Interleukin 21 in cancer and immunotherapy20S%F8ndergaard.pdf · 2011. 1. 14. · 5 Summary Interleukin (IL)-21 is a recently discovered cytokine with pleiotropic immunomodulatory

Faculty of Science Novo Nordisk A/S University of Copenhagen Denmark Denmark

Interleukin 21 in cancer and immunotherapy

PH.D. THESIS Henrik Søndergaard, M.Sc. 2009

Interleukin 21 in cancer and immunotherapyPh.D. Thesis 2009 © Henrik Søndergaard

ISBN 978-87-7611-388-4Printed by SL grafik, Frederiksberg C, Denmark (www.slgrafik.dk)

1

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.

3

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

4

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

γc Common IL-2 receptor γ chain

GC Germinal center

IBD Inflammatory Bowel disease

IBF IRF-4-binding protein

iDC Immature dendritic cells

IDO Indoleamine 2,3-dioxygenase

IFN Interferon

IL 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.

Tfh Follicular helper T cells

Th Helper T cells

Th17 IL-17-producing helper T cells

TILs Tumor infiltrating lymphocytes

TRAIL Tumour necrosis factor–related

apoptosis-inducing ligand

Tregs Regulatory T cells

UC Ulcerative colitis

WT Wild type

5

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

6

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).

7

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

8

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-21R-

deficiente 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).

9

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 anti-

tumor 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.

10

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?

11

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 ageing1. 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

12

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 (Tregs) 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)

13

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 (Tregs) 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

14

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.

15

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.

17

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 anti-

tumor 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, tumor-

infiltrating 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-21R-

deficient mice subjected to various experimental tumors controlled by NK, NKT or CD8+ T cells

or with responsiveness to IL-21 therapy.

18

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.

19

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)

21

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 therapyH. Søndergaard & K. Skak

Immunopharmacology, Novo Nordisk A/S, Måløv, Denmark

Key wordsautoimmunity; cancer; cytokine;interleukin-21

CorrespondenceHenrik SøndergaardNovo Nordisk A/SDepartment of ImmunopharmacologyNovo Nordisk Park F6.2.30DK-2760 MåløvDenmarkTel: +45 4443 1376Fax: +45 4443 4537e-mail: [email protected]

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 novelcytokine produced by activated CD4+ T cells and natural killer T (NKT) cells. IL-21is part of a family of cytokines which include IL-2, -4, -7, -9 and -15 that allshare the common IL-2 receptor γ chain (γc) in their individual receptor complexes.IL-21 receptor (IL-21R) is widely expressed on both myeloid and lymphoid celllineages and IL-21 actions include co-stimulation of B cell differentiation andimmunoglobulin (Ig) production, co-mitogen of T cells, and stimulation of NK andCD8+ T cell cytotoxic function. Initially, IL-21 was recognized for its anti-tumoureffects in several preclinical tumour models, warranting its currently ongoing clinicaldevelopment as a cancer immunotherapeutic. More recently, IL-21 has been associatedwith the development of a panel of autoimmune and inflammatory diseases, whereneutralization 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 implicationsin immunopathologies.

Introduction

Cytokines are secreted polypeptides used primarily by theimmune system for intercellular communication. Cytokines arevital in all aspects of immunology from early haematopoiesisto the generation, maintenance and contraction of bothinnate and adaptive immune responses. Therefore, the timelyintroduction of cytokines or blockade of their signallingpathway can have decisive effects on immune processes.

Historically, identification of novel cytokines, and under-standing of their production and function have been funda-mental for the development of new clinical strategies. Theuse of recombinant cytokines have pioneered in the field ofcancer immunotherapy where interleukin (IL)-2 and inter-feron (IFN)-α have been used for more than two decadesand still represent effective treatments for certain cancers, e.g.melanoma and renal cell carcinoma (RCC). The blockade ofendogenous cytokines and their signalling pathways representnovel approaches in the fight against many autoimmune dis-eases; particularly the neutralization of tumour necrosis factor(TNF)-α and its signalling have revolutionized the treatmentof rheumatoid arthritis.

The discovery of IL-21 adds to a still growing panel ofhuman cytokines, and this cytokine has not only shown poten-tial as cancer therapy, but also as an attractive target forseveral autoimmune diseases. Here, we will briefly review

the immunobiology of IL-21 and cover the advances of thisinteresting cytokine in cancer therapy and its emerging rolein autoimmune and inflammatory diseases.

IL-21 and IL-21 receptor biology, signallingand expression

In 2000, a new type 1 cytokine receptor was discovered anddenoted novel orphan interleukin receptor (NILR) (1). Soonafter, a functional cloning approach identified a four-helix-bundle cytokine with structural relation to IL-2, IL-4 andIL-15 as the natural ligand to NILR, naming the new enti-ties IL-21 and IL-21 receptor (IL-21R) (2). The IL-21R geneis located directly downstream of IL-4R-α on human chro-mosome 16, and IL-21R has an amino acid sequence withgreatest homology to the IL-2 receptor beta-chain (IL-2R-β).The IL-21 gene is located on human chromosome 4q26-q27approximately 180 kb from the IL-2 gene and the matureIL-21 polypeptide consists of 131 amino acid residues mostsimilar to IL-15 (2).

Human and murine IL-21 and IL-21R show approximately60% overall amino acid sequence homology with significantconservation in regions of cytokine–receptor interaction (2),and to a large extent the function of IL-21 has been shown tobe similar in mouse and human, although several discrepancieshave also been described.

© 2009 John Wiley & Sons A/S 1

22

IL-21 in cancer and immunopathology H. Søndergaard & K. Skak

Figure 1 The common gamma chain (γc) cytokine family and interleukin (IL)-21 intracellular signalling. IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 all sharethe common IL-2 receptor (IL-2R)γc in their individual receptor complexes. IL-21 signals through its unique IL-21 receptor (IL-21R) in a heterodimericcomplex with γc. Upon ligand interaction, the IL-21R/γc complex recruits and phosphorylates janus kinases (JAK)1 and JAK3, which downstreamactivates signal transducers and activators of transcription (STAT). IL-21 primarily activates STAT3, but also STAT1 and more transiently STAT5a and5b. The phosphatidylinositol-3-kinase (PI-3K)-AKT and mitogen-activated protein kinase (MAPK) pathways are also involved in IL-21 signalling. Targetgenes positively regulated by IL-21 are indicated.

The IL-21R signals as a heterodimeric complex with thecommon IL-2 receptor gamma chain (γc, CD132) (3), mak-ing IL-21 the newest member of the γc cytokine family, whichalso includes IL-2, IL-4, IL-7, IL-9 and IL-15 (Figure 1).Like other type 1 cytokine receptors, the IL-21R/γc com-plex is a receptor tyrosine kinase, which upon ligand inter-action recruits and phosphorylates janus kinases (JAK) thatsubsequently activates signal transducers and activators oftranscription (STAT). Similar to its other family membersIL-21R recruits and activates JAK1 and JAK3 (3). Down-stream, IL-21 signalling primarily activates STAT3, but alsoSTAT1 and more transiently STAT5a and STAT5b (3, 4).The phosphatidylinositol-3-kinase (PI-3K)-AKT and mitogen-activated protein kinase (MAPK) pathways have also beensuggested to transmit IL-21 signals (4). The target genes forIL-21 signalling still remains to be fully elucidated, but tar-get genes identified so far include cyclin A/B/E, granzymeA/B, IFN -γ, CXCR3, CXCR6, Bcl-3, JAK3, IL-21 and IL-21R (5–7), indicating that IL-21 plays a role in cell cycleprogression, cellular activation, trafficking, cell survival andpositively 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), whereasthe IL-21R is much more widely expressed, includingB 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 crit-ical for normal haematopoiesis (11, 12). In the T cell lineage,IL-21R is not expressed until thymocytes differentiate intoCD4 and CD8 double positive cells. Low levels of IL-21R arefound on mature CD4+ and CD8+ T cells which increase inresponse to T cell receptor (TCR) stimulation (13). In contrast,NKT cells express IL-21R ex vivo without prior activation. Inthe B cell lineage the earliest detection of IL-21R is on pre-B cells, and mature follicular B cells express higher levelscompared with T cells, and this is further increased upon Bcell activation. Mature marginal zone B cells have lower IL-21R expression compared with follicular B cells, and plasmacells express very low IL-21R levels (13, 14). Overall, IL-21Rshows the highest expression on activated lymphocytes, indi-cating that IL-21 mainly acts as a co-stimulant of activatedlymphocytes.

IL-21 in immunobiology

Humans deficient of the γc receptor suffer from X-linkedsevere combined immunodeficiency (XSCID) syndrome, acondition where T cells and NK cells are completely absentand B cells are present but functionally impaired. This showsthe sum of actions and impact that the γc-dependent cytokineshave on normal immunobiology. IL-21 is no exception and itspleiotropic effects correspond to the cellular distribution of itsreceptor. These are summarized in Figure 2.

2 © 2009 John Wiley & Sons A/S

23

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 ofco-stimulation determine the cellular response: B cells proliferate and differentiate in response to B cell receptor (BCR) and CD40 co-stimulation, butundergo apoptosis without; CD8+ T cells primarily expand and increase their cytotoxicity together with IL-15 or TCR co-stimulation; resting NK cellsexpress 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 ownproduction, activation of NKT cells and regulation of CD4+ T cell differentiation and proliferation. IL-21 does not directly modulate regulatory T cells(Tregs), but it has been suggested to reduce Treg differentiation. Dendritic cells (DC) remain immature in response to IL-21, whereas macrophagesbecome activated.

B cells

B cells express the highest levels of IL-21R, making B cellsprime responders to IL-21. Initially, IL-21 was found toaugment anti-CD40-induced B cell proliferation but inhibitproliferation induced by anti-IgM and IL-4 (2). IL-21 alsoinhibited B cell proliferation and induced apoptosis whenB cells were activated via innate toll-like receptors (TLR)recognizing the bacterial component lipopolysaccaride (LPS)or viral DNA cytosine-guanine motifs (CpG) (13). Furtherstudies have clarified that IL-21 mainly inhibits B cell responsesand induces apoptosis of resting B cells in the absence ofproper co-stimulation, whereas cross-linking of both the B cellreceptor (BCR) and CD40 induces extensive proliferation anddifferentiation into immunoglobulin (Ig) producing plasmacells 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 timeof immunization reduces switching to IgE, but not IgG by spe-cific inhibition of germline C(ε) transcription in B cells (16).However, the regulation of Ig class switching by IL-21 isalso context dependent, because IL-21 inhibits IgE switch-ing induced by IL-4 and PHA stimulation, whereas it pro-motes IgE switching induced by IL-4 and anti-CD40 stimula-tion (17).

IL-21 co-stimulation is a strong inducer of B lymphocyte-induced maturation protein-1 (BLIMP-1), a transcriptionalmaster switch controlling the terminal differentiation ofB cells into plasma cells, and IL-21 can directly induce plasmacell differentiation of anti-IgM-stimulated B cells. In addi-tion, IL-21 induces Bcl-6, which has been hypothesized to be


24


involved in the differentiation of germinal center (GC) B cellsinto memory B cells (15), suggesting a more complex role ofIL-21 in B cell differentiation.

Taken together, IL-21 is a critical regulator of B cellresponses; B cells faced with IL-21 in the context of antigen-specific BCR stimulation and T cell co-stimulation willundergo class switch recombination and differentiate into anti-body producing plasma cells. In contrast, B cells encounteringIL-21 during unspecific TLR stimulation or without properT cell help will undergo apoptosis.

CD4+ T cells

CD4+ T cells stimulated via their TCR are main producers ofIL-21, and TCR stimulation increases CD4+ T cell expressionof IL-21R, giving IL-21 an autocrine role in CD4+ T cellresponses.

