Journal: Scientific Reports

Article Title: Dual activity of Minnelide chemosensitize basal/triple negative breast cancer stem cells and reprograms immunosuppressive tumor microenvironment

doi: 10.1038/s41598-024-72989-6

Figure Lengend Snippet: Minnelide suppresses TGFβ-induced upregulation of signaling pathways. ( A-C ) Protein expression of HSP70/72, A20, Vimentin, MYC and BRD4 was shown in human MDA-MB-231 and murine 4T1 and EMT6 cell lines treated with increased dose of Minnelide in the presence and absence of recombinant human TGFβ. Experiments were done in duplicates. ( D) TCGA data showing the genetic alterations of myc in Basal-like, ER- and ER + breast cancer subtypes. ( E–G) Kaplan–Meier TCGA data showing the association between the Myc expression levels and survival rates among the patients with high gene expression (red) in Basal-like, ER- and ER + breast cancer compared to patients with low expression (blue), respectively.

Article Snippet: Antibodies against cMyc (ab32072, Abcam), vimentin (5741, Cell Signaling Technology), A20/TNFAIP3 (NBP1-77,533, Novus), HSP70/72 (ADI-SPA-810-F, Enzo Life Sciences), Brd4 (ab128874, Abcam), and ß-actin (664803, BioLegend).

Techniques: Expressing, Recombinant

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A-B) Scatter plots of silicon beads (180 nm, 240 nm, 300 nm, 590 nm, and 880 nm) and 488-conjugated polystyrene beads (100 nm and 500 nm). C) Scatter plot of total plasma EVs. D-F) PBS in 647-, 405, and dual-gated channels. G-I) Plasma in 647-, 405-, and dual-gated channels. J-L) Plasma + TMEM119-647 antibody in 647-, 405-, and dual-gated channels. M-O) Plasma + TREM2-405 in 647-, 405-, and dual-gated channels. LALS, long angle light scatter; SALS, short angle light scatter.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques: Clinical Proteomics

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A) Example nanoscale flow cytometry scatter plots of TMEM119, TREM2 and dual-positive EVs. B) TMEM119 + /TREM2 + events/µl labelled in 3-, 9-, and 15-month wildtype and APP/PS1 rat plasma assessed using a 2-way ANOVA (genotype ⍰ sex). C) Size distribution of TMEM119 +/ TREM2 + EVs based on standardized reference beads. D) Western blot of TMEM119 and TREM2 from isolated rat plasma EVs. E) Western blot of TREM2 from immunoprecipitated TMEM119 + EVs. F) Immunofluorescent 20 ⍰ images of TMEM119 and TREM2 in 15-month-old APP/PS1 hippocampus. Scale bar indicates 25 µm. * Indicates statistical significance ( p < 0.05) Data represent the group mean ± SEM. n = 10-12 males and females per group. LALS, long angle light scatter; SALS, short angle light scatter.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques: Flow Cytometry, Clinical Proteomics, Western Blot, Isolation, Immunoprecipitation

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A) Coronal section for the hippocampal region of interest. B) qPCR measurement of TMEM119 and TREM2 in 3-, 9-, and 15-month WT and APP/PS1 hippocampal RNA isolates assessed using 2-way ANOVAs (age ⍰ genotype). C-E) Western blot of TMEM119 and TREM2 and corresponding quantification in hippocampal protein isolates from 3-, 9-, and 15-month WT and APP/PS1 rats assessed using t -tests. * Indicates statistical significance ( p < 0.05). Data represent the group mean ± SEM. n = 2-3 males and 2-3 females per group.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A) Coronal section for the corpus callosum region of interest. B) qPCR measurement of TMEM119 and TREM2 in 3-, 9-, and 15-month WT and APP/PS1 corpus callosum RNA isolates assessed using 2-way ANOVAs (age ⍰ genotype). C-E) Western blot of TMEM119 and TREM2 and corresponding quantification in corpus callosum protein isolates from 3-, 9-, and 15-month WT and APP/PS1 rats assessed using t -tests. * Indicates statistical significance ( p < 0.05). Data represent the group mean ± SEM. n = 2-3 males and 2-3 females per group.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A) Quantification of TREM2 cell density in the hippocampus of 3-, 9-, and 15-month WT and APP/PS1 rats assessed using t -tests. B) Representative 20 ⍰ images of TREM2 + cells in the hippocampus of 15 month WT and APP/PS1 rats. C) Quantification of TREM2 cell density in the corpus callosum of 3-, 9-, and 15-month WT and APP/PS1 rats assessed using t -tests. D) Representative 20 ⍰ images of TREM2 + cells in the corpus callosum of 15-month WT and APP/PS1 rats. Scale bar indicates 25 µm. * Indicates statistical significance ( p < 0.05). Data represent the group mean ± SEM. n = 4 males and 4 females per group.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques:

Journal: bioRxiv

Article Title: Increased circulating TREM2 + microglial extracellular vesicles in aged APP/PS1 Alzheimer’s disease rats

doi: 10.1101/2025.06.02.657443

Figure Lengend Snippet: A) Simple linear regression between TMEM119 + /TREM2 + events/µl and total RAWM errors in 3-, 9-, and 15-month WT and APP/PS1 rats. n = 10-12 males and females per group.

Article Snippet: Plasma aliquots were thawed at room temperature prior to incubating 10 µl with anti-TMEM119 Coralite-647 (200 ng/sample, Proteintech 66948) and anti-TREM2 Alexa Fluor-405 (400 ng/sample, Novus Biologicals 07101) for 30 mins at room temperature in the dark.

Techniques:

Journal: Brain Pathology

Article Title: TREM 2 Protein Expression Changes Correlate with A lzheimer's Disease Neurodegenerative Pathologies in Post‐Mortem Temporal Cortices

doi: 10.1111/bpa.12190

Figure Lengend Snippet: Information of the primary antibodies used in this study.Abbreviations: GFAP = glial fibrillary acidic protein; IHC = immunohistochemistry; WB = Western blot

Article Snippet: Three commercially available TREM2 antibodies were compared in our antibody characterization study: a goat polyclonal antibody from R&D Systems (AF1828, Minneapolis, MN, USA), raised against recombinant TREM2 molecule spanning amino acids 19–174; a goat polyclonal antibody from Novus Biologicals (NBP1‐06095, Littleton, CO, USA), raised against the amino acids 199–212; and a mouse monoclonal antibody from Fitzgerald (10R‐1115, Acton, MA, USA) raised against amino acids 19–161.

Techniques: Immunohistochemistry, Western Blot

Journal: Brain Pathology

Article Title: TREM 2 Protein Expression Changes Correlate with A lzheimer's Disease Neurodegenerative Pathologies in Post‐Mortem Temporal Cortices

doi: 10.1111/bpa.12190

Figure Lengend Snippet: S pearman's correlation coefficients R ho and significant levels P between microglial markers and neurodegenerative markers in middle temporal cortices

Article Snippet: Three commercially available TREM2 antibodies were compared in our antibody characterization study: a goat polyclonal antibody from R&D Systems (AF1828, Minneapolis, MN, USA), raised against recombinant TREM2 molecule spanning amino acids 19–174; a goat polyclonal antibody from Novus Biologicals (NBP1‐06095, Littleton, CO, USA), raised against the amino acids 199–212; and a mouse monoclonal antibody from Fitzgerald (10R‐1115, Acton, MA, USA) raised against amino acids 19–161.

Techniques:

Journal: Brain Pathology

Article Title: TREM 2 Protein Expression Changes Correlate with A lzheimer's Disease Neurodegenerative Pathologies in Post‐Mortem Temporal Cortices

doi: 10.1111/bpa.12190

Figure Lengend Snippet: Semiquantitative assessment of microglial TREM 2 expression pattern

Article Snippet: Three commercially available TREM2 antibodies were compared in our antibody characterization study: a goat polyclonal antibody from R&D Systems (AF1828, Minneapolis, MN, USA), raised against recombinant TREM2 molecule spanning amino acids 19–174; a goat polyclonal antibody from Novus Biologicals (NBP1‐06095, Littleton, CO, USA), raised against the amino acids 199–212; and a mouse monoclonal antibody from Fitzgerald (10R‐1115, Acton, MA, USA) raised against amino acids 19–161.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic representation of TREM2 processing by ADAM10/17. Cleavage occurs C‐terminal of residue His 157. The entire ectodomain (residues 19–171) was used for immunization of rats to generate TREM2 antibodies. CTF, C‐terminal fragment; sTREM2, soluble TREM2. Immunoblot analysis of membrane fractions of HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) = 0.0011; P (DMSO vs isotype) = 0.992; P (isotype vs 4D9) = 0.0005; n.s., not significant. Immunoblot analysis of conditioned media from HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) < 0.0001; P (DMSO vs isotype) = 0.6372; P (isotype vs 4D9) < 0.0001; n.s., not significant. 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti‐HA antibody was used as a positive control, while empty vector‐transfected HEK293 Flp‐In cells were used as a negative control. Scale bar = 10 μm. Peptide ELISAs detect anti‐mouse TREM2 antibody binding to tiled stalk region peptides, full‐length stalk peptide, or a truncated ADAM cleavage site peptide. The binding epitope of 4D9 antibody is located 12‐amino acids N‐terminal of the ADAM cleavage site at His 157. Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel). 4D9 binding to the mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3 μM. Data represent the mean ± SEM ( n = 3). ECD, extracellular domain. Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ECD evaluated by Biacore, k on = 5.9 × 10 5 M −1 s −1 , k off = 4.0 × 10 −5 s −1 , K D = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable. Data information: Statistical evaluations are displayed as follows: ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Western Blot, Stable Transfection, Inhibition, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Transfection, Binding Assay, Sequencing, Titration, SPR Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry dose–response curve for cell binding of 4D9 mAb (EC50 = 0.29 nM), 4D9 Fab (EC50 = 0.17 nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM ( n = 2). In vitro ADAM17 sheddase activity is blocked by 4D9‐effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM‐conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab, and isotype control. Data represent the mean ± SEM ( n = 6). One‐way ANOVA, Tukey's post hoc test; P (4D9 Fab vs 4D9 mAb) = 0.8855; P (4D9 Fab vs uncleaved) < 0.0001; P (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant. ELISA‐mediated quantification of sTREM2 in conditioned media from HEK293 cells stably overexpressing mouse TREM2 treated with a dose titration of 4D9 mAb (EC50 = 2.3 nM), 4D9 Fab, or isotype for 18 h. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab, or isotype for 5 min. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1 mg/ml POPC/POPS liposomes and 20 μg/ml 4D9 mAb, 4D9 Fab, or isotype for 5 min. p‐SYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA, Tukey's post hoc test (cell line effect: F 1,16 = 365.7, P ≤ 0.0001; treatment effect: F 3,16 = 39.35, P < 0.0001; cell line × treatment effect: F 3,16 = 38.75, P < 0.0001); P (no Ab vs isotype) = 0.6218; P (no Ab vs 4D9 mAb) < 0.0001; P (no Ab vs 4D9 Fab) = 0.7301; P (isotype vs 4D9 mAb) < 0.0001; P (isotype vs 4D9 Fab) > 0.9999; P (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant. Schematic representation of the proposed mechanism of action of antibody 4D9. Binding of 4D9 to TREM2 leads to receptor clustering on the cell surface, thereby driving downstream p‐SYK signaling. At the same time, cell‐surface levels are enhanced by inhibition of ectodomain shedding, potentially because of the inability of proteases to cleave dimeric substrates. Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Stable Transfection, In Vitro, Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Titration, Plasmid Preparation, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry detection of cell‐surface binding of 4D9 (blue) and isotype (green) to wild‐type and Trem2 − / − mouse bone marrow‐derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM ( n = 3). Student's t ‐test (two‐tailed); Trem2 +/+ : P = 0.0005; Trem2 −/− : P = 0.4435; n.s., not significant. Immunoblot analysis of membrane fractions of bone marrow‐derived macrophages (BMDMs) upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 relative to an isotype antibody. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 5–6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc, antibody heavy chain; Ab lc, antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Wild‐type, Trem2 +/− , and Trem2 −/− BMDMs were plated in low concentration of M‐CSF on 4D9 or isotype‐coated wells for 5 days, and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1, respectively). Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Derivative Assay, Two Tailed Test, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: A Flow cytometry detection of cell‐surface 4D9 (red) binding and isotype control (blue) on primary wild‐type and Trem2 − / − mouse microglia. Data are shown as mean fluorescent intensity (MFI). B Uptake assay for fluorescently labeled myelin, Aβ(1–42), and inactivated Escherichia coli particles. The top row shows that myelin and Aβ (pseudocolored in white) accumulate within the plasma membrane of primary microglia (labeled with DyLight 649 isolectin and pseudocolored in red). Overnight, pre‐treatment with antibody 4D9 significantly increased the percentage of substrate‐positive cells (middle row). Pre‐treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E. coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar = 20 μm. C–E Quantification of the change in uptake of myelin (C), Aβ(1–42) (D), and E. coli (E) upon antibody treatment relative to the uptake of the respective substrate in the absence of antibody. The number of substrate‐positive cells relative to the total number of cells was quantified in each condition. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA (myelin), Sidak's multiple comparisons test (time effect: F 1,4 = 0.01123, P = 0.9207; treatment effect: F 1,4 = 27.98, P = 0.0061; time x treatment effect: F 1,4 = 0.5948, P = 0.4836); P (1 h) = 0.1362; P (2 h) = 0.0185; unpaired t ‐test (two‐tailed) with Welch's correction (Aβ(1–42) and E. coli ); P (Aβ(1–42)) = 0.0151; P ( E. coli ) = 0.5751; n.s., not significant. Data information: Statistical evaluations are displayed as follows: * P < 0.05.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Labeling, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG‐effectorless backbone compared with an isotype control were determined in wild‐type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 h, and 4 and 7 days after 10 mg/kg intravenous injection of antibody. Brain antibody concentration was measured 24 h post‐intravenous dosing of isotype hIgG at 100 mg/kg, and 4D9‐hIgG dosed intravenously at 100, 50, and 10 mg/kg. Detection of hIgG levels by ELISA demonstrated dose‐dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM ( n = 5). Schematic depicting the TE assay setup for bound and total sTREM2 detection. For the bound assay, a secondary anti‐human IgG detects 4D9 antibody bound to soluble TREM2 in CSF and plasma. For the total assay, a saturating amount of 4D9 antibody is added to eliminate unbound sTREM2. The ratio of 4D9‐bound sTREM2 to total sTREM2 is calculated to determine the level of TE achieved by the concentration of antibody present. Target engagement time course demonstrated near 100% 4D9‐bound sTREM2 in CSF of wild‐type mice at 24 h post‐dose. Over a 10‐day time course with time points at days 1, 2, 4, and 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ˜ 50% by day 10. Animals were dosed intravenously with 50 mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM ( n = 5). Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10 mg/kg with > 50% bound sTREM2 at 1 mg/kg of antibody. 4D9‐bound sTREM2 was undetectable at 0.1 mg/kg and lower. 4D9 was IV‐dosed at 50, 10, 1, 0.1, 0.01, 0.001, and 0.0001 mg/kg, and isotype‐dosed at 50 mg/kg. CSF bound: total sTREM2 in wild‐type mice was measured at 24 h. Data represent the mean ± SEM ( n = 5). 4D9 antibody demonstrates a dose‐dependent increase in total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wild‐type mice dosed with 4D9 or isotype control was performed by a MSD‐platform‐based ELISA. Data represent the mean ± SEM ( n = 5) and are shown as pg TREM2 per ug of total protein. One‐way ANOVA, Dunnett's post hoc test, P (isotype vs 4D9 [100 mg/kg]) < 0.0001; P (isotype vs 4D9 [50 mg/kg]) = 0.0007. Data information: Only male mice were used. Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic outlining study design and timeline of intraperitoneal injections of either isotype control of 4D9 antibody in 6‐month‐old APP‐NL‐G‐F and age‐matched WT mice. Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9‐injected WT and APP‐NL‐G‐F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10 μm; scale bar (inset) = 2.5 μm. Quantification of cortical TREM2 stainings shown in (B). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 70.63, P ≤ 0.0001; treatment effect: F 1,23 = 14.33, P = 0.0010; genotype × treatment effect: F 1,23 = 14.51, P = 0.0009); P (WT isotype vs WT 4D9) > 0.9999; P (WT isotype vs APP isotype) = 0.02; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0001; n.s., not significant. Quantification of cortical IBA1 staining shown in B. Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,21 = 215.4, P ≤ 0.0001; treatment effect: F 1,21 = 0.5994, P = 0.4475; genotype × treatment effect: F 1,21 = 0.2992, P = 0.5902); P (WT isotype vs APP isotype) < 0.0001; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.9986; n.s., not significant. Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the areas in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1, and Aβ costainings at a larger magnification. Of note, we did not observe a complete co‐localization of P2RY12 and IBA1 suggesting different microglial populations. Scale bar = 10 μm; scale bar (insets) = 5 μm. Quantification of P2RY12‐stained cells in the cortex shown in (E). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 71.99, P ≤ 0.0001; treatment effect: F 1,23 = 9.029, P = 0.0063; genotype × treatment effect: F 1,23 = 5.93, P = 0.0231); P (WT isotype vs APP isotype) = 0.0018; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0051. Data information: Each data point for all quantifications represents an average of n = 3 replicates per mouse. All data represent the mean ± SEM ( n = 6–7). No gender‐specific differences could be observed. Statistical evaluations are displayed as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Immunohistochemical staining, Injection, Staining

