Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a&b) Neutrophils (4 × 10 5 /ml) from healthy donors were incubated with SARS-CoV-2 (MOI = 1) with or without autologous platelets (4 × 10 6 /ml) for 5 h at 37 °C. The scale bar is 10 μm. The detailed structure of SARS-CoV-2-induced NET formation was observed under a confocal microscope (Leica). NET formation was visualized by fluorescent staining of DNA (blue), histone (green), and MPO (red) (a) . NETs level was measured by MetaMorph software and presented as Cit-H3 area (mm 2 ) (b) . (c) Human neutrophils (4 × 10 5 /ml) were pretreated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 (MOI = 0.1 and 1) in the presence or absence of platelets (4 × 10 6 /ml) for 5 h and 20 h. The level of NET formation was determined by histone area (μm 2 ). (d) Neutrophils (4 × 10 5 /ml) from WT, clec5a -/- tlr2 -/- , and clec5a -/- tlr2 -/- mice were incubated with SARS-CoV-2 (MOI = 1) in the presence or absence of WT platelets (4 × 10 6 /ml) for 5 h at 37 °C. (e) Human neutrophils were pre-treated with anti-hCLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or combination of both antibodies for 30 min at room temperature, followed by incubation with SARS-CoV-2 spike pseudotyped virus (MOI = 0.1) in the presence or absence of autologous platelets (4 × 10 6 /ml) for 3 h. Data are mean ± SEM and repeats of 3 to 5 independent experiments. *p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t-test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Incubation, Microscopy, Staining, Software, Virus

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: (a) EVs from healthy controls (HCs-EVs, n=5) and COVID-19 patients (COVID19-EVs, n=5) were harvested by ultracentrifugation, then lysed in RIPA solution before subjected to mass spectrometry analysis. Proteins expressed in COVID-19 EVs, but not in HCs EVs, were further analyzed using the QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA) software. Proteins which were expressed in all the COVID19-EVs were displayed. (b&c) HCs-EVs (n=10) and COVID19-EVs (n=10) were analyzed by flow cytometry, and markers highly activated in COVID-19 platelets were expressed as a heat map (b) or by mean fluorescence intensity (c) . (d) Neutrophils were pre-incubated with anti-CLEC5A mAb (3E12A2, 100 μg/ml), anti-TLR2 mAb (# MAB2616, 100 μg/ml), or both anti-CLEC5A mAb (3E12A2, 100 μg/ml) and anti-TLR2 mAb (# MAB2616, 100 μg/ml), for 30 min at room temperature, followed by incubation with EVs (1 μg/ml) from COVID-19 patients (n=6) at 37°C for 3 h. Data are mean ± sd and repeats of at least three independent experiments. * p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Mass Spectrometry, Software, Flow Cytometry, Fluorescence, Incubation

Journal: bioRxiv

Article Title: CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation

doi: 10.1101/2022.02.01.478701

Figure Lengend Snippet: C57BL/6 mice (WT) (n=3) and clec5a -/- tlr2 -/- mice (n=3)were inoculated with AAV-hACE2 for 14 days, followed by intranasal inoculation of SARS-CoV-2 (8 × 10 4 PFU/per mice). Tissues were collected at 3 days and 5 days post-infection. (a) The level of proinflammatory cytokines and chemokines were measured by real-time PCR and presented as fold change (compared to AAV-hACE2 uninfected mice/mock). ( b-d) NET structure and thrombus were detected by Hoechst. 33342 (blue), anti-MPO antibody (green), anti-citrullinated histone H3 (red), anti-CD42b antibody (yellow) (b) , and images were captured by a confocal microscope and subjected to determine the area of MPO (c) and CD42b (d) using MetaMorph TM software. (e) Cell infiltrated to lung. Interstitial macrophage (interstitial MΦ) was defined as CD11b + CD64 + F4/80 + cells; monocyte-derived dendritic cell (DC)/macrophage (MΦ) was defined as CD11b + CD64 + Ly6C + ; Ly6C + monocyte was defined as Ly6C + . The cell number of each cell population was calculated using the multiple fluorescent staining image and analyzed by software MetaMorph TM , and the data was presented as cell number/ per 664225 (815 × 815). Scale bar is 200 μm. Data are represented as mean ± SEM. * p <0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t -test).

Article Snippet: For blocking assay, neutrophils were preincubated with isotype (100 μg/ml), anti-CLEC5A mAb (100 μg/ml, clone 3E12A2), anti-TLR2 mAb (100 μg/ml, R&D system), or a mixture of anti-CLEC5A mAb and anti-TLR2 mAb for 30 min at room temperature before incubation with SARS-CoV-2.

