Bone Marrow-derived Dendritic Cells Research Articles

Glioblastoma-derived exosomes promote lipid accumulation and induce ferroptosis in dendritic cells via the NRF2/GPX4 pathway.

Glioblastoma-derived exosomes (GDEs), containing nucleic acids, proteins, fatty acids and other substances, perform multiple important functions in glioblastoma microenvironment. Tumor-derived exosomes serve as carriers of fatty acids and induce a shift in metabolism towards oxidative phosphorylation, thus driving immune dysfunction of dendritic cells (DCs). Lipid peroxidation is an important characteristic of ferroptosis. Nevertheless, it remains unclear whether GDEs can induce lipid accumulation and lipid oxidation to trigger ferroptosis in DCs. In our study, we investigate the impact of GDEs on lipid accumulation and oxidation in DCs by inhibiting GDEs secretion through knocking down the expression of Rab27a using a rat orthotopic glioblastoma model. The results show that inhibiting the secretion of GDEs can reduce lipid accumulation in infiltrating DCs in the brain and decrease mature dendritic cells (mDCs) lipid peroxidation levels, thereby suppressing glioblastoma growth. Mechanistically, we employed in vitro treatments of bone marrow-derived dendritic cells (BMDCs) with GDEs. The results indicate that GDEs decrease the viability of mDCs compared to immature dendritic cells (imDCs) and trigger ferroptosis in mDCs via the NRF2/GPX4 pathway. Overall, these findings provide new insights into the development of immune-suppressive glioblastoma microenvironment through the interaction of GDEs with DCs.

Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus.

Dendritic cells (DCs) are professional antigen-presenting cells, which are key components of the immune system and involved in early immune responses. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with avian influenza virus (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death. For this study, chicken bone marrow-derived DC (ckBM-DC)s were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphologies, and evaluate virus replication. A strong proinflammatory response was observed at 8 hours post infection, via upregulation of chicken interleukin-1β and stimulation of the interferon response pathway. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The increase in activation of infected DCs may be indicative of a dysregulated immune response typically seen with HPAI infections.

Microevolution during Chronic Infection May Lead T. asahii to Coexist with the Host.

Trichosporon asahii (T. asahii) is part of the cutaneous fungal microbiota in humans and can cause lethal opportunistic infection. During infection, microorganisms can adapt to their environment by adjusting gene expression and cellular activities. Investigation of the microevolutionary changes in T. asahii during chronic infection. Two T. asahii strains were isolated from a chronic trichosporonosis patient between a 15-year interval, and the microevolutionary changes were compared by the immune response of dendritic cell (DC), mice survival model, and transcriptome sequencing analysis. Compared with the primary T. asahii strain, the microevolved strain induced much lower expression of TNF-α by mice bone marrow-derived DC and had a much superior survival rate, a total of 2212 significantly differentially expressed genes were identified in the microevolved strain, and functional analysis showed significance in the downregulated transcription and metabolic process, especially the valine, leucine, and isoleucine degradation pathways, which were associated with pathogenicity and virulence; hence, the results were highly consistent with the decreased immunogenicity and virulence of the microevolved strain. These results demonstrated that the microevolution during chronic infection could induce changes in immunogenicity, virulence, and transcriptome, which might lead T. asahii to coexist with the host.

Hypofractionated radiotherapy combined with lenalidomide improves systemic antitumor activity in mouse solid tumor models.

