Monocyte-derived Dendritic Cells Research Articles

Effect of immune-modulating metronomic capecitabine as an adjuvant therapy in locoregionally advanced nasopharyngeal carcinoma

IntroductionMetronomic capecitabine used as an adjuvant therapy improves survival in patients with locoregionally advanced nasopharyngeal carcinoma (LA-NPC). This therapeutic approach may also contribute to improving immune function, consequently enhancing overall therapeutic efficacy.AimWe aimed to evaluate the effect of metronomic capecitabine as adjuvant therapy on immune function and survival in cases of LA-NPC.Subjects and methods28 patients with LA-NPC were enrolled in the study and equally assigned to two groups of 14 each: experimental and control group. The experimental group received induction chemotherapy + concurrent chemotherapy + adjuvant chemotherapy as well as oral capecitabine at a dose of 650 mg/m² of body surface area twice daily for 1 year, with the option to discontinue in case of intolerance. The control group did not receive additional chemotherapy or targeted drugs after the induction chemotherapy + concurrent chemoradiotherapy; however, they were followed up regularly. Changes in immune function and survival were compared between the two groups.ResultsThe median follow-up time was 43.5 months. One year after adjuvant chemotherapy, the experimental group showed higher levels of CD8 + cells, CD28 + CD8 + cells, and activated CD8 + cells compared to the control group (P < 0.05). The CD4/CD8 ratio and proportion of monocyte-derived dendritic cells were also higher in the experimental group than in the control group, but the difference was not statistically significant (P ≥ 0.05). Comparisons of 3-year overall survival, local-regional recurrence-free survival, progression-free survival, and distant metastasis-free survival between the two groups showed percentages of 92.9% vs. 78.6%, 92.9% vs. 92.9%, 78.6% vs. 71.4%, and 85.7% vs. 0.78 0.6% respectively, but these differences were not significant (P > 0 0.05 ).ConclusionMetronomic capecitabine chemotherapy was observed to induce an immunomodulatory effect in LA-NPC.Trial registrationNCT02958111, date of registration 04-11-2016.

High production of IL-12 by human dendritic cells stimulated with combinations of pattern-recognition receptor agonists

The cytokine IL-12p70 is crucial for T helper 1 (Th1) polarization and the generation of type 1 immunity required to fight cancer and pathogens. Therefore, strategies to optimize the production of IL-12p70 by human dendritic cells (DCs) may significantly improve the efficacy of vaccines and immunotherapies. However, the rules governing the production of IL-12p70 remain obscure. Here, we stimulated pattern recognition receptors (PRRs) representing five families of PRRs, to evaluate their ability to elicit high production of IL-12p70 by monocyte-derived DCs. We used ten well-characterized agonists and stimulated DCs in vitro with either single agonists or 27 different combinations. We found that poly(I:C), which engages the RNA-sensing PRRs TLR3 and MDA5, and LPS which stimulates TLR4, were the only agonists that could elicit notable IL-12p70 production when used as single ligands. We identified six different combinations of PRR agonists, all containing either the TLR3/MDA5 agonist poly(I:C) or the TLR7/8 agonist R848, that could synergize to elicit high production of IL-12p70 by human DCs. Five of the six combinations also triggered high production of the antiviral and antitumor cytokine IFNβ. Overall, the tested PRR ligands could be divided into three groups depending on whether they triggered production of both IL-12p70 and IFNβ, only one of the two, or neither. Thus, combinations of PRR agonists were found to increase the production of IL-12p70 by human DCs in a synergistic manner, and we identified six PRR agonist combinations that may represent strong adjuvant candidates, in particular for therapeutic cancer vaccines.

