Th1 cytokine-based immunotherapy for cancer

Oxford,UK

Introduction

Cancer immunotherapy aims to manipulate the immune system to effectively fight against cancer.A variety of immunotherapeutic approaches,including monoclonal antibodies,cancer vaccines and adoptive cell transfer,have been exploited to enhance antitumor immune response.In many cases these immunotherapies show potential antitumor activities in animal models but their clinical efficacy is not as good as animal study,mostly because of the inadequate activation of immune effector cells and/or the strong immunosuppressive response in tumors.

Cytokines are pleiotropic proteins that can effectively activate immune cells and/or counteract immunosuppression in tumors,and have been extensively explored in cancer immunotherapy.The first cytokine successfully used in clinical cancer therapy is IL-2,but it is only effective in certain types of cancers and a complete clinical response is rare.[1]Recent years,numerous studies have been attempted to further increase the efficacy and safety of cytokine therapy as well as to develop novel cytokinebased immunotherapy strategies,among which Th1-type cytokines show potent antitumor activities in animal models and in patients.This review focuses mainly on recent advances of Th1-type cytokines including IL-1,IL-2 and IL-12 family cytokines and their applications in cancer therapy.The author reviews the antitumor functions of cytokines either used cytokine alone or in combination with other immunotherapy regimens such as cancer vaccine and adoptive cell transfer,or with classic chemotherapy to effectively eliminate tumors.Furthermore,some immune adjuvants that enhance the Th1-type response to kill tumors are also reviewed.

IL-1 family cytokines

IL-1 was the first described IL-1 family member which includes 11 members:IL-1 family member 1 (IL-1F1,IL- 1α),IL-1β (IL-1F2),IL-1 receptor antagonist (IL- 1Ra,IL-1F3),IL-18 (IL-1F4),IL-1F5,IL-1F6,IL-1F7,IL-1F8,IL-1F9,IL-1F10 and IL-33 (IL-1F11).They are produced by a wide range of cell types,mainly by macrophages and dendritic cells (DCs) in response to pathogen infection or TLR activation.[2]Most IL-1 family cytokines are synthesized as precursor peptides,which need to be cleaved to generate mature cytokines.For example,precursor IL-1β is cleaved by caspase-1 to produce mature IL-1β.Most IL-1 family members function through binding to cell surface receptors,but IL-1α and IL-33 can also bind to DNA in the nucleus and thus regulate the downstream pathway.[3]

IL-1 is a costimulator of T cell.It can upregulate IL-2R expression on T cells and enhance the proliferation and survival of CD4+T cells in response to antigen.[4]IL-1 can effectively induce the proliferation of effector T cells even in the presence of Treg cells[5]and enhance maturation and activation of DCs triggered by Fas signaling.[6]More recent work[7]demonstrated that IL-1,but not IL-6,plays a predominant role in promoting Th17 differentiation.Given the critical role of Th17 cells in in flammatory and autoimmune diseases,[8]IL-1 is also considered as a proin flammatory cytokine.Accordingly,overproduction of IL-1β contributes to many in flammatory diseases and the in flammatory property of IL-1 precludes its application in tumor immunotherapy.In fact,IL-1 signaling inhibitors,such as recombinant IL-1R antagonist (IL-1Ra) and anti-IL-1β antibody,have been used in patients with in flammatory diseases such as rheumatoid arthritis.[9]

IL-18 was initially described as an IFN-γ inducing factor[10]and a key stimulator of Th1 response.Macrophages,DCs,epithelial cells,as well as a variety of tumor cells express IL-18.Like IL-1,IL-18 is synthesized as an inactive precursor that requires cleavage by caspase-1 to produce mature IL-18.[11]Mice deficient of in flammasome or caspase-1 show decreased resistance to cancers due to a marked reduction of mature IL-18.[12]IL-18 receptor(IL-18R) belongs to the IL-1R/TLR family.[13]After binding to its receptor,IL-18 triggers the recruitment of adaptor molecules such as MyD88 and then activates IL-1R-associated kinases (IRAKs) and TNFR-associated factor 6(TRAF6),thus leading to activation of mitogen-activated protein kinase (MAPK) and NF-κB.This pathway results in transcription of related genes,especially Th1-type cytokines like IFN-γ,IL-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF).

