Antigen Presentation Research Articles

The Neurolipid Atlas: a lipidomics resource for neurodegenerative diseases uncovers cholesterol as a regulator of astrocyte reactivity impaired by ApoE4.

Lipid changes in the brain have been implicated in many neurodegenerative diseases including Alzheimer's Disease (AD), Parkinson's disease and Amyotrophic Lateral Sclerosis. To facilitate comparative lipidomic research across brain-diseases we established a data commons named the Neurolipid Atlas, that we have pre-populated with novel human, mouse and isogenic induced pluripotent stem cell (iPSC)-derived lipidomics data for different brain diseases. We show that iPSC-derived neurons, microglia and astrocytes display distinct lipid profiles that recapitulate in vivo lipotypes. Leveraging multiple datasets, we show that the AD risk gene ApoE4 drives cholesterol ester (CE) accumulation in human astrocytes recapitulating CE accumulation measured in the human AD brain. Multi-omic interrogation of iPSC-derived astrocytes revealed that cholesterol plays a major role in astrocyte interferon-dependent pathways such as the immunoproteasome and major histocompatibility complex (MHC) class I antigen presentation. We show that through enhanced cholesterol esterification ApoE4 suppresses immune activation of astrocytes. Our novel data commons, available at neurolipidatlas.com, provides a user-friendly tool and knowledge base for a better understanding of lipid dyshomeostasis in neurodegenerative diseases.

Respiratory infection- and asthma-prone, low vaccine responder children demonstrate distinct mononuclear cell DNA methylation pathways

BackgroundInfants with frequent viral and bacterial respiratory infections exhibit compromised immunity to routine immunizations. They are also more likely to develop chronic respiratory diseases in later childhood. This study investigated the feasibility of epigenetic profiling to reveal endotype-specific molecular pathways with potential for early identification and immuno-modulation. Peripheral blood mononuclear cells from respiratory infection allergy/asthma-prone (IAP) infants and non-infection allergy/asthma prone (NIAP) were retrospectively selected for genome-wide DNA methylation and single nucleotide polymorphism analysis. The IAP infants were enriched for the low vaccine responsiveness (LVR) phenotype (Fisher's exact p-value = 0.02).ResultsAn endotype signature of 813 differentially methylated regions (DMRs) comprising 238 lead CpG associations (FDR < 0.05) emerged, implicating pathways related to asthma, mucin production, antigen presentation and inflammasome activation. Allelic variation explained only a minor portion of this signature. Stimulation of mononuclear cells with monophosphoryl lipid A (MPL), a TLR agonist, partially reversed this signature at a subset of CpGs, suggesting the potential for epigenetic remodeling.ConclusionsThis proof-of-concept study establishes a foundation for precision endotyping of IAP children and highlights the potential for immune modulation strategies using adjuvants for future investigation.

Photodynamic Therapy Synergizes CD47 Blockade Strategy for Enhanced Antitumor Therapy.

The antitumor strategies based on innate immunity activation have become favored by researchers in recent years. In particular, strategies targeting antiphagocytic signaling blockade to enhance phagocytosis have been widely reported. For example, the addition of prophagocytic signals such as calreticulin could make the strategy significantly more effective. In this study, an antitumor strategy that combines photodynamic therapy (PDT) with CD47 blockade has been reported. This approach promotes the maturation of dendritic cells and the presentation of tumor antigens by PDT-mediated tumor immunogenic cell death, as well as the enhancement of cytotoxic T lymphocyte infiltration in tumor areas and the phagocytic activity of phagocytes. Furthermore, the downregulation and blockage of CD47 protein could further promote phagocytic activity, strengthen the innate immune system, and ultimately elevate the antitumor efficacy and inhibit tumor metastasis.

