LILRB2-mediated TREM2 signaling inhibition suppresses microglia functions

Cell lines

HEK293T and HMC-3 cell lines were acquired from the American Type Culture Collection (ATCC) and cultured in DMEM+ 10% FBS. BV2 was cultured in DMEM+ 10% FBS. The 2B4 NFAT-GFP reporter cell line was cultured in RPMI-1640 + 10% FBS.

Immunostaining of human brain tissue

Human brain tissues were purchased from Novusbio as prepared slides. For normal subject brain tissues, catalog# NBP2-77565 was used. For AD patient brain tissues, catalog# NBP2-78018 was used. Before immunostaining, the slides were deparaffinized and rehydrated according to a previously published protocol [22]. Antigen retrieval was done using 88% formic acid following a previously published protocol [23].

After antigen retrieval, slides were first blocked in 1% BSA PBS with 0.3% Triton X-100 for 2 hours, then stained with corresponding antibodies: IBA1 (1:1000, Abcam, ab5076), 6E10 (1:500, Biolegend), GFAP (1:1000, Thermo Fisher PA1-10004), LILRB2 (1:500, Thermo Fisher 16-5149-85) and TREM2 (1:1000, Thermo Fisher, 27,599-1-AP) in 1% BSA PBS with 0.3% Triton X-100 overnight at 4 °C in a humidity chamber. After washing in PBS 0.3% Triton X-100, corresponding secondary antibodies with fluorescent labeling were incubated with brain slices for 2 hours at 4 °C in a humidity chamber. The slides were then mounted using ProLong Gold Antifade Mountant (Thermo Scientific). The slides were imaged using a Leica TCS SP5 confocal microscope.

Fc fusion proteins and oAβ

LILRB2- and TREM2-Fc fusion proteins were constructed by fusing the extracellular domain (ECD) of the receptors to human IgG1 Fc. The fusion protein gene was cloned into a vector that drives protein expression by a CMV promoter. Proteins were expressed in Expi 293 cells and purified by protein A agarose to purity > 95% confirmed by SDS-PAGE.

Oligomer Aβ (oAβ) was prepared according to protocols previously reported [24, 25]. Briefly, unlabeled β-amyloid (1-42), 5-FAM-labeled β-amyloid (1-42), or biotin-β-amyloid (1-42) were purchased from AnaSpec as a lyophilized powder. The lyophilized powder was dissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol) and then dried under a vacuum to dissolve pre-existing aggregates. The dried β-amyloid peptide was then reconstituted in DMSO to prepare β-amyloid monomers and stored at − 80 °C until use. To prepare oligomer β-amyloid, the DMSO-dissolved β-amyloid monomers were diluted to DPBS and incubated at 37 °C overnight.

Ligands binding to LILRB2 and TREM2 as measured by ELISA

For oAβ ELISA, 2 μg/mL protein A (Sino Biological) was coated on a high-binding ELISA plate by incubation overnight at 4 °C. After blocking with 1% BSA PBS, LILRB2- or TREM2-Fc fusion protein was captured by incubation at 2 μg/mL for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, biotinylated oAβ was added at designated concentrations and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, streptavidin-HRP (R&D Systems) was added at 1:200 concentration and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before the reaction was stopped by the addition of 1 N H2SO4. Optical density (OD) values were read at 450 nm.

For the PS (L-α-phosphatidylserine, purchased from Avanti Polar Lipids) ELISA, 0.1 mg/mL of PS dissolved in methanol was coated onto a high-binding ELISA plate by evaporating at room temperature for 4 hours. After blocking with 1% BSA PBS, LILRB2- or TREM2-Fc fusion protein was allowed to bind by incubating at designated concentrations for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, anti-human Fc-HRP (Jackson ImmunoResearch) was added at 1:10,000 dilution and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before the reaction was stopped by the addition of 1 N H2SO4. OD values were read at 450 nm.

Ligands binding to LILRB2 and TREM2 as measured by BLI

LILRB2- or TREM2-Fc fusion proteins were diluted to 30 μg/mL in kinetics buffer. PS liposomes were prepared as previously reported with modifications [26]. Briefly, PS was dissolved in chloroform and dried under a vacuum to form a thin layer. DPBS was added to re-hydrate PS, and the liposomes were formed by sonication on ice until the solution became translucent. Octet Red 96 instrument (Fortebio) was used in the bio-layer interferometry (BLI) assay. Protein A sensors (Fortebio) were used to capture Fc fusion proteins. During all incubation steps, samples were kept at room temperature with 1000 rpm shaking.

