Fusion Of Dendritic Cells Research Articles

Safety Evaluation and Preliminary Flow Cytometrical Analysis in Dogs With Oral Melanoma Treated with An Immunotherapeutic Dendritic Cell Fusion Vaccine and Concurrent Radiotherapy

Safety Evaluation and Preliminary Flow Cytometrical Analysis in Dogs With Oral Melanoma Treated with An Immunotherapeutic Dendritic Cell Fusion Vaccine and Concurrent Radiotherapy

IMMU-12. PHASE I/II TRIAL OF IMMUNOTHERAPY WITH FUSIONS OF DENDRITIC CELLS AND TUMOR CELLS FOR RELAPSED OR REFRACTORY BRAIN TUMORS IN CHILDREN AND YOUNG ADULTS

BACKGROUND/OBJECTIVESRelapsed or refractory brain tumors in childhood continue to have a dismal prognosis in spite of intensive multidisciplinary treatment. Cancer immunotherapy is newly developed to be expected as next promising treatment for highly aggressive pediatric cancer. This trial was designed to evaluate the safety and effectiveness of an immunotherapy with fusions of dendritic cells (DCs) and tumor cells in patients with malignant brain tumors.METHODSPatients with histopathologically confirmed malignant and recurrent/refractory brain tumor were eligible for this immunotherapy trial. Autologous cultured tumor cells obtained from surgical specimens were fused with autologous DCs using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region and repeated 3–10 times in each 28–84 days cycle. Treatment-related toxicity, progression-free survival (PFS), and overall survival (OS) were evaluated.RESULTSSix patients were enrolled, three with high grade glioma and three with ependymoma. Median age at first course of immunotherapy was 10 years (range 8–25 years) and median follow-up time from the first course of immunotherapy was 13.5 months (range 3–33 months). All patients with immunotherapy were well tolerated to this treatment with no adverse events except local erythema in injected site. Median progression free survival and overall survival were 18 months and 18.5 months, respectively.CONCLUSIONSFC immunotherapy with autologous DCs and tumor cells for brain tumor in children and young adults were extremely well tolerated and showed encouraging responses in this series. Further phase II study of FC immunotherapy is planned to improve survival and reduce treatment related morbidity.

PEDT-03 A clinical trial of dendritic cell-based immunotherapy for refractory brain tumors in children

Background/Objectives: Relapse or refractory brain tumor in childhood continue to have a dismal prognosis in spite of developing multidisciplinary treatment. Cancer immunotherapy is newly expected as next promising treatment for highly aggressive pediatric cancer. This trial was designed to evaluate the safety and clinical responses to an immunotherapy with fusions of dendritic cells (DCs) and tumor cells in patients with malignant brain tumors. Design/Methods: Patients with histopathologically confirmed high-grade or recurrent brain tumor were eligible for our immunotherapy. Autologous cultured tumor cells obtained from surgical specimens were fused with autologous DCs using polyethylene glycol. The fusion cells (FC) were inoculated intradermally in the cervical region and repeated 3–10 times in each 28–84 days cycle. Toxicity, progression-free survival (PFS), and overall survival (OS) of this trial were evaluated. Results: Six patients were enrolled, three with high grade glioma and three with ependymoma. Median age at first course of immunotherapy was 10 years (range 8–25 years) and median time of follow-up from first course of immunotherapy was 13.5 months (range 3–33 months). All patients with immunotherapy were well tolerated and no adverse event without local erythema in injected site. Median progression free survival and overall survival were 18 months and 18.5 months, respectively. Conclusions: FC immunotherapy with autologous DCs and tumor cells for brain tumor in children and young adults were extremely well tolerated and encouraging. Further phase II study of FC immunotherapy is planned to improve prognosis and overcome treatment related neurological sequelae for highly malignant tumors.

Fusion of Dendritic Cells Activating Rv2299c Protein Enhances the Protective Immunity of Ag85B-ESAT6 Vaccine Candidate against Tuberculosis.

In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens.

