moDC Maturation Research Articles

Mono-palmitoyl-N-alkylurea ligands as specific activators of human Toll-like receptor 2/6 heterodimer.

Ligands for Toll-like-receptor 2 (TLR2) have demonstrated significant potential as immune-stimulating components in synthetic vaccines. Activation of TLR2 relies on the formation of dimeric complexes with either TLR1 or TLR6 and the nature of these dimers can impact therapeutic outcomes. The lipopeptide-based TLR2 ligands Pam3CysSK4 and Pam2CysSK4 have been extensively studied, and their recognition by different TLR-receptor heterodimers, TLR2/TLR1 and TLR2/TLR6, respectively, has been established. However, the high lipophilicity of these ligands, containing multiple palmitoyl residues, can result in solubility issues when used as vaccine adjuvants. To address this, we previously synthesized a less lipophilic ligand containing a single palmitoyl chain called mini-UPam, which effectively stimulates human moDC maturation. We here probe the receptor-dimer specificity of several mini-Upam derivatives and reveal that these mini-UPam are hTLR2/TLR6 selective ligands and that the introduction of longer urea alkyl chains does not shift the binding specificity to hTLR2/TLR1 heterodimers, in contrast to their Pam2CysSK4 and Pam3CysSK4 counterparts, pointing to a different binding mode of the UPam ligands.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. Epithelial activation and/or barrier effects upon exposure to 2-50 μg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 μg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Abstract 1363: Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC

Abstract Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

Abstract 2478: Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity

Abstract Extracellular ATP (eATP) is a potent immunostimulatory signal in the tumor microenvironment (TME), but it is rapidly degraded into AMP and immunosuppressive adenosine. Therefore, ATP metabolism is an attractive anti-tumor target. Several enzymes are involved in the immunosuppressive process used by the TME to convert ATP to AMP and adenosine. Among them, CD39 (gene: ENTPD1), an extracellular ATPase, is a promising target. Analyzing mRNA expression from the TCGA, we show that CD39 is consistently expressed at high levels in comparison to the other extracellular ATP-converting enzymes, ENPP1 and TNAP. We further demonstrate that CD39 has greater enzymatic activity at the slightly acidic physiological pH (pH = 6.8) of the TME than either ENPP1 or TNAP. Flow cytometry profiling of dissociated primary human gastric tumors showed that CD39 was highly expressed on both myeloid and lymphoid cells, including several populations of T cells present in the tumors. This is distinct from findings in healthy primary human PBMCs that show high expression of CD39 on myeloid subsets and relatively few subsets of T cells with high CD39 expression. AB598 is a novel, humanized, Fc-silent anti-CD39 therapeutic antibody that potently binds to CD39 and inhibits its enzymatic activity with sub-nanomolar potency. Treatment of dissociated primary human gastric tumor cells with AB598 resulted in a decrease in the rate of ATP degradation, indicating both the effectiveness of AB598 and the relevance of CD39 as a main contributor to the ATPase activity of the TME. Although eATP is low in healthy tissues, high levels of ATP can be released when cancer cells are treated with immunogenic cell death (ICD)-inducing agents, increasing the eATP in the TME, providing a rationale for the combination of ICD-inducing chemotherapy and AB598. Thus, cancer cells were co-cultured with monocyte-derived dendritic cells (moDCs) and treated with chemotherapy and AB598 to preserve the eATP released by dying cancer cells. The increased expression of CD83 and CD86 on moDCs demonstrated that the combination treatment enhanced the maturation of moDCs, compared to single or no treatment. To further elucidate the anti-tumor effects of the combination treatment, autogenous T cells were introduced into this system. AB598 significantly enhanced T cell activation in the presence of chemotherapy. These data demonstrate that using AB598 results in elevated eATP, which boosts immune cell activation to promote anti-tumor immunity. Citation Format: Ke Jin, Julie Clor, Kaustubh Parashar, Rebecca Fuchs, LC Stetson, Sean Cho, Matthew J. Walters, Ester Fernandez-Salas, Christine E. Bowman. Inhibition of CD39 by AB598 increases extracellular ATP resulting in activation of myeloid cells and T cells to enhance anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2478.

