped to protect against all cancers.
Immune recognition of transformed cells during early cancer formation is suggested to shape
an emerging lesion by deleting particularly abnormal and immunogenic cell-clones in a process
called immunoediting. Since cancers are genetically instable and consist of a heterogenic
population of cells, this process eventually promotes the outgrowth of immune-escape variants
(Dunn et al., 2002; Dunn et al., 2004).
1 According to the World Health Organization, Fact sheet N°297, February 2009, www.who.int/cancer
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The existence of immunoediting implies a period of equilibrium where immunoediting
outbalances tumor-growth, and evidence of this was recently shown in experimental animals
(Koebel et al., 2007). Whether emerging human cancers occasionally are eliminated during
periods of immunoediting is difficult to show, but clinically detectable tumors that have
escaped show signs of an immunological selection process; loss or downregulation of
molecules in the antigen-processing machinery is a typical finding in human cancers (Cabrera
et al., 1996; Hicklin et al., 1998; Garcia-Lora et al., 2003), and loss of immunogenicity has
been found in trials with specific antigen targeting (Khong and Restifo, 2002; Yee et al., 2002).
Similar shaping and generation of treatment-resistant clones within established tumors is often
the reason that chemotherapy fails to control cancers (Mellor and Callaghan, 2008).
These inherent problems outline the challenge in modern cancer therapy, where inadequate
host responses and poor therapeutic measures result in treatment-refractory tumors that
eventually progress. Because cancer immunotherapy targets the host response, it represents
an entirely new frontline to combine with conventional therapies, and if properly applied, it
might facilitate additional selection pressure to eradicate cancers.
However, in addition to the generation of immune-tolerated variants, cancer cells also exploit a
panel of immunosuppressive mechanisms, generally disabling immune reactivity (Gajewski et
al., 2006; Rabinovich et al., 2007). These include alterations in the antigen presentation
machinery, poor immune cell chemoattraction, lack of activating co-stimulatory signals,
secretion of immunosuppressive factors, activation of negative regulatory pathways, and
specific recruitment of immunosuppressive cell populations. Thus, successful cancer
immunotherapy needs to circumvent these many evasive mechanisms, which are illustrated in
Figure 1.
The defining goal in cancer immunotherapy is to increase the interaction and reactivity
between the immune system and cancers. Tumor infiltrating lymphocytes (TILs) are a common
term for immune-effector cells in the defence against cancer and several reports show that
TILs are beneficial in human cancers. Particularly, the number of CD8 + cytotoxic T cells and an
increased ratio of CD8 + cytotoxic T cells/CD4 + regulatory T cells (T regs ) are independent
prognostic factors for improved overall survival (Clemente et al., 1996; Galon et al., 2006;
Pages et al., 2005; Gao et al., 2007; Naito et al., 1998; Piersma et al., 2007; Sato et al.,
2005; Sharma et al., 2007; Schumacher et al., 2001; Zhang et al., 2003)
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Figure 1. Tumor-intrinsic barriers for successful cancer immunotherapy. Tumor cells exploit several
immunosuppressive mechanisms to evade immune responses. Generally, tumors do not induce significant
inflammation, which is needed for proper immune cell chemotaxis. Secretion of soluble factors from tumor cells or
from resident regulatory cells i.e. natural killer T (NKT) cells, works to maintain immature dendritic cells (iDC), and
promote development and recruitment of regulatory dendritic cells (rDC), regulatory T cells (T regs) and myeloid-derived
suppressor cells (MDSC), which further contribute to the suppressive environment. Increased activity of catabolic
enzymes i.e. indoleamine 2,3-dioxygenase (IDO) increases depletion of essential amino acids required for effector cell
activity. Defects in the antigen presentation machinery lead to low levels of tumor antigen presentation, restricting
immune recognition. Increased expression of negative co-stimulatory receptors such as programmed death receptor
ligand-1 (PD-L1) on tumors and cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD8 + T cells limit effector cell funtions
and expression of apoptosis-inducing ligands by tumors can terminate effector cells. TRAIL, tumour necrosis factor–
related apoptosis-inducing ligand; FasL, Fas ligand; TGFβ, transforming growth factor β. (Drawn with inspiration from
(Gajewski et al., 2006; Rabinovich et al., 2007))
For that reason, many cancer immunotherapeutic approaches focus on boosting the amount
and functionality of TILs (reviewed in Blattman and Greenberg, 2004; Dougan and Dranoff,
2009). The passive infusion of monoclonal antibodies (mAb) is the new paradigm in cancer
therapy; several are approved for clinical use and are either targeting tumor-associated
surface proteins or growth factor receptors overexpressed by tumors. The anti-tumor function
of these antibodies is not completely understood, but includes steric inhibition of target
receptors, complement activation, and activation of cell-mediated cytotoxicity. The next
generation of mAb mainly focuses on re-generating immune co-stimulation or blocking of
regulatory pathways as outlined in figure 1. Tumor-specific vaccines based on attenuated
tumor cells, viral vectors expressing tumor-associated antigens or modified dendritic cells (DC)
have all been explored to boost the pool of tumor reactive cells, but so far only prophylactic
vaccines against virally-induced cancer has been approved. Adoptive cell transfer (ACT) is
another approach that relies on ex vivo expansion of cancer-specific T cells isolated from
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patient TIL populations. While this type of therapy is restricted to highly specialized
institutions, it has shown response rates of ~50% in traditionally unmanageable cancers e.g.
metastatic melanoma, emphasizing the potential of approaches that modulate TILs (Rosenberg
et al., 2008).
Cytokines are secreted proteins able to systemically modulate immune responses. Interleukin
(IL)-2 and interferon (IFN)-α are approved cytokine-therapies for metastatic melanoma (MM)
and advanced renal cell carcinoma (RCC) (Dougan and Dranoff, 2009). IFN-α has important
anti-viral activities and IL-2 is a potent T cell growth factor, and while both therapies show
evidence of immune-mediated anti-tumor activity their mechanism of action remains unclear.
Although these therapies offer moderate response rates, they still represent the only effective
and potentially curative treatment for patients with MM and RCC. However, the side effects of
these therapies are considerable and resemble symptoms of systemic infections with
hypotension, fever and malaise. IL-2 can induce a potentially lethal vascular leak syndrome,
which requires intensive care, and this has limited its general use.
In addition to their therapeutic effects, cytokines are important tools for many of the new
approaches outlined above; cytokines are used as adjuvants boosting anti-tumor vaccines and
are critical for the ex vivo expansion of TILs for ACT.
Clearly, cytokines are attractive candidates for cancer immunotherapy and the exploration of
new cytokine-therapies is warranted. Interleukin (IL)-21 is a novel cytokine related to IL-2,
with profound immunomodulatory effects and putative anti-tumor activity, which is reviewed in
Paper I of this thesis.
The mouse as experimental system
The mouse was used as the model organism in all experiments throughout this thesis due to
its biological similarity, practicality and pedigree in studies of cancer, immunology and IL-21.
Generally, the mouse reflects human immunobiology remarkably well and although the mouse
and human immune systems clearly are not identical, the mouse is the model of choice in
experimental immunology (Mestas and Hughes, 2004).
In IL-21 research, the mouse is almost exclusively used as the experimental animal and for
several reasons. Mouse and human IL-21 and IL-21R show great overall sequence homology
with specific conservation in areas of cytokine-receptor interactions (Parrish-Novak et al.,
2000). Furthermore, the tissue distribution of the IL-21R seems to be analogous between mice
and humans, and although some discrepancies have been described, most of the cellular
responses to IL-21 are similar in mice and humans (Spolski and Leonard, 2008). Thus, it is
reasonable to assume that results from mice using the murine orthologue protein mIL-21 will
be guidelines for what to expect in the human organism.
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The mouse has a long history in models of cancer (Frese and Tuveson, 2007) and for many
reasons. Mice are easy to handle, require low amounts of drug for pharmacologic activity and
are practical for larger group sizes desired for statistical comparisons. The use of
transplantable tumors where cancer cells are injected e.g. subcutaneously or intravenously
gives a very reproducible onset of disease and homogenous disease development, which is
critical for controlled pharmacological drug testing. Measureable endpoints are also a
necessity; subcutaneous injection of tumor cells allows objective monitoring of disease
progression through direct measurement of tumor size and intravenous injection results in
quantifiable lung metastasis development. Furthermore, the existence of numerous mutant
mouse strains with specific immunodeficiencies and the wide use of mice for gene-targeting
are both very helpful tools to clarify the mechanism of action of immunotherapies and also to
determine the contribution of endogenous proteins in disease development and control, which
are both objectives in this thesis.
However, translation of results from mice to humans should always be done with careful
considerations for the different size, metabolism, pharmacokinetics and varying biology that
exist between these species and importantly how well the model depicts the development and
course of the human disease. But generally, the mouse seems to be a suitable animal model to
study the anti-tumor effects of IL-21 and evaluate the role of IL-21-deficiency in tumor
immunity.
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Chapter 3 – Manuscripts
The work in this thesis is covered by 3 original manuscripts preceded by a review manuscript
which will add to the introduction of the thesis. Papers II and III will cover the objectives of
part 1 and Paper IV will cover the objectives of part 2.
Paper I. Interleukin 21: roles in immunopathology and cancer therapy (Review)
This review will introduce the biology of IL-21 and IL-21R, including the intracellular signaling
properties, identified target genes and expression pattern of the IL-21R. The cellular
immunobiology of IL-21 is reviewed describing the cell types that produce IL-21 and the
effects of IL-21 on the major immune cell subsets. Finally, the review covers the most recent
advances of IL-21 in cancer immunotherapy and the emerging role of IL-21 in various
immunopathologies.
Paper II. Interleukin 21 therapy increases the density of tumor infiltrating CD8 + T
cells and inhibits syngeneic tumor growth
This manuscript examines the anti-tumor effect of IL-21 protein by intraperitoneal and
subcutaneous administration in B16 melanomas and RenCa renal cell carcinomas. The antitumor
effect of early and delayed administration of IL-21 is explored along with the
pharmacokinetics and biodistribution of IL-21. The responsible effector cells for the anti-tumor
effect of IL-21 are determined and the effect of IL-21 on the density of tumor infiltrating T
cells is evaluated.
Paper III. Intratumoral interleukin 21 increases anti-tumor immunity, tumorinfiltrating
CD8 + T cell density and activity, and enlarges draining lymph nodes
This manuscript compares the anti-tumor effect of intratumoral and subcutaneous
administration of IL-21 in B16 melanomas and RenCa renal cell carcinomas. The effect of
intratumoral administration of IL-21 is examined on the density and activity of tumor
infiltrating T cells, on local versus systemic tumor-growth, and on the number and proliferation
of T cells in tumor draining lymph nodes.
Paper IV. Endogenous IL-21 restricts CD8 + T cell expansion and is not required for
tumor immunity
This manuscript examines the role of endogenous IL-21 signaling during natural tumor
immunosurveillance, and primary and secondary tumor immunity using IL-21- and IL-21Rdeficient
mice subjected to various experimental tumors controlled by NK, NKT or CD8 + T cells
or with responsiveness to IL-21 therapy.
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During the course of this work, contributions have also been made to two separate original
manuscripts, which are not included in this thesis and will only be referenced in the text:
1. Eriksen, K.W., Søndergaard H, Woetmann A, Krejsgaard T, Skak K, Geisler C, Wasik
M.A., Ødum N., The combination of IL-21 and IFN-α boosts STAT3 activation,
cytotoxicity and experimental tumor therapy. Mol Immunol. 2009 Feb;46(5):812-20.
Epub 2008 Oct 22.
2. Skak K, Søndergaard H, Frederiksen K.S., Ehrnrooth E, In vivo antitumor efficacy of
interleukin-21 in combination with chemotherapeutics. Cytokine. 2009 Dec;48(3):231-
8. Epub 2009 Aug 25.
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Paper I:
Interleukin 21: roles in immunopathology and cancer therapy (Review)
Søndergaard H. and Skak K., Tissue Antigens. 2009 Oct. 21, (Epub ahead of print)
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Tissue Antigens ISSN 0001-2815
R E V I E W A R T I C L E
IL-21: roles in immunopathology and cancer therapy
H. Søndergaard & K. Skak
Immunopharmacology, Novo Nordisk A/S, Måløv, Denmark
Key words
autoimmunity; cancer; cytokine;
interleukin-21
Correspondence
Henrik Søndergaard
Novo Nordisk A/S
Department of Immunopharmacology
Novo Nordisk Park F6.2.30
DK-2760 Måløv
Denmark
Tel: +45 4443 1376
Fax: +45 4443 4537
e-mail: hris@novonordisk.com
Received 21 August 2009;
accepted 21 August 2009
doi: 10.1111/j.1399-0039.2009.01382.x
Abstract
Cytokines are secreted signalling molecules with decisive effects on haematopoiesis,
innate and adaptive immunity, and immunopathology. Interleukin (IL)-21 is a novel
cytokine produced by activated CD4 + T cells and natural killer T (NKT) cells. IL-21
is part of a family of cytokines which include IL-2, -4, -7, -9 and -15 that all
share the common IL-2 receptor γ chain (γ c ) in their individual receptor complexes.
IL-21 receptor (IL-21R) is widely expressed on both myeloid and lymphoid cell
lineages and IL-21 actions include co-stimulation of B cell differentiation and
immunoglobulin (Ig) production, co-mitogen of T cells, and stimulation of NK and
CD8 + T cell cytotoxic function. Initially, IL-21 was recognized for its anti-tumour
effects in several preclinical tumour models, warranting its currently ongoing clinical
development as a cancer immunotherapeutic. More recently, IL-21 has been associated
with the development of a panel of autoimmune and inflammatory diseases, where
neutralization of IL-21 has been suggested as a potential new therapy. In this review,
we will cover the latest discoveries of IL-21 as a cancer therapy and its implications
in immunopathologies.
Introduction
Cytokines are secreted polypeptides used primarily by the
immune system for intercellular communication. Cytokines are
vital in all aspects of immunology from early haematopoiesis
to the generation, maintenance and contraction of both
innate and adaptive immune responses. Therefore, the timely
introduction of cytokines or blockade of their signalling
pathway can have decisive effects on immune processes.
Historically, identification of novel cytokines, and understanding
of their production and function have been fundamental
for the development of new clinical strategies. The
use of recombinant cytokines have pioneered in the field of
cancer immunotherapy where interleukin (IL)-2 and interferon
(IFN)-α have been used for more than two decades
and still represent effective treatments for certain cancers, e.g.
melanoma and renal cell carcinoma (RCC). The blockade of
endogenous cytokines and their signalling pathways represent
novel approaches in the fight against many autoimmune diseases;
particularly the neutralization of tumour necrosis factor
(TNF)-α and its signalling have revolutionized the treatment
of rheumatoid arthritis.
The discovery of IL-21 adds to a still growing panel of
human cytokines, and this cytokine has not only shown potential
as cancer therapy, but also as an attractive target for
several autoimmune diseases. Here, we will briefly review
the immunobiology of IL-21 and cover the advances of this
interesting cytokine in cancer therapy and its emerging role
in autoimmune and inflammatory diseases.
IL-21 and IL-21 receptor biology, signalling
and expression
In 2000, a new type 1 cytokine receptor was discovered and
denoted novel orphan interleukin receptor (NILR) (1). Soon
after, a functional cloning approach identified a four-helixbundle
cytokine with structural relation to IL-2, IL-4 and
IL-15 as the natural ligand to NILR, naming the new entities
IL-21 and IL-21 receptor (IL-21R) (2). The IL-21R gene
is located directly downstream of IL-4R-α on human chromosome
16, and IL-21R has an amino acid sequence with
greatest homology to the IL-2 receptor beta-chain (IL-2R-β).
The IL-21 gene is located on human chromosome 4q26-q27
approximately 180 kb from the IL-2 gene and the mature
IL-21 polypeptide consists of 131 amino acid residues most
similar to IL-15 (2).
Human and murine IL-21 and IL-21R show approximately
60% overall amino acid sequence homology with significant
conservation in regions of cytokine–receptor interaction (2),
and to a large extent the function of IL-21 has been shown to
be similar in mouse and human, although several discrepancies
have also been described.
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IL-21 in cancer and immunopathology
H. Søndergaard & K. Skak
Figure 1 The common gamma chain (γ c) cytokine family and interleukin (IL)-21 intracellular signalling. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 all share
the common IL-2 receptor (IL-2R)γ c in their individual receptor complexes. IL-21 signals through its unique IL-21 receptor (IL-21R) in a heterodimeric
complex with γ c. Upon ligand interaction, the IL-21R/γ c complex recruits and phosphorylates janus kinases (JAK)1 and JAK3, which downstream
activates signal transducers and activators of transcription (STAT). IL-21 primarily activates STAT3, but also STAT1 and more transiently STAT5a and
5b. The phosphatidylinositol-3-kinase (PI-3K)-AKT and mitogen-activated protein kinase (MAPK) pathways are also involved in IL-21 signalling. Target
genes positively regulated by IL-21 are indicated.
The IL-21R signals as a heterodimeric complex with the
common IL-2 receptor gamma chain (γ c , CD132) (3), making
IL-21 the newest member of the γ c cytokine family, which
also includes IL-2, IL-4, IL-7, IL-9 and IL-15 (Figure 1).
Like other type 1 cytokine receptors, the IL-21R/γ c complex
is a receptor tyrosine kinase, which upon ligand interaction
recruits and phosphorylates janus kinases (JAK) that
subsequently activates signal transducers and activators of
transcription (STAT). Similar to its other family members
IL-21R recruits and activates JAK1 and JAK3 (3). Downstream,
IL-21 signalling primarily activates STAT3, but also
STAT1 and more transiently STAT5a and STAT5b (3, 4).
The phosphatidylinositol-3-kinase (PI-3K)-AKT and mitogenactivated
protein kinase (MAPK) pathways have also been
suggested to transmit IL-21 signals (4). The target genes for
IL-21 signalling still remains to be fully elucidated, but target
genes identified so far include cyclin A/B/E, granzyme
A/B, IFN -γ, CXCR3, CXCR6, Bcl-3, JAK3, IL-21 and IL-
21R (5–7), indicating that IL-21 plays a role in cell cycle
progression, cellular activation, trafficking, cell survival and
positively regulates its own expression (Figure 1).
IL-21 production is restricted to activated CD4 + T cells (2),
and activated natural killer T (NKT) cells (8), whereas
the IL-21R is much more widely expressed, including
B cells, T cells, NK cells, NKT cells, dendritic cells (DC),
macrophages, keratinocytes and intestinal fibroblasts (9, 10),
indicating a broad range of actions of IL-21.
Studies in IL-21R-deficient mice show that IL-21 is not critical
for normal haematopoiesis (11, 12). In the T cell lineage,
IL-21R is not expressed until thymocytes differentiate into
CD4 and CD8 double positive cells. Low levels of IL-21R are
found on mature CD4 + and CD8 + T cells which increase in
response to T cell receptor (TCR) stimulation (13). In contrast,
NKT cells express IL-21R ex vivo without prior activation. In
the B cell lineage the earliest detection of IL-21R is on pre-
B cells, and mature follicular B cells express higher levels
compared with T cells, and this is further increased upon B
cell activation. Mature marginal zone B cells have lower IL-
21R expression compared with follicular B cells, and plasma
cells express very low IL-21R levels (13, 14). Overall, IL-21R
shows the highest expression on activated lymphocytes, indicating
that IL-21 mainly acts as a co-stimulant of activated
lymphocytes.
IL-21 in immunobiology
Humans deficient of the γ c receptor suffer from X-linked
severe combined immunodeficiency (XSCID) syndrome, a
condition where T cells and NK cells are completely absent
and B cells are present but functionally impaired. This shows
the sum of actions and impact that the γ c -dependent cytokines
have on normal immunobiology. IL-21 is no exception and its
pleiotropic effects correspond to the cellular distribution of its
receptor. These are summarized in Figure 2.
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H. Søndergaard & K. Skak IL-21 in cancer and immunopathology
Figure 2 The major actions of IL-21. IL-21 is produced by CD4 + T cells and natural killer (NK) T cells in response to T cell receptor (TCR) activation,
and modulates a broad range of both myeloid and lymphoid immune cells. The effects of IL-21 are generally context dependent and various forms of
co-stimulation determine the cellular response: B cells proliferate and differentiate in response to B cell receptor (BCR) and CD40 co-stimulation, but
undergo apoptosis without; CD8 + T cells primarily expand and increase their cytotoxicity together with IL-15 or TCR co-stimulation; resting NK cells
express very little IL-21R, but in concert with IL-2 or IL-15, IL-21 drives terminal NK cell differentiation. IL-21 shows autocrine stimulation of its own
production, activation of NKT cells and regulation of CD4 + T cell differentiation and proliferation. IL-21 does not directly modulate regulatory T cells
(T regs), but it has been suggested to reduce T reg differentiation. Dendritic cells (DC) remain immature in response to IL-21, whereas macrophages
become activated.
B cells
B cells express the highest levels of IL-21R, making B cells
prime responders to IL-21. Initially, IL-21 was found to
augment anti-CD40-induced B cell proliferation but inhibit
proliferation induced by anti-IgM and IL-4 (2). IL-21 also
inhibited B cell proliferation and induced apoptosis when
B cells were activated via innate toll-like receptors (TLR)
recognizing the bacterial component lipopolysaccaride (LPS)
or viral DNA cytosine-guanine motifs (CpG) (13). Further
studies have clarified that IL-21 mainly inhibits B cell responses
and induces apoptosis of resting B cells in the absence of
proper co-stimulation, whereas cross-linking of both the B cell
receptor (BCR) and CD40 induces extensive proliferation and
differentiation into immunoglobulin (Ig) producing plasma
cells with enhanced IgG production (15).
IL-21R knockout mice had reduced serum levels of IgG,
but increased levels of IgE (12), and IL-21 given at the time
of immunization reduces switching to IgE, but not IgG by specific
inhibition of germline C(ε) transcription in B cells (16).
However, the regulation of Ig class switching by IL-21 is
also context dependent, because IL-21 inhibits IgE switching
induced by IL-4 and PHA stimulation, whereas it promotes
IgE switching induced by IL-4 and anti-CD40 stimulation
(17).
IL-21 co-stimulation is a strong inducer of B lymphocyteinduced
maturation protein-1 (BLIMP-1), a transcriptional
master switch controlling the terminal differentiation of
B cells into plasma cells, and IL-21 can directly induce plasma
cell differentiation of anti-IgM-stimulated B cells. In addition,
IL-21 induces Bcl-6, which has been hypothesized to be
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IL-21 in cancer and immunopathology
H. Søndergaard & K. Skak
involved in the differentiation of germinal center (GC) B cells
into memory B cells (15), suggesting a more complex role of
IL-21 in B cell differentiation.
Taken together, IL-21 is a critical regulator of B cell
responses; B cells faced with IL-21 in the context of antigenspecific
BCR stimulation and T cell co-stimulation will
undergo class switch recombination and differentiate into antibody
producing plasma cells. In contrast, B cells encountering
IL-21 during unspecific TLR stimulation or without proper
T cell help will undergo apoptosis.
CD4 + T cells
CD4 + T cells stimulated via their TCR are main producers of
IL-21, and TCR stimulation increases CD4 + T cell expression
of IL-21R, giving IL-21 an autocrine role in CD4 + T cell
responses.
IL-21 is not a classical helper T cell (T h )1 or T h 2 cytokine,
but can be produced by CD4 + T cells during both highly T h 1
and T h 2 skewed immune responses in vivo (18). CD4 + follicular
helper T cells (T fh ), identified by their expression of
the chemokine receptor CXCR5, express high levels of IL-
21 required for T fh cell development, cognate B cell help
and GC formation (19). The recently characterized IL-17-
producing CD4 + helper T cells (T h 17) also produce IL-21
and are distinct from the classical T h 1 and T h 2 cells. T h 17
cells are involved in the clearance of certain pathogens and
in autoimmune pathogenesis (20). Regulatory CD4 + T cells
(T regs ), known for their immunosuppressive effects and characterized
by high expression of IL-2Rα (CD25) and the transcription
factor forkhead box P3 (FoxP3), have not yet shown
evidence of IL-21 production.
In response to anti-CD3/CD28 stimulation, IL-21 potently
co-stimulates CD4 + T cell proliferation and IFN-γ production,
which counteracts T reg suppression (21, 22), but IL-21
has not shown any direct effects on T regs (21, 23). Although,
during CD4 + T cell differentiation IL-21 has been suggested
to inhibit FoxP3 expression and in turn increase T h 17 cell
development (24). IL-21 can drive the differentiation of T h 17
cells in concert with transforming growth factor (TGF)β, but
in the presence of other proinflammatory cytokines such as
IL-6, IL-21 is dispensable for T h 17 differentiation and rather
serves as an auto-amplification loop for T h 17 cell expansion
(25). In addition, IL-21 regulates its own production in
an autocrine fashion through STAT3 activation (6).
Overall, IL-21 is produced by several different CD4 + T cell
subtypes, it is essential in T fh cell development and GC
formation, and serves as an autocrine amplification loop for
its own production and for CD4 + T cell proliferation and
survival.
CD8 + T cells
In addition to B cells, CD8 + T cells are the primary responders
to IL-21. CD8 + T cells also increase their expression
of the IL-21R in response to TCR stimulation, showing that
IL-21 primarily affects activated CD8 + T cells. Alone, IL-21
does not induce significant proliferation of CD8 + T cells,
but in response to antigen-independent stimulation IL-21 costimulates
proliferation and expansion together with IL-7 or
IL-15 of both naïve and activated CD8 + T cells (7, 26). IL-
21 increases IFN-γ and IL-2 production and sustains the
expression of CD62L and CD28 on IL-15-stimulated CD8 +
T cells. CD28 is an important co-receptor engaged by DC
ligands during TCR stimulation, which is lost on senescent
T cells (26).
IL-21R-deficient mice showed reduced antigen-specific
CD8 + T cell expansion and cytotoxicity compared with WT in
response to viral stimulation (7), highlighting a role for IL-21
also in antigen-specific CD8 + T cell expansion and function.
Similarly, in vitro expansion of antigen-specific CD8 +
T cells in blood from melanoma patients was substantially
increased by IL-21 in concert with autologous DCs pulsed
with a melanoma-specific antigen (27). This effect was caused
by increased proliferation and survival, and could not be
mimicked by IL-2, IL-7 or IL-15. Consistent with antigenindependent
stimulation, IL-21-co-stimulated antigen-specific
CD8 + T cells showed high CD28 expression, increased IL-
2 production, high-avidity TCRs and increased cytotoxic
activity.
Taken together, IL-21 co-stimulates antigen-dependent and
-independent proliferation, expansion, survival, and cytotoxicity
of CD8 + T cells. Furthermore, IL-21 maintains CD8 + T
cell expression of CD28 and increases their IFN-γ and IL-2
production, creating a more robust and independent CD8 + T
cell response.
NK cells
Mature resting NK cells show very low expression of IL-
21R, but IL-21R expression is up-regulated on both mature
NK cells and on NK cell precursors upon activation. IL-21
enhances NK cell differentiation from bone-marrow-derived
precursors (2), but IL-21R knockout mice have normal development
and activity of mature NK cells (11), showing that
IL-21 is redundant for normal NK cell development and
maturation. This is because NK precursor cells do not
express IL-21R, but IL-15, which is critical for normal
NK cell development, induces IL-21R expression (28) and
subsequently IL-21 can accelerate the NK cell maturation
process (29).
Stimulation of NK cells with viral particles, IL-2 or IL-15
greatly enhances responsiveness to IL-21, which then induces
a large granular phenotype with increased cytolytic activity,
IFN-γ production and perforin expression (11, 30). However,
despite the co-stimulation of NK cell maturation and function,
IL-21 inhibits proliferation of NK cells induced by IL-2 and
IL-15 and increases NK cell apoptosis (11, 30).
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H. Søndergaard & K. Skak IL-21 in cancer and immunopathology
NKT cells
NKT cells express inhibitory and activating NK cell receptors
together with a restricted repertoire of TCRs. In contrast
to conventional T cells, the NKT cell TCR recognizes
lipid antigens such as the agonist alpha-galactosylceramide (α-
GC), presented by the MHC-like molecule CD1d expressed
on DCs. Resting NKT cells express the IL-21R and produce
IL-21 in response to anti-CD3 or α-GC stimulation (8). Following
anti-CD3 stimulation, the level of IL-21 production