IL-21 is not a classical helper T cell (Th)1 or Th2 cytokine,but can be produced by CD4+ T cells during both highly Th1and Th2 skewed immune responses in vivo (18). CD4+ fol-licular helper T cells (Tfh), identified by their expression ofthe chemokine receptor CXCR5, express high levels of IL-21 required for Tfh cell development, cognate B cell helpand GC formation (19). The recently characterized IL-17-producing CD4+ helper T cells (Th17) also produce IL-21and are distinct from the classical Th1 and Th2 cells. Th17cells are involved in the clearance of certain pathogens andin autoimmune pathogenesis (20). Regulatory CD4+ T cells(Tregs), known for their immunosuppressive effects and char-acterized by high expression of IL-2Rα (CD25) and the tran-scription factor forkhead box P3 (FoxP3), have not yet shownevidence of IL-21 production.

In response to anti-CD3/CD28 stimulation, IL-21 potentlyco-stimulates CD4+ T cell proliferation and IFN-γ produc-tion, which counteracts Treg suppression (21, 22), but IL-21has not shown any direct effects on Tregs (21, 23). Although,during CD4+ T cell differentiation IL-21 has been suggestedto inhibit FoxP3 expression and in turn increase Th17 celldevelopment (24). IL-21 can drive the differentiation of Th17cells in concert with transforming growth factor (TGF)β, butin the presence of other proinflammatory cytokines such asIL-6, IL-21 is dispensable for Th17 differentiation and ratherserves as an auto-amplification loop for Th17 cell expan-sion (25). In addition, IL-21 regulates its own production inan autocrine fashion through STAT3 activation (6).

Overall, IL-21 is produced by several different CD4+ T cellsubtypes, it is essential in Tfh cell development and GCformation, and serves as an autocrine amplification loop forits own production and for CD4+ T cell proliferation andsurvival.

CD8+ T cells

In addition to B cells, CD8+ T cells are the primary respon-ders to IL-21. CD8+ T cells also increase their expression

of the IL-21R in response to TCR stimulation, showing thatIL-21 primarily affects activated CD8+ T cells. Alone, IL-21does not induce significant proliferation of CD8+ T cells,but in response to antigen-independent stimulation IL-21 co-stimulates proliferation and expansion together with IL-7 orIL-15 of both naı̈ve and activated CD8+ T cells (7, 26). IL-21 increases IFN-γ and IL-2 production and sustains theexpression of CD62L and CD28 on IL-15-stimulated CD8+

T cells. CD28 is an important co-receptor engaged by DCligands during TCR stimulation, which is lost on senescentT cells (26).

IL-21R-deficient mice showed reduced antigen-specificCD8+ T cell expansion and cytotoxicity compared with WT inresponse to viral stimulation (7), highlighting a role for IL-21also in antigen-specific CD8+ T cell expansion and func-tion. Similarly, in vitro expansion of antigen-specific CD8+

T cells in blood from melanoma patients was substantiallyincreased by IL-21 in concert with autologous DCs pulsedwith a melanoma-specific antigen (27). This effect was causedby increased proliferation and survival, and could not bemimicked by IL-2, IL-7 or IL-15. Consistent with antigen-independent stimulation, IL-21-co-stimulated antigen-specificCD8+ T cells showed high CD28 expression, increased IL-2 production, high-avidity TCRs and increased cytotoxicactivity.

Taken together, IL-21 co-stimulates antigen-dependent and-independent proliferation, expansion, survival, and cytotoxi-city of CD8+ T cells. Furthermore, IL-21 maintains CD8+ Tcell expression of CD28 and increases their IFN-γ and IL-2production, creating a more robust and independent CD8+ Tcell response.

NK cells

Mature resting NK cells show very low expression of IL-21R, but IL-21R expression is up-regulated on both matureNK cells and on NK cell precursors upon activation. IL-21enhances NK cell differentiation from bone-marrow-derivedprecursors (2), but IL-21R knockout mice have normal devel-opment and activity of mature NK cells (11), showing thatIL-21 is redundant for normal NK cell development andmaturation. This is because NK precursor cells do notexpress IL-21R, but IL-15, which is critical for normalNK cell development, induces IL-21R expression (28) andsubsequently IL-21 can accelerate the NK cell maturationprocess (29).

Stimulation of NK cells with viral particles, IL-2 or IL-15greatly enhances responsiveness to IL-21, which then inducesa 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 andIL-15 and increases NK cell apoptosis (11, 30).


25


NKT cells

NKT cells express inhibitory and activating NK cell recep-tors together with a restricted repertoire of TCRs. In con-trast to conventional T cells, the NKT cell TCR recognizeslipid antigens such as the agonist alpha-galactosylceramide (α-GC), presented by the MHC-like molecule CD1d expressedon DCs. Resting NKT cells express the IL-21R and produceIL-21 in response to anti-CD3 or α-GC stimulation (8). Fol-lowing anti-CD3 stimulation, the level of IL-21 productionis even higher in NKT cells compared with splenic CD4+

T cells, suggesting that NKT cells may represent an importantsource of IL-21. NKT cells also respond to IL-21 stimulation;IL-21 increases survival and proliferation of NKT cells andenhances α-GC-stimulated proliferation and expansion. Fur-thermore, IL-21 co-stimulation of NKT cells increases theirgranular morphology, granzyme B expression and cytokineproduction (8). These data suggest that NKT cells represent animportant source of IL-21 and that IL-21 generally increasesNKT cell activation.

Dendritic cells

IL-21 has mainly shown inhibitory effects on DCs. IL-21maintains bone-marrow-derived DCs in a more immaturestate characterized by increased phagocytotic activity anddecreased antigen presentation, thereby limiting the activationof antigen-specific T cell responses (31, 32). This indicates apotential role for IL-21 also in the restriction or terminationof immune responses. However, DCs pretreated with IL-21 and α-GC increases stimulation of NKT cell IFN-γproduction (33), indicating that the role of IL-21 on DCs ismore complex and needs further investigation.

