DC-based cancer vaccines (original) (raw)
The era of ex vivo DC vaccines was ushered in by the pioneering work of Inaba, Steinman, and colleagues, demonstrating that mouse DCs can be cultured ex vivo from bone marrow precursors (6). In a similar fashion, human DCs can be generated in culture from CD34+ hematopoietic progenitors and, more commonly, from peripheral blood–derived monocytes (reviewed in refs. 7–9). For cancer vaccination, the goal is to generate ex vivo a population of antigen-loaded DCs that stimulates robust and long-lasting CD4+ and CD8+ T cell responses in the patient with cancer, with the emphasis on “long-lasting”. What seems to be the rate-limiting step at present is the inability to fully recapitulate ex vivo the development of immunocompetent DCs, in particular the process of DC activation. In what is undoubtedly an oversimplification, DC activation can be divided into two stages (Figure 1). In the periphery, quiescent (immature) DCs undergo a maturation process in response to inflammatory stimuli originating from pathogens (pathogen-associated molecular patterns [PAMPs]) or from dying cells, collectively referred to as “danger signals” or “danger-associated molecular patterns (DAMPs)” (10). One important consequence of the maturation process is that DCs acquire the capacity to home to lymph nodes. DCs receiving the appropriate maturation stimuli upregulate expression of CC chemokine receptor 7 (CCR7) and become responsive to CC chemokine ligand 19 (CCL19) and CCL21, chemoattractants produced in the afferent lymphatics and the lymph node. DC migration is also controlled by leukotrienes (such as LTD4 and LTE4), which act downstream of CCR7 signaling (11, 12). When reaching the lymph node, antigen-loaded mature DCs undergo an additional activation step, termed “licensing,” in response to various stimuli, notably CD40 ligand (CD40L) which is expressed on cognate CD4+ T cells. In addition to antigen loading, which will be discussed in the next section, DCs need to be generated in vitro such that they undergo optimal maturation but not licensing because full activation of DCs ex vivo might be counterproductive, as discussed below. The goal, therefore, is to differentiate antigen-loaded DCs only to the point that they have acquired lymph node migratory capacity and become responsive to licensing stimuli when they reach the lymph node and encounter cognate T cells (Figure 1).
Ex vivo differentiation and activation of DCs for cancer immunotherapy. (A) The most common method used to generate DCs for clinical trials is to culture CD14+ monocytes in serum-free media in the presence of GM-CSF and IL-4. Following 5–7 days in culture, the monocytes differentiate into immature DCs, which lose CD14 expression and express moderate to low levels of CD40 and the costimulatory ligands B7-1 and B7-2. DC maturation is accomplished by culturing the immature DCs for an additional 24–48 hours in the presence of several biological agents, the most popular combination being TNF, IL-6, IL-1β, and PGE2 (41). Mature DCs further upregulate CD40, B7-1, and B7-2 and induce the de novo expression of the lymph node homing receptor CC chemokine receptor 7 (CCR7). Antigen loading occurs at either the immature or mature DC stage. (B) Mature antigen-loaded DCs are injected into patients subcutaneously, intradermally, or intravenously. They migrate to the draining lymph node, where they encounter and present antigen (not shown) to cognate CD4+ T cells. Cross-linking CD40 on the DCs by CD40L, which is expressed on the antigen-activated CD4+ T cell, induces the mature DCs to differentiate further, a process known as licensing. Licensed DCs upregulate additional cell surface products, notably the ligands for OX40 and 4-1BB (OX40L and 4-1BBL, respectively). The licensed DCs present antigen to cognate CD8+ T cells. 4-1BBL–mediated costimulation through 4-1BB on the antigen-activated CD8+ T cells enhances the survival and proliferative capacity of the activated CD8+ T cells. Likewise, OX40L-mediated costimulation enhances the survival and proliferation of the activated CD4+ T cells (not shown).
