Optimizing Dendritic Cell-Based Approaches for Cancer Immunotherapy

Dendritic cells (DC) are professional antigen-presenting cells uniquely suited for cancer immunotherapy. They induce primary immune responses, potentiate the effector functions of previously primed T-lymphocytes, and orchestrate communication between innate and adaptive immunity. The remarkable diversity of cytokine activation regimens, DC maturation states, and antigen-loading strategies employed in current DC-based vaccine design reflect an evolving, but incomplete, understanding of optimal DC immunobiology. In the clinical realm, existing DC-based cancer immunotherapy efforts have yielded encouraging but inconsistent results. Despite recent U.S. Federal and Drug Administration (FDA) approval of DC-based sipuleucel-T for metastatic castration-resistant prostate cancer, clinically effective DC immunotherapy as monotherapy for a majority of tumors remains a distant goal. Recent work has identified strategies that may allow for more potent “next-generation” DC vaccines. Additionally, multimodality approaches incorporating DC-based immunotherapy may improve clinical outcomes.


INTRODUCTION
As professional antigen-presenting cells (APC), dendritic cells (DC) function at the interface of the innate and adaptive immune systems. DCs serve as sentinel members of the innate immune arm, responding to "danger" signals by elaborating protective cytokines (e.g., IL-6, IL-12). In their role as master APCs, DCs induce adaptive responses by processing and presenting antigens to naïve T-lymphocytes at lymphoid organs in the context of major histocompatibility (MHC) molecules [1]. Due to these bridging functions, significant effort has been invested in targeting DCs directly or indirectly for the induction of tumor-specific immune responses in cancer patients.
Following the initial promise of DCbased vaccines in lymphoma and melanoma patients in the 1990s [2,3], autologous DCs have been employed in immunotherapy for several tumor types, including prostate cancer (PC), malignant glioma, and renal cell carcinoma (RCC) with varying success [4]. FDA approval of sipuleucel-T -prostatic acid phosphatase GM-CSF fusion proteinpulsed blood DCs -for metastatic castration-resistant PC [5] has energized efforts to replicate and improve upon the success of such DC-based strategies in other tumor types. However, clinically effective DCbased immunotherapy as monotherapy for a majority of tumors remains a distant goal.
In this review, we summarize the rationale for recruiting DCs in immunotherapy, discuss relevant DC immunobiology, critically evaluate DC-based vaccine design and clinical efficacy of DC vaccines in human trials, and explore multimodality strategies to optimize the benefit of current DC-based immunotherapy.

Effective APCs
DCs are "master" APCs. Their potency for inducing T-cell proliferation is 10 to 100 times that of B-cells or monocytes [6,7].
DCs sensitize antigen-specific responses in both CD4 + [8] and CD8 + T-cells [9]. CD8 + T-cells differentiate into cytotoxic T-lymphocytes (CTLs) and contribute to direct tumoricidality. CD8 + T-cell memory is more effectively maintained when stimulated by DCs [6]. While CD8 + T-cells have historically been valued as the primary effectors of anticancer immunity, increasing evidence supports the importance of CD4 + T-cell help in potentiating CTL responses [10], facilitating immunologic memory and displaying direct cytotoxicity of their own [11].

Cross-Presentation
DCs induce CD8 + T-cell responses, in part, due to their ability to cross-presentre-route exogenous antigens, typically presented on MHC class II molecules, into pathways for class I presentation [6]. Although evidence initially suggested that certain activated human blood DC subsets (i.e., CD141 + /BDCA3 + ) are specialized for crosspresentation [12], emerging data indicate that all lymphoid organ-resident DCs (CD141 + /BDCA3 + , CD1c + /BDCA1 + , or plasmacytoid) cross-present efficiently [13]. Two intracellular pathways are utilized for cross-presentation: a) cytosolic (proteasome-dependent), whereby internalized proteins escape intracellular trafficking and are transported to endoplasmic reticulum by TAP1/2 transporters for class I loading; and b) vacuolar (proteasome/TAP-independent), wherein exogenous antigens are degraded in endocytic compartments by lysosomal proteases, cathepsins, or IRAP, and loaded onto class I molecules [14]. Augmentation of cross-presentation is increasingly utilized in DC-based vaccine design [15].