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: A, B Overview of the cortex immunohistochemically stained for Aβ plaques in 6‐month‐old APP‐NL‐G‐F mice treated with isotype control (A) and 4D9 antibody (B). Scale bar = 100 μm. C Higher magnification images of cortical Aβ plaques in APP isotype and 4D9‐injected mice. Scale bar = 10 μm. D Quantification of cortical plaque area from stainings shown in (C). Mann–Whitney U ‐test, P = 0.0082. E Quantification of total plaque coverage of Aβ stainings in the cortex shown in (C). Mann–Whitney U ‐test, P = 0.014. F Immunoblot analysis of soluble (DEA fraction, top) and insoluble Aβ levels (FA fraction, bottom) in brains of APP‐NL‐G‐F mice treated with either isotype control or 4D9 antibody. While a reduction in levels of soluble Aβ is evident from the DEA fractions, no change in levels of insoluble Aβ could be detected in the FA fractions. Note that the higher background in the DEA immunoblot is due to a much longer exposure time, which was needed to visualize soluble Aβ. G MSD ELISA quantification of soluble Aβ42 in DEA extracts as shown in (F). Mann–Whitney U ‐test, P = 0.026. H MSD ELISA quantification of insoluble Aβ42 in FA extracts as shown in (F). Unpaired t ‐test (two‐tailed) with Welch's correction; P = 0.0877. Data information: Each data point for both quantifications represents an average of n = 3 replicates per mouse. All data represent the mean ± SEM ( n = 6–7). No gender‐specific differences could be observed. Statistical evaluations are displayed as follows: * P < 0.05; ** P < 0.01. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Staining, Injection, MANN-WHITNEY, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Quantification of AR + cells in CD4 + T cells, CD8 + T cells, monocytes, and neutrophils from the peripheral blood of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples) by flow cytometry. b Quantification of AR + cells in intraprostatic CD4 + T cells, CD8 + T cells, macrophages, and neutrophils was conducted in healthy prostates and prostate cancer tissues by flow cytometry ( n = 6 biologically independent samples). c , d The RNA-seq analysis of BMDMs cultured in RM1 CM and treated with ASC-J9 or DMSO. c A heatmap of DEGs in macrophages, where gene counts for the DMSO group have been normalised, and gene expression values are coloured based on upregulation (red) or downregulation (blue). DMSO treatment is represented in black, while ASC-J9 treatment is depicted in red. d A volcano plot displaying the gene expression of selected TREM family members ( Trem2 and Trem1 ), macrophage polarisation markers ( Cd163, Arg1, Cd86 , and Tnf ), and pro-migration factors ( Ccl2 and Ccl8 ), with gene expression values coloured according to upregulation (red) or downregulation (blue). e Quantification of TREM2 + cells in CD4 + T cells, CD8 + T cells, monocytes, and neutrophils from peripheral blood of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples) by flow cytometry. f Quantification of TREM2 + cells in intraprostatic CD4 + T cells, CD8 + T cells, macrophages, and neutrophils was conducted in healthy prostates and prostate cancer tissues by flow cytometry ( n = 6 biologically independent samples). g Pearson correlation analysis of AR and TREM2 protein levels in peripheral blood monocytes of patients with prostate cancer ( n = 53 biologically independent samples). h Representative dot plots of TREM2 expression levels in peripheral blood mononuclear cells classified as TREM2 high (TREM2 high ), TREM2 low (TREM2 low ), and TREM2 negative (TREM2 neg ) (left). Representative dot plots of AR expression in peripheral blood TREM2 neg , TREM2 low , and TREM2 high mononuclear cells (middle). Quantification of AR expression in peripheral blood TREM2 neg , TREM2 low , and TREM2 high mononuclear cells of prostate cancer patients ( n = 53 biologically independent samples) (right). i Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in peripheral monocytes of healthy individuals ( n = 31 biologically independent samples) and patients with prostate cancer ( n = 53 biologically independent samples). j Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in intraprostatic macrophages of healthy prostate and prostate cancer tissues ( n = 6 biologically independent samples). k Representative immunoblot analysis of AR and TREM2 in CD68 + macrophages of tumour regions and adjacent normal prostate of prostate cancer patients. Experiment was repeated three times independently with similar results. l Representative multiplex immunofluorescence staining images of AR, TREM2, and CD206 in prostate tumour regions and adjacent normal prostate tissues. Nuclei were stained with DAPI. Scale bar: 10 μm. m Quantification of co-expression, singular expression, and non-expression of AR and TREM2 in CD206-expressing cells in tumour regions and distant normal prostate tissues ( n = 5 biologically independent samples) from multiplex immunofluorescence in (Fig. 1l). n Multiplex fluorescent immunohistochemistry (using TSA technology) analysis. Representative tumour regions of FFPE prostatectomy specimens were stained for CD68, CD206, CD86, AR, and TREM2. Each triangle or pentagon represents the CD68 + CD206 + cells or CD68 + CD86 + cells, respectively. Scale bar: 20 µm. o Percentage of TREM2 - AR - , TREM2 + AR - , TREM2 - AR + , and TREM2 + AR + cells in CD68 + CD206 + macrophages or CD68 + CD86 + macrophages in multiplex immunofluorescence image of the tumour regions of FFPE prostatectomy specimens, respectively ( n = 6 biologically independent samples). For ( l , n ) experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-way ANOVA with Sidak’s multiple comparisons for ( a − o ); by the Wald test under a negative binomial generalized linear model, and adjusted for multiple testing via the Benjamini-Hochberg method for ( d ); by two-sided Pearson correlation analysis ( g ); and by one-way ANOVA with Tukey’s multiple comparisons for ( h ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Flow Cytometry, RNA Sequencing, Cell Culture, Gene Expression, Migration, Expressing, Western Blot, Multiplex Assay, Immunofluorescence, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Representative immunofluorescence staining images of AR and F4/80 in WT BMDMs and TREM2 KO BMDMs. Scale bar: 5 μm. b Representative flow plots and quantification of AR expression in WT BMDMs and TREM2 KO BMDMs ( n = 5 biologically independent samples). c Representative immunofluorescence staining images of AR in WT BMDMs treated with RM1 CM, RM1 CM + DMSO or RM1 CM + ENZA, respectively. Scale bar: 10 μm. d , e Protein expression levels of AR ( d ) and TREM2 ( e ) in WT BMDMs treated with DMSO or 10 μM ENZA for 48 h were quantified by flow cytometry ( n = 5 biologically independent samples). f , g Quantification of AR ( f ) and TREM2 ( g ) expression in WT BMDMs treated with RM1 CM for 48 h ( n = 5 biologically independent samples). h , i Relative protein expression of AR ( h ) and TREM2 ( i ) in THP1 cells treated with 22Rv1 CM for 48 h ( n = 5 biologically independent samples). j , k Protein expression levels of AR ( j ) and TREM2 ( k ) in THP1 cells treated with Lncap CM for 48 h ( n = 5 biologically independent samples). l , m Representative flow plots and quantification of TREM2 ( l ) and AR ( m ) in TAMs of Pten PC-/- TREM2 f/f mice and Pten PC-/- TREM2 f/f -Lyz2-cre mice ( n = 5 biologically independent samples). n The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways following ASC-J9 treatment in WT BMDMs. o The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways in RM1 CM treated TREM2 KO BMDMs compared to WT BMDMs. p Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, p-Syk, and p-JAK1 in WT BMDMs, TREM2 KO BMDMs and DAP12 KO BMDMs treated with RM1 CM for 48 h. Experiment was repeated three times independently with similar results. q Representative immunoblot analysis of AR and ROR-γ in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 5 μM SR2211 (ROR-γ inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. r Representative immunoblot analysis of AR and p-STAT3 in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 10 μM C188-9 (p-STAT3 inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. For a and c, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-sided Mann-Whitney U test for ( b , d − m ); and by Hypergeometric Test for ( n , o ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Immunofluorescence, Staining, Expressing, Flow Cytometry, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme of mass spectrometry. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . Briefly, proteins from human prostate tumour tissues were extracted, followed by immunoprecipitation using anti-TREM2 antibody or IgG and agarose beads, and then the enriched proteins were lysed for peptide identification. b Venn diagram and the table showing the secretory proteins in the anti-TREM2-enriched complex. c Pearson correlation analysis of APOE and TREM2 mRNA levels in TCGA database of prostate cancer ( n = 498 biologically independent samples). d Representative immunoblot analysis of TREM2 and APOE in macrophages isolated from human prostate cancer tissues which was immunoprecipitated with anti-TREM2 antibody. Sample processing controls (lysate input, run on the same separate gel) are shown in panel (1 & 2). Experiment was repeated three times independently with similar results. e Representative multiplex immunofluorescence staining images of TREM2, APOE, and CD206 in prostate tumour tissues and adjacent normal prostate tissues. Nuclei were stained with DAPI. Scale bar: 10 μm. f Quantification of co-expression, singular expression, and non-expression of TREM2 and APOE in CD68-expressing cells in tumour regions and distant normal prostate tissues ( n = 5 biologically independent samples) from multiplex immunofluorescence of (Fig. 3e). g The concentration of APOE in the serum of both healthy individuals ( n = 12 biologically independent samples) and patients ( n = 28 biologically independent samples) with prostate cancer was detected using ELISA. h The concentration of APOE in the homogenates of normal prostate and prostate cancer tissues was detected using ELISA ( n = 6 biologically independent samples). i The concentration of APOE in normal cell culture media and RM1 CM was measured using ELISA ( n = 6 biologically independent samples). j Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, and p-Syk in WT BMDMs, TREM2 KO BMDMs and DAP12 KO BMDMs treated with RM1 CM or 100 nM recombinant APOE protein for 48 h. Experiment was repeated three times independently with similar results. k Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, and p-Syk in WT BMDMs treated with RM1 CM or RM1 CM plus 1 ng/ml anti-APOE for 48 h. Experiment was repeated three times independently with similar results. l , m RT-qPCR analysis of in WT BMDMs and TREM2 KO BMDMs treated with 100 nM recombinant APOE protein ( l ) or RM1 CM plus 1 ng/ml anti-APOE ( m ) for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. For e, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P -values were determined by two-sided Pearson correlation analysis for ( c ); by two-way ANOVA with Sidak’s multiple comparisons for ( f ); by two-sided Mann-Whitney U test for ( g − i ); and by two-way ANOVA with Tukey’s multiple comparisons for ( l , m ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Mass Spectrometry, Immunoprecipitation, Western Blot, Isolation, Multiplex Assay, Immunofluorescence, Staining, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Recombinant, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Schematic representation of TREM2 +/+ and TREM2 -/- BMDMs induction. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . BMDMs were treated with RM1 CM plus 10 μM ENZA, and gene expression was analysed by RT-qPCR and protein expression by flow cytometry. b RT-qPCR analysis of M2-like macrophage markers Il10 and Tgfb1 in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. c ELISA analysis of IL-10 and TGF-β in the supernatant of WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). d , e Representative flow plots and quantification of CD206 ( d ) and CD86 ( e ) in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). f A genomic view of AR enrichment on IL10 and TGFB1 promoters in THP-1 cells was analysed from published ChIP-seq data . AR peaks under DMSO and 10 nM R1881 conditions are depicted in blue and red, respectively. g Prediction of Ar binding sites on the Il10 (left) and Tgfb1 (right) promoters through the JASPAR database. h The specific binding of Ar to the predicted binding site in Il10 and Tgfb1 promoters in WT BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). i The binding of Ar to the Il10 and Tgfb1 promoters in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and WT BMDM group, respectively. j The binding of Ar to the Il10 and Tgfb1 promoters in WT BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and CTL group, respectively. All the data are presented as mean ± SD. The P -values were determined by two-way ANOVA with Tukey’s multiple comparisons for ( b − d ); by two-way ANOVA with Sidak’s multiple comparisons for ( h ); and by two-sided Mann-Whitney U test for ( i , j ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a , b Assessment of the indirect antitumour effects of BMDMs. WT BMDMs and TREM2 KO BMDMs were co-cultured with RM1 cells at a ratio of 1:1 in Transwell systems with 10 μM ENZA. a RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells after 48 h of co-culture ( n = 6 biologically independent samples). b Representative images and quantification of migrated RM1 cells were obtained after 24 h of co-culture ( n = 5 biologically independent samples). c , d WT BMDMs and TREM2 KO BMDMs were co-cultured with RM1 cells at a ratio of 1:1 in Transwell systems with 100 nM recombinant APOE protein. c RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells after 48 h of co-culture ( n = 6 biologically independent samples). d Representative images and quantification of migrated RM1 cells were obtained after 24 h of co-culture ( n = 5 biologically independent samples). e RT-qPCR analysis of pro-migratory cytokines Ccl2 and pro-proliferative cytokine Il23a in WT BMDMs and TREM2 KO BMDMs co-cultured with RM1 cells plus 10 μM ENZA for 48 h ( n = 6 biologically independent samples). Gene expression was normalized to Actb expression. f Representative images of Transwell migration assays (with three technical replicates) of RM1 cells cultured in normal medium alone or co-cultured with WT BMDMs at a ratio of 1:1, or co-cultured with WT BMDMs and treated with 1 ug/ml blocking antibodies against CCL2, CCL7, and CCL13. Cells that penetrated the membrane after 24 h of culture were stained using crystal violet. The migration ability of the cells was quantified using the optical density (OD) of the crystal violet-stained cells ( n = 3 independent experiments). The schematics in ( a − d , f ) are created in Biorender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . g WT BMDMs were co-cultured with RM1 cells at a ratio of 1:1 plus blocking antibody against Ki67 in Transwell systems for 48 h, RT-qPCR was performed to detect the mRNA levels of Ki67 in RM1 cells ( n = 6 biologically independent samples). h ELISA analysis of CCL2 and IL-23 in the supernatant of WT BMDMs and TREM2 KO BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h ( n = 5 biologically independent samples). i A genomic view of AR enrichment on IL23A and CCL2 promoters in THP-1 cells was analysed from published ChIP-seq data . AR peaks under DMSO and 10 nM R1881 conditions are depicted in blue and red, respectively. j Prediction of AR binding sites on the Ccl2 (left) and Il23a (right) promoters through the JASPAR database. k The specific binding of Ar to the predicted binding site in Ccl2 and Il23a promoters in WT BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). l The binding of Ar to the Ccl2 and Il23a promoters in WT BMDMs and TREM2 KO BMDMs treated with RM1 CM was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and WT BMDM group, respectively. m The binding of Ar to the Ccl2 and Il23a promoters in WT BMDMs treated with RM1 CM plus 10 μM ENZA for 48 h was detected by ChIP-qPCR ( n = 6 biologically independent samples). Relative fold enrichment was normalised relative to IgG and CTL group, respectively. All the data are presented as mean ± SD. The P -values were determined by two-way ANOVA with Tukey’s multiple comparisons for ( a − e , h ); by two-way ANOVA with Sidak’s multiple comparisons for ( k ); by one-way ANOVA with Tukey’s multiple comparisons for ( f ); and by two-sided Mann-Whitney U test for ( g − m ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Cell Culture, Quantitative RT-PCR, Co-Culture Assay, Recombinant, Gene Expression, Expressing, Migration, Blocking Assay, Membrane, Staining, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Box plot depicting AR (left), TREM2 (medium) and APOE (right) expression in TCGA bulk RNA-seq samples of human normal prostate ( n = 152 biologically independent samples) and prostate cancer samples (PRAD, n = 492 biologically independent samples) from GEPIA database. Box plots show the median (centre line), 25th and 75th percentiles (box bounds), and whiskers extend to the 5th and 95th percentiles which represent minima and maxima. b Disease-free survival based on AR, TREM2 and APOE expression in primary prostate tumours (TCGA) from GEPIA database. the High AR (left) /TREM2 (medium) /APOE (right) group (red) ( n = 246 biologically independent samples) corresponds to the first 50% of expression, and the Low AR (left) /TREM2 (medium) /APOE (right) group (blue) ( n = 246 biologically independent samples) corresponds to the last 50% of expression. c Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice were subjected to subcutaneous injections of RM1-Luc cells in the inguinal region. Surgical castration (CTX) was performed after 3 days. ENZA treatment commenced on day five. Mice were euthanized on day 15 for analysis and data collection. The sham group for each genotype serves as its own control. RM1-Luc: luciferase-tagged RM1, s.c.: subcutaneous, CTX: Castration, ENZA: Enzalutamide. d − f Representative prostate tumour size ( n = 5 mice) ( d ), tumour weight ( n = 6 mice) ( e ), and tumour growth curve ( n = 5 mice) ( f ) of each group. g The proportion of CD45 + CD11b + F4/80 + macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). h The proportion of CD11b + F4/80 + CD206 + (left) macrophages and CD11b + F4/80 + CD86 + (right) macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). i ELISA assay was employed to quantify the protein levels of IL-10 and TGF-β in the tumour griding supernatant of tumour-bearing mice ( n = 5 mice). j The proportion of CD45 + CD3 + CD8 + T cells in the tumour was quantified by flow cytometry ( n = 5 mice). k The number of PD1 + CD8 + T cells mg -1 in the tumour ( n = 5 mice). l The number of IFN-γ + CD8 + T cells mg -1 (left) and TNF + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). m The proportion of granzyme B (left) and perforin (right) in the intraprostatic CD8 + T cells of tumour-bearing mice ( n = 5 mice). (n) Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice were subjected to subcutaneous injections of RM1 cells in the inguinal region. Surgical castration (CTX) was performed after 3 days. ENZA and anti-PD1 treatment commenced on day five. Mice were euthanized on day 15 for analysis and data collection. o , p Representative prostate tumour size ( n = 6 mice) ( o ), and tumour weight ( n = 6 mice) ( p ) of each group. All the data are presented as mean ± SD. The P -values were determined by two-sided Mann-Whitney U test for ( a ); by Log-rank (Mantel-Cox) test for ( b ); by two-way ANOVA with Tukey’s multiple comparisons for ( f ); and by one-way ANOVA with Tukey’s multiple comparisons for ( e , g − m , p ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Expressing, RNA Sequencing, Control, Luciferase, Flow Cytometry, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . RM1-Luc cells were intra-prostatically injected into C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice. Surgical castration was performed after 3 days. ENZA treatment commenced on day five. IVIS analysis of mice was performed every 3 days starting from day eight. Mice were euthanized on day 17 for analysis and data collection. b Survival curve of mice intra-prostatically injected with RM1-Luc cells ( n = 10 mice). c Representative IVIS fluorescence images and ROI quantification of prostate tumours in mice of each group ( n = 3 mice). d Representative IVIS fluorescence images and ROI quantification of the lymph node, lung, and liver in tumour-bearing mice ( n = 3 mice). e Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . RM1-Luc cells were subcutaneously injected into C57BL/6 TREM2 f/f and TREM2 f/f -Lyz2-cre mice. Radical resection was performed to remove the tumour on day three. Surgical castration was performed on day five. IVIS analysis of mice was conducted every 5 days starting from day eight. Mice were euthanized on day 18 for analysis and data collection. f , g Representative recurrent prostate tumour images ( f ) and tumour weight ( g ) of each group ( n = 5 mice). h Representative IVIS fluorescence images and ROI quantification of recurrent prostate tumours in mice ( n = 3 mice). i Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . BMDMs from C57BL/6 TREM2 +/+ CD45.2 mice and C57BL/6 TREM2 -/- CD45.2 mice were injected into CD45.1 mice via the tail vein, and RM1-Luc cells were subcutaneously injected into the inguinal region of the mice 24 h later. Surgical castration was performed after 3 days. ENZA treatment commenced on day five. IVIS analysis of mice was performed every 4 days starting from day seven. Mice were euthanized on day 15 for analysis and data collection. j , k Representative prostate tumour images ( j ) and tumour weight ( k ) of each group ( n = 5 mice). RM1-Luc: luciferase-tagged RM1, s.c.: subcutaneous, CTX: Castration, ENZA: Enzalutamide, IVIS: In vivo imaging system, BMDM: Bone marrow-derived macrophages. All the data are presented as mean ± SD. The P -values were determined by Log-rank (Mantel-Cox) test for ( b ); by two-way ANOVA with Tukey’s multiple comparisons for ( c , h ); and by one-way ANOVA with Tukey’s multiple comparisons for ( d − k ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Injection, Fluorescence, Luciferase, In Vivo Imaging, Derivative Assay

