IL2

IL-2 Kendall A. Smith* The Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA * corresponding author tel: 212-746-4608, fax: 212-746-8167, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03001.

SUMMARY Interleukin 2 (IL-2), originally termed T cell growth factor (TCGF), was the first cytokine to be isolated, purified, and characterized at the molecular level. Structurally, IL-2 is prototypic of the interleukins, hematopoietic cytokines, and some more classic hormones, such as growth hormone and prolactin, in that it is a small globular glycoprotein, composed of four amphipathic antiparallel  helices. Functionally, IL-2 is somewhat atypical of the interleukins, in that its production is restricted to only antigen or mitogenactivated T lymphocytes (T cells), and its action is restricted to antigen-activated T cells and natural killer (NK) cells. IL-2 promotes the proliferation, differentiation, and survival of these target cells, such that it is obligatory for a physiologic antigen-specific acquired cellular immune response, and it also augments the innate host defenses through its effects on NK cells. IL-2 was discovered to mediate these biological activities by binding to IL-2-specific receptors, which are expressed transiently on antigenactivated T cells and continuously by NK cells. In vitro, IL-2 regulates the tempo, magnitude, and duration of the proliferative expansion of antigenselected T cells. In vivo, IL-2 is responsible for the clonal expansion of antigen-selected CD4+ and CD8+ T cells. Subsequent to their expansion, the differentiated function of both T helper cells and T cytolytic cells is dependent upon an adequate supply of IL-2. Moreover, the maintenance of the expanded clones is supported by IL-2. Consequently, T cell memory is dependent upon IL-2, in that the size, function, and maintenance of the pool of memory T cells is determined by IL-2. In addition to these positive influences attributable to IL-2, there are negative feedback regulatory effects of IL-2 that function to limit the ultimate immune response. These

observations are supported by data derived from IL-2 and IL-2R gene deletion experiments. IL-2 is presently in use therapeutically for the augmentation of the immune system for the treatment of cancer and infectious diseases. Various doses and regimens are currently popular, some based upon empiric observations and some based upon pharmacodynamics and pharmacokinetics. By comparison, commonly used immunosuppressive agents, such as glucocorticoids, cyclosporin, and rapamycin are focused on inhibiting either the production or action of IL-2. Accordingly, IL-2 plays a central role in the therapeutic manipulation of the immune system.

BACKGROUND

Discovery Despite its designation as the second interleukin, IL-2 was actually the first interleukin to be identified and characterized at the molecular level. Within 5 years of P.C. Nowell's seminal report that lymphocytes could be stimulated to proliferate by phytohemagglutinin (PHA) (Nowell, 1960), mitogenic `factors' were reported to be present in the medium `conditioned' by proliferating lymphocytes (Gordon and MacLean, 1965; Kasakura and Lowenstein, 1965). However, more than a decade passed before Doris Morgan, with coworkers Frank Ruscetti and Robert Gallo, reported that PHA-stimulated lymphocyte-conditioned medium contained a mitogenic activity that could promote the long-term growth of T cells (Morgan et al., 1976). The discovery of IL-2 proceeded in three phases: the discovery of the activity, the discovery of the molecule, and the discovery of the gene. The first discovery was Morgan's observation that the

114 Kendall A. Smith lymphocyte-conditioned medium contained an activity that appeared unique, in that it promoted the continuous proliferation of T cells (Morgan et al., 1976). This observation opened the way for the creation of the first antigen-specific T cell clones (Gillis and Smith, 1977; Baker et al., 1979), and the demonstration that a soluble T cell growth factor (TCGF) activity was responsible for both initiating and maintaining long-term T cell growth after antigen stimulation (Baker et al., 1978; Gillis et al., 1978). Prior to this demonstration, T cell proliferation was considered by most immunologists to be mediated solely by the antigen. The second phase in the discovery process was the biochemical identification, isolation, characterization, and purification of the molecule now known as IL-2 (Robb and Smith, 1981). These studies were aided considerably by the generation of the first monoclonal antibodies reactive to IL-2, which permitted a one-step affinity purification of the molecule to homogeneity (Smith et al., 1983). At the time, the demonstration that the TCGF activity could all be ascribed to a single molecule set a new precedent; previously most workers had assumed that the activity resulted from a combination of several molecules. The third phase of discovery was made by Tadamitsu Taniguchi's group, who were the first to isolate a cDNA that encoded a protein with activity detected by the TCGF assay (Taniguchi et al., 1983). The identification of the IL-2 gene also rested upon the demonstration that the protein specified by the cDNA satisfied the biochemical criteria that we had described for purified, homogeneous natural IL-2 molecules, derived either from normal or from leukemic human T cells (Robb and Smith, 1981; Smith et al., 1983).

Alternative names It is difficult in retrospect to be sure that IL-2 is the cytokine responsible for the plethora of mitogenic factors reported by alternative names before 1983. However, given the data at hand now, it is fairly certain that the `blastogenic factor' activity described in 1965 (Gordon and MacLean, 1965; Kasakura and Lowenstein, 1965) was actually due to IL-2. In the almost 20 years between 1965 and 1983, many other mitogenic factors were described as present in lymphocyte-conditioned media. However, these activities were never purified to homogeneity, so that it is impossible now to ascribe them to IL-2. However, given the methods used to produce the activities from mitogen-stimulated lymphocytes, it is fairly certain

that IL-2 was present in these conditioned media, and was responsible for the mitogenic activities detected.

