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: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Imaging

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: Deep spatial profiling of human palatine tonsil tissues. (A) Hematoxylin and eosin (H&E) staining after 92 MICS cycles, including the marked epithelium, germinal center (GC), and T cell zone of the lymphoid follicle. MICS DAPI and stroma staining depicting the composition and structure of the tonsil. Markers: Collagen III, collagen IV, fibronectin (all extracellular matrix (ECM), cytokeratin (epithelium), podoplanin (lymphatic vessels), CD105/SM Actin (blood vessels). (B) Immune cell content of a human palatine tonsil comprising T cells (CD3), B cells (CD19/CD20), plasma cells (PCs) (CD38/CD138), NK cells (CD56), granulocytes (CD15/CD66b), mast cells (CD117), macrophages (MΦ) (CD163/CD169/CD206), myeloid dendritic cells (mDCs) (CD11c), and plasmacytoid dendritic cells (pDCs) (CD123). (C) Detailed view on the T cell zone, mainly composed of CD4 + helper T cells (T h ) and CD8 + cytotoxic T cells (T c ), mDCs (CD11c), and PCs (CD38/CD138). (D) Detailed view on the GC-mantle zone border, showing different B cells (CD11b, CD21, CD22), mDCs (CD11c), and PCs (CD38/CD138). (E) Cell annotations of three different tonsil samples plus respective bar graphs of gated cell populations, comparing the cell content between the three tonsil samples. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: 976 x 640 µm, zoomed-in subregions in (C, D) : 334 µm x 219 µm. Scale bar: 100 µm.

Article Snippet: CD206 , DCN228 , 50 , 130-123-671 , FITC (PE) , Miltenyi Biotec.

Techniques: Staining, Clinical Proteomics

Journal: PLoS ONE

Article Title: CD73 Activity is Dispensable for the Polarization of M2 Macrophages

doi: 10.1371/journal.pone.0134721

Figure Lengend Snippet: Purified human monocytes were polarized with LPS+TNF or IL-4+M-CSF in the presence or absence of CD73 inhibitor AMPCP for 3 days. (A) Flow cytometric analyses of CD14 and CD206 surface expression. (B) qPCR analyses of CCL19 and MRC1 expression. Results are shown as boxplots with 5–95 percentiles (n = 8 different donors) from 4 different experiments. None of the differences between the AMPCP-treated and control cells were statistically significant under any condition.

Article Snippet: For human samples, MRC1 (Hs00267207_m1), B2M (Hs00984230_m1), NT5E (Hs00159686_m1) and CCL19 (Hs00171149_m1) were used. qPCR was done on a 7900HT Fast Sequence Detection System (Applied Biosystems, Stockholm Sweden).

Techniques: Purification, Expressing, Control

Journal: PLoS ONE

Article Title: CD73 Activity is Dispensable for the Polarization of M2 Macrophages

doi: 10.1371/journal.pone.0134721

Figure Lengend Snippet: (A) Gene expression (mean ± SEM) of Nos2 (a pro-inflammatory macrophage marker) in peritoneal cells after the indicated in vivo polarization was determined using qPCR. (B) The concentrations of the cytokines IL-6, IL-12 (p40) and KC (pro-inflammatory macrophage markers) in the peritoneal lavage fluid of wild-type (WT) and CD73-deficient (KO) mice after the in vivo polarizations. (C) Gene expression (mean ± SEM) of Mrc1 and Arg1 (both anti-inflammatory macrophage marker) in peritoneal cells after in vivo polarization was determined using qPCR. (D) Intracellular expression of Ym-1, (E) intracellular expression of Relm alpha and ( F ) surface expression of CD206 (all anti-inflammatory macrophage markers) was determined on total peritoneal leukocytes using flow cytometry and is shown together with representative histograms (specific stainings in white, isotype control stainings in grey). All data are from 6–9 mice/group (except in M(-) control groups where n = 2–4 and in F where n = 3–8). In (B, D, E, F) each dot represents an individual experimental value, the vertical line the median and the whiskers the interquartile ranges. Data are from at least 4 different experiments. NA = not available.