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Experimental scheme. Created in BioRender. Qiaohua, W. (2025) https://BioRender.com/ly3agho . Pten PC-/- TREM2 f/f and Pten PC-/- TREM2 f/f -Lyz2-cre mice were surgically castrated at week eight. ENZA was administrated 12 weeks after surgical castration when castration resistance was observed. Prostate tumour volume was monitored by magnetic resonance imaging (MRI) at weeks 12 and 24. Mice were euthanized at week 24 for analysis and data collection. CTX: Castration, ENZA: Enzalutamide, MRI: Magnetic resonance imaging. b Representative MRI scans of mice in each group at weeks 12 and 24. The red circle highlights the area of prostate carcinoma. c Waterfall plots depicting the proportional change in tumour response at weeks 12 and 24 ( n = 3 mice). d Representative haematoxylin and eosin (H&E) and Ki67 staining at the observation endpoint. Scale bar: 40 μm. e Quantitative analysis of adenocarcinoma, prostatic intraepithelial neoplasia (PIN), or normal-like glands at the observation endpoint in each group ( n = 3 mice). f Percentage of Ki-67 + cell area relative to total area ( n = 3 mice). g ELISA assay was employed to quantify the protein levels of IL-10 (left) and TGF-β (right) in tumour griding supernatant of mice ( n = 5 mice). h The proportion of CD45 + CD11b + F4/80 + macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). i The proportion of CD11b + F4/80 + CD206 + (up) macrophages and CD11b + F4/80 + CD86 + (down) macrophages in the tumour were quantified by flow cytometry ( n = 5 mice). j The proportion of CD45 + CD3 - NK1.1 + NK cells in the tumour was quantified by flow cytometry ( n = 5 mice). k The proportion of CD45 + CD3 + CD8 + T cells in the tumour was quantified by flow cytometry ( n = 5 mice). l The number of PD1 + CD8 + T cells mg -1 in the tumour ( n = 5 mice). m The number of IFN-γ + CD8 + T cells mg -1 (left) and TNF + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). n The number of Granzyme B + CD8 + T cells mg -1 (left) and Perforin + CD8 + T cells mg -1 (right) in the tumour ( n = 5 mice). For b and d, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-way ANOVA with Tukey’s multiple comparisons for (c, e); and by one-way ANOVA with Tukey’s multiple comparisons for ( f – n ). Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence (IF) staining: 1 × 10 5 WT or TREM2 KO BMDMs were seeded in confocal dishes (SAINING, 1051001), and cells were incubated overnight at 4 °C stained with anti-AR antibody (Abcam, ab108341, 1:100), anti-F4/80 antibody (Thermo Fisher Scientific, 14-4801-82, 1:100), or anti-TREM2 antibody (Sino Biological, 50149-R014-100, 1:100).

