Tremendous scientific advances have been made in the last decade in therapeutic cancer vaccine development, with many entering phase II and phase III clinical trials (2). Most cancer vaccines in development aim to promote tumor-associated antigens to be presented by antigen presenting cells (APCs) to generate long-lasting T cell immunity against cancer (3). Because dendritic cells (DCs) are the most efficient APCs, effective presentation of tumor antigens by DCs is considered a key determinant for cancer vaccine development (4).

Usually, the immune system identifies and destroys neoplastically-transformed cells. This immune surveillance mechanism functions as the body's primary defense against cancer. CD8⁺ T lymphocytes are the primary player in the recognition and destruction of cancer cells (5, 6). Following stimulation through tumor antigen recognition presented by DCs, naive CD8⁺ T cells are stimulated to proliferate and differentiate into effector cells, namely cytotoxic T lymphocytes (CTLs). Following recognition of major histocompatibility complex (MHC) class I-antigen complexes on tumor cell surface, activated CTLs induce tumor cell lysis by secreting perforin, granulysin and granzyme, as well as producing the death ligands including Fas Ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) (7). A subset of antigen-specific T cells will differentiate into memory cells for long-lived anti-tumor protection. DCs also activate CD4⁺ T helper (Th) cells, that are critical for CD8⁺ T cell activation (8). This cross-priming is required to produce effective and durable CTL responses by breaking cross-tolerance and providing protection of CTLs from activation-induced cell death (AICD) (8, 9). Additionally, Th cells are also capable of eradicating tumor cells following activation (10, 11).

Several conditions, however, result in the failure of the immune system to destroy malignant cells (Figure 1). These include having a low number of tumor-specific T cells, suppression of T cell infiltration into tumor microenvironments, and T cell dysfunction/exhaustion (5, 6, 12–14). A low number of tumor-specific T cells results in a reduced number of cells capable of recognizing and killing neoplastic cells, hence tumor immune escape (6). Both failure in tumor antigen presentation and the development of immune tolerance contribute to this condition (5, 6). As tumor cells develop into a solid tumor mass, they create an immunosuppressive local microenvironment by secretion of specific factors that may restrict T cell infiltration, inactivate CTLs, and induce T cell apoptosis (13), further hampering cancer elimination. Due to chronic antigen exposure, T cells can also become dysfunctional and exhausted (12, 14). These T cells exhibit loss of the effector functions and upregulation of their immune checkpoint receptors such as PD1 and LAG3, the receptors that promote tolerance induction that subsequently prevents T cell activation upon stimulation.

To create neoplastic immunity, patients need to increase both the number and functionality of their cancer-specific T cells. This currently can be achieved by de novo generation of T cell-mediated immunity (15–18), through presentation by DCs (19, 20). One strategy utilizes a patient's own DCs as the therapeutic vaccine. DCs are maturated ex vivo using stimulatory cytokines and toll-like receptor (TLR) agonists, such as a combination of interferon (IFN)γ and lipopolysaccharide (LPS), and then loaded with patient-specific tumor antigens or proteins (21). The cells are then intradermally injected back into the patient together with adjuvants with the aim of generating a prolonged host immune response (22). In 2010, this strategy resulted in the first US Food and Drug Administration (FDA)-approved cancer vaccine, called Sipuleucel-T for prostate cancer patients (23). Increased survival in patients who received this personalized DC vaccine was achieved, suggesting successful long-lasting T cell immunity (24). Whilst this strategy has been successful in some patients, it has generally been inefficient. This is because the ex vivo DC vaccine preparation alters DC viability and functionality, is laborious and the output is of variable quality (19, 20). Moreover, the autologous DC generated from the patient's peripheral blood DC precursors, may have already been the subject of epigenetic imprinting by chemotherapy, radiation, immunotherapy or immune dysregulation by cancer cells, as such therapies have been shown to induce phenotypic alterations in immune cells (25). Understanding and modifying the epigenetic imprint of DC ex vivo (26), for example by the use of epigenetic modulators during tumor antigen loading, offers an intriguing avenue for future therapeutic exploration. Another strategy that currently holds promise in cancer vaccine development includes the injection of antigenic peptides or genetic material encoding for these peptides, in combination with adjuvants, to target DCs in vivo. However, despite appropriate antigen and adjuvant selections, many therapeutic cancer vaccines still fail to provide sustained T cell immunity, due to the many immune escape mechanisms available to neoplastic cells.