IL-18 promotes IFN-γ secretion from Th1 cells and natural killer (NK) cells,therefore playing an important role in resistance to intracellular pathogen infection and tumors.[14]A recent study[15]showed that IL-18-activated NK cells produce high levels of chemokines,and then recruit CD8+T cells into tumor sites tofight against cancer.IL-18 has been well studied in mouse tumor models and demonstrated significant antitumor effects.As a single agent,high-dose IL-18 elicits antitumor activity in tumor-bearing mice but with severe toxicity.[16]IL-18 was also investigated as an adjuvant in combination with suicide gene therapy,[17]tumor vaccine,[18,19]DC-based vaccine,[20-22]DNA vaccine,[23]antibody-superantigen fusion protein vaccine[24]and tumor-derived exosome vaccine[25]to enhance their antitumor efficacy in mouse models.These effects were associated with the increased activation of DCs,Th1-cell mediated response,CD8+cytotoxic T lymphocytes(CTL) cytotoxicity and production of Th1-type cytokines including IFN-γ and IL-12.

Although first discovered as an IFN-γ inducer,IL-18 alone induces only a small amount of IFN-γ.However,IL-12 can increase IL-18R expression on Th1 cells and synergize with IL-18 to secrete a large amount of IFN-γ.[26]Coughlin and colleagues[27]found that the combination of IL-12 with IL-18 synergistically induced murine tumor regression.Intratumoral injection with DCs engineered to secrete both IL-12 and IL-18 resulted in complete rejection of tumors with a strong Th1 response.[28]Similarly,immunization of DC-tumor fusion cells co-transduced with IL-12 and IL-18 genes significantly inhibited the growth and metastasis of tumors,which were associated with an increased Th1 response,IFN-γ production and enhanced cytotoxicities of NK cells and CTLs.[29]Some data showed that IL-18 administration attenuated the liver toxicity of IL-12 therapy without affecting the antitumor capacity of IL-12.[30]IL-18 therapy showed significant antitumor activity in animal studies but its clinical efficacy was limited.A phase I study of recombinant human IL-18 in patients with advanced cancer showed that IL-18 was safe and well-tolerated.[31]However,a phase II study of IL-18 in 64 patients with metastatic melanoma showed that IL-18 therapy had no apparent clinical efficacy,and the study was subsequently terminated.[32]

Despite its dominant role in the Th1 immune response,IL-18 also induced the production of Th2-type cytokines such as IL-13 by NK cells and mast cells,and was involved in a Th2 immune response.[33]Moreover,an increased serum IL-18 level was related to tumor progression in cancer patients,[34]and IL-18 accelerated tumor growth and metastasis.[35]In addition,tumorderived IL-18 suppressed NK activity and facilitated the metastasis of NK cell-dependent tumors.[36]A further study[37]revealed that tumor-derived IL-18 converted Kit-CD11b-NK cells into regulatory Kit+NK cells,which promoted tumor progression in tumor-bearing mice.

Taken together,IL-18 has been explored as an adjuvant therapy against cancer because of its strong immuno-stimulatory effects.However,IL-18 may also have an immunosuppressive effect and play a role in cancer progression and metastasis.Further studies are needed to elucidate the biological as well as pathological activities of IL-18 before we apply IL-18 in tumor immunotherapy.

