Antigen-pulsed Dendritic Cells Research Articles

Chlamydia muridarum T Cell Antigens and Adjuvants That Induce Protective Immunity in Mice

Major impediments to a Chlamydia vaccine lie in discovering T cell antigens and polarizing adjuvants that stimulate protective immunity. We previously reported the discovery of three T cell antigens (PmpG, PmpF, and RplF) via immunoproteomics that elicited protective immunity in the murine genital tract infection model against Chlamydia infection after adoptive transfer of antigen-pulsed dendritic cells. To expand the T cell antigen repertoire necessary for a Chlamydia vaccine, we evaluated 10 new Chlamydia T cell antigens discovered via immunoproteomics in addition to the 3 antigens reported earlier as a molecular subunit vaccine. We first tested five adjuvants, including three cationic liposome formulations (dimethyldioctadecylammonium bromide-monophosphoryl lipid A [DDA-MPL], DDA-trehalose 6,6'-dibehenate [DDA-TDB {CAF01}], and DDA-monomycolyl glycerol [DDA-MMG {CAF04}]), Montanide ISA720-CpG-ODN1826, and alum using the PmpG protein as a model T cell antigen in the mouse genital tract infection model. The results showed that the cationic liposomal adjuvants DDA-MPL and DDA-TDB elicited the best protective immune responses, characterized by multifunctional CD4(+) T cells coexpressing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), and reduced infection by more than 3 logs. Using DDA-MPL as an adjuvant, we found that 7 of 13 Chlamydia T cell antigens (PmpG, PmpE, PmpF, Aasf, RplF, TC0420, and TC0825) conferred protection better than or equal to that of the reference vaccine antigen, major outer membrane protein (MOMP). Pools of membrane/secreted proteins, cytoplasmic proteins, and hypothetical proteins were tested individually or in combination. Immunization with combinations protected as well as the best individual protein in that combination. The T cell antigens and adjuvants discovered in this study are of further interest in the development of a molecularly defined Chlamydia vaccine.

Gene silencing of TGF-β1 enhances antitumor immunity induced with a dendritic cell vaccine by reducing tumor-associated regulatory T cells.

Active immunotherapy and cancer vaccines that promote host antitumor immune responses promise to be effective and less toxic alternatives to current cytotoxic drugs for the treatment of cancer. However, the success of tumor immunotherapeutics and vaccines is dependent on identifying approaches for circumventing the immunosuppressive effects of regulatory T (Treg) cells induced by the growing tumor and by immunotherapeutic molecules, including Toll-like receptor (TLR) agonists. Here, we show that tumors secrete high concentrations of active TGF-β1, a cytokine that can convert naive T cells into Foxp3+ Treg cells. Silencing TGF-β1 mRNA using small interfering RNA (siRNA) in tumor cells inhibited active TGF-β1 production in vitro and restrained their growth in vivo. Prophylactic but not therapeutic administration of TGF-β1 siRNA reduced the growth of CT26 tumors in vivo. Furthermore, suppressing TGF-β1 expression at the site of a tumor, using siRNA before, during and after therapeutic administration of a TLR-activated antigen-pulsed dendritic cell vaccine significantly reduced the growth of B16 melanoma in mice. The protective effect of co-administering TGF-β1 siRNA with the DC vaccine was associated with suppression of CD25+ Foxp3+ and CD25+ IL-10+ T cells and enhancement of tumor infiltrating CD4 and CD8 T cells. Our findings suggest that transient suppression of TGF-β1 may be a promising approach for enhancing the efficacy of tumor vaccines in humans.

Antigen-specific immature dendritic cell vaccine ameliorates anti-dsDNA antibody-induced renal damage in a mouse model

