Overall clinical course of indeterminate dendritic cell tumor patients without skin lesions:A rare case report

BACKGROUND Indeterminate dendritic cell tumor(IDCT)is a rare tumor of immune cells,and IDCT patients without skin lesions are rarely reported.Therefore,the clinical course in this type of patient is unclear,and further research on the underlying pathological mechanisms and appropriate treatments is needed.CASE SUMMARY This study describes a female IDCT patient with bile duct lesions.The strong mimicry of IDCT lesions confused doctors,and consequently,this patient,who had no skin lesions,was first diagnosed with cholangiocarcinoma.Then,she presented with persistent abdominal distension without jaundice.Enlarged mesenteric lymph nodes along with massive ascites were observed in the subsequent imaging examination.However,no tumor cells or pathogens were found in the three subsequent ascites analyses.It took 2 years to reach the correct diagnosis,which was eventually obtained by performing surgery for biopsy of the patient’s abdominal lymph nodes.However,by then,she was already in a cachexic state.Finally,she received a cycle of cyclophosphamide therapy and was advised to visit a hospital specializing in rare diseases.CONCLUSION For IDCT patients without skin lesions,early biopsy is the key to obtaining a correct diagnosis.Moreover,the collective management of IDCT patients is important.Further histological and molecular biology studies based on human specimens are critical for understanding the pathological mechanism of dendritic cell tumors in the future.

Tumor-derived DEFB1 induces immune tolerance by inhibiting maturation of dendritic cell and impairing CD8+T cell function in esophageal squamous cell carcinoma

Objective:CD8+T cells are the key effector cells in the anti-tumor immune response.The mechanism underlying the infiltration of CD8+T cells in esophageal squamous cell carcinoma(ESCC)has not been clearly elucidated.Methods:Fresh ESCC tissues were collected and grouped according to the infiltration density of CD8+T cells.After the transcriptome sequencing on these samples and the combined analyses with The Cancer Genome Atlas(TCGA)ESCC data,a secreted protein DEFB1 was selected to explore its potential role in the infiltration of CD8+T cells.Bioinformatics analyses,histological verification and in vitro experiments were then performed.Results:DEFB1 was highly expressed in ESCC,and the high expression of DEFB1 was an independent risk factor for overall survival.Since the up-regulation or down-regulation of DEFB1 did not affect the proliferation,migration and apoptosis of ESCC cells,we speculated that the oncogenic effect of DEFB1 was achieved by regulating microenvironmental characteristics.Bioinformatics analyses suggested that DEFB1 might play a major role in the inflammatory response and anti-tumor immune response,and correlate to the infiltration of immature dendritic cell(imDC)in ESCC.Histological analyses further confirmed that there were less CD8+T cells infiltrated,less CD83+mature DC(mDC)infiltrated and more CD1a+imDC infiltrated in those ESCC samples with high expression of DEFB1.After the treatment with recombinant DEFB1 protein,the maturation of DC was hindered significantly,followed by the impairment of the killing effects of T cells in both 2D and 3D culture in vitro.Conclusions:Tumor-derived DEFB1 can inhibit the maturation of DC and weaken the function of CD8+T cells,accounting for the immune tolerance in ESCC.The role of DEFB1 in ESCC deserves further exploration.

Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA

Objective: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. Methods: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. Results: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccination with DC/extract or DC/RNA, either prior to G422 tumor challenge or in tumor-harboring mice, significantly prolonged survival compared with other control groups (P<0.01). Conclusion: DCs pulsed with tumor extracts or RNA derived from autologous tumors has potential antitumor effects via activation of cell-mediated immunity. Our results suggest a useful therapeutic strategy against gliomas.

Revolutionizing tumor immunotherapy:unleashing the power of progenitor exhausted T cells

In exploring persistent infections and malignancies, a distinctive subgroup of CD8^(+) T cells, progenitor exhausted CD8^(+) T(Tpex) cells, has been identified. These Tpex cells are notable for their remarkable self-renewal and rapid proliferation abilities. Recent strides in immunotherapy have demonstrated that Tpex cells expand and differentiate into responsive exhausted CD8^(+) T cells, thus underscoring their critical role in the immunotherapeutic retort. Clinical examinations have further clarified a robust positive correlation between the proportional abundance of Tpex cells and enhanced clinical prognosis. Tpex cells have found noteworthy applications in the formulation of inventive immunotherapeutic approaches against tumors. This review describes the functions of Tpex cells in the tumor milieu, particularly their potential utility in tumor immunotherapy. Precisely directing Tpex cells may be essential to achieving successful outcomes in immunotherapy against tumors.