Everolimus treatment enhances inhibitory immune checkpoint molecules’ expression in monocyte-derived dendritic cells

BackgroundAntigen-specific T-cell immunity is provided by dendritic cells (DCs), which are specialized antigen-presenting cells. Furthermore, they establish a link between innate and adaptive immune responses. Currently, DC modification is a new approach for the therapy of several disorders. During solid organ transplantation, Everolimus, which is a mammalian target of rapamycin (mTOR) inhibitor, was initially utilized to suppress the immune system's functionality. Due to the intervention of Everolimus in various signaling pathways in cells and its modulatory properties on the immune system, this study aims to investigate the effect of treatment with Everolimus on the maturation and expression of immune checkpoint genes in monocyte-derived DCs. MethodsTo isolate monocytes from PBMCs, the CD14 marker was used via the MACS method. Monocytes were cultured and induced to differentiate into monocyte-derived DCs by utilizing GM-CSF and IL-4 cytokines. On the fifth day, immature DCs were treated with Everolimus and incubated for 24 h. On the sixth day, the flow cytometry technique was used to investigate the effect of Everolimus on the phenotypic characteristics of DCs. In the end, the expression of immune checkpoint genes in both the Everolimus-treated and untreated DCs groups was assessed using the real-time PCR method. ResultsThe findings of this research demonstrated that the administration of Everolimus to DCs led to a notable rise in human leukocyte antigen (HLA)-DR expression and a decrease in CD11c expression. Furthermore, there was a significant increase in the expression of immune checkpoint molecules, namely CTLA-4, VISTA, PD-L1, and BTLA, in DCs treated with Everolimus. ConclusionThe findings of this study show that Everolimus can target DCs and affect their phenotype and function in order to shift them toward a partially tolerogenic state. However, additional research is required to gain a comprehensive understanding of the precise impact of Everolimus on the activation status of DCs.

The Dysregulation of the Monocyte–Dendritic Cell Interplay Is Associated with In-Hospital Mortality in COVID-19 Pneumonia

Background: The monocyte–phagocyte system (MPS), including monocytes/macrophages and dendritic cells (DCs), plays a key role in anti-viral immunity. We aimed to analyze the prognostic value of the MPS components on in-hospital mortality in a cohort of 58 patients (M/F; mean age ± SD years) with COVID-19 pneumonia and 22 age- and sex-matched healthy controls. Methods: We measured frequencies and absolute numbers of peripheral blood CD169+ monocytes, conventional CD1c+ and CD141+ (namely cDC2 and cDC1), and plasmacytoid CD303+ DCs by means of multi-parametric flow cytometry. A gene profile analysis of 770 immune-inflammatory-related human genes and 20 SARS-CoV-2 genes was also performed. Results: Median frequencies and absolute counts of CD169-expressing monocytes were significantly higher in COVID-19 patients than in controls (p 0.04 and p 0.01, respectively). Conversely, percentages and absolute numbers of all DC subsets were markedly depleted in patients (p &lt; 0.0001). COVID-19 cases with absolute counts of CD169+ monocytes above the median value of 114.68/μL had significantly higher in-hospital mortality (HR 4.96; 95% CI: 1.42–17.27; p = 0.02). Interleukin (IL)-6 concentrations were significantly increased in COVID-19 patients (p &lt; 0.0001 vs. controls), and negatively correlated with the absolute counts of circulating CD1c+ cDC2 (r = −0.29, p = 0.034) and CD303+ pDC (r = −0.29, p = 0.036) subsets. Viral genes were upregulated in patients with worse outcomes along with inflammatory mediators such as interleukin (IL)-1 beta, tumor necrosis-α (TNF-α) and the anticoagulant protein (PROS1). Conversely, surviving patients had upregulated genes related to inflammatory and anti-viral-related pathways along with the T cell membrane molecule CD4. Conclusions: Our results suggest that the dysregulated interplay between the different components of the MPS along with the imbalance between viral gene expression and host anti-viral immunity negatively impacts COVID-19 outcomes. Although the clinical scenario of COVID-19 has changed over time, a deepening of its pathogenesis remains a priority in clinical and experimental research.

Comparing the efficacy of combined versus single immune cell adaptive therapy targeting colorectal cancer.

