Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Hepatic MTH1+CD3+ T cells correlated with disease activity in AIH. (A) Representative confocal staining of CD3 (red), MTH1 (green), and DAPI (for nuclei in blue) (magnification ×400) in the liver of patients with AIH. Scale bar, 20 μm. (B) Number of MTH1+CD3+ T cells in portal areas was positively correlated with degree of hepatic inflammation but showed no clear link with fibrosis stages in AIH. (C) Number of MTH1+CD3+ T cells in portal areas had a significant positive correlation with levels of serum ALT and AST in patients with AIH. (D) Number of MTH1+CD3+ T cells in portal areas was positively correlated with levels of serum ALP and GGT. Bars reflect the mean ± SEM. Abbreviations: DAPI, 4 0, 6‐diamidino‐2‐phenylindole; hpf, high‐power field.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: Activity Assay, Staining

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib significantly inhibited T‐cell proliferation in human T cells in vitro . Isolated human CD3+ T cells were cultured with/without anti‐CD3/CD28 beads for 72 hours. Representative western blot analyses of MTH1 72 hours after anti‐CD3/CD28 beads stimulation. (B,C) Isolated T cells were activated with anti‐CD3/CD28 beads with or without 2 μM karonudib for 72 hours. The percentage of CD25+ and CD69+ T cells was determined on day 3 by flow cytometry. (D) Analysis of T‐cell number treated with karonudib for 72 hours after anti‐CD3/CD28 beads stimulation. (E) Statistical analysis of the T‐cell proliferation assay treated with/without 2 μM karonudib for 72 hours from HCs and patients with AIH who were treatment naive. (F) Representative western blot analyses of P53, P21, P27, CDK2, and cyclin E 72 hours after anti‐CD3/CD28 beads stimulation. The GAPDH blot was used as a loading control. Data are from one experimental representative of at least three independent experiments and represent triplicate wells. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Cell Culture, Western Blot, Flow Cytometry, Proliferation Assay, Control

Journal: Hepatology Communications

Article Title: MutT Homolog 1 Inhibitor Karonudib Attenuates Autoimmune Hepatitis by Inhibiting DNA Repair in Activated T Cells

doi: 10.1002/hep4.1862

Figure Lengend Snippet: Karonudib rendered activated T cells more susceptible to DNA damage in vitro . (A) Representative fields corresponding to each treatment were photographed. Isolated human T cells were activated with/without anti‐CD3/CD28 beads in the presence of DMSO or 2 μM karonudib for 72 hours. The alkaline comet assay was conducted and nucleoids were visualized by epifluorescence microscopy using a fluorescein isothiocyanate filter. (B) Quantification of comet tail moment. Values represent mean ± SEM from three independent experiments (100 comets per experiment). (C) Levels of cleaved‐PARP and phosphorylated histone H2AX (γ‐H2AX) were determined by immunoblot analysis. The GAPDH blot was used as a loading control. (D,E) Intracellular flow cytometry assessment of annexin V and propidium iodide expression in anti‐CD3/CD28 beads‐stimulated CD4+ and CD8+ T lymphocytes treated with 2 μM karonudib or DMSO after 72 hours. Data are from one experimental representative of at least three independent experiments. Bars reflect the mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Abbreviations: DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

Article Snippet: Confocal laser scanning microscopy was used for the detection of costaining markers, as described. ( ) Briefly, after antigen retrieval, liver samples were incubated with donkey serum for 30 minutes before being incubated with primary antibodies MTH1 (ab200832; Abcam), CD3 (60181‐1‐Ig; Proteintech), CD4 (ab67001; Abcam), and CD8 (ab17147; Abcam) at 4°C overnight.

Techniques: In Vitro, Isolation, Alkaline Single Cell Gel Electrophoresis, Epifluorescence Microscopy, Western Blot, Control, Flow Cytometry, Expressing

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 1 week in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) 7–9 days before harvest (n = 5 pairs from four experiments). (A–D) Response curves. Restriction response to various concentrations of PGF2α (A) and relaxation response curves for nitroprusside (B), bradykinin (C), or substance P (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. mN, milliNewtons. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD45, and HLA-DR. The staining for human CD45 is indistinguishable from that for human CD3 (data not shown). Original magnification, ×200.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of allogeneic T cells on arterial graft function at 2 weeks in vivo. Transplanted human arterial segments were recovered from mice injected with saline (open squares) or PBMCs (filled squares) for 13–15 days (n = 4–7 pairs from four to six experiments). (A–D) Response curves. Constriction response to various concentrations of PGF2α (A) and relaxation response to bradykinin (B), nitroprusside (C), or YC-1 (D) after preconstriction with PGF2α. *P < 0.05; **P < 0.01; #P < 0.001 vs. saline control. (E) Immunohistochemistry of graft sections stained for human and murine (inset) CD31, human CD3, and iNOS. iNOS expression was highly localized to infiltrating T cells (arrows). Original magnification: CD31 and CD3 panels, ×200; iNOS panels, ×400.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Expressing

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of IFN-γ neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti–IFN-γ mAb (filled triangles) or control mAb (IgG2a) (open triangles) for 2 weeks (n = 5 matched triplicates from three experiments). (A and B) Endothelium-independent responses to various concentrations of PGF2α (A) and nitroprusside (B). (C and D) Endothelium-dependent response to bradykinin before (C) and after (D) treatment with L-NAME. *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG2a group, n = 3–5. (E) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. The luminal HLA-DR–positive cells are also human CD31–positive (data not shown). Goat anti-mouse secondary Ab alone did not stain the grafts. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (F and G) RNA transcript levels of iNOS (F) and eNOS (G) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Immunohistochemistry, Staining, Quantitative RT-PCR

Journal:

Article Title: T cell-mediated vascular dysfunction of human allografts results from IFN-? dysregulation of NO synthase

doi: 10.1172/JCI200421767

Figure Lengend Snippet: Effects of TNF neutralization on T cell–mediated graft dysfunction in vivo. Transplanted human arterial segments were recovered from mice injected with saline alone (open squares) or PBMCs together with anti-TNF mAb (filled triangles) or control mAb (IgG1) (open triangles) for 2 weeks (n = 4 matched triplicates from two experiments). (A–C) Concentration-dependent responses to PGF2α (A), nitroprusside (B), and bradykinin (C). *P < 0.05; **P < 0.01; #P < 0.001 vs. PBMC + IgG1 group; n = 3–4. (D) Immunohistochemistry of graft sections stained for human CD3 and HLA-DR. Original magnification: CD3 panels, ×200; HLA-DR panels, ×400. (E and F) RNA transcript levels of iNOS (E) and eNOS (F) in whole tissue sections analyzed by quantitative RT-PCR.

Article Snippet: Immunostaining was performed as described ( 14 ) using the following primary mAb’s: mouse anti–human CD31, mouse anti–human HLA-DR (α-chain), and mouse anti–human CD45 (all from DAKO Corp.); mouse anti–human CD3 (R&D Systems Inc.); mouse anti-iNOS (Transduction Laboratories); and rat anti–mouse CD31 (BD Biosciences).

Techniques: Neutralization, In Vivo, Injection, Saline, Control, Concentration Assay, Immunohistochemistry, Staining, Quantitative RT-PCR

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: 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: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: Ten-day expansion of NK cells from PBMCs with NK Cloudz and GMP human platelet lysate cultured in the G-Rex6M. (A) Flow cytometer density plots at 0, 7, and 10 days compare CD3 and CD56 expression of the expanded cell population. Black arrows indicate CD3 − CD56 + (NK) cells, which correspond with the green boxes in (B) the plot of cell distribution of viable CD45 + cells. (C) Viable NK cell fold change relative to day 0. (D) The viability of two populations: the light gray bars represent the viability of all CD45 + cells (including NK cells) and the dark gray bars represent the viability of only the CD45 + CD3 − CD56 + (NK) population. The graphs are broken out by donor (Dn A, Dn B, Dn C, and Dn D) to highlight the donor variability in the process.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques: Cell Culture, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: Comparison of the xeno-free 10% GMP human platelet lysate/NK Cloudz protocol to the existing FBS/NK Cloudz protocol in the G-Rex6M. The results show (A) the cell distribution on day 10, (B) NK (CD3 − CD56 + ) fold change between days 0 and 10, (C) the viability of either all CD45 + cells or NK cells alone on day 10, and (D) the percent of target K562 cells killed on day 10 with a 1:1 effector:target (E:T) ratio and a 4 h incubation. Data represent the mean ± SD from 4 separate donors. The GMP human platelet lysate resulted in similar purity, expansion, viability, and cytotoxicity as the FBS protocol.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques: Comparison, Incubation

Journal: Frontiers in Immunology

Article Title: Feeder-Cell-Free and Serum-Free Expansion of Natural Killer Cells Using Cloudz Microspheres, G-Rex6M, and Human Platelet Lysate

doi: 10.3389/fimmu.2022.803380

Figure Lengend Snippet: The G-Rex6M/NK Cloudz™ “low-touch” protocol compared with the flask-based “high-touch” feeder cell protocol. The feeder cell protocol used the same donors but was otherwise different: (See methods in (A) or in the Materials and Methods section). The results show the following: (B) the cell distribution on day 10, (C) the NK (CD3 − CD56 + ) fold change between days 0 and 10, (D) the viability of either all CD45 + cells (light gray bars) or NK cells alone (dark gray bars) on day 10, and (E) the percent of target K562 cells killed with a 1:1 effector:target (E:T) ratio over 4 h. Data represent the mean ± SD from 3 separate donors.

Article Snippet: Samples were washed with flow buffer (PBS supplemented with 1% BSA (BP9700-100, ThermoFisher)), then stained with CD3-AlexaFluor 405 (FAB100V, Bio-Techne), CD56-APC (318310, Biolegend, San Diego, CA, USA), CD45-PE-cy7 (557748, BD Biosciences, Franklin Lakes, NJ, USA), CD16-PerCP (302030, Biolegend), NKp46-BB515 (564536, BD), NKG2D-PE (557940, BD), and CD2-AlexaFluor750 (FAB18561S, Bio-Techne).

Techniques: