Human antagonistic TREM2 antibodies rescue elevated membrane-bound TREM2 levels and reduce p-Syk in primary human macrophages isolated from PGRN mutation carriersA.Figure 3A. Schematic presentation of human TREM2 with the identified binding site of antagonistic antibodies Ab1 and Ab2 (purple) in the Ig-like V-type domain. Light purple indicates the overlapping amino acid sequence of the two peptides, which are bound by Ab1 and Ab2 (see also EV1B). The disease associated Y38C and R47H mutations are indicated in yellow. Created with BioRender.com B. AlphaLISA-mediated quantification of p-Syk in human macrophages with a dose titration treatment of Ab1 and Ab2 with or without liposomes for 5 min. IC50 and maximal inhibition (max) are indicated by a dotted line. Data represent the mean ± SEM (n=3 independent experiments). Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant C. ELISA-mediated quantification confirms reduced PGRN serum levels in GRN mutation carriers vs. healthy controls. PGRN was measured by ELISA in technical triplicates and normalized to serum levels of healthy controls. Data points indicate individual patients (GRN-FTLD) and healthy controls (HC). Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant D. Western blot of TREM2 in lysates and conditioned media of cultured human macrophages isolated from GRN-FTLD patients and HC. Mature (mTREM2), immature (imTREM2), and soluble TREM2 (sTREM2) are indicated. Calnexin was used as loading control. E. Quantification of mTREM2 expression levels in lysates of cultured human macrophages isolated from GRN-FTLD patients (data shown in D). mTREM2 levels were normalized to HC (n=4). Data points indicate individual patients. Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant F. ELISA-mediated quantification of sTREM2 in conditioned media of human macrophages isolated from GRN-FTLD patients and HC (n=3). sTREM2 could not be measured in conditioned media of human macrophages isolated from patient #3 due to low overall cell yield. Data points indicate individual patients and HC. Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant G. Western blot of TREM2 in lysates and media of cultured human macrophages isolated from GRN-FTLD #1 and HC #1 upon treatment with Ab1 and Ab2. An isotype antibody was used as a negative control. ADAM protease inhibition (GM) does not further increase mTREM2 levels in GRN-FTLD patients. Equal amounts of protein were loaded. GAPDH was used as loading control. H. Quantification of mTREM2 expression normalized to HC (n=4) (data shown in G). Data points indicate individual patients. Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant I. ELISA-mediated quantification of sTREM2 in conditioned media of human macrophages isolated from GRN-FTLD patients (n=3). sTREM2 could not be measured in conditioned media of human macrophages isolated from patient #3 due to low overall cell yield. Data points indicate individual patients. Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant J. AlphaLISA-mediated quantification of p-Syk levels in human macrophages upon treatment with Ab1 and Ab2 with liposomes for 60 min (n=3). An isotype antibody was used as a negative control. Data points indicate individual patients. Isolated material from patient #3 did not yield enough cells to perform this experiment. Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant