Journal: bioRxiv

Article Title: Longitudinal single-cell epitope and RNA-sequencing reveals the immunological impact of type 1 interferon autoantibodies in critical COVID-19

doi: 10.1101/2021.03.09.434529

Figure Lengend Snippet: a) Experimental set-up. Frozen PBMCs are thawed, multiplexed, stained and processed using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). b) Marker genes for each of the 11 cell types identified including CD4+ T cells (T4), CD8+ T cells (T8), gamma delta T cells (Tgd), natural killer cells (NK), B cells (B), plasmablasts (PB), classical monocytes (cM), non-classical monocytes (ncM), conventional dendritic cells (cDC), plasmacytoid dendritic cells (pDC), and CD34+ hematopoietic progeneitors (Progen). c) UMAP projections of PBMCs from donors separated by COVID-19 status and severity. Cells are colored by type. d) Boxplots (showing median, 25 th and 75 th percentile) of the percentages of T8, PB, cMs and ncMs (y-axis) by COVID-19 status and severity level on day of hospital admission (D0). Each dot represents the percentage of a specific cell type per donor. Shown statistical comparisons are between cells from all C19+ critical donors (including the anti-IFN-α2 autoantibody donors) and healthy controls, C19− donors or combined C19+ Moderate-Severe donors. Other cell types can be found in . e) Boxplots of the percentages of T8, PB, cMs, ncMs (y-axis) in COVID-19 patients over day 0, 4, 7 and 14 since hospitalization (D0, D4, D7, D14). Other cell types can be found in . f) Scatterplot of SARS-CoV2 viral titer as measured by qRT-PCR in tracheal aspirates (inverse dCT, x-axis) and percentage of plasmablasts (PB) (y-axis) as quantified in donor-matched single-cell PBMC data (R = Pearson correlation). Holm’s multiple-testing corrected, permutation-based p-values: *** p < 0.001, ** p < 0.01, * p < 0.05, ns = not significant.

Article Snippet: A DNA plasmid containing full-length cDNA sequence with a Flag-Myc tag (Origene #RC221091) was verified by Sanger sequencing and used as template in T7-promoter-based in vitro transcription/translation reactions (Promega, Madison, WI: #L1170) using [S35]-methionine (PerkinELmer, Waltham, MA; #NEG709A).

Techniques: Staining, Sequencing, Marker, Quantitative RT-PCR

Journal: Cell Reports Methods

Article Title: Generation, expansion, gene delivery, and single-cell profiling in rhesus macaque plasma B cells

doi: 10.1016/j.crmeth.2024.100878

Figure Lengend Snippet:

Article Snippet: IFN alpha 2B , Proteintech , HZ-1072.

Techniques: Enzyme-linked Immunospot, Isolation, Enzyme-linked Immunosorbent Assay, Recombinant, Software

Journal: International Journal of Molecular Sciences

Article Title: Targeting ADAR1 Restores Interferon Signaling and Enhances Immunotherapy Response in Multiple Myeloma

doi: 10.3390/ijms27156602

Figure Lengend Snippet: Single-cell transcriptomic analysis showing ADAR1 expression patterns and its association with IFN signaling in multiple myeloma. ( A ) UMAP visualization of single-cell RNA-seq data from the GSE124310 dataset showing major cell populations in the MM bone marrow microenvironment. ( B ) Feature plot showing the distribution of ADAR1 expression across different cell clusters. ( C ) Violin plot comparing ADAR1 expression levels among different immune cell populations. ( D ) Comparison of ADAR1 expression between malignant plasma cells and plasma cells from healthy donors. ( E ) Analysis of T-cell exhaustion signatures in MM samples. ( F ) Type I IFN pathway activity in malignant plasma cells stratified by ADAR1 expression levels.

Article Snippet: For IFN stimulation, recombinant human IFNα (10 ng/mL; MedChemExpress, HY- P78672 , Monmouth Junction, NJ, USA) was added to the co-culture system and incubated for 48 h. For immune checkpoint inhibition, pembrolizumab (20 μg/mL; MedChemExpress, Cat. No. HY-P9902) was applied for 48 h. Control groups received PBS or isotype control antibodies.