Study inhibition and apoptosis of peripheral blood dendritic cells hybridize tumor vaccine induced systematic immune effector cells to the BxPC-3 in vitro

Objective To observe the inhibition and apoptosis effects of systematic immune effector cells (SIECs) induced by increased expansion of normal human peripheral venous blood dendritic cells (DCs) hybridoma vaccine on orthotopic pancreatic cancer cell line (BxPC-3) in vitro. Methods The peripheral blood mononuclear cells (PBMCs) of healthy human were obtained by gradient centrifugation of lymphocyte separation fluid, and DCs and DCs derived monocytes cells (SIECs) were obtained by adherent method. DCs and SIECs were respectively cultured with granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-4, tumor necrosis factor-α (TNF-α), IL-2, BxPC-3 tumor specific antigen (pc-3tsa). After five days, DCs were collected and counted. BxPC-3TAA-sensitized DCs (referred to as sensitive DCs), and DCs were fused with BxPC-3 by PEG+ 10% dimethylsurfoxide (DMSO) with 10∶1, 5∶1, 1∶5 to obtain sensitive DCs-BxPC-3 and DCs-BxPC-3 fusion tumor vaccines. On the 7th day, SIECs were respectively mixed with sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs, and DCs for 1 days in a 40∶1 mixture. The mixed cultured cells were collected, and the Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay and cell counting kit-8 (CCK-8) toxicity assay were performed on BxPC-3 in the mixed target SIECs∶BxPC3=10∶1. Results The sensitives DCs, DCs and BxPC-3 were fused, and the fusion effect was the best when the ratio was 1∶1, and the fusion rate was 40.0%. The CCK-8 assay showed that the inhibition rates of BxPC-3 induced by SIECs and SIECs by sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs and DCs were 53.1%, 45.3% and 78.8%, respectively. The Annexin V-FITC/PI assay showed that apoptosis rates of sensitive DCs-BxPC-3, DCs-BxPC-3, sensitive DCs, DCs and SIECs were 24.17%, 11.43%, 85.87% and 29.94%. Conclusion Tumor antigen sequential sensitization DCs have a superimposed effect on antigen acquisition, which can play a better anti-tumor effect. Early DCs fusion tumor vaccine can induce SIECs to inhibit tumor cells to a certain extent. Key words: Dendritic cells; Sequential sensitization; Pancreatic cancer; Tumor vaccine; Cell inhibition; Apoptosis

Fusion of Dendritic Cells and Cancer-Associated Fibroblasts for Activation of Anti-Tumor Cytotoxic T Lymphocytes

Here we explored the fusion of dendritic cells (DCs), potent antigen-presenting cells that initiate primary immune responses, with cancer-associated fibroblasts (CAFs), which are a stromal component needed for tumor progression, with the aim of stimulating T cells to inhibit tumor growth. Dendritic cells from the bone marrow of BALB/c mice were co-cultured with CAFs from H22 mouse hepatoma cells. CAFs were found to express fibroblast activation protein and α-smooth muscle actin by flow cytometry, Western blotting and immunofluorescence. Polyethylene glycol was added to the co-culture medium to encourage fusion, and the ability of the resulting fusion cells to produce TNF-α, IL-1β, IL-6, and IL-12p70 was confirmed using ELISA. These fusion cells efficiently stimulated T lymphocytes in vitro, causing them to generate IFN-α and IFN-γ. T cells activated by DC/CAF fusion cells led to strong CTL response against CAFs in vitro. The activated T cells also inhibited growth of H22 xenografts in vivo. These results indicate that DC/CAF fusion cells show potential for stimulating T cells as a novel anti-tumor vaccine.

Optimizing dendritic cell preparation for fusion with melanoma cells.

Fusion of dendritic cells (DCs) with melanoma cells could reinforce the antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach. To define the dosage of the stimulating factors as well as the induction condition for the optimal DCs preparation and cell fusion. DCs were generated from murine bone marrow cells, and cultured with four different concentrations of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were confirmed to be mature by detecting the expression of MHC-II, CD11c, CD80, and CD83 by flow cytometry. DCs-melanoma fusion cells were generated using polyethylene glycols (PEG) with different molecular weights and the fusion efficiency was detected by fluorescence-activated cell sorter (FACS). The largest quantity of DCs was found when cells were cultured with 1000 U/ ml of GM-CSF and 500 U/ml of IL-4 (1.69 ± 0.04 ×10⁶ ml⁻¹, p<0.001 when compared with the other three groups). The expression levels of MHC-II and CD83 on day 7 after incubation were significantly lower than those on day 3 (MHC-II: p<0.001; CD83: p<0.001). The efficiency of cell fusion under induction of PEG-3000 was significantly higher than that of PEG-4000 (15.4 ± 0.56% vs. 11.1 ± 0.45%, p<0.001). The largest quantity for mature DCs was stimulated with 1000 U/ml of GM-CSF and 500 U/ml of IL-4 and the highest fusion efficiency was under induction of PEG-3000.

