Monocyte-derived Dendritic Cells Research Articles

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [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 5272.

Abstract 6639: Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy

Abstract Activation of immune receptors, such as Toll-like (TLR), NOD-like (NLR) and Stimulator of Interferon Genes (STING) is required for efficient innate and adaptive immunity. Gram-negative bacteria (G-NB) contain multiple TLR, NOD and STING agonists. Potential utility of G-NB for cancer immunotherapy is supported by observations of tumor regression in the setting of infection and Coley’s Toxins. Coley reported that intravenous (i.v.) administration was likely most effective but produced toxicity. The discovery of TLRs and their agonists, particularly the potent/multi-functional TLR4 agonist lipopolysaccharide (LPS)-endotoxin, comprising ~75% of the outer membrane of G-NB, suggests that it may be both a critical active ingredient and responsible for dose-limiting toxicity of i.v. G-NB. We have produced killed, intact bacteria products from non-pathogenic G-NB with ~90% reduction of LPS activity. One product, Decoy10, contained TLR2,4,8,9, NOD2 and STING agonist activity and exhibited reduced i.v. toxicity in mice and rabbits relative to unprocessed cells. Decoy10 and closely related Decoy20 produced single agent activity or combination-mediated durable regressions, with immunological memory, in mice with syngeneic or human breast, colorectal, hepatocellular, pancreatic carcinomas or non-Hodgkin’s lymphoma. Regressions were observed in combination with chemotherapy, a non-steroidal anti-inflammatory drug, anti-PD-1, or rituximab, were associated with induction of 18-26 plasma cytokines/chemokines, activation of innate and adaptive immune pathways in tumors, and were dependent on NK, CD4+ and CD8+ T cells (Newman, Cancer Res 2023;83(7 Suppl):Abstract nr 4165). These and preliminary clinical results demonstrating rapid clearance of Decoy20 with transient induction of >50 plasma cytokines/chemokines (Newman et al., J Immunother Cancer 2023;11(Suppl 2)782-E:A1770) are supportive of a “pulse-prime” mechanism, whereby Decoy bacteria produce transient, but broad innate and adaptive immune activation. We have now analyzed immune activation by Decoy bacteria in vitro using human peripheral blood mononuclear cells (PBMCs). Decoy10 enhanced polarization of CD14+ monocytes to M1 macrophages and inhibited Treg polarization. Decoy10 enhanced monocyte-derived dendritic cell maturation and enhanced Th1, Th2 and Th17 CD4+ polarization, with Th1 at 10-100-fold lower Decoy10 concentrations than Th2 or Th17. Decoy10 also activated NKT and T cells, and enhanced PBMC-mediated tumor cell killing. The results demonstrate that Decoy bacteria stimulate polarization, maturation, or activation of cellular mediators of innate and adaptive anti-tumor immune responses, inhibit an immune-suppressive mechanism, and enhance immune cell killing of tumor cells. The results are consistent with and significantly extend our previously reported in vivo data. Citation Format: Michael J. Newman, Deepak Singh. Mechanisms of action of a killed, bacteria-based, multiple immune receptor agonist in development for pulsed anti-tumor immunotherapy [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 6639.

Abstract 741: Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive anti-tumor immunity

