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Cell surface phenotype of bone marrow and PBMC-derived DCs. Bone marrow and PBMC-derived DC precursors were cultured for 5 days in the presence of GM-CSF and IL-4, and then stained for flow cytometry. Cells demonstrate high levels of CD80, CD86, CD40, and MHC molecules characteristic of DCs.
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Dendritic cells (DCs) primed with tumor antigens can effectively mediate the regression of a variety of established solid malignancies in both murine and human models. Despite such clinical efficacy, the optimal means of DC priming is unknown. The goal of this study was to compare three methods of tumor preparation: irradiation, boiling, or freeze...
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To identify prognostic factors associated with survival beyond 4 years and overall response in patients with metastatic melanoma treated with high-dose bolus i.v. interleukin-2 (IL-2) given either alone or in combination with a variety of melanoma vaccines.
684 consecutive patients with metastatic melanoma received high-dose bolus i.v. IL-2 either...
In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predic...
Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser...
DNA-based immunization strategies designed to elicit cellular antitumor immunity offer an attractive alternative to protein- or peptide-based approaches. In the present study we have evaluated the feasibility of DNA vaccination for the induction of CTL reactivity to five different melanoma Ags in vitro. Cultured, monocyte-derived dendritic cells (D...
Purpose:
Electroporation of dendritic cells (DC) with mRNA encoding tumor-associated antigens (TAA) has multiple advantages compared to peptide loading. We investigated the immunologic and clinical responses to vaccination with mRNA-electroporated DC in stage III and IV melanoma patients.
Experimental design:
Twenty-six stage III HLA*02:01 melan...
Citations
... Several approaches have been devised to overcome this drawback. It has been reported that the use of cytokines like GM-CSF, IL-4, TNF-α, and IL-6 can boost the immunogenicity of immature DCs that are loaded with tumor antigens and facilitate the proper presentation of DCs to major histocompatibility complex (MHC) class 1 and 2 molecules [32,40]. Some studies have explored the use of hypochlorous acid as a possible solution [41][42][43]. ...
Glioblastoma multiforme (GBM), a highly aggressive tumor, poses significant challenges in achieving successful treatment outcomes. Conventional therapeutic modalities including surgery, radiation, and chemotherapy have demonstrated limited efficacy, primarily attributed to the complexities associated with drug delivery to the tumor site and tumor heterogeneity. To address this critical need for innovative therapies, the potential of cancer vaccines utilizing tumor cells and dendritic cells has been explored for GBM treatment. This article provides a comprehensive review of therapeutic vaccinations employing cell-based vaccine strategies for the management of GBM. A meticulous evaluation of 45 clinical trials involving more than 1500 participants revealed that cell-based vaccinations have exhibited favorable safety profiles with minimal toxicity. Moreover, these vaccines have demonstrated modest improvements in overall survival and progression-free survival among patients. However, certain limitations still persist. Notably, there is a need for advancements in the development of potent antigens to evoke immune responses, as well as the optimization of dosage regimens. Consequently, while cell-based vaccinations show promise as a potential therapeutic approach for GBM, further research is imperative to overcome the current limitations. The ultimate objective is to surmount these obstacles and establish cell-based vaccinations as a standard therapeutic modality for GBM.
... Two days after bacterial injection (on day 0), OT-I T cells were intravenously injected into the adoptive transfer mice and tumor volumes were recorded twice a week. Arabinose (100 mg) was injected IP at 48 injected with ID-OVA had no significant tumor growth over eighteen days of observation ( Figure 4C). In comparison, all tumors grew in control ID-GFP mice over the same period ( Figure 4D). ...
... (3) Presentation of the delivered antigen activates antigen-specific vaccine CD8 T cells (12,20), which kill the presenting cancer cells (36,(39)(40)(41)(42). (4) Cancer cell death and T cell activation induce antigen presenting cells (APCs) to cross-present tumor associated antigens (TAAs, brown) (43-46). (5) Activation of tumor-specific CD8 T cells (23,24,(47)(48)(49) leads to the formation of antitumor immunity (50-53). ...
