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摘要： Agonistic anti-CD40 with anti-PD-1 can elicit objective responses in a small number of patients with pancreatic ductal adenocarcinoma (PDA). Better understanding of their individual effects on the pancreatic tumor microenvironment will help inform new strategies to further improve outcomes. In this study, we map tumor-specific CD8+ T-cell differentiation following agonistic anti-CD40 and/or anti-PDL1 in PDA. Rare Tcf1+Slamf6+ tumor-specific CD8+ T cells are identified and likely seed memory precursors that transition into exhausted or effector T cells. In tumors, anti-PDL1 drove the clonal expansion of Gzmk+ progenitor exhausted CD8+ T cells, whereas anti-CD40 promoted CD4+ T-cell clonal expansion and accumulation of Tcf1+Slamf6+ CD8+ T cells. Cloning the most frequent intratumoral T-cell receptors revealed identical neoepitope specificity, yet the top T-cell receptors from anti-PDL1 +/- anti-CD40 cohorts lacked tetramer binding, suggesting lower affinity. Anti-CD40 + anti-PDL1 markedly drove the clonal hyperexpansion of a unique exhausted T-cell subset in the spleen. Exhausted T cells were enriched for IL-2R beta, and provision of IL-15 complex (IL-15C) mitigated systemic and intratumoral T-cell exhaustion when combined with anti-CD40 + anti-PDL1, resulting in enhanced antitumor effects, prolongation of animal survival, and resistance to orthotopic tumor rechallenge. Mechanistically, anti-CD40 + anti-PDL1 decreased Tox, whereas IL-15C + anti-CD40 + anti-PDL1 increased T-bet, thereby conferring a higher T-bet:Tox ratio in tumor-specific CD8+ T cells. Collectively, agonistic anti-CD40 + anti-PDL1 drove systemic and intratumoral CD8+ T-cell clonal expansion and acquisition of exhaustion features in a tissue-specific manner. Provision of IL-15C altered the trajectory of T-cell differentiation induced by immunotherapy, resulting in PDA eradication and long-lived antitumor memory T cells.

关键词： IVT-mRNA   epitope   presentation   purification   mass spectrometry  

摘要： Presentation of human leukocyte antigen (HLA)-class I-restricted neo-epitopes is key for inducing an adaptive cytotoxic T-lymphocyte response against cancer. Validating presentation of these cancer-specific neo-epitopes following delivery to antigen-presenting cells (APCs) is critical to advance personalized therapeutic cancer vaccines. Current workflows for neo-epitope identification are often laborious and depend on computational deconvolution to determine the correct peptide sequence and its corresponding restriction element. We evaluated an mRNA-based workflow for more precise purification of HLA-I-peptide (pHLA)-complexes, facilitating peptide identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach uses mRNA encoding a specific HLA-I-molecule fused to a Twin-Strep-Tag (HLA-TST), allowing affinity-based purification and downstream analysis of pHLA-complexes. As a proof-of-concept, we co-electroporated mRNA encoding TST-HLA-A*02:01 and mRNA encoding an HLA-A*02:01-restricted epitope in HLA-A*02:01-negative APCs. We demonstrated successful purification and detection of the delivered epitope via LC-MS/MS. These findings highlight the potential of the mRNA-based workflow to verify neo-epitope presentation by APCs. Still, further investigation is necessary to fully understand the technical variables that can influence peptide identification by LC-MS/MS.

