Interleukin-2

To avoid immune responses against self-antigens, most self-reactive T cells are eliminated during selection in the thymus. However, this selection process is ‘leaky’, and potentially harmful self-specific T cells with pos-sible effector activity are released from the thymus. Self-specific T cells do not usually mediate autoimmune diseases because, in a normal immune system, they are suppressed in the periphery by thymus-derived regu-latory T (TReg) cells1,2. This TReg cell-mediated control is exemplified by three observations: first, humans and mice with a genetically determined TReg cell deficit develop multiple organ-specifi c autoimmune diseases3,4; second, a quantitative or qualitative defect in TReg cells is present in many mouse and human autoimmune diseases5; and third, efficient depletion of TReg cells in healthy individuals at any time in life induces a rapid autoimmune and inflammatory response that leads to multi-organ autoimmunity6. These observations have led to the concept of a balance between TReg cells and effector T cells in health that is dysregulated in auto-immune diseases and inflammatory disorders, which have a TReg cell in sufficiency.

The discovery of TReg cells has opened up a new thera-peutic possibility in terms of restoring a healthy balance between TReg cells and effector T cells through a quali-tative and/ or quantitative increase in TReg cell function. Owing to a lack of drugs that were capable of enhanc-ing TReg cell activity in vivo, initial efforts at therapeu-tic intervention used adoptively transferred TReg cells. Clinical improvements after TReg cell transfer have been

seen in many animal models of autoimmune diseases and, recently, in humans with type 1 diabetes7.

The discovery that interleukin-2 (IL-2) is a key cytokine for TReg cell differentiation, survival and function8–10 has led to new opportunities for tipping the balance of TReg cells and effector T cells towards TReg cells. Proof-of-concept clinical trials have shown that at low doses, IL-2 specifically and safely activates TReg cells in humans, and also improves autoimmune and allo immune inflammatory conditions11,12. These data inspired a broad investigation of the effects of IL-2 in diseases with an immune component, even beyond the field of autoimmunity.

Here, we review historical and current developments in IL-2 therapy, discussing evidence from mechanistic studies and clinical trials that has led to a shift in our understanding of IL-2 from a cytokine that activates effector T cells against cancer when used at a high dose, to a cytokine that activates TReg cells to control autoimmunity and inflammation at a low dose.

IL‑2 and homeostasisIL-2 was the first cytokine to be molecularly cloned, and based on its originally discovered action, it was named ‘T cell growth factor’ (BOX 1). Many studies in the 1970s and 1980s convincingly showed that IL-2 is an autocrine survival and proliferation signal for T cells, and stimulates the differentiation of naive T cells into effector T cells13. Thus, IL-2 was established as the essential soluble media-tor of T cell-dependent effector immune responses and

1Sorbonne Université, UPMC Univ Paris 06, UMRS 959, Immunology-Immunopathology-Immunotherapy (i3), F-75651 Paris, France.2INSERM, UMRS 959, Immunology-Immunopathology-Immunotherapy (i3), F-75005 Paris, France.3Assistance Publique – Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Biotherapy and Département Hospitalo-Universitaire Inflammation-Immunopathology-Biotherapy (i2B), F-75651 Paris, France.4Department of Pathology, University of California San Francisco, California 94143–0511, USA.e-mails: [email protected]; [email protected]:10.1038/nri3823Published online 17 April 2015

The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseasesDavid Klatzmann1–3 and Abul K. Abbas4

Abstract | Depletion of regulatory T (TReg) cells in otherwise healthy individuals leads to multi-organ autoimmune disease and inflammation. This indicates that in a normal immune system, there are self-specific effector T cells that are ready to attack normal tissue if they are not restrained by TReg cells. The data imply that there is a balance between effector T cells and TReg cells in health and suggest a therapeutic potential of TReg cells in diseases in which this balance is altered. Proof-of-concept clinical trials, now supported by robust mechanistic studies, have shown that low-dose interleukin-2 specifically expands and activates TReg cell populations and thus can control autoimmune diseases and inflammation.

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T follicular regulatory cells(TFR cells). Cells that are derived from thymus-derived regulatory T cells and share phenotypic characteristics with T follicular helper (TFH) cells, including the expression of the B cell follicle-homing receptor CXC-chemokine receptor 5. TFH cells control germinal centre B cells undergoing somatic hypermutation and selection that results in antibody affinity maturation. TFR cells reduce TFH cell and germinal centre B cell numbers.

Type 2 innate lymphoid cells(ILC2s). ILCs are rare cells from the lymphoid lineage — comprising the ILC1, ILC2 and ILC3 subsets — that produce many of the same cytokines as T cells but lack T cell antigen receptors. They have diverse roles in immune responses and inflammation. ILC2s produce type 2 cytokines, such as interleukin-5 (IL-5) and IL-13, and have key roles in anthelminthic responses and allergic lung inflammation.

was thought to be required for the development of all such responses.

The birth of an IL‑2 paradox. The aforementioned dogma was not challenged until 1993, when it was dis-covered that knockout of the gene encoding IL-2 in mice led to severe lymphoproliferation and auto immunity, rather than the predicted immune deficiency14. This finding was soon followed by the description of simi-lar phenotypes of mice with a knockout of the gene encoding the β-chain of the IL-2 receptor (IL-2Rβ; hereafter referred to as CD122)15 or IL-2Rα (here after referred to as CD25)16. The description of CD25 as the canonical phenotypic marker of TReg cells17 sug-gested that IL-2 signalling defects might affect TReg cells and lead to autoimmunity. This was confirmed by showing that TReg cells are absent in IL-2-deficient or IL-2R-deficient mice, and that adoptive transfer of these cells from normal mice could prevent auto-immunity in the deficient mice18. The surprising con-clusion of these knockout mouse studies is that the dominant role of IL-2 is the maintenance of TReg cells, and not the development of effector and memory T cells, because loss of IL-2 signalling leads to a break-down of immune tolerance and homeostasis. Thus, an agent that had been used for decades to stimulate effec-tor T cells turned out to be dispensable for these cells, and instead is required for activation of the TReg cells that suppress effector T cells.

Pleiotropic effects of IL‑2. IL-2 is predominantly pro-duced by activated CD4+ T cells and, to a lesser extent, by activated CD8+ T cells, activated dendritic cells, natural killer (NK) cells and NKT cells10. IL-2 production fol-lowing activation of CD4+ T cells is transient; it involves increased transcription and stability of IL2 mRNA, fol-lowed by transcriptional silencing and degrad ation of IL2 mRNA8.

