Presentation Of Exogenous Antigens Research Articles

A recombinant pseudorabies virus surface - displaying the classical swine fever E2 protein induces specific antibodies rapidly

Pseudorabies virus (PRV) and classical swine fever virus (CSFV) are both economically important pathogens threatening the pig industry in many countries. The triple-gene-deleted variant of PRV, herein referred to as rPRVTJ-delgE/gI/TK, has exhibited pronounced efficacy and safety profiles. This underscores its viability as a prospective vaccine vector. However, the generation of specific anti-E2 antibodies necessitates elevated immunization doses and extended durations when the extracellular domain of the E2 protein of CSFV is secreted via the recombinant rPRVTJ-delgE/gI/TK vector. To enhance the presentation of exogenous antigens by antigen-presenting cells (APCs), we engineered the E2 protein expressed on the surface of PRV particles in this study. The recombinant virus expressing the E2 protein with a heterogonous transmembrane domain was generated in the backbone of rPRVTJ-delgE/gI/TK and designated as rPRVTJ-UL44-E2. The E2 gene was fused to the 3′ terminus of the UL44 gene utilizing P2A, a self-cleaving peptide sequence. The electron microscopy showed that the E2 protein was anchored on the surface of the viral particles of rPRVTJ-delgE/gI/TK-E2. The insertion of the E2 gene did not alter the native biological characteristics of the viral vector. Rabbits immunized with 107 median tissue culture infective doses (TCID50) of rPRVTJ-UL44-E2 exhibited a rapid seroconversion to anti-E2 specific antibodies within 7 days post-immunization (dpi). All the rabbits immunized with the rPRVTJ-UL44-E2 had generated antibodies specific to E2 prior to the administration of the booster immunization. However, the immunized rabbits were not protected from the CSFV C-strain challenge. Nevertheless, this strategy has notably achieved rapid induction of E2-specific non-neutralizing antibodies. These findings provide insights that the design of rPRVTJ-UL44-E2 requires optimization, thereby indicating a promising avenue for augmenting vaccine-induced immune responses.

Abstract 2457: Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma

Abstract The median survival time for newly diagnosed patients with glioblastoma (GBM) is 15 months, highlighting the need for improved therapies. Current treatment for newly diagnosed and recurrent GBM consists of tumor resection and chemoradiation therapy. The immunosuppressive tumor microenvironment (TME) is a central obstacle in the treatment of GBM. Lack of NK cell and CD8+ T cell infiltration prevents efficacy with immunotherapies like immune checkpoint blockade (ICB).One approach our lab is pursuing to enhance tumor infiltrating lymphocytes is through harnessing the anti-viral cGAS-STING pathway. Upon cGAS activation by cytosolic double stranded DNA, the TBK1 kinase is phosphorylated, initiating a subsequent type I interferon (IFN) response and the recruitment of immune cells. This pathway is commonly altered in many cancer types to evade immune activation. Our lab has demonstrated survival benefits with STING agonist treatment in immunocompetent GBM mouse models. We have also shown strong STING localization in the vasculature, implicating endothelial cells as a potential treatment target for anti-tumor efficacy. Here we investigated the potential role of tumor vasculature in mediating the effects of STING agonists in GBM therapy.We have shown robust cGAS-STING activation in brain endothelial cell lines through enhanced secretion of CXCL10, IFN-β, and RANTES upon STING agonist treatment in vitro. Western blotting also shows pathway activation in response to treatment through increased phospho-TBK1 levels. Activation was further potentiated by manganese.To investigate the effects of STING activation in brain endothelial cells in detail, we performed RNA sequencing. This showed 262 differentially expressed genes in response to pharmacologic cGAS-STING activation, with 183 of these being upregulated. The top gene ontology terms enriched include type I interferon signaling pathway, defense response to virus, and antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-independent. In addition, gene expression of tight junction protein Claudin 5 was also enhanced. We have also shown increased resistance in response to activation, through transendothelial electrical resistance (TEER) assays. Taken together, these results suggest that cGAS-STING activation in endothelial cells leads to tight junction remodeling in addition to immunomodulatory changes, implicating roles for endothelial cells in barrier function and immune cell interactions respectively. Current research in our lab focuses on elucidating the mechanisms behind these changes. Our goal is to identify beneficial endothelial cell changes which promote anti-tumor immunity in order to inform future treatment strategies. We are currently performing studies aimed at demonstrating that cGAS-STING activation in endothelial cells is critical for the efficacy of STING agonist treatment. Citation Format: Andrea Schmidt, Sandra Remson, Philippa Vaughn-Beaucaire, Jorge L. Jimenez-Macias, Praveen Srinivasan, Maximillian Pinho-Schwermann, Sean Lawler. Brain derived endothelial cells as a mediator of cGAS-STING driven anti-tumor efficacy in glioblastoma [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 2457.

