Fig 2: 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 oAß 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 oAß (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 oAß-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 oAß or PS. f-g. Titration of Ab29 in rescuing of oAß or PS-LILRB2-induced inhibition of TREM2 signaling. Plate-coated oAß (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 oAß-lipoprotein complex phagocytosis by HMC3 human microglial cell line. HMC3 cells were incubated with FAM-oAß-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 oAß-lipoprotein complex with indicated treatments for 1 hour. Immunoblot of phosphorylated SYK (pSYK), total SYK of HMC3 upon treatment, with ß-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 oAß-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)

Fig 3: The shared ligands oAß and PS induce co-ligation of LILRB2 and TREM2 and inhibit TREM2 signaling. a. Schematic illustration of the cell line LILRB2-TREM2 showing the design of BiFc assay with LILRB2 and TREM2 fusion constructs. b-c. oAß or PS-induced co-ligation of TREM2 and LILRB2 as measured by the BiFc assay. HEK293T cells co-expressing LILRB2-N173 Venus and TREM2-C155 Venus were incubated with plate-coated oAß (b) or PS (c). Y-axis shows the MFI signals from complemented Venus. Data are presented as mean ± SD (n = 3 independent experiments). d. Representative immunofluorescence images showing oAß-lipid-induced co-ligation of TREM2 and LILRB2. Scale bar represents 5 µm. e. Schematic diagram showing co-ligation of LILRB2 and TREM2 by oAß or PS inhibits TREM2 signaling in reporter cell assays. The ITIM motifs of LILRB2 attenuate signaling generated by ITAM motifs of TREM2 upon cross-linking by shared ligands oAß or PS. f-g. oAß or PS-induced TREM2 signaling of reporter cells co-expressing LILRB2 and TREM2. Reporter cells expressing corresponding receptors (x-axis) were incubated with oAß (f) or PS (g). Y-axis is TREM2 signaling of treatment groups 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. oAß-lipoprotein complex phagocytosis profile of BV2 cells expressing the LILRB2 transgenes (x-axis). BV2 cells were incubated with FAM-oAß-lipoprotein complex. 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)

Fig 4: LILRB2 targeting antibody Ab29 showed potent blocking activity, high affinity, and specificity. a-b. Titration of blocking the activity of Ab29 against oAß or PS-LILRB2 interactions. Plate-coated oAß (a) or PS (b) was incubated with LILRB2-chimeric reporter cells under the presence of increasing concentrations of Ab29 or control IgG. The activation of LILRB2-chimeric reporter cells was observed as a percentage of GFP+ cells. Data are presented as mean ± SD (n = 3 independent experiments). c. Representative immunostaining images showing Ab29 blocks oAß and PS from binding to LILRB2 expressed on the cell surface. Scale bar represents 5 µm. d. Ab29 blocks LILRB2 binding with oAß as measured by BLI. LILRB2 was loaded onto protein A sensors via binding with sensor-captured Ab29. The LILRB2-loaded sensors were then incubated with biotinylated oAß (1 µM) and the binding signals were amplified with streptavidin. The amount of oAß bound onto the sensors was presented as wavelength shift in nanometers (nm). The red dotted vertical line marks the transit from incubation with biotinylated oAß to signal amplification by streptavidin. e. Ab29 blocks LILRB2 binding with PS liposomes as measured by BLI. LILRB2 was loaded onto protein A sensors via binding with sensor-captured Ab29. The LILRB2-loaded sensors were incubated with PS liposomes (1 mM) (association stage) and then the sensors were dipped into kinetics buffer without PS allowing the bound PS to freely dissociate (dissociation stage). The amount of PS 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. f. Cross-reactivity of Ab29 against other LILRB and LILRA family receptors as measured by ELISA. Plate-coated LILRB and LILRA family receptors (shown in the x-axis) were incubated with Ab29 (4 nM). The amount of bound antibody was detected by anti-human F(ab)2 HRP, and the values are shown as OD450. LILRB2-Fc was included as the positive control. Data are presented as mean ± SD (n = 4 independent experiments)

Fig 5: LILRB2 and TREM2 are expressed on human microglia and they share ligands oAß and PS. a. Immunofluorescence staining of AD patient brain tissue for LILRB2, IBA1 (microglia marker), amyloid plaques (6E10), GFAP (astrocyte marker), and TREM2. Scale bar = 20 µm. b. Titration curves of oAß binding to LILRB2 and TREM2 Fc fusion proteins by ELISA. Data are presented as mean ± SD (n = 3 independent experiments). c. Titration curves of PS binding to LILRB2 and TREM2 Fc fusion proteins by ELISA. Data are presented as mean ± SD (n = 3 independent experiments). d. Representative immunostaining images of oAß and PS binding to LILRB2 and TREM2 expressed on 293 T cell surface. Scale bar represents 5 µm. e-h. oAß or PS binding to LILRB2 or TREM2 as measured by BLI. In the association stage, protein A sensor-captured Fc fusion proteins (LILRB2-Fc: e and f, TREM2-Fc: g and h) were incubated with oAß (1 µM, e and g) or PS liposomes (1 mM, f and h), and the amount of oAß (e and g) or PS (f and h) 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 ligands allowing free dissociation