Integrative Analysis Identified rs907091 at the miRNA Binding Region of the IKZF3 Gene as a Potential Functional Variant for Rheumatoid Arthritis

Objective: Previous studies have identified a large number of associations between genetic variants and rheumatoid arthritis (RA), but the functional mechanisms underlying the associations are largely unknown. Methods: Based on publicly available datasets and results, we explored the functional mechanisms underlying the associations between regulatory polymorphisms in miRNA target sites (poly-miRTSs) and RA by combining integrative analyses (expression quantitative trait locus analysis, eQTL, and gene enrichment analysis) with in-house molecular experiments (allelic expression imbalance experiment, AEI, and dual-luciferase reporter gene assay). Results: By integrating the results from the MirSNP and eQTL databases, a total of 114 pairs between poly-miRTSs and target genes were identified, which correspond to 70 unique poly-miRTSs and 28 genes. Most of the 28 genes were located in the HLA region, and they tended to be enriched in multiple immune-related GO terms or pathways, e.g., “antigen processing and presentation” and “immune response”. Among these poly-miRTSs, rs907091 in the 3’UTR of IKZF3 (non-HLA region) was highlighted. Further AEI experiments showed that the C allele had a higher expression of ~18% than the T allele, suggesting an obvious allelic expression imbalance. Dual-luciferase reporter gene assays showed that miR-326 and miR-330-5p could regulate IKZF3 expression (P&lt;0.05) mediated by rs907091 in the 3’UTR binding site. Conclusion: Our study explored some functional mechanisms underlying the associations between poly-miRTSs and target genes and highlighted rs907091 at the miRNA binding region of the IKZF3 gene as a potential functional variant for rheumatoid arthritis.

Advanced Strategies for Strengthening the Immune Activation Effect of Traditional Antitumor Therapies.

The utilization of traditional therapies (TTS), such as chemotherapy, reactive oxygen species-based therapy, and thermotherapy, to induce immunogenic cell death (ICD) in tumor cells has emerged as a promising strategy for the activation of the antitumor immune response. However, the limited effectiveness of most TTS in inducing the ICD effect of tumors hinders their applications in combination with immunotherapy. To address this challenge, various intelligent strategies have been proposed to strengthen the immune activation effect of these TTS, and then achieve synergistic antitumor efficacy with immunotherapy. These strategies primarily focus on augmenting the tumor ICD effect or facilitating the antigen (released by the ICD tumor cells) presentation process during TTS, and they are systematically summarized in this review. Finally, the existing bottlenecks and prospects of TTS in the application of tumor immune regulation are also discussed.

Intranasal Immunization of Pneumococcal pep27 Mutant Attenuates Allergic and Inflammatory Diseases by Upregulating Skin and Mucosal Tregs.

Conventional immunization methods such as intramuscular injections lack effective mucosal protection against pathogens that enter through the mucosal surfaces. Moreover, conventional therapy often leads to adverse events and compromised immunity, followed by complicated outcomes, leading to the need to switch to other options. Thus, a need to develop safe and effective treatment with long-term beneficial outcomes to reduce the risk of relapse is mandatory. Mucosal vaccines administered across mucosal surfaces, such as the respiratory or intestinal mucosa, to prompt robust localized and systemic immune responses to prevent the public from acquiring pathogenic diseases. Mucosal immunity contains a unique immune cell milieu that selectively identify pathogens and limits the transmission and progression of mucosal diseases, such as allergic dermatitis and inflammatory bowel disease (IBD). It also offers protection from localized infection at the site of entry, enables the clearance of pathogens on mucosal surfaces, and leads to the induction of long-term immunity with the ability to shape regulatory responses. Regulatory T (Treg) cells have been a promising strategy to suppress mucosal diseases. To find advances in mucosal treatment, we investigated the therapeutic effects of intranasal pep27 mutant immunization. Nasal immunization protects mucosal surfaces, but nasal antigen presentation appears to entail the need for an adjuvant to stimulate immunogenicity. Here, a novel method is developed to induce Tregs via intranasal immunization without an adjuvant to potentially overcome allergic diseases and gut and lung inflammation using lung-gut axis communication in animal models. The implementation of the pep27 mutant for these therapies should be preceded by studies on Treg resilience through clinical translational studies on dietary changes.