Recently several studies, as discussed in (27–31) show that epigenetic mechanisms drive phenotypic changes in both immune and cancer cells during their interactions. Epigenetics examines chemical modifications to a cell's deoxyribonucleic acid (DNA) that alters gene expression and thus the properties and behavior of cells, without changing their DNA sequence. These modifications include DNA methylation, histone modifications and ribonucleic acid (RNA)-associated mechanisms, via microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) which mediate alterations in chromatin accessibility at regulatory regions that determine cell fate (32–35). For example, DNA methylation results in a closed conformation of the chromatin, inhibiting binding of the transcription machinery and thus preventing gene expression (32). Various histone modifications, on the other hand, regulate cellular gene expression by modifying the polarity of the nucleosome particle, and/or by recruiting protein complexes, to result in either a closed or open chromatin conformation (33). Similarly, lncRNAs regulate gene expression by direct binding to chromatin remodeling complexes and targeting them to specific genomic loci to alter DNA methylation or histone marks (35). Additionally, miRNAs are able to regulate gene expression post-transcriptionally (34). In the following section we will discuss epigenetic changes in both immune and cancer cells that may be induced by cancer vaccine therapy.

Therapeutic cancer vaccines commonly utilize tumor-associated antigens presented by DCs to expand naive CD8⁺ T cells and drive their differentiation into both effector and memory cells. Activation of CTLs requires three signals (Figure 2): the first originates from the engagement of the T cell receptors (TCRs) with antigens as complexes with the MHC class I molecules on the surface of DCs; the second is the interactions of costimulatory molecules of DCs with cognate receptors of T cells including interactions between CD80/CD86 and CD28, CD70 and CD27, 41IBBL and 41IBB, OX40L and OX40, as well as GITRL and GITR (8, 36); and the third derives from cytokines including interleukin (IL)2 and IL12 secreted by DCs (37). Additionally, the tumor specific DCs activate Th cells through the interactions between TCRs and MHC class II-antigen complexes as well as the binding between their costimulatory molecules, such as the binding between CD80/CD86 and CD28. The activated Th cells in turn license DCs by upregulating their CD40L and LTαβ to interact with CD40 and LTαβR on DCs, respectively (36). The licensed DCs then produce polarizing factors such as IL12 to further differentiate CD4⁺ helper cells. The licensed DCs also increase the expression of CD80, CD70, OX40L, 41BBL, and GITRL, and secrete stimulatory cytokines such as IL2, IL12 and IFNγ, to generate CTLs with prolonged life-span with more effective effector function as reviewed in (8, 9, 36) (Figure 2).

Existing effector memory T cells can rapidly expand upon effective vaccination and differentiate into effector T cells to further mediate specific tumor destruction (15, 16). The vaccine-induced generation of antigen-specific T cells with distinct cellular phenotypes from genetically identical naive cells is mostly mediated by global epigenetic reprogramming. Recent work shows that epigenetic mechanisms control gene expression during CD8⁺ T cell differentiation following activation (27, 31). Epigenetic profiles also provide heritable maintenance of the phenotype of the differentiated T cells, following signal withdrawal (27, 31, 38, 39).

DNA methylation plays a significant role in CD8⁺ T cell differentiation into both effector and memory cells. In mammals, DNA methylation occurs mostly on CG dinucleotides (CpG). DNA methylation in CpG islands, short regions in the genome with high frequency of CpGs, is associated with transcriptional repression (32). During CD8⁺ differentiation, CpG islands become highly methylated at the promoters of silenced genes, and demethylated at the promoters of expressed genes (40–42). This alteration in methylation pattern dictates lineage-specific changes during differentiation following antigen-induced activation (43).

Like DNA methylation, promoters and other regulatory regions in the genome also undergo histone modifications during CD8⁺ T cell differentiation. Multiple studies show that in effector cells at the gene loci that are reduced in expression such as the memory cell-associated genes, activating histone marks including acetylation at lysine 9 on the histone 3 tail (H3K9Ac) and trimethylation at lysine 4 on the histone 3 tail (H3K4me3) are lost (41, 44–52). At the same gene loci, repressive marks including DNA methylation and trimethylation at lysine 27 on the histone 3 tail (H3K27me3) are gained. On the other hand, in the same cells, the effector cell-associated genes are upregulated and demonstrate decreased repressive and increased activating epigenetic marks (41, 44–52).

Importantly, in the absence of antigen presentation, memory cell subsets maintain their epigenetic patterns in order to retain their cellular phenotype (53). DNA methylation patterns of memory cells for example are preserved after antigen is withdrawn. This indicates involvement of epigenetic regulation in the maintenance of cellular phenotype to promote long-lasting vaccine-induced immunity. Similarly, di-acetylated histone H3 (diAcH3) is highly present in the expressed gene loci of activated effector CTLs, and this epigenetic mark remains present in the acquired memory cells (54). Additionally, several gene loci in naive and memory T cells remain poised in a resting state by the presence of bivalent epigenetic marks; the activating H3K4me3 and the repressive H3K27me3. This bivalency has been shown to be a crucial mechanism in regulating T cell faith, since following antigen stimulation, the activated gene loci are readily resolved into a monovalent H3K4me3 state subsequently allowing rapid differentiation into effector cells (45).

Recently, epigenetic enhancers have been shown to regulate CD8⁺ T cell differentiation in response to antigen presentation. The activation of the enhancers during differentiation was mapped based on genome-wide analysis of several epigenetic marks including H3K4me3, H3K27ac, H3K4me1 and the binding of histone acetyltransferase p300 (49, 55). These regions display striking epigenetic specificity in naive, effector, and memory T cells. Distinct transcription factors have also been shown to bind specifically to the enhancers of different subsets of CD8⁺ T cells (40). Similarly, chromatin accessibility profiles indicate unique regulatory regions in different CD8⁺ T cell subsets that also correspond to the expression of subset-specific genes (56, 57).

Furthermore, the levels of epigenetic modifier and transcription factor expression are distinct amongst T cell subsets. These may influence the capacity of T cells to react upon antigen stimulation. Indeed, the lack of DNA methyltransferase 3A (DNMT3A), a de novo methylating enzyme, promotes bias toward memory cell differentiation (58). Absence of the epigenetic modifier methyl-CpG-binding domain protein 2 (MBD2) causes impaired T cell differentiation into the effector phenotype (59). The epigenetic modifier BMI1, a reader of H3K27me3, and EZH2, a writer of H3K27me3 are both highly expressed upon T cell stimulation and differentiation into effector cells (60, 61). Histone deacetylases, SIRT1 (50) and HDAC7 (54) as well as BRD4, a reader of acetylated lysines (62) epigenetically repress gene expression and have been shown essential in directing differentiation of CD8⁺ T cells to gain their effector function.

In effector T cells, transcription factor PRDM1/Blimp1 (63), TBX21/Tbet (64, 65), and ID2 (66) are highly expressed to control CTL function via epigenetic regulations. PRDM1 for example, has been shown to recruit the repressive epigenetic modifier G9A and HDAC2 to both IL2RA and CD27 loci, promoting differentiation of CD8⁺ T cells into effector cells (51). TBX21 is necessary to induce the expression of IFNγ, granzyme B, and perforin, by inducing rapid DNA demethylation and histone acetylation at the promoters of these gene loci (67–69). Furthermore, in both naive and memory cells, EOMES (65, 70), TCF1 (71), and FOXO1 (72–75) are highly expressed and have been shown to readily promote differentiation of these cells into CTLs, although their mode of action in regulating epigenetic changes in T cells remains unexplored.

Another benefit of therapeutic cancer vaccines is their potential to revitalize exhausted T cells, by promoting de novo generation of T cell-mediated immunity (15–18). Exhausted T cells are a hallmark of cancer and the result of persistent antigen stimulation (76). They exhibit defective proliferation capacities, impaired stimulatory cytokine secretion, increased checkpoint receptor expression, and impaired effector functions (76). Recent studies show direct involvement of epigenetic mechanisms in T cell exhaustion. For example, compared to functional T cells, exhausted T cells exhibit reorganization of chromatin accessibility and activation of the exhaustion-specific enhancers (77, 78). Exhausted T cells also exhibit lower levels of diacetylated histone H3 (diAcH3) in comparison to functional T cells (79).