Background: Hypofractionated radiotherapy (hRT) can induce a T cell-mediated abscopal effect on non-irradiated tumor lesions, especially in combination with immune checkpoint blockade (ICB). However, clinically, this effect is still rare, and ICB-mediated adverse events are common. Lenalidomide (lena) is an anti-angiogenic and immunomodulatory drug used in the treatment of hematologic malignancies. We here investigated in solid tumor models whether lena can enhance the abscopal effect in double combination with hRT. Methods: In two syngeneic bilateral tumor models (B16-CD133 melanoma and MC38 colon carcinoma), the primary tumor was treated with hRT. Lena was given daily for 3 weeks. Besides tumor size and survival, the dependence of the antitumor effects on CD8+ cells, type-I IFN signaling, and T cell costimulation was determined with depleting or blocking antibodies. Tumor-specific CD8+ T cells were quantified, and their differentiation and effector status were characterized by multicolor flow cytometry using MHC-I tetramers and various antibodies. In addition, dendritic cell (DC)-mediated tumor antigen cross-presentation in vitro and directly ex vivo and the composition of tumor-associated vascular endothelial cells were investigated. Results: In both tumor models, the hRT/lena double combination induced a significant abscopal effect. Control of the non-irradiated secondary tumor and survival were considerably better than with the respective monotherapies. The abscopal effect was strongly dependent on CD8+ cells and associated with an increase in tumor-specific CD8+ T cells in the non-irradiated tumor and its draining lymph nodes. Additionally, we found more tumor-specific T cells with a stem-like (TCF1+ TIM3- PD1+) and a transitory (TCF1- TIM3+ CD101- PD1+) exhausted phenotype and more expressing effector molecules such as GzmB, IFNγ, and TNFα. Moreover, in the non-irradiated tumor, hRT/lena treatment also increased DCs cross-presenting a tumor model antigen. Blocking type-I IFN signaling, which is essential for cross-presentation, completely abrogated the abscopal effect. A gene expression analysis of bone marrow-derived DCs revealed that lena augmented the expression of IFN response genes and genes associated with differentiation, maturation (including CD70, CD83, and CD86), migration to lymph nodes, and T cell activation. Flow cytometry confirmed an increase in CD70+ CD83+ CD86+ DCs in both irradiated and abscopal tumors. Moreover, the hRT/lena-induced abscopal effect was diminished when these costimulatory molecules were blocked simultaneously using antibodies. In line with the enhanced infiltration by DCs and tumor-specific CD8+ T cells, including more stem-like cells, hRT/lena also increased tumor-associated high endothelial cells (TA-HECs) in the non-irradiated tumor. Conclusions: We demonstrate that lena can augment the hRT-induced abscopal effect in mouse solid tumor models in a CD8 T cell- and IFN-I-dependent manner, correlating with enhanced anti-tumor CD8 T cell immunity, DC cross-presentation, and TA-HEC numbers. Our findings may be helpful for the planning of clinical trials in (oligo)metastatic patients.

Matrix metalloproteinase-8 regulates dendritic cell tolerance in late polymicrobial sepsis via the nuclear factor kappa-B p65/β-catenin pathway.

Tolerogenic dendritic cells (DCs) are associated with poor prognosis of sepsis. Matrix metalloproteinases (MMPs) have been shown to have immunomodulatory effects. However, whether MMPs are involved in the functional reprogramming of DCs is unknown. The study aims to investigate the role of MMPs in sepsis-induced DCs tolerance and the potential mechanisms. A murine model of late sepsis was induced by cecal ligation and puncture (CLP). The expression levels of members of the MMP family were detected in sepsis-induced tolerogenic DCs by using microarray assessment. The potential roles and mechanisms underlying MMP8 in the differentiation, maturation and functional reprogramming of DCs during late sepsis were assessed both in vitro and in vivo. DCs from late septic mice expressed higher levels of MMP8, MMP9, MMP14, MMP19, MMP25 and MMP27, and MMP8 levels were the highest. MMP8 deficiency significantly alleviated sepsis-induced immune tolerance of DCs both in vivo and in vitro. Adoptive transfer of MMP8 knockdown post-septic bone marrow-derived DCs protected mice against sepsis-associated lethality and organ dysfunction, inhibited regulatory T-cell expansion and enhanced Th1 response. Furthermore, the effect of MMP8 on DC tolerance was found to be associated with the nuclear factor kappa-B p65/β-catenin pathway. Increased MMP8 levels in septic DCs might serve as a negative feedback loop, thereby suppressing the proinflammatory response and inducing DC tolerance.

Antigen-Specific Stimulation of CD8+ T Cells by Murine Bone Marrow-Derived Dendritic Cells After Treatment with Nanoparticles.