IL-2 family cytokines

First discovered in 1976 as a T cell growth factor,IL-2 played an important role in T cell activation and proliferation.Currently,IL-2 family members include IL-4,IL-7,IL-9,IL-15 and IL-21,which share the common cytokine receptor chain (chain) with IL-2.These cytokines increased CD8+T cell-mediated antitumor activity,which might be useful novel immunotherapeutic agents.[38]

IL-2 is a pleiotropic cytokine that drives T cell growth and promotes NK cell activation.These findings have led to its approval by the FDA for the treatment of patients with renal cell cancer and metastatic melanoma.IL-2 is mainly produced by T cells and to a lesser extent by DCs and NK cells.IL-2 receptor is made up of IL-2Rα (CD25),IL-2Rβ and IL-2R.IL-2Rα is the "low-affinity" IL-2R; IL-2Rβ in combination with IL-2R forms the mid-affinity IL-2R; all the 3 subunits together form the high-affinity IL-2R.[39]Naive T cells and NK cells express mid-affinity IL-2R,but IL-2Rα is rapidly upregulated after activation by T cell receptor(TCR) or IL-2 signaling,with increasing responsiveness to IL-2.[40]Treg cells express high-affinity IL-2R and have much higher affinity for IL-2 than naive T cells,especially in low levels of IL-2.Thus,low levels of IL-2 favor Treg expansion to maintain self tolerance and host homeostasis.[41]IL-2 binds to IL-2R to stimulate the phosphorylation of JAK/STAT,PI3-K/Akt and MAPK signaling pathways and subsequently to induce the expression of downstream genes.Recently,it was revealed that IL-2 also activated T cells through "transpresent" activation.IL-2Rα positive DCs bind to capture and then deliver IL-2 to naive T cells that only express mid-affinity IL-2Rβ and IL-2R,thereby effectively transactivating naive T cells.[42]

IL-2 promotes T cell growth and effector T cell differentiation.Recent data have shown that IL-2 promotes T cell proliferation by upregulating microRNA miR-182 expression,which abrogates Foxo1-mediated suppression on resting T cells.[43]IL-2 also induces the differentiation of naive CD8+T cells into effector CTL and memory T cells.[44]IL-2 induces expression of IL-12Rβ2 and T-bet in T cells and is therefore necessary for Th1 differentiation.[45]However,IL-2 is also important for Th2[45]and Treg development,[46]and it can suppress Th17 and Tfh development through the downregulation of RORγt[47]or activation STAT5 pathway,respectively.[48]A study[49]found that IL-2 modulated the differentiation of Th1,Th2,Treg and Th17 cells by regulating the expression of IL-12Rβ,IL-4Rα and IL-2Rβ.

IL-2 has been used in cancer treatment for many years but its clinical response is limited partially due to the immunosupression mediated by Treg and myeloid-derived suppressor cells (MDSCs) in tumors.Considering low levels of IL-2 favors the expansion of Treg cells,[50]high levels of IL-2 immunotherapy have been tested in patients.In addition,the combination of IL-2 with anti-CD40 has been proved to reduce the proportion of Tregs and MDSCs in tumor tissues.[51]In patients,IL-2-diphtheria toxin conjugate selectively eliminated Tregs,thus enhancing the efficacy of DC vaccine.[52]A study[53]reported a "IL-2 superkine",which had an increased binding affinity for IL-2Rβ and effectively activated naive T cells independent of IL-2Ra.IL-2 superkine induced less expansion of Treg compared with IL-2 and had great potential in clinical treatment of cancer.

Furthermore,IL-2 has been investigated in combination with other approaches such as DC-tumor fusion cell vaccine[54]and exosome-based tumor vaccine[55]to further increase its therapeutic effects.Compared with IL-2 alone,the combined vaccine of IL-2 with gp100 peptide significantly increased the overall response rate in patients with metastatic melanoma.[56]A recent phase III trial involving 185 patients with advanced melanoma verified that IL-2 combined with tumor peptide vaccine significantly improved the clinical response and prolonged progression-free survival of patients with melanoma.[57]Moreover,IL-2 combined with chimeric antigen receptors (CARs)-modified T cells remarkably improved the clinical response in patients with metastatic melanoma or B cell lymphoma,making it a rapidly developing strategy for cancer immunotherapy.[58]

In conclusion,IL-2 has been used for the treatment of renal cell cancer and melanoma cancer for more than two decades,but its clinical response is limited.IL-2 in combination with DC or tumor vaccines could further increase its efficacy in cancer therapy; however,adoptive cell transfer (ACT),especially CAR-T cell transfer,remarkably improved the cure rates of cancer patients,demonstrating the power and potential of IL-2 combined with CAR-T in tumor immunotherapy.