Impact on Humoral Immunity
The role of the humoral system in antitumor immunity is increasingly appreciated, and several mechanisms via which antibodies mediate these effects have been elucidated. These include interfering or altering transmembrane signaling, inducing antibodydependent cellular cytotoxicity, or participating in complement-mediated cytotoxicity [16]. DCs indirectly facilitate humoral im-munity by activating follicular CD4 + T-helper (Th) cells, which contribute to germinal center formation and regulate differentiation of B-cells into plasma cells and memory B-cells [17]. DCs directly influence differentiation and survival of B-cells, generation of antibody-secreting plasma cells, and stimulation of memory B-cells via subset-specific cytokine production [18]. Moreover, using a non-degradative intracellular pathway via FcγRIIB, DCs directly present antigen to Bcell receptors, resulting in antibody production [19,20].
DCs can potentiate antitumor humoral immune effects. In a BALB neuT-transgenic murine model, vaccination with bone marrow-derived DCs modified by a recombinant adenovirus-expressing truncated neu oncoprotein (DCAd.Neu) induced potent serum antineu antibodies and IFN-γ secretion by CD4 + and CD8 + T-cells. More importantly, DCAd.Neu prevented autochthonous breast cancers and inhibited growth of transplantable neu-expressing breast cancers [21]. In a separate study, HER2-expressing recombinant adenoviral vaccination alone in the BALB neuTtransgenic model also induced anti-neu antibodies, which were both necessary and sufficient for antitumor protection. Antibody effectiveness was also subtype-dependent, with IgG2a being most effective [22].

Induction of Natural Killer (NK) and NK T-Cell (NKT) Responses
DCs favorably condition the tumor microenvironment (TME) via their interactions with NK and NKT-cells. DCs attract NK cells to the TME by secreting CXCR3 ligands, thereby stimulating NK effector functions [23]. Once NKs are recruited, interactions between NKs and DCs reciprocally enhance antitumor immunity. NK cells can induce DC activation, facilitate DC maturation to a type 1-polarizing phenotype (DC1), and edit DCs by eliminating tolerogenic subtypes [24].
While NKT-cells mediate direct tumor lysis, their antitumor effects depend in large part on their ability to activate NK cells and DCs [25]. Targeting NKT-DC interactions have clinical implications: Activating NKT cells with α-galactosylceramide-loaded DCs (with low-dose lenalidomide) resulted in clinical regression and broad immune activation in myeloma [26].

Direct DC Tumoricidality
Evidence supports DCs' capacity for direct antitumor cytotoxicity [27]. This is achieved when DCs take up apoptotic tumor cells and present tumor antigens to other effector elements, thereby eliciting a tumorspecific immune response.

DC IMMUNOBIOLOGY
DC-based vaccines differ from conventional (peptide, protein, DNA) vaccines in that a dynamic component of the immune system is harnessed to affect immunization [16]. DCs are governed by a pre-programmed life cycle as well as a range of constitutive and inducible functions that have been exploited for vaccine development. This section briefly explores the immunobiology of DCs pertinent to their use in immunotherapy.

DC Activation and Function
DCs primarily exist in immature (nonactivated) and mature (activated) states. Immature DCs (iDC) are responsible for capture, transport, and processing of antigens [28] while awaiting infectious/inflammatory signals, which commences maturation. Upon maturation, DCs lose their phagocytic and antigen-processing capabilities [28,29] and upregulate chemokine receptors, allowing migration to sites of eventual activity [30]. The ability of DCs to induce T-cell responses is augmented in a number of ways: increased expression of surface MHC [31,32] and costimulatory [33] molecules and elaboration of soluble factors that influence polarization of the ensuing immune response [34,35].

DC Subsets and Plasticity
Two major subsets of DCs are described: classical (cDC; myeloid or mDC) and plasmacytoid (pDC) DCs. cDCs have historically been distinguished from pDCs on the basis of CD11c expression [36] and myeloid markers [37]. cDCs highly express class II molecules and are efficient at inducing T-cell prolifera-tion [38]. Although cDCs are referred to as lymphoid-organ "resident" due to their frequent occurrence in the thymus, spleen, and lymph nodes, a subpopulation was discovered in circulating blood and are termed migratory DCs [38]. Migratory DCs are further subdivided on the basis of reciprocal CD141/BDCA3 and CD1c/BDCA1 expression [1]. CD1c + /BDCA1 + DCs are predominantly found in the blood compartment, are similar to murine CD11b + DCs, and are potent activators of CD4 + T-cells [1]. Human CD141 + /BDCA3 + DCs are similar to murine CD8α + DCs in their ability to generate robust CD8 + T-cell responses and cross-present exogenous antigens on MHC class I [12,39]. pDCs differ from cDCs by virtue of CD303 + and CD11cstatus, low class II expression, and relatively poor ability to stimulate T-cells [36]. Despite these shortcomings, pDCs' ability to respond to viral infections, via increased Toll-like receptor (TLR)-7/-9 expression and vigorous IFN-α/β production [40,41], may be harnessed in DC vaccine design.
Classification schemes of DC lineage have proven remarkably complex to define. Early classification attempts based on surface marker expression or transcription factors assumed that myeloid-and lymphoid-derived progenitor populations developed into distinct DC subsets [42][43][44][45][46]. However, there is substantial plasticity in these populations; it is now clear that both myeloid-and lymphoid-derived progenitors can develop into cDC/mDC or pDC subsets via intermediary progenitors under the influence of Flt3 ligand [38,47,48]. This plasticity has been exploited by various activation protocols in DC vaccine design [6].