Structure Human IL-2 is a small (15.5 kDa) globular glycoprotein of 133 amino acids (Robb and Smith, 1981; Taniguchi et al., 1983). The primary sequences of IL-2 from 31 species are now known. The fundamental structure is prototypic for many of the interleukins, and consists of four antiparallel amphipathic  helices. There is one intrachain disulfide bond, between residues 58 and 105, and the molecule is remarkably stable to extremes of pH, salt concentration, and temperature. The secondary and tertiary protein structures are necessary for biologic activity, but the carbohydrate components are dispensable (Robb and Smith, 1981).

Main activities and pathophysiological roles The IL-2 molecule was first identified and characterized as a growth factor for CD8+ T cell clones, which were derived from long-term IL-2-dependent cytolytic T lymphocyte lines (CTLL) (Baker et al., 1979; Gillis et al., 1978; Gillis and Smith, 1977). Now, after more than 20 years of research, it has been confirmed and become established that one of the major activities of IL-2 is to promote the clonal expansion of antigenactivated CD8+ T cells. Although IL-2 also serves as a growth factor for CD4+ T cells (Gullberg and Smith, 1986), as well as natural killer (NK) cells (Caligiuri et al., 1990), the expansion of cytolytic T lymphocytes (CTLs) is undoubtedly one of its most important functions within the host defense system. This conclusion is based on recent experiments performed with IL-2 knockout mice (Cousens et al., 1995). In these mice, the antigen-specific expansion of CD8+ T cells is attenuated by more than 90%. This specific activity of IL-2 for CTL expansion is remarkable, in that these mice have all of the other potentially redundant cytokines with TCGF activity, including IL-4, IL-7, and IL-15, as well as any cytokines yet to be discovered. The differentiation of both T helper (TH) cells and CTLs is also influenced by IL-2. IL-2 is obligatory for the generation of both TH1 and TH2 cells, and it potentiates the production of the characteristic cytokines released by these differentiated TH cells (Seder et al., 1994; Seder and Paul, 1994; Swain, 1994). In a similar fashion, the production of

IL-2 115 cytokines by CTLs, particularly IFN and TNF, is markedly augmented by IL-2. IL-2 also promotes CTL activity by activating the expression of the cytolytic molecules, such as perforin and the serine esterases found in the cytolytic granules of CTLs. NK cells are influenced in a similar way by IL-2, such that it stimulates their proliferative expansion and production of the NK-derived cytokines, IFN , TNF, and GM-CSF. IL-2 also synergizes with both IFN and IL-12 in augmenting NK cell cytolytic activity (Khatri et al., 1998). In addition to promoting the generation of both innate and acquired immunity by stimulating the expansion and differentiation of antigen-selected T cells and NK cells, IL-2 also appears ultimately to trigger the feedback downregulation of the expanded, differentiated effector cells. There are several ways that IL-2 accomplishes such an effect, which initially appeared paradoxical for such a prototypic growth factor. First, IL-2 is produced only transiently after antigen activation (Cantrell and Smith, 1983, 1984; Meuer et al., 1984; Smith, 1980, 1988). Consequently, the positive effects of IL-2 are only transient. In addition, the withdrawal of IL-2 results in apoptosis, so that many of the antigen-specific effector cells are removed by programmed cell death as soon as there is no longer an antigen-induced production of IL-2 (Smith, 1980). In addition to this passive mechanism, there are more positive ways in which IL-2 gives negative signals. One is via the IL-2-promoted expression of CTLA-4, a negative regulator of TCR signals, which competes with the positive, costimulatory molecule, CD28 (Waterhouse et al., 1996; Perez et al., 1997). Thus, instead of leading to continuous cytokine production, the IL-2-promoted expression of CTLA-4 actually suppresses IL-2 gene expression. Also, IL-2 promotes the expression of Fas ligand (FasL), which activates programmed cell death via Fas, which is expressed to a greater extent on antigenactivated CD4+ T cells (Singer and Abbas, 1994; Kneitz et al., 1995; Fournel et al., 1996; Refaeli et al., 1998).

GENE AND GENE REGULATION

Accession numbers Human cDNA: E02018; genomic DNA: K02056 (GenEMBL)

Chromosome location Human chromosome 4, band q26-28.

Regulatory sites and corresponding transcription factors The  350 bp immediately upstream from the transcription start site contain regulatory elements for several transcription factors, the most important of which are AP-1, NFB, and NF-AT. These three families of transcription factors synergize to promote maximal transcriptional activation of the IL-2 gene (Garrity et al., 1994). Exclusion of any one of these factors results in a marked attenuation of IL-2 gene expression.

Cells and tissues that express the gene The only cells known to express the IL-2 gene are T cells.

