Immunoglobulin idiotype (Id) is a tumor-specific antigen that is produced by each B cell tumor clone. Id protein has been used for immunotherapy in patients with MM [23, 24]. Id vaccination could induce responses by both antibodies and Id-specific T cells, including CD4⁺ and CD8⁺ T cells, through the presentation of Id protein on the surface of professional APCs. Id-specific CTL lines that kill autologous primary myeloma cells in vitro have been generated. In one report, killing activity was induced by only MHC class I-restriction [25], while another report described both class I- and class II-restriction [26]. Autologous DCs that were generated from MM patients have been shown to efficiently endocytose different classes of Id proteins, and autologous Id-specific CTLs containing both CD4⁺ and CD8⁺ T cells that were generated by Id-pulsed DCs were able to recognize and kill autologous primary myeloma cells in vitro [26, 27]. Various studies of DC-based Id vaccination in MM have been reported [28-35]. Although Id-specific CTLs and immune responses could be induced in some patients, clinical responses have rarely been observed after vaccination [30], possibly because Id protein is a weak antigen and immature DCs have been used in some studies [28].

A variety of myeloma-associated antigens that may induce immune responses from DC-based vaccines have been identified in MM patients. Many potential tumor-associated antigens (TAAs) in MM have been investigated, including polymorphic epithelial mucin (MUC1), human telomerase reverse transcriptase (hTERT), preferentially expressed antigen of myeloma (PRAME), Sperm protein 17 (Sp17), Wilms' tumor-I (WTI), Dickkopf-1 (DKK-1), and members of the cancer germ-like family (MAGE, GAGE, BAGE, LAGE, and NY-ESO-1). MUC1 was expressed in all MM cell lines and primary myeloma cells. MUC1-specific CTLs that were induced in vitro using peptide-pulsed DCs or plasma cell RNA-loaded DCs efficiently killed not only target cells pulsed with the antigenic peptide but also MM cells [36, 37]. NY-ESO-1 is the most immunogenic of the cancer testis antigens, proteins that are expressed in a variety of tumors, but whose expression in normal tissue is limited to the testis and placenta [38]. Spontaneous humoral and CD8⁺ T cell-mediated responses to NY-ESO-1 have been identified in patients with advanced disease [38]. In addition, in vitro monocyte-derived DCs transfected with PTD-NY-ESO-1 protein can induce CD8⁺ cellular antitumor immunity superior to that achieved with NY-ESO-1 protein alone [39]. Sp17-specific HLA class I-restricted CTLs were successfully generated by DCs that had been loaded with recombinant Sp17 protein and were able to kill autologous tumor cells that expressed Sp17 [40]. The overexpression of hTERT on MM compared to the expression levels in normal cells indicated that this telomerase also could be used as a myeloma-associated antigen. hTERT was capable of triggering antitumor-CTL responses and killing hTERT+ tumor cells [41]. Recently, a report demonstrated that activated T lymphocytes were able to successfully kill myeloma cells after stimulation by DCs loaded with hTERT- and MUC1-derived nonapeptides [42]. DKK1, a novel protein that is not expressed in most normal tissues but is expressed in almost all myeloma cells, may be an important antigenic target for antimyeloma immunotherapy. DKK1-specific CTLs that were generated by DCs pulsed with DKK1 peptides were specifically lysed by autologous primary myeloma cells and DKK1-positive cell lines [43]. In general, DCs loaded with TAAs may be promising agents for use in immunotherapy against MM.