Techniques: Infection, Real-time Polymerase Chain Reaction, Microscopy, Software, Derivative Assay, Staining

Journal: Chemical Reviews

Article Title: ImmunoPET: Concept, Design, and Applications

doi: 10.1021/acs.chemrev.9b00738

Figure Lengend Snippet: Figure 20. ImmunoPET imaging of ovarian cancers with a bispecific radiotracer 89Zr-DFO-REGN4018. (a) 89Zr-DFO-REGN4018 immu- noPET/CT imaging of humanized tumor-bearing mice showed the distribution of the tracer to the spleen (yellow arrow), lymph nodes (green arrow), and tumor (red arrow). (b) Blocking with a MUC16 parental antibody reduced the tumor uptake of 89Zr-DFO-REGN4018 without influencing the spleen and lymph node uptake. (c) Blocking with an anti-CD3 antibody substantially reduced the spleen and lymph node uptake of 89Zr-DFO-REGN4018 without influencing the tumor uptake. Reproduced with permission from ref 604. Copyright 2019 American Association for the Advancement of Science.

Article Snippet: ImmunoPET imaging with a 89Zr-labeled antimouse CD3 antibody (clone 17A2; R&D Systems) revealed a correlation between high tumor uptake of the radiotracer and better therapeutic response of anticytotoxic T lymphocyte antigen 4 (CTLA-4) therapy in a preclinical colorectal cancer model.788 However, significant liver accumulation of the radiotracer was found on immunoPET images, which was explained as liver clearance of the radiolabeled antibody.

Techniques: Imaging, Blocking Assay

Journal: Science translational medicine

Article Title: Human pluripotent stem cell-derived erythropoietin-producing cells ameliorate renal anemia in mice.

doi: 10.1126/scitranslmed.aaj2300

Figure Lengend Snippet: Fig. 8. Therapeutic effects of the transplantation of hiPSC- EPO–producing cells on renal anemia in adenine-treated mice. Renal anemia was in- duced using adenine treatment (50 mg/kg body weight daily for5weeks)inimmunodeficient mice (NOD.CB17-Prkdcscid/J mice). Twenty aggregates of hiPSC-EPO cells (5.0 × 105 cells per aggregate) were trans- planted into the kidney sub- capsules of mice with renal anemia. (A) Hematocrit was ex- amined during the first 4 weeks after transplantation using glass capillary tubes. (B) Human EPO concentrations in mouse serum at 4 weeks after transplanta- tionweremeasuredusingELISA. (C) Hematocrit was examined for up to 28 weeks after trans- plantation. The gray shaded areas in (A) and (C) indicate the normal hematocrit range in NOD.CB17-Prkdcscid/J mice. (D) Human EPO concentrations in mouse serum after trans- plantation were measured using ELISA. (E) The hiPSC-EPO– producing cell grafts were eval- uated using immunohisto- chemistry for EPO (green), AFP (red) and ALBUMIN (red) and using H&E staining. (F) The new vasculature in the grafts derived from host mice was examined by anti-mouse CD31/ PECAM-1 immunostaining and H&E staining. (G) The human EPO concentrations in host mouse serum after phlebotomy were measured using ELISA. The data from three independent experiments are means ± SEM; n = 6 for hiPSC-EPO–producing cells and saline in (A) and (B). The data from two independent experiments are means ± SEM; n = 4 for hiPSC-EPO cells and sa- line in (C), (D), and (G). *P < 0.05 versus control; ANOVA with Bonferroni’s test (A, C, D, and G) and Student’s t test (B). Scale bars, 40 mm (E) and 20 mm (F).

Article Snippet: The samples were incubated overnight at 4°C with the following primary antibodies: anti-EPO (Santa Cruz Biotechnology), AFP (Sigma-Aldrich), albumin (Bethyl Laboratories), HNF-1b (Santa Cruz 12 of 15 by guest on S eptem ber 27, 2017 http://stm .sciencem ag.org/ D ow nloaded from Biotechnology), HNF-4 (Santa Cruz Biotechnology), SALL4 (Abcam), GATA4 (Santa Cruz Biotechnology), CK19 (Dako), Ki67 (BD Biosciences), CD31 (Dianova), HIF-1a (Sigma-Aldrich), and HIF-2a (Novus Biologicals).

