Author Affiliations: Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, United Kingdom (Xu HM)

Corresponding Author: Hong-Mei Xu, MD, PhD, Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, United Kingdom (Tel: +44-1865-85652; Fax: +44-1865-275501; Email: hongmei.xu@path.ox.ac.uk)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60305-2

Published online September 25, 2014.

 

 

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.

 

 

ABSTRACT: Cytokine-based immunotherapy is executed by harnessing cytokines to activate the immune system to suppress tumors. Th1-type cytokines including IL-1, IL-2, IL-12 and granulocyte-macrophage colony-stimulating factor are potent stimulators of Th1 differentiation and Th1-based antitumor response. Many preclinical studies demonstrated the antitumor effects of Th1 cytokines but their clinical efficacy is limited. Multiple factors influence the efficacy of immunotherapy for tumors. For instance immunosuppressive cells in the tumor microenvironment can produce inhibitory cytokines which suppress antitumor immune response. Most studies on cytokine immunotherapy focused on how to boost Th1 response; many studies combined cytokine-based therapy with other treatments to reverse immunosuppression in tumor microenvironment. In addition, cytokines have pleiotropic functions and some cytokines show paradoxical activities under different settings. Better understanding the physiological and pathological functions of cytokines helps clinicians to design Th1-based cancer therapy in clinical practice.

 

(Hepatobiliary Pancreat Dis Int 2014;13:482-494)

 

KEY WORDS: cytokine; gene therapy; adjuvant therapy

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 cytokine-based 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 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 inflammatory and autoimmune diseases,^[8] IL-1 is also considered as a proinflammatory cytokine. Accordingly, overproduction of IL-1β contributes to many inflammatory diseases and the inflammatory 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 inflammatory 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 inflammasome 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 to fight 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, tumor-derived 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.

 

 

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 "trans-present" 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 trans-activating 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 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 inflammatory 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 inflammatory 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-transfected fibroblasts,^[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-modified fibroblasts,^[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 peptide-pulsed, 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 was first discovered as a colony stimulator of granulocyte and monocytes/macrophage differentiation. It causes a dramatic increase in the number of colony-forming 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. GM-CSF-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 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-phosphate-guanosine oligodeoxynucleotides (CpG ODN).

 

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 proinflammatory 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 reflecting 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 antigen-specific 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]

 

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 tumor-derived 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 tumor-bearing 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 well-tolerated 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 recurrence-free survival.^[131]

 

Increasing data have shown that HSPs were over- expressed 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 anti-cancer agents, either used alone or together with other antitumor reagents.^[132]

 

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 co-administered 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 antigen-specific 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.

 

 

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 influence 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 GM-CSF-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 gene-engineered T cells.^[82]

 

It should be noted that cytokines have pleiotropic functions and are capable of influencing 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, it also 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 antigen-specific 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]

 

In summary, many cytokines are well-characterized as pro-inflammatory cytokines, but they also have suppressive and anti-inflammatory 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. 

 

 

1 Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula US, et al. High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006. Cancer 2008;113:293-301. PMID: 18457330

2 Dinarello CA. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 2009;27:519-550. PMID: 19302047

3 Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK, et al. IL-33, an interleukin-1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005;23:479-490. PMID: 16286016

4 Ben-Sasson SZ, Hu-Li J, Quiel J, Cauchetaux S, Ratner M, Shapira I, et al. IL-1 acts directly on CD4 T cells to enhance their antigen-driven expansion and differentiation. Proc Natl Acad Sci U S A 2009;106:7119-7124. PMID: 19359475

5 O'Sullivan BJ, Thomas HE, Pai S, Santamaria P, Iwakura Y, Steptoe RJ, et al. IL-1 beta breaks tolerance through expansion of CD25⁺ effector T cells. J Immunol 2006;176: 7278-7287. PMID: 16751371

6 Guo Z, Zhang M, An H, Chen W, Liu S, Guo J, et al. Fas ligation induces IL-1beta-dependent maturation and IL-1beta-independent survival of dendritic cells: different roles of ERK and NF-kappaB signaling pathways. Blood 2003;102:4441-4447. PMID: 12920043

7 Chung Y, Chang SH, Martinez GJ, Yang XO, Nurieva R, Kang HS, et al. Critical regulation of early Th17 cell differentiation by interleukin-1 signaling. Immunity 2009;30:576-587. PMID: 19362022

8 Sutton C, Brereton C, Keogh B, Mills KH, Lavelle EC. A crucial role for interleukin (IL)-1 in the induction of IL-17-producing T cells that mediate autoimmune encephalomyelitis. J Exp Med 2006;203:1685-1691. PMID: 16818675

9 Gabay C, Lamacchia C, Palmer G. IL-1 pathways in inflammation and human diseases. Nat Rev Rheumatol 2010;6:232-241. PMID: 20177398

10 Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming MA, et al. Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science 1997;275:206- 209. PMID: 8999548

