EBV + B cell-derived exosomes promote EBV-associated T/NK-cell lymphoproliferative disease immune evasion by STAT3/IL-10/PD-L1 pathway.

EBV-associated T/NK-cell lymphoproliferative diseases (EBV-T/NK-LPDs) are characterized by the clonal proliferation of EBV-positive ( +) T/NK cells. EBV is typically latent in B cells and the mechanism by which the EBV genome invades T/NK cells remains unknown. Recent studies have demonstrated that exosomes derived from EBV + B cells play a pivotal role in immunosuppressive microenvironment remodeling. Moreover, the existence of an immunosuppressive microenvironment is known to be critical in the development of EBV-T/NK-LPDs. Hence, we hypothesized that exosomes derived from EBV + B cells might promote the development of EBV-T/NK-LPDs by stimulating immune evasion. In this study, we utilized paraffin sections to clarify the STAT3/IL-10/PD-L1-associated immunosuppressive microenvironment in EBV-T/NK-LPDs. Further, we extracted exosomes from BL2009 (EBV + B cell lymphoma) and CA46 (EBV- B cell lymphoma) cell lines to co-culture with cutaneous T-cell lymphoma (CTCL) cell lines, to verify the changes in the above immune evasion pathway. The paraffin sections of EBV-T/NK-LPDs showed high-expression levels of IL-10/PD-L1, which might be related to the phosphorylation of STAT3. Exosomes derived from EBV + B cells could significantly activate the STAT3/IL-10/PD-L1 pathway. After being treated with C188-9, EBV + B cell-derived exosomes were no longer able to stimulate the expression of IL-10/PD-L1 in CTCL cells. EBV-T/NK-LPDs have a STAT3/IL-10/PD-L1 overactivation-associated immunosuppressive microenvironment. Our study elucidated part of this mechanism. Exosomes derived from EBV + B could induce phosphorylation of STAT3 in CTCL cells, leading to the overexpression of IL-10/PD-L1. Our findings might shed light on new directions for understanding immune evasion in EBV-T/NK-LPDs.

Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites.

Introduction: Intra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells. Methods: B lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry. Results: Antibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients' sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells. Discussion: These studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.

Participant-derived cell line transcriptomic analyses and mouse studies reveal a role for ZNF335 in plasma cholesterol statin response.

BACKGROUND: Statins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained. METHODS: To identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335). RESULTS: The statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR = 5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho = 0.237, FDR-adj p = 0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho = 0.233, FDR-adj p = 0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild-type C57BL/6J mice in a sex combined model (p = 0.04). Furthermore, male (but not female) mice carrying the Zfp335R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43 ± 18% and -23 ± 19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335R1092W allele(s) exhibited a significantly blunted LDL statin response. CONCLUSIONS: Our in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy.

Enriching iPSC research diversity: Harnessing human biobank collections for improved ethnic representation.

Biobanks of human biosamples and cell lines are indispensable for biomedical research on human health and disease and for drug development projects. Many human cell line biobanks worldwide hold collections of lymphoblastoid cell lines (LCLs), representing thousands of affected and control donors from diverse ethnic/ancestry groups. In recent years, induced human pluripotent stem cells (iPSCs) and differentiated human cells derived from these iPSCs have become indispensable for applied biomedical research. Establishing iPSCs remains a laborious and costly step towards generating differentiated human cells. To address this research need, several non-profit and commercial biobanks have established iPSC collections for distribution to researchers, thereby serving as a resource for generating differentiated human cells. The most common starting materials for generation of iPSCs are a skin biopsy for harvesting fibroblasts, or a blood sample for collection of peripheral blood mononuclear cells. However untapped resources include the large established collections of biobanked human LCLs which can be reprogrammed to iPSCs using a variety of published protocols including the use of non-integrating episomal vectors. Many biobanks curate LCLs from diverse ethnic/ancestry populations, an aspect largely absent in most established iPSC biobanks which tend to primarily reflect populations from developed countries. Here, we call upon researchers across the breadth of iPSC research to tap the unique resource of existing and diverse human LCL collections for establishing biobanked iPSC panels that better represent the varied human ethnic (and hence genomic) diversity, thereby benefiting precision medicine and drug development research on a global scale.

Genome-Wide DNA Methylation in Early-Onset-Dementia Patients Brain Tissue and Lymphoblastoid Cell Lines.

Epigenetics, a potential underlying pathogenic mechanism of neurodegenerative diseases, has been in the scope of several studies performed so far. However, there is a gap in regard to analyzing different forms of early-onset dementia and the use of Lymphoblastoid cell lines (LCLs). We performed a genome-wide DNA methylation analysis on sixty-four samples (from the prefrontal cortex and LCLs) including those taken from patients with early-onset forms of Alzheimer's disease (AD) and frontotemporal dementia (FTD) and healthy controls. A beta regression model and adjusted p-values were used to obtain differentially methylated positions (DMPs) via pairwise comparisons. A correlation analysis of DMP levels with Clariom D array gene expression data from the same cohort was also performed. The results showed hypermethylation as the most frequent finding in both tissues studied in the patient groups. Biological significance analysis revealed common pathways altered in AD and FTD patients, affecting neuron development, metabolism, signal transduction, and immune system pathways. These alterations were also found in LCL samples, suggesting the epigenetic changes might not be limited to the central nervous system. In the brain, CpG methylation presented an inverse correlation with gene expression, while in LCLs, we observed mainly a positive correlation. This study enhances our understanding of the biological pathways that are associated with neurodegeneration, describes differential methylation patterns, and suggests LCLs are a potential cell model for studying neurodegenerative diseases in earlier clinical phases than brain tissue.

Analysis of CD20 and PD-L1 levels on small extracellular vesicles (sEV) produced by DLBCL cells and EBV-transformed B cells, and potential role in T cell inhibition.

