Journal: Theranostics

Article Title: Secreted Clever-1 modulates T cell responses and impacts cancer immunotherapy efficacy

doi: 10.7150/thno.110544

Figure Lengend Snippet: Circulating soluble forms of Clever-1 are enriched in cancer patients. A , Frequency distribution and B , median of sClever-1 concentration in plasma of healthy donors (HD) (n = 32, blue), patients with treatment naïve breast cancer (BC) (n = 138, magenta) and patients with various advanced solid tumors participating in the MATINS trial (n = 193, orange). One-way ANOVA with Dunnett's multiple comparison test. C , ROC-curves of plasma sClever-1 concentration in BC and MATINS patients. AUC for sClever-1 was 0.84 (CI 0.77-0.90) in BC and 0.85 (CI 0.80-0.90) in MATINS. Specificity for both cohorts at 48.7 ng/mL was 100%. D , Quantification (ELISA) of sClever-1 in mouse serum of tumor-bearing wild type (WT, n = 5), full Clever-1 knock-out (Full KO, n = 4) and macrophage specific Clever-1 knock-out (Macro KO, n = 5) mice. The signal was normalized to a pool of normal mouse serum. E , sClever-1 concentration in plasma of breast cancer patients before (pre) and three weeks after (post) mastectomy (n = 24). F , sClever-1 levels in macrophages stimulated with IFN/LPS or dexamethasone/IL4. Repeated measures One-way ANOVA (n = 3). G , sClever-1 levels in primary human liver sinusoidal endothelial cells (LSEC) treated with or without TNFα and IFNγ. Paired Student's t-test (n = 8). H , Volcano plot showing the difference in cytokines between breast cancer patients (BC, n = 42) and healthy donors (HD, n = 32). Significant differences are shown in red. Multiple Unpaired Student's t-test with Benjamini-Hochberg FDR method. I , Violin plots of the most significant cytokines that differed between BC and HD. * P < 0.05, **** P < 0.0001.

Article Snippet: The macrophages were then polarized to M1 by first adding 20 ng/mL of recombinant IFNγ for 24 h and then 100 ng/mL of LPS (Invivogen) for another 24 h. For M2 polarization, the macrophages were incubated with recombinant human IL-4 (Peprotech) and 100 nM of dexamethasone (Merck) for 48 h. Human lymphatic endothelial cells (HLEC, No.2500) were purchased from ScienCell and cultured in Endothelial Cell Medium (ECM, No.1001).

Techniques: Concentration Assay, Clinical Proteomics, Comparison, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Theranostics

Article Title: Secreted Clever-1 modulates T cell responses and impacts cancer immunotherapy efficacy

doi: 10.7150/thno.110544

Figure Lengend Snippet: sClever-1 is truncated at the C-terminus and released by serine protease activity. A-B , Electron microscopy images of immunogold labelling of Clever-1 in KG-1 cells showing outward membrane budding (A) and a tunneling nanotube-like structure (B) between two cells. Arrows point to gold particles. C-D , Western blot showing sClever-1 in conditioned media of PMA-differentiated KG-1 cells after 24 h inhibitor treatments: (E) bafilomycin (Baf), concanamycin (Con), GW4896 (GW), P1860, marimastat (Mari), untreated (Ctrl), bexmarilimab (Bex), and isotype control (hIgG4); (F) leupeptin (Leu), Bestatin (Bes), aprotinin (Apr), pepstatin A (Pep A), SID 26681509 (SID), CA-074 and E-64. E , Quantification (ELISA) of sClever-1 in vesicle fractions in KG-1 cell culture supernatant, blood and lymph separated by ultracentrifugation. Samples were normalized to 1 µg/µL of protein. The graphs represent one independent assay with three technical replicates. F , Western blot analysis of sClever-1 detected with 3-372 mouse anti-human Clever-1 antibody (parent of Bex) in vesicle and liquid fractions of plasma and cell culture supernatants of primary human macrophages (M0, no polarization; M1, IFNγ + LPS; M2, IL-4 + dexamethasone), endothelial cells (HLEC, human lymphatic endothelial cells; HPMEC, human pulmonary microvascular endothelial cells) and cell lines (KG-1 with/without PMA and HEK293). Equal loading of 10 µg of protein sample per lane was ensured by Qubit measurement of protein concentration. G , Western blot showing the abundance of a ~200 kDa sClever-1 species in healthy donors (n = 3) and MATINS patients (n = 4). # denotes the vesicle fraction. CD63 was used as a loading control. H , Mass spec analysis of Coomassie stained bands immunoprecipitated from human serum with Bex or 9-11 anti-Clever-1 antibodies and relative isotype controls hIgG4 and rIgG2a, respectively. The cartoons show Clever-1-specific peptide hits mapped to the full-length Clever-1 protein for Bex (orange) and 9-11 (magenta) pulldown. Venn diagrams depict shared peptides identified by Bex and 9-11.