Although a single TAA can induce antitumor immune response against MM, tumors may escape immune recognition via down-regulation of specific antigen expression. In contrast, DCs loaded with antigens derived from whole tumor cells can improve the antitumor response, limiting the risk of immunological escape. Numerous reports have described these alternative approaches, such as DCs pulsed with myeloma lysates [44, 45], DCs pulsed with myeloma apoptotic bodies [46, 47], DCs transfected with myeloma-derived RNA [37], DCs pulsed with myeloma-derived heat shock protein (HSP) gp96 [48, 49], and DC-myeloma cell hybrids [50, 51]. These techniques have the advantage of allowing the presentation of multiple epitopes to MHC on DCs, therefore inducing polyclonal T cell responses from many potentially unknown TAAs and reducing the probability of immune escape by a single TAA. DCs loaded with myeloma cell lysates induced much more potent cytotoxicity in autologous plasma cells than Id protein-pulsed DCs did, suggesting the superiority of the myeloma cell as a source of a tumor antigen [45]. The apoptotic bodies derived from myeloma cells have also been used as tumor antigens for loading onto DCs. Interestingly, these apoptotic bodies were shown to be more effective than cell lysates at inducing CTLs against autologous myeloma cells [52]. HSPs are a class of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stresses. Tumor-derived HSPs, such as HSP70 and gp96, are immunogenic, potent stimulators of tumor-specific CTLs. The specific CTLs that were stimulated by myeloma-derived gp96-loaded DCs were able to lyse myeloma cells but not normal blood cells in a MHC class I-restricted manner [48, 49]. Moreover, fusion of autologous DCs with patient-derived tumors stimulated both helper and cytotoxic T cell responses through the presentation of internalized and newly synthesized antigens [51]. In mouse MM models, vaccination with DCs fused with either plasmacytoma cells or tumor cells resulted in eradication of tumors and protective immunity against subsequent tumor challenge [50]. In general, the production of DC vaccines using whole tumor antigens has become a promising tool for immunotherapy against MM.

The initial success of DC-based vaccines involving immature or partially-mature "first-generation" DCs has been reported [55, 56]. The first therapeutic DC-based cancer vaccine against prostate cancer (Sipuleucel-T/Provenge) received US FDA approval and demonstrated the ability of the vaccine to prolong overall survival in patients with highly advanced cancer [57]. However, such DCs express suboptimal levels of costimulatory molecules and constitute weaker immunogens than subsequently implemented mature DCs, the "second generation" of clinically applied standard DCs (sDCs). sDC vaccines induced by cytokine cocktails containing IL-1β/TNF-α/IL-6/prostaglandin E₂ (PGE₂) have been developed [58]. Such DCs are fully mature and exhibit increased expression costimulatory molecules and CCR7 and high migratory responsiveness to lymph node-associated chemokines; they have been widely tested in clinical trials. However, to date, sDC vaccines still have limitations, including the mediation of Th2 polarization, promotion of secretion of the immunosuppressive cytokine IL-10 from DCs, inability to induce the Th1-type response (because PGE₂ abolishes the secretion of IL-12p70), and high activity of such DCs in activating Tregs [59, 60]. In addition, the induction of antitumor CTL responses by DC-based vaccines benefits from high expression of costimulatory molecules, CCR7 responsiveness, and migration in response to lymphoid chemokines from mature DCs [61]. At the same time, high IL-12p70 secretion has been shown to dramatically enhance the ability of DCs to induce tumor-specific Th1 cells and CTLs and to promote tumor rejection in therapeutic mouse models [62]. Unfortunately, both first- and second-generation DCs were unable to induce effective CTL responses. Therefore, several investigators, including our group, have tried to develop potent DCs that possess all required features for inducing effective tumor-specific immune responses. We demonstrated the feasibility of inducing mature but "nonexhausted" DCs, by exposing immature DCs to type 1 and type 2 interferons (IFNs) combined with TNFα or Toll-like receptor (TLR) ligands. In particular, α-type 1-polarized DCs (αDC1s) that have been induced to mature using an αDC1-inducing cytokine cocktail containing IL-1β, TNF-α, IFN-α, IFN-γ, and polyinosinic:polycytidylic acid [poly(I:C)] have been developed to generate strong functional CTLs in several diseases and are on average 20-fold higher than sDCs [63, 64]. The resulting αDC1s showed high expression of several costimulatory molecules or high secretion of IL-12p70, improving the induction of tumor-specific and functional CTLs in comparison with that in sDCs in vitro and in vivo [64-67]. Recently, we successfully generated αDC1s from MM patients with high expression of costimulatory molecules, significant production of IL-12p70, and potent generation of myeloma-specific CTLs [46, 47]. Such a novel strategy would provide improved potency of ex vivo-generated DCs for cancer therapy.

The other strategy to induce potent DCs from patients with MM was the use of helper cells to promote type 1-polarization of DCs. NK cells rapidly migrate to sites of infection and control the immune response in viral infections. Indeed, it has been demonstrated that NK cells play a major immunoregulatory role in the development of protective T cell-mediated immunity against intracellular pathogens and cancers [68]. Such helper activity of NK cells is at least partially mediated by the functional modulation of DCs. This phenomenon depended on the production of IFN-γ and TNF-α by activated NK cells [68] and was associated with enhanced tumor antigen cross-presentation and the induction of Th1 and CTL responses [69-71]. Recent data from our laboratory and other groups has demonstrated that such NK-DC interactions promote the subsequent induction of tumor-specific responses in CD4⁺ and CD8⁺ T cells, allowing NK cells to act as helper cells in the development of type-1 polarized DCs in responses against cancer [69-71]. Resting NK cells that are activated in the presence of TLR agonists, IL-2, and IFN-α can induce potent DCs with enhanced IL-12p70 production in vitro, generating stronger functional CTLs against myeloma cells than sDCs [71].