Colorectal cancer (CRC) is the most frequent cancer with limited therapeutic achievements. Recently, adoptive cellular immunotherapy has been developed as an antitumor therapy. However, its efficacy has not been tested in CRC. This study investigated the ability of an immune cell cocktail of dendritic cells (DCs), T cells, and natural killer (NK) cells to overcome immunological hurdles and improve the therapeutic efficacy of cell therapy for CRC. CRC lysate-pulsed monocyte-derived DCs (Mo-DCs), CRC antigen-specifically expanded T cells (CTL), and in vitro-expanded NK cells were cultured from patient peripheral blood mononuclear cells (PBMC). The ability of the combined immune cells to kill autologous tumor cells was investigated by co-culturing the combined immune cells with patient-derived tumor cells. The Mo-DCs produced expressed T cell co-stimulating molecules like CD80, CD86, human leukocyte antigen (HLA)-DR and HLA-ABC, at high levels and were capable of activating naive T cells. The expanded T cells were predominantly CD8 T cells with high levels of CD8 effector memory cells and low levels of regulatory T cells. The NK cells expressed high levels of activating receptors and were capable of killing other cancer cell lines (K562 and HT29). The immune cell cocktail demonstrated a higher ability to kill autologous tumor cells than single types. An in vivo preclinical study confirmed the safety of the combined immune cell adaptive therapy showing no therapy-related death or general toxicity symptoms. The results suggested that combined immune cell adaptive therapy could overcome the limited efficacy of cell immunotherapy.

PCV2 Induced Endothelial Derived IL-8 Affects MoDCs Maturation Mainly via NF-κB Signaling Pathway.

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.

Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1β, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1β, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

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.

Impaired reprogramming of the autophagy flux in maturing dendritic cells from crohn disease patients with core autophagy gene-related polymorphisms

ABSTRACT Crohn disease (CD) is an inflammatory bowel disease whose pathogenesis involves inappropriate immune responses toward gut microbiota on genetically predisposed backgrounds. Notably, CD is associated with single-nucleotide polymorphisms affecting several genes involved in macroautophagy/autophagy, the catabolic process that ensures the degradation and recycling of cytosolic components and microorganisms. In a clinical translation perspective, monitoring the autophagic activity of CD patients will require some knowledge on the intrinsic functional status of autophagy. Here, we focused on monocyte-derived dendritic cells (DCs) to characterize the intrinsic quantitative features of the autophagy flux. Starting with DCs from healthy donors, we documented a reprogramming of the steady state flux during the transition from the immature to mature status: both the autophagosome pool size and the flux were diminished at the mature stage while the autophagosome turnover remained stable. At the cohort level, DCs from CD patients were comparable to control in term of autophagy flux reprogramming capacity. However, the homozygous presence of ATG16L1 rs2241880 A>G (T300A) and ULK1 rs12303764 (G/T) polymorphisms abolished the capacity of CD patient DCs to reprogram their autophagy flux during maturation. This effect was not seen in the case of CD patients heterozygous for these polymorphisms, revealing a gene dose dependency effect. In contrast, the NOD2 rs2066844 c.2104C>T (R702W) polymorphism did not alter the flux reprogramming capacity of DCs. The data, opening new clinical translation perspectives, indicate that polymorphisms affecting autophagy-related genes can differentially influence the capacity of DCs to reprogram their steady state autophagy flux when exposed to proinflammatory challenges.

Abstract CT093: Vaccine immunotherapy by homologous antigenic loading as adjuvant therapy for glioblastoma: Ongoing phase I analysis