11 Reddy P. Interleukin-18: recent advances. Curr Opin Hematol 2004;11:405-410. PMID: 15548995

12 Zaki MH, Vogel P, Body-Malapel M, Lamkanfi M, Kanneganti TD. IL-18 production downstream of the Nlrp3 inflammasome confers protection against colorectal tumor formation. J Immunol 2010;185:4912-4920. PMID: 20855874

13 O'Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: 10 years of progress. Immunol Rev 2008;226:10- 18. PMID: 19161412

14 Takeda K, Tsutsui H, Yoshimoto T, Adachi O, Yoshida N, Kishimoto T, et al. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 1998;8:383-390. PMID: 9529155

15 Wong JL, Berk E, Edwards RP, Kalinski P. IL-18-primed helper NK cells collaborate with dendritic cells to promote recruitment of effector CD8⁺ T cells to the tumor microenvironment. Cancer Res 2013;73:4653-4662. PMID: 23761327

16 Jonak ZL, Trulli S, Maier C, McCabe FL, Kirkpatrick R, Johanson K, et al. High-dose recombinant interleukin-18 induces an effective Th1 immune response to murine MOPC-315 plasmacytoma. J Immunother 2002;25:S20-27. PMID: 12048347

17 Ju DW, Yang Y, Tao Q, Song WG, He L, Chen G, et al. Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity. Gene Ther 2000;7:1672-1679. PMID: 11083476

18 Yoshimura K, Hazama S, Iizuka N, Yoshino S, Yamamoto K, Muraguchi M, et al. Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igkappa leader sequence. Cancer Gene Ther 2001;8:9-16. PMID: 11219497

19 Zhang LH, Pan JP, Yao HP, Sun WJ, Xia DJ, Wang QQ, et al. Intrasplenic transplantation of IL-18 gene-modified hepatocytes: an effective approach to reverse hepatic fibrosis in schistosomiasis through induction of dominant Th1 response. Gene Ther 2001;8:1333-1342. PMID: 11571570

20 Ju DW, Tao Q, Lou G, Bai M, He L, Yang Y, et al. Interleukin 18 transfection enhances antitumor immunity induced by dendritic cell-tumor cell conjugates. Cancer Res 2001;61:3735-3740. PMID: 11325846

21 Tatsumi T, Gambotto A, Robbins PD, Storkus WJ. Interleukin 18 gene transfer expands the repertoire of antitumor Th1-type immunity elicited by dendritic cell-based vaccines in association with enhanced therapeutic efficacy. Cancer Res 2002;62:5853-5858. PMID: 12384548

22 Xia D, Zheng S, Zhang W, He L, Wang Q, Pan J, et al. Effective induction of therapeutic antitumor immunity by dendritic cells coexpressing interleukin-18 and tumor antigen. J Mol Med (Berl) 2003;81:585-596. PMID: 12937899

23 Marshall DJ, Rudnick KA, McCarthy SG, Mateo LR, Harris MC, McCauley C, et al. Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine. Vaccine 2006;24:244-253. PMID: 16135392

24 Wang Q, Yu H, Ju DW, He L, Pan JP, Xia DJ, et al. Intratumoral IL-18 gene transfer improves therapeutic efficacy of antibody-targeted superantigen in established murine melanoma. Gene Ther 2001;8:542-550. PMID: 11319621

25 Dai S, Zhou X, Wang B, Wang Q, Fu Y, Chen T, et al. Enhanced induction of dendritic cell maturation and HLA-A*0201-restricted CEA-specific CD8(+) CTL response by exosomes derived from IL-18 gene-modified CEA-positive tumor cells. J Mol Med (Berl) 2006;84:1067-1076. PMID: 17016692

26 Robinson D, Shibuya K, Mui A, Zonin F, Murphy E, Sana T, et al. IGIF does not drive Th1 development but synergizes with IL-12 for interferon-gamma production and activates IRAK and NFkappaB. Immunity 1997;7:571-581. PMID: 9354477

27 Coughlin CM, Salhany KE, Wysocka M, Aruga E, Kurzawa H, Chang AE, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis. J Clin Invest 1998;101: 1441-1452. PMID: 9502787

28 Tatsumi T, Huang J, Gooding WE, Gambotto A, Robbins PD, Vujanovic NL, et al. Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res 2003;63: 6378-6386. PMID: 14559827

29 Iinuma H, Okinaga K, Fukushima R, Inaba T, Iwasaki K, Okinaga A, et al. Superior protective and therapeutic effects of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma. J Immunol 2006;176:3461-3469. PMID: 16517714

30 Rodriguez-Galan MC, Reynolds D, Correa SG, Iribarren P, Watanabe M, Young HA. Coexpression of IL-18 strongly attenuates IL-12-induced systemic toxicity through a rapid induction of IL-10 without affecting its antitumor capacity. J Immunol 2009;183:740-748. PMID: 19535628