In the association stage of the BLI assay, protein A sensor-captured Fc fusion proteins were incubated with oAβ (1 μM in kinetics buffer) or PS liposome (1 mM in kinetics buffer) for the designated time. After the association stage, the sensors were dipped into a ligand-free kinetics buffer to allow the bound ligand oAβ or PS to freely dissociate for the designated time.

For measuring the kinetics of LILRB2 or TREM2 binding with oAβ, protein A sensor-captured Fc fusion proteins were incubated with oAβ at multiple concentrations for the designated time. After the association stage, the sensors were dipped into a kinetics buffer to allow the bound ligand oAβ to freely dissociate for the designated time. The binding kinetics parameters were calculated by analysis software from Fortebio (version 11) using a 1:1 binding model. KD was calculated as kdis/kon.

Panning of phage-displayed scFv libraries for LILRB2 specific antibodies

A phage-displayed scFv antibody library was prepared previously [27]. Panning of the library for LILRB2 specific antibodies was carried out as described previously with modifications [27]. Briefly, MaxiSorp Nunc-Immuno tubes (Thermo Fisher Scientific) were coated with 20 μg/mL LILRB2-Fc in DPBS overnight at 4 °C. Unbound antigen was removed after washing with DPBS. After blocking the surface with 5% milk in DPBS, the phage library was incubated with the coated-LILRB2 for 2 hours at room temperature in 5% milk with 0.1 mg/mL D3-D4-Fc. After washing with PBS + 0.05% tween-20 to remove unbound phage, captured phage was eluted by incubating with 100 mM TEA for 20 min. Eluted phage-infected log-phase growing E. coli TG1 and then were amplified on 2x YTAG agar 500cm2 square plate (Corning) at 30 °C overnight. The amplified phage-infected TG1 was used to prepare the phage for the next round of panning using the M13KO7 helper phage. The enrichment process was done in three rounds using the output from the previous round as the input for the next round.

After three rounds of panning, the output titer was measured and single colonies were used to prepare phage for ELISA. High-binding ELISA plates (Corning) were coated with LILRB2-Fc at 2 μg/mL overnight at 4 °C. After blocking with 5% milk in PBS, phage prepared from single TG1 colonies in 5% milk PBS was incubated with coated LILRB2 for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, anti-M13-HRP (Santa Cruz Biotechnology) was added at 1:2000 concentration and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before being stopped by 1 N H2SO4. OD values were read at 450 nm. Top 20% high-binding clones were selected. Phagemids were extracted using Qiagen BioRobot Universal System in 96-well format. After DNA sequencing, sequences were analyzed using the IMGT V-quest service to identify antibody sequences with unique CDR3 regions.

Conversion of phage scFv to IgG

Unique scFv clones were converted into human IgG1 using mixed universal primers with degeneracy [27]. Individual heavy and light variable chains were amplified using PrimeStar GXL polymerase (Takara Bio). Gel-purified variable chain fragments were cloned into digested vectors using In-fusion HD cloning enzyme mix (Takara Bio). After the converted plasmid was sequenced, sequences verified IgG plasmids were transfected into Expi 293 cells at the 2-mL scale. After culturing for 5 days, cells were removed and antibody-containing supernatant was collected for screening assay.

For milligram-scale antibody purification, Expi293-produced antibodies were purified using CaptivA Protein A affinity resin (Repligen) and eluted with 0.1 M glycine (pH = 2.5) and then neutralized with 1/20 volume 1 M Tris-HCl (pH = 9). Buffer exchange to DPBS was done using Amicon Ultra-15 ultrafiltration units (Mw cutoff = 30 k) (MilliporeSigma).

NFAT-GFP reporter assay

The chimeric LILRB2 reporter construct was generated by fusing LILRB2 (aa22-461) with helical and cytoplasmic regions of human TREM2 (aa175-230). The original signal peptide of LILRB2 was replaced by leader sequence from mouse immunoglobulin κ light chain. A HA tag was introduced to the N′ of LILRB2. For mutant LILRB2 reporter constructs, LILRB2 with designated mutations were prepared using In-fusion HD cloning enzyme mix with the WT LILRB2 reporter construct as the template.