Dendritic cells (DCs) can inhibit immune response by clonal anergy when immature. Recent studies have shown that immature DCs (iDCs) may serve as a live cell vaccine after specific antigen pulse based on its potential of blocking antibody production. In this study, we aimed to investigate the effects of nuclear antigen-pulsed iDCs in the treatment of lupus-like renal damages induced by anti-dsDNA antibodies. iDCs were generated from haemopoietic stem cells in bone marrow and then pulsed in vitro with nuclear antigen. The iDC vaccine and corresponding controls were injected into mice with lupus-like renal damages. The evaluation of disease was monitored by biochemical parameters and histological scores. Anti-dsDNA antibody isotypes and T-lymphocyte-produced cytokines were analysed for elucidating therapeutic mechanisms. RESULTS; The mice treated with antigen-pulsed iDCs had a sustained remission of renal damage compared with those injected with non-pulsed iDCs or other controls, including decreased anti-dsDNA antibody level, less proteinuria, lower blood urea nitrogen and serum creatinine values, and improved histological evaluation. Analysis on isotypes of anti-dsDNA antibody showed that iDC vaccine preferentially inhibited the production of IgG3, IgG2b and IgG2a. Furthermore, administration of antigen-treated iDCs to mice resulted in significantly reduced IL-2, IL-4 and IL-12 and IFN-γ produced by T-memory cells. Conversely, the vaccination of antigen-pulsed mature DCs led to increased anti-dsDNA antibody production and an aggravation of lupus-like disease in the model. CONCLUSIONS; These results suggested the high potency of iDC vaccine in preventing lupus-like renal injuries induced by pathogenic autoantibodies.

Role of Interleukin-23-Dependent Antifungal Immune Responses in Dendritic Cell-Vaccinated Mice

CD40 ligand (CD40L) transduction of antigen-pulsed dendritic cells (DCs) can result in antigen-specific humoral immune responses even in CD4(+) T-cell-depleted settings. Here, we show that CD40L transduction of DCs results in the induction of interleukin-12p40 (IL-12p40), IL-12p70, and IL-23. Using DCs that were deficient in IL-12p40, IL-12p35, or IL-23p19, we show that these molecules are dispensable for primary IgG1 responses to Pneumocystis, but IgG2c was dependent on IL-12p40 and IL-23p19 but not IL-12p35. Antigen-specific recall responses in CD4-deficient mice were critically dependent on IL-12p40 and IL-23p19 expression in DCs and were not affected by the lack of IL-12p35. To confirm that this defect in recall was due to IL-23, transduction of IL-12p40(-/-) DCs with a recombinant adenovirus expressing functional IL-23 restored recall responses in DC-vaccinated CD4-deficient mice. These data show that DC-produced IL-23 is critical for vaccine-induced antigen-specific IgG2c and recall antibody responses in the setting of CD4(+) T-cell depletion.

Mature autologous dendritic cell vaccines in advanced non-small cell lung cancer: a phase I pilot study

BackgroundOverall therapeutic outcomes of advanced non-small-cell lung cancer (NSCLC) are poor. The dendritic cell (DC) immunotherapy has been developed as a new strategy for the treatment of lung cancer. The purpose of this study was to evaluate the feasibility, safety and immunologic responses in use in mature, antigen-pulsed autologous DC vaccine in NSCLC patients.MethodsFive HLA-A2 patients with inoperable stage III or IV NSCLC were selected to receive two doses of 5 × 107 DC cells administered subcutaneous and intravenously two times at two week intervals. The immunologic response, safety and tolerability to the vaccine were evaluated by the lymphoproliferation assay and clinical and laboratorial evolution, respectively.ResultsThe dose of the vaccine has shown to be safe and well tolerated. The lymphoproliferation assay showed an improvement in the specific immune response after the immunization, with a significant response after the second dose (p = 0.005). This response was not long lasting and a tendency to reduction two weeks after the second dose of the vaccine was observed. Two patients had a survival almost twice greater than the expected average and were the only ones that expressed HER-2 and CEA together.ConclusionDespite the small sample size, the results on the immune response, safety and tolerability, combined with the results of other studies, are encouraging to the conduction of a large clinical trial with multiples doses in patients with early lung cancer who underwent surgical treatment.Trial RegistrationCurrent Controlled Trials: ISRCTN45563569

A High-Affinity T-Helper Epitope Enhances Peptide-Pulsed Dendritic Cell–Based Vaccine

The NV epitope, a dominant helper determinant from the circumsporozoite antigen of Plasmodium falciparum, is strongly immunogenic and can provide help for cytotoxic T-lymphocyte (CTL) activation. In this study, we evaluated whether the addition of NV peptide can augment the efficacy of peptide-pulsed dendritic cell (DC) immunization in vivo. Using B16 melanoma as tumor model, we demonstrated that DCs pulsed with both NV and gp100 (a melanoma-specific antigen) peptide enhanced immune priming and protection from tumor challenge in vivo. Further, we showed the mechanisms of the NV epitope that help CTL activation; MHC-II-restricted NV peptide induced dramatically more effective helper cells, with a higher level of CD40L expression and IFN-γ production, which, in turn, more effectively conditioned DCs for CTL activation. The improved helper cells also induced greater IL-12 production by DCs, accounting for the reciprocal T-helper polarization to Th1, and increased the expression of costimulatory molecules. Collectively, these findings demonstrate that NV peptide in addition to tumor antigen-pulsed DC immunizations augment helper cell activation, which in turn promotes maturation of DC, and enhance in vivo antitumor activity.