Immunotherapy of Radioresistant Mammary Tumors with Early Metastasis Using Molecular Chaperone Vaccines Combined with Ionizing Radiation

In the current study, exposure of mammary tumor cells derived from mice transgenic for the polyomavirus middle T oncogene to ionizing radiation resulted in the generation of a tumor cell population that preferentially expressed cancer stem cell markers. In addition, these cells were more resistant to subsequent radiation treatments and appeared to acquire an enhanced capacity for dissemination to the lungs of mice. Therefore, we tested an immunotherapy approach to the treatment of local and disseminated mammary tumor cells in a murine model using a recently developed molecular chaperone-based vaccine that specifically targets the radioresistant subpopulation of tumor cells. Heat shock protein 70-peptide complexes (Hsp70.PC-F) were extracted from fusions of dendritic cells and radiation-enriched tumor cells, and the resulting chaperone vaccines were used to treat mice with pre-existing lung metastases. Immunization of mice with the Hsp70.PC-F vaccine resulted in a T cell-mediated immune response, including a significant increase in CD4 and CD8 T cell proliferation and the induction of effector T cells capable of targeting radioresistant tumor cells. Importantly, the growth of primary tumors was inhibited, and the number of tumor cells metastasizing to lung was reduced significantly by combining chaperone vaccine with radiotherapy. These results indicate that Hsp70.PC-F vaccine can induce specific immunity to radioresistant populations of mammary tumor cells and, thus, can complement radiotherapy, leading to synergistic killing.

Inhibitory effect of mmunoeffector cells on growth of PC3 induced by dendritic cell fusion hybrids

Objective To observe the inhibition of immunoeffector cells on the.growth PC3 cells induced by dendritic cell fusion hybrids.Methods DCs were fused with PC3 cells and fusion cells (DC fusion hybrids) were marked with CD11c MicroBeads.In vitro,cytotoxin study,the immunoeffector cells were divided into A1 group (without DC stimulated),A2 group (DC stimulated),A3 group (DC fusion hybrids stimulated).During the in vivo study,18 nude mice were allocated to 3 groups:preventive group (Ⅱ),1-2 days before inoculating PC3,giving DC fusion hybrids activated immunoeffector cells; treated group (Ⅲ),giving DC fusion hybrids activated immunoeffector cells after emergence of implanted tumor in all nude mice; control group (Ⅰ),giving equivalent amount of 1640 cultured liquid.Results In vitro,the maximal killing rate was A3 group (76.6％).In vivo,12 mice developed implanted tumor on the day 12 in group Ⅰ and Ⅲ,separately; In group Ⅱ,on the 30th day,1 of the 6 mice developed implanted tumor.On the 45th day after development of implanted tumor,all the nude mice were.sacrificed and the tumor body weighed.There was significant difference among group Ⅰ,Ⅱ,Ⅲ (P ＜ 0.05).Conclusion DC fusion hybrids may play an important role in anti-tumor therapy. Key words: Dendritic cell; Pancreaticcarcinoma; Immunotherapy; Fusion hybrids

In vitro evaluation of human hybrid cell lines generated by fusion of B-lymphoblastoid cells and ex vivo tumour cells as candidate vaccines for haematological malignancies

