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com/locate/jnlabr/ycyto Cytokine 28 (2004) 109e123

Overview of interleukin-2 function, production and clinical applications

Sarah L. Gaena,*, Kathleen D. Liub
University at Bualo, State University of New York, Department of Oral Biology and Department of Microbiology, 3435 Main Street, Bualo, NY 14214, USA b University 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
a

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 eects 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 rst 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 specic receptors that presumably mediated its internalization [2]. Because IL-2 exerts a striking array of pleiotropic eects on numerous target cells, a number of dierent activities were described and named prior to
* Corresponding author. Tel.: C1 716 829 2786; fax: C1 716 829 3942. E-mail address: sgaen@bualo.edu (S.L. Gaen). 1043-4666/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2004.06.010

its purication 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 of w15 kDa. It exists in a globular structure with four a-helices folded in a conguration typical of the Type I cytokine family (Table 1).

1.2. Main activities and pathophysiological roles IL-2 exerts its eects 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 anity IL-2 receptor (Table 2), thus permitting rapid and selective expansion of eector T cell populations activated by antigen [6]. Accordingly, a major function

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Table 1 Main biological activities of IL-2 (IL-2 induces a myriad of eects on cells of the immune system; some of its major eects are outlined here) Cell type CD4C T cells Primary activities of IL-2 Induces expansion of antigen-specic clones via both proliferative and anti-apoptotic mechanisms Augments production of other cytokines Required for dierentiation to Th1 and Th2 subsets Induces apoptosis of activated T cells via Fas/FasL signaling (activation-induced cell death) Involved in development of CD4CCD25C T regulatory cells (?) Induces expansion of antigen-specic clones Augments cytokine secretion Augments cytolytic activity Induces proliferation of memory CD8C cells Enhances antibody secretion Initiates immunoglobulin J chain transcription and synthesis Promotes proliferation Promotes proliferation Augments cytokine production Enhances cytolytic activity

CD8C T cells

B cells

NK cells

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 antiapoptotic signals, IL-2 also exerts eects on cellular metabolism and glycolysis that are necessary for longterm 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 eects 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. Second, 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 act during thymic development to prevent autoimmunity, probably by inuencing the development of CD4CCD25C T regulatory cells [13e15]. In addition to its eects 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 GMCSF. 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 identied, 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 immunoglobulin 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, suggesting that IL-2 corresponds to the Idd3 gene (Figs. 1 and 2) [24]. 2.2. Regulatory sites and corresponding transcription factors

Table 2 Binding anities and subunit compositions of IL-2 receptor complexes (Kd: dissociation constant) IL-2 anity High Intermediate IL-2Rb gc Low IL-2Ra Subunit IL-2Ra composition IL-2Rb gc Dissociation constant Ability to signal

Kd Z 10e75 pM Kd Z 0.5e2 nM Kd Z 10e20 nM Complete Complete None

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

S.L. Gaen, K.D. Liu / Cytokine 28 (2004) 109e123 Table 3 Phenotypes of mice with targeted deletions in IL-2 or IL-2 receptor subunits Targeted Major cause gene of death IL-2 Cytokines Eects on T cells aected directly Eects on B cells Eects on NK cells References

111

Anemia, IL-2 ulcerative colitis

Normal lineage Normal lineage Normal lineage development development development Increased serum Ig levels Slightly reduced After birth, CD4C cells develop activated phenotype activity Normal lineage development Normal lineage None reported development Increased serum Ig levels After birth, CD4C cells develop activated phenotype Normal lineage development After birth, CD4C cells develop activated phenotype Absence of CD4CCD25C T regulatory cells Development severely impaired Normal lineage development Increased serum Ig levels Fail to develop

Schorle et al., 1991 [72] Kundig et al., 1993 [70]; Sadlack et al., 1993 [77] Willerford et al., 1995 [79]