DC Activation via TLR Signaling
Although the innate immune system is considered more non-specific compared to the adaptive system, it possesses the ability to respond to countless microbial threats while discriminating them from self-antigens [49]. This was explained by the discovery of receptors such as TLRs on immune cells that recognized molecular patterns common to many pathogens, termed pathogen-associated molecular patterns [49]. Eleven TLRs with homology to invertebrate counterparts have been identified in humans [50]. Examples include TLR2 (ligand: lipoteichoic acid [LTA]); TLR3 (double-stranded RNA); TLR4 (lipopolysaccharide [LPS]); TLR7/8 (singlestranded RNA); and TLR9 (unmethylated CpG-containing DNA) [51]. TLRs initiate downstream molecular events by recruiting MyD88 and TRAF6, thereby inducing expression of cytokine genes relevant to inflammation via two disparate pathways: a) NF-κB and AP-1 (involving the canonical IκB-kinase complex [IKK-α, IKK-β, and IKK-γ]); and b) MAP kinases (including ERK, JNK, p38) [52].
TLRs are found on various DC subsets and direct their respective immunogenic capacities. For instance, pDCs (expressing TLR7/9) and cDC/mDCs (TLR8) respond to certain infections but not others [53]. Furthermore, different signaling pathways are activated depending on the TLR(s) stimulated and adaptor proteins involved. For example, simultaneous activation of MyD88-dependent and MyD88-independent (TRIF-mediated) pathways synergizes to generate DC phenotypes that secrete IL-12 more potently than when either pathway is activated individually [54]. Finally, TLR-primed DCs induce antigen-specific high-avidity CD8 + [55,56] and type 1-polarized CD4 + Th (Th1) responses [57], providing a compelling rationale for TLR agonism in DC-based immunization.

DC1-Mediated Th1
The plasticity of DC lineage and influence of external signals impacts DC maturation to disparate phenotypes. In turn, these phenotypes polarize the immune response (i.e., induce diverse Th subsets) via cytokine elaboration [6]. The DC1 phenotype, so named because it induces CD4 + Th1 immunity [58], is the primary phenotype utilized in vaccination for several reasons. DC1-secreted IL-12p70 polarizes naïve CD4 + T-cells to IFN-γ-secreting Th1; IFN-γ is critically important for tumor rejection in a number of models [59]. Importantly, Th1-driven CTLs detect class I-tumor antigen complexes with higher affinity than Th2-driven counterparts [60]. Finally, Th1 subsets are instrumental in B-cell responses by inducing antibody classswitching and IgG production [6]. IL-12p70 contributes to host anti-tumor responses by promoting NK cell activation [61] and displaying anti-angiogenic properties by hindering tumor neovascularization [62]. In our studies, CD8 + T-cells could only recognize HLA-A2 pos cancer cells if the sensitizing DCs secreted IL-12p70. Moreover, IL-12p70-sensitized T-cells recognized antigen at significantly lower concentrations during recall responses compared with non-IL-12p70-sen-sitized T-cells [55]. Taken together, incorporation of IL-12p70-producing DC1 in vaccine design appears warranted.