Increasing evidence supports a role for small extracellular vesicles (sEV, including exosomes) in Diffuse Large B-cell lymphoma (DLBCL) progression and resistance to treatment. CD20 and PD-L1 are found on DLBCL-derived sEV, but little is known about their patient-level heterogeneity. Moreover, the capacity of PD-L1+ sEV to modulate T cells needs to be clarified. Herein we analyzed sEV produced by human DLBCL cell lines and EBV-transformed B cell-lymphoblastoid cell lines (LCLs), a model allowing autologous T cell co-cultures. We determined CD20 and PD-L1 levels on plasma sEV from patient samples vs healthy volunteers (HV). sEV functional relevance was also investigated on CD4+ and CD8+ T cells. sEV derived from all cell lines showed an enrichment of CD20 and a high glycosylated PD-L1 expression when compared to cell lysates. High PD-L1 expression on LCL-derived sEV was associated with higher CD4+ and CD8+ T cell apoptosis. In patients, plasma sEV concentration was higher vs HV. Compared to sEV-CD20 level that seemed higher in patients, PD-L1 level in sEV was not different from those of HV. A high glycosylated PD-L1 level was shown in sEV from both patients and HV plasma samples, that was associated with the same inhibiting effect on activated T cells. We conclude that sEV derived from EBV-transformed B cells realize an immunosuppressive role that involved cell-cell interaction and probably at least PD-L1. Furthermore, our findings suggest the potential of circulating sEV as a source of biomarkers in DLBCL, notably to have information on immunotherapeutic target levels of parental tumor cells.

Human gene regulatory evolution is driven by the divergence of regulatory element function in both cis and trans.

Gene regulatory divergence between species can result from cis-acting local changes to regulatory element DNA sequences or global trans-acting changes to the regulatory environment. Understanding how these mechanisms drive regulatory evolution has been limited by challenges in identifying trans-acting changes. We present a comprehensive approach to directly identify cis- and trans-divergent regulatory elements between human and rhesus macaque lymphoblastoid cells using assay for transposase-accessible chromatin coupled to self-transcribing active regulatory region (ATAC-STARR) sequencing. In addition to thousands of cis changes, we discover an unexpected number (∼10,000) of trans changes and show that cis and trans elements exhibit distinct patterns of sequence divergence and function. We further identify differentially expressed transcription factors that underlie ∼37% of trans differences and trace how cis changes can produce cascades of trans changes. Overall, we find that most divergent elements (67%) experienced changes in both cis and trans, revealing a substantial role for trans divergence-alone and together with cis changes-in regulatory differences between species.

Establishment of novel cell lines that maintain the features of B cells derived from patients with neuromyelitis optica spectrum disorder.

B cells that produce anti-aquaporin-4 (AQP4) antibodies play a crucial role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis. We previously reported that naïve B (NB) cells from patients with NMOSD, unlike those from healthy controls, exhibit transcriptional changes suggesting the adoption of an antibody-secreting cell (ASCs) phenotype. CD25+ NB cells, whose numbers are increased in NMOSD patients, have a greater capacity to differentiate into ASCs than do CD25- NB cells. Here, we attempted to establish novel B cell subset cell lines from patients with NMOSD to enable molecular analysis of their abnormalities. We generated Epstein-Barr virus-immortalized lymphoblastoid cell lines (LCLs) from CD25+ NB, CD25- NB, and switched memory B (SMB) cells. All LCLs largely maintained the features of the original cell type in terms of cell surface marker expression and could differentiate into ASCs. Notably, CD25+ NB-LCLs derived from patients with NMOSD exhibited a greater capacity to differentiate into SMB-LCLs than did CD25- NB-LCLs derived from patients with NMOSD, suggesting that the established LCLs maintained the characteristics of cells isolated from patients. The LCLs established in this study are likely to be useful for elucidating the mechanism by which cells that produce anti-AQP4 antibodies develop in NMOSD.

Network pharmacology analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer's disease. / çŸ¥æ¯æ²»ç–—é˜¿å°”èŒ¨æµ·é»˜ç— ç½‘ç»œè¯ç†å­¦ç ”ç©¶åŠå®žéªŒéªŒè¯.

OBJECTIVES: To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD). METHODS: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aß1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aß deposition in the pharynx was detected by Thioflavin S staining. RESULTS: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aß1-42 (both P<0.01), inhibit the expression of ß-secretase 1 (BACE1), APP and Aß1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3ß/GSK3ß in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aß deposition. CONCLUSIONS: Anemarrhenae Rhizoma may reduce the production of Aß in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3ß pathway.

Janus kinase inhibitors ameliorate clinical symptoms in patients with STAT3 gain-of-function.

Germline gain-of-function (GOF) variants in the signal transducer and activator of transcription 3 (STAT3) gene is an inborn error of immunity presenting with autoimmunity and lymphoproliferation. Symptoms can vary widely, and no effective treatment has been established. This study investigated the efficacy of Janus kinase (JAK) inhibitors (JAKi) in patients with STAT3-GOF. Four patients were enrolled and their clinical symptoms before and after the initiation of treatment with JAKi were described. A cell stimulation assay was performed using Epstein-Barr virus transformed lymphoid cell lines (EBV-LCLs) that were derived from the patients with STAT3-GOF. The patients presented with various symptoms, and these symptoms mostly improved after the initiation of JAKi treatment. Upon interleukin-6 stimulation, the EBV-LCLs of patients showed enhanced STAT3 phosphorylation compared with those of the EBV-LCLs of healthy controls. In conclusion, four Japanese patients with STAT3-GOF were successfully treated with JAKi. JAKi ameliorated various symptoms and therefore, the use of JAKi could be an effective treatment option for patients with STAT3-GOF.