Three polypeptide chains — CD25, CD122 and IL-2Rγ (CD132) — together form the high-affinity IL-2R (dissociation constant (Kd) = 10−11 M)10. The IL-2-induced oligomerization of IL-2R initiates sig-nal transduction, which is mediated only by the β-chain and γ-chain, through Janus kinase 1 (JAK1)-mediated and JAK3-mediated activation of signal transducer and acti-vator of transcription 5 (STAT5), as well as the phos-phoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The CD122–CD132 dimer can also signal in the absence of CD25, but has a lower binding affinity for IL-2 (Kd = 10−9 M)10. The high-affinity trimeric IL-2R is constitutively expressed by TReg cells, whereas other types of T cell only acquire CD25 upon activation10.

IL-2 has pleiotropic functions (FIG. 1). It supports the development of TReg cells in the thymus, although it may be dispensable for this role19. It is a key survival factor for TReg cells in the periphery, and is required for the functional competence and stability of TReg cell populations20,21. The molecular mechanism of IL-2-induced TReg cell stability involves, at least in part, epigenetic changes in the FOXP3 locus (which encodes forkhead box P3)22,23. One canonical feature of FOXP3+ TReg cells is their inability to produce IL-2 (REFS 22,23).

Aside from its role in supporting TReg cells, the other main functions of IL-2 are to support the proliferation of CD4+ T cells, and to support the terminal differenti-ation of CD8+ T cells. In the absence of IL-2 signalling, CD8+ T cells respond to a primary infection but fail to respond to secondary antigen exposure24. Prolonged CD25 expression on CD8+ T cells favours terminal effector differentiation in vivo25,26. However, IL-2 neu-tralization induces autoimmune diseases such as gas-tritis, diabetes, thyroiditis and neuropathy, which could be adoptively transferred by CD4+ and CD8+ T cells27, suggesting that the differentiation of effector T cells can occur even in the absence of IL-2.

IL-2 has been shown to promote the development of naive CD4+ T cells into peripherally derived TReg cells28, T helper 2 (TH2) cells29, TH9 cells30 and, in some studies, TH1 cells31. By contrast, IL-2 suppresses the differentia-tion of CD4+ T cells into TH17 cells32 and T follicular helper (TFH) cells33 by a STAT5-dependent mechanism. Thus, IL-2 may suppress germinal centre formation and create a regulatory environment in germinal cen-tres by altering the balance of T follicular regulatory cells (TFR cells)34–36 and TFH cells.

NK cells express high levels of the CD122–CD132 dimeric IL-2R8. IL-2 mainly stimulates the proliferation of NK cells, and also increases their cytotoxic activity and cytokine production, although to a lesser extent than does IL-12. Type 2 innate lymphoid cells (ILC2s) express high levels of CD25 (REF. 37) and therefore pro-liferate in response to IL-2 through activation of the trimeric IL-2R38. This proliferation is accompanied by the production of IL-5, which can, in turn, expand eosinophil populations39. Following stimulation with IL-2, γδ T cells acquire interferon-γ production and cytolytic activity40.

Box 1 | The discovery of interleukin‑2 as a T cell growth factor

In the 1950s and 1960s, immunological studies were mostly focused on humoral immune responses and on antibody function and structure, in part because such studies were facilitated by the availability of assays for measuring antibody responses. T cells were studied in more detail from the 1960s onwards and much effort was put into finding ways to culture them. Molecules capable of activating T cells were sought from the supernatant of cultured T cells; they were first detected in supernatants of mixed lymphocyte cultures115,116. It was another 10 years before it was shown that media from cultures of mitogen-activated cells could support the growth of T cell clones, indicating the presence of a growth factor in these cultures117. Of note, the authors concluded their report of this discovery by stating that the possibility to grow T cells in vitro “may be relevant to immunotherapy”. This protocol was then used to support the proliferation of T cell clones specific for tumour antigens that retained their functional cytolytic activity118. Lymphocytes were rapidly identified as the source of what was known at the time as T cell growth factor. The interplay between T cell growth factor and another molecule produced by non-T cells that could activate T cells, termed lymphocyte activating factor (LAF), led to the development of the interleukin terminology. LAF became known as interleukin-1 (IL-1) and T cell growth factor became known as IL‑2, as a result of the hypothesis — now known to be largely incorrect — that T cell growth factor was produced in response to LAF, which was thus put at the top of the cytokine cascade. IL‑2 was the first interleukin to be cloned, in 1983 (REF. 64). The discovery of IL‑2 has been outlined in detail elsewhere119.

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In summary, although many studies show multiple effects of IL-2 on a range of immune cell populations, the most consistent finding of studies in which IL-2 is administered or inhibited is an effect on TReg cells, and this is the main focus of the remainder of this Review.

High sensitivity of TReg cells to IL‑2. One of the most important results that has contributed to our under-standing of the biology and therapeutic effects of IL-2 is the demonstration that IL-2-induced signalling and downstream gene activation occur at markedly lower

Nature Reviews | Immunology

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Figure 1 | Pleiotropic effects of interleukin‑2 in controlling autoimmunity. Upon T cell receptor ligation and co-stimulation, naive CD4+ or CD8+ T cells transiently express CD25 and respond to interleukin-2 (IL-2) through the high-affinity trimeric IL-2 receptor (IL-2R). The differentiation fate of CD4+ T cells depends on the cytokine milieu: IL-12 and IL-18 favour the differentiation of T helper 1 (TH1) cells; IL-4 and IL-33 favour TH2 cells; transforming growth factor-β (TGFβ) and IL-4 favour TH9 cells; TGFβ, IL-1, IL-6 and IL-23 favour TH17 cells; TGFβ favours peripherally induced regulatory T (pTReg) cells; and IL-6 and IL-21 favour T follicular helper (TFH) cells. As part of the fate-orienting milieu, IL-2 favours the differentiation of TH1, TH2, TH9 and pTReg cells, and inhibits the differentiation of TH17 and TFH cells. The differentiation fate of CD4+ and CD8+ T cells is influenced by the strength of IL-2 signalling, with greater signal strength favouring

differentiation into pTReg cells and effector memory T cells, respectively. TReg cells constitutively express CD25 and respond vigorously to IL-2 by upregulating CD25 and becoming more potent (activated) suppressor cells. Some of these cells migrate to lymph node germinal centres where they are known as T follicular regulatory (TFR) cells. Together, these changes contribute to tipping the immune balance towards regulation rather than inflammation, notably by: favouring the differentiation of naive CD4+ T cells into pTReg cells rather than TH17 cells (part a); helping to control autoantibody production by favouring the differentiation of TFR cells over TFH cells (part b); and helping to control autoreactive CD8+ effector T cells by increasing the number and function of TReg cells (part c). The mechanisms of the suppressive activity of CD8+ TReg

cells in vivo remain to be determined.