Safety and immunogenicity of hepatitis B surface antigen-pulsed dendritic cells in patients with chronic hepatitis B

The immune modulator capacity of antigen-pulsed dendritic cells (DC) has been documented in patients with cancers and in animal models of chronic viral infections. Cancer antigen-pulsed DC are now used for treating patients with cancer. But viral antigen-pulsed DC are not used in chronic viral-infected patients because safety of antigen-pulsed DC has not been evaluated in these patients. DC were isolated from human peripheral blood mononuclear cells by culturing with human-grade granulocyte-macrophage colony stimulating factor and interleukin-4. Human blood DC were cultured with hepatitis B surface antigen (HBsAg) for 8h to prepare HBsAg-pulsed DC. After immunogenicity assessment of HBsAg-pulsed DC in vitro, five million HBsAg-pulsed DC were administered intradermally to five patients with chronic hepatitis B (CHB) 1-3 times. HBsAg-pulsed DC were immunogenic in nature because they produced significantly higher levels of interleukin-12 and interferon-γ compared to unpulsed DC (P<0.05). Also, HBsAg-pulsed DC induced proliferation of HBsAg-specific T lymphocytes in vitro. CHB patients injected with HBsAg-pulsed DC did not exhibit generalized inflammation, exacerbation of liver damage, abnormal kidney function, or features of autoimmunity. Administration of HBsAg-pulsed DC induced anti-HBs in two patients and HBsAg-specific cellular immunity in 1 patient. This is the first study about preparation of antigen-pulsed DC using human consumable materials for treating patients with CHB. Because HBsAg-pulsed DC were safe for all patients with CHB and had immune modulation capacity in some patients, phase I and phase II clinical trials with antigen-pulsed DC in CHB and other chronic infections are warranted.

Immunology in the heart of the High Tatra mountains

Immunology in the heart of the High Tatra mountains

Dendritic cells stimulated with outer membrane protein A (OmpA) of Salmonella typhimurium generate effective anti-tumor immunity

Gram-negative bacterial outer membrane proteins (Omps) have an important role in pathogenesis and signal reception. We previously reported that Acinetobacter OmpA ( AbOmpA) induced maturation of bone marrow-derived dendritic cells (BMDCs) and that AbOmpA-primed DCs produced IL-12 which generated Th1 CD4 + T-cells. We analyzed the effects of Salmonella typhimurium OmpA (OmpA-Sal) on dendritic cell (DC) maturation in the present study, and determined that tumor antigen-pulsed DCs stimulated with OmpA-Sal induced anti-tumor responses in a mouse model. OmpA-Sal activated BMDCs by augmenting expression of MHC class II and of the co-stimulatory molecules CD80 and CD86. RT-PCR revealed that IL-12(p40) gene expression is highly augmented in OmpA-Sal-stimulated BMDCs. DNA (CRT/E7) vaccination combined with OmpA-Sal stimulation generated more antigen-specific CD8 + T-cells in the present study. Certain antigen-pulsed BMDCs stimulated with OmpA-Sal induced strong PADRE-specific CD4 + and E7-specific CD8 + T-cell responses. In addition, BMDCs stimulated with OmpA-Sal (OmpA-Sal-BMDCs) and pulsed with both E7 and PADRE peptide generated greater numbers of E7-specific CD8 + effector and memory T-cells than those pulsed with E7 peptide alone. E7- and PADRE-expressing OmpA-Sal-BMDC vaccines resulted in significant long-term protective anti-tumor effects in vaccinated mice. Our data suggested that E7- and PADRE-expressing BMDCs that were matured in the presence of OmpA-Sal might enhance anti-tumor immunity and support the therapeutic use of OmpA-Sal in DC-based immunotherapy.

