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摘要： OBJECTIVE:More than 10% of patients with type 1 diabetes (T1D) do not have high-risk HLA-DR3 or -DR4 haplotypes with distinct clinical features, such as later onset and reduced insulin dependence. We aimed to identify genetic drivers of T1D in the absence of DR3/DR4 and improve prediction of T1D risk in these individuals. RESEARCH DESIGN AND METHODS:We performed T1D association and fine-mapping analyses in 12,316 non-DR3/DR4 samples. Next, we performed heterogeneity tests to examine differences in T1D risk variants in individuals without versus those with DR3/DR4 haplotypes. We further assessed genome-wide differences in gene regulatory element and biological pathway enrichments between the non-DR3/DR4 and DR3/DR4 cohorts. Finally, we developed a genetic risk score (GRS) to predict T1D in individuals without DR3/DR4 and compared with an existing T1D GRS. RESULTS:A total of 18 T1D risk variants in non-DR3/DR4 samples were identified. Risk variants at the MHC and multiple other loci genome wide had heterogeneity in effects on T1D dependent on DR3/DR4 status, and non-DR3/DR4 T1D had evidence for a greater polygenic burden. T1D-assocated variants in non-DR3/DR4 were more enriched for regulatory elements and pathways involved in antigen presentation, innate immunity, and β-cells and depleted in T cells compared with DR3/DR4. A non-DR3/DR4 GRS outperformed an existing risk score GRS2 in discriminating non-DR3/DR4 T1D from no diabetes (area under the curve 0.867; P = 7.48 × 10-32) and type 2 diabetes (0.907; P = 4.94 × 10-44). CONCLUSIONS:In total, we identified heterogeneity in T1D genetic risk dependent on high-risk HLA-DR3/DR4 haplotype, which uncovers disease mechanisms and enables more accurate prediction of T1D across the HLA background.

关键词： AIM2   Head and neck squamous cell carcinoma   IL17   MAPK pathway   STAT1  

摘要： Chronic inflammation has been recognized as one of the hallmarks of head and neck squamous cell carcinoma (HNSCC), Absent In Melanoma 2(AIM2) has emerged as important regulators of chronic inflammatory, and participated in initiation, progression of kinds of human cancers. Nonetheless, the biological functions and underlying mechanisms of AIM2 in HNSCC remain inadequately understood. Based on the bioinformatics analysis of public databases, we identified elevated AIM2 expression in HNSCC, which positively correlates with disease stage and HPV infection, thereby possessing both diagnostic and prognostic significance. Immunohistochemistry on clinical samples revealed that AIM2 expression was frequently upregulated in cancerous tissues compared to paracancerous tissues, exhibiting a significant association with Ki-67 expression. Modulating AIM2 expression in HNSCC cell lines through transfection with inhibitors or mimics demonstrated that ectopic AIM2 expression enhances cell growth, migration, tumorigenesis, and metastasis both in vitro and in vivo. A dual luciferase reporter assay indicated that the transcription factor STAT1 can bind directly to the AIM2 promoter region and activate its transcription. The STAT1 inhibitor, fludarabine, reduces AIM2 expression and subsequently diminishes cell proliferation. Mechanistically, AIM2 exerts its tumor-promoting effects through the IL-17-MAPK signaling pathway. Collectively, our data demonstrate that AIM2, transcriptionally activated by STAT1, exhibits oncogenic functions by promoting the IL-17-MAPK signaling pathway, suggesting that AIM2 may be a new intervention targets for the diagnostic and treatment of HNSCC.

关键词： 淋巴细胞活化基因3(LAG3)   真核细胞抗原   活体骨髓瘤细胞   杂交瘤   单克隆抗体(mAb)  

摘要： 目的 制备小鼠抗人淋巴细胞活化基因3（LAG3）单克隆抗体（mAb）并进行抗体免疫学鉴定。方法 以构建的稳定表达人LAG3胞外区和跨膜区的小鼠3T3单克隆细胞（LAG3-mLumin-3T3）免疫Balb/c小鼠，流式细胞术和免疫荧光法检测小鼠血清中是否含有抗人LAG3抗体。SP2/0细胞皮下注射Balb/c小鼠形成实体骨髓瘤，体外分离的小鼠骨髓瘤细胞，与免疫小鼠的脾细胞融合建立杂交瘤，有限细胞稀释法分离出杂交瘤单克隆细胞。收集杂交瘤单克隆细胞培养液，流式细胞术检测细胞分泌LAG3 mAb。以构建的表达LAG3胞外区4个独立结构域的3T3细胞株（LAG3-胞外结构域1/2/-3/-4-3T3）流式细胞术区分mAb结合抗原表位。流式细胞术检测LAG3 mAb与活化状态下的人外周血单核细胞（PBMC）共同培养前后细胞LAG3表达。结果 重组真核细胞抗原免疫后小鼠能产生特异性抗人LAG3抗体。杂交瘤融合获得杂交瘤单克隆细胞能够分泌小鼠抗人LAG3 mAb；不同单克隆细胞株分泌的mAb识别的LAG3抗原结构域存在差异。结论 成功制备小鼠抗人LAG3 mAb，不同杂交瘤细胞克隆分泌的mAb识别表位不同，为进一步研究LAG3生物学特性及其检测试剂与肿瘤治疗阻断抗体的研发奠定基础。