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concentrations of IL-2 in TReg cells than in effector T cells or NK cells41 (FIG. 2). TReg cells have a 10–20-fold lower activation threshold for IL-2 than effector T cells when measured in terms of the level of phosphorylated STAT5 (pSTAT5). Moreover, downstream of pSTAT5, the activation of numerous genes that are important for cell function requires IL-2 doses that are 100-times lower for TReg cells than for effector T cells42.

Importantly, this greater sensitivity to IL-2 is not solely due to a higher level of expression of CD25 on TReg cells compared with effector T cells, as T cell blasts that have even higher levels of CD25 are less sensitive to IL-2-mediated activation than are TReg cells42. The exqui-site sensitivity of TReg cells to IL-2 could thus also be due to IL-2R signalling specificity. Whereas the MAPK, PI3K–AKT and STAT5 pathways are all activated in effector T cells in response to antigen, co-stimulation and IL-2, TReg cells — in which high PTEN expression levels inhibit the PI3K-dependent signalling path-way43,44 — may be more dependent on IL-2–STAT5 signalling (FIG. 2).

In vivo experiments in mice confirmed these find-ings. At doses of IL-2 of approximately 20,000–50,000 IL-2 international units (IU) per day, TReg cell populations are expanded compared with those from control mice, and they express higher levels of activation markers and are more suppressive. At these doses of IL-2, neither clonal expansion nor activation of CD4+ or CD8+ effector T cells or of NK cells can be detected (see below).

IL‑2 in pathophysiologyThe inflammatory disease that develops in mice lack-ing IL-2 or functional IL-2R is characterized by colitis and the production of multiple autoantibodies14–16, but the specific effects can vary depending on the mouse strain. On the BALB/c background, the domi-nant pheno type is autoimmune haemolytic anaemia caused by erythrocyte-specific antibodies, whereas in C57BL/6 mice, the dominant phenotype is colitis. Over time, all Il2-knockout mouse strains produce autoanti-bodies that are specific for nucleoproteins and other self-antigens. Interestingly, the autoantibodies are pro-duced even in germ-free Il2-knockout mice, whereas the colitis is largely abolished when these knockout mice are made germ free45,46. These data indicate that the defect in IL-2-dependent FOXP3+ TReg cells results in immune responses against self-antigens (leading to autoantibody production and ‘true’ autoimmunity), and perhaps also responses against commensal micro-organisms in the gut (which may lead to colitis). In all of these models, the disease can be corrected by providing normal TReg cells.

Humans with rare mutations in CD25 also develop autoimmunity47. The disease caused by IL-2 or IL-2R deficiency is not as severe as that caused by muta-tions in FOXP3 in humans with immune dysregulation polyendocrinopath y enteropathy X-linked syndrome3 (IPEX syndrome) or in scurfy mice4. This might imply that some TReg cells are relatively IL-2 independent48,49. In line with this, it has been shown that IL-15 can substitute for IL-2 to support and expand TReg cell populations50.


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Figure 2 | Effector T cells and regulatory T cells show differential use of signalling pathways and differential sensitivity to interleukin‑2. The activation of effector T cells and regulatory T (TReg) cells is mediated by combined signalling through the T cell receptor (TCR) and co-stimulatory molecules such as CD28, and through the interleukin-2 receptor (IL-2R). a | It is hypothesized that TReg cells have evolved to depend more heavily on signal transducer and activator of transcription 5 (STAT5)-dependent IL-2R signalling than on TCR and CD28 signalling compared with effector T cells. Dashed arrows and thick arrows indicate reduced and increased signalling, respectively. b | Effector T cells are poorly sensitive to in vitro IL-2 stimulation as measured by downstream STAT5 phosphorylation and they do not proliferate following in vivo treatment with low-dose IL-2. By contrast, TReg cells are highly sensitive to in vitro IL-2 stimulation and proliferate following low-dose IL-2 treatment in vivo. Data generated from cells obtained from patients with type 1 diabetes treated with low-dose IL-2 (REFS 82,85). Error bars indicate standard error of the mean. AP-1, activator protein 1; APC, antigen-presenting cell; IU, international unit; JAK, Janus kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated T cells; PI3K, phosphoinositide 3-kinase.

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IL-2 international units(IL-2 IU). The interleukin-2 (IL-2) IU is a biological activity determined by the ability to support the growth of an IL-2-dependent cell line. In practice, as the manufacture of IL-2 is standardized, 1.1 mg of IL-2 corresponds to 18 million IU.

Immune dysregulation polyendocrinopathy enteropathy X-linked syndrome(IPEX syndrome). This rare genetic autoimmune disease is caused by mutations in the FOXP3 gene (which encodes forkhead box P3). Affected males present with early-onset severe enteropathy that is usually accompanied by insulin-dependent diabetes (type 1). Other autoimmune manifestations include hypothyroidism, anaemia, thrombocytopenia and neutropenia. Increased serum IgE levels and dermatitis are often also present.

IL-2–antibody complex(IL-2c). A complex of interleukin-2 (IL-2) and IL-2-specific antibody that has a prolonged half-life compared with IL-2. Its biological activity depends on the antibody. Some complexes preferentially stimulate effector T cells, whereas others preferentially stimulate regulatory T cells.

Vascular leak syndrome(VLS). VLS (also known as capillary leak syndrome, systemic capillary leak syndrome or Clarkson disease) is characterized by a leakage of fluid from the circulatory system into the interstitial space, resulting in hypotension, haemoconcentration and hypoalbuminaemia.

Cytokine stormAlso known as hypercytokinaemia. The systemic expression of a vigorous immune response resulting in the release of inflammatory mediators into the bloodstream, including cytokines, oxygen free radicals and coagulation factors. The primary symptoms of a cytokine storm are high fever, swelling and redness caused by vascular leak, extreme fatigue and nausea.

In many individuals with autoimmune disease, IL-2 production is low compared with healthy individuals, and this could result in the instability and defective func-tion of TReg cells. This is the case for autoimmune dis-eases such as type 1 diabetes51, rheumatoid arthritis52 and systemic lupus erythematosus (SLE)53. However, these findings are not typical of all patients with these dis-eases, and decreased IL-2 production or IL-2R expres-sion is not considered to be a hallmark of most human autoimmune diseases.

Nonetheless, most autoimmune diseases involve a dysregulated balance between TReg cells and effector T cells, and thus, providing more IL-2-dependent sig-nalling may help to correct the disease by increasing TReg cell number and function, even in the absence of a pre-existing TReg cell defect. This idea is highlighted by the therapeutic efficacy of IL-2 in mouse models in which there is not necessarily a measurable IL-2 or IL-2R defect. IL-2 and IL-2–antibody complex (IL-2c)54 have been administered in various experimental auto-immune diseases55–60 or other inflammatory settings61–63, and in all cases showed some (often remarkable) efficacy.

