Active research on cancer immunotherapy has shown that T cells play the most important role in anti-cancer immunity. To eliminate cancer cells, T-cell responses proceed step by step along the cancer-immunity cycle (1). Initially, tumor antigens need to be properly presented by dendritic cells (DCs) to prime and activate tumor-specific T cells. The activated T cells then move in the tumor bed followed by recognition of cognate antigens. After an encounter with an antigen, the tumor-specific T cell should properly induce effector functions. Finally, if this cycle lasts long enough to keep its optimal condition, patients may be able to avoid cancers.
Indeed, not only T cells but also almost all types of immune cells can be found within a distinct tumor microenvironment (TME) that differs between individual patients depending on the cancer types, disease progression, and conditions of immunity (2). Nevertheless, meta-analyses of clinical studies have demonstrated that strong tumor infiltration of effector immune cells, especially CD8+ T cells, are clearly associated with good prognosis and clinical outcomes. The problem is that not all patients have a good immunological status with enough effector T cells. Some clinical data have epidemiologically shown that T-cell counts are highly associated with cancer incidence (3). Furthermore, lymphopenia has been frequently observed in cancer patients with poor prognosis, although it is still not clear whether the lymphopenia is a cause or an effect.
The common cytokine-receptor gamma chain (γc) cytokines are important regulators of the immune system by exhibiting pleiotropic roles in both innate and adaptive immune cells (4). These cytokines consist of diverse interleukins (ILs): IL-2, IL-4, IL-7, IL-9, IL-15. and IL-21. Among them, IL-2 and IL-7 have been long and widely used for inducing the development and proliferation of T cells. In general, IL-2 acts as a ‘stimulus’ to induce activation of T-cell responses, whereas IL-7 acts as an ‘expander’ to increase the number of T cells, which have been more widely used to overcome the lymphopenia derived from chronic viral infections rather than from cancer. In this review, we summarize the biology of the cytokines and their clinical uses against cancer.
IL-2 is a 15.5-kDa glycoprotein that was first identified as a T-cell growth factor in 1976 (5). IL-2 is predominantly produced by activated CD4+ helper T (TH) cells in secondary lymphoid organs and to a lesser extent by activated CD8+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and DCs. IL-2 stimulates downstream signals in target immune cells by binding to the IL-2 receptor (IL-2R) that has either 2 or 3 subunits, including the cytokine-specific IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132 or more frequently as γc). IL-2Rβ is shared with IL-15, and γc is shared with IL-4, IL-7, IL-9, IL-15, and IL-21. IL-2Rβ and γc constitute an intermediate-affinity dimeric IL-2Rβγ (dissociation constant (Kd) ≈ 10−9 M), whereas all 3 subunits make up a high-affinity trimeric IL-2Rαβγ (Kd ≈ 10−11 M) (6). Although IL-2Rα by itself is also a low-affinity monomeric IL-2R (Kd ≈ 10−8 M), it does not induce a signal but seems to support the binding of IL-2Rβγ for IL-2 (6, 7). Although the IL-2Rβ is expressed on memory CD8+ T cells at higher levels than on memory CD4+ T cells, these cells are relatively unresponsive to the physiological levels of IL-2, but become sensitive to the high amounts of IL-2 that are generally induced by the administration of exogenous IL-2 (8, 9). The high-affinity trimeric IL-2Rαβγ is predominantly expressed on immunosuppressive CD4+Foxp3+ regulatory T (TREG) cells via constitutive expression of IL-2Rα at high levels, whereas other T subsets transiently upregulate IL-2Rα after activation following T-cell receptor (TCR) stimulation (10).
IL-2 plays a critical role in the regulation of CD4+ T-cell immunity. It promotes polarization and survival of the TH1, TH2, TH9, and TREG cells, but inhibits generation of TH17 and follicular helper T (TFH) cells by regulating STAT5 downstream signaling (11-15). Mice lacking IL-2, IL-2Rα, or IL-2Rβ develop systemic autoimmunity and enlargement of peripheral lymphoid organs associated with the polyclonal expansions of T and B cells (16-18). The autoimmune disorders can be prevented by the adoptive transfer of TREG cells, suggesting that IL-2 signaling contributes greatly to the immune regulation of TREG cells (19, 20). Indeed, IL-2 signaling is essential for the homeostasis of TREG cells but not for the early development and suppressor function of the cells (21). IL-2 also controls the differentiation and expansion of CD8+ T cells. After TCR stimulation, antigen-specific CD8+ T cells rapidly upregulate IL-2Rα expression within 3 days and then become effector T cells expressing different levels of IL-2Rα coupled with the reciprocal signal strength of IL-2. Whereas cells receiving strong IL-2 signaling differentiate toward short-lived terminal-effector cells mediated by the induction of B-lymphocyte-induced maturation protein 1 (BLIMP1), cells receiving low IL-2 signaling upregulate IL-7Rα (also known as CD127) and CD62L expressions displaying a phenotype of long-lived memory cells (22, 23). Persistent stimulation of TCR with IL-2 signals not only increases cytotoxic activities of activated CD8+ T cells but also promotes apoptosis of the cells by activation-induced cell death via the expression of the death receptor FAS (also known as CD95) and its ligand, CD95L (24). This process is very important in maintaining the self-tolerance of overactive T cells. Since IL-2 plays the pleiotropic roles in T-cell homeostasis, the administration of recombinant IL-2 was evaluated in several clinical trials for the treatment of cancers in diverse dose ranges and therapeutic schedules.