Introduction
Immunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice.
Methods
To test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion.
Results
We showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation.
Discussion
This response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients.
... Other immune cells, such as NK cells [84], DC cells [85], and neutrophils [86], also affect the anti-tumor immune responses. However, the role of NK cells, DC cells, and neutrophils at relapse after BCMA CAR-T cell therapy is still unclear. ...
Background
BCMA CAR-T is highly effective for relapsed/refractory multiple myeloma(R/R-MM) and significantly improves the survival of patients. However, the short remission time and high relapse rate of MM patients treated with BCMA CAR-T remain bottlenecks that limit long-term survival. The immune microenvironment of the bone marrow (BM) in R/R-MM may be responsible for this. The present study aims to present an in-depth analysis of resistant mechanisms and to explore potential novel therapeutic targets for relapse of BCMA CAR-T treatment via single-cell RNA sequencing (scRNA-seq) of BM plasma cells and immune cells.
Methods
This study used 10X Genomic scRNA-seq to identify cell populations in R/R-MM CD45 ⁺ BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. Cell Ranger pipeline and CellChat were used to perform detailed analysis.
Results
We compared the heterogeneity of CD45 ⁺ BM cells before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We found that the proportion of monocytes/macrophages increased, while the percentage of T cells decreased at relapse after BCMA CAR-T treatment. We then reclustered and analyzed the alterations in plasma cells, T cells, NK cells, DCs, neutrophils, and monocytes/macrophages in the BM microenvironment before BCMA CAR-T treatment and relapse after BCMA CAR-T treatment. We show here that the percentage of BCMA positive plasma cells increased at relapse after BCMA CAR-T cell therapy. Other targets such as CD38, CD24, SLAMF7, CD138, and GPRC5D were also found to be expressed in plasma cells of the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Furthermore, exhausted T cells, TIGIT ⁺ NK cells, interferon-responsive DCs, and interferon-responsive neutrophils, increased in the R/R-MM patient at relapse after BCMA CAR-T cell treatment. Significantly, the proportion of IL1β hi Mφ, S100A9 hi Mφ, interferon-responsive Mφ, CD16 hi Mφ, MARCO hi Mφ, and S100A11 hi Mφ significantly increased in the R/R-MM patient at relapse after BCMA CAR-T cell therapy. Cell–cell communication analysis indicated that monocytes/macrophages, especially the MIF and APRIL signaling pathway are key players in R/R-MM patient at relapse after BCMA CAR-T cell therapy.
Conclusion
Taken together, our data extend the understanding of intrinsic and extrinsic relapse of BCMA CAR-T treatment in R/R-MM patient and the potential mechanisms involved in the alterations of antigens and the induced immunosuppressive microenvironment, which may provide a basis for the optimization of BCMA CAR-T strategies. Further studies should be performed to confirm these findings.
... For treating GBM with DC vaccine, DCs are isolated from the peripheral blood CD-14 positive monocytes and GM-CSF and IL-4 are used to induce the differentiation of immature DCs [21]. The tumor antigens (including polypeptide, RNA, DNA, and tumor lysates) are loaded into the immature DCs, which are then presented on MHCs, and the various cytokines (for example, of GM-CSF, of IL-.4, of TNF-α, and IL-6 under) action to maturity [21,22]. The USA, Europe, and Japan have published a lot of respect for the use of DC vaccine therapy in glioma clinical research paper [23]. ...
Glioblastoma (GBM) is the most common primary malignancy of the central nervous system in adults. The prognosis for late-stage glioblastoma (World Health Organization grade IV astrocytic glioma) is very poor. Novel treatment options are sought after and evaluated by clinicians and researchers, and remarkable advances have been made in surgical techniques, radiotherapy, and chemotherapy. However, the treatment of glioblastoma remains extremely difficult and it can extend the lives of patients by only a few months. There has been notable progress in the field of immunotherapy, particularly with the use of tumor vaccines, for treating glioblastoma; especially peptide vaccines and cell-based vaccines such as dendritic cell vaccines and tumor cell vaccines. However, the results of the current clinical trials for vaccination are not satisfactory. This article reviews the progress in the development of vaccines for glioblastoma.