The assessment of antigen presentation by dendritic cells and subsequent antigen-dependent activation of T lymphocytes is a critical step underlying the efficacy of nanoparticle-based therapeutic vaccines. Since nanoparticle physicochemical properties determine their interactions with the immune system, the early stages of nanotechnology-based vaccine development commonly involve optimizing the particles' properties to create a formulation with desired stability, antigen release, targeting of desired cell populations, and efficacy. To accelerate this process, in vitro models suitable for the rapid assessment of a novel vaccine candidate's efficacy are highly desirable. One such model is described in this protocol. Herein, nanoparticles are formulated to deliver a model antigen, SIINFEKL (OVA257-264), the immunodominant class I peptide derived from ovalbumin. These nanoparticles are added to the culture of murine bone marrow-derived dendritic cells, which are subsequently co-incubated with CD8+ T cells from OT-I transgenic mice. The efficient antigen presentation by dendritic cells results in the antigen-dependent proliferation of CD8+ T cells, which is detected by flow cytometry.

Mycobacterium smegmatis enhances shikonin-induced immunogenic cell death-an efficient in situ tumor vaccine strategy.

Tumor vaccines are a promising avenue in cancer immunotherapy. Despite the progress in targeting specific immune epitopes, tumor cells lacking these epitopes can evade the treatment. Here, we aimed to construct an efficient in situ tumor vaccine called Vac-SM, utilizing shikonin (SKN) to induce immunogenic cell death (ICD) and Mycobacterium smegmatis as an immune adjuvant to enhance in situ tumor vaccine efficacy. SKN showed a dose-dependent and time-dependent cytotoxic effect on the tumor cell line and induced ICD in tumor cells as evidenced by the CCK-8 assay and the detection of the expression of relevant indicators, respectively. Compared with the control group, the in situ Vac-SM injection in mouse subcutaneous metastatic tumors significantly inhibited tumor growth and distant tumor metastasis, while also improving survival rates. Mycobacterium smegmatis effectively induced maturation and activation of bone marrow-derived dendritic cells (DCs), and in vivo tumor-draining lymph nodes showed an increased maturation of DCs and a higher proportion of effector memory T-cell subsets with the Vac-SM treatment, based on flow cytometry analysis results. Collectively, the Vac-SM vaccine effectively induces ICD, improves antigen presentation by DCs, activates a specific systemic antitumor T-cell immune response, exhibits a favorable safety profile, and holds the promise for clinical translation for local tumor immunotherapy.

Detection of Antigen Presentation by Murine Bone Marrow-Derived Dendritic Cells After Treatment with Nanoparticles.

Nanoparticles are frequently considered in vaccine applications due to their ability to co-deliver multiple antigens and adjuvants to antigen-presenting cells. Some nanoparticles also have intrinsic adjuvant properties that further enhance their ability to stimulate immune cells. The delivery of tumor-specific antigens to antigen-presenting cells (APCs) with subsequent antigenic peptide presentation in the context of class I major histocompatibility complex (MHC-I) molecules represents an essential effort in developing nanotechnology-based cancer vaccines. Experimental models are, therefore, needed to gauge the efficiency of nanotechnology carriers in achieving peptide antigen delivery to APCs and presentation in the context of MHC-I. The assay described herein utilizes a model antigen ovalbumin and model APCs, murine bone marrow-derived dendritic cells. The 25-D1.16 antibody, specific to the ovalbumin (OVA) MHC-I peptide SIINFEKL, recognizes this peptide presented in the context of the murine H2-Kb class I MHC molecule, allowing the presentation of this antigen on APCs to be detected by flow cytometry after nanoparticle delivery.

Ectromelia Virus Affects the Formation and Spatial Organization of Adhesive Structures in Murine Dendritic Cells In Vitro.

Ectromelia virus (ECTV) is a causative agent of mousepox. It provides a suitable model for studying the immunobiology of orthopoxviruses, including their interaction with the host cell cytoskeleton. As professional antigen-presenting cells, dendritic cells (DCs) control the pericellular environment, capture antigens, and present them to T lymphocytes after migration to secondary lymphoid organs. Migration of immature DCs is possible due to the presence of specialized adhesion structures, such as podosomes or focal adhesions (FAs). Since assembly and disassembly of adhesive structures are highly associated with DCs' immunoregulatory and migratory functions, we evaluated how ECTV infection targets podosomes and FAs' organization and formation in natural-host bone marrow-derived DCs (BMDC). We found that ECTV induces a rapid dissolution of podosomes at the early stages of infection, accompanied by the development of larger and wider FAs than in uninfected control cells. At later stages of infection, FAs were predominantly observed in long cellular extensions, formed extensively by infected cells. Dissolution of podosomes in ECTV-infected BMDCs was not associated with maturation and increased 2D cell migration in a wound healing assay; however, accelerated transwell migration of ECTV-infected cells towards supernatants derived from LPS-conditioned BMDCs was observed. We suggest that ECTV-induced changes in the spatial organization of adhesive structures in DCs may alter the adhesiveness/migration of DCs during some conditions, e.g., inflammation.

