Molecular Pathways

Interleukin-21 Signaling: Functions in Cancer and Autoimmunity Ian D. Davis,1Kresten Skak,3 Mark J. Smyth,2 Paul E.G. Kristjansen,3 Dennis M. Miller,4 and Pallavur V. Sivakumar4

Abstract -21 (IL-21) is a with structural and to IL-2 and IL-15, yet possesses several biological properties distinct from these . IL-21is produced mainly by activated CD4+ T cells and natural killer T cells and mediates its activity by binding to the IL-21 receptor (IL-21R), consisting of an IL-21^ specific a chain (IL-21Ra; JAK/STAT) that heterodimerizes with the common g chain (CD132). Intracellular signaling occurs through the Janus-activated kinase/signal transducer and activator of transcription pathways. Physiologic expression of IL-21R is restricted to lymphoid tissues and peripheral blood mononuclear cells; however, other tissues such as epithelium, synovium, or transformed cells can acquire expression of both components of IL-21R heterodimer. IL-21has complex activities on a wide variety of cell types, leading to enhancement of adaptive T-cell immunity, antibody production, activation of subtypes, and opposition to suppressive effects mediated by regulatoryTcells. Functionally, these activities promote immune responses and point to a physiologic role of IL-21in autoimmunity and immune enhancement. Therapeutic manipulation of IL-21 activity may allow improved immunotherapy for cancer as well as insights into autoimmune disease. Recently conducted phase1trials in metastatic and have shown that recom- binant IL-21 has a favorable safety profile and support its continued investigation as a potential anticancer drug.

Interleukin-21 (IL-21) is a four-helix bundle cytokine that is IL-21RSignaling Pathways produced by activated CD4+ Tcells and natural killer T(NKT) cells (1–3). IL-21 was initially discovered by functional cloning IL-21R is a heterodimer, consisting of the IL-21–specific after expression of the IL-21 receptor (IL-21R) a-chain IL-21R a-chain (which has no intrinsic signaling ability) plus (IL-21Ra) in BaF3 cells and a library from activated Tcells to the common g-chain (gc; CD132; Fig. 1). IL-21 is thus a screen for ligands (1). IL-21 was shown to have structural member of a family of cytokines defined by the use of gc, which homology to IL-2 and IL-15, and an initial report described also includes IL-2, IL-4, IL-7, IL-9, and IL-15. Whereas members effects of IL-21 on the proliferation and function of natural of the gc family of cytokines share some activities, signaling killer (NK) cells, B cells, and Tcells (1). Subsequent studies have through IL-21R has several important differences in compari- shown important effects of IL-21 on myeloid-derived cells, son with other gc family members. Unlike the IL-21R complex, epithelium, and other tissues such as synovium or transformed which is a heterodimeric receptor, the receptor for IL-2 is a cells. Within 4 years of its initial description, human clinical trimer consisting of gc plus the IL-2R a-chain (CD25) and IL-2R trials of recombinant IL-21 had commenced and anticancer h-chain (CD122); the a and h chains are responsible for high- efficacy was shown (4). Further clinical development is currently affinity binding of IL-2 whereas the h and gc chains are under way with IL-21 either as a single agent or in combination responsible for signal transduction (5). IL-15 signaling is with other treatment modalities in multiple cancers. This unique: expression of the receptor a-chain is widespread; review will highlight the signaling pathways associated with however, only when IL-15 and the IL-15R a-chain are IL-21R, the nature of the cellular interactions affected by IL-21 coexpressed can the complex be presented in trans to signaling, and the functional implications of this activity. responding cells bearing the IL-15Rh and gc complex (6). IL-21Ra contains six tyrosine residues in the cytoplasmic domain. The specific functions of each residue remain unclear; however, mutation studies have shown that Y510 is sufficient for transduction of a full proliferative signal (7). After binding Authors’Affiliations: 1Ludwig-Austin Joint Medical Oncology Unit, Austin of IL-21 to the IL-21Ra/g complex, Janus-activated kinase 2 3 c Health; Peter MacCallum Cancer Centre, Melbourne, Australia; Novo Nordisk 1 and Janus-activated kinase 3 undergo autophosphorylation A/S, Copenhagen, Denmark; and 4ZymoGenetics, Seattle,Washington Received 5/21/07; revised 7/9/07; accepted 7/27/07. and subsequent phosphorylation of signal transducer and Grant support: National Health and Medical Research Council Program Grant and activator of transcription (STAT)-1 and STAT3 and, to a lesser Research Fellowship (M.J. Smyth). extent, STAT4 and STAT5 (refs. 7–10; Fig. 1). This is different Requests for reprints: Ian D. Davis, Ludwig-AustinJoint Medical Oncology Unit, from IL-2 or IL-15, both of which induce strong activation of Austin Hospital, Studley Road, Heidelberg,Victoria 3084, Australia. Phone: 61-3- 9496-5726; Fax: 61-3-9457-6698; E-mail: [email protected]. the STAT5, but not STAT3, pathway. These differences directly F 2007 American Association for Cancer Research. influence the differential effects of these cytokines on immune doi:10.1158/1078-0432.CCR-07-1238 cell subsets. IL-21R signaling leads to up-regulation of SOCS-1

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Fig. 1. IL-21R signaling pathway. IL-21 mainly signals via the Janus-activated kinase (Jak)/STAT pathway with STAT1 and STAT3 as primary targets. Signaling through the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated kinase (MAPK) pathways may also be relevant in certain physiologic or pathologic conditions.

in CD8+ T cells, which is a likely consequence of STAT1 and nature of the costimulatory signal involved. Important mech- STAT3 phosphorylation (11). SOCS-1 is also critical to anisms for stimulation of B cells include ligation of surface modulation of the signal because it affects the rate of loss of CD40 (physiologically mediated by CD40 ligand expressed on phosphorylated STAT3 (11). In addition, IL-21R signaling leads activated T cells) or signaling via Toll-like receptor (TLR) to activation of both the mitogen-activated protein kinase and subtypes (receptors involved in recognition of pathogens). phosphatidylinositol 3-kinase pathways, which are shown to be IL-21 augments proliferation induced by CD40 ligation; important for IL-21–mediated cell proliferation (7, 10). IL-21 however, it augments apoptosis and inhibits proliferation also reduces expression of Eomesodermin (Eomes), a factor driven by TLR-4 or TLR-9 agonists or by stimulation with that plays a role in regulating IFN-g production in CD4+ Tcells anti-IgM cell-surface receptor (1, 15, 16). In resting or naBve B (12). In B cells and plasma cells, IL-21 signaling induces BIM, cells and in B-cell chronic lymphocytic leukemia, IL-21R BLIMP, and Bcl-6, depending on the costimulatory signal signaling is associated with a shift toward proapoptotic signals. applied, and may be a key factor in survival, differentiation, The proapoptotic effect is mediated both by down-regulation of and apoptosis of these lymphocytes (13, 14). Although antiapoptotic mediators such as Bcl-XL and up-regulation of signaling pathways in different lymphocyte subsets have been proapoptotic factors including Bim, JunD/activator protein-1, examined, the predominantly affected by IL-21 signal and Bcl-6; activation of caspase-3 and caspase-8; and increased transduction remain to be elucidated fully. cleavage of Bid, poly(ADP-ribose) polymerase, and p27Kip-1 (17–19). In some respects, this is the opposite of what might Interactions of IL-21onVarious CellTypes be expected with signaling through STAT3 (20). In combination with activation via CD40, IL-21 induces B cells. IL-21 has profound effects on B-cell differentiation isotype switching to production of IgG1 and IgG3 (13, 21, 22) and antibody production (Fig. 2). These effects depend on the and directly inhibits IL-4–induced Cq transcription and IgE

www.aacrjournals.org 6927 Clin Cancer Res 2007;13(23) December 1, 2007 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2007 American Association for Cancer Research. Molecular Pathways production in mice (23, 24). In contrast, IL-21 enhances IgE antigen via the T-cell receptor. CD3 and associated molecules production in human B cells costimulated with IL-4 and CD40 are important in this process, and T-cell activation can be activation (25). Mice deficient in IL-21R show normal B-cell mimicked by cross-linking CD3 using antibodies. IL-21 numbers but have enhanced IgE titers and significant defects in costimulates proliferation of Tcells stimulated with anti-CD3 antibody responses to antigenic stimulus (21). In vitro, IL-21 or antigen (1, 26). IL-21 has effects on both CD8+ Tcells and promotes differentiation of plasma cells through increased CD4+ Tcells. IL-21 synergizes with IL-15 in inducing optimal Blimp-1 expression but does not induce somatic hypermuta- and sustained antigen-specific CD8+ T-cell responses in vitro tion (13, 14). Taken together, these data indicate that IL-21 and in vivo (27). IL-21 prevents IL-15–induced down- plays a key physiologic role in promoting T-cell–mediated modulation of CD28 (a molecule involved in mediating humoral immune responses and antibody production and in costimulatory signals to Tcells) on CD8 + Tcells and leads to the maturation of B cells into primary antibody-producing higher production of IL-2 and IFN-g after triggering via the plasma cells. These properties of IL-21 suggest that it could also T-cell receptor/CD3 complex in the presence of IL-15 (28), be a causative factor in multiple autoimmune diseases suggesting that IL-21 may be important in promoting T-cell involving B cells and autoantibody production. activation and memory. In addition, IL-21 promotes prolifer- T cells. CD8+ (‘‘effector’’) T-cell responses are not only ation and expansion of antigen-specific Tcells in vitro and required for elimination of intracellular viral infections but also in vivo (refs. 26, 29; reviewed in ref. 30). play key roles in antitumor immunity and autoimmunity, Recently, IL-21 was shown to play a key role in Thelper (Th) whereas CD4+ (‘‘helper’’) Tcells are thought to orchestrate cell differentiation. Th cells can be divided into three distinct different arms of the immune response, including CD8 and lineages: Th1 T cells are involved in supporting CD8+ effector B-cell responses, leading to efficient immune and pathologic T-cell function; Th2 T cells tend to promote antibody responses; responses. Signaling via IL-21R can lead to multiple effects on and the recently described Th17 cells promote inflammatory Tcells, including cellular proliferation, differentiation, activa- responses and play a pivotal role in autoimmunity. The tion, and changes in and cytokine production and hallmark of Th1 responses is production of IFN-g whereas effector function. Tcells are activated through recognition of Th2 responses are characterized by IL-4 and IL-5 production

Fig. 2. Role of IL-21in autoimmunity and cancer. IL-21is involved in reciprocal cross talk between disparate cell types, leading to effects on innate immune responses and adaptive antibody and cellular immune responses.This, in turn, may lead to autoimmune responses against normal or malignant tissues.

