Overview of Interleukin-2 Function, Production

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Overview of interleukin-2 function, productionand clinical applications

Sarah L. Gaffena,*, Kathleen D. Liub

aUniversity at Buffalo, State University of New York, Department of Oral Biology and Department of Microbiology,

3435 Main Street, Buffalo, NY 14214, USAbUniversity of California, San Francisco, Department of Medicine, Division of Nephrology and Critical Care Medicine,

Box 0624, San Francisco, CA 94143, USA

Received 28 June 2004; accepted 28 June 2004

Abstract

The existence of interleukin (IL)-2 has been recognized for over 25 years, and it remains one of the most extensively studied

cytokines. Here we present a broad overview of IL-2 history, functional activities, biological sources, regulation and applications to

disease treatment. IL-2 exerts a wide spectrum of effects on the immune system, and it plays crucial roles in regulating both immune

activation and homeostasis. Both IL-2 and its multipartite receptor are prototypical of the Type I receptor superfamily, and both

have been exploited in numerous ways in the clinic. Despite the wealth of information generated about IL-2 from in vitro culture

systems, in vivo mouse knockout models, and clinical trials in humans, fascinating new aspects of its functions in the immune system

continue to emerge.

2004 Elsevier Ltd. All rights reserved.

1. Background

1.1. Discovery

IL-2 was discovered in 1975 as a growth-promoting

activity for bone marrow-derived T lymphocytes [1], and

was among the first cytokines to be characterized at the

molecular level. Subsequent experiments showed it to be

a soluble activity present in conditioned medium derived

from cells stimulated with mitogens, and its discovery

made it possible to generate and culture T lymphocytes.

It was also demonstrated that this T cell growth factor(TCGF) activity declined over time, indicating the

existence of specific receptors that presumably mediated

its internalization [2]. Because IL-2 exerts a striking array

of pleiotropic effects on numerous target cells, a number

of different activities were described and named prior to

its purification and cloning. While it is likely that many

of these activities can be attributed at least in part to

IL-2, such conditioned media almost certainly included

additional cytokines. The gene for IL-2 was cloned in

1983 [3,4], and its crystal structure was solved in 1992

[5]. IL-2 is a monomeric, secreted glycoprotein with

a molecular weight ofw15 kDa. It exists in a globular

structure with four a-helices folded in a configuration

typical of the Type I cytokine family (Table 1).

1.2. Main activities and pathophysiological roles

IL-2 exerts its effects on many cell types, the most

prominent of which is the T lymphocyte. Indeed, one

of the most rapid consequences of T cell activation

through its antigen receptor is the de novo synthesis of

IL-2. This is quickly followed by expression of a high

affinity IL-2 receptor (Table 2), thus permitting rapid

and selective expansion of effector T cell populations

activated by antigen [6]. Accordingly, a major function

* Corresponding author. Tel.: C1 716 829 2786; fax: C1 716 829

3942.

E-mail address: [email protected] (S.L. Gaffen).

1043-4666/$ - see front matter 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cyto.2004.06.010

Cytokine 28 (2004) 109e123

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of IL-2 is to promote proliferation of both CD4C and

CD8C T cells. IL-2-induced proliferation occurs via

pro-proliferative signals through the proto-oncogenes

c-myc and c-fos, in combination with anti-apoptotic

signals through Bcl-2 family members [7]. More

recently, it has become clear that, in addition to anti-

apoptotic signals, IL-2 also exerts effects on cellular

metabolism and glycolysis that are necessary for long-

term survival of T cells [8,9].

Paradoxically, studies in IL-2 knockout mice (Table

3) have revealed that perhaps the most important

activity of IL-2 is to downregulate immune responses

in order to prevent autoimmunity. These inhibitory

effects of IL-2 create a negative feedback loop that is

achieved by several mechanisms. First, IL-2 production

is quite transient; thus, in the absence of continued

antigenic stimulation, activated T cells die due to

cytokine deprivation in their microenvironments. Sec-

ond, IL-2 initiates a pro-apoptotic pathway through

enhancing FasL expression on activated T cells [10,11].

Since T cells also express Fas/CD95, this event leads to

programmed cell death (apoptosis) of activated T

lymphocytes. In this regard, IL-2/ mice exhibit

a strikingly similar autoimmune phenotype to the

Fas/ (gld) or FasL/ (lpr) strains of mice [12]. In

addition, there is compelling evidence that IL-2 may actduring thymic development to prevent autoimmunity,

probably by influencing the development of

CD4CCD25C T regulatory cells [13e15].

In addition to its effects on T cells, IL-2 is also

a growth factor for natural killer (NK) cells (together

with IL-15, which signals through an essentially

identical receptor) [16e19]. IL-2 promotes production

of NK-derived cytokines such as TNFa, IFNg and GM-

CSF. Furthermore, IL-12 and IL-2 act synergistically to

enhance NK cytotoxic activity [20].

A number of functions for IL-2 in B cells have been

identified, mostly pertaining to antibody secretion. In

IgM-expressing B cells, IL-2 (in synergy with IL-5)

upregulates expression of heavy and light chain genes as

well as inducing de novo synthesis of the immunoglob-

ulin J chain gene [21]. The latter is required for

oligomerization of the IgM pentamer, and represents

a tightly controlled stage in B cell activation [22]. As in T

cells, IL-2 increases expression of IL-2Ra in B cells, thus

enhancing their responsiveness to IL-2 [23].