摘要： Type 2 diabetes mellitus (T2D) is defined by chronic hyperglycemia due to insufficient insulin secretion or activity and decreased insulin sensitivity, known as insulin resistance (IR). This condition leads to oxidative stress and inflammation, increasing the risk of systemic inflammatory diseases. Obesity and a sedentary lifestyle are major risk factors for IR and T2D. Various metabolites act as mediators of IR by disrupting communication between organs. Lipids, including free fatty acids and short-chain fatty acids, along with intracellular lipotoxins, impair insulin function and mitochondrial activity, contributing to IR through direct and indirect mechanisms such as oxidative stress and inflammation. Our research explores the role of TNF alpha and CXCR1/2 in IR conditions, emphasizing their interactions and potential as therapeutic targets. In this study we selected two models of IR, adipocytes and hepatocytes, since are key players in glucose and lipid metabolism. To develop IR model, TNF alpha was used as challenge and we focused on investigating the role of CXCR1/2 inhibition. We assessed glucose uptake, insulin signaling pathways, and gene expression related to IR. Cells treated with TNF alpha showed reduced p-Akt and increased p-JNK levels, indicative of IR. In contrast, CXCR1/2 inhibition restored p-Akt levels and reduced p-JNK levels, suggesting improvements in insulin signaling and glucose uptake. Furthermore, CXCR1/2 inhibition counteracted the TNF alpha-induced decrease in IGF expression and restored GLUT2 expression, indicating enhanced insulin sensitivity. These results underscore the pivotal role of CXCR1/2 in modulating the inflammatory response and insulin signaling in IR conditions in both IR models. CXCR1/2 inhibition can mitigate IR and improve glucose metabolism. Thus, targeting the TNF alpha-CXCR1/2 pathway presents a promising therapeutic approach for managing IR and T2D. Further investigation is necessary to understand the clinical implicat

关键词： PBLD   MAVS   USP4   RNA viruses   Type   IFN  

摘要： Phenazine biosynthesis- like domain- containing protein (PBLD) has been reported to be involved in the development of many cancers. However, whether PBLD regulates innate immune responses and viral replication is unclear. In this study, although it was found that the activity of PBLD extends to other PRRs, we focused on the RLR pathway activated via the p53-USP4-MAVS axis in response to virus infections. We found that PBLD deubiquitinates and stabilizes MAVS through ubiquitin- specific protease transcription of USP4 via the upregulation of p53. USP4, which is a MAVS- interacting antiviral innate immunity through the p53-USP4-MAVS signaling, providing a preliminary basis for research on PBLD as a target molecule for treating RNA virus infection.

关键词： Scophthalmus maximus   IL4/13A   Th2 immunity   immune response   gene identification  

摘要： Th2 immunity is a primary host defense against extracellular pathogens, and different IL4/13 paralogues are involved in this immune response in fish. Here, we identified IL4/13A for further Th2 immune response providing information in turbot. The results showed that the full length of the IL4/13A gene is 1,333 bp, containing a 432-bp open reading frame (ORF) that encoded 144 amino acids. Phylogenetic analysis recently showed that turbot IL4/13A has a relationship with Dicentrarchus labrax. Moreover, syntenic analysis revealed similar neighboring genes associated with turbot IL4/13A, compared with other teleosts and mammals. In addition, IL4/13A was widely expressed in all examined tissues with the highest expression level in skin, followed by liver and gill. Finally, IL4/13A showed a general trend of upregulation in immune tissues following bacterial challenge. The significant quick induction of IL4/13A indicated its key roles to prevent pathogens. Characterizations of IL4/13A will probably contribute to understanding of Th2 immunity in fish.