31 Robertson MJ, Mier JW, Logan T, Atkins M, Koon H, Koch KM, et al. Clinical and biological effects of recombinant human interleukin-18 administered by intravenous infusion to patients with advanced cancer. Clin Cancer Res 2006;12: 4265-4273. PMID: 16857801

32 Tarhini AA, Millward M, Mainwaring P, Kefford R, Logan T, Pavlick A, et al. A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer 2009;115:859-868. PMID: 19140204

33 Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol 2001;19:423-474. PMID: 11244043

34 Vidal-Vanaclocha F, Mendoza L, Telleria N, Salado C, Valcárcel M, Gallot N, et al. Clinical and experimental approaches to the pathophysiology of interleukin-18 in cancer progression. Cancer Metastasis Rev 2006;25:417-434. PMID: 17001512

35 Carrascal MT, Mendoza L, Valcárcel M, Salado C, Egilegor E, Tellería N, et al. Interleukin-18 binding protein reduces b16 melanoma hepatic metastasis by neutralizing adhesiveness and growth factors of sinusoidal endothelium. Cancer Res 2003;63:491-497. PMID: 12543807

36 Terme M, Ullrich E, Aymeric L, Meinhardt K, Desbois M, Delahaye N, et al. IL-18 induces PD-1-dependent immunosuppression in cancer. Cancer Res 2011;71:5393-5399. PMID: 21724589

37 Terme M, Ullrich E, Aymeric L, Meinhardt K, Coudert JD, Desbois M, et al. Cancer-induced immunosuppression: IL-18-elicited immunoablative NK cells. Cancer Res 2012;72:2757- 2767. PMID: 22427351

38 Markley JC, Sadelain M. IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T cell-mediated rejection of systemic lymphoma in immunodeficient mice. Blood 2010;115:3508-3519. PMID: 20190192

39 Takeshita T, Asao H, Ohtani K, Ishii N, Kumaki S, Tanaka N, et al. Cloning of the gamma chain of the human IL-2 receptor. Science 1992;257:379-382. PMID: 1631559

40 Depper JM, Leonard WJ, Drogula C, Krönke M, Waldmann TA, Greene WC. Interleukin 2 (IL-2) augments transcription of the IL-2 receptor gene. Proc Natl Acad Sci U S A 1985;82: 4230-4234. PMID: 2987968

41 Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 2010;33: 153-165. PMID: 20732639

42 Wuest SC, Edwan JH, Martin JF, Han S, Perry JS, Cartagena CM, et al. A role for interleukin-2 trans-presentation in dendritic cell-mediated T cell activation in humans, as revealed by daclizumab therapy. Nat Med 2011;17:604-609. PMID: 21532597

43 Stittrich AB, Haftmann C, Sgouroudis E, Kühl AA, Hegazy AN, Panse I, et al. The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nat Immunol 2010;11:1057-1062. PMID: 20935646

44 Feau S, Arens R, Togher S, Schoenberger SP. Autocrine IL-2 is required for secondary population expansion of CD8(+) memory T cells. Nat Immunol 2011;12:908-913. PMID: 21804558

45 Liao W, Lin JX, Wang L, Li P, Leonard WJ. Modulation of cytokine receptors by IL-2 broadly regulates differentiation into helper T cell lineages. Nat Immunol 2011;12:551-559. PMID: 21516110

46 Cote-Sierra J, Foucras G, Guo L, Chiodetti L, Young HA, Hu-Li J, et al. Interleukin 2 plays a central role in Th2 differentiation. Proc Natl Acad Sci U S A 2004;101:3880-3885. PMID: 15004274

47 Davidson TS, DiPaolo RJ, Andersson J, Shevach EM. Cutting Edge: IL-2 is essential for TGF-beta-mediated induction of Foxp3⁺ T regulatory cells. J Immunol 2007;178:4022-4026. PMID: 17371955

48 Laurence A, Tato CM, Davidson TS, Kanno Y, Chen Z, Yao Z, et al. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 2007;26:371-381. PMID: 17363300

49 Johnston RJ, Choi YS, Diamond JA, Yang JA, Crotty S. STAT5 is a potent negative regulator of TFH cell differentiation. J Exp Med 2012;209:243-250. PMID: 22271576

50 Ahmadzadeh M, Rosenberg SA. IL-2 administration increases CD4⁺ CD25(hi) Foxp3⁺ regulatory T cells in cancer patients. Blood 2006;107:2409-2414. PMID: 16304057

51 Weiss JM, Back TC, Scarzello AJ, Subleski JJ, Hall VL, Stauffer JK, et al. Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment. Proc Natl Acad Sci U S A 2009;106:19455-19460 PMID: 19892741

52 Dannull J, Su Z, Rizzieri D, Yang BK, Coleman D, Yancey D, et al. Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells. J Clin Invest 2005;115:3623-3633. PMID: 16308572

53 Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L, et al. Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'. Nature 2012;484:529-533. PMID: 22446627