Abstract The expression of ILT (LILRB) family members and their major ligand HLA-G within solid tumors are associated with immune suppression and poor patient survival. Blocking multiple ILT family members might be more potent than targeting individual members by overcoming compensatory resistances within tumor microenvironment. Based on this hypothesis, we developed a monoclonal antibody (mAb) LITCHI 7 which reacts to three major ILT family members, ILT2, ILT4 and ILT5, and blocks ligand binding to these receptors. LITCHI 7 exhibits a broad binding capability to a wide range of human myeloid and T cell subsets that express ILT2, ILT4, and/or ILT5. Binding to monocyte-derived dendritic cells (moDC), LITCHI 7 released the inhibition on activating Fcγ receptor. On LPS-treated macrophages, LITCHI 7 enhanced inflammatory macrophage differentiation. The effect is primarily mediated by blocking ILT4 since ILT4 mono-specific mAb is as potent as cross-reactive LITHI 7 in modulating myeloid differentiation although both ILT2 and ILT5 are highly expressed by different myeloid subsets. The result was further confirmed by CRISPR Cas9 knock down of ILT2 or ILT5. In the absence of either ILT2 or ILT5, the accumulation of CD14+ monocytes and inflammatory macrophages were not changed during differentiation. While different ILT family members are generally enriched on myeloid cells, ILT2 is also expressed on NK cells and effector memory CD45RA+ T cells (Temra). Blocking ILT2 by LITCHI 7 enhanced TCR signaling when co-culturing the MART-1 specific T cell reporter line with MART-1/HLA-G positive melanoma cells. The effect of LITCHI 7 is mediated by ILT2 blockade since knocking out ILT2, but not ILT4 and ILT5, reduced accumulation of Temra in activated PBMC. To assess whether immune modulation of LITCHI 7 can be translated into anti-tumor activity by blocking different ILT family members, tumor killing assays were performed using innate and adaptive immune effector cells. LITCHI7 enhanced NK cell-mediated cytotoxicity on HLA-G+ tumor cells by blocking ILT2. Both ILT2 and ILT4 blockade play a role in macrophage phagocytosis on lymphoma cells since LITCHI 7 as well as individual anti-ILT2 or ILT-4 mAbs increased macrophage phagocytosis to a similar level. Finally, LITCHI 7 could boost in vitro T cell-mediated killing of CD19+ lymphoma by CD19 specific CAR-T cells in the presence of CD33+ myeloid cells. In summary, LITCHI 7 can enhance both innate and adaptive anti-tumor immunity through ILT2 or ILT4 blockade directly on effector cells or indirectly through myeloid cell reprogramming. As clinical trials are ongoing by targeting ILT2 and ILT4, blocking multiple ILT family members through a single cross-reactive mAb may provide broad anti-tumor effects and advantages when combining with additional oncology treatments. Citation Format: Shiming Ye, Dong Zhang, Brenal Singh, Jaishree Bankoti, Diane Cohen, Siusze Tan, Donghee Choi, Jonathan Hickson, Min-Zu Wu, Urvi Kolhatkar, Mikhail Binnewies, Wei Jin, Jason Hall, John Engelhardt, Alexander Shoemaker. Distinct functional effects of ILT family members in mediating the activity of an ILT2/ILT4/ILT5 cross-reactive monoclonal antibody in releasing the suppression on innate and adaptive 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 741.

Immunomodulatory and Anti-Inflammatory Effects of Vitamin A and Tryptophan on Monocyte-Derived Dendritic Cells Stimulated with Gliadin in Celiac Disease Patients.

Celiac Disease (CeD) is an autoimmune disorder with various symptoms upon gluten exposure. Dendritic cells (DCs) play a crucial role in gliadin-induced inflammation. Vitamin A (retinol; Ret) and its metabolite, retinoic acid (RA), along with tryptophan (Trp) and its metabolite, kynurenic acid (KYNA), are known to influence the immune function of DCs and enhance their tolerogenicity. This research aims to assess the impact of gliadin on DC maturation and the potential of vitamin A and tryptophan to induce immune tolerance in DCs. The monocyte cells obtained from peripheral blood mononuclear cells (PBMCs) of celiac disease patients were differentiated into DCs in the absence or presence of Ret, RA, Trp, KYNA, and then stimulated with peptic and tryptic (PT) digested of gliadin. We used flow cytometry to analyze CD11c, CD14, HLA-DR, CD83, CD86, and CD103 expression. ELISA was carried out to measure TGF-β, IL-10, IL-12, and TNF-α levels. qRT-PCR was used to assess the mRNA expression of retinaldehyde dehydrogenase 2 (RALDH2) and integrin αE (CD103). The mRNA and protein levels of Indoleamine 2, 3-dioxygenase (IDO) was analyzed by qRT-PCR and Western blot assays, respectively. Our findings demonstrate that PT-gliadin enhances the expression of maturation markers, i.e. CD83, CD86 and HLA-DR and promote the secretion of TNF-α and IL-12 in DCs. Interestingly, vitamin A, tryptophan, and their metabolites increase the expression of CD103, while limiting the expression of HLA-DR, CD83, and CD86. They also enhance RALDH2 and IDO expression and promote the secretion of TGF-β and IL-10, while limiting IL-12 and TNF-α secretion. These findings suggest that vitamin A and tryptophan have beneficial effects on PT-gliadin-stimulated DCs, highlighting their potential as therapeutic agents for celiac disease. However, further research is needed to fully understand their underlying mechanisms of action in these cells.

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.