Macrophages

IL-21R is expressed on both mouse and human macrophagesand in contrast to DCs, there is limited evidence that IL-21has proinflammatory effects on macrophages. IL-21 signallingis not required for macrophage development, but increasesmacrophage production of the neutrophil chemoattractantCXCL8/IL-8, increases phagocytosis and protease activity,and also the ability to stimulate antigen-specific CD4+ T cellproliferation (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 activitywas shown: murine colon carcinoma cells transduced withthe 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). Sincethen, several groups have studied IL-21 anti-tumour effectsby this approach, using genetically modified tumour cellsfrom both mouse and humans (see Table 1). In general, thesestudies show very potent anti-tumour activity of IL-21 withreports of complete tumour rejection in most studies. In thesestudies, NK cells and CD8+ T cells were responsible for theanti-tumour activity, with involvement of both IFN-γ andperforin, but with no role for CD4+ T cells (see Table 1).Although these data reflect the potential of IL-21 therapy andpoint out possible effector mechanisms of IL-21, tumour cellssecreting IL-21 are not clinically translatable.

A more relevant approach came with therapeutic admin-istration of IL-21-expressing plasmids. Using this approach,Wang et al. showed significant anti-tumour activity againstB16 melanomas and MCA205 fibrosarcomas mediated by NKcells, with a minor role for CD8+ T cells but not CD4+

T cells (39). Brady et al. showed that IL-21-expressing plas-mids injected at the time of tumour cell inoculation medi-ated NK cell- and perforin-dependent reduction in lung andliver metastasis, whereas IFN-γ, Fas ligand or TNF-relatedapoptosis-inducing ligand (TRAIL) did not contribute (30).Nakano et al. showed significant tumour growth inhibition ofsubcutaneous head and neck squamous cell carcinomas usingIL-21 plasmids, dependent on T cells, NK cells and with gen-eration of tumour-specific Ig (42). Together, these data showthat IL-21 administration by a more therapeutic approach hasa similar mechanism of anti-tumour activity even adding apossible role for B cells, but perhaps with less dramatic anti-tumour effects compared with the IL-21-secreting tumours.

Moroz et al. were the first to use recombinant IL-21 pro-tein and showed significant tumour growth inhibition andprolonged survival in an ovalbumin (OVA)-expressing lym-phoma 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). Thiswas a CD8+ T cell-dependent effect where IL-21 increasedthe expansion and cytotoxicity of OVA-specific CD8+ T cells,and generated a more durable response compared with IL-2and IL-15 leading to concomitant immunity towards tumourrechallenge. Interestingly, pretreatment with IL-21 (day −4to day 6 post-tumour inoculation) completely abrogated theanti-tumour effect of IL-21, indicating that the timing ofIL-21 is critical for the outcome. In a fully syngeneic sys-tem, we have shown significant tumour growth inhibition ofB16 melanomas and RenCa RCCs in response to therapeu-tic administration of IL-21 protein, particularly by subcuta-neous administration (43). The anti-tumour activity was CD8+

T cell-dependent, and IL-21 increased the density of tumourinfiltrating CD8+ T cells, a finding associated with improvedprognosis in human cancers. Using stereotactical injections ofIL-21 protein, Daga et al. showed significant tumour rejectionof intracranially implanted gliomas, mediated by NK cells and


26


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-γproduction (spleen)

(36)

B16F1 melanoma, MethAfibrosacoma (syngeneic)

IL-21-secreting tumour Complete tumour rejection CTL, NK-, not CD4+ T cellspartly perforin mediated, notIFN-γ dependent

(37)

AsPC1 pancreatic carcinoma(human xenograft)

IL-21-secreting tumour Significant tumour growthinhibition

Partly NK cells, ↑ IFN-γproduction (spleen cells)

(38)

B16 melanoma, MCA205fibrosarcoma (syngeneic)

IL-21-expressing plasmids(d5 and 12 PTI)

Significant tumour growthinhibition and increasedsurvival

NK cells, minor CTL role notCD4+ T cells, ↑ NK cellcytotoxicity, no IFN-γ increase(serum)

(39)

B16F10 melanoma, RenCa renalcell carcinoma, DA3 mammarycarcinoma (syngeneic)

IL-21-expressing plasmids(d -2 or 1 PTI)

Reduced number of lung andliver metastasis

NK cells, perforin dependent,not IFN-γ, Fas ligand or TRAIL

(30)

OVA-expressing, E.G7 thymoma(semi-syngeneic)

IL-21 protein 20–100 μg i.p.early (d2–12 PTI) 1×/daylate (d12–22 PTI) 1×/day

Significant tumour growthinhibition and increasedsurvival compared with IL-2and IL-15

CTL, early moderate role for NKcells, not CD4+ T cells, ↑ andsustained Ag-specific CTLresponse, ↑ cytotoxicity

(40)

Several carcinomas withendogenous or transfectedNKG2D ligands (syngeneic)

IL-21 protein 50 μg i.p.i.v. metastasis: (d0–3 PTI)spontaneous: (d10–12 PTI)

Reduced number of lungmetastasis

NK cells, NKG2D and perforindependent, not IFN-γ and Fasligand

(41)

SCCVII head and necksquamous cell carcinoma(syngeneic)

IL-21-expressing plasmids(d5, 12, 19 and 26 PTI)

Significant tumour growthinhibition and increasedsurvival

T cells, NK cells andtumour-specificimmunoglobulins

(42)

B16F0 melanoma, RenCa renalcell carcinoma (syngeneic)

IL-21 protein 50 μg i.p. or s.c.d3 or 8 PTI, 1×/day, B16d7 or 12 PTI, 3×/week, RenCa

Significant tumour growthinhibition

CTL, not NK cells↑ density of tumour infiltratingCD8+ T cells

(43)

GL261 glioma (syngeneic) IL-21-secreting tumour or IL-21protein 1 μg i.t.

Significant tumour rejection B cells and NK cells↑ tumour-specific IgG,↑ serum-induced ADCC andcomplement-mediated lysis

(44)

PTI, post tumour injection; NK, natural killer; IFN, interferon; IL, interleukin; CTL, CD8+ cytotoxic T cells; OVA, ovalbumin; TRAIL, tumour necrosisfactor-related apoptosis-inducing ligand; ADCC, antibody-dependent ceullular cytotoxicity.