The ups and downs of IL‑2 therapyTherapeutic use of IL-2 in humans is often adjusted to weight (dose per kg) or body surface area (dose per m2). For ease of comparison of different studies, we have con-verted all dosing to a fixed dose corresponding to an adult of 70 kg and 1.8 m2 in the following discussion70.

Initial clinical development: why high doses? The field of IL-2 therapy started with the use of IL-2 purified from cell culture supernatant and gained pace after the clon-ing of IL-2 in 1983 (REF. 64). Recombinant IL-2 was ini-tially used at high doses, probably because of its short half-life, which was thought to explain the poor clinical response in the early clinical trials carried out with puri-fied IL-2 (REFS 65,66). The first clinical trial reporting efficacy of IL-2 in human cancer67 used escalating doses of up to ~120 million IU (MIU), in three bolus infu-sions every 8 hours, to ensure that “serum levels were continuously maintained at concentration necessary to stimulate high affinity IL-2 receptors” (REF. 70). Severe side effects were noted, including a generalized vascular leak syndrome (VLS), and increased serum creatinine and bilirubin levels (indicating kidney and liver damage). The maximum tolerated dose of IL-2 was found to be 42–50 MIU every 8 hours (126–150 MIU per day) and at this dosage, some complete clinical responses were observed in patients with renal cell carcinoma or mela-noma68. Subsequent attempts to lower the IL-2 dose to reduce side effects resulted in decreased efficacy69.

Thus, the paradigm was established that (very) high doses of IL-2 were necessary to achieve a clinical response in patients with cancer, with the drawback of severe side effects.

High‑dose IL‑2 in cancer: lessons learnt. High doses of IL-2 were subsequently extensively used in patients with cancer. Importantly, they brought the first proof of concept that stimulation of immune responses could be

a therapeutic modality in cancer. Large studies showed an ~15% response rate to IL-2 treatment, with 7–9% of patients with melanoma or renal cell carcinoma hav-ing complete and often long-lasting responses70. These results led to the approval of IL-2 therapy by the US Food and Drug Administration for renal cell carcinoma and metastatic melanoma in 1992 and 1998, respectively.

It is striking that more than 20 years later, the mecha-nisms of the successful immune responses induced by IL-2 therapy in patients with cancer are still not under-stood, and it is not clear why only some patients respond to high-dose IL-2. The discovery that IL-2 markedly expands TReg cell populations could, in part, explain treatment failures in some patients71–74.

The clinical efficacy of high-dose IL-2 in some patients with cancer is with the drawback of a high tox-icity of the treatment. One of the main complications is VLS, which has recently been attributed to a direct effect of IL-2 on CD25-expressing endothelial cells75, and which leads to multi-organ dysfunction. Initial rates of mortality with high-dose IL-2 therapy were up to 4%, with deaths mostly occurring as a result of severe bacte-rial infections; these were later prevented by antibiotic therapy. Other common side effects of high-dose IL-2 are malaise, nausea and a flu-like syndrome, which are common manifestations of a cytokine storm, as is seen in settings such as acute infection76 and graft-versus-host disease (GVHD)77.

In summary, high-dose IL-2 has a poor safety pro-file but a robust efficacy in only a fraction of patients with a restricted set of cancers. High doses of IL-2 are still used in patients with these cancers, although the severe side effects and the inability to identify potential responders have severely restrained the use of high-dose IL-2 (REF. 70).

The resurrection of IL‑2: proof of concept. IL-2 has been available as an approved drug (Proleukin, Novartis; generic name aldesleukin) since 1992, and was thus available for clinical investigations. The effect of IL-2 on TReg cells was known since the mid-1990s, but it was more than 10 years before the first trials investigating the ability of IL-2 to promote TReg cell activity in humans with autoimmune and inflammatory diseases were initi-ated11,12. The common perception of IL-2 among clini-cians was that of a very toxic drug that could be useful only in some late-stage cancers. In addition, IL-2 was considered to be a cytokine that primarily stimulates the effector arm of the immune response, and thus could aggravate autoimmune diseases. In fact, the Proleukin drug brochure specifically warns that “Proleukin has been associated with exacerbation of pre-existing or initial presentation of autoimmune disease and inflam-matory disorders” (REF. 121). Thus, most clinicians were not prepared to reconsider IL-2 as a therapy that could suppress immune responses in autoimmune diseases.

In 2006, researchers were searching for means by which to stimulate TReg cells in patients with hepa titis C virus-induced vasculitis (HCV-induced vasculitis). It had previously been shown that patients with HCV-induced vasculitis had a deficiency in the number

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Graft-versus-host disease(GVHD). A common complication of allogeneic stem cell transplantation, in which immune cells from the graft recognize the recipient cells as foreign and attack them. The main target tissues are the liver, skin and gastrointestinal tract. It can be acute (within 100 days of transplantation) and life-threatening, or chronic.

Hepatitis C virus-induced vasculitis(HCV-induced vasculitis). 10–15% of patients with chronic HCV infection develop systemic cryoglobulinaemic vasculitis. Cryoglobulins are cold-insoluble immune complexes containing rheumatoid factor and polyclonal IgG that deposit on vascular endothelium, causing vasculitis in organs such as the skin and kidneys, and in peripheral nerves.

of TReg cells78 and that the resolution of the vasculitis after treatment with antivirals or rituximab was asso-ciated with a normalization of TReg cell numbers79,80. IL-2 was thus suggested as a potential therapy for HCV-induced vasculitis, on the basis of a major TReg cell population expansion that had been observed in patients with cancer who were treated with high-dose IL-2 (REF. 73). To avoid the severe side effects of high-dose IL-2 and also prevent, as far as possible, the activation of effector T cells, it was thought that using low-dose IL-2 could be the solution. Indeed, low-dose IL-2 had been well tolerated in other indications81 and the constitutively high levels of expression of CD25 on TReg cells could make them more sensitive than effector T cells to IL-2 (although this had not been definitively shown at the time).

Patients with HCV-induced vasculitis were treated with low-dose IL-2, with doses ranging from 1.5 MIU to 3 MIU per day. Patients received a cumulative dose of 50 MIU administered over 2 months11, which corresponds to one-third of the daily dose admin-istered to some patients with cancer. The treatment was well tolerated, and there was a greater than two-fold expansion of activated TReg cell populations with no detectable activation of effector T cells. A marked anti-inflammatory activity of IL-2 was also observed. Of note, a significant clinical improvement was seen in eight out of ten patients11. IL-2 was also shown to have beneficial effects for the treatment of chronic GVHD, an alloimmune inflammatory disease occur-ring after allogeneic haematopoietic stem cell trans-plantation (aHSCT)12. The results of these two trials

showed that low-dose IL-2 could be a novel class of immunoregulatory drug that functions by inducing the expansion and/or activation of TReg cells. The door was thus opened for IL-2 therapy to be ‘resurrected’ into clinical use in the form of low-dose IL-2 to control autoimmunity and inflammation.