PROTEIN

Accession numbers Human IL-2: AAA9879

Discussion of crystal structure The initial structure reported for IL-2 as a 4-fold bundle of antiparallel  helices was essentially correct (Brandhuber et al., 1987), but after comparison with the structures of IL-4 and GM-CSF, was found to be in error (Bazan, 1992). However, it is now recognized that many of the molecules with an interleukin designation, as well as the hematopoietic growth factors, and growth hormone and prolactin all have the same basic structure, even though they have no significant primary structural homologies.

Posttranslational modifications The only posttranslational modification of IL-2 is glycosylation (Robb and Smith, 1981). Although the exact nature of the carbohydrate components remain to be determined, the glycosylation accounts for variability in size as monitored by SDS-PAGE, and of charge as determined by isoelectric focusing. In fact, this variability led to the erroneous conclusion that the biologic activity was due to a family of similar

116 Kendall A. Smith molecules rather than a single protein with variable size and charge variation due to glycosylation. The functions of the carbohydrate components are unknown. However, like many secreted proteins, it is assumed that at least one function is the enhancement of water solubility. Recombinant IL-2 derived from E. coli lacks any carbohydrate and is relatively insoluble in aqueous solutions. The carbohydrate components are unnecessary for the biologic activities of IL-2. Detailed experiments with rIL-2 compared with natural IL-2 have shown coincident dose-response curves for both IL-2R binding and growth promotion. Accordingly, there is a significant portion of the molecule that contains no carbohydrates, thereby allowing the amino acid residues to determine IL-2R binding. Since natural IL-2 molecules that are carbohydratefree do not exist, it is possible that nonglycosylated IL-2 may be recognized as foreign by the immune system, provoking an immune response to the therapeutic administration of rIL-2. This would be even more likely if the rIL-2 molecules also contained amino acid substitutions, which might serve as altered peptides that could be presented to and recognized by T cells.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce T cells are the only cells that have been found to express the IL-2 gene, and IL-2 gene expression is strictly controlled by the T cell antigen receptor (TCR) (Smith, 1980; Taniguchi et al., 1983; Meuer et al., 1984). Upon activation of the TCR, IL-2 gene expression is transient, with mRNA detectable by RNase protection assay within 45 minutes, and peak levels occur at  6 hours (Shaw et al., 1988). Thereafter, by 12 hours the IL-2 mRNA is barely detectable. The secretion of IL-2 protein follows this time course, with peak concentrations detectable in the media at  12±24 hours (Smith, 1980).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators When assayed by flow cytometry, about 60% of human CD4+ T cells produce IL-2 when activated polyclonally with phorbol myristic acetate (PMA)

and a calcium ionophore. By comparison, only  30% of CD8+ T cells produce detectable intracellular IL-2. Because there are normally twice as many CD4+ T cells as CD8+ T cells, most ( 80%) of the IL-2-producing capacity is derived from CD4+ T cells. In large part, this discrepancy explains the `help' that CD8+T cells must receive from CD4+ T cells. The `help' is manifested by the IL-2-promoted expansion of the number of antigen-specific CD8+ T cells. Activation of the CD28 `costimulatory molecule' markedly enhances IL-2 production (Verweij et al., 1991). Most evidence indicates that this effect is mediated via activation of NFB, which synergizes with the transcription factors activated by the TCR (Verweij et al., 1991; Ghosh et al., 1993). In addition, CD28 also signals a stabilization of the IL-2 mRNA, which markedly prolongs its half-life (June et al., 1987; Powell et al., 1998; Thompson et al., 1989). These effects of CD28 have led to its designation as the `second signal' in T cell activation, the first signal being supplied by the TCR. However, the contribution of the CD28-augmented production of IL-2 to the subsequent events has not been clearly delineated in most instances, although in some instances it has proven to be the most important of the `second signals' (Seder et al., 1994). Therefore, if the CD28 contributes the `second signal', then IL-2 should be considered to contribute the `third signal' (Powell et al., 1999). Some endogenous modulators of IL-2 production have already been mentioned, such as CTLA-4. However, another important class of negative regulatory molecules is the glucocorticoid hormones produced by the adrenal gland. It has been known since the time of Nowell that glucocorticoids suppress lymphocyte blastic transformation and proliferation (Nowell, 1961). Early on we traced this effect to a suppression of IL-2 production (Smith et al., 1977; Gillis et al., 1979a,b). More recently, it was shown that the effects of glucocorticoids can be attributed to an inhibition of activation of the AP-1 transcription factor c-Jun (Yang-Yen et al., 1990), and NFB (Auphan et al., 1995; Ray and Prefontaine, 1994). There appears to be a feedback regulatory loop between the immune system and the hypothalamicpituitary-adrenal (HPA) axis (Chrousos, 1995). With the introduction of antigen and activation of both innate and acquired host defenses, the production of cytokines, especially the proinflammatory cytokines, such as IL-1, IL-6, and TNF, leads to the activation of the HPA axis and the production of glucocorticoids by the adrenal gland. These hormones then function to attenuate the antigen-driven activation of the immune system, by suppressing the output of IL-2 and other cytokines, particularly the proinflammatory

IL-2 117 cytokines, that initially elicited the release of the glucocorticoids.