IL-2 immunotherapy was evaluated in several clinical trials because of its ability to expand and activate cytotoxic effector cells. The administration of high-dose IL-2 in bolus doses achieved effective objective regressions of metastatic cancers; however, severe side effects were seen in patients, including fluid retention, organ dysfunction, hypotension, and treatment-related deaths. The toxicities were directly related to the dose of IL-2 administered (25-27). In 255 patients with metastatic renal-cell carcinoma (mRCC), IL-2 therapy achieved 14% of overall objective response rate (ORR) with 5% complete responses (CRs) and 9% partial responses (PRs). The median response duration of partial responders was 19 months. Although the severe treatment-associated toxicities were still observed in the patients, the U.S. Food and Drug Administration approved high-dose bolus IL-2 as a single agent in 1992 after the demonstration of durable responses in mRCC patients (28). In 1998, IL-2 was also approved for the treatment of metastatic melanoma (MM) by demonstrating durable responses of 16% ORR with 6% CRs, 10% PRs, and 5.9 months median response duration among 270 patients with MM (29). Continuous infusions of low-dose IL-2 were also evaluated for up to 3 months. This regimen showed positive responses by showing selective differentiation and expansion of NK cells without severe toxicities, although cell activation was not observed (30-33). IL-2 administration has also been widely tested in adoptive T-cell therapy with autologous tumor-infiltrating lymphocytes and chimeric antigen receptor T cells (34).
Despite its considerable antitumor efficacy during several clinical trials, IL-2 therapy has still been done carefully because of the severe toxicities induced by the high-dose administration needed to achieve an immune-regulatory effect in tissues. Furthermore, the general limitation of immunotherapy with recom-binant proteins is the short half-life and their low, unstable bioactivity. The direct binding of IL-2 to IL-2Rα-expressing endothelial cells is considered to induce vascular leak syndrome (VLS), which is associated with the severe vascular failures of high-dose IL-2 immunotherapy (35). Moreover, the expansion of immunosuppressive TREG cells might also interfere with the efficacy of high-dose IL-2 immunotherapy (36).
To avoid the strong binding of IL-2 to high-affinity IL-2Rs, one group of researchers generated IL-2 mutants called IL-2 ‘superkines’ (also called super-IL-2) by increasing their binding affinities for IL-2Rβ (37, 38). The IL-2 superkines showed improved antitumor responses
In addition to the regulation of IL-2 binding specificity, other approaches were tried to improve the serum half-life and bioactivity of IL-2. One method is to use cytokine complexes that are formed by combining a cytokine with an anti-cytokine antibody or a respective soluble cytokine receptor. Although why the cytokine complexes show increased bioactivity
Another group of researchers developed a super mutant IL-2-Fc (also called sumIL-2Fc) by conjugating Fc fragments and introducing mutations to make a stable IL-2 with increased IL-2Rβ binding. SumIL-2Fc showed increased antitumor activity to native IL-2 therapy showing a selective increase of CD8+ T cells but not of TREG cells (46). Polyethylene glycol (PEG) conjugated IL-2 (PEG-IL-2) was also administered to mRCC and MM patients to increase IL-2 persistence; however, it did not increase antitumor activity more than did high-dose IL-2 (47). In 2016, Nektar Therapeutics, a biopharmaceutical company in CA, developed another form of PEGylated IL-2 by conjugating six releasable PEG linkers (also known as NKTR-214 or Bempegaldesleukin) (48). The NKTR-214 was designed as a prodrug showing increased persistence with an inhibited IL-2Rα binding because of the location of the PEG chain at the binding interface. Treatment with NTKR-214 induced superior antitumor responses by inducing an increase of CD8+ T cells and their functionality as a single agent or as combination therapies with vaccination and with checkpoint inhibitors (48, 49). The recent approaches using IL-2 in cancer therapy are summarized in Fig. 1.
