Gene-modified Dendritic Cells Research Articles

Engineered Exosomes Containing microRNA-29b-2 and Targeting the Somatostatin Receptor Reduce Presenilin 1 Expression and Decrease the β-Amyloid Accumulation in the Brains of Mice with Alzheimer's Disease.

Exosomes are membrane vesicles secreted by various cells and play a crucial role in intercellular communication. They can be excellent delivery vehicles for oligonucleotide drugs, such as microRNAs, due to their high biocompatibility. MicroRNAs have been shown to be more stable when incorporated into exosomes; however, the lack of targeting and immune evasion is still the obstacle to the use of these microRNA-containing nanocarriers in clinical settings. Our goal was to produce functional exosomes loaded with target ligands, immune evasion ligand, and oligonucleotide drug through genetic engineering in order to achieve more precise medical effects. To address the problem, we designed engineered exosomes with exogenous cholecystokinin (CCK) or somatostatin (SST) as the targeting ligand to direct the exosomes to the brain, as well as transduced CD47 proteins to reduce the elimination or phagocytosis of the targeted exosomes. MicroRNA-29b-2 was the tested oligonucleotide drug for delivery because our previous research showed that this type of microRNA was capable of reducing presenilin 1 (PSEN1) gene expression and decreasing the β-amyloid accumulation for Alzheimer's disease (AD) in vitro and in vivo. The engineered exosomes, containing miR29b-2 and expressing SST and CD47, were produced by gene-modified dendritic cells and used in the subsequent experiments. In comparison with CD47-CCK exosomes, CD47-SST exosomes showed a more significant increase in delivery efficiency. In addition, CD47-SST exosomes led to a higher delivery level of exosomes to the brains of nude mice when administered intravenously. Moreover, it was found that the miR29b-2-loaded CD47-SST exosomes could effectively reduce PSEN1 in translational levels, which resulted in an inhibition of beta-amyloid oligomers production both in the cell model and in the 3xTg-AD animal model. Our results demonstrated the feasibility of the designed engineered exosomes. The application of this exosomal nanocarrier platform can be extended to the delivery of other oligonucleotide drugs to specific tissues for the treatment of diseases while evading the immune system.

Abstract CT153: Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections

Abstract Background: Effective immunotherapy options are lacking for patients with advanced non-small cell lung cancer (NSCLC) who progress on a programmed cell death-(ligand)1 [PD-(L)1] inhibitor and for those who are epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement positive after progression on tyrosine kinase inhibitor (TKI) therapy. One potential approach to improve immune checkpoint efficacy in these patient populations is to promote cytolytic T cell infiltration into tumors. This can be accomplished via in situ vaccination with chemokine gene-engineered functional antigen presenting cells (APCs) which can take advantage of the full repertoire of tumor antigens and convert the tumor into a lymph node-like environment to promote both local and systemic antitumor responses. The chemokine CCL21 promotes co-localization of naive T cells and antigen-experienced dendritic cells (DCs) to facilitate T cell activation. Our preclinical studies and phase I trial of intratumoral (IT) administration of DC genetically modified to overexpress CCL21 (CCL21-DC) revealed augmentation of tumor antigen presentation in situ, resulting in systemic antitumor immunity. However, increased PD-L1 expression was observed in some patient tumors, suggesting that tumor-mediated impairment of T cell function may be forestalling a more robust CCL21-DC mediated antitumor response. Similarly, improved PD-(L)1 inhibitor efficacy may be possible with enhanced T cell infiltration and augmented APC function following IT CCL21-DC. Therefore, we are conducting a phase I trial, combining IT CCL21-DC with pembrolizumab in patients with advanced NSCLC. Methods: Phase I, dose-escalating, multi-cohort trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either (1) EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or (2) EGFR/ALK mutant after progression on TKI therapy. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by pembrolizumab once every three weeks for up to 1 year. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and determination of drug target activity by immune monitoring studies. This trial, NCT03546361, is currently open for enrollment, and this updated abstract will highlight the feasibility of repeated IT injections in treated patients. Citation Format: Bin Liu, Aaron Lisberg, Ramin Salehi-Rad, Michael Oh, Jay M. Lee, Linh M. Tran, Kostyantyn Krysan, Raymond J. Lim, Camelia Dumitras, Zhe Jing, Fereidoun Abtin, Robert D. Suh, Scott J. Genshaft, Scott S. Oh, Gregory A. Fishbein, Anita Kaul, Kanwarpal S. Kahlon, Shahryar A. Ashouri, Jonathan W. Goldman, David A. Elashoff, Edward Garon, Steven M. Dubinett. Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT153.