... The culture medium containing cytokines was refreshed every two days. The cells were harvested on day 5. DCs were identified by a FACScan Flow Cytometer (Becton Dickinson) using anti-CD86, anti-CD11c and anti-CD83 antibodies (BD Pharmingen; Strome et al. 2002). ...
Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best conditions including voltage, the pulse length or number, buffer, incubation time and DNA form. In this study, the delivery of pEGFP-N1 vector into two adherent cell lines (HEK-293 T and COS-7) with the same origin (epithelial cells), and also mouse bone marrow-derived dendritic cells (DCs) was evaluated using electroporation under different conditions alone and along with heat treatment. Our data showed that the highest green fluorescent protein (GFP) expression in HEK-293 T and COS-7 cells was observed in serum-free RPMI cell culture medium as electroporation buffer, voltage (200 V), the pulse number (2), the pulse length (15 ms), the circular form of DNA, and 48 h after electro-transfection. In addition, the highest GFP expression in DCs was detected in serum-free RPMI, voltage (300 V), the pulse number (1), the pulse length (5 ms), and 48 h after electro-transfection. The use of sucrose as electroporation buffer, the pulse number (2), and the pulse length (25 ms) led to further cytotoxicity and lower transfection in HEK293T and COS-7 cells than other conditions. Moreover, the high voltage (700 V) increased the cell cytotoxicity, and decreased electro-transfection efficiency in DCs. On the other hand, the best conditions of electroporation along with heat treatment could significantly augment the transfection efficiency in all the cells. These data will be useful for gene delivery in other cells with the same properties using physical methods.
Supplementary information:
The online version contains supplementary material available at 10.1007/s10616-022-00524-4.
... The lysis procedure mostly assures that tumor cells die before antigen loading, thus guaranteeing the safety of these cells. Freeze-thaw processing, UVB irradiation to induce cellular necrosis and apoptosis, manual and/or mechanical breakdown of the tumor, and whole tumor Hypoclorous acid Oxidation (HOCl) are the most common procedures to prepare tumor lysates [39][40][41][42][43]. Loading DCs with tumor lysates has the advantage off reducing the possibility of escape tumors from immunological control, bypassing the need to identify specific antigens, which in many cases have not yet been identified, allowing this methodology to be applied to a wider range of tumor types. ...
Dendritic cells (DCs) are immune specialized cells playing a critical role in promoting immune response against antigens, and may represent important targets for therapeutic interventions in cancer. DCs can be stimulated ex vivo with pro-inflammatory molecules and loaded with tumor-specific antigen(s). Protocols describing the specific details of DCs vaccination manufacturing vary widely, but regardless of the employed protocol, the DCs vaccination safety and its ability to induce antitumor responses is clearly established. Many years of studies have focused on the ability of DCs to provide overall survival benefits at least for a selection of cancer patients. Lessons learned from early trials lead to the hypothesis that, to improve the efficacy of DCs-based immunotherapy, this should be combined with other treatments. Thus, the vaccine's ultimate role may lie in the combinatorial approaches of DCs-based immunotherapy with chemotherapy and radiotherapy, more than in monotherapy. In this review, we address some key questions regarding the integration of DCs vaccination with multimodality therapy approaches for cancer treatment paradigms.
... It was reported that subjecting cancer cells to hyperthermia compared with irradiation was more effective for pulsing DC and provided stronger tumor-specific T cell responses against laryngeal cancer probably through enhancing heat shock protein expression, which might improve tumor-associated antigen delivery to DCs (8). Another research group reported that pulsing DCs with irradiated tumor cells exhibited greater in vivo protection against melanoma than other strategies (i.e., freezethawed or boiled TCLs), likely due to a different mechanism of tumor cell death (10). They concluded that irritated TCL-loading strategy might have superior efficacy for developing a DC-based vaccine for solid malignancies. ...