IL-7 is a 25-kDa glycoprotein that was first discovered in the 1980s and plays pleiotropic roles in the homeostasis of lymphocytes, especially T cells. In general, non-hematopoietic stromal cells in diverse tissues are known as the main producers of IL-7 (50). Most recently, a subset of fibroblastic reticular cells (FRCs) in T-cell zones of lymph nodes was identified as an important cellular source of IL-7 for the homeostasis of peripheral T cells (51). However, the identification of exact sources, whether they are tissues or cell types, needs to be further elucidated, since the expression profiles examined so far remain controversial depending on the study models (52). Moreover, a subset of DCs is also known to produce small amounts of IL-7, whereas the lymphocytes, including T cells, B cells, and NK cells, do not (53).
IL-7R is a heterodimer consisting of 2 subunits, IL-7Rα (CD127) complexed with the γc. Whereas IL-2Rα constructs a high-affinity receptor specific for IL-2, IL-7Rα is not specific for IL-7 but is also shared with thymic stromal lymphopoietin (TSLP), which is also important for the thymic development of T cells (54). Therefore, IL-7Rα-deficient mice seem to develop the severe tendency of lymphopenia more than IL-7-deficient mice, and mice without both molecules show the phenotype of severe combined immunodeficiency (SCID) (55, 56). IL-7Rα is broadly expressed throughout the lymphoid system. IL-7 is a critical factor for the development and maintenance of the diverse range of lymphoid cells, since common-lymphoid progenitor in the bone marrow (BM) also expresses the IL-7Rα and develops into the diverse lymphoid populations. Although mature B cells in the periphery lose the expression of IL-7Rα, IL-7 is still an important factor for B-cell development by regulating the proliferation and survival of B-cell progenitors in the BM (57). De-spite its essential role in mouse B cells, the requirement of IL-7 in human B-cell development is controversial, since some patients with SCID expressing the mutant IL-7R showed a normal number of B cells (58). Nevertheless, IL-7 has also been implicated as a homeostatic mediator of B-cell progenitors. The importance of IL-7 signaling on T-cell homeostasis is well defined. In the thymus, thymopoiesis proceeds through, in order, the double-negative (DN; CD4−CD8−), double-positive (DP; CD4+CD8+), and single-positive (SP) CD4+ or CD8+ progenitor cells. IL-7Rα is highly expressed in the early stage of DN cells and then downregulated on DP cells followed by re-expression on SP cells. Severe loss of DN cells and immunodeficiency in the absence of IL-7 signaling showed a pivotal requirement of IL-7 for T-cell development (59). Although IL-7 plays a major role in the development of both αβ and γδ T cells, αβ T cells but not γδ T cells can be partially restored through the transgenic expression of anti-apoptotic molecules, such as B-cell lymphoma 2 (Bcl2), suggesting that IL-7 signaling is essential for the survival of thymocytes (60-64). Furthermore, IL-7 signaling also regulates β-selection and TCR rearrangement during the DN stage (65). Development of γδ T cells seems to depend more on the source of IL-7, since IL-7 deficiency specific for the thymic epithelial cells, but not for intestinal intraepithelial cells, showed a decrease in γδ T cells (66, 67). Unlike mature B cells, IL-7Rα is highly expressed on the recent thymic emigrants (RTEs), naïve T cells, and memory T cells in the periphery. IL-7Rα is also expressed on mouse thymic NK cells (CD11b− CD16−CD69hiLy49lo), which are similar to human CD56hiCD16− NK cells, showing reduced cytotoxicity but increased production of inflammatory cytokines (68). For DCs, IL-7Rα is expressed only on migratory DCs
Following thymic export, RTEs induce prolonged survival and proliferate through IL-7 signaling without TCR stimulation (70, 71). However, the survival and differentiation of non-RTE mature T cells depend on signals through TCR as well as cytokines (72, 73). IL-7 would regulate naïve T cells directly more than IL-2 does since the expression level of IL-7Rα is higher than that of IL-2Rα, which is relatively low in naïve T cells. Although IL-7 signaling is required for both survival and proliferation of naïve T cells, the homeostatic proliferation is more remarkable under lymphopenic conditions (74-76). IL-7 is known to regulate the quantity of CD8 co-receptor expressed in a feedback loop to control TCR engagement to self-antigens. Since the only ligands available for T cells are the self-antigens during homeostatic proliferation, IL-7 seems to regulate the proliferation by increasing the weak TCR signaling (77). After TCR stimulation, the recently activated T cells not only increase the expression of IL-2Rα but also become a major source of IL-2 themselves (22). With regard to the polarization of CD4+ TH subset differentiation, low expression of IL-7Rα defines immunosuppressive TREG cells by inversely correlating with the Foxp3 expression, whereas the IL-2 signaling induces the Foxp3 (12, 78, 79). As previously mentioned, the cell-fate decision of CD8+ T cells is regulated in a contrary direction between IL-2 and IL-7 signaling (Fig. 2). Whereas the strong IL-2 signaling mediates the differentiation toward terminal effector cells, a subset of activated CD8+ T cells expressing a high level of IL-7Rα display a phenotype of memory T cells (22). In addition to the expression of anti-apoptotic molecules, IL-7 signaling also mediates the long-term survival of T cells by increasing glucose uptake (80, 81). More recently, it has also been reported that IL-7 specifically controls the longevity of CD8+ memory T cells by inducing triglyceride synthesis followed by sustained ATP levels (82). Although IL-7 is closer to a ‘homeostatic’ cytokine than the stimulatory IL-2 is, it still seems to affect the effector functions of T cells. Continuous IL-7 signaling not only generates cells expressing memory markers but also induces production of IFNγ followed by IFNγ-induced cell death during homeostasis (83). Therefore, elucidation of T-cell responses after IL-7-mediated immunotherapy might be therapeutically more significant.