RECEPTOR UTILIZATION The IL-2 receptor (IL-2R) was the first cytokine receptor to be discovered and characterized. As such, IL-2Rs are prototypic and satisfy the description of true hormone receptors, which can bind the ligand with high affinity, and specificity, and as well, they deliver signals to the cell as a consequence of binding.

IN VITRO ACTIVITIES

In vitro findings In vitro, IL-2 promotes the proliferation of T cells and NK cells, and augments the differentiation of T cells, B cells, and NK cells.

Bioassays used Five bioassays are used for IL-2: (1) IL-2 CTLL proliferation assay; (2) generation of CTLs; (3) generation of TH1 and TH2 cells; (4) activation of NK cell cytolytic activity and cytokine production; and (5) activation of antibody production. The IL-2 bioassay is the prototypic in vitro cytokine assay (Gillis et al., 1978). The IL-2 activity in a sample is quantified by titration, in the same way that antibody activity in a serum sample is quantified by titration. Routinely, serial 2-fold dilutions of an IL-2 standard and experimental samples are added to 96well microtiter plates, followed by the addition of IL2-responsive CTLL cells. The plate is cultured overnight at 37 C, then the proportion of cells synthesizing DNA is monitored by [3 H]thymidine incorporation, or the number of cells is quantified by vital dye uptake. The data from the experimental samples are assigned an IL-2 concentration by comparison with the standard curve, using the EC50 for comparison. The assay is sensitive to a concentration of 1 pM, and the IL-2 dose-response curve occurs between 1 and 100 pM. Before IL-2 was available in pure form the biologic activity of a sample was defined in biological units/mL. A great deal of confusion ensued regarding these units, in that the definition of a unit varied from lab to lab. A unit was originally defined as the reciprocal of the dilution that yielded 50% of maximal proliferation of CTLL cells. The

International Standard Specific Activity of purified, homogeneous IL-2 is now defined as 15 million IU/ mg IL-2 protein. Therefore, a solution of IL-2 at 1 mg/mL (66.7 mM) should yield 50% of maximal biologic activity at a dilution of 1 : 15 million. This calculates as 1 IU/mL=4.5 pM, which is the specific activity that we calculated initially based upon our IL-2-radiolabeled binding assay as compared with the CTLL bioassay (Robb et al., 1981). One of the main reasons that the IL-2 bioassay is so rapid, requiring only 24 hours to complete, relates to the effect of IL-2 withdrawal from IL-2-dependent cells, such as the CTLLs. Withdrawal of IL-2 results in rapid programmed cell death, such that the cells are irreversibly damaged within 12 hours, and become fragmented between 12 and 18 hours. Therefore, the assay background in the wells that contain no IL-2 is very low, and the maximum versus minimum signal is large. Endogenous IL-2 is necessary for the in vitro generation of CTLs, and when supplied exogenously, IL-2 potentiates the magnitude of cytolytic activity generated (Baker et al., 1978). The relative contribution of the proliferative expansion of antigen-selected cells versus a differentiative effect of IL-2 on the generation of cytolytic activity still remains obscure, although it is clear that IL-2 also facilitates the differentiation of CTLs. IL-2 signals the transcriptional activation of the genes involved in cytolysis, such as perforin, and the granzymes (Liu et al., 1992). In addition, IL-2 stimulates the expression of the Fas ligand, which is necessary for Fas-mediated apoptosis (Ju et al., 1995; Fournel et al., 1996; Refaeli et al., 1998). IL-2 is also necessary for the optimal generation of both TH1 and TH2 cells (Croft et al., 1992; Seder et al., 1994; Seder and Paul, 1994; Swain, 1994, 1999). Thus, TH1 cells will predominate when activated in the presence of IL-12 and IFN , but IL-2 augments the differentiative process. Similarly, the addition of IL-2 to TH cells in the presence of IL-4 leads to the optimal generation of TH2 cells, which are capable of marked IL-4 production when reactivated. Again, there is undoubtedly a proliferative component to the generation of the optimal number of differentiated TH1 and TH2 cells, in that IL-2 is the principal growth factor for T cells (Rogers et al., 1998). However, there is also a differentiative contribution of IL-2, which has yet to be explained on a molecular level. The effect of IL-2 on B cells has been controversial for many years (Nakanishi et al., 1984; Muraguchi et al., 1985). In contrast to T cells, IL-2 does not play a major role as a growth factor for B cells. However, detailed studies have shown the IL-2 is involved in the