Preparation of glycopeptide-modified pH-sensitive liposomes for promoting antigen cross-presentation and induction of antigen-specific cellular immunity.

Cross-presentation, exogenous antigen presentation onto major histocompatibility complex class I molecules on antigen presenting cells, is crucially important for inducing antigen-specific cellular immune responses for cancer immunotherapy and for the treatment of infectious diseases. One strategy to induce cross-presentation is cytosolic delivery of an exogenous antigen using fusogenic or endosomolytic molecule-introduced nanocarriers. Earlier, we reported liposomes modified with pH-responsive polymers to achieve cytosolic delivery of an antigen. Polyglycidol-based or polysaccharide-based pH-responsive polymers can provide liposomes with delivery performance of antigenic proteins into cytosol via membrane fusion with endosomes responding to acidic pH, leading to induction of cross-presentation. Mannose residue was introduced to pH-responsive polysaccharides to increase uptake selectivity to antigen presenting cells and to improve cross-presentation efficiency. However, direct introduction of mannose residue into pH-responsive polysaccharides suppressed cytoplasmic delivery performance of liposomes. To avoid such interference, for this study, mannose-containing glycans were incorporated separately into pH-responsive polysaccharide-modified liposomes. Soybean agglutinin-derived glycopeptide was used as a ligand for lectins on antigen presenting cells. Incorporation of glycopeptide significantly increased the cellular uptake of liposomes by dendritic cell lines and increased cross-presentation efficiency. Liposomes incorporated both glycopeptide and pH-responsive polysaccharides exhibited strong adjuvant effects in vitro and induced the increase of dendritic cells, M1 macrophages, and effector T cells in the spleen. Subcutaneous administration of these liposomes induced antigen-specific cellular immunity, resulting in strong therapeutic effects in tumor-bearing mice. These results suggest that separate incorporation of glycopeptides and pH-responsive polysaccharides into antigen-loaded liposomes is an effective strategy to produce liposome-based nanovaccines to achieve antigen cross-presentation and induction of cellular immunity towards cancer immunotherapy.

MicroRNA transcriptome analysis reveals the immune regulatory mechanism of Crassostrea hongkongesis against Vibrio harveyi infection

MicroRNAs (miRNAs) are small non-coding RNA molecules that modulate target-genes expression and play crucial roles in post-transcriptional regulation and immune system regulation. The Hong Kong oyster (Crassostrea hongkongesis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen host interaction. Vibrio harveyi, a Gram negative luminescent marine bacterium, is widely distributed in coastal water environments and can cause large-scale death of C. hongkongesis. However, little in formation is available on the immune regulatory mechanisms of C. hongkongesis infected with V. harveyi. Therefore, we performed microRNA transcriptome analysis for elucidating the immunoregulation mechanism of C. hongkongesis infected with V. harveyi. The results show that a total of 308468208 clean reads and 288371159 clean tags were obtained. 222 differentially expressed miRNAs were identified. A total of 388 target genes that were differentially expressed and negatively correlated with miRNA expression were predicted by 222 DEmiRs. GO enrichment analysis of 388 DETGs showed that they were mainly enriched in the immune-related term of membrane-bounded vesicle, endocytic vesicle lumen, antigen processing and presentation of exogenous peptide antigen via MHC class I, antigen processing and presentation of peptide antigen via MHC class I, and other immune-related term. KEGG enrichment analysis showed that DETGs were mainly enriched in the Complement and coagulation cascades, Herpes simplex virus 1 infection, Bacterial invasion of epithelial cells, Antigen processing and presentation and NOD-like receptor signaling pathway. The 16 key DEmiRs and their target genes form a regulatory network for seven immune-related pathways. These results suggest that V. harveyi infection induces a complex miRNA response with wide-ranging effects on immune gene expression in the C. hongkongesis. This study explored the immune response of C. hongkongesis to V. harveyi infection at the level of miRNAs, which provides new ideas for the healthy culture and selective breeding of C. hongkongesis.

Controlling Antigen Fate in Therapeutic Cancer Vaccines by Targeting Dendritic Cell Receptors.