Clinical development of low‑dose IL‑2The terminology ‘low-dose IL-2’ requires clarifica-tion. Of note, mouse models are not useful for defining low doses of IL-2 as applicable to humans because the IL-2 doses that stimulate TReg cells without stimulat-ing effector T cells in mice (~20,000 IU) correspond to very high equivalent doses by weight for humans. In the cancer field, the term low-dose IL-2 has been used for doses of ~5 MIU per injection. However, these were given three times a day for long treatment periods, with the total amount of IL-2 administered to each patient being in excess of 100 MIU. In evaluating the safety and biological effects of low-dose IL-2, it is therefore impor-tant to consider both single and cumulative dosage. In the trials of low-dose IL-2 reviewed here, the single dose varies from 0.09 to 5.4 MIU and the cumulative dose ranges from 1.5 to 52.5 MIU, except for a few patients with chronic GVHD who received up to 320 MIU (see Supplementary information S1 (table)).

Safety. The safety of low-dose IL-2 was initially an important concern, but several trials have now estab-lished that low doses of IL-2 are well tolerated. Local reactions at injection sites and flu-like syndromes were the most frequent mild to moderate adverse events in patients or healthy volunteers receiving between 0.18 MIU and 3 MIU of IL-2 per day11,82–84 (BOX 2; FIG. 3). Of note, in a double-blind, randomized, placebo-con-trolled trial, the total number of days on which adverse events were reported during the study was higher in the placebo group than in the IL-2-treated groups82. Together, these studies show that at doses that stimulate TReg cells, IL-2 is well tolerated.

Main biological findings. The trials of low-dose IL-2 that have been reported so far have used different doses and different schedules of administration. However, the main conclusions are that IL-2 induces robust expansion of the TReg cell population, and that the maximum magni-tude82 and duration85 of TReg cell population expansion are dose dependent. Some effects could be detected at an IL-2 dose as low as 0.18 MIU per day in healthy vol-unteers86. Although there are so far no descriptions of patients who do not respond to low-dose IL-2, one study has shown significant variations in the TReg cell response, with some patients who received the lowest dose of IL-2 (0.33 MIU per day) responding better than patients who received the highest dose (3 MIU per day)82. Of note, the minimum dose of IL-2 necessary to stimulate TReg cells has not yet been established, and may vary according to the patient and the disease.

The TReg cell response to IL-2 has mostly been stud-ied in peripheral blood mononuclear cells (PBMCs). In one study, IL-2 induced a marked increase in the

Box 2 | The main side effects of low‑dose interleukin‑2

Any evaluation of the safety and clinical tolerance of low‑dose interleukin‑2 (IL‑2) should bear in mind that relatively few patients have been treated for a long period of time, and that studies have mainly been carried out with no placebo control and often in patients receiving additional potentially toxic treatments. The only serious adverse events that have been reported so far for low‑dose IL‑2 occurred during the treatment of patients with chronic graft‑versus‑host disease, who received relatively high cumulative doses of IL‑2 together with other immunosuppressants and corticosteroids (see Supplementary information S1 (table)). Most reported adverse events of IL‑2 treatment are of mild or moderate severity (grade 1 or 2)120. The most frequent adverse events reported in all trials of low‑dose IL‑2 are injection site reactions and constitutional symptoms (such as fatigue, fever, malaise and arthralgia).

In the only double‑blind, placebo‑controlled study of low‑dose IL‑2 reported so far (see Supplementary information S1 (table)), 12, 12, 13 and 19 adverse events were reported in patients who received 0, 0.3, 1 and 3 MIU of IL‑2 per day, respectively. Injection site reactions occurred in approximately 30% of the IL-2-treated patients, but these were irrespective of the dose and were not seen for all injections in a given patient. This remains unexplained and might be related to the propensity of IL-2 to form aggregates. Constitutional symptoms seemed to be more dose dependent. However, fatigue was observed more frequently in the placebo group. In total, the number of days during which patients declared adverse events was higher for patients receiving placebo than for patients receiving any dose of IL‑2.

In another study, IL-2 was well tolerated in 18 children (7–12 years old) with type 1 diabetes who were treated with IL‑2 at a dose of 0.25–1 MIU per day for a mean treatment duration of more than 6 months (D.K., unpublished observations).

No significant abnormalities in patient biochemistry have been reported as a consequence of low‑dose IL‑2 therapy. Eosinophilia seems to be dependent on the IL‑2 dose and scheme of administration. It does not occur in all patients and, for those patients with eosinophilia, it does not occur during all courses of treatment.

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http://www.nature.com/nri/journal/v15/n5/full/nri3823.html#supplementary-information

Adverse eventsMedical occurrences that are temporally (but not necessarily causally) associated with the use of a medicinal product. The severity of adverse events is graded from 1 to 4, with the grades representing mild, moderate, severe and potentially life-threatening events, respectively. Any adverse event that causes death, is life threatening, requires hospitalization, or results in persistent or significant disability or incapacity is considered a serious adverse event.

number of TReg cells that was accompanied by a marked decrease in the number of infiltrating CD8+ effector T cells in scalp biopsies of patients with autoimmune alopecia areata (see below); these patients markedly improved during treatment83. Thus, in line with obser-vations made in animal models, the IL-2-dependent increase in the number of TReg cells that is mostly docu-mented in peripheral blood is also seen at the site of the autoimmune manifestation.

The expansion of TReg cell populations is accompa-nied by a marked increase in their expression of acti-vation markers, such as CD25, glucocorticoid-induced TNFR-related protein (GITR; also known as TNFRSF18) and cytotoxic T lymphocyte antigen 4 (CTLA4)85,87. In addition, the suppressive activity of these activated TReg cells is greater than that of control TReg cells11,12,88. Thus, we conclude that IL-2 induces the robust proliferation and activation of TReg cells, but its dose–response rela-tionship in health and disease seems to be complex and needs to be investigated further to optimize IL-2 use.

A major proliferative response of CD8+CD25+FOXP3+ T cells to IL-2 has also been observed11,82. This increase in the number of CD8+ regulatory T cells is twofold to five-fold greater than the corresponding increase in the num-ber of CD4+ TReg cells11,82; similar observations have been made in mice122 and macaques88. As CD8+CD25+FOXP3+ T cells are highly suppressive in vitro88–90,122, further studies are warranted to evaluate whether they have therapeutic effects in the context of disease.