Abstract CT219: Phase I trial of in situ vaccination with autologous CCL21 gene-modified dendritic cells (CCL21-DC) combined with pembrolizumab for advanced non-small cell lung cancer (NSCLC)

Abstract Background: Although immune checkpoint blockade (ICB) targeting Programmed Death-1/Programmed Death-Ligand 1 (PD-1/PD-L1) alone or in combination with chemotherapy is now a first-line option for patients with advanced NSCLC, the majority of patients do not benefit from anti-PD-1/PD-L1 monotherapy, and the combination with chemotherapy is often associated with toxicity. For patients who initially respond to PD-1/PD-L1 inhibition, many relapse after an initial response and are in need for innovative strategies to overcome the resistance to ICB. One possible approach is to utilize in situ vaccination with functional chemokine gene-modified antigen presenting cells (APCs) that take advantage of the full repertoire of tumor antigens in the tumor microenvironment (TME) in order to restore tumor antigen presentation, promote tumor infiltration of immune cells driven by chemokine gradient, and facilitate tumor-specific T cell activation, both locally and systemically. The chemokine CCL21 is a therapeutic candidate due to its ability to promote infiltration and co-localization of naïve T cells and antigen-experienced dendritic cells (DCs) and facilitate T cell activation. In preclinical studies as well as a phase I clinical trial, we observed that intratumoral (IT) injection of CCL21-DC induces the infiltration of autologous DC and T cells into the TME, and generates systemic tumor-specific immune responses against multiple tumor antigens. We also observed tumoral PD-L1 upregulation following CCL21-DC injection, which may hinder T cell function. Similarly, the lack of efficacy of PD-1/PD-L1 inhibitors could potentially be combated by enhanced T cell infiltration and augmented APC function in the TME following in situ vaccination with CCL21-DC. Therefore, we are currently evaluating the safety and efficacy of combining IT CCL21-DC and intravenous (IV) pembrolizumab in advanced NSCLC patients following progression on ICB or tyrosine kinase inhibitor (TKI) therapy in a phase I trial (NCT03546361). Methods: Phase I, dose-escalating, multi-cohort trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or EGFR/ALK mutant after progression on TKI. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by q3wk pembrolizumab up to 1 year. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and immune monitoring studies. [B.L. and A.L. contributed equally to this work as first authors.] Citation Format: Bin Liu, Aaron Lisberg, Ramin Salehi-Rad, Jay Lee, Linh M. Tran, Kostyantyn Krysan, Raymond Lim, Camelia Dumitras, Zhe Jing, Michael Oh, Fereidoun Abtin, Robert Suh, Scott Genshaft, Scott Oh, Gregory Fishbein, Ciara M. O'Higgins, Anita Kaul, Kanwarpal Kahlon, Shahryar Ashouri, Jonathan Goldman, David Elashoff, Edward Garon, Steven Dubinett. Phase I trial of in situ vaccination with autologous CCL21 gene-modified dendritic cells (CCL21-DC) combined with pembrolizumab for advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT219.

Abstract 4206: In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory

Abstract Decresased responses to immune checkpoint inhibitors (ICIs) in NSCLC are associated with low tumor mutational burden (TMB), impaired tumor antigen presentation, decreased baseline CD8+ T lymphocytes tumor infiltration and low PD-L1 expression in the tumor microenvironment (TME). One approach to augment the efficacy of ICIs in NSCLC is to enhance anti-tumor T cell responses by utilizing in situ vaccination (ISV) with professional antigen-presenting dendritic cells (DCs). In preclinical studies as well as a phase I clinical trial, we have shown that ISV with gene-modified DCs expressing CCL21 (CCL21-DC) promotes effector T lymphocyte infiltration into the tumor, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could promote T cell priming and activation, thereby sensitizing non-responsive NSCLC tumors to ICI. Utilizing multiple syngeneic murine models of NSCLC with distinct driver mutations [KrasG12D/P53+/-/Lkb1-/- (KPL); KrasG12D/P53+/- (KP); and KrasG12D (K)] and varying TMB, we observed that CCL21-DC ISV synergizes with anti-PD-1 therapy resulting in the eradication of a subset of tumors. Evaluation of KPL tumors by flow cytometry and scRNA-seq revealed that combination therapy reprogrammed the myeloid compartment to be less immunosuppressive, and induced T cell infiltration, Th1 polarization, as well as the enrichment of stem-like TCF1+ T cells within the TME. Intratumoral (IT) CCL21-DC enhanced CD103+DC migration to the local lymph node as determined by flow cytometry. Addition of ICI to IT CCL21-DC resulted in enhanced T cell proliferation and INF-γ secretion within the TME and the spleen. Whole exome sequencing (WES) of tumors revealed immunoediting of tumor subclones after IT CCL21-DC and ICI combination therapy. Tumor rechallenge studies demonstrated the establishment of systemic tumor-specific immunity in a subset of mice that were cured of disease.In conclusion, tumor vaccination with CCL21-DC sensitizes immune-resistant murine NSCLC to anti-PD-1 therapy. Combination therapy enhances anti-tumor T cell responses, resulting in immunoediting of tumor subclones and the generation of systemic tumor-specific immunity. In an ongoing clinical trial, we are evaluating the safety and efficacy of ISV with CCL21-DC in combination with anti-PD-1, pembrolizumab, in patients with advanced NSCLC who have progressed on front-line therapy. Citation Format: Ramin Salehi-Rad, Raymond J. Lim, Linh M. Tran, Yushen Du, Camelia Dumitras, Tianhao Zhang, William Crosson, Jensen Abascal, Kostyantyn Krysan, Bin Liu, Steven M. Dubinett. In situ vaccination with CCL21-DC sensitizes non-responsive murine NSCLCs to anti-PD-1 therapy and generates systemic tumor-specific immune memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4206.

