Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine- 13 C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D , E , F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of Neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: The recombinant human TREM2 protein was obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Western Blot, Labeling

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Fracture-associated behavioral allodynia and spinal over-expressions of CCL21, TREM2, and DAP12 after orthopedic surgery in mice. The development of mechanical allodynia was assessed by paw withdrawal mechanical threshold (A) and paw withdrawal mechanical frequency to 0.16 g filament (B) in the von Frey test after sham surgery and tibial fracture ( n = 8). The development of cold allodynia was assessed by cold response scoring (C) in acetone test after sham surgery and tibial fracture ( n = 8). The spinal dorsal horn L 4 - 5 segments were collected for biochemical experiments. (D–F) ELISA identified the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture ( n = 5). All the data are expressed as mean ± SEM and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. group sham surgery.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Intrathecal pre-administration of artesunate reduces fracture-associated spinal over-expressions of CCL21, TREM2, and DAP12 and microglia activation. Intrathecal artesunate (100 μg) was administered daily for three consecutive days on days 4, 5, and 6 after tibial fracture with orthopedic surgery. All biochemical data were collected on day 7 after sham and fracture surgeries. (A–C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal CCL21, TREM2, and DAP12 proteins after tibial fracture. (D,E) Immunohistochemistry staining showed representative photomicrographs of the marker of microglia activation (Iba1) in the spinal dorsal horn after fracture intervention and artesunate treatment (scale bar, 50 μm). All biochemical results are expressed as mean ± SEM ( n = 3–5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group sham + DMSO, * p < 0.05 vs. group fracture + DMSO.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Marker

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Induction of acute pain by exogenous CCL21 and attenuation of allodynia by artesunate. Artesunate (i.t., 100 μg, indicated by a red arrow) was injected 60 min prior to recombinant CCL21 (i.t., 0.1 μg) application. (A,B) Exogenous CCL21-induced acute inflammatory pain was alleviated by the pre-application of artesunate. All behavioral results are mean ± SEM ( n = 8) and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. All biochemical data were collected at hour 12 after intrathecal injection. (C) ELISA identified that pretreatment with artesunate downregulated the increased levels of spinal TREM2 and DAP12 proteins after exogenous CCL21 exposure. All biochemical results are expressed as mean ± SEM ( n = 5) and analyzed by one-way ANOVA with Bonferroni post hoc comparisons. # p < 0.05 vs. group DMSO, * p < 0.05 vs. group recombinant CCL21 (0.1 μg) + DMSO.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Injection, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Artesunate Therapy Alleviates Fracture-Associated Chronic Pain After Orthopedic Surgery by Suppressing CCL21-Dependent TREM2/DAP12 Inflammatory Signaling in Mice

doi: 10.3389/fphar.2022.894963

Figure Lengend Snippet: Spinal neutralization of TREM2 reduces fracture-associated chronic pain and CCL21-induced acute pain. (A,B) A neutralizing antibody against TREM2 (anti-TREM2, i.t., 2 μg, indicated by a red arrow) was injected on day 14 after tibial fracture. Behavioral test showed the attenuation of the established fracture-associated mechanical allodynia and cold allodynia by anti-TREM2. (C,D) Anti-TREM2 (i.t., 2 μg, indicated by a red arrow) was injected 60 min prior to recombinant CCL21 (i.t., 0.1 μg). Exogenous CCL21-induced acute inflammatory pain was alleviated by the pre-application of anti-TREM2. All results are mean ± SEM ( n = 8) and analyzed by two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant CCL21 (Abcam, ab201361, United Kingdom), a neutralizing antibody against CCL21 (anti-CCL21, R&D Systems, AF457, United States) and a neutralizing antibody against TREM2 (anti-TREM2, R&D Systems, 1729-T2, United States), was dissolved in normal saline or 10% DMSO for i.t.

Techniques: Neutralization, Injection, Recombinant

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 1 TREM2 is a potential marker of colorectal cancer (CRC). (A) Kaplan-Meier analysis representing the association of TREM2 expression with overall survival in CRC patients from The Cancer Genome Atlas cohort; ≥ 50% quantile of TREM2 expression is defined as the high-expressing group, and others are considered in the low-expressing group. (B) Immunohistochemistry staining representing TREM2 (brown) expression in CRC tissues. (C) Bar plots summarizing the immunohistochemistry semi-quantitative analysis; score 1, ≤ 25%; 2, 26–50%; 3, 51–75%; 4, > 75%. Scale bar: 100 μm; magnification: 200× (n = 4). (D) Relative TREM2 mRNA levels in CRC patients (n = 4). (E) CRC tissues or normal tissues were double stained with anti-CD206 (marked macrophages) (green) and anti-TREM2 (red) antibodies, and then observed by fluorescent microscopy. DAPI, blue (n = 10). Scale bars: 20 μm. Data are expressed as means ± SD; * P < 0.05, **P < 0.01, by unpaired Student’s t test (C-D). TREM2, triggering-receptor-expressed on myeloid cells 2; CD, cluster of differentiation; DAPI, 4′,6-diamidino-2-phenylindole

