Antigen-loaded Dendritic Cells Research Articles

An Organoid Approach to Target Her2/PD‐L1 Positive Gastric Cancers

BackgroundTumors can evade immune surveillance by expressing ligands such as programmed cell death 1 (PDL‐1) which interacts with PD‐1 or CTLA‐4, respectively, on CD8+ cytotoxic T lymphocytes (CTLs). These interactions inhibit CTL proliferation, survival, and effector function subsequently leading to immune evasion and cancer persistence. Despite that 40% of gastric cancers express PD‐L1, the response rate to immunotherapy is only 30%. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several metastatic cancers, including gastric. Half of PD‐L1‐positive gastric cancers co‐express HER2, but whether HER2‐induced PD‐L1 sustains tumor cell immune evasion has not been determined.HypothesisThe current research tests the hypothesis that targeted anti‐HER2 and immune checkpoint inhibition may provide synergistic effects in gastric cancer treatment.MethodsPatient‐derived gastric cancer organoids, generated from resected tumor tissues or biopsies were co‐cultured with cytotoxic T lymphocytes (CTLs) and tumor antigenloaded dendritic cells and myeloid‐derived suppressor cells (MDSCs).ResultsWhile PD‐L1‐positive tumor organoids co‐expressed HER2, pharmacological inhibition and gene knockdown of HER2 resulted in decreased PD‐L1 expression in tumor organoids. Within the organoid/immune cell co‐culture, decreased PD‐L1 expression in patient‐derived gastric cancer organoids correlated with a significant increase in CTL proliferation and survival, leading to cancer cell death. Similar results were within the co‐culture were observed in response to Nivolumab treatment. MDSCs within the co‐culture counteracted the inhibitory effects of Her2 inhibition by Mubritinib and Nivolumab.ConclusionThus, HER2‐induced PD‐L1 may contribute to tumor immune evasion. Organoid/immune cell co‐cultures may be used to effectively screen for targeted therapy in individual gastric cancer patients. Therapeutic anti‐HER2 antibody and, immune checkpoint inhibition, as well as depletion of MDSCs, may provide synergistic effects in gastric cancer treatment.Support or Funding Information• NIH NIDDK R01DK083402‐08 (Zavros)NIH 1U19AI116491‐01, (PIs: Weiss and Wells, Zavros Project Leader 1

Abstract P5-04-10: Immune and transcriptional signatures of dendritic cell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients

Abstract Women with triple-negative breast cancer (TNBC) have an increased pathologic complete response rate (pCR; residual disease after neoadjuvant chemotherapy) as compared to women with non-TNBC, and those with pCR have a 90% disease-free survival. However, women with TNBC who do not achieve a pCR have an increased risk of recurrence, decreased overall survival, and post-recurrence survival as compared to women with non-TNBC who do not achieve a pCR. A high priority for clinical research is therefore to increase the pCR rate in breast and axilla with preoperative therapy, as therapeutic options for patients who do not have a pCR are limited. Immunotherapy is an attractive strategy as human BCs can be immunogenic and enhancing the immune effector function may augment the cytotoxic effects of standard therapies. Immunity against tumor antigens can be boosted in cancer patients by vaccination with ex vivo-generated tumor antigen-loaded DCs. Here, we report the extended analysis of a clinical trial on 10 TNBC pts assessing the feasibility of combining cyclin B1/WT1/CEF (antigen)-loaded DC vaccination with preoperative chemotherapy in patients with locally advanced TNBC. Combination of preoperative chemotherapy and intratumoral and subcutaneous autologous DC vaccination led to 70% of combined rate of pCR and residual cancer burden 1 (RCB1). To assess expansion of antigen-specific T cell responses, IFN-g-ELISpot was carried out with PBMCs from baseline (BL) and several time points during vaccine treatment that were cultured with control peptides or with peptide libraries covering vaccine antigens. The expansion of antigen-specific immune responses could be detected at various time points post treatment. Transciptional profiling on blood (Nanostring) and tumors (RNAseq) revealed profound changes in immune transcription signatures. T cell and DC signatures in blood and T cell, inflammation, cytotoxic and cell cycle signatures in pre-chemotherapy breast cancer biopsies were linked with pathological responses in definitive surgery specimens. Taken together, differential gene and immunologic signatures of the pre-treatment breast cancer biopsies distinguish pts who have a pCR vs no pCR and can identify potential therapeutic targets for pts with TNBC. Citation Format: Te-Chia Wu, Joyce O’Shaughnessy, Lee K. Roberts, Jennifer L. Smith, Susan B. Burkeholder, Jennifer Finholt, Jessica Tarnowski, Tuoc Dao, Jeffrey Lamont, Sandra M. Zurawski, Phuong Nguyen, Yuanyuan Wang, Kyung In Kim, Derek Blankenship, Jacob Turner, Xuan Wang, Florentina Marches, Maren K. Levin, Michael Grant, Gerard Zurawski, Virginia Pascual, Jacques Banchereau, Karolina Palucka. Immune and transcriptional signatures of dendritic cell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-10.