Unlike IL-2, clinical trials with IL-7 as cancer immunotherapy have reported no objective responses, whereas preclinical studies have shown the potential antitumor activity of IL-7 therapy. However, dramatic changes in pharmacodynamics associated with beneficial immunological phenotypes have been widely announced through clinical trials. The Cytheris Corp., now RevImmune, Inc., has mainly developed immunotherapy with recombinant human IL-7 (rhIL-7). The first clinical trial was carried out with the rhIL-7 produced in
The antitumor potency of IL-7 therapy has been demonstrated in preclinical studies. Intratumoral delivery of IL-7-transduced DCs induced superior antitumor responses with increased production of granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFNγ (90). Treatment of IL-7 with GM-CSF-secreting tumor vaccines also improved the survival of tumor-bearing mice by increasing activated DCs and T cells within draining lymph nodes with the increased cytolytic T cells within tumors (91). These results further demonstrated the importance of both innate and adaptive immunity against cancer immunotherapy with IL-7. Adjuvant treatment of IL-7 with a vaccination regimen improved the survival of tumor-bearing mice by augmenting the vaccine-induced tumor-specific CD8+ T-cell responses. In this setting, adjuvant treatment with IL-7 not only increased the pathogenic properties of the CD8+ T cells but also made them refractory to the TGFβ-mediated inhibitory network (92).
In addition to the use of native IL-7 cytokine, efforts to increase the bioactivity of IL-7 have also been developed. One group of researchers showed that the treatment of an IL-7 complex formed with an IL-7Rα-Fc induced antitumor responses by increasing tumor infiltration of T cells through CXCR3 chemokine signaling (93). An IL-7 complex with a neutralizing antibody clone M25 is also known to have increased bioactivity. Treatment of the IL-7 complexes induced a massive increase of naïve and memory T cells in normal mice and also restored the impaired thymopoiesis in IL-7-deficient mice. Unlike the IL-2 neutralizing antibody S4B6, treatment with M25 did not induce cell proliferation either by itself or by separately injecting it even one minute after IL-7. Furthermore, IL-7 complexes with an M25 antigen-binding fragment (Fab) also did not show mitogenic activity. These results suggest that both the pre-mixture form of the complexes
The current understanding of cancer immunotherapy indicates that most anticancer therapies regulate the immune response of the cancer patients, especially toward increasing the tumor-specific T-cell responses, whether the therapeutic agent directly targets T cells or not. In general, prolonged survival of patients correlates with the number of cytotoxic/memory T cells, TH1 cells, and other effector cells. In contrast, an increased number of immunosuppressive cells, including TREG cells and myeloid-derived suppressor cells, are associated with poor prognosis. It has also been discovered that the densities, phenotypes, and characteristics of individual immune cells in TME may affect the efficacy of immunotherapy. Given that the immune response in TME has chaotic complexity, it is not surprising that the currently emerging prime immunotherapies with checkpoint inhibitors (CPIs) cannot conquer cancers yet, but they are taking priority in most clinical trials nowadays. Epidemiological studies have demonstrated that it is crucial to choose the right pair for combined immunotherapy rather than focusing on the single therapeutic agent that regulates only a fraction of immune response in TME. Since IL-2 and IL-7 have been well defined to increase the T-cell responses, they could be the best partners for most immunotherapies, including the CPIs. Although the augmented T-cell responses by the cytokine therapy have been observed, it remains to be seen how these cytokines may change the heterogeneous phenotypes of T cells and their functional characteristics in TME. Indeed, since the single-cell analysis technique is necessary for revealing the conditional change of TME, it is becoming important not only to understand the detailed mechanism but also to interpret it for the development of successful therapeutic strategies. Overall, we believe that therapeutic challenges with the T-cell regulators, IL-2 and IL-7, will be the promising strategies for anticancer immunotherapy if the detailed mechanisms of T-cell responses in TME are followed.
This work has supported by the Research Institute of NeoImmune Tech, Inc., and by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (2017M3A9C8033570).
The authors have no conflicting interests.