118 Kendall A. Smith transcriptional activation of the J chain, and promotes RNA splicing necessary for the switch from membranous to secretory IgM (Blackman et al., 1986; Tigges et al., 1989; Kang et al., 1998). Therefore, IL-2 is obligatory for the primary, IgM response to antigens. IL-2 plays a novel role in stimulating NK cells. NK cells differ from T cells by their constitutive expression of IL-2 receptors without any obvious activating signal (Smith, 1989). As already discussed, only 10% of circulating NK cells express heterotrimeric, highaffinity IL-2Rs, the remainder express the intermediate-affinity IL-2R, comprising heterodimers (Caligiuri et al., 1990). In vitro, the addition of IL-2 to NK cells augments many of the known NK cell functions, including their proliferation, their cytolytic capacity, and their ability to produce cytokines. NK cells express a restricted repertoire of cytokines when stimulated by IL-2, including IFN , TNF, and GMCSF. Since antigen-activated T cells are the only cells capable of producing IL-2, presumably the IL-2 effect on NK cells and the innate host defenses only comes into play later in an immune response, after the activation of TH cells and CTLs by their specific antigens. Therefore, the cytokines produced by T cells, especially IL-2 and IFN , which are known to be potent stimuli for NK cells, connect the innate and acquired host defenses once the acquired, antigenspecific cells have proliferated and differentiated. It is important to emphasize that lymphocytes are the only cells known to be capable of responding to IL-2. In particular, vascular endothelial cells do not express detectable IL-2Rs, nor do they respond in vitro to physiologic concentrations of IL-2. Therefore, many of the well-known effects of IL-2 on the circulation of leukocytes and their transmigration between capillary endothelial cells has been traced to effects of other cytokines, notably TNF and chemokines, rather than IL-2 itself.

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles In vivo, IL-2 is obligatory for the proliferative expansion of antigen-activated T cells, both CD4+ TH1 and TH2 cells and CD8+ CTLs, and for the peripheral expansion of NK cells. Moreover, IL-2 is necessary for the differentiation of all of these cells into effector cells. Accordingly, IL-2 determines the magnitude of a primary cellular immune response,

and as well, it determines the magnitude of the pool of memory cells that survive after the primary immune response has occurred. These conclusions are based primarily upon experiments performed with IL-2 knockout mice, as detailed below. In this regard, it is important to note that these experiments have provided the first proof that IL-2 is not redundant with regard to its role in the generation of normal immune response, and other cytokines and/or surface ligands cannot substitute for IL-2.

Species differences Due to subtle differences in the structures of both the ligands and receptors, there is not always crossreactivity between species. For example, human IL-2 has activity for murine cells, but murine IL-2 has no activity for human cells.

Knockout mouse phenotypes The most definitive data regarding the biological and immunological significance of IL-2 has come from studies of IL-2 knockout mice (Schorle et al., 1991). Deletion of the IL-2 gene has no effect on lymphocyte development, so that at birth, IL-2 knockout mice have normal numbers of T cells, B cells, and NK cells in both primary and secondary lymphoid compartments. However, during postnatal development a progressive lymphoid hyperplasia occurs, and cells accumulate in the secondary lymphoid tissues with an activated surface phenotype (Schorle et al., 1991). Concomitantly, an autoimmune hemolytic anemia ensues, and mice begin to die from anemia within the first few weeks of life. Mice surviving beyond this initial phase subsequently develop colonic enteritis (Sadlack et al., 1993, 1995). However, if kept in a pathogen-free environment, the enteritis is circumvented. Therefore, antigenic activation appears to contribute to the pathology. It is pertinent that CTLA-4 knockout mice develop an identical syndrome (Waterhouse et al., 1996). Since the expression of CTLA-4 is IL-2-dependent, and CTLA-4 functions to provide inhibitory signals that suppress lymphocyte activation, it appears that the lack of either IL-2 or CTLA-4 leads to the absence of normal feedback downregulatory effects. Based upon the activity of IL-2 in vitro as a lymphocyte growth factor it was anticipated that the phenotype of the IL-2 knockout mouse would be an immunodeficiency, rather than a lymphoproliferative syndrome. Actually, when first tested in infectious

IL-2 119 disease models the immune responses of IL-2 knockout mice were reported to be still detectable, although decreased compared with wild-type mice (Kundig et al., 1993). This led to the erroneous conclusion that the cytokines responsible for T cell clonal expansion in vivo are redundant, and that several cytokines could perform the TCGF role. However, subsequently it was found that there is a marked attenuation of the expansion of antigen-activated CD8+ T cells in IL-2 knockout mice (Cousens et al., 1995). Therefore, these experiments substantiated that IL-2 is the principal growth factor for CD8+ T cells, both in vitro and in vivo. Accordingly, rather than a redundancy of cytokines in the T cell immune response, it appears that the other cytokines with TCGF activity cannot substitute for IL-2. Therefore, IL-3, IL-4, IL-7, IL-9, IL-15, etc. do not compensate for the absence of IL-2.

Transgenic overexpression The construction of IL-2 transgenic mice that overexpress human IL-2 in all tissues does not result in a distinctive phenotype (Ishida et al., 1989). Infiltration of dendritic epithelial cells into the skin and infiltration of inflammatory cells into the cerebellum have been described (Katsuki et al., 1989). Most noteworthy, the chronic overexpression of IL-2 has not been reported to lead to the development of leukemias or lymphomas, or to the development of autoimmune diseases.