Experimental study on dendritic cells pulsed with brain tumor stem cells for immunotherapy of glioma

Objective To investigate the effect of dendritic cells pulsed with brain tumor stem cells which are used to treat on intracranial glioma. Methods We obtained murine brain tumor stem cells by grow ing C6 cells in epidermal grow th factor/basic fibroblast grow th factor w ithout serum.Dendritic cells isolated from rat bone marrow w ere pulsed w ith BTSCs. Rat brain

EXPRESSION AND SIGNIFICANCE OF TUMOR INFILTRATING DENDRITIC CELLS IN RENAL CELL CARCINOMA

Objective: To study the expression of dendritic cells in human renal cell carcinoma and explore the cause, so to reveal the mechanism of escaping immune surveillance in RCC. Methods: The expressions of CD83+DCS, CD1a+DCS,VEGF and TGF-β1 in tumoral, peritumoral and normal kidney tissues of RCC in 30 cases were detected by immunohistochemistry using streptavidin/peroxidese(SP) Results: CD83+DCS were mainly located in the peritumoral areas; whereas CD1a+DCS、were mainly retained within the cancer nests. The number of CD83+DCS was inversely correlated with the clinical stage(P<0.05); but there were no significant correlations between the number of CD1a+DCS、and the clinical stage(P>0.05). The expressions of CD83+DCS and CD1a+DCS have significant difference between the tumoral, peritumoral and normal kidney tissues(P<0.001). The expression of VEGF and TGF-β1 were significantly lower in samples with highly infiltrating CD83+DCS(P<0.05); Whereas CD1a+DCS were not (P>0.05). Conclusion: DC has the tendency to gathering in tumor, but because of the immunosuppressive cytokins, for example VEGF and TGF-β1, inhibits the maturation of DC, there are less mature TIDCS(CD83+TIDCS) in the tumoral tissues, they are mainly located in the peritumoral areas. This may contribute to the mechanism of escaping immune surveillance in RCC.

Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-ulsed dendritic cells and cytokine-induced killer cells

AIM： To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells （DCs） and cytokine-induced killer cells. METHODS： Freshly collected hepatocellular carcinoma （HCC） tumor tissues were incubated with a mixture of neuraminidase and recombinant αl,3-galactosyltrans- ferase （αI,3GT） to synthesize α-Gal epitopes on car- bohydrate chains of the glycoproteins of tumor mem- branes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage 111 primary HCC were randomly chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS： The evaluation demonstrated that the pro- cedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly pro- longed the survival of treated patients as compared with the controls （17.1 ± 2.01 mo vs 10.1 ±4.5 mo, P = 0.00121）. After treatment, all patients in the study group had positive delayed hypersensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-y-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO- and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the se- rum. CONCLUSION： This new tumor-specific immunotherapy is safe, effective and has a great potential for the treat- ment of tumors.

Follicular dendritic cell sarcoma of the liver:unusual presentation of a rare tumor and literature review

BACKGROUND:Hepatic follicular dendritic cell (FDC) sarcoma is an extremely rare neoplasm.Most commonly,FDC sarcoma presents as a solitary mass in lymph nodes,however,several extra-nodal locations have been identified.METHODS:We report a case of a 53-year-old female who presented with symptoms of abdominal pain,fever,anemia,and jaundice.After an extensive review of the literature,we have found only 12 cases of hepatic FDC sarcoma.RESULTS:The tumor was 11.5 cm in diameter and composed of spindle and epithelioid cells with ovoid nuclei and associated with mixed inflammatory infiltrate.Immunohistochemical stains were positive for CD35 and CD21.The patient underwent a left hepatic lobectomy.CONCLUSIONS:Liver follicular dendritic cell sarcoma is a very rare tumor.Most cases present with abdominal pain and weight loss,and most of them can be managed by hepatic resection with excellent short-term outcomes.