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Marker, Expressing, Immunohistochemistry, Staining, Microscopy

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Scheme 1 Schematic illustration of targeted delivery of a PD1-TREM2 scFv by CAR-T cells to enhances anti-tumor efficacy in colorectal cancer. First, the lentivirus encoding CART-CEA.sBsAb was generated using a three-plasmid lentiviral packing system. Mouse T cells were transduced with the encoding CART-CEA.sBsAb, and the successful preparation of autocrine PD-1-TREM2 scFv CAR-T cells (CART-CEA.sBsAb) was achieved. The anti-tumor mechanism of CART-CEA.sBsAb was evaluated in a subcutaneous CRC mouse model. The PD-1-TREM2 scFv secreted by CAR-T cells remained localized within tumors and blocked the PD-1/PD-L1 pathway and the binding of ligands to TREM2 receptors present on MDSCs and TAMs; the percentages of MDSCs and TAMs were decreased, and the levels of TNF-α, IFN-γ, IL-2, granzyme B, and perforin were enhanced. Finally, the intratumoral proportion and cytotoxic ability of CART-CEA.sBsAb increased

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Generated, Plasmid Preparation, Transduction, Binding Assay

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 2 Construction and characterization of the bi-specific scFv antibody (BsAb). (A) Schematic representation of PD-1-scFv, TREM2-scFv, and BsAb. (B) SDS-PAGE analysis of PD-1 scFv, TREM2 scFv, and BsAb derived from the supernatants of CHO cells using nickel column purification and concentration. (C) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to 293T stably expressing PD-1 (293T-PD-1). The 293T-PD-1 cells were treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (D) Flow cytometric histograms demonstrating His-tag detection of PD-1 scFv, TREM2 scFv, and BsAb binding to TREM2 on the RAW 264.7 cells. PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL and MFI of His was detected by flow cytometry. Bar plots summarizing the data are shown on the right. (E-F) Relative viability of MC38 cells, mouse primary hepatocytes, and mouse primary intestinal cells treated with PD-1 scFv, TREM2 scFv, and BsAb at 200 µg/mL for 24 h analyzed using the CCK-8 kit. Data are expressed as means ± SD; * P < 0.05, **P < 0.01 (n = 3), by unpaired Student’s t test (C-D). PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary; RAW 264.7, Mouse Mononuclear Macrophages Cells; 293T, human embryonic kidneys; CCK, cell counting kit; SD, standard deviation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: SDS Page, Derivative Assay, Nickel Column, Purification, Concentration Assay, Binding Assay, Stable Transfection, Expressing, Flow Cytometry, CCK-8 Assay, Polyacrylamide Gel Electrophoresis, Cell Counting, Standard Deviation

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 3 The bi-specific scFv antibody (BsAb) delays tumor growth and modulates intratumoral cytokines and the proportions of infiltrating immune cells, MDSCs, and M2 TAMs. (A) Schematic representation of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 106 MC38 cells expressing CEA. On day 7, mice received intravenous injection of PD-1 scFv, TREM2 scFv, or the BsAb at 200 µg/mouse daily; the PBS-treated group served as the control. (B) Bioluminescence images of mice treated with PD-1 scFv, TREM2 scFv, and BsAb at days 7, 14, and 21 (n = 4). (C) Biolumi nescence signals of individual groups at day 7, 14, and 21 are shown on the Y-axis. The PBS group is indicated as black, the PD-1 scFv group is in orange, the TREM2 scFv group is in blue, and the BsAb group is in red. (D-E) Tumor volume and body weight of mice subjected to the indicated treatments (n = 4). (F-H) MC38-CEA tumors excised from individual groups of sacrificed mice on day 23. Tumor volume and weight of mice subjected to the indicated treat ments (n = 4). (I) Effect of different treatments on the survival of tumor-bearing mice. (J-L) Effect of different treatment modalities on the infiltration of CD8-positive T cells (J), MDSCs (K), and M2 TAMs (L). Proportions of tumor-infiltrating CD8-positive T cells, MDSCs, and M2 TAMs were detected by flow cytometry on day 23. All cells gated on CD45, CD45 + CD11b Gr1 + were marked as MDSCs, whereas cells gated at CD45 + CD11b F4/80 CD206 were marked as M2 TAMs. Representative plots are shown on the right (n = 4). Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test and one-way ANOVA. scFv, single-chain fragment variable; CEA, carcinoembryonic antigen; PD-1, programmed death-1; TREM2, triggering-receptor- expressed on myeloid cells 2; PBS, phosphate buffered saline; MDSCs, myeloid-derived suppressor cells; TAMs, tumor associated macrophages; CD, cluster of differentiation