关键词： 赤羽病病毒   Gn蛋白   昆虫杆状病毒表达系统   单克隆抗体  

摘要： 为制备针对赤羽病病毒（Akabane virus， AKAV）Gn蛋白的单克隆抗体，本研究将目的蛋白Gn的基因序列构建至pFastBac HTB载体，通过昆虫杆状病毒表达系统（BEVS）制备重组AKAV Gn蛋白。将纯化的重组蛋白免疫小鼠，取其脾细胞和骨髓瘤细胞（SP2/0）进行细胞融合及亚克隆，使用间接ELISA法筛选出能稳定分泌AKAV Gn蛋白的单克隆抗体（mAb）。经过Western Blot、IFA以及中和效价分析，证实获得的5株单抗均能分泌AKAV Gn蛋白，并能与AKAV感染的BHK-21细胞产生特异性反应，且中和效价均高于1︰27。单抗亚类鉴定结果表明5株单抗均为IgG亚类，轻链类型均为κ链。本试验制备的AKAV Gn单克隆抗体为深入探究AKAV Gn蛋白功能以及AKAV检测技术的完善奠定了基础。

关键词： Fulminant myocarditis   Plasma proteomic   S100A8/A9   Biomarker   Therapeutic target   FM   fulminant myocarditis   MS   mass spectrometry   DAMP   damage-associated molecular pattern   TLR4   Toll-like receptor 4   RAGE   receptor for advanced glycosylation end products   CVB3   Coxsackievirus B3   NFM   acute nonfulminant myocarditis   AMI   acute myocardial infarction   ADHF   acute decompensated heart failure   ELISA   enzyme-linked immunosorbent assay   IL   interleukin   CXCL2   CXC motif chemokine ligand 2   TNF-α   tumor necrosis factor-alpha   TGF-β   transforming growth factor-beta   NLRP3   NOD-like receptor protein 3   IQR   interquartile range   ROC   receiver operating characteristic   AUC   area under the curve   K-M   Kaplan–Meier   LVEF   left ventricular ejection fraction   hs-cTnI   high-sensitivity cardiac troponin I   NT-proBNP   N-terminal pro-brain natriuretic peptide   DEP   differentially expressed protein   BP   biological process   CC   cellular components   MF   molecular function   HW/BW   heart weight to body weight ratio   PPI   protein–protein interaction   PCA   principal component analysis  

摘要： Introduction Fulminant myocarditis (FM) is a lethal inflammatory myocardial disease characterized by rapid progression and poor prognosis. Investigating plasma proteins linked to FM may elucidate underlying pathological mechanisms, identify novel biomarkers for early detection in high-risk populations, and advance the development of targeted therapeutic strategies. Objectives This study aimed to investigate the proteomic profiles of FM patients and identify potential biomarkers and therapeutic targets, focusing on S100A8/S100A9. Methods We conducted a comprehensive proteomic analysis of an extensive plasma sample collection derived from FM patients across three clinical cohorts. Our approach included screening for proteins correlated with cardiac dysfunction and disease severity. Key findings were validated in an independent cohort. In parallel, we employed a CVB3-induced FM mouse model to evaluate the expression of these biomarkers in myocardial tissue and assessed the effects of targeted interventions. Specifically, we investigated the therapeutic impacts of blocking the S100A8/A9 heterodimer using the inhibitor ABR-238901. Results The analysis revealed significant innate immune activation and severe metabolic disturbances in FM patients. Through proteomic profiling and correlation analysis, proteins S100A8 and S100A9 emerged as significant biomarkers linked to cardiac dysfunction and FM severity. S100A8/A9 heterodimer was validated as a crucial diagnostic marker for FM, with elevated plasma levels correlating with increased rates of cardiac death, heart transplantation, and hospitalizations due to heart failure or myocarditis. In CVB3-induced FM mouse models, myocardial tissues showed increased levels of S100A8 and S100A9. Treatment with ABR-238901 significantly reduced mortality by alleviating acute inflammation and improving cardiac function. Conclusion S100A8/A9 emerged as an essential biomarker and a promising therapeutic target for FM. These findings offer n