Abstract 6687: Evaluation of a new co-cultured microbiome ecosystem therapy candidate (MaaT034) for clinical testing as adjuvant/neoadjuvant to immune checkpoint inhibitors in solid tumors

Abstract Background: Increasing evidence suggests that gut microbiome composition modulates tumor response to therapies, including immune checkpoint inhibitors (ICI). Clinical proofs of concept were obtained in pilot studies using healthy or ICI-responder donor-derived fecal matter transplants to modulate the gut microbiome of non-responding cancer patients and improve their response to ICI. These results support the development of Microbiome Ecosystem TherapiesTM (MET) replicating at large industrial scale the effects of ICI-responders gut microbiome. MaaT Pharma, a clinical-stage biotech, has developed a unique, ground-breaking, patented co-culture process (MET-C). MaaT034, the first-in-class co-cultured microbiome product, has been selected for optimizing intestinal homeostasis and to activate immune functions alone and in combination with an ICI treatment. Method: We combined metagenomic analysis, metabolite quantification by LC-MRM/MS, Caco-2/THP-1 leaky gut model, PBMC assay and mixed lymphocyte reaction (MLR) to assess the impact of MaaT034 on gut homeostasis and immune activation. Results: Metagenomic analysis demonstrates that MaaT034 replicates the richness and diversity of native-based microbiome ecosystems. Metabolite quantification also revealed that MaaT034 microbes secrete anti-inflammatory metabolites such as short chain fatty acids and indoles, as well as metabolites associated with ICI response such as secondary biliary acids. Using a Caco-2/THP-1 leaky gut model, we then demonstrate that MaaT034 conditioned medium was able to restore the integrity of a THP-1-induced damaged gut barrier. This was associated with and a reduction of inflammatory signals. We further performed a human PBMC assay to evaluate the impact of MaaT034 on immune cell activation. We observed the secretion of immunostimulatory cytokines associated with myeloid cell activation in response to MaaT034 conditioned medium. Notably, anti-CD3 mediated T-cells activation was further increased in response to MaaT034 conditioned medium as evidenced by increased IFNγ release associated with increased Granzyme B and CD25. Finally, MLR was performed to determine the immunomodulatory potential of MaaT034 through the interaction between T-cells and monocytes-derived dendritic cells (moDC). Interestingly, MaaT034 conditioned medium improved moDC maturation as well as moDC-induced T-cell activation. MaaT034 conditioned medium further increased anti-PD-1 mediated T-cell activation, as evidenced by IFNγ release. Conclusion: Altogether, these results highlight the potential of MaaT034 to produce metabolites involved in the restoration of gut barrier integrity and stimulation of immune cell response to ICI therapy, with potential benefit for solid tumors treatment. Citation Format: Bastien Laperrousaz, Julie Reygner, Cyrielle Gasc, Charlotte Petitjean, Aurore Duquenoy, Lora Martin, Vincent Bonnaud, Marianne Robin, Sophie Declomesnil, Carole Schwintner, Nathalie Corvaïa. Evaluation of a new co-cultured microbiome ecosystem therapy candidate (MaaT034) for clinical testing as adjuvant/neoadjuvant to immune checkpoint inhibitors in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6687.

Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes

BackgroundStreptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections.MethodsHuman primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined.ResultsIn vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations.ConclusionsOur data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.

Human papillomavirus infection affects the immune microenvironment and antigen presentation in penile cancer.

Penile squamous cell carcinoma (PSCC) is a largely neglected condition, predominantly affecting underdeveloped regions, and is associated with risk factors such as low socioeconomic status, phimosis, and human papillomavirus (HPV) infection. Unlike other urogenital cancers, its pathophysiology and therapeutic targets remain poorly understood, particularly regarding the immune response to the tumor microenvironment. This study aims to investigate immune cell infiltration profiles, dendritic cell maturation, and lymphocyte apoptosis in both HPV-positive and HPV-negative PSCC. Clinical and histopathological data, along with peripheral blood and tumor tissue samples, were collected from 30 patients (66.6% were HPV-positive and 33.3% HPV-negative), with an additional 19 healthy donors serving as controls. Tumor-infiltrating immune cells were analyzed following enzymatic digestion of tumor tissue, enabling detailed phenotypic characterization. A simulated tumor microenvironment was created using supernatants derived from primary cultures of HPV-positive PSCC tumors. Peripheral blood mononuclear cells were isolated and differentiated into dendritic cells (Mo-DCs) for further phenotyping and lymphoproliferation assays. Lymphocytes from healthy donors and patients were exposed to tumor culture supernatants to evaluate apoptosis induced by the tumor microenvironment. Results showed that HPV-positive tumors exhibited lower T lymphocyte frequencies compared to HPV-negative tumors. Additionally, patients infected with high-risk HPV demonstrated reduced maturation rates of Mo-DCs and decreased expression of co-stimulatory molecules on these cells compared to healthy donors. Furthermore, Mo-DCs from hrHPV-positive patients showed impaired lymphoproliferation capacity relative to controls, while HPV-negative patients exhibited a trend towards reduced lymphoproliferative ability. Regarding the simulated tumor microenvironment, lymphocytes from healthy donors underwent apoptosis, contrasting with patients' lymphocytes, which showed increased viability when cultured with tumor supernatants. These results underscore the impact of HPV infection on T lymphocyte infiltration, Mo-DC maturation, and lymphocyte survival in PSCC, offering critical insights for advancing our understanding of the tumor microenvironment and guiding the development of immunotherapy strategies.