DC2-Mediated Th2
DCs that promote CD4 + Th type-2 (Th2) differentiation are referred to as DC2. Cytokines that favor production of Th2 subsets include IL-4 and anti-IFN-γ; Th2, in turn, produce IL-4, IL-5, IL-6, and IL-10 [6]. Th2, primarily involved in promoting allergic reactions, defending against parasitic infection, and inducing B-cell differentiation, are considered less effective than their Th1 counter- Antigen-specific T-cell responses can be induced by traditional ex vivo-manipulated DCs or DC receptor targeting in vivo. In ex vivo manipulation, monocyte or CD34 + precursors are sequentially matured with proinflammatory cytokines, loaded with antigen, and injected (either IN or ID/SC). Liposomes or nanoparticles comprising monoclonal DC receptor-targeting antibody-antigen chimera (± adjuvant) can also be delivered to lymph nodes via IV routes. Within the lymph node, DCs present antigen to CD8 + /CD4 + T-cells in the context of MHC Class I/II molecules, triggering antigen-specific CTLs. Natural DC subsets, such as mDCs and pDCs, can be targeted with DC targeting liposomes/nanoparticles, which in turn stimulate antigen-specific CD8 + /CD4 + T-cell production. Cross-talk between DC-delivering mechanisms can induce other immune cells, such as NK cells. These effector populations migrate to the tumor bed, where they directly attack tumor cells. Multimodality optimization of DC immunotherapy involves preventing antigen-specific CTL exhaustion and depleting tumor-elaborated Treg and MDSCs. Anti-CTLA-4 and anti-PD-1 mAb are immunostimulatory therapies aimed at recovering T-cell cytotoxicity. A variety of agents, including IL-2, chemotherapy, and TKIs can mute Treg/MDSCs. COX-2 inhibitors, ATRA, and vitamin D3 can specifically target MDSCs, whereas anti-CD25 mAbs and denileukin diftitox target CD25 on Treg. Non-CD25-based alternatives, such as 1-MT, inhibit Treg-generated IDO. parts in combating cancer. While earlier reports posited an antitumor effect for Th2 [63], more recent evidence suggests that Th2 may be pro-tumorigenic. In a murine mammary carcinoma model, DC2-derived Th2 facilitated tumor development by generation of IL-4 and IL-13 [64]. In another study, IL-4-expressing Th2 promoted pulmonary metastasis of mammary carcinoma by enhancing TGF-β and EGF production by tumor-associated macrophages [65]. These data have engendered a bias against the use of DC2 in vaccine design.

DC-BASED VACCINE DESIGN STRATEGIES
Given the diverse anticancer armamentarium offered by DC immunobiology, an intense search for the optimal vaccine construction strategy has emerged in recent years. Two strategies ( Figure 1) are widely accepted: a) direct targeting of antigens to DC receptors in vivo; and b) ex vivo-generated antigen-loaded DCs.

DC-Targeting In Vivo
In this approach, chimeric antigen-antibody complexes targeted to DC surface molecules are internalized into endosomic compartments for MHC loading and stimulation of T-cell responses [76]. While a comprehensive review of DC receptors is presented elsewhere [76,77], a discussion of the most promising molecules follows.
A few salient features of in vivo DC-targeting deserve mention. First, antigen must be delivered to mature/activated DCs, since antigen presentation by immature DCs induces tolerance rather than immunity [18]. Vaccine constructs must rely on the retained ability of mature DCs to present antigen taken up via endocytic receptors [90]. Functionally, this is achieved by accompanying antigen-mAb conjugates with molecular adjuvants (e.g., anti-CD40 mAb, poly-I:C, CpG) [77]. Second, targeting receptors on distinct DC subsets may bias the immunologic outcome. CD8α + DCs targeted using anti-DEC-205ovalbumin (OVA) favored the generation of CD8 + immunity, whereas CD8α -DCs targeted using anti-DCIR-OVA or anti-Dectin-1-OVA more efficiently induced CD4 + immunity [91]. Third, engaging different DC receptors may deliver distinct signals to the same DC, polarizing their function accordingly. Targeting DC-ASGPR (without adjuvant) generated IL-10-secreting regulatory T-cells, whereas targeting DCs with anti-LOX1 mAb resulted in Th1 polarization [92]. Fourth, the choice of adjuvant may be critical in skewing immune responses to a desired phenotype. Potent Th1 responses were induced by anti-Clec9A-OVA mAb delivered to CD8α + DCs in the presence of poly-I:C (TLR3 agonist). When curdlan (β-glucan) was used as adjuvant, immunization with anti-Clec9A induced Th17 responses [93]. Finally, the ubiquitous expression of some molecules (e.g., DEC-205, MR) on non-DC APCs (monocytes, B-cells) may dissipate the antigen-targeting efficiency of this approach. Given these complexities, and the uncertainty associated with translating preclinical evidence into clinical success, moving in vivo DC-targeting to human trials requires considerable work yet.