Both DNA methylating enzymes, DNMT1 and DNMT3B are upregulated in exhausted T cells (80), whilst DNMT3A has been demonstrated to functionally establish de novo exhaustion-specific DNA methylation patterns (81). Indeed, by blocking de novo DNA methylation, exhausted T cells retained their effector function (81). In exhausted T cells however, the expression levels of checkpoint/coinhibitory receptors, including PD1 and LAG3 were highly elevated (78, 82), which correlated with demethylation (83) and binding of transcription factor GATA3, BLIMP1, IRF4, BATF, and NFATc1 to the gene loci (51, 82, 84).

Additionally, lncRNAs including lncRNA-CD244 and lncRNA-Tim3 (85, 86) and miRNAs including miR-720, miR-31, miR-92a-3p, miR-21-5p, miR-16-5p, miR-126, and miR-182-5p (87–89) are capable of inducing exhaustion phenotypic changes by targeting specific pathways that impair T cell effector function. Therapies targeting these regulatory RNAs therefore may help restore T cell anti-tumor functions.

Therapeutic cancer vaccines are able to direct the proliferation and differentiation of naive and memory CD8⁺ T cells into CTLs through epigenetic modifications. As previously discussed, the involvement of epigenetic modifiers and transcription factors have been observed in directing T cell differentiation. This knowledge could potentially be used to improve the efficacy of therapeutic cancer vaccines.

For instance, BRD4 and SIRT1 are known to regulate differentiation of naive T cells into CTLs (50, 62). The absence of these two epigenetic modifiers promotes T cell differentiation into memory cells. Inhibition of these two epigenetic modifiers using the pharmacological inhibitor JQ1, results in the differentiation of naive CD8⁺ T cells into memory T cells that are long-lived, self-renewing and provide maintenance of acquired functional immunity, indicating that this pharmacological agent can be used to help create long-lasting immune response (62).

Another example is histone acetylation in Tbet-mediated IFNγ expression in CTLs. An HDAC inhibitor, trichostatin-A (TSA), can bypass the control of Tbet in inducing IFNγ expression (90). As IFNγ is critical for CTLs to exert their tumor killing activities, this pharmacological epigenetic modifier could potentially be used to enhance the efficacy of cancer vaccines.

Recently generation of CTLs was shown to depend on T cell receptor-mediated let-7 miRNAs downregulation. Decrease of let-7 miRNAs is necessary for the acquisition of effector function through derepression of the let-7 targets (91). On the other hand, miR-155 is necessary to generate effector CD8⁺ T cells (92). Therefore, it has been suggested since that modulation of let-7 miRNAs or miR-155 can be used to potentiate immunotherapies for cancer.

Furthermore, as previously mentioned, therapeutic vaccines can reverse systemic exhaustion by promoting de novo generation of functional T cells. This T cell exhaustion phenomenon is dependent on the host DNA methylation profile. Recently, in mice, T cell exhaustion was successfully reversed by inhibition of de novo methylation using Decitabine, an FDA-approved DNA demethylating agent (81). Moreover, as exhausted T cells overexpress checkpoint receptors that prevent them from killing tumor cells, the use of checkpoint inhibitors has proven useful to remove such molecular breaks. Thus, these pharmacological agents could potentially be used in combination with therapeutic cancer vaccines to rejuvenate exhausted T cells, whilst effectively promoting new T cell-mediated immunity.

The magnitude of T cell activation and the accompanying epigenetic modulations dictate the efficacy of a vaccine being developed. The strength of the immune response elicited by vaccines is also highly dependent on the chosen antigens. Several strategies have been recently implemented to optimize this selection. These include personalized peptide vaccines that utilize multiple cancer peptides to complement pre existing host immunity (93). Another strategy is using neoantigens, that is, antigens that arise because of mutations in tumor cell DNA. Once identified, patient's T cells are used to screen which neoantigens harness the potential for effective antitumor responses. Vaccines are then developed based on these screening results. Recently, cancer-specific epigenetic marks have been explored to be used as therapeutic target antigens in vaccines. For instance, several miRNAs have been used in cancer vaccine development (94). Such strategies may provide significant additional resources for individualized cancer treatment.