Frontiers in Tumor Immunity: Grand Challenge “Cancer and Immunity: A Family Drama”

The twentieth century has been a time of intense debate on the role of the immune system in controlling cancer initiation, growth, and final victory. At the beginning of the century, according to the common sense generated by the very recent Pasteur discoveries, physicians and scientists have considered a growing cancer as an internal infection involving a fight with the patient's immune system. They were so convinced of the similarities between cancer and infections that William B. Coley proposed to treat patients with bacterial toxins, hypothesizing that the anti-bacterial immune response would clear the tumor. Although infrequent but undisputable therapeutic successes of this approach, together with epidemiologic studies reporting high incidence of cancer in immunodeficient individuals, yielded to the launching of the theory of immunosurveillance of cancer in the late 1950s. This theory proposed that, as for infectious agents, nascent cancerous cells, although originating from self, bore neo-antigens recognized by the host's immune system and are therefore in most cases rejected. Only in immuno-impaired individuals (primary or induced by viruses or chemicals) or when developing in immunoprivileged sites, would cancers grow, become clinically detectable and kill the patient despite the efforts of his immune system, i.e., a family story inside the body, very badly controlled, finishing in a very unhappy ending. Contenders of this theory were however numerous. Most cancers originating in immunodeficient individuals were found to be of viral origin, so that immune surveillance seemed antiviral and not antitumoral, and despite the discovery of tumor-associated antigens in the 1990s, cancer immunotherapy yielded scarce successes. With the exception of Interleukin-2 for the treatment of metastatic renal cell cancer, no immunotherapeutic drugs have been approved. Accordingly, with the recent triumphant march of cancer genomics, and the discovery of targeted chemotherapies, cancer immunology has been consigned to the dark once again. However, the beginning of the twenty-first century witnessed a renaissance of the field. Basic studies demonstrated that tumors develop more easily and rapidly in immunodeficient animals, even without oncogene activation or chemical promotion. Clinical studies established that, whereas chronic inflammation promotes cancer growth and metastasis, intratumoral Th1/CD8 memory T cell infiltration is a major prognostic factor for disease-free as well as overall survival in cancer patients. Even more practically important, efficient immunotherapies mostly based on the use of monoclonal antibodies targeted to the tumor cells or modulating the immune and stromal status, are being approved, which has changed the management of many cancers. A first therapeutic vaccine, using antigen-pulsed dendritic cells, has become a new treatment modality for metastatic prostatic cancer; others will come. In this regard, it is noteworthy that the efficacy of classical treatments, such as chemotherapies, seems to depend on their capacity to activate an anti-tumor immune reaction. Even so, major questions have not yet been resolved, among which are: Is an immune reaction efficient because it recognizes cancer cell antigens or because it modifies the tumor microenvironment? How do the cancer cells and the plasticity of their genome influence the immune contexture? What tumor-associated driver molecules should be targeted by immunotherapies? What is the complexity of the tumor-associated immune reaction in terms of immune cell populations and targets, how is it modified during tumor evolution, and how is it in primary sites compared with different metastatic sites? Should all efficient therapies modulate the immune reaction and how? The era of large libraries of human specimens linked to systems biology and tailored mouse models will allow us to address these questions in an effective manner, and to propose an immunological management of the diseases, taking place in the growing field of targeted therapies based on the cancerous cells but also on the genetics of the patient, as well as the environment of the tumor cells.

Burkholderia pseudomallei proteins presented by monocyte-derived dendritic cells stimulate human memory T cells in vitro.

Melioidosis is a severe infectious disease caused by the saprophytic facultative intracellular pathogen Burkholderia pseudomallei. The disease is endemic in Southeast Asia and Northern Australia, and no effective vaccine exists. To describe human cell-mediated immune responses to B. pseudomallei and to identify candidate antigens for vaccine development, the ability of antigen-pulsed monocyte-derived dendritic cells (moDCs) to trigger autologous T-cell responses to B. pseudomallei and its products was tested. moDCs were prepared from healthy individuals exposed or not exposed to B. pseudomallei, based on serological evidence. These were pulsed with heat-killed B. pseudomallei or purified antigens, including ABC transporters (LolC, OppA, and PotF), Bsa type III secreted proteins (BipD and BopE), tandem repeat sequence-containing proteins (Rp1 and Rp2), flagellin, and heat shock proteins (Hsp60 and Hsp70), prior to being mixed with autologous T-cell populations. After pulsing of cells with either heat-killed B. pseudomallei, LolC, or Rp2, coculturing the antigen-pulsed moDCs with T cells elicited gamma interferon production from CD4(+) T cells from seropositive donors at levels greater than those for seronegative donors. These antigens also induced granzyme B (cytotoxic) responses from CD8(+) T cells. Activation of antigen-specific CD4(+) T cells required direct contact with moDCs and was therefore not dependent on soluble mediators. Rp peptide epitopes recognized by T cells in healthy individuals were identified. Our study provides valuable novel data on the induction of human cell-mediated immune responses to B. pseudomallei and its protein antigens that may be exploited in the rational development of vaccines to combat melioidosis.