Ex Vivo-Generated Antigen-Loaded DCs
Pioneering studies describing the ability to culture murine DCs ex vivo from bone marrow precursors galvanized DC vaccine development in the 1990s [94]. Human applications followed soon thereafter. Human DCs could be generated from peripheral blood-derived monocytes or CD34 + hematopoietic progenitors [95]. Ex vivo-manipulated autologous DCs could be expanded, loaded with antigen, and administered back to patients to generate antitumor immunity [1]. This approach had two ostensible advantages: a) bypassing endogenous DC dysfunction in cancer-bearing patients; and b) streamlining immune responses to recognize and eliminate tumor cells in an antigen-specific fashion.
Although a majority of clinical trials have utilized ex vivo-generated DC vaccines, several controversies linger. First, the maturation state of DCs has been a matter of debate. Despite moderate clinical benefit in trials using IL-4-immature DCs [4], a metaanalysis revealed a significant association between DC maturation and improved clinical responses in PC [96]. These findings have been reproduced in melanoma [97] and glioblastoma [98]. These disparities in clinical efficacy reflect lessons learned in the laboratory. By virtue of their low co-stimulatory and class II molecule expression (and intermediate class I expression), iDCs induce suboptimal T-cell priming and generate T-cell tolerance. Fully activated DCs (e.g., matured with TLR agonists) can abrogate such tolerance [6]. Furthermore, maturation can be performed rapidly in 2 to 3 day protocols (versus ≥1 week) [99]. These "rapid-activation" systems obviate longer culture times without compromising DC functionality [16].
Third, the ideal strategy for DC antigenloading is not universally agreed upon. The most common approach has been loading with tumor-associated peptides or whole recombinant tumor proteins [1]. While these non-mutated self-antigens may break self-tolerance at the cellular level, they rarely do so at the host level [108], accounting for disappointing clinical results. Putative reasons for this phenomenon include negative selection of high-avidity clones with ensuing self-tolerance to low-avidity clones [109] and maintenance of host level self-tolerance via pre-programmed immunosuppressive elements (e.g., Treg) [108]. Strategies have been proposed to overcome these pitfalls: a) co-administration of TLR agonists, as discussed earlier [6]; b) silencing of antigen presentation "attenuators" (e.g., SOCS1) may enhance in vivo DC function by augmenting immunogenicity [108]; c) using homologous xenogeneic (e.g., murine) antigens to break self-tolerance [110]; and d) engineering DCs loaded with mutated neo-antigens from patient-specific tumors, which may activate a Tcell repertoire without pre-programmed Treg [111]. Other modalities of DC loading (engineered fusion proteins, autologous/allogeneic tumor cells, tumor cell-lysate, DC-tumor hybrids, and DNA-or mRNA-transfected) have emerged and are reviewed elsewhere [99].
Fourth, the optimal route for DC administration remains controversial. Historically, with intradermal/subcutaneous injection techniques, DC trafficking to regional lymph nodes was considered critically important to their function. Indeed, maturation cocktails (e.g., PGE2-containing) were designed to optimize trafficking ability [112]. The growing popularity of IFNγ/LPS maturation regimens, with their incident lack of CCR7/CXCR-4 ("trafficking" chemokines) expression [99], dictated a search for alternative techniques to over-come this limitation. Ultrasound-guided intranodal injection, which co-localizes DC1derived IL-12p70 "burst" with the anatomic site of T-cell sensitization, has emerged as a feasible solution [113].
Finally, the opportunity for DC vaccination in early disease settings remains underexplored, with most trials focusing on locally advanced/metastatic settings [4]. Immunization in early disease or reducedtumor states may circumvent tumor-and patient-induced immune dysfunction inher-ent in advanced disease settings [6]. Indeed, our group has conducted phase I/II neoadjuvant HER2/neu-pulsed autologous DC1 trials in early (Stage I) HER2 pos -invasive breast cancer and HER2 pos -ductal carcinoma in situ (DCIS) with encouraging results. In the initial phase I study, 5/27 (18.5 percent) subjects had no evidence of residual DCIS (complete response [CR]) at surgery, with a substantial loss of target antigen in the remainder of patients [114]. In the subsequent phase I/II study, nearly 25 percent of Table 1 In another study, a Wilms' tumor-1 (WT1) mRNA-electroporated DC vaccine was effective in acute myeloid leukemia patients with minimal residual disease, but not in those with relapsed or progressive disease [115]. Finally, ongoing trials in resected glioma (NCT00045968) and RCC (NCT01582672) are exploiting reduced-tumor states to evaluate DC vaccine efficacy [4].