Exogenous α-ketoglutarate Modulates Redox Metabolism and Functions of Human Dendritic Cells, Altering Their Capacity to Polarise T Cell Response.

Alpha-ketoglutarate (αKG) emerged as a key regulator of energetic and redox metabolism in cells, affecting the immune response in various conditions. However, it remained unclear how the exogenous αKG modulates the functions of dendritic cells (DCs), key cells regulating T-cell response. Here we found that non-toxic doses of αKG display anti-inflammatory properties in human APC-T cell interaction models. In a model of monocyte-derived (mo)DCs, αKG impaired the differentiation, and the maturation of moDCs induced with lipopolysaccharide (LPS)/interferon (IFN)-γ, and decreased their capacity to induce Th1 cells. However, αKG also promoted IL-1β secretion by mature moDCs, despite inflammasome downregulation, potentiating their Th17 polarizing capacity. αKG induced the expression of anti-oxidative enzymes and hypoxia-induced factor (HIF)-1α in moDCs, activated Akt/FoxO1 pathway and increased autophagy flux, oxidative phosphorylation (OXPHOS) and glycolysis. This correlated with a higher capacity of immature αKG-moDCs to induce Th2 cells, and conventional regulatory T cells in an indolamine-dioxygenase (IDO)-1-dependent manner. Additionally, αKG increased moDCs' capacity to induce non-conventional T regulatory (Tr)-1 and IL-10-producing CD8+T cells via up-regulated immunoglobulin-like transcript (ILT3) expression in OXPHOS-dependent manner. These results suggested that exogenous αKG-altered redox metabolism in moDCs contributed to their tolerogenic properties, which could be relevant for designing more efficient therapeutic approaches in DCs-mediated immunotherapies.

Infliximab aggregates produced in severe and mild elevated temperature stress conditions induce an extended specific CD4 T-cell response

Aggregation has been widely described as a factor contributing to therapeutic antibody immunogenicity. Although production of high-affinity anti-drug antibodies depends on the activation of CD4 T lymphocytes, little is known about the T-cell response induced by antibody aggregates, especially for aggregates produced in mild conditions resulting from minor handling errors of vials. Large insoluble infliximab (IFX) aggregates produced in severe elevated temperature stress conditions have been previously shown to induce human monocyte-derived dendritic cell (moDC) maturation. We here showed that large IFX aggregates recruit in vitro a significantly higher number of CD4 T-cells compared to native IFX. Moreover, a larger array of T-cell epitopes encompassing the entire variable regions was evidenced compared to the native antibody. We then compared the responses of moDCs to different types of aggregates generated by submitting IFX to mild conditions of various times of incubation at an elevated temperature. Decreasing stress duration reduced aggregate size and quantity, and subsequently altered moDC activation. Of importance, IFX aggregates generated in mild conditions and not altering moDC phenotype generated an in vitro T-cell response with a higher frequency of CD4 T cells compared to native IFX. Moreover, cross-reactivity studies of aggregate-specific T cells showed that some T cells could recognize both native and aggregated IFX, while others responded only to IFX aggregates. Taken together, our results suggest that aggregation of antibodies in mild elevated temperature stress conditions is sufficient to alter moDC phenotype in a dose-dependent manner and to increase T-cell response.