Enhanced Induction of HIV-specific Cytotoxic T Lymphocytes by Dendritic Cell-targeted Delivery of SOCS-1 siRNA

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

Immunotherapy for Epithelial Ovarian Cancer - Immunological Background, Critical Review of Methods and Future Perspectives

Ovarian cancer is one of the most fatal female neoplasms due to its aggressive behavior and late diagnosis. Despite improvments of the surgical approach and the use of chemotherapy, the survival rate observed in patients with advanced ovarian cancer is still unsatisfactory. The presence of cancer modulates adversely the immunological response of the host. Immunosurveillance over the tumor could be at least partly restored by the use of immunotherapy based either on techniques which allow to recognize and directly inhibit tumor cells, or on methods which mobilize and augment an anti-tumor host response. The first kind of strategy uses monoclonal antibodies directed against cancer specific antigens (CA-125) and molecules responsible for tumor growth and spreading (HER-2, EGFR-1, VEGF). The second kind of strategy modifies the host immune status by use of different techniques. Some of them are based on the administration of cytokines given in order to facilitate anti-tumor immunity. Recent studies have explored novel techniques which use hybrid cytokine/monoclonal antibodies targeted to specific cancer antigens, cancer antigen-pulsed and cancer cell-fused dendritic cells, or modification of regulatory or cytotoxic T cells. New methods of immunotherapy appear on the horizon, however, their efficacy still awaits confirmation in clinical trials. Keywords: Ovarian cancer, immunotherapy, monoclonal antibody, cytokines, DC, Treg cells

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca(2+)) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca(2+) signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca(2+)-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.

Reduction of major histocompatibility complex class I expression on bladder carcinoma following tumor antigen‐pulsed dendritic cell vaccine: Implications for immunoresistance in therapy

To clarify the relationship between a decreased major histocompatibility complex class I (MHC-I) expression on bladder tumors and decreased immunological efficacy of tumor antigen-pulsed dendritic cell vaccine in a rat bladder carcinoma model induced by N-methyl-N-nitrosourea irrigation. Enzyme-linked immunosorbent assay was used to evaluate interferon-gamma concentration in the serum and colorimetric lactate dehydrogenase release assay in vitro was used to test the cytotoxicity capability of T lymphocytes. MHC-I expression on tumor cells was detected by flow cytometry and analyzed with CellQuest software. The tumor antigen sensitized dendritic cell vaccine group showed decreased hyperplastic formations, lower pathological stages in rat bladders and more potent cytotoxicity activity (P < 0.001) than the dendritic cell vaccine group. Additionally, immunization with pulsed dendritic cell vaccine induced higher specific cytokine production of interferon-gamma. Nevertheless, a decreased MHC-I expression on bladder tumors was tested after immunotherapy by pulsed dendritic cell vaccine on week 15. As expected, the cytotoxic activity of T lymphocytes from rats on tumor cells with low MHC-I expression was also decreased to 19.70 +/- 4.82% as compared with tumor cells with high MHC-I (52.10 +/- 8.66%, P = 0.005). Tumor antigen sensitized dendritic cell vaccine has beneficial activity on N-methyl-N-nitrosourea-induced bladder cancer in situ in rats, but therapeutic responses are accompanied by decreased MHC-I expression on tumors, possibly suggesting poor long-term therapeutic outcomes.