CLINICAL EFFICACY OF DC-BASED IMMUNOTHERAPY
As mentioned earlier, DC-based immunotherapy has produced inconsistent clinical results. In this section, the clinical efficacy of "traditional" ex vivo antigenloaded DC immunotherapy is summarized.
In a pilot study, four B-cell lymphoma patients administered autologous idiotypespecific DCs demonstrated promising clinical responses [2]. Soon thereafter, a randomized phase III trial comparing autologous DC vaccination with dacarbazine as first-line therapy for advanced melanoma was initiated but closed prematurely due to the lack of meaningful objective response rates (ORR) on interim analysis [3]. Its failure was attributed to several factors, including suboptimal DC maturation, inadequate dosage, and subcutaneous route of injection. Despite this setback, DCbased approaches have been adopted for a wide range of tumor types, including PC, lung cancer, melanoma, RCC, non-Hodgkin's lymphoma, breast cancer, and malignant glioma ( Table 1, Supplement). Encouragingly, the FDA recently approved sipuleucel-T -autologous APCs (including DCs) activated with prostatic acid phosphatase-GM-CSF fusion protein -for metastatic castration-resistant PC. In two placebo-controlled randomized trials, sipuleucel-T prolonged overall survival (OS) by nearly four months compared with placebo, with a 22 percent relative mortality risk reduction [5,116].
Clinical experience with DC-based vaccination underscores a few distinct advantages. First, DC immunotherapy is safe; injection site reaction, fever, and fatigue are the most commonly reported adverse effects in phase I trials; systemic grade 3-4 toxicity is rare [4]. Additionally, concerns regarding immunotherapy-induced autoimmunity appear less worrisome for DC-based approaches compared with mAb (e.g., anti-CTLA-4) or cytokine (e.g., IL-2) therapies [4,114]. Moreover, DC therapies are expected to adequately preserve quality of life in immunized patients [117]. Finally, regardless of the immune monitoring technique (ELISPOT, tetramer assays, skin biopsy of delayed-type hypersensitivity reactions), a majority of DC-based trials indicate that this approach is highly immunogenic, even in advanced malignancy [4]. In metastatic PC and RCC, antigen-specific immunity was induced in 77 percent and 61 percent of patients, respectively [96]. DCs' immunogenicity in early disease settings is more readily discernible. Our phase I/II HER2-pulsed DC1 trial demonstrated durable (up to 48 months post-immunization) anti-HER2 Th1 immunity in a majority of HER2 pos -DCIS patients [114].
While tolerable and immunogenic, DCbased approaches have been criticized for their disappointing ORRs (partial/complete response by WHO/RECIST criteria) [118]; a recent systematic review concluded that ORR in melanoma, PC, glioma, and RCC were 8.5 percent, 7.1 percent, 15.6 percent, and 11.5 percent, respectively [4]. A more longitudinal endpoint of treatment efficacy (e.g., OS) may be more appropriate than measuring short-term tumor regression. This paradigm is exemplified by the IMPACT trial, wherein a survival benefit for sipuleucel-T was observed over placebo despite its lack of improvement in time to biochemical failure or progression-free survival [5]. OS is increasingly utilized as an end-point in newer DC-based vaccine trials (Supplement).
This discordance between robust immunogenicity, poor ORR, and measurable survival benefit seen with DC-based approaches can be explained by the fact that immune potentiation against advanced cancer is an indolent, not immediate, process. Furthermore, an initial increase in radi-ographic tumor burden, which would be considered progressive disease by RECIST, often reflects a protective vaccine-induced peritumoral immune infiltrate [119]. As such, tumor regression following immunotherapy has been documented even after initial progression or appearance of new lesions [120]. In an effort to capture these atypical response patterns, novel surrogates for vaccine-induced efficacy were proposed -so-called immune-related response criteria (IRRC). IRRC includes index as well as new lesion(s) in measuring tumor burden and emphasizes the need for longitudinal surveillance to confirm progression [121]. Adoption of IRRC-defined endpoints in future clinical trials may uncover the true merit of DC-based vaccination.

OPTIMIZING DC-BASED APPROACHES
The maximal benefit of DC-based immunotherapy may be realized in combinatorial approaches with other anticancer therapies that synergistically enhance DC function. This section will illustrate the rationale for such approaches (Table 2, Figure  1).

"Next Generation" DCs
Our growing understanding of DC biology sheds light on strategies to optimize vaccine efficacy. First, exploiting the diversity of DC lineage may prove advantageous. For instance, although pDCs have a proclivity toward Th2 polarization, their ability to produce IFN-α/β during viral infection can activate other DCs, augment T-cell crosspriming, and generate potent CTL responses [122]. CD141 + /BDCA3 + DCs, when activated by poly-I:C, produce abundant amounts of IL-12p70 and IFN-β, excel at antigen cross-presentation, and result in stronger Th1 induction than CD1c + DCs [123]. Second, manipulating ex vivo culture conditions may generate more immunogenic DCs. Langerhans cell-type DCs -derived from CD34 + progenitors or IL-15-monocytes [124] -are more efficient at priming antigen-specific CTLs than GM-CSF/IL-4-DCs [125]. Trials employing Langerhans-type DCs are under way in melanoma [4]. Third, modified expression of co-stimulatory molecules could enhance DC potency. CD40L overexpression in murine DCs via mRNA-electroporation enhanced B7/IL-12p70 production, critical for Th1 immunity. Moreover, mRNA-electroporated DCs encoding CD40L, CD70, and TLR4 generated durable tumor responses in chemorefractory metastatic melanoma [126].