is even higher in NKT cells compared with splenic CD4 +
T cells, suggesting that NKT cells may represent an important
source of IL-21. NKT cells also respond to IL-21 stimulation;
IL-21 increases survival and proliferation of NKT cells and
enhances α-GC-stimulated proliferation and expansion. Furthermore,
IL-21 co-stimulation of NKT cells increases their
granular morphology, granzyme B expression and cytokine
production (8). These data suggest that NKT cells represent an
important source of IL-21 and that IL-21 generally increases
NKT cell activation.
Dendritic cells
IL-21 has mainly shown inhibitory effects on DCs. IL-21
maintains bone-marrow-derived DCs in a more immature
state characterized by increased phagocytotic activity and
decreased antigen presentation, thereby limiting the activation
of antigen-specific T cell responses (31, 32). This indicates a
potential role for IL-21 also in the restriction or termination
of immune responses. However, DCs pretreated with IL-
21 and α-GC increases stimulation of NKT cell IFN-γ
production (33), indicating that the role of IL-21 on DCs is
more complex and needs further investigation.
Macrophages
IL-21R is expressed on both mouse and human macrophages
and in contrast to DCs, there is limited evidence that IL-21
has proinflammatory effects on macrophages. IL-21 signalling
is not required for macrophage development, but increases
macrophage production of the neutrophil chemoattractant
CXCL8/IL-8, increases phagocytosis and protease activity,
and also the ability to stimulate antigen-specific CD4 + T cell
proliferation (34, 35).
IL-21 as cancer therapy
Preclinical data
The stimulatory effects of IL-21 on NK cells and CD8 +
T cells suggest that IL-21 may possess anti-tumour activity.
In 2003, the first evidence of IL-21 anti-tumour activity
was shown: murine colon carcinoma cells transduced with
the IL-21 gene were completely rejected in syngeneic mice.
Rejection was dependent on both NK and T cells and with
concomitant immunity to parental cell rechallenge (36). Since
then, several groups have studied IL-21 anti-tumour effects
by this approach, using genetically modified tumour cells
from both mouse and humans (see Table 1). In general, these
studies show very potent anti-tumour activity of IL-21 with
reports of complete tumour rejection in most studies. In these
studies, NK cells and CD8 + T cells were responsible for the
anti-tumour activity, with involvement of both IFN-γ and
perforin, but with no role for CD4 + T cells (see Table 1).
Although these data reflect the potential of IL-21 therapy and
point out possible effector mechanisms of IL-21, tumour cells
secreting IL-21 are not clinically translatable.
A more relevant approach came with therapeutic administration
of IL-21-expressing plasmids. Using this approach,
Wang et al. showed significant anti-tumour activity against
B16 melanomas and MCA205 fibrosarcomas mediated by NK
cells, with a minor role for CD8 + T cells but not CD4 +
T cells (39). Brady et al. showed that IL-21-expressing plasmids
injected at the time of tumour cell inoculation mediated
NK cell- and perforin-dependent reduction in lung and
liver metastasis, whereas IFN-γ, Fas ligand or TNF-related
apoptosis-inducing ligand (TRAIL) did not contribute (30).
Nakano et al. showed significant tumour growth inhibition of
subcutaneous head and neck squamous cell carcinomas using
IL-21 plasmids, dependent on T cells, NK cells and with generation
of tumour-specific Ig (42). Together, these data show
that IL-21 administration by a more therapeutic approach has
a similar mechanism of anti-tumour activity even adding a
possible role for B cells, but perhaps with less dramatic antitumour
effects compared with the IL-21-secreting tumours.
Moroz et al. were the first to use recombinant IL-21 protein
and showed significant tumour growth inhibition and
prolonged survival in an ovalbumin (OVA)-expressing lymphoma
model when IL-21 was injected intraperitoneally (i.p.)
early (from day 2 to day 12 post-tumour inoculation) or late
(from day 12 to day 22 post-tumour inoculation) (40). This
was a CD8 + T cell-dependent effect where IL-21 increased
the expansion and cytotoxicity of OVA-specific CD8 + T cells,
and generated a more durable response compared with IL-2
and IL-15 leading to concomitant immunity towards tumour
rechallenge. Interestingly, pretreatment with IL-21 (day −4
to day 6 post-tumour inoculation) completely abrogated the
anti-tumour effect of IL-21, indicating that the timing of
IL-21 is critical for the outcome. In a fully syngeneic system,
we have shown significant tumour growth inhibition of
B16 melanomas and RenCa RCCs in response to therapeutic
administration of IL-21 protein, particularly by subcutaneous
administration (43). The anti-tumour activity was CD8 +
T cell-dependent, and IL-21 increased the density of tumour
infiltrating CD8 + T cells, a finding associated with improved
prognosis in human cancers. Using stereotactical injections of
IL-21 protein, Daga et al. showed significant tumour rejection
of intracranially implanted gliomas, mediated by NK cells and
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IL-21 in cancer and immunopathology
H. Søndergaard & K. Skak
Table 1 Preclinical anti-tumour activity and mechanisms of IL-21 monotherapy
Tumour model Treatment Anti-tumour effect Mechanism of action References
Colon 26 carcinoma (syngeneic) IL-21-secreting tumour Complete tumour rejection NK- and T cells, ↑ IFN-γ
production (spleen)
B16F1 melanoma, MethA IL-21-secreting tumour Complete tumour rejection CTL, NK-, not CD4 + T cells
fibrosacoma (syngeneic)
partly perforin mediated, not
IFN-γ dependent
AsPC1 pancreatic carcinoma IL-21-secreting tumour
Significant tumour growth Partly NK cells, ↑ IFN-γ
(human xenograft)
inhibition
production (spleen cells)
B16 melanoma, MCA205
fibrosarcoma (syngeneic)
B16F10 melanoma, RenCa renal
cell carcinoma, DA3 mammary
carcinoma (syngeneic)
OVA-expressing, E.G7 thymoma
(semi-syngeneic)
Several carcinomas with
endogenous or transfected
NKG2D ligands (syngeneic)
SCCVII head and neck
squamous cell carcinoma
(syngeneic)
B16F0 melanoma, RenCa renal
cell carcinoma (syngeneic)
GL261 glioma (syngeneic)
IL-21-expressing plasmids
(d5 and 12 PTI)
IL-21-expressing plasmids
(d -2 or 1 PTI)
IL-21 protein 20–100 μg i.p.
early (d2–12 PTI) 1×/day
late (d12–22 PTI) 1×/day
IL-21 protein 50 μg i.p.
i.v. metastasis: (d0–3 PTI)
spontaneous: (d10–12 PTI)
IL-21-expressing plasmids
(d5, 12, 19 and 26 PTI)
IL-21 protein 50 μg i.p. or s.c.
d3 or 8 PTI, 1×/day, B16
d7 or 12 PTI, 3×/week, RenCa
IL-21-secreting tumour or IL-21
protein 1 μg i.t.
Significant tumour growth
inhibition and increased
survival
Reduced number of lung and
liver metastasis
Significant tumour growth
inhibition and increased
survival compared with IL-2
and IL-15
Reduced number of lung
metastasis
Significant tumour growth
inhibition and increased
survival
Significant tumour growth
inhibition
Significant tumour rejection
NK cells, minor CTL role not
CD4 + T cells, ↑ NK cell
cytotoxicity, no IFN-γ increase
(serum)
NK cells, perforin dependent,
not IFN-γ, Fas ligand or TRAIL
CTL, early moderate role for NK
cells, not CD4 + T cells, ↑ and
sustained Ag-specific CTL
response, ↑ cytotoxicity
NK cells, NKG2D and perforin
dependent, not IFN-γ and Fas
ligand
T cells, NK cells and
tumour-specific
immunoglobulins
CTL, not NK cells
↑ density of tumour infiltrating
CD8 + T cells
B cells and NK cells
↑ tumour-specific IgG,
↑ serum-induced ADCC and
complement-mediated lysis
(36)
(37)
(38)
(39)
(30)
(40)
(41)
(42)
(43)
(44)
PTI, post tumour injection; NK, natural killer; IFN, interferon; IL, interleukin; CTL, CD8 + cytotoxic T cells; OVA, ovalbumin; TRAIL, tumour necrosis
factor-related apoptosis-inducing ligand; ADCC, antibody-dependent ceullular cytotoxicity.
B cells, with increases in tumour-specific antibodies, seruminduced
antibody-dependent cellular cytotoxicity (ADCC) and
complement-dependent tumour cell lysis (44). Taken together,
these results show that therapeutic use of recombinant IL-
21 protein is effective in the treatment of various preclinical
tumours.
Taken as a whole, IL-21 anti-tumour activity depends
on NK cells, CD8 + T cells or both, with a possible role
for B cell-derived tumour-specific Ig. CD4 + T cells seem
less important, but so far total CD4 + T cell depletions
have been made, and more specific depletion of CD4 + T
cell subsets, e.g. T regs and T h 17 cells is needed to fully
clarify their role. Perforin, which is critical for both NK
and CD8 + T cell cytotoxicity, is up-regulated by IL-21
and in contrast to Fas ligand or TRAIL, perforin seems
to be important for IL-21 anti-tumour activity. IL-21 also
increases IFN-γ production, but the need for IFN-γ is more
varied than perforin, probably depending on specific tumour
cell susceptibility to this molecule. IL-21 generally increases
tumour infiltrating CD8 + T cells, but it remains to be shown
whether this is secondary to increases in CD8 + T cell
expansion or whether IL-21 also has direct effects on T cell
trafficking. Generally, it remains to be clarified at what stage
during a tumour immune response IL-21 is beneficial; does
IL-21 mainly shape the initiation and expansion processes in
tumour draining lymph nodes or does it primarily modulate
tumour infiltrating lymphocytes during the effector phase.
In addition to its immune-mediated anti-tumour mechanisms,
IL-21 has direct cytotoxic effects on certain B cell lymphomas
(45), and potential anti-angiogenic effects of IL-21
has been suggested (46). Table 1 summarizes the main preclinical
in vivo anti-tumour data of IL-21 monotherapy.
In addition to having anti-tumour effects when given as
monotherapy, IL-21 also shows significant additive effects
together with a range of other biologicals modifying both
innate and adaptive responses. IL-21 protein alone enhanced
the anti-tumour effect against B16 melanomas treated with
adoptively transferred transgenic CD8 + T cells recognizing
a melanoma antigen and an antigen-specific vaccine (7). But
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H. Søndergaard & K. Skak IL-21 in cancer and immunopathology
in concert with IL-15, IL-21 significantly boosted the antitumour
effect by increasing the circulating level of adoptively
transferred CD8 + T cells and augmenting their IFN-γ
production. The sequential stimulation of NKT cells with
the agonist α-GC followed by IL-21 stimulation synergistically
inhibited B16 melanoma lung metastasis formation
by boosting the concomitant NK cell activation induced by
NKT cells (47). In combination with a potent triple antibody
cocktail (Trimab), IL-21 helped to completely eradicate
established tumours (48). Trimab consists of anti-DR5,
anti-CD40 and anti-CD137 (4-1BB), which together induce
a powerful T cell-dependent anti-tumour response through
DR5-mediated tumour cell apoptosis and generation of antigen,
co-stimulation of DCs via CD40, and T cell activation
through CD137 stimulation.
Several other interesting combination partners for IL-21
have recently been reviewed (49), expanding the potential of
IL-21 in cancer therapy even further. Still, many of the combination
partners so far tested with IL-21 are only in early
clinical testing or otherwise difficult to clinically translate.
However, we have recently published that IL-21 has additive
anti-tumour effects in combination with certain chemotherapies,
provided that IL-21 treatment is delayed compared to
chemotherapy (50). These data indicate that IL-21 is feasible
in combination with conventional therapies.
Another interesting aspect of IL-21 in cancer therapy is its
use in the ex vivo generation of antigen-specific CD8 + T cells
for adoptive cell therapy. IL-21 conditioning in vitro induces
a unique differentiation program in CD8 + T cells yielding a
highly effective anti-tumour response upon adoptive transfer,
which cannot be mimicked with IL-2 or IL-15 (51).
In conclusion, IL-21 alone or in combination with other
biological response modifiers stimulates both the innate and
adaptive arm of the immune system which shows significant
anti-tumour activity in several different preclinical tumour
models. These findings have paved the way for IL-21 clinical
trials, which are currently ongoing.
Clinical data
IL-2, which is closely related to IL-21, is approved for the
treatment of metastatic melanoma (MM) and RCC. Although
IL-2 shows encouraging responses in groups of patients with
these generally unmanageable diseases, its use is limited
because of severe toxicities, e.g. vascular leak syndrome,
requiring intensive care (52).
In phase I clinical trials IL-21 safety was tested in patients
with MM and RCC (53, 54). IL-21 was well tolerated;
the most common dose-related adverse effects were flu-like
symptoms such as fever, fatigue, chills and myalgia, and
dose-limiting toxicities included lymphopenia, neutropenia,
thrombocytopenia and hepatotoxicity with increases in liver
enzymes. Forty-seven MM and 19 RCC patients were treated
with IL-21. Of these, two complete responses (MM) and four
partial responses (RCC) were observed according to response
evaluation criteria in solid tumours (RECIST). IL-21 also
showed evidence of immune activation, with increases in
granzyme B, perforin, IFN-γ and chemokine C-X-C motif
receptor (CXCR)3 mRNA expression in peripheral blood
NK cells and CD8 + T cells (5, 53), suggestive of increased
effector cell function. Overall, phase I results endorsed
progress to phase II trials.
To date, two phase II trials have been completed, where IL-
21 efficacy was evaluated in patients with stage IV MM (55)
and in combination with a novel tyrosine kinase inhibitor
(sorafinib) in RCC [Bhatia et al., J Clin Oncol 27:15s, 2009
(suppl; abstr 3023), ASCO Annual Meeting]. Out of 24 MM
patients treated by i.v. administration of IL-21, one partial
and one complete response were observed per RECIST, and
adverse effects were similar to those observed in the phase
I trails. In the combination of IL-21 and sorafinib, 14 out
of 29 previously treated RCC patients completed 21 weeks
treatment with stable disease or better and with an acceptable
safety profile.
Mild lymphocytopenia is a frequently observed adverse
effect of IL-21, but interestingly IL-21 increased the frequency
of lymphocytes expressing CD62L and CCR7 (55), indicating
that IL-21-induced lymphocytopenia might be caused by a
redistribution of lymphocytes to secondary lymphoid compartments.
Consistent with the phase I trials, NK cells and CD8 +
T cells in phase II trials showed significant increases in perforin,
granzyme B and IFN-γ expression following IL-21 (55).
Also, CD8 + T cells and NK cells increased their expression of
the activation markers CD25 and CD69 in response to IL-21,
whereas CD4 + T cells did not, indicating biologically different
effects of IL-21 on these lymphocytes. These results
confirm that IL-21 is a well-tolerated drug that has objective
anti-tumour activity in human cancer patients associated with
signs of relevant immune activation.
Further trials are currently ongoing, evaluating IL-21 alone
and in combination with other compounds. Preliminary reports
from a trial of IL-21 in combination with rituximab (anti-
CD20 antibody) for the treatment of non-hodgkin’s lymphoma
(ClinicalTrials.gov identifier: NCT00347971) is encouraging
(55). The final results of these and future clinical trials
will be very interesting to follow.
IL-21 in immunopathology
Potent stimulation of immune responses inherently risks the
development of autoimmunity. Given that IL-21 stimulates
NK and T cell-mediated tumour immunity, plays a central
role in B cell differentiation and antibody production, and
amplifies the expansion of proinflammatory T h 17 cells, it is
not difficult to appreciate that host-derived IL-21 could also
be a key player in immunopathologies (see Table 2).
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H. Søndergaard & K. Skak
Table 2 The role of IL-21 in autoimmune diseases
Disease Preclinical data References Human data References
Systemic lupus erythematosus Lupus-prone BXSB-Yaa, Sanroque and
MRL-Fas lpr mice overexpress IL-21
(56–58) Polymorphisms in IL-21 and IL-21R genes
associate with SLE
IL-21R.Fc ameliorates lupus and IL-21R (58, 59) Low IL-21R expression on B cells in SLE
deficiency protects from lupus
patients correlates with disease activity
Type 1 diabetes
NOD mice overexpress IL-21, IL-21R (63, 66, 67) Polymorphisms in the IL-2/IL-21 locus and
deficiency protects from diabetes, and
in the IL-21 and IL-21R genes associate
transgenic expression of IL-21 induces
with T1D
diabetes C57BL/6 mice
Ag-specific CD4 + T cells in RIP-OVA mice (65)
overexpress IL-21 counteracting T reg
suppression of diabetes
Rheumatoid arthritis IL-21R.Fc ameliorates CIA in DBA/1 mice (70) Inflamed synovial tissue, synovial
lymphocytes and PBL from RA patients
overexpress IL-21R
Inflammatory bowel disease
Multiple sclerosis
IBF-deficient mice overexpress IL-21 and
develop spontaneous arthritis
IL-21R-deficient K/BxN mice are protected
from arthritis
IL-21 co-stimulation of TGFβ-treated
CD4 + CD25 − T cells inhibits colitis
suppression in SCID mice receiving
untreated CD4 + CD25 − T cells
IL-21 is overexpressed in gut mucosa from
DSS- and TNBS-induced colitis and IL-21
deficiency protects from colitis
IL-21 administration prior to, but not post
MOG immunization increases EAE
severity
IL-21R-deficient mice are protected from
MOG-EAE
IL-21/IL-21R-deficient mice are not
protected from MOG-EAE and IL-21R.Fc
increases severity of PLP-EAE
(71) IL-21R.Fc blocks inflammatory
cytokine-release in RA synovial cell
cultures
(72) Polymorphisms in IL-2/IL-21 locus
associate with RA
(77) Polymorphisms in IL-2/IL-21 locus
associate with UC and CD, and inflamed
gut mucosa from UC and CD patients
overexpress IL-21
(78) IL-21 from LPM cells increases MMP
release from intestinal fibroblasts in CD
patients
IL-21R.Fc inhibits CCL20 secretion and T
cell chemotaxis by IBD mucosa
(82) IL-21 drives secondary autoimmunity in MS
patients treated with
lymphocyte-depleting Ab (alemtuzumab)
(24)
(83)
(60, 61)
(62)
(68, 69)
(73, 74)
(75)
(76)
(78, 79, 81)
(10)
(80)
(85)
Ag, antigen; SLE, systemic lupus erythematosus; NOD, non-obese diabetic; RIP, rat insulin promoter; T1D, type 1 diabetes; OVA, ovalbumin; RA,
rheumatoid arthritis; CIA, collagen induced arthritis; PBL, peripheral blood lymphocytes; IBF, IRF-4-binding protein; TGF, transforming growth factor;
UC, ulcerative colitis; CD, Crohn’s disease; LPM, lamina propria monocytes; TNBS, trinitrobenzene sulfonic acid; MMP, matrix metalloproteinase; DSS,
dextran sulfate sodium; MOG, myelin oligodendrocyte glycoprotein; PLP, myelin proteolipid protein; EAE, experimental autoimmune encephalomyelitis
Systemic lupus erythematosus
Systemic lupus erythematosus (SLE) is a chronic autoimmune
disease that can affect connective tissues throughout the body.
Hallmarks of SLE are high levels of circulating autoantibodies
thought to arise from aberrant apoptosis, characteristic rashes,
and glomerulonephritis with associated renal dysfunction.
BXSB-Yaa mutant mice, which spontaneously develop a
condition similar to SLE, with lymphadenopathy, hypergammaglobulinemia,
and severe immune-complex-mediated
glomerulonephritis, have elevated serum levels of IL-21 (56).
In another mutant mouse strain, the sanroque mouse, a mutation
in the roquin protein negatively regulates T fh cell development.
This mouse developed a similar lupus-like syndrome
associated with increased T fh cell levels and overproduction
of IL-21 (57). The lupus-prone MRL-Fas lpr mouse showed
increased IL-21 production from CD4 + T cells and IL-21R.Fc
administration ameliorated disease severity (58). And, underlining
the role of IL-21 in experimental SLE, homozygous
IL-21R −/− BXSB-Yaa mice failed to develop renal disease
and mortality (59). These studies outline a potential benefit
of IL-21 neutralization in SLE patients, particularly given the
critical role for IL-21 in T fh cell and GC development, plasma
cell differentiation and antibody production.
In humans, polymorphisms in the human IL-21 and IL-
21R gene show association with SLE (60, 61) and peripheral
B cells from SLE patients have decreased IL-21R expression
correlating with increased disease activity (62). Taken
together, these data indicate a putative role for IL-21 in the
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H. Søndergaard & K. Skak IL-21 in cancer and immunopathology
pathogenesis of SLE, but future studies are required to better
clarify its role in human disease.
Type 1 diabetes
Type 1 diabetes (T1D) is a condition caused by specific
autoimmune destruction of insulin-producing β-cells in the
pancreas, resulting in loss of blood glucose control and is
fatal without insulin-replacement therapy.
In non-obese diabetic (NOD) mice, a commonly used model
of T1D, the insulin-dependent diabetes susceptibility locus
Idd3 on chromosome 3 has major impact on the spontaneous
disease development and spans the region for the IL-2 and
IL-21 genes. It was initially shown that NOD mice overexpress
IL-21 and it was postulated that IL-21 could drive
homeostatic expansion in NOD mice leading to the generation
of autoreactive T cells (63). This theory was questioned
by findings of genetic variations in the IL-2 gene, leading
to impaired T reg suppressive functions that associated with
diabetes development, and this was instead proposed as the
causal link of Idd3 (64). T regs can protect against diabetes
development as shown in another diabetes model; however,
here, diabetic mice had normal T reg suppressive capacity, but
instead showed overexpression of IL-21 which counteracted
T reg -mediated suppression (65). In support, two recent studies
independently show that IL-21R-deficient NOD mice completely
fails to develop diabetes, correlating with reduced
insulitis, decreased infiltration of CD4 + and CD8 + T cells
in the pancreas, and no increases in T reg levels (66, 67). Furthermore,
in contrast to IL-21R +/+ NOD splenocytes, adoptive
transfer of IL-21R −/− NOD splenocytes does not induce diabetes
in NOD/scid mice and transgenic expression of IL-21
induces spontaneous diabetes in diabetes-resistant C57BL/6
mice (67). These data strongly advocate for a central role of
IL-21 in diabetes development in NOD mice and suggest that
IL-21 is a relevant susceptibility gene in the Idd3 locus. Much
effort has been put into identifying the one gene that confers
the diabetes association of the NOD Idd3 locus, but it appears
that IL-2 and IL-21 are not mutually exclusive diabetes susceptibility
genes.
In a recent large genome-wide association study of human
T1D, the chromosome region 4q27 was identified as one
of six new robust disease-associated loci (68). This locus is
the human orthologue of the Idd3 containing both the IL-2
and IL-21 gene. In another analysis of IL-21 and IL-21R
gene-sequence variants, polymorphisms in both genes were
association with diabetes (69). These data indicate that IL-21
could have a role in human T1D development, but the strong
set of experimental data still lack supportive human data to
confirm this hypothesis.
Rheumatoid arthritis
Rheumatoid arthritis (RA) is a chronic, inflammatory disorder
of idiopathic etiology that affects many tissues and organs. It
principally involves autoimmune attacks of the joints producing
inflammatory synovitis that often progresses to destruction
of the articular cartilage and joint deformities.
In animal models of RA, IL-21R.Fc reduced the histological
and clinical signs of collagen-induced arthritis (CIA)
in DBA/1 mice immunized with bovine collagen (70). In
another study, interferon regulatory factor 4 (IRF-4)-binding
protein (IBF)-deficient mice showed increased IL-21 and
IL-17 production leading to spontaneous development of a
RA-like condition (71). K/BxN mice spontaneously develop
autoantibody-dependent arthritis, but IL-21R-deficient K/BxN
mice were completely refractory to disease (72). Here, IL-21
mainly had a pathogenic role in T fh development and autoantibody
production, whereas T h 17 cells and T regs seemed to be
uninvolved (72).
In RA patients, IL-21R expression was increased in
inflamed synovial tissue and lymphocytes, and in peripheral
blood lymphocytes compared with osteoarthritis (OA)
patients (73, 74). Also, IL-21 production was detected in RA
synovial tissue cultures, and IL-21R.Fc inhibited secretion of
proinflammatory cytokines from the synovial tissue (75). In
a recent study, it was proposed that genetic polymorphisms
in the IL-2/IL-21 locus were associated with the development
of RA (76). Overall, increasing evidence suggests that IL-21
could be involved in the pathogenesis of RA.
Inflammatory bowel disease
Inflammatory bowel disease (IBD) mainly comprises ulcerative
colitis (UC) and Crohn’s disease (CD), where both
involve idiopathic autoimmune destruction of the gut mucosa,
but differ in the areas of the gut they affect.
In experimental colitis, CD4 + CD25 − T cell transfer to
SCID mice results in colitis development, co-transfer of
TGFβ-treated CD4 + CD25 − T cells ameliorates disease,
whereas co-transfer of TGFβ and IL-21-treated CD4 + CD25 −
T cells fails to suppress colitis development (77). This is
thought to occur because IL-21 inhibits TGFβ-induced FoxP3
expression and T reg differentiation of CD4 + CD25 − T cells
and instead promotes differentiation of T h 17 cells producing
high levels of IL-17 as well as IL-21 (77). In experimental
colitis induced by dextran sulfate sodium (DSS) or
trinitrobenzene sulfonic acid (TNBS), IL-21 expression was
increased in the gut mucosa, and IL-21-deficient mice were
highly protected from disease correlating with reduced T h 17
cell activity (78).
In humans, IL-21 expression is increased in inflamed, but
not unaffected gut mucosa from both UC and CD patients (78,
79). Isolated lamina propria T cells from CD patients showed
reduced IL-17 production when stimulated in the presence of
anti-IL-21 antibody (78), indicating that IL-21 is present in
IBD and has potential impact on IL-17 responses, which are
believed to be pathogenic in CD. Intestinal fibroblasts isolated
from colitis patients and healthy controls express IL-21R, and
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H. Søndergaard & K. Skak
IL-21 alone or in concert with TNF-α significantly induced their
secretion of matrix metalloproteinases (MMP), thought to play
a key role in tissue degradation (10). Also, IL-21R.Fc treatment
reduced the MMP production from intestinal fibroblasts
induced by supernatants from CD lamina propria mononuclear
cells (10). Moreover, IL-21 stimulation induced secretion of
the T cell attractant, CCL20/MIP-3α from colon epithelial
cells, and anti-IL-21 blocked T cell chemotaxis induced by
supernatants from IBD mucosal explants (80). In genome-wide
association studies single nucleotide polymorphisms (SNPs)
within the IL-2/IL-21 locus 4q27 associated with both UC
and CD (81). Overall, evidence is accumulating that IL-21 is
a very relevant cytokine in the pathogenesis of IBD with a
role in both tissue remodelling and T cell trafficking.
Multiple sclerosis
Multiple sclerosis (MS) is an autoimmune disease of unknown
etiology, which attacks the central nervous system (CNS),
leading to demyelination and loss of physical and cognitive
function.
Experimental autoimmune encephalomyelitis (EAE) is the
classical animal model of MS, where immunization with
myelin-derived peptides or proteins induces CNS inflammation
mimicking human disease. In EAE, IL-21 protein
administration prior to myelin oligodendrocyte glycoprotein
(MOG)-peptide immunization increased the severity of disease,
whereas IL-21 administration during disease progression
did not increase severity (82). Initially, MOG immunization
of IL-21-deficient mice showed reduced EAE disease activity
ascribed to a lack of T h 17 cell differentiation (24). However,
more recent findings have challenged this, showing no reduction
or even exacerbated EAE in IL-21- and IL-21R-deficient
mice and competent T h 17 cell differentiation (83). This discrepancy
may arise from genetic variations in the mice used
in the different experiments (83). Also, in EAE induced by
myelin proteolipid protein (PLP), administration of IL-21R.Fc
before and after peptide immunisation increased the severity
of disease associated with decreased T reg numbers and Foxp3
expression (84).
Altogether, the role of IL-21 in EAE is controversial, and
a direct link between IL-21 and human MS remains to be
shown. However, a recent publication shows that MS patients
who developed secondary autoimmunity resulting from treatment
with a lymphocyte-depleting monoclonal antibody,
alemtuzumab, had greatly elevated serum IL-21, which was
genetically predetermined. It is possible that IL-21 drives
excess homeostatic T cell expansion and apoptosis leading
to secondary autoimmunity (85). Clearly, these data warrant
future studies of IL-21 in the pathogenesis of MS.
Chronic viral infections
Very recently, a trio of papers showed that host IL-21 was critical
for the control of chronic viral infections by subjecting
IL-21- and IL-21R-deficient mice to chronic lymphocytic
choriomeningitis virus (LCMV) (86–88). In these studies, IL-
21 was needed neither during the acute phase of viral infections,
nor for the maintenance of memory CD8 + T cells after
resolved infections, but without IL-21 chronic presence of
virus antigen resulted in exhaustion of CD8 + T cell responses
and poor viral control. These studies highlight yet another
important aspect of IL-21 with potential ramifications for
novel therapies. Here it is also worth noticing that the role
of endogenous IL-21 in cancer control, which also includes
chronic persistence of antigen, remains to be determined.
Concluding remarks
In less than a decade, IL-21 has advanced from being a
novel member of the γ c receptor family to a cytokine in
clinical trials for the treatment of cancer, with implications
in several autoimmune pathogeneses. As cancer therapy,
IL-21 monotherapy shows moderate clinical responses, but so
far clinical trials have been limited to pretreated, end-stage
patients and it would be very interesting to evaluate IL-21 in
patients with less advanced disease. The manageable toxicity
of IL-21 encourages combination with other drugs, and such
options ought to be pursued in future trials. Continued research
into IL-21 anti-cancer biology will be essential to further clarify
its immunotherapeutic effects, support future clinical trials
and perhaps identify novel interesting combination partners.
The data reviewed here clearly suggest that neutralization
of IL-21 could hold therapeutic value in several major
immunopathologies, where particularly IBD, RA and SLE
seem to be relevant candidates. However, disclosing the role
of IL-21 in human immunopathologies will be essential to
warrant the development of new IL-21-blocking compounds.
Hopefully, the next few years of IL-21 research will clarify
its full potential in cancer therapy and its intriguing role in
immunopathologies.
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© 2009 John Wiley & Sons A/S 13
33