Curcumin and Berberine Arrest Maturation and Activation of Dendritic Cells Derived from Lupus Erythematosus Patients.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease recognized by elevated activity of autoimmune cells, loss of tolerance, and decreased regulatory T cells producing inhibitory cytokines. Despite many efforts, the definitive treatment for lupus has not been fully understood. Curcumin (CUR) and berberine (BBR) have significant immunomodulatory roles and anti-inflammatory properties that have been demonstrated in various studies. This study aimed to investigate the anti-inflammatory properties of CUR and BBR on human monocyte-derived dendritic cells (DCs) with an special focus on the maturation and activation of DCs. Human monocytes were isolated from the heparinized blood of SLE patients and healthy individuals, which were then exposed to cytokines (IL-4 and GM-CSF) for five days to produce immature DCs. Then, the obtained DCs were characterized by FITC-uptake assay and then cultured in the presence of CUR, BBR, or lipopolysaccharide (LPS) for 48 h. Finally, the maturation of DCs was analyzed by the level of maturation using flow cytometry or real-time PCR methods. The results showed promising anti-inflammatory effects of CUR and BBR in comparison with LPS, supported by a significant reduction of not only co-stimulatory and antigen-presenting factors such as CD80, CD86, CD83, CD1a, CD14, and HLA-DR but also inflammatory cytokines such as IL-12. CUR and BBR could arrest DC maturation and develop a tolerogenic DC phenotype that subsequently promoted the expression of inhibitory cytokines and reduced the secretion of proinflammatory markers.

Itaconate as a potential target for antimalarial therapy

In a recent publication, Ramalho et al. investigated monocyte-derived dendritic cell (MODC) mobilization in response to Plasmodium infection. The authors showed that elevated levels of itaconate in MODCs results in reduced CD8 T cell activation and that the absence of itaconate is associated with enhanced parasite control.

The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells

OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.

Retinoic acid-loaded liposomes induce human mucosal CD103+ dendritic cells that inhibit Th17 cells and drive regulatory T-cell development in vitro.

The active vitamin A metabolite, all-trans-retinoic acid (RA), primes precursor dendritic cells (DCs) into a mucosal phenotype with tolerogenic properties characterized by the expression of integrin CD103. CD103+ DCs can counteract pathogenic Th1 and Th17 in inflammatory bowel disease (IBD) or celiac disease (CD). Tolerogenic manipulation of DCs using nanoparticles carrying tolerogenic adjuvants and disease-specific antigens is a valuable treatment strategy to induce antigen-specific mucosal tolerance in vivo. Here, we investigated the effects of RA-loaded liposomes on human DC phenotype and function, including DC-driven T-cell development, both during the generation of monocyte-derived DCs (moDCs) as well as by priming immature moDCs. RA liposomes drove CD103+ DC differentiation as well as ALDH1A2 expression in DCs. Neutrophil-dependent Th17 cell development was reduced by RA-liposome-differentiated and RA-liposome-primed DCs. Moreover, RA liposome treatment shifted T-cell development toward a Th2 cell profile. Importantly, RA liposomes induced the development of IL-10-producing and FoxP3+ regulatory T cells (Tregs) of various Treg subsets, including ICOS+ Tregs, that were potent inhibitors of bystander memory T-cell proliferation. Taken together, RA-loaded liposomes could be a novel treatment avenue for IBD or CD patients.

Involvement of autologous myeloid dendritic cells in the evaluation of immediate hypersensitivity reactions to betalactams

BackgroundAmoxicillin (AX) and clavulanic acid (CLV) are the betalactam antibiotics (BLs) most used to treat bacterial infections, although they can trigger immediate hypersensitivity reactions (IDHRs). The maturation analysis of monocyte-derived dendritic cells (moDCs) and their capacity to induce proliferative response of lymphocytes are useful to test the sensitisation to a drug, although without optimal sensitivity. Nevertheless, this can be improved using directly isolated DCs such as myeloid DCs (mDCs). MethodsmDCs and moDCs were obtained from 28 allergic patients (AP), 14 to AX, 14 to CLV and from 10 healthy controls (HC). The expression of CCR7, CD40, CD80, CD83, and CD86 was analysed after stimulation with both BLs. We measured the capacity of these pre-primed DCs to induce drug-specific activation of different lymphocyte subpopulations, CD3+, CD4+, CD8+, CD4+Th1, and CD4+Th2, by flow cytometry. ResultsHigher expression of CCR7, CD40, CD80, CD83, and CD86 was observed on mDCs compared to moDCs from AP after stimulating with the culprit BL. Similarly, mDCs induced higher proliferative response, mainly of CD4+Th2 cells, compared to moDCs, reaching up to 67% of positive results with AX, whereas of only 25% with CLV. ConclusionsmDCs from selective AP efficiently recognise the culprit drug which trigger the IDHR. mDCs also trigger proliferation of lymphocytes, mainly those with a Th2 cytokine pattern, although these responses depend on the nature of the drug, mimicking the patient's reaction.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells.