In most cancer vaccines, adjuvants and immunostimulants are chosen to facilitate generation of CD8⁺ T cell responses to MHC class I-presented tumor antigens. For this reason, the adjuvant should activate APCs such as DCs, promote antigen presentation and subsequent presentation to induce secretion of stimulatory cytokines, such as IFNγ, IL12, and IL2 (Figure 2). Adjuvants that promote cytokine production and Th1 differentiation (95) are desired as Th1 cells costimulate native CD8⁺ T cells to differentiate into CTLs (8) (Figure 2). Moreover, following the stimulation, Th1 cells produce IFNγ that in turn increase antigen presentation on cancer cells (10), to enhance direct killing of tumor cells (11) as well as create an immunogenic tumor microenvironment (96), thus further helping tumor control. Adjuvants additionally can be selected based on their ability to induce specific innate cells such as natural killer (NK) cell-mediated tumor killing. NK cells are the effector cells of the innate immune system that upon stimulation can directly lyse tumor cells via perforin and granzyme (7). They also have a main role as rapid and potent cytokine producing cells, such as IFNγ and TNFα, that stimulate killing through the death receptor pathways (7, 96). Moreover, NK cells induce DC maturation and amplify T cell anti-tumor responses (97).

One of the main antigen recognition and activation pathways utilized by APCs are TLRs. TLRs are receptors expressed by APCs that can recognize conserved structures derived from pathogens, namely MAMPs (microbe-associated molecular patterns). The same receptors can also recognize DAMPs (damage-associated molecular patterns) that are expressed by cells under conditions of stress. TLR ligands/agonists are widely used to stimulate innate immune responses. TLR agonists, especially those targeting endosomal TLRs, have been shown to generate anti-tumor immunity (98). Thus, cancer vaccines targeting TLR activation could result in the generation of a range of cytokines that stimulate a Th1 bias, as well as promote CTL induction and NK cell-mediated killing that can then be utilized for directed tumor treatment strategies (99).

TLR3, TLR7, TLR8, and TLR9 are predominantly endosomal. It is known that different subsets of DCs have been shown to express distinct arrays of TLRs (100). TLR3 for example is predominantly expressed in conventional DCs (101). This subset of DCs are especially efficient in activating CD8⁺ T cells and inducing adaptive immune responses against tumor cells (100). Additionally, several cancer cells have been shown to express TLR3 at various levels, including hepatocellular carcinoma (102), breast cancer (103), and neuroblastoma (104). Polyinosinic:polycytidylic acid (Poly I:C) and polyadenylic:polyuridylic acid (Poly A:U) are synthetic analogs of viral dsRNAs which are recognized by TLR3 (105) that have been extensively used as an adjuvant in many clinical trials for cancer vaccines (106). The agonists of TLR3 are capable of activating APCs and cancer cells to induce secretion of inflammatory cytokines including type1 interferons that in turn activate T cell responses against cancer cells (107, 108). Poly I:C is also capable of reversing the pro-cancer innate immune response to anti-cancer immunity, especially within the tumor microenvironment (109). In clinical trials, albeit with limited numbers of patients, both Poly ICLC and Poly I:C12C, modified versions of Poly I:C, were shown to boost anti-tumor activity by inducing potent tumor-specific CTL and NK responses (110, 111).

TLR8 is expressed by conventional DCs and monocytes, whereas TLR7 is expressed predominantly in plasmacytoid DCs (101). Plasmacytoid DCs are a major producer of stimulatory cytokines in response to many viral infections (100). The ligands of TLR7 and TLR8 have been exploited as adjuvants. Their receptors are similar in structure but promote secretion of distinct sets of proinflammatory cytokines by APCs. TLR7 induces the secretion of type I interferons such as IFNα, while TLR8 promotes secretion of TNFα and IL12 (112). Both receptors are endosomal and recognize viral ssRNA (105) and also bind their synthetic analogs, including imiquimod and resiquimod (113, 114). In clinical studies, TLR7/8 agonists enhanced CD8⁺ T cell responses of a vaccine to prostate-specific peptide and NY-ESO-1, an tumor-specific antigen (115). Additionally, imiquimod has been approved for the treatment of basal cell carcinoma by the FDA (116).