Article Snippet: For human samples, MRC1 (Hs00267207_m1), B2M (Hs00984230_m1), NT5E (Hs00159686_m1) and CCL19 (Hs00171149_m1) were used. qPCR was done on a 7900HT Fast Sequence Detection System (Applied Biosystems, Stockholm Sweden).

Techniques: Gene Expression, Marker, In Vivo, Expressing, Flow Cytometry, Control

Journal: Frontiers in Immunology

Article Title: Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors

doi: 10.3389/fimmu.2020.01412

Figure Lengend Snippet: Polarization of M0 and M2 macrophages after treatment with free and nanocomplexed poly(I:C). Expression of the M2 markers (A–D) CD206 and (E–H) CD163 in ENCP-treated M0 and M2 macrophages, in comparison to the prototypic phenotypes evaluated by FACS. M2 M1 represents M2 macrophages that were treated with LPS + IFN-γ for their M1 polarization. Macrophages were incubated with the treatments for 48 h, and the poly(I:C) dose used was 5 μg/mL. Each symbol shape represents a different donor. Values are shown as mean ± SD ( n ≥ 3). Statistical comparison was done using an ordinary one-way ANOVA followed by a Tukey's comparison test between groups; or a paired t -test for (C,G) . Statistically significant differences are represented as ** p < 0.01. C12r8, laurate-octaarginine; HA, hyaluronic acid; pArg, poly-arginine; PEG–PGA, pegylated polyglutamic acid; pIC, poly(I:C).

Article Snippet: They were then stained with APC-mouse anti-human HLA-DR (552764, BD Biosciences, CA, USA), FITC-mouse anti-human mannose receptor CD206 (551135), anti-human CD163-BV421 (562643), CD80 APC-H7-mouse anti-Human CD80 (Clone L307.4; 561134) and anti-human CD68-PE (556078) (all from BD Biosciences, CA, USA).

Techniques: Expressing, Incubation

Journal: Frontiers in Immunology

Article Title: Arginine-Based Poly(I:C)-Loaded Nanocomplexes for the Polarization of Macrophages Toward M1-Antitumoral Effectors

doi: 10.3389/fimmu.2020.01412

Figure Lengend Snippet: Schematic illustration of the in vitro effects of the poly(I:C) ENCPs developed in this study. (A) Upon interaction with macrophages, poly(I:C) nanocomplexes are taken up. (B) This process allows poly(I:C) to reach its target receptor TLR3, found in the endosomes. It is expected that this interaction activates the TLR3 and the TRIF pathway, stimulating the upregulation of type I IFN genes. (C) The expression of M2 (CD206 and CD163) surface markers was slightly decreased, while M1 (CD80 and MHC II) markers are not substantially modified. Nevertheless, (D) CXCL10 and CCL5 chemokines, involved in the attraction of CD8 T cells to the tumor microenvironment, are secreted. (E) The direct cytotoxicity of macrophages toward cancer cells is also enhanced. Images were reproduced from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License https://creativecommons.org/licenses/by/3.0 .

Article Snippet: They were then stained with APC-mouse anti-human HLA-DR (552764, BD Biosciences, CA, USA), FITC-mouse anti-human mannose receptor CD206 (551135), anti-human CD163-BV421 (562643), CD80 APC-H7-mouse anti-Human CD80 (Clone L307.4; 561134) and anti-human CD68-PE (556078) (all from BD Biosciences, CA, USA).