Article Snippet: The macrophages were then polarized to M1 by first adding 20 ng/mL of recombinant IFNγ for 24 h and then 100 ng/mL of LPS (Invivogen) for another 24 h. For M2 polarization, the macrophages were incubated with recombinant human IL-4 (Peprotech) and 100 nM of dexamethasone (Merck) for 48 h. Human lymphatic endothelial cells (HLEC, No.2500) were purchased from ScienCell and cultured in Endothelial Cell Medium (ECM, No.1001).

Techniques: Activity Assay, Electron Microscopy, Membrane, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Clinical Proteomics, Protein Concentration, Mass Spectrometry, Staining, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcriptional Profiling of Human Brain Endothelial Cells Reveals Key Properties Crucial for Predictive In Vitro Blood-Brain Barrier Models

doi: 10.1371/journal.pone.0038149

Figure Lengend Snippet: ( A ) Expression levels of total IFN, IFNα/β and IFNγ signaling pathway genes were determined by microarray analysis for the two BEC lines. Each circle represents one gene of the indicated reactome pathway and the difference in expression between hCMEC/D3 and hpBEC cells. To compare IFN signaling reactomes between BEC lines, the values of the four replicates were averaged, and the probe-set with the highest value was used to represent each gene. The distributions of the genes within each gene set were compared with a one-sample t-test, to test whether the mean of the distributions were different from zero. ( B ) HLA class I, b2-microtublin and β actin gene expression in resting hCMEC/D3 (red bars) and hpBECs (blue bars) determined by microarray analysis. All displayed HLA class I and b2-microtublin genes were significantly (p<0.001) higher expressed in the hCMEC/D3 cells. ( C–E ) Flow cytometry analysis of surface expression of ( C & D ) HLA class I and ( E ) II molecules on hCMEC/D3 cells (red histograms, red bars) and hpBECs (blue histograms, blue bars). ( C ) The HLA class I expression levels and the mean fluorescence intensities (MFI) on both resting BEC lines, in one of four similar experiments, is presented. ( D ) The effect of IFNα upon HLA class I surface expression and ( E ) the effect of IFNγ upon HLA class II surface expression on the two BEC lines are displayed as average MFIs of three similar experiments. *p<0.05, **p<0.01 and ***p<0.001 (Student's t -test).

Article Snippet: Confluent hpBECs and hCMEC/D3 were treated with either human recombinant IFNγ (R&D Systems) or human recombinant IFNα (Roferon-A, Roche, Switzerland) (100 U/ml each) for 16 hours at 37°C before they were Flow Cytometry analysed.

Techniques: Expressing, Microarray, Gene Expression, Flow Cytometry, Fluorescence

