Additional file 3 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 3: Supplementary Figure 3. LILRB2 targeting antibodies showed potent blocking activity, high affinity, and specificity. a-b. Screening of antibodies inhibiting oAβ or PS-induced activation of LILRB2-chimeric reporter. Plate-coated oAβ (a) or PS (b) was incubated with LILRB2-chimeric reporter cells under the presence of unpurified antibody supernatant (1:20 dilution, antibody name is shown in x-axis). The activation of LILRB2-chimeric reporter cells was observed as percentages of GFP+ cells. Data are presented as mean ± SD (n = 3 independent experiments). c-d. Titration of blocking activities of purified LILRB2 antibodies against oAβ- or PS-LILRB2 interactions. Plate-coated oAβ (c) or PS (d) was incubated with LILRB2-chimeric reporter cells under the presence of increasing concentrations of purified LILRB2 antibodies (antibody names are shown in the figure legend). The activation of LILRB2-chimeric reporter cells was observed as percentages of GFP+ cells. Data are presented as mean ± SD (n = 3 independent experiments). e-f. Maximum wavelength shifts of the oAβ- (e) or PS (f)-LILRB2 binding curve in association stage presented in g and h, respectively. g-h. Antibodies blocking LILRB2 binding with oAβ or PS as measured by BLI. LILRB2 was loaded onto protein A sensors via binding with sensor-captured LILRB2 antibodies. The LILRB2-loaded sensors were then incubated with biotinylated oAβ (1 μM, g) or PS (1 mM, h). The amount of oAβ (g) or PS (h) bound onto the sensors is presented as wavelength shift in nanometers (nm). i. Binding kinetics profile between Ab29 and LILRB2 as measured by BLI. In the association stage, protein A sensor-captured Ab29 was incubated with LILRB2-His at indicated concentrations for the designated time presented on the x-axis. The amount of LILRB2-His bound onto the sensors is presented as a wavelength shift in nanometers (nm). The red dotted vertical line marks the transit from association stage to dissociation stage, where the sensors were dipped into kinetics buffer without LILRB2-His allowing free dissociation. j. Titration profiles of purified antibodies binding to plate-coated LILRB2 as measured by ELISA. Data are presented as mean ± SD (n = 3 independent experiments). k. Binding of purified LILRB2 antibodies to LILRB2 expressed on HEK293T cells. The percentage of positive staining was gated based on control IgG-treated cells. Data are presented as mean ± SD (n = 3 independent experiments). l. Cross-reactivity of all LILRB2 blocking antibodies against other LILRB and LILRA family receptors as measured by ELISA. LILRB2-Fc was included as the positive control. Data are presented as mean ± SD (n = 3 independent experiments). m. Cross-reactivity of Ab29 to LILRB and LILRA family receptors as measured by BLI. In the association stage, protein A sensor-captured Ab29 was incubated with Fc fusion proteins of LILRB and LILRA family receptors. The amount of Fc fusion bound onto the sensors was presented as wavelength shift in nanometers (nm). The red dotted vertical line marks the transit from association stage to dissociation stage, where the sensors were dipped into kinetics buffer without Fc fusion receptors allowing free dissociation. LILRB2-Fc was included as the positive control. n. Maximum wavelength shifts of the Ab29-LILR binding curve in association stage as presented in m.