Chaetocin enhances dendritic cell function via the induction of heat shock protein and cancer testis antigens in myeloma cells.

Dendritic cells (DC)-based vaccines are considered useful in cancer immuno-therapy, and the interactions of DC and dying tumor cells are important and promising for cancer immunotherapy. We investigated whether chaetocin could be used to induce death of myeloma cells, for loading onto DCs can affect DCs function. In this study, we show that the dying myeloma cells treated with chaetocin resulted in the induction of heat shock protein (HSP) 90, which was inhibited by antioxidant N-acetyl cysteine, and showed an increase in the expression of MAGE-A3 and MAGE-C1/CT7. DCs loaded with chaetocin-treated dying myeloma cells produced low levels of IL-10 and enhanced the cross presentation of DCs. Additionally, these DCs most potently inhibited regulatory T cells, induced Th1 polarization and activated myeloma-specific cytotoxic T lymphocytes compared with DCs loaded with UVB-irradiated dying myeloma cells. These results suggest that the pretreatment of myeloma cells with chaetocin can enhance DC function through the up-regulation of HSP90 and cancer testis antigens in dying myeloma cells and can potently induce the Th1 polarization of DCs and myeloma-specific cytotoxic T lymphocytes.

Sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) reduces the migratory capacity of CCL21-treated monocyte-derived dendritic cells.

The migration of dendritic cells (DCs) to secondary lymphoid organs depends on chemoattraction through the interaction of the chemokine receptors with chemokines. However, the mechanism of how lymphoid chemokines attract DCs to lymphoid organs remains unclear. Here, we demonstrate the mechanism of DC migration in response to the lymphoid chemokine CCL21. CCL21-mediated DC migration is controlled by the regulation of sarcoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) expression rather than through the activation of mitogen-activated protein kinases CCL21-exposed mature DCs (mDCs) exhibited decreased SERCA2 expression but not decreased phospholamban (PLB) or Hax-1 expression, which are known to be SERCA2-interacting proteins. In addition, CCL21 did not affect the mRNA levels of SERCA2 or its interacting protein Hax-1. Interestingly, SERCA2 expression was inversely related to DC migration in response to chemokine stimulation. The migratory capacity of CCL21-treated mDCs was decreased by the phospholipase C inhibitor U73122 and by the protein kinase C inhibitor BAPTA-AM. The migratory capacities of mDCs were increased in response to SERCA2 siRNA expression but were decreased by SERCA2 overexpression. In addition, DCs treated with a SERCA2-specific inhibitor (cyclopiazonic acid) had significantly increased migratory capacities as mDCs regardless of SERCA2 expression. Moreover, SERCA2 expression was dependent on DC maturation induced by cytokines or Toll-like receptor agonists. Therefore, the migratory capacities differed in differentially matured DCs. Taken together, these results suggest that SERCA2 contributes to the migration of CCL21-activated DCs as an important feature of the adaptive immune response and provide novel insights regarding the role of SERCA2 in DC functions.

Lenalidomide Synergistically Enhances the Effect of Dendritic Cell Vaccination in a Model of Murine Multiple Myeloma.

We investigated the efficacy of lenalidomide (LEN) in combination with dendritic cell (DC) vaccination in the MOPC-315 murine myeloma model. After tumor growth, LEN was injected intraperitoneally for 4 consecutive days in combination with DC vaccination. The combination of LEN and vaccination efficiently inhibited tumor growth compared with the single agents alone. A cytotoxic assay revealed that the anticancer effects of DC vaccination plus LEN involved not only generation of antigen-specific cytotoxic T lymphocytes but also NK cells. Vaccinated mice had reduced numbers of suppressor cells, including both myeloid-derived suppressor cells and regulatory T cells, in the spleen. The proportions of CD4+ and CD8+ T cells increased in the spleen, and a Th1 cytokine (interferon-γ) rather than a Th2 cytokine (interleukin-10) was synthesized in response to tumor antigens. LEN enhanced the innate immune response by modulating NK cell numbers and function. In addition, LEN reduced the production levels of angiogenesis-inducing factors in tumor-bearing mice. Together, these results suggest that a combination of LEN and DC vaccination may synergistically enhance anticancer immunity in the murine myeloma model, by inhibiting immunosuppressor cells and stimulating effector cells, as well as effectively polarizing the Th1/Th2 balance in favor of a Th1-specific immune response.