Antigen-presenting cells (APCs) orchestrate immune responses and are therefore of interest for the targeted delivery of therapeutic vaccines. Dendritic cells (DCs) are professional APCs that excel in presentation of exogenous antigens toward CD4+ T helper cells, as well as cytotoxic CD8+ T cells. DCs are highly heterogeneous and can be divided into subpopulations that differ in abundance, function, and phenotype, such as differential expression of endocytic receptor molecules. It is firmly established that targeting antigens to DC receptors enhances the efficacy of therapeutic vaccines. While most studies emphasize the importance of targeting a specific DC subset, we argue that the differential intracellular routing downstream of the targeted receptors within the DC subset should also be considered. Here, we review the mouse and human receptors studied as target for therapeutic vaccines, focusing on antibody and ligand conjugates and how their targeting affects antigen presentation. We aim to delineate how targeting distinct receptors affects antigen presentation and vaccine efficacy, which will guide target selection for future therapeutic vaccine development.

Salmonella delivers H9N2 influenza virus antigens via a prokaryotic and eukaryotic dual-expression vector and elicits bivalent protection against avian influenza and fowl typhoid

The H9N2 avian influenza virus significantly affects the health of poultry and humans. We identified a prokaryotic and eukaryotic dual-expression vector system, pJHL270, that can provide simultaneous MHC class I and II stimulation of the host immune system, and we designed vaccine antigens by selecting the consensus HA1 sequence and M2e antigens from H9N2 virus circulating in South Korea from 2000 to 2021. The genes were cloned into the pJHL270 vector, and the cloned plasmid was delivered by a live-attenuated Salmonella Gallinarum (SG) strain. The immunity and protective efficacy of the SG-based H9N2 vaccine construct, JOL2922, against avian influenza and fowl typhoid (FT) were evaluated. The Ptrc and CMV promoters conferred antigen expression in prokaryotic and eukaryotic cells to induce balanced Th-1/Th-2 immunity. Chickens immunized with JOL2922 yielded high antigen-specific humoral and mucosal immune responses. qRT-PCR revealed that the strain generated polyfunctional IFN-γ and IL-4 secretion in immunized chickens. Furthermore, a FACS analysis showed increased CD3CD4+ and CD3CD8+ T-cell subpopulations following immunization. Peripheral Blood Mononuclear Cells (PBMCs) harvested from the immunized chickens significantly increased MHC class I and II expression, 3.5-fold and 2.5-fold increases, respectively. Serum collected from the immunized groups had an evident hemagglutinin inhibition titer of ≥6 log2. Immunization reduced the lung viral titer by 3.8-fold within 5 days post-infection. The strain also generated SG-specific humoral and cellular immune responses. The immunized birds all survived a virulent SG wild-type challenge. In addition, the bacterial burden was reduced by 2.7-fold and 2.1-fold in spleen and liver tissue, respectively, collected from immunized chickens. Our data indicate that an attenuated SG strain successfully delivered the dual-expression vector system and co-stimulated MHC class I and II antigen presentation pathways via exogenous and endogenous antigen presentation, thereby triggering a balanced Th-1/Th-2-based immune response and conferring effective protection against avian influenza and FT.

Gene signatures for predicting the response to immune checkpoint inhibitor by integrating bulk and single-cell sequence data in patients with bladder cancer.

535 Background: Although immune checkpoint inhibitor (ICI) is regarded as a breakthrough in bladder cancer (BC) therapy, only a limited fraction of patients benefit from it. Thus, we aim to establish gene signature (iGS) and develop a model that predicts the response of ICI for the patients with BC. Methods: We collected bulk RNA expression data from BC cohort (GSE176307) receiving ICI and single-cell sequencing data from patients with BC (GSE135337). Single-sample gene set enrichment (ssGSEA) analysis was used to develop predictive iGS. Clinical benefit of ICI was defined as a complete or partial response or stable disease at week 24. The gene ontology (GO) and pathway enrichment analysis were established through DAVID database. Logistic regression model was constructed with iGS and clinical factors. The final model was validated by the K-fold cross validation technique. Results: We integrated bulk RNA expression data and single-cell sequencing data, and identified subset of cells (N= 157) that were associated with the overall survival (OS) of patients receiving ICI. These cells were compared with all other cells, and 85 up-regulated differentially expressed genes (DEGs) were identified. DEGs were mainly enriched in 'antigen processing and presentation of exogenous peptide antigen via MHC class II', 'peptide antigen assembly with MHC class II protein complex' and 'immune response' in the biological process enrichment analysis. High iGS score, that was derived from ssGSEA, was significantly associated with longer OS in multivariate Cox regression analysis (Hazard ratio: 0.40, P = 0.02) Final prediction model for clinical benefit included iGS score [odds ratio (OR) = 3.89], tumor mutation burden (OR = 9.8) and age (OR = 0.93) with accuracy of 0.80 and an area under the receiver operator characteristic curve (AUROC) of 0.77. Conclusions: We showed the potential of iGS from bulk and single cell RNA sequencing for predicting clinical benefit of ICI in BC.