54 Ogawa F, Iinuma H, Okinaga K. Dendritic cell vaccine therapy by immunization with fusion cells of interleukin-2 gene-transduced, spleen-derived dendritic cells and tumour cells. Scand J Immunol 2004;59:432-439. PMID: 15140052

55 Yang Y, Xiu F, Cai Z, Wang J, Wang Q, Fu Y, et al. Increased induction of antitumor response by exosomes derived from interleukin-2 gene-modified tumor cells. J Cancer Res Clin Oncol 2007;133:389-399. PMID: 17219198

56 Smith FO, Downey SG, Klapper JA, Yang JC, Sherry RM, Royal RE, et al. Treatment of metastatic melanoma using interleukin-2 alone or in conjunction with vaccines. Clin Cancer Res 2008;14:5610-5618. PMID: 18765555

57 Schwartzentruber DJ, Lawson DH, Richards JM, Conry RM, Miller DM, Treisman J, et al. gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 2011;364:2119-2127. PMID: 21631324

58 Rosenberg SA. Raising the bar: the curative potential of human cancer immunotherapy. Sci Transl Med 2012;4:127ps8. PMID: 22461638

59 Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 2003;3: 133-146. PMID: 12563297

60 Oppmann B, Lesley R, Blom B, Timans JC, Xu Y, Hunte B, et al. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 2000;13:715-725. PMID: 11114383

61 Pflanz S, Timans JC, Cheung J, Rosales R, Kanzler H, Gilbert J, et al. IL-27, a heterodimeric cytokine composed of EBI3 and p28 protein, induces proliferation of naive CD4⁺ T cells. Immunity 2002;16:779-790. PMID: 12121660

62 Langowski JL, Zhang X, Wu L, Mattson JD, Chen T, Smith K, et al. IL-23 promotes tumour incidence and growth. Nature 2006;442:461-465. PMID: 16688182

63 Kastelein RA, Hunter CA, Cua DJ. Discovery and biology of IL-23 and IL-27: related but functionally distinct regulators of inflammation. Annu Rev Immunol 2007;25:221-242. PMID: 17291186

64 Rogge L, Barberis-Maino L, Biffi M, Passini N, Presky DH, Gubler U, et al. Selective expression of an interleukin-12 receptor component by human T helper 1 cells. J Exp Med 1997;185:825-831. PMID: 9120388

65 Brunda MJ, Luistro L, Warrier RR, Wright RB, Hubbard BR, Murphy M, et al. Antitumor and antimetastatic activity of interleukin 12 against murine tumors. J Exp Med 1993;178: 1223-1230. PMID: 8104230

66 Tahara H, Zeh HJ 3rd, Storkus WJ, Pappo I, Watkins SC, Gubler U, et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res 1994;54:182-189. PMID: 7903204

67 Adris S, Chuluyan E, Bravo A, Berenstein M, Klein S, Jasnis M, et al. Mice vaccination with interleukin 12-transduced colon cancer cells potentiates rejection of syngeneic non-organ-related tumor cells. Cancer Res 2000;60:6696-6703. PMID: 11118055

68 Nishioka Y, Hirao M, Robbins PD, Lotze MT, Tahara H. Induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12. Cancer Res 1999;59:4035- 4041. PMID: 10463604

69 Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, et al. Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice. J Exp Med 2001;194:1195-1205. PMID: 11696586

70 Okada N, Iiyama S, Okada Y, Mizuguchi H, Hayakawa T, Nakagawa S, et al. Immunological properties and vaccine efficacy of murine dendritic cells simultaneously expressing melanoma-associated antigen and interleukin-12. Cancer Gene Ther 2005;12:72-83. PMID: 15389286

71 Suzuki T, Fukuhara T, Tanaka M, Nakamura A, Akiyama K, Sakakibara T, et al. Vaccination of dendritic cells loaded with interleukin-12-secreting cancer cells augments in vivo antitumor immunity: characteristics of syngeneic and allogeneic antigen-presenting cell cancer hybrid cells. Clin Cancer Res 2005;11:58-66. PMID: 15671528

72 Lasek W, Basak G, Switaj T, Jakubowska AB, Wysocki PJ, Mackiewicz A, et al. Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice. Cancer Immunol Immunother 2004;53:363-372. PMID: 14605763

73 Atkins MB, Robertson MJ, Gordon M, Lotze MT, DeCoste M, DuBois JS, et al. Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 1997;3:409-417. PMID: 9815699

74 Leonard JP, Sherman ML, Fisher GL, Buchanan LJ, Larsen G, Atkins MB, et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood 1997;90:2541-2548. PMID: 9326219

75 Kang WK, Park C, Yoon HL, Kim WS, Yoon SS, Lee MH, et al. Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. Hum Gene Ther 2001;12:671-684. PMID: 11426466

76 Mazzolini G, Alfaro C, Sangro B, Feijoó E, Ruiz J, Benito A, et al. Intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas. J Clin Oncol 2005;23:999-1010. PMID: 15598979