2. Gene and gene regulation

2.1. Relevant linkages

IL-2 is located on human chromosome 4 and mouse

chromosome 3. Interestingly, in mice, the IL-2 genes lies

within a 0.35 cM of Idd3, a susceptibility locus for

insulin-dependent diabetes in the non-obese diabetic

(NOD) mouse. Moreover, a polymorphism in IL-2 (a

serine to proline substitution at position 6 of the mature

IL-2 protein) consistently segregates with Idd3, suggest-

ing that IL-2 corresponds to the Idd3 gene (Figs. 1 and

2) [24].

2.2. Regulatory sites and corresponding

transcription factors

Like many cytokines, expression of the IL-2 gene is

controlled at multiple levels. In particular, a great deal is

known about transcriptional control of IL-2, because its

upregulation is the major endpoint of signaling by the T

cell antigen receptor (TCR). TCR recognizes the MHC/

antigen complex on antigen-presenting cells, and the

TCR signal can be mimicked in vitro by crosslinking

TCR with antibodies to CD3. An intricate array of

signals is triggered by TCR, which ultimately lead

to transcription of genes encoding IL-2 and other

Table 1

Main biological activities of IL-2 (IL-2 induces a myriad of effects on

cells of the immune system; some of its major effects are outlined here)

Cell type Primary activities of IL-2

CD4C T cells Induces expansion of antigen-specific clones via

both proliferative and anti-apoptotic mechanisms

Augments production of other cytokines

Required for differentiation to Th1 and Th2 subsetsInduces apoptosis of activated T cells via Fas/FasL

signaling (activation-induced cell death)

Involved in development of CD4CCD25C T

regulatory cells (?)

CD8C T cells Induces expansion of antigen-specific clones

Augments cytokine secretion

Augments cytolytic activity

Induces proliferation of memory CD8C cells

B cells Enhances antibody secretion

Initiates immunoglobulin J chain transcription

and synthesis

Promotes proliferation

NK cells Promotes proliferationAugments cytokine production

Enhances cytolytic activity

Table 2

Binding affinities and subunit compositions of IL-2 receptor complexes

(Kd: dissociation constant)

IL-2 affinity High Intermediate Low

Subunit

composition

IL-2Ra IL-2Rb IL-2Ra

IL-2Rb gc

gc

Dissociation

constant

KdZ 10e75 pM KdZ 0.5e2 nM KdZ 10e20 nM

Ability

to signal

Complete Complete None

110 S.L. Gaffen, K.D. Liu / Cytokine 28 (2004) 109e123


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cytokines (Figs. 3 and 4) [25]. While new details of these

pathways continue to emerge, a simplified picture

indicates that signaling through TCR triggers a phos-

pholipase C (PLC)g-dependent pathway, which in turn

activates three major classes of transcription factors:

nuclear factor of activated T cells (NFAT), NF-kB,

and AP-1. Alone, however, TCR signaling does nottrigger maximal IL-2 secretion. Rather, optimal IL-2

production requires additional signals from co-stimula-

tory molecules such as CD28 [26]. Importantly, signals

derived from co-stimulatory receptors greatly enhance

the activation of AP-1 and NF-kB, although the precise

mechanisms by which co-stimulation occurs is still

the subject of much research. Collectively, these

transcription factors, together with constitutively ex-

pressed Oct-1, act in a concerted fashion to drive

transcription of the IL-2 gene.

The major regulatory sites that confer T cell-specific,

inducible transcription of a reporter gene in T cell lines

are located within a w300 base pair (bp) regionupstream of the IL-2 start site [27]. As would be

expected, a high degree of sequence homology between

the mouse and human promoters occurs across this

region [28]. This proximal IL-2 promoter includes

binding sites for Oct, NFAT, AP-1, and NF-kB, and

each of these transcription factors plays an important

role in control of IL-2 expression, as outlined below.

2.2.1. Oct

The IL-2 proximal promoter contains two binding

sites for the Oct family proteins, which are both

important for transcription. While mutation of either

site reduces promoter function, mutation of both sites

completely blocks promoter activity. Oct-1 is constitu-

tively expressed in T cells, and Oct-2 is upregulated after

T cell activation. Both Oct-1 and Oct-2 probably

participate in gene activation [27].

2.2.2. NFAT

The IL-2 promoter also contains two sites for NFAT

family members, and in vivo footprinting studies

indicate that both sites are indeed occupied in stimulated

T cells [29]. The specific NFAT family members

involved in IL-2 gene regulation are NFATc1 and

NFATc2 [30]. Prior to T cell activation, these proteins

are located in the cytoplasm, and signals through the

Ca2C-dependent phosphatase calcineurin result in

NFAT de-phosphorylation and subsequent transloca-

tion to the nucleus. Interestingly, calcineurin is the

target of several potent immunosuppressive drugs

(including cyclosporin A and rapamycin), which sup-

press T cell activity by inhibiting IL-2 secretion (see

Section 8.5).

2.2.3. AP-1

The AP-1 transcription factor is a dimer, typically

composed of the c-Jun and c-Fos proteins. The Ras-

Raf-Erk pathway leads to production of c-Fos. The

JNK signaling pathway leads to formation of AP-1 by

causing phosphorylation of c-Jun, thus permitting its

Table 3

Phenotypes of mice with targeted deletions in IL-2 or IL-2 receptor subunits

Targeted

gene

Major cause

of death

Cytokines

affected

directly

Effects on T cells Effects on B cells Effects on NK cells R eferences

IL-2 Anemia,

ulcerative colitis

IL-2 Normal lineag e

development

Normal lineage

development

Normal lineage

development

Schorle et al., 1991 [72]

After birth, CD4C cellsdevelop activated phenotype

Increased serum Ig levels Slightly reducedactivity

Ku ndig et al., 1993 [70];Sadlack et al., 1993 [77]