Long-term survival and long-lived immune memory in patients with metastatic melanoma vaccinated with melanoma-antigen loaded dendritic cells.

e19018 Background: Clinical trials have demonstrated the safety of cancer antigen-loaded dendritic cells (DCs) as well as clinical and immune responses. We showed that antigen-pulsed DCs administered to patients with metastatic melanoma are immunogenic and can induce durable objective clinical responses even in patients who failed previous therapy. Here we analyzed long term immunological outcomes in our cohort of vaccinated patients. Methods: Between March 1999 and February 2005 sixty four patients with metastatic melanoma were treated with DCs vaccines in the course of four phase I/IIa clinical trials. DCs were generated either from CD34+ hematopoietic progenitors or from blood monocytes. Forty nine (49) HLA-A*0201+ patients received vaccines pulsed with melanoma antigen derived peptides (MART-1, MAGE-3, TYR and gp100). Twenty one patients received DCs loaded with killed allogeneic tumors regardless of their HLA type. KLH was used as a helper antigen and, in HLA- A*0201+ patients, Flu-M1 peptide was used as the control antigen. Patients received up to eight vaccinations with antigen-loaded DCs over a maximum of 7 months vaccination schedule. Results: DC vaccinations were safe and tolerable. Nine (14%) of 64 patients are alive as of January 2010. Median survival was 17 (95% CI 12-26) months. Preliminary analysis of long-term immunological outcomes demonstrated the induction of long-lived melanoma-antigen specific CD8+T cells in a patient who obtained durable complete regression of in-transit melanoma. There, DC vaccination expanded circulating MART-1-specific CD8+ T cells. These could be detected after the fourth and eighth DC vaccination as well as 6 years after the last vaccination with DCs. The T cells had a predominantly effector memory phenotype. A broad repertoire of CD4+ T cells recognizing multiple tumor antigens was also induced in the same patient. These induced tumor-specific CD4+ T cells primarily secreted IL-2. Conclusions: DC vaccines can bring clinical benefit and elicit durable responses in a fraction of patients with metastatic cancer. DC vaccines have the potential to expand melanoma- antigen specific memory T cells. No significant financial relationships to disclose.

Neem leaf glycoprotein enhances carcinoembryonic antigen presentation of dendritic cells to T and B cells for induction of anti-tumor immunity by allowing generation of immune effector/memory response

Vaccination with neem leaf glycoprotein matured carcinoembryonic antigen (CEA) pulsed dendritic cells (DCs) enhances antigen-specific humoral and cellular immunity against CEA and restricts the growth of CEA + murine tumors. NLGP helps better CEA uptake, processing and presentation to T/B cells. This vaccination (DCNLGPCEA) elicits mitogen induced and CEA specific T cell proliferation, IFNγ secretion and induces specific cytotoxic reactions to CEA + colon tumor cells. In addition to T cell response, DCNLGPCEA vaccine generates anti-CEA antibody response, which is principally IgG2a in nature. This antibody participates in cytotoxicity of CEA + cells in antibody-dependent manner. This strong anti-CEA cellular and humoral immunity protects mice from tumor development and these mice remained tumor free following second tumor inoculation, indicating generation of effector memory response. Evaluation of underlying mechanism suggests vaccination generates strong CEA specific CTL and antibody response that can completely prevent the tumor growth following adoptive transfer. In support, significant upregulation of CD44 on the surface of lymphocytes from DCNLGPCEA immunized mice was noticed with a substantial reduction in L-selectin (CD62L).

Abstract 4761: The iCD40. MyD88 combo-vector: A new platform for enhanced DC tumor immunotherapy

Abstract Introduction: Given the pre-eminent role of dendritic cells (DCs) as antigen-presenting cells, their exploitation as natural adjuvants in vaccination protocols for the treatment of various malignancies and infectious diseases is not surprising. Nevertheless, inadequate or unsustained activation has likely limited response rates in clinical trials. We previously reported a potent synthetic dimerizer drug-inducible CD40 (iCD40) receptor that permits temporally controlled DC-specific activation within the context of an immunological synapse (Hanks et al, Nat Med, 2005). We showed that when combined with Toll-like Receptor (TLR) adjuvants, iCD40-modified DCs (iCD40-DCs) have enhanced survival, co-stimulatory marker expression, migration, IL-12 production, antigen (Ag)-specific T cell stimulatory capacity and anti-tumor responses (Lapteva et al, Cancer Res, 2007). A phase I/II clinical trial based on iCD40-DCs to treat castration-resistant, metastatic prostate cancer began enrolling patients in August 2009. Procedures: To simplify this approach, we have developed a unified, inducible DC switch, called “iCD40. MyD88”, which combines signaling elements from costimulatory molecule, CD40, and TLR adapter, MyD88, fused to tandem FK506-binding binding proteins (FKBP12v36). Activation is initiated in vitro or in vivo by lipid-permeable dimerizing ligands (i.e. AP1903 or AP20187). Results: Both murine and human iCD40. MyD88-DCs secrete high (ng) levels of IL-12 and maturation markers in a ligand-dependent fashion without the need for exogenous adjuvants. iCD40. MyD88 activation is associated with synergistic activation of IKK&amp;#945;/&amp;#946; and p38. Moreover, tumor-bearing mice treated with antigen-pulsed, iCD40. MyD88-DCs show a potent anti-tumor immune response, consistent with expansion of antigen-specific CTLs in vivo. Bicistronic vectors combining iCD40. MyD88 along with tumor-associated antigens are underway. Conclusion: This “combo-switch” permits potent, targeted activation of antigen-pulsed DCs in vivo, leading to improved anti-tumor immunity with a high likelihood for reduced side effects. Production of a single vector that combines a potent DC switch with tumor antigen is the first requisite step leading to economically viable, “off-the-shelf”, vaccine therapies for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4761.