Enhancing DC maturation. Recent insights in DC biology have provided some guidelines as to how optimally matured DCs might be generated ex vivo so that when administered to a patient with cancer they have the ability to migrate to a lymph node and respond to licensing stimuli. These include new information regarding the role of TLRs in sensing danger signals, the identity of molecular mediators of feedback mechanisms that attenuate DC function, the identity of DC-derived costimulatory signals that potentiate T cell activation, and the recognition that DC viability can affect their immunogenicity.
Pathogen-mediated maturation of DCs is mediated mainly through the TLRs that are expressed on immature DCs and activated in response to distinct microbial compounds, PAMPs (10, 13). Culture of DCs with such compounds or their pharmacological analogs (such as the TLR4 ligand LPS, the TLR3 ligand polyinosinic-polycytidylic acid [polyI:C], the TLR9 ligand oligodeoxynucleotide containing one or more unmethylated CpG dinucleotides [CpG ODN], and the TLR7/8 ligands R848 and imiquimod) can induce the phenotypic and functional maturation of cultured DCs. Importantly, functional maturation of DCs is markedly augmented by using certain combinations of TLR agonists (14–16). Cytokines, such as TNF, IL-1, and IL-6, are also capable of promoting DC maturation but cannot substitute for TLR stimulation (17). This should raise concern because most cancer DC vaccine clinical trials use cytokine-only maturation protocols that do not include TLR ligands (18). The remarkable discovery of two phagosome autonomous uptake mechanisms that discriminate in their ability to process antigens for MHC class II presentation depending on the presence or absence of TLR ligands in the phagosome (19) strongly argues, as was indeed shown (20), that fusion of antigens with TLR ligands should enhance the presentation of antigens in the context of MHC class II and thereby potentiate CD4+ T cell immunity. Optimal ex vivo DC maturation might therefore require a combination of both cytokines and TLR ligands, with enhanced antitumor immunity and clinical efficacy being achieved by physically linking TLR ligands with the tumor antigen. In a recent study, Gavin et al. have raised some questions regarding the physiological role of TLRs, introducing the possibility that engaging other pathogen-sensing receptors might be more useful in DC vaccination (21).
Ex vivo DC maturation protocols attempt to recapitulate a complex biological process that has evolved in response to infection with pathogens, but these have so far had limited success in generating DCs that elicit effective antitumor immunity. An alternative and complementary approach is to inhibit negative regulatory pathways that attenuate DC maturation (22). This approach was pioneered by Shen and colleagues, who showed that siRNA inhibition of the function of SOCS1 (which negatively regulates cytokine signaling in DCs and T cells) in DCs potentiates DC immunogenicity (23). Another attractive target is glucocorticoid-induced leucine zipper (GILZ), which is the common effector of suppressive signals mediated by glucocorticoids, IL-10, and TGF-β (24).
Survival and proliferative signals are provided to activated T cells through costimulatory molecules such as OX40 and 4-1BB, which are cross-linked by ligands expressed on activated DCs (25, 26). Current strategies to enhance costimulation include the systemic administration of agonist antibodies or soluble ligands at the time of DC vaccination (25). An alternative approach, transfecting the genes encoding the corresponding ligands into the antigen-loaded DCs, is a simple procedure using readily available reagents that provides added specificity by limiting costimulation to cognate T cells. For example, transfection of mRNA encoding OX40 into DCs has been shown to potentiate the ability of mouse DCs to induce antitumor immunity in vivo and to enhance the activation of human DCs in vitro (27).
Recent studies have also shown that extending the persistence and presentation of antigen by DCs in the lymph node enhances the ensuing immune response (28, 29). However, DCs that are presenting antigen in the lymph node are prone to elimination by their cognate T cells (30). Therefore methods to enhance DC viability, such as generating DCs expressing antiapoptotic proteins (31, 32) or using siRNA to decrease the expression of proapoptotic proteins (33) should also potentiate their immunogenicity.