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Control, Flow Cytometry, Saline, Derivative Assay

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 4 Construction and characterization of bi-specific scFv antibody (BsAb)- secreting CAR-T cells. (A) Schematic representation of Mock, PD-1 scFv, TREM2 scFv, and BsAb-secreting CAR constructs targeting CEA. CART-CEA was constructed as a control. (B) Efficient transduction of all constructs in mouse primary T cells as observed by fluorescence microscopy. Scale bar: 50 μm. (C, D) Flow cytometric assay analysis (C) and bar plot summary (D) indicating the transduction efficiency of all constructs in mouse primary T cells. (E) Western blot detecting the His-tag for CAR protein in mouse primary T cells. (F) Cytotoxicity of all constructed CAR-T cells against MC38 cells. All groups of CAR-T cells were co-cultured with MC38 cells at 1:1, 4:1, 8:1, and 16:1, ratios. MC38 cell lysis was detected by an LDH cytotoxicity kit. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by one-way ANOVA. CAR-T, chimeric antigen receptor-modified-T; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; CEA, carcinoembryonic antigen; LDH, lactate dehydrogenase

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Construct, Control, Transduction, Fluorescence, Microscopy, Flow Cytometry, Western Blot, Cell Culture, Lysis, Modification

Journal: Molecular cancer

Article Title: Target delivery of a PD-1-TREM2 scFv by CAR-T cells enhances anti-tumor efficacy in colorectal cancer.

doi: 10.1186/s12943-023-01830-x

Figure Lengend Snippet: Fig. 5 Anti-tumor effects of armed CAR-T cells in tumor-bearing mice. (A) Time-course diagram of the animal experiment. Six-week-old C57BL/6J mice were subcutaneously injected with 2 × 106 MC38 cells expressing CEA. On day 7, mice from all groups were individually injected with 1 × 107 constructed CAR-T cells through the tail vein. Tumor imaging was performed once a week. (B) Bioluminescence images of mice treated with CART-CEA, CART-CEA. sPD-1scFv, CART-CEA.sTREM2 scFv, and CART-CEA.sBsAb at days 7, 14, and 21; PBS treatment served as the control (n = 4). (C) Bioluminescence signals of mice in each group at days 7, 14, and 21 are shown on the Y-axis. The PBS group is shown in black, the CART-CEA group in purple, the CART-CEA.sPD-1 scFv group in orange, the CART-CEA.sTREM2 scFv group in blue, and the CART-CEA.sBsAb group in red. (D) Comparison of tumor volume over time in each group of tumor-bearing mice. (E) Weight development of mice during treatment in each group. (F) Effect of different treatments on the survival of tumor-bearing mice. Data are expressed as means ± SD; * P < 0.05; **P < 0.01, by unpaired Student’s t test. CAR-T, chimeric antigen receptor-modified-T; CEA, carcinoembryonic antigen; PD-1, programmed death-1; scFv, single-chain fragment variable; TREM2, triggering-receptor-expressed on myeloid cells 2; PBS, phosphate-buffered saline

Article Snippet: The binding ability of PD-1 scFv, TREM2 scFv, and bispecific scFv to PD-1 and TREM2 protein were evaluated by sandwich ELISA, wherein recombinant mouse PD-1-Fc (GenScript) or TREM2 protein (R&D Systems) (200ng/mL) were coated on the plates, followed by purified PD-1 scFv, TREM2 scFv, and bi-specific scFv detected by His-tag antibody-HRP (1:500 diluted; Abcam).

Techniques: Injection, Expressing, Construct, Imaging, Control, Comparison, Modification, Saline

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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

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 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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

Techniques: Flow Cytometry, Binding Assay, Stable Transfection, In Vitro, Activity Assay, Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Titration, Plasmid Preparation, Liposomes, 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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

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: 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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

Techniques: Flow Cytometry, Binding Assay, Control, Labeling, Clinical Proteomics, Membrane, 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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

Techniques: Drug discovery, In Vivo, Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, 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: Eight‐month‐old female Wistar rats were immunized subcutaneously (s.c.) and intraperitoneally (i.p.) with a mixture of 70 μg recombinant his‐tagged mouse Trem2 protein (aa19–171, Creative BioMart) in 500 μl PBS, 5 nmol CpG2006 (TIB MOLBIOL, Berlin, Germany), and 500 μl Incomplete Freund's adjuvant.

Techniques: Control, Immunohistochemical staining, Injection, Staining, Comparison