Techniques: In Vitro, Expressing, Modification

Journal: Nature Communications

Article Title: IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population

doi: 10.1038/ncomms14919

Figure Lengend Snippet: ( a – e ) Lung tissue and peritoneal cells from C57BL/6 J ( a , c , d ) and BALB/c ( b , e ) mice undergoing CLP and antibiotic treatment were harvested at the indicated time points. ( a ) mRNA expression of Cebpb (C/EBPβ) , Arg1 (arginase-1), Mrc1 (MR) and Igf1 in the total lungs were determined by RT-qPCR at indicated times after CLP ( n ≥4 mice per group). ( b ) Lungs obtained from six mice, either naive or CLP (day 10 after CLP), were pooled from two independent samples and CD11b + cells were isolated. mRNA expression of Arg1 (arginase-1), Mrc1 (MR), Rentla (encoding Fizz1) in isolated CD11b + cells were determined by RT-qPCR. ( c ) IGF-1, CCL17 and CCL22 concentrations in the lungs were determined by ELISA at indicated times after CLP ( n ≥3 mice per group). ( d ) Representative FACS plots and frequency of peritoneal CD206 + F4/80 + macrophages at indicated times after CLP ( n ≥3 mice per group). ( e ) Representative western blot of Arg1 (Arginase-1) expression in the peritoneal cells at day 10 after CLP ( n =9 for naive group and n =5 for CLP group). ND, not detected. * P <0.05, ** P <0.01 and *** P <0.001 (one-way ANOVA result with Dunnett posthoc tests in a , c , two-tailed unpaired Student's t -test in b , d ). Data are from one ( b ) and representative of two ( a , c – e ) independent experiments (mean±s.e.m. in a – d ).

Article Snippet: To stain for surface antigens, cells were incubated with specific antibodies to F4/80 (BM8, eBioscience), CD206 (mannose receptor C type 1, MR; MR5D3, AbD Serotec), CD4 (GK1.5, eBioscience; H129.19, BD Biosciences), CD4 (RPA-T4, BD Biosciences, for human), CD45 (30-F11, BD Biosciences), Ly-6A/E (Sca-1; E13-161.7, BioLegend), CD3e (145-2C11, BD Biosciences), CD8a (53-6.7, eBioscience), CD19 (eBIO1D3, 1D3, eBioscience; 6D5, BioLegend), CD11b (M1/70, BioLegend), CD11c (N418, BioLegend; Hl3, BD Biosciences), CD49b/Pan-NK (DX5, BD Biosciences), FcɛRI Alpha (MAR-1, eBioscience), T1/St2 (IL-33R, DJ8, MD Biosciences) or the appropriate isotype controls for 30 min. To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with mouse Foxp3 Buffer Set (BD Biosciences), according to the manufacturer's recommendations.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: IL-33 contributes to sepsis-induced long-term immunosuppression by expanding the regulatory T cell population

doi: 10.1038/ncomms14919

Figure Lengend Snippet: ( a ) Peritoneal cells were collected from C57BL/6 J or Rag1 −/− mice 15 days after CLP and antibiotic treatment and the frequency of F4/80 + CD206 + macrophages determined by FACS ( n ≥3 mice per group). ( b ) Peritoneal cells of BALB/c, Il1rl1 −/− and Stat6 −/− mice were harvested in ‘ a ' above and the frequency of F4/80 + CD206 + macrophages determined by FACS ( n ≥5 mice per group). ( c ) Representative FACS plots of IL-4Rα + F4/80 + peritoneal macrophages of C57BL/6 J mice at day 15 after CLP ( n =5 per group). ( d , e ) Peritoneal macrophages harvested in b above were stimulated with IL-4 in vitro . ( d ) STAT6 phosphorylation in the cells was determined by FACS ( n =4 per group). ( e ) Concentrations of CCL22 in the 18 h culture supernatants determined by ELISA ( n =4 mice per group). ( f ) Peritoneal cells of BALB/c, Il1rl1 −/− and Stat6 −/− mice were harvested in a above. The number of viable bacteria recovered from lysates of peritoneal macrophages exposed, in vitro , to L. pneumophila for 72 h in the presence or absence of arginase inhibitor BEC. ( g ) BALB/c macrophages from BMDM were cultured for 2 days with M-CSF (M0) or IL-4, IL-13 and IL-33 (M2). The number of viable bacteria recovered from lysates of M2-polarized macrophages exposed, in vitro , to L. pneumophila for 72 h in the presence or absence of BEC. ( h ) M0 or M2 macrophages were adoptively transferred (4 × 10 6 cells, i.v.) into naive BALB/c mice, which were challenged 12 days later with L. pneumophila . Survival of mice was recorded ( n =10 mice for M0 group and n =11 for M2 group). ND, not detected. * P <0.05, ** P <0.01 and *** P <0.001 (two-tailed unpaired Student's t- test in a , b , one-way ANOVA result with Bonferroni's posthoc tests in d , Mantel-Cox log-rank test in e ). Data are representative of two ( a , c – g ) independent experiments; or pooled from two ( b , h ) experiments (mean±s.e.m. in a , b , e – h ).