Heat-shocked tumor cell lysate-pulsed dendritic cells induce effective anti-tumor immune response in vivo

AIM. To study whether heat-shocked tumor cells could enhance the effect of tumor cell lysate-pulsed dendritic cells （DCs） in evoking anti-tumor immune response in vivo. METHODS： Mouse undifferentiated colon cancer cells （CT-26） were heated at 42℃ for 1 h and then frozenthawed. The bone marrow-derived DCs pulsed with heatshocked CT-26 cell lysate （HSCT-26 DCs） were recruited to immunize syngeneic naive BALB/c mice. The cytotoxic activity of tumor specific cytotoxic T lymphocytes （CTLs） in mouse spleen was evaluated by IFN-enzyme-linked immunospot （ELISpot） and LDH release assay. The immunoprophylactic effects induced by HSCT-26 DCs in mouse colon cancer model were compared to those induced by single CT-26 cell lysate-pulsed DCs （CT-26 DCs） on tumor volume, peritoneal metastasis and survival time of the mice. RESULTS： Heat-treated CT-26 cells showed a higher hsp70 protein expression. Heat-shocked CT-26 cell lysate pulsing elevated the co-stimulatory and MHC-Ⅱ molecule expression of bone marrow-derived DCs as well as interleukin-12 p70 secretion. The IFN-y secreting CTLs induced by HSCT-26 DCs were significantly more than those induced by CT-26 DCs （P=0.002）. The former CTLs＇ specific cytotoxic activity was higher than the latter CTLs＇ at a serial E/T ratio of 10：1, 20：1, and 40：1. Mouse colon cancer model showed that the tumor volume of HSCT-26 DC vaccination group was smaller than that of CT-26 DC vaccination group on tumor volume though there was no statistical difference between them （24 mm^3 vs 8 mm^3, P=0.480）. The median survival time of mice immunized with HSCT-26 DCs was longer than that of those immunized with CT-26 DCs （57 d vs 43 d, P = 0.0384）. CONCLUSION： Heat-shocked tumor cell lysate-pulsed DCs can evoke anti-tumor immune response in vivo effectively and serve as a novel DC-based tumor vaccine.

Dendritic cell-based immunotherapy for malignant glioma

The immunotherapy for malignant glioma faces unique difficult, due to some anatomical and immunological characteristics including the existence of blood brain barrier, the absence of lymphatic tissues and dendritic cells （DCs） in the central nervous system （CNS） parenchyma, and the presence of an immunosuppressive microenvironment. Therefore, immunothera-peutic approaches will not be beneficial unless the compromised immune status in malignant glioma patients is overcome. DC-based immunotherapy, vaccinating cancer patients with DCs pulsed with various tumor antigens, is one of the most promising immunotherapeutic approaches for treatment of malignantglioma because it seems able to overcome, at least partially, the immunosuppressive state associated with primary malignancies. The preparation of DCs, choice of antigen, and route and schedule of administration are improving and optimizing with rapid development of molecular biology and gene engineering technology. DC vaccination in humans, after a number of pre-clinical models and clinical trials, would increase the clinical benefits for malignant glioma immunotherapy.

Antitumor immunity by a dendritic cell vaccine encoding secondary lymphoid chemokine and tumor lysate on murine prostate cancer

Aim： To investigate the antitumor immunity by a dendritic cell （DC） vaccine encoding secondary lymphoid chemokine gene and tumor lysate on murine prostate cancer. Methods： DC from bone marrow of C57BL/6 were transfected with a plasmid vector expressing secondary lymphoid chemokine （SLC） cDNA by Lipofectamine2000 liposome and tumor lysate. Total RNA extracted from SLC＋lysate-DC was used to verify the expression of SLC by reverse transcriptase-polymerase chain reaction （RT-PCR）. The immunotherapeutic effect of DC vaccine on murine prostate cancer was assessed. Results： We found that in the prostate tumor model of C57BL/6 mice, the adminstration of SLC＋lysate-DC inhibited tumor growth most significantly when compared with SLC-DC, lysate-DC, DC or phos- phate buffer solution （PBS） counterparts （P 〈 0.01）. Immunohistochemical fluorescent staining analysis showed the infiltration of more CD4＋, CD8＋ T cell and CD11c＋ DC within established tumor treated by SLC＋lysate-DC vaccine than other DC vaccines （P 〈 0.01）. Conclusion： DC vaccine encoding secondary lymphoid chemokine and tumor lysate can elicit significant antitumor immunity by infiltration of CD4＋, CD8＋ T cell and DC, which might provide a potential immunotherapy method for prostate cancer.

Dendritic cell-based cancer immunotherapy for colorectal cancer

Colorectal cancer(CRC) is one of the most common cancers and a leading cause of cancer-related mortality worldwide. Although systemic therapy is the standard care for patients with recurrent or metastatic CRC, the prognosis is extremely poor. The optimal sequence of therapy remains unknown. Therefore, alternative strategies, such as immunotherapy, are needed for patients with advanced CRC. This review summarizes evidence from dendritic cell-based cancer immunotherapy strategies that are currently in clinical trials. In addition, we discuss the possibility of antitumor immune responses through immunoinhibitory PD-1/PD-L1 pathway blockade in CRC patients.

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency

AIM： To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells （DC） and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 （EC109）. METHODS： The fusion vaccine was produced by fusing traditional polyethyleneglycol （PEG）, inducing cytokine, sorting CD34＋ magnetic microbead marker and magnetic cell system （MACS）. The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution （PBS）, inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups： P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells.RESULTS： Flow cytometry showed that the expression of folate receptor （FR）, EC109 （C）, Des （D） in human nasopharyngeal carcinoma cell line （HNE1） （B） was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells （C） were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes （PBL）. In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups. CONCLUSION： Fusion cells are highly expressed not only in FR but also in CD80. The fusion vaccine has a distinctive protective effect against tumor EC109 and can inhibit the growth of tumor in mice, and its immune protection against tumor attack is more significant.

The current role of dendritic cells in the progression and treatment of colorectal cancer

Colorectal cancer(CRC)is the third most common cancer and the second leading cause of cancer-related deaths worldwide.Dendritic cells(DCs)constitute a heterogeneous group of antigen-presenting cells that are important for initiating and regulating both innate and adaptive immune responses.As a crucial component of the immune system,DCs have a pivotal role in the pathogenesis and clinical treatment of CRC.DCs cross-present tumor-related antigens to activate T cells and trigger an antitumor immune response.However,the antitumor immune function of DCs is impaired and immune tolerance is promoted due to the presence of the tumor microenvironment.This review systematically elucidates the specific characteristics and functions of different DC subsets,as well as the role that DCs play in the immune response and tolerance within the CRC microenvironment.Moreover,how DCs contribute to the progression of CRC and potential therapies to enhance antitumor immunity on the basis of existing data are also discussed,which will provide new perspectives and approaches for immunotherapy in patients with CRC.

Enhancing Antitumor by Immunization with Fusion of Dendritic Cells and Engineered Tumor Cells

A novel approach for a dentritic cells (DCs) based tumor vaccine was developed for the formation of hybrid engineered J558 after fusion with DCs. To make the hybrid tumor vaccine generate more efficient specific CTL cytotoxicity against wild type tumor cells, we genetically engineered tumor cells with mIL 12 gene prior to the cell fusion. mIL 12 was detected at 870±60 pg/(10 5 cells/ml) in the culture supernatants and the fusion ratio was about 30 % by the co focal microscopic analysis. Vaccination of mice with DCs fused with engineered J558 induced more efficient tumor specific CTL cytotoxicity against wild type tumor cells in vitro and with efficient antitumor immunity in vivo . These results suggest that this approach of using DCs fused with engineered tumor cells could be applied in clinical settings of DCs based cancer vaccines.

Emerging roles of plasmacytoid dendritic cell crosstalk in tumor immunity

Plasmacytoid dendritic cells(pDCs)are a pioneer cell type that produces type I interferon(IFN-I)and promotes antiviral immune responses.However,they are tolerogenic and,when recruited to the tumor microenvironment(TME),play complex roles that have long been a research focus.The interactions between p DCs and other components of the TME,whether direct or indirect,can either promote or hinder tumor development;consequently,p DCs are an intriguing target for therapeutic intervention.This review provides a comprehensive overview of p DC crosstalk in the TME,including crosstalk with various cell types,biochemical factors,and microorganisms.An in-depth understanding of p DC crosstalk in TME should facilitate the development of novel p DC-based therapeutic methods.