Paper II:
Interleukin 21 therapy increases the density of tumor infiltrating CD8 + T cells and inhibits
syngeneic tumor growth
Søndergaard H., Frederiksen K.S., Thygesen P.,Galsgaard E.D., Skak K. Kristjansen P.E.G.
and Kragh M., Cancer Immunol Immunother. 2007, Sep;56(9):1417-28. Epub. 2007 Feb.
35

Cancer Immunol Immunother (2007) 56:1417–1428
DOI 10.1007/s00262-007-0285-4
ORIGINAL ARTICLE
Interleukin 21 therapy increases the density of tumor infiltrating
CD8 + T cells and inhibits the growth of syngeneic tumors
Henrik Søndergaard Æ Klaus S. Frederiksen Æ Peter Thygesen Æ
Elisabeth D. Galsgaard Æ Kresten Skak Æ Paul E. G. Kristjansen Æ
Niels Ødum Æ Michael Kragh
Received: 13 October 2006 / Accepted: 24 December 2006 / Published online: 7 February 2007
Ó Springer-Verlag 2007
Abstract Interleukin (IL)-21 is a recently discovered
cytokine in early clinical development, which has
shown anti-tumor activity in various animal models. In
the present study, we examine the anti-tumor activity
of IL-21 protein therapy in two syngeneic tumor
models and its effect on the density of tumor infiltrating
T cells. We treated mice bearing established subcutaneous
B16 melanomas or RenCa renal cell
carcinomas with intraperitoneal (i.p.) or subcutaneous
(s.c.) IL-21 protein therapy and subsequently scored
the densities of tumor infiltrating CD4 + and CD8 + T
H. Søndergaard (&) E. D. Galsgaard
K. Skak M. Kragh
Department of Cancer Pharmacology,
Biopharmaceuticals Research Unit, Novo Nordisk A/S,
Novo Nordisk Park F6.2.30, DK 2760 Måløv, Denmark
e-mail: hris@novonordisk.com
K. S. Frederiksen
Department of Molecular Genetics,
Biopharmaceuticals Research Unit, Novo Nordisk A/S,
Novo Alle, DK 2880 Bagsværd, Denmark
P. Thygesen
Department of Exploratory ADME,
Biopharmaceuticals Research Unit, Novo Nordisk A/S,
Novo Nordisk Park, DK 2760 Måløv, Denmark
P. E. G. Kristjansen
Department of Development Projects 05,
Novo Nordisk A/S, Novo Alle,
DK 2880 Bagsværd, Denmark
N. Ødum
Department of Molecular Biology and Physiology
and Department of Medical Microbiology and Immunology,
University of Copenhagen, Copenhagen, Denmark
cells by immunohistochemistry. Whereas both routes
of IL-21 administration significantly inhibited growth
of small, established RenCa and B16 tumors, only s.c.
therapy significantly inhibited the growth of large,
established tumors. We found a greater bioavailability
and significant drainage of IL-21 to regional lymph
nodes following s.c. administration, which could account
for the apparent increase in anti-tumor activity.
Specific depletion of CD8 + T cells with monoclonal
antibodies completely abrogated the anti-tumor activity,
whereas NK1.1 + cell depletion did not affect tumor
growth. In accordance, both routes of IL-21 administration
significantly increased the density of tumor
infiltrating CD8 + T cells in both B16 and RenCa tumors;
and in the RenCa model s.c. administration of
IL-21 led to a significantly higher density of tumor
infiltrating CD8 + T cells compared to i.p. administration.
The densities of CD4 + T cells were unchanged
following IL-21 treatments. Taken together, these data
demonstrate that IL-21 protein has anti-tumor activity
in established syngeneic tumors, and we show that
IL-21 therapy markedly increases the density of tumor
infiltrating CD8 + T cells.
Keywords Interleukin-21
Tumor infiltrating lymphocytes Cancer
Melanoma Renal cell carcinoma
Abbreviations
IL-21 Interleukin 21
i.v. Intravenous
i.p. Intraperitoneal
s.c. Subcutaneous
TILs Tumor infiltrating lymphocytes
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37

1418 Cancer Immunol Immunother (2007) 56:1417–1428
NK cells Natural killer cells
CTLs Cytotoxic T lymphocytes
AOI Area of interest
AUC Area under the curve
WT Wild type
LN Lymph node
IP-10 Interferon-inducible protein 10
MIG Monokine induced by interferon gamma
I-TAC Interferon-inducible T cell alpha
chemoattractant
Introduction
Successful immune-based cancer therapy needs to enhance
the interaction and/or reactivity between the
immune system and the cancer cells. In tumor immunity,
tumor infiltrating lymphocytes (TILs) are regarded
as the primary effector cells, and the number of
TILs has previously been correlated with prolonged
survival in cancer patients [6,18]. The number of tumor
infiltrating CD8 + T cells and the CD8 + /CD4 + T cell
ratio have been shown to be independent prognostic
factors for improved survival in several different human
cancers [21,22,28,30]. Thus, novel treatments able
to increase the number of tumor-specific, tumor-infiltrating
immune effector cells could hold a promising
clinical future.
IL-21 is the latest member of the common c-chaindependent
cytokine family and is currently in early
clinical development for the treatment of cancer. IL-21
was discovered as a product of activated CD4 + T
helper cells, and its unique receptor IL-21R has been
identified on a broad range of immune cells including
B, T, NK, and dendritic cells [3,23]. IL-21 has pleiotropic
immune modulatory activity, which has shown
encouraging anti-tumor effects in several different
animal models [16]. CD8 + cytotoxic T lymphocytes
(CTLs), NK cells, or both have been identified as the
main mediators of IL-21 anti-tumor activity [16], and in
this respect IL-21 has been suggested to play a role in
the transition from innate to adaptive immunity [14].
However, it remains to be shown whether its anti-tumor
activity in vivo is mediated by modulation of the
tumor infiltrating effector cell populations. In vitro, IL-
21 stimulation induced the differentiation and maturation
of human NK cells from progenitor cells [23,31]
and also increased the cytolytic activity of mature
activated human NK cells [23]. Murine NK cells have
shown more biphasic responses to IL-21 stimulation
in vitro, depending on IL-21 concentration, co-stimuli,
and cell maturation stage [14,24,36]. In mice and
humans, IL-21 has been shown to increase the expansion
of antigen stimulated CD8 + cytotoxic T cells as
well as enhance their cytolytic activity in vitro
[14,17,39]. In vivo, IL-21 has been shown to enhance
the expansion, activity, and long-term survival of
ovalbumin-specific CD8 + T cells detected in lymph
nodes (LNs) in mice bearing ovalbumin-expressing
E.G7 lymphomas [20]. Based on these results we
anticipate that IL-21 in vivo is able to increase the
number and/or reactivity of TILs.
In previous studies of IL-21-mediated anti-tumor
activity, the cytokine was primarily administered via
plasmid gene delivery [38], tumor cell secretion
[7,8,19,37], or used in systems where the immune response
was enhanced by introduction of foreign antigens
or adoptive transfer of tumor specific lymphocytes
[13,20,26,39]. Clearly the effects of IL-21 protein
therapy also need to be investigated in simple
syngeneic models using more conventional routes of
administration, which are more clinically relevant. The
use of IL-21 protein therapy in a native syngeneic
model could help to determine whether IL-21 is able to
modulate TILs in vivo. The evaluation of s.c. administration
of IL-21 is of major interest for the clinical
application of IL-21 because of patient convenience
and since increased tolerability and sustained efficacy
has been shown in clinical trials with IL-2 by this route
of administration [11].
In this study, we demonstrate the anti-tumor effects
of s.c. and i.p. IL-21 protein therapy in two syngeneic
tumor models. Our data indicate that IL-21 protein via
both routes of administration can inhibit established
tumor growth and that s.c. administration could be
applicable in the clinic. Furthermore, we show that IL-
21 therapy strongly increases the density of CD8 + TILs
without changing the CD4 + TILs, and that the CD8 + T
cells are essential for the IL-21-induced anti-tumor
activity.
Materials and methods
Mice
Wild type (WT) female C57BL/6 and BALB/c mice
were purchased from Taconic Europe A/S, Lille
Skensved, Denmark, whereas female C57BL/6 nude
mice (B6.Cg/Ntac-Foxn1 nu N9) were acquired from
Taconic, Hudson, NY, USA. The animals were 6 weeks
old on arrival and were allowed to acclimatize for at
least one week before start of experiments. WT mice
were housed in a standard animal facility whereas nude
mice were isolated in an immunodeficient facility separated
by a barrier. Light was controlled on a 12-h
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Cancer Immunol Immunother (2007) 56:1417–1428 1419
light–dark cycle, and the animals were given free access
to food and drinking water. The animals were observed
daily for clinical signs and their body weights were recorded
regularly. All experiments were conducted in
accordance with corporate and governmental policies.
Cell lines
C57BL/6 derived B16 (F0) melanoma cells (American
Type Culture Collection (ATCC), CRL-6322) and
BALB/c derived RenCa renal cell carcinoma cells
(kindly provided by Dr. Robert H. Wiltrout, NCI at
Frederick, MD, USA) were cultured in RPMI 1640 with
GlutaMAX TM supplemented with 10% heat-inactivated
FCS, sodium pyruvate (RenCa only), non-essential amino
acids (RenCa only), and 5% penicillin-streptomycin
(all from GIBCO Cell Culture, Invitrogen, Denmark).
Reagents and antibodies
Recombinant murine IL-21 (IL-21) protein was provided
by Novo Nordisk A/S, Denmark and Zymogenetics,
Inc., WA, USA and used in all experiments. The
stock solutions contained IL-21 in a concentration of
5.5–10 mg/ml and working preparations were diluted in
PBS. Radioactive 125 I conjugated IL-21 was produced
at Novo Nordisk A/S, Denmark. The depleting antimouse
CD8 (clone 2.43, TIB-210 from ATCC) and
anti-mouse NK1.1 (clone PK136, HB191 from ATCC)
monoclonal antibodies were obtained from supernatants
of hybridomas cultured at Novo Nordisk A/S,
Denmark. The antibodies were purified in-house by
affinity chromatography. For the histological examination
we used rat anti-mouse CD4 (L3T4, clone
RM4–5) (BD Pharmingen, CA, USA), rat anti-mouse
CD8 (Ly-2, clone 53–6.7) (BD Pharmingen, CA,
USA), rat serum IgG2a (Serotec, UK), and biotinconjugated
donkey anti-rat IgG (Jackson ImmunoResearch
Laboratories, Inc., PA, USA).
In vivo tumor models
On day 0, C57BL/6 or BALB/c mice were inoculated
s.c. in the right flank with 10 5 B16 melanoma or RenCa
renal cell carcinoma cells, respectively. All mice were
randomized and ear-tagged prior to treatment. The
tumor volume was measured as two perpendicular
diameters approximately three times per week, and
calculated by the following formula:
Volume ¼ p 6 d2 1 d 2 if d 1 \d 2 ;
where d represents the two diameters:
Treatment with IL-21 was either initiated early with
~5 mm 3 mean tumor volume or late with ~50 mm 3
mean tumor volume. Fifty lg of IL-21 or PBS in a
dosing volume of 200 lL was administered either
intraperitoneally (i.p.) or subcutaneously (s.c.) in the
contralateral flank 1x/daily in the C57BL/6-B16 model
and 3x/week in the BALB/c-RenCa model. The 50 lg
dose was chosen on the basis of dose-titration experiments
prior to this work (data not shown). Termination
criteria were a tumor volume of 1,000 mm 3 or more
than 20% weight loss from time of cell inoculation.
In vivo immune cell depletion
In vivo CD8 + and NK1.1 + cell depletion was performed
by using anti-mouse CD8 (clone 2.43) and anti-mouse
NK1.1 (clone PK136) monoclonal antibodies, respectively,
as previously reported [33]. C57BL/6 mice
inoculated s.c. in the right flank with 10 5 B16 melanoma
cells were injected with antibodies (100 lg/
mouse) i.p. on day –1, 0, 6, and 12 in relation to the
onset of treatment. Treatment with 50 lg IL-21
administered s.c. was initiated when mean tumor
volume had reached ~5 mm 3 corresponding to early
treatment. Prior to the experiment, the dose and
schedule of the depleting antibodies were verified by
flow cytometry analysis showing complete depletion of
CD8 + and NK1.1 + cells throughout the course of the
experiment (data not shown).
In vitro tumor cell proliferation assay
Potential effects of IL-21 on the growth of B16 melanoma
cells and RenCa carcinoma cells in vitro were
examined in a colorimetric assay using the proliferation
reagent 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-
5-tetrazolio]-1,3 benzene disulfonate (WST-1) (Roche
diagnostics GmbH, Germany). Briefly, 3000 B16 or
RenCa cells/well were incubated in 96-well plates in
their appropriate media and stimulated with increasing
concentrations of IL-21 protein. After 48 h cell growth,
cells were incubated with WST-1 for 2 h at 37C.
Absorbance at 450 nm was used to determine the relative
proliferation of cells.
IL-21 pharmacokinetics
IL-21 was administered intravenous (i.v.), i.p. and s.c.
(50 lg/animal) to BALB/c mice. Mice were anesthetized
with FORENE Isoflurane (Abbot Scandinavia
AB, Sweden) after 5 min, 15 min, 30 min, 1 h, 2 h, 4 h,
and 8 h post injections, blood was collected from the
retro-orbital sinus, and a DuoSet sandwich ELISA kit
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1420 Cancer Immunol Immunother (2007) 56:1417–1428
was used to detect IL-21 protein in the serum (R&D
systems, Inc., MN, USA). Briefly, goat anti-mouse IL-
21 antibodies were coated overnight onto a 96-well
plate at RT. Serum samples were diluted 1:10 or 1:100
in PBS with 1% BSA and IL-21 standards were likewise
supplemented with normal mouse serum. A biotin-labeled
goat anti-mouse IL-21 antibody was used as
detection antibody, and for the color reaction streptavidin-HRP
with tetramethylbenzidine (TMB) was used
as substrate (Sigma–Aldrich Chemie GmbH, Germany).
The result was measured as absorbance at
450 nm. The lower limit of detection was ~0.06 ng/ml
with a dynamic range of 0.06–100 ng/ml. The serum
concentration-time data were analyzed by a non-compartmental
pharmacokinetic analysis using WinNonlin
Professional (Pharsight, Inc., CA, USA) based on a
sparse blood sampling schedule, where the mean serum
concentration-time profiles of blood samples from
three animals per time point were performed. The area
under the curve (AUC), a measure of drug exposure
and the bioavailability are reported.
IL-21 biodistribution
Mice were injected i.p. with 0.05 ml of 125 I-IL-21
(~2 lCi/animal) or Na 125 I as control in the lower right
abdominal quadrant or subcutaneously in the right foot
pad in order to use the popliteal LN as an exclusive
lymph drainage site. After 15 min, 30 min, 1 h, 2 h,
4 h, 6 h, and 8 h animals were sacrificed and the following
tissues/organs were collected and analyzed:
heart, lungs, liver, spleen, kidneys, thyroidal gland,
small and large intestines, mesenteric LNs, right and
left popliteal LNs, and skin at injection site after s.c.
administration, i.e. the foot. Na 125 I solution was used
to monitor how free 125 I would behave contra protein
bound 125 I in order to detect if the 125 I from the protein
was detached in vivo (data not shown). The c-radiation
was measured in all samples by a Cobra Auto-Gamma
gamma-counter (PerkinElmer, Inc., MA, USA) and
related to the total c-radiation of the initial injected
dose of 125 I-IL-21 in %.
Immunohistochemistry of tumor infiltrating T cells
Six lm cryo-sections were made from tumor biopsies
taken out at termination of the therapeutic studies.
Sections were immunohistochemically stained with rat
anti-mouse CD4 (clone RM4-5) or rat anti-mouse CD8
(clone 53-6.7) antibodies (5 lg/ml), whereas rat serum
IgG2a was used as matching isotype control. Biotinconjugated
donkey anti-rat IgG (diluted 1:3,000) was
used as secondary antibody. Sections were fixed in 4%
paraformaldehyde at 4C and endogenous biotin
activity was blocked using Biotin blocking system from
Dako A/S, Denmark. Prior to the antibody stainings
non-specific binding was blocked by incubation in TBS
with 3% skim milk, 3% BSA, and 7% donkey serum.
Incubation with the primary antibodies was made at
4C over night followed by 1 h incubation at RT with
the secondary antibody both diluted in TBS with 0.5%
skim milk, 3% BSA, and 7% donkey serum. Streptavidin
conjugated alkaline phosphatase and Liquid
Permanent Red Chromogen (Dako A/S, Denmark)
was used to visualize positive cells and sections were
counterstained with Mayer’s hematoxylin to reveal
nuclei morphology.
Quantification of tumor infiltrating T cells
A stereological method was established to quantify
the density of tumor infiltrating T cells. Images from
immunohistochemically stained tumor sections were
analyzed via online light microscopy at 20· magnification
using C.A.S.T. grid software ver. 2.3.1.3 from
Olympus Denmark A/S, Denmark. In all tumor sections
(one from each tumor) the density of tumor
infiltrating lymphocytes was blindly scored counting all
positive cells intratumorally and relating them to an
area of interest (AOI), representing the total tumor
area measured stereologically, excluding necrotic tumor
tissue and non-tumor tissue such as peritumoral
connective tissue. The C.A.S.T. grid software and a
motorized stage system enabled side by side imaging to
ensure that no area was evaluated twice or omitted.
Statistics
Student’s t-test (two-tailed, assuming equal variance)
was used for statistical evaluations of differences between
treated and control groups. Data are shown as
mean ± SEM and a P value less than 0.05 was considered
statistically significant.
Results
IL-21 does not inhibit B16 or RenCa tumor cell
growth in vitro
To determine whether IL-21 protein had any direct
inhibitory effects on B16 or RenCa tumor cells we
performed a tumor cell proliferation assay with increasing
concentrations of IL-21 (0–5,000 ng/ml). We found
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Cancer Immunol Immunother (2007) 56:1417–1428 1421
no effects on B16 or RenCa cell growth after 48 h
incubation (Fig. 1). Consistent with these results, no IL-
21 receptor mRNA expression in either tumor cell line
examined by quantitative RT-PCR analysis was found
(data not shown).
Absorbance 450 nm
+/- SEM
3
2.5
2
1.5
1
0.5
0
B16
RenCa
0 1 10 100 1000 5000
IL-21 concentration (ng/mL)
Fig. 1 IL-21 does not inhibit proliferation of B16 and RenCa
cells in vitro. B16 cells (filled square) or RenCa cells (square)
(3,000/well) were grown in appropriate media and stimulated
with the indicated concentrations of IL-21 for 48 h. Absorbance
at 450 nm was measured after 2 h incubation with WST-1
proliferation reagent. Mean ± SEM of duplicates
Subcutaneous IL-21 protein therapy significantly
inhibits B16 and RenCa tumor growth
Subcutaneous syngeneic B16 and RenCa tumors were
established in their respective hosts, C57BL/6 and
BALB/c mice, by injection of 10 5 cells/animal. In both
models IL-21 treatment was initiated either early, when
tumors were just palpable (~5 mm 3 mean tumor volume),
or late, when tumors were more established
(~50 mm 3 mean tumor volume).
Early treatment with IL-21 using either route of
administration showed significant (P < 0.001) growth
inhibition of B16 melanoma tumors (Fig. 2a). In the
early treatment of RenCa carcinomas only s.c. administration
of IL-21 showed significant growth inhibition
(P < 0.001), whereas i.p. administration showed a
borderline significant growth inhibition (P = 0.06)
(Fig. 2b). The difference between s.c. and i.p. administration
in the early treatment of RenCa carcinomas
was likewise close to statistical significance (P = 0.09).
In the late treatment regimens s.c. administration of
IL-21 showed significant (P < 0.05) growth inhibition
in both models, whereas i.p. administration was
unable to significantly inhibit tumor growth (Fig 2c, d).
Generally, the late treatment regimen showed less
growth inhibition than the early treatment, and s.c.
a
3
Mean tumor volume (mm )
+/- SEM
1100
1000
900
800
700
600
500
400
300
200
100
0
b
600
PBS i.p.
IL-21 i.p.
IL-21 s.c.
500
PBS i.p.
IL-21 i.p.
IL-21 s.c.
400
300
200
Treatment start
100
Treatment start
0
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
3 )
(mm
Mean tumor volume
+/-SEM
Days post B16 tumor inoculation
Days post RenCa tumor inoculation
c
Mean tumor volume (mm 3
)
+-/ SEM
1000
900
800
700
600
500
400
300
200
100
0
PBSi.p.
IL-21 i.p.
IL-21 s.c.
Treatment start
7 8 9 10 11 12 13 14 15 16
Days post B16 tumor inoculation
d
Mean tumor volume (mm 3
)
+-/ SEM
900
800
700
600
500
400
300
200
100
0
PBS i.p.
IL-21 i.p.
IL-21 s.c.
Treatment start
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Days post RenCa tumor inoculation
Fig. 2 IL-21 therapy inhibits growth of syngeneic B16 melanomas
and RenCa carcinomas. All animals were injected with
10 5 cells s.c. in the right flank and randomized prior to treatment
start as indicated. Treatment was started either early (a and b)
with ~5 mm 3 mean tumor volume or late (c and d) with ~50 mm 3
mean tumor volume. Fifty lg IL-21 or PBS was injected i.p. or s.c.
(contralateral to the tumor site) daily in the B16 model (a and c)
and 3·/week in the RenCa model (b and d). Mean ± SEM, a
n = 12, b n = 15, c and d n = 10, P = 0.06, *P < 0.05, **P < 0.01,
***P < 0.001, compared to PBS controls by Student’s t-test
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1422 Cancer Immunol Immunother (2007) 56:1417–1428
administration appeared to be more efficient than i.p.
administration, although this difference did not reach
statistical significance (Fig. 2).
Alongside these results, we monitored animal
weight and general health in response to IL-21
administration and found no treatment associated effects
on animal health (data not shown), suggesting
that the treatment was well tolerated.
CD8 + T cells are essential for the anti-tumor
activity of IL-21
In order to determine which immune effector cells
were important for the anti-tumor activity of IL-21
protein in our models, C57BL/6 nude mice and wild
type (WT) mice specifically depleted of CD8 + T cells
or NK1.1 + cells were used. As shown in Fig. 3a, early
s.c. IL-21 protein therapy was unable to inhibit B16
tumor growth in C57BL/6 nude mice. C57BL/6 nude
3 )
a
Mean tumor volume (mm
+/-SEM
3 )
b
Mean tumor volume (mm
+/-SEM
1600
1400
1200
1000
800
600
400
200
0
1000
900
800
700
600
500
400
300
200
100
0
Vehicle s.c.
IL-21 s.c.
Treatment start
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Days post B16 tumor inoculation
Vehicle s.c.
IL-21 s.c. + ∆NK1.1 + ∆CD8
IL-21 s.c. + ∆CD8
IL-21 s.c. + ∆NK1.1
IL-21 s.c.
Treatment start
3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Days post B16 tumor inoculation
Fig. 3 The anti-tumor effect of IL-21 is CD8 + T cell dependent.
C57BL/6 nude (a) or C57BL/6 WT mice (b) were injected with
10 5 B16 melanoma cells s.c. in the right flank and randomized
prior to treatment start as indicated. WT animals were depleted
of NK1.1 + cells, CD8 + cells or both using monoclonal Ab
administrated i.p. on day –1, 0, 6 and 12 compared to treatment
start. Fifty lg IL-21 or vehicle was injected s.c. (contralateral to
the tumor site) daily from day 3 after tumor inoculation.
Mean ± SEM, n = 10, *P < 0.05, **P < 0.01, compared to vehicle
control, CD8-depleted and NK1.1 + CD8-depleted groups by
Student’s t-test
mice are fully NK cell competent and T cell deficient,
verified by flow cytometry (data not shown). This result
suggests that T cells and not NK cells are essential for
the anti-tumor activity of IL-21 in this model. To further
examine the specific cell types involved in the antitumor
activity, we depleted B16 tumor-bearing C57BL/
6 WT mice with anti-CD8 and/or anti-NK1.1 antibodies
prior to treatment start with s.c. IL-21 again initiated
early. As shown in Fig. 3b, significant anti-tumor
activity was still exhibited in NK.1.1 + cell-depleted
animals (P < 0.01) compared to vehicle controls,
whereas CD8 + T cell depletion completely abrogated
the tumor growth inhibition of IL-21. Together, these
results show that CD8 + T cells are the essential cells for
the anti-tumor activity of IL-21 in our model.
IL-21 significantly increases the density of tumor
infiltrating CD8 + T cells
Based on the finding that CD8 + T cells were responsible
for the anti-tumor activity in our models we
examined whether IL-21 increased the number of tumor
infiltrating CD8 + T cells as a possible mechanism
of action. Immunohistochemistry was used to stain
CD4 + and CD8 + T cells in tumor biopsies from our
in vivo therapeutic studies. One section from each tumor
biopsy obtained at the end of the late treatment
experiments was stained (Fig. 2c, d). The density of
TILs was scored by counting all positive cells in a
stereologically defined area of interest (AOI) in which
necrotic areas and peritumoral connective tissue were
excluded. Thus, our results reflect the density of intratumoral
infiltrating lymphocytes, which are in direct
contact with tumor cells and have the potential of
performing cytotoxic effects. Representative pictures
of anti-CD8 stained B16 melanomas and RenCa carcinomas
with and without IL-21 treatments are shown
in Fig. 4. In B16 melanomas the density of tumor
infiltrating CD4 + T cells was unchanged following IL-
21 treatments (Fig. 5a), whereas the density of CD8 + T
cells showed a significant 7–10-fold increase (P < 0.05)
following i.p. and s.c. administration of IL-21 (Fig. 5b).
In RenCa carcinomas the density of CD4 + T cells was
likewise unchange after IL-21 treatments (Fig. 5c) and
the density of CD8 + T cells was increased 3-fold after
i.p. administration (P < 0.05) and 8-fold after s.c.
administration (P < 0.01) of IL-21 (Fig 5d). Moreover,
the difference between i.p. and s.c. IL-21 administration
was significant (P < 0.05) reflecting the improved
efficacy obtained in the RenCa carcinomas with s.c.
administration (Fig. 2d). Together these data show that
IL-21 strongly increases the density of tumor infiltrating
CD8 + T cells without affecting the CD4 + T cell
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42