77 Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I, et al. Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 2004;22:1389-1397. PMID: 15084613

78 Heinzerling L, Burg G, Dummer R, Maier T, Oberholzer PA, Schultz J, et al. Intratumoral injection of DNA encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy. Hum Gene Ther 2005;16:35-48. PMID: 15703487

79 Carreno BM, Becker-Hapak M, Huang A, Chan M, Alyasiry A, Lie WR, et al. IL-12p70-producing patient DC vaccine elicits Tc1-polarized immunity. J Clin Invest 2013;123:3383-3394. PMID: 23867552

80 Rudman SM, Jameson MB, McKeage MJ, Savage P, Jodrell DI, Harries M, et al. A phase 1 study of AS1409, a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma. Clin Cancer Res 2011;17: 1998-2005. PMID: 21447719

81 Lutsiak ME, Semnani RT, De Pascalis R, Kashmiri SV, Schlom J, Sabzevari H. Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood 2005;105:2862-2868. PMID: 15591121

82 Kerkar SP, Muranski P, Kaiser A, Boni A, Sanchez-Perez L, Yu Z, et al. Tumor-specific CD8⁺ T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts. Cancer Res 2010;70:6725-6734. PMID: 20647327

83 Pegram HJ, Lee JC, Hayman EG, Imperato GH, Tedder TF, Sadelain M, et al. Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning. Blood 2012;119:4133-4141. PMID: 22354001

84 Gianni AM, Siena S, Bregni M, Tarella C, Stern AC, Pileri A, et al. Granulocyte-macrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 1989;2:580-585. PMID: 2570283

85 Nemunaitis J, Singer JW, Buckner CD, Durnam D, Epstein C, Hill R, et al. Use of recombinant human granulocyte-macrophage colony-stimulating factor in graft failure after bone marrow transplantation. Blood 1990;76:245-253. PMID: 2194592

86 Caux C, Dezutter-Dambuyant C, Schmitt D, Banchereau J. GM-CSF and TNF-alpha cooperate in the generation of dendritic Langerhans cells. Nature 1992;360:258-261. PMID: 1279441

87 Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176: 1693-1702. PMID: 1460426

88 Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc Natl Acad Sci U S A 1993;90:3539-3543. PMID: 8097319

89 Cao X, Ju DW, Tao Q, Wang J, Wan T, Wang BM, et al. Adenovirus-mediated GM-CSF gene and cytosine deaminase gene transfer followed by 5-fluorocytosine administration elicit more potent antitumor response in tumor-bearing mice. Gene Ther 1998;5:1130-1136. PMID: 10326037

90 Tso CL, Zisman A, Pantuck A, Calilliw R, Hernandez JM, Paik S, et al. Induction of G250-targeted and T-cell-mediated antitumor activity against renal cell carcinoma using a chimeric fusion protein consisting of G250 and granulocyte/monocyte-colony stimulating factor. Cancer Res 2001;61:7925-7933. PMID: 11691814

91 Simons JW, Mikhak B, Chang JF, DeMarzo AM, Carducci MA, Lim M, et al. Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer. Cancer Res 1999;59:5160-5168. PMID: 10537292

92 Soiffer R, Hodi FS, Haluska F, Jung K, Gillessen S, Singer S, et al. Vaccination with irradiated, autologous melanoma cells engineered to secrete granulocyte-macrophage colony-stimulating factor by adenoviral-mediated gene transfer augments antitumor immunity in patients with metastatic melanoma. J Clin Oncol 2003;21:3343-3350. PMID: 12947071

93 Hodi FS, Mihm MC, Soiffer RJ, Haluska FG, Butler M, Seiden MV, et al. Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients. Proc Natl Acad Sci U S A 2003;100:4712-4717. PMID: 12682289

94 Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, et al. Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med 2010;363:411-422. PMID: 20818862

95 Alexandroff AB, Jackson AM, O'Donnell MA, James K. BCG immunotherapy of bladder cancer: 20 years on. Lancet 1999;353:1689-1694. PMID: 10335805

96 Morales A, Eidinger D, Bruce AW. Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Urol 1976;116:180-183. PMID: 820877

97 Lamm DL, Thor DE, Harris SC, Reyna JA, Stogdill VD, Radwin HM. Bacillus Calmette-Guerin immunotherapy of superficial bladder cancer. J Urol 1980;124:38-40. PMID: 6997513

98 Sylvester RJ, van der Meijden AP, Lamm DL. Intravesical bacillus Calmette-Guerin reduces the risk of progression in patients with superficial bladder cancer: a meta-analysis of the published results of randomized clinical trials. J Urol 2002;168:1964-1970. PMID: 12394686