Sitagliptin Induces Tolerogenic Human Dendritic Cells

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Oxidant-modified amylin fibrils and aggregates alter the inflammatory profile of multiple myeloid cell types, but are non-toxic to islet β cells

Diabetes mellitus currently affects ∼10% of the population worldwide, with Type 2 predominating, and this incidence is increasing steadily. Both Type 1 and 2 are complex diseases, involving β-cell death and chronic inflammation, but the pathways involved are unresolved. Chronic inflammation is characterized by increased oxidant formation, with this inducing protein modification, altered function and immunogenicity. Amylin, a peptide hormone co-secreted with insulin by β-cells, has attracted considerable interest for its amyloidogenic properties, however, the effects that oxidants have on amylin aggregation and function are poorly understood. Amylin was exposed in vitro to hypochlorous acid, hydrogen peroxide and peroxynitrous acid/peroxynitrite to investigate the formation of post-translational oxidative modifications (oxPTMs, via mass spectrometry) and fibril formation (via transmission electron microscopy). Amylin free acid (AFA) was also examined to investigate the role of the C-terminal amide in amylin. Oxidant exposure led to changes in aggregate morphology and abundance of oxPTMs in a concentration-dependent manner. The toxicity and immunogenic potential of oxidant-modified amylin or AFA on pancreatic islet cells (INS-1E), human monocyte cell line (THP-1) and monocyte-derived dendritic cells (moDCs) were examined using metabolic activity and cytokine assays, and flow cytometry. No significant changes in vitality or viability were detected, but exposure to oxidant-modified amylin or AFA resulted in altered immunogenicity when compared to the native proteins. THP-1 and moDCs show altered expression of activation markers and changes in cytokine secretion. Furthermore, oxidant-treated amylin and AFA promoted maturation of THP-1 and pre-mature moDCs, as determined by changes in size, and maturation markers.

Sulforaphane Reduces the Chronic Inflammatory Immune Response of Human Dendritic Cells.

Sulforaphane (SFN) is an isothiocyanate of vegetable origin with potent antioxidant and immunomodulatory properties. The characterization of its pleiotropic activity in human dendritic cells (DCs) is poorly summarized. The aim of this work was to study the immunomodulatory power of SFN in response to an inflammatory microenvironment on human monocyte-derived DCs (moDCs). We studied the immunological response induced by SFN. Apoptosis and autophagy assays were performed using flow cytometry on moDCs and a cancer cell line (THP-1). These included moDC maturation, lymphocyte proliferation and cytokine production under different experimental conditions. We investigated whether these results were associated with an inflammatory microenvironment induced by lipopolysaccharides (LPSs). Our results demonstrated that SFN could interact with moDCs, significantly reducing the autophagy process and enhancing apoptosis similarly to cancer cell line THP-1 cells in a chronic inflammatory microenvironment. Under chronic inflammation, SFN modulated the phenotypical characteristics of moDCs, reducing the expression of all markers (CD80, CD83, CD86, HLA-DR and PD-L1). SFN significantly reduced the Th2 proliferative response, with a decrease in the IL-9 and IL-13 levels. Although we did not observe any changes in the regulatory proliferative response, we noted an increase in the IL-10 levels. These findings demonstrate that SFN exerts protective effects against LPS-induced inflammation via the modulation of moDCs/T cells towards a regulatory profile. SFN may be a potential candidate for the treatment of pathologies with an inflammatory profile.

Harnessing the Full Potential of Regulatory T Cells by mRNA-induced Antigen Specificity

Abstract Regulatory T cells (Tregs) are crucial in inducing and maintaining tolerance, and already have shown clinical potential. In this study, we describe an efficient non-viral engineering method by TCR-encoding mRNA electroporation, while also exploring the potential of further promoting on-target effects by disrupting the endogenous TCR using a fully RNA-based CRISPR/Cas9 technology. Endogenous TCR disruption prior to TCR-transfection resulted in more than 90% blockade of the endogenous TCR expression and a similar high (>85%) but more homogenous expression of the transgenic TCR. Importantly, following antigen-specific TCR-induced stimulation, both engineered Tregs significantly increased the expression of multiple activation and suppression markers. Moreover, the engineered Tregs outcompeted polyclonal Tregs in suppressing antigen-specific effector T cell proliferation and performed better in blocking moDC maturation, even in the presence of antigen-activated effector T cells. Secretome screening revealed a distinct anti-inflammatory profile for TCR-Tregs. On the other hand, TCR-engineered KO Tregs co-expressed anti-inflammatory cytokines together with some pro-inflammatory cytokines, but qualitatively different from their effector Th counterparts. Hence, an anti-inflammatory profile with Th cell features is seen with TCR-engineered KO Tregs. In conclusion, we show that this versatile approach can be used to induce the functional expression of a transgenic TCR, but also for CRISPR/Cas9-mediated disruption of endogenous TCRs with high efficiency using an RNA-based platform. Comparison between polyclonal Tregs and TCR engineered Tregs in EAE using an HLA-transgenic mouse model is now being investigated. National Multiple Sclerosis Society (RG-1612-26484)

Abstract 1802: Personalization of oncolytic virus therapy: Heterogenous anti-tumor responses in preclinical prostate and bladder cancer models

Abstract Clinical studies revealed that single-agent immunotherapies yield limited efficacy in the management of prostate and bladder cancer (PCa, BCa). PCa and BCa often induce immunosuppressive and immune evasion mechanisms that restrict anti-tumor responses. Oncolytic viruses (OVs) offer promising and safe therapeutic options in the treatment of cancer as OVs target and or preferentially replicate in tumor cells, potentially evoking systemic anti-tumor immune responses. Combination of immunotherapy with OVs may, therefore, unleash the full potential of immunotherapy for these cancers. To achieve beneficial anti-tumor responses a more personalized OV therapy approach seems crucial. Strikingly, only few studies describe head-to-head comparison of the effects of different OVs. Together with our partners in the oncolytic viro-immunotherapy (OVIT) consortium we are developing and evaluating multiple OVs for their abilities to induce oncolysis and subsequent immunomodulation. Furthermore, we are developing tools to predict oncolytic virus effectiveness and design strategies to personalize oncolytic virus therapy. The OVs that were generated by our consortium encompass the spontaneous Orthoreovirus mutant (jin-3), a non-human primate Adenovirus (AdV-lumc007dRbD), and a Newcastle Disease Virus variant (NDV-F0).In this study we determined the anti-tumor and immunomodulatory effects of selected OVs in multiple ‘near-patient and patient-derived human PCa and BCa models; 1) tumor cell line cultures, 2) cocultures of tumor cell lines and monocyte-derived dendritic cells (mo-DCs), and 3) ex-vivo cultured tumor tissue slices and explants from patient material or patient-derived xenograft (PDX) models. The selected OVs infected a panel of PCa and BCa cell lines and displayed heterogenous effects on tumor cell viability and expression of immunogenic cell death markers (HSP90, Calreticulin, and HMGB1). For instance, the jin-3 virus showed enhanced tumor tropism in most PCa cells, while AdV-lumc007dRbD was more efficient in BCa cells. Virus infection of tumor cells resulted in maturation and activation of moDCs. Furthermore, OVs were able to infect and replicate in ex-vivo cultured tumor tissue slices and viability of ex-vivo tumor tissue explants was reduced following viral inoculation. Transcriptomic analysis of infected patient tissues revealed heterogenous responses to the tested OVs.Taken together, we established and compared the oncolytic properties of various OVs in multiple preclinical models of PCa and BCa. The data indicate heterogeneity of response for the tested OVs in urological cancers. In order for OV therapy to reach robust clinical efficacy, better appreciation of underlying molecular and cellular mechanisms is crucial for the personalization of oncolytic viro-immunotherapy. Citation Format: Thijs J. Janssen, Geertje van der Horst, Arjanneke F. van der Merbel, Maaike van der Mark, Martine L. Lamfers, Bernadette G. van den Hoogen, Sjoerd S. van der Burg, Dana A. Mustafa, Niven Mehra, Tilly Aalders, Nadine van Montfoort, Jack Schalken, Rob C. Hoeben, Gabri van der Pluijm. Personalization of oncolytic virus therapy: Heterogenous anti-tumor responses in preclinical prostate and bladder cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1802.

Leonurine promotes the maturation of healthy donors and multiple myeloma patients derived-dendritic cells via the regulation on arachidonic acid metabolism.

Objective: Leonurine is a bioactive alkaloid compound extracted from Leonurus japonicus Houtt, which potentially has immunomodulatory effects. The immunomodulatory effect and mechanism of leonurine on monocyte derived dendritic cells (moDCs) from healthy donors (HDs) and multiple myeloma (MM) patients were investigated for the first time. Methods: Peripheral blood from HDs and MM patients was isolated for peripheral blood mononuclear cells (PBMCs). The generation of moDCs was conducted by the incubation of monocytes from PBMCs in the medium consisting of RPMI 1640 medium, 2mmol/L L-glutamine, 5% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin and 0.1mg/mL streptomycin. During the incubation of 7days, the cells were administrated with 1μM leonurine or 1 × PBS as the control group. On the 8th day, cells were harvested. The expression of maturation associated surface markers CD40, CD83, and HLA-DR on moDCs was analyzed by flow cytometry. Moreover, moDCs with or without 1μM leonurine administration were evaluated by LC-MS/MS for metabolomics which was further analyzed for the potential mechanism of leonurine on moDCs. Results: The proportion of moDCs in the harvested cells was significantly higher in the HD group (n = 14) than in the MM patient group (n = 11) (p = 0.000). Leonurine significantly enhanced the median fluorescence intensity of CD83, HLA-DR and CD40 expression on HD-moDCs (n = 14; p = 0.042, p = 0.013, p = 0.084) as well as MM paitent-moDCs (n = 11; p = 0.020, p = 0.006, p = 0.025). The metabolomics data showed that in moDCs (HD, n = 15), 18 metabolites in the pathway of arachidonic acid metabolism showed significant differences between the leonurine group and the control group (VIP all >1 and P all <0.05). To be specific, 6-Keto-PGE1, 8,9-DHET, 11 (R)-HETE, 12-Keto-LTB4, 12-OxoETE, 15 (S)-HETE, 15-Deoxy-Delta12,14-PGJ2, 15-Keto-PGF2a, 20-COOH-LTB4, Lecithin, PGA2, PGB2, PGE2, PGF2a, PGG2, Prostacyclin were significantly upregulated in the leonurine group than in the control group, while Arachidonic Acid and TXB2 were significantly downregulated in the leonurine group than in the control group. Conclusion: Leonurine significantly promotes the maturation of moDCs derived from HDs and MM patients, the mechanism of which is related to arachidonic acid metabolism.

Pomalidomide enhances the maturation of dendritic cells derived from healthy donors and multiple myeloma patients.

Objective: To explore the effect of pomalidomide on the maturation of monocyte-derived dendritic cells (moDCs) from healthy donors (HDs) and multiple myeloma (MM) patients. Methods: MoDCs were generated by the incubation of monocytes from peripheral blood mononuclear cells (PBMCs) for 7days in a medium consisting of 800U/ml granulocyte-macrophage colony stimulating factor (GM-CSF), 500U/ml interleukin-4 (IL-4), RPMI 1,640 medium, 5% human serum, 100U/ml penicillin and 0.1mg/ml streptomycin. Meanwhile, the incubation system was administrated with 10µM pomalidomide or 1 × PBS as the control group. On the eighth day, cells were harvested and analyzed by flow cytometry. The CD80+CD86+ cell population in total cells was gated as moDCs in the FACS analyzing system. After that, the expression of CD40 and HLA-DR on moDCs was analyzed. Meanwhile, the supernatant from the incubation system was evaluated for the secretion of cytokines interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and macrophage inflammatory protein 1α (MIP-1α) by enzyme-linked immunosorbent assay (ELISA). Results: When analyzing all the HD-moDCs together (n = 15), pomalidomide significantly increased the mean fluorescence intensity (MFI) of CD40 expression and HLA-DR expression on moDCs compared with the control group (p = 0.003, p = 0.040). Meanwhile, the proportion of CD40+ moDCs and HLA-DR+ moDCs in total moDCs was significantly higher in the pomalidomide group than in the control group (p = 0.008, p = 0.032). When analyzing all MM patient-moDCs together (n = 11), pomalidomide significantly increased the MFI of CD40 expression and HLA-DR expression on moDCs compared with the control group (p = 0.047, p = 0.006). Meanwhile, the proportion of HLA-DR+ moDCs in total DCs was significantly higher in the pomalidomide group than in the control group (p < 0.001). Moreover, HD-moDCs (n = 8) treated with pomalidomide secreted 192% IL-12, 110% TNF-α, and 112% MIP-1α of the untreated moDCs (p = 0.020, p = 0.006, p = 0.055). However, when analyzing MM patient-moDCs (n = 10) together, the secretion of IL-12, TNF-α and MIP-1α from moDCs showed no significant difference between the pomalidomide group and the control group (p = 0.458, p = 0.377, p = 0.248). Conclusion: In vitro, 10µM pomalidomide enhances the maturation of moDCs derived from both HDs and MM patients. Pomalidomide shows potential to be applied as a DC adjuvant for DC-based immunotherapy, such as the DC vaccine and DC cell therapy in MM.