Fusions of dendritic cells (DCs) and tumour cells have been shown to induce protective immunity to tumour challenge in animal models, and to represent a promising approach to cancer immunotherapy. The broader clinical application of this approach, however, is potentially constrained by the lack of replicative capacity and limited standardisation of fusion cell preparations. We show here that fusion of ex vivo tumour cells isolated from patients with a range of haematological malignancies with the human B-lymphoblastoid cell line (LCL), HMy2, followed by chemical selection of the hybridomas, generated stable, self-replicating human hybrid cell lines that grew continuously in tissue culture, and survived freeze/thawing cycles. The hybrid cell lines expressed HLA class I and class II molecules, and the major T-cell costimulatory molecules, CD80 and CD86. All but two of 14 hybrid cell lines generated expressed tumour-associated antigens that were not expressed by HMy2 cells, and were therefore derived from the parent tumour cells. The hybrid cell lines stimulated allogeneic T-cell proliferative responses and interferon-gamma release in vitro to a considerably greater degree than their respective parent tumour cells. The enhanced T-cell stimulation was inhibited by CTLA4-Ig fusion protein, and by blocking antibodies to MHC class I and class II molecules. Finally, all of five LCL/tumour hybrid cell lines tested induced tumour antigen-specific cytotoxic T-cell responses in vitro in PBL from healthy, HLA-A2+ individuals, as detected by HLA-A2-peptide pentamer staining and cellular cytotoxicity. These data show that stable hybrid cell lines, with enhanced immunostimulatory properties and potential for therapeutic vaccination, can be generated by in vitro fusion and chemical selection of B-LCL and ex vivo haematological tumour cells.

Dendritic/Tumor Fusion Cells as Cancer Vaccines

A promising cancer vaccine involves the fusion of dendritic cells (DCs) with tumor cells such that a broad array of tumor antigens are presented in the context of DC-mediated costimulation and stimulatory cytokines. In diverse animal models, vaccination with DC/tumor fusions results in protection from an otherwise lethal challenge of tumor cells and eradication of established disease. In phase I clinical studies, vaccination with DC/tumor fusions was well tolerated, and induced immunologic responses in the majority of patients and clinical responses in a subset. Vaccine efficacy may be blunted by the immunosuppressive milieu characteristic of patients with malignancy, including the increased presence of regulatory T cells, and inhibitory pathways such as the PD-1/PDL-1 pathway. A current focus of research interest lies in enhancing response to cancer vaccines, by combining vaccination with tumor cytoreduction, regulatory T-cell depletion, and blockade of critical inhibitory pathways.

Induction of cytotoxic T lymphocytes against ovarian cancer‐initiating cells

The majority of patients with stage III/IV ovarian carcinoma that respond initially to standard therapies ultimately undergo relapse due to the survival of small populations of cells with tumor-initiating potential. These ovarian cancer (OVCA)-initiating cells (OCIC) are sometimes called cancer stem cells (CSC) because they express stem cell markers, and can survive conventional therapies such as chemotherapy, which usually target rapidly replicating tumor cells, and give rise to recurrent tumors that are more chemo-resistant and more aggressive. Thus, it would be desirable to develop a therapy that could selectively target OCIC and be used to complement the conventional therapies. In this study, we isolated a subset of OVCA cells with a CD44(+) phenotype in samples from patients with OVCA that possess CSC properties including the formation of spheroids in culture, self-renewal and the ability to be engrafted in immune-compromised mice. We next explored the use of immunotherapy using fusions of dendritic cells and OCIC to specifically target the OCIC subpopulations. Fusion cells (FCs) prepared in this way activated T cells to express elevated levels of IFN-γ with enhanced killing of CD44(+) OVCA cells. We envision a combined approach where conventional therapies such as chemotherapy kill the bulk of tumor cells, whereas OCIC-reactive cytotoxic T lymphocytes target the resistant OCIC fraction. A combined therapy such as this may represent a promising approach for the treatment of OVCA.

Dendritic cell–tumor cell hybrids and immunotherapy: what's next?

Dendritic cells (DC) are professional antigen-presenting cells currently being used as a cellular adjuvant in cancer immunotherapy strategies. Unfortunately, DC-based vaccines have not demonstrated spectacular clinical results. DC loading with tumor antigens and DC differentiation and activation still require optimization. An alternative technique for providing antigens to DC consists of the direct fusion of dendritic cells with tumor cells. These resulting hybrid cells may express both major histocompatibility complex (MHC) class I and II molecules associated with tumor antigens and the appropriate co-stimulatory molecules required for T-cell activation. Initially tested in animal models, this approach has now been evaluated in clinical trials, although with limited success. We summarize and discuss the results from the animal studies and first clinical trials. We also present a new approach to inducing hybrid formation by expression of viral fusogenic membrane glycoproteins.