Exosomes with FOXP3 from gene-modified dendritic cells ameliorate the development of EAE by regulating the balance of Th/Treg.

Objective: To investigate the efficiency and potential mechanisms of exosomes from dendritic cells (DCs) transfected with Forkhead box protein P3 (FOXP3) in the development of experimental autoimmune encephalomyelitis (EAE).Method: Mouse bone marrow-derived immature DCs were loaded with adenovirus carrying FOXP3 gene, and exosomes were generated. Then the exosomes with FOXP3 (FOXP3-EXOs) were co-cultured with CD4+T cell in vitro to evaluate their potential on CD4+T cell proliferation and differentiation, and injected into EAE mice to assess their effects on the development of EAE.Result: FOXP3-EXOs were effective to inhibit the CD4+T cell proliferation and the production of Interferon gamma (IFN-γ), interleukin (IL)-6, and IL-17, while they promoted the production of IL-10 in vitro. Moreover, FOXP3-EXOs treatment significantly decreased the neurological scores, reduced the infiltration of inflammatory cells into the spinal cord, and decreased demyelination in comparison to saline and Con-EXOs treated EAE mice. Moreover, the FOXP3-EXOs treatment resulted in obvious increases in the levels of regulatory T (Treg) cells and IL-10, whereas levels of T helper 1 (Th1) cells, Th17 cells, IFN-γ, IL-6, and IL-17 decreased significantly in the splenocyte culture of EAE mice.Conclusion: The present study preliminarily investigated the effects and potential mechanisms of FOXP3-EXOs in EAE and revealed that the FOXP3-EXOs could inhibit the production of Th1 and Th17 cells and promote the production of Treg cells as well as ameliorate the development of EAE. The neuroprotective effects of FOXP3-EXOs on EAE are likely due to the regulation of Th/Treg balance.

Technical Feasibility and Safety of Repeated Computed Tomography–Guided Transthoracic Intratumoral Injection of Gene-Modified Cellular Immunotherapy in Metastatic NSCLC

IntroductionTo assess the technical feasibility and safety of repeated percutaneous computed tomography (CT)–guided transthoracic biopsies and intratumoral injections of gene-modified dendritic cells in metastatic NSCLC. MethodsA total of 15 patients with 15 NSCLC lesions measuring greater than 1.0 cm underwent two cycles of intratumoral biopsies and CCL21 dendritic cell injections separated by 7 days. All needle placements and injections were done under CT guidance. Clinical and imaging follow-up was done approximately 4 weeks after the first procedure. Safety and feasibility were determined as: (1) safety and feasibility similar to that of single-needle biopsy, and (2) an absence of serious adverse events defined as grade greater than or equal to three according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. ResultsA total of 30 percutaneous, transthoracic intratumoral biopsies and injections into the lung cancer were performed, two cycles (at d 0 and 7) received by each patient (311 biopsies and 96 intratumoral injections). All percutaneous cases achieved technical success with respect to needle placement for both biopsy and injection of CCL21 dendritic cells. Only minor complications were observed (grade <3), including pneumothorax (n = 10, 33%) and small postbiopsy hemorrhage (n = 2, 7%). Pneumothorax was moderate (n = 1) or trace (n = 9), with resolution of the moderate pneumothorax after manual aspiration without chest tube placement. No patient required chest tube placement. No other complications or serious adverse effects related to the biopsy or dendritic cell injection were noted. All patients were in stable condition after up to 4 hours in the recovery unit and were discharged home on the same day. No procedure-related complications were observed on imaging or clinical follow-up at 4 weeks. ConclusionsRepeated percutaneous, transthoracic CT-guided biopsies and intratumoral gene-modified cell-based immunotherapy injections into lung cancers are technically feasible, safe, and reproducible. There were no procedure-related serious (defined as grade ≥3) adverse events.

