Extended Data Fig. 4: ATV promotes TfR-TREM2 receptor complex formation and internalization and endosomal TREM2 signaling.

(a) Representative Western blot of co-IP of TREM2 with TfR. hTREM2-DAP12 HEK293 cells were treated with 100 nM ATV:TREM2, anti-TREM2, or isotype controls for 5 min at 37 °C. (b) Co-IP quantification of Western blot from (A) (n = 6 independent experiments; two-tailed paired t-test for ISO vs anti-TREM2; two tailed Wilcoxon test ATV:ISO vs ATV:TREM2, mean ± SEM). (c) Schematic illustration of cis- and trans-activation models that could mediate pSyk enhancement by ATV:TREM2. (d) Western blot validation of TfR knockdown in the TfR^RNAi cell line. (e) Cell based cis/trans assay indicates ATV:TREM2 enhances pSyk activity in cis. Relative pSYK signal is expressed as raw pSYK AlphaLisa value normalized to ATV:TREM2 treated control (n = 3 independent experiment, mean ± SEM). (f) Normalized pSyk signal measured by AlphaLisa assay. TfR^RNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of recombinant TfR protein for 5 min at 37 °C (n = 3 independent experiment, mean ± SEM). (g) Normalized pSyk signal detected by AlphaLisa. TfR^RNAi cells were treated with 10 nM anti-TREM2 or ATV:TREM2 pre-incubated with a dose titration of a secondary anti-human IgG Fc antibody for 5 min at 37 °C (n = 3 independent experiment, mean ± SEM) (h) Immunofluorescence microscopy of hTREM2-DAP12 HEK293 cells demonstrates reduction of surface TREM2 levels with ATV:TREM2 vs anti-TREM2, no changes in total TREM2 levels, consistent with re-distribution of the receptor from the plasma membrane to endosomes (n = 4 independent experiments except for anti-TREM2 MV (n = 3), Tukey’s multiple comparisons test, mean ± SEM). (i) hTREM2-DAP12 HEK293 cells dosed with antibody for 10 minutes shows that at similar amounts of bound antibody detected by anti-IgG (representing 5 nM of ATV:TREM2 and 10 nM of anti-TREM2, n = 4 independent experiments, Tukey’s multiple comparisons test, mean ± SEM(j) Images depicting masking algorithm used to identify whether TfR-Alexa-647 labeled recycling endosomes (rainbow spots in middle images) either contain (green spots in right-most images) or do not contain (red spots in right-most images) IgG spots (white spots in left-most image) upon dosing with anti-TREM2 (top row) or ATV:TREM2 (bottom row) for 10 minutes with 10 nM antibody. (k) Representative images for hTREM2-DAP HEK293 cells dosed with 10 nM antibody for 10 minutes including 20 ug mL⁻¹ TfR-Alexa-647, fixed, permeabilized, and stained with anti-IgG and anti-pSyk. IF shows ATV increased colocalization of antibody with pSyk in early endosomes. (l) Quantification of percent of IgG or pSyk spots localized within Tf-positive endosomes (n = 3 independent experiments, Tukey’s multiple comparisons test, mean ± SEM).