Techniques: Magnetic Resonance Imaging, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Cancers

Article Title: TREM2 Is Associated with Advanced Stages and Inferior Prognosis in Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers14194635

Figure Lengend Snippet: Figure 1. Representative images of tissue microarrays showing (A) strong and (B) low TREM2 expression in primary tumors of oral squamous cell carcinoma patients. Zoom images of selected regions from the cores are shown underneath.

Article Snippet: PBMCs at a density of 1 × 106/tube were incubated with the 100-fold diluted APC-Cy7 anti-TREM2 antibody (clone 2B5; Novus Biologicals, Centennial, CO, USA) for 15 min at RT in the dark.

Techniques: Expressing

Journal: Cancers

Article Title: TREM2 Is Associated with Advanced Stages and Inferior Prognosis in Oral Squamous Cell Carcinoma.

doi: 10.3390/cancers14194635

Figure Lengend Snippet: Figure 2. Kaplan–Meier curves of progression-free survival (PFS) and overall survival (OS) for 171 patients with OSCC according to TREM2 expression in primary tumor. (A) Patients with high TREM2 expression showed significantly inferior PFS (p = 0.038). (B) Patients with high TREM2 expression showed significantly inferior OS (p = 0.011).

Article Snippet: PBMCs at a density of 1 × 106/tube were incubated with the 100-fold diluted APC-Cy7 anti-TREM2 antibody (clone 2B5; Novus Biologicals, Centennial, CO, USA) for 15 min at RT in the dark.

