Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: Bone tissues surgically removed from PJI patient with S. aureus osteomyelitis were processed for histology and immunohistochemistry. (A) Representative 100x image (bar =100 μm) of a H&E-stained section is shown to illustrate the inflammatory cells within the region of interest (box). ( B-D) Parallel histology sections containing the region of interest were immunostained with labelled antibodies against CD3, PD1, S. aureus, TIM-3 (green), LAG-3, and CD66b, counter stained with DAPI, and representative fluorescent microscopy images are shown at 200x (bar = 100 μm). ( B ) Note CD3 + /PD1 + T cells detected in areas of S. aureus infection (white arrows). (C) Note CD3 + /TIM-3 + (white arrows) and CD3 + /LAG-3 + (yellow arrows) T cells at the site of S. aureus infection. (D) Note TIM-3 + /CD66b+ neutrophils at the site of infection (white arrows).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Immunohistochemistry, Staining, Microscopy, Infection

Journal: Cancer Research

Article Title: Human Tumor–Associated Macrophages and Neutrophils Regulate Antitumor Antibody Efficacy through Lethal and Sublethal Trogocytosis

doi: 10.1158/0008-5472.CAN-23-2135

Figure Lengend Snippet: The ability of blood and tumor myeloid cells to perform ADT and kill EGFR hi A431 cells in the presence of anti-EGFR Abs under conditions representing solid human tumors. A, The frequencies of CD45 − EpCam + cells, CD14 + CD206 + HLA-DR hi TAM, and CD11b + CD66b + CD14 − TAN were analyzed by flow cytometry in digested lung tumors. Paired t test. B and C, Representative dot plots showing the levels of trogocytosis and killing activity mediated by blood monocytes ( B ) and PBNs ( C ) cocultured with PKH67 + A431 cells at the indicated E:T ratios in the presence of cetuximab and IgA anti-EGFR Abs, respectively, for 12 hours. Nonspecific human IgG1 or IgA2 isotype control Abs were used as a control. D and E, Cumulative flow cytometry results showing the ability of indicated FcR + effectors to kill PKH67 + A431 tumor cells in the presence of cetuximab (IgG) or IgA anti-EGFR (IgA) Abs at a 2:1 E:T ratio in a 12-hour FACS-based assay. One-way ANOVA with Tukey multiple comparisons tests. Summary graphs represent the total tumoricidal activity of effectors calculated as described in Materials and Methods. F–H, Representative images of GFP + A431 tumor cells cocultured with blood monocytes (patient LC441) at different E:T ratios and cetuximab for 48 hours in the IncuCyte Live Cell Analysis System. Image of A431+Ab, where Ab is a cetuximab, is intentionally the same in both and . Scale bar, 400 μm. Representative experiments showing the kinetics of A431 tumor cell growth when cocultured with blood monocytes ( F ), PBNs ( G ), and TAMs ( H ) in the presence of anti-EGFR Abs at the indicated E:T ratios in the IncuCyte Live Cell Analysis System. I and J, Summary results showing the kinetics of GFP + A431 tumor cell growth when cocultured with FcR + effectors in the presence of cetuximab or IgA anti-EGFR Ab at 2:1 E:T ratio in the IncuCyte Live Cell System. The percentage of tumor cell growth inhibition/stimulation was calculated at 48 hours. One-way ANOVA with Tukey multiple comparisons tests. K, Representative dot plots and cumulative flow cytometry data demonstrating the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue. Paired t test. MFI, mean fluorescence intensity. L, Representative flow cytometry histograms showing the expression of EGFR on the surface of EpCam + cells in tumor and distant lung tissue in comparison with A431 and A549 tumor cells. Number of patients is indicated on the graphs. All data represented as mean ± SEM. FcR + effectors were freshly isolated for all experiments.

Article Snippet: Tumor-associated neutrophils (TAN) were isolated from tumor single-cell suspensions using positive selection with CD15 or CD66b antibody–conjugated magnetic microbeads (130–046–601 and 130–111–552, Miltenyi Biotec Inc.) according to the manufacturer's protocol as previously described ( ).

Techniques: Flow Cytometry, Activity Assay, Control, Cell Analysis, Inhibition, Expressing, Fluorescence, Comparison, Isolation