Abstract Glioblastoma (GBM) is a devastating tumor of the central nervous system (CNS) for which median survival remains 14-18 months despite aggressive standard of care. Early clinical studies of dendritic cell (DC) vaccination for the adjuvant treatment of GBM previously suggested mild to moderate clinical benefit, but results were both inconsistent and inconclusive. Homologous antigenic loading is an ex vivo technique that leverages p38MAPK and mTORC1 signaling cascades to initiate powerful cDC1-like skewing in monocyte-derived DC, leading to downstream induction of CD161int CD8+ cytolytic memory effectors. Here we report ongoing results of a completed phase I clinical trial with minimal exclusion criteria in which dendritic cell vaccines were prepared through homologous antigenic loading and administered to newly-diagnosed patients bilaterally in the vicinity of the deep cervical lymph nodes, assisted by ultrasound sonography. Patients were additionally adjuvanted with six weeks of concurrent type I interferon. Four dose levels from 3.5 x 106 to 3.6 x 107 total vaccine cells were tested, none of which resulted in AEs &amp;gt; grade 2 attributable to the investigational regimen. Immunohistochemistry of tumors derived from early post-vaccination second resections displayed enhanced CD8+ T-cell infiltration and pathologic findings consistent with residual rather than relapsed GBM in 2/3 patients. Radiologic pseudoprogression was also routinely observed among patients of all cohorts. Analysis of post-vaccination circulating PBMC indicated expansion of both CD4+ and CD8+ central memory T-cell compartments (p&amp;lt;0.05 for each by student’s two-tailed t-test) as well as expansion of CD8+CD127+ T-cells (p&amp;lt;0.05 by student’s two tailed t-test) in 4/4 patients analyzed. With an average of 10.9 months follow-up, median survival of this largely MGMT promoter unmethylated cohort was not yet reached and was statistically greater (p&amp;lt;0.05 by log-rank[Mantle-Cox]) than that of matched historical controls. At the time of submission, 13/15 trial patients remained alive with ECOG &amp;lt;/= 2. The results suggest that modern dendritic cell vaccines are safe, potentially efficacious, and can be effectively integrated within existing standards of care. Citation Format: Joseph F. Georges, Mazen Zaher, Corey M. Mossop, Christopher J. Bilbao, Christina M. Clay, Sabina Amin, Hamza A. Shaikh, Akshar Trivedi, Wei Liu, Madhuri Namekar, Colby J. Hofferek, Keenan Ernste, Eva H. Schumann, Mia Vu, Damilola Oyewole-Said, Nalini Bisht, Jonathan Vazquez-Perez, Ashley M. Goralczyk, Lindsey Colman, Lynne S. Kohler, Thuy V. Cao, Ngozi Okereke, Benjamin L. Musher, Vinod Ravi, Jan O. Kemnade, Rodrick C. Zvavanjanja, Jay-Jiguang Zhu, Sigmund H. Hsu, Yoshua Esquenazi-Levy, Nitin Tandon, Alan Turtz, Vanaja Konduri, William Karl Decker. Vaccine immunotherapy by homologous antigenic loading as adjuvant therapy for glioblastoma: Ongoing phase I analysis [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 CT093.

Abstract LB125: Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells

Abstract Chemotherapy does not sufficiently eliminate tumor-initiating cells (TICs) of hepatocellular carcinoma (HCC). To remove TICs and achieve personalized cancer therapy, dendritic cells (DC) were vaccinated against tumor germline mutations, but metastatic cancers recurred in many cases. Emerging data suggest that nanoparticle-based siRNA delivered targeting of stemness transcription factor NANOG (pluripotency transcription factor) potentiates anti-tumor immunity. We hypothesized that pre-treatment of selective TIC inhibitors targeting stemness genes sensitized the efficacy of immunotherapy targeting of liver TICs. Novel TIC-targeting immunoglobulin, variable chain fragments were identified by phage-display library screening. The top single chain camelid antibody (scFv) candidates were identified and individually cloned into mammalian expression vectors to test binding to TICs. A Tri-specific T cell engager (TriTE) was constructed to connect TIC-targeting scFv with anti-dendritic cell (DC) scFv (anti-CD14) and CD3. Such scFv vectors were tested for target cell lysis. Candidate ligands of anti-TIC ScFvs present on the tumor cell surface were identified through a series of bioinformatic analysis., The latter provided the preconditions for one of our aims to identify the target, tumor surface ligand(s) of anti-TIC ScFv to better understand the mechanism of TriTE action upon a tumor cell. IP-Mass spectrometry analyses demonstrated that two target surface antigens, TBCE and CEP170, along with their respective mRNAs were highly expressed in CD133+ TIC Huh7 cells when compared to CD133- non-TIC Huh7 cells. This accounted for specific TriTE binding to TIC cells, but not to CD133(-) Huh7 cells or primary hepatocytes. These data demonstrated that these unique tumor antigens, primarily arising during neoplastic transformation, elicited T and/or monocyte immunity capable of protecting the host from cancer progression. These conjunctive with PD1 antibody therapeutic modality leads to the personalized immune/chemotherapy of HCC patients. Human monocyte-derived DCs, TIC and T cells were incubated with TriTE to measure IFNγ production during CTL killing effects against TICs. TriTE treatment significantly reduced tumor growth of PDX tissues in NSG-SG3 mice with humanized immunity. Other novel surface antigens on TIC were identified by mass spectroscopy analyses. These data demonstrated that the presence of unique tumor antigens, primarily arising during neoplastic transformation, which elicited T and/or myeloid cell immunity capable of protecting the host from cancer progression. Self-adapting antigen-targeting machinery with Trite therapy is adaptable and fine-tuning next-generation immunotherapy. Citation Format: Wei Ma, Nuan Wang, Hye Yeon Choi, Lin Ziying, Nicolette Fajardo, Royce Ilaga, Risa Machida, Zhou Lin, Ambika Ramrakhiani, Yvonne Y. Chen, Linda Sher, Stanley M. Tahara, Keigo Machida. Tri-specific T cell engager targeting of selective surface cancer-selective ligands recruits both T cells and APC targeting of cancer neoantigens of tumor-initiating stem-like cells [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 LB125.

Abstract LB120: Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors

Abstract Despite the remarkable success of chimeric antigen receptor T cell (CAR-T) therapies in treating hematological malignancies, their use in solid tumor indications has been met with distinct challenges. The immunosuppressive tumor microenvironment (TME) and tumor antigen heterogeneity are two significant factors that may adversely impact the efficacy of these ground-breaking therapies. Engineered natural killer T (NKT) cells harbor biological features distinct from CAR-T cell or CAR-NK cell therapies and thus represent a novel therapeutic approach to deploy “off the shelf” to patients. Appia Bio’s ACUA platform is capable of generating large quantities of highly pure CAR-NKT cells from cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) for clinical use. We have demonstrated that multiple distinct mechanisms of anti-tumor activity are operative in CAR-NKT cells, including CAR-mediated killing, NK receptor-mediated killing, adjuvant effects on antigen-specific T cells and immature dendritic cells, as well as the ability to deplete of pro-tumorigenic macrophages.When transduced with a CAR, ACUA-derived CAR-NKT cells show potent CAR-mediated tumor cell killing, similar to CAR-T cells. In contrast to conventional T cells, CAR-NKT cells also demonstrate potent CAR antigen-independent tumor cell killing, mediated partially through NKG2D and DNAM-1 activating NK receptors. CAR-NKT cells, but not conventional CAR-T cells, enhanced the function of NY-ESO specific T cells in controlling multiple rounds of tumor rechallenge, through the secretion of soluble components. In regard to other adjuvant effects which may provide additional benefit in vivo, ACUA-derived NKT cells enhanced the activation of peripheral blood monocyte-derived dendritic cells, which can regulate beneficial Th1 type immune responses. To explore how ACUA-derived NKT cells could reshape the TME, we generated immunosuppressive M2-polarized macrophages and observed that ACUA-derived NKT cells can either kill or reprogram these cells to a pro-inflammatory phenotype following co-culture. Notably, these immune cell adjuvant mechanisms are specific to ACUA-derived NKT cells and are not observed with conventional CAR-T cells. In summary, these mechanistic studies underscore the advantages of ACUA-derived NKT cells over conventional CAR-T cells and highlight their potential as a promising differentiated allogeneic therapy for the treatment of solid tumors. Citation Format: Xi Wang, Andrew J. Hou, Michael A. Christopher, Yannick Bulliard, Jason S. Damiano. Stem cell-derived ACUA CAR-NKT allogeneic cell therapies harness orthogonal mechanisms of action for the treatment of solid tumors [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 LB120.