To license or not to license? Licensing of antigen-loaded DCs in the T cell zone of lymph nodes is mediated by local stimuli, notably CD40L expressed by cognate CD4+ T cells, IFN-γ, and surely other stimuli yet to be identified (34). Signaling through CD40 has multiple effects on DCs, including inducing the upregulation of costimulatory molecules, the secretion of cytokines (notably IL-12), and the upregulation of several antiapoptotic molecules, all of which cumulatively potentiate the ability of DCs to optimally activate cognate T cells, especially CD8+ T cells (35, 36). However, premature licensing of DCs prior to their encounter with cognate T cells in the lymph node might be counterproductive. IL-12, an important licensing cytokine that mediates the polarization of activated CD4+ T cells to a Th1 phenotype such that they provide help for the generation of potent CD8+ CTL responses, is a case in point. DCs can be induced in vitro and in vivo to secrete IL-12, but IL-12 expression is transient and DCs become refractive to subsequent induction of IL-12, a phenomenon termed “exhaustion” or “paralysis” (37, 38). These observations strongly suggest that ex vivo DC maturation protocols should avoid conditions that induce DCs to express IL-12, and should instead use conditions that induce the DCs to acquire responsiveness to IL-12 induction (39). But can one enhance the licensing potential of ex vivo–generated DCs, that is, specifically augment CD40 signaling after they have been injected into the patient? An ingenious solution has been offered by the work of Hanks et al. (40), who developed a drug-inducible CD40 expression system whereby the trimerization-dependent activation of ectopically expressed engineered CD40 molecules in ex vivo–generated DCs is delayed until they reach the lymph node. This was achieved by fusing a membrane-localized cytoplasmic domain of CD40 to a drug-binding domain and injecting the appropriate bivalent drug into mice once the DCs arrived at the lymph node, thereby mediating CD40 trimerization.
DC generation and maturation protocols. The most widely used maturation protocol for human monocyte–derived DCs consist of four reagents, TNF, IL-1β, IL-6, and PGE2, also known as monocyte-conditioned media mimic or cytokine cocktail (41). A recent phase III clinical trial failed to show that vaccinating melanoma patients with cytokine cocktail–matured DCs provided benefit over standard dacarbazine (DTIC) chemotherapy (42). It is not inconceivable that the suboptimal nature of the maturation conditions, and hence the suboptimal immunogenicity of the DCs, was a primary reason for the failure. It is tempting to speculate that the main culprit in the cytokine cocktail formula was PGE2. The rationale for including PGE2 in the maturation protocol is to endow the ex vivo–generated DCs with the capacity to migrate (43, 44), but PGE2, in the context of the tumor microenvironment, can mediate Th2 polarization and promote the differentiation of DCs secreting the immunosuppressive cytokine IL-10 (45). Therefore, the key negative impact of PGE2 on the function of ex vivo–generated DCs is probably that PGE2 abolishes both the responsiveness of mature DCs to stimulation through CD40 and their ability to synthesize IL-12 when they reach the lymph node and encounter cognate T cells (44). PGE2 notwithstanding, the elegant study of Sporri and Reis e Sousa has shown that optimal activation of DCs requires TLR signaling, which this maturation protocol does not provide (17). If all that is not enough, a recent study comparing several maturation protocols found that cytokine cocktail–matured DCs were most effective, even more than immature DCs, at expanding a population of immunosuppressive Tregs expressing the forkhead box transcription factor FOXP3 (46).