Cancer Immunol Immunother (2007) 56:1417–1428 1423
Fig. 4 Pictures of tumor
infiltrating CD8 + T cells in
B16 melanomas and RenCa
carcinomas. Representative
pictures of cryo-sections at
·20 magnification showing
tumor infiltrating CD8 + T
cells in B16 melanomas: PBS
(a), i.p. IL-21 (b), s.c. IL-21
(c) and RenCa carcinomas:
PBS (d), i.p. IL-21 (e), s.c. IL-
21 (f). Tumor biopsies were
obtained at the end of the
experiments shown in Fig. 2c,
d. CD8 + T cells were stained
with liquid permanent red by
immunohistochemistry
according to ‘‘Materials and
methods’’ and they are
indicated by arrows. Sections
have been counterstained
with Mayer’s hematoxylin
yielding a blue nucleus stain
Mean no. of cells/10 6 µm 2 (AOI)
+/- SEM
16
14
12
10
8
6
4
2
0
a
CD4 + T cells
Mean no. of cells/10 6 µm 2 (AOI)
+/- SEM
16
14
12
10
8
6
4
2
0
b
CD8 + T cells
Mean no. of cells/10 6 µm 2 (AOI)
+/- SEM
160
140
120
100
80
60
40
20
0
c
PBS i.p. IL-21 i.p. IL-21 s.c
Mean no. of cells/10 6 µm 2 (AOI)
+/- SEM
160
140
120
100
80
60
40
20
0
d
PBS i.p. IL-21 i.p. IL-21s.c.
Fig. 5 IL-21 increases the density of tumor infiltrating CD8 + T
cells. The bar plots show the density of tumor infiltrating CD4 + T
cells (a and c) and CD8 + T cells (b and d) in B16 melanomas (a
and b) and the RenCa carcinomas (c and d). Tumor biopsies
were obtained at the end of the experiments shown in Fig. 2c, d
and stained for CD8 + and CD4 + T cells by immunohistochemistry.
All positive cells located intratumorally were counted in
one section from each biopsy and related to a stereologically
measured area of interest (AOI) excl. necrotic areas and nontumor
tissue, such as connective tissue. Bars represent mean ±
SEM, n = 7–10, *P < 0.05, **P < 0.01, Student’s t-test. AOI area
of interest
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1424 Cancer Immunol Immunother (2007) 56:1417–1428
density, consequently increasing the CD8 + /CD4 + T cell
ratio in both tumor models.
Subcutaneous administration of IL-21 results
in a slow release from the injection site
and significant draining to regional lymph nodes
In order to examine whether differences in the
biodistribution of IL-21 could account for the observed
efficacy after s.c. versus i.p administration, the kinetics
of the two different routes of administration was
compared. Particularly, we wanted to investigate the
degree of regional LN drainage of IL-21 after s.c.
administration, since IL-21 in this compartment
could be relevant during the generation of immune
responses. 125 I labeled IL-21 was used to measure the
kinetics of IL-21 distribution in several major organs
and tissues as listed in Materials and methods. Figure 6
shows the distribution over time at the s.c. injection
site, i.e the right foot (Fig. 6a), and in the popliteal LNs
(Fig. 6b). The results show that upon s.c. injection
125 I-IL-21 is released slowly from the injection site and
a considerable amount is drained through the regional
LN (C max ~ 4.7% of injected dose). In contrast, very
low levels was detected in the popliteal LN node after
i.p. injection and in the contralateral LN after s.c.
injection (C max < 0.1% of injected dose). We found no
specific retention or major differences in the biodistribution
between i.p. and s.c. over time in the major
organs: heart, lungs, liver, spleen, kidneys, and small
and large intestines (data not shown). The thyroid
gland generally showed an increased radioactivity over
time after both routes of administration due to its
retention of free 125 I.
IL-21 has a greater bioavailability and higher peak
serum concentration after subcutaneous
administration
Next, we examined the serum concentration-time
profiles of IL-21 after i.v., i.p. and s.c. administration to
examine whether the pharmacokinetics of IL-21 contributes
to the observed efficacy differences. IL-21 was
detectable in serum after 5 min with all administration
routes. A peak serum concentration of 113 ng/ml of
IL-21 was reached 1 h post i.p administration, whereas
s.c. administration provided a higher peak IL-21 concentration
of 230 ng/ml 2 h post injection (Fig. 7). S.c.
administration resulted in a more sustained concentration
of IL-21 in serum and 2.5-fold higher AUC
compared to i.p administration (665 h·ng/ml vs.
242 h·ng/ml after s.c. and i.p. administration, respectively).
The AUC after i.v. administration was even
a 90
125 I-IL-21 s.c.
80
70
60
50
40
30
20
10
0
0 1 2 3 4 5 6 7 8
Hours post injection
Radioactivity/Dose %
b
Radioactivity/Dose %
6
5
4
3
2
1
125 I-IL-21 s.c., right node (injection side)
125 I-IL-21 s.c., left node
125 I-IL-21 i.p., right node
0
0 1 2 3 4 5 6 7 8
Hours post injection
Fig. 6 125 I-IL-21 shows slow release and significant lymph
drainage following s.c. administration.
125 I-IL-21 was injected
either i.p. or s.c. in the right footpad in BALB/c mice, and
animals were sacrificed at indicated time points and c-radiation
at the subcutaneous injection site (a) and in popliteal LNs (b)
was measured. Each data point represents the mean ± SEM of
triplicate mice
Serum IL- 21 conc. (ng/mL)
+/-SEM
10000
1000
100
10
1
0.1
IL-21 i.p.
IL-21 s.c.
IL-21 i.v.
0 1 2 3 4 5 6 7 8
Hours post injection
Fig. 7 Greater bioavailability and peak serum conc. of IL-21
after s.c. administration. Fifty lg IL-21 was injected i.p. in the
lower right abdominal quadrant, s.c. in the right flank or i.v. in
the tail vein of BALB/c mice. Blood samples were drawn at
indicated time points and the serum concentration of IL-21 was
measured by ELISA. Each data point represents mean ± SEM
of triplicate mice
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Cancer Immunol Immunother (2007) 56:1417–1428 1425
higher (1,010 h·ng/ml) due to the very high initial
IL-21 concentration that within 1 h decreased to a level
approximately 10-fold lower than both i.p. and s.c
administrations (Fig. 7). The bioavailability was 24%
and 66% for i.p. and s.c. administration, respectively.
These results indicate that IL-21 has a prolonged
pharmacokinetic profile following s.c. administration
with a higher peak serum concentration and greater
bioavailability compared to i.p. administration.
Discussion
Previous studies of IL-21 anti-tumor activity have been
conducted with IL-21 secreting tumors [7,8,19,37], IL-
21 expression plasmids [38], in model systems immunogenically
enhanced with foreign antigens, or by the
use of adoptive transfer of antigen specific lymphocytes
[13,20,26,39]. Although these studies support the concept
that IL-21 aids in the destruction of cancer, these
methods of IL-21 administration are not clinically
applicable. More recently, a number of studies have
shown anti-tumor effects of intraperitoneal administration
of recombinant IL-21 protein [13,32,35]. In
these studies IL-21 therapy was given either in combination
with other therapies, as prophylactic therapy
at or before tumor inoculation, and only for very few
days duration. Here, we have used intraperitoneal and
subcutaneous administration of IL-21 protein to mice
bearing established syngeneic tumors in order to study
the effects of IL-21 alone under more therapeutically
relevant conditions, and IL-21 was administered
throughout the experiments to maximize the treatment
effect. The use of s.c. administration of cytokines for
cancer therapy has previously been applied successfully,
as s.c. administration of IL-2 resulted in less adverse
events yet maintaining efficacy and enabling
outpatient treatments [11]. In addition, s.c. administration
is believed to be more convenient for the
patients. We report that IL-21 protein therapy significantly
inhibits tumor growth in two syngeneic tumor
models. The effect was not due to a direct inhibition of
tumor cell proliferation, as also shown previously with
B16 tumor cells [38]. Our data indicate that s.c.
administration of IL-21 in the RenCa model is at least
as effective as or perhaps more effective than i.p.
administration with early as well as late therapy initiation.
This might be due to prolonged availability of
IL-21, since s.c. administration of IL-21 resulted in a
more prolonged presence of the protein in serum and
higher bioavailability compared to i.p. administration.
Furthermore s.c. administration also resulted in a significant
passage of IL-21 through regional lymphatics,
which may also have contributed to an improved antitumor
immune response. IL-21 has been shown to expand
antigen stimulated CD8 + T cells [17,39] and this
effect might be further improved by the direct stimulation
of CD8 + T cells in LNs during an immune response.
This notion is supported by our finding that
CD8 + T cells were indispensable for the reduced tumor
growth observed after IL-21 treatment. Interestingly, it
has also been shown that the site of immunization
produces a subsequent site-specific homing of T cells
and accompanying anti-tumor activity [5]. Thus, s.c.
administration of IL-21 might also mount an immune
response targeted more specifically against the s.c.
compartment, where the tumors in this study were
located. In the B16 model the efficacy difference
between s.c. and i.p. was less pronounced compared to
the more immunogenic RenCa model. It is not clear at
this point whether this difference is caused by differences
between the two tumor cell lines or differences
between their hosts (BALB/c and C57BL/6 mice for
RenCa and B16 tumors, respectively). It is possible
that different drug delivery methods may affect tumors
with different immunogenicity differently, as recently
described [4]. Altogether, our results suggest that s.c.
administration of IL-21 could be advantageous and
deserves clinical evaluation.
In our experiments we found that depletion of
NK1.1 + cells did not reduce the anti-tumor effect of
IL-21, whereas depletion of CD8 + cells or growth
of tumors in athymic mice completely abrogated the
effect of IL-21. Taken together, these data show that
CD8 + T cells are required for the anti-tumor activity in
our model, whereas NK cells played an insignificant
role under these settings. In the literature either CD8 +
T cells, NK cells, or both cell types have been described
as required for the IL-21 anti-tumor activity depending
on the models used [16]. Most of the studies demonstrating
NK cell-mediated anti-tumor activity of IL-21
have been performed in i.v. metastasis models where
IL-21 was administered at the time of tumor establishment
or in models using IL-21-expressing tumors
[2,19,34,35,37]. Two major differences might explain
these different effector mechanisms. First, we have
treated subcutaneous tumors, which are less accessible
to NK cells compared to intravenous tumors. Second, in
our experiments treatment was initiated only after the
tumors became established. The larger tumor burden in
this setting might overwhelm the effect of NK cells as
they are unable to clonally expand. In the i.v. metastasis
model where IL-21 therapy is given at the time of tumor
inoculation, NK-mediated killing of the tumor cells
may eradicate the tumor cells almost completely before
an adaptive immune response can evolve, consistent
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1426 Cancer Immunol Immunother (2007) 56:1417–1428
with the notion that the efficacy in this model was
sustained in Rag-1 -/- mice [2]. In a study by Ma et al.
[19] rejection of IL-21 secreting B16F1 tumor cells
inoculated subcutaneously required both NK cells and
CD8 + T cells. In this model, NK cell-depletion resulted
in rapid growth of the tumors, suggesting an early role
of NK cells, whereas CD8 + T cell-depletion resulted
in delayed tumor growth, suggesting that adaptive
immunity might have played a role later on to kill
remaining tumor cells, as suggested by the authors [19].
By contrast Wang et al. showed that depletion of NK
cells completely abrogated the anti-tumor effect of
IL-21 expressing plasmids administered day 5 and 12
post tumor inoculation with MCA205 fibrosarcomas
[38]. Although these results apparently contradict the
hypothesis suggested by us and others [4,19] that NK
cells primarily play a role early in the anti-tumor
response, it is possible that the much slower growth rate
of MCA205 cells compared to B16 cells allows NK cells
to kill MCA205 cells before the tumor burden becomes
too large, even though treatment is initiated day 5.
Also, differences in immunogenicity and expression of
ligands for activating and inhibitory NK cell receptors
between the two tumor lines might contribute to these
different results.
Several studies have shown that IL-21 can stimulate
CD8 + T cells to proliferate, increase their cytotoxicity,
and sustain survival [1,14,17,20,39]. In order to elicit
tumor cytotoxicity, CD8 + T cells must infiltrate the tumor
to come in close contact with the tumor cells. The
benefit of TILs and specifically CD8 + TILs and the
CD8 + /CD4 + TIL ratio have also been shown in several
different human cancers where they were predictive of
improved survival [9,12,21,22,28,30]. In this study, we
demonstrate that IL-21 protein given therapeutically
significantly increased the density of tumor infiltrating
CD8 + T cells without changing the CD4 + T cell density,
consequently increasing the CD8 + /CD4 + T cell ratio in
both models. Interestingly, the density of CD8 + TILs
was significantly higher after s.c. administration of IL-21
compared to i.p. administration in the RenCa carcinomas,
supporting our notion that s.c. administration
might be favorable. Generally, the RenCa carcinomas
also showed approximately 10-fold higher density of
infiltrating T cells compared to B16 tumors reflecting
the higher inherent immunogenicity of RenCa as previously
reported [15,29]. In a recently published article
a similar increase in CD8 + T cell infiltration was observed
in a small number of tumors following challenge
with an IL-21 expressing mouse bladder cancer [10].
There are several possible explanations for the
increased density of CD8 + TILs after IL-21 stimulation.
First, IL-21 has been shown to increase the proliferation
of antigen-stimulated CD8 + T cells [17,20,39], which in
turn could yield a greater pool of tumor specific T cells
infiltrating the tumor. Another possibility is that IL-21
sustains the survival of CD8 + T cells infiltrating the
tumor, which has been supported by both in vitro and
in vivo data [1,20]. Particularly, it has been shown that
IL-21 is able to sustain CD28 expression on CD8 + T
cells and increase their IL-2 production [1], both of
which could enhance their survival in a challenging
tumor environment [25]. In this context, it should be
noted that our results represent the conditions about
8–10 days after the first IL-21 dose, and one day after
the last dose, thus the effects of IL-21 have been sustained
for more than a week from the initial stimulation.
Finally, it is possible that IL-21 increases the
specific homing of CD8 + T cells into tumors. IL-21 has
been shown to increase CXC chemokines such as IP-10,
MIG and I-TAC in IL-21-secreting tumors [8], which
could work as T cell attractants [27], but to date there
are no data demonstrating that systemic IL-21 delivery
improves chemokine-mediated homing of CD8 + T cells
to tumors. Presently, the mechanism of IL-21 antitumor
activity remains to be fully elucidated, but the
finding that IL-21 increased tumor infiltration of CD8 +
T cells is a step forward that encourages the use of IL-21
in oncology.
In conclusion, we have shown that IL-21 protein
mono-therapy inhibited established syngeneic tumor
growth in two preclinical models of melanoma and
renal cell carcinoma. Furthermore, we have shown that
IL-21 markedly increased the density of tumor-infiltrating
CD8 + T cells, which are essential for the antitumor
effect. These findings support the use of IL-21 as
a promising new anti-cancer drug.
Acknowledgement We would like to thank Heidi Winther,
Bodil Andreasen, Birte Jørgensen and Kirsten Meeske for
technical assistance with the experiments, and Mark Smyth for
valuable discussion of the manuscript.
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48

Paper III:
Intratumoral interleukin 21 increases anti-tumor immunity, tumor-infiltrating CD8 + T cell
density and activity, and enlarges draining lymph nodes
Søndergaard H., Galsgaard E.D., Bartholomæussen M., Ødum N. and Skak K.,
J Immunother in press
49

Intratumoral interleukin 21 increases anti-tumor immunity, tumor-infiltrating
CD8 + T cell density and activity, and enlarges draining lymph nodes *
Henrik Søndergaard 1 , Elisabeth D. Galsgaard 1 , Monica Bartholomæussen 1 , Per Thor
Straten 2 , Niels Ødum 3 , Kresten Skak 1
1 Biopharmaceuticals Research Unit, Novo Nordisk A/S, Måløv Denmark, 2 Center for Cancer
Immunotherapy, Department of Hematology, Herlev University Hospital, Denmark, 3 Institute of
Biology, University of Copenhagen, Denmark
Corresponding author:
Henrik Søndergaard, M.Sc.
Novo Nordisk A/S
Department of Immunopharmacology
Novo Nordisk Park F6.2.30
DK-2760, Måløv, Denmark
Phone (direct): +45 44431376
Fax: +45 44434537
E-mail: hris@novonordisk.com
* This work was funded by Novo Nordisk A/S.
Short running title: Intratumoral IL-21 increases anti-tumor immunity, TIL activity and density,
and enlarges LNs
Key words: intratumoral, interleukin-21, tumor-infiltrating lymphocytes, T cells, draining lymph
nodes, cancer, melanoma, renal cell carcinoma
Abbreviations: IL-21, interleukin 21; SC, subcutaneous; IT, intratumoral; TILs, tumor-infiltrating
lymphocytes; NK cells, natural killer cells; Tregs, regulatory T cells; AOI, area of interest; WT,
wild type; LN, lymph node; CXCL, chemokine CXC motif ligand; CCL, C-C motif ligand
51

Abstract
Interleukin (IL)-21 is a novel cytokine in clinical development for the treatment of cancer. In this
study, we have compared the efficacy of subcutaneous (SC) and intratumoral (IT) administration of
IL-21 protein in two syngeneic mouse tumor models, RenCa renal cell carcinoma and B16
melanoma, and investigated the mechanisms by which IL-21 enhances CD8 + T cell-mediated antitumor
immunity.
We found that in comparison to SC administration, IT administration of IL-21 more potently
inhibited tumor growth and increased survival. This correlated with increased densities of tumorinfiltrating
CD8 + and CD4 + CD25 - T cells, but not CD4 + CD25 + FoxP3 + T cells. Furthermore, IT
administration of IL-21 increased degranulation, and expression of IFNγ and granzyme B in tumorinfiltrating
CD8 + T cells. Tumors injected with IL-21 grew slower than contralateral tumors,
suggesting that the increased efficacy of IT administration of IL-21 was due to a local rather than
systemic effect. IT administration of IL-21 led to enlarged tumor-draining lymph nodes (LNs), with
increased naïve lymphocyte numbers and proliferation of activated lymphocytes, suggesting that
local administration of IL-21 generally benefits the tumor microenvironment and activates tumordraining
LNs.
Overall, our data suggest that IL-21 augments CD8 + T cell-mediated anti-tumor immunity through
increased proliferation and effector function and acts both on tumor-infiltrating CD8 + T cells as
well as on the draining lymph nodes. IT administration led to superior CD8 + T cell proliferation,
effector function and anti-tumor efficacy, suggesting that IT administration of IL-21 may be
clinically useful in patients with unresectable tumors.
Introduction
The finding of tumor-infiltrating lymphocytes
(TILs) in human cancers shows that the
immune system to some degree recognizes
cancers. Several reports have shown the
benefit of TILs in human cancers, particularly
the number of CD8 + T cells and an increased
ratio of CD8 + /regulatory T cells (Tregs) are
associated with improved patient prognosis
(1-10). Adoptive cell therapy (ACT) with ex
vivo expanded autologous TILs has shown
encouraging response rates, indicating the
potential of trying to enhance TIL numbers
(11). However, far from all cancers contain
adequate amounts of TILs, and the anti-tumor
activity of TILs are rarely sufficient to control
the disease. This is mainly due to a variety of
inherent mechanisms that facilitate tumor
immune escape (12): Insufficient T cell
chemotaxis, lack of co-stimulation and
increased inhibitory ligand expression, coinfiltration
of suppressive immune cells such
as Tregs, the release of immune suppressive
soluble mediators e.g. IL-10 and TGFβ, and
generation of antigen-loss variants. Clearly,
TILs are faced with many hurdles in the
tumor microenvironment that hinder
successful immunotherapy. Therefore, novel
measures are needed that can boost both the
number and reactivity of TILs and at the same
time avoid infiltration of suppressive immune
cells.
Interleukin 21 (IL-21) is the latest member of
the common γ-chain cytokine family and is
currently in clinical development for the
treatment of cancer. IL-21 is produced by
activated CD4 + T cells and is particularly
found in follicular helper T cells and Th17
cells, and activated NKT cells (13-19). The
IL-21 receptor (IL-21R) is widely expressed
throughout the immune system including
macrophages, B, T, NK, NKT and dendritic
cells (20). Hence, IL-21 has pleiotropic
actions on the immune system which include
co-stimulation of B cell maturation and Ig
52

production, increased NK cell cytotoxicity
and bone marrow recruitment, co-stimulation
of T cell proliferation as well as CD8 + T cell
expansion and cytotoxicity (20). In vivo, IL-
21 has shown encouraging anti-tumor activity
in several different animal models, where the
anti-tumor activity was mainly dependent on
NK cells, CD8 + T cells or both (21).
Currently, IL-21 is in phase II clinical trials
for the treatment of stage IV melanoma and
renal cell carcinoma (22). So far, clinical
results have shown that the drug is generally
well tolerated (23), and immune activation
and signs of clinical activity have also been
observed (22;24). To support the further
development of IL-21 as an oncology drug it
will be important to fully understand the
pleiotropy of this cytokine to yield the best
clinical outcome, hereby understand the
therapeutic functions of the cytokine in vivo,
establish the most optimal route of
administration and possibly identify potential
biological markers of efficacy.
Previously, we have shown that subcutaneous
administration of IL-21 protein was more
effective than intraperitoneal administration
and led to increased densities of tumorinfiltrating
CD8 + T cells in syngeneic mouse
tumor models (25). However, it still remains
to be shown whether a local administration of
IL-21 can further augment the anti-tumor
activity compared to systemic treatment, and
whether it would be even more potent in
modulating the density and activity of tumorinfiltrating
T cells. IL-2, which is closely
related to IL-21 and approved as therapy for
stage IV melanoma and renal cell carcinoma,
has previously shown improved patient
outcome and less severe adverse effects after
local administration compared to systemic
treatment (26).
In this study, we have compared intratumoral
and subcutaneous administration of IL-21 in
two syngeneic subcutaneous mouse tumor
models. Our data show potent anti-tumor
efficacy and increased survival after
intratumoral administration, enlargement of
tumor-draining lymph nodes (LNs) and
increased density and activation of tumorinfiltrating
CD8 + T cells, suggesting that local
administration of IL-21 could improve patient
outcome and is warranted for clinical
investigation.
Materials and Methods
Mice
Wild type (WT) C57BL/6 and BALB/c
female mice were purchased from Taconic
Europe A/S, Lille Skensved, Denmark,
whereas female C57BL/6 nu/nu (nude) mice
(B6.Cg/Ntac-Foxn1 nu N9) and female
C57BL/6 β2m -/- (B6.129-B2m tm1Jae N12) were
acquired from Taconic, Hudson, NY, USA.
The animals were 6-8 weeks old on arrival
and were allowed to acclimatize for at least
one week before start of experiments. WT
mice were housed in a standard animal
facility whereas nude and β2m -/- mice were
isolated in a facility for immunodeficient
animals. Light was controlled on a 12 hr lightdark
cycle, and the animals were given free
access to food and drinking water. The
animals were observed daily for clinical signs
and their body weights were recorded
regularly. All experiments were approved by
the relevant ethical committees and conducted
in accordance with corporate policies on
animal welfare.
Cell lines
C57BL/6-derived B16 (F0) melanoma cells
(American Type Culture Collection (ATCC),
CRL-6322) and BALB/c-derived RenCa renal
cell carcinoma cells (kindly provided by Dr.
Robert H. Wiltrout, NCI at Frederick, MD,
USA) were cultured in RPMI 1640 with
GlutaMAX TM supplemented with 10% heatinactivated
FCS, sodium pyruvate (RenCa
only), nonessential amino acids (RenCa only),
and 5% penicillin-streptomycin (all from
GIBCO Cell Culture, Invitrogen, Denmark).
Reagents and antibodies
Recombinant murine IL-21 (IL-21) protein
provided by Novo Nordisk A/S, Denmark was
53

