Additional file 4 of LILRB2-mediated TREM2 signaling inhibition suppresses microglia functions
Creators: name: Zhao, Peng
name: Xu, Yuanzhong
name: Jiang, Lu-Lin
name: Fan, Xuejun
name: Ku, Zhiqiang
name: Li, Leike
name: Liu, Xiaoye
name: Deng, Mi
name: Arase, Hisashi
name: Zhu, Jay-Jiguang
name: Huang, Timothy Y.
name: Zhao, Yingjun
name: Zhang, Chengcheng
name: Xu, Huaxi
name: Tong, Qingchun
name: Zhang, Ningyan
name: An, Zhiqiang
Publisher: name: figshare
Year: 2022
Subjects: subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Additional file 4: Supplementary Figure 4. Screening of LILRB2 antibodies for rescuing LILRB2-mediated TREM2 signaling inhibition. a-b. LILRB2 antibodies rescue oAβ or PS-LILRB2-mediated inhibition of TREM2 signaling. Plate-coated oAβ (a) or PS (b) was incubated with LILRB2/TREM2 reporter cells in the presence of 10 μg/mL purified LILRB2 antibodies. The activation of LILRB2/TREM2 reporter cells was observed as percentage GFP+ cells. TREM2 signaling in the treatment groups was normalized based on the percentage of GFP+ reporter cells expressing only TREM2 (set to 100%). Data are presented as mean ± SD (n = 4 independent experiments). c. Surface expression of TREM2 and LILRB2 on HMC cells. Indicated antibodies were used to stain surface receptors on HMC cells after Fc blocking by human Fc fragment. The antibody was detected by Alexa Fluor 488-streptavidin, and the fluorescent signals (x-axis) were plotted as a histogram with normalized cell percentage on the y-axis. d. MFI of HMC3 cells stained by indicated antibodies (x-axis) as shown in c. Data are presented as mean ± SD (n = 4 independent experiments). e. Immunoblot of phosphorylated SHP1 (pSHP1), SHP1 of HMC3 upon incubation with oAβ-lipoprotein complex with indicated treatments for 1 hour. Results are from total input cell lysate with β-actin as the loading control. f-g. oAβ or PS activation of TREM2 GFP reporter cells. Plate-coated oAβ (f) or PS (g) was incubated with reporter cells expressing either human or mouse TREM2, and the percentage of GFP+ cells is presented. Data are presented as mean ± SD (n = 4 independent experiments). h-i. Blocking of oAβ or PS-TREM2 signaling by a TREM2 antagonist antibody. Plate-coated oAβ (h) or PS (i) was incubated with reporter cells expressing either human or mouse TREM2 in the presence of TREM2 antagonist antibody or control IgG, the percentage of GFP+ cells are shown with an antibody treatment. Data are presented as mean ± SD (n = 4 independent experiments). j. The LILRB2 antibody clone 42D1 was validated for not blocking either oAβ- or PS-induced LILRB2 signaling. Plate-coated oAβ or PS was incubated with LILRB2-GFP reporter cells in the presence of soluble 42D1 or control IgG, the percentage of GFP+ cells is shown with an antibody treatment. Data are presented as mean ± SD (n = 4 independent experiments). k. Plate-coated 42D1 activates LILRB2 signals. Plate-coated anti-LILRB2 clone 42D1 or control IgG was incubated with LILRB2-GFP reporter cells. The percentages of GFP+ cells are shown. Data are presented at mean ± SD (n = 4 independent experiments). l. Antibody 42D1 showed no recuse of LILRB2-mediated TREM2 signaling inhibition in reporter cells. LILRB2-TREM2 2B4 reporter cells were stimulated with either oAβ or PS co-incubated with Ctrl Ig, 42D1, or Ab29. The TREM2 signaling was detected as increased GFP expression. Data are presented at mean ± SD (n = 3 independent experiments). m. Validation of negative controls and quenching method used in the hMGL phagocytosis assay. hMGLs were incubated with oAβ-lipid (fluorescence-labeled) under the conditions labeled on the x-axis. The temperatures are incubation temperature. CytoD means co-incubated with CytoD during phagocytosis. TB means trypan blue, which was added after phagocytosis to quench cell surface FAM signals as described in the method section. Data are presented at mean ± SD (n = 3 independent experiments).