A35 Dendritic Cell in Situ Vaccination Potentiates Anti-PD-1 Efficacy and Induces Immunoediting in a Murine Model of NSCLC

Studies reveal that responses to checkpoint blockade in non-small cell lung cancer (NSCLC) are associated with high tumor mutational burden (TMB), preexisting CD8+ T-cell infiltration, and high baseline PD-L1 expression within the tumor microenvironment (TME). In contrast, co-occurring KRAS/LKB1 mutation is associated with primary resistance to PD-1 blockade and decreased overall survival. In preclinical studies as well as a phase I clinical trial, we have discovered that intratumoral (IT) vaccination with gene-modified dendritic cells expressing CCL21 (CCL21-DC) promotes tumor effector T-lymphocyte infiltration, PD-L1 upregulation, and systemic tumor-specific immune responses. We hypothesized that in situ vaccination with CCL21-DC could restore tumor antigen presentation and promote T-cell priming and activation, thereby sensitizing nonresponsive NSCLC tumors to checkpoint blockade. Although genetically engineered murine models (GEMMs) of NSCLC bear driver mutations of the disease, recent studies reveal that these GEMMs possess low mutational burden. We established novel GEMMs of NSCLC [KrasG12D (K), KrasG12DP53-/- (KP), KrasG12DP53+/-Lkb1-/- (KPL)] bearing common driver mutations and varying mutational loads by in vitro exposure of tumor cell lines to the carcinogen N-methyl-N-nitrosourea (MNU). Our preclinical KPL model with high TMB recapitulates the immunologic phenotype of human disease, and contains a predominance of myeloid-derived suppressor cells (MDSC), low tumor-infiltrating lymphocytes (TILs), and low PD-L1 expression within the TME. As anticipated, the KPL tumors are resistant to anti-PD-1 therapy, even with increased mutational load. We evaluated IT CCL21-DC combined with anti-PD-1 therapy in immunocompetent mice bearing KPL tumors with high TMB, and observed that IT CCL21-DC vaccination induces infiltration of autologous T lymphocytes and conventional type I DCs (cDC1s) into the TME and sensitizes the tumors to anti-PD-1 therapy. Combination therapy also reprogrammed the myeloid compartment, resulting in a significant reduction of MDSCs and a concurrent increase in CD11b+Ly6GhiLy6Clo monocyte/myeloid population. Whole-exome sequencing (WES) of tumors revealed immunoediting and selective depletion of tumor subclones post IT CCL21-DC and anti-PD1 combination therapy. Future studies will evaluate the evolution of the T-cell receptor (TCR) repertoire in response to the combination treatment and define functional responses to neoepitopes. These studies will enhance our understanding of the molecular mechanisms of tumor vaccination and facilitate the development of rational combination strategies.

IL-10 gene-modified dendritic cells-induced type 1 T regulatory cells inhibit graft-versus-host disease while preserving graft-versus-leukemia effect

To explore the role of type 1 regulatory T (Tr1) cells in allogeneic bone marrow transplantation (allo-BMT), we generated Tr1 cells from naïve CD4+ T cells in donor mice that were subjected to stimulation of recipient interleukin (IL)-10 gene-modified dendritic cells (DCs). In animal models, the severity of graft-versus-host disease (GVHD) in the group co-infused with DC-induced Tr1 cells was markedly milder than that in the groups without infusion of Tr1 cells. Moreover, the co-infusion Tr1 cells significantly prolonged the leukemia-free survival in tumor-bearing mice. Besides, Tr1-induced cells mediated the shift from T helper (Th)1 to Th2 cytokines in allo-BMT recipients. In Conclusions, our findings suggest that IL-10 gene-modified DC-induced Tr1 cells could inhibit GVHD while maintaining Graft-versus-leukemia (GVL) through mediating the shift of Th1/Th2 cytokines in an allo-BMT mice model.

The therapeutic effects of interleukin 10 gene-modified bone marrow-derived dendritic cells in a murine model of liver fibrosis