IFN-γR/STAT1 signaling in recipient hematopoietic antigen-presenting cells suppresses graft-versus-host disease.

The absence of IFN-γ receptor (IFN-γR) or STAT1 signaling in donor cells has been shown to result in reduced induction of acute graft-versus-host disease (GVHD). In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFN-γR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. LPS-matured, BM-derived Ifngr1-/- Stat1-/- DCs (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human antigen-presenting cells (APCs) with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFN-γR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens, while promoting the presentation of endogenous antigens. Thus, the indirect presentation of host antigens to donor lymphocytes was defective in IFN-γR/STAT1-deficient, donor-derived APCs in fully donor chimeric mice. The differential effects of IFN-γR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signaling pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.

KIF5A upregulation in hepatocellular carcinoma: A novel prognostic biomarker associated with unique tumor microenvironment status.

Liver hepatocellular carcinoma (LIHC) is one of the most common liver malignancies with high mortality and morbidity. Thus, it is crucial to identify potential biomarker that is capable of accurately predicting the prognosis and therapeutic response of LIHC. Kinesin family member 5A (KIF5A) is a microtubule-based motor protein involved in the transport of macromolecules such as organelle proteins in cells. Recent studies have illustrated that the high expression of KIF5A was related to poor prognosis of solid tumors, including bladder cancer, prostate cancer, and breast cancer. However, little is currently known concerning the clinical significance of KIF5A expression in LIHC. Herein, by adopting multi-omics bioinformatics analysis, we comprehensively uncovered the potential function and the predictive value of KIF5A in stratifying clinical features among patients with LIHC, for which a high KIF5A level predicted an unfavorable clinical outcome. Results from KIF5A-related network and enrichment analyses illustrated that KIF5A might involve in microtubule-based process, antigen processing and presentation of exogenous peptide antigen via MHC class II. Furthermore, immune infiltration and immune function analyses revealed upregulated KIF5A could predict a unique tumor microenvironment with more CD8+T cells and a higher level of anti-tumor immune response. Evidence provided by immunohistochemistry staining (IHC) further validated our findings at the protein level. Taken together, KIF5A might serve as a novel prognostic biomarker for predicting immunotherapy response and could be a potential target for anti-cancer strategies for LIHC.

Endothelial Cells Activate Alloreactive CD4+ T Cells to Initiate Acute Graft-Versus-Host Disease

