Fig. 2: ATV:TREM2 is a novel activating antibody that potentiates receptor clustering and endocytosis to enhance TREM2 signaling.
a, Antibody schematic of ATV:TREM2 with human TREM2 Fab affinity and ATV binding site in the Fc domain and effectorless Fc mutations. b, Fluorescence polarization (FP)-based detection of TREM2 stalk peptide cleavage by recombinant ADAM17 (n = 4 independent experiments; two-tailed unpaired t-test, mean ± s.e.m.). c, ATV:TREM2 and PS liposome co-treatment enhanced pSyk in WT iMG (n = 3 independent experiments; Tukey’s multiple comparisons test, mean ± s.e.m.). d, Schematic illustrating ATV and TREM2 Fab valency effects on pSyk signaling. Antibodies include anti-TREM2 and ATV:TREM2 with MV and BV Fabs. e, hTREM2-DAP12 HEK293 cells treated with a dose response of antibodies for 5 minutes, followed by pSyk detection (n = 3 independent experiments; mean ± s.e.m.). f, pSyk is blocked by co-treatment of ATV:TREM2 and anti-TfR. hTREM2-DAP12 HEK293 cells were dosed with 100 nM TREM2 antibodies and a titration anti-TfR. pSyk was detetced 5 minutes after treatment (n = 3 independent experiments; mean ± s.e.m.). g, TfR and TREM2 co-IP. hTREM2-DAP12 HEK293 cells were treated with 100 nM per antibody for 5 minutes, followed by IP on cell lysates with anti-TREM2. TfR was detected by western blot. h, Co-IP quantification of western blot data in g (n = 6 independent experiments; Wilcoxon test for ISO versus anti-TREM2, two-tailed paired t-test for ATV:ISO versus ATV:TREM2, mean ± s.e.m.). i, Schematic of BioID TREM2 receptor clustering assay strategy. j, Representative western blot detection of biotinylated TREM2 after streptavidin IP. TREM2-BioID expression was induced 24 hours before the assay with 2 ng ml−1 of Dox. Cells were treated with 100 nM antibody and 2 µM biotin. k, Quantification of western blot from j (n = 4 independent experiments; ratio of two-tailed paired t-test, mean ± s.e.m.). l, Representative immunofluorescence images of hTREM2-DAP12 HEK293 cells stained for IgG (green), pSyk (yellow) and EEA1 (red). Cells were treated with 10 nM per antibody for 10 min. m, Quantification of spot intensity for IgG and pSyk immunofluorescence per cell (n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test, mean ± s.e.m.). n, Quantification of percent of IgG or pSyk spots localized within EEA1+ endosomes (n = 3 independent experiments with 3,000–5,000 cells per condition; Tukey’s multiple comparisons test, mean ± s.e.m.).