99 Sylvester RJ, Brausi MA, Kirkels WJ, Hoeltl W, Calais Da Silva F, Powell PH, et al. Long-term efficacy results of EORTC genito-urinary group randomized phase 3 study 30911 comparing intravesical instillations of epirubicin, bacillus Calmette-Guérin, and bacillus Calmette-Guérin plus isoniazid in patients with intermediate- and high-risk stage Ta T1 urothelial carcinoma of the bladder. Eur Urol 2010;57:766-773. PMID: 20034729

100 Babjuk M, Oosterlinck W, Sylvester R, Kaasinen E, Böhle A, Palou-Redorta J, et al. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update. Eur Urol 2011;59:997-1008. PMID: 21458150

101 Seow SW, Rahmat JN, Bay BH, Lee YK, Mahendran R. Expression of chemokine/cytokine genes and immune cell recruitment following the instillation of Mycobacterium bovis, bacillus Calmette-Guérin or Lactobacillus rhamnosus strain GG in the healthy murine bladder. Immunology 2008;124:419-427. PMID: 18217952

102 Prescott S, James K, Hargreave TB, Chisholm GD, Smyth JF. Intravesical Evans strain BCG therapy: quantitative immunohistochemical analysis of the immune response within the bladder wall. J Urol 1992;147:1636-1642. PMID: 1593713

103 Suttmann H, Jacobsen M, Reiss K, Jocham D, Böhle A, Brandau S. Mechanisms of bacillus Calmette-Guerin mediated natural killer cell activation. J Urol 2004;172:1490-1495. PMID: 15371877

104 Higuchi T, Shimizu M, Owaki A, Takahashi M, Shinya E, Nishimura T, et al. A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth. Cancer Immunol Immunother 2009;58:1245-1255. PMID: 19139883

105 Kemp TJ, Ludwig AT, Earel JK, Moore JM, Vanoosten RL, Moses B, et al. Neutrophil stimulation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) results in the release of functional soluble TRAIL/Apo-2L. Blood 2005;106:3474- 3482. PMID: 16037389

106 Jinesh GG, Chunduru S, Kamat AM. Smac mimetic enables the anticancer action of BCG-stimulated neutrophils through TNF-α but not through TRAIL and FasL. J Leukoc Biol 2012;92:233-244. PMID: 22517918

107 Breban R, Bisiaux A, Biot C, Rentsch C, Bousso P, Albert ML. Mathematical model of tumor immunotherapy for bladder carcinoma identifies the limitations of the innate immune response. Oncoimmunology 2012;1:9-17. PMID: 22720207

108 Biot C, Rentsch CA, Gsponer JR, Birkhäuser FD, Jusforgues-Saklani H, Lemaître F, et al. Preexisting BCG-specific T cells improve intravesical immunotherapy for bladder cancer. Sci Transl Med 2012;4:137ra72. PMID: 22674550

109 Pieraerts C, Martin V, Jichlinski P, Nardelli-Haefliger D, Derre L. Detection of functional antigen-specific T cells from urine of non-muscle invasive bladder cancer patients. Oncoimmunology 2012;1:694-698. PMID: 22934261

110 Elsäßer J, Janssen MW, Becker F, Suttmann H, Schmitt K, Sester U, et al. Antigen-specific CD4 T cells are induced after intravesical BCG-instillation therapy in patients with bladder cancer and show similar cytokine profiles as in active tuberculosis. PLoS One 2013;8:e69892. PMID: 24039703

111 Williams SK, Hoenig DM, Ghavamian R, Soloway M. Intravesical therapy for bladder cancer. Expert Opin Pharmacother 2010;11:947-958. PMID: 20205607

112 O'Donnell MA. Optimizing BCG therapy. Urol Oncol 2009;27:325-328. PMID: 19414123

113 O'Donnell MA, Luo Y, Chen X, Szilvasi A, Hunter SE, Clinton SK. Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guérin. J Immunol 1999;163:4246-4252. PMID: 10510362

114 Arnold J, de Boer EC, O'Donnell MA, Böhle A, Brandau S. Immunotherapy of experimental bladder cancer with recombinant BCG expressing interferon-gamma. J Immunother 2004;27:116-123. PMID: 14770083

115 Luo Y, Henning J, O'Donnell MA. Th1 cytokine-secreting recombinant Mycobacterium bovis bacillus Calmette-Guérin and prospective use in immunotherapy of bladder cancer. Clin Dev Immunol 2011;2011:728930. PMID: 21941579

116 Brandau S, Suttmann H. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomed Pharmacother 2007;61:299-305. PMID: 17604943

117 Srivastava P. Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses. Annu Rev Immunol 2002;20:395-425. PMID: 11861608

118 Basu S, Binder RJ, Ramalingam T, Srivastava PK. CD91 is a common receptor for heat shock proteins gp96, hsp90, hsp70, and calreticulin. Immunity 2001;14:303-313. PMID: 11290339

119 Binder RJ, Srivastava PK. Peptides chaperoned by heat-shock proteins are a necessary and sufficient source of antigen in the cross-priming of CD8⁺ T cells. Nat Immunol 2005;6:593-599. PMID: 15864309