Journal: Communications Biology

Article Title: Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing

doi: 10.1038/s42003-023-05409-6

Figure Lengend Snippet: a JunB overexpression in NCOR2-013 overexpressing THP-1 macrophages was confirmed by RT-qPCR and WB ( n = 4 biological replicates, data are presented as mean values ± SD). b , c The JunB overexpressing THP-1 macrophages and control cells were infected with T. marneffei conidia (MOI = 10) for 24 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( b ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in JunB overexpressing THP-1 macrophages and control cells ( c ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 9 biological replicates, data are presented as mean values ± SD). d , e The NCOR2-013 overexpressing THP-1 macrophages and control cells were stimulated with IFN-γ (100 ng/mL) for 24 h, and then infected with T. marneffei conidia (MOI = 10) for 24 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( d ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in IFN-γ-stimulated NCOR2-013 overexpressing THP-1 macrophages and control cells ( e ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 8 biological replicates, data are presented as mean values ± SD). f , g The NCOR2-013 overexpressing THP-1 macrophages and control cells were stimulated with LPS (500 ng/mL) for 24 h, and then were infected with T. marneffei conidia (MOI = 10) for 24 h and 48 h. The expression of TNF-α and IL-1β was detected by RT-qPCR ( f ) ( n = 3 biological replicates, data are presented as mean values ± SD). T. marneffei CFUs were detected in LPS-stimulated NCOR2-013 overexpressing THP-1 macrophages and control cells ( g ). Four gradient serial dilutions (10 0 , 10 −1 , 10 −2 , 10 −3 ) were performed ( n = 9 biological replicates, data are presented as mean values ± SD). All data are shown as mean ± SD from three independent experiments. WB were shown were the representative blot. Two-tailed Student’s t test was used to determine significance, denoted by *( P < 0.05), **( P < 0.01), and ns (not significant). Supplementary material is available (Supplementary Figs. , ; Supplementary Data ). MOI multiplicity of infection, NC negative control, NCOR2-013 OE NCRO2-013 overexpression, JunB OE JunB overexpression, Tm T. marneffei .

Article Snippet: Human recombinant IFN-γ and GM-CSF antibodies were purchased from Solarbio Science & Technology (China), and the LPS (from E. coli ) was from Sigma-Aldrich (USA).

Techniques: Over Expression, Quantitative RT-PCR, Control, Infection, Expressing, Two Tailed Test, Negative Control

Journal: Stem Cells Translational Medicine

Article Title: Safety Profile of Good Manufacturing Practice Manufactured Interferon γ‐Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

doi: 10.1002/sctm.16-0485

Figure Lengend Snippet: Residual interferon γ in cell washings and Infusion medium a

Article Snippet: Two days before the expansion was complete, cells were fed with fresh D4 culture media containing 500 U/ml GMP‐grade recombinant human IFNγ (R&D Systems, Minneapolis, MN, http://www.rndsystems.com ).

Techniques: Control

Journal: Stem Cells Translational Medicine

Article Title: Safety Profile of Good Manufacturing Practice Manufactured Interferon γ‐Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

doi: 10.1002/sctm.16-0485

Figure Lengend Snippet: Clustering of global transcriptional responses of MSCs following priming with IFNγ. (A): Principal Component Analysis (PCA) of differential gene expression of MSCs (open shapes) and γMSC (filled shapes) from each donor (same color). (B): Euclidean distance between samples to assess overall similarity among the samples. The darker blue indicates a shorter distance, more similar; lighter blue indicates greater distance, less similar. (C): Heat map analysis of RNA‐Seq gene expression data from MSCs and γMSC. The heatmap was generated using the normalized expression values for the 20 most upregulated and downregulated genes following interferon γ priming. Color code indicates relative expression; red highest, blue lowest. Abbreviation: MSCs, mesenchymal stem/stromal cells.

Article Snippet: Two days before the expansion was complete, cells were fed with fresh D4 culture media containing 500 U/ml GMP‐grade recombinant human IFNγ (R&D Systems, Minneapolis, MN, http://www.rndsystems.com ).

Techniques: Gene Expression, RNA Sequencing, Generated, Expressing

Journal: Stem Cells Translational Medicine

Article Title: Safety Profile of Good Manufacturing Practice Manufactured Interferon γ‐Primed Mesenchymal Stem/Stromal Cells for Clinical Trials

doi: 10.1002/sctm.16-0485

Figure Lengend Snippet: Soft agar colony forming assay to detect malignant transformation. Representative photographs of triplicate agar plates for each condition are shown. The positive control was a human ES‐2 ovarian clear cell carcinoma cell line. The negative control was saline. Original magnification was ×46. Abbreviations: IFNγ, interferon γ; MSCs, mesenchymal stem/stromal cells.

Article Snippet: Two days before the expansion was complete, cells were fed with fresh D4 culture media containing 500 U/ml GMP‐grade recombinant human IFNγ (R&D Systems, Minneapolis, MN, http://www.rndsystems.com ).

Techniques: Transformation Assay, Positive Control, Negative Control, Saline