Exploring the immunomodulatory properties of glucan particles in human primary cells

The immunomodulatory properties of β-glucans have sparked interest among various medical fields. As vaccine adjuvants, glucan particles offer additional advantages as antigen delivery systems. This study reported the immunomodulatory properties of glucan particles with different size and chemical composition. The effect of glucan microparticles (GPs) and glucan nanoparticles (Glu 130 and 355 NPs) was evaluated on human immune cells. While GPs and Glu 355 NPs demonstrated substantial interaction with Dectin-1 receptor on monocytes, Glu 130 NPs exhibited reduced activation of this receptor. This observation was substantiated by blocking Dectin-1, resulting in inhibition of reactive oxygen species production induced by GPs and Glu 355 NPs. Notably, monocyte-derived dendritic cells (moDCs) stimulated by Glu 355 NPs exhibited phenotypic and functional maturation, essential for antigen cross-presentation. The immunomodulatory efficacy was investigated using an autologous mixed lymphocyte reaction (AMLR), resulting in considerable rates of lymphocyte proliferation and an intriguing profile of cytokine and chemokine release. Our findings highlight the importance of meticulously characterizing the size and chemical composition of β-glucan particles to draw accurate conclusions regarding their immunomodulatory activity. This in vitro model mimics the human cellular immune response, and the results obtained endorse the use of β-glucan-based delivery systems as future vaccine adjuvants.

The effect of probiotic Bifidobacterium lactis Bl-04 on innate antiviral responses invitro

Consumption of certain probiotic strains may be beneficial for reducing the risk of acute upper respiratory tract infections (URTIs), however, underlying immunological mechanisms are elusive. Bifidobacterium lactis Bl-04™ has been reported in humans to significantly reduce the risk of URTIs, affect the innate immunity in the nasal mucosa, and reduce nasal lavage virus titer after a rhinovirus (RV) challenge. To study the immunological mechanisms, we investigated the effect of Bl-04 on cytokine production and transcriptomes of human monocyte-derived macrophages (Mfs) and dendritic cells (DCs), and further on RV replication and cytokine production in MRC-5 fibroblasts. The results showed that Bl-04 modulates antiviral immune responses and potentiates cytokine production during viral challenge mimic in immune cells. However, effect of Bl-04 on RV replication and cytokine production in fibroblasts was negligible. Overall, the findings suggest that Bl-04 mildly stimulates antiviral immunity in Mfs and DCs, and potentially influences viral replication in fibroblasts that however warrants further investigations.

Bispecific antibodies tethering innate receptors induce human tolerant-dendritic cells and regulatory T cells.

There is an urgent need for alternative therapies targeting human dendritic cells (DCs) that could reverse inflammatory syndromes in many autoimmune and inflammatory diseases and organ transplantations. Here, we describe a bispecific antibody (bsAb) strategy tethering two pathogen-recognition receptors at the surface of human DCs. This cross-linking switches DCs into a tolerant profile able to induce regulatory T-cell differentiation. The bsAbs, not parental Abs, induced interleukin 10 and transforming growth factor β1 secretion in monocyte-derived DCs and human peripheral blood mononuclear cells. In addition, they induced interleukin 10 secretion by synovial fluid cells in rheumatoid arthritis and gout patients. This concept of bsAb-induced tethering of surface pathogen-recognition receptors switching cell properties opens a new therapeutic avenue for controlling inflammation and restoring immune tolerance.

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.