IL-2Ra Anemia,

ulcerative colitis

IL-2 Normal lineage development Normal lineage

development

None reported Willerford et al., 1995 [79]

After birth, CD4C cells

develop activated phenotype

Increased serum Ig levels

IL-2Rb Anemia,

ulcerative colitis

IL-2 Normal lineag e

development

Normal lineage

development

Fail to develop Suzuki et al.,

1995, 1997 [80,81]IL-15

After birth, CD4C cells

develop activated phenotype

Increased serum

Ig levels

Malek et al., 2002 [13]

Absence of CD4CCD25C

T regulatory cells

gc Mice survive in

pathogen-freeenvironments

IL-2 Development severely

impaired

Lineage development

severely impaired

Fail to develop DiSanto et al., 1995 [83];

Cao et al., 1995 [82]IL-4Diminished serum

Ig levels

IL-7

IL-9

IL-15

IL-21

111S.L. Gaffen, K.D. Liu / Cytokine 28 (2004) 109e123
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association with c-Fos. AP-1 cooperates with multiple

transcription factors in composite DNA binding sites,

including NF-kB and Oct [27].

2.2.4. NF-kB

Although detectable levels of IL-2 are secreted in

response to TCR alone, optimal activation of T cells

occurs only when TCR-derived signals are accompanied

by signals from co-stimulatory receptors. The canonical

co-stimulator is CD28, which binds to B7 expressed on

antigen-presenting cells. A major signaling pathway

enhanced by CD28 leads to nuclear import of NF-kB

[25]. NF-kB is composed of a heterodimer of two

subunits, p50 and p65. In the absence of stimulation,

NF-kB is tethered in the cytoplasm by an inhibitor

molecule, termed IkB. CD3/CD28 signaling leads to

phosphorylation of IkB on two serine residues, which

causes it to be ubiquitinated and targeted for degrada-

tion. Consequently, NF-kB is released and its nuclear

localization signal exposed, allowing for rapid nuclear

translocation. There are two NF-kB sites within the IL-2

promoter, one of which is a composite element

containing an AP-1 site (termed the CD28RE/AP site

[31]).

In addition to the combinatorial activity of tran-

scription factors, there is also involvement of chromatin

structure and nuclear dynamics in IL-2 gene regulation.

For example, nucleosome positioning in the proximal

IL-2 promoter is affected by TCR signaling [32], and

controls access of transcription factors to the promoter.

Moreover, it is clear that the 5# minimal promoter does

not contain the entire spectrum of regulatory elements

necessary to direct tissue- and temporal-specificity of IL-

2 expression in vivo. Thus, when the 5# promoter region

encompassing 600 bp upstream of the IL-2 start site was

used to drive expression of a transgene in mice, only 1 in

17 lines showed correct expression patterning [33]. This

finding is not surprising, since chromatin structure and

distal locus controlling regions are involved in

regulation of many genes, including other cytokines

[34]. More recently, a regulatory region located in

a 6.4 kb region upstream of the IL-2 gene was found to

confer position-independent transgene expression, in-

dicative of a locus controlling element [35].

Another intriguing feature of cytokine gene regula-

tion is that it sometimes occurs in a monoallelic manner.

Single cell analyses performed on CD4C T cells from

mice heterozygous for the IL-2 null mutation indicated

1 31 46/1cac tct ctt taa tca cta ctc aca gta acc tca act cct gcc aca atg tac agg atg caa

M Y R M Q61/6 91/16ctc ctg tct tgc att gca cta agt ctt gca ctt gtc aca aac agt gca cct act tca agtL L S C I A L S L A L V T N S A P T S S

121/26 151/36tct aca aag aaa aca cag cta caa ctg gag cat tta ctg ctg gat tta cag atg att ttgS T K K T Q L Q L E H L L L D L Q M I L

181/46 211/56aat gga att aat aat tac aag aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tacN G I N N Y K N P K L T R M L T F K F Y

241/66 271/76atg ccc aag aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa cctM P K K A T E L K H L Q C L E E E L K P

301/86 331/96ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta aga ccc agg gac ttaL E E V L N L A Q S K N F H L R P R D L

361/106 391/116atc agc aat atc aac gta ata gtt ctg gaa cta aag gga tct gaa aca aca ttc atg tgtI S N I N V I V L E L K G S E T T F M C

421/126 451/136gaa tat gct gat gag aca gca acc att gta gaa ttt ctg aac aga tgg att acc ttt tgtE Y A D E T A T I V E F L N R W I T F C

481/146 511

caa agc atc atc tca aca cta act tga taa tta agt gct tcc cac tta aaa cat atc aggQ S I I S T L T *

541 571cct tct att tat tta aat att taa att tta tat tta ttg ttg aat gta tgg ttt gct acc

601 631tat tgt aac tat tat tct taa tct taa aac tat aaa tat gga tct ttt atg att ctt ttt

661 691gta agc cct agg ggc tct aaa atg gtt tca ctt att tat ccc aaa ata ttt att att atg

721 751ttg aat gtt aaa tat agt atc tat gta gat tgg tta gta aaa cta ttt aat aaa ttt gat

781aaa tat aaa aaa

Fig. 1. Nucleotide and amino acid sequence of human interleukin-2. Leader peptide is in blue and underlined. The * symbol indicates the stop codon.



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that the relative frequency of IL-2-producing cells was

reduced to approximately half, suggesting monoallelic

expression of IL-2 [36]. Similar findings were made for

the IL-4 gene [37]. However, other studies have called

this finding into question. For example, mice expressing

the green fluorescent protein (GFP) in place of one of

the IL-2 loci were shown to co-express GFP and IL-2

[38], arguing in favor of biallelic expression of IL-2. It is

possible that both modes exist, depending on cellular

context or magnitude of stimulation.