A Combination Therapy with Dendritic Cells, Pomalidomide and Programmed Death-Ligand 1 Blockade Exerts a Potent Antitumor Immunity in a Murine Model of Multiple Myeloma

Multiple myeloma (MM) is the second-most-common hematologic malignancy, and develops from clonal malignant plasma cells within bone marrow. Despite tremendous improvements in therapeutic strategies (e.g. stem cell transplantation, immune-modulatory drugs (IMiDs), proteasome inhibitors, and, more recently, immunotherapy), which have led to improved responses to treatment and overall survival, most patients eventually relapse. We have previously shown that the immunization with tumor antigen-loaded dendritic cells (DCs) and pomalidomide/dexamethasone synergistically potentiates the enhancing the antitumor immunity in a myeloma mouse model. In the present study, we investigated whether a DC-based vaccine combined with pomalidomide and PD-L1 blockade has a synergistic effect in a murine MM model. MOPC-315 cell lines were injected subcutaneously to establish MM-bearing mice. Four test groups were used to mimic the clinical protocol: (1) PBS control, (2) DCs + pomalidomide/dexamethasone, (3) pomalidomide/dexamethasone + PD-L1 blockade, and (4) DCs + pomalidomide/dexamethasone + PD-L1 blockade. After treatment, preclinical response and in vitro immunological responses were evaluated. The study was designed to closely mimic the clinical MM treatment protocol and clearly demonstrated that combination treatment with DCs + pomalidomide with dexamethasone + PD-L1 blockade more strongly inhibited MM tumor growth. Consequently, the mice treated with DCs + pomalidomide with dexamethasone + PD-L1 blockade displayed markedly induced tumor regression and significantly prolonged survival, as well as very strong anti-myeloma CTL responses and increased numbers of effector cells (such as CD4+ T cells, CD8+ T cells, memory T cells, NK cells and M1 macrophages) associated with antitumor effects. This treatment also effectively decreased the proportions of suppressor cells, including MDSCs, Tregs and M2 macrophages, in the spleen and tumor microenvironment of treated mice. Tregs, MDSCs and M2 macrophages play crucial roles in immunosuppression and tolerance, which are mediated by tumor-secreted cytokines. The inhibition of Tregs, MDSC and M2 macrophage accumulation may enhance systemic cell-mediated immunity through the activation of DCs or CTLs. Importantly, treatment with pomalidomide with dexamethasone + PD-L1 blockade led to decreased expression of PD-L1 and CTLA-4 in treated mice, which further induced effector cell infiltration of the tumor microenvironment. Moreover, DCs + pomalidomide with dexamethasone + PD-L1 blockade induced the activation of cell-mediated immunity by increasing Th1-specific immune responses, as evidenced by the increased production of IFN-γ and a decrease in the regulatory-specific immune response, as evidenced by the decreased production of TGF-β, IL-10 and VEGF in the spleen and tumor microenvironment. These findings show that inducing the systemic immune response represent a means of treating myeloma. Immunotherapy clearly represents a revolution in cancer care, and promising responses have been shown to various treatments, particularly immune checkpoint inhibitors, IMiDs, DCs and CAR T cells. However, not all patients are responsive to current immunotherapies, and among those patients who do respond, the effects are not always long-lasting. Thus, combination approaches are a cornerstone of cancer therapy for improving patient outcomes in MM. This study demonstrated that the combination of DC vaccination + pomalidomide with dexamethasone + PD-L1 blockade synergistically enhances myeloma immune responses to inhibit tumor growth, restores and enhances host immune effector cells, and reduces the generation of immune suppressor cells in MM. This study provides a framework for developing and understanding the role of immunotherapeutic modalities employing DCs, pomalidomide and PD-L1 blockade to inhibit tumor growth and restore immune function in myeloma-bearing mice. Figure Disclosures No relevant conflicts of interest to declare.