Article Snippet: To stain for surface antigens, cells were incubated with specific antibodies to F4/80 (BM8, eBioscience), CD206 (mannose receptor C type 1, MR; MR5D3, AbD Serotec), CD4 (GK1.5, eBioscience; H129.19, BD Biosciences), CD4 (RPA-T4, BD Biosciences, for human), CD45 (30-F11, BD Biosciences), Ly-6A/E (Sca-1; E13-161.7, BioLegend), CD3e (145-2C11, BD Biosciences), CD8a (53-6.7, eBioscience), CD19 (eBIO1D3, 1D3, eBioscience; 6D5, BioLegend), CD11b (M1/70, BioLegend), CD11c (N418, BioLegend; Hl3, BD Biosciences), CD49b/Pan-NK (DX5, BD Biosciences), FcɛRI Alpha (MAR-1, eBioscience), T1/St2 (IL-33R, DJ8, MD Biosciences) or the appropriate isotype controls for 30 min. To stain for intracellular murine antigens, cells were first stained for surface antigens, then fixed and permeabilized with mouse Foxp3 Buffer Set (BD Biosciences), according to the manufacturer's recommendations.

Techniques: In Vitro, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Bacteria, Cell Culture, Two Tailed Test

Journal: Journal of Nanobiotechnology

Article Title: Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration

doi: 10.1186/s12951-024-02507-3

Figure Lengend Snippet: A-PUAT membranes stimulate macrophage polarization to M2-phenotype. ( A ) SEM results revealing the morphology of RAW264.7 cells cultured on different membranes. ( B ) The viability of RAW264.7 cells seeded on each membrane at indicated time points. ( C ) Relative transcription levels of pro-inflammatory gene and anti-inflammatory gene in each group. ( D ) The contents of TNF-α and IL-10 secreted by RAW264.7 cells cocultured on different substrates. ( E ) Representative immunofluorescent images revealing the cytoskeleton of RAW264.7 cells (FITC, green) and M2-phenotype macrophages (CD206, red) after culturing on different membranes for 3 days. ( F ) Quantitative analysis of orientation of macrophages cultured on different membranes. ( G ) Quantitative analysis of CD206 positive cells in RAW264.7 cells seeded on different membranes. ( H ) Flow cytometry showing the expression of CD80 and CD206 in RAW264.7 cells cultured on different substrates.

Article Snippet: Then samples were cultured overnight at 4 ℃ with primary antibodies to detect CD206 (1:500, 24595, CST).

Techniques: Cell Culture, Membrane, Flow Cytometry, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Electrical aligned polyurethane nerve guidance conduit modulates macrophage polarization and facilitates immunoregulatory peripheral nerve regeneration

doi: 10.1186/s12951-024-02507-3

Figure Lengend Snippet: Relevance between macrophages and SCs at 7 and 14 days after grafting. ( A ) Representative immunofluorescence images of the transverse sections of different NGCs showing the distribution of macrophages (M0, CD68, green) and SCs (S100β, red). ( B ) Quantitative analyses showed the number of infiltrated macrophages and SCs in each group. ( C ) Representative immunofluorescence images of M1-phenotype macrophages (iNOS, red), and M2-phenotype macrophages (CD206, red).

Article Snippet: Then samples were cultured overnight at 4 ℃ with primary antibodies to detect CD206 (1:500, 24595, CST).