Techniques: Single Cell, Expressing, RNA Sequencing, Comparison, Clinical Proteomics, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting ADAR1 Restores Interferon Signaling and Enhances Immunotherapy Response in Multiple Myeloma

doi: 10.3390/ijms27156602

Figure Lengend Snippet: ADAR1 suppresses MDA5-mediated type I interferon signaling in multiple myeloma. ( A ) Comparison of A-to-G RNA editing frequency between ADAR1-high and ADAR1-low samples in the GSE110486 dataset. ( B ) RIP-qPCR analysis showing the enrichment of Alu dsRNA associated with ADAR1 and MDA5 following ADAR1 knockdown. ( C ) Western blot validation of MDA5 immunoprecipitation in the RIP assay. ( D ) Western blot analysis of IFNα and IFNβ expression after ADAR1 knockdown in U266 and H929 cells co-cultured with HS-5 stromal cells. ( E ) ELISA measurement of IFNα and IFNβ secretion following ADAR1 knockdown. ( F ) Comparison of IFNα levels in peripheral blood samples from healthy donors and MM patients. ( G ) Spearman correlation analysis between ADAR1 expression and IFNα levels in MM patient samples. ( H ) Western blot analysis of IFNα, IFNβ, and ISG15 expression following simultaneous knockdown of ADAR1 and MDA5. ( I ) ELISA measurement of IFNα and IFNβ secretion after co-silencing ADAR1 and MDA5. ( J ) Western blot analysis of ISG15 expression following combined knockdown of ADAR1 and STAT1. ( K ) ELISA analysis of IFNα and IFNβ secretion after STAT1 knockdown. ns, not significant; * p < 0.05; *** p < 0.001.

Article Snippet: For IFN stimulation, recombinant human IFNα (10 ng/mL; MedChemExpress, HY- P78672 , Monmouth Junction, NJ, USA) was added to the co-culture system and incubated for 48 h. For immune checkpoint inhibition, pembrolizumab (20 μg/mL; MedChemExpress, Cat. No. HY-P9902) was applied for 48 h. Control groups received PBS or isotype control antibodies.

Techniques: Comparison, Knockdown, Western Blot, Biomarker Discovery, Immunoprecipitation, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Targeting ADAR1 Restores Interferon Signaling and Enhances Immunotherapy Response in Multiple Myeloma

doi: 10.3390/ijms27156602

Figure Lengend Snippet: The ADAR1–MDA5–IFNα axis regulates CD8 + T-cell activity and affects the response of multiple myeloma cells to PD-1 blockade. ( A ) ELISA analysis of IFNα and IFNβ secretion in U266 monoculture and U266–HS-5 co-culture systems. ( B ) CCK-8 assay measuring the relative CD8 + T-cell proliferation in the triple co-culture system. T-cell proliferation in the NC group was normalized to 100%, and values in the remaining groups are presented relative to the NC control. ( C ) ELISA analysis of PF1 and GZMB secretion in the triple co-culture system. ( D ) Spearman correlation analysis between IFNα levels and CD8 + T-cell proliferation and cytotoxic molecule expression. ( E ) CCK-8 assay evaluating U266 cell proliferation under different experimental conditions. ( F ) Flow cytometric analysis of U266 cell apoptosis using Annexin V/PI staining. ( G ) Flow cytometric analysis of U266 cell apoptosis, including early, late, and total apoptosis. ns, not significant; * p < 0.05.

Article Snippet: For IFN stimulation, recombinant human IFNα (10 ng/mL; MedChemExpress, HY- P78672 , Monmouth Junction, NJ, USA) was added to the co-culture system and incubated for 48 h. For immune checkpoint inhibition, pembrolizumab (20 μg/mL; MedChemExpress, Cat. No. HY-P9902) was applied for 48 h. Control groups received PBS or isotype control antibodies.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, CCK-8 Assay, Control, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting ADAR1 Restores Interferon Signaling and Enhances Immunotherapy Response in Multiple Myeloma

doi: 10.3390/ijms27156602

Figure Lengend Snippet: ADAR1 inhibition enhances the antitumor efficacy of PD-1 blockade in a multiple myeloma mouse model. ( A ) Endpoint tumor volumes in the MOPC315/BALB/c mouse model across different treatment groups. ( B ) Tumor growth curves of mice treated with NC, 8-azaadenosine, PD-1 blockade, or combination therapy. ( C ) Representative TUNEL staining of tumor sections showing apoptotic cells (red) with DAPI nuclear counterstaining (blue). ( D ) Western blot analysis of IFNα protein expression in tumor tissues. ( E ) Immunohistochemical staining showing CD8 + T-cell infiltration in tumor tissues. ** p < 0.01.