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

BackgroundThe Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod’s immune response on a molecular level.ResultsA comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5’RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.ConclusionsThis study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Activation of the hypoxia response in the aging cerebrovasculature protects males against cognitive impairment.

Alzheimer's disease (AD) is a neurodegenerative disorder with a distinct sex bias. Age-related vascular alterations, a hallmark of AD onset and progression, are consistently associated with sexual dimorphism. Here, we conducted an integrative meta-analysis of 335,803 single-nucleus transcriptomes and 667 bulk transcriptomes from the vascular system in AD and normal aging to address the underlying sex-dependent vascular aging in AD. All vascular cell types in male AD patients exhibited an activated hypoxia response and downstream signaling pathways including angiogenesis. The female AD vasculature is characterized by increased antigen presentation and decreased angiogenesis. We further confirmed that these sex-biased alterations in the cerebral vascular emerged and were primarily determined in the early stages of AD. Sex-stratified analysis of normal vascular aging revealed that angiogenesis and various stress-response genes were downregulated concurrently with female aging. Conversely, the hypoxia response increased steadily in males upon aging. An investigation of upstream driver transcription factors (TFs) revealed that altered communication between estrogen receptor alpha (ESR1) and hypoxia induced factors during menopause contributes to the inhibition of angiogenesis during normal female vascular aging. Additionally, inhibition of CREB1, a TF that targets estrogen, is also related to female AD. Overall, our study revealed a distinct cerebral vascular profile in females and males, and revealed novel targets for precision medicine therapy for AD.

Human erythroid progenitors express antigen presentation machinery.

Early-life immune exposures can profoundly impact lifelong health. However, functional mechanisms underlying fetal immune development remain incomplete. Erythrocytes are not typically considered active immune mediators, primarily because erythroid precursors discard their organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Erythroid progenitors and precursors circulate in human fetuses and neonates. Although there is limited evidence that erythroid precursors are immunomodulatory, our understanding of the underlying mechanisms remains inadequate. To define the immunobiological role of fetal and perinatal erythroid progenitors and precursors, we analyzed single cell RNA-sequencing data and found that transcriptomics support erythroid progenitors as putative immune mediators. Unexpectedly, we discovered that human erythroid progenitors constitutively express Major Histocompatibility Complex (MHC) class II antigen processing and presentation machinery, which are hallmarks of specialized antigen presenting immune cells. Furthermore, we demonstrate that erythroid progenitors internalize and cleave foreign proteins into peptide antigens. Unlike conventional antigen presenting cells, erythroid progenitors express atypical costimulatory molecules and immunoregulatory cytokines that direct the development of regulatory T cells, which are critical for establishing maternal-fetal tolerance. Expression of MHC II in definitive erythroid progenitors begins during the second trimester, coinciding with the appearance of mature T cells in the fetus, and is absent in primitive progenitors. Lastly, we demonstrate physical and molecular interaction potential of erythroid progenitors and T cells in the fetal liver. Our findings shed light on a unique orchestrator of fetal immunity and provide insight into the mechanisms by which erythroid cells contribute to host defense.

Unlocking the secrets of Hofbauer cells in placental (patho-) physiology: Isolation and quality assessment in human term placenta

