Journal: Cancer Research

Article Title: A Novel Small Molecule Inhibitor of Signal Transducers and Activators of Transcription 3 Reverses Immune Tolerance in Malignant Glioma Patients

doi: 10.1158/0008-5472.can-07-1243

Figure Lengend Snippet: Figure 4. WP1066 enhances phosphorylation of intracellular signaling molecules. Monocytes and Tcells isolated from the PBMCs of GBM patients were incubated with either the medium or medium supplemented with 5 Amol/L WP1066. After 2 h, monocytes and T cells were stimulated for 5 min with 2 Ag/mL LPS and 5 Ag/mL anti-CD3 antibody, respectively. Subsequently, cells were lysed, electrophoretically fractionated in 8% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with anti-phosphotyrosine monoclonal antibody 4G10. A, for monocyte membranes, subsequently the same membrane was stripped and reblotted with antibodies to phosphorylated p72Syk (Tyr352), Syk, phosphorylated Hck, Hck, and Lyn. B, for the T-cell membranes, after stripping, the membrane was reblotted with antibodies to phosphorylated ZAP-70 (Tyr319), ZAP-70, p56-Lck, and h-actin.

Article Snippet: T cells from the same patients were isolated with human CD3 microbeads and a magnetic separation column (LS MACS column, Miltenyi Biotec) with the exception of one patient who lacked sufficient PBMCs for both monocyte and T-cell isolations.

Techniques: Phospho-proteomics, Isolation, Incubation, Membrane, Stripping Membranes

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: Journal for Immunotherapy of Cancer

Article Title: TIM-3 blockade enhances ex vivo stimulated allogeneic NK cell therapy for relapsed murine neuroblastoma after hematopoietic cell transplant

doi: 10.1136/jitc-2024-010239

Figure Lengend Snippet: 15–4P Stimulation increases NKG2D expression and murine NK cell proliferation compared with IL-15/IL-15Rα stimulation alone. ( A ) Schematic showing AgN2a-4P and NK cell receptor-ligand interactions. B6 NK cells are isolated and cultured with IL-15/IL-15Rα conjugate and with or without irradiated AgN2a-4P cells at a 1:1 ratio for 12 days. All expression markers were examined on CD3 − NK1.1 + NK cells. ( B ) Dot plots of NKG2D (top) and NKp46 (bottom) on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with percentage of gated cells. ( C ) Per cent of NK cells positive for NKG2D (left) and NKp46 (right) and MFI below. ( D ) Fold change of IL-15 NK cells and 15–4P stimulated NK cell expansion at day 12 compared with day 0. ( E ) Dot plots of proliferation marker Ki-67 on IL-15 NK cells (left) and 15–4P stimulated NK cells (right) are shown with ( F ) percentage and MFI. ( G ) Percentage and MFI of TRAIL (top) and Fas-L (bottom) were assessed for each group. Data are representative of an experiment that was replicated two times. Representative dot plots examples of one replicate out of a minimum of three replicates. All bar graphs show individual experimental replicates plotted with mean and SEM (n=3–5). Two-sided two-sample t-tests were performed where indicated (*p<0.05, **p<0.01, ***p<0.001, ns=not significant). AgN2a, aggressive variant of the Neuro-2a murine neuroblastoma cell line; MFI, median fluorescence intensity; NK, natural killer.

Article Snippet: T cells were depleted using a CD3 MicroBead Kit (Miltenyi Biotec) by negative selection on the AutoMACs Pro Separator (Miltenyi Biotec) at day+0.

Techniques: Expressing, Isolation, Cell Culture, Irradiation, Marker, Variant Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD3 , REA1151 , 50 , 130-120-267 , FITC (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Journal of Translational Medicine

Article Title: Niraparib exhibits a synergistic anti-tumor effect with PD-L1 blockade by inducing an immune response in ovarian cancer

doi: 10.1186/s12967-021-03073-0

Figure Lengend Snippet: The information of antibodies used for western blot, immunohistochemistry, and flow cytometry

Article Snippet: CD3 and isotype , Human , 5 μl of antibody per test , Elabscience, E-AB-F1001E , Flow cytometry.

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Association of postpartum thyroid dysfunction with antepartum hormonal and immunological changes.

doi: 10.1210/jc.2002-021219

Figure Lengend Snippet: FIG. 1. Graphical output from the FACScan analyzer (Becton Dickinson and Co.). Before analysis lymphocytes were stained with R-phyco- erythrin-Cy5 mouse antihuman CD3 fluorescein isothiocyanate-conjugated mouse antihuman CD4 together with either phycoerythrin- conjugated rat antihuman IL-4 or phycoerythrin-conjugated mouse antihuman IFN-. The output was gated on the basis of side scatter and CD3 staining. Shown are control cells in the left panels and stimulated cells in the right panels. Lymphocytes gated for right-angle light scatter and CD3 staining (a and b), CD4 expression (c and d), IL-4 expression (c–f), and IFN- expression (e and f) are shown.

Article Snippet: The culture plate was placed in a humidified incubator at 37 C, gas phase 5% CO2 in air, for 4 h. Aliquots (350 l) from the stimulated and unstimulated wells were transferred into fluorescenceactivated cell sorter (FACS) tubes and the surface antigen (CD3 and CD4) was stained by adding 5 l conjugated antibody (R-phycoerythrin-Cy5 conjugated mouse antihuman CD3, DAKO Corp. Ltd., Ely, UK; fluorescein isothiocyanate-conjugated mouse antihuman CD4, Serotec Ltd., Oxford, UK) and incubated in the dark for 30 min.

Techniques: Staining, Control, Expressing