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A role for IL-21 in this expression through STAT3 activation; thus, IL-21 induces process has recently been shown: NKT-cell–induced apoptosis transcription of the IL-21 in an autocrine feedback loop and inhibition of IgE production in Bq cells was shown (33, 34). Other studies have shown that IL-21 stimulates genes to depend on IL-21 produced by NKTcells (2). Such linked to Th1 responses, such as T-bet and IFN-c (35). In an interaction may be another link between innate and contrast, IL-21 acts on naBve Th cell precursors to decrease IFN- adaptive immunity, a distinction that is becoming increasingly g production in a specific fashion without decreasing other Th1 blurred (47). and Th2 cytokines and reduces responsiveness of developing T Dendritic cells and monocytes/macrophages. Dendritic cells cells to IL-12 by means of reduction of STAT4 expression (36). play a central role in initiating and maintaining immune In agreement with these findings, studies in IL-21R–deficient responses and are involved in cross talk between Tcells, B cells, mice have indicated that IL-21 is important for development of and NK cells (48). Several different types of dendritic cells have Th2 responses but is not essential for Th1 responses (37). It is been described and IL-21 has effects on each. In monocyte- likely that the end result of IL-21 signaling on Th1/Th2/Th17 derived dendritic cells, IL-21 strongly induces expression of differentiation is dependent on the context in which it is found. SOCS-1 and SOCS-3 genes, which are known to interfere with Regulatory T cells. An important distinction between IL-21 TLR-4 signaling, although weak up-regulation of TLR-4 and other gc cytokines, such as IL-2 and IL-15, is their effects on expression itself is also induced (49). IL-21 decreases the regulatory T cells (Treg). Tregs are a population of lipopolysaccharide-induced production of tumor necrosis CD4+CD25+Foxp3+IL-7Rlo cells that have profound suppressive factor-a, IL-12, CCL5, and CXCL10 (49). Murine bone effects on T-cell proliferation and function (38). Tregs are marrow–derived dendritic cells generated in the presence of involved in constitutive suppression of autoreactive Tcells; IL-21 exhibited an immature phenotype with high antigen- reduction of Treg numbers and function is associated with uptake ability and low expression of CD86 and MHC class II various autoimmune disorders including inflammatory bowel and could not induce antigen-specific T-cell–mediated contact disease. Both IL-2R and IL-21R complexes are expressed on hypersensitivity (50). The opposite effect was seen with Tregs. Recent studies have shown that IL-21 inhibits trans- dendritic cells cultured in the presence of IL-15. This suggests forming -h–driven differentiation of naBve Th that IL-21 may be involved in the negative regulation of the cells into Foxp3+ Tregs (32, 33). In contrast to IL-2, IL-21 does functions of myeloid dendritic cells, particularly in relation to not enhance the proliferation of Tregs (39, 40). IL-21 allows interactions with Tcells, and points to an immunomodulatory CD4+ Th cells to become resistant to the suppressive effects of role for IL-21 in a broader sense. Tregs without directly reversing the function of CD4+CD25+ Macrophage function is also dependent on IL-21 signaling. Tregs (41). In contrast, IL-2, IL-7, and IL-15 have direct effects In mice lacking IL-21R or treated with an IL-21R-Fc fusion on Tregs either by enhancing their proliferation or by reversing protein as an IL-21 ligand trap, infection with pathogens such their anergic effects (41). These contrasting effects of IL-21 as Schistosoma mansoni and Nippostrongylus brasiliensis was versus IL-2 on Tregs could be due to differences in their ability associated with reduced granuloma formation and hepatic to induce (IL-2) or not induce (IL-21) phosphorylation of fibrosis and reduced expression of Th2 cytokines and other STAT5 because it has been shown that STAT5a/b signaling is relevant mediators (51). IL-21 was associated with up- essential for the up-regulation of Foxp3, the transcription factor regulation of IL-4Ra and IL-13Ra expression (51). These that drives generation of Tregs (42). IL-21 may therefore findings suggest a role for IL-21 in mediating chronic promote a propensity toward autoimmunity and therefore also inflammatory responses. to antitumor immunity, and these effects may help differentiate Epithelial cells and other cells. IL-21R is expressed and IL-21 IL-21 responses in cancer patients. has biological activity on many other nonimmune cell types, NK and NKT cells. IL-21 induces maturation and activation including intestinal epithelium (52), intestinal fibroblasts in and thereby increases the cytolytic potential of NK cells (43), the setting of inflammatory bowel disease (53), gastric with induction of various genes involved in activation of innate epithelium in Helicobacter pylori infection (54), rheumatoid immune responses. These include cytokines such as IFN-c, synovium (55), and hematopoietic progenitors (56). It is likely IL-10, T-bet, and granulocyte macrophage colony-stimulating factor that such effects of IL-21 in physiologic immune responses or (GM-CSF; refs. 35, 43, 44) and gene products involved in pathologic autoimmune responses in these tissues are mediated interactions with cells of the adaptive , both through immune effector cells as well as directly on the including MyD88, IL-2Ra/CD25, IL-12Rh2, and IL-18R cellular components of the target tissues. However, analysis of (10, 35). IL-21 also enhances antibody-dependent cellular IL-21R– or IL-21–deficient mice has shown that basal cytotoxicity by NK cells, leading to secretion of IFN-g, tumor homeostasis and development of various cell types and tissues necrosis factor-a, IL-8, macrophage inflammatory protein-1a, are not affected in these mice, suggesting that the IL-21 pathway and regulated on activation, normal T-cell expressed and is not crucial for these aspects (21).5 secreted (RANTES), and to an enhanced antitumor effect (45). IL-21 both is produced by and acts on NKTcells, enhancing their proliferation in combination with IL-2 or IL-15, particu- larly in the context of the NKT-cell–activating CD1d ligand 5 P.V.Sivakumar and M. Anderson, unpublished observations.

