Journal: JCI Insight

Article Title: Tumor-infiltrating exhausted CD8 + T cells dictate reduced survival in premenopausal estrogen receptor–positive breast cancer

doi: 10.1172/jci.insight.153963

Figure Lengend Snippet: CD8 + T cells from 8 ER + BC and 2 TNBC patient tissues were single-cell index sorted for whole-transcriptome analysis in the context of several cell surface proteins. ( A ) t-Distributed stochastic neighbor embedding (t-SNE) projection of 4 major clusters of CD8 + T cells identified and annotated as exhausted T cells, resident effector memory T cells, effector memory T cells, and central memory T cells. ( B ) Top 10 most differentially expressed genes for each CD8 + T cell cluster. ( C ) t-SNE overlay of CD8 + T cells identified as PD-1 – CD39 – (blue), PD-1 + CD39 – (orange), PD-1 + CD39 + (red), or PD-1 – CD39 + (gray). ( D ) Genes most significantly differentially expressed by PD-1 + CD39 + CD8 + T cells. ( E ) Overlay of cell surface protein expression onto t-SNE cluster projections. Protein expression for PD-1, CD103, CD69, CD39, CD137, and CCR7 acquired from index sort information and shown here as positively (purple) or negatively (yellow) expressed for each cell. Gene Set Enrichment Analysis (GSEA) of PD-1 + CD39 + CD8 + T cell differentially expressed genes as compared with T EX signatures identified from ( F ) lung cancer and ( G ) melanoma publications. Gene rank shown is derived from the current data set. ( n = 10 BC patients; 9 tumors; 2 T + LNs; 3 NCBTs; 7 matched PBMCs.)

Article Snippet: The GSEA was implemented by GSEA software (v4.0.3) with the latest Hallmark Molecular Signatures Database from Broad Institute ( , ).

Techniques: Expressing, Derivative 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 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: Concentrations of sTREM2 and membrane‐bound TREM2 were finally calculated using the MSD Discovery Workbench software (v4.0.12).

Techniques: Residue, Western Blot, Membrane, Stable Transfection, Inhibition, Negative Control, Control, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Transfection, Binding Assay, Sequencing, Comparison, 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 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: Concentrations of sTREM2 and membrane‐bound TREM2 were finally calculated using the MSD Discovery Workbench software (v4.0.12).

Techniques: Flow Cytometry, Binding Assay, Derivative Assay, Two Tailed Test, Western Blot, Membrane, Knock-Out, Control, 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: 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: Concentrations of sTREM2 and membrane‐bound TREM2 were finally calculated using the MSD Discovery Workbench software (v4.0.12).

Techniques: Drug discovery, In Vivo, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Injection, Concentration Assay