Allogeneic donor T cells can exert an anti-leukemic response (GvL effect) after hematopoietic cell transplantation (allo-HCT). However, allogeneic donor T cells can also cause life-threatening acute graft-versus-host disease (aGvHD). Therefore, donor T cell priming is considered as a crucial event determining allo-HCT outcome. In aGvHD, donor CD4+ T cells are activated by antigen presenting cells (APCs), proliferate, differentiate and migrate to cause tissue damage. However, non-hematopoietic APCs have been shown to drive aGvHD pathology, making hematopoietic APCs dispensable in the initiation of aGvHD (Toubai et al., Blood 2012, Koyama et al., Nat Med 2012). During the aGvHD initiation phase allogenic T cells exclusively migrate to secondary lymphoid organs (SLOs) before they enter target tissues in the aGvHD effector phase (Beilhack et al., Blood 2005, Beilhack et al., Blood 2008). These observations led us to investigate the role of MHC class II mediated antigen presentation by non-hematopoietic lymph node (LN) stromal (SCs) and endothelial cells (ECs) in aGvHD. Recently we learned that alloreactive CD4+ T cells are activated in SLOs by non-hematopoietic APCs independently from Ccl19 expressing SCs, while these SCs provide protective niches to donor Tregs mitigating GvHD. Consequently, here we explored MHCII mediated antigen presentation by SLO blood endothelial cells (BECs) and lymph endothelial cells (LECs). BECs and LECs expressed endogenous MHCII, costimulatory receptors CD80/CD86 and acquired MHCII from dendritic cells via trogocytosis. Upon myeloablative irradiation, BECs and LECs upregulated CD80 and CD86 whereas MHCII expression decreased upon loss of radiosensitive host DCs. scRNA-seq revealed, BECs and LECs subsets expressing genes involved in MHCII antigen presentation (e.g. Cd74, Ctss, Ctsl, Tap2, H2-Aa, H2-Ab1, H2-Eb1) leading to exogenous antigen presentation (DQ-OVA) by EC subsets. To elucidate MHCII antigen presentation in ECs we crossed Cdh5-Cre to H2-Ab1fl mice to conditionally knockout MHCII on ECs (MHCIIΔCdh5). Allo-HCT in MHCIIΔCdh5 recipients utilizing an MHC major mismatched model (FVB→B6) did not alter allogeneic CD4+ T cell activation during aGvHD initiation. However, MHCIIΔCdh5 showed marginally reduced aGvHD and better survival. Notably, MHCIIΔCdh5 recipients retained functional hematopoietic APCs presenting alloantigens via MHCII. Therefore, we generated bone marrow (BM) chimeras by syngeneic (syn) HCT from MHCII-deficient (MHCIIΔ) donors into B6.WT mice. After allo-HCT of these BM chimeras, donor CD4+ T cells got activated in SLOs during the aGvHD initiation phase. However, to our surprise also syngeneic BM chimeric animals spontaneously developed GvHD-like symptoms making them unsuitable for studying MHCII mediated non-hematopoietic antigen presentation after allo-HCT. This striking phenotype could be explained by a defective CD44hiCD62Llow CD4+ T cell memory development in MHCIIΔ BM chimeras (Huang et al., J Immunol. 2014). Utilizing our scRNA seq dataset we identified Tie2 and Vav1 to be expressed on cells of hematopoietic lineage, however Tie2 expression was observed on ECs of the LNs. Therefore, we generated MHCIIΔVav1 mice by crossing Vav1-Cre to H2-Ab1fl. CD45- LNs cells from these animals activated CD4+ T cell in mixed lymphocyte reaction as well as in vivo. MHCIIΔVav1 mice developed less aGvHD with delayed kinetics. Subsequently, we surgically transplanted LNs from syngeneic MHCIIΔVav1 mice into complete MHCIIΔ mice. After MHCIIΔVav1 LN engraftment, these MHCIIΔ recipients underwent allo-HCT with allogenic CD4+ T cells. Alloreactive donor CD4+ T cells expressed more IFNγ and TNFα compared to controls. These experiments proved that non-hematopoietic cells in LNs can activate alloreactive CD4+ T cells to initiate aGvHD. Ultimately, we crossed MHCIIΔCdh5 to MHCIIΔVav1, resulting in mice deficient for MHCII in the entire hematopoietic and endothelial compartments. After allo-HCT MHCIIΔVav1ΔCdh5 were significantly better protected from aGvHD than littermate controls, revealing a previously unidentified role of ECs in initiating CD4+ T cell mediated aGvHD. Based on these findings we propose ECs as important non-hematopoietic APCs to activate alloreactive CD4+ T cells to trigger aGvHD suited for therapeutic interventions after allo-HCT.

Identification of candidate blood biomarkers for the diagnosis of septicaemic melioidosis based on WGCNA

Melioidosis is an infectious disease caused by Burkholderia pseudomallei (Bp), a gram-negative bacillus. Sepsis is the most prevalent type of melioidosis. Due to factors such as lack of precision and slow presentation of bacterial culture tests, the misdiagnosis rate could exceed 100 per cent. Therefore, more reliable, and adaptable diagnostic methods are urgently needed. Weighted gene co-expression network analysis (WGCNA) was employed to screen the featured modules specially expressed in sepsis patients caused by Bp. Two representative co-expression modules were selected to perform gene ontology(GO) and KEGG analysis using ClusterProfiler package based on R language. We found that antigen processing and presentation of exogenous peptide antigen via MHC class I pathway, cytosol to ER transport and cell killing related pathways enriched in darkmagenta module which significantly correlated with the sepsis caused by Bp. Eventually, a diagnostic 6-mRNA signature consisting of ASPHD2, LAP3, SEPT4, FAM26F, WARS and LGALS3BP was identified, which could discern the sepsis caused by Bp compared with other organisms. This will provide a new insight in screening markers for early detection of sepsis caused by Bp, and the interaction between pathogens and hosts. This should shed light on the early detection of Bp-caused infectious diseases.

Effects of RAGE Deletion on the Cardiac Transcriptome during Aging.