Dendritic cell (DC) migration from peripheral tissues via afferent lymphatic vessels to draining lymph nodes (dLNs) is important for the organism's immune regulation and immune protection. Several lymphatic endothelial cell (LEC)-expressed adhesion molecules have thus far been found to support transmigration and movement within the lymphatic vasculature. In this study, we investigated the contribution of CD112, an adhesion molecule that we recently found to be highly expressed in murine LECs, to this process. Performing in vitro assays in the murine system, we found that transmigration of bone marrow-derived dendritic cells (BM-DCs) across or adhesion to murine LEC monolayers was reduced when CD112 was absent on LECs, DCs, or both cell types, suggesting the involvement of homophilic CD112-CD112 interactions. While CD112 was highly expressed in murine dermal LECs, CD112 levels were low in endogenous murine dermal DCs and BM-DCs. This might explain why we observed no defect in the in vivo lymphatic migration of adoptively transferred BM-DCs or endogenous DCs from the skin to dLNs. Compared to murine DCs, human monocyte-derived DCs expressed higher CD112 levels, and their migration across human CD112-expressing LECs was significantly reduced upon CD112 blockade. CD112 expression was also readily detected in endogenous human dermal DCs and LECs by flow cytometry and immunofluorescence. Upon incubating human skin punch biopsies in the presence of CD112-blocking antibodies, DC emigration from the tissue into the culture medium was significantly reduced, indicating impaired lymphatic migration. Overall, our data reveal a contribution of CD112 to human DC migration.

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 cytomegalovirus exploits STING signaling and counteracts IFN/ISG induction to facilitate infection of dendritic cells

Human cytomegalovirus (HCMV) is a widespread pathogen that in immunocompromised hosts can cause life-threatening disease. Studying HCMV-exposed monocyte-derived dendritic cells by single-cell RNA sequencing, we observe that most cells are entered by the virus, whereas less than 30% of them initiate viral gene expression. Increased viral gene expression is associated with activation of the stimulator of interferon genes (STING) that usually induces anti-viral interferon responses, and with the induction of several pro- (RHOB, HSP1A1, DNAJB1) and anti-viral (RNF213, TNFSF10, IFI16) genes. Upon progression of infection, interferon-beta but not interferon-lambda transcription is inhibited. Similarly, interferon-stimulated gene expression is initially induced and then shut off, thus further promoting productive infection. Monocyte-derived dendritic cells are composed of 3 subsets, with one being especially susceptible to HCMV. In conclusion, HCMV permissiveness of monocyte-derived dendritic cells depends on complex interactions between virus sensing, regulation of the interferon response, and viral gene expression.

Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn‑glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Mass cytometry revealed the circulating immune cell landscape across different Suzuki stages of Moyamoya disease

Moyamoya disease (MMD) is a cerebrovascular disorder marked by progressive arterial narrowing, categorized into six stages known as Suzuki stages based on angiographic features. Growing evidence indicates a pivotal role of systemic immune and inflammatory responses in the initiation and advancement of MMD. This study employs high-dimensional mass cytometry to reveal the immunophenotypic characteristics of peripheral blood immune cells (PBMCs) at various Suzuki stages, offering insights into the progression of MMD. PBMC samples from eight patients with early-stage MMD (Suzuki stages II and III) and eight patients with later-stage MMD (Suzuki stages IV, V, and VI) were analyzed using high-dimensional mass cytometry to evaluate the frequency and phenotype of immune cell subtypes. We identified 15 cell clusters and found that the immunological features of early-stage MMD and later-stage MMD are composed of cluster variations. In this study, we confirmed that, compared to later-stage MMD, the early-stage MMD group exhibits an increase in non-classical monocytes. As the Suzuki stage level increases, the proportions of plasmacytoid DCs and monocyte-derived DCs decrease. Furthermore, T cells, monocytes, DCs, and PMN-MDSCs in the early-stage MMD group show activation of the canonical NF-κB signaling pathway. We summarized and compared the similarities and differences between early-stage MMD patients and later-stage MMD patients. There is a potential role of circulating immune dysfunction and inflammatory responses in the onset and development of MMD.

Allogeneic NK cells induce the in vitro activation of monocyte-derived and conventional type-2 dendritic cells and trigger an inflammatory response under cancer-associated conditions.