IntroductionHofbauer cells (HBCs) are macrophages of fetal origin that reside in the villous tissue. They are the only immune cells within healthy villi. While HBCs perform innate immune functions such as phagocytosis and antigen presentation, they are increasingly recognized for their diverse roles in placental physiology e.g. vascular functionality, tissue homeostasis, tolerance. Consequently, HBCs are of utmost interest in a variety of non-physiological placental conditions. IsolationVillous tissue is collected freshly after delivery and finely minced. The resulting tissue is digested in a two-step process, using Trypsin/DNase to separate cytotrophoblasts and collagenase/DNase to penetrate deeper into the villous stroma, containing HBCs, and obtain a single cell suspension. After a density gradient centrifugation, the corresponding cell layer is collected and subjected to negative immune selection of HBCs, yielding unaffected cells that have not been activated during the isolation process. Quality controlIn addition to a classical immunocytochemistry (ICC) approach including macrophage markers, and markers for potentially contaminating cell types (e.g. fibroblasts, muscle, mesenchymal cells), we have developed a multi-color flow cytometry (FC) panel. This panel assesses Hofbauer cell purity and polarization states more accurately and comprehensively than qualitative ICC, using percentage analysis of parent cells to estimate the expression levels of specific markers. DiscussionThe presented protocol allows us to isolate HBCs in significant numbers and high purity, even from placentae compromised by preeclampsia (PE) with limited placental volume. We have successfully developed and implemented this protocol to study healthy, diabetic and PE macrophages, aiding a better understanding of the underlying placental pathophysiology at the cellular level.

Assessing the implications of sentinel lymph node removal in cervical cancer: an immunogenetic perspective – a SENTICOL ancillary study

BackgroundCervical cancer’s lymphatic spread primarily begins from the sentinel lymph nodes (SLNs), underlining their pivotal role in disease metastasis. However, these nodes’ immune gene expression profiles and immunoregulation mechanisms have...

Allostimulation leads to emergence of a human B cell population with increased expression of HLA class I antigen presentation–associated molecules and the immunoglobulin receptor FcRL5

In the extensive literature characterizing lymphocyte contributions to transplant-related pathologies including allograft rejection and graft-versus-host disease, T cell–focused investigation has outpaced investigation of B cells. Most B cell–related reports describe regulatory and antibody-producing functions, with less focus on the potential role of antigen-presenting capacity. Using in vitro human mixed lymphocyte reactions (MLRs) to model allostimulation, we analyzed responder B cells using transcriptional analysis, flow cytometry, and microscopy. We observed emergence of an activated responder B cell subpopulation phenotypically similar to that described in individuals with graft-versus-host disease or allograft rejection. This population had markedly increased expression of FcRL5 (Fc receptor like 5) and molecules associated with human leukocyte antigen class I antigen presentation. Consistent with this phenotype, these cells demonstrated increased internalization of irradiated cell debris and dextran macromolecules. The proportion of this subpopulation within MLR responders also correlated with emergence of activated, cytotoxic CD8+ T cells. B cells of similar profile were quite infrequent in unstimulated blood from healthy individuals but readily identifiable in disaggregated human splenocytes and increased in both cases upon allostimulation. Further characterization of the emergence and function of this subpopulation could potentially contribute to identification of novel biomarkers and targeted therapeutics relevant to curbing transplant-related pathology.

Homologous Tumor Targeting Molybdenum‐Doped Prussian Blue for Enhancing Immunotherapy via PTT/CDT and Remodeled Tumor Immune Microenvironment

AbstractImmunotherapy offers a promising avenue for reducing tumor metastasis and recurrence but faces challenges from the tumor immunosuppressive microenvironment (TIME) and restricted antigen presentation. To address these challenges, this study have developed an innovative approach utilizing molybdenum (Mo)‐doped Prussian blue nanoparticles coated with a cancer cell membrane (CCM), referred to as PMo@CCM. This novel nanoplatform excels in performing photothermal therapy (PTT), while the Mo and Fe components effectively deplete glutathione (GSH) and generate reactive oxygen species (ROS), thereby significantly enhancing chemodynamic therapy (CDT) and remodeling the TIME. The synergistic PTT/CDT approach not only induces tumor immunogenic cell death (ICD) but also facilitates antigen presentation. The CCM coating further supplies antigens and prompts dendritic cell (DC) maturation. This comprehensive strategy markedly enhances the effectiveness of immunotherapy, as evidenced by a significant increase in T cell activation. Moreover, the use of programmed cell death protein 1 antibodies (anti PD‐1) effectively blocks the PD‐1 immune checkpoint pathway. RNA sequencing analysis has identified genes associated with the observed substantial reduction in tumor growth. In conclusion, the PMo@CCM nanoplatform enables homologously targeted tumor synergistic therapy, guided by photothermal and magnetic resonance imaging (PTI&amp;MRI), significantly impeding the progression of both primary and metastatic tumors.