The Potential Role of Moringa oleifera Lam. Leaf Proteins in Moringa Allergy by Functionally Activating Murine Bone Marrow-Derived Dendritic Cells and Inducing Their Differentiation toward a Th2-Polarizing Phenotype.

Moringa oleifera leaves are an inexpensive substitute for staple foods. Despite limited data, Moringa oleifera leaf protein (Mo-Pr) may be allergenic in BALB/c mice. In mouse models and allergic patients, dendritic cells (DCs) may be involved in food allergy. In addition, some allergens, including food allergens, can directly activate DCs and induce Th2 polarization. We investigated whether Mo-Pr can modulate the functional profile of murine bone marrow-derived dendritic cells (BMDCs) in vitro. BMDCs were obtained from mouse bone marrow cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days and then treated with lipopolysaccharide (LPS) or Mo-Pr. BMDC phenotypes were evaluated via flow cytometry, cytokine production was assessed using ELISA, the expression of key genes was studied using qRT-PCR, the effects on T-cell differentiation were investigated using mixed lymphocyte reaction (MLR), and transcriptional changes in BMDCs were investigated using RNA-Seq. Mo-Pr-specific IgE was investigated in recipient serum after BMDC transfer. Mo-Pr treatment significantly induced BMDC maturation, increased the expression of CD80/86 and MHC II, resulted in the production of IL-12 and TNF-α, and induced T-cell differentiation. Mo-Pr treatment stimulated BMDCs' expression of the Th2 promoters OX40L and TIM-4, induced the production of the Th2-type chemokines CCL22 and CCL17, and decreased the Th1/Th2 ratio in vitro. Healthy recipients of Mo-Pr-treated BMDCs produced Mo-Pr-specific IgE.

IFN-γ inhibitory molecules derived from Eimeria maxima inhibit IL-12 secretion by modulating MAPK pathways in chicken macrophages

IFN-γ plays a crucial role in resisting intracellular parasitic protozoa, such as Eimeria species. In our previous study, we identified 4 molecules derived from Eimeria maxima (E. maxima) that significantly inhibited IFN-γ production. However, the mechanism underlying this inhibitory effect remains unknown. In this study, we first investigated the effects of these 4 IFN-γ inhibitory molecules on the expression levels of chicken Toll-like receptors (chTLRs), IL-12, IL-10, TGF-β, and TNF-α in chicken macrophage HD11 and bone marrow-derived dendritic cells (BMDCs). The results demonstrated that these 4 inhibitory molecules significantly downregulated the mRNA levels of chTLR-2, chTLR-4, chTLR-21, and both mRNA and protein levels of IL-12. Subsequently, to clarify the effects of these 4 inhibitory molecules on the IL-12 secretion-related signaling pathways in chicken macrophages, qRT-PCR and Western blot were used to detect the changes of key molecules involved in the signaling pathways of IL-12 secretion (NF-κB, ERK1/2, p38, JNK, STAT3) following coincubation with these inhibitory molecules. Finally, RNAi was employed to verify the function of key molecules in the signaling pathway. The results revealed a significant upregulation in the expression of ERK1/2 phosphorylated protein induced by the 4 inhibitory molecules. Knockdown of the ERK1/2 gene significantly reduced the inhibitory effect of the 4 E. maxima inhibitory molecules on IL-12. These findings indicate that the 4 inhibitory molecules can inhibit the secretion of IL-12 by upregulating the expression of ERK1/2 phosphorylated protein, which is a key molecule in the ERK-MAPK pathway. Our study may contribute to elucidating the mechanisms underlying immune evasion during E. maxima infections, thereby providing new insights for the control of chicken coccidiosis.

Optimizing the generation of mature bone marrow-derived dendritic cells in vitro: a factorial study design

BackgroundFactorial design is a simple, yet elegant method to investigate the effect of multiple factors and their interaction on a specific response simultaneously. Hence, this type of study design reaches the best optimization conditions of a process. Although the interaction between the variables is widely prevalent in cell culture procedures, factorial design per se is infrequently utilized in improving cell culture output. Therefore, we aim to optimize the experimental conditions for generating mature bone marrow-derived dendritic cells (BMDCs). Two different variables were investigated, including the concentrations of the inducing factors and the starting density of the bone marrow mononuclear cells. In the current study, we utilized the design of experiments (DoE), a statistical approach, to systematically assess the impact of factors with varying levels on cell culture outcomes. Herein, we apply a two-factor, two-level (22) factorial experiment resulting in four conditions that are run in triplicate. The two variables investigated here are cytokines combinations with two levels, granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or with interleukin-4 (IL4). The other parameter is cell density with two different concentrations, 2 × 106 and 4 × 106 cells/mL. Then, we measured cell viability using the trypan blue exclusion method, and a flow cytometer was used to detect the BMDCs expressing the markers FITC-CD80, CD86, CD83, and CD14. BMDC marker expression levels were calculated using arbitrary units (AU) of the mean fluorescence intensity (MFI). ResultsThe current study showed that the highest total viable cells and cells yield obtained were in cell group seeded at 2 × 106 cells/mL and treated with GM-CSF and IL-4. Importantly, the expression of the co-stimulatory molecules CD83 and CD80/CD86 were statistically significant for cell density of 2 × 106 cells/mL (P < 0.01, two-way ANOVA). Bone marrow mononuclear cells seeded at 4 × 106 in the presence of the cytokine mix less efficiently differentiated and matured into BMDCs. Statistical analysis via two-way ANOVA revealed an interaction between cell density and cytokine combinations. ConclusionThe analysis of this study indicates a substantial interaction between cytokines combinations and cell densities on BMDC maturation. However, higher cell density is not associated with optimizing DC maturation. Notably, applying DoE in bioprocess designs increases experimental efficacy and reliability while minimizing experiments, time, and process costs.

Bile acids induce IL-1α and drive NLRP3 inflammasome-independent production of IL-1β in murine dendritic cells.

Bile acids are amphipathic molecules that are synthesized from cholesterol in the liver and facilitate intestinal absorption of lipids and nutrients. They are released into the small intestine upon ingestion of a meal where intestinal bacteria can modify primary into secondary bile acids. Bile acids are cytotoxic at high concentrations and have been associated with inflammatory diseases such as liver inflammation and Barrett's Oesophagus. Although bile acids induce pro-inflammatory signalling, their role in inducing innate immune cytokines and inflammation has not been fully explored to date. Here we demonstrate that the bile acids, deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) induce IL-1α and IL-1β secretion in vitro in primed bone marrow derived dendritic cells (BMDCs). The secretion of IL-1β was found not to require expression of NLRP3, ASC or caspase-1 activity; we can't rule out all inflammasomes. Furthermore, DCA and CDCA were shown to induce the recruitment of neutrophils and monocytes to the site of injection an intraperitoneal model of inflammation. This study further underlines a mechanistic role for bile acids in the pathogenesis of inflammatory diseases through stimulating the production of pro-inflammatory cytokines and recruitment of innate immune cells.

Salmonella enterica serovar Choleraesuis vector outperforms alum as an adjuvant, increasing a cross-protective immune response against Glaesserella parasuis