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic representation of TREM2 processing by ADAM10/17. Cleavage occurs C‐terminal of residue His 157. The entire ectodomain (residues 19–171) was used for immunization of rats to generate TREM2 antibodies. CTF, C‐terminal fragment; sTREM2, soluble TREM2. Immunoblot analysis of membrane fractions of HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) = 0.0011; P (DMSO vs isotype) = 0.992; P (isotype vs 4D9) = 0.0005; n.s., not significant. Immunoblot analysis of conditioned media from HEK293 Flp‐In cells stably overexpressing both mouse TREM2 and mouse DAP12 upon treatment with 4D9 antibody reveals decreased levels of sTREM2 similar to what can be achieved by ADAM protease inhibition using the GM6001 inhibitor. An isotype antibody was used as a negative control. sAPPα served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated. Levels of sTREM2 were quantified by MSD ELISA. Data represent the mean ± SEM ( n = 3). One‐way ANOVA, Tukey's post hoc test; P (DMSO vs GM) < 0.0001; P (DMSO vs isotype) = 0.6372; P (isotype vs 4D9) < 0.0001; n.s., not significant. 4D9 antibody selectively detects TREM2 on the cell surface of HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12. An anti‐HA antibody was used as a positive control, while empty vector‐transfected HEK293 Flp‐In cells were used as a negative control. Scale bar = 10 μm. Peptide ELISAs detect anti‐mouse TREM2 antibody binding to tiled stalk region peptides, full‐length stalk peptide, or a truncated ADAM cleavage site peptide. The binding epitope of 4D9 antibody is located 12‐amino acids N‐terminal of the ADAM cleavage site at His 157. Sequence comparison of mouse TREM2 and human TREM2 shows substantial sequence conservation around the 4D9 epitope (upper panel). Immunoblot analysis demonstrates that antibody 4D9 is highly specific for mouse TREM2 and does not detect human TREM2 or mouse TREM1 (lower panel). 4D9 binding to the mouse TREM2 ECD is competed off by a stalk region peptide. A competition ELISA demonstrates that a dose titration of stalk peptide reduces binding of 4D9 to TREM2 ECD with an EC50 of 1.3 μM. Data represent the mean ± SEM ( n = 3). ECD, extracellular domain. Surface plasmon resonance binding kinetics of increasing concentrations of 4D9 antibody to mouse TREM2 ECD evaluated by Biacore, k on = 5.9 × 10 5 M −1 s −1 , k off = 4.0 × 10 −5 s −1 , K D = 68 pM. 4D9 binding to human TREM2 or mouse TREM1 was undetectable. Data information: Statistical evaluations are displayed as follows: ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Western Blot, Stable Transfection, Inhibition, Negative Control, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Transfection, Binding Assay, Sequencing, Titration, SPR Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry dose–response curve for cell binding of 4D9 mAb (EC50 = 0.29 nM), 4D9 Fab (EC50 = 0.17 nM), and isotype to HEK cells stably overexpressing mouse TREM2. Data represent the mean ± SEM ( n = 2). In vitro ADAM17 sheddase activity is blocked by 4D9‐effectorless mAb and 4D9 Fab fragment but not an isotype control. Fluorescence polarization of FAM‐conjugated TREM2 stalk peptide was detected in the presence or absence of ADAM17 and 4D9 mAb, 4D9 Fab, and isotype control. Data represent the mean ± SEM ( n = 6). One‐way ANOVA, Tukey's post hoc test; P (4D9 Fab vs 4D9 mAb) = 0.8855; P (4D9 Fab vs uncleaved) < 0.0001; P (4D9 mAb vs uncleaved) < 0.0001; n.s., not significant. ELISA‐mediated quantification of sTREM2 in conditioned media from HEK293 cells stably overexpressing mouse TREM2 treated with a dose titration of 4D9 mAb (EC50 = 2.3 nM), 4D9 Fab, or isotype for 18 h. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 treated with a dose titration of 4D9 mAb, 4D9 Fab, or isotype for 5 min. Data represent the mean ± SEM ( n = 3). AlphaLISA‐mediated quantification of p‐SYK levels in HEK293 Flp‐In cells stably overexpressing mouse TREM2 and mouse DAP12 or empty vector treated with 1 mg/ml POPC/POPS liposomes and 20 μg/ml 4D9 mAb, 4D9 Fab, or isotype for 5 min. p‐SYK levels were also determined for cells treated with liposomes only. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA, Tukey's post hoc test (cell line effect: F 1,16 = 365.7, P ≤ 0.0001; treatment effect: F 3,16 = 39.35, P < 0.0001; cell line × treatment effect: F 3,16 = 38.75, P < 0.0001); P (no Ab vs isotype) = 0.6218; P (no Ab vs 4D9 mAb) < 0.0001; P (no Ab vs 4D9 Fab) = 0.7301; P (isotype vs 4D9 mAb) < 0.0001; P (isotype vs 4D9 Fab) > 0.9999; P (4D9 mAb vs 4D9 Fab) < 0.0001; n.s., not significant. Schematic representation of the proposed mechanism of action of antibody 4D9. Binding of 4D9 to TREM2 leads to receptor clustering on the cell surface, thereby driving downstream p‐SYK signaling. At the same time, cell‐surface levels are enhanced by inhibition of ectodomain shedding, potentially because of the inability of proteases to cleave dimeric substrates. Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Stable Transfection, In Vitro, Activity Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Titration, Plasmid Preparation, Inhibition