In addition to transcriptional regulation, IL-2 ex-

pression is controlled at the mRNA level. Indeed,

Fig. 2. Nucleotide and amino acid sequence of mouse interleukin-2. Leader peptide is in blue and underlined. The * symbol indicates the stop codon.

The polymorphism associated with Idd3 (susceptibility locus for insulin-dependent diabetes) in the NOD mouse is indicated in red.

NF- B AP-1

CD28RE/AP

CREB Oct-1AP-1NFATNF- BNFAT AP-1

NFAT/AP-1

CD28 SignalsTCR/CD3 Signals

Oct-1

~-300 ~ -60

Fig. 3. Proximal promoter of the human IL-2 gene. Schematic diagram of the 5# upstream region of the IL-2 gene, including binding sites for the Oct-

1, Ap-1, NFAT, CREB and NF-kB transcription factors.



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control of message stability is a characteristic feature of

multiple cytokines including IL-6, GM-CSF and IL-3

[39]. The IL-2 message contains several AU-rich

elements (AREs) that target transcripts for rapid

degradation [40]. The half life of IL-2 mRNA is only

30e60 min, but this doubles in response to T cell

signaling. The IL-2 gene contains at least two cis

elements that regulate transcript stability, located in

both the 3# and 5# untranslated regions (UTRs) [41,42].

The stability element in the 5# UTR appears to be

a target of the JNK pathway, and both act in

a combinatorial manner to regulate message turnover.

2.3. Cells and tissues that express the gene

By far the majority of IL-2 is derived from activated

CD4C T cells. Flow cytometry studies analyzing IL-2

production by intracellular staining indicate that ap-

proximately 60% of activated CD4C T cells secrete IL-2

following non-specific stimulation (i.e., treatment with

phorbol 12-myristate 13 acetate (PMA) and a calcium

ionophore or antibodies that crosslink CD3 and CD28).

Whereas most or all T cells produce IL-2 immediately

following antigen stimulation, only the Th1 subset

produces it in large amounts after T helper cell

differentiation [43]. In addition, CD8C T cells also

secrete substantial quantities of IL-2 after stimulation of

their T cell receptors.

Minor amounts of IL-2 are also produced by certain

antigen-presenting cells. For example, several B cell lines

have been shown to produce small amounts of this

cytokine [23,44]. More recently, dendritic cells (DCs)

were found to produce IL-2 transiently following

microbial challenge [45]. In these cases, IL-2 may serve

to enhance T cell activation, a hypothesis supported by

the finding that DCs derived from IL-2/ mice are

impaired in the ability to promote T cell proliferation. In

contrast, however, macrophages apparently do not

produce IL-2 upon bacterial activation [46], so not all

modes of T cell activation require IL-2 from APC.

3. Protein

3.1. Description

The primary translation product of human IL-2

contains 153 amino acids, and is processed to a mature

form by cleavage of a 20 amino acid, hydrophobic

leader sequence. The N-terminal 20 amino acids are

essential for interaction with the IL-2 receptor, and an

IL-2 mimetic peptide has been developed that is

comprised of its N-terminal 30 amino acids (Fig. 5) [47].

From a structural standpoint, IL-2 is typical of

the short-chain Type I cytokines, despite a lack of

major sequence homology among these proteins [5,48].

PKC

CD4

PLC

NF-B

Calcineurin

NF-AT

DAG

Ins(1,4,5)P3

PtdIns(4,5)P2

LAT

Ca2+

Lck

Raf

Ras

MEK

Vav

Rac cdc42

ERK

TCR

CD3CD3

ZAP70

LAT

Gads

SLP76

Grb2

Sos

AP-1

JNK

IKK

Fig. 4. Signaling pathways activated by the T cell receptor and CD28 molecules that lead to IL-2 production in T helper cells. After engaging MHC

Class II and antigen (not shown), the T cell receptor (TCR)/CD3 complex recruits CD4C and its associated kinase p56-Lck. Subsequently, the

cytoplasmic tails of various CD3 components become phosphorylated by p56-Lck, leading to recruitment of the kinase ZAP70, which proceeds to

phosphorylate various adaptors (e.g., LAT, SLP-76, Gads, and Vav) and also phospholipase C (PLC) g. LAT engages the Ras-Raf pathway, which

contributes to AP-1 formation. PLCg activity leads to production of diacylglycerol (DAG) and intracellular calcium (Ca2C), which in turn activates

protein kinase C (PKC) and calcineurin, respectively. PKC is upstream of both the JNK and NF- kB pathways, whereas calcineurin is upstream of

NFAT. Together, NFAT, AP-1, NF-kB and Oct-1 regulate the IL-2 proximal promoter (see Fig. 3). Figure kindly provided by Dr. Xin Lin,

University at Buffalo, State University of New York.



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Specifically, Type I cytokines are described as four a-

helical bundles, as their three-dimensional structures

contain 15-amino acid a-helices in a characteristic

arrangement. The first and last of the helices are

connected by long overhand loops, resulting in an

upeupedownedown topology in which the first two

helices are oriented in an up position (as viewed from

the N-terminal direction) and the last two are orientedin a down position. In addition, the N- and C-termini

are closely positioned to one another. Major contact

sites with the three subunits of IL-2R have also been

defined [49]. A single, essential disulfide bond between

cysteines 58 and 105 connects the second helix to the

inter-helical region between the third and fourth helices,

which probably provides crucial stability to the cyto-

kines structure.