Objective: To investigate the therapeutic effects and mechanisms of interleukin-10 (IL-10) gene-modified dendritic cells (DC-IL-10) in mice with liver fibrosis. Methods: DC-IL-10 was constructed in vitro, the phenotype and function of which were evaluated by flow cytometry. BALB/c mice were treated with intraperitoneal injection of carbon tetrachloride (CCl4) to establish liver fibrotic model. DC-IL-10 was administrated via tail vein. Animals were divided into 4 groups including normal dendritic cell(DC) control, liver fibrosis only, negative lentiviral transfection DC (DC-mock) and DC-IL-10. Liver function, cytokine secretion, T lymphocyte differentiation and liver histomorphology were tested. Real-time PCR and western blot were used to analyze the effect of DC-IL-10 on Wnt/β-catenin signaling pathway and its role in liver fibrosis. Results: When compared with DC control and DC-mock, the expression of DC-IL-10 surface stimulating molecules (major histocompatibity complex-Ⅱ, CD(80), CD(86)) were significantly decreased (F=14.708, 22.503, 12.595, respectively, all P<0.05), and DC-IL-10 significantly inhibited T lymphocyte proliferation (F=50.295, P<0.05). When compared with liver fibrosis group, serum alanine aminotransferase and aspartate transaminase were decreased in DC-IL-10 treated group (all P<0.05), other parameters including inflammatory factors (tumor necrosis factor α, IL-6, IL-1β) reduced (all P <0.05), the proportion of regulatory T cells (Treg) increased (F=6.742, P<0.05), pathological damage improved, the expression of Wnt3a, α-SMA and β-catenin mRNA and protein significantly reduced in DC-IL-10 treatment group (all P<0.001) . Conclusions: DC-IL-10 induces elevation of Treg for immune tolerance, as well as inhibition of inflammatory response, block of Wnt/β-catenin signaling pathway, which translates into improvement of liver fibrosis.

The immunoprotection of transplant mice allograft by suppressor of cytokine signaling 1 gene-modified dendritic cells induces T cell anergy

Objective To study the regulation of high expression of suppressor of cytokine signaling 1 (SOCS1) molecule in dendritic cells (DC), and to verify the effect of DC-SOCS1 on T cell function and allografts immune protective effects in vitro and in vivo. Methods DC were modified with high affinity replication-defective adenoviral vector Ad5F35 over-expressing SOCS1. The phenotype of transduced mature DC and the function were analyzed, and the mechanism of inducing donor-specific CD4+ T cells hyporesponsiveness was explored. The mouse allogenic islet transplantation model was established, and 24 h prior to transplantation each recipient mouse was given DC cells, DC-SOCS1 cells or equal amount of phosphate buffer (PBS), named DC group, DC-SOCS1 group and control group respectively. The survival of islet grafts of transplanted recipients was observed, and on the day 7 and the rejection day post transplantation, the islet graft glucose tolerance, pathological changes, the percentage of T cell subsets in the spleen and draining lymph nodes as well as related cytokines were observed. The possible associated mechanisms of SOCS1 gene-modified mature DC mitigating immune rejection and inducing transplant tolerance in vivo were explored. Results Genetic modified Ad5F35 dramatically increased the transduction efficiency of DC compared to regular Ad. The experimental data demonstrated there was a tendency that DC overexpressing SOCS1 can induce DC low down the express of MHC and costimulatory molecules, potentiate the ability of DC-SOCS1 to induce donor specific T cell anergy in mixed lymphocytes reaction (MLR) assay. DC-SOCS1 significantly prolonged the survival time of the islet allograft with a median survival time of (37.00±2.19) days, which is significantly longer than control [(20.00±0.55) d, P=0.008] and DC group [(10.00±0.45) d, P=0.001]. Flow cytometric analysis revealed that the DC-SOCS1 dramatically reduced the population of Th1 and Tc1 [(9.92±1.57)%, P=0.027], CD4 [(17.77±2.80)%, P=0.007] and CD8 [(14.92±2.18)%, P=0.004] in spleen lymphocytes compared to control group and DC group on day 7, and a reduction of CD4 [(26.00±2.02)%, P=0.034] was observed in draining lymph nodes compared to DC group. Conclusion SOCS1 genetic modification efficiently confers mDC with low express of MHC and costimulatory molecules, and allows them to induce T cell hyporesponsiveness in vitro. The DC-SOCS1 pretreated recipients exhibit immune tolerance and prolong islet allograft survival in vivo. Key words: Dendritic cells; Gene transduction; Suppressor of cytokine signaling 1; Islet transplantation; Immune tolerance

CD38 gene-modified dendritic cells inhibit murine asthma development by increasing IL-12 production and promoting Th1 cell differentiation.

