Journal: PLOS One

Article Title: The combination of LILRB4-targeting NK cell engagers and cGAS–STING agonists enhances the anti–multiple myeloma immune activity of NK cells

doi: 10.1371/journal.pone.0339375

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) dimensionality reduction clustering identifies multiple cell subpopulations of multiple myeloma cells. (B) The Volcano plots display the gene expression profiles of each tumor cell subpopulation with significant differences compared to other subpopulations. Log2 (fold change) > 1 and adjusted P < 0.05 were considered as significantly differentially expressed genes. (C) Heatmap showing precise differences in LILRB4 expression across tumor cell subsets. (D) Density plot of LILRB4 expression in tumor cells from UMAP. Color intensity indicates the expression level. (E) Copy Number Variation Score for Tumor Subpopulation Cells. (F) Metabolic pathway enrichment analysis of differentially expressed genes in tumor cell subsets, where color intensity reflects the degree of correlation. (G) Dotplot diagrams display the expression of ligand–receptor pairs between LILRB4 + MM cells (the initiating point of communication) and other cells. (H) LILRB4-related survival analysis in MM was performed using the TCGA database, with P values calculated by the log-rank test. (I) Western blot analysis was used to detect LILRB4 protein expression levels in MM1.S and RPMI-8226 cells. (J) Flow cytometry was employed to assess the expression of LILRB4 protein in MM1.S and RPMI-8226 cells.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 hours, incubated overnight at 4°C with primary antibodies (1:1000 dilution, rabbit anti-human, Proteintech), Rabbit anti-human LILRB4 antibody was used to detect the expression of LILRB4 in MM cell lines, which used GAPDH as an internal reference.

Techniques: Gene Expression, Expressing, Western Blot, Flow Cytometry

Journal: PLOS One

Article Title: The combination of LILRB4-targeting NK cell engagers and cGAS–STING agonists enhances the anti–multiple myeloma immune activity of NK cells

doi: 10.1371/journal.pone.0339375

Figure Lengend Snippet: (A) Schematic representation of the BiKE structure. (B) Schematic representation of BiKE-mediated NK cells killing of tumors. (C) BiKE SDS-PAGE of non-reduced and reduced proteins, stained with Coomassie blue. (D) ELISA test measuring the affinity of different concentrations of BiKE to the LILRB4 antigen. (E) Elisa tests the affinity of different concentrations of BiKE to the CD16A antigen. (F) Indirect labeling Flow cytometry was used to assess the binding ability of BiKE to MM1.S, NK, and K562 cells. Goat anti human IgG (H + L) antibody labeled with FITC as the secondary antibody. (G) PBMCs were co-cultured with LILRB4 + MM1.S cells for 4 hours. The regulation of CD69 expression in NK cells by monoclonal antibodies and BiKE in the co-culture system was analyzed by flow cytometry. Data are presented as mean ± SD, *P < 0.05, ***P < 0.001, ****P < 0.0001, n = 4 compared to each other group by one-way ANOVA.

Article Snippet: The membranes were blocked with 5% nonfat dry milk for 2 hours, incubated overnight at 4°C with primary antibodies (1:1000 dilution, rabbit anti-human, Proteintech), Rabbit anti-human LILRB4 antibody was used to detect the expression of LILRB4 in MM cell lines, which used GAPDH as an internal reference.

Techniques: SDS Page, Staining, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry, Binding Assay, Cell Culture, Expressing, Bioprocessing, Co-Culture Assay

Journal: Clinical and Experimental Immunology

Article Title: Early exposure of interferon-? inhibits signal transducer and activator of transcription-6 signalling and nuclear factor ?B activation in a short-term monocyte-derived dendritic cell culture promoting 'FAST' regulatory dendritic cells

doi: 10.1111/j.1365-2249.2011.04537.x

Figure Lengend Snippet: Interferon (IFN)-γ in combination with the FAST-dendritic cell (DC) protocol generates phenotypically tolerogenic DC. Monocytes were isolated from peripheral blood and cultured without (UT-DC) or with IFN-γ (IFN-γ-DC) in the presence of interleukin (IL)-4 and granulocyte–macrophage colony-stimulating factor (GM-CSF) for 24 h. DC were then either matured with tumour necrosis factor (TNF)-α (10 ng/ml) and prostaglandin E2 (PGE2) (1 µM) for another 24 h (a: plus TNF-α and PGE2, representative of six independent experiments) or were cultured for another 24 h in the absence (b: minus TNF-α and PGE2, representative of three independent experiments. Fluorescence activated cell sorter (FACS) analysis was used to determine phenotypic markers. The following co-stimulatory and linage markers were examined: CD83, CD80, CD86, immunoglobulin (Ig)-like transcript 4 (ILT4), CD11c, DC-SIGN, CD14, human leucocyte antigen D-related (HLA-DR). Line indicates the isotype control and solid histogram shows the tested monoclonal antibody. Negative gate was set to <0·5%.

Article Snippet: Antibodies The phenotypic profile of DC was defined using the following directly conjugated monoclonal antibodies (mAb): anti-CD83-fluorescein isothiocyanate (FITC) (HB15e), anti-CD86-FITC (FUN1), anti-CD80-FITC (L307·4), anti-DC-SIGN-FITC (DCN46), anti-human leucocyte antigen D-related (HLA-DR)-phycoerythrin-cyanin-5 (PE-Cy5) (G46-6) (BD Bioscience, San Jose, CA, USA) and rat anti-human immunoglobulin (Ig)-like transcript 4 (ILT4) (42D1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a primary mAb and FITC-conjugated anti-rat IgG was used for detection.

Techniques: Isolation, Cell Culture, Fluorescence, Control