3.2. Posttranslational modifications

IL-2 exhibits O-linked glycosylation at threonine 3,

but this modification is not essential for its biological

activity [50], nor does it change its activity in standard

bioassays. The functional significance of glycosylation

of IL-2 is not known, but it is likely that it enhances

solubility in aqueous environments. In addition, recent

data indicate another possible role for glycosylation.

Namely, one of the susceptibility alleles for diabetes in

the NOD mouse, Idd3, is closely linked to (and may in

fact be identical to) the IL-2 gene [24]. The IL-2

allotypes in susceptible and resistant mice exhibit

differential electrophoretic migrations that correlate

with changes in glycosylation [51].

4. Cellular sources and tissue expression

4.1. Eliciting and inhibitory stimuli

As outlined above, IL-2 is made by CD4C T cells,

CD8C T cells, some B cells and dendritic cells.

Activation of IL-2 production in T cells requiressignaling from two distinct pathways (Fig. 3). Accord-

ingly, anything that impacts these pathways may serve

to regulate IL-2 production and function. The so-called

signal 1 is initiated from the TCR/CD3 complex,

which engages specific antigen in the context of Class II

MHC on antigen-presenting cells (APCs). Unlike

antigeneantibody interactions, the binding affinity of

TCR/CD3 for antigen/MHC is extremely low. Thus,

a variety of accessory molecules are required to create

a productive interaction between the T cell and APC

[52]. In order to trigger significant levels of IL-2, T cells

also require a signal from a co-stimulatory molecule

(signal 2). The canonical co-stimulator is CD28,

which engages B7-1 and B7-2, but a variety of others

have been identified [25]. A number of pharmacological

enhancers and inhibitors have been identified that

promote TCR signaling. First, T cells can be stimulated

non-specifically with PMA and ionomycin, which results

in potent IL-2 production. PMA mimics the signal

through the TCR/CD3 complex. PMA is an analog of

diacylglycerol, a second messenger normally produced

by PLCg. DAG causes release of calcium from in-

tracellular stores, activates the phosphatase calcineurin,

and ultimately triggers nuclear import of the NFAT

transcription factor. Ionomycin is a calcium ionophorethat efficiently shuttles CaCC ions into the cell and

further augments signaling. In the laboratory, agonistic

antibodies to CD3 and CD28 are also routinely used to

mimic TCR/CD28 signaling and potentiate IL-2 secre-

tion [53].

A number of drugs act at various points in the TCR

signaling pathway to block IL-2 production. For

example, cyclosporin A (CsA) is a cyclic oligopeptide

and a potent immunosuppressant that blocks the

activity of calcineurin, and thus prevents NFAT from

gaining access to the nucleus. Rapamycin and FK506,

other common immunosuppressants in clinical use, also

block calcineurin, although by a different mechanism

[54e56] (see Section 8.5). Inhibitors of the NF-kB

pathway such as PDTC [57] and SN50 [58] also block

IL-2 secretion and may eventually be useful clinically.

5. IL-2 receptor

The IL-2 receptor (IL-2R) is a remarkably complex,

multipartite receptor that has been intensively studied

with respect to its binding characteristics, signaling and

subunit dynamics [59,60]. Early IL-2 binding studies

Fig. 5. Crystal structure of human IL-2. Three-dimensional model of

human IL-2, determined from secondary structure predictions and

comparisons to other members of the cytokine receptor superfamily.Figure reprinted with permission from Ref. [5]. Copyright 1992,

American Association for the Advancement of Science. Other

structural information is at PDB id: 1M47 (Protein Data Bank [128],

crystal structure at 1.99 A resolution).

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revealed the existence of three classes of IL-2 binding

complexes that exhibited low, intermediate and high

affinities for ligand, respectively. It is now recognized

that IL-2R is composed of three subunits. IL-2Ra (also

known as CD25C or Tac) constitutes the low affinity

receptor, and is homologous to a similar affinity-

modulating subunit in the IL-15 receptor complex (IL-15Ra). While IL-2Ra enhances the affinity of IL-2R for

ligand by approximately 100-fold, it does not contribute

to signal transduction in any way. In contrast, the IL-

2Rb (p75) and gc (IL-2Rg, p65) subunits are necessary

and sufficient for effective signaling [61e63]. Alone,

neither IL-2Rb nor gc bind IL-2 detectably, but the IL-

2Rb/gc complex comprises the intermediate affinity IL-2

receptor complex, and is capable of mediating the full

spectrum of IL-2-dependent activities if exposed to IL-2

in sufficient quantities. IL-2Rb and gc are members of

the Type I cytokine receptor superfamily [64,65], and

activate a variety of signaling pathways common to this

family [59,66].

One striking feature of IL-2R is the remarkable

degree to which other cytokine receptors employ its

subunits, and thus the IL-2 family of cytokines has

been defined to include receptors that share its subunits.

Whereas IL-2Ra is used exclusively by IL-2R, the IL-

2Rb chain forms an essential part of the trimeric IL-15

receptor. Moreover, the gc subunit forms part of the IL-

4, IL-7, IL-9, IL-15 and IL-21 receptor complexes [65].

Indeed, inherited mutations in the human gc gene cause

X-linked immunodeficiency syndromes due to a pheno-

typic loss of these cytokine activities (particularly IL-7

and IL-15) [67,68]. It is important to emphasize that,since the IL-15 receptor uses both the IL-2Rb and gc

subunits, IL-15 elicits highly similar or identical

signaling pathways in target cells [16,18,69]. Despite

the redundant use of subunits, however, knockout

studies have indicated unique functions for each of the

IL-2-family cytokines.