Cardiac aging is characterized by increased cardiomyocyte hypertrophy, myocardial stiffness, and fibrosis, which enhance cardiovascular risk. The receptor for advanced glycation end-products (RAGE) is involved in several age-related diseases. RAGE knockout (Rage−/−) mice show an acceleration of cardiac dimension changes and interstitial fibrosis with aging. This study identifies the age-associated cardiac gene expression signature induced by RAGE deletion. We analyzed the left ventricle transcriptome of 2.5-(Young), 12-(Middle age, MA), and 21-(Old) months-old female Rage−/− and C57BL/6N (WT) mice. By comparing Young, MA, and Old Rage−/− versus age-matched WT mice, we identified 122, 192, and 12 differently expressed genes, respectively. Functional inference analysis showed that RAGE deletion is associated with: (i) down-regulation of genes involved in antigen processing and presentation of exogenous antigen, adaptive immune response, and cellular responses to interferon beta and gamma in Young animals; (ii) up-regulation of genes related to fatty acid oxidation, cardiac structure remodeling and cellular response to hypoxia in MA mice; (iii) up-regulation of few genes belonging to complement activation and triglyceride biosynthetic process in Old animals. Our findings show that the age-dependent cardiac phenotype of Rage−/− mice is associated with alterations of genes related to adaptive immunity and cardiac stress pathways.

Resting and Activated DCs Present Exogenous Antigens to CD4 T Cells by Distinct Antigen Processing Pathways

Activation of immature DCs by pathogens results in an APC that is a highly efficient stimulator of naïve CD4 T cells. Curiously, even fully-mature DCs can efficiently process and present antigenic peptide to CD4 T cells, however the mechanism used by mature DCs to function as APCs and whether these mechanisms are identical in immature DC and mature DC is not known. We now show that that immature and mature DCs differ use different mechanisms to present distinct epitopes of the influenza virus hemagglutinin (HA) protein to T cells. Immature DCs use newly-synthesized MHC-II targeted to DM+ late endosomal antigen processing compartments for presentation to HA-specific T cells. By contrast, mature DCs use recycling MHC-II that enters Rab11a+ early endosomes to process and present a distinct HA epitope to different HA-specific T cells. Rab11a knock-down inhibits recycling of pMHC-II and inhibits presentation of a “recycling endosome” epitope of HA from influenza virus internalized in mature DCs. These studies highlight a “division of labor” in DCs in which immature DCs preferentially present antigens acquired in late endosomes on MHC-II but mature DCs use recycling pMHC-II to efficiently present antigenic peptides acquired in early endosomes for CD4 T cell activation.

Bioinspired vaccines to enhance MHC class-I antigen cross-presentation.

Cross-presentation of exogenous antigen on MHC class-I is a crucial process for generating a CD8+ T cell response, and is therefore an important design consideration in the development of T-cell-engaging vaccines against viruses, intracellular bacteria, and cancers. Here, we briefly summarize known cross-presentation pathways and highlight how synthetic vaccines can be engineered to enhance MHC-I presentation of exogenous peptide and protein antigens by professional antigen-presenting cells (APCs). In particular, we summarize how molecular engineering and nanotechnology are being harnessed to enhance antigen delivery to lymph nodes and to cross-presenting dendritic cells, to bypass endosomal trafficking of exogenous antigen to promote delivery of antigen to the cytosol of APCs, and to coordinate the delivery of antigen withimmune-stimulating adjuvants that can act synergistically to augment antigen cross-presentation.

Pyroptosis-Related Gene to Construct Prognostic Signature and Explore Immune Microenvironment and Immunotherapy Biomarkers in Bladder Cancer.

Bladder cancer is known to be the most common malignant tumor in the urinary system and has a poor prognosis; thus, new targets for drug treatment are urgently needed. Pyroptosis is defined as programmed cell death in the inflammatory form mediated by the gasdermin protein. It has therapeutic potential due to the synergistic effect of radiotherapy and chemotherapy, can reverse chemotherapy resistance, is able to regulate the body environment to alter tumor metabolism, and may enhance the response rate of the immune checkpoint inhibitor. Accordingly, this study attempted to explore the role of pyroptosis in bladder cancer. A prognostic model based on five pyroptosis-related genes was constructed by conducting univariate Cox survival and LASSO regression analyses using The Cancer Genome Atlas (TCGA) cohort. Patients were divided into high- and low-risk groups according to the median risk score, with all five PRGs having downregulated expression in the high-risk group. The high-risk group was shown to have a worse prognosis than the low-risk group, and survival differences between the two groups were then validated in the Gene Expression Omnibus (GEO) cohort. Moreover, the ROC curves demonstrated the model’s moderate predictive ability. The univariate and multivariate Cox regression analyses indicated that risk scores were found to serve as an independent prognosis factor for OS in bladder cancer patients. In addition, the high-risk group was observed to be associated with advanced N and TNM stages. A nomogram combining risk scores and clinical features was then established, with the ROC curve indicating that the AUC of TCGA training cohort in 3 and 5 years was 0.789 and 0.775, respectively. The calibration curve exhibited a high consistency between the actual survival rate and the predicted rate. Furthermore, the GO and KEGG analyses found that antigen processing and presentation of exogenous antigen, exogenous peptide antigen, and peptide antigen were enriched in the low-risk group. A higher abundance of tumor-infiltrating immune cells and additional active immune pathways were also noted in the low-risk group. In addition, immunotherapy biomarkers, including TMB, PD1, PD-L1, CTLA4, and LAG3, were shown to have higher levels in the low-risk group. Therefore, patients in the low-risk group may be potential responders to immune checkpoint inhibitors.