Muting Immunosuppressive Phenotypes
Tumors create immunosuppressive networks (Treg, MDSCs) that mediate escape from immune surveillance. Two broad strategies to mute Treg/MDSCs are plausible and may improve DC potency. First, DCs can be harnessed to directly target immunosuppressive elements. We recently demonstrated that TLR4-activated DC1 not only inhibits Treg effects but also converts regulatory cells into IFN-γ-secreting Th1 [127]. Alternatively, loading DCs with immunogenic FoxP3 epitopes may generate FoxP3specific CTLs capable of eliminating Treg. Melanoma-bearing mice vaccinated with FoxP3 mRNA-transfected DCs reduced intratumoral FoxP3 + Treg by 85 percent and augmented TRP2-specific CTL responses following co-vaccination with TRP2-DCs [128]. While promising, such approaches must consider the risk of depleting Treg systemically, which may promote irreversible autoimmunity.
The inhibition of Treg functional activity may complement DC vaccination. In a murine model of graft-versus-host disease, mAbs targeting OX40 or GITR (TNF family receptors influencing Treg function) abrogated Treg-mediated suppression [134]. Anti-GITR mAb in conjunction with HER2/neu-expressing DC vaccines displayed potent anti-tumor immunity in a tolerogenic murine model [135]. A synthetic  peptide inhibiting nuclear translocation of FoxP3 protected mice undergoing CD8 + peptide immunization against tumor implantation [136]. Finally, disrupting Treg trafficking to tumors/lymph nodes may boost DC function at these sites. CCR4 antagonists, which block CCL22/CCL17-mediated Treg recruitment, induced antigen-specific CTLs when combined with peptide vaccination in murine models [137].
MDSCs have emerged as key tumor-induced suppressors of T-cell responses.

Targeting Immune Checkpoint Pathways
CTLA-4 and PD-1 are the best understood immune checkpoint receptors that negatively regulate activated CTL function, resulting in an "exhausted" T-cell phenotype. Monoclonal antibodies targeting CTLA-4/PD-1 are immunostimulatory therapies aimed at recovering T-cell cytotoxicity [141]. Anti-CTLA-4 is tumor non-specific, preventing downregulation of CTL function by inhibiting CTLA-4:B7 interaction [142]. Preliminary clinical evidence suggests that combination of DC immunotherapy and anti-CTLA-4 may be synergistic in their benefit. In advanced melanoma patients, co-administration of MART1-pulsed DCs and anti-CTLA-4 mAb (tremelimumab) yielded durable antitumor responses at a higher rate than with either agent alone [143]. The non-specific mechanism of CTLA-4 blockade, however, manifests as dose-limiting toxicity in many patients. Conversely, anti-PD-1 antibodies, which impair the inhibitory CTL:PD-1 ligand interaction on tumors, potentiate tumorspecific immunity and demonstrate a more favorable toxicity profile [144]. Administration of anti-PD-1 antibody (pidilizumab) enhanced activated-CTL responses following stimulation with an autologous myeloma-DC fusion vaccine [145]. Pidilizumab is currently being investigated in combination with DC vaccination in hematologic, renal, and prostate malignancies [4].

Cytokines and TLR Agonists
Cytokines and TLR agonists are attractive adjuncts for DC vaccines due to their critical role in regulating lymphocyte homeostasis and potentiating CTL function. Concomitant administration of systemic IL-2 with DCs proved effective in preclinical studies. In a murine sarcoma model, IL-2 potentiated antitumor effects of tumor lysate-pulsed DCs in vivo and induced protective immunity to lethal tumor challenge; this combination also mediated regression of established pulmonary metastases [146], suggesting its applicability in advanced malignancy. In advanced melanoma patients, however, tumor lysate-pulsed DCs plus IL-2, albeit well tolerated and variably immunogenic, failed to induce meaningful clinical responses [147,148]. Despite these results, several trials employing adjunctive cytokine therapy (GM-CSF, IL-2, IFN-γ, pegylated-IFN-α, IL-12) with DC vaccination are under way [4].