The adjuvant and/or vector significantly affect a vaccine's efficacy. Although traditional adjuvants such as alum have contributed to vaccine development, deficiencies in the induction of cellular and mucosal immunity have limited their further promotion. Salmonella vectors have unique advantages for establishing cellular and mucosal immunity due to mucosal pathways of invasion and intracellular parasitism. In addition, Salmonella vectors can activate multiple innate immune pathways, thereby promoting adaptive immune responses. In this work, the attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) vector rSC0016 was used to deliver the conserved protective antigen HPS_06257 of Glaesserella parasuis (G. parasuis), generating a novel recombinant strain rSC0016(pS-HPS_06257). The rSC0016(pS-HPS_06257) can express and deliver the HPS_06257 protein to the lymphatic system of the host. In comparison to HPS_06257 adjuvanted with alum, rSC0016(pS-HPS_06257) significantly increased TLR4 and TLR5 activation in mice as well as the levels of proinflammatory cytokines. In addition, rSC0016 promoted a greater degree of maturation in bone marrow-derived dendritic cells (BMDCs) than alum. The specific humoral, mucosal, and cellular immune responses against HPS_06257 in mice immunized with rSC0016(pS-HPS_06257) were significantly higher than those of HPS_06257 adjuvanted with alum. HPS_06257 delivered by the S. Choleraesuis vector induces a Th1-biased Th1/Th2 mixed immune response, while HPS adjuvanted with alum can only induce a Th2-biased immune response. HPS_06257 adjuvanted with alum only causes opsonophagocytic activity (OPA) responses against a homologous strain (G. parasuis serotype 5, GPS5), whereas rSC0016(pS-HPS_06257) could generate cross-OPA responses against a homologous strain and a heterologous strain (G. parasuis serotype 12, GPS12). Ultimately, HPS_06257 adjuvanted with alum protected mice against lethal doses of GPS5 challenge by 60 % but failed to protect mice against lethal doses of GPS12. In contrast, mice immunized with rSC0016(pS-HPS_06257) had 100 % or 80 % survival when challenged with lethal doses of GPS5 or GPS12, respectively. Altogether, the S. Choleraesuis vector rSC0016 could potentially generate an improved innate immune response and an improved adaptive immunological response compared to the traditional alum adjuvant, offering a novel concept for the development of a universal G. parasuis vaccine.

Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection.

Notch signaling manipulates the function and phenotype of dendritic cells (DCs), as well as the interaction between DCs and CD4+ T cells. However, the role of Notch signaling in Helicobacter pylori (H. pylori) infection remains elusive. Murine bone marrow-derived dendritic cells (BMDCs) were pretreated in the absence or presence of Notch signaling inhibitor DAPT prior to H. pylori stimulation and the levels of Notch components, cytokines and surface markers as well as the differentiation of CD4+ T cells in co-culture were measured using quantitative real-time PCR (qRT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Compared with the control, the mRNA expression of all Notch receptors and Notch ligands Dll4 and Jagged1 was up-regulated in H. pylori-stimulated BMDCs. The blockade of Notch signaling by DAPT influenced the production of IL-1β and IL-10 in H. pylori-pulsed BMDCs, and reduced the expression of Notch1, Notch3, Notch4, Dll1, Dll3 and Jagged2. In addition, DAPT pretreatment decreased the expression of maturation markers CD80, CD83, CD86, and major histocompatibility complex class II (MHC-II) of BMDCs, and further skewed Th17/Treg balance toward Treg. Notch signaling regulates the function and phenotype of DCs, thus mediating the differentiation of CD4+ T cells during H. pylori infection.

PEI-Based Nanoparticles for Tumor Immunotherapy via In Situ Antigen-Capture Triggered by Photothermal Therapy.

Activating a tumor antigen-specific immune response is key to the success of tumor immunotherapy and the development of personalized antitumor therapy. Nanocarriers can capture, enrich, and protect in situ produced tumor antigens due to immunogenic cell death (ICD), thus enhancing the tumor-specific immune response. Developing multifunctional nanocarriers that combine multiple antigen capturing mechanisms is crucial to the activation of tumor-specific immune responses. In this study, polyethylenimine (PEI) was employed as a main building block to construct a series of multifunctional indocyanine green (ICG)-loaded nanoparticles to capture antigens via multiple mechanisms: electrostatic interactions with PEI, hydrophobic interactions with the thermosensitive segment (POEGMA300), and covalent bonding with the pyridyl disulfide (PDS) groups, respectively. Their capacity of ICD induction, tumor antigen-capture, and antitumor immune responses were evaluated. Both the intrinsic toxicity of PEI and the ICG-mediated photothermal effect were responsible for inducing ICD. The positively charged PEI segment exhibited the best antigen-capturing ability via electrostatic interactions, promoted bone marrow-derived dendritic cell maturation and CD8+ T cell proliferation, and elicited antitumor immune responses in vivo. PDS groups bonded antigens covalently and significantly contributed to the suppression of distant tumor growth. Although the thermosensitive hydrophobic polymer segment did not contribute positively to antigen capture or tumor growth inhibition, NPs containing all of the functional modules prolonged the survival of tumor-bearing mice more than other treatments. This study provides more chemical insights into the design of polymer-based in situ nanovaccines against cancer.