B cells, with increases in tumour-specific antibodies, serum-induced antibody-dependent cellular cytotoxicity (ADCC) andcomplement-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 preclinicaltumours.

Taken as a whole, IL-21 anti-tumour activity dependson NK cells, CD8+ T cells or both, with a possible rolefor B cell-derived tumour-specific Ig. CD4+ T cells seemless important, but so far total CD4+ T cell depletionshave been made, and more specific depletion of CD4+ Tcell subsets, e.g. Tregs and Th17 cells is needed to fullyclarify their role. Perforin, which is critical for both NKand CD8+ T cell cytotoxicity, is up-regulated by IL-21and in contrast to Fas ligand or TRAIL, perforin seemsto be important for IL-21 anti-tumour activity. IL-21 alsoincreases IFN-γ production, but the need for IFN-γ is morevaried than perforin, probably depending on specific tumourcell susceptibility to this molecule. IL-21 generally increasestumour infiltrating CD8+ T cells, but it remains to be shown

whether this is secondary to increases in CD8+ T cellexpansion or whether IL-21 also has direct effects on T celltrafficking. Generally, it remains to be clarified at what stageduring a tumour immune response IL-21 is beneficial; doesIL-21 mainly shape the initiation and expansion processes intumour draining lymph nodes or does it primarily modulatetumour infiltrating lymphocytes during the effector phase.

In addition to its immune-mediated anti-tumour mecha-nisms, IL-21 has direct cytotoxic effects on certain B cell lym-phomas (45), and potential anti-angiogenic effects of IL-21has been suggested (46). Table 1 summarizes the main pre-clinical in vivo anti-tumour data of IL-21 monotherapy.

In addition to having anti-tumour effects when given asmonotherapy, IL-21 also shows significant additive effectstogether with a range of other biologicals modifying bothinnate and adaptive responses. IL-21 protein alone enhancedthe anti-tumour effect against B16 melanomas treated withadoptively transferred transgenic CD8+ T cells recognizinga melanoma antigen and an antigen-specific vaccine (7). But


27


in concert with IL-15, IL-21 significantly boosted the anti-tumour effect by increasing the circulating level of adop-tively transferred CD8+ T cells and augmenting their IFN-γproduction. The sequential stimulation of NKT cells withthe agonist α-GC followed by IL-21 stimulation synergis-tically inhibited B16 melanoma lung metastasis formationby boosting the concomitant NK cell activation induced byNKT cells (47). In combination with a potent triple anti-body cocktail (Trimab), IL-21 helped to completely eradi-cate established tumours (48). Trimab consists of anti-DR5,anti-CD40 and anti-CD137 (4-1BB), which together inducea powerful T cell-dependent anti-tumour response throughDR5-mediated tumour cell apoptosis and generation of anti-gen, co-stimulation of DCs via CD40, and T cell activationthrough CD137 stimulation.

Several other interesting combination partners for IL-21have recently been reviewed (49), expanding the potential ofIL-21 in cancer therapy even further. Still, many of the com-bination partners so far tested with IL-21 are only in earlyclinical testing or otherwise difficult to clinically translate.However, we have recently published that IL-21 has additiveanti-tumour effects in combination with certain chemothera-pies, provided that IL-21 treatment is delayed compared tochemotherapy (50). These data indicate that IL-21 is feasiblein combination with conventional therapies.

Another interesting aspect of IL-21 in cancer therapy is itsuse in the ex vivo generation of antigen-specific CD8+ T cellsfor adoptive cell therapy. IL-21 conditioning in vitro inducesa unique differentiation program in CD8+ T cells yielding ahighly 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 otherbiological response modifiers stimulates both the innate andadaptive arm of the immune system which shows significantanti-tumour activity in several different preclinical tumourmodels. These findings have paved the way for IL-21 clinicaltrials, which are currently ongoing.

Clinical data

IL-2, which is closely related to IL-21, is approved for thetreatment of metastatic melanoma (MM) and RCC. AlthoughIL-2 shows encouraging responses in groups of patients withthese generally unmanageable diseases, its use is limitedbecause of severe toxicities, e.g. vascular leak syndrome,requiring intensive care (52).

In phase I clinical trials IL-21 safety was tested in patientswith MM and RCC (53, 54). IL-21 was well tolerated;the most common dose-related adverse effects were flu-likesymptoms such as fever, fatigue, chills and myalgia, anddose-limiting toxicities included lymphopenia, neutropenia,thrombocytopenia and hepatotoxicity with increases in liverenzymes. Forty-seven MM and 19 RCC patients were treatedwith IL-21. Of these, two complete responses (MM) and four

partial responses (RCC) were observed according to responseevaluation criteria in solid tumours (RECIST). IL-21 alsoshowed evidence of immune activation, with increases ingranzyme B, perforin, IFN-γ and chemokine C-X-C motifreceptor (CXCR)3 mRNA expression in peripheral bloodNK cells and CD8+ T cells (5, 53), suggestive of increasedeffector cell function. Overall, phase I results endorsedprogress 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 MMpatients treated by i.v. administration of IL-21, one partialand one complete response were observed per RECIST, andadverse effects were similar to those observed in the phaseI trails. In the combination of IL-21 and sorafinib, 14 outof 29 previously treated RCC patients completed 21 weekstreatment with stable disease or better and with an acceptablesafety profile.

Mild lymphocytopenia is a frequently observed adverseeffect of IL-21, but interestingly IL-21 increased the frequencyof lymphocytes expressing CD62L and CCR7 (55), indicat-ing that IL-21-induced lymphocytopenia might be caused by aredistribution of lymphocytes to secondary lymphoid compart-ments. Consistent with the phase I trials, NK cells and CD8+

T cells in phase II trials showed significant increases in per-forin, granzyme B and IFN-γ expression following IL-21 (55).Also, CD8+ T cells and NK cells increased their expression ofthe activation markers CD25 and CD69 in response to IL-21,whereas CD4+ T cells did not, indicating biologically dif-ferent effects of IL-21 on these lymphocytes. These resultsconfirm that IL-21 is a well-tolerated drug that has objectiveanti-tumour activity in human cancer patients associated withsigns of relevant immune activation.

Further trials are currently ongoing, evaluating IL-21 aloneand in combination with other compounds. Preliminary reportsfrom 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 encourag-ing (55). The final results of these and future clinical trialswill be very interesting to follow.

IL-21 in immunopathology

Potent stimulation of immune responses inherently risks thedevelopment of autoimmunity. Given that IL-21 stimulatesNK and T cell-mediated tumour immunity, plays a centralrole in B cell differentiation and antibody production, andamplifies the expansion of proinflammatory Th17 cells, it isnot difficult to appreciate that host-derived IL-21 could alsobe a key player in immunopathologies (see Table 2).


28


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 andMRL-Faslpr mice overexpress IL-21

(56–58) Polymorphisms in IL-21 and IL-21R genesassociate with SLE

(60, 61)

IL-21R.Fc ameliorates lupus and IL-21Rdeficiency protects from lupus

(58, 59) Low IL-21R expression on B cells in SLEpatients correlates with disease activity

(62)

Type 1 diabetes NOD mice overexpress IL-21, IL-21Rdeficiency protects from diabetes, andtransgenic expression of IL-21 inducesdiabetes C57BL/6 mice

(63, 66, 67) Polymorphisms in the IL-2/IL-21 locus andin the IL-21 and IL-21R genes associatewith T1D

(68, 69)

Ag-specific CD4+ T cells in RIP-OVA miceoverexpress IL-21 counteracting Tregsuppression of diabetes

(65)

Rheumatoid arthritis IL-21R.Fc ameliorates CIA in DBA/1 mice (70) Inflamed synovial tissue, synoviallymphocytes and PBL from RA patientsoverexpress IL-21R

(73, 74)

IBF-deficient mice overexpress IL-21 anddevelop spontaneous arthritis

(71) IL-21R.Fc blocks inflammatorycytokine-release in RA synovial cellcultures

(75)

IL-21R-deficient K/BxN mice are protectedfrom arthritis

(72) Polymorphisms in IL-2/IL-21 locusassociate with RA

(76)

Inflammatory bowel disease IL-21 co-stimulation of TGFβ-treatedCD4+CD25− T cells inhibits colitissuppression in SCID mice receivinguntreated CD4+CD25− T cells

(77) Polymorphisms in IL-2/IL-21 locusassociate with UC and CD, and inflamedgut mucosa from UC and CD patientsoverexpress IL-21

(78, 79, 81)

IL-21 is overexpressed in gut mucosa fromDSS- and TNBS-induced colitis and IL-21deficiency protects from colitis

(78) IL-21 from LPM cells increases MMPrelease from intestinal fibroblasts in CDpatients

(10)

IL-21R.Fc inhibits CCL20 secretion and Tcell chemotaxis by IBD mucosa

(80)

Multiple sclerosis IL-21 administration prior to, but not postMOG immunization increases EAEseverity

(82) IL-21 drives secondary autoimmunity in MSpatients treated withlymphocyte-depleting Ab (alemtuzumab)

(85)

IL-21R-deficient mice are protected fromMOG-EAE

(24)

IL-21/IL-21R-deficient mice are notprotected from MOG-EAE and IL-21R.Fcincreases severity of PLP-EAE

(83)

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 autoimmunedisease that can affect connective tissues throughout the body.Hallmarks of SLE are high levels of circulating autoantibodiesthought to arise from aberrant apoptosis, characteristic rashes,and glomerulonephritis with associated renal dysfunction.

BXSB-Yaa mutant mice, which spontaneously develop acondition similar to SLE, with lymphadenopathy, hyper-gammaglobulinemia, and severe immune-complex-mediatedglomerulonephritis, have elevated serum levels of IL-21 (56).In another mutant mouse strain, the sanroque mouse, a muta-tion in the roquin protein negatively regulates Tfh cell devel-opment. This mouse developed a similar lupus-like syndromeassociated with increased Tfh cell levels and overproduction

of IL-21 (57). The lupus-prone MRL-Faslpr mouse showedincreased IL-21 production from CD4+ T cells and IL-21R.Fcadministration ameliorated disease severity (58). And, under-lining the role of IL-21 in experimental SLE, homozygousIL-21R−/−BXSB-Yaa mice failed to develop renal diseaseand mortality (59). These studies outline a potential benefitof IL-21 neutralization in SLE patients, particularly given thecritical role for IL-21 in Tfh cell and GC development, plasmacell differentiation and antibody production.

In humans, polymorphisms in the human IL-21 and IL-21R gene show association with SLE (60, 61) and peripheralB cells from SLE patients have decreased IL-21R expres-sion correlating with increased disease activity (62). Takentogether, these data indicate a putative role for IL-21 in the


29


pathogenesis of SLE, but future studies are required to betterclarify its role in human disease.

Type 1 diabetes

Type 1 diabetes (T1D) is a condition caused by specificautoimmune destruction of insulin-producing β-cells in thepancreas, resulting in loss of blood glucose control and isfatal without insulin-replacement therapy.

In non-obese diabetic (NOD) mice, a commonly used modelof T1D, the insulin-dependent diabetes susceptibility locusIdd3 on chromosome 3 has major impact on the spontaneousdisease development and spans the region for the IL-2 andIL-21 genes. It was initially shown that NOD mice over-express IL-21 and it was postulated that IL-21 could drivehomeostatic expansion in NOD mice leading to the genera-tion of autoreactive T cells (63). This theory was questionedby findings of genetic variations in the IL-2 gene, leadingto impaired Treg suppressive functions that associated withdiabetes development, and this was instead proposed as thecausal link of Idd3 (64). Tregs can protect against diabetesdevelopment as shown in another diabetes model; however,here, diabetic mice had normal Treg suppressive capacity, butinstead showed overexpression of IL-21 which counteractedTreg-mediated suppression (65). In support, two recent studiesindependently show that IL-21R-deficient NOD mice com-pletely fails to develop diabetes, correlating with reducedinsulitis, decreased infiltration of CD4+ and CD8+ T cellsin the pancreas, and no increases in Treg levels (66, 67). Fur-thermore, in contrast to IL-21R+/+NOD splenocytes, adoptivetransfer of IL-21R−/−NOD splenocytes does not induce dia-betes in NOD/scid mice and transgenic expression of IL-21induces spontaneous diabetes in diabetes-resistant C57BL/6mice (67). These data strongly advocate for a central role ofIL-21 in diabetes development in NOD mice and suggest thatIL-21 is a relevant susceptibility gene in the Idd3 locus. Mucheffort has been put into identifying the one gene that confersthe diabetes association of the NOD Idd3 locus, but it appearsthat IL-2 and IL-21 are not mutually exclusive diabetes sus-ceptibility genes.