TLR9 agonists are also potent adjuvants. TLR9 itself is predominantly endosomal, and present abundantly in DCs, especially plasmacytoid DCs. It binds microbial DNA, recognizing in particular the unmethylated CpG motifs in viral and bacterial genomes (105). The synthetic TLR9 ligand, CpG oligodeoxynucleotide (CpG-ODN), a short unmethylated ssDNA, activates DCs to secrete type I interferons, and promotes a strong CTL response (117). When used in combination with DC-based cancer vaccines, CpG-ODN enhances CD8⁺ T cell activity. In combination with tumor-specific-peptide-based vaccines, such as NY-ESO-1 and MART1, CpG-ODN resulted in elevated CD8⁺ T cell responses, however tumor eradication was rarely achieved (115).

Unlike their endosomal counterpars, TLRs expressed on the cell surface typically recognize extracellular foreign microbes. TLR4, one of the surface TLRs, recognizes LPS molecules of gram-negative bacteria (105). In humans, LPS can cause septic shock syndrome, due to its potent immune stimulatory activity (118). A derivative of LPS, monophosphoryl lipid A (MPL) in combination with the classical adjuvant alum, was licensed by the FDA for use as part of the human papillomavirus vaccine in 2009 (119). In clinical trials, MPL has also been used as an adjuvant for cancer vaccines to promote Th1-specific immune responses (120, 121).

Other adjuvants that have been used to induce T cell responses have included classic formulations/emulsions including oil or saponin. QS-21 is a potent saponin-based adjuvant that is isolated from Quillaja Saponaria (122). Although its mechanism of action is largely unknown, QS-21 has been shown to activate the secretion of IL2 and IFNγ, stimulate the proliferation of CTLs and induce Th1 bias (123). Formulations of QS-21 has been tested in human clinical trials for various cancer vaccines (124, 125). Another strong adjuvant that has been trialed for cancer vaccines is Montanide. The aim of this classical adjuvants is to allow sustained antigen release from the immunization site. This strategy is used to create a prolonged and higher amplitude of CTL-mediated immune response. Montanide-based adjuvants are water-in-oil emulsions that promote slow release of antigens and thus prolong antigen presentation to the immune system (126). In clinical trials, montanide ISA720 and ISA51 promote Th1 immune responses and significant CTL activation (127, 128).

Several delivery systems, including virosomes, liposomes, viral-like proteins (VLPs), and immune-stimulating complexes (ISCOMs) have been developed and used in clinical trials to improve the efficacy of cancer vaccines. Virosomes are empty viral particles that can carry tumor-specific antigens as vaccines (129). In metastatic breast cancer patients, the modified influenza virosomes containing the breast cancer peptide (Her/neu⁺) are well tolerated and not only promote secretion of proinflammatory cytokines, including IL2, TNFα and IFNγ but also promote T cell immunity (130). Liposomes are synthetic phospholipid vesicles that work similarly to virosomes. They are often used to deliver messenger RNA (mRNA) encoding for a specific antigen (131). They have shown promise in delivering mRNA to APCs in clinical trials for non-small cell lung cancer, prostate cancer and follicular lymphoma patients (132, 133), and inducing antigen-specific CD8⁺ T cell responses. Liposomes that carry DNA have also been developed to stimulate TLR9, activate DCs and subsequently CTLs (134). VLPs are multimeric structures of viral proteins devoid of viral genetic material. Similar to native viruses, specific epitopes on VLPs can be recognized and presented by APCs to promote immune responses as reviewed in Ong et al. (135). VLPs have been developed for use in vaccines for various forms of cancer, including liver, cervix, lung, skin, breast, and prostate, as they not only promote antigen-specific immunity, but also counteract the immunosuppressive microenvironment created by a tumor mass (135). Finally, ISCOMs are composed of saponin, cholesterol and phospholipid. They are regularly used as a vaccine delivery system, however saponin can also stimulate the immune system by activating DCs and inducing robust antigen-specific T cell responses (136). Furthermore, ISCOMATRIX® has been used with the recombinant NY-ESO-1 protein in cancer patients to induce T cell immune responses (137, 138), however this vaccine failed to promote an adequate immune response in advanced melanoma patients (139).