Natural Adjuvants for in situ Vaccination Lymphoma Immunotherapy

Abstract BACKGROUND In situ vaccination (ISV) against lymphoma can be achieved with Flt3-ligand treatment to recruit dendritic cells (DC), radiotherapy to load DC with tumor antigens, and intratumoral injection of a pattern-recognition- receptor agonist (PRRa) to activate antigen-loaded DC. While clinical trials of ISV using synthetic PRRa have yielded remissions, the optimal approach to activate DC is unknown. We hypothesize that ‘natural’ PRRa, such as the attenuated pathogens in common prophylactic vaccines, could target multiple PRR, leading to more robust activation of DC as compared to synthetic PRRa. METHODS 20 FDA-approved vaccines (BCG, MMR, etc.) were screened in vitro, for their effect on DC phenotype and function. DC tumor antigen cross-presentation was assessed using CRISPR gene-edited β2m−/−GFP-lymphoma cells and a GFP-specific CD8 T cell system. Mechanisms of immune activation were interrogated using MyD88, TRIF, MAVS, and IFNAR knockout mice, as well as a library of CRISPR-edited PRR-null Raw264 macrophages. Potent vaccines functioning through distinct mechanisms were evaluated in vivo in an ISV using the A20 murine lymphoma model. RESULTS Several vaccines induced robust DC activation, cross-presentation and increases in T cell activation, proliferation, and tumor killing. Some vaccines were more effective than synthetic PRRa in activating DCs to induce a T cell response. Distinct PRR and signaling pathways were engaged by different vaccines. In vivo, vaccine combination therapy induced tumor regression in a majority of animals. CONCLUSIONS Prophylactic vaccines, used alone or in combination, are effective DC activators and can be repurposed for use in ISV, with immediate translation into the clinic.

Th17 responses to autologous dendritic cell vaccine.

132 Background: Dendritic cells (DC) loaded with autologous tumor antigens in vitro, can induce a Th17 phenotype preceding a Th1 type response. We examined this in patients with metastatic melanoma who had been treated in a randomized trial with either DC loaded with autologous tumor antigens (ATA) from self-renewing tumor cells (DCV) or irradiated tumor cells (TCV). Methods: Blood samples were obtained at baseline, one week before three weekly vaccinations (week-0), and at week-4, (one week after the third vaccination). They were analyzed for growth factors, tumor markers and biomarkers using a quantitative, multiplex ELISA. Monocytes, lymphocytes, loaded and unloaded DC were analyzed for cytokine production and intercellular responses using mixed lymphocyte co-cultures (MLC). Results: When compared to baseline or the TCV arm, DCV-treated patients showed reductions in tumor markers, inflammation and angiogenesis in parallel with Th17 pathway activation, IL12 p70 heterodimer expression was detected in monocytes and DC only after antigen loading. The IL12 p40 fraction was present after differentiation and was enhanced after antigen loading. Similarly, the p19 component of IL23 was expressed before DC loading and later enhanced. MLC showed induction of Th17, Th1 and CD8 subpopulations. In the presence of antigen-loaded DCs, the FOXp3 cells were reduced in number, an effect that was canceled by neutralizing the IL12p40 subunit or by the adding tumor cells alone in the MLC. Conclusions: DC produced ex vivo can activate multiple cytotoxic pathways depending on IL12 subunits. Expression of IL23p19 and the shared IL12p40 fraction are responsible for Th1 priming (IL12 incomplete) and Th17 induction (IL23 complete), determinants of the reactive phenotype of naïve DCs. IL12 p70 heterodimer expression after DC loading enables a targeted Th1 type response while neutralizing the IL12 subunit p40, enhances Foxp3 positive T-regs. DC vaccines produced ex vivo could induce a Th1/Th17 cytotoxic response against tumors. In the absence of antigens, however, The Th17 phenotype can become tolerizing in the microenvironment. The findings are important for applications in settings in which tumor infiltration with Th17 correlates with survival, such as in advanced ovarian cancer. Clinical trial information: NCT00436930.