Ficus carica polysaccharide extraction via ultrasound-assisted technique: Structure characterization, antioxidant, hypoglycemic and immunomodulatory activities

In this research, the ultrasound-assisted extraction (UAE) conditions of the water-soluble polysaccharide (FCPS) from Ficus carica fruits were optimized using the response surface methodology. The optimal FCPS yield was 7.97 % achieved by conducting ultrasound-assisted extraction four times at a solid–liquid ratio of 1:20 (g/mL) and an ultrasound temperature of 70 °C. Then, the structure, antioxidant properties, hypoglycemic effects, and immunomodulatory activities of FCPS were evaluated. FCPS was characterized as irregular, rough-surfaced, flaky materials consisting of pyran-type polysaccharides with α- and β-glycosidic linkages, and composed of multiple monosaccharides and only one homogeneous concentrated polysaccharide component (FCPS1) with a molecular weight of 4.224 × 104 Da. The results suggested FCPS exhibited remarkable antioxidant activity in vitro, as evidenced by improved cell viability and reduced the reactive oxygen species (ROS) levels. Meanwhile, FCPS effectively improved liver-related insulin resistance by promoting glucose consumption in hepatocytes and activated the immune response through activation of murine bone marrow-derived dendritic cells (DCs) and upregulation of interleukin 6 (IL6) and interleukin 12 (IL-12) expression. The findings demonstrate the efficacy of the UAE technique in isolating FCPS with biological functionality and FCPS could potentially serve as a beneficial organic antioxidant source and functional food, carrying important implications for future studies.

GPC3 and PEG10 peptides associated with placental gp96 elicit specific T cell immunity against hepatocellular carcinoma

The placenta and tumors can exhibit a shared expression profile of proto-oncogenes. The basis of placenta-derived heat shock protein gp96, which induces prophylactic and therapeutic T cell responses against cancer including hepatocellular carcinoma (HCC), remains unknown. Here, we identified the associated long peptides from human placental gp96 using matrix-assisted laser desorption/ionization-time-of-flight and mass spectrometry and analyzed the achieved proteins through disease enrichment analysis. We found that placental gp96 binds to numerous peptides derived from 73 proteins that could be enriched in multiple cancer types. Epitope-harboring peptides from glypican 3 (GPC3) and paternally expressed gene 10 (PEG10) were the major antigens mediating anti-HCC T cell immunity. Molecular docking analysis showed that the GPC3- and PEG10-derived peptides, mainly obtained from the cytotrophoblast layer of the mature placenta, bind to the lumenal channel and client-bound domain of the gp96 dimer. Immunization with bone marrow-derived dendritic cells pulsed with recombinant gp96-GPC3 or recombinant gp96-PEG10 peptide complex induced specific T cell responses, and T cell transfusion led to pronounced growth inhibition of HCC tumors in nude mice. We demonstrated that the chaperone gp96 can capture antigenic peptides as an efficient approach for defining tumor rejection oncoantigens in the placenta and provide a basis for developing GPC3 and PEG10 peptide-based vaccines against HCC. This study provides insight into the underlying mechanism of the antitumor response mediated by embryonic antigens from fetal tissues, and this will incite more studies to identify potential tumor rejection antigens from placenta.

Identification of the allergenic sensitizing potential of bisphenol A substitutes used in the industry.