Techniques: Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: Immunotherapy landscape analyses of necroptosis characteristics for breast cancer patients

doi: 10.1186/s12967-022-03535-z

Figure Lengend Snippet: Validation of the NRG signature in an external clinical cohort. The expression of 6 selected NRGs (IPMK, SLC39A7, FASLG, HSP90AA1, FLT3, LEF1) ( A ) and immune checkpoints (PD-1, PD-L1, and CTLA-4) ( B ) in the two groups were detected using qRT-PCR. Immunohistochemistry was used to compare the expression of PD-1 ( C ), PD-L1 ( D ), CTLA-4 ( E ), and IPMK ( F ) in the two risk groups. G The co-expression of CD206 and IPMK was detected using immunofluorescence. ns, not significant; *P < 0.5; **P < 0.01; ***P < 0.001.

Article Snippet: For immunofluorescence (IF), anti-IPMK (1:200, Proteintech, USA) and anti-CD206 (1:100, Proteintech, USA) were used as primary antibodies to show the coexpression of CD206 and IPMK in the BC samples.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemistry, Immunofluorescence

Journal: The FASEB Journal

Article Title: A muscle cell‐macrophage axis involving matrix metalloproteinase 14 facilitates extracellular matrix remodeling with mechanical loading

doi: 10.1096/fj.202100182rr

Figure Lengend Snippet: FIGURE 2 Representative images of skeletal muscle immunohistochemistry. (A) Skeletal muscle macrophages were identified using antibodies against CD11b (green) and CD206 (red); CD11b+/CD206+ cells were counted (white arrows). (B) Satellite cells were identified using a Pax7 antibody (red) within the laminin border (green); satellite cells were expressed per fiber. Muscle fiber cross-sectional area was measured in these images. (C) Myonuclei (gold arrows) were identified as DAPI+ (blue) nuclei within the dystrophin border (green). (D) Capillaries were identified using fluorescently conjugated lectin (pink) with a laminin antibody to outline fibers (green) and measured per fiber (yellow arrows). Scale bar = 100 μm for (A), (B), and (D); scale bar = 20 μm in (C)

Article Snippet: Human skeletal muscle macrophage identification and quantification were performed according to a detailed and validated method.19 Briefly, macrophages were identified by stepwise incubation with antibodies against the pan macrophage marker CD11b (Cat# MON1019- 1, Cell Sciences, Newburyport, MA, USA) followed by CD206 (Cat# AF2535, R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemistry

Journal: The FASEB Journal

Article Title: A muscle cell‐macrophage axis involving matrix metalloproteinase 14 facilitates extracellular matrix remodeling with mechanical loading

doi: 10.1096/fj.202100182rr

Figure Lengend Snippet: FIGURE 5 Assessing Mmp14 expression across cell types in Sham and 4-day mechanical overloaded (4DMOV) muscles and macrophage subclusters. (A) Under Sham conditions (red), FAPs are the predominant source of Mmp14. Following 4DMOV (blue) macrophages present as the majority of Mmp14 expressing cells. (B) Representative images of mouse skeletal muscle staining of pan macrophage marker CD206 (red), Mmp14 (green), and DAPI (blue) under Sham or 4DMOV conditions. Scale bar = 20 μm. (C) K-means clustering of macrophages from both sham and 4DMOV muscles reveals 3 sub clusters with Sham clustering separately (cluster 3, gold) and 4DMOV macrophage clusters consisting of cluster 1 (blue) and cluster 2 (red). (D) Feature plot of Mmp14 expression across the macrophage cluster. Endo, endothelial; FAPs, fibro- adipogenic progenitors; Macs, macrophages; Mmp14, matrix metalloproteinase 14; MN, myonuclei; Neutro, Neutrophils

Article Snippet: Human skeletal muscle macrophage identification and quantification were performed according to a detailed and validated method.19 Briefly, macrophages were identified by stepwise incubation with antibodies against the pan macrophage marker CD11b (Cat# MON1019- 1, Cell Sciences, Newburyport, MA, USA) followed by CD206 (Cat# AF2535, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Muscles, Staining, Marker