Abstract B110: Proinflammatory allogeneic dendritic cells promote activation of bystander immune cells and indirectly license antigen-specific T-cells

Abstract Autologous patient-derived dendritic cells (DCs) modified ex vivo to present tumor-associated antigens have frequently been utilized as cancer vaccines. However, apart from the stringent logistics in producing DCs on a patient basis, accumulating evidences indicate that such DCs are poor in migration and in fact do not directly interact with naïve T-cells in vivo. Instead, it is the bystander host-DCs taking up material from apoptotic vaccine DCs and subsequently initiate antitumor T-cell responses. We examine the possibility of harnessing allogeneic pro-inflammatory DCs (alloDCs) as immune enhancers which may affect the activation of bystander immune cells and promote antigen-specific T-cell responses. We have verified the inflammatory state of DCs matured with a maturation cocktail in combination with an infection-enhanced adenovirus. Such proinflammatory alloDCs expressed co-stimulatory and activation molecules and secreted Th-1 type cytokines in vitro. T and NK cells up-regulated activation markers after co-culture with such alloDCs and created an immunostimulatory environment which was found to promote maturation of antigen-loaded DCs and the subsequent activation of antigen-specific T-cells by cross-presentation. Our findings were evaluated as a therapeutic strategy in murine models with success. We are proposing that vaccination with proinflammatory alloDCs in combination with the use of Ad5M as possible antigen delivery vehicle can be a cost-effective strategy to induce antitumor immune responses. Citation Format: Grammatiki Fotaki, Chuan Jin, Iliana Kyriaki Kerzeli, Mohanraj Ramachandran, Alex Karlsson-Parra, Di Yu, Magnus Essand. Proinflammatory allogeneic dendritic cells promote activation of bystander immune cells and indirectly license antigen-specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B110.

Dendritic cell extracellular vesicles.

In addition to direct cell-to-cell contact, dendritic cells (DCs) can regulate the onset of adaptive immunity through the secretion of nano-sized membrane structures, called extracellular vesicles (EVs). This novel mode of communication between cells has added a new layer of complexity to the regulation of immune responses. DCs secrete into their environment different types of EVs containing immunomodulatory molecules that have distinct structural and biochemical properties depending on their intracellular site of origin. Exosomes are generated inside multivesicular bodies and are secreted when these compartments fuse with the plasma membrane, whereas microvesicles are formed and released by budding from the cells' plasma membrane. Once outside the cell of origin, these vesicles can reach target cells through membrane receptor-ligand interactions, modifying their physiological state by the transfer of the EV content or by triggering cell signaling at the cells' surface. Particularly, EVs released by DCs contain major histocompatibility complex (MHC) class I and class II molecules able to activate cognate T cells and promote humoral responses. These activities motivated the use of DC-derived EVs in the treatment of cancer, infectious diseases and autoimmune disorders. The therapeutic potential of these vesicles led to the use of EVs from tumor antigen-loaded DCs in cancer clinical trials, although with limited clinical effects. In this review we will focus on the different EVs released by DCs, their composition and biogenesis, together with their proposed functions as immune regulators.

Combination Strategies to Optimize Efficacy of Dendritic Cell-Based Immunotherapy.

Dendritic cells (DCs) are antigen-presenting cells (APCs) that are essential for the activation of immune responses. In various malignancies, these immunostimulatory properties are exploited by DC-therapy, aiming at the induction of effective anti-tumor immunity by vaccination with ex vivo antigen-loaded DCs. Depending on the type of DC-therapy used, long-term clinical efficacy upon DC-therapy remains restricted to a proportion of patients, likely due to lack of immunogenicity of tumor cells, presence of a stromal compartment, and the suppressive tumor microenvironment (TME), thereby leading to the development of resistance. In order to circumvent tumor-induced suppressive mechanisms and unleash the full potential of DC-therapy, considerable efforts have been made to combine DC-therapy with chemotherapy, radiotherapy or with checkpoint inhibitors. These combination strategies could enhance tumor immunogenicity, stimulate endogenous DCs following immunogenic cell death, improve infiltration of cytotoxic T lymphocytes (CTLs) or specifically deplete immunosuppressive cells in the TME, such as regulatory T-cells and myeloid-derived suppressor cells. In this review, different strategies of combining DC-therapy with immunomodulatory treatments will be discussed. These strategies and insights will improve and guide DC-based combination immunotherapies with the aim of further improving patient prognosis and care.

