Upregulation of Myc promotes the evasion of NK cell­mediated immunity through suppression of NKG2D ligands in K562 cells.

c­Myc is a characteristic oncogene with dual functions in cell proliferation and apoptosis. Since the overexpression of the c­Myc proto­oncogene is a common event in the development and growth of various human types of cancer, the present study investigated whether oncogenic c­Myc can alter natural killer (NK) cell­mediated immunity through the expression of associated genes, using PCR, western blotting and flow cytometry assays. Furthermore, whether c­Myc could influence the expression levels of natural killer group 2 member D (NKG2D) ligands, which are well known NK activation molecules, as well as NK cell­mediated immunity, was investigated. c­Myc was inhibited by 10058­F4 treatment and small interfering RNA transfection. Upregulation of c­Myc was achieved by transfection with a pCMV6­myc vector. The inhibition of c­Myc increased MHC class I polyeptide­related sequence B and UL16 binding protein 1 expressions among NKG2D ligands, and the overexpression of c­Myc suppressed the expression of all NKG2D ligands, except MHC class I polyeptide­related sequence A. Furthermore, the alteration of c­Myc activity altered the susceptibility of K562 cells to NK cells. These results suggested that the overexpression of c­Myc may contribute to the immune escape of cancer cells and cell proliferation. Combined treatment with NK­based cancer immunotherapy and inhibition of c­Myc may achieve improved therapeutic results.

Combination Treatment of Stereotactic Body Radiation Therapy and Immature Dendritic Cell Vaccination for Augmentation of Local and Systemic Effects.

PURPOSE: The purpose of this study was to investigate the efficacy of stereotactic body radiation therapy (SBRT) as a tumor-associated antigen (TAA) presentation method for dendritic cell (DC) sensitization and evaluate its effect in combination with immunotherapy using an intratumoral injection of immature DCs (iDCs). METHODS AND MATERIALS: CT-26 colon carcinoma cell was used as a cancer cell line. Annexin V staining and phagocytosis assays were performed to determine the appropriate radiation dose and incubation time to generate TAAs. BALB/c mice were used for in vivo experiments. Cancer cells were injected into the right legs and left flanks to generate primary and metastatic tumors, respectively. The mice were subjected to radiation therapy (RT) alone, intradermal injection of electroporated DCs alone, or RT in combination with iDC intratumoral injection (RT/iDC). Tumor growth measurement and survival rate analysis were performed. Enzyme-linked immunospot and cytotoxicity assays were performed to observe the effect of different treatments on the immune system. RESULTS: Annexin V staining and phagocytosis assays showed that 15 Gy radiation dose and 48 hours of incubation was appropriate for subsequent experiments. Maximum DC sensitization and T-cell stimulation was observed with RT as compared to other TAA preparation methods. In vivo assays revealed statistically significant delay in the growth of both primary and metastatic tumors in the RT/iDC group. The overall survival rate was the highest in the RT/iDC group. CONCLUSION: The combination of SBRT and iDC vaccination may enhance treatment effects. Clinical trials and further studies are warranted in the future.

Expansion of cytotoxic natural killer cells using irradiated autologous peripheral blood mononuclear cells and anti-CD16 antibody.

Natural killer (NK) cells are considered a promising strategy for cancer treatment. Various methods for large-scale NK cell expansion have been developed, but they should guarantee that no viable cells are mixed with the expanded NK cells because most methods involve cancer cells or genetically modified cells as feeder cells. We used an anti-CD16 monoclonal antibody (mAb) and irradiated autologous peripheral blood mononuclear cells (PBMCs) (IrAPs) to provide a suitable environment (activating receptor-ligand interactions) for the NK cell expansion. This method more potently expanded NK cells, and the final product was composed of highly purified NK cells with lesser T-cell contamination. The expanded NK cells showed greater upregulation of various activation receptors, CD107a, and secreted larger amounts of interferon gamma. IrAPs expressed NKG2D ligands and CD48, and coengagement of CD16 with NKG2D and 2B4 caused potent NK cell activation and proliferation. The expanded NK cells were cytotoxic toward various cancer cells in vitro and in vivo. Moreover, irradiation or a chemotherapeutic drug further enhanced this antitumor effect. Therefore, we developed an effective in vitro culture method for large-scale expansion of highly purified cytotoxic NK cells with potent antitumor activity using IrAPs instead of cancer cell-based feeder cells.