Antigen-Specific Polyclonal Cytotoxic T Lymphocytes Induced by Fusions of Dendritic Cells and Tumor Cells

The aim of cancer vaccines is induction of tumor-specific cytotoxic T lymphocytes (CTLs) that can reduce the tumor mass. Dendritic cells (DCs) are potent antigen-presenting cells and play a central role in the initiation and regulation of primary immune responses. Thus, DCs-based vaccination represents a potentially powerful strategy for induction of antigen-specific CTLs. Fusions of DCs and whole tumor cells represent an alternative approach to deliver, process, and subsequently present a broad spectrum of antigens, including those known and unidentified, in the context of costimulatory molecules. Once DCs/tumor fusions have been infused back into patient, they migrate to secondary lymphoid organs, where the generation of antigen-specific polyclonal CTL responses occurs. We will discuss perspectives for future development of DCs/tumor fusions for CTL induction.

A Heat Shock Protein 70-Based Vaccine with Enhanced Immunogenicity for Clinical Use

In previous studies, we have shown that heat shock protein 70-peptide complexes (HSP70.PCs) derived from the fusion of dendritic cells (DCs) to tumor cells (HSP70.PC-F) possess superior properties compared with HSP70.PCs from tumor cells. HSP70.PC-F are more effective in stimulation of DC maturation and induction of CTL that are able to provide protection of mice against challenge with tumor cells. To develop an improved formulation of HSP70.PC-based tumor vaccine for patient use, we extracted HSP70.PC-F from DCs fused to patient-derived ovarian cancer cells or established human breast cancer cells and examined their properties as tumor vaccines. HSP70.PC-F induced T cells that expressed higher levels of IFN-gamma and exhibited increased levels of killing of tumor cells, compared with those induced by HSP70.PC derived from tumor cells. Enhanced immunogenicity of HSP70.PC-F was associated with improved composition of the vaccine, including increased content of tumor Ags and their processed intermediates, and the detection of other heat shock proteins (HSPs) such as HSP90 and HSP110. The present study has therefore provided an alternative approach to preparation of HSP-based vaccines using DC/tumor fusion technology and gentle and rapid isolation of HSP peptide complexes.

Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response

Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response.

Increase of In Vivo Antitumoral Activity by CD40L (CD154) Gene Transfer Into Pancreatic Tumor Cell-Dendritic Cell Hybrids

Fusion of dendritic cells (DC) with tumor cells is an approach in immunotherapy combining antigenicity and capacity of antigen presentation to activate T cells for the induction of tumor-specific cytotoxic immunity. Although there have been reports of clinical benefit, response rates have been limited and further improvements are warranted. We used murine DC and a novel protocol for an effective fusion of those cells with the murine pancreatic cell line Panc02. We observed 2 events: only moderate in vitro and in vivo cytotoxicity of tumor cell/DC hybrids and a down-regulation of costimulatory molecules on fused cells. Therefore, we transfected tumor cell/DC hybrids with an adenovirus expressing CD154 to improve DC activation and generating antitumor immune response without the need of CD4 T cells. High CD154 expression could be obtained by transfection of DC and Panc02 cells prior fusion. Furthermore, vaccination with CD154-transfected tumor cell/DC hybrid led to a significantly increased induction of cytotoxic T cells in vitro and to an improved antitumoral effect in an orthotopic in vivo mouse model. CD154-transfected tumor cell/DC hybrids are a promising approach to increase the efficiency of antitumoral response.

Enhancement of anti-tumor immunity by high levels of Th1 and Th17 with a combination of dendritic cell fusion hybrids and regulatory T cell depletion in pancreatic cancer