We also found that the selected combinations of TLR agonists synergistically triggered a Th1-polarizing capacity through production of high amounts of IL-12p70 [72]. However, the major limitation of this combination was the decreased ability of these cells to migrate into lymph nodes compared to that of conventional DCs. When DCs are activated by individual TLR agonists, such as LPS or poly(I:C), or by a combination of 2 TLR agonists, all cells mature and produce high levels of bioactive IL-12p70 in early phase of maturation and after subsequent stimulation with T cell-related DC activating signal CD40L. In addition, the phenotyes of these matured DCs were markedly enhanced when a combination of type I and type II IFN was added. These combinations of stimuli also regulated the expression of CD38 and CD74, markers related to the full activation of DCs [73, 74]. We demonstrated that at the optimal concentration used to stimulate DCs, the combination of 2 TLR agonists with type I and II IFNs can be used to generate fully mature DCs that have high migratory capacity and can maintain IL-12p70-producing capacity. The regulation of CD38 and CD74 in DCs could in turn enhance the migratory activity of DCs in the presence of a combination of 2 TLR agonists and IFNs [75].

Ursolic acid (URC) is isolated from Uncaria rhynchophylla and phytochemically classified as a triterpene. Triterpene compounds have been identified as a unique class of natural products possessing diverse biological activities. Recently, we reported that URC activates human DCs in a fashion that favors Th1 polarization via the activation of TLR2-and/or TLR40 dependent IL-12p70 and induces the production of IFN-γ by CD4⁺ naïve T cells [76]. In addition, combination URC and IFN-γ enhanced the activation of DCs via promotion of IFN-γ-induced Th1-cell polarization that was dependent on the activation of IL-12p70 and independent of TLR4 [77]. The potential of natural products to enhance DC maturation and activation has important implications for the use of DCs as cancer vaccines.

The clinical responses of DC vaccines using Id-pulsed DCs have been disappointing. The use of TAAs can induce more significant immune responses than Id. Although a single TAA can induce antitumor immune responses against MM, tumors may escape immune recognition via down-regulation of specific antigen expression. In addition, only a small number of tumor samples from MM patients showed a similar level of TAA expression, limiting the usefulness of TAAs in MM. Therefore, to overcome the effects of TAA-based immunotherapy, our group has attempted to use other tumor antigens that improve the cross-presentation of the DC vaccine in patients with MM.

The selected antigen should possess the best characteristics for induction of cross-presentation and should be tumor specific and easily available, but should not induce immune suppression. Whole tumor antigens, including myeloma cell lysates and apoptotic bodies from myeloma cells, are the best tumor antigens and have been selected by many investigators. In practical terms, a number of patients with MM have less than 50% myeloma cells in the BM at diagnosis or during progression of the disease. When mononuclear cells from the BM are used as a source of tumor antigens, contamination with normal cells, especially lymphocytes, may occur. Thus, it is necessary to use purified, optimized myeloma cells, if possible, as a source of tumor antigens for the generation of myeloma-specific CTLs stimulated by DCs [44]. We have shown that the function of DCs was affected by the concentration of myeloma cell lysates (i.e., higher concentrations of lysates suppressed T cell stimulatory capacities) [44]. CTLs that were generated by purified, optimized myeloma cell lysates pulsed with DCs demonstrated much stronger cytotoxicity against autologous plasma cells. These findings indicate that it is important to optimize the concentration of myeloma cell lysates that were loaded onto DCs to potentiate their function.