Enhancement of antitumor immunity by combination of anti-CTLA-4 antibody and radioimmunotherapy through the suppression of Tregs.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is expressed during cluster of differentiation (CD)4+ T-cell activation and terminates immune responses by interrupting CD28-enhanced activation. In addition, CTLA-4 is known to be constitutively expressed in regulatory T-cells (Tregs) and to contribute to immune suppression by enhancing the suppressive function of Tregs. However, the molecular mechanisms underlying CTLA-4-mediated Treg suppression remains incompletely understood. Furthermore, it is uncertain whether the in vivo immune suppressive functions of CTLA-4 are mediated only by a reduction in the level of conventional T-cell activity, or enhancement of Treg function. The present study demonstrated that combination therapy with an anti-CTLA-4 monoclonal antibody and dendritic cell-mediated radioimmunotherapy (IR/DC) was able to promote an antitumor response and influence Treg function in a mouse model of lung cancer. Cell surface markers, including CTLA-4, CD25 and CD4, were analyzed using flow cytometry, and T-cell activities were measured using ELISPOT and cytotoxicity assays. It was revealed that anti-CTLA-4 combined treatment with IR/DC immunotherapy may execute a more powerful and effective anti-tumor immunity through the inhibition of Treg function.

Combination treatment with decitabine and ionizing radiation enhances tumor cells susceptibility of T cells.

Decitabine has been found to have anti-metabolic and anti-tumor activities in various tumor cells. Recently, the use of decitabine in combination with other conventional therapies reportedly resulted in improved anti-tumor activity against various tumors. Ionizing radiation (IR) is widely used as a cancer treatment. Decitabine and IR improve immunogenicity and susceptibility of tumor cells to immune cells by up-regulating the expression of various molecules such as major histocompatibility complex (MHC) class I; natural-killer group 2, member D (NKG2D) ligands; and co-stimulatory molecules. However, the effects of combining decitabine and IR therapies are largely unknown. Our results indicate that decitabine or IR treatment upregulates MHC class I, along with various co-stimulatory molecules in target tumor cells. Furthermore, decitabine and IR combination treatment further upregulates MHC class I, along with the co-stimulatory molecules, when compared to the effect of each treatment alone. Importantly, decitabine treatment further enhanced T cell-mediated cytotoxicity and release of IFN- γ against target tumor cells which is induced by IR. Interestingly, decitabine did not affect NKG2D ligand expression or NK cell-mediated cytotoxicity in target tumor cells. These observations suggest that decitabine may be used as a useful immunomodulator to sensitize tumor cells in combination with other tumor therapies.

Enhanced dendritic cell-based immunotherapy using low-dose cyclophosphamide and CD25-targeted antibody for transplanted Lewis lung carcinoma cells.

Regulatory T cells (Tregs) is one of the main obstacles to the success of cancer immunotherapy. The effect of dendritic cell (DC)-based immunotherapy can be attenuated by immune suppressive functions of Tregs. We used a CD25-targeted antibody and low-dose cyclophosphamide (CTX) as immunomodulators to increase the antitumor effect of intratumoral injection of immature DCs into the irradiated tumor cells (IR/iDC). CTX or CD25-targeted antibody alone showed a significant reduction in the number of Tregs within the tumor microenvironment. When they are combined with IR/iDC, the number of Tregs was further reduced. Although IR/IDC showed strong antitumor effects such as reduction in tumor growth, increase in Th1 immune response, and improvement of survival, the therapeutic effect was further improved by combining treatments with immunomodulators. CTX and CD25-targeted antibody showed no significant difference in tumor growth when combined with IR/iDC, but CTX further increased the number of interferon (IFN)-γ-secreting T cells, cytotoxicity, and survival rate. Although irradiation induced depletion of T lymphocytes, administration of DCs recovered this depletion. Particularly, the lymphocytes were more significantly increased when CTX and IR/iDC were combined. Low-dose CTX has already been used as an immunomodulator in clinical trials, and it offers several advantages, including convenience, low-cost, and familiarity to clinicians. However, CD25-targeted antibody cannot only deplete Tregs, but also may affect IL-2-dependent effector T lymphocytes. Therefore, CTX is an effective means to inhibit Tregs, and an effective immunomodulatory agent for multimodality therapy such as combination treatment of conventional cancer therapy and immunotherapy.

Combination effect of regulatory T-cell depletion and ionizing radiation in mouse models of lung and colon cancer.

PURPOSE: To investigate the potential of low-dose cyclophosphamide (LD-CTX) and anti-CD25 antibody to prevent activation of regulatory T cells (Tregs) during radiation therapy. METHODS AND MATERIALS: We used LD-CTX and anti-CD25 monoclonal antibody as a means to inhibit Tregs and improve the therapeutic effect of radiation in a mouse model of lung and colon cancer. Mice were irradiated on the tumor mass of the right leg and treated with LD-CTX and anti-CD25 antibody once per week for 3 weeks. RESULTS: Combined treatment of LD-CTX or anti-CD25 antibody with radiation significantly decreased Tregs in the spleen and tumor compared with control and irradiation only in both lung and colon cancer. Combinatorial treatments resulted in a significant increase in the effector T cells, longer survival rate, and suppressed irradiated and distal nonirradiated tumor growth. Specifically, the combinatorial treatment of LD-CTX with radiation resulted in outstanding regression of local and distant tumors in colon cancer, and almost all mice in this group survived until the end of the study. CONCLUSIONS: Our results suggest that Treg depletion strategies may enhance radiation-mediated antitumor immunity and further improve outcomes after radiation therapy.

Radiation-induced matrix metalloproteinases limit natural killer cell-mediated anticancer immunity in NCI-H23 lung cancer cells.

Radiotherapy has been used to treat cancer for >100 years and is required by numerous patients with cancer. Ionizing radiation effectively inhibits the growth of cancer cells by inducing cell death and increasing anticancer immunity, through the induction of natural killer group 2 member D ligands (NKG2DLs); however, adverse effects have also been reported, including the promotion of metastasis. Matrix metalloproteinases (MMPs) are induced by ionizing radiation and have an important role in the invasion and metastasis of cancer cells. Previously, MMPs were demonstrated to increase the shedding of NKG2DLs, which may reduce the surface expression of NKG2DLs on cancer cells. As a consequence, the cancer cells may escape natural killer (NK)­mediated anticancer immunity. In the present study, NCI­H23 human non­small cell lung cancer cells were used to investigate the combined effects of ionizing radiation and MMP inhibitors on the expression levels of NKG2DLs. Ionizing radiation increased the expression of MMP2 and ADAM metalloproteinase domain 10 protease, as well as NKG2DLs. The combined treatment of ionizing radiation and MMP inhibitors increased the surface expression levels of NKG2DLs and resulted in the increased susceptibility of the cancer cells to NK­92 natural killer cells. Furthermore, soluble NKG2DLs were increased in the media by ionizing radiation and blocked by MMP inhibitors. The present study suggests that radiotherapy may result in the shedding of soluble NKG2DLs, through the induction of MMP2, and combined treatment with MMP inhibitors may minimize the adverse effects of radiotherapy.

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells.

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.

Antitumor effect of dendritic cell loaded ex vivo and in vivo with tumor-associated antigens in lung cancer model.

Various ex vivo or in vivo loading protocols have been developed or evaluated for the delivery of tumor antigens to dendritic cells (DCs). We compared the antitumor effect of mature DCs electroporation-pulsed (EP/mDC) ex vivo with tumor cell lysate and immature DCs (iDCs) injected into the tumor apoptosed by ionizing radiation (IR/iDC) in lung cancer model. DCs were generated from bone marrow of C57BL/6 mice. Ionizing radiation (IR) was applied at a dose of 10 Gy to the tumor on the right thigh. iDCs were intratumorally injected into the irradiated tumor and EP/mDC was injected subcutaneously in the right flank. DC injection induced strong tumor-specific immunity against Lewis lung carcinoma, as compared with the tumor-bearing control and IR only treated mice. The growth of a distant tumor on the right and left flank was inhibited by IR/iDC and EP/mDC. Particularly, IR/iDC resulted in a more significant inhibition of tumor growth and prolonged survival time. It was related to increase of tumor-specific interferon-gamma, cytotoxicity, and decrease of regulatory T-cells. The results indicate that DCs electroporation-pulsed with tumor cell lysate induce a potent antitumor effect, but that iDCs intratumoral injected into the irradiated tumor induce a more potent antitumor effect.

Synergistic enhancement of NK cell-mediated cytotoxicity by combination of histone deacetylase inhibitor and ionizing radiation.

BACKGROUND: The overexpression of histone deacetylase (HDAC) and a subsequent decrease in the acetylation levels of nuclear histones are frequently observed in cancer cells. Generally it was accepted that the deacetylation of histones suppressed expression of the attached genes. Therefore, it has been suggested that HDAC might contribute to the survival of cancer cells by altering the NKG2D ligands transcripts. By the way, the translational regulation of NKG2D ligands remains unclear in cancer cells. It appears the modulation of this unclear mechanism could enhance NKG2D ligand expressions and the susceptibility of cancer cells to NK cells. Previously, it was reported that irradiation can increase the surface expressions of NKG2D ligands on several cancer cell types without increasing the levels of NKG2D ligand transcripts via ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related (ATM-ATR) pathway, and suggested that radiation therapy might be used to increase the translation of NKG2D ligands. METHODS: Two NSCLC cell lines, that is, A549 and NCI-H23 cells, were used to investigate the combined effects of ionizing radiation and HDAC inhibitors on the expressions of five NKG2D ligands. The mRNA expressions of the NKG2D ligands were quantitated by multiplex reverse transcription-PCR. Surface protein expressions were measured by flow cytometry, and the susceptibilities of cancer cells to NK cells were assayed by time-resolved fluorometry using the DELFIA® EuTDA cytotoxicity kit and by flow cytometry. RESULTS: The expressions of NKG2D ligands were found to be regulated at the transcription and translation levels. Ionizing radiation and HDAC inhibitors in combination synergistically increased the expressions of NKG2D ligands. Furthermore, treatment with ATM-ATR inhibitors efficiently blocked the increased translations of NKG2D ligands induced by ionizing radiation but did not block the increased ligand translations induced by HDAC inhibitors. The study confirms that increased NKG2D ligand levels by ionizing radiation and HDAC inhibitors could synergistically enhance the susceptibilities of cancer cells to NK-92 cells. CONCLUSIONS: This study suggests that the expressions of NKG2D ligands are regulated in a complex manner at the multilevel of gene expression, and that their expressions can be induced by combinatorial treatments in lung cancer cells.

CTLA-4 blockade enhances antitumor immunity of intratumoral injection of immature dendritic cells into irradiated tumor in a mouse colon cancer model.

Dendritic cells (DCs)-based cancer immunotherapy has been used various strategies to inhibit immune suppressive mechanisms. CD25 antibodies and cyclophosphamide are well-studied immunomodulators through inhibition of regulatory T cells (Treg) and a blockade the immune-checkpoint molecule, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) was recently targeted for immunomodulation. We used anti-CTLA-4 antibody, which is known to induce effective antitumor immunity by facilitating tumor-specific T-cell activation and suppressing Treg cells, as useful immunomodulator to provide a potentiating effect in the intratumoral injection of immature DCs (iDCs) into the irradiated tumor (IR/iDC). Ionizing radiation (IR) was applied at a dose of 10 Gy to the tumor on the right thigh of mice. Then, iDCs were intratumorally injected into the irradiated tumor. Anti-CTLA-4 antibody (100 µg/mouse) was administered intraperitoneally to mice on the same day with every iDCs injection. The growth of distant tumors was inhibited by IR/iDC and this effect was significantly augmented by combination treatment of anti-CTLA-4 antibody. Furthermore, the survival rate of tumor-bearing mice improved more by the combination treatment of anti-CTLA-4 antibody and IR/iDC compared with other groups. It was related to the increased tumor-specific interferon-γ-secreting T cells and CTL activity. Therefore, our results demonstrated that immunomodulator such as anti-CTLA-4 antibody enhances antitumor immunity of intratumoral injection of iDCs into irradiated tumor and suggested a new strategy to get more clinical benefits for cancer treatment.

Enhanced maturation and function of dendritic cells using hydrogel coated plate and antigen electroporation.

Dendritic cells (DCs) are potent antigen-presenting cells that can be matured in vitro from immature dendritic cells (iDCs) in the presence of several biological agents such as cytokine cocktail, CD40L, TNF-a and antigen loading, which are necessary and achieved using various protocols, such as lipofection, passive pulse or electroporation. However, these DCs maturation protocols may cause with a significant loss of cells because of cellular attachment and spreading during culturing. Some biomaterials that influence adhesion and development of cells have been used in cell culture techniques, and it was thought that they might be applied on the culture of DCs. In this study, we used polyHEMA, which is a hydrogel coating biomaterial that prevents DCs from adherence, and investigated whether hydrogel coating affects the maturation of iDCs. The efficiency in the generation of mDCs was improved through hydrogel coating procedure and a dendritic cell maturation marker, CD83, was significantly increased in hydrogel-coated culture condition. The antigen-loaded mDCs from electroporation were further expressed the CD83. The mDCs generated in the hydrogel-coated culture condition showed more, longer and thicker dendrites, and produced more amounts of cytokines such as IL-12 and IFN-γ. Therefore, it was suggested that the hydrogel-coated culture condition could improve function of mDCs. Cheol-Hun Son and Jae-Ho Bae contributed equally to this work.

The novel resveratrol analogue HS-1793 induces apoptosis via the mitochondrial pathway in murine breast cancer cells.

Resveratrol (3,4',5 tri-hydroxystilbene), a natural plant polyphenol, has gained interest as a non-toxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. Several studies were undertaken to obtain synthetic analogues of resveratrol with potent anticancer activity. The aim of the present study was to investigate the effect of HS-1793 as a new resveratrol analog on apoptosis via the mitochondrial pathway in murine breast cancer cells. A pharmacological dose (1.3-20 µM) of HS-1793 exerted a cytotoxic effect on murine breast cancer cells resulting in apoptosis. HS-1793-mediated cytotoxicity in FM3A cells by several apoptotic events including mitochondrial cytochrome c release, activation of caspase-3 and PARP occurred. In addition, HS-1793 induced collapse of ∆Ψm and enhanced AIF and Endo G release from mitochondria while undergoing apoptosis. These results demonstrate that the cytotoxicity by HS-1793 in FM3A cells can mainly be attributed to apoptosis via a mitochondrial pathway by caspase activation or contributions of AIF and Endo G.

Improvement of antitumor effect of intratumoral injection of immature dendritic cells into irradiated tumor by cyclophosphamide in mouse colon cancer model.

Recently, chemotherapy and radiotherapy are known to directly affect some immunosuppressive barriers within a tumor microenviroment. We used cyclophosphamide (CTX), which is known to enhance the immune response by suppressing CD4+CD25+ regulatory T cells (Treg cells) when used at a low dose, as a chemotherapeutic agent to provide a synergic effect in the irradiation and dendritic cells (DC) combination therapy. Some previous studies observed that a single-dose CTX treatment significantly reduced the number of Treg cells in 3-5 days, however, the reduced Treg cells increased rapidly after 5 days. To overcome the disadvantages of a single-dose CTX, we used 30 mg/kg dose of CTX, which was treated intraperitoneally to mice 3 days before every immature DC (iDC) injection (known as "metronomic schedule CTX"). Irradiation was applied at a dose of 10 Gy to the tumor on the right thigh by a linear accelerator. Then, iDC was intratumorally injected into the irradiated tumor site. Growth of a distant tumor on the right and left flank was suppressed by an injection of iDC into the irradiated tumor, and this effect was increased by the metronomic schedule CTX. Also, combinations treated with the metronomic schedule CTX and ionizing radiation (IR)/iDC, showed the longest survival time compared with other groups. This antitumor immune response of IR/iDC was improved by metronomic schedule CTX and this result was associated with decreasing the proportion of CD4+CD25+ Treg cells and increasing the number of tumor-specific interferon-γ-secreting T cells. Our results demonstrated that metronomic schedule CTX improves the antitumor effect of immunization with an injection of DC s into the irradiated tumor.

Cyclophosphamide potentiates the antitumor effect of immunization with injection of immature dendritic cells into irradiated tumor.

Growth of a tumor on the left flank was suppressed by direct injection of immature DCs (iDCs) into the irradiated tumor on the right thigh (IR/DC). This antitumor immune effect of IR/DC was enhanced by pretreatment with CTX (CTX+IR/DC) and this effect was related with increased number of tumor-specific IFN-γ secreting T cells and decreased ratio of CD4(+)CD25(+)/CD4(+) T cells. The treatment with CTX+IR/DC increased or decreased the levels of IL-2 or IL-10, respectively. These results demonstrated that antitumor effect of IR/DC could be augmented by pretreatment with low-dose CTX, suggesting a new antitumor therapeutic modality of chemoradioimmunotherapy.