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Functional Interactions observed in animal models including melanoma, sarcoma, and bladder and renal cell carcinoma (61–64). Through depletion The properties of IL-21 as described above suggest several studies, the antitumor activity of IL-21 was shown to depend on important functional interactions. IL-21 is involved with NK cells or CD8+ Tcells, or both, depending on the tumor interactions between B cells, Tcells, NK cells, and dendritic model. Tumor rejection requires but not IFN-g, IL-12, cells and may act as a key cytokine linking innate and adaptive IL-4, or IL-10 (62), and in some models, rejection occurs via an immune responses (Fig. 2). IL-21 may be regarded as a CD4+ T- NKG2D-dependent mechanism (65). Other models have cell–derived helper cytokine that enhances antigen-specific shown that antitumor responses are associated with tumor immune responses by driving generation and survival of long- infiltration by CD8+ Tcells and with induction of central and term CD8+ Tcells and by differentiating B cells into potent effector memory cells (63, 64, 66) that can be sustained over antibody-producing plasma cells. IL-21 also mediates effects on long periods in vivo (29) or, in immunodeficient mice, by nonimmune cells and plays a role in various inflammatory activation of NK cells. This disparity may be due to conditions probably through its ability to drive lineage unanticipated variables such as the route of injection or the specification of Th17 cells. IL-21R is expressed on intestinal nature of the vaccine (e.g., in the Renca renal cell carcinoma epithelial cells and is functionally active (52). Expression of and B16 melanoma model, s.c. injection of IL-21 was superior IL-21R on intestinal epithelial cells is higher in patients with to i.p. treatment; ref. 64). Tumor immunity induced by local inflammatory bowel disease than in controls. Increased IL-21 high concentrations of IL-21 due to gene transfer techniques production was also shown in gut Tcells isolated from can lead to regression of distant tumor deposits (63), raising inflammatory bowel disease patients in these studies. Signaling the possibility that intratumoral administration of IL-21 may be through IL-21R on these cells leads to phosphorylation of an alternative to systemic therapy. The ability of IL-21 to downstream intermediaries and increased production of enhance endogenous tumor-specific antibody responses may macrophage inflammatory protein-3a (MIP-3a), a chemokine also be important in models in which the relevant tumor involved in attraction of Tcells to sites of inflammation. A antigen is on the cell surface (67). IL-21 can therefore induce neutralizing anti–IL-21 antibody reduced MIP-3a production antitumor responses not only by activating Tand NK cells but (52), suggesting that IL-21 may have direct effects on gut also by facilitating tumor-specific antibody production and epithelial cells in the promotion of inflammatory bowel enhancing antibody-dependent cellular cytotoxicity. disease. Other studies showing effects of IL-21 on expression of metalloproteases by gut fibroblasts (53) support the hypothesis that IL-21 mediates these effects, at least in part, Clinical/Translational Advances by acting on nonimmune cells. IL-21 seems to have a defined role in the maintenance and The unique biology of IL-21 provides many opportunities for possibly initiation of autoimmunity and mediates these effects its manipulation in different clinical conditions. In the setting through activation of Tand B cells. In animal models of of autoimmune disease, manipulations to decrease the effect of disease, such as mouse models of systemic lupus erythemato- IL-21 may lead to improved clinical outcomes, especially in sus, a disease largely dependent on autoantibody production, diseases that are driven by enhanced autoantibody responses, increased IL-21 has been shown in serum (13) and depletion of like systemic lupus erythematosus, scleroderma, rheumatoid IL-21 improves outcomes (57). IL-21–deficient mice are less arthritis, or multiple sclerosis. Because Treg numbers and prone to the development of experiment autoimmune enceph- function are integral to the pathogenesis of some, if not all, alomyelitis, a murine model of multiple sclerosis (33). In a autoimmune conditions, it will be important to achieve a murine model of rheumatoid arthritis, which is dependent on greater understanding of the interactions of IL-21 with Tregs. both T-cell and B-cell functions, IL-21 depletion improved Nevertheless, it is possible that neutralization of IL-21 by physical and histologic signs and was associated with a means of specific antibodies or soluble IL-21R ligand trap reduction in IL-6 and an increase in IFN-g (58). This is constructs may lead to meaningful clinical remissions in some consistent with observations that IL-21 is increased in animals autoimmune diseases, particularly those in which inflammato- recovering from induced lymphopenia or with constitutive ry cytokines are known to have a key role. lymphopenia (59), both conditions in which autoimmunity In the setting of vaccination, it may be predicted that occurs. There is increasing evidence that IL-21 is involved in concurrent administration of IL-21 might increase the magni- human autoimmune conditions. Tcells derived from periph- tude, affinity, and duration of T-cell responses. Other strategies eral blood or synovial fluid of humans with rheumatoid aimed at improving T-cell priming or recall responses, or arthritis secrete higher levels of -a and decreasing Treg numbers or function, may also be improved in IFN-g in response to IL-21, compared with controls (55). In combination with IL-21. The differential effects of the gc patients with Crohn’s disease, IL-21 enhances Th1 responses cytokines might be used to advantage in other conditions such and IFN-g expression (60). However, considering the recent as HIV infection. Culture of HIV-infected CD8+ Tcells with IL- discovery of the function of IL-21 in induction of Th17 cells 21 leads to an increase in perforin expression of both memory and the possible inhibition of Tregs, IL-21 may also promote and effector subsets, including HIV-specific Tcells, without autoimmunity through its effects on Th17 cells. Therefore, causing T-cell activation or proliferation or increasing degran- targeting IL-21 may benefit autoimmune conditions driven by ulation induced by ligation of the T-cell receptor (68). In both T-cell and B-cell activation and enhanced autoantibody contrast, IL-15 increases TCR-mediated degranulation (68). responses. This suggests that IL-21 treatment may improve T-cell function Considering its potential for immune activation, it is not in HIV-infected patients without inducing nonspecific T-cell surprising that IL-21 has antitumor effects. These have been activation (69).

