A reporter cell system for the triggering receptor expressed on myeloid cells 2 reveals differential effects of disease‐associated variants on receptor signaling and activation by antibodies against the stalk region

Abstract

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on myeloid‐derived cell types. The extracellular immunoglobulin‐like domain of TREM2 binds anionic ligands including Apolipoprotein E and Amyloid‐β. The transmembrane domain interacts with its adaptor protein DAP12/TYROBP that is responsible for propagation of downstream signaling upon ligand interaction. Several sequence variants of TREM2 have been linked to different neurodegenerative diseases including Alzheimer's disease. Here, we generated HEK 293 Flp‐In cell lines stably expressing human TREM2 and DAP12 using a bicistronic construct with a T2A linker sequence allowing initial expression of both proteins in stoichiometric amounts. Cell biological and biochemical analyses revealed transport of TREM2 to the cell surface, and canonical sequential proteolytic processing and shedding of TREM2 (sTREM2). The functionality of this cell system was demonstrated by detection of phosphorylated spleen tyrosine kinase (SYK) upon stimulation of TREM2 with the anionic membrane lipid phosphatidylserine or anti‐TREM2 antibodies. Using this cell model, we demonstrated impaired signaling of disease associated TREM2 variants. We also identified a monoclonal antibody against the stalk region of TREM2 with agonistic activity. Activation of TREM2‐DAP12 signaling with the monoclonal antibody and the partial loss of function of disease associated variants were recapitulated in induced pluripotent stem cell derived microglia. Thus, this reporter cell model represents a suitable experimental system to investigate signaling of TREM2 variants, and for the identification of ligands and compounds that modulate TREM2‐DAP12 signaling. MAIN POINTS Disease associated variants impair the signaling activity of TREM2 by distinct mechanisms. Targeting the stalk region of TREM2 with bivalent antibodies activates TREM2 signaling.

Differential interaction with TREM2 modulates microglial uptake of modified Aβ species

“…The negatively stained sample was then examined by using Talos L120C (Thermo Fischer Scientific) at an acceleration voltage of 120 kV imaged with 4 k  4 K Ceta CMOS camera at x45,000. Representative images are shown in Figure S1 high glucose [4.5 g/L], phenol red, sodium pyruvate additive, 10 mM HEPES), supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% penicillin and streptomycin (P/S) solution, and 100 μg/ml hygromycin in incubators maintained at 37 C and 5% CO 2 (Ibach et al, 2021). Using the QuikChange-II kit (Agilent Technologies), site directed mutagenesis was carried out according to the manufacturer's instructions in order to introduce the stop codon at aa158 of full length TREM2 construct.…”

“…Using the QuikChange-II kit (Agilent Technologies), site directed mutagenesis was carried out according to the manufacturer's instructions in order to introduce the stop codon at aa158 of full length TREM2 construct. The generation of stable cell line was described previously (Ibach et al, 2021).…”

“…Samples prepared in 5x SDS sample buffer and boiled at 95 C for 5 min were separated according to their molecular weight on a precasted discontinuous Bis-Tris NuPAGE Novex 4%-12% gel (Invitrogen™, #V601020). Separation was achieved by using 1x NuPAGE™ MES SDS Running Buffer and a constant voltage of 120 V for 1 h. Gels were subsequently analyzed by western immunoblot analysis (Ibach et al, 2021;Kumar et al, 2021).…”

“…Proteins separated by SDS-PAGE were transferred to NC membranes as described previously (Ibach et al, 2021), at constant current of 400 mA and for 2 h. The membranes were then blocked for 1 h at constant agitation with a 5% milk solution, followed by incubation of the membrane over night at 4 C and constant agitation (orbital shaker) with a solution of the primary antibody in 1x TBS-T. The next day, the membranes were washed (3 Â 5 min) followed by incubation with respective secondary antibody conjugated either to horseradish peroxidase or a fluorophore for 1 h at RT.…”

Selection and structural characterization of anti-TREM2 scFvs that reduce levels of shed ectodomain

“…Because of the potential therapeutic impact of targeting TREM2, we decided to generate single-chain variable antibody fragments (scFvs) against the human TREM2 ectodomain with which to study TREM2 structure and function. Agonist antibodies have already been reported in recently published work (Ellwanger et al, 2021;Fassler et al, 2021;Ibach et al, 2021;Price et al, 2020;Schlepckow et al, 2020;Wang et al, 2020). The group of Schlepckow have shown that a full-length antibody specific to mouse TREM2 can decrease shedding and activate TREM2 signaling in vitro, and also lead to a significant reduction in amyloid plaques in 6-month-old amyloid-beta precursor protein knockin mice (Schlepckow et al, 2020).…”