Background and purpose:
One of the most effective methods for the development of dendritic cell (DC)-based cancer immunotherapy is ex vivo pulsing of DCs with tumor cell lysates (TCLs). However, antitumor immune responses of DCs are significantly influenced by how TCLs were prepared. Here, we compared four strategies of TCL preparation derived from colon cancer cells, HT-29, for ex vivo maturation of DCs.
Experimental approach:
Peripheral blood monocytes were isolated from healthy volunteers and incubated with granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 to differentiate into DCs in 10 days. Morphological properties, phenotype characteristics (i.e. CD83 and CD86), and cytokine production (i.e. IL-10 and interferon gamma) of DCs loaded with four different TCLs (i.e. freeze-thaw, hypochlorous acid (HOCl), hyperthermia, and UV irradiation) were evaluated.
Findings/results:
HOCl preparations led to the generation of DCs with higher surface expression of maturation biomarkers (particularly CD83), while UV preparations resulted in DCs with lower levels of surface biomarkers compared to freeze-thawed preparations. The supernatant of DCs pulsed with HOCl preparation showed significantly higher levels of interferon gamma and lower levels of IL-10 compared with the other groups.
Conclusion and implications:
Our results suggest that pulsing DCs with HOCl preparation may be superior to other TCLs preparation strategies, possibly due to induction of rapid necrotic cell death.
... The superior immune-activating ability that chemotherapy and radiotherapy induce in cancer cells is highlighted in DC vaccination studies of cancer mouse models exploiting this strategy. In these studies, DC vaccination of DCs loaded with radiation-treated or chemotherapy-treated cancer cells, resulted in reduced tumor volume compared to mice vaccinated with DCs loaded with untreated tumor cells (40)(41)(42)(43). Moreover, chemotherapy and radiotherapy were reported to stimulate human leukocyte antigen I (HLA-I) expression of the tumor, making tumor cells more sensitive to cytotoxic killing by CD8 + T cells (38). ...
Current treatment for patients with non-small-cell lung cancer (NSCLC) is suboptimal since therapy is only effective in a minority of patients and does not always induce a long-lasting response. This highlights the importance of exploring new treatment options. The clinical success of immunotherapy relies on the ability of the immune system to mount an adequate anti-tumor response. The activation of cytotoxic T cells, the effector immune cells responsible for tumor cell killing, is of paramount importance for the immunotherapy success. These cytotoxic T cells are primarily instructed by dendritic cells (DCs). DCs are the most potent antigen-presenting cells (APCs) and are capable of orchestrating a strong anti-cancer immune response. DC function is often suppressed in NSCLC. Therefore, resurrection of DC function is an interesting approach to enhance anti-cancer immune response. Recent data from DC-based treatment studies has given rise to the impression that DC-based treatment cannot induce clinical benefit in NSCLC by itself. However, these are all early-phase studies that were mainly designed to study safety and were not powered to study clinical benefit. The fact that these studies do show that DC-based therapies were well-tolerated and could induce the desired immune responses, indicates that DC-based therapy is still a promising option. Especially combination with other treatment modalities might enhance immunological response and clinical outcome. In this review, we will identify the possibilities from current DC-based treatment trials that could open up new venues to improve future treatment.
... As previously described, the extraction of DCs was performed from the bone marrow of male BALB/c mice provided by the animal center of Pasteur Institute of Iran (IPI) [19]. DCs were identified by staining with conjugated antibodies for CD11c, CD40, CD86, and MHCII markers [20]. FACS analysis was conducted on a FACScan flow cytometer (Becton Dickinson). ...