摘要： Osteoarthritis (OA) is a major global health problem with no disease-modifying therapies. Interleukin-1 (IL-1) is a critical cytokine associated with the pathophysiology of OA and can be inhibited by IL-1 receptor antagonist (IL-1Ra). Here, we tested the delivery of a gene therapeutic encoding the human IL-1Ra to the knee in a phase 1, open-label clinical trial that enrolled nine patients with radiographic knee OA. The IL-1Ra gene was delivered by a self-complementary (sc)-recombinant adeno-associated virus (rAAV) serotype 2.5 (sc-rAAV2.5IL-1Ra) by intra-articular injection into an index knee at one of three doses: low [1 x 1011 viral genomes (vg)], mid (1 x 1012 vg), or high (1 x 1013 vg). The primary outcome was safety. There were no serious adverse events (AEs) related to sc-rAAV2.5IL-1Ra. Two AEs occurred that were possibly related to the vector. Both were effusions with increased pain and resolved with conservative treatment. sc-rAAV2.5IL-1Ra did not cause changes in vital signs, physical findings, or clinical laboratory measures. Less than 1% of the injected dose of sc-rAAV2.5IL-1Ra vg was detected in circulation after 1 day and was cleared within a week. Titers of neutralizing antibodies to AAV2.5 rose in serum and synovial fluid. In all cases, IL-1Ra concentration increased in the synovial fluid, and IL-1Ra concentrations remained elevated after 1 year. Baseline pain and function scores improved during the study. Therefore, we found that intra-articular gene therapy with sc-rAAV2.5IL-1Ra was safe. The sustained increase in local IL-1Ra in human knee joints supports the further clinical examination of this therapy to provide therapeutic benefit in OA.

关键词： Autophagy   Interleukin-22 (IL-22)   IL-22   Autophagy-IL-22 interplay   Immune responses  

摘要： Autophagy, a critical cellular process for maintaining homeostasis, involves the degradation and recycling of cellular components through double-membraned vesicles that are transported to lysosomes. This mechanism plays a pivotal role in the immune system by influencing cell fate decisions and functional differentiation. Emerging evidence indicates that autophagy significantly impacts the differentiation and function of T cells and group 3 innate lymphoid cell (ILC3), which are the primary producers of Interleukin-22 (IL-22). IL-22, a key cytokine involved in modulating immune responses and maintaining tissue integrity, is particularly important in combating inflammatory diseases, infections, and cancer. It exerts its effects through a signaling pathway that involves the IL-22R1 and IL-10R2 receptors. Studies have demonstrated that IL-22 can promote autophagy by activating the AMPK pathway and that its intervention can upregulate the expression of autophagy-related genes, underscoring its significant role in the regulation of autophagy. These findings reveal a complex relationship and bidirectional relationship between autophagy and IL-22, highlighting their multifaceted interactions under both physiological and pathological conditions. This review aims to provide a detailed exploration of the dynamic interplay between autophagy and IL-22, with a focus on their mutual regulatory mechanisms, functional significance, and potential for therapeutic interventions. By performing a comprehensive analysis, we sought to clarify the intricate cross talk between autophagy and IL-22, thereby advancing our understanding of their roles in cellular and immune homeostasis and their potential as targets for clinical interventions.

关键词： Glucocorticoids   T cells   Immunosuppression   IL-2 signaling   TCR signaling   Jak/STAT pathway   Erk1/2 activation   I kappa B activation  

摘要： BackgroundGlucocorticoids (GCs) are highly effective anti-inflammatory drugs that suppress T-cell activation, cytokine production, and T-cell proliferation. Nevertheless, at which molecular level and how fast GCs exert their immunosuppressive effect in T cells still remains elusive, as inconsistent genomic and non-genomic mechanisms of action have been proposed. One model postulates that GCs quickly inhibit proximal T-cell receptor (TCR) signaling via a non-genomic mechanism, whereas others have shown a strong inhibition of interleukin-2 (IL-2) transcription at later stages of T-cell activation. Due to their therapeutic significance, we have decided to shed light onto this issue and investigated how fast and at which level GCs inhibit T-cell activation by analyzing TCR and IL-2 *** utilized primary human T cells isolated from healthy donors, which were stimulated with immobilized CD3/CD28 antibodies. These cells were treated with three different GCs, diflorasone, dexamethasone, and *** of signaling kinetics revealed that GCs did not affect early TCR signaling as suggested by the normal phosphorylation levels of lymphocyte-specific protein tyrosine kinase (Lck), zeta-chain-associated protein kinase 70 (Zap70), linker for activation of T cells (LAT), and unchanged Ca2+ influx. Conversely, we found that GCs strongly and rapidly suppressed the activation of the Janus kinase (Jak)/ signal transducer and activator of transcription (STAT) pathway within 4-6 h upon CD3/CD28 stimulation in primary human T cells. This observation was in line with a strong inhibition of cytokine production and with the impaired upregulation of the IL-2 receptor (IL-2R) upon GC treatment, thus resulting in the abrogation of T-cell *** study, by showing that GCs rapidly suppress the IL-2/IL-2R expression and signaling without significantly affecting proximal TCR signaling, has highlighted a clear mechanism of action of GCs that co

关键词： Huangqi fuling decoction   Gastric cancer   Invasion   Apoptosis   Network pharmacology  

摘要： Gastric cancer (GC) is a prevalent digestive tract malignancy, and Huangqi Fuling decoction (HF) has shown potential in enhancing immune function and exhibiting anti-GC activity. However, its mechanisms remain unclear. This study utilized network pharmacology, molecular docking, and in vitro experiments to preliminarily explore the mechanisms by which HF inhibits gastric cancer invasion and metastasis while promoting apoptosis. Public databases identified differentially expressed genes (DEGs), HF targets, and GC-related genes. GO and KEGG analyses revealed signaling pathways. Clinical relevance, immune infiltration, immunotherapy, and molecular docking of hub genes were analyzed. Eight hub genes-PTGS2, MMP9, SELE, CCL2, VCAM1, ICAM1, CXCL2, and CXCL10-associated with the TNF signaling pathway were identified. HF inhibits the invasion and metastasis of GC cells by down-regulating MMP9 and PTGS2 expression, while inducing apoptosis by suppressing BCL-2 expression and promoting BAX expression. Additionally, HF can arrest the cell cycle, blocking AGS cells in the S phase and HGC-27 cells in the G0/G1 phase. This study confirms that HF promotes apoptosis and inhibits metastasis and invasion in GC cells, primarily by modulating the TNF signaling pathway. Additionally, the anti-tumor effects of HF on GC may involve immune regulatory mechanisms, but the mechanism require further experimental verification.

摘要： Interferon-gamma (IFN-gamma) plays complex and, sometimes, contradictory roles in cancer, which can affect patient responses to treatments such as immunotherapies. We recently demonstrated that IFN-gamma production by chimeric antigen receptor (CAR) T cells is not required for efficacy in hematologic tumor models, whereas IFN-gamma receptor (IFN-gamma R) signaling in solid tumor cells facilitates CAR T cell adhesion and antigen-specific cytotoxicity. Here, we show that IFN-gamma induces apoptosis of CAR T cells bearing a CD28 intracellular signaling domain, which can be reduced through targeting of IFN-gamma or IFN-gamma R. In hematologic malignancies, knockout of IFN-gamma R (IFN-gamma RKO) in CAR T cells increased their persistence without compromising efficacy. In xenograft and syngeneic solid tumor models, IFN-gamma R knockout CAR T cells displayed more potent tumor control, prolonged survival, and improved T cell memory that conferred protection from tumor rechallenge. RNA sequencing of tumor-infiltrating IFN-gamma RKO CAR T cells derived from tumor-bearing mice revealed increased cell death in tumor cells. Collectively, these data show that inhibition of IFN-gamma signaling can increase the expansion and antitumor activity of CD28-based CAR T cells in liquid and solid tumors.