NK cells also respond to low-dose IL-2, although much less so than TReg cells. In patients with chronic GVHD who received continuous treatment with IL-2, the number of blood NK cells doubled over the course of 8 weeks, whereas the number of TReg cells increased sixfold to eightfold12. In patients with type 1 diabetes, there was only a non-significant trend for a moderate increase in the number of NK cells at the highest dose of IL-2 used (3 MIU per day)82,85. In healthy volunteers, no effect of IL-2 on total NK cell number was reported. The non-cytotoxic CD56hi NK cells seemed to be most sensitive to IL-2 (REFS 11,86,87). Thus, low-dose IL-2 may in some cases expand NK cell populations, but in a daily and cumulative dose-dependent manner, and mostly for doses of more than 1 MIU per day.

In patients receiving continuous low-dose IL-2, a sig-nificant but asymptomatic eosinophilia peaked at 10% of white cells in the blood after 4 weeks of treatment, and subsequently declined12. In patients with HCV-induced vasculitis, there was a non-significant increase in the mean number of eosinophils after IL-2 treatment, with only some patients having increased values, and often not for all courses of IL-2 (REF. 39). A modest but significant increase in the number of eosinophils was also seen in healthy volunteers who received IL-2. In summary, IL-2 does expand eosinophil populations, but in a daily and cumulative dose-dependent manner. At doses of less than or equal to 1 MIU per day, the effect seems to be minimal.

An intriguing and significant dose-dependent decrease in the number of CD19+ B cells, which also affected marginal zone B cells, has been reported in two trials of low-dose IL-2 (REFS 11,82), with a strong unex-pected correlation between an increase in the number of TReg cells and a decrease in the number of B cells82. This effect could be linked to an inhibition of TFH cells and/or a stimulation of TFR cells. The clinical relevance of these findings remains to be clarified, particularly because B cell inhibition could be advantageous in the case of antibody-mediated autoimmune diseases for which B cell-depleting agents can be efficacious. Along these lines, three ongoing trials of low-dose IL-2 in patients with SLE have noted decreased levels of DNA-specific antibodies following IL-2 administration (see Supplementary information S1 (table)).

Immunomonitoring of patients with type 1 diabe-tes receiving increasing doses of low-dose IL-2 showed the dose-dependent induction of a regulatory milieu, as assessed by TReg cell to effector T cell ratios, TReg cell activation markers, plasma cytokine and chemokine levels, and transcriptomics85. This study also showed that low-dose IL-2 therapy did not induce IL-2-specific antibodies.


Potential adverse effects Potential beneficial effects

Mild to moderate constitutional symptoms such as asthenia, myalgia, fever and arthralgia

Local reaction at injection site

AtherosclerosisBy boosting TReg cells, IL-2 could help to control local inflammation to reduce plaque formation

Type 1 diabetesBy boosting TReg cells, IL-2 could suppress effector T cell-mediated killing of insulin-producing cells

Systemic lupus erythematosusBy blocking TFH cells and stimulating TFR cell differentiation, IL-2 could reduce autoantibody formation and immune complex deposition

Rheumatoid arthritisBy boosting TReg cells, blocking TH17 cells and favouring pTReg cells, IL-2 could help to control inflammation-dependent joint destruction

Figure 3 | Effects of interleukin‑2 in patients with autoimmune or inflammatory diseases. At low doses, there are only mild to moderate adverse reactions to interleukin-2 (IL-2), including reactions of unknown cause at the site of injection and constitutional symptoms that are possibly triggered by cytokine release. The potential beneficial effects are indicated relative to paradigmatic diseases: atherosclerosis as an inflammatory disease; type 1 diabetes as a T cell-mediated autoimmune disease; systemic lupus erythematosus as an autoantibody-mediated autoimmune disease; and rheumatoid arthritis as an autoimmune inflammatory disease. pTReg, peripherally induced regulatory T; TFH, T follicular helper; TFR, T follicular regulatory;T

H, T helper.

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Alopecia areataA prevalent autoimmune disease (affecting 1.7% of the general population) that leads to hair loss on the scalp and other areas of the body. Infiltration of CD4+ and CD8+ T cells around hair follicles is associated with the condition.

TRANSREG clinical trialAn open-label Phase II clinical trial investigating the stimulation of regulatory T cells by low-dose interleukin-2 in 11 autoimmune diseases: rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, psoriasis, Behcet disease, Wegener granulomatosis, Takayasu disease, Crohn disease, ulcerative colitis, autoimmune hepatitis and sclerosing cholangitis (ClinicalTrials.gov identifier: NCT01988506).

Altogether, doses of IL-2 below or equal to 1 MIU per day seem to specifically induce TReg cell expansion and activation, and a decrease in the number of B cells, both of which are desirable effects for the treatment of autoimmune diseases. At doses of more than 1 MIU per day, the effects on TReg cells are more marked, but some expansion of NK cell and eosinophil populations can be observed, which might be unwanted in some clinical settings.

Efficacy in human autoimmune and inflammatory diseases. Patients with HCV-induced vasculitis have a set of symptoms including fatigue, arthralgia, purpura, kidney involvement and neuropathy. In eight out of ten patients treated with low-dose IL-2, these symp-toms progressively disappeared11. In most cases, clinical improvements started to be observed after the second or third course of IL-2 therapy11. In patients with alopecia areata, regrowth of the scalp and/or body hair was noted in all of the five patients treated with IL-2, and four had a regrowth of scalp hair83. Recently, early clinical results of low-dose IL-2 in patients with SLE that have been presented at meetings have reported marked improve-ment in clinical scores (see Supplementary information S1 (table)). In one patient with refractory SLE, a rapid induction of clinical remission was reported90.

In chronic GVHD, of the 23 patients who were evaluated — all of whom were refractory to systemic glucocorticoid therapy — 12 had a partial response to IL-2 and 11 had stable disease after IL-2 therapy12. In responding patients, extended therapy led to sustained clinical responses and a lowering of the corticosteroid doses administered, which is an indication of treatment efficacy. Low-dose IL-2 was also investigated for the prevention of GVHD in patients receiving aHSCT84. The IL-2 treatment not only reduced the frequency and severity of acute GVHD, but was also accompanied by a reduction in the number of infectious episodes, which are a major post-transplantation complication84.

Thus, although the dosing and timing of IL-2 admin-istration may not have been optimal in these prelimi-nary studies, they concordantly show some biological and clinical efficacy of low-dose IL-2 that correlates with the expansion and activation of TReg cell populations.

The promise of low‑dose IL‑2By demonstrating the specificity of the effects of low-dose IL-2 on TReg cells and by reporting data that indi-cate a clinical benefit for patients, the preliminary studies discussed above have given optimism for further inves-tigation. This has also been reinforced by mechanistic studies showing that, at least in theory, low-dose IL-2 should have therapeutic effects on both T cell-mediated and antibody-mediated autoimmune diseases.