An effective therapeutic vaccine to eradicate HIV-1 infection does not exist yet. Among different vaccination strategies, cell-based vaccines could achieve in clinical trials. Cell viability and low nucleic acid expression are the problems related to dendritic cells (DCs) and mesenchymal stem cells (MSCs), which are transfected with plasmid DNA. Thus, novel in vitro strategies are needed to improve DNA transfection into these cells. The recent study assessed immune responses generated by MSCs and DCs, which were derived from mouse bone marrow and modified with Nef antigen using novel methods in mice. For this purpose, an excellent gene transfection approach by mechanical methods was used. Our data revealed that the transfection efficacy of Nef DNA into the immature MSCs and DCs was improved by the combination of chemical and mechanical (causing equiaxial cyclic stretch) approaches. Also, chemical transfection performed two times with 48-hour intervals further increased gene expression in both cells. The groups immunized with Nef DC prime/rNef protein boost and then Nef MSC prime/rNef protein boost were able to stimulate high levels of IFN-γ, IgG2b, IgG2a, and Granzyme B directed toward Th1 responses in mice. Furthermore, the mesenchymal or dendritic cell-based immunizations were more effective compared to protein immunization for enhancement of the Nef-specific T-cell responses in mice. Hence, the use of chemical reagent and mechanical loading simultaneously can be an excellent method in delivering cargoes into DCs and MSCs. Moreover, DC- and MSC-based immunizations can be considered as promising approaches for protection against HIV-1 infections.
... Recently, it has been reported that cancer cells exposed to gamma irradiation have immunostimulatory effects on the innate and adaptive immune responses [33][34][35]. Kim et al. showed that gamma (10 to 100 Gy)-irradiated colon cancer cells can enhance DC maturation when co-cultured with human dendritic cells, and these cells are apparently able to drive the differentiation of Th1 and cytotoxic CD8 T cells [33]. Strome et al. also reported that BMDCs loaded with irradiated E.G7 tumors (100 Gy), which express the OVA antigen, can promote the activation of the adaptive immunity by specifically generating OVA-specific IFN-γ-producing CD8 + T cells; treatment with these cells imparted significant protection in two analogous human melanoma models [34]. ...
... Kim et al. showed that gamma (10 to 100 Gy)-irradiated colon cancer cells can enhance DC maturation when co-cultured with human dendritic cells, and these cells are apparently able to drive the differentiation of Th1 and cytotoxic CD8 T cells [33]. Strome et al. also reported that BMDCs loaded with irradiated E.G7 tumors (100 Gy), which express the OVA antigen, can promote the activation of the adaptive immunity by specifically generating OVA-specific IFN-γ-producing CD8 + T cells; treatment with these cells imparted significant protection in two analogous human melanoma models [34]. Similar to these experimental methods, the exosomes used in this study were isolated after 100 Gy irradiation. ...
Cancer cells can secrete exosomes under various stressful conditions, whose functions are involved in the delivery of various biologically active materials into host cells and/or modulation of host immune responses. Therefore, an improved understanding of the immunological interventions that stress-induced tumor exosomes have may provide novel therapeutic approaches and more effective vaccine designs. Here, we confirmed the phenotypical and functional alterations of dendritic cells (DCs), which act as a bridge between the innate and adaptive arms of immunity, following non-irradiated (N-exo) and gamma-irradiated melanoma cancer cell-derived exosome (G-exo) stimulation, and evaluated the N-exo- and G-exo-stimulated DCs as therapeutic cancer vaccine candidates. We demonstrated that G-exo-stimulated DCs result in DC maturation by the upregulation of surface molecule expression, pro-inflammatory cytokine release, and antigen-presenting ability, and the downregulation of endocytic capacity. In addition, these cells promoted T cell proliferation and the generation of T helper type 1 (Th1) and interferon (IFN)-γ-producing CD8+ T cells. However, N-exo-stimulated DCs induced semi-mature phenotypes and functions, eventually inhibiting T cell proliferation, decreasing IFN-γ, and increasing IL-10-producing CD4+ T cells. In addition, although N-exo and G-exo stimulations showed similar levels of antigen-specific IFN-γ production, which served as tumor antigen sources in melanoma-specific T cells, G-exo-stimulated DC vaccination conferred a stronger tumor growth inhibition than N-exo-stimulated DC vaccination; further, this was accompanied by a high frequency of tumor-specific, multifunctional effector T cells. These results suggest that gamma irradiation could provide important clues for designing and developing effective exosome vaccines that can induce strong immunogenicity, especially tumor-specific multifunctional T cell responses.