IL-12 family cytokines

IL-12 family cytokines include IL-12,IL-23,IL-27 and IL-35.[59]Most IL-12 family members are Th1-promoting cytokines that can induce the IFN-γ and Th1 response.IL-12 family cytokines are heterodimeric proteins with two subunits:IL-12 is composed of p35 and p40,IL-23 of p40 and p19 (homologous to p35),[60]IL-27,a heterodimeric cytokine,of Epstein-Barr virus-induced gene 3 (EBI3,homologous to p40)and p28 (homologous to p35),[61]and IL-35,of p35 and EBI3.IL-12,IL-23,IL-27 and IL-35 are important mediators of in flammatory response.However,they have distinct expression patterns and functions in the immune system.For example,IL-12 and IL-27 are involved in Th1 differentiation,while IL-23 is critical for Th17 differentiation by inducing IL-17.In addition,IL-23 mediates tumor-related in flammatory response and upregulates the production of Foxp3 and IL-10 in tumor-infiltrating Treg cells,thus contributing to tumor development.[62]IL-27 synergizes with IL-12 to increase IFN-γ production and exerts strong antitumor effects,but it can also suppress Th2,Th17 and Treg differentiation.[63]

IL-12 is a potent Th1 cytokine mainly produced by macrophages and DCs in response to microbial products or TLR signaling.IL-12 receptor has two subtypes,IL-12R1 and IL-12R2.After binding to its receptor,IL-12 activates the JAK/STAT pathway,especially STAT4 signaling,to induce IFN-γ,the later then stimulates macrophages and DCs to produce more IL-12.IL-12 and IFN-γ also increase the expression of IL-12Rβ2 on Th1 cells,thus maintaining IFN-γ synthesis and serving as a survival signal for Th1 lineage.[64]

IL-12 plays a central role in Th1 differentiation and NK cell activation,indicating that IL-12 could be a powerful Th1-therapeutic agent or could be used as an adjuvant to boost antitumor immunity.It was demonstrated that administration of IL-12 alone induces effective Th1-type response against tumors,but with dose-limiting toxicity.[65]To overcome this obstacle,efforts have been directed to increase exogenous IL-12 gene expression in different cell types,including IL-12 gene-transfectedfibroblasts,[66]IL-12 gene-transduced tumor cells,[67]and IL-12 gene-modified DCs.[68]IL-12-engineered cells secrete high levels of IL-12 and efficiently eliminate tumors or suppress tumor growth.In addition,IL-12,as an adjuvant,can significantly increase the antitumor efficacy of cancer vaccine,[69]DC vaccine,[70]and DC-tumor fusion cell vaccine.[71]These treatments lead to increased antigen-specific CTL response and IFN-γ secretion,reduced tumor incidence,and prolonged survival of mice.Improved antitumor effects were also observed when IL-12 was coadministered with other cytokine-based immunotherapies,such as IL-18[28]and IL-15.[72]