Regulatory T cells (Tregs) play an important role in immunological self-tolerance and protect the host from autoimmune disease. However, in cancer immunity, Tregs might block anti-tumor immune responses. Therefore, the depletion of Tregs using a specific agent that suppresses its function or population, such as an anti-CD25 antibody, could promote anti-tumor immune responses. In the present study, a cytotoxicity assay, enzyme-linked immunosorbent spot (ELISPOT) assay and measuring cytokine secretion, were used to study the efficacy of Treg depletion by anti-CD25 antibody added to a dendritic cell/tumor cell (DC/TC) fusion hybrid vaccine in a murine pancreatic cancer model. All the mice treated with the combined therapy of fusion hybrid vaccine and Treg depletion rejected tumor growth in a challenging test, although the rejection rate was 20% both for mice that received the fusion hybrids alone or Treg depletion alone. In addition, combined therapy showed a significantly improved survival in comparison to other treatment or control groups. The NK cell activity for DC/TC fusion + Treg depletion was significantly higher than that for the other treatment groups. Cytotoxic T lymphocyte (CTL) activity for DC/TC could potentially be enhanced by the addition of Treg depletion therapy. The treatments including DC/TC fusion induced IFN-gamma secreting effector cells in ELISPOT assays. Furthermore, a cytometric beads array assay used to measure cytokine secretion showed that DC/TC fusion + Treg depletion stimulated the highest levels of IFN-gamma Th1/Th2 ratios and Th17. These results demonstrate that Treg depletion combined with DC/TC fusion hybrid vaccine enhanced the efficacy of immunotherapy in pancreatic cancer by activating CTLs and NK cells.

Fusion of Dendritic Cells and CD34+CD38− Acute Myeloid Leukemia (AML) Cells Potentiates Targeting AML-initiating Cells by Specific CTL Induction

Distinct leukemia-initiating cells (L-ICs) represent a critical target for therapeutic intervention of acute myeloid leukemia (AML). A potential strategy to eradicate L-ICs is to generate L-IC-specific cytotoxic T lymphocytes (CTLs). However, owing to rarity and immortality of L-ICs, it is difficult for antigen-presenting cells to capture L-ICs for specific antigen presentation. Here, we report a novel approach by fusing allogeneic dendritic cells (DCs) and CD34CD38 AML progenitor cells, through which specific CTLs were effectively induced, leading to the cytolysis to AML-initiating cells. Fusion of either DC/CD34CD38 AML cell or DC/CD34 AML cell could effectively induce the proliferation and activation of CTLs. However, only the former CTLs could effectively attack AML progenitor cells, and result in the unkilled progenitor/initiating cells losing the abilities of active proliferation in vitro and engraftment in NOD-SCID mice. These findings suggest that AML progenitor/initiating cell-specific CTLs may be generated based on allogeneic DC/progenitor cell fusion strategy; the induced CTLs may potentially eradicate AML by targeting L-ICs directly or indirectly.

Assessment of telomerase inactivation in T cell responses and antitumor immunity (40.28)

Abstract Telomerase knockout (mTerc-/-) mice expressing polyoma middle T oncogene (PyMT) in 4 generations were immunized with fusions of dendritic cells and MUC1-positive mammary tumor cells (FC/MMT). Both CD4 and CD8 T cells from immunized MMT mice divided vigorously as measured by CFSE labeling. Comparable cell division was observed in T cells from immunized G0 mTerc+/-MMT, G1 and G2 mTerc-/-MMT mice. In contrast, cell division decreased in T cells from G3 mTerc-/-MMT mice. CTL against mammary tumor cells were induced in LNC and Splenocytes from immunized but not non-immunized early generations of mTerc-/-MMT mice. However, CTL in the late generations of immunized mTerc-/-MMT mice decreased. Finally, the efficacy of immunotherapy in the prevention and treatment of spontanenous mammary carcinomas was assessed. In the prevention study, the time required for the mammary tumors in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice immunized with FC/MMT were 124.7±3.8, 118.6±10.8, 139.9±25.3, 138±12.1 and 146.8±21.2 days, respectively. The time required for the mammary tumor development in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice immunized with FC/MMT in the treatment study were 112.4±10.4, 113.6±8.4, 136.3±10, 144.3±12.7 and 158.7±15.7 days, respectively. In contrast, the latency time for tumor development in MMT, G0 mTerc+/-MMT, G1, G2 and G3 mTerc-/-MMT mice without immunization were 73.07±28.03, 96.07±28.79, 115.6±8.88, 117.14±32.1 and 127.11±35.84 days, respectively. The difference of time required for tumor development between mice with or without vaccination in both prevention and treatment studies is statistically significant.