used in all experiments. The stock solutions
contained IL-21 in a concentration of 10.5
mg/mL and working preparations were
diluted in PBS, IL-21-vehicle diluted
similarly was used as control. For the
histological examination we used rat antimouse
CD4 (clone RM4-5), rat anti-mouse
CD8a (clone 53-6.7) (both from BD
Pharmingen, CA, USA), rat serum IgG2a
(Serotec, UK), and biotin-conjugated donkey
anti-rat IgG (Jackson ImmunoResearch
Laboratories Inc., PA, USA). For flow
cytometry analysis the following antibodies
were used: Rat anti-mouse CD45
Allophycocyanin (APC)-Cy7 (clone 30-F11),
rat anti-mouse CD8a APC (clone 53-6.7), rat
anti-mouse CD4 Peridinin Chlorophyll
Protein Complex (PerCP) (clone RM4-5), rat
anti-mouse CD19 PE-Cy7/PerCPCy5.5 (clone
1D3), rat anti-mouse CD25 Fluorescein
isothiocyanate (FITC) (clone 7D4), rat antimouse
CD62L PE (clone MEL-14), rat antimouse
IFNγ APC (clone XMG1.2), rat IgG1,
κ (isotype), rat anti-mouse CD107a FITC
(clone 1D4B), rat IgG2a, κ FITC (isotype), 7-
amino-actinomycin D (7-AAD) staining
solution (all from BD Pharmingen, CA,
USA), hamster anti-mouse TCRβ FITC (clone
H57-597), hamster anti-mouse TCRβ Alexa
Flour 750 (clone H57-597), rat anti-mouse
CD8 Pacific Blue (clone 53-6.7), rat antimouse
CD44 FITC/APC (clone IM7), rat antimouse
FoxP3 PE (clone FJK-16s), rat antimouse
granzyme B PE (clone 16G6), rat
IgG2b, κ PE (isotype) (all from eBioscience,
CA, USA), rat anti-mouse CD62L APC
(clone MEL-14) (Beckman Coulter, CA,
USA), rat anti-mouse CD4 Pacific Orange
(clone R2a30), rat anti-mouse CD45 Pacific
Orange (clone 30-F11) (both from CALTAG
laboratories, Invitrogen, CA, USA), rabbit
anti-rat/human KI67 FITC (clone SP6) and
rabbit IgG FITC (isotype) (both from Abcam
plc, UK).
In vivo tumor models
On day 0, C57BL/6 WT, C57BL/6 nu/nu and
C57BL/6 β2m -/- were inoculated
subcutaneously in the right flank with 10 5
B16 melanoma cells. BALB/c mice were
inoculated in the right or in both flanks with
2x10 5 RenCa renal cell carcinoma cells. All
mice were randomized and ear-tagged prior to
treatment. The tumor volume was measured
as two perpendicular diameters with a digital
caliper approximately three times per week,
and calculated by the following formula:
2
Volume = 0.5 × d
1
× d
2
, if d
1
< d
2
, where d represents
the two diameters.
Treatment with IL-21 was initiated when
tumors reached a mean volume between 40
mm 3 to above 100 mm 3 . Fifty µg IL-21 or
vehicle in a dosing volume of 30 µL using a
0.3 ml BD Micro-Fine+ insulin syringe with a
8 mm 30G needle (BD Pharmingen, CA,
USA) was administered intratumorally (IT) in
the center of the tumor nodule or in a volume
of 200 µL subcutaneously (SC) 3x/week in
the BALB/c-RenCa model and 1x/daily in the
C57BL/6-B16 model. This dose was chosen
on the basis of experiments in our previous
work (25). Termination criteria were a tumor
volume of 1000 mm 3 , which was used as a
surrogate survival endpoint in Kaplan-Meyer
analysis, or more than 20% weight loss from
time of cell inoculation.
Immunohistochemistry of tumorinfiltrating
T cells
Six µm cryo-sections were made from tumorbiopsies
taken out at termination of the
therapeutic studies. Sections were
immunohistochemically stained with rat antimouse
CD4 or rat anti-mouse CD8 antibodies
(5 µg/ml), whereas rat serum IgG2a was used
as matching isotype control. Biotinconjugated
donkey anti-rat IgG (diluted
1:3000) was used as secondary antibody.
Sections were fixed in 4% paraformaldehyde
at 4°C and endogenous biotin activity was
blocked using Biotin blocking system from
Dako A/S, Denmark. Prior to the antibody
staining non-specific binding was blocked by
54

incubation in TBS with 3% skim milk, 3%
BSA, and 7% donkey serum. Incubation with
the primary antibodies was made at 4°C over
night followed by 1 h incubation at RT with
the secondary antibody, both diluted in TBS
with 0.5% skim milk, 3% BSA, and 7%
donkey serum. Streptavidin conjugated
alkaline phosphatase and Liquid Permanent
Red Chromogen (Dako A/S, Denmark) was
used to visualize positive cells and sections
were counterstained with Mayer’s
hematoxylin to reveal nuclei morphology.
Stereological quantification of tumorinfiltrating
T cells
A stereological method was used to quantify
the density of tumor-infiltrating T cells.
Images from immunohistochemically stained
tumor sections were analyzed via online light
microscopy at 20× magnification using
C.A.S.T. grid software ver. 2.3.1.3 from
Olympus Denmark A/S, Denmark. In all
tumor sections (one from each tumor) the
density of tumor-infiltrating lymphocytes was
blindly scored counting all positive cells
intratumorally and relating them to an area of
interest (AOI), representing the total tumor
area measured stereologically, excluding
necrotic tumor tissue and non-tumor tissue
such as peritumoral connective tissue. The
C.A.S.T. grid software and a motorized stage
system enabled side by side imaging to ensure
that no area was omitted or evaluated twice.
Flow cytometric analysis of tumorinfiltrating
lymphocytes
Tumor-biopsies were taken out at the
termination of experiments or on day 1, 5, and
10 post treatment-start and weighed.
Mechanical dissection with scalpel blades
followed by enzymatic digestion for 1 h at
37°C with collagenase type IV (Sigma-aldrich
GmbH, Germany) and DNase I (Roche
Diagnostics GmbH, Germany) were used to
dissociate the tumor tissue. Single cell
suspensions were made by passage through a
40µm BD Falcon cell strainer (BD
Biosciences, CA, USA). Cells were surface
stained with appropriate antibodies for 30-40
min and washed. A FoxP3 staining kit
(eBioscience, CA, USA) was used for
intracellular staining of cells according to the
manufactures instructions. Briefly, cells were
fixed for 60 min using the Fix/Perm buffer,
washed once and stained for 30 min in
permeabilization buffer. The stained cells
were transferred to BD TruCOUNT tubes
(BD Biosciences, CA, USA) for
quantification of cells. An LSR II flow
cytometer (BD Biosciences, CA, USA) was
used for acquisition and data were analyzed
using BD FACSDiva software (BD
Biosciences, CA, USA). Live lymphocytes
were indentified based on FSC/SSC
properties and gated as 7AAD - CD45 + . The
absolute number of TCRβ + CD8 + ,
TCRβ + CD4 + , TCRβ + CD4 + CD25 - ,
TCRβ + CD4 + CD25 + ,
TCRβ + CD4 + CD25 + FoxP3 + and TCRβ - CD19 +
cells were calculated based on the number of
acquired TruCOUNT beads related to the
total number of beads per tube as provided by
the manufacturer. The absolute number of the
different cell populations in each biopsy was
related to the biopsy weight. CD8 + T cell
activation was analyzed directly ex vivo
detecting the percentage of CD107a + and
IFNγ + TCRβ + CD8 + T cells as well as the
median fluorescence intensity of granzyme B
in TCRβ + CD8 + T cells, all compared to
matching isotype controls.
Quantification of tumor-draining lymph
node lymphocytes
On day 0, BALB/c mice were inoculated
subcutaneously in the right hind foot pad with
1x10 6 RenCa renal cell carcinoma cells. Mice
were randomized and ear-tagged prior to
treatment and tumor volume was measured
three times per week as the perpendicular
diameters and calculated as described above.
Intratumoral treatment with 50 µg IL-21 was
initiated approximately on day 12 post tumor
inoculation when the mean tumor volume >
40 mm 3 and given 5x/week for 8 days. One
day after the last IL-21 injection the popliteal
55

lymph nodes (LNs) in the tumor-bearing and
contralateral legs were removed. LNs were
forced through a 40µm BD Falcon cell
strainer (BD Biosciences, CA, USA) using a
pestle, and total viable cell numbers were
counted on a ViCell automated cell viability
counter (Beckman Coulter, CA, USA). Cells
were stained with appropriate antibodies for
30 min. and washed. Intracellular staining
was done using a FoxP3 staining kit
(eBioscience, CA, USA) according to
manufactures instructions described above.
Acquisition was performed on a LSR II flow
cytometer (BD Biosciences, CA, USA) and
data were analyzed using BD FACSDiva
software (BD Biosciences, CA, USA). Cell
frequencies obtained by flow cytometry were
used to calculate the absolute number of cell
subpopulations based on the total cell count.
The following gating strategy was used: FSC-
H/FSC-A was used to eliminate doublets and
FSC-A/SSC-A to gate live lymphocytes.
TCRβ, CD4, CD25, CD8, and CD19 were
used to discriminate main T cell populations
and B cells. CD62L and CD44 were used to
further discriminate between activated and
naive T cells. KI67 expression was used to
identify proliferating cells compared to a
matching isotype control..
Statistics
Student’s t-test (two-tailed, assuming equal
variance), two-tailed Mann-Witney U-test or
One-way ANOVA with Tukey’s post test was
used for statistical evaluations of differences
between treated and control groups as
indicated in figure legends. Mantel Cox Log
Rank test was used to evaluate statistical
differences in Kaplan-Meyer analyses and
correlation data were compared by the
Spearman non-parametric correlation
coefficient. Data are generally shown as
mean±SEM unless otherwise noted.
Bonferroni correction was used to correct for
mass significance and a P value less than 0.05
was considered statistically significant.
Results
Intratumoral administration of IL-21
augments anti-tumor efficacy
Previously, we have shown superior antitumor
efficacy of subcutaneous (SC)
administration of IL-21 compared to
intraperitoneal therapy in mice (25). In this
study we wanted to investigate whether
equivalent doses of IL-21 given locally could
further augment the anti-tumor effect of this
cytokine. Initially, we inoculated RenCa renal
cell carcinoma cells subcutaneously in
syngeneic BALB/c mice. Intratumoral (IT)
IL-21 treatment was initiated when tumors
were established with a mean tumor volume
above 40 mm 3 . Our results showed that 50 µg
IL-21 administered intratumorally potently
inhibited growth of RenCa tumors compared
to vehicle treatment (p

in the local tumor environment provides
increased anti-tumor activity.
Intratumoral IL-21 increases long-term
anti-tumor effect compared to
subcutaneous administration
In light of the increased efficacy on tumorgrowth
inhibition of intratumoral
administration of IL-21 compared to
subcutaneous administration we next
investigated whether this effect would
translate into improved long-term survival.
Domestic ethical guidelines on animal
experiments prohibit use of death as an
endpoint, thus Kaplan-Meyer survival
analysis was conducted with the surrogate
survival endpoint ‘time to tumor size >1000
mm 3 ’. BALB/c mice bearing subcutaneous
RenCa tumors were treated with subcutaneous
or intratumoral administration of 50 µg IL-21.
Here, significantly increased survival time
was observed in mice injected intratumorally
with IL-21 compared to both vehicle
treatment (p

One section from each tumor-biopsy was
stained and the density of tumor-infiltrating T
cells was scored by counting positively
stained cells in a stereologically measured
area of interest (AOI), which excluded
peritumoral connective tissue and necrotic
tissue. Representative pictures are shown of
CD8 + (Figure 4a and b) and CD4 + (Figure 4c
and d) stained cells in IL-21 and vehicle
treated tumors. The density of CD8 + T cells
was increased 9.3 fold following intratumoral
IL-21 treatment compared to controls
(p

We also quantified the density of
CD4 + CD25 + FoxP3 + regulatory T cells (Tregs)
on day 1 and 10 post treatment-start (Figure
5d). On day 1, there was no difference in the
density of Tregs between treatments.
Interestingly, on day 10 the density of Tregs
decreased after intratumoral administration of
IL-21 despite the increased CD4 + CD25 - T cell
density, whereas the density of Tregs after
subcutaneous administration correlated more
with the increased CD4 + CD25 - T cell density.
Although these differences did not reach
statistical significance they indicate that
intratumoral administration of IL-21 can
selectively increase the density of certain
tumor-infiltrating T cell subsets in RenCa
tumors, but not Tregs, considerably increasing
the ratio of CD8 + T cells/Tregs.
Intratumoral IL-21 increases IFNγ and
granzyme B expression, as well as
degranulation of tumor-infiltrating CD8 + T
cells
The significant effects on the densities of
tumor-infiltrating T cells led us to examine
whether these infiltrating T cells also showed
increased activation following intratumoral
administration of IL-21. In a similar
experiment to the one described in figure 1b,
we stained TILs from RenCa tumors for IFNγ,
granzyme B, and the degranulation-associated
molecule CD107a on day 1, 5 and 10 post
treatment-start and analyzed the expression of
these molecules by flow cytometry. TILs
were gated into CD45 + TCRβ + CD8 + T cells
and in figure 6a representative FACS plots
from day 5 post treatment-start show the
expression of the three molecules after
vehicle, subcutaneous and intratumoral IL-21
treatment. On day 1, CD107a expression was
similar between treatments and minor
increases were observed in the expression of
granzyme B and IFNγ after intratumoral IL-
21, but no significant differences (Figure 6b).
On day 5, subcutaneous administration of IL-
21 showed minor increases in CD107a,
granzyme B and IFNγ expression, however
compared to both vehicle and subcutaneous
administration, intratumoral administration of
IL-21 significantly increased the percentage
of tumor-infiltrating CD8 + T cells expressing
CD107a and IFNγ (p

compared to LNs draining vehicle-treated
tumors (Figure 7a). The average number of
cells in the tumor-draining LNs increased
from 2.1x10 6 in vehicle to 5.7x10 6 in IL-21
treated mice (p

Previously, we have used IL-21 protein
administered by conventional routes and
shown that subcutaneous administration of
IL-21 had anti-tumor activity and was able to
increase the density of tumor-infiltrating
CD8 + T cells (25). Here, we have investigated
whether local administration of IL-21 further
increases the anti-tumor effects of IL-21
compared to systemic treatment. The clinical
applicability of local therapy is more limited
than systemic treatment, but is valid e.g. in
unresectable metastasis in melanoma or as
adjuvant therapy in association with surgery,
and local therapy lowers the risk of systemic
adverse effects. Results with IL-2, which is
approved for the treatment of renal cell
carcinoma and metastatic melanoma, have
shown improved responses after intratumoral
administration in metastatic skin disease of
melanoma (28) and less severe side effects
than systemic treatment (26). This indicates
that local therapy is valid in certain clinical
settings and points out the rationale for
exploring this route of administration also for
IL-21.
In this study, we demonstrate the first results
using local administration of IL-21 protein in
two different preclinical solid tumor models –
RenCa renal cell carcinoma and B16
melanoma. Our results show that intratumoral
administration of IL-21 potently inhibits B16
and RenCa tumor growth leading to
prolonged long-term survival, particularly in
the RenCa model, compared to a similar dose
by subcutaneous administration. These results
indicate that IL-21 has improved anti-tumor
effects when present locally in the tumor
environment and that local IL-21 therapy
could improve patient outcome, where it is
applicable.
Generally, our data showed that RenCa
carcinomas had increased responses to IL-21
therapy compared to B16 melanomas, which
is consistent with the literature where RenCa
cells have generally been found to be more
immunogenic compared to B16 cells (29;30).
Specifically, B16 melanomas have been
described to have very poor immunogenic
characteristics with low MHC expression and
no transporters associated with antigen
processing (TAP)-1 expression (reviewed in
31). We have previously shown that the
density of tumor-infiltrating CD8 + T cells was
approximately 10-fold higher in RenCa
compared to B16 tumors (25), and theses
findings are supported by the difference in
MHC class I expression on our B16 and
RenCa cells (Supplementary figure 1). Thus,
adaptive immune responses are likely to have
a greater role in immune responses against
RenCa tumors compared to B16 tumors.
So far, the anti-tumor activity of IL-21 has
mainly been assigned to either NK cells,
CD8 + T cells or both depending on the
specific models used (21). Previously we have
shown that CD8 + T cells were the main
mediators of IL-21 anti-tumor activity using
B16 melanomas, whereas NK cells were of
less importance (25). Here, we have extended
these data to intratumoral injection of IL-21
where the anti-tumor activity against B16
melanomas was lost in athymic mice and in
β2m-deficient mice. The mechanism of the
CD8 + T cell-dependent anti-tumor activity of
IL-21 is not understood in detail; IL-21
increases cytotoxicity and IFNγ production
from CD8 + T cells in concert with other
stimuli (32-34), and co-stimulates CD8 + T
cell proliferation and antigen-specific CD8 + T
cell expansion and survival (35-38). However,
these data have mainly been generated in vitro
by stimulation of CD8 + T cells from e.g.
spleen or blood, and it remains to be shown
whether IL-21 similarly can activate CD8 + T
cells in vivo and more importantly those
infiltrating tumors. Here, we found that IL-21
increased the surface expression of CD107a,
and intracellular expression of granzyme B
and IFNγ in RenCa tumor-infiltrating CD8 + T
cells analyzed directly ex vivo. Increased
expression of granzyme B and IFNγ in CD8 +
T cells have both been found in IL-21 clinical
trials (22;24), suggesting that these molecules
are very relevant markers of IL-21 activity.
CD107a residing in cytotoxic granules is
exposed on the cell surface upon
61

degranulation and is associated with tumor
cytolytic activity (39). Therefore, our findings
suggest that IL-21 stimulates cytolytic activity
directly in tumors. Seeing that IL-21 activates
tumor-infiltrating CD8 + T cells in vivo is
encouraging, and that it does so even more
when given locally supports the use of
intratumoral administration.
Although IL-21 can augment expansion,
activity and survival of CD8 + T cells, these
cells still need to get into contact with tumor
cells and be present in proper numbers to
mediate effective killing. Previously, we have
shown that subcutaneous administration of
IL-21 increased the density of tumorinfiltrating
CD8 + T cells, which also have
been indicated by others (40;41). In this
study, we found that intratumoral IL-21
therapy increased the density of tumorinfiltrating
CD8 + T cells even further
compared to subcutaneous administration in
both B16 and RenCa tumors and increased the
density of CD4 + CD25 - T cells, suggesting that
part of the increased anti-tumor activity of
intratumoral IL-21 may be ascribed to
increased TIL numbers. This notion is further
supported by our finding that the density of
tumor-infiltrating CD8 + T cells and to less
extent CD4 + T cells correlated with tumor
inhibition. Furthermore, these data suggest
that the density of tumor-infiltrating CD8 + T
cells might be a relevant biological marker of
IL-21 anti-tumor activity.
The mechanism by which IL-21 increases the
density of tumor-infiltrating T cells is still not
clear, and could have several explanations.
First, IL-21 may increase the expansion of
antigen-specific CD8 + T cells (33;35;37;38),
which could produce a greater number of
tumor-reactive cells infiltrating the tumor.
Our findings that tumor-draining LNs were
enlarged in response to intratumoral IL-21
treatment with increased proliferation of
activated T cells is supportive of this option,
but the increased LN activation and tumor
infiltration remains to be directly linked.
Second, the ability of IL-21 to sustain
survival of both CD4 + and CD8 + T cells in
vitro (36) and CD8 + T cells in vivo (37) could
mediate an increase in the long-term tumor T
cell densities. However, we observed a
striking increase in T cell densities already 5
days post treatment-start, making it less likely
that increased survival was the main
mechanism, and rather points to an active
response. Third, IL-21 has been shown to
mediate the release of soluble factors involved
in lymphocyte trafficking such as chemokine
CXC motif ligand (CXCL)9, CXCL10 and
CXCL11 (40) and C-C motif ligand (CCL)20
(42), and shown up-regulation of relevant
chemokine receptors in cancer patients (24),
indicating that IL-21 could increase T cell
chemotaxis, but direct evidence of this still
remains. Finally, whether or not IL-21 in it
self could act as a T cell attractant is another
possibility but this remains speculative.
Interestingly, despite increased densities of
tumor-infiltrating CD8 + and CD4 + CD25 - T
cells, the density of tumor-infiltrating Tregs
rather decreased following intratumoral IL-21
treatment in RenCa tumors. This indicates
that IL-21 can selectively increase the density
of certain T cells in tumors without increasing
the density of Tregs. These findings are
supported by studies showing that IL-21 has
no direct effects on Tregs (43;44). Also,
recent data suggest that the percentage of
tumor-infiltrating Tregs is decreased in IL-21
secreting tumors (45). It is not known what
the mechanism behind this selectivity is, but
IL-21 has been suggested to suppress the
expression of FoxP3 (46), and the number of
FoxP3 + cells in culture (38). Although we did
observe a lower FoxP3 expression in the
tumor-infiltrating CD4 + CD25 + T cell
population following intratumoral IL-21
treatment, this was not significant (data not
shown).
Nonetheless, our finding that intratumoral
administration of IL-21 increased the density
of CD8 + T cells in RenCa tumors, while the
density of Tregs decreased, suggests a highly
increased tumor-infiltrating CD8 + T cell/Treg
ratio. In the clinic, prognostic evaluations of
TILs have shown that not only the level of
62

CD8 + T cell infiltration, but especially a high
tumor-infiltrating CD8 + T cell/Treg ratio was
a significant independent prognostic factor of
improved overall survival (4;7).
In general, our data suggest that IL-21 has
improved anti-tumor activity when present in
the local tumor environment. This is
supported by studies of IL-21-expressing
tumors where tumors are unable develop
(40;47-49). Also, we found that in animals
bearing subcutaneous tumors on both flanks,
the tumors injected intratumorally with IL-21
grew slower than contralateral tumors. Our
finding that local administration of IL-21
enlarged tumor-draining LNs, increased the
number of naïve CD4 + and CD8 + T cells and
the proliferation of activated T cells, is
additional evidence that IL-21 benefits the
local tumor environment.
Although IL-21 can co-stimulate proliferation
of naïve T cells (33;50), we mainly found
increased proliferation of activated T cells,
pointing to non-proliferative effects of IL-21
on LN T cells. IL-21-induced chemokine
secretion (40;42) could locally increase the
recruitment of T cells to the draining LN.
Also, IL-21 has been shown to maintain T cell
expression of CD62L and CCR7 (51;52),
thereby keeping T cells in a more precursorlike
state which may favor their retention in
secondary lymphoid organs or simply skew
the distribution toward more CD62L + cells.
Still, it remains to be fully clarified what the
causative mechanism is and whether this
increase in naïve T cells in draining LNs has
any impact on the anti-tumor response.
Similarly, B cell numbers also significantly
increased in tumor-draining LNs following
IL-21 treatment, and particularly naïve B cells
(data not shown). But again, proliferation was
mainly seen in activated B cells, showing that
IL-21 also has both proliferative and nonproliferative
effects on LN B cells. IL-21 costimulates
B cell proliferation, but induces B
cell apoptosis without proper co-stimulation
(53), indicating the presence of B cellactivating
antigens in the RenCa tumor
model. This also points to a possible role for
B cells in the anti-tumor activity of IL-21,
which has previously been suggested (54-56),
but this remains to be clarified in the B16 and
RenCa models and was beyond the scope of
this paper.
In conclusion, we have shown that compared
to subcutaneous administration, intratumoral
administration of IL-21 protein has superior
anti-tumor activity, which may be ascribed to
increased activation of tumor-infiltrating
CD8 + T cells as well as increased density of
tumor-infiltrating CD8 + T cells, with the latter
being strongly associated with tumor growth
inhibition. Overall, our data warrant the
clinical investigation of local IL-21 therapy
and suggest that the density of tumorinfiltrating
CD8 + T cell is a relevant
biological marker of IL-21 anti-tumor effect.
Acknowledgements
This work was funded by Novo Nordisk A/S
and we would like to thank Birte Jørgensen,
Heidi Winther and Tonja Lyngse Jørgensen
for technical assistance with the experiments.
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66

Figures
a
Mean tumor volume (mm 3 )
±SEM
b
Mean tumor volume (mm 3 )
±SEM
1100 Vehicle IT
1000 IL-21 IT
900
800
700
600
500
400
300
200
100
0
* ** ** ***
8 10 12 14 16 18 20 22
Days post RenCa inoculation
***
900 Vehicle IT
800
700
600
500
50μg IL-21 SC
2μg IL-21 IT
10μg IL-21 IT
50μg IL-21 IT
#
400
300
200
100
* * * *
0
8 10 12 14 16 18 20 22 24 26
Days post RenCa inoculation
Figure 1. Intratumoral IL-21 administration
increases anti-tumor efficacy in RenCa
carcinomas compared to subcutaneous
administration. BALB/c mice were injected with
2x10 5 RenCa cells SC in the right (a and b) or in
both flanks (c) and randomized prior to treatmentstart
as indicated by arrows. Treatment was started
with a mean tumor volume > 40 mm 3 (a) >100 mm 3
(b) >50 mm 3 (c). Fifty μg or indicated doses of IL-
21 or vehicle was injected IT or SC 3x/week. Mean
± SEM, n = 9 (a), n = 8, representative of two
independent experiments (b), n = 10 (c), # P=0.09,
*P