Dendritic cell vaccination with a toll-like receptor agonist derived from mycobacteria enhances anti-tumor immunity.

Dendritic cell (DC)-based vaccines are considered useful in cancer immunotherapy, and the interaction of DC and adjuvants is important in the design of the next generation vaccines. In this study, whether DC combined with Rv2299c derived from mycobacteria could improve anti-tumor immune responses in a colon cancer mouse model was evaluated. MC38 cell lines were injected subcutaneously to establish colon-cancer-bearing mice and the following four groups were evaluated: PBS control, tumor antigen (TA) loaded-DC, Rv2299c, and a combination of TA-loaded-DC and Rv2299c. The combination treatment with TA-loaded-DC and Rv2299c exhibited greater inhibition of tumor growth compared to other groups. These effects were associated with the reduction of suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, and the induction of effector cells, such as CD4+ T cells and CD8+ T cells, in spleen, and with the activation of cytotoxic T Lymphocytes and NK cells. These results suggest that TA-loaded-DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting the generation of immune-suppressive cells and recovering numbers of effector cells, and demonstrated superior polarization of the Th1/Th2 balance in favor of the Th1 immune response.

Branched Polyethylenimine-Superparamagnetic Iron Oxide Nanoparticles (bPEI-SPIONs) Improve the Immunogenicity of Tumor Antigens and Enhance Th1 Polarization of Dendritic Cells.

Nanoparticles in the field of dendritic cell (DC) research are emerging as a promising method of enhancing the efficacy of cancer immunotherapy. We investigated the effect of branched polyethylenimine-superparamagnetic iron oxide nanoparticles (bPEI-SPIONs) on tumor cells loaded onto DCs. The tumor antigens were prepared as follows: (1) apoptotic U266 cells with ultraviolet B (UVB) irradiation followed by a 2 h incubation in the absence (2 h postirradiated cells) or (2) presence of bPEI-SPIONs (bPEI-SPION 2 h postirradiated cells) and (3) apoptotic U266 cells with UVB irradiation followed by an overnight 16 h incubation (16 h postirradiated cells). bPEI-SPIONs render U266 cells sensitive to UVB irradiation through reactive oxygen species production to accelerate apoptotic death. The 2 h postirradiated cells and bPEI-SPION 2 h postirradiated cells released immunogenic proteins, including Hsp70, Hsp90, and HMGB1. The DCs loaded with bPEI-SPION 2 h postirradiated cells showed the highest IL-12p70 production and Th1 polarization compared with other DCs. These results suggest that bPEI-SPIONs are a promising method of enhancing the immunogenicity of tumor cells and promoting Th1 polarization of DCs loaded with these tumor cells.

Generation of potent dendritic cells with improved migration ability through p-cofilin and sarco/endoplasmic reticulum Ca(2+) transport ATPase 2 regulation.

BACKGROUND AIMS: It is important to improve the migratory ability of dendritic cells (DCs) and to increase DC potency for successful DC-based cancer immunotherapy. The intracellular Ca(2+) signaling pathway has an important role on the regulation of DC migration. Our preliminary studies revealed that sarco/endoplasmic reticulum Ca(2+) transport ATPase 2 (SERCA2) expression was inversely related to DC migratory capacity, and the expression level of p-cofilin and SERCA2 on mature DCs showed a counter-trend. METHODS: We selected the appropriate six maturation cocktails on the basis of the expression levels of SERCA2 and p-cofilin and investigated the functional characteristics and migratory capacity of mature DCs. Among the these six maturation cocktails, DCIFN-γ/IL-1ß/Poly-I:C showed potent type 1 immune response with interleukin (IL)-12p70 production and strong Th1-polarization, and this DC elicited strong antigen-specific cytotoxic T-lymphocyte responses. RESULTS: Interestingly, DCIFN-γ/IL-1ß/Poly-I:C showed lower expression of SERCA2 and higher expression of p-cofilin compared with those matured with the use of other cocktails. In vitro migration assay showed that DCs matured with the use of this maturation cocktail had significantly increased migratory ability compared with αDC1s and other DCs. CONCLUSIONS: Interferon-γ, IL-1ß and Poly-I:C maturation cocktail may be used in the field of cancer immunotherapy to generate potent immune-stimulatory DCs with improved type 1 immune response and migration capacity.