IL-2Ra

Anemia, IL-2 ulcerative colitis

IL-2Rb

Anemia, IL-2 ulcerative colitis IL-15

Suzuki et al., 1995, 1997 [80,81] Malek et al., 2002 [13]

gc

Mice survive in pathogen-free environments

IL-2 IL-4 IL-7 IL-9 IL-15 IL-21

Lineage development severely impaired Diminished serum Ig levels

Fail to develop

DiSanto et al., 1995 [83]; Cao et al., 1995 [82]

cytokines (Figs. 3 and 4) [25]. While new details of these pathways continue to emerge, a simplied picture indicates that signaling through TCR triggers a phospholipase 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 not trigger maximal IL-2 secretion. Rather, optimal IL-2 production requires additional signals from co-stimulatory 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 expressed Oct-1, act in a concerted fashion to drive transcription of the IL-2 gene. The major regulatory sites that confer T cell-specic, inducible transcription of a reporter gene in T cell lines are located within a w300 base pair (bp) region upstream 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 constitutively 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 specic 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 translocation to the nucleus. Interestingly, calcineurin is the target of several potent immunosuppressive drugs (including cyclosporin A and rapamycin), which suppress 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 RasRaf-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

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1 31 46/1 cac tct ctt taa tca cta ctc aca gta acc tca act cct gcc aca atg tac M Y 61/6 91/16 ctc ctg tct tgc att gca cta agt ctt gca ctt gtc aca aac agt gca cct A P L L S C I A L S L A L V T N S

agg atg caa R M Q act tca agt T S S

121/26 151/36 tct aca aag aaa aca cag cta caa ctg gag cat tta ctg ctg gat tta cag atg att ttg S T K K T Q L Q L E H L L L D L Q M I L 181/46 211/56 aat gga att aat aat tac aag aat ccc aaa ctc acc agg atg ctc aca ttt aag ttt tac N G I N N Y K N P K L T R M L T F K F Y 241/66 271/76 atg ccc aag aag gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa ctc aaa cct M P K K A T E L K H L Q C L E E E L K P 301/86 331/96 ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt cac tta aga ccc agg gac tta L E E V L N L A Q S K N F H L R P R D L 361/106 391/116 atc agc aat atc aac gta ata gtt ctg gaa cta aag gga tct gaa aca aca ttc atg tgt I S N I N V I V L E L K G S E T T F M C 421/126 451/136 gaa tat gct gat gag aca gca acc att gta gaa ttt ctg aac aga tgg att acc ttt tgt E 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 agg Q S I I S T L T * 541 571 cct tct att tat tta aat att taa att tta tat tta ttg ttg aat gta tgg ttt gct acc 601 631 tat tgt aac tat tat tct taa tct taa aac tat aaa tat gga tct ttt atg att ctt ttt 661 691 gta agc cct agg ggc tct aaa atg gtt tca ctt att tat ccc aaa ata ttt att att atg 721 751 ttg aat gtt aaa tat agt atc tat gta gat tgg tta gta aaa cta ttt aat aaa ttt gat 781 aaa 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.

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 degradation. 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 transcription 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 aected 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-specicity of IL2 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 nding 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, indicative of a locus controlling element [35]. Another intriguing feature of cytokine gene regulation 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

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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.

that the relative frequency of IL-2-producing cells was reduced to approximately half, suggesting monoallelic expression of IL-2 [36]. Similar ndings were made for the IL-4 gene [37]. However, other studies have called this nding into question. For example, mice expressing the green uorescent protein (GFP) in place of one of
TCR/CD3 Signals

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 expression is controlled at the mRNA level. Indeed,
CD28 Signals

NFAT AP-1 NFAT/AP-1

~-300

Oct-1

NF- B

CREB

NF- B AP-1 CD28RE/AP

NFAT

AP-1 Oct-1

~ -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 Oct1, Ap-1, NFAT, CREB and NF-kB transcription factors.

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TCR CD4 CD3 LAT

Grb2 Sos Lck ZAP70 Ras Gads SLP76 Vav Raf Rac MEK IKK JNK ERK cdc42
PKC

CD3

LAT
PLC

PtdIns(4,5)P2 Ins(1,4,5)P3 DAG Ca2+

Calcineurin

NF-AT NF-B

AP-1
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 Bualo, State University of New York.

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

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 nding 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 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 approximately 60% of activated CD4C T cells secrete IL-2 following non-specic 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 dierentiation [43]. In addition, CD8C T cells also secrete substantial quantities of IL-2 after stimulation of their T cell receptors. 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].

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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 requires signaling from two distinct pathways (Fig. 3). Accordingly, 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 specic antigen in the context of Class II MHC on antigen-presenting cells (APCs). Unlike antigeneantibody interactions, the binding anity 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 signicant 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 identied [25]. A number of pharmacological enhancers and inhibitors have been identied that promote TCR signaling. First, T cells can be stimulated non-specically 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 intracellular stores, activates the phosphatase calcineurin, and ultimately triggers nuclear import of the NFAT transcription factor. Ionomycin is a calcium ionophore that eciently 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 secretion [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 dierent 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.