Preclinical studies demonstrated that coadministration of IL-12 with vaccine showed remarkable antitumor effects,supporting the clinical application of IL-12 in cancer patients.The therapeutic effect of IL-12 had been evaluated in several clinical cancer trials,but showed limited efficacy in most instances.A phase I trial of systemic administration of recombinant human IL-12 (rhIL-12) in cancer patients showed limited clinical response with tolerable side effects.[73]However,the following phase II study demonstrated that administration of IL-12 at the same dosage caused severe dose-limiting toxicities and some patients were unable to tolerate.[74]Later,intratumoral injection of IL-12 gene-modifiedfibroblasts,[75]IL-12-modified DCs,[76]recombinant IL-12 adenoviral vector,[77]or IL-12 plasmid DNA[78]demonstrated that IL-12 adjuvant therapy was well-tolerated and effectively activated an immune response,but exerted only mild clinical response.Recently,a clinical trial using gp100 peptidepulsed,IL-12-producing DCs for the treatment of melanoma patients showed that IL-12 was critical for antigen-specific CD8+T cell activation and that serum IL-12 levels were positively correlated with the patients'response.[79]These findings highlighted the potential value of IL-12 in cancer immunotherapy.Moreover,patients with melanoma showed partial response to the treatment of IL-12-antibody fusion protein.[80]Chemotherapeutic agent cyclophosphamide reduced the number and suppressed the capability of Treg cells and effectively eradicated tumors.[81]A recent work[82]reported that IL-12 gene-engineered T cells combined with cyclophosphamide effectively suppressed established tumors.Moreover,CAR-modified T cells engineered to secrete IL-12 could eradicate established tumors even without prior cyclophosphamide conditioning,[83]demonstrating a novel strategy to treat tumor.

GM-CSF

GM-CSF was first discovered as a colony stimulator of granulocyte and monocytes/macrophage differentiation.It causes a dramatic increase in the number of colonyforming progenitor cells in the peripheral blood of cancer patients,and has been used in patients with chemotherapy-induced neutropenia.[84]It is also used to enhance hematopoietic regeneration in bone marrow transplantation.[85]Furthermore,CSF-mobilized peripheral blood mononuclear cells (PBMCs) are the dominant cells for transplantation in cancer patients.

GM-CSF is essential for the differentiation and functional activity of macrophages.In 1992,Caux and Inaba et al[86,87]reported that GM-CSF cooperation with IL-4 or TNF-α was crucial for the generation and expansion of DCs in vitro.Given the important role of DCs in the activation of naive T cells and the following antigen-specific T cell response,GM-CSF has been heavily studied as an adjuvant therapy for tumors.GMCSF-gene modified tumor cells activated antigen specific antitumor immunity,[88]and GM-CSF greatly increased the antitumor effects of cytosine deaminase (CD) gene therapy.[89]A study[90]also demonstrated that tumor antigen and GM-CSF fusion protein effectively activated DCs to elicit antigen specific Th1 response.

A phase I trial of patients with prostate cancer showed that autologous GM-CSF gene-transduced cancer vaccines could induce the infiltration of DCs and macrophages into injection sites.[91]Similarly,vaccination with GM-CSF engineered melanoma cell vaccine induced the infiltration of T cells into tumor sites,with enhanced antitumor immunity.[92]Another trial combining GM-CSF-modified tumor vaccine with anti-CTLA4 antibody achieved objective cancer regression in patients with metastatic melanoma or ovarian carcinoma.[93]In 2010,a DC-based vaccine,Sipuleucel-T,was approved by the FDA for treating patients with metastatic prostate cancer.Sipuleucel-T consists of autologous DCs pulsed with a fusion protein of GM-CSF and prostatic acid phosphatase.Sipuleucel-T could prolong the survival of prostate cancer patients by approximately 4 months,confirming that cytokines in combination with DC-based vaccines improve therapeutic outcomes.[94]However,there are no clinically complete response in these patients.Despite this progress,research is still needed to further improve the therapeutic effect of GM-CSF in cancer patients.

Th1 response enhanced by immune adjuvants

Th1 response is essential for antitumor immunity.Many immune modulators have shown strong capability to enhance Th1 cytokine production and potentiate Th1-immunity in response to cancer vaccines.We focus mainly on three important Th1 adjuvants:bacillus Calmette-Guérin (BCG),heat-shock proteins (HSPs)and TLR9 agonist,unmethylated cytosine-phosphateguanosine oligodeoxynucleotides (CpG ODN).