6. Biological activities in vivo

6.1. Normal physiological roles

IL-2 is crucial for the maintenance of immune

homeostasis, made strikingly evident from studies in

IL-2 and IL-2 receptor knockout mice (Table 3). First,

IL-2 is an important expansion factor for most or all

types of activated T cells. Although other cytokines

appear to be partially redundant with IL-2 in this

regard, this cytokine is vital for determining the

magnitude and duration of primary and memory

immune responses. Second, IL-2 plays a central role in

downregulating immune responses. Its absence results in

severe autoimmunity due to a failure to eliminate

activated T cells [70e72]. Third, IL-2 opposes IL-15 in

maintaining CD8C T cell memory responses [73,74].

Finally, recent studies have indicated that a major

function of the IL-2/IL-2 receptor system lies in

directing development and function of T regulatory

cells [15].

6.2. Species differences

While human recombinant IL-2 (hIL-2) effectively

activates signaling in both human and murine T

lymphocytes, murine IL-2 (mIL-2) promotes prolifera-

tion far more effectively in mouse cells than in human

cells [75]. Mechanistically, the IL-2Ra subunit is re-

sponsible for conferring species specificity in IL-2

binding [76].

6.3. Knockout mouse phenotypes

IL-2 was originally defined as a T cell growth factor,

and it clearly plays an important role in mediating

expansion of newly activated T cells following TCR

stimulation. Thus, it was contrary to all expectations

that the most profound defect in mice with targeted

deletions in the IL-2 gene was not immunodeficiency,

but rather a lethal, autoimmune inflammatory disease

affecting multiple target organs [70,72,77]. At birth, IL-

2/ mice have normal numbers of T, B, and NK cells.

Although the kinetics of the IL-2 deficiency syndrome

vary depending on genetic background, these mice show

an increase in activated CD4C T cells (CD44C) and

a corresponding decrease in T cells with a nave

phenotype (CD45RBlow

/Mel-14high

). Shortly thereafter,massive activation of B and CD8C T cells occurs,

accompanied by hyperplasia of lymph nodes and spleen

[71]. The mice experience autoimmune hemolytic anemia

early in life, followed by ulcerative colitis, both of which

are thought to be the primary cause of death [77]. An

intact T cell compartment is necessary for development

of inflammatory bowel disease (IBD) in these mice.

Interestingly, IBD is not observed in mice kept in

pathogen-free conditions, indicating a role for antigen in

this process. Infiltrations of mononuclear cells are

observed in many other organs as well, including lung,

pancreas, heart, and liver [71]. CD4C T cells are

required for the development of the IL-2-deficiency

syndrome, as neither nude mice nor IL-2/:Rag-2/

mice develop disease [78].

Mice deficient in the various subunits of the IL-2

receptor have also been generated. A similar autoim-

mune syndrome is observed in mice deficient for IL-2Ra

[79], consistent with the hypothesis that physiological

levels of IL-2 can only be detected by high affinity IL-2

receptors. However, IL-2Rb-deficient mice exhibit

additional defects related to a lack of IL-15 responsive-

ness. In addition to suffering severe autoimmunity, they

also fail to develop NK cells or intestinal epithelial



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lymphocytes (IELs) [80,81]. Finally, gc knockout mice

exhibit an X-linked form of severe combined immuno-

deficiency syndrome (SCID) similar to the human

disease, characterized by a lack of T, B and NK cells

[82,83].

6.4. Transgenic overexpression

Transgenic mice that express human IL-2 under the

control of the constitutive murine MHC Class I (H-2Kd)

promoter have been described [84]. Development of

lymphocyte subsets was normal in these mice, and they

did not demonstrate signs of autoimmunity. However,

these mice did exhibit immune dysfunction, character-

ized by severe lung and skin lesions, with infiltration of

Thy-1C dendritic epithelial cells into skin and brain. In

addition, mice that express IL-2 under the control of the

rat insulin promoter were generated in order to de-

termine whether constitutive IL-2 could cause a loss of

immune tolerance and trigger diabetes in vivo. Although

the transgene caused inflammation and insulitis, it did

not consistently induce diabetes [85,86]. Moreover, even

in mice where diabetes was detected, there was no

evidence for antigen-specificity [86]. In these cases, IL-2

augmented recruitment and activation of inflammatory

cells, but apparently did not cause a breakdown in

specific T cell tolerance.

6.5. Interactions with cytokine network

Because IL-2 is a crucial growth- and expansion

factor for T helper cells, it indirectly influences theproduction of virtually all T cell-derived cytokines.

Moreover, since the IL-2 receptor subunits and/or

intracellular signaling intermediates are used by other

cytokine receptors, there is considerable potential for

antagonism based on competition for limited factors.

There is a particularly intricate interplay between IL-2

and IL-15. Although IL-2 and IL-15 use identical

receptor subunits to deliver signals, these cytokines

nonetheless exhibit contrasting effects in vivo [16]. IL-2

also influences expression of many cytokines and

chemokines or their receptors. In consequence, the net

effect of IL-2-dependent signaling depends on the

concentration of IL-2, concentration of other cytokines,

and the relative levels and types of target cells.

6.6. Endogenous inhibitors and enhancers

There are a number of endogenous inhibitors of IL-2

production, which act by antagonizing signaling

through the T cell receptor. In particular, activated T

cells upregulate expression of an inhibitory signaling

receptor, CTLA-4, which antagonizes the action of the

co-stimulator CD28. Like CD28, CTLA-4 binds to B7-1

and B7-2 on antigen-presenting cells. However, CTLA-4

causes a rapid downregulation of TCR signaling and

thereby shuts off IL-2 transcription [87]. The adrenal

glucocorticoids also negatively regulate IL-2 produc-

tion, at least in part by suppressing the NF-kB and AP-1

transcription factors [88,89]. Furthermore, the activities

of cytokines are frequently modulated in vivo by

decoy receptors that compete with the cytokinereceptor to inhibit signaling. In the case of IL-2, soluble

IL-2Ra receptors (sIL-2R) have been identified in

a number of autoimmune and inflammatory conditions

[90e93]. However, it is not clear to what extent sIL-2R

blocks the effects of IL-2 under physiological conditions.