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).

Specically, Type I cytokines are described as four ahelical bundles, as their three-dimensional structures contain 15-amino acid a-helices in a characteristic arrangement. The rst and last of the helices are connected by long overhand loops, resulting in an upeupedownedown topology in which the rst two helices are oriented in an up position (as viewed from the N-terminal direction) and the last two are oriented in 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 dened [49]. A single, essential disulde 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 cytokines structure. 3.2. Posttranslational modications IL-2 exhibits O-linked glycosylation at threonine 3, but this modication is not essential for its biological activity [50], nor does it change its activity in standard bioassays. The functional signicance 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 dierential electrophoretic migrations that correlate with changes in glycosylation [51].

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

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revealed the existence of three classes of IL-2 binding complexes that exhibited low, intermediate and high anities 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 anity receptor, and is homologous to a similar anitymodulating subunit in the IL-15 receptor complex (IL15Ra). While IL-2Ra enhances the anity of IL-2R for ligand by approximately 100-fold, it does not contribute to signal transduction in any way. In contrast, the IL2Rb (p75) and gc (IL-2Rg, p65) subunits are necessary and sucient for eective signaling [61e63]. Alone, neither IL-2Rb nor gc bind IL-2 detectably, but the IL2Rb/gc complex comprises the intermediate anity IL-2 receptor complex, and is capable of mediating the full spectrum of IL-2-dependent activities if exposed to IL-2 in sucient 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 dened to include receptors that share its subunits. Whereas IL-2Ra is used exclusively by IL-2R, the IL2Rb chain forms an essential part of the trimeric IL-15 receptor. Moreover, the gc subunit forms part of the IL4, IL-7, IL-9, IL-15 and IL-21 receptor complexes [65]. Indeed, inherited mutations in the human gc gene cause X-linked immunodeciency syndromes due to a phenotypic 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.

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 dierences While human recombinant IL-2 (hIL-2) eectively activates signaling in both human and murine T lymphocytes, murine IL-2 (mIL-2) promotes proliferation far more eectively in mouse cells than in human cells [75]. Mechanistically, the IL-2Ra subunit is responsible for conferring species specicity in IL-2 binding [76]. 6.3. Knockout mouse phenotypes IL-2 was originally dened 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 immunodeciency, but rather a lethal, autoimmune inammatory disease aecting multiple target organs [70,72,77]. At birth, IL2/ mice have normal numbers of T, B, and NK cells. Although the kinetics of the IL-2 deciency 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 na ve 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 inammatory 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. Inltrations 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-deciency syndrome, as neither nude mice nor IL-2/:Rag-2/ mice develop disease [78]. Mice decient in the various subunits of the IL-2 receptor have also been generated. A similar autoimmune syndrome is observed in mice decient for IL-2Ra [79], consistent with the hypothesis that physiological levels of IL-2 can only be detected by high anity IL-2 receptors. However, IL-2Rb-decient mice exhibit additional defects related to a lack of IL-15 responsiveness. In addition to suering severe autoimmunity, they also fail to develop NK cells or intestinal epithelial