The human TREM2-DAP12 reporter construct was generated by fusing human TREM2 (aa19-169) with huDAP12 (aa28-113) with D50A mutation. The original signal peptide of TREM2 was replaced by leader sequence from mouse immunoglobulin κ light chain. A HA tag was introduced to the N-terminus of TREM2. The mouse TREM2-DAP12 reporter construct was generated in a similar design except for mouse TREM2 (aa19-171) replaced the human TREM2.

The co-ligation reporter cell lines LILRB2-TREM2 or LILRB2_muITIM-TREM2 were generated by transducing co-ligation reporter constructs into the parental 2B4 NFAT-GFP reporter cells. The co-ligation reporter construct enabling WT LILRB2 and TREM2 co-expression was generated by fusing WT LILRB2 (aa1-598), IRES, and human TREM2-DAP12 (Igk-HA-huTREM2 (19-169)-huDAP12 (aa28-113, D50A)). IRES enables co-expression of both receptors (LILRB2 and TREM2) in the same reporter cell. Similarly, the co-ligation reporter construct enabling LILRB2_muITIM and TREM2 co-expression was generated by fusing LILRB2_muITIM (LILRB2 with mutations Y533F, Y562F, and Y592F), IRES, and TREM2-DAP12 (Igk-HA-huTREM2 (aa19-169)-huDAP12 (aa28-113, D50A)).

All chimeric reporter genes were cloned into pCDH-CMV-MCS-EF1α-Puro. The 2B4 reporter cells transduced with individual reporter constructs were generated by lentivirus transduction. To prepare lentivirus particles, pCMV-VSV-G (Addgene 8454), pCMV delta R8.2 (Addgene 12,263), and individual pCDH transfer plasmids containing GOI were transfected into HEK293T. The 2B4 NFAT-GFP parental reporter cells were transduced with lentivirus supernatant (1:1 diluted in RPMI-1640) overnight under the presence of 10 μg/mL polybrene (Santa Cruz Biotechnology). After 48 hours of transduction, cells were selected with 1 μg/mL puromycin until a sufficient number of cells with transgene emerged.

For the reporter assay, ligands were coated onto 96-well cell culture plates at their optimal concentrations determined in preliminary experiments: oAβ (1 μM in DPBS, overnight, 4 °C), PS (0.1 mg/mL in methanol, room temperature until fully evaporated), PC (L-α-phosphatidylcholine, purchased from Avanti Polar Lipids, 0.03 mg/mL in methanol, room temperature until fully evaporated), and antibodies (10 μg/mL, DPBS, overnight, 4 °C). After ligand coating, unbound ligands were removed by washing with DPBS 3 times. A total of 100,000 reporter cells were seeded into individual wells (96-well plate) in 0.1 mL complete medium with 1 μg/mL puromycin with designated treatments. After overnight culturing, GFP positive populations were read using an iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected.

Blocking of ligand-receptor interaction by LILRB2 antibodies as measured by BLI

Protein A sensors (Fortebio) were used to capture Fc fusion proteins. During all incubation steps, samples were kept at room temperature with 1000 rpm shaking. For assays with oAβ as the ligand, individual purified LILRB2 antibodies were loaded onto sensors at 30 μg/mL. After antibody capture, unbound Protein A was blocked by incubating in 0.1 mg/mL human Fc fragment. The sensor-captured antibodies were then incubated with LILRB2-Fc at 200 nM in kinetics buffer for 120 seconds to load LILRB2. The LILRB2-loaded sensors were incubated with biotin-oAβ at 1 μM in kinetics buffer for the designated time. The sensors were incubated with 200 nM streptavidin to further amplify oAβ binding signals. For assays with PS as the ligand, the only difference is that PS liposomes (1 mM PS concentration) in kinetics buffer were incubated with LILRB2-loaded sensors instead of oAβ, and there was no streptavidin-based signal amplification step. After PS binding to LILRB2, the sensors were incubated in a blank kinetics buffer to allow free dissociation of PS.

Antibody LILRB2 binding affinity measured by ELISA

High-binding ELISA plates (Corning) were coated with LILRB2-Fc at 2 μg/mL overnight at 4 °C. After blocking with 1% BSA PBS, individual purified LILRB2 antibodies (at designated concentrations) in 1% BSA PBS were incubated with coated LILRB2 for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, anti-human F(ab)2-HRP (Jackson ImmunoResearch) was added at 1:5000 concentration and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before being stopped by 1 N H2SO4. OD values were read at 450 nm.