Finally, there are a number of mediators in the IL-2

signaling pathway that serve to attenuate signaling. For

example, at least two suppressors of cytokine signaling

(SOCS) family members are induced after IL-2R

stimulation, which act to inhibit activity of the

JAKeSTAT pathway [94]. Also, the tyrosine phospha-

tases Shp-1 and Shp-2 have been linked to IL-2R [95,96].

Apart from its initial stimulation by the T cell

receptor, the most striking endogenous enhancer of

IL-2 activity in T cells is IL-2 itself, which stimulates

expression of IL-2Ra and thus promotes efficient

autocrine signaling. Likewise, in B cells, both IL-2 and

IL-5 upregulate IL-2Ra [23], thereby sensitizing B cells

to physiological levels of IL-2.

7. Clinical applications

7.1. Normal levels and effects

Information on serum IL-2 levels in humans, both in

health and disease states, remains relatively limited.

However, a correlation has been demonstrated between

elevated IL-2 levels and progression of gastric and non-

small cell lung cancer [97,98]. In addition, high serum

IL-2 levels are associated with progression of autoim-

mune conditions such as scleroderma and rheumatoid

arthritis [99,100], and IL-2 levels are also elevated in

chronic hepatitis B infection [101]. Interleukin-2 pro-

duction by peripheral blood lymphocytes is reduced in

patients infected with the human immunodeficiency

virus (HIV) [102]. Of note, soluble IL-2 receptor levels

(sIL-2R) have been much more extensively studied in

a variety of disease states, including lymphoproliferative

and autoimmune disorders. In many of these, elevated

sIL-2R levels correlate with severity of illness and can be

used to predict disease relapse [103].

7.2. Role in experiments of nature and disease states

No known human disease is directly attributable to

a deficiency or excess of IL-2. However, HIV infection

leads to a progressive immunodeficiency characterized

by a reduction of CD4C T cells and a consequent



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susceptibility to opportunistic infections. It has been

demonstrated that not only are the total number of

CD4C T cells directly affected by HIV infection, there is

a selective deficiency in production of IL-2 by surviving

CD4C and CD8C T cells [102]. This lack of IL-2 leads

to an inability of the immune system to activate antigen-

specific CD8C

CTLs, and may lead to the paradoxicalhypergammaglobulinemia often observed due to a lack

of IL-2-mediated negative feedback.

8. In therapy

8.1. Preclinical

IL-2 has been studied in a number of animal models

of cancer, including melanoma, prostate cancer, neuro-

blastoma, hepatocellular carcinoma (reviewed in

Ref. [104]). In addition, using the severe combined

immunodeficient (SCID) mouse engrafted with human

peripheral blood lymphocyte (PBL), Caligiuri and

colleagues have demonstrated that low dose IL-2

prevents EpsteineBarr virus-mediated lymphoprolifer-

ative disorders [105]. More recent animal studies have

focused on newer techniques of IL-2 delivery, such as

intratumoral injection of cells secreting IL-2 or gene

therapy with adenoviral vectors [106].

8.2. Effects of therapy

As detailed below, IL-2 currently has two major

clinical uses: as an anti-tumor therapy for renal cellcarcinoma and melanoma, and as an immune therapy in

patients with HIV infection. The commercially available

preparation (Aldesleukin, Chiron Corporation) is a re-

combinant protein with a single amino acid modification

at residue 125 and no amino-terminal alanine. While it is

not clear precisely how IL-2 works as an anti-cancer

therapy, it is thought that the exogenous IL-2 may

promote a CTL-mediated anti-tumor response [107].

This has been indirectly substantiated in animal models,

where a quantitative increase in tumor-specific CTL

precursors occurs in mice cured of their tumors by IL-2

therapy, compared to either nave mice or mice that

failed to achieve tumor regression (Ref. [106] and

references therein). In patients with HIV infection, IL-

2 therapy leads to an increased number of CD4C

T lymphocytes. Recent studies characterizing this

expanded population have demonstrated a selective

peripheral expansion of a nave CD4C/CD25C T cell

subset [108,109].

8.3. Pharmacokinetics

After intravenous injection, the kinetics of serum IL-

2 levels are consistent with a 2-compartment model of

distribution. The initial rapid distribution phase (half

life of 7e13 min) is followed by a slower elimination

phase (half life of 70e85 min) [110,111]. The calculated

volume of distribution of IL-2 is approximately equal to

the extracellular fluid volume. IL-2 is cleared by the

kidney. With subcutaneous injection, peak plasma levels

are approximately 0.1e0.01% of those seen withintravenous administration. While an intravenous dose

of 4.4! 106 IU/m2 results in a peak serum concentra-

tion of approximately 2! 106 IU/ml, a dose of

4.2! 106 IU/m2 administered subcutaneously results

in a peak serum concentration of approximately 40 IU/

ml [110]. Therefore, different routes and doses of IL-2

dosing may selectively enhance effects on high or low

affinity IL-2 receptors.