BZLF1-DEC205 Fusion Protein Enhances EBV-Protective Immunity in a Spontaneous Model of EBV-Driven Lymphoproliferative Disease

Epstein-Barr virus (EBV) is a human herpes virus that infects over 90% of the world's population and is linked with cancer development. In immune-competent individuals, EBV-infection is controlled by a highly efficient virus-specific T cell response. Following primary infection, the virus achieves lifelong persistence within the human host. Risk of EBV-driven cancers increases with immune suppression (IS). Solid organ transplant recipients receive IS medications to prevent graft rejection and are at highest risk of developing EBV-associated lymphomas known as post-transplant lymphoproliferative disease (PTLD). PTLD represents a serious complication of organ transplantation, associated with poor prognosis. Currently, no standard approach for prevention or treatment exists. Reducing the level of IS medication may control PTLD but often leads to graft-rejection.In order to promote long-term protection from EBV-driven cancers, we have developed a vaccine to bolster EBV-specific immunity by targeting the EBV immediate early protein, BZLF1. BZLF1 initiates the activation of lytic stage in EBV-infected cells and promotes B-cell transformation. Work by our group has shown BZLF1-specific T cell expansion following reduction in IS medications correlates with PTLD tumor regression and improved patient survival.Here we specifically delivered the protein (BZLF1) to dendritic cells (DCs) through its endocytic receptor DEC205. DCs were generated from HLA-B8+ donor monocytes incubated with interleukin-4 (IL4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Mature DCs were then loaded with DEC205-BZLF1 fusion protein or control protein (DEC205-Human Chorionic Gonadotropin (DEC205-HCG)). Antigen-loaded DCs were co-cultured with autologous peripheral blood mononuclear cells (PBMCs) in the presence of IL-2 for 10 days. Cells were analyzed by flow cytometry using HLA-tetramers to detect and quantify antigen-specific cytotoxic T leukocyte (CTL) response. To test the EBV vaccine in-vivo, we utilized a human-murine chimeric model of EBV-driven lymphoproliferative disease (EBV-LPD). Severe combined immune deficient (SCID) mice were engrafted with PBMCs from EBV+ donors (Hu-PBL-SCID model). The spontaneous EBV-LPD that develops in this model is comprised of human CD20+, EBV+ lymphoblasts that closely resembles PTLD. Mice were immunized with DCs loaded with DEC205-BZLF1 or DEC205-HCG at the time of PBMC transplant and received booster doses at day 14 and 28. Splenocyte from vaccinated mice were stimulated with autologous tumor (lymphoblastoid cells line, (LCL)) pulsed with BZLF1 pepmix, BZLF1 pepmix alone, and anti-CD3. Secretion of IFNg by stimulated splenocytes was detected using Human IFNg Enzyme-Linked Immunosorbent Spot (ELISpot).In vitro co-cultures treated with DEC205-BZLF1-loaded DCs showed increased expansion of EBV-specific CTLs (p-value: 0.0002) capable of abundant IFNg production and potent cytotoxicity against autologous tumor. This vaccine significantly improved survival in vaccinated mice (p-value: 0.035). Splenocytes from mice in the DEC205-BZLF1 vaccination group revealed higher responsiveness to autologous LCLs and BZLF1 pepmix compared to controls as determined by ELISpot. Human cells recovered from mouse spleen will be analyzed by mass cytometry using a multi-parametric antibody panel to evaluate central/effector memory status, CD4+ Th, CD8+ CTL, NK, and monocyte subsets.These results further support pre-clinical and clinical development of vaccine approaches utilizing the BZLF1 protein as an immunogen to harness adaptive cellular responses to prevent EBV-associated LPD in vulnerable patient populations. DisclosuresNo relevant conflicts of interest to declare.

Antigen-loaded dendritic cells triggers a specific cytotoxic Tlymphocytes immune response against hepatocellular carcinoma: in vitro study.