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The preclinical activity of IL-21 in various tumor models colorectal cancer (Eudract no. 2006-004231-30). It is likely that points to its obvious application as an anticancer agent. Clinical IL-21 will enhance responses to active vaccination against cancer development for this indication has already commenced and antigens; however, no such treatments or studies are currently results from two phase 1 trials have been reported using two under investigation. Possible initial candidates for such vaccine treatment schedules: daily intravenous dosing for 5 days combination studies might be in the setting of virus-related followed by a 9-day break (‘‘5 + 9’’) or continuous thrice- cancers, such as human papillomavirus vaccine or hepatitis B weekly dosing (‘‘3/wk’’; refs. 4, 70). These two trials involved surface antigen vaccine. Combinations of IL-21 with cytotoxic patients with advanced melanoma or renal cell carcinoma. The chemotherapy or small-molecule targeted therapies could be objectives of the trials were to determine the safety of IL-21, to justified on the basis that chemotherapy-induced cell death may define a maximum tolerated dose and optimal treatment enhance access of tumor antigens to antigen-presenting cells, regimen, to document the effects of IL-21 on various bio- with subsequent improvement of T-cell responses due to markers, and to describe any antitumor effects. These trials have concurrent IL-21 administration. A phase 1/2 trial combining shown that IL-21 has mild toxicity and is generally well IL-21 with sorafenib in renal cell carcinoma is now recruiting tolerated. In particular, capillary leak syndrome has not been (clinicaltrials.gov identifier NCT00389285). More novel combi- observed even with dose levels near the maximum tolerated nations, such as concurrent treatment with IL-21 and agonistic intravenous dose of 30 Ag/kg in either treatment schedule. antibodies against tumor necrosis factor–related apoptosis- Biological effects have been observed even at the lowest doses inducing ligand receptors (71, 72), may also provide further tested (1-3 Ag/kg), and some patients with melanoma or renal insights into the anticancer mechanisms of IL-21, but presently cell carcinoma have had clinical responses (4, 70). A phase 2 no such clinical studies are planned. trial of IL-21 monotherapy in melanoma has now been IL-21 is a complex cytokine with pleiotropic activities. A completed (clinicaltrials.gov identifier NCT00336986). Other greater understanding of the network of interactions mediated treatment regimens are being tested, including s.c. dosing by IL-21 signaling has revealed a wealth of potential clinical (Eudract no. 2006-000376-32). The ability of IL-21 to enhance conditions in which manipulation of IL-21–related responses antibody-dependent cellular cytotoxicity preclinically provides may lead to benefit for patients. It is remarkable that a the justification for current studies in combination with cytokine with such a plethora of activities has such little in CD20+ B-cell non-Hodgkin’s lymphoma (clinical- toxicity. The results of further clinical trials are awaited with trials.gov identifier NCT00347971) or with cetuximab in interest.

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