Pharmacological effects In vitro, the tempo, magnitude, and duration of T cell proliferation after antigen stimulation is determined by the availability of IL-2 (Cantrell and Smith, 1984). Thus, if supplied exogenously, IL-2 will continue to promote clonal expansion of antigen-reactive cells, and IL-2 will circumvent the apoptosis that occurs when the proliferating cells consume IL-2. In vivo, after the massive proliferation, which results in an increase of 10,000±100,000 in the number of antigenreactive cells, there is a loss of  90% of these expanded cells (Gallimore et al., 1998; MuraliKrishna et al., 1998). Just as the supply of IL-2 in vitro circumvents apoptosis, IL-2 administration in vivo after antigen prevents the loss of the expanded antigen-reactive cells (Kuroda et al., 1996). Accordingly, these findings are important when considering the use of IL-2 as an immunotherapeutic, or as an adjuvant for vaccines.

Interactions with cytokine network IL-2 augments the production of almost all of the other cytokines presently identified, either directly via the transcriptional activation of the various cytokine genes, or indirectly, through the activation of the secondary cytokines activated. The magnitude of these effects is strictly IL-2 concentration-dependent, and as well, dependent upon the number of IL-2R+ cells present. In addition, the relative proportions of antigen-activated T cells and NK cells will determine the ultimate outcome of the endogenous release or the exogenous administration of IL-2.

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects IL-2 is the principal growth factor responsible for the expansion of antigen-activated, mature peripheral CD8+ T cells. For example, after a systemic infection with a virus such as Epstein-Barr virus (EBV), a marked proliferative expansion of antigenspecific CD8+ T cells occurs, which ultimately successfully kills the EBV-infected B cells (Callan et al., 1996). This expansion is dependent upon `help' from CD4+ T cells, a conclusion derived from experiments where CTL clones have been expanded in vitro, then reinfused. Without antigen-specific CD4+ T cells, the infused CD8+ T cell clones rapidly undergo apoptosis. Although it has yet to be shown conclusively, the data available support the interpretation that the `help' derived from CD4+ T cells is IL-2. This same IL-2 help promotes the proliferation and differentiation of TH1 and TH2 cells, as well as the proliferation and differentiation of NK cells. Since IL-2 promotes the expansion of antigenselected clones, IL-2 is responsible, in large part, for the size of the population of memory cells that persist after the primary immune response ceases (Ehlers and Smith, 1991). The IL-2-withdrawal apoptosis that occurs after the antigenic stimulus is cleared also ultimately determines the size of the memory cell pool. Therefore, the extent and duration of T cell memory is determined by the availability of IL-2 during and immediately after the primary immune response.

120 Kendall A. Smith

Role in experiments of nature and disease states AIDS represents the prototypic disease state that is manifested by a deficiency of T cell help. There is both a quantitative and a qualitative deficiency of CD4+ T cells in AIDS. Thus, in addition to a decline in the concentration of circulating CD4+ T cells, their functional capacity to produce cytokines, especially IL-2, becomes compromised. In experiments to determine the ability of T cells to produce cytokines after an in vitro polyclonal stimulation, we found that there is a selective deficiency in IL-2 production by both CD4+ T cells and CD8+ T cells from HIV+ individuals, as compared with normals. The end result of this deficiency in cytokine-producing capacity is the persistence of HIV, and ultimately, the susceptibility to opportunistic infections. The deficiency of IL-2 production by cells from HIV+ individuals may well account for the paradoxical hypergammaglobulinemia often seen. Thus, the normal IL-2-mediated negative feedback regulation of the immune system is also compromised. Concomitantly, an inability to promote the expansion of antigen-selected CD8+ CTLs because of the lack of IL-2 creates a situation that resembles a so-called `TH2 phenotype', whereby the humoral immune response predominates over the cellular immune response (Clerici et al., 1991, 1993).

IN THERAPY

Pharmacokinetics The pharmacodynamics of IL-2, or how IL-2 affects the cells and tissues of the body, are based on the type and distribution of IL-2 receptors (Smith, 1989, 1993). High-affinity IL-2Rs, comprised of heterotrimeric  chains are expressed only transiently on antigenactivated T cells and B cells, and  10% of NK cells. Because the affinity of heterotrimeric IL-2Rs is very high, 5±10 pM, only very low concentrations of IL-2, <100 pM, saturate these receptors. By comparison, the intermediate-affinity IL-2Rs, which are expressed by most NK cells, require 100-fold higher IL-2 concentrations ( 10 nM) for saturation. This 100fold difference in affinity, combined with the difference in the number of antigen-activated T and B cells ( 1 million) versus the number of circulating NK cells ( 1000 million), accounts for the main difference in the effects of high doses versus low doses