Predominant T helper (Th)2 and impaired Th1 cell polarization has a crucial role in the development of asthma. Cluster of differentiation (CD)38 is associated with the increased release of interleukin (IL)‑12 from dendritic cells (DCs) and DC‑induced Th1 cell polarization. However, whether CD38 expression affects DC function in asthma development remains unknown. In the current study, adenoviruses were constructed containing the murine CD38 gene. Overexpression of CD38 protein level in DCs induced from bone‑marrow derived DCs (BMDCs) by recombinant mouse granulocyte macrophage colony‑stimulating factor and IL‑4 was achieved through 24h adenovirus infection. The results demonstrated that BMDCs with CD38 overexpression exhibited no phenotypic change; however, following stimulation with lipopolysaccharide (LPS), maturation and IL‑12 secretion were increased. In addition, CD38‑overexpressing BMDCs stimulated with LPS exhibited more effective Th1 cell differentiation. Mice that were administered CD38‑overexpressing BMDCs exhibited milder symptoms of asthma. Furthermore, decreased IL‑4, IL‑5 and IL‑13 levels were detected in bronchoalveolar lavage fluid (BALF), reduced immunoglobulinE levels were measured in the sera, and increased interferon‑γ was detected in BALF from the recipients of CD38‑overexpressing BMDCs. Increased phosphorylated‑p38 expression was also detected in LPS-stimulated CD38-overexpressing BMDCs, whereas pretreatment with a p38‑specific inhibitor was able to abolish the effects of LPS stimulation and CD38 overexpression on IL‑12 release and Th1 cell differentiation in BMDCs. These results suggested that CD38 may be involved in the DC function of alleviating asthma via restoration of the Th1/Th2 balance, thus providing a novel strategy for asthma therapy.

IL-36 is required for the induction of therapeutic immunity by Tbet gene-modified dendritic cells. (TUM10P.1024)

Abstract Our lab has developed a dendritic cell (DC) vaccine as a means of overcoming immune dysfunction in the tumor microenvironment (TME) and generating a therapeutic anti-tumor immune response. As protective Type 1 immune responses are characterized by intrinsic expression of the Tbet transactivator in T cells and DC, we engineered DC with Tbet cDNA (ie, DC.Tbet) and injected these cells into the TME of syngeneic tumor-bearing mice as a therapy. Treatment of MCA205 sarcomas with DC.Tbet promoted the rapid (hours) and sustained (days-weeks) infiltration of Type 1-polarized CD4+ and CD8+ TIL into the TME and led to increased TME expression of factors characteristic of ectopic lymphoid structures (ELS), such as TNFSF14, CCL19, and CCL21. DC.Tbet cells revealed a 60-fold increase in the level of IL-36gamma mRNA over mock-transduced DC and inhibition of signaling through the IL-36 receptor (IL-36R) using natural antagonist IL-1F5 abrogated the ability of DC.Tbet therapy to inhibit tumor growth in vivo, concurrent with reduced recruitment of therapy-induced CD4+ and CD8+ TIL. The efficacy of DC.Tbet therapy was similarly negated in MCA205-bearing IL-36R-/- hosts. These data suggest a mechanism whereby treatment with DC.Tbet leads to recruitment of anti-tumor TIL via an IL-36R signaling-dependent mechanism that supports the development of ELS in the TME. These findings foreshadow the clinical use of IL-36gamma in cancer vaccine formulations as a means to promote therapeutic immunity.

A Novel Modular Polymer Platform for the Treatment of Head and Neck Squamous Cell Cancer

Objectives: Evaluate the therapeutic efficacy of a novel modular polymer platform in the treatment of head and neck squamous cell carcinoma (HNSCC). 50% of HNSCC patients fail primary management, and salvage of the recurrent disease patient is of paramount importance. Methods: Study Design: In vivo study. Setting: Academic research laboratory. C3H/HeJ mice were randomized to receive implantation of 1) no polymer; 2) plain polymer; 3) plain polymer containing gene-modified dendritic cells over-expressing CCL21 (DC-AdCCL21); and 4) plain polymer with concurrent daily CCL21 injections. Tumor size was measured until the mice were euthanized. At necropsy, the tumors were excised and weighed. Results: Our results using this novel polymer platform demonstrate a remarkable reduction in tumor growth. The dendritic cell CCL21 secreting polymer effectively reduced SCCVII/SF tumors in the C3H/HeJ mice by over 16-fold ( P &lt; 0.01) as compared to control, plain polymer, and plain polymer + intratumoral CCL21 injection groups. We also demonstrate that we can effectively grow dendritic cells in the polymer that can actively secrete CCL21. Treatment with the dendritic cell CCL21 secreting polymer led to a marked reduction in tumor burden with extensive mononuclear cell infiltration of the tumors. Conclusions: Our promising results indicate that this polymer may represent a new therapeutic modality for patients with HNSCC. Our data provide a strong rationale for further evaluation of this DC-AdCCL21 polymer in regulation of tumor immunity and genetic immunotherapy for HNSCC. Once this polymer platform is further optimized, we will plan for the ultimate validation in the context of a prospective trial in patients with unresectable advanced or recurrent HNSCC.