The issue of dose and schedule. The current assumption is that the therapeutic potential of low-dose IL-2 is in large part linked to the stimulation of TReg cells, which are thus a surrogate marker of efficacy. Therefore, the question is how much TReg cell stimulation is required, and for how long, to have significant therapeutic effects

in diseases that are often chronic in nature? The initial development of IL-2 therapy in patients with cancer was based on the assumption that constant detection of IL-2 in the serum was necessary for efficacy70, but it is not known whether constant TReg cell stimulation would be required in autoimmune diseases, or for how long.

By modelling the pharmacokinetics of TReg cells dur-ing and after a 5-day treatment of IL-2, we defined a 5-day induction course of 1 MIU of IL-2 per day as a scheme that will approximately double the percentage of TReg cells in PBMCs, to be followed by a fortnightly injection of 1 MIU of IL-2 as a maintenance treatment that should support an increased number of TReg cells at 20–60% above baseline values. This treatment modality is used in our current studies, and preliminary results from 36 patients in the TRANSREG clinical trial have con-firmed these predictions. Importantly, we found that this treatment course is well tolerated in the long term (more than 6 months) and does not significantly modify the number of effector T cells, NK cells or eosinophils.

Further clinical trials will be required to better deci-pher the IL-2 dose–response relationship in terms of the specificity of TReg cell stimulation and treatment efficacy (FIG. 4). It should also be mentioned that continuous administration of IL-2 achieved by gene transfer is a possible treatment modality; this has been a convenient approach to use in experimental models91–93 but it would be less easy to adjust treatment over time if applied to humans.

Immunoregulation without immunosuppression. The main complications of immunosuppression are related to infection. As there is normally a balance between TReg cells and effector T cells, and because TReg cells also control immune responses to infection that are medi-ated by effector T cells, the question thus arises as to whether protective antimicrobial immunity would be compromised when enhancing TReg cell function in a non-antigen-specific manner. Preclinical and clinical data available so far indicate that this is not the case. The administration of an IL-2-expressing adeno-asso-ciated virus vector in mice maintained IL-2 production and the associated increase in the number of TReg cells for more than a year93. Under such conditions, which permanently increase the proportion of TReg cells to 150–200% of baseline values, no effects were observed on mouse lifespan, clinical signs or tissue histology. Furthermore, when challenged with an influenza virus vaccine or a lethal dose of a mouse-adapted influenza virus, mice receiving long-term administration of IL-2 mounted normal protective immune responses93.

In patients with chronic HCV infection, no increase in viral load was observed during IL-2 treatment despite increased TReg cell numbers, and there was even a small but significant decrease in the viral load at the end of the follow-up period11. In the GVHD-prevention trial, the recovery of in vitro immune responses against recall anti-gens was identical in the IL-2-treated and control groups, and, importantly, a marked decrease in the number of infectious episodes was observed in the treated group as compared with controls84. Nevertheless, given the effects

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http://clinicaltrials.gov/

of IL-2 on TFH cells and TFR cells, and the striking decrease in the number of B cells that has been reported in some trials of IL-2 (REFS 11,82), further studies of humoral responses in individuals treated with IL-2 are warranted.

In the trial of low-dose IL-2 therapy for chronic GVHD, three patients had bacterial infections of grade 3 or higher12, but these findings are difficult to interpret in light of the other immunosuppressive drugs or corti-costeroids that these patients received. Of note, in a trial investigating the adoptive transfer of ex vivo-expanded TReg cells in children with type 1 diabetes, one patient contracted an influenza virus infection immediately after the TReg cell infusion. The infection resolved normally

within a few days, indicating that increased TReg cell number did not induce systemic immune suppression7.

The apparent preservation of a normal immune response to infection during IL-2 therapy is, in fact, not surprising. Inflammation is controlled by TReg cells but it also controls TReg cells94. At high levels of inflamma-tion, TReg cells become less efficient95. It is thus expected that a moderate increase in the number of TReg cells or their functionality should not affect effector immune responses that are triggered by highly inflammatory events, such as infection or vaccination, as the inflam-mation would be expected to constrain the TReg cell response.

Fold

incr

ease

in th

e nu

mbe

r of T

Reg

cel

ls

Time

a Repeated courses

b Daily continuous administration

Baseline number of TReg cells

AUC

c Induction and maintenance

d Repeated single injections

e Biomarker-defined administration

The dose of IL-2 and the timing of courses determine the AUC and effects on other cells

IL-2 dose determines the AUC (with a possible plateau effect) and effects on other cells; with time, this scheme may induce significant effects on NK cells, effector T cells and eosinophils

IL-2 dose determines the amplitude of the induction effect (Emax); the repetition frequency determines the AUC

IL-2 dose determines the amplitude of the effect, which rapidly returns to baseline

IL-2 dose and spacing are defined based on biomarkers of efficacy (possibly, but not necessarily, related to TReg cells)


Emax

IL-2 administration

MaintenanceInduction

Figure 4 | Possible administration schemes for interleukin‑2. The number of regulatory T (TReg) cells and their expression of activation markers are currently the best surrogate markers of the biological activity of interleukin-2 (IL-2). The efficacy of low-dose IL-2 treatment will probably depend on the extent and duration of these effects on TReg cells. Treatment efficacy can be characterized by the maximum effect gained (Emax) and the overall effect over time, which is defined by the area under the curve (AUC; compared with baseline). The dose and schedule of IL-2 administration will have a major influence on these parameters, as well as on the specificity of the effect for TReg cells as opposed to effector T cells, natural killer (NK) cells and eosinophils. a | Repeated courses of IL-2 (indicated by the red arrows) at defined intervals11,83,90 will produce an increase in the number of TReg cells at the end of each course, followed by a decrease in their number to a level that depends on the dose administered85 and the timing of the next course11. b | Daily continuous administration of IL-2 will produce the greatest overall effects (highest AUC), but with the side effect of some expansion of NK cell, effector T cell and eosinophil populations12. c | A short induction course followed by maintenance treatment with IL-2 will enable a desired Emax to be reached, and then a desired AUC to be maintained by repeated single injections, the timing of which will determine the long-term AUC. d | Repeated single administration of IL-2 will provide transient boosts to TReg cell numbers. e | Personalized biomarker-defined administration will be the ultimate treatment modality when (or if) biomarkers of efficacy are identified. At present (in the absence of an ideal personalized biomarker- controlled administration schedule), the induction course followed by maintenance treatment with IL-2, or repeated courses of IL-2, might be appropriate for settings in which the disease is moderate and there is a need for long-term support of TReg cells (for example, in patients with recently diagnosed type 1 diabetes). In more severe or more inflammatory settings, daily continuous administration of IL-2 might be favourable (for example, in patients with chronic graft-versus-host disease). Finally, repeated single administration of IL-2 might be more appropriate for treatments that aim to support the suppressive activity of TReg cells over long periods of time in conditions in which this activity may be defective owing to IL-2 deprivation (for example, for the prevention of type 1 diabetes).