of IL-2. At high doses, IL-2 concentrations high enough to bind to and activate most of the NK cells are achieved. Consequently, the proinflammatory cytokines produced by NK cells result in severe systemic toxicity. In contrast, if the doses of IL-2 are lower, only the high-affinity IL-2Rs are saturated, and a correspondingly lower number of cells are activated. Therefore, the systemic toxicities can be markedly attenuated or avoided altogether. Because IL-2 is a small (15 kDa) globular glycoprotein, it passes freely between capillary endothelial cells, so that after i.v. bolus administration it distributes into the total extracellular space, which in a normal adult, is  15 L. This  decay phase, due to distribution, occurs with a half-time of  10 minutes, so that in four half-times, > 94% of the initial peak plasma IL-2 concentration is dissipated due to distribution. Subsequently, there is a decay due to renal excretion that has a half-time of 3 hours. Accordingly, within 16 hours, the majority of the administered IL-2 is eliminated. Hepatic metabolism does not appear to play a role in IL-2 pharmacokinetics, at least where nonglycosylated rIL-2 is concerned. As well, the binding and internalization of IL-2 by IL-2 receptors on target cells has not been found to account for a significant effect of the distribution and removal of IL-2. The pharmocokinetics of both the ultra-high-dose and the high-dose IL-2 therapies are similar, in that renal clearance rates are exceeded, and there is a progressive increase in plasma IL-2 concentrations over the 3±5 day treatment intervals. This accounts for the progressive increases in the severity of systemic inflammation over the treatment interval, which effectively precludes longer treatment intervals at these high doses. After a subcutaneous injection of IL-2, there is a relatively rapid appearance of IL-2 within the plasma, with a half-time of 1 hour calculated for absorption. Absorption is assumed to occur via the lymphatics, based upon studies performed with other cytokines with a similar size and structure. Thereafter, the halftime for clearance from the plasma follows a similar time course as observed after i.v. administration. Accordingly, the subcutaneous injection every 12±24 hours results in continuous detectable plasma IL-2 concentrations that vary according to the IL-2 dose. We have found that the low-dose daily subcutaneous administration results in peak plasma IL-2 concentrations at 2±3 hours of  15±30 pM. There is no accumulation of IL-2, and there are no symptoms of systemic inflammation at these low doses (1.2 million U/m2 ). Characteristically, after the subcutaneous injection of IL-2, there is a classic delayed-type hypersensitivity

IL-2 121 (DTH) reaction that develops within 24 hours at the injection site. The size of the inflammatory response is IL-2 dose-dependent, as is its duration. This DTH reaction necessitates changing the location of injection in a rotating fashion, from the trunk, to the upper arms and legs.

Clinical results History: Ultra-high-dose IL-2 Therapy in Cancer IL-2 was the first interleukin to be used therapeutically. Pioneered by Steven Rosenberg in attempts to boost immune reactivity in cancer patients, the principles of dose-intensification until toxicity, derived from cytotoxic chemotherapy, were used to establish the IL-2 dose and regimen used (Rosenberg et al., 1985, 1987). Therefore, the doses of IL-2 and regimens used were established empirically, without consideration of IL-2 pharmacodynamics. IL-2 was administered in very high doses intravenously (i.v.) in bolus injections every 8 hours. This kind of treatment regimen resulted in severe systemic toxicity, clinical grade III±IV (Lotze et al., 1985), which subsequently came to be termed the `cytokine syndrome'. The IL-2 doses used, amounting to  50 million units (3.3 mg) per injection, resulted in plasma IL-2 concentrations of > 10 nM, more than enough to saturate the highaffinity IL-2 receptors, and as well, the intermediateaffinity IL-2 receptors on the majority of NK cells. The end result is stimulation of a massive secondary release of proinflammatory cytokines, IFN , TNF, and GM-CSF, which produce the `capillary leak' characteristic of a systemic inflammatory response syndrome (SIRS). When given for 3±5 days, this ultra-high dose IL-2 therapy results in an antitumor response in about 15% of individuals suffering from renal cell carcinoma, with about 10% of individuals achieving a long-term, complete response (Rosenberg et al., 1987). However, the mechanisms responsible for this antitumor response still remain obscure, even after 15 years of IL-2 therapy. Those who have used this treatment suggest that the severe toxicity is necessary to achieve the antitumor response. However, as 100% of subjects experience the toxicity, but only 15% of individuals achieve a response, it does not appear to be this simple. High-dose IL-2 therapy in HIV infection More recently, IL-2 has been used in the treatment of individuals infected with HIV. Two different dosing regimens have been tried, a high-dose, intermittenttreatment regimen (Kovacs et al., 1996) and a

low-dose, continuous-treatment regimen (Jacobson et al., 1996). The high-dose, intermittent-treatment regimen has been patterned after the ultra-high-dose regimen used for cancer. The dose has been lowered substantially from the ultra-high cancer dose, so that 15 million units (1 mg) are given, either as a continuous 24-hour i.v. infusion, or in two divided subcutaneous injections every 12 hours. This regimen is somewhat better tolerated than the ultra-high-dose regimen, but there is still considerable systemic inflammation, and most patients must be hospitalized. As in the ultra-high doses, the high-dose intermittent IL-2 therapy results in a marked leukopenia of all of the white blood cells during the treatment interval. Subsequently, after discontinuation of treatment, there is a rebound in the concentration of circulating leukocytes, such that their levels rapidly return and exceed by more than 10-fold their pretreatment values. Thereafter, over the ensuing 2 months, the concentration of CD4+ T cells, CD8+ T cells, NK cells, B cells, and monocytes gradually return toward their baseline values. Evaluation of the efficacy of the high-dose IL-2 therapy on the concentrations of circulating lymphocytes has been arbitrarily calculated by averaging the concentration of cells from 30 days and 60 days post-IL-2 treatment, compared with the baseline concentrations. Using this analysis, the data from trials reveal that there is an  50% increase in the concentration of circulating CD4+ T cells compared with baseline levels after six cycles of therapy given every 2 months. However, from the data available, it still remains unclear as to whether there is a net increase in the number of CD4+ T cells, or whether there is simply a shift of cells from the circulation to the tissues and back again as a result of the cytokine syndrome. In this regard, the changes in all of the other lymphocyte subsets in parallel with the CD4+ T cells suggests that there are profound shifts in both plasma and cells as a result of this kind of IL-2 therapy. Daily Low-dose IL-2 Therapy in HIV Infection to Accelerate Immunologic Recovery InHIV+individuals,daily,low-dose(1.2millionU/m2 ) IL-2 therapy accelerates the recovery of circulating CD4+ T cell concentrations  2±3-fold compared with antiviral therapy alone. Thus, CD4+ T cell concentrations increase at a rate of 10 cells/mm3 per month. Therefore, over the course of a year of combination antiviral chemotherapy and IL-2 immunotherapy, the concentrations of circulating lymphocytes can be normalized in most individuals, so that the risk of opportunistic infections is minimized. Simultaneously with the increase in CD4+ T cells,