The cytokine cocktail protocol is not the only human DC maturation protocol used. Kalinski and colleagues have designed a “megacytokine cocktail” protocol consisting of 5 reagents, TNF, IL-1β, PolyI:C, IFN-α, and IFN-γ (47). In vitro megacytokine cocktail–matured DCs exhibited superior immunogenicity to cytokine cocktail–matured DCs, that is, they stimulated more potent CTL responses (43). Furthermore, the megacytokine cocktail–matured DCs were responsive to stimulation through CD40, able to produce IL-12, and, notably, despite the absence of PGE2 in the megacytokine cocktail the DCs retained lymph node migratory capacity in vitro. Promising as this might be, judging from in vitro analysis, clinical trials are necessary to determine the value of this novel approach in vivo. It is thought, based on in vitro studies, that maturing DCs in TNF alone or omitting PGE2 from the cytokine cocktail will not generate DCs able to induce therapeutically effective antitumor immunity (41). However, the results of a study in which rhesus macaques infected with SIV were vaccinated with DCs matured using TNF and a follow-up clinical trial in which patients infected with HIV were vaccinated with DCs matured using IL-1β, IL-6, and TNF (but in the absence of PGE2) were nothing short of spectacular, resulting in the induction of T cell responses and substantial reductions in viral titer in most vaccinees (48, 49). In addition, several rapid, two-day “fast-DC” protocols have been developed that generate DCs able to stimulate T cell responses in vitro as effectively as DCs generated by standard protocols, which usually require 7–9 days of culture. (50–52). In a recently published clinical trial, HER2/neu-positive breast cancer patients vaccinated with peptide-loaded DCs generated in a two-day culture of monocytes incubated with IFN-γ and LPS induced HER2/neu-specific CD4+ and CD8+ T cell responses and measurable decreases in tumor volume (53). Importantly, in vitro analysis suggests that DCs generated in such a manner are mature, as judged by phenotypic analysis, and transiently secrete IL-12, but are not “exhausted” because they are able to respond to CD40 signaling by producing more IL-12 (50, 53). Clearly, our understanding of the immunobiology of DCs is still evolving, and in vitro observations, notwithstanding their critical importance in guiding the development of improved DC-based vaccines, as well as encouraging data from early clinical trials need to be interpreted with caution.
An alternative to optimizing the ex vivo DC maturation process is to circumvent this step altogether and mature the DCs in situ. This can be achieved by injecting antigen-loaded ex vivo–generated immature DCs into sites that have been pretreated with adjuvant to induce a local inflammatory reaction (54, 55). In addition to simulating more closely the maturation process that occurs following infection with a pathogen, in situ maturation dispenses with the need to use reagents that are expensive and often hard to get, especially for clinical applications. For example, my group has shown that immature ex vivo–generated DCs injected into the skin (the ear pinna) of mice pretreated with the adjuvant imiquimod (a TLR7/8 ligand) showed lymph node migration and CTL and antitumor immunity induction comparable, if not superior, to that of DCs generated and matured ex vivo. In addition, in cancer patients, in situ–matured, ex vivo–generated DCs acquired lymph node migratory capacity comparable to that of DCs generated and matured ex vivo (54). Clinical trials are currently ongoing to assess the therapeutic efficacy of this simplified method of DC vaccination.
Lastly, an approach that does not pertain directly to the maturation issue but represents a radical departure from the standard vaccination protocols using ex vivo–generated and ex vivo–matured DCs is the use of subcellular vesicles, known as exosomes or dexosomes, derived from antigen-loaded ex vivo–generated DCs (56). In mice, injection of exosomes derived from antigen-loaded immature DCs has been shown to stimulate protective antitumor immunity (57). One concern with using exosomes is that because they are derived from immature DCs (as opposed to mature DCs) they might induce tolerance instead of immunity if they are not subjected to additional manipulation. Apart from this concern and the practical problems of using exosomes, these vesicles might hide an important biological phenomenon — exosomes might be a (or the) conduit of antigens in instances in which antigen-capturing DCs transfer antigen to lymph node–resident DCs for presentation to cognate T cells (58). Of note, in a recent publication, Kovar et al. have described a different type of vesicle, derived from mature DCs by sonication, that seems to be more potent than exosomes in stimulating immune responses (59). The bottom line is that strategies such as using in situ–matured, ex vivo–generated DCs or subcellular vesicles derived from ex vivo–generated DCs represent fertile avenues for further exploration that could improve the potency of the DC vaccination approach and simplify this otherwise complex protocol.