Dendritic Cell-Based Cancer Immunotherapy against Multiple Myeloma: From Bench to Clinic.

Although the introduction of stem cell transplantation and novel agents has improved survival, multiple myeloma (MM) is still difficult to cure. Alternative approaches are clearly needed to prolong the survival of patients with MM. Dendritic cell (DC) therapy is a very promising tool immunologically in MM. We developed a method to generate potent DCs with increased Th1 polarization and migration ability for inducing strong myeloma-specific cytotoxic T lymphocytes. In this review, we discuss how the efficacy of cancer immunotherapy using DCs can be improved in MM.

Generation of multiple peptide cocktail-pulsed dendritic cells as a cancer vaccine.

Cancer immunotherapy based on dendritic cell (DC) vaccination has promising alternatives for the treatment of cancer. A central tenet of DC-based cancer immunotherapy is the generation of antigen-specific cytotoxic T lymphocyte (CTL) response. Tumor-associated antigens (TAA) and DC play pivotal roles in this process. DCs are well known to be the most potent antigen-presenting cells and have the most powerful antigen-presenting capacity. DCs pulsed with various TAA have been shown to be effective in producing specific antitumor effects both in vitro and in vivo. Several types of tumor antigens have been applied in cancer treatment including tumor RNA, lysates, apoptotic bodies, heat shock protein, peptides from TAA, and allogeneic tumor cells. Among them, the use of immunogenic HLA-A*0201-specific epitopes from multiple TAA enhances induction of antigen-specific CTL and associated therapeutic efficacy in HLA-A*0201(+) cancer patients. The current chapter provides a detailed protocol of generating multiple peptide cocktail-pulsed DC to elicit CTL with a broad spectrum of immune responses against the related tumor antigens.

Dendritic cells loaded with myeloma cells pretreated with a combination of JSI-124 and bortezomib generate potent myeloma-specific cytotoxic T lymphocytes in vitro.

Signal transducer and activator of transcription 3 (STAT3) is highly activated in multiple myeloma. Activated STAT3 promotes survival and proliferation of cancer cells, suppresses Th1 immune response, and induces dysfunction of immune cells. We investigated whether pretreating myeloma cells with a phosphor (p)-STAT3 inhibitor (JSI-124) and/or bortezomib before loading into dendritic cells (DCs) can affect DC function. The combination treatment with JSI-124 and bortezomib resulted in the highest expression of heat shock protein (HSP) 90 and the lowest expression of p-STAT3 in dying myeloma cells. DCs loaded with dying myeloma cells treated by JSI-124 and bortezomib produced the least amount of p-STAT3 compared to other treatments. The DCs were recovered from abnormal cytokine secretions of interleukin (IL)-10, IL-6, and IL-23 without any effect on production of IL-12p70. DCs loaded with JSI-124 and bortezomib treated, dying myeloma cells most potently generated myeloma-specific cytotoxic T lymphocytes (CTLs). The data suggest that pretreatment of myeloma cells with JSI-124 and bortezomib can recover DC function through the up-regulation of HSP90 and the down-regulation of p-STAT3 and inhibitory cytokines, and that these DCs can potently generate myeloma-specific CTLs.

Presence of dendritic cells in chicken spleen cell preparations and their functional interaction with the parasite Toxoplasma gondii.

Toxoplasmosis is a worldwide epizootic disease of mammals. Chickens, albeit being less susceptible, can be contaminated in free-range flocks and may have an important role in parasite transmission. Plastic adherence selection of chicken spleen cells enriched 8F2+ (putative chicken CD11c) MHC II+ cells of the myeloid type; however, we did not succeed to separate dendritic cells from macrophages using their feature to become loosely adherent after culture as in mammals. Still we clearly identified dendritic-like cells being morphologically distinguishable from macrophages in the KUL01 (macrophage marker) negative fraction, exhibiting responsiveness to LPS and parasite extracts by developing characteristic cellular protrusions as well as a minor phagocytic incorporation of dead parasites. Live T. gondii tachyzoites were able to invade the two different types of myeloid adherent cells, to replicate, and to induce an overall decrease in the expression of MHC II and co-stimulatory molecules, CD80 and CD40. Our data indicate that dendritic cells in addition to macrophages may have a role in hiding viable replicating T. gondii tachyzoites from the immune system and in shuttling them to different organs in the chicken as previously described for different Apicomplexa infecting mammals.