Fig 2: 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

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 blocking antibody Ab29 promoted hMGL migration, phagocytosis, and cytokine responses. a. Surface expression of TREM2 and LILRB2 on hMGL cells. The fluorescent signals (x-axis) were plotted as a histogram with normalized cell percentage in the y-axis. b. MFI of hMGL cells labeled by indicated antibodies (x-axis) as shown in a. Data are presented as mean ± SD (n = 3 independent experiments). c. Ab29 promotes hMGL cell migration. hMGL cells were plated onto transwell chamber inserts, and hMGL cell migration toward the oAß-lipoprotein complex in the receiving chamber was measured with treatments shown. For quantification, crystal violet-stained hMGL cells were quantified by eluting crystal violet in 33% acetic acid and measured the absorbance at 590 nm using a plate reader. Percentages of migrated cells over total are plotted. Data are presented as mean ± SD (N = 3 independent experiments). d. Representative immunostaining images showing Ab29 promotes hMGL migration induced by oAß-lipid. The hMGLs were pre-labeled with CFSE to visualize the cells under the fluorescence microscope. Scale bar = 100 µm. e. Ab29 promotes oAß-lipoprotein complex phagocytosis by hMGLs. hMGL cells were incubated with FAM-oAß-lipoprotein complex and indicated antibodies. 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 = 3 independent experiments). f. Representative immunostaining images showing Ab29 promotes hMGL phagocytosis of oAß-lipid. Scale bar represents 5 µm. g. PLA assay indicates that oAß-lipid induces clustering of LILRB2 and TREM2, while Ab29 blocks the clustering. hMGLs were incubated with oAß-lipid (100 nM) and antibodies (Ctrl IgG or Ab29, 10 µg/mL), and the LILRB2 and TREM2 clustering were detected by PLA assay. The red color indicates the PLA signals detected, and the blue color represents the cell nucleus. Scale bar = 20 µm. h-k. Ab29 promotes oAß-lipoprotein complex-induced inflammatory cytokine responses in hMGLs. hMGL cells were incubated with oAß-lipoprotein complex with indicated treatments. The relative mRNA levels of TNF-a (g), IL-6 (h), CCL3 (i), and IL-1ß (j) were determined using qRT-PCR with GAPDH as the internal control. Data are presented as mean ± SD (n = 3 independent experiments)

Fig 5: Key domains and amino acid residues of LILRB2 for ligands and antibody binding. a. Schematic diagram showing the design of domain-swapping mutants of LILRB2. B1D1 and B1D2 mutants were generated by replacing the D1 and D2 domains of LILRB2 with their corresponding domains from LILRB1, respectively. b-d. oAß, PS, and Ab29 binding to LILRB2 mutants as measured on chimeric reporter cells. Chimeric NFAT-GFP reporter cells expressing individual mutants of LILRB2 (listed in x-axis) were incubated with oAß (b) PS (c), and Ab29 (d). The activation of reporter cells is shown as the percentage of GFP+ cells. Data are presented as mean ± SD (n = 3 independent experiments). e. Alignment of D1 and D2 domains of LILRB2 and LILRB1. Amino acid residues are numbered according to Uniprot entry Q8N423. Identical residues are shown in white text with red background. Similar residues are shown in black text with a yellow background. Non-conserved residues are in white background. Individual domains of LILRB2 are also labeled. The red arrows indicate the single-amino acid mutations designed for testing. f. Crystal structure of LILRB2 (2gw5) visualized in UCSF Chimera showing the D1 and D2 domains as ribbons. Selected residues for mutation studies are shown in ball-and-stick with side chains displayed. Three-letter code names and residue numbers are also labeled following Uniprot entry Q8N423. g. Schematic diagram showing the distribution of single-amino acid mutations in LILRB2 D1 and D2 domains