120 Udono H, Srivastava PK. Heat shock protein 70-associated peptides elicit specific cancer immunity. J Exp Med 1993;178:1391-1396. PMID: 8376942

121 Wang XY, Li Y, Manjili MH, Repasky EA, Pardoll DM, Subjeck JR. Hsp110 over-expression increases the immunogenicity of the murine CT26 colon tumor. Cancer Immunol Immunother 2002;51:311-319. PMID: 12111119

122 Chen X, Tao Q, Yu H, Zhang L, Cao X. Tumor cell membrane-bound heat shock protein 70 elicits antitumor immunity. Immunol Lett 2002;84:81-87. PMID: 12270543

123 Gong J, Zhang Y, Durfee J, Weng D, Liu C, Koido S, et al. A heat shock protein 70-based vaccine with enhanced immunogenicity for clinical use. J Immunol 2010;184:488- 496. PMID: 19949080

124 Huang C, Yu H, Wang Q, Yang G, Ma W, Xia D, et al. A novel anticancer approach: SEA-anchored tumor cells expressing heat shock protein 70 onto the surface elicit strong anticancer efficacy. Immunol Lett 2005;101:71-80. PMID: 15908014

125 Wu Y, Wan T, Zhou X, Wang B, Yang F, Li N, et al. Hsp70-like protein 1 fusion protein enhances induction of carcinoembryonic antigen-specific CD8⁺ CTL response by dendritic cell vaccine. Cancer Res 2005;65:4947-4954. PMID: 15930317

126 Dai S, Wan T, Wang B, Zhou X, Xiu F, Chen T, et al. More efficient induction of HLA-A*0201-restricted and carcinoembryonic antigen (CEA)-specific CTL response by immunization with exosomes prepared from heat-stressed CEA-positive tumor cells. Clin Cancer Res 2005;11:7554- 7563. PMID: 16243831

127 Chen W, Wang J, Shao C, Liu S, Yu Y, Wang Q, et al. Efficient induction of antitumor T cell immunity by exosomes derived from heat-shocked lymphoma cells. Eur J Immunol 2006;36:1598-1607. PMID: 16708399

128 Belli F, Testori A, Rivoltini L, Maio M, Andreola G, Sertoli MR, et al. Vaccination of metastatic melanoma patients with autologous tumor-derived heat shock protein gp96-peptide complexes: clinical and immunologic findings. J Clin Oncol 2002;20:4169-4180. PMID: 12377960

129 Pilla L, Patuzzo R, Rivoltini L, Maio M, Pennacchioli E, Lamaj E, et al. A phase II trial of vaccination with autologous, tumor-derived heat-shock protein peptide complexes Gp96, in combination with GM-CSF and interferon-alpha in metastatic melanoma patients. Cancer Immunol Immunother 2006;55:958-968. PMID: 16215718

130 Testori A, Richards J, Whitman E, Mann GB, Lutzky J, Camacho L, et al. Phase III comparison of vitespen, an autologous tumor-derived heat shock protein gp96 peptide complex vaccine, with physician's choice of treatment for stage IV melanoma: the C-100-21 Study Group. J Clin Oncol 2008;26:955-962. PMID: 18281670

131 Wood C, Srivastava P, Bukowski R, Lacombe L, Gorelov AI, Gorelov S, et al. An adjuvant autologous therapeutic vaccine (HSPPC-96; vitespen) versus observation alone for patients at high risk of recurrence after nephrectomy for renal cell carcinoma: a multicentre, open-label, randomised phase III trial. Lancet 2008;372:145-154. PMID: 18602688

132 Jego G, Hazoumé A, Seigneuric R, Garrido C. Targeting heat shock proteins in cancer. Cancer Lett 2013;332:275-285. PMID: 21078542

133 Adams S. Toll-like receptor agonists in cancer therapy. Immunotherapy 2009;1:949-964. PMID: 20563267

134 Walker PS, Scharton-Kersten T, Krieg AM, Love-Homan L, Rowton ED, Udey MC, et al. Immunostimulatory oligodeoxynucleotides promote protective immunity and provide systemic therapy for leishmaniasis via IL-12- and IFN-gamma-dependent mechanisms. Proc Natl Acad Sci U S A 1999;96:6970-6975. PMID: 10359823

135 Xu H, An H, Yu Y, Zhang M, Qi R, Cao X. Ras participates in CpG oligodeoxynucleotide signaling through association with toll-like receptor 9 and promotion of interleukin-1 receptor-associated kinase/tumor necrosis factor receptor-associated factor 6 complex formation in macrophages. J Biol Chem 2003;278:36334-36340. PMID: 12867418

136 Shirota Y, Shirota H, Klinman DM. Intratumoral injection of CpG oligonucleotides induces the differentiation and reduces the immunosuppressive activity of myeloid-derived suppressor cells. J Immunol 2012;188:1592-1599. PMID: 22231700