8.4. Toxicity

In early clinical trials, IL-2 administration led to

significant toxicity [112,113], likely due to an inflamma-

tory response mediated by the exogenously administered

IL-2, leading to a systemic inflammatory response

syndrome. Common toxicities included hypotension,

nausea, vomiting, diarrhea, confusion, shortness of

breath, pulmonary edema, abnormal liver function tests,

renal failure, pancytopenia, rash, fever, chills and

malaise, and infection [114e117]. Interestingly, in

a retrospective review of 1241 patients treated with IL-

2, with improvements in dose reduction protocols based

on toxicity and with prophylactic therapy (such as

antibiotics to prevent infection), there was a substantial

reduction in Grade 3 and 4 toxicities with no significantchange in response rates to therapy [118]. With long-

term therapy there have been reports of both hypo- and

hyperthyroidism [119], but it is not clear if this is due to

direct effects of IL-2 on the thyroid gland or production

of anti-thyroid antibodies. However, there is no pre-

dictive relationship between thyroid dysfunction and

response to therapy [120]. Thus, response does not

correlate with severity of side effects. With subcutaneous

injection of IL-2, erythema and tenderness at the

injection site has been reported. Not surprisingly (given

the lower peak serum levels), there is a lower incidence

of severe toxicity with subcutaneous IL-2 administration

when compared with intravenous IL-2, but a substantial

number of patients nonetheless experience moderate

toxicity, including fever, malaise and nausea [121].

8.5. Clinical results

IL-2 is approved by the Federal Drug Administration

for the treatment of renal cell carcinoma (RCC) and

melanoma and is currently undergoing large-scale

clinical trials for HIV infection. IL-2 has been tried for

a variety of other conditions, including breast, ovarian,

colorectal, bladder, gastric, liver, lung, prostate, and



11/15

head and neck squamous cell cancers, hematologic

malignancies, as well as EBV and hepatitis B infection.

Despite its promise in animal models, there is no clear

role for IL-2 in the treatment of non-Hodgkins

lymphoma. There may be a role of IL-2 in immuno-

therapy to prevent relapse following bone marrow

transplantation in acute myelocytic leukemia. Althoughthe effects of high and low doses of IL-2 may be

mediated by high versus intermediate affinity receptors

(see Table 2), it should be noted that the dosing

regimens for IL-2 as a cancer treatment were empirically

derived prior to the discovery of the IL-2Rb and gc

receptor subunits. IL-2 monotherapy has a reported

tumor regression rate of 20% and a complete response

rate of 9% for renal cell carcinoma. IL-2 was sub-

sequently approved for the treatment of metastatic

melanoma in 1998. For melanoma, the tumor regression

rate is 17%, with a complete response rate of 7%. In

these treatment regimens, IL-2 is administered at a dose

of 600,000e720,000 IU/kg every 8 h until dose-limiting

toxicity or a total of 12e15 doses have been adminis-

tered; this constitutes one cycle of therapy. In the initial

trials, a maximum of 5 courses of therapy were

administered.

For RCC, concurrent administration of lymphokine

activated killer (LAK) cell immunotherapy has not been

shown to have any survival benefit and significantly

increases the side effects of treatment [122]. In contrast

to LAK cells, which are primarily NK and T cells

isolated from the peripheral blood, TIL cells are

composed of T, B and NK cells isolated directly from

the original tumor. It is not yet clear if co-administra-tion of tumor infiltrating lymphocytes (TIL) has any

benefit over IL-2 monotherapy (reviewed in Ref. [123]).

For melanoma, combination therapy with other immu-

nomodulators such as interferon-a and chemotherapeu-

tic agents may be more effective than IL-2 monotherapy.

However, the optimal regimen has yet to be identified

and is complicated by the large number of agents used in

combination in any given clinical trial. The Intergroup

trial, comparing conventional chemotherapy to chemo-

therapyC IL-2/IFNa, is ongoing. It does not appear,

however, that adoptive immunotherapy with TIL or

LAK has any survival benefit.

In the era prior to the advent of highly active

antiretroviral therapy (HAART) for HIV, IL-2 was

shown to substantially increase CD4C T cell counts in

patients who started therapy with CD4C T cell counts

greater than 200 cells/mm3. Although this was associat-

ed with a small rise in HIV viral load, this effect did not

appear to be clinically significant or meaningful [124].

Subsequent studies demonstrated similar efficacy of

intravenous and subcutaneous dosing regimens, with

shorter duration of side effects with the subcutaneous

regimens, which allowed for the outpatient administra-

tion of IL-2 [125]. However, just as the studies

demonstrating clinical efficacy of IL-2 were reported,

HAART was introduced. Thus, more recent protocols

have demonstrated that high and intermediate doses of

IL-2 (7.5 and 4.5 million units injected subcutaneously

twice a day, respectively) are efficacious in increasing

CD4C T cells in HIVC patients with greater than 200

CD4C

T cells/mm3

without causing significant increasesin viral load. Two larger studies [126] are underway to

validate these results and to determine if IL-2 therapy

affects morbidity and mortality (ESPRIT and SIL-

CAAT, in patients with greater than and less than 200

CD4C T cells/mm3, respectively).

Finally, a number of agents in clinical use in solid

organ and bone marrow transplantation target IL-2

production and/or the IL-2 signaling cascade, such as

cyclosporine A, tacrolimus (FK506) and sirolimus

(rapamycin). Anti-IL-2Ra antibodies (anti-Tac) are also

currently conditioning and anti-rejection regimens for

kidney transplantion, and they likely function by

specifically blocking IL-2-mediated signaling through

high affinity receptors [121,127].

Acknowledgements

We thank Drs. Xin Lin, J. Fernando Bazan, and

James Clements for helpful suggestions and critical

comments. SLG is supported by the National Institutes

of Health (AI49329) and the Immune Deficiency

Foundation.

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Overview of Interleukin-2 Function, Production

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