摘要： Proteasomes generate antigenic peptides that are presented on the tumor surface to cytotoxic T-lymphocytes. Immunoproteasomes are highly specialized proteasome variants that are expressed at higher levels in antigen-presenting cells and contain replacements of the three constitutive proteasome catalytic subunits to generate peptides with a hydrophobic C-terminus that fit within the groove of MHC class I (MHC-I) molecules. A hallmark of cancer is the ability to evade immunosurveillance by disrupting the antigen presentation machinery and downregulating MHC-I antigen presentation. High-throughput screening was performed to identify compound A, a novel molecule that selectively increased immunoproteasome activity and expanded the number and diversity of MHC-I-bound peptides presented on multiple myeloma cells. Compound A increased the presentation of individual MHC-I-bound peptides by >100-fold and unmasked tumor-specific neoantigens on myeloma cells. Global proteomic integral stability assays determined that compound A binds to the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28 alpha/beta (PSME1/PSME2) with immunoproteasomes. CRISPR/Cas9 silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of compound A on antigen presentation. Treatment of multiple myeloma cell lines and patient bone marrow-derived CD138+ cells with compound A increased the anti-myeloma activity of allogenic and autologous T cells. Compound A was well-tolerated in vivo and co-treatment with allogeneic T cells reduced the growth of myeloma xenotransplants in NOD/SCID gamma mice. Taken together, our results demonstrate the paradigm shifting impact of immunoproteasome activators to diversify the antigenic landscape, expand the immunopeptidome, potentiate T-cell-directed therapy, and reveal actionable neoantigens for personalized T-cell immunotherapy.

摘要： γδ T-cells represent key players in the immune-surveillance after TCR alpha/beta (αβ)/CD19-depleted HLA-Haploidentical Stem Cell Transplantation (haplo-HSCT). Although encouraging data are available on the impact of Vδ2-targeting-therapy in improving HSCT-clinical outcomes, their role in providing antimicrobial immunity is largely unexplored. This study aimed to investigate the antiviral protective profile of Vδ2 T-cells in pediatric patients given this type of allograft. The characterization of γδ T-cells was performed by flow cytometry in haplo-HSCT-pediatric patients (n=26) in the donor graft and after 30, 60 and 120 days post-HSCT. The antiviral activity of Vδ2 T-cells was assessed on CMV-infected-fibroblasts and the CMV-specific αβ T-cell immunity was analyzed by flow cytometry. Early after HSCT, frequency of Vδ2 T-cells was significantly higher in patients who did not experience viral reactivation (No-VR) than in patients with CMV reactivation (CMV-R). Interestingly, this difference was already present in the grafts. Clustering analysis identified a protective subset of Vδ2 T-cells in No-VR patients, which expresses CD16, NKG2D, and CD107a and produces high levels of IFN-γ. This subset directly correlated with IL-15 and inversely with the CMV-DNA level. Stimulated Vδ2 T-cells inhibit CMV replication, acquired CD86/HLA-DR molecules, induced HLA-DR on monocytes, and improved the αβ CMV-specific T-cell response. Altogether, these results identify an antiviral protective profile displayed by Vδ2 T-cells early after HSCT, and define their ability to inhibit CMV replication, to induce antigen-presenting cell maturation and to improve αβ virus-specific T-cell response, opening a new application of Vδ2-targeting immunotherapy after HSCT, adding the antiviral to the antitumor potential.

关键词： CD8(+) T cells   Fusokine   IL-15   Immunotherapy   Sushi domain  

摘要： BACKGROUND:To overcome the limitations of IL-15 and to improve the efficacy of IL-15 in immunotherapy, several strategies have been introduced. OBJECTIVE:The objective of this study was to generate and evaluate a novel anti-CD8/IL-15 (N72D)/Sushi fusion protein with the potential to target CD8 T cells and enhance functionality of CD8 T cells against tumor cells. METHODS:In this connection, a novel fusokine that contains IL-15(N72D), a Sushi domain, and anti-CD8 single-chain fragment variable (scFv) was designed. The size accuracy and binding potency of the isolated protein were assessed using western blotting and indirect surface staining. Following purification, the potential function of the anti-CD8/IL-15(N72D)/Sushi fusion protein in the induction of proliferation and cytotoxicity of CD8 T cells was evaluated. RESULTS:In-silico analysis revealed that fusokine is structurally stable, correctly folded and can interact with the CD8 co-receptor. Both fusokine and IL-15(N72D)/Sushi were produced in CHO-S cell line with a final concentration of 18.43 mg/l and 12.64 mg/l respectively. Fusokine bound to 97.6 % of CD8 T cells and significantly induced T cell proliferation and cytotoxic potential in peripheral blood mononuclear cells (PBMCs) in a time dependent manner. Compared to both the control and the IL-15 (N72D)/sushi treated groups, fusokine showed superior potential in CD8 T cell functionality. CONCLUSION:Anti-CD8/IL-15(N72D)/Sushi has the ability to effectively target CD8 T cells, promote lymphocyte proliferation and induce cytotoxicity against tumor cells. Due to its promising properties, it could be considered as a new potential immunotherapy approach.