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Flow cytometry detection of cell‐surface binding of 4D9 (blue) and isotype (green) to wild‐type and Trem2 − / − mouse bone marrow‐derived macrophages. Representative histograms are shown as MFI (mean fluorescent intensity). Graphs represent the median signal intensity ± SEM ( n = 3). Student's t ‐test (two‐tailed); Trem2 +/+ : P = 0.0005; Trem2 −/− : P = 0.4435; n.s., not significant. Immunoblot analysis of membrane fractions of bone marrow‐derived macrophages (BMDMs) upon treatment with 4D9 antibody reveals increased levels of membrane‐bound TREM2 relative to an isotype antibody. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Calnexin served as a loading control. Levels of membrane‐bound TREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 5–6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Immunoblot analysis of sTREM2 in conditioned media from BMDM upon treatment with 4D9 and isotype antibodies. BMDMs from TREM2 knockout mice were included to show the specificity of the anti‐TREM2 antibody. Soluble APP served as a loading control. Note that heavy and light chains of the antibodies used for treatment are also detected and annotated as follows: Ab hc, antibody heavy chain; Ab lc, antibody light chain. Levels of sTREM2 were quantified by MSD ELISA. TREM2 antibody clone 5F4 was used for detection. Data represent the mean ± SEM ( n = 6). Unpaired t ‐test (two‐tailed) with Welch's correction; P < 0.0001. No gender‐specific differences could be observed. Wild‐type, Trem2 +/− , and Trem2 −/− BMDMs were plated in low concentration of M‐CSF on 4D9 or isotype‐coated wells for 5 days, and cellular ATP levels were measured by luminescence detection to indicate cell viability. 4D9 and isotype data for each genotype represent the mean ± SEM ( n = 2 and n = 1, respectively). Data information: Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Derivative Assay, Two Tailed Test, Western Blot, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: A Flow cytometry detection of cell‐surface 4D9 (red) binding and isotype control (blue) on primary wild‐type and Trem2 − / − mouse microglia. Data are shown as mean fluorescent intensity (MFI). B Uptake assay for fluorescently labeled myelin, Aβ(1–42), and inactivated Escherichia coli particles. The top row shows that myelin and Aβ (pseudocolored in white) accumulate within the plasma membrane of primary microglia (labeled with DyLight 649 isolectin and pseudocolored in red). Overnight, pre‐treatment with antibody 4D9 significantly increased the percentage of substrate‐positive cells (middle row). Pre‐treatment with the isotope control did not affect the uptake rate (bottom row). No changes in the uptake of E. coli particles were observed. Substrate uptake in the absence of antibody is shown in the top row. Hoechst 33342 was used to counter stain the nuclei (in cyan) and to assess cell density. Scale bar = 20 μm. C–E Quantification of the change in uptake of myelin (C), Aβ(1–42) (D), and E. coli (E) upon antibody treatment relative to the uptake of the respective substrate in the absence of antibody. The number of substrate‐positive cells relative to the total number of cells was quantified in each condition. Data represent the mean ± SEM ( n = 3). Two‐way ANOVA (myelin), Sidak's multiple comparisons test (time effect: F 1,4 = 0.01123, P = 0.9207; treatment effect: F 1,4 = 27.98, P = 0.0061; time x treatment effect: F 1,4 = 0.5948, P = 0.4836); P (1 h) = 0.1362; P (2 h) = 0.0185; unpaired t ‐test (two‐tailed) with Welch's correction (Aβ(1–42) and E. coli ); P (Aβ(1–42)) = 0.0151; P ( E. coli ) = 0.5751; n.s., not significant. Data information: Statistical evaluations are displayed as follows: * P < 0.05.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Flow Cytometry, Binding Assay, Labeling, Staining, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: 4D9 demonstrates standard IgG pharmacokinetics in vivo . Peripheral clearance rates of 4D9 on a human IgG‐effectorless backbone compared with an isotype control were determined in wild‐type mice by hIgG ELISA detection of plasma antibody concentrations 1 and 24 h, and 4 and 7 days after 10 mg/kg intravenous injection of antibody. Brain antibody concentration was measured 24 h post‐intravenous dosing of isotype hIgG at 100 mg/kg, and 4D9‐hIgG dosed intravenously at 100, 50, and 10 mg/kg. Detection of hIgG levels by ELISA demonstrated dose‐dependent brain concentrations of 4D9 in the single digit nM range. Animals were perfused to minimize IgG contribution from the plasma. Data represent the mean ± SEM ( n = 5). Schematic depicting the TE assay setup for bound and total sTREM2 detection. For the bound assay, a secondary anti‐human IgG detects 4D9 antibody bound to soluble TREM2 in CSF and plasma. For the total assay, a saturating amount of 4D9 antibody is added to eliminate unbound sTREM2. The ratio of 4D9‐bound sTREM2 to total sTREM2 is calculated to determine the level of TE achieved by the concentration of antibody present. Target engagement time course demonstrated near 100% 4D9‐bound sTREM2 in CSF of wild‐type mice at 24 h post‐dose. Over a 10‐day time course with time points at days 1, 2, 4, and 7 and study termination at day 10, the bound/total sTREM2 reduces gradually to reach ˜ 50% by day 10. Animals were dosed intravenously with 50 mg/kg of isotype and 4D9 antibodies. Data represent the mean ± SEM ( n = 5). Target engagement dose response demonstrated saturated bound sTREM2 at 50 and 10 mg/kg with > 50% bound sTREM2 at 1 mg/kg of antibody. 4D9‐bound sTREM2 was undetectable at 0.1 mg/kg and lower. 4D9 was IV‐dosed at 50, 10, 1, 0.1, 0.01, 0.001, and 0.0001 mg/kg, and isotype‐dosed at 50 mg/kg. CSF bound: total sTREM2 in wild‐type mice was measured at 24 h. Data represent the mean ± SEM ( n = 5). 4D9 antibody demonstrates a dose‐dependent increase in total brain TREM2 levels. Quantification of total TREM2 in brain lysates from wild‐type mice dosed with 4D9 or isotype control was performed by a MSD‐platform‐based ELISA. Data represent the mean ± SEM ( n = 5) and are shown as pg TREM2 per ug of total protein. One‐way ANOVA, Dunnett's post hoc test, P (isotype vs 4D9 [100 mg/kg]) < 0.0001; P (isotype vs 4D9 [50 mg/kg]) = 0.0007. Data information: Only male mice were used. Statistical evaluations are displayed as follows: *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Concentration Assay

Journal: EMBO Molecular Medicine

Article Title: Enhancing protective microglial activities with a dual function TREM 2 antibody to the stalk region

doi: 10.15252/emmm.201911227

Figure Lengend Snippet: Schematic outlining study design and timeline of intraperitoneal injections of either isotype control of 4D9 antibody in 6‐month‐old APP‐NL‐G‐F and age‐matched WT mice. Immunohistochemical costainings of TREM2 (magenta) and IBA1 (green) microglia in the cortex of isotype control and 4D9‐injected WT and APP‐NL‐G‐F mice. Side panel shows images from each staining at a larger magnification indicated by the dotted white boxes. Scale bar = 10 μm; scale bar (inset) = 2.5 μm. Quantification of cortical TREM2 stainings shown in (B). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 70.63, P ≤ 0.0001; treatment effect: F 1,23 = 14.33, P = 0.0010; genotype × treatment effect: F 1,23 = 14.51, P = 0.0009); P (WT isotype vs WT 4D9) > 0.9999; P (WT isotype vs APP isotype) = 0.02; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0001; n.s., not significant. Quantification of cortical IBA1 staining shown in B. Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,21 = 215.4, P ≤ 0.0001; treatment effect: F 1,21 = 0.5994, P = 0.4475; genotype × treatment effect: F 1,21 = 0.2992, P = 0.5902); P (WT isotype vs APP isotype) < 0.0001; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.9986; n.s., not significant. Confocal images of P2RY12 (red), IBA1 (green), and Aβ (gray) costainings from cortex. Top panel: Dotted white boxes indicate the areas in P2RY12 and IBA1 costainings that are magnified as inset. Bottom panel: Insets show P2RY12, IBA1, and Aβ costainings at a larger magnification. Of note, we did not observe a complete co‐localization of P2RY12 and IBA1 suggesting different microglial populations. Scale bar = 10 μm; scale bar (insets) = 5 μm. Quantification of P2RY12‐stained cells in the cortex shown in (E). Two‐way ANOVA, Tukey's multiple comparison test (genotype effect: F 1,23 = 71.99, P ≤ 0.0001; treatment effect: F 1,23 = 9.029, P = 0.0063; genotype × treatment effect: F 1,23 = 5.93, P = 0.0231); P (WT isotype vs APP isotype) = 0.0018; P (WT 4D9 vs APP 4D9) < 0.0001; P (APP isotype vs APP 4D9) = 0.0051. Data information: Each data point for all quantifications represents an average of n = 3 replicates per mouse. All data represent the mean ± SEM ( n = 6–7). No gender‐specific differences could be observed. Statistical evaluations are displayed as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Specificity of antibody 4D9 for mouse TREM2 was demonstrated by immunoblotting of 10 ng mouse TREM2‐(His) 6 as purchased from Sino Biological.

Techniques: Immunohistochemical staining, Injection, Staining