Hepatocellular carcinoma (HCC) is one of the common malignancies, characterized by poor response to conventional therapeutic options. Immunotherapy with dendritic cells (DCs)-vaccines is one of the most successful strategies used for the treatment of HCC. However, the methods applied in the preparation of antigen-loaded DCs are important factors for optimization of DCs vaccines. The present study was conducted to investigate the effect of HCC-whole tumor cell lysate prepared using rapid repetitive freeze-thaw cycles on the immunogenicity of DCs and evaluate the ability of whole tumor cell lysate-pulsed DCs vaccine to induce a specific cytotoxic Tlymphocytes (CTLs) response against HepG2 cell line. Immature DCs generated from peripheral blood monocytes were randomized into two groups: control DCs and whole tumor cell lysate-pulsed DCs. Phenotypic analysis of the DCs' cell maturation marker CD83 and co-stimulatory molecule CD86 was performed. HCC-specific cytotoxic activity of CD8+ CTLs was measured in vitro. Loading of DCs with necrotic whole cell lysate resulted in non-significant changes in DCs' expression of CD83, but a significant increase in expression of CD86. In addition, CD8+ CTLs stimulated with whole tumor cell lysate-pulsed DCs showed a high cytotoxic activity that specifically attack HepG2 cells. Our findings indicated that pulsation of DCs with whole tumor cell lysate prepared by repetitive freeze-thaw cycles could efficiently enhance the ability of DCs to induce proliferation and clonal expansion of CD8+ CTLs. Data herein, also indicated that whole tumor cell lysate-pulsed DCs triggers a specific CD8+ CTLs against HCC tumor cells.

Synergistic Antimyeloma Activity of Dendritic Cells and Pomalidomide in a Murine Myeloma Model.

We have previously shown that immunization with tumor antigen-loaded dendritic cells (DCs) and the immunomodulating drug, lenalidomide, synergistically potentiates the enhancing antitumor immunity in a myeloma mouse model. In this study, we investigated the immunogenicity of DCs combined with pomalidomide and dexamethasone in a myeloma mouse model. MOPC-315 cells were injected subcutaneously to establish myeloma-bearing mice. Four test groups were used to mimic clinical protocol: (1) PBS control, (2) DCs, (3) pomalidomide + dexamethasone, and (4) DCs + pomalidomide + dexamethasone. The combination of DCs plus pomalidomide and dexamethasone displayed greater inhibition of tumor growth compared to the other groups. This effect was closely related with reduced numbers of immune suppressor cells including myeloid-derived suppressor cells, M2 macrophages, and regulatory T cells, with the induction of immune effector cells such as CD4+ and CD8+ T cells, memory T cells, natural killer (NK) cells, and M1 macrophages, and with the activation of T lymphocytes and NK cells in the spleen. Moreover, the level of the immunosuppressive factor vascular endothelial growth factor was significantly reduced in the tumor microenvironment. The collective findings in the murine myeloma model suggest that tumor antigen-loaded DCs combined with pomalidomide and dexamethasone synergistically enhance antitumor immunity by skewing the immune-suppressive status toward an immune-supportive status.

Immunotherapeutic strategies for treatment of hepatocellular carcinoma with antigen-loaded dendritic cells: in vivo study.

Hepatocellular carcinoma (HCC) is one of the major health problems in the world. DCs-based vaccines are a promising immunotherapeutic strategy that aims at the optimal for induction of a specific antitumor immune response and destruction of tumor cells. The present study was conducted to investigate the immunogenic characters of whole tumor lysate-pulsed DCs vaccine and its ability to induce a specific antitumor immune response in HCC mice model. We also evaluate the effectiveness of prophylactic and therapeutic immunization strategies against HCC in mice models. Mice-derived DCs were in vitro loaded with whole tumor lysate prepared from liver tissue of HCC mice and evaluated for expression of surface maturation markers CD83 and CD86. In vivo immunization of mice with whole tumor lysate-pulsed DCs was performed in two strategies; prophylactic (pre-exposure to HCC) and therapeutic (post-exposure to HCC). Effectiveness of both protocols was investigated in terms of histopathological examination of liver sections and measurement of serum levels of immune cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2). Loading of DCs with whole tumor cell lysate exhibited a significant increase in expression of CD83 and CD86. In vivo administration of prophylactic doses of whole tumor lysate-pulsed DCs in mice before induction of HCC evokes a strong antitumor immune response presented by absence of malignant cells in liver sections and the significant increase in IFN-γ and IL-2. Data herein indicated that prophylactic vaccination with whole tumor lysate-pulsed DCs exhibited an effective antitumor immune response against HCC more than therapeutic protocol.

Expansion and Antitumor Cytotoxicity of T-Cells Are Augmented by Substrate-Bound CCL21 and Intercellular Adhesion Molecule 1.