Exploring the Action Mechanism of Yadanzi (Brucea javanica) in the Treatment of Glioblastoma Based on Bioinformatics and Network Pharmacology

Abstract Objective The aim of the study is to explore the molecular mechanism of Yadanzi (Brucea javanica) in the treatment of glioblastoma (GBM) by using the methods of bioinformatics and network pharmacology. Methods The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature retrieval method were applied to obtain the active ingredients of Yadanzi (Brucea javanica), and to predict the relevant targets of the active ingredients. The GBM-related targets were retrieved and screened through the Gene Expression Profiling Interactive Analysis (GEPIA) database, and mapped to each other with the targets of the components of Yadanzi (Brucea javanica) to obtain the intersection targets. The GBM differentially expressed gene targets were imported into the String database to obtain the protein interaction relationship, the Cytoscape software was used to draw the protein interaction network, the Cytobba and MCODE plug-ins were used to screen the core genes and important protein interaction modules, and the GEPIA database was applied to make survival analysis of the core genes. The network map of “active ingredients-targets” was constructed through the Cytoscape 3.6.1 software. Gene Ontology (GO) biological function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis for GBM differentially expressed genes were performed through the DAVID database. Results Through TCMSP and literature retrieval, 23 potential active ingredients and 129 related targets were obtained from Yadanzi (Brucea javanica). In the GEPIA database, 247 GBM differentially expressed genes were screened, including 113 up-regulated genes and 134 downregulated genes. After mapping with the targets related to the active ingredients of Yadanzi (Brucea javanica), six intersection targets were obtained, that is, the potential action targets of Yadanzi (Brucea javanica) in treating GBM, including MMP2, HMOX1, BIRC5, EGFR, CCNB2, and TOP2A. Cytoscape software was applied to build an “active ingredient-action target” network. Two active ingredients and five action targets of β-sitosterol (BS) and luteolin were found, and the targets were mainly concentrated in BS. It was found by KEGG pathway enrichment analysis that GBM differentially expressed genes were mainly involved in signaling pathways related to Staphylococcus aureus infection, phagosome formation, tuberculosis and systemic lupus erythematosus and other infectious and autoimmune diseases. It was found by GO enrichment analysis that the GBM differentially expressed genes mainly involved such biological processes (BP) as the processing and presentation of exogenous antigenic peptides and polysaccharide antigens through MHC II molecules, γ-interferon-mediated signaling pathways, extracellular matrix composition, and chemical synapses transmission; it involved cellular components such as cell junctions, axon terminal buttons, extracellular space, vesicle membranes for endocytosis, and MHC II protein complexes; molecular functions such as calcium-mediated ionic protein binding, MHC II molecular receptor activity, immunoglobulin binding, and phospholipase inhibitor activity were also involved. Survival analysis was conducted by GEPIA on the top 37 core targets in degree value, and a total of five genes related to GBM prognosis were obtained. Among them, FN1 and MMP2 were highly expressed while GABRD (γ-aminobutyric acid A receptor delta subunit), RBFOX1, and SLC6A7 were expressed at a low level in cancer patients. Conclusion The pathogenesis of GBM is closely related to the human immune system, and BS and luteolin may be the main material basis of Yadanzi (Brucea javanica) for the treatment of GBM and the improvement of prognosis. The molecular mechanism may be related to the physical barrier formed by destroying the tumor cell stromal molecules and its involvement in tumor immune response.

