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: Journal for immunotherapy of cancer

Article Title: PARP inhibitors enhance antitumor immune responses by triggering pyroptosis via TNF-caspase 8-GSDMD/E axis in ovarian cancer.

doi: 10.1136/jitc-2024-009032

Figure Lengend Snippet: Figure 5 Deficiency of GSDME blunts the immune response triggered by PAPR inhibitor in vivo. (A–L) Immunocompetent C57BL/6 mice were transplanted with wild type (WT) or Gsdme-deficient ID8 cells and challenged with niraparib for about 4 weeks. Tumors were harvested and subjected to bulk T-cell receptor (TCR) sequencing and evaluation of immune status. The frequency of T-cell clonotypes in the top 25% of TCR repertoires was shown using pie charts ((A) n=4) and quantified (B). The clonal expansion was evaluated using clonality (C). The TCR diversity was calculated by normalized Shannon diversity entropy (D). The proportion of dendritic cell differentiation (CD11c+MHCIIHigh) and maturation (CD11c+MHCIIHighCD86High) in tumors (E), lymph nodes (F), and spleens (G) was determined by flow cytometry. The proportion of CD3+ T cells and NK cells among CD45+ immune cells in tumors was determined by flow cytometry (H). The production of IFN-γ in tumor-infiltrated CD4/8+ T and NK cells was examined by flow cytometry (I). The expression of granzyme B in tumor-infiltrated CD8+ T and NK cells was evaluated by mean fluorescence intensity (J). Representative images of GSDME, CD4, CD8, and GZMB IHC staining in tumor sections (K) and numbers of indicated immune cells in a ×20 field of a microscope (L). Scale bars: 200 µm. WT or Gsdme-deficient ID8 cells were intrabursally transplanted into C57BL/6 mice and received niraparib and/or anti-PD-1 treatment. Representative bioluminescent images of mice bearing WT and Gsdme-KO ID8 tumors at the endpoint (M). The tumor weight was quantified in each mouse treated with niraparib and/or anti-PD-1 antibodies ((N) n=7). Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (B–J, L, and N).

Article Snippet: Subsequently, these isolated T cells were stimulated with anti- CD3 (BioXCell, BE0002) and anti- CD28 antibodies (BioXCell, BE0328) in the presence or absence of olaparib/niraparib.

Techniques: In Vivo, Sequencing, Cell Differentiation, Flow Cytometry, Expressing, Fluorescence, Immunohistochemistry, Microscopy

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: 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