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

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lymphocytes (IELs) [80,81]. Finally, gc knockout mice exhibit an X-linked form of severe combined immunodeciency 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, characterized by severe lung and skin lesions, with inltration 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 determine whether constitutive IL-2 could cause a loss of immune tolerance and trigger diabetes in vivo. Although the transgene caused inammation and insulitis, it did not consistently induce diabetes [85,86]. Moreover, even in mice where diabetes was detected, there was no evidence for antigen-specicity [86]. In these cases, IL-2 augmented recruitment and activation of inammatory cells, but apparently did not cause a breakdown in specic 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 inuences the production 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 eects in vivo [16]. IL-2 also inuences expression of many cytokines and chemokines or their receptors. In consequence, the net eect 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 o IL-2 transcription [87]. The adrenal glucocorticoids also negatively regulate IL-2 production, 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 cytokine receptor to inhibit signaling. In the case of IL-2, soluble IL-2Ra receptors (sIL-2R) have been identied in a number of autoimmune and inammatory conditions [90e93]. However, it is not clear to what extent sIL-2R blocks the eects 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 phosphatases 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 ecient 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 eects 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 nonsmall cell lung cancer [97,98]. In addition, high serum IL-2 levels are associated with progression of autoimmune conditions such as scleroderma and rheumatoid arthritis [99,100], and IL-2 levels are also elevated in chronic hepatitis B infection [101]. Interleukin-2 production by peripheral blood lymphocytes is reduced in patients infected with the human immunodeciency 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 deciency or excess of IL-2. However, HIV infection leads to a progressive immunodeciency 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 aected by HIV infection, there is a selective deciency 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 antigenspecic CD8C CTLs, and may lead to the paradoxical hypergammaglobulinemia 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, neuroblastoma, hepatocellular carcinoma (reviewed in Ref. [104]). In addition, using the severe combined immunodecient (SCID) mouse engrafted with human peripheral blood lymphocyte (PBL), Caligiuri and colleagues have demonstrated that low dose IL-2 prevents EpsteineBarr virus-mediated lymphoproliferative 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. Eects of therapy As detailed below, IL-2 currently has two major clinical uses: as an anti-tumor therapy for renal cell carcinoma and melanoma, and as an immune therapy in patients with HIV infection. The commercially available preparation (Aldesleukin, Chiron Corporation) is a recombinant protein with a single amino acid modication 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-specic CTL precursors occurs in mice cured of their tumors by IL-2 therapy, compared to either na ve mice or mice that failed to achieve tumor regression (Ref. [106] and references therein). In patients with HIV infection, IL2 therapy leads to an increased number of CD4C T lymphocytes. Recent studies characterizing this expanded population have demonstrated a selective peripheral expansion of a na ve CD4C/CD25C T cell subset [108,109]. 8.3. Pharmacokinetics After intravenous injection, the kinetics of serum IL2 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 uid volume. IL-2 is cleared by the kidney. With subcutaneous injection, peak plasma levels are approximately 0.1e0.01% of those seen with intravenous administration. While an intravenous dose of 4.4 ! 106 IU/m2 results in a peak serum concentration 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, dierent routes and doses of IL-2 dosing may selectively enhance eects on high or low anity IL-2 receptors. 8.4. Toxicity In early clinical trials, IL-2 administration led to signicant toxicity [112,113], likely due to an inammatory response mediated by the exogenously administered IL-2, leading to a systemic inammatory 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 IL2, 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 signicant change in response rates to therapy [118]. With longterm therapy there have been reports of both hypo- and hyperthyroidism [119], but it is not clear if this is due to direct eects of IL-2 on the thyroid gland or production of anti-thyroid antibodies. However, there is no predictive relationship between thyroid dysfunction and response to therapy [120]. Thus, response does not correlate with severity of side eects. 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 immunotherapy to prevent relapse following bone marrow transplantation in acute myelocytic leukemia. Although the eects of high and low doses of IL-2 may be mediated by high versus intermediate anity 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 subsequently 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 administered; 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 benet and signicantly increases the side eects 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-administration of tumor inltrating lymphocytes (TIL) has any benet over IL-2 monotherapy (reviewed in Ref. [123]). For melanoma, combination therapy with other immunomodulators such as interferon-a and chemotherapeutic agents may be more eective than IL-2 monotherapy. However, the optimal regimen has yet to be identied and is complicated by the large number of agents used in combination in any given clinical trial. The Intergroup trial, comparing conventional chemotherapy to chemotherapy C IL-2/IFNa, is ongoing. It does not appear, however, that adoptive immunotherapy with TIL or LAK has any survival benet. 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 associated with a small rise in HIV viral load, this eect did not appear to be clinically signicant or meaningful [124]. Subsequent studies demonstrated similar ecacy of intravenous and subcutaneous dosing regimens, with shorter duration of side eects with the subcutaneous regimens, which allowed for the outpatient administration of IL-2 [125]. However, just as the studies

demonstrating clinical ecacy 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 ecacious in increasing CD4C T cells in HIVC patients with greater than 200 CD4C T cells/mm3 without causing signicant increases in viral load. Two larger studies [126] are underway to validate these results and to determine if IL-2 therapy aects morbidity and mortality (ESPRIT and SILCAAT, 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 specically blocking IL-2-mediated signaling through high anity 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 Deciency Foundation.

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