Dendritic cells can prime anti-tumor CD8+ T cell responses through major histocompatibility complex cross-dressing

Antigen cross-presentation, wherein dendritic cells (DCs) present exogenous antigen on major histocompatibility class I (MHC-I) molecules, is considered the primary mechanism by which DCs initiate tumor-specific CD8+ Tcell responses. Here, we demonstrate that MHC-I cross-dressing, an antigen presentation pathway in which DCs acquire and display intact tumor-derived peptide:MHC-I molecules, is also important in orchestrating anti-tumor immunity. Cancer cell MHC-I expression was required for optimal CD8+ Tcell activation in two subcutaneous tumor models. Invivo acquisition of tumor-derived peptide:MHC-I molecules by DCs was sufficient to induce antigen-specific CD8+ Tcell priming. Transfer of tumor-derived human leukocyte antigen (HLA) molecules to myeloid cells was detected invitro and in human tumor xenografts. In conclusion, MHC-I cross-dressing is crucial for anti-tumor CD8+ Tcell priming by DCs. In addition to quantitatively enhancing tumor antigen presentation, MHC cross-dressing might also enable DCs to more faithfully and efficiently mirror the cancer cell peptidome.

The Role of Type-2 Conventional Dendritic Cells in the Regulation of Tumor Immunity.

Simple SummaryRecent studies revealed that type-2 conventional dendritic cells (cDC2s) play an important role in antitumor immunity by promoting cytotoxic T-cell responses and helper T-cell differentiation. This review outlines the role of cDC2s in tumor immunity and summarizes the latest progress regarding their potential in cancer vaccination and cDC2-targeted cancer immunotherapy.Conventional dendritic cells (cDCs) orchestrate immune responses to cancer and comprise two major subsets: type-1 cDCs (cDC1s) and type-2 cDCs (cDC2s). Compared with cDC1s, which are dedicated to the activation of CD8+ T cells, cDC2s are ontogenically and functionally heterogeneous, with their main function being the presentation of exogenous antigens to CD4+ T cells for the initiation of T helper cell differentiation. cDC1s play an important role in tumor-specific immune responses through cross-presentation of tumor-derived antigens for the priming of CD8+ T cells, whereas little is known of the role of cDC2s in tumor immunity. Recent studies have indicated that human cDC2s can be divided into at least two subsets and have implicated these cells in both anti- and pro-tumoral immune responses. Furthermore, the efficacy of cDC2-based vaccines as well as cDC2-targeted therapeutics has been demonstrated in both mouse models and human patients. Here we summarize current knowledge about the role of cDC2s in tumor immunity and address whether these cells are beneficial in the context of antitumor immune responses.

Efficient Cholesterol Transport in Dendritic Cells Defines Optimal Exogenous Antigen Presentation and Toxoplasma gondii Proliferation.

Dendritic cells are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway to stimulate CD4+ T cells, or with MHC-I to activate CD8+ T lymphocytes through the cross-presentation pathway. DCs represent one of the main cellular targets during infection by Toxoplasma gondii. This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, T. gondii modulates the host immune response through multiple interactions with proteins and lipids. Cholesterol is an important cellular component that regulates cellular physiology at the structural and functional levels. Although different studies describe the relevance of cholesterol transport for exogenous antigen presentation, the molecular mechanism underlying this process is not defined. Here, we focus our study on the inhibitor U18666A, a drug widely used to arrest multivesicular bodies biogenesis that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of T. gondii-associated antigens and parasite proliferation were hampered in treated cells. However, neither antigen uptake nor BMDC viability was significantly affected by the U18666A treatment. By contrast, this drug altered the transport of MHC-I and MHC-II molecules to the plasma membrane. Since U18666A impairs the formation of MVBs, we analyzed in T. gondii infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by T. gondii. Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. Altogether, our results highlight the importance of cholesterol trafficking and MVBs formation in DCs for optimal antigen presentation and T. gondii proliferation.

The ABCs of Antigen Presentation by Stromal Non-Professional Antigen-Presenting Cells.

Professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, are known for their ability to present exogenous antigens to T cells. However, many other cell types, including endothelial cells, fibroblasts, and lymph node stromal cells, are also capable of presenting exogenous antigens to either CD8+ or CD4+ T cells via cross-presentation or major histocompatibility complex (MHC) class II-mediated presentation, respectively. Antigen presentation by these stromal nonprofessional APCs differentially affect T cell function, depending on the type of cells that present the antigen, as well as the local (inflammatory) micro-environment. It has been recently appreciated that nonprofessional APCs can, as such, orchestrate immunity against pathogens, tumor survival, or rejection, and aid in the progression of various auto-immune pathologies. Therefore, the interest for these nonprofessional APCs is growing as they might be an important target for enhancing various immunotherapies. In this review, the different nonprofessional APCs are discussed, as well as their functional consequences on the T cell response, with a focus on immuno-oncology.