Percent < 1000 mm 3
100
80
60
40
20
Treatment (day 13-29, 3x/w eek)
*
**
0
0 10 20 30 40 50
Days post RenCa inoculation
Vehicle IT
IL-21 IT
IL-21 SC
Figure 2. Intratumoral IL-21 therapy increases survival compared to subcutaneous administration. BALB/c mice
were injected with 2x10 5 RenCa cells SC in the right flank and randomized prior to treatment-start as indicated.
Treatment was started with a mean tumor volume > 40 mm 3 . Fifty µg IL-21 or vehicle was injected IT or SC 3x/week
for the indicated period of time. The chart shows Kaplan-Meyer survival analysis of mice with tumors < 1000 mm 3 , n =
10, representative of two independent experiments *P

a
Mean tumor volume (mm 3 )
±SEM
1000 Vehicle IT
900 IL-21 IT
800
700
600
500
400
300
200
100
***
***
** *
0
2 4 6 8 10 12 14 16
Days post B16 tumor inoculation
b
Mean tumor volume (mm 3 )
±SEM
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
0
Vehicle IT
50μg IL-21 SC
2μg IL-21 IT
10μg IL-21 IT
50μg IL-21 IT
8 10 12 14 16 18 20 22
Days post B16 tumor inoculation
*
c
Mean tumor volume (mm 3 )
±SEM
1100 Vehicle IT
1000
900
800
700
600
500
400
300
200
50μg IL-21 IT
C57BL/6 nu/nu
100
0
6 7 8 9 10 11 12 13 14 15
Days post B16 tumor inoculation
d
Mean tumor volume (mm 3 )
±SEM
1000 Vehicle IT
900
800
700
600
500
400
300
200
100
50μg IL-21 IT
C57BL/6 β2m -/-
0
6 7 8 9 10 11 12 13 14 15 16
Days post B16 tumor inoculation
Figure 3. Intratumoral IL-21 increases CD8 + T cell-dependent anti-tumor effect in B16 melanomas. C57BL/6 WT
(a and b), C57BL/6 nu/nu (c) and CD57BL/6 β2m-/- (d) mice were injected with 1x10 5 B16 melanoma cells SC in the
right flank and randomized prior to treatment-start as indicated. Treatment was started with a mean tumor volume > 50
mm 3 (a) > 75 mm 3 (b), > 60 mm 3 (c and d). Fifty μg or indicated doses of IL-21 or vehicle was injected IT or SC
1x/day. Mean ± SEM, n = 15, representative of two independent experiements (a), n = 7 (b), n = 10 (c and d), *P

a
c
Vehicle IT
Vehicle IT
b
d
e
CD8 + T cells
15 ***
IL-21 IT
f
CD4 + T cells
15 **
IL-21 IT
Cells / mm 2 AOI
10
5
Cells / mm 2 AOI
10
5
0
Vehicle IT
IL-21 IT
0
Vehicle IT
IL-21 IT
Figure 4. Intratumoral IL-21 increases the density of tumor-infiltrating T cells. Cryo-sections from tumor-biopsies
obtained at the end of the experiment shown in figure 3a were stained for CD4 + and CD8 + T cells by
immunohistochemistry according to ‘Materials and methods’. Positive cells are visualized with liquid permanent red
and indicated by arrows. Nuclei are blue by Mayer’s hematoxylin staining. All positive cells located intratumorally
were counted in one section from each biopsy and related to a stereologically measured area of interest (AOI) excl.
necrotic areas and non-tumor tissue, such as connective tissue. Representative pictures of cryo-sections at 20×
magnification showing tumor-infiltrating CD8 + (a and c) and CD4 + T cells (b and d) in B16 melanomas treated as
shown. The bar plots show the density of tumor-infiltrating CD8 + T cells (e) and CD4 + T cells (f) scored from the
immunohistochemically stained sections. Bars represent mean±SEM, n=15, **P

a
CD8 + T cells
b
Cells/ g tumor tissue
c
150000
100000
50000
0
5000000
*
Vehicle
IL-21 SC
CD8 + T cells
**
IL-21 IT
800000
CD8 + T cells/ g tumor tissue
1000000
100000
10000
1000
***Spearman correlation p=0.0005
100
10 100 1000
Tumor volume (mm 3 )
CD4 + CD25 - T cells
d
Vehicle
IL-21 SC
IL-21 IT
Day 10 Day 5 Day 1
Cells / g tumor tissue
Cells / g tumor tissue
Cells / g tumor tissue
4000000
3000000
2000000
1000000
0
5000000
4000000
3000000
2000000
1000000
0
5000000
4000000
3000000
2000000
1000000
0
Vehicle
Vehicle
Vehicle
IL-21 SC
IL-21 SC
p=0.06
IL-21 SC
IL-21 IT
**
***
IL-21 IT
*
IL-21 IT
Cells / g tumor tissue
Cells / g tumor tissue
Cells / g tumor tissue
600000
400000
200000
0
1000000
800000
600000
400000
200000
0
800000
600000
400000
200000
0
Vehicle
Vehicle
Vehicle
IL-21 SC
IL-21 SC
IL-21 SC
IL-21 IT
*
*
IL-21 IT
IL-21 IT
Cells / g tumor tissue
Cells / g tumor tissue
500000
400000
300000
200000
100000
0
250000
200000
150000
100000
50000
0
Vehicle
Tregs
IL-21 SC
IL-21 IT
Figure 5. The density of tumor-infiltrating CD8 + and CD4 + CD25 - T cells, but not Tregs increases after
intratumoral IL-21 correlating with tumor-growth inhibition. BALB/c mice bearing SC RenCa tumors were treated
with vehicle, 50 µg IL-21 SC or IT similar to the experiment in figure 1b. Tumor-biopsies were taken out and weighed
at the end of the experiment and the absolute numbers of tumor-infiltrating CD8 + T cells were quantified by flow
cytometry and related to biopsy weight (a), and the CD8 + T cell density was related to final tumor volume (b). In a
separate but similar experiment tumor-biopsies were taken out on day 1, 5 and 10 post treatment-start and the densities
of tumor-infiltrating CD8 + and CD4 + CD25 - T cells were quantified (c), also on day 1 and day 10 post treatment-start the
density of tumor-infiltrating CD4 + CD25 + FoxP3 + T cells (Tregs) were quantified (d). Bars represent mean±SEM, n=7
(a) and n=4 (c and d), *P

a
Day 5 post treatment-start
Isotype
Vehicle
IL-21 SC
IL-21 IT
b
CD107a
Granzyme B
IFNγ
Day 5 Day 1
%CD107a+CD8+ T cells
%CD107a+CD8+ T cells
8
6
4
2
0
20
15
10
5
0
*
Granzyme B Median FI
of CD8+ T cells
Granzyme B Median FI
of CD8+ T cells
550
500
450
400
350
300
250
2000
1500
1000
500
0
*
%IFNγ+CD8+ T cells
%IFNγ+CD8+ T cells
3
2
1
0
20
15
10
5
0
*
Day 10
%CD107a+CD8+ T cells
2.5
2.0
1.5
1.0
0.5
0.0
Vehicle
IL-21 SC
*
IL-21 IT
Granzyme B Median FI
of CD8+ T cells
2000
1500
1000
500
0
Vehicle
IL-21 SC
IL-21 IT
%IFNy+CD8+ T cells
15
10
5
0
Vehicle
IL-21 SC
*
IL-21 IT
Figure 6. Intratumoral IL-21 increases IFNγ and granzyme B expression and degranulation in tumorinfiltrating
CD8 + T cells. BALB/c mice bearing SC RenCa tumors were treated with vehicle, 50 µg IL-21 SC or IT
similar to the experiment in figure 1b. Tumor-biopsies were taken out on day 1, 5 and 10 post treatment-start and
stained for intracellular IFNγ and granzyme B, and for surface expression of CD107a. Representative FACS plots are
shown from day 5 post treatment-start (a) and bar plots show the percent of CD107a + , median fluorescence intensity
(MFI) of granzyme B expression and percent of IFNγ expressing CD8 + T cells from day 1, 5 and 10 post treatment-start
(b). Bars depict mean±SEM, n=4, *p

a
IL-21
Vehicle
b
Number of cells (10 6 )
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
**
*
* * *
IL-21
Vehicle
*
c
Number of cells (10 6 )
1.5
1.2
0.9
0.6
0.3
0.0
**
*
**
IL-21
Vehicle
Vehicle
IL-21
% KI67+ cells
15 IL-21
Vehicle
10
5
**
*
**
*
0
TCRβ + CD8 + T cells
TCRβ + CD4 + T cells
TCRβ+CD8+
TCRβ+CD4+
TCRβ - CD19 + B cells
TCRβ+CD19+
Number of KI67+ cells (10 6 )
0.08
0.06
0.04
0.02
0.00
*
IL-21
Vehicle
**
*
Number of KI67+ cells (10 6 )
0.15
0.10
0.05
0.00
IL-21
Vehicle
** *
0.20
0.15
0.10
0.05
0.00
IL-21
Vehicle
*
CD62L+CD44+
CD62L-CD44+
CD62L+CD44+
CD62L-CD44+
CD62L+CD44+
CD62L+CD44-
CD62L-CD44-
CD62L+CD44-
CD62L-CD44-
CD62L+CD44-
CD62L-CD44-
CD62L-CD44+
Total cells
Lymphocytes
TCRβ+CD4+
TCRβ+CD8+
CD4+CD25+
TCRβ-CD19+
CD62L+CD44+
CD62L-CD44+
CD62L+CD44+
CD4+CD25-
CD62L+CD44-
CD62L-CD44-
CD62L+CD44-
CD62L-CD44-
CD62L-CD44+
d
CD8 + T cells CD4 + T cells
e
f
Number of KI67+ cells (10 6 )
Figure 7. IL-21 enlarges tumor-draining lymph nodes by increasing naïve lymphocyte numbers and proliferation
of activated lymphocytes. BALB/c mice were inoculated with 1x10 6 RenCa tumor cells SC in the hind foot pad and
treated on day 12 post inoculation with vehicle or 50 µg IL-21 IT 5x/week. On day 20 popliteal LNs were taken out and
the absolute number of LN lymphocytes was counted and analyzed by flow cytometry. Macroscopic picture of IL-21
and vehicle treated draining LNs (a), the absolute number of the indicated lymphocyte populations in tumor-draining
LNs (b), and the absolute number of the indicated T cell subpopulations (c). In an identical experiment LN lymphocytes
were stained for intracellular KI67 and analyzed by flow cytometry. Representative FACS plots show KI67 staining in
CD8 + and CD4 + T cells from vehicle and IL-21 treated mice with the distribution of CD62L and CD44 expression (d),
the percent of KI67 + CD8 + and CD4 + T cells, and CD19 + B cells are shown (e), and the number KI67 + CD8 + and CD4 +
T cells and CD19 + B cells are shown based on their CD62L and CD44 expression as indicated (f). Bars represent
mean±SEM, n=5, *P

B16
RenCa
Supplementary figure 1. Greater MHC class I expression on RenCa compared to B16 cells. B16 melanoma and
RenCa renal cell carcinoma cells were stained with primary unlabelled mouse anti-H-2Kb and H-2Db (B16) and mouse
anti-H-2Kd and H-2Dd (RenCa). Primary staining with mouse IgG2a was used as an isotype control. Secondary
staining with goat anti-mouse IgG PE was used for detection by flow cytometry. Histogram X-axes depict the level of
MHC class I expression as fluorescence intensity of B16 (left) and RenCa (right) after secondary staining.
Representative of two independent experiments.
74

a
SC in lower leg
IT in foot tumor
b
4.0
3.5
IL-21
Vehicle
Cell count (10 6 cells)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Total cells
Lymphocytes
CD4+
CD8+
CD19+
CD4+CD25-
CD4+CD25+
Supplementary figure 2. IL-21 increases lymphocyte numbers in naïve lymph nodes draining the foot pad.
BALB/c mice inoculated with 1x10 6 RenCa tumor cells SC in the foot pad were injected once with 30µL of Pantent
Blue V dye 12 days post inoculation either SC in the lower leg or IT and popliteal LNs were taken out 30 min. later.
The macroscopic pictures show two representative popliteal LNs after injection with Pantent Blue V dye SC in the
lower leg or IT in foot pad tumor as indicated (a). BALB/c mice bearing no tumors were injected in the foot pad with
50 µg IL-21 on days 0, 2 and 4, popliteal LNs were taken out on day 7 and LN lymphocytes were quantified by flow
cytometry and the bar plot shows the absolute number of the indicated lymphocyte populations (b). Bars represent
mean±SEM, n=5, P

Supplementary figure 3.
Cell count (10 6 cells)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
IL-21
Vehicle
Total cells
Lymphocytes
TCRb-CD19+
TCRb+CD8+
TCRb+CD4+
CD4+CD25-
CD4+CD25+
Supplementary figure 3. Intratumoral IL-21 does not modulate contralateral lymph nodes. BALB/c mice were
inoculated with 1x10 6 RenCa tumor cells SC in the right hind foot pad and treated on day 12 post inoculation with
vehicle of 50 µg IL-21 IT 5x/week (from experiment in figure 6). On day 20 the left popliteal LNs (contralateral to the
tumor site) were taken out and the absolute number of indicated LN lymphocytes was quantified by flow cytometry.
76

Paper IV:
Endogenous interleukin 21 restricts CD8 + T cell expansion and is not required for tumor
immunity
Søndergaard H., Coquet J.M., Uldrich A.P., McLaughlin N, Godfrey D.I., Sivakumar P.V. Skak
K. and Smyth M.J., J Immunol 2009 Dec 1;183(11):7326-36. Epub 2009 Nov 13.
77

Published November 13, 2009, doi:10.4049/jimmunol.0902697
The Journal of Immunology
Endogenous IL-21 Restricts CD8 T Cell Expansion and Is not
Required for Tumor Immunity 1
Henrik Søndergaard,* ‡ Jonathan M. Coquet, † Adam P. Uldrich,* Nicole McLaughlin,*
Dale I. Godfrey, † Pallavur V. Sivakumar, § Kresten Skak, ‡ and Mark J. Smyth 2 *
IL-21 has antitumor activity through actions on NK cells and CD8 T cells, and is currently in clinical development for the
treatment of cancer. However, no studies have addressed the role of endogenous IL-21 in tumor immunity. In this study, we have
studied both primary and secondary immune responses in IL-21 / and IL-21R / mice against several experimental tumors. We
found intact immune surveillance toward methylcholanthrene-induced sarcomas in IL-21 / and IL-21R / mice compared with
wild-type mice and B16 melanomas showed equal growth kinetics and development of lung metastases. IL-21R / mice showed
competent NK cell-mediated rejection of NKG2D ligand (Rae1) expressing H-2b RMAS lymphomas and sustained transition
to CD8 T cell-dependent memory against H-2b RMA lymphomas. -Galactosylceramide stimulation showed equal expansion
and activation of NKT and NK cells and mounted a powerful antitumor response in the absence of IL-21 signaling, despite reduced
expression of granzyme B in NKT, NK, and CD8 T cells. Surprisingly, host IL-21 significantly restricted the expansion of
Ag-specific CD8 T cells and inhibited primary CD8 T cell immunity against OVA-expressing EG7 lymphomas, as well as the
secondary expansion of memory CD8 T cells. However, host IL-21 did not alter the growth of less immunogenic MC38 colon
carcinomas with dim OVA expression. Overall, our results show that endogenous IL-21/IL-21R is not required for NK, NKT, and
CD8 T cell-mediated tumor immunity, but restricts Ag-specific CD8 T cell expansion and rejection of immunogenic tumors,
indicating novel immunosuppressive actions of this cytokine. The Journal of Immunology, 2009, 183: 7326–7336.
The novel class I cytokine IL-21 is a member of the common
-chain receptor family. IL-21 is primarily produced
by activated CD4 T cells and NKT cells (1, 2) and signals
through its unique IL-21R. IL-21R is expressed by most leukocytes
including B, T, NK, NKT, macrophages, and dendritic
cells (DCs), 3 giving IL-21 substantial effects in both humoral and
cellular immune responses; these effects include costimulation of
B cell proliferation, differentiation, and isotype switching, Th17
cell differentiation of CD4 T cells, increased CD8 T cell expansion
and effector function, and activation of NK and NKT cells
(3). These profound immunomodulatory effects of IL-21 regulates
immune responses in a variety of diseases, including infections,
autoimmunity, and cancer (3–5).
The antitumor effects of IL-21 have been extensively investigated
in mouse tumor models using a range of different sources of
IL-21 delivery such as, IL-21-transfected tumor cell lines, IL-21-
*Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne,
† Department of Microbiology and Immunology, The University of Melbourne,
Parkville, Victoria, Australia; ‡ Immunopharmacology, Novo Nordisk A/S, Måløv,
Denmark; and § Zymogenetics, Seattle, WA 98102
Received for publication August 17, 2009. Accepted for publication October 6, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grant 454569 from the National Health and Medical
Research Council of Australia Program (to M.J.S., and D.I.G.), by a Cancer Research
Institute Postgraduate Scholarship (to J.M.C.), a Doherty Fellowship (to A.P.U.), and
by National Health and Medical Research Council Research Fellowships (to M.J.S.
and D.I.G.). D.I.G. and M.J.S. have received research support from Novo Nordisk
A/S.
2 Address correspondence and reprint requests to Dr. Mark J. Smyth, Peter MacCallum
Cancer Centre, St Andrews Place, East Melbourne, 3002, Victoria, Australia.
E-mail address: mark.smyth@petermac.org
3 Abbreviations used in this paper: DC, dendritic cell; MCA, methylcholanthrene;
GC, alpha-galactosylceramide; MSCV, murine stem cell virus; WT, wild type.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
expressing plasmids and recombinant mouse IL-21. In this study,
antitumor effects have been demonstrated both on established s.c.
tumors, on lung and liver metastases from i.v. injected tumors, and
on disseminated tumors (4). In addition to its antitumor effects as
monotherapy, IL-21 shows additional efficacy when used in combination
with several other therapies (5). The antitumor activity of
IL-21 is mainly mediated by NK cells (6–8) and CD8 T cells
(9–11) with requirement of IFN-, perforin, or both (7, 8, 12–14)
depending on specific model conditions. Consistently, IL-21 has
been found to sustain Ag-specific CD8 T cell responses (9), augmenting
both naive and memory CD8 T cell expansion (11, 15)
and boosting both CD8 T cell and NK cell cytotoxicity in vitro
(11, 16). In a few reports, B cells have also been suggested to be
involved in IL-21 antitumor responses with increased production
of tumor-specific IgG (17, 18). Moreover, IL-21 has been found to
modulate the activity and cytokine production of NKT cells and
enhance the antitumor effects mediated by NKT cell stimulation
(2, 8, 19). Based on these data, IL-21 is currently in clinical trials
for the treatment of metastatic melanoma and renal cell carcinoma
where it has shown an acceptable safety profile with reports of
responding patients along with several indications of in vivo immune
activity (20–22).
The majority of studies on IL-21 in tumor immunology have
used exogenous sources of IL-21. However, IL-21 also plays an
important role in the pathogenesis of several autoimmune diseases
and here the role of endogenous IL-21/IL-21R has been widely
studied. Particularly, crossing of IL-21R-deficient (IL-21R / )
mice onto diabetes prone NOD mice, spontaneous arthritis susceptible
K/XbN mice, and BXSB-Yaa mice that develop a systemic
lupus erythematosus-like syndrome all showed significantly reduced
disease activity (23–25). Furthermore, collagen-induced arthritis
were reduced in DBA/1 mice treated with IL-21R.Fc (26)
and IL-21 / mice showed resistance to DSS- and TNBS-induced
colitis (27). To this end, blockade of the host IL-21/IL-21R axis
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902697
79

The Journal of Immunology
has been suggested as a potential therapeutic opportunity in several
of these autoimmune disorders.
To date, no studies have investigated the role of endogenous
IL-21/IL-21R signaling in the control of tumor initiation, growth
and metastasis. Based on the evident antitumor effect of exogenous
IL-21, we wanted to explore the contribution of host signaling via
the endogenous IL-21/IL-21R axis in both primary and secondary
tumor immune responses. In fully backcrossed IL-21 and IL-21R
gene-targeted mice, we investigated the response to several different
experimental tumors controlled by different effector cells or
with responsiveness to IL-21 therapy. We show in this study that
endogenous IL-21 is not required for tumor immunity but reveal a
novel suppressive effect of IL-21 on CD8 T cell immunity.
Materials and Methods
Mice
C57BL/6 (B6) wild-type (WT) mice were purchased from the Walter and
Eliza Hall Institute of Medical Research (Melbourne, Victoria, Australia),
or bred in-house at the Peter MacCallum Cancer Centre Animal Facility
(Melbourne, Victoria, Australia). B6 IL-21-deficient (IL-21 / ) mice,
originally provided by Dr. P. Sivakumar (Zymogenetics, Seattle, WA), and
B6 IL-21R / mice, originally provided Dr. W. J. Leonard (National
Heart, Blood and Lung Institute, Bethesda, MD) were backcrossed to B6
for 8–12 generations at the Peter MacCallum Cancer Centre Animal Facility
(Melbourne, Victoria, Australia). B6 TCR.J18 / mice obtained
from Dr. M. Taniguchi (Chiba, Japan) were backcrossed for 12 generations
to B6 at the Peter MacCallum Cancer Centre Animal Facility (Melbourne,
Victoria, Australia). Mice age 6–14 wk were used in all experiments in
accord with animal ethics guidelines of the Peter MacCallum Cancer
Centre.
Cell lines and culture conditions
B16 (F10) melanoma cells (American Type Culture Collection (CRL-
6322; ATCC), RMA (H-2b ), RMAS (H-2b ), RMAS murine stem cell
virus (MSCV) (empty vector transfected) and stable transfectant RMAS-
Rae1, and MC38 OVA dim (28) were all maintained in RPMI 1640 supplemented
with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 g/ml
streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2
mM glutamax, and 15 mM HEPES buffer (all from Invitrogen). Chicken
OVA transfected EL-4 T cell lymphoma cells EG7 (CRL-2113; ATCC)
and a more immunogenic variant of EG7 were grown in similar medium
supplemented with 0.4 g/ml geneticin G418 (Invitrogen).
Reagents and Abs
-Galactosylceramide (GC), a marine sponge glycolipid that activates
CD1d-restricted NKT cells (29), was provided by the Pharmaceutical Research
Laboratories (Kirin Brewery), and working concentrations were prepared
in PBS. Methylcholanthrene (MCA) was purchased from Sigma-
Aldrich. For flow cytometric analysis, the following anti-mouse Abs were
used: FITC-conjugated, FoxP3 (FJK-16s) and CD44 (IM7) (eBioscience);
PE-conjugated CD25 (PC61.5) and TCR- (H57-597) (eBioscience); PE-
Cy5.5-conjugated TCR- (H57-597) (eBioscience); PE-Cy7-conjugated,
CD3 (145-2C11), CD8 (53-6.7), and NK1.1 (PK136) (BD Pharmingen);
allophycocyanin-conjugated TCR- (H57-597), CD62L (MEL-14), CD4
(GK1.5), B220 (RA3-6B2) (eBioscience), allophycocyanin-Cy7-conjugated
CD3 (145-2C11), CD8 (53-6.7) and B220 (RA3-6B2) (BD Pharmingen);
and Pacific blue-conjugated CD4 (RM4-5) (eBioscience). PE-conjugated
SIINFEKL-loaded MHC class I tetramer from Dr. A. Brooks
(University of Melbourne, Parkville, Australia) and GC-loaded CD1d tetramer
produced in house by K. Kyparissoudis (University of Melbourne,
Parkville, Australia), using a construct originally provided by M. Kronenberg
(La Jolla Institute for Allergy and Immunology, La Jolla, CA) were
used to detect OVA-specific CD8 T cells and NKT cells, respectively.
Furthermore, Fc receptor block (clone 2.4G2; grown in-house) was used in
all experiments to prevent nonspecific binding of Abs. Cell suspensions
were stained in FACS tubes or 96-well U-bottom plates for 30 min at 4°C
in the dark and washed between incubations. Flow cytometric acquisition
was performed on a FACSCanto-II or LSR-II (BD Biosciences). Nonviable
lymphocytes were excluded on the basis of staining with 7-aminoactinomycin
D (eBioscience) or hydroxystilbamidine methanesulfonate/Fluoro-
Gold (FG) (Molecular Probes). Analysis was performed using FlowJo software
(Tree Star) and FACSDiva software (BD Biosciences).
MCA-induced fibrosarcoma model
Male B6 WT, IL-21 / , or IL-21R / mice were injected s.c. with 100 l
of corn oil containing MCA (100 g or 25 g) on the left hind flank and
were monitored for the onset and progression of tumors on a weekly basis
until 50 wk of age (30). Survival was plotted using a Kaplan-Meier curve
in which the time when tumors exceeded 150 mm 2 was used as endpoint.
Other experimental tumor models
B6 WT, IL-21 / , or IL-21R / mice were inoculated s.c. in the right
flank with 10 5 B16F10 melanoma, 5 10 6 RMAS (H-2b-negative), 5
10 6 RMA-S MSCV (empty vector transfected), 5 10 6 RMAS-Rae1,
5 10 5 MC38 OVA dim , or 3 10 6 immunogenic EG7 cells. Rechallenges
were made more than 40 days post tumor regression with 10 6 RMA or 3
10 6 EG7 cells. In these experiments, tumor size was calculated as a product
of two perpendicular diameters measured with a digital caliper approximately
three times per week. The termination criterion was a tumor volume
of 150 mm 2 , which was used as a surrogate survival endpoint in Kaplan-
Meier analysis. In other experiments, B6 WT, IL-21 / , or IL-21R /
mice were injected i.v. in the tail vein on day 0 with 2 10 5 B16 melanoma
cells either unpulsed or pulsed for 2 days in vitro with 500 ng/ml
GC. On day 14 postinoculation, mice were sacrificed, lungs were harvested,
and the number of surface metastasis per lung was counted using a
dissecting microscope.
In vivo stimulation of NKT cells
B6 WT, IL-21 / , IL-21R / , or TCR.J18 / mice were injected i.p.
with 2 g of GC prepared in PBS at 200-l doses. After 2, 24, 72, or
144 h postinjection, livers and spleens were harvested to assay for NK,
NKT, and CD8 T cell activation by flow cytometry. NKT cells were
identified as TCR GC/CD1dtetramer cells, NK cells as
NK.1.1 TCR GC/CD1dtetramer and CD8 T cells as TCR CD8
cells. For intracellular staining, cells were cultured in GolgiStop (BD
Biosciences) for 2–4 h before being surface stained and subsequently fixed
and permeabilized using the BD Cytofix/Cytoperm Plus Fixation/Permeabilization
kit (BD Biosciences). In experiments in which GC was used
in conjunction with chicken OVA, B6 WT, IL-21 / , or TCR.J18 /
were injected with OVA (400 g/mouse) and GC (1 g/mouse) i.v. in
200 l of PBS. On days 7 and 35 postinjection, livers and spleens were
harvested and assayed for CD8 T cell activation by staining with SIIN
FEKL-loaded MHC class I tetramers. For intracellular IFN- staining, cells
were restimulated with SIINFEKL peptide for 12 h in vitro in the presence
of GolgiStop for the last 4 h, and subsequently stained for CD8 and intracellular
IFN- and analyzed by flow cytometry.
Statistics
7327
Student’s t test (two-tailed, assuming equal variance), two-tailed Mann-
Whitney U test or Kruskal-Wallis test with Dunn’s posttest was used for
statistical evaluations of differences between WT B6 mice and IL-21 / /
IL-21R / mice as indicated in each experiment. Mantel Cox Log Rank
test was used to evaluate statistical differences in Kaplan-Meier analyses.
Data are generally shown as individual observations or as mean SEM
unless otherwise noted, and p 0.05 was considered statistically
significant.
Results
Endogenous IL-21 does not protect from MCA-induced
carcinogenesis
To date no studies have evaluated the effect of endogenous IL-21/
IL-21R signaling in tumor immunity or tumor immune surveillance.
Initially we wanted to study the involvement of the IL-21/
IL-21R axis in tumor immune surveillance and here we used the
chemical carcinogen 3-MCA, which is known for its ability to
promote fibrosarcoma carcinogenesis (31) and illustrate natural antitumor
immunity (32). Groups of B6 WT, IL-21 / , and IL-
21R / mice were inoculated s.c. with 25 or 100 g of MCA and
observed for fibrosarcoma development over a period of 50 wk
(Fig. 1). At 100 g of MCA, 80% of WT mice were sacrificed
over the course of the experiment, whereas 50% were sacrificed
at the 25-g dose. However, at the doses examined in this study,
neither the onset nor incidence of MCA-induced fibrosarcomas
were significantly different between the IL-21 / and IL-21R /
80