BCG

BCG is an attenuated strain of Mycobacterium bovis and was initially developed as a vaccine against tuberculosis.A study[95]found that mice infected with BCG demonstrated resistance to tumors.In 1976,Morales and colleagues[96]first used BCG for patients with superficial bladder cancer.Since then,numerous clinical trials have confirmed that intravesical BCG therapy reduced recurrence and progression of bladder cancer after transurethral resection.[97-99]Now,intravesical BCG immunotherapy remains the standard treatment of non-muscle invasive bladder cancer and one of the most successful examples of cancer immunotherapy.[100]

As a potent Th1 immune stimulant,BCG activates the local immune system characterized by the upregulation of various Th1 cytokines and chemokines(such as IL-12,IL-18,IFN-γ,IL-8 and TNF-α) in the urine and bladder tissues,[101]as well as by the infiltration of neutrophils,mononcytes/macrophages,γδT,NKT cells,NK cells and T cells,all of which are critical for the treatment of bladder cancer.[102-104]In addition to secretion of proin flammatory cytokines such as IL-1,IL-6,IL-8 and TNF-α to recruit immune cells into the bladder,BCG-activated neutrophils also release tumor necrosis factor (TNF-α)-related apoptosis-inducing ligand (TRAIL) to mediate tumor cell death.[105]Moreover,inhibitor of apoptosis protein (IAP) antagonists can effectively increase the tumor-killing activity of BCG-stimulated neutrophils,highlighting the potential of the two being combined in immunotherapy for bladder cancer.[106]

As mentioned above,local innate immunity is important for the antitumor efficacy of BCG therapy.However,using a mathematical model re flecting the interactions between innate immune cells,tumor cells and BCG in bladder cancer patients during BCG therapy,Breban et al[107]found that innate immune response alone did not mediate the observed antitumor efficacy of BCG.In fact,pre-immunization of BCG markedly improved the therapeutic effect of subsequent intravesical BCG therapy with an increased infiltration of IFN-γ-producing T cells into the bladder,indicating a robust acquired immune response after repeated BCG administration.[108]Recently,reports[109,110]further showed that purified protein derivate (PPD)-specific T cells were increased in the urine and blood of bladder cancer patients after BCG therapy,and PPD-specific T cells contributed to the antitumor effect of BCG.In summary,intravesical BCG therapy activates the local innate immune response and the systemic antigenspecific T cell response to effectively eliminate bladder cancer cells.

Intravesical BCG therapy is effective in reducing recurrence and progression of non-muscle invasive bladder cancer patients,but a high percentage of patients may fail to respond to BCG immunotherapy and almost half of the responders relapse within 5 years.[111]The reasons for BCG ineffectiveness and recurrence are unknown.In addition,BCG immunotherapy has side effects including cystitis,fever,hematuria and even sepsis.A study[112]was conducted to improve the efficacy and/or to reduce the toxicity of BCG therapy by using different BCG dosages,different administration routes and different schedule regimens.In addition,given that the Th1-immune response is crucial for antitumor activity of BCG,strategies that combine BCG therapy with Th1 cytokines have been extensively investigated to further enhance the efficacy of BCG adjuvant therapy.[113]Similarly,the genetic manipulation of BCG to secrete Th1 cytokines has also been explored to further enhance the antitumor efficacy of BCG in animal models.[114,115]Furthermore,it was reported that both innate and acquired immune responses are critical for the antitumor efficacy of BCG,pre-immunization of BCG might improve the clinical efficacy of BCG immunotherapy,[107]and that BCG therapy combined with chemotherapy or regimens that counteract immunosuppressive cells such as MDSCs,Treg cells and M2 macrophages may further improve clinical efficacy whilst decreasing adverse events of BCG immunotherapy.[116]

HSP

HSPs are a highly conserved family of molecular chaperones involved in protein folding and transport.HSPs are grouped into several subfamilies according to their molecular weight:HSP100,HSP90,HSP70,HSP60,HSP40 and small HSPs (HSP27 etc.).HSP levels are increased by stress stimuli including heat,oxidative stress,hypoxia and viral infection.HSPs are also potent stimulators of the innate and acquired immune systems,inducing activation of DCs and NK cells and augmenting T cell and humoral immune responses.[117]Tumor-derived HSPs function as chaperones of tumor antigen to transfer their chaperoned antigen peptides to DCs and cross-prime antigen specific CD8+T cells.[118,119]