Bisphenol (BP-)A is a chemical used in Europe to produce polycarbonate plastics and epoxy resin or as colour developer in thermal paper. Due to its toxicity, BPA presence was restricted by European regulations. Therefore, substitute chemicals are replacing BPA. To assess the allergenic sensitizing potential of 27 substitutes to BPA used in the industry. The expression of two costimulatory molecules and six cytokines were analysed by flow cytometry in mouse bone marrow-derived dendritic cells (BMDCs) exposed to the chemicals. All substances except one induced overexpression of at least one receptor and were thus identified as having allergenic sensitizing potential. Based on the BMDC model, they were classified as extreme (1 out of 27), strong (20 out of 27) and moderate (5 out of 27) sensitizers. BPA was classified as a moderate sensitizer and BPF was the only substitute classified as a non-sensitizer. The more potent substitutes induced more than 2-fold secretion of CCL3, CCL4 and/or CCL5 by dendritic cells. Most of the BPA substitutes tested in this study have an allergenic sensitizing potential; 24 of them being more potent than BPA itself. Only BPE, BPF and 2,4-BPS appeared to be weaker sensitizers than BPA.

A Novel High-Throughput Method for Identifying T-Cell Receptor: Minor Histocompatibility Antigen Interactions in Mouse Models of Graft-Vs-Host Disease

Graft-vs-host disease is a complication of allogeneic stem cell transplant (alloSCT) in which donor T cells target alloantigens expressed by healthy recipient tissues. In major-histocompatibility-matched alloSCT, CD8-mediated GVHD is caused by T cell receptor (TCR) allorecognition of class-I presented minor histocompatibility antigens (miHAs). The vast majority of miHAs remain unknown between any given donor-recipient pairing. However, determining the identities of these miHAs and their cognate TCRs would prove useful towards the development of allografts that minimize GVHD yet preserve the therapeutic benefits of alloSCT. Here, we implement a high-throughput TCR cloning method, TCXpress, to screen alloreactive TCRs from a mouse model of MHC-matched alloSCT against putative miHA targets determined informatically. 129 (H-2 b) mice, which express the immunodominant miHA H60, were lethally irradiated and reconstituted with bone marrow and CD4 + and CD8 + T cells from B6 (H-2 b) donors. At day +10, recipient spleens were harvested and CD44 +PD-1 + donor CD8 + cells were single-cell sorted according to H-2Kb:H60 tetramer (Tet H60) staining into unknown miHA-reactive (Tet H60-) and control H60-reactive (Tet H60+) cohorts for TCR cloning using the TCXpress platform (BlueSphere Bio). Clonotyping was performed by sequencing the CDR3 region of TCRs. Putative miHAs were identified informatically by filtering non-synonymous variants between B6 and 129 reference exomes on predicted class I binding affinities. To interrogate TCR target specificity, Jurkat reporter cells, which express GFP and CD69 upon TCR activation, were transduced with selected alloreactive TCRs and reacted in a multiplexed fashion against a panel of B6WT3 antigen-presenting cells (H-2 b) transduced with tandem minigenes (TMG) of predicted miHAs. To test TCR reactivity against hematopoietically-expressed miHAs independent of our calling strategy, Jurkat reporters were also reacted against bone-marrow derived dendritic cells (BMDCs) from control B6 and 129 mice. From 3 recipient mice, 966 alloreactive TCRs were cloned and sequenced (735 Tet H60- and 231 Tet H60+) corresponding to 553 unique clonotypes (437 Tet H60- and 116 Tet H60+). Sixty-seven percent (N=668) of TCRs sequenced had clonotype occurrences of ≥ 2, indicating clonal expansion post-alloSCT, whereas there were no repeated clonotypes among donor B6 CD8 + cells pre-transplant (N=173 TCRs). A total of 47 Tet H60- TCR clones and 3 Tet H60+ control TCR clones were selected for transduction into Jurkat reporters and screened against a limited TMG library of 158 putative B6→129 miHAs, as well as H60. Expectedly, all TCR clonotypes isolated from the Tet H60+ sort gate reacted against H60-expressing B6WT3 cells and 129 BMDCs. From the Tet H60- TCRs, one clone was validated against another documented 129 miHA, H4, while an additional 4 clones were found to react against novel B6→129 miHAs (Serpinb8 and Cdt1). Interestingly, 2 highly over-represented clones were autoreactive against B6 tissues (B6WT3 cells and BMDCs), but not 129 BMDCs. In summary, we demonstrate the capacity of a novel TCR cloning platform to validate informatically-predicted miHAs and their cognate TCRs in a mouse model of GVHD. This approach may be readily adapted to develop new TCR:miHA therapeutics and gain a deeper understanding of GVH responses targeting miHAs.