7328 HOST IL-21 IN TUMOR IMMUNITY
FIGURE 1. Endogenous IL-21 does not protect against MCA-induced
carcinogenesis. Cohorts of mice were challenged with 25 or 100
g of the chemical carcinogen 3 MCA s.c. and were monitored for
sarcoma incidence. Data depict survival of B6 WT, IL-21 / , and IL-
21R / mice. Survival was defined as when tumor size 150 mm 2 for
n 16 –21 mice per group. Results are representative of two independent
experiments.
mice compared with WT, suggesting that natural immune surveillance
against chemically induced fibrosarcomas does not require
IL-21 signaling.
IL-21/IL-21R signaling does not protect against B16F10 tumor
growth or lung metastasis
In previous studies of IL-21 antitumor activity, we and others have
found that the B16 melanoma model was sensitive to treatments
with IL-21 alone or in different combinations (6, 10, 11, 14, 33). In
this study, we used the B16 melanoma model to examine whether
host-derived IL-21 or IL-21R signaling might play a role in the
control of B16 tumor growth. WT, IL-21 / , and IL-21R / mice
were injected with B16F10 melanoma cells s.c. and monitored for
tumor growth and survival (Fig. 2A) or i.v. and evaluated 14 days
later for development of lung metastases (Fig. 2B). The results of
the s.c. experiment showed equal growth kinetics of B16F10 in
both WT, IL-21 / , and IL-21R / with similar survival measured
as the individual time when tumor size exceeded 150 mm 2 .
Furthermore, after i.v. injection of B16F10, the number of metastases
observed in the lungs 14 days later was also equivalent between
WT and IL-21 / or IL-21R / mice. These results suggest
that endogenous IL-21/IL-21R signaling does not protect against
B16 melanoma growth or metastasis development.
Primary tumor rejection by NK cells via NKG2D and transition
to CD8 T cell immunity does not require IL-21R signaling
IL-21 has been suggested to mediate tumor rejection through NK
cells and more specifically via the activating receptor NKG2D (6,
7). To specifically investigate NK cell-mediated tumor immunity
and particularly NKG2D rejection mechanisms, we used the TAPdeficient
T cell lymphoma cell line RMAS, which expresses very
FIGURE 2. B16 melanoma growth and lung metastases are not controlled
by IL-21/IL-21R signaling. B6 WT, IL-21 / , and IL-21R / mice
were injected with B16F10 melanoma cells either s.c. with 1 10 5 cells
on the right flank and monitored for tumor growth and survival defined as
time when tumor size 150 mm 2 (A) or 2 10 5 cells i.v. and harvesting
of lungs 14 days later for evaluation of lung metastases (B). Tumor growth
curves represent mean SEM and Kaplan-Meier survival curves depict
survival defined as time when tumor size 150 mm 2 for n 9–12 mice
in A. Data show mean SEM for n 6–7 mice in B.
low levels of H-2b and a variant transfected to express the NKG2D
ligand retinoic acid inducible-1 (Rae1). RMAS lymphomas are
well known for their NK cell sensitivity both in vitro and in vivo
(34) and Rae1-transfected variants have been shown to enhance
the NK cell-mediated rejection (35). In this study, we inoculated
IL-21R / and WT mice s.c. with parental RMAS cells, RMAS
MSCV (RMAS cells transfected with an empty vector), and
RMAS-Rae1 (Fig. 3A). WT and IL-21R / showed equal outgrowth
of the RMAS and RMAS MSCV, whereas RMAS-Rae1
showed equal rejection in both strains (9 of 10 tumors were rejected
in WT and 9 of 11 tumors were rejected in IL-21R / ).
These results suggest competent NKG2D-dependent NK cell-mediated
tumor rejection despite IL-21R deficiency.
To further explore the functional properties of this NK cellmediated
rejection we next addressed whether or not endogenous
IL-21/IL-21R signaling plays a role in the transition from innate to
adaptive immunity, which has previously been suggested (16). In
this experiment, we rechallenged WT and IL-21R / mice that
initially had rejected RMAS-Rae1 over 40 days postregression
with Ag-processing competent and H-2b-positive cells RMA (Fig.
3B). As a control, RMA tumors were inoculated in naive WT mice
and these tumors all grew out. The RMAS-Rae1 immunized
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FIGURE 3. Primary NKG2D-dependent tumor rejection by NK cells
and transition to CD8 T cell memory is intact in IL-21R / mice. A, B6
WT and IL-21R / mice were challenged with 5 10 6 RMAS, RMAS
MSCV (empty vector), or RMAS-Rae1 s.c. on the right flank and monitored
for tumor growth. B, Naive B6 WT mice and WT and IL-21R /
mice that rejected their primary RMAS-Rae1 tumor challenge were rechallenged
40 days postrejection with 10 6 RMA cells s.c. on the contralateral
flank and monitored for tumor growth. Data in A depict mean
SEM for n 8–11 mice per group. Data in B show individual tumor
growth curves for n 6 mice (naive WT), n 10 mice (immune WT), and
n 9 mice (immune IL-21R / ).
mice, however, showed potent memory responses regardless of the
lack of IL-21R signaling with initial tumor growth followed by
complete rejection already within 10 days post-rechallenge in both
strains. These results suggest that IL-21R signaling is redundant in
the transition from an initial NK cell-mediated immunization to a
successful CD8 T cell-mediated response.
7329
NKT cell activation primes NK cells and mediates antitumor
response in the absence of IL-21/IL-21R function
Recently, our lab showed that NKT cells produce IL-21 following
GC stimulation and show activation in response to IL-21 stimulation
(2). In this experiment, we used GC stimulation as a way
to induce IL-21 production and to investigate whether the activation
and antitumor response of NKT cells could in part be mediated
by IL-21. We injected WT, IL-21 / , and IL-21R / with 2
g of GC i.p. and quantified the expansion of NKT cells after 2,
24, 72, and 144 h in liver and spleen using flow cytometry where
NKT cells were identified as TCR GC/CD1dtetramer lymphocytes
(Fig. 4A). Two hours after GC stimulation the detection
of NKT cells decreased compared with unstimulated mice and almost
disappeared after 24 h due to down-regulation of their TCR.
However, after 72 h a substantial expansion (4- to 7-fold in both
liver and spleen) of the NKT cell population was observed followed
by a contraction of the NKT cell population by 144 h (Fig.
4A). NKT cell expansion was found to be similar between WT and
IL-21 / /IL-21R / mice, suggesting a normal proliferative response
of NKT cells without IL-21 signaling. In addition, NKT
cells were stained for CD4 and intracellular IFN- expression after
GC stimulation (Fig. 4B). These data showed a rapid induction
of, and increased proportion of, IFN- expressing NKT cells after
2 and 24 h in both CD4 /CD4 subsets, which declined again at
72 h and almost returned to baseline by 144 h. FACS plots are only
shown for liver NKT cells but similar results were obtained in the
spleen (data not shown). No difference was seen between WT and
IL-21 / or IL-21R / NKT cell IFN- responses after GC
stimulation, suggesting that cytokine production from NKT cells in
response to GC does not require endogenous IL-21.
Because GC also stimulates potent IFN- production by NK
cells (36), NK cell activation was also examined by gating on NK
cells (GC/CD1d tetramer NK1.1 TCR ) and detecting their
intracellular IFN- expression (Fig. 4, C and D). Representative
FACS plots of gated liver NK cells are shown, but similar results
were found in spleen NK cells (data not shown). The results show
that NK cell proportions and intracellular IFN- expression was
comparable between NK cells from WT, IL-21 / and IL-21R /
mice following GC stimulation at all time points (Fig. 4, C and
D). Thus, IL-21 did not appear to be important for NK cell IFN-
production following GC stimulation.
The granularity/cytotoxicity of NK cells (33), CD8 T cells (11)
and NKT cells (2) can also be augmented by IL-21. In this experiment,
we analyzed the expression of intracellular granzyme B on
gated NKT, NK, and CD8 T cells following GC administration
(Fig. 4E). Intracellular granzyme B expression was most strikingly
up-regulated in NK cells within 24 h of stimulation. NKT, NK, and
CD8 T cells from WT mice expressed slightly more intracellular
granzyme B than IL-21 / mice at the 72 h time point. IL-21 also
appeared to be involved in the maintenance of NKT cell granzyme
B expression at 144 h as well as NK cell granzyme B expression
at 24 h. These results suggest that endogenous IL-21 is produced
after GC stimulation and may play a role in GC-mediated granzyme
B up-regulation and possibly cytotoxicity of NKT, NK, and
CD8 T cells. Therefore, to explore the capacity of IL-21 / NKT
cell-mediated tumor immune responses, we injected WT and IL-
21 / mice i.v. with B16F10 melanoma cells either unpulsed or
pulsed in vitro for 2 days with 500 ng/ml GC and 14 days later
lungs were harvested to count metastases (Fig. 4F). This experimental
protocol, based on a previously published approach (37),
affected neither the in vitro growth nor viability of the tumor cells
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7330 HOST IL-21 IN TUMOR IMMUNITY
FIGURE 4. Activated NKT cells prime NK cells and mediate antitumor response in the absence of IL-21 or IL-21R. B6 WT (n 4 mice), IL-21 /
(n 4 mice), and IL-21R / (n 2–4 mice) were administered 2 g of GC i.p. Liver and spleen specimens were harvested 2, 24, 72, or 144 h later
to assay for NKT and NK cell activation by flow cytometry. A, NKT cells were gated as TCR GC/CD1dtetramer and the absolute number of NKT
cells in the liver (left) and spleen (right) of mice after administration of GC are shown. B, Representative profiles of CD4 vs intracellular IFN- expression
of gated NKT cells from the liver following GC. Number represents the percentage of cells in that quadrant. C, Representative density profiles of TCR
vs NK1.1 expression on non-NKT cells in the liver following GC administration where gated region depicts NK cells (GC/
CD1dtetramer NK1.1 TCR ). The number represents the percentage of cells in that gate. D, Representative histograms of intracellular IFN- expression
by gated NK cells from C. WT and IL-21 / mice were administered 2 g of GC i.p. and liver and spleen specimens were harvested at the specified time
points and cells were stained for TCR-, GC/CD1dtetramer, NK1.1, CD8, and intracellular granzyme B. E, Representative histograms of n 4 mice at
each time point of granzyme B expression on gated NKT cells (GC/CD1d tetramer TCR ), NK cells (GC/CD1d tetramer NK1.1 TCR ) and CD8
T cells (GC/CD1d tetramer TCR CD8 ) from WT and IL-21 / mice after GC administration. F, B6 WT and IL-21 / mice were injected i.v. with
2 10 5 B16F10 melanomas either unpulsed or pulsed in vitro for 2 days with 500 ng/ml GC, 14 days later lungs were harvested and lung metastasis
were counted. Data depict mean SEM for n 7–8 mice. Representative experiment is of three separate experiments, and Kruskal-Wallis test with Dunn’s
posttest was used to compare B16 GC in WT and IL-21 / . , p 0.01; , p 0.001.
(data not shown). The results showed a significant reduction in the
number of lung metastases in mice that had received the GCpulsed
B16F10 cells compared with mice receiving unpulsed cells,
but this reduction was similar in WT and IL-21 / , suggesting that
IL-21 did not contribute to the NKT cell-mediated antitumor response
in this model. Similar results, but with increasing number
of lung metastases, were obtained when cells were pulsed with
lower concentrations of GC (data not shown).
CD8 T cell expansion, but not IFN- production is enhanced
in IL-21 / mice
Stimulation of NKT cells with GC have also been shown to enhance
CD8 T cell responses (38–40). To test whether IL-21 production
from NKT cells might contribute to the generation of
CD8 T cell-based immune responses, we injected WT, IL-21 / ,
or TCR.J18 / mice with chicken OVA protein i.p. 1 g of
GC and detected the Ag-specific CD8 T cell response after 7 or
35 days in spleens and livers via SIINFEKL-loaded MHC class I
tetramers (Fig. 5A). Immunization with OVA alone did not produce
a very strong Ag-specific response, whereas addition of GC
produced a marked increase in SIINFEKL-specific CD8 T cells
in both WT and IL-21 / on day 7. Intriguingly, CD8 T cells
expanded to a greater proportion in livers of IL-21 / mice ( p
0.05), suggesting that IL-21 inhibited the expansion of Ag-specific
CD8 T cells. As a control NKT cell-deficient TCR.J18 / mice
showed no increased response to OVA plus GC. On day 35, the
level of OVA-specific CD8 T cells were reduced to the levels
seen with OVA immunization alone in both strains. For a more
functional readout, splenocytes from WT or IL-21 / mice were
also re-stimulated on day 7 or 35 with SIINFEKL peptide for 12 h
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7331
FIGURE 6. OVA dim expressing MC38 tumor growth is similar in WT
and IL-21 / mice. B6 WT and IL-21 / mice were challenged with 5
10 5 MC38-OVA dim cells s.c. on the right flank and monitored for tumor
growth. Survival was defined as time when tumors 150 mm 2 . Tumor
growth curves represent mean SEM and Kaplan-Meier survival curves
depict survival defined as time when tumor size 150 mm 2 for n 12
mice.
FIGURE 5. CD8 T cell expansion, but not IFN- response is enhanced
in IL-21 / mice. B6 WT, TCR.J18 / , or IL-21 / mice were injected
i.v. with either 400 g of OVA alone or 400 g of OVA plus 1 g/mouse
of GC i.p. A, On days 7 or 35, spleens and livers of mice were harvested
to detect the Ag-specific CD8 T cell response by SIINFEKL-loaded MHC
class I tetramer staining. B, On day 7 or day 35 after OVA plus GC
injection, splenocytes from WT or IL-21 / mice were restimulated with
SIINFEKL peptide for 12 h in vitro, stained for CD8 and intracellular
IFN-, and the percentages of CD8 IFN- were obtained by flow cytometry.
Each data point represents individual mice and Mann-Whitney U
test was used to compare WT and IL-21 / groups. , p 0.05.
in vitro, stained for CD8 and intracellular IFN- expression and
analyzed by flow cytometry (Fig. 5B). On day 7 a detectable
amount of IFN- was found (0.1%–0.2% of lymphocytes), but
with similar levels in both WT and IL-21 / and on day 35 the
levels of IFN- had dropped to very low levels. Taken together,
these results suggest that an Ag-specific CD8 T cell response can
be mounted by NKT cell stimulation without IL-21 production, but
that endogenous IL-21 has suppressive effects on Ag-specific
CD8 T cell expansion at least in liver.
Primary antitumor response against OVA dim expressing MC38
colon carcinoma is normal in IL-21 / mice
Seeing that both NK cell- and NKT cell-mediated tumor immune
responses were largely unchanged by the lack of endogenous
IL-21 and IL-21R, but that a CD8 T cell response increased in
IL-21 / mice, we next wanted to explore the functionality of
CD8 T cell-dependent antitumor immune responses without host
IL-21 signaling. Initially, we used a MC38 colon carcinoma cell
line transfected to express low levels of OVA (MC38 OVA dim ).
This cell line was recently established in our laboratory and found
to give 100% outgrowth in WT mice, but with a period of tumor
growth inhibition (our unpublished observations). Another MC38
variant established with high expression of OVA (MC38
OVA bright ) showed complete CD8 T cell-dependent rejection in
WT (28), suggesting that MC38 OVA dim tumors generate an insufficient
immune response for tumor rejection with the potential
for modulation in IL-21 / mice. We injected WT and IL-21 /
mice with MC38 OVA dim cells s.c. and monitored tumor growth
and survival, defined as time when tumor size 150 mm 2 (Fig. 6).
The results showed equal tumor growth and survival in WT and
IL-21 / mice with a period of tumor growth delay observed in
both strains. These results suggest that MC38 OVA dim tumor
growth is not controlled by IL-21-dependent mechanisms.
IL-21 / and IL-21R / mice show potent CD8 T cell
mediated antitumor response against immunogenic EG7 tumors
To investigate the CD8 T cell response in another and more
immunogenic tumor model we used an immunogenic variant of the
OVA-expressing EL-4 lymphoma cell line EG7 grown in our laboratory.
EG7 lymphomas have previously been found to respond to
IL-21 therapy (9). Our EG7 variant gives a minor fraction of spontaneous
rejections in WT mice (our unpublished observations), so
this model has an active CD8 T cell driven tumor immune response
that possibly could be modulated by IL-21/IL-21R deficiency.
WT and IL-21 / mice (Fig. 7A) or IL-21R / mice (Fig.
7B) were inoculated with EG7 cells s.c. and monitored for tumor
growth and survival defined as the time when individual tumor size
exceeded 150 mm 2 . The results showed, as previously observed,
spontaneous complete rejection in WT mice of 18% (3/17) and
33% (5/15) in the two separate experiments shown. Interestingly,
IL-21 / and IL-21R / mice both showed increased rejection
rates of 53% (9/17) and 94% (15/16), respectively. In Kaplan-
Meier survival analysis this difference was significant for both IL-
21 / and IL-21R / when compared with WT ( p 0.01). Furthermore,
we detected the OVA-specific CD8 T cell response in
blood using SIINFEKL-loaded MHC class I tetramers on day 13
postinjection of tumor in the IL-21 / experiment (Fig. 7C) and
84

7332 HOST IL-21 IN TUMOR IMMUNITY
FIGURE 7. IL-21 / and IL-21R / mice show powerful immune response against immunogenic EG7 tumors. In two separate experiments, B6 WT
and IL-21 / (A) or IL-21R / (B) mice were challenged with 3 10 6 EG7 cells s.c. on the right flank and monitored for tumor growth and survival,
which was defined as time when tumors 150 mm 2 . On day 13 postinjection of tumor, blood was drawn from WT and IL-21 / mice and stained with
SIINFEKL-loaded MHC class I tetramers (C) and similarly on day 10 postinjection tumor in WT and IL-21R / mice (D). Data are individual tumor growth
curves showing number of mice in which tumors have grown out or regressed out of total mice in A and B. Kaplan-Meier survival curves depict time when
individual tumors exceeded 150 mm 2 , Mantel Cox Log Rank test was used to evaluate statistical differences in survival between WT and IL-21 / or
IL-21R / . , p 0.01. Dot plots represent the percentage of SIINFEKL-specific cells of total CD8 T cells in blood from individual mice. Mann-
Whitney U test was used to compare differences between WT and IL-21 / or IL-21R / . , p 0.05.
day 10 postinjection of tumor in the IL-21R / experiment (Fig.
7D). In general, more than 90% of the detected OVA-specific
CD8 T cells had the phenotype CD62L CD44 (data not
shown), indicating that they were activated effector-memory cells.
In the IL-21 / experiment the results on day 13 showed a robust
OVA-specific CD8 T cell response with a significant increase in
the percentage of OVA-specific CD8 T cells in IL-21 / mice
compared with WT mice with a mean of 9.6% vs 2.6% ( p 0.05),
respectively. Similar results were found in IL-21R / mice, where
OVA-specific CD8 T cells were increased already on day 10
postinjection of tumor with generally lower percentages as expected
at this earlier time point (mean of 1.1% in IL-21R / mice
vs 0.7% in WT mice, ( p 0.05). Furthermore, we found that the
level of OVA-specific CD8 T cells showed significant inverse
correlation with the tumor size at the time of sampling and it was
also significantly predictive of complete rejection (data not
shown). Together, these observations suggest a suppressive role of
endogenous IL-21 and IL-21R signaling in CD8 T cell-dependent
immunity toward immunogenic tumors, restricting the expansion
of Ag-specific CD8 T cells.
CD8 T cell-mediated memory responses are intact in IL-21 /
and IL-21R / mice
Seeing the suppressive effect of IL-21 on primary tumor immune
responses we next explored whether IL-21 / or IL-21R / mice
might have altered long-term CD8 T cell-based memory responses.
To test this, cohorts of WT, IL-21 / , and IL-21R /
mice that initially had rejected their primary tumor challenge with
the immunogenic EG7 tumors were rechallenged with conventional
EG7 tumor cells. In naive WT, IL-21 / , and IL-21R /
mice, these tumors all grew out (data not shown). The rechallenged
mice were injected between 40 days and up to 6 mo after their
primary tumor rejection and monitored for tumor growth (Fig. 8A).
Initial tumor growth was observed in most mice followed by complete
rejection of all tumors in both WT, IL-21 / , and IL-21R /
mice, within 10–15 days post injection, suggesting that IL-21 /
and IL-21R / mice have competent CD8 T cell-mediated memory
responses. To evaluate the magnitude of the adaptive memory
response in the absence of IL-21, blood samples were collected
pre-rechallenge and on day 3 post-rechallenge to detect the level of
OVA-specific CD8 T cells using SIINFEKL-loaded MHC class
I tetramers (Fig. 8B). Before rechallenge all mice displayed a significant
level of circulating SIINFEKL-specific CD8 T cells but
with no difference between strains (WT: 1.4%, IL-21 / : 1.5%,
IL-21R / : 2.3%, mean % of total CD8 T cells), suggesting that
IL-21/IL-21R deficiency does not alter the circulating effectormemory
CD8 T cell pool. On day 3 post-rechallenge the level of
OVA-specific CD8 T cells generally increased in all three strains
compared with pre-rechallenge (WT: 1.8%, IL-21 / : 2.7%, IL-
21R / : 3.7%, mean % of total CD8 T cells). For comparison,
naive WT and IL-21 / or IL-21R / mice challenged with EG7
showed on average 0.25% OVA-specific CD8 T cells, indicating
that the level of de novo generated OVA-specific CD8 T cells
was very low on day 3 postchallenge of tumor as expected. The
average absolute changes of OVA-specific CD8 T cells on day 3
relative to pre-rechallenge was 0.4% in WT mice, but was increased
to 1.2% in IL-21 / mice and 1.3% in IL-21R / mice.
This difference was statistically significant in IL-21 / mice compared
with WT mice ( p 0.05), but not for IL-21R / mice.
Overall, these data suggest that CD8 T cell memory is intact in
mice deficient for IL-21 and IL-21R, but that IL-21 also suppresses
the secondary expansion of Ag-specific CD8 T cells.
Discussion
In this study, we present the first set of data examining both primary
and secondary tumor immune responses in mice deficient of
IL-21 or IL-21R. Our results show that these mice have intact
tumor immune surveillance, primary and secondary tumor immunity,
but, in contrast to the literature, reveal a suppressive role for
endogenous IL-21 during Ag-specific CD8 T cell expansion and
reactivity to immunogenic tumors. Exogenous IL-21 has shown
significant antitumor activity in numerous experimental mouse tumor
studies, either secreted from artificial tumor cells, expressed
by plasmids, or injected as recombinant protein (4). Studies of
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7333
FIGURE 8. CD8 T cell-mediated memory responses are intact in IL-21 / and IL-21R / mice. A, Groups of B6 WT, IL-21 / , and IL-21R / mice
that had initially rejected a primary tumor challenge with 3 10 6 EG7 (immunogenic) cells s.c. on the right flank were rechallenged 40 days and up
to 6 mo postrejection with 3 10 6 EG7 (conventional) s.c. on the flank contralateral to the primary challenge and monitored for tumor growth. B,
Pre-rechallenge and on day 3 post-rechallenge blood was drawn and stained with SIINFEKL-loaded MHC-I tetramers. Data in A depict individual tumor
growth curves, for n 8 (WT), n 13 (IL-21 / ), and n 14 (IL-21R / ) mice and are representative of three independent experiments. B, Dot plots
represent percentage of SIINFEKL-specific cells of total CD8 T cells in blood from individual mice pre-rechallenge and day 3 post-rechallenge and the
absolute percentage of change from pre-rechallenge to day 3 post-rechallenge. Kruskal-Wallis test with Dunn’s posttest was used to compare the absolute
percentage of change in WT, IL-21 / , and IL-21R / mice , p 0.05 compared with WT.
endogenous IL-21 have established that the cytokine and its
signaling pathway play a significant role in the pathogenesis of
several experimental autoimmune diseases, including colitis (27),
diabetes (25), arthritis (24), and systemic lupus erythematosus
(23), with experimental autoimmune encephalomyelitis being debated
(41, 42). These results highlight that IL-21 is primarily a
proinflammatory cytokine. However, our data now extend these
results and propose that endogenous IL-21 might also have potentially
immunosuppressive effects.
IL-21 therapy has shown antitumor activity through effects on
NK cells and CD8 T cells (4), and augmented antitumor responses
by NKT cells (8). Based on these data we anticipated that
IL-21 / and IL-21R / mice might have reduced immunity to
tumors particularly mediated by these effector cells. We found intact
tumor immune surveillance toward MCA-induced sarcomas
despite the lack of functional IL-21 or IL-21R. In contrast to transplantable
tumor models, the MCA-induced sarcoma model reflects
a more biologically relevant carcinogenesis process, which is subjected
to natural tumor immune surveillance mainly conducted by
NKT cells and NK cells in a perforin- and IFN--dependent manner
(43, 44). So, during the course of this model, activation of NK
and NKT cells must be occurring. This seems to be a suitable
model to test for potential IL-21 involvement as IL-21 is reported
to increase IFN- production and perforin-dependent antitumor
responses by NK cells as well as stimulate granularity and IFN-
production by NKT cells, which can also produce IL-21 upon activation
(2, 33).
We used the B16 melanoma model and H-2b RMAS-Rae1
tumors to detect potential IL-21 involvement in primary tumor
responses with different degrees of immune activity. B16 melanomas
showed equal s.c. growth kinetics and development of lung
metastases and RMAS-Rae1 tumors were equally rejected in the
absence of IL-21 signaling. Recombinant IL-21 has previously
shown antitumor responses in both tumor models; in B16 melanoma
through actions on either NK cells or CD8 T cells (6, 10,
33), and in RMAS-Rae1 tumors through NK cells in an NKG2Ddependent
manner (7). B16 melanomas are well known for being
aggressive and poorly immunogenic tumors, which might not generate
a sufficient host immune response for endogenous IL-21 to
play a role and the level of endogenous IL-21 production could in
this study be insignificant or otherwise redundant. RMAS-Rae1
tumors clearly show an active immune response as seen previously
(35), but in this study it might be that NKG2D stimulation alone is
sufficient for tumor rejection and because this response is mainly
NK cell-mediated it could also be that IL-21-producing cells are
not properly engaged. However, when we rechallenged mice postrejection
of RMAS-Rae1 tumor with H-2b RMA cells, which
are controlled by T cells but not NK cells, we found powerful but
equal rejection compared with WT. Although these results suggest
involvement of T cells, under these conditions IL-21 signaling was
not essential for the successful transition from innate NK cellmediated
immunity to adaptive T cell-mediated immunity, which
has previously been suggested (16).
A critical factor in these experiments is whether IL-21 is produced
to any significant amount. To address this proposal, we used
the CD1d-restricted ligand GC, which induce IL-21 production
from NKT cells as recently described (2). NKT cells secondarily
enhance IFN- production and antitumor activity of NK cells (45),
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7334 HOST IL-21 IN TUMOR IMMUNITY
IL-21 can activate NKT cells (2), and similarly enhance IFN-
production and antitumor activity of NK cells (16, 33). So, it could
be that IL-21 was a potential link in the direct effects of GC on
NKT cells and in the sequential effects on NK cells. However, we
found that NKT cell expansion and IFN- production in response
to GC stimulation were not influenced by host IL-21 nor was the
concomitant IFN- production from NK cells. These data suggest
that IL-21 does not have a major autocrine role during GC activation
of NKT cells or in paracrine stimulation of NK cells. Our
finding that IL-21 was required to some extent for increased granzyme
B expression in NKT, NK and CD8 T cells following GC
stimulation indicates that IL-21 is produced following GC stimulation
and possibly plays a role in the optimal development of
effector functions in these cells. This finding is supported by several
studies in both mice and humans showing that IL-21 up-regulates
granzyme B in NK cells, CD8 T cells and B cells (2, 21,
22, 46, 47). However, our finding that GC pulsed B16 melanomas
were also strongly rejected in the absence of IL-21 suggests
that host IL-21 is not a critical factor in GC-mediated NKT celldependent
tumor rejection. In contrast, we have previously shown
that the sequential activation of NKT cells and NK cells with GC
followed by recombinant IL-21 stimulation was a very effective
treatment of experimental tumors (8). Interestingly, this study
showed that the additional effects of IL-21 were seen only if the
treatment was given 3 days after GC administration and not if
IL-21 treatment was started together with or 6 days after GC
administration, showing that the timing or context of IL-21 is essential
for the antitumor response. So, although the IL-21 production
we achieve following GC stimulation might be insignificant
to add to the antitumor effect, another explanation might be that
IL-21 is produced at a suboptimal time point to add to the antitumor
effect in the models tested.
IL-21 mediates antitumor effects through stimulation of CD8 T
cells, and is a potent costimulant of Ag-specific CD8 T cell expansion
and cytotoxicity (9–11, 48), suggesting that such responses
might be impaired without functional IL-21 signaling. In
contrast to what is presented in the literature with exogenous IL-
21, we found that endogenous IL-21 has suppressive activity on
CD8 T cell responses, restricting Ag-specific CD8 T cell expansion
and antitumor activity against immunogenic tumors.
These results suggest that IL-21 is produced during T cell sensitive
tumor immune responses, but unexpectedly has suppressive rather
than stimulatory effects. In support of these findings, we have previously
reported that IL-21 / and IL-21R / showed exacerbated
responses to experimental autoimmune encephalomyelitis
(41). Our results are also supported by studies showing that IL-21
can maintain DCs in an immature state inhibiting Ag presentation
and T cell activation (49, 50). Furthermore, they are supported by
results showing that the antitumor activity of IL-21 is lost by treating
mice around the time of tumor inoculation as compared with
treatment started just 2 days posttumor inoculation (9). A recent
trio of studies suggests that IL-21 signaling is essential for maintaining
CD8 T cell responses during chronic viral infections (51–
53). Interestingly, they also show that host IL-21 limits Ag-specific
CD8 T cell expansion during the acute phase of infections,
whereas IL-21 is strictly needed to sustain virus-specific CD8 T
cells during chronic infections (52). Together with our results,
these findings support our notion that the timing or context of
IL-21 is critical for the outcome of an immune response; whereas
endogenous IL-21 produced during the priming phase might limit
expansion of Ag-specific CD8 T cells and antitumor activity,
IL-21 delivered after the initial priming works to sustain or boost
CD8 T cell activity against both viruses and tumors.
We mainly found increased CD8 T cell expansion in the liver
in response to OVA and GC immunization, which is most likely
due to the increased proportion of NKT cells in this organ compared
with the spleen. However, we only observed an increase in
OVA-specific CD8 T cells on day 7 after GC and OVA immunization,
whereas similar levels were found on day 35 postimmunization.
And, the circulating level of effector/memory CD8 T
cells more than 40 days postrejection of EG7 was similar between
WT, IL-21 / and IL-21R / mice. These results indicate that
host IL-21 is not involved in the long-term maintenance of effector/memory
CD8 T cells. This suggestion seems to contrast findings
with recombinant IL-21 (9), however, in this study tumors
were not completely rejected as in our model, but instead showed
chronic growth inhibition maintaining the presence of Ag. Furthermore,
in studies of viral infections IL-21 was not needed to
maintain circulating effector/memory CD8 T cells after a resolved
infection, only for maintaining effector CD8 T cells during
chronic infections (53). Taken together, these results indicate
that IL-21 is not required to maintain circulating memory CD8 T
cells in the absence of Ag, but can sustain CD8 T cell responses
during Ag persistency. This is also consistent with the findings that
TCR stimulation is required for IL-21 production (1, 2).
In addition to its effects during primary CD8 T cell-mediated
tumor immunity, IL-21 also has a putative role in secondary CD8
T cell memory responses (9, 11, 15, 54). However, we found that
secondary CD8 T cell memory responses were normal or even
increased in IL-21 / and IL-21R / mice, indicating that endogenous
IL-21 might also limit secondary effector CD8 T cell expansion.
Consistently, IL-21 was not required for viral recall responses
(53). However, in this study IL-21 did not limit secondary
Ag-specific T cell expansion and although differences might have
been missed due to small group sizes, more work is warranted to
confirm the role of endogenous IL-21 during secondary CD8 T
cell expansion. At present, the mechanism behind the suppressive
effect of IL-21 is unknown. Recently, it was found that TCR priming
in the presence of IL-21 results in IL-10-producing immunosuppressive
T cells (55). So, the production of IL-21 in response to
Ag could mediate suppression either through inhibition of DCs as
stated above or by induction of IL-10-producing immunosuppressive
T cells. In contrast with this scenario, we did not see any
suppressive effects of host IL-21 on the growth of less immunogenic
MC38-OVA dim tumors. This may be due to an insufficient
Ag-induced immune response or other redundant immune suppressive
mechanisms by this tumor.
In this study, we anticipated that endogenous IL-21 would to
some degree be involved in tumor immunity. However, based on
the data presented in this study, we conclude that endogenous
IL-21 and IL-21R is not required for immune surveillance or tumor
immunity mediated by NK, NKT, or CD8 T cells, at least in the
range of tumor models tested. We have not investigated the role of
endogenous IL-21 in the development of B cell lymphomas or in
B cell-dependent tumor rejection, in which IL-21 could have a role
(18, 56). However, our findings suggest that anti-IL-21 treatment,
which has been proposed as a potential treatment strategy for many
autoimmune diseases (57), would not compromise tumor immune
surveillance. Indeed, given the enhanced CD8 T cell response,
short-term blockade of host IL-21 signaling during the early priming
phase followed by stimulation with recombinant IL-21 therapy
might even enhance tumor immunity. We have highlighted that the
actions of IL-21 are potentially very context-dependent, but it is
still unknown which cells produce IL-21 during a tumor immune
response, in which anatomical context, and when does this happen.
Injection of recombinant IL-21 into mice results in high nanogram
per milliliter serum concentrations that are maintained for
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The Journal of Immunology
many hours (10), and generally endogenous serum cytokine concentrations
are only in the picogram range even after stimulation.
Thus it is perhaps not surprising that our study highlights the great
difference between studying the function of an administered recombinant
cytokine and the endogenous cytokine by gene targeting.
CD4 T cells are the most likely producers of IL-21 in our
tumor model and the need for TCR stimulation suggests that secondary
lymphoid organs are the primary location for IL-21 production.
This suggests that IL-21 could be produced during the
early priming phase, which could be the context of its suppressive
actions. However, we believe the generation of reporter mice coexpressing,
e.g., GFP along with IL-21 will be an essential tool to
formally answer these questions.
In summary, we have demonstrated that endogenous IL-21 signaling
is not required for tumor immune surveillance, NK, NKT,
and CD8 T cell-dependent primary tumor immunity, or for
CD8 T cell-dependent secondary memory responses. However,
we found that endogenous IL-21 restricts CD8 T cell expansion
and immunity toward immunogenic tumors. These results reveal
an unexpected suppressive role for IL-21 in Ag-specific immunity
in contrast to its general perception as a proinflammatory cytokine
in both cancer immunotherapy and autoimmunity.
Acknowledgments
We thank Michelle Stirling for the breeding and maintenance of the mice
in these studies and Charlene Guan and Suzanne Medwell for the genotyping
of mice. We also thank Ralph Rossi for flow cytometry support and
Konstantinos Kyparissoudis for providing GC-loaded CD1d tetramer.
Disclosures
H.S. and K.S. are employed by Novo Nordisk, and P.V.S. is employed by
Zymogenetics. D.I.G. and M.J.S. have received research support from
Novo Nordisk. The remaining authors have no financial conflict of interest.
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Chapter 4 – Discussion
The primary objective of this thesis was to evaluate the anti-tumor effects of IL-21 protein
therapy in relevant preclinical cancer models and secondarily investigate the role of
endogenous IL-21 in tumor immunity.
In Paper II and III, we have demonstrated that IL-21 significantly inhibited growth of B16
melanoma and RenCa renal cell carcinoma. These models were chosen based on their wide
usage in cancer therapy studies and responsiveness to immunotherapy (Sayers et al., 1998;
Wigginton et al., 1996; DeMatos et al., 1998; van et al., 1999). In order to mimic conditions in
humans, where the immune system poorly recognizes tumors, B16 and RenCa cells were
inoculated in their respective syngeneic hosts, C57BL/6 and BALB/c mice, providing low
intrinsic immune reactivity. Furthermore, these mouse strains are fully immunocompetent in
contrast to e.g. xenograft models allowing the entire immune repertoire to come in to play as
it would in humans. However, the syngeneic interaction between the tumor and host also
makes it challenging to mount an immune response and these models therefore represent
reasonable models for the testing of tumor immunotherapy.
In Paper II and III RenCa tumors were generally found to be more responsive to IL-21 therapy
compared to B16 tumors and showed higher densities of tumor infiltrating T cells. These
observations were expected based on the literature showing increased immunogenicity of
RenCa compared to B16 (Krup et al., 1999; Scheffer et al., 2003), and in Paper III these
observations were extended to our models, where increased expression of MHC class I was
found on RenCa compare to B16 cells. Nevertheless, B16 and RenCa are both aggressive
tumors; in Paper II and III vehicle treated tumors reached a tumor size of ~1000 mm 3 within
16-22 days and both tumors have been found to kill their host within approximately 25 days
post intraperitoneal injection (Krup et al., 1999). Thus, B16 and RenCa are aggressive
preclinical tumor models with different inherent immunogenicity and it was encouraging that
significant anti-tumor effect was obtained in both models despite their limited therapeutic
window. The use of therapeutic administration of IL-21 in both Paper II and III, where
treatment of even larger established tumors results in significant tumor-growth inhibition,
further strengthens the results. Based on these model characteristics, we consider the results
obtained in Paper II and III clinically relevant.
However, several limitations to these models should be acknowledged. First, both models rely
on transplantation of 10 5 or more tumor cells, which poorly reflect the slow development of
human cancer, where the immune system gradually tolerates a tumor. Instead, the injection of
tumor cells could generate considerable cell death, which abruptly exposes the immune system
91