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IL‑2 and inflammation. On the one hand, TReg cells tend to lose their functional capacity in highly inflammatory environments94. They might even become unstable, los-ing FOXP3 expression and converting to a phenotype that is more characteristic of effector CD4+ T cells (these converted cells have been termed ‘ex-TReg cells’), although such a process is much debated96. Different mechanisms can regulate the susceptibility of TReg cells to inflamma-tion-induced FOXP3 destabilization: FOXP3 expression is downregulated by polyubiquitylation97 mediated by the E3 ubiquitin ligase STUB1, which is induced by pro-inflammatory cytokines and lipopolysaccharides98; PIM1 kinase phosphorylates a serine residue of human FOXP3, thereby impairing its function99; and binding of methyl CpG-binding protein 2 (MeCP2) to FOXP3 stabilizes its expression100.

On the other hand, TReg cells suppress inflammation by multiple mechanisms, including reducing co-stimulation to activate effector T cells, consuming IL-2 and secreting immunosuppressive cytokines such as IL-10. The anti-inflammatory effects of TReg cells have been observed in various models of inflammatory diseases in mice. In a model of atherosclerosis that spontaneously devel-ops in low-density lipoprotein receptor-knockout mice, TReg cell depletion aggravated atherosclerosis and TReg cell repletion improved it61. In line with these observa-tions, treatment with IL-2 also improved atherosclerotic lesions101,102. Recent reports confirmed that TReg cells can improve other inflammatory conditions, such as acute lung injury103, muscular dystrophy63 or beryllium-induced granulomatous inflammation62. In the muscular dystro-phy model, treatment with IL-2c increased the number of TReg cells and the IL-10 concentration in muscle, result-ing in decreased myofibre injury. In humans treated with IL-2, the analysis of the entire transcriptome of PBMCs suggested a major anti-inflammatory role of low-dose IL-2 (REF. 11).

These results warrant the investigation of low-dose IL-2 in inflammatory diseases or conditions that are not caused by autoimmune or alloimmune reactions (FIG. 3).

IL‑2 in other indications. TReg cells have a role in control-ling allergy, as indicated by the presence of this symptom in the IPEX syndrome. IL-2c has been shown to improve an experimental form of allergy in mice104. Thus, IL-2 also has some potential for the treatment of allergy. However, the dose-dependent eosinophilia that can be induced by IL-2 should be carefully considered in terms of the appli-cation of this therapy to asthma, in which the harmful role of eosinophils is still debated105. Furthermore, ILC2s can respond to IL-2 by producing IL-13, which is detrimental in allergy and asthma106. IL-2 has already been evaluated, with some success, in the fields of HSCT12,84 and solid organ transplantation107.

Ageing of the immune system is associated with abnormalities of the T cell repertoire, with the appearance of oligoclonal expansions108. Injection of TReg cells into old mice or the treatment of these mice with IL-2 prevented such oligoclonal T cell expansions109. The role of IL-2 in preserving T cell homeostasis during ageing should be more thoroughly investigated.

Second‑generation IL‑2 and combination therapies. Fusions of IL-2 with carrier proteins, such as the Fc domain of IgG, have improved its short half-life110. In addition, several mutant forms of IL-2 have been pro-duced in attempts to alter its ability to bind to different components of the IL-2R, and thus to change its range of activity. One of the earliest such mutant forms of IL-2 was designed to reduce binding to the CD122–CD132 IL-2R dimer (to reduce activation of NK cells and cytokine-mediated toxicity), while preserving its abil-ity to bind to CD25 and hence the trimeric high-affinity IL-2R111. This mutant IL-2 protein retained the ability to activate T cells and mediate antitumour effects, but its effect on TReg cells has not yet been reported. A different IL-2 mutant has been produced that shows increased binding to CD122 and thus has an increased ability to activate CD8+ T cells and NK cells112. Complexes of specific monoclonal antibodies bound to IL-2 have modified the interaction with IL-2R complexes. Depending on the antibody-binding site on IL-2, the IL-2c preferentially activates cells expressing high levels of CD122, such as memory CD8+ T cells and NK cells, or CD25-expressing cells such as TReg cells54,113.

Although this field is extremely active and worth pur-suing, it should be kept in mind that modified IL-2 or IL-2c with preferential activity for TReg cells has not yet been evaluated in patients. It will be many years before an optimized TReg cell-focused form of IL-2 is at the same stage of development as ‘plain’ IL-2, with its 30 years of clinical use. Thus, it is reasonable to propose that clini-cal development of plain IL-2 should be pursued with-out waiting for the promises of more effective variants; also, the experience gained with plain IL-2 will help to develop second-generation versions of IL-2.

Combination therapy is often superior to single therapy for complex diseases. High-dose IL-2 is already being tested in combination with checkpoint inhibitors for the immunotherapy of cancer114. Similarly, combi-nations of low-dose IL-2 with other immune modula-tors will be worth evaluating in autoimmune diseases. For example, combining low-dose IL-2 with antibodies targeting inflammatory cytokines such as IL-1, IL-6, IL-17 or IL-23 could prove to have synergistic effects in controlling immune-triggered inflammation.

Finally, the possibility to combine the use of IL-2 with antigens to induce specific tolerance is extremely attractive and should be explored.

Conclusions and perspectivesIndependent clinical trials have now shown the safety of low-dose IL-2, at least for treatment lengths rang-ing from several months to a year, which overcomes the most frequent barrier in the development of novel therapies. Moreover, these trials have provided prelim-inary indications of significant biological and clinical efficacy. Without waiting for a comprehensive under-standing of its mechanisms of action, the promises of low-dose IL-2 therapy already warrant broad clinical investigations. These should be aimed at exploring the large field of potential target diseases, but also at transforming current indications of efficacy into proof

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of efficacy that can only be obtained from state of the art, double-blind, placebo-controlled randomized tri-als. Comprehensive ancillary studies embedded in these

clinical trials should help to harness IL-2 as the next revolution in immunotherapy — immunoregulation without immunosuppression.

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http://dx.doi.org/10.2337/db14-1322

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AcknowledgementsThe work of D.K. is funded by French state funds within the Investissements d’Avenir programme (ANR-11-IDEX-0004-02; LabEx Transimmunom); the European Research Council Advanced Grant (ERC-2012-AdG, TRiPoD, Agreement num-ber 322856); the Assistance Publique – Hopitaux de Paris, France; the Sorbonne University, Pierre and Marie Curie Medical School, Paris, France; the Institut National de la Santé et de la Recherche Médicale (INSERM); and Le Centre National de la Recherche Scientifique (CNRS).

Competing interests statementThe authors declare competing interests: see Web version for details.

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