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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
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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
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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
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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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