Techniques: Transplantation Assay, Capsules, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Derivative Assay, Immunostaining, Saline, Control

Journal: bioRxiv

Article Title: Dysregulated lymphocyte localization in idiopathic multicentric Castleman disease

doi: 10.64898/2025.12.12.693846

Figure Lengend Snippet: (A) UMAP of B cell sub-cluster highlighting GC B cells. (B) Quantification and comparison of total GC B cells in Infl. CTRL (n=4) and iMCD-TAFRO (n=4). (C) IF images of GCs using B cell marker CD20 and (D) quantification of cells within the GC. p-value is as indicated. (E) UMAP projection of T/NK cell sub-cluster indicating Tfh-like cells in Infl CTRL and iMCD-TAFRO and (F) quantification. (G) Within the Tfh-like cell cluster, differential gene expression comparing iMCD-TAFRO to Infl. CTRL indicates a down-regulation of GC-Tfh genes (grey) and up-regulation of anti-migration-associated genes (red) in iMCD-TAFRO Tfh-like cells. (H) GC-Tfh enrichment scores were applied to the Tfh-like subcluster in both groups. (I) Quantification of the average GC-Tfh activity scores per patient. (J) Representative IF images of GC-Tfh cells that express CD3, PD1, and BCL6 within GCs and (K) quantification. Dots in bar graphs represent individual patient samples. *p<0.05.

Article Snippet: Histology sections (5 um) of FFPE tissue from each sample was cut fresh and stained with antibodies against CD3 (Novus # NBP2-54392AF532) and CD20 (Novus # NBP2-47840AF594) and nuclear dye Sytox13.

Techniques: Comparison, Marker, Gene Expression, Migration, Activity Assay

Journal: Frontiers in immunology

Article Title: Intracellular Lipid Accumulation Drives the Differentiation of Decidual Polymorphonuclear Myeloid-Derived Suppressor Cells via Arachidonic Acid Metabolism.

doi: 10.3389/fimmu.2022.868669

Figure Lengend Snippet: FIGURE 3 | Induced lipid accumulation and the enrichment of fatty acid metabolism after exposure to decidual explant supernatant (DES). To evaluate the functional characteristics of DES-conditioned circulating neutrophils, CD3/CD28-stimulated T cells were cocultured with circulating neutrophils from healthy donors either untreated or treated with 50% (v/v) DES at a ratio of 2:1 for 3.5 days. The proportion of proliferative CD4+ T cells or CD8+ T cells (n = 6) (A) and the percentage of IFN-g-expressing CD3+ T cells or CD8+ T cells (n = 6) (B) were analyzed. (C, D) Increased free fatty acid uptake (BODIPY FL C16) (n = 6) and intracellular lipid accumulation (BODIPY 493/503) (n = 5) were detected after exposure to DES. (E) GO terms associated with fatty acid metabolism in circulating neutrophils were upregulated after DES treatment (n = 3). (F) GSEA showing enrichment of the fatty acid metabolism gene set in the DES-condition. (G) Heatmap showing the differentially expressed genes related to lipid metabolism. All data are represented as mean ± SD. Significance was determined by the unpaired Student’s t-test. ****p < 0.0001, *p < 0.05. FMO, fluorescent minus one.

Article Snippet: An Fc receptor blocking solution (BioLegend, San Diego, CA, USA) was added prior to staining with the following antibodies: CD45, CD11b, CD66b, pSTAT3 (BD Biosciences, San Jose, CA, USA), CD33, HLA-DR, CD15, CD3, CD4, CD8, arginase 1 (ARG1), IL6Ra, IFN-g (BioLegend), inducible nitric oxide synthase (iNOS) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and FABP5 (R&D Systems).

Techniques: Functional Assay, Expressing

Journal: Frontiers in immunology

Article Title: Intracellular Lipid Accumulation Drives the Differentiation of Decidual Polymorphonuclear Myeloid-Derived Suppressor Cells via Arachidonic Acid Metabolism.

doi: 10.3389/fimmu.2022.868669

Figure Lengend Snippet: FIGURE 5 | Immunosuppressive induction of circulating neutrophils after IL6 + AA treatment. (A) Flow cytometry was used to detect the expression of CD11b on neutrophils from healthy donors after stimulation with IL6 and AA (n = 8). (B) The expression of CD11b in CD15+ dMDSCs and autologous pN (n = 13). (C) The expression of immunosuppressive regulatory molecules, including iNOS (n = 10), ROS (n = 9), and ARG1 (n = 7), in neutrophils from healthy donors after IL6 + AA treatment. Neutrophils were previously treated with control medium, IL6+AA, or 50% (v/v) DES for 16 h. CD3/CD28-stimulated T cells were cocultured with treated neutrophils at a ratio of 2:1 for 3.5 days. Functional characteristics were then evaluated through the proportion of proliferative CD4+ T cells or CD8+ T cells (n = 6) (D) and the percentage of IFN-g-expressing CD3+ T cells or CD8+ T cells (n = 6). (E) All data are represented as mean ± SD. Significance was determined by the unpaired Student’s t-test or by one-way ANOVA with Tukey’s post-hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, NS, not significant. FMO, fluorescent minus one.

