Extended Data Fig. 6: ATV:TREM2 increases phagocytosis and CSF1R in human TREM2 tg;TfR^mu/hu mice.

(a) Antibody levels detected in brain lysates by ELISA shows increased brain exposure of ATV:TREM2 (30 mg kg⁻¹) compared anti-TREM2 (30 mg kg⁻¹) and comparable brain exposure for ATV:TREM2 at 10 mg kg^-1 and anti-TREM2 at 30 mg kg⁻¹ at day 4 post-dose. (n = 5 mice/group except for ATV:TREM2 3 mg kg⁻¹ (n = 4)). (b) ATV:TREM2 increases CSF1R in the brain compared to brain exposure matched anti-TREM2 day 2 post dose (n = [8, 9, 8, 9, 10]mice/group, Kruskal-Wallis test, mean ± SEM). (c) CSF1R analysis in CSF same as in B. (n = [8, 9, 8, 9, 10]mice/group, Tukey’s multiple comparisons test, mean ± SEM) (b-c) Circles represents male mice and triangle represents female mice. (d) Detection of antibody concentrations show matched brain exposure of three ATV:TREM2 molecules with different ATV affinities 1-day post-dose (n = 5 mice/group). (e-f) High and mid-affinity ATV:TREM2 molecules induce comparable increase of CSF1R in the brain (e) n = 5 mice/group except for Vehicle (Veh) and day 4 10 mg kg⁻¹ 8,000 nM (n = 4, Kruskal-Wallis test, compared to vehicle group) and CSF (f) (n = 5 mice/group, except for Veh and day 4 5 mg kg⁻¹ 110 nM n = 4), Dunnett’s multiple comparisons test for day 1, Kruskal-Wallis test for day 4) day 1 and 4 post dose whereas low-affinity ATV:TREM2 induces a weak elevation of CSF1R in CSF at day 4 (n = 4-5 mice/group, mean ± SEM). (g) Schematic of experimental approach to evaluate in vivo dosed antibody impact to microglial phagocytosis ex vivo. A single dose of ATV:TREM2, anti-TREM2, or ATV:ISO was administrated to human TREM2 tg; TfR^mu/hu KI mice and brain microglia were isolated 2 days post dose for ex vivo myelin phagocytosis analysis. The same method was used to assess amyloid phagocytosis. (h) FACS gating strategy to quantify pHrodo-myelin positive microglia. After treatment with pHrodo-green myelin and staining, cell suspensions containing microglia were analyzed using BD FACS Aria III. Single cells were separated from debris by FSC and SSC characteristics. Live microglia were identified as a population of CD11b⁺ and propidium iodide^negative cells. pHrodo-myelin uptake was then quantified in 20,000 microglia recorded from each sample. (i) Antibody concentrations were detected in brain lysates by ELISA which shows comparable levels of ATV:TREM2 and anti-TREM2 day 2 post dose (n = [9, 10, 10, 9, 10] mice/group). (j) ATV:TREM2 induces transcription of genes associated with phagocytosis. Axl, Itgax, Lgals3 mRNA levels were detected in isolated brain microglia after peripheral administration of 10 mg kg⁻¹ ATV:TREM2 1-, 4-, and 7-days post dose. Graphs represent bulk mRNA measured by RNA-seq. Data shown as log₂ counts per million (with a pseudocount of 1 added) in each biological replicate (n = 8 mice/group, each group compared to ATV:ISO group, Kruskal-Wallis test for Axl, Dunnett’s multiple comparisons for Itgax and Lgals3, mean ± SEM).