Surgical treatment of liver inflammatory pseudotumor-like follicular dendritic cell sarcoma: A case report

BACKGROUND Inflammatory pseudotumor-like follicular dendritic cell sarcoma(IPT-like FDCS)is rare with a low malignant potential.Hepatic IPT-like FDCS has similar clinical features to hepatocellular carcinoma(HCC),making it extremely difficult to distinguish between them in clinical practice.We describe the case of a young female patient diagnosed with HCC before surgery,which was pathologically diagnosed as IPT-like FDCS after the left half of the liver was resected.During 6 mo of follow-up,the patient recovered well with no signs of recurrence or metastasis.CASE SUMMARY A 23-year-old female patient with a 2-year history of hepatitis B presented to the Affiliated Hospital of Guizhou Medical University.She was asymptomatic at presentation,and the findings from routine laboratory examinations were normal except for slightly elevated alpha-fetoprotein levels.However,ultrasonography revealed a 3-cm diameter mass in the left hepatic lobe,and abdominal contrastenhanced computed tomography revealed that the tumor had asymmetrical enhancement during the arterial phase,which declined during the portal venous phase,and had a pseudo-capsule appearance.Based on the findings from clinical assessments and imaging,the patient was diagnosed with HCC,for which she was hospitalized and had undergone laparoscopic left hepatectomy.However,the tumor specimens submitted for pathological analyses revealed IPT-like FDCS.After surgical removal of the tumor,the patient recovered.In addition,the patient continued to recover well during 6 mo of follow-up.CONCLUSION Hepatic IPT-like FDCS is difficult to distinguish from HCC.Hepatectomy may provide beneficial outcomes in non-metastatic hepatic IPT-like FDCS.

Tumor Antigen-pulsed CD8α^+ Dendritic Cells Induce T Cell-mediated Graft-versus-tumor Effect In Vitro

The graft-versus-tumor （GVT） effect of T cells induced by tumor antigen-pulsed CD8α＋ dendritic cells （DCs） in vitro was investigated in this study. Immature CD8c（DCs were prepared from C57BL/6 （H-2b） bone marrow cells by using a cytokine cocktail. On the 3rd day of culture, CD8α＋ DCs were pulsed by allogeneic （Balb/c, H-2d） EL9611 leukemia antigen, or RM-1 syngeneic prostate cancer antigen, with the concentration series of 0, 2.5, 5.0, 10.0, 20.0 μg/mL, respectively, then antigen-loaded immature CD8α＋ DCs were co-cultured with syngeneic T cells according to the DC/T ratio of 1：1, 2：1 and 4：1. T cell proliferation was measured by MTT assay. Cytokines including interferon gamma （IFN-γ） and interleukin-10 （IL-10） in CD8α＋ DCs and T co-culture supernatant were detected by using ELI SA. Cytotoxic effect of antigen-specific T cells was tested by LDH release assay. Conventional mature DCs （mDCs） induced, from C57BL/6 （H-2b） bone marrow cells by using granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-4 （IL-4） served as a control. The results showed that the proliferative activity of T cells stimulated by CD8α＋ DCs loaded with allogeneic or syngeneic tumor antigen was augmented with the CD8＆ DC/T ratio increased （P〈0.05）. When antigen concentration 〈 5 μg/mL and CD8a＋ DC/T ratio 〈 2：1, the ability of CD8α＋ DCs to stimulate T cell proliferation was higher than mDC control in allogeneic tumor antigen-pulsed groups （P〈0.05）, but not in syngeneic tumor antigen-pulsed groups （P〉0.05）. The level of IFN-γ and IL-10 in CD8α＋DCs and T cell co-culture supernatant were increased in both allogeneic and syngeneic antigen-pulsed groups （P〈0.05）, and the cytokine level was higher in allogeneic antigen-pulsed groups than in syngeneic antigen groups when the CD8α＋ DC/T was 1：1 or 2：1 （P〈0.05）. There existed a negative correlation between the level of IL-10 and T cell proliferation. T cell cytotoxieity assay showed that when CD8α＋ DCs were pulsed with allogeneic tumor antigen, the maximal T cell killing efficiency could reach （100±7.7）%, whereas syngeneic tumor antigen-pulsed group had only （65.0±3.4）%. It was concluded that syngeneic and allogeneic tumor antigen-pulsed immature CD8α＋ DCs could stimulate T cells to exert the GVT effect in vitro, and the GVT effect was more obvious with allogeneic tumor antigen than with syngeneic tumor antigen. The optimal condition was low allogeneic tumor antigen pulsation （〈 5 μg/mL） and low CD8α＋ DC/T ratio （1：1 and 2：1）.

