Fig. 5

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 oAβ-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 oAβ-lipid. The hMGLs were pre-labeled with CFSE to visualize the cells under the fluorescence microscope. Scale bar = 100 μm. e. Ab29 promotes oAβ-lipoprotein complex phagocytosis by hMGLs. hMGL cells were incubated with FAM-oAβ-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 oAβ-lipid. Scale bar represents 5 μm. g. PLA assay indicates that oAβ-lipid induces clustering of LILRB2 and TREM2, while Ab29 blocks the clustering. hMGLs were incubated with oAβ-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 oAβ-lipoprotein complex-induced inflammatory cytokine responses in hMGLs. hMGL cells were incubated with oAβ-lipoprotein complex with indicated treatments. The relative mRNA levels of TNF-α (g), IL-6 (h), CCL3 (i), and IL-1β (j) were determined using qRT-PCR with GAPDH as the internal control. Data are presented as mean ± SD (n = 3 independent experiments)