HSPs have been extensively explored as immune adjuvants in mouse tumor models.Vaccination of tumorderived HSP70,[120]HSP110-transduced tumor cells,[121]tumor cell membrane-bound HSP70,[122]HSP70-peptide complexes derived from DC-tumor fusion cells,[123]or HSP70 and superantigen-anchored tumor vaccine[124]could elicit significant protective antitumor immunity to tumor cell re-challenge and prolong survival of tumorbearing mice.HSP70 also could remarkably enhance the efficacy of DC vaccines in activating Th1-based response and antigen-specific CD8+CTLs.[125]In addition,exosomes derived from heat-stressed tumor cells contain high levels of HSPs and tumor antigens,and are significantly efficient in activating DC and inducing antitumor response.[126,127]In short,HSP-peptide complexes derived from tumors elicit specific T cell response and are safe adjuvants in animal models.

Some clinical trials investigated the efficacy of HSP adjuvant therapy.Phase I and phase II trials demonstrated that HSP-peptide vaccines were welltolerated and showed increased immune response in some patients,which correlated with a positive clinical response in melanoma and colorectal cancer.[128,129]In 2008,a phase III trial of tumor-derived HSPgp96-peptide complexes (Vitespen) in 322 patients with stage IV melanoma showed a positive response and suggested the usefulness of Vitespen in the treatment of melanoma patients.[130]However,another phase III trial of 818 patients with advanced renal cell carcinoma indicated that Vitespen failed to improve recurrencefree survival.[131]

Increasing data have shown that HSPs were overexpressed in cancer cells,contributing to tumor progression and chemotherapy resistance.HSP90 inhibitors could degrade its oncogenic "client" proteins and had therefore been exploited as potential anticancer agents,either used alone or together with other antitumor reagents.[132]

CpG ODN

TLRs are pattern-recognition receptors recognizing conserved molecules in pathogens.[133]TLR activation initiates innate and adaptive immune responses.Many adjuvants are ligands of TLRs that activate DCs and macrophages to produce Th1 cytokine and to activate T cells.Therefore,they are potentially immunostimulators or vaccine adjuvants to enhance antitumor immunity.TLR7 agonist imiquimod has already been used in patients with basal cell carcinomas.[134]

CpG ODN binds to TLR9 on macrophages and DCs to stimulate Th1 cytokines like IL-12,thus promoting a Th1-dominant immune response.[135]Recent work[136]showed that intratumoral injection of CpG ODN induced MDSCs to differentiate into macrophages with increased tumoricidal activity,thus reducing the immunosuppression in tumors.CpG ODN elicits significant antitumor activity when given alone or coadministered with various forms of tumor vaccines.[137]When coinjected with tumor antigen peptides such as HPV16/E7 vaccine[138]or GM-CSF tumor vaccine,[139]CpG ODN showed enhanced activation of DCs and antigen specific CTLs to protect against tumor re-challenge or to suppress established tumors.It also remarkably enhances the antitumor response of antigen-pulsed DC vaccine,resulting in the increase of Th1 cytokines and antigenspecific CTL response,leading to longer survival in murine models of colon carcinoma.[140]

CpG ODN demonstrates significant antitumor adjuvant activities,and several CpG ODNs have been investigated for the treatment of cancer,such as CPG 7909 (PF-3512676),ISS 1018,IMO-2055 and CpG-28.[141,142]Early phase I and phase II trials indicated that CpG ODN therapy was well-tolerated and could improve the antitumor immune response of cancer vaccine.[143]Recently,a phase III clinical trial with NSCLC,the combination of CpG ODN with chemotherapy,failed to improve overall survival and progression-free survival,and was accompanied with serious side effects.[144]This led to the halt of the clinical trial with CpG ODN in cancer patients.