Adoptive immunotherapy is based on ex vivo expansion and stimulation of T-cells, followed by their transfer into patients. The need for the ex vivo culturing step provides opportunities for modulating the properties of transferred T-cells, enhancing their antitumor abilities, and increasing their number. Here, we present a synthetic immune niche (SIN) that increases the number and antitumor activity of cytotoxic CD8+ T-cells. We first evaluated the effect of various SIN compositions that mimic the physiological microenvironment encountered by T-cells during their activation and expansion in the lymph node. We found that substrates coated with the chemokine CCL21 together with the adhesion molecule intercellular adhesion molecule 1 significantly increase the number of ovalbumin-specific murine CD8+ T-cells activated by antigen-loaded dendritic cells or activation microbeads. Notably, cells cultured on these substrates also displayed augmented cytotoxic activity toward ovalbumin-expressing melanoma cells, both in culture and in vivo. This increase in specific cytotoxic activity was associated with a major increase in the cellular levels of the killing-mediator granzyme B. Our results suggest that this SIN may be used for generating T-cells with augmented cytotoxic function, for use in cancer immunotherapy.

Poly-gamma glutamic acid/alum enhances the efficacy of vaccine antigen by modulating antigen transport to lymph nodes and the adaptive immune responses

Abstract Vaccination is the most effective strategy to protect against infectious diseases and adjuvants are proven to be the key component in vaccines capable of improving antigen-specific immune responses. Conventional vaccine adjuvants, however, still have several limitations such as safety concerns and insufficient induction of vaccine efficacy. Here, we developed a novel adjuvant by combining poly-gamma glutamic acid (γ-PGA) with alum that is already approved for human use worldwide. The physiochemical properties, efficacy, and underlying mechanism of the PGA/Alum as an adjuvant were investigated using ovalbumin (OVA) as a model antigen in vivo and in vitro. PGA/Alum-OVA administered mice showed the robust increase of antigen-specific humoral and cellular immune responses compared to mice administered with γ-PGA-OVA or alum-OVA. Importantly, the PGA/Alum-OVA-induced antibodies had significantly higher antibody-dependent cellular cytotoxicity (ADCC) activity. The enhanced activation and antigen processing of dendritic cells (DCs) and the facilitated migration of antigen-loaded DCs from the injection sites to draining lymph nodes were observed in PGA/Alum-OVA administered mice. Finally, the efficacy of the PGA/Alum as a vaccine adjuvant was evaluated using pandemic H1N1 influenza vaccine antigen. The PGA/Alum drastically enhanced protective efficacy of the pandemic H1N1 vaccine against influenza virus infection. Taken together, PGA/Alum may be a promising adjuvant to improve the influenza vaccine efficacy.

Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling.

Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of 'kiss-and-run' interactions between immune cells 'Labelling Immune Partnerships by SorTagging Intercellular Contacts' (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4+ T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

Clinical-Scale Rapid Autologous BK Virus-Specific T Cell Line Generation From Kidney Transplant Recipients With Active Viremia for Adoptive Immunotherapy.

Polyomavirus-associated nephropathy (PVAN) after BK virus reactivation in kidney transplant recipients (KTR) can compromise graft survival. Lowering immunosuppression is the only established approach to prevent or treat PVAN but nonspecifically increasing host immune competence also augments rejection risk. Ex vivo T cell stimulation/expansion offers the possibility to generate BK-specific T cell lines for adoptive immunotherapy. The objective of this study was to develop and characterize a clinical-scale protocol to generate BK-specific T cell lines from viremic KTR. Peripheral blood mononuclear cells from healthy controls and viremic KTR were stimulated using BK virus peptide libraries loaded or not on monocytes-derived dendritic cells. Cell counts, flow cytometry, and next-generation sequencing were used to assess T cell expansion, differentiation, and clonal diversity. Enzyme-linked immunospots, cytotoxicity assays as well as adoptive transfer in NOD/SCID/IL2Rγ mice were used to assess for pathogen-specificity and evidence of nonspecific alloreactivity. T cell lines from KTR and healthy control showed similar characteristics, implying that ongoing immunosuppression and chronic virus exposure do not compromise the differentiation, specificity, or clonal diversity of T cell lines after ex vivo production. Using antigen-loaded dendritic cells improved T cell expansion and favored central memory T cell differentiation. The T cell lines were antigen-specific and showed no nonspecific alloreactivity in vitro and in vivo. Using a rapid, clinically compliant culture system, we show that autologous BK virus-specific T cell lines can be reliably generated from viremic KTR. Our results pave the way for the treatment or prevention of PVAN with adoptive immunotherapy.