Antibody LILRB2 binding kinetics measured by BLI

Protein A sensors (Fortebio) were used to capture individual Fc fusion proteins. During all incubation steps, sample temperature was set to room temperature with 1000 rpm shaking. Individual purified LILRB2 antibodies were loaded onto sensors at 30 μg/mL. After antibody capture, the antibody-loaded sensors were incubated with LILRB2-His at designated concentrations for the defined time. After LILRB2 binding, the sensors were incubated in a blank kinetics buffer to allow free dissociation of LILRB2 for the designated time. The binding kinetics parameters were calculated by the Fortebio software (version 11) using a 1:1 binding model with global fitting. KD is calculated by kdis/kon.

Antibody binding to LILRB2 expressed on the cell surface

To prepare LILRB2-expressing cells, HEK293T cells were transfected with pcDNA3.1 carrying LILRB2 (aa1-598) with CMV-driven expression. After transfection for 72 hours, purified LILRB2 antibody (at designated concentration) in 1% BSA DPBS was incubated with transfected cells for 30 min on ice. Unbound antibody was removed by centrifuging cells at 350 rpm for 5 min. Then, Alexa Fluor 488-anti-human IgG (H + L) in 1% BSA DPBS (Thermo Fisher Scientific) at 2 μg/mL was incubated for 30 min on ice. After centrifuging cells at 350 rpm for 5 min to remove unbound secondary antibody, cells were analyzed using the iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected. The control cell line was transfected with a blank pcDNA3.1 plasmid.

Antibody LILRB2 specificity measured by BLI

We measured the cross-reactivity of LILRB2 antibodies to members of the LILRB and LILRA family of proteins by the BLI assay. Protein A sensors (Fortebio) were used to capture individual LILRB2 antibodies. During all incubation steps, sample temperature was set to room temperature with 1000 rpm shaking. Individual purified LILRB2 antibodies were loaded onto sensors at 30 μg/mL. After antibody capture, unbound protein A was blocked by incubating in 0.1 mg/mL human Fc fragment. Then the antibody-loaded sensors were incubated with purified Fc fusion proteins of ECD LILRB and LILRA receptor family members at 200 nM in kinetics buffer for the defined time. After LILRB2 binding, the sensors were incubated in a blank kinetics buffer to allow free dissociation of Fc fusion proteins for the designated time.

Antibody LILRB2 specificity measured by ELISA

High-binding ELISA plates (Corning) were coated with purified ECD-Fc fusion proteins of LILRB and LILRA receptor family members at 2 μg/mL overnight at 4 °C. After blocking with 1% BSA PBS, individual purified LILRB2 antibodies at 4 nM in 1% BSA PBS were incubated with coated antigens for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, anti-human F(ab)2-HRP (Jackson ImmunoResearch) was added at 1:5000 concentration and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before being stopped by 1 N H2SO4. OD values were read at 450 nm.

Preparation of oAβ-lipoprotein complexes

L-α-phosphatidylserine (PS) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids as powder. PS and DMPC were dissolved in chloroform at 10 mg/mL and mixed at 1:4. Chloroform was evaporated under vacuum and formed a thin layer containing the mixture of PS and DMPC. DPBS was added to re-hydrate the lipid mixture to 5 mg/mL, and the liposomes were formed by sonication on ice until the solution becomes translucent. To prepare oAβ-lipoprotein complexes, PS/DMPC liposomes and APOE3 were mixed at final concentrations of 1 mg/mL for PS/DMPC liposomes and 0.25 mg/mL for APOE3. The mixture was incubated at 18 °C 15 min and 30 °C 15 min for 3 cycles [28]. Then FAM-labeled oAβ was added into the lapidated APOE at a final concentration of 1 μM and incubated at room temperature for 1 hr.