Cytotoxic T-lymphocytes response in vitro activated by dendritic cells pulsed with heat shock protein 70 derived from human bladder tumor cell lines of EJ

Objective： To investigate whether human dendritic cells （DC） derived from peripheral blood mononuclear cells （PBMC）, which were pulsed by heat shock protein 70 （HSP70） isolated from human bladder tumor cell lines of E J, were able to induce peptide specific cytotoxic T-lymphocytes （CTL） response in vitro and give the experimental foundation for the future clinical trials of immunotherapy in bladder tumor. Methods： The E J-derived HSP70 co-cultured with DC from the healthy volunteers＇ PBMC, along with the crude lysate （the supematant before HSP70 purification） from EJ cells were used as the experimental groups and DC not pulsed by any tumor cells antigen were the blank control. The autologous T-lymphocytes were added into the above various DC groups, and after incubation, the stimulation indexes （SI） and interferon-y （IFN-γ） were detected to evaluate the immune activities of various DC groups. The killing effects of CTL to target cells, EJ and Hela cells, were determined with 51^Cr releasing test. Results： Both DC/HSP70 and DC/the crude lysate could effectively activate CTL in vitro and kill target cells EJ. The killing effect of DC/HSP70 to EJ was much stronger than DC/the crude lysate （the supernatant before HSP70 purification） （P 〈 0.05）. DC without any tumor cell antigens had a lower killing power to EJ. Meanwhile, DC/ HSP70 had little killing power to Hela non-relevant to bladder tumor histopathologically as compared with EJ cells （P 〈 0.05）. Conclusion： The DC pulsed by HSP70 derived from the autologous tumor cells could induce a peptide complexes specific CTL response to tumor cells, and the CTL response induced by the DC/HSP70 was stronger, which display the basis of the possible clinical application of DC/HSP70 for bladder tumor.

Differential centrifugation enhances the anti-tumor immune effect of tumor lysate-pulsed dendritic cell vaccine against glioblastoma

Objective This study aimed to improve the antitumor immunocompetence of a tumor lysate-pulsed dendritic cell(DC)vaccine through differential centrifugation and provide a theoretical basis for its clinical application in glioblastoma.Methods Peripheral blood mononuclear cells were extracted using Ficoll-Paque PLUS and induced into mature DCs in vitro with a cytokine cocktail.The modified tumor lysate was generated by differential centrifugation.The maturity markers of DCs in each group,namely the modified tumor lysate,tumor lysate,and negative and positive control groups,were assessed using flow cytometry.Furthermore,their ability to stimulate lymphocyte proliferation and in vitro antitumor effects were assessed using Cell Trace TM CFSE.IFN-γsecretion levels were measured with ELISA.Intracellular reactive oxygen species were measured using 2’,7’-dichlorofluorescein diacetate(DCFDA)staining.The results were statistically analyzed using an unpaired Student’s t-test and were considered significant at P<0.05.Results Compared with tumor lysate-pulsed DCs,modified tumor lysate-pulsed DCs had a higher expression of maturity markers:CD1a(7.38±0.53%vs.4.47±0.75%)and CD83(19.81±4.09%vs.9.64±1.50%),were better capable of stimulating lymphocyte proliferation[proliferation index(PI):8.54±0.16 vs.7.35±0.05],secreting IFN-γ,and inducing stronger in-vitro cytotoxic T lymphocyte(CTL)cytotoxicity against glioblastoma cells.In addition,we found that the level of ROS in modified tumor lysate-pulsed DCs was lower than that in tumor lysate-pulsed DCs.Conclusion Differential centrifugation of tumor lysates can improve the antitumor immunocompetence of DC vaccines,and reactive oxygen species may be the key to affecting DC function in the whole tumor lysate.