Leonurine Promotes the Maturation of Healthy Donors and Multiple Myeloma Patients Derived-Dendritic Cells

Abstract: Objective Leonurine is an agent from Leonurus japonicus Houtt , which potentially has immunomodulatory effects. The immunomodulatory effect of leonurine on dendritic cells (DCs) is not clear up to date. In the current study, the effect of leonurine on the maturation of monocyte derived dendritic cells (moDCs) from healthy donors (HDs) and multiple myeloma (MM) patients will be investigated for the first time. Method Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of HDs and MM patients. The generation of moDCs from monocytes in PBMCs was conducted by the incubation of 7 days in a medium consisting of RPMI 1640 medium, 5% human serum, 800U/mL GM-CSF, 500U/mL IL-4, 100U/mL penicillin and 0.1mg/mL streptomycin. During this period, the incubation system was administrated with leonurine at 1 µM or 1×PBS as the control group. On the 8 th day, cells were harvested, and the immunophenotyping of cells were analyzed by the flow cytometry. The CD80+CD86+ cell population in total cells is gated as DCs in the FACS analyzing system. Then, the median fluorescence intensity (MFI) of surface markers CD40, CD83 and HLA-DR on DCs were analyzed respectively. Results The proportion of DCs in the harvested cells was significantly higher in HD group (n=14) than that in MM patient group (n=11) (75.55%±9.69% vs 61.39%±4.86%, P=0.000). When analyzing all the HD-derived moDCs (n=14), leonurine significantly enhanced the MFI of CD40 (2.11×105±0.92×105 vs 1.81×105±0.72×105, P = 0.049), CD83 (2.41×105±2.06×105 vs 2.14×105±1.71×105, P=0.042) and HLA-DR (12.21×105±4.20×105 vs 11.11×105±3.94×105, P=0.013) on moDCs when compared with the control group. When analyzing all the MM patients-derived moDCs (n=11), leonurine also significantly enhanced the MFI of CD40 (1.99×105±0.98×105 vs 1.72×105±0.78×105, P = 0.025), CD83 (0.30×105±0.18×105 vs 0.27×105±0.16×105, P = 0.020) and HLA-DR (7.49×105±2.58×105 vs 6.80×105±2.18×105, P = 0.006) on moDCs when compared with the control group. Conclusions Regarding the moDCs generated in vitro, a smaller number of MM patients-derived moDCs can be generated compared with HD-derived moDCs. Leonurine at 1 µM in vitro can promote the maturation of both HD-derived moDCs and MM patients-derived moDCs. Leonurine shows potential to be a DC adjuvant for DC-based therapeutic strategies, such as DC vaccine and DC cell-therapy in MM. Key words: Leonurine; Monocyte derived Dendritic Cells; Multiple Myeloma; DC Adjuvant