Detection of ABCB5 tumour antigen-specific CD8+ T cells in melanoma patients and implications for immunotherapy.

ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Ex Vivo Induction of Multiple Myeloma-specific Immune Responses by Monocyte-derived Dendritic Cells Following Stimulation by Whole-tumor Antigen of Autologous Myeloma Cells

The introduction of novel agents has significantly expanded treatment options for multiple myeloma (MM), albeit long-term disease control cannot be achieved in the majority of patients. Vaccination with MM antigen-loaded dendritic cells (DCs) represents an alternative strategy that is currently being explored. The aim of this study was to assess the immunogenic potential of ex vivo-generated monocyte-derived DCs (moDCs), following stimulation with the whole-antigen array of autologous myeloma cells (AMC). MoDCs were loaded with antigens of myeloma cells by 2 different methods: phagocytosis of apoptotic bodies from γ-irradiated AMC, or transfection with AMC total RNA by square-wave electroporation. Twenty patients with MM were enrolled in the study. Following stimulation and maturation, moDCs were tested for their capacity to induce T-helper 1 and cytotoxic T lymphocyte responses in vitro. Both strategies were effective in the induction of myeloma-specific cytotoxic T lymphocyte and T-helper 1 cells, as demonstrated by cytotoxicity and ELISpot assays. On the whole, T-cell responses were observed in 18 cases by either method of DC pulsing. We conclude that both whole-tumor antigen approaches are efficient in priming autologous antimyeloma T-cell responses and warrant further study aiming at the development of individualized DC vaccines for MM patients.

Intratumoral delivery of tumor antigen-loaded DC and tumor-primed CD4+ T cells combined with agonist α-GITR mAb promotes durable CD8+ T-cell-dependent antitumor immunity

ABSTRACT The progressive tumor microenvironment (TME) coordinately supports tumor cell expansion and metastasis, while it antagonizes the survival and (poly-)functionality of antitumor T effector cells. There remains a clear need to develop novel therapeutic strategies that can transform the TME into a pro-inflammatory niche that recruits and sustains protective immune cell populations. While intravenous treatment with tumor-primed CD4+ T cells combined with intraperitoneal delivery of agonist anti-glucocorticoid-induced TNF receptor (α-GITR) mAb results in objective antitumor responses in murine early stage disease models, this approach is ineffective against more advanced tumors. Further subcutaneous co-administration of a vaccine consisting of tumor antigen-loaded dendritic cells (DC) failed to improve the antitumor efficacy of this approach. Remarkably, these same three therapeutic agents elicited significant antitumor benefits when the antitumor CD4+ T cells and tumor antigen-loaded DC were co-injected directly into tumors along with intratumoral or intraperitoneal delivery of α-GITR mAb. This latter protocol induced the production of an array of antitumor cytokines and chemokines within the TME, supporting increased tumor-infiltration by antitumor CD8+ T cells capable of mediating tumor regression and extended overall survival.

Combination therapy with dendritic cells and lenalidomide is an effective approach to enhance antitumor immunity in a mouse colon cancer model.

In this study, we investigated efficacy of lenalidomide in combination with tumor antigen-loaded dendritic cells (DCs) in murine colon cancer model. MC-38 cell lines were injected subcutaneously to establish colon cancer-bearing mice. After tumor growth, lenalidomide (50 mg/kg/day) was injected intraperitoneally on 3 consecutive days in combination with tumor antigen-loaded DC vaccination on days 8, 12, 16, and 20. The tumor antigen-loaded DCs plus lenalidomide combination treatment exhibited a significant inhibition of tumor growth compared with the other groups. These effects were associated with a reduction in immune suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, with the induction of immune effector cells, such as natural killer cells, CD4+ T cells and CD8+ T cells in spleen, and with the activation of cytotoxic T lymphocytes and NK cells. This study suggests that a combination of tumor antigen-loaded DC vaccination and lenalidomide synergistically enhanced antitumor immune response in the murine colon cancer model, by inhibiting the generation of immune suppressive cells and recovery of effector cells, and demonstrated superior polarization of Th1/Th2 balance in favor of Th1 immune response. This combination approach with DCs and lenalidomide may provide a new therapeutic option to improve the treatment of colon cancer.