A major obstacle to tumor therapy is the immunosuppressive environment in established tumors,including immunosuppressive cells such as tolerogenic DCs,[145,146]Treg cells,MDSCs and a variety of inhibitory molecules such as CTLA4.Therefore,CpG combined with immunosuppressive blockage strategy may achieve a more efficient therapeutic effect.Krieg[147]demonstrated that the combination of CpG ODN with anti-CTLA4 antibody improved response with no adverse side-effects in cancer patients.In addition,CpG ODN combined with chemotherapy or other immunotherapies,such as antibody therapy,also enhanced the antitumor effects.[148]Furthermore,nanoparticle delivery was also exploited for CpG ODN to enhance its Th1-based response.[149]Conroy et al[150]found that CpG ODN increased Tregs and the activated STAT3 in DCs,thus inhibiting the Th1-type cytokines and chemokines.[151]Further elucidation of the activities and signaling mechanisms of CpG ODN are needed before we exploit it in tumor immunotherapy.

Conclusion

Th1-cytokine based immunotherapy plays an important role in tumor treatment.Th1 cytokines alone or as adjuvants enhance antitumor effects in animal models(Table).However,clinical trials do not always show a positive response,[32,74]despite mounting significant immune response such as activation of DCs and macrophages,upregulation of Th1-type cytokines and chemokines,and increased cytotoxicities of CTLs and NK cells.[80,91]

At present,only a few cytokines are effective in patients.Multiple factors in fluence the effectiveness of tumor immunotherapy.Tumors can induce immunosuppressive cells such as Tregs,MDSCs,regulatory DCs and some NK subsets.[47]These cells express inhibitory molecules like CTLA4 and PD1,[152]and secrete multiple inhibitory cytokines,such as IL-10,TGF-β,IL-27 and vascular endothelial growth factor (VEGF),thereby inhibiting antitumor response.[153]Most studies of tumor immunotherapy focused on how to stimulate potent Th1 and CD8+CTL responses,more research combines cytokine-based therapy with other strategies to reverse immunosuppression or immune tolerance in the tumor microenvironment.For example,combining GMCSF-secreting tumor vaccines with CTLA4 antibody demonstrated synergistic antitumor effects.[93,154]In addition,some chemotherapies and radiotherapies exhibited immunogenicity or selectively depleted Treg cells or MDSCs,[81]indicating that combination of immunotherapy with chemotherapy or radiotherapy improves patient outcomes.For example,depletion of Treg cells through a cyclophosphamide conditioning regimen effectively increased the efficacy of IL-12 geneengineered T cells.[82]

It should be noted that cytokines have pleiotropic functions and are capable of in fluencing many aspects of the immune system.Some cytokines show paradoxical effects under different settings.As mentioned above,IL-2 is necessary for Th1 differentiation;[46]however,italso promotes Treg development.[47]In addition,low levels of IL-2 favors expansion of Treg cells,[41]which may account for the disappointing clinical efficacy of low-dose IL-2 regimens in cancer therapy.On the contrary,high-dose IL-2 administration significantly enhanced therapeutic response in patients with metastatic renal cell carcinoma.[155]Similarly,IL-12 is a potent Th1 cytokine to induce IFN-γ production by T and NK cells.It significantly increases the antitumor efficacy of tumor vaccines with increased antigenspecific CTL response and IFN-γ secretion.[69]However,long-term culture of T cells with IL-12 induced the expression of an immunoinhibitory protein,TIM-3,and impaired Th1 cell function that contributed to the insufficient antitumor immunity of IL-12 therapy in lymphoma patients.[156]In addition,tumor-derived IL-18 converted Kit-CD11b-NK cells into regulatory Kit+NK cells,thus promoting tumor progression.[37]

Table.Th1-cytokine family members and their antitumor activities

In summary,many cytokines are well-characterized as pro-in flammatory cytokines,but they also have suppressive and anti-in flammatory activities in certain settings.A better understanding of the roles and mechanisms of cytokines in the immune response is important for the design of cytokine-based tumor immunotherapy.

Acknowledgment:I thank Prof.Matthew Freeman and Dr.Adam Grieve for the critical reading of the manuscript.

Contributors:XHM wrote the whole article.XHM is the guarantor.

Funding:None.

Ethical approval:Not needed.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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