Article Snippet: An Fc receptor blocking solution (BioLegend, San Diego, CA, USA) was added prior to staining with the following antibodies: CD45, CD11b, CD66b, pSTAT3 (BD Biosciences, San Jose, CA, USA), CD33, HLA-DR, CD15, CD3, CD4, CD8, arginase 1 (ARG1), IL6Ra, IFN-g (BioLegend), inducible nitric oxide synthase (iNOS) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and FABP5 (R&D Systems).

Techniques: Flow Cytometry, Expressing, Control, Functional Assay

Journal: Frontiers in immunology

Article Title: Intracellular Lipid Accumulation Drives the Differentiation of Decidual Polymorphonuclear Myeloid-Derived Suppressor Cells via Arachidonic Acid Metabolism.

doi: 10.3389/fimmu.2022.868669

Figure Lengend Snippet: FIGURE 6 | Arachidonic acid metabolism mediated intracellular lipid accumulation and the differentiation of PMN-MDSCs. (A) The increased lipid accumulation in circulating neutrophils stimulated by IL6+AA was blocked by a COX-2 inhibiter, rofecoxib (n = 6). (B) PGE2 secretion from the neutrophils of healthy donors was also inhibited by rofecoxib (n = 8). (C) Neutrophils were treated with control medium, IL6 + AA, IL6 + AA with rofecoxib, 50% (v/v) DES, or 50% (v/v) DES with rofecoxib for 16 h. Then, CD3/CD28-stimulated T cells were cocultured with treated neutrophils at a ratio of 2:1 for 3.5 days. The induced suppressive activity by IL6 + AA and DES were both blocked by rofecoxib (n = 6). All data are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s post-hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. NS, not significant. FMO, fluorescent minus one.

Article Snippet: An Fc receptor blocking solution (BioLegend, San Diego, CA, USA) was added prior to staining with the following antibodies: CD45, CD11b, CD66b, pSTAT3 (BD Biosciences, San Jose, CA, USA), CD33, HLA-DR, CD15, CD3, CD4, CD8, arginase 1 (ARG1), IL6Ra, IFN-g (BioLegend), inducible nitric oxide synthase (iNOS) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and FABP5 (R&D Systems).

Techniques: Control, Activity Assay

Journal: Frontiers in immunology

Article Title: Intracellular Lipid Accumulation Drives the Differentiation of Decidual Polymorphonuclear Myeloid-Derived Suppressor Cells via Arachidonic Acid Metabolism.

doi: 10.3389/fimmu.2022.868669

Figure Lengend Snippet: FIGURE 8 | IL6 promoted the differentiation of PMN-MDSCs via STAT3 phosphorylation. (A) Lipid accumulation in circulating neutrophils from healthy donors treated with control medium, IL6 + AA, IL6 + AA with STAT3 inhibitor, 50% (v/v) DES, or 50% (v/v) DES with STAT3 inhibitor (n = 6). (B) The uptake of fatty acid in circulating neutrophils treated with control medium, IL6 + AA, or IL6 + AA with STAT3 inhibitor was evaluated by flow cytometry C16 (n = 5). (C) IL6 stimulated the STAT3 phosphorylation in neutrophils. (D) The upregulation of FABP5 expression stimulated by IL6 + AA was blocked by a STAT3 inhibitor (n = 5). (E) The secretion of PGE2 stimulated by IL6 + AA was inhibited after STAT3 blockade (n = 7). (F) Neutrophils were previously treated with control medium, IL6 + AA, and IL6 + AA with a STAT3 inhibitor for 16 h. Then, CD3/CD28-stimulated T cells were cocultured with treated neutrophils at a ratio of 2:1 for 3.5 days. The proliferation of CD4+ T cells or CD8+ T cells was restored after STAT3 blockade (n = 6). (G) The ability to produce IFN-g in CD3+ T cells or CD8+ T cells increased under the action of a STAT3 or FABP5 inhibitor (n = 6). All data are represented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s post-hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant. FMO, fluorescent minus one.

Article Snippet: An Fc receptor blocking solution (BioLegend, San Diego, CA, USA) was added prior to staining with the following antibodies: CD45, CD11b, CD66b, pSTAT3 (BD Biosciences, San Jose, CA, USA), CD33, HLA-DR, CD15, CD3, CD4, CD8, arginase 1 (ARG1), IL6Ra, IFN-g (BioLegend), inducible nitric oxide synthase (iNOS) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and FABP5 (R&D Systems).

Techniques: Phospho-proteomics, Control, Cytometry, Expressing