In a recent large genome-wide association study of humanT1D, the chromosome region 4q27 was identified as oneof six new robust disease-associated loci (68). This locus isthe human orthologue of the Idd3 containing both the IL-2and IL-21 gene. In another analysis of IL-21 and IL-21Rgene-sequence variants, polymorphisms in both genes wereassociation with diabetes (69). These data indicate that IL-21could have a role in human T1D development, but the strongset of experimental data still lack supportive human data toconfirm this hypothesis.

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory disorderof idiopathic etiology that affects many tissues and organs. It

principally involves autoimmune attacks of the joints produc-ing inflammatory synovitis that often progresses to destructionof the articular cartilage and joint deformities.

In animal models of RA, IL-21R.Fc reduced the histo-logical and clinical signs of collagen-induced arthritis (CIA)in DBA/1 mice immunized with bovine collagen (70). Inanother study, interferon regulatory factor 4 (IRF-4)-bindingprotein (IBF)-deficient mice showed increased IL-21 andIL-17 production leading to spontaneous development of aRA-like condition (71). K/BxN mice spontaneously developautoantibody-dependent arthritis, but IL-21R-deficient K/BxNmice were completely refractory to disease (72). Here, IL-21mainly had a pathogenic role in Tfh development and autoan-tibody production, whereas Th17 cells and Tregs seemed to beuninvolved (72).

In RA patients, IL-21R expression was increased ininflamed synovial tissue and lymphocytes, and in periph-eral blood lymphocytes compared with osteoarthritis (OA)patients (73, 74). Also, IL-21 production was detected in RAsynovial tissue cultures, and IL-21R.Fc inhibited secretion ofproinflammatory cytokines from the synovial tissue (75). Ina recent study, it was proposed that genetic polymorphismsin the IL-2/IL-21 locus were associated with the developmentof RA (76). Overall, increasing evidence suggests that IL-21could be involved in the pathogenesis of RA.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) mainly comprises ulcer-ative colitis (UC) and Crohn’s disease (CD), where bothinvolve 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 toSCID mice results in colitis development, co-transfer ofTGFβ-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 isthought to occur because IL-21 inhibits TGFβ-induced FoxP3expression and Treg differentiation of CD4+CD25− T cellsand instead promotes differentiation of Th17 cells produc-ing high levels of IL-17 as well as IL-21 (77). In experi-mental colitis induced by dextran sulfate sodium (DSS) ortrinitrobenzene sulfonic acid (TNBS), IL-21 expression wasincreased in the gut mucosa, and IL-21-deficient mice werehighly protected from disease correlating with reduced Th17cell activity (78).

In humans, IL-21 expression is increased in inflamed, butnot unaffected gut mucosa from both UC and CD patients (78,79). Isolated lamina propria T cells from CD patients showedreduced IL-17 production when stimulated in the presence ofanti-IL-21 antibody (78), indicating that IL-21 is present inIBD and has potential impact on IL-17 responses, which arebelieved to be pathogenic in CD. Intestinal fibroblasts isolatedfrom colitis patients and healthy controls express IL-21R, and


30


IL-21 alone or in concert with TNF-α significantly induced theirsecretion of matrix metalloproteinases (MMP), thought to playa key role in tissue degradation (10). Also, IL-21R.Fc treatmentreduced the MMP production from intestinal fibroblastsinduced by supernatants from CD lamina propria mononuclearcells (10). Moreover, IL-21 stimulation induced secretion ofthe T cell attractant, CCL20/MIP-3α from colon epithelialcells, and anti-IL-21 blocked T cell chemotaxis induced bysupernatants from IBD mucosal explants (80). In genome-wideassociation studies single nucleotide polymorphisms (SNPs)within the IL-2/IL-21 locus 4q27 associated with both UCand CD (81). Overall, evidence is accumulating that IL-21 isa very relevant cytokine in the pathogenesis of IBD with arole in both tissue remodelling and T cell trafficking.

Multiple sclerosis

Multiple sclerosis (MS) is an autoimmune disease of unknownetiology, which attacks the central nervous system (CNS),leading to demyelination and loss of physical and cognitivefunction.

Experimental autoimmune encephalomyelitis (EAE) is theclassical animal model of MS, where immunization withmyelin-derived peptides or proteins induces CNS inflam-mation mimicking human disease. In EAE, IL-21 proteinadministration prior to myelin oligodendrocyte glycoprotein(MOG)-peptide immunization increased the severity of dis-ease, whereas IL-21 administration during disease progressiondid not increase severity (82). Initially, MOG immunizationof IL-21-deficient mice showed reduced EAE disease activityascribed to a lack of Th17 cell differentiation (24). However,more recent findings have challenged this, showing no reduc-tion or even exacerbated EAE in IL-21- and IL-21R-deficientmice and competent Th17 cell differentiation (83). This dis-crepancy may arise from genetic variations in the mice usedin the different experiments (83). Also, in EAE induced bymyelin proteolipid protein (PLP), administration of IL-21R.Fcbefore and after peptide immunisation increased the severityof disease associated with decreased Treg numbers and Foxp3expression (84).