The use of whole tumor cells instead of single antigens may enhance antitumor effects, but may also target multiple tumor variants and counteract tumor immune evasion. However, it is impractical to obtain sufficient amounts of purified autologous myeloma cells for tumor antigens in the clinical setting of MM. As an alternative source of tumor-relevant antigens, allogeneic tumor cells or established cancer cell lines have been used to overcome this limitation in various tumors [46, 47]. In clinical practice, allogeneic myeloma cell lines may be an effective source of universal tumor antigens that could be used to load DCs for the generation of myeloma-specific CTLs in MM patients. Tumor antigens derived from DC-loaded irradiated allogeneic myeloma cells could generate myeloma-specific CTLs against autologous myeloma cells in patients with MM [46, 71]. The success of using an allogeneic myeloma cell line as tumor antigens led to the possibility that allogeneic myeloma cells could also be used as a viable source of tumor antigens in the context of appropriate major MHC alleles to autologous CTLs. We investigated the possibility of using autologous DCs loaded with an apoptotic allogeneic matched monoclonal immunoglobulin subtype of myeloma and showed that the CTL generated by these tumor antigen-loaded DCs could generate myeloma-specific CTLs against autologous myeloma cells in MM patients [47]. These findings suggested that allogeneic myeloma cell lines and allogeneic matched monoclonal immunoglobulin subtype of myeloma were effective tumor antigens capable of inducing functional CTLs against patients' own myeloma cells.

The suppressive effects of tumor cells during DC generation have been explained previously by the ability of the tumor microenvironment to suppress DC differentiation [78]. This suppression is due to the activation of STAT3 and the production of immunosuppressive factors. These factors can influence STAT3 and extracellular signal-regulated kinase (ERK) phosphorylation, resulting in hyperactivation of STAT3 and ERK, which may be responsible for defective DC differentiation [79]. In addition to generate potent and specific tumor antigen-loaded DCs for vaccination, alternative methods have attempted to restore defective DC function and to enhance DC function in MM. The immune-mediated antitumor effects of DCs are enhanced by inhibition of the janus-activated kinase 2 (JAK2)/STAT3 pathway [80], inhibition of p38 or activation of the MEK/ERK or mitogen-activated protein kinase (MAPK) pathways, and neutralization of IL-6 [81]. Recently, we found that when MM-specific DCs were generated by loading tumor lysates from myeloma cells collected at diagnosis or during the relapsed/progressive state, these DCs showed lower phenotypic maturation, less T cell stimulatory capacity, less CTL activity, and highly abnormal IL-6 and IL-12 secretion compared to the secretion by unloaded DCs. Moreover, the levels of VEGF, phospho-STAT3, and phospho-ERK1/2 in DCs were significantly higher than in unloaded DCs. After neutralization of VEGF activity, DC functions, signal transduction, and cytokine production returned to normal. Therefore, inhibitory factors and abnormal signaling pathways during maturation with tumor antigens in DCs may be responsible for the defective activity of DCs in MM, and these abnormalities may be overcome by neutralizing the signaling that would lead to a suppressed immune response [82]. Currently, we are working to develop strategies that recover dysfunction of DCs caused from loading tumor antigens through treatment of myeloma cells with a combination of the selective JAK/STAT3 inhibitor JSI-124 and proteasome inhibitor bortezomib. We observed that production of inhibitory cytokines, such as IL-10, IL-23, and especially IL-6, which is known as the main plasma cell growth factor that induces DC dysfunction in MM patients, was down-regulated in DCs loaded with JSI-124- and bortezomib-induced dying myeloma tumor cells. Furthermore, phospho-STAT3 was also down-regulated in these DCs. Consistent with their recovery functions, these DCs displayed a superior ability to induce myeloma-specific responses of CTLs (unpublished data). We found that pretreatment of myeloma cells with a combination of JSI-124 and bortezomib recovered DC dysfunction caused by loading of dying myeloma cells through the up-regulation of Hsp90 and down-regulation of STAT3 phosphorylation and inhibitory cytokine production. These DCs could generate potent myeloma-specific CTLs.

In vitro-generated DCs that are used for targeting local lymph nodes in vaccination protocols are highly sought after, but difficult to achieve in practice. Type 1-polarized DCs with higher levels of IL-12p70 and potent CTL generation targeting are limited by their migratory capacity to primary lymph nodes due to the relatively lower expression of CCR7 in these DCs than in sDCs. The first important mediator in the mobilization of DCs to lymph nodes is CCR7. However, upregulation of CCR7 alone is insufficient to drive DC migration toward CCL19 and CCL21. Up-regulation of CD38 and down-regulation of CD74 regulate DC migration in vitro and in vivo [73, 74]. We recently reported that the migratory capacity of DCs is enhanced by the regulation of CD38, CD74, and CCR7 expression on DCs, resulting from the TLR-agonist-mediated synergistic effects of type I and II IFNs on the regulation of DC migration and providing a novel approach to improving vaccination efficacy [75].