122 Kendall A. Smith there is a more rapid rise in NK cells, such that they increase by 100 cells/mm3 per month for the first 2 months, then level off and remain elevated as long as IL-2 therapy is continued. By comparison, in the absence of viral antigenic stimulation, elevated circulating CD8+ T cell concentrations gradually decrease to the normal ranges in most subjects, despite the administration of daily IL-2. Low-dose IL-2 Therapy to Generate Protective Immunity Given that the extent of IL-2-dependent expansion of antigen-selected clones determines the size of the memory pool, and that IL-2 administration, especially during the phase after clearance of antigen, prevents apoptosis of the expanded clones, it follows that IL-2 therapy will serve as an effective adjuvant to vaccines. Such an approach should augment the efficacy of vaccines so that repetitive vaccine doses would be unnecessary. Also, a similar approach may well prove useful when vaccines are administered in therapeutic settings, for example in HIV infection when viral replication is suppressed by antiviral chemotherapy. As IL-2 augments both the number and function of antigen-stimulated cells, IL-2 adjuvant immunotherapy is logical in the treatment of latent infections, to boost immunity so that microbial latency is maintained, even after the initial infection has been cleared. Thus, HIV infection may ultimately be contained with such an approach. Moreover, hepatitis C virus (HCV) and cytomegalovirus (CMV) infections may also benefit from combined vaccine/ IL-2 therapy. Immunosuppressive Therapy: Inhibition of IL-2 Production and/or Action The mechanism of action of the most commonly used immunosuppressive drugs is focused on the prevention of the production or action of IL-2. As already described, glucocorticoids are immunosuppressive because they prevent T cell proliferation (Nowell, 1961) by blocking IL-2 production (Gillis et al., 1979a,b) via the specific inactivation of the transcription factors necessary for IL-2 gene expression (YangYen et al., 1990; Auphan et al., 1995; Ray and Prefontaine, 1994). In a similar fashion, the immunosuppressive drug cyclosporin A (CSA) also inhibits TCR-activated proliferation by preventing IL-2 production. However, the mechanism of action, though similar to that of the glucocorticoid hormones (GCHs), involves the inactivation of NF-AT, rather than NFB (McCaffery et al., 1993; Northrop et al., 1994). CSA prevents the

activation of the phosphatase calcineurin, which is required for the dephosphorylation of NF-AT, thereby allowing its nuclear translocation and the transcriptional activation of the IL-2 gene. The macrolide FK-506 works similarly. Rapamycin, another macrolide structurally similar to FK-506, prevents IL-2 action rather than IL-2 production (Powell et al., 1999). The mechanism involves the prevention of the degradation of the cyclin-dependent kinase inhibitor (CKI) p27, which is required for IL-2-promoted progression through the G1 phase of the cell cycle. The combination of GCHs, CSA, and rapamycin is very effective in immunosuppression, in that they all prevent either the production or action of IL-2, but do so by different mechanisms. The use of the Tac monoclonal antibody to prevent IL-2 binding to its high-affinity receptor, has recently been shown to be an effective immunosuppressant therapy for the prevention of allograft rejection (Vincenti et al., 1998; Waldmann and O'Shea, 1998). Accordingly, all of these approaches underscore the central role of IL-2 in the generation of an effective immune response, and the effectiveness of immunosuppressive therapies that block either IL-2 production or action.

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LICENSED PRODUCTS Recombinant IL-2 is available as des-Ala, 125-Ser IL-2 as Proleukin from the Chiron Corporation, Emeryville, CA, USA. It is approved for the treatment of renal cell carcinoma and malignant melanoma. This product is produced in E. coli and differs from natural IL-2 in being nonglycosylated, missing the N-terminal amino acid, alanine, and by the substitution of serine for cysteine at position 125. It is formulated in 150 mM phosphate buffer, pH 7.4, with sodium dodecyl sulfate, in a lyophilized cake, containing 1.1 mg of IL-2 protein. It may also be present in aggregates rather than monomers.