Abstract 3568: Robust and potent dendritic cells for cancer immunotherapy

Abstract Background: Dendritic cells (DCs) are fundamental to adaptive immunity by detecting, engulfing and presenting foreign or changed antigenic peptides to lymphocytes that thereafter initiate antigen-specific immune attacks. DC-based immunotherapy against cancer has been promising for a long time, but has not yet met expectations. Cancer cell heterogeneity and numerous immune evasion mechanisms represent challenges. According to textbook renderings, DCs may induce either tolerance or immune activation depending upon the nature of the encountered antigen and the status of the microenvironment. Recently, we have discovered a more complicated scenario according to which monocyte-derived DCs (moDCs) exhibit dual pro- and anti-inflammatory features, suggesting that selected conditions that enhance pro-inflammatory features and counteract tolerogenic features could generate more robust and potent DCs for cancer therapy. Materials and Methods: Healthy blood donor buffy coats were used for the isolation of monocytes and conventional CD141+ and CD1c+ natural circulating DCs. IL-4 and GM-CSF were used to differentiate monocytes into immature moDCs. Toll-like receptor agonists were used to induce DC maturation, and specific agonists and inhibitors were tested in different DC variants. Flow cytometry, ELISA determination of cytokine secretion, HPLC-based quantitation of kynurenine secretion, genome-wide gene expression analyses and functional assays, such as antigen uptake, migration and the mixed leukocyte reaction were used for evaluation of treated DCs. Results: Conventional DCs were more susceptible to apoptosis, and dependent upon culture conditions, than moDCs. Inhibition of β-catenin signaling stimulated several pro-inflammatory features, e.g. increased IL-12 and decreased IL-10 secretion, with reciprocal effects caused by β-catenin stimulation. Maturation of moDCs was accompanied by very strong induction of indoleamine 2, 3-dioxygenase 1 (IDO1) transcription and paralleled by increased kynurenine secretion, but this was partially counteracted by β-catenin stimulation. Conclusions: Preparation of more potent DCs for cancer therapy needs to take into account DC longevity and conflicting activation patterns of pro- and anti-inflammatory abilities. Citation Format: Waqas Azeem, Seyed Mohammad Lellahi, Yaping Hua, Anne Margrete Øyan, Benjamin Gabriel, Karl-Henning Kalland. Robust and potent dendritic cells for cancer immunotherapy [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 3568.

Effects of Dasatinib on the Maturation of Monocyte-Derived Dendritic Cells Derived from Healthy Donors and Chronic Myelogenous Leukemia Patients

To investigate the effects of dasatinib on the maturation of monocyte-derived dendritic cells (moDCs) derived from healthy donors (HDs) and chronic myelogenous leukemia (CML) patients. Peripheral blood mononuclear cells (PBMCs) were isolated from HDs (n=10) and CML patients (n=10) who had got the remission of MR4.5 with imatinib treatment. The generation of moDCs from PBMCs was completed after 7 days of incubation in DC I culture medium, and another 3 days of incubation in DC II culture medium with or without 25 nmol/L dasatinib. On the 10th day, cells were harvested and expression of molecules of maturation related marker were assessed by flow cytometry. The CD80+CD86+ cell population in total cells was gated as DCs in the fluorescence-activated cell storting (FACS) analyzing system, then the expression of CD83, CD40 or HLA-DR in this population was analyzed respectively. The proportion of CD80+CD86+ cells in total cells didn't show a statistical difference between HD group and patient group (89.46%±9.70% vs 87.39%±9.34%, P=0.690). Dasatinib significantly enhanced the expression of the surface marker CD40 (P=0.008) and HLA-DR (P=0.028) on moDCs derived from HDs compared with the control group, while the expression of CD83 on moDCs didn't show a significant difference between dasatinib group and the control group (P=0.428). Meanwhile, dasatinib significantly enhanced the expression of the surface marker CD40 (P=0.023), CD83 (P=0.038) and HLA-DR (P=0.001) on moDCs derived from patients compared with the control group. For CML patients, the same high proportion of moDCs as HDs can be induced in vitro, which provides a basis for the application of DC-based immunotherapy strategy. Dasatinib at the concentration of 25 nmol/L can efficiently promote the maturation of moDCs derived from HDs and CML patients in vitro. Dasatinib shows potential as a DC adjuvant to be applied in DC-based immunotherapy strategies, such as DC vaccine and DC cell-therapy.

Fucodendropeptides induce changes in cells of the immune system in food allergic patients via DC-SIGN receptor

Food allergy induced by lipid transfer proteins (LTPs) of Rosacea fruit family, such as peach, is becoming an important health problem in the Mediterranean area. Current treatments, such as allergen specific immunotherapy (AIT) with allergenic extracts show promising, but in many cases, they need an improvement in homogeneity, availability and induction of tolerant responses. Peptide-based vaccines containing adjuvants, such as carbohydrates for C-type lectin receptors (CLRs) are presented as an alternative approach. In this work, we have prepared fucosylated glycodendropeptides (GDPs) functionalized with Pru p 3 peptides via click chemistry. These GDPs, DnFuc9Prup3, induced changes in moDC maturation and lymphocyte proliferation in food allergic patients, indicating specific recognition via DC-SIGN receptor. From these data, D4Fuc9Prup3 can be considered a promising candidate for specific immunotherapy development.