Abstract 6722: A CD33 antigen targeted Gamma Delta T-cell engager in combination with zoledronate promotes Vg9Vd2+ T cell proliferation and cytotoxicity against acute myeloid leukemia

Abstract Vγ9Vδ2+ T cell (GDT) targeted immunotherapy is of interest to harness its MHC-independent cytotoxic potential to promote anti-tumor immunity. However, it remains unclear whether GDT (1-5% of total T cells) are present at sufficient numbers to be therapeutically relevant. Accumulation of intracellular phosphoantigens promotes heterodimerization of butyrophilin (BTN) 2A1 and BTN3A1 on the cell surface, leading to TCR-mediated activation of GDT. To date, clinical trials in cancer patients have failed to demonstrate monotherapy activity using a variety of GDT activating agents, including aminobisphosphonates such as zoledronic acid (ZA), Vδ2+ targeted T cell engagers and BTN3A1 targeted antibodies. These data leave fundamental questions regarding the therapeutic potential of GDT unanswered, including whether they are present in sufficient numbers, whether the agents in question are providing adequate GDT activation, and whether GDT are being directed to tumor cell targets. The studies herein were thus designed to further understand the role of phosphoantigens, butyrophilins and antigen presenting cells (APCs) in activating GDT. Specifically, APC mediated GDT activation was studied in an in vitro co-culture system in the presence and absence of ZA or a CD33-directed GDT engager incorporating a heterodimer of BTN2A1 and BTN3A1 to dissect the contribution of phosphoantigens, butyrophilins and other costimulatory molecules on GDT proliferation and activation. To this end, while BTN2A1/3A1-Fc-CD33scFv treatment alone in T and monocyte-derived dendritic cell (mDC) co-cultures or in unfractionated PBMCs increased the frequency of GDT by 2 to 10-fold after 7-12 days, the combination with low dose ZA (0.5 to 2 μM) synergistically increased GDT proliferation by 40 to 100-fold. These results indicate that GDT proliferation relies both on the abundance of BTN2A1/3A1 heterodimers and other costimulatory signals activated by ZA. Synergistic effects of ZA and BTN2A1/3A1-Fc-CD33scFv in GDT proliferation was similarly observed in human PBMC-engrafted NSG-IL-15tg mice 12 days after treatment. Importantly, the addition of BTN2A1/3A1-Fc-CD33scFv to T (or PBMC) and CD33+ AML co-cultures enhanced both GDT proliferation and anti-leukemic activity while sparing healthy CD33+ immune cells. Collectively, these results demonstrate that GDT proliferation and effector function is dependent both upon the abundance of butyrophilin heterodimers, and other cell surface molecules which are modulated by intracellular phosphoantigen accumulation. Citation Format: Anne Y. Lai, Chunyan Wang, Dana C. Baiu, Kelsey Smith, Arpita Patel, Kinsley Evans, Noah Murr, Derek Franklin, Mahmud Hussain, Joseph Pate, Nathan Oien, Vishruti Makani, Abhinav Shukla, Keith Wilson, George Fromm, Taylor H. Schreiber, Jenny E. Gumperz, Suresh De Silva. A CD33 antigen targeted Gamma Delta T-cell engager in combination with zoledronate promotes Vg9Vd2+ T cell proliferation and cytotoxicity against acute myeloid leukemia [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 6722.

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 4115: Unleashing the power of MHC associated peptide proteomics: Immunogenicity assessment of oncology drugs

Abstract Bispecifics targeting multiple antigens or epitopes have been showing great promise for treatment of cancer. However, managing unwanted immunogenicity has become a challenge in the development cycle of these promising therapeutics as there is a trend towards higher unwanted immune responses compared with classical monoclonal antibodies. A thorough assessment of their immunogenic potential leads to safe biotherapeutics with high treatment efficacy. MAPPS assays (MHC associated peptide proteomics) enable the precise identification of all the regions of a protein that may evoke an immune response. This information is key for correct risk assessment and for modulating biotherapeutics. High-sensitive MAPPS workflow leads to higher numbers of identified peptides (self + non-self peptides) and gives the deepness of analysis required for confident immunogenicity derisking and modulation. We show the importance of peptides presented by HLA-DP and HLA-DQ (next to the standard HLA-DR presented peptides). Next, we also show the first ever comparison of presented peptides in true immune cells: dendritic cells (as compared to monocyte derived dendritic cells) and B cells. Citation Format: Sofie Pattyn. Unleashing the power of MHC associated peptide proteomics: Immunogenicity assessment of oncology drugs [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 4115.

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.