Natural killer (NK) cells are innate lymphocytes capable to recognize and kill virus-infected and cancer cells. In the past years, the use of allogeneic NK cells as anti-cancer therapy gained interest due to their ability to induce graft-versus-cancer responses without causing graft-versus-host disease and multiple protocols have been developed to produce high numbers of activated NK cells. While the ability of these cells to mediate tumor kill has been extensively studied, less is known about their capacity to influence the activity of other immune cells that may contribute to a concerted anti-tumor response in the tumor microenvironment (TME). In this study, we analyzed how an allogeneic off-the-shelf cord blood stem cell-derived NK-cell product influenced the activation of dendritic cells (DC). Crosstalk between NK cells and healthy donor monocyte-derived DC (MoDC) resulted in the release of IFNγ and TNF, MoDC activation, and the release of the T-cell-recruiting chemokines CXCL9 and CXCL10. Moreover, in the presence of prostaglandin-E2, NK cell/MoDC crosstalk antagonized the detrimental effect of IL-10 on MoDC maturation leading to higher expression of multiple (co-)stimulatory markers. The NK cells also induced activation of conventional DC2 (cDC2) and CD8+ T cells, and the release of TNF, GM-CSF, and CXCL9/10 in peripheral blood mononuclear cells of patients with metastatic colorectal cancer. The activated phenotype of MoDC/cDC2 and the increased release of pro-inflammatory cytokines and T-cell-recruiting chemokines resulting from NK cell/DC crosstalk should contribute to a more inflamed TME and may thus enhance the efficacy of T-cell-based therapies.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ Tcells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ Tcells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Impaired monocyte-derived dendritic cell phenotype in prostate cancer patients: A phenotypic comparison with healthy donors.

Dendritic cells (DCs) play a crucial role in immunity. Research on monocyte-derived DCs (Mo-DCs) cancer vaccines is in progress despite limited success in clinical trials. This study focuses on Mo-DCs generated from prostate cancer (PCA) patients, comparing them with DCs from healthy donors (HD-DCs). Mo-DCs were isolated from PCA patient samples, and their phenotype was compared to HD-DCs. Key parameters included monocyte count, CD14 expression, and the levels of maturation markers (HLA-DR, CD80, CD86) were assessed. PCA samples exhibited a significantly lower monocyte count and reduced CD14 expression compared to healthy samples (p ⟨ 0.0001). Additionally, PCA-DCs expressed significantly lower levels of maturation markers, including HLA-DR, CD80, and CD86, when compared to HD-DCs (p = 0.123, p = 0.884, and p = 0.309, respectively). The limited success of DC vaccines could be attributed to impaired phenotypic characteristics. These observations suggest that suboptimal characteristics of Mo-DCs generated from cancer patient blood samples might contribute to the limited success of DC vaccines. Consequently, this study underscores the need for alternative strategies to enhance the features of Mo-DCs for more effective cancer immunotherapies.

RWTC-MBTA Vaccine Induces Potent Adaptive Immune Responses Against Glioblastomas via Dynamic Activation of Dendritic Cells.

Despite strides in immunotherapy, glioblastoma multiforme (GBM) remains challenging due to low inherent immunogenicity and suppressive tumor microenvironment. Converting "cold" GBMs to "hot" is crucial for immune activation and improved outcomes. This study comprehensively characterized a therapeutic vaccination strategy for preclinical GBM models. The vaccine consists of Mannan-BAM-anchored irradiated whole tumor cells, Toll-like receptor ligands [lipoteichoic acid (LTA), polyinosinic-polycytidylic acid (Poly (I:C)), and resiquimod (R-848)], and anti-CD40 agonistic antibody (rWTC-MBTA). Intracranial GBM models (GL261, SB28 cells) are used to evaluate the vaccine efficacy. A substantial number of vaccinated mice exhibited complete regression of GBM tumors in a T-cell-dependent manner, with no significant toxicity. Long-term tumor-specific immune memory is confirmed upon tumor rechallenge. In the vaccine-draining lymph nodes of the SB28 model, rWTC-MBTA vaccination triggered a major rise in conventional dendritic cell type 1 (cDC1) 12h post-treatment, followed by an increase in conventional dendritic cell type 2 (cDC2), monocyte-derived dendritic cell (moDC), and plasmacytoid dendritic cell (pDC) on Day 5 and Day 13. Enhanced cytotoxicity of CD4+ and CD8+ T cells in vaccinated mice is verified in co-culture with tumor cells. Analyses of immunosuppressive signals (T-cell exhaustion, myeloid-derived suppressor cells (MDSC), M2 macrophages) in the GBM microenvironment suggest potential combinations with other immunotherapies for enhanced efficacy. In conclusion, the authors findings demonstrate that rWTC-MBTA induces potent and long-term adaptive immune responses against GBM.