Whereas a number of whole-pathogen-based vaccines against infectious diseases have been shown to modulate the epigenetic landscape of immune cells, much less is known about the adjuvants and carriers used in cancer vaccines and patients. For example, vaccination with the bacillus Calmette-Guérin (BCG) vaccine for tuberculosis, has been shown to specifically alter epigenetic profiles of monocytes and broadly enhance protection against multiple infectious pathogens (beyond tuberculosis) in humans (140). This suggests that vaccines could leave stable epigenetic marks in certain immune cell populations and alter how the immune system reacts toward subsequent diverse challenges after vaccination, perhaps including cancer. In fact, the non-specific beneficial effects of the BCG vaccine are used in the clinic to treat bladder cancer (141). Modulation of T cell immunity using vaccines in combination with specific adjuvants may provoke changes in epigenetic profiles of immune cells and improve anti-tumor immunity. These beneficial epigenetic profiles may be further potentiated by the use of epigenetic modulating drugs. Indeed, epigenetic potentiation of the NY-ESO-1 protein vaccine with montanide-based adjuvant using decitabine, a DNMT inhibitor, has been successful in treating epithelial ovarian cancer (28).

Several adjuvants currently used in cancer vaccines are indeed capable of altering immune cell interactions with cancer cells by inducing stable epigenetic modifications in both host immune and cancer cells. These adjuvants could therefore be harnessed as promising candidates to promote beneficial epigenetic modulations in vaccine-based therapies. The use of TLR-ligand adjuvants could indeed be promising, as studies have shown that epigenetic reprogramming can be achieved via TLR stimulation. For instance, stimulating TLR3 with Poly I:C activates the epigenetic machinery causing a global change in the expression of epigenetic modifiers that in turn promotes chromatin remodeling and nuclear reprogramming (142). In addition, TLR3 receptor combined with Poly I:C directly promoted global DNA methylation in peripheral blood mononuclear cells (PBMCs) in pigs (143). Poly I:C promotes the expression of pro-inflammatory cytokines IL23 and IL33 by direct modification of the epigenetic marks on the promoters of these gene loci (144, 145). Furthermore, it reactivates the expression of several silenced miRNAs in tumor cells that subsequently leads to its direct anti-tumor activity (146). Such direct epigenetic modifications by Poly I:C are highly beneficial to improve the efficacy of therapeutic cancer vaccines.

Another advantageous cancer vaccine adjuvant candidate could be CpG-ODN, a ligand for TLR-9. Although there is less data available regarding the effects of CpG-ODN on global epigenetic reprogramming, it has been shown to promote chromatin changes in specific gene loci. For example CpG DNA induced production of IL12 due to its ability to elicit epigenetic modifications on the IL12p40 promotor, including histone acetylation and nucleosomal remodeling, which leads to gene activation (147). In cancer cells, CpG-ODN has been shown to directly exert its anticancer potential. For instance, in chronic lymphocytic leukemia (CLL), CpG-ODN promotes epigenetic changes associated with active transcription, namely, H3K9/K14 acetylation and H3K4 trimethylation at the promoter of PRDM1 (148). PRDM1 expression promotes terminal differentiation of CLLs (149, 150), which is established as a potent therapy for CLL.

Epigenetic-modulating activites of TLR4 ligand adjuvants may mimic those exerted by LPS. This classical TLR4 ligand promotes innate immune responses by reprogramming monocytes to accumulate active histone marks such as H4Ac, at promoters of genes involved in inflammation and phagocytic pathways (151). However, further stimulation of innate immune cells by LPS can promote tolerance, by removal of H4Ac at promoters of inflammatory gene loci, such as IL6 and TNFα (152, 153). It was further identified that Trichostatin A, a deacetylase inhibitor could reverse the repression of IL6 and restore H4Ac (152).

Additionally, adjuvants that deliver genetic materials can also potentially be used to promote beneficial epigenetic modulations during vaccine-based cancer therapies. RNA-LPX, a liposome-based adjuvant for example, has been shown to efficiently target DCs and promote strong antigen-specific T cell responses in melanoma patients (131). Since the expression of many miRNAs are altered in various cancer cells, such form of adjuvant could potentially be used to target miRNAs to both alter epigenetic imprinting in the cancer cells and promote cancer elimination.

Despite progression in the knowledge of adjuvants for cancer therapy, their mode of action and the precise epigenetic mechanisms involved are still largely unmapped. As discussed earlier, all types of adjuvants may exert direct and indirect effects, which might result in epigenetic modifications in the cells of the immune system and the associated cancer cells. The accumulating evidence highlighted above provides a rationale to investigate more broadly the potential use of epigenetic modifications by vaccine-adjuvants in the context of cancer therapy.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.