CX3CL1 release during immunogenic apoptosis is associated with enhanced anti-tumour immunity.

Immunogenic cell death (ICD) has emerged as a novel option for cancer immunotherapy. The key determinants of ICD encompass antigenicity (the presence of antigens) and adjuvanticity, which involves the release of damage-associated molecular patterns (DAMPs) and various cytokines and chemokines. CX3CL1, also known as neurotactin or fractalkine, is a chemokine involved in cellular signalling and immune cell interactions. CX3CL1 has been denoted as a "find me" signal that stimulates chemotaxis of immune cells towards dying cells, facilitating efferocytosis and antigen presentation. However, in the context of ICD, it is uncertain whether CX3CL1 is an important mediator of the effects of ICD. In this study, we investigated the intricate role of CX3CL1 in immunogenic apoptosis induced by mitoxantrone (MTX) in cancer cells. The Luminex xMAP technology was used to quantify murine cytokines, chemokines and growth factors to identify pivotal regulatory cytokines released by murine fibrosarcoma MCA205 and melanoma B16-F10 cells undergoing ICD. Moreover, a murine tumour prophylactic vaccination model was employed to analyse the effect of CX3CL1 on the activation of an adaptive immune response against MCA205 cells undergoing ICD. Furthermore, thorough analysis of the TCGA-SKCM public dataset from 98 melanoma patients revealed the role of CX3CL1 and its receptor CX3CR1 in melanoma patients. Our findings demonstrate enhanced CX3CL1 release from apoptotic MCA205 and B16-F10 cells (regardless of the cell type) but not if they are undergoing ferroptosis or accidental necrosis. Moreover, the addition of recombinant CX3CL1 to non-immunogenic doses of MTX-treated, apoptotically dying cancer cells in the murine prophylactic tumour vaccination model induced a robust immunogenic response, effectively increasing the survival of the mice. Furthermore, analysis of melanoma patient data revealed enhanced survival rates in individuals exhibiting elevated levels of CD8+ T cells expressing CX3CR1. These data collectively underscore the importance of the release of CX3CL1 in eliciting an immunogenic response against dying cancer cells and suggest that CX3CL1 may serve as a key switch in conferring immunogenicity to apoptosis.

Separable nanocomposite hydrogel microneedles for intradermal and sustained delivery of antigens to enhance adaptive immune responses

Vaccines play a critical role in combating infectious diseases and cancers, yet improving their efficacy remains challenging. Here, we introduce a separable nanocomposite hydrogel microneedle (NHMN) patch designed for intradermal and sustained delivery of ovalbumin (OVA), a model antigen, to enhance adaptive immune responses. The NHMN patch consists of an array of OVA-loaded microneedles made from photo-cross-linked methacrylated hyaluronic acid and laponite (LAP), supported by a hyaluronic acid backing. The incorporation of LAP not only enhances the mechanical strength of the pure hydrogel microneedles but also significantly prolongs OVA release. Furthermore, in vitro cell experiments demonstrate that NHMNs effectively activate dendritic cells without compromising cell viability. Upon skin penetration, NHMNs detach from the backing as the hyaluronic acid rapidly dissolves upon contact with the skin interstitial fluid, thereby acting as antigen reservoirs to release antigens to abundant skin dendritic cells. NHMNs containing 0.5% w/v LAP achieved a 15-day OVA release in vivo. Immunization studies demonstrate that the intradermal and sustained release of OVA via NHMNs elicited stronger and longer-lasting adaptive immune responses compared to conventional bolus injection. Given its easy to use, painless and minimally invasive features, the NHMN patch shows promise in improving vaccination accessibility and efficacy against a range of diseases. Statement of significanceThe study introduces a separable nanocomposite hydrogel microneedle (NHMN) patch. This patch consists of an array of ovalbumin (OVA, a model antigen)-loaded microneedles made from photo-cross-linked methacrylated hyaluronic acid and laponite, with a hyaluronic acid backing, designed for intradermal and sustained delivery of antigens. This patch addresses several key challenges in traditional vaccination methods, including poor antigen uptake and presentation, and rapid systematic clearance. The incorporation of laponite enhances mechanical strength of microneedles, promotes dendritic cell activation, and significantly slows down antigen release. NHMN-based vaccination elicits stronger and longer-lasting adaptive immune responses compared to conventional bolus injection. This NHMN patch holds great potential for improving the efficacy, accessibility, and patient comfort of vaccinations against a range of diseases.