Article Snippet: For IFN stimulation, recombinant human IFNα (10 ng/mL; MedChemExpress, HY- P78672 , Monmouth Junction, NJ, USA) was added to the co-culture system and incubated for 48 h. For immune checkpoint inhibition, pembrolizumab (20 μg/mL; MedChemExpress, Cat. No. HY-P9902) was applied for 48 h. Control groups received PBS or isotype control antibodies.

Techniques: Inhibition, TUNEL Assay, Staining, Western Blot, Expressing, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Targeting ADAR1 Restores Interferon Signaling and Enhances Immunotherapy Response in Multiple Myeloma

doi: 10.3390/ijms27156602

Figure Lengend Snippet: Proposed model of ADAR1-mediated immune evasion in multiple myeloma. ADAR1 promotes A-to-I editing of endogenous dsRNA, thereby limiting its recognition by the innate immune sensor MDA5. Suppression of the MDA5–MAVS pathway attenuates type I interferon signaling, resulting in reduced IFN-α production and impaired downstream JAK/STAT activation. Consequently, CD8 + T-cell function is compromised, contributing to the establishment of an immunosuppressive bone marrow microenvironment.

Article Snippet: For IFN stimulation, recombinant human IFNα (10 ng/mL; MedChemExpress, HY- P78672 , Monmouth Junction, NJ, USA) was added to the co-culture system and incubated for 48 h. For immune checkpoint inhibition, pembrolizumab (20 μg/mL; MedChemExpress, Cat. No. HY-P9902) was applied for 48 h. Control groups received PBS or isotype control antibodies.

Techniques: Activation Assay, Cell Function Assay

Journal: Cell Communication and Signaling : CCS

Article Title: STING-STAT1-ZBP1 axis orchestrates PANoptotic signaling in ischemia-reperfusion induced acute kidney injury

doi: 10.1186/s12964-026-02830-2

Figure Lengend Snippet: STING and IFNAR Pathways Cooperatively Promote STAT1 Activation Drives PANoptosis in Renal Tubular Epithelial Cells . A - B Primary renal tubular epithelial cells (PRTCs) were treated with DMSO (control), diABZI (STING agonist), or diABZI and Anifrolumab (IFNAR inhibitor). Representative Western blot analysis and quantification of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) and ZBP1 in PRTCs (n=3 α-Tubulin loading control). Red separator lines indicate different experimental conditions. C Primary renal tubular epithelial cells (PRTCs) were treated with DMSO, diABZI, or diABZI and Anifrolumab. Flow cytometry quantification of cell death (PI⁺, V610). D - E PRTCs were treated with DMSO, diABZI, or diABZI (10 μM) and IFN-α (1 ng/ml). Western blot analysis and quantification of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) in each group (n=3; α-Tubulin loading control). Red separator lines indicate different experimental conditions. F Proposed mechanism of the STING-STAT1-ZBP1 axis in renal tubular epithelial cells promoting pan-apoptosis in IRI-AKI. Following IRI-AKI, STING interacts with and activates STAT1, facilitating its nuclear translocation. STAT1 binds to the promoter region of the ZBP1 gene, initiating its transcription. The upregulated ZBP1 subsequently triggers the activation of PANoptosis, which drives the progression of AKI

Article Snippet: Primary cells were treated with diABZI (medchemexpress-HY-112921A), Cocl2 (sigma-232696), Fludarabine (medchemexpress-HY-B0069), Anifrolumab (medchemexpress-HY- P99168 ), IFN-α (medchemexpress-HY-P7023), Necrostatin-1 (medchemexpress-HY-15760), Z-VAD-FMK (medchemexpress-HY-16658B), MCC950 (medchemexpress-HY-12815), 2’3’-cGAMP (c-GAMP: tlrl-nacga23-02) for 24–48 h.

Techniques: Activation Assay, Control, Western Blot, Flow Cytometry, Translocation Assay