Altogether, the role of IL-21 in EAE is controversial, anda direct link between IL-21 and human MS remains to beshown. However, a recent publication shows that MS patientswho developed secondary autoimmunity resulting from treat-ment with a lymphocyte-depleting monoclonal antibody,alemtuzumab, had greatly elevated serum IL-21, which wasgenetically predetermined. It is possible that IL-21 drivesexcess homeostatic T cell expansion and apoptosis leadingto secondary autoimmunity (85). Clearly, these data warrantfuture studies of IL-21 in the pathogenesis of MS.

Chronic viral infections

Very recently, a trio of papers showed that host IL-21 was crit-ical for the control of chronic viral infections by subjecting

IL-21- and IL-21R-deficient mice to chronic lymphocyticchoriomeningitis virus (LCMV) (86–88). In these studies, IL-21 was needed neither during the acute phase of viral infec-tions, nor for the maintenance of memory CD8+ T cells afterresolved infections, but without IL-21 chronic presence ofvirus antigen resulted in exhaustion of CD8+ T cell responsesand poor viral control. These studies highlight yet anotherimportant aspect of IL-21 with potential ramifications fornovel therapies. Here it is also worth noticing that the roleof endogenous IL-21 in cancer control, which also includeschronic persistence of antigen, remains to be determined.

Concluding remarks

In less than a decade, IL-21 has advanced from being anovel member of the γc receptor family to a cytokine inclinical trials for the treatment of cancer, with implica-tions in several autoimmune pathogeneses. As cancer therapy,IL-21 monotherapy shows moderate clinical responses, but sofar clinical trials have been limited to pretreated, end-stagepatients and it would be very interesting to evaluate IL-21 inpatients with less advanced disease. The manageable toxicityof IL-21 encourages combination with other drugs, and suchoptions ought to be pursued in future trials. Continued researchinto IL-21 anti-cancer biology will be essential to further clar-ify its immunotherapeutic effects, support future clinical trialsand perhaps identify novel interesting combination partners.

The data reviewed here clearly suggest that neutralizationof IL-21 could hold therapeutic value in several majorimmunopathologies, where particularly IBD, RA and SLEseem to be relevant candidates. However, disclosing the roleof IL-21 in human immunopathologies will be essential towarrant the development of new IL-21-blocking compounds.

Hopefully, the next few years of IL-21 research will clarifyits full potential in cancer therapy and its intriguing role inimmunopathologies.

References

1. Ozaki K, Kikly K, Michalovich D et al. Cloning of a type Icytokine receptor most related to the IL-2 receptor beta chain.Proc Natl Acad Sci U S A 2000: 97: 11439–44.

2. Parrish-Novak J, Dillon SR, Nelson A et al. Interleukin 21 andits receptor are involved in NK cell expansion and regulationof lymphocyte function. Nature 2000: 408: 57–63.

3. Asao H, Okuyama C, Kumaki S et al. Cutting edge: thecommon gamma-chain is an indispensable subunit of the IL-21receptor complex. J Immunol 2001: 167: 1–5.

4. Zeng R, Spolski R, Casas E et al. The molecular basis ofIL-21-mediated proliferation. Blood 2007: 109: 4135–42.

5. Frederiksen KS, Lundsgaard D, Freeman JA et al. IL-21induces in vivo immune activation of NK cells and CD8(+)T cells in patients with metastatic melanoma and renal cellcarcinoma. Cancer Immunol Immunother 2008: 57: 1439–49.


31


6. Caprioli F, Sarra M, Caruso R et al. Autocrine regulation ofIL-21 production in human T lymphocytes. J Immunol 2008:180: 1800–7.

7. Zeng R, Spolski R, Finkelstein SE et al. Synergy of IL-21 andIL-15 in regulating CD8+ T cell expansion and function. J ExpMed 2005: 201: 139–48.

8. Coquet JM, Kyparissoudis K, Pellicci DG et al. IL-21 isproduced by NKT cells and modulates NKT cell activation andcytokine production. J Immunol 2007: 178: 2827–34.

9. Spolski R, Leonard WJ. Interleukin-21: basic biology andimplications for cancer and autoimmunity. Annu Rev Immunol2008: 26: 57–79.

10. Monteleone G, Caruso R, Fina D et al. Control of matrixmetalloproteinase production in human intestinal fibroblasts byinterleukin 21. Gut 2006: 55: 1774–80.

11. Kasaian MT, Whitters MJ, Carter LL et al. IL-21 limits NKcell responses and promotes antigen-specific T cell activation:a mediator of the transition from innate to adaptive immunity.Immunity 2002: 16: 559–69.

12. Ozaki K, Spolski R, Feng CG et al. A critical role for IL-21 inregulating immunoglobulin production. Science 2002: 298:1630–4.

13. Jin H, Carrio R, Yu A et al. Distinct activation signalsdetermine whether IL-21 induces B cell costimulation, growtharrest, or Bim-dependent apoptosis. J Immunol 2004: 173:657–65.

14. Good KL, Bryant VL, Tangye SG. Kinetics of human B cellbehavior and amplification of proliferative responses followingstimulation with IL-21. J Immunol 2006: 177: 5236–47.

15. Konforte D, Simard N, Paige CJ. IL-21: an executor of B cellfate. J Immunol 2009: 182: 1781–7.

16. Suto A, Nakajima H, Hirose K et al. Interleukin 21 preventsantigen-induced IgE production by inhibiting germ lineC(epsilon) transcription of IL-4-stimulated B cells. Blood2002: 100: 4565–73.

17. Wood N, Bourque K, Donaldson DD et al. IL-21 effects onhuman IgE production in response to IL-4 or IL-13. CellImmunol 2004: 231: 133–45.

18. Pesce J, Kaviratne M, Ramalingam TR et al. The IL-21receptor augments Th2 effector function and alternativemacrophage activation. J Clin Invest 2006: 116: 2044–55.

19. Silver JS, Hunter CA. With a little help from th

Documents

Interleukin 21 in cancer and immunotherapy20S%F8ndergaard.pdf · 2011. 1. 14. · 5 Summary Interleukin (IL)-21 is a recently discovered cytokine with pleiotropic immunomodulatory