The efficacies of 3 features, namely, fully mature status, high migratory responsiveness to lymph-node associated chemokines (CCR7 ligands), and ability to produce high levels of IL-12p70, determine whether DCs are appropriate carriers for anticancer vaccines [63]. However, although α-type 1-polarized DCs possess all of the above features, the expression of CCR7 and the migration of DCs in response to the CCR7 ligand (CCL19 and CCL21) were lower in these DCs than in conventional sDCs [63, 64] due to the lack of PGE₂, a critical component of the standard cytokine cocktail that is important for the expression of CCR7 and the responsiveness of DCs to CCR7 ligands [83]. We also investigated whether migration could be enhanced by increasing the expression of CCR7 or the migration of DCs and found that after stimulation of monocyte-derived DCs by CCL21 2 hr before the end of maturation by α-type 1-polarized cytokine cocktail, the migration of harvested DCs through transwells was faster than that of unstimulated DCs. Interestingly, phenotypes and cytokine production levels were similar in both DCs. Furthermore, we also found that CCL21-treated DCs could induce the ability to stimulate tumor-specific responses of CTLs by the enhancement of CXCL10 (IP-10) secretion (unpublished data). We are now investigating this phenomenon to generate monocyte-derived DCs in MM patients and in vivo mouse models.

Therapeutic DC vaccines need to be highly effective at inducing the expansion of tumor-specific T cells and need to avoid interaction with and induction of immunosuppressor cells, such as Tregs and MDSCs. Recently, type 1-polarized DCs were shown to suppress the secretion of CCL22 (Tregand Th2-type attracting chemokines), enhance the secretion of CCL5 and CXCL10 (Th1- and effector T cell-attracting chemokines), and suppress the induction of Tregs compared to sDCs [84]. In addition, to enhance the antitumor effects of DC-based vaccines in preclinical in vivo mouse models, we have developed several models of combination therapy using DCs and immunomodulatory drugs, including cyclophosphamide and lenalidomide. Cyclophosphamide is frequently used to enhance or augment antitumor effects in cancer immunotherapy [85]. Low-dose cyclophosphamide may enhance the antitumor efficacy of DC vaccines by increasing the proportion of IFN-γ secreting lymphocytes and suppressing the proportion of CD4⁺CD25⁺FoxP3⁺ Tregs in tumor-bearing mice [85]. The result of a clinical trial using allogeneic DC vaccines combined with low-dose cyclophosphamide has revealed that the combination therapy could induce stronger antitumor responses compared to the DC vaccine alone [86]. Recently, we developed a cancer model of combination therapy in mice showing that a single administration of low-dose cyclophosphamide before the first DC vaccination augmented the antitumor effects of DC vaccines to completely eradicate the tumor, prolonging the survival of vaccinated mice [70].

We are developing a mouse myeloma combination therapy model, in which the myeloma tumor-bearing mice were vaccinated with a combination of DCs and lenalidomide. Lenalidomide is a thalidomide analog that is more potent and has less adverse effects [11]. Lenalidomide can induce apoptosis; inhibit the production of cytokines (IL-6, VEGF, and TNF-α) in BM; and stimulate T cell and NK cell proliferation, cytotoxicity, and cytokine (IL-2, IFN-γ) production [11]. In addition, lenalidomide can inhibit the frequency and function of Tregs, resulting in inhibition of Treg expansion and forkehad box P3 (FOXP3) expression in cancer patients [87]. Interestingly, this drug can also induce the activation of APC function, resulting in upregulation of CD40, CD80, and CD86, in chronic lymphocytic leukemia [88]. Therefore, lenalidomide could be used as an immunomodulatory drug in order to enhance immune responses against cancer. Our in vitro study showed that lenalidomide enhanced the maturation and function of DCs in synergistic stimulation with LPS to increase phenotype expression, IL-12p70 production, T cell stimulation capacities, and cytotoxic activities against myeloma cells. In addition, lenalidomide combined with LPS induced type 1-polarization of naïve T cells and suppressed the generation of Tregs (unpublished data). Moreover, our in vivo mouse myeloma model showed that a treatment combining the immunomodulatory drug lenalidomide with a DCs vaccine improved antitumor immunity by inhibiting immunosuppressor cells and recovering effector cells and inducing superior polarization of the Th1/Th2 balance in favor of the Th1 response. This immunomodulatory effect may be a crucial component of the enhancer-like properties of lenalidomide in the context of antitumor immunity against MM (unpublished data).