to potentially immunogenic antigens. Second, tumor cells were injected subcutaneously, which
may represent a site with enhanced immunosurveillance because of its critical junction
between the host and the environment (Girardi, 2007), and although this site has some
anatomical relevance for melanomas, it has much less so for renal cancer. Third, the
transplantable tumors used here have a low frequency of spontaneous metastasis, and the
effect of IL-21 on spontaneous metastasis was therefore not evaluated, although this is the
predominant cause of death in humans. An alternative to transplantable models could have
been transgenic cancer models, which more closely mimic the slow process of human cancer
development and now include many organ-specific models (Ostrand-Rosenberg, 2004).
However, cancers in these models often take months to develop with differential onset and
disease penetrance, making controlled pharmacological testing of drugs very difficult. Since
there are pros and cons for all animal models results should preferably be reproduced in more
than one model. Thus, it was encouraging to show qualitatively similar anti-tumor effect and
similar effect on the density of tumor infiltrating T cells in both B16 and RenCa tumors,
although of different magnitudes.
In Paper II the anti-tumor efficacy of subcutaneous administration appeared greater than
intraperitoneal administration particularly in the RenCa model and in Paper III intratumoral
administration showed superior long-term tumor-growth inhibition compared to subcutaneous
administration in RenCa. To compare the anti-tumor efficacies in Paper II and III across the
different administration routes and various starting tumor sizes the classical T/C% value has
been used (see Table 1 and 2). The T/C% value describes the ratio of the mean endpoint
tumor sizes of treated over controls, hence a lower T/C% value equals better efficacy. It has
traditionally been used to compare the efficacy of chemotherapies in preclinical studies and
shown to be predictive of their response rates in clinical trials against certain cancers
(Voskoglou-Nomikos et al., 2003).
T/C% values support the notion that intraperitoneal administration is less efficacious compared
to subcutaneous administration and that intratumoral administration has the greatest efficacy
of the evaluated administration routes. These data suggest that intratumoral administration is
a more effective administration route for IL-21. Unfortunately, intratumoral administration is
not very practical, since metastatic cancer rarely is available for intratumoral injections.
However, intratumoral injections could help increase immune responses toward primary
tumors, if such are accessible, and results from IL-2 clinical trials have shown that
intratumoral administration could increase the response rates and lower side effects in soft
tissue skin metastasis of melanoma (Radny et al., 2003).
The T/C% values also show that subcutaneous administration is a feasible administration route
for IL-21. Thus, subcutaneous administration of IL-21 deserves clinical investigation since it
92

could potentially lower adverse events while sustaining efficacy as previously shown with IL-2
(Geertsen et al., 2004; Negrier et al., 2008). Also, subcutaneous administration might allow
outpatient treatment given the moderate toxicity of IL-21 (Davis et al., 2007; Davis et al.,
2009).
Table 1. Summary of efficacy in B16 melanomas
Treatment start (~mean tumor size) Paper/Figure Dose & schedule T/C% * P-value $
& route of administration
Early treatment (~5mm 3 )
I.p. II/2A 50 μg daily 30

suggest that earlier treatment start with smaller tumor burdens and continued presence of IL-
21 benefit the anti-tumor effect. Unfortunately, this notion contrasts the general practice in
modern cancer therapy, where new experimental treatments must initially show efficacy in
pre-treated patients with end-stage disease in order to be continued, and this has also been
the case for IL-21 (Davis et al., 2009). Undoubtedly, many experimental treatments would
benefit from trials in patients with less advanced disease. Cytokines are no exception, since
they function to boost weak inherent immune responses, which might become progressively
more exhausted as disease progresses caused by the tumor-intrinsic immunosuppression as
presented in figure 1 of this thesis.
In Paper II and III, subcutaneous and intratumoral administration of IL-21 significantly
increased the density of tumor infiltrating CD8 + T cells, which were required for and correlated
with the anti-tumor effect. Furthermore, the increased effect of intratumoral administration of
IL-21 was associated with further increased density and activity of tumor infiltrating CD8 + T
cells.
These results suggest that the density of tumor infiltrating CD8 + T cells might represent a
relevant biological marker of IL-21 efficacy that could prove clinically useful. Although the
mechanism behind this increase in CD8 + T cell infiltration still needs to be clarified, these
results also suggest that IL-21 is able to overcome a critical barrier for successful tumor
immunotherapy, namely low immune cell infiltration and function as outlined in figure 1 of this
thesis. Furthermore, in Paper III intratumoral IL-21 adminitration showed an increase in the
density of tumor infiltrating CD4 + T cells but not T regs , suggesting that IL-21 also is able to
selectively increase certain T cells subsets in the tumor environment, without increasing
suppressive T cells.
Results from cancer patients have clearly shown what benefit increased CD8 + T cell infiltration
in tumors and particularly an increased CD8 + T cell/T reg ratio could have, if these findings are
translatable to humans (Clemente et al., 1996; Galon et al., 2006; Pages et al., 2005; Gao et
al., 2007; Naito et al., 1998; Piersma et al., 2007; Sato et al., 2005; Sharma et al., 2007;
Schumacher et al., 2001; Zhang et al., 2003). However, this remains an open question since
the evaluation of tumor infiltrating lymphocytes in tumor biopsies remains to be included in IL-
21 clinical trials. But, other signs of relevant immune activation from Paper II and III seem to
be translatable. In Paper III we show that the expression of granzyme B and IFNγ was
increased in tumor infiltrating CD8 + T cells. Consistently, IL-21 administration in clinical trials
increased granzyme B and IFNγ expression in CD8 + T cells from PBMCs (Davis et al., 2009;
Frederiksen et al., 2008). In addition, IL-21 administration in clinical trials increased the
fractions of CD62L + CD4 + and CD8 + T cells in PBMCs (Davis et al., 2009). This was thought to
be associated with the frequent finding of lymphopenia in treated patients caused by increased
94

secondary lymphoid redistribution. In Paper III, intratumoral administration of IL-21 resulted
in increased numbers of CD62L + CD4 + and CD8 + T cells in tumor-draining lymph nodes caused
primarily by non-proliferative effects, indicating analogous activities in our model. Still, none of
these clinical biomarkers were associated with anti-tumor responses showing the need for new
predictive markers of IL-21 anti-tumor efficacy.
In Paper IV, the quest to unveil the role of endogenous IL-21 in cancer control surprisingly led
to the conclusion that endogenous IL-21 is not required for tumor immunity, but rather
restricted CD8 + T cell expansion and the control of immunogenic tumors. Considering the
literature, these results were counterintuitive, since endogenous IL-21 mainly has been shown
to promote proinflammatory conditions and autoimmunity as described in Paper I. However, as
it was highlighted in Paper I, the actions of IL-21 are very context dependent and both the
timing of IL-21 administration and the level of co-stimulation can greatly influence the
outcome of IL-21 stimulated immune responses. This notion is exemplified by the effect IL-21
has on B cells; IL-21 stimulation of unstimulated B cells induces apoptosis, whereas the
addition of B cell receptor and CD40 co-stimulation enables IL-21 to drive significant B cell
differentiation (Ettinger et al., 2005). Very recently, it was found that endogenous IL-21 has a
critical role in CD8 + T cell control of chronic viral infections (Elsaesser et al., 2009; Frohlich et
al., 2009; Yi et al., 2009), but in one of these studies it was also shown that endogenous IL-21
restricted antigen-specific CD8 + T cell expansion during the acute phase of infections
(Elsaesser et al., 2009). These results are further evidence of the complex role IL-21 has on
immune responses and support a dichotomous role of IL-21 in CD8 + T cell immunity as
suggested here in Paper IV. However, they also suggest that endogenous IL-21 could play a
role in a more chronic CD8 + T cell-controlled tumor model, perhaps not thoroughly addressed
in Paper IV. Still, the MCA sarcoma model used in Paper IV is a more chronic tumor model that
apart from the generation of NK and NKT cell-controlled tumors (Crowe et al., 2002; Smyth et
al., 2001) also has been shown to give rise to occult cancers contained by adaptive immunity
(Koebel et al., 2007). Despite the unchanged incidence and tumor-growth of MCA sarcomas in
IL-21-deficient mice, focus on more chronic tumor models controlled by CD8 + T cells in future
studies could contain a role for IL-21.
The results in Paper IV suggest that neutralization of IL-21, which has been suggested as a
potential treatment for several autoimmune diseases as reviewed in Paper I, does not overtly
risk more frequent cancer development or compromised immunity toward cancers. Rather,
based on the findings in this thesis, short-term and well-timed neutralization of host IL-21
followed by administration of IL-21 protein might even enhance tumor immunity.
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Dichotomous features are not unique to IL-21. IL-2, which is approved as therapy for
metastatic melanoma and advanced renal cell carcinoma, shows a very overt paradox in its
actions. Originally, IL-2 was identified as a critical T cell growth factor in vitro and therefore
assumed to be an important driver of T cell expansion in vivo, able to kick start tumor immune
responses. However, this theory was questioned when the main phenotype found in IL-2-
deficent mice was lymphoproliferation with lethal multi-organ autoimmunity. The discovery of
T reg cell biology has now clarified that IL-2 is indispensable for T reg cell development, but
dispensable for immunity, and that T reg deficiency is responsible for the phenotype in IL-2-
deficient mice (reviewed by Malek and Bayer, 2004). Although the in vivo biology of IL-2 is still
incompletely understood, IL-2 has also shown important functions for CD8 + T cell immunity in
vivo (Antony et al., 2006), which could explain the anti-tumor effects seen in patients.
In a few selected patients, IL-2 shows remarkable clinical responses, while most do not seem
to respond at all (Atkins et al., 1999; McDermott and Atkins, 2006; Klapper et al., 2008). The
opposing actions of IL-2 combined with the incompletely understood immune status of patients
prior to treatment are perhaps the reason for these inconsistencies. Arguably, IL-2 is beneficial
for certain cancer patients, but as a cytokine for cancer immunotherapy it may be far from
optimal and the exploration of alternatives is clearly warranted.
IL-2 has been known for more than 25 years, and despite extensive studies, its critical activity
on T regs was just realized in the last decade. IL-21 is only 9 years old and has so far shown
pleiotropic actions mainly of proinflammatory nature. But, beside the findings in this thesis
immunosuppressive activities of IL-21 are now beginning to emerge (Spolski et al., 2009), and
it is likely that more are awaiting discovery. As presented in this thesis, endogenous IL-21-
signaling limits CD8 + T cell expansion and tumor immunity whereas the administration of
recombinant IL-21 protein enhances CD8 + T cell-mediated anti-tumor immune responses.
Obviously, these paradoxical functions of IL-21 should be the focus of future studies, but
acknowledging that IL-21 has opposing activities will also increase the need to carefully
determine when and who to treat with IL-21. Clarification of the biology behind the
immunosuppressive activities of IL-21 will be detrimental to its use with consideration for both
the timing and duration of IL-21 administration. Because immunotherapy is relatively new in
cancer therapy, traditional evaluations of predictive factors for treatment efficacy have mainly
focused on non-immunological parameters (Motzer et al., 2004). However, greater focus on
clarifying patient immune status prior to treatment with identification of immunological factors
predictive of treatment efficacy might help to better select patients that would benefit from
immunotherapy including IL-21. Recently, such efforts have been employed and shown to be
relevant for the outcome of IL-2 therapy (Donskov and von der, 2006; Jensen et al., 2009).
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Based on the findings in this thesis, characterizing the level and proportions of different tumor
infiltrating lymphocytes might be interesting to explore for this purpose.
Collectively, the findings in this thesis outline the main challenge for the use of cytokines as
therapies or as therapeutic targets – their pleiotropic and dichotomous actions. A more
thorough understanding of the biology of cytokines is essential to yield therapeutic benefit of
their administration or neutralization in human disease. This thesis has contributed with new
insights into IL-21 anti-tumor biology and shown the role of IL-21 as a cytokine in host tumor
immunity. Specifically, IL-21 showed significant anti-tumor effects in preclinical models with
clinically relevant immunomodulatory effects, whereas host IL-21 was not required for tumor
immunity but showed a suppressive role during CD8 + T cell-dependent tumor immunity. These
results support the future development of IL-21 in oncology, but highlight the need for
continued studies of IL-21 anti-tumor biology to fully understand its pleiotropic actions.
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Chapter 5 – Conclusion
From part 1 in this thesis it can be concluded that:
1. IL-21 protein monotherapy therapy can inhibit established syngeneic tumor growth in two
aggressive subcutaneous tumor models with different immunogenicity: B16 melanoma
and RenCa renal cell carcinoma.
2. Subcutaneous administration of IL-21 has improved pharmacokinetics and at least as
good or better efficacy compared to intraperitoneal administration, suggesting that
subcutaneous administration of IL-21 could be applicable in future clinical trials.
3. CD8 + T cells are essential for the anti-tumor effect of IL-21 in the models used and IL-21
increases the density of tumor infiltrating CD8 + T cells, which correlates with tumorgrowth
inhibition, suggesting that the density of tumor infiltrating CD8 + T cells could be a
relevant biological marker of IL-21 anti-tumor activity.
4. Compared to subcutaneous administration, intratumoral administration of IL-21 increases
long-term tumor-growth inhibition, and increases the density, exocytotic activity, and
granzyme B and IFNγ expression of tumor infiltrating CD8 + T cells. Intratumoral
administration of IL-21 selectively increases the density of tumor infiltrating CD4 + T cells
and not T regs , and increases the number of naïve T cells and proliferation of activated T
cells in tumor draining lymph nodes. Together, these data suggest that local IL-21
administration benefits the tumor environment, can overcome tumor infiltrating CD8 + T
cell disability and deserves to be clinically investigated when this administration route is
accessible.
From part 2 in this thesis it can be concluded that:
1. Endogenous IL-21 is not required for tumor immunosurveillance, NK, NKT and CD8 + T
cell-dependent primary tumor immunity, or for secondary CD8 + T cell memory responses.
2. Rather, endogenous IL-21 restricts CD8 + T cell expansion and immunity toward
immunogenic tumors, indicating an unexpected immunosuppressive role of IL-21 in CD8 +
T cell immunity.
Taken together, the results presented in this thesis support the clinical investigation of IL-21
protein therapy for the treatment of cancer, but reveal a dichotomous role for IL-21 in tumor
immunity.
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Chapter 6 – Future perspectives
The ultimate goal of any cancer therapy is to cure the disease. While the finding in this thesis
that IL-21 significantly inhibited tumor growth and increased tumor infiltrating CD8 + T cells is
encouraging, IL-21 was not curative in our models. In early clinical trials, IL-21 has shown
moderate response rates, and although a couple of complete responses were achieved, IL-21
alone was not curative (Davis et al., 2007; Davis et al., 2009). A likely way forward for IL-21
is exploration of combination strategies. For this purpose, a better understanding of IL-21
biology and its dichotomous actions as highlighted in this thesis will be essential to ensure the
basis for a rational selection of combination partners. Specific questions generated by this
thesis include; what is the causative mechanism behind the IL-21-induced increase in tumor
infiltrating CD8 + T cells? What circumstances determine whether IL-21 suppresses or promotes
tumor immunity? Where and when is IL-21 produced and by what cells during tumor immune
responses? Answers to these questions may help to clarify the best way to apply IL-21, expand
the knowledge about what triggers IL-21 production physiologically and the context in which it
is produced, and potentially identify new targets or combination options to pursue.
Results from the treatment of human immunodeficiency virus (HIV), another highly mutating
human threat, clearly show the shortage of single targeted therapies and the success obtained
by multi-combinations (Palella, Jr. et al., 1998). These findings indicate that to restrain a
mutating enemy it is best to attack from several fronts. For this reason, it is very likely that
the ultimate goal in cancer therapy will be achieved through multi-combination therapy.
An important feature of a drug to be used for combination therapy is that the drug is well
tolerated and so far this has been the case in the early clinical trials of IL-21 (Davis et al.,
2007; Davis et al., 2009). In cancer, most novel treatments are initially tested in end-stage
and pre-treated patients, so it is likewise important that a new drug can potentially work in
concert with conventional drugs. Here, we have recently shown in experimental models that
IL-21 has additive effects in combination with IFNα and is feasible in combination with several
chemotherapies (Eriksen et al., 2009; Skak et al., 2009 (Cytokine) in press
doi:10.1016/j.cyto.2009.07.039). Obviously, the ability to provide additive or synergistic
effects to other therapies is vital for a combination drug, and here IL-21 has shown additional
anti-tumor effects together with several interesting combination partners as recently reviewed
(Skak et al., 2008).
Since IL-21 does not rely on a specific target for its actions, it represents an attractive partner
to many targeted therapies. Experimental evidence of this has been shown using a triple
immune-stimulating antibody cocktail together with IL-21, which resulted in cures of large
101

established tumors (Smyth et al., 2008). Such results show that clinical investigation of multicombination
therapy should be prioritized and that IL-21 is an attractive partner for this
purpose.
In humans, the currently most successful immunotherapeutic protocol relies on a combination
of non-myeloablative radio- or chemotherapy followed by adoptive transfer of ex vivo
expanded antigen-specific tumor infiltrating CD8 + T cells, which has shown remarkable
response rates and cures in selected patients with metastatic melanoma (Rosenberg et al.,
2008). Traditionally, IL-2 has been used for the ex vivo expansion of tumor-reactive T cells,
but interestingly, culture experiments with IL-21 resulted in less differentiated CD8 + T cells
with a more robust in vivo expansion capacity and anti-tumor effect (Hinrichs et al., 2008).
These results highlight another interesting aspect of IL-21 biology that deserves future
attention.
The implications of IL-21 in autoimmune diseases as outlined in Paper I and its emerging
immunosuppressive activities, further emphasize the multifaceted research needed to
comprehend IL-21. Only the future will tell whether IL-21 ends up as a cancer therapy, will be
part of a multi-combination strategy or perhaps be targeted for neutralization in autoimmune
diseases. Continued research to fully understand the biology of this fascinating cytokine will be
essential to make the most of it.
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