137 Miconnet I, Koenig S, Speiser D, Krieg A, Guillaume P, Cerottini JC, et al. CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide. J Immunol 2002;168:1212-1218. PMID: 11801657

138 Kim TY, Myoung HJ, Kim JH, Moon IS, Kim TG, Ahn WS, et al. Both E7 and CpG-oligodeoxynucleotide are required for protective immunity against challenge with human papillomavirus 16 (E6/E7) immortalized tumor cells: involvement of CD4⁺ and CD8⁺ T cells in protection. Cancer Res 2002;62:7234-7240. PMID: 12499264

139 Sandler AD, Chihara H, Kobayashi G, Zhu X, Miller MA, Scott DL, et al. CpG oligonucleotides enhance the tumor antigen-specific immune response of a granulocyte macrophage colony-stimulating factor-based vaccine strategy in neuroblastoma. Cancer Res 2003;63:394-399. PMID: 12543793

140 Saha A, Bhattacharya-Chatterjee M, Foon KA, Celis E, Chatterjee SK. Stimulatory effects of CpG oligodeoxynucleotide on dendritic cell-based immunotherapy of colon cancer in CEA/HLA-A2 transgenic mice. Int J Cancer 2009;124:877- 888. PMID: 19035460

141 Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Adv Drug Deliv Rev 2009;61:195-204. PMID: 19211030

142 Hofmann MA, Kors C, Audring H, Walden P, Sterry W, Trefzer U. Phase 1 evaluation of intralesionally injected TLR9-agonist PF-3512676 in patients with basal cell carcinoma or metastatic melanoma. J Immunother 2008;31: 520-527. PMID: 18463532

143 Pashenkov M, Goëss G, Wagner C, Hörmann M, Jandl T, Moser A, et al. Phase II trial of a toll-like receptor 9-activating oligonucleotide in patients with metastatic melanoma. J Clin Oncol 2006;24:5716-5724. PMID: 17179105

144 Gnjatic S, Sawhney NB, Bhardwaj N. Toll-like receptor agonists: are they good adjuvants? Cancer J 2010;16:382-391. PMID: 20693851

145 Liu Q, Zhang C, Sun A, Zheng Y, Wang L, Cao X. Tumor-educated CD11bhighIalow regulatory dendritic cells suppress T cell response through arginase I. J Immunol 2009;182:6207- 6216. PMID: 19414774

146 Han Y, Chen Z, Yang Y, Jiang Z, Gu Y, Liu Y, et al. Human CD14⁺ CTLA-4⁺ regulatory dendritic cells suppress T-cell response by cytotoxic T-lymphocyte antigen-4-dependent IL-10 and indoleamine-2,3-dioxygenase production in hepatocellular carcinoma. Hepatology 2014;59:567-579. PMID: 23960017

147 Krieg AM. Toll-like receptor 9 (TLR9) agonists in the treatment of cancer. Oncogene 2008;27:161-167. PMID: 18176597

148 Friedberg JW, Kelly JL, Neuberg D, Peterson DR, Kutok JL, Salloum R, et al. Phase II study of a TLR-9 agonist (1018 ISS) with rituximab in patients with relapsed or refractory follicular lymphoma. Br J Haematol 2009;146:282-291. PMID: 19519691

149 Erikçi E, Gursel M, Gürsel I. Differential immune activation following encapsulation of immunostimulatory CpG oligodeoxynucleotide in nanoliposomes. Biomaterials 2011;32:1715- 1723. PMID: 21112627

150 Conroy H, Marshall NA, Mills KH. TLR ligand suppression or enhancement of Treg cells? A double-edged sword in immunity to tumours. Oncogene 2008;27:168-180. PMID: 18176598

151 Kortylewski M, Kujawski M, Herrmann A, Yang C, Wang L, Liu Y, et al. Toll-like receptor 9 activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy. Cancer Res 2009;69:2497-2505. PMID: 19258507

152 de Visser KE, Eichten A, Coussens LM. Paradoxical roles of the immune system during cancer development. Nat Rev Cancer 2006;6:24-37. PMID: 16397525

153 Flavell RA, Sanjabi S, Wrzesinski SH, Licona-Limón P. The polarization of immune cells in the tumour environment by TGFbeta. Nat Rev Immunol 2010;10:554-567. PMID: 20616810

154 Hodi FS, Butler M, Oble DA, Seiden MV, Haluska FG, Kruse A, et al. Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients. Proc Natl Acad Sci U S A 2008;105:3005-3010. PMID: 18287062

155 Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula US, et al. High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006. Cancer 2008;113:293-301. PMID: 18457330

156 Yang ZZ, Grote DM, Ziesmer SC, Niki T, Hirashima M, Novak AJ, et al. IL-12 upregulates TIM-3 expression and induces T cell exhaustion in patients with follicular B cell non-Hodgkin lymphoma. J Clin Invest 2012;122:1271-1282. PMID: 22426209