Potential Mechanism and Therapeutic Targets of Polymyositis.

To investigate the variability in the expression profile of genes associated with polymyositis (PM), explore the potential molecular mechanisms underlying PM, and predict novel targets for intervention. Descriptive study. Place and Duration of the Study: Department of Rheumatology, Taizhou Municipal Hospital, Taizhou, China, from August to November 2023. Three microarray datasets (GSE3112, GSE39454, and GSE128470) were extracted from the gene expression omnibus (GEO). The analysis of this research involved identifying the differentially expressed genes (DEGs) in PM compared to normal samples. Enrichment analysis, gene-microRNA, gene-transcription factor (TF), and protein-protein interaction (PPI) network studies were conducted to identify hub genes and relevant pathways. Additionally, the drug-gene interaction database (DGIdb) was used to predict therapeutic medications. Eighty-eight DEGs were identified. The enrichment analysis results highlighted the significant involvement of downregulated DEGs in antigen processing and presentation. Based on the PPI networks, seven hub genes with high connectivity degrees were selected including a cluster of differentiation 74 (CD74), human leukocyte antigen (HLA)-DPA1, HLA-B, guanylate-binding protein 1 (GBP1), recombinant 2', 5'-oligoadenylate synthetase 1 (OAS1), HLA-C, and HLA-E. This research screened-out core genes, projected prospective therapeutic medications, discovered DEGs between PM and normal samples, and offered fresh perspectives for additional research into the possible mechanism and therapeutic targets of PM. Polymyositis, DEGs, Hub genes, Bioinformatics, Potential therapeutic agents.

Combined analysis of single-cell sequencing and bulk transcriptome sequencing reveals new mechanisms for non-healing diabetic foot ulcers.

Diabetic foot ulcers (DFUs) pose a significant challenge in diabetes care. Yet, a comprehensive understanding of the underlying biological disparities between healing and non-healing DFUs remains elusive. We conducted bioinformatics analysis of publicly available transcriptome sequencing data in an attempt to elucidate these differences. Our analysis encompassed differential analysis to unveil shifts in cell composition and gene expression profiles between non-healing and healing DFUs. Cell communication alterations were explored employing the Cellchat R package. Pseudotime analysis and cytoTRACE allowed us to dissect the heterogeneity within fibroblast subpopulations. Our findings unveiled disruptions in various cell types, localized low-grade inflammation, compromised systemic antigen processing and presentation, and extensive extracellular matrix signaling disarray in non-healing DFU patients. Some of these anomalies partially reverted in healing DFUs, particularly within the abnormal ECM-receptor signaling pathway. Furthermore, we distinguished distinct fibroblast subpopulations in non-healing and healing DFUs, each with unique biological functions. Healing-associated fibroblasts exhibited heightened extracellular matrix (ECM) remodeling and a robust wound healing response, while non-healing-associated fibroblasts showed signs of cellular senescence and complement activation, among other characteristics. This analysis offers profound insights into the wound healing microenvironment, identifies pivotal cell types for DFU healing promotion, and reveals potential therapeutic targets for DFU management.