Quercetin enhances susceptibility to NK cell-mediated lysis of tumor cells through induction of NKG2D ligands and suppression of HSP70.

It is known that treatments with heat shock, some anticancer drugs, and ionizing radiation increase the expression of heat-shock proteins (HSPs) and natural killer group 2D (NKG2D) ligands in tumor cells. The increased HSPs may make the tumor cells resistant to apoptosis and reduction of HSPs may make the tumor cells more susceptible to natural killer (NK)-cell mediated lysis of tumor cells. In this study, we investigated whether quercetin which has inhibitory activities against heat-shock factor, protein kinase C, nuclear factor-kappaB, and phosphatidyl inositol 3-kinase, can modulate the expression of NKG2D ligands and suppress the HSPs in tumor cells. The results of this study showed that quercetin significantly induced the expression of several NKG2D ligands including major histocompatibility complex class I-related chain B, UL16-binding protein 1, and UL16-binding protein 2 in K562, SNU1, and SNU-C4 cells. The quercetin-treated K562, SNU1, and SNU-C4 cells showed an enhanced susceptibility to NK-92 cells through induction of NKG2D ligands. This increased expression of NKG2D ligands seemed to be due to the inhibition of the nuclear factor-kappaB and phosphatidyl inositol 3-kinase pathways. The findings of this study suggest that the induced NKG2D ligands with the decrease of HSP70 protein by quercetin may provide an attractive strategy to improve the effectiveness of NK cell-based cancer immunotherapy.

Phase I study of autologous dendritic cell tumor vaccine in patients with non-small cell lung cancer.

BACKGROUND: A dendritic cell vaccine has been developed as a novel strategy for generating antitumor immunity in the treatment of cancer. The purpose of this study was to assess the maximal tolerated dose, safety, and immunologic response of a new dendritic cell vaccine (DC-Vac) into which tumor lysate was loaded by electroporation and pulse in patients with advanced non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Fifteen patients with inoperable stage III or IV NSCLC were assigned to cohorts that received 3, 6, or 12 × 10(6) DC-Vac intradermally 3 times at 2 week intervals. We also evaluated immunologic and tumor responses. RESULTS: The maximum dose of DC-Vac (12 × 10(6)) was shown to be safe. In 5 of 9 patients, the vaccine resulted in increased interferon (IFN)-γ production by CD8+ cells after exposure to tumor lysate. Additionally, there were mixed responses which do fulfill progressive disease definition but demonstrate some clinical benefit in two patients. CONCLUSION: The administration of tumor lysate-loaded autologous dendritic cells by electroporation and pulse was non-toxic and induced immunologic responses to tumor antigens. The two mixed tumor responses which were achieved may represent a potential benefit of this new DC-Vac.

The cryopreservation of high concentrated PBMC for dendritic cell (DC)-based cancer immunotherapy.

Peripheral blood mononuclear cells (PBMC) have been accepted as a unique material for cancer immunotherapy using dendritic cells (DC) or activated lymphocytes that are being developed as an alternative or adjuvant to conventional therapies such as surgery, chemotherapy and radiation treatment. Although successful cryopreservation of large numbers of PBMC is critical for the immunotherapy, subsequent functional study of the effects of PBMC cryopreservation on differentiation into immune cells has not been well defined. In this study, over 1.0 x 10(8)cells/ml PBMC were cryopreserved as long as 52 weeks using a controlled-rate freezer (CRF) and stored in a vapor phase of liquid nitrogen tank. The effect of PBMC cryopreservation on differentiation into DC was studied by comparing the phenotypic and functional properties of immature DC (iDC) and mature DC (mDC) derived from cryopreserved PBMC to those from fresh PBMC. The results show that cryopreservation of PBMC at a fairly high cell concentration does not significantly affect cell recovery, viability, or phenotypes of PBMC. After differentiation into DC, iDC and mDC derived from cryopreserved PBMC had their typical phenotypes and function equivalent to those derived from fresh PBMC. Therefore, the improved cryopreservation process of PBMC described in this study is available for DC-based cancer immunotherapy.