Extranodal induction of therapeutic immunity in the tumor microenvironment after intratumoral delivery of Tbet gene-modified dendritic cells

Murine dendritic cells (DC) transduced to express the Type-1 transactivator T-bet (i.e. mDC.Tbet) and delivered intratumorally (i.t.) as a therapy are superior to control wild-type DC in slowing the growth of established subcutaneous (s.c.) MCA205 sarcomas in vivo. Optimal anti-tumor efficacy of mDC.Tbet-based gene therapy was dependent on host NK cells and CD8+ T cells, and required mDC.Tbet expression of MHC class I molecules, but was independent of the capacity of the injected mDC.Tbet to produce pro-inflammatory cytokines (IL-12 family members or IFN-γ) or to migrate to tumor-draining lymph nodes (TDLN) based on CCR7 ligand chemokine recruitment. Conditional (CD11c-DTR) or genetic (BATF3−/−) deficiency in host antigen crosspresenting DC did not diminish the therapeutic action of i.t.-delivered wild-type mDC.Tbet. Interestingly, we observed that i.t delivery of mDC.Tbet (versus control mDC.Null) promoted the acute infiltration of NK cells and naïve CD45RB+ T cells into the tumor microenvironment (TME) in association with elevated expression of NK- and T cell-recruiting chemokines by mDC.Tbet. When taken together, our data support a paradigm for extranodal (cross)priming of therapeutic Type-1 immunity in the TME after i.t. delivery of mDC.Tbet-based gene therapy.

Novel mechanisms of anti-tumor action for therapeutic T-bet gene-modified dendritic cells (P4228)

Abstract Clinical implementation of Type-1 polarized dendritic cell (DC) in vaccine formulations holds significant promise in the cancer therapy setting. We have recently demonstrated that DC engineered to express the T cell Type-1 transactivator protein T-bet (TBX21) promote robust Type-1 T cell-mediated immunity via a unique mechanism that is largely interleukin (IL)-12p70-independent. In the current study, we investigated the anti-tumor mechanisms of action associated with intratumoral administration of DC.Tbet developed from syngenic IL-12p35 -/-, IL-12p40 -/-, IFNγ -/- or β2m -/- mice. We observed that Type-1 anti-tumor immunity associated with slowed tumor growth was effectively developed in animals receiving DC.Tbet deficient in their ability to produce IL-12 family cytokines (i.e. IL-12, IL-23, IL-35) or IFN-γ. Surprisingly, we observed that the ability of DC.Tbet to present MHC class I/peptide complexes (i.e. lost in DC developed from β2m -/- mice) was not important for early priming of anti-tumor CD8+ T cells, but was essential for the sustained therapeutic efficacy of DC.Tbet (and treatment-induced anti-tumor CD8+ T cells) within the tumor microenvironment (TME). In order to best translate this cancer treatment modality into the clinic, we are currently evaluating how DC.Tbet conditions the TME for improved function of Ag-primed Type-1 T effector cells and how these APC collaborate with host DC to diversify the therapeutic T cell repertoire.

Improvement of HBsAg Gene-Modified Dendritic Cell-Based Vaccine Efficacy by Optimizing Immunization Method or the Application of β-glucosylceramide

Hepatocellular carcinoma (HCC) in China is mostly Hepatitis B virus infection related. The antitumor efficacy of HBsAg gene-modified dendritic cells (DC) has been widely tested both in vitro and in vivo. In this study, we analyzed whether adenoviral vector mediated HBsAg expression would alter cell surface phenotype or autologous T cell stimulating function of mature DCs. Further, the anti-tumor efficacy of pAd-HBsAg-DC-based vaccine was evaluated in mice bearing HBsAg expressing HCC. We also tested whether β-glucosylceramide (β-GC) would enhance the anti-tumor activity of pAd-HBsAg-DC. Results revealed that pAd-HBsAg-DC expressed and secreted HBsAg, while maintaining phenotypic characteristics of mature DCs. Vaccination with pAd-HBsAg-DC conferred specific therapeutic antitumor immunity to animal model bearing HBsAg expressing HCC. The application of β-GC activated mice hepatic NKT cells and enhanced the antitumor activity of pAd-HBsAg-DC. Most importantly, in vivo results showed that the inhibiting effect of pAd-HBsAg-DC vaccination on tumor growth was more significant when applied before tumor inoculation, suggesting that genetically modified DC based therapeutic cancer vaccine may achieve the most optimized antitumor effect when applied before tumor onset, and β-GC may serve as a potent innate immune enhancer for augmenting the antitumor effect of pAd-HBsAg-DC vaccine.