Chemotherapy
The traditional view of chemotherapy as immunosuppressive has been challenged, prompting a re-evaluation of its utility as an adjunct to immunotherapy. In recent years, successful "chemoimmunotherapy" combinations have emerged [152]. Such clinical effects may be explained by the mechanistic synergism between these two modalities, the heightened sensitization of tumor cells to chemotherapy during vaccination-invoked immune siege [153] or provocation of immune responses induced by chemotherapy-induced cell death [154]. The impact of chemotherapeutic agents on antitumor immunity varies by their unique immunologic repercussions. Three effects are recognized: a) increasing Teff stimulation (e.g., cyclophosphamide, paclitaxel); b) enhancing tumor immunogenicity (e.g., doxorubicin, cisplatin, 5-FU); and c) decreasing tumor-induced immunosuppression (e.g., gemcitabine, cyclophosphamide, paclitaxel/carboplatin) [152]. Ultimately, chemotherapy-specific immune effects should guide selection of optimal agent(s) for chemoimmunotherapy.
While the immune benefits of various chemotherapeutic agents are increasingly recognized, optimal sequencing of chemoimmunotherapy is yet to be conclusively established. Although patients heavily pretreated with chemotherapy are less responsive to subsequent immune manipulations [158], less aggressive regimens administered prior to immunization may potentiate antitumor immunity. Dacarbazine treatment before peptide vaccination broadened the T-cell receptor diversity of melan-A-specific CTL clones in melanoma patients, with a trend toward longer survival [159]. Conversely, DC vaccination may effectively prime the immune system before cytotoxic insult. Indeed, patients with advanced small cell lung cancer demonstrated notable clinical responses to second-line chemotherapy following vaccination with DCs transduced with adenoviral-delivered wild-type p53 [160]. To confound matters, concurrent chemotherapy and DC vaccination may be a viable strategy in certain tumor types; a majority of colon cancer patients concomitantly receiving adjuvant oxaliplatin/capecitabine and KLH/CEA-pulsed DCs demonstrated CEAspecific T-cell responses [161]. Several trials attempting to elucidate the optimal dosing/timing of chemoimmunotherapy are under way [4,99].

Radiotherapy
There has been a recent paradigm shift from viewing radiotherapy as merely cytoreductive to appreciating its varied immunomodulatory effects. While these effects are quite complex [51], a simplified rationale for combining DC immunotherapy and radiotherapy follows. Irradiation of tumor cells enhances their immunogenicity via upregulating class I molecules (e.g., in melanoma [162]) or tumorassociated antigen expression (e.g., CEA on gastric adenocarcinoma cells [163]). Radiation-induced release of proinflammatory cytokines (TNF-α, IL-1β) or endogenous TLR agonists (HMGB1 [TLR4]) activate DCs and prime antigen-specific T-cell responses [51]. Moreover, radiation exposure alters the TME favorably, selectively inhibiting Treg [164] and inducing CTL-mediated targeting of tumor stroma [165]. In light of this dynamic interplay between irradiated tumor, DCs, and effector/suppressive immune fractions, combinatorial approaches of DC vaccination with radiotherapy are currently being explored in several tumor types [4].

Targeted Therapies
Targeted molecular therapies can be utilized in combination with DC immunotherapy. A promising agent is sunitinib, a TKI targeting c-KIT, VEGFR, PDGFR, and Flt-3, primarily applied in GIST/RCC patients [166]. In preclinical models, sunitinib effectively decreases TME accumulation of MDSC, restores Th1/CTL functionality, inhibits PD-1L expression on pDCs/MDSCs, depletes CTLA-4/PD-1 expression on activated-CTLs, and mutes the expression of inhibitory IL-10, TGFβ, and FoxP3 from tumor-infiltrating leukocytes [167,168]. Likewise, the immune effects of vemurafenib, a BRAF V600E -targeting TKI in melanoma, are increasingly appreciated. In conjunction with its inhibitory effects on Treg and MDSCs, vemurafenib reduces tumor-induced CCL2 expression and enhances TME Teff infiltration [169]. Interestingly, vemurafenib reversed BRAF V600E melanoma-induced DC dysfunction without deleterious effects on DC viability or ability to prime Tcell responses, making it an exciting candidate for combination immunotherapy [4,170]. Indeed, we are actively examining the efficacy of this combination in a murine model of BRAF V600E /PTEN -/melanoma.

CONCLUSIONS AND OUTLOOK
Realistically, tangible benefits with DC immunotherapy will likely be realized by employing a multifaceted strategy of DC delivery (e.g., uniting in vivo DC-targeting and ex vivomanipulated DCs in individual trials), rationally combining multivalent DC-based vaccines with established anticancer agents, and utilizing these multimodality approaches in early disease or reduced-tumor settings. Furthermore, the repeatedly proven safety of DC vaccination places the onus on regulatory agencies to allow investigators to bypass resource-intensive phase I testing and focus the majority of efforts in evaluating DCs' therapeutic efficacy. These developments might expedite the availability of clinically effective DC approaches for cancer immunotherapy.
Supplement. Clinical trials employing dendritic cell-based immunization approaches. Trials are grouped by phase of development (I-III) and malignancy type. Trial endpoints are indicated (i.e., safety, immunogenicity, tumor/disease response, and overall survival). Trials that were withdrawn or terminated were excluded from this list. Data was obtained using the search terms "dendritic cells" and "cancer" on www.clinicaltrials.gov.