Dendritic cell-related hub genes in head-and-neck squamous cell carcinoma: implications for prognosis and immunotherapy.

In the context of head-and-neck squamous cell carcinoma (HNSCC), dendritic cells (DCs) assume pivotal responsibilities, acting as architects of antigen presentation and conductors of immune checkpoint modulation. In this study, we aimed to identify hub genes associated with DCs in HNSCC and explore their prognostic significance and implications for immunotherapy. Integrated clinical datasets from The Cancer Genome Atlas (TCGA)-HNSCC and GSE65858 cohorts underwent meticulous analysis. Employing weighted gene co-expression network analysis (WGCNA), we delineated candidate genes pertinent to DCs. Through the application of random survival forest and least absolute shrinkage and selection operator (LASSO) Cox's regression, we derived key genes of significance. Lisa (epigenetic Landscape In Silico deletion Analysis and the second descendent of MARGE) highlighted transcription factors, with Dual-luciferase assays confirming their regulatory role. Furthermore, immunotherapeutic sensitivity was assessed utilizing the Tumor Immune Dysfunction and Exclusion online tool. This study illuminated the functional intricacies of HNSCC DC subsets to tailor innovative therapeutic strategies. We leveraged clinical data from the TCGA-HNSCC and GSE65858 cohorts. We subjected the data to advanced analysis, including WGCNA, which revealed 222 DC-related candidate genes. Following this, a discerning approach utilizing random survival forest analysis and LASSO Cox's regression unveiled seven genes associated with the prognostic impact of DCs, notably ACP2 and CPVL, associated with poor overall survival. Differential gene expression analysis between ACP2 + and ACP2 - DC cells revealed 208 differential expressed genes. Lisa analysis identified the top five significant transcription factors as STAT1, SPI1, SMAD1, CEBPB, and IRF1. The correlation between STAT1 and ACP2 was confirmed through quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Dual-luciferase assays in HEK293T cells. Additionally, TP53 and FAT1 mutations were more common in high-risk DC subgroups. Importantly, the sensitivity to immunotherapy differed among the risk clusters. The low-risk cohorts were anticipated to exhibit favorable responses to immunotherapy, marked by heightened expressions of immune system-related markers. In contrast, the high-risk group displayed augmented proportions of immunosuppressive cells, suggesting a less conducive environment for immunotherapeutic interventions. Our research may yield a robust DC-based prognostic system for HNSCC; this will aid personalized treatment and improve clinical outcomes as the battle against this challenging cancer continues.

Normothermic liver perfusion derived extracellular vesicles have concentration-dependent immunoregulatory properties.

Extracellular vesicles (EVs) are major contributors to immunological responses following solid organ transplantation. Donor derived EVs are best known for their role in transplant rejection through transferring donor major histocompatibility complex proteins to recipient antigen presenting cells, a phenomenon known as ‛cross-decoration'. In contrast, donor liver-derived EVs are associated with organ tolerance in small animal models. Therefore, the cellular source of EVs and their cargo could influence their downstream immunological effects. To investigate the immunological effects of EVs released by the liver in a physiological and transplant-relevant model, we isolated EVs being produced during normothermic ex vivo liver perfusion (NEVLP), a novel method of liver storage prior to transplantation. We found EVs were produced by the liver during NEVLP, and these EVs contained multiple anti-inflammatory miRNA species. In terms of function, liver-derived EVs were able to cross-decorate allogeneic cells and suppress the immune response in allogeneic mixed lymphocyte reactions in a concentration-dependent fashion. In terms of cytokine response, the addition of 1×109 EVs to the mixed lymphocyte reactions significantly decreased the production of the inflammatory cytokines TNF-α, IL-10 and IFN-γ. In conclusion, we determined physiologically produced liver-derived EVs are immunologically regulatory, which has implications for their role and potential modification in solid organ transplantation.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1 (AP1), we have developed a fluorescence polarization-based assay for inhibitor screening against Nef’s activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of DMSO, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.