Fig. 4

LILRB2 blocking antibody Ab29 completely rescued LILRB2-mediated TREM2 signaling inhibition and promoted microglial phagocytosis by blocking LILRB2/TREM2 co-ligation. a. Schematic diagram showing the design of a BiFc assay studying the effect of LILRB2 and TREM2 blocking antibodies in preventing the ligand-mediated receptor co-ligation. b and c. LILRB2 blocking antibodies abolish oAβ or PS-induced co-ligation of LILRB2 and TREM2. Antibodies at 10 μg/mL were incubated with HEK293T cells co-expressing LILRB2-N173 Venus and TREM2-C155 Venus plus plate-coated oAβ (b) or PS (c). The MFI signals from complemented Venus are shown. Data are presented as mean ± SD (n = 3 independent experiments). d. Representative immunofluorescence images showing Ab29 blocks oAβ-lipid from inducing LILRB2-TREM2 co-ligation. Scale bar represents 5 μm. e. schematic diagram showing LILRB2 blocking antibody rescues TREM2 signaling inhibition by blocking LILRB2-ligand interactions. The assay is depicted as an NFAT-GFP reporter assay. The ITIM motifs of LILRB2 block signaling generated by ITAM motifs of TREM2 upon cross-linking of the two receptors by shared ligands oAβ or PS. f-g. Titration of Ab29 in rescuing of oAβ or PS-LILRB2-induced inhibition of TREM2 signaling. Plate-coated oAβ (f) or PS (g) was incubated with LILRB2/TREM2 reporter cells under the presence of increasing concentrations of Ab29 or control IgG. The activation of LILRB2/TREM2 reporter cells was observed as percentages of GFP⁺ cells. TREM2 signaling of 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). h. Ab29 promotes oAβ-lipoprotein complex phagocytosis by HMC3 human microglial cell line. HMC3 cells were incubated with FAM-oAβ-lipoprotein complex and indicated antibodies (treatment table). Phagocytosis was quantified by flow cytometry, with the MFI shown. Negative phagocytosis control included 10 μM CytoD together with indicated treatments. Data are presented as mean ± SD (n = 4 independent experiments). i. Ab29 improves TREM2 pathway signaling as indicated by the increased pSYK level. HMC3 cells were incubated with oAβ-lipoprotein complex with indicated treatments for 1 hour. Immunoblot of phosphorylated SYK (pSYK), total SYK of HMC3 upon treatment, with β-actin as the loading control. Data are presented as mean ± SD (n = 3 independent experiments). j. Ab29 blocks LILRB2 pathway signaling as indicated by the decreased pSHP1 level. Immunoblot of phosphorylated SHP1 (pSHP1), SHP1 of HMC3 upon incubation with oAβ-lipoprotein complex with indicated treatments for 1 hour. Results are from anti-LILRB2 immunoprecipitation with LILRB2 as the loading control. Data are presented as mean ± SD (n = 3 independent experiments)