SLC gene-modified dendritic cells mediate T cell-dependent anti-gastric cancer immune responses in vitro

Dendritic cells (DCs) are potent professional antigen-presenting cells (APCs) with the ability to prime naïve T cells, and play an important role in the initiation and regulation of immune responses. In this study, we constructed a recombinant adenovirus carrying the SLC gene (Ad-SLC), and detected the biological effects of Ad-SLC-modified DCs as an adjuvant for the initiation of gastric cancer immune responses. Human DCs were transfected with Ad-SLC and the recombinant adenovirus carrying the β-galactosidase gene, Ad-LacZ, respectively. Modified DCs were pulsed with the cell lysate antigen of SGC-7901 cells (a type of gastric cancer cell line) and co-cultured with autologous T cells. The T cells were harvested and incubated with SGC-7901 cells and the cytotoxic function of the T cells was detected. Based on the data, the expression of mature DC phenotypes CD83 and CCR7 was upregulated after transfection with Ad-SLC and the chemotaxis function of DCs was augmented after transfection with Ad-SLC. Moreover, the expression of RANTES in DCs was upregulated by Ad-SLC transfection, while expression levels of IL-12p70 and IL-10 were not significantly altered. When co-cultured with autologous T cells, DCs modified with the SLC gene and pulsed with SGC-7901 cell lysates significantly promoted the proliferation of autologous T cells and induced Th1 differentiation, and displayed a strong cytotoxicity to SGC-7901 cells. In conclusion, Ad-SLC promoted DC maturation, enhancing the ability of DCs for T-cell chemotaxis and T-cell stimulation, and induced specific anti-gastric cancer cellular immunity. Recombinant Ad-SLC-modified DCs may be used as an adjuvant to induce an effective anti-gastric cancer immune response.

Immunosuppressive exosomes from TGF-β1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells

Immunosuppressive exosomes from TGF-β1 gene-modified dendritic cells attenuate Th17-mediated inflammatory autoimmune disease by inducing regulatory T cells

Cytotoxic T lymphocyte antigen 4 immunoglobulin modified dendritic cells attenuate allergic airway inflammation and hyperresponsiveness by regulating the development of T helper type 1 (Th1)/Th2 and Th2/regulatory T cell subsets in a murine model of asthma

T helper type 2 (Th2) and regulatory T cells (T(reg) ) have been postulated to have critical roles in the pathogenesis of allergic asthma. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified dendritic cells (DC-CTLA4Ig) have the potential to reduce Th2 cells and induce T(reg) cells. In the present study, we evaluated the therapeutic effects and potential mechanisms of the adoptive transfer of DC-CTLA4Ig into mice in an experimental model of asthma. BALB/c mice were sensitized with ovalbumin (OVA) and challenged with aerosolized OVA for 7 days. Just prior to the first challenge, DC-CTLA4Ig, DCs or DCs infected with DC-green fluorescent protein (GFP) were injected intravenously into mice. The administration of DC-CTLA4Ig reduced airway hyperresponsiveness, relieved asthmatic airway inflammation and decreased the numbers of esosinophils in the BALF in OVA-sensitized/challenged mice. In addition, DC-CTLA4Ig altered the balance of Th1/Th2 cytokine production in the lungs with increased interferon (IFN)-γ levels and decreased interleukin (IL)-4 levels, decreased the percentage of Th2 and increased both the percentage of Th1 and T(reg) cells in the lungs of OVA-sensitized/challenged mice. This research demonstrates that DC-CTL4Ig reduces airway hyperresponsiveness effectively and prevents airway inflammation in OVA-sensitized/challenged mice, which is due most probably to attenuated secretion of Th2 cytokines and increased secretion of Th1 cytokines in the local airway, and the correction of the pulmonary imbalance between Th1/Th2 cells and Th2/T(reg) cells.

Enhanced cytotoxicity of IL-24 gene-modified dendritic cells co-cultured with cytokine-induced killer cells to hepatocellular carcinoma cells

As a novel human cytokine found recently, IL-24 could selectively kill tumor cells by multiple ways. Dendritic cells (DCs) are the major antigen-presenting cells. Recent studies have revealed that IL-24 can promote the antigen-presenting function of DCs. In this study, we evaluated the antitumor effect and mechanism of co-cultured cytokine-induced killer (CIK) cells and autologous DCs modified with IL-24 gene on hepatocellular carcinoma (HCC) cells. DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells. Recombinant adenovirus AdVGFP/IL-24 (Ad-IL24) was constructed expressing IL-24. IL-24 gene was transduced into DCs via Ad-IL24, the cells obtained were named DC-IL-24. We demonstrated that the expression rates of CD80, CD83, CD1a, HLA-DR, CD40, CXCR4 on DC-IL-24 were significantly increased compared with those of the control group. DC-IL-24 produced markedly higher levels of IL-24, IL-12 and TNF-alpha as compared with those of DCs. On comparison with non-transfected DCs co-cultured with CIK cells, transfected DCs co-cultured with CIK cells had a significant higher lytic activity against SMMC7721 cells, a HCC cell line.