The specific and non-specific cytotoxic activity of killer cells against gastric cancer cells induced by cytokines

Objective To observe the roles of specific and non-specific cytotoxic activity in cyto-kine-induced killer cells (CIK) against gastric cancer (SGC-7901 ) cells in vitro. Methods Dendritic cells (DC) and CIK were induced from peripheral blood mononuclear cells (PBMC) from healthy donors. The DC were sensitized with tumor antigen extracted from gastric cancer SGC-7901 cells. The CIK were in-duced with DC to form DC-CIK,or induced with gastric cancer antigen-pulsed DC to form antigen-DC-CIK. On the 14th day of culture,CIK, DC-CIK and antigen-DC-CIK phenotypes (CD3 ,CD3/CD8 and CD3/CD56) ware analyzed by flow cytometry. CIK, DC-CIK and antigen-DC-CIK were used as the effector cells, and the SGC-7901 cells with or without blockade of major histocompatibility complex (MHC) molecule as the target cells. At 5: 1,10:1 and 20:1 effector-target ratio,the cytotoxic activity was detected by MTT. Results On the 14th day of culture,the proportion of CD3~+ CD8~+ and CD3~+ CD56~+ double positive cells in antigen-DC-CIK was significantly higher than that in DC-CIK and pure CIK (P <0.01 ). At 20:1 effector-target ratio in vitro,the cytotoxic activity of CIK, DC-CIK and antigen-DC-CIK against SGC-7901 cells was (46.6± 2.2) %, (52.6 ± 1.6) % and (72.5±2.1 ) % (P < 0.01 ), and after MHC molecule was blocked in SGC-7901 cells, the cytotoxic activity was (44.3 ± 1.1 ) %, (49.9 ±0. 9) % and (50.4 ± 1.9) % respectively. The proportion of specific cytotoxic activity in antigen-DC-CIK was higher than in DC-CIK and CIK. Conclusion The major eytotoxic activity of CIK against gastric cancer cells is non-specific, and specific cytotoxic activity only accounts for 4.9%. After induction of CIK by DC ,the total cytotoxic activity was enhanced and the ma-jor cytotoxic activity is non-specific, and specific cytotoxie activity was enhanced slightly to 5.1%. After in-duction of CIK by antigen-pulsed DC, the major cytotoxic activity is still non-specific, and the specific cyto-toxic activity was largely increased to 30.5%. Key words: Stomach neoplasm; Dendric cells; Killer cells; Antigen

Vaccination with immature dendritic cells combined with CD40 mAb induces protective immunity against B lymphoma in hu-SCID mice

Dendritic cells (DCs) pulsed by tumor antigens have been widely used as tumor vaccines to specifically trigger the cytotoxicity of CD8+ T cells. But the tumor microenviroment with enriched immunosuppressants hampered DC maturation and co-stimulation. CD40/CD40L signaling, one of the most important co-stimulatory molecules is capable of effectively skewing the immune response by promoting DCs maturation and co-stimulation. To establish a novel specific immunotherapeutic approach for the use of DC vaccine in the treatment of B lymphoma, hu-SCID mice bearing B lymphoma were vaccinated by different combination of tumor antigen pulsed DC or imDC vaccines and immune-enhancing agencies such as agonist CD40 mAb and T cells. The results of immature DCs combined with agonistic CD40 mAb were encouraging with achievement of tumor regression and induction of antigen-specific immune responses. These findings demonstrated the potential utility of imDC-based tumor vaccination combining with agonistic CD40 mAb in the treatment of malignant lymphoma.