Immunoblot assays

HMC3 cells were seeded into a 6-well plate at 1 × 106 cells/well in EMEM without FBS. After 1-hour incubation with designated treatments, the supernatant was removed, and cells were washed three times by DPBS. The cell lysate was obtained by lysing cells using NP-40 lysis buffer (1% NP40, 50 mM Tris-HCl, pH = 8, 150 mM NaCl) with Halt™ Protease and Phosphatase Inhibitor Cocktail (100X) (ThermoFisher). After removing debris by centrifugation, the total protein amount normalized by Pierce BCA Protein Assay Kit (ThermoFisher). Protein samples were resolved by 10% SDS-polyacrylamide gels (Biorad) and later transferred onto Immun-Blot PVDF membranes (Biorad). Proteins were probed with specific primary antibodies and secondary antibodies diluted in 5% BSA TBST [21, 24, 29]. The primary antibodies used are: SYK (1:1000, Cell Signaling 13198S), Phospho-Syk (Tyr525/526) (1:1000, Thermo Fisher MA5-14918), β-Actin (1:1000, Cell Signaling 4970S), SHP1 (1:1000, Cell Signaling 3759S), Phospho-SHP-1 (Tyr564) (1:1000, Cell Signaling 8849S), and LILRB2 (1:1000, Thermo Fisher PA5-46983).

The immunoreactive bands were visualized with the West Pico PLUS Chemiluminescent Substrate (ThermoFisher). The immunoreactive bands were quantified using ImageJ. Three independent treatment replicates were conducted with the representative immunoblot shown.

Imaging of 293 T cells for oAβ and PS binding

LILRB2 or TREM2 expressing HEK293T cells were seeded into 8-well chamber slides (Lab-Tek) at 20,000 cells/well in DMEM+ 10% FBS overnight. The cells were then washed with DPBS and fixed with 4% PFA for 10 min at room temperature. For oAβ binding, Hilyte Fluor 488 labeled oAβ (prepared as described above using monomer Anaspec AS-60479-01) was added to 0.2 μM. For PS binding, PS liposomes (prepared as described above) were added to 1 μM. During incubation, 1% BSA PBS was used as the diluent to avoid non-specific binding. After incubation for 1 hour at room temperature, the solution containing ligands was removed, and cells were then washed twice with DPBS. For detecting the PS binding, FITC Annexin V (1:20, Biolegend) was incubated with cells for 30 min before nucleus staining. The nucleus was stained with TO-PRO-3 (1 μM) in DPBS for 30 min and then mounted using ProLong Gold Antifade Mountant (Thermo Scientific). Slides were imaged using a Leica TCS SP5 confocal microscope.

Microglia phagocytosis assay

A total of 10,000 cells/well (BV2 with transgene or HMC3) were seeded in 96-well plates in serum-free DMEM medium overnight. oAβ-lipoprotein complex was diluted in serum-free DMEM medium with 1% BSA to a concentration equivalent to 100 nM FAM-oAβ. The medium in the cell culture plate was replaced with the diluted oAβ-lipoprotein complex and incubated at 37 °C for 2 hours. After phagocytosis, cells were detached by trypsin for 5 min, and cell surface-bound FAM-oAβ was quenched by adding trypan blue to 0.2% and incubated for 5 min. Cells were then transferred into a V-bottom 96-well plate and washed twice by 350 g 5 min centrifugation. When antibody treatment is needed, 10 μg/mL final antibody concentration was diluted together with the oAβ-lipoprotein complex. With CytoD treatment, serum-free DMEM medium (with 1% BSA) containing 10 μM CytoD was pre-incubated with cells for 30 min at 37 °C. CytoD was also included together with the oAβ-lipoprotein complex during phagocytosis. The phagocytosis was quantified using the iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected. Anti-LILRB2 42D1 was purchased from Thermo Fisher with catalog number 16-5149-85. The TREM2 blocking antibody was purchased from Millipore Sigma with catalog number MABN755.

For phagocytosis experiments using hMGL, cryopreserved iCell Microglia (R1131, FujiFilm) [30, 31] were seeded in a poly-D-lysine coated 96-well plate at 10,000 cells/well in basal medium supplied in the kit overnight. oAβ-lipoprotein complex was diluted to a concentration equivalent to 100 nM FAM-oAβ with 1% BSA. The medium in the cell culture plate was replaced with the diluted oAβ-lipoprotein complex and incubated at 37 °C for 2 hours. After phagocytosis, cells were detached by trypsin for 5 min, and cell surface-bound FAM-oAβ was quenched by adding trypan blue to 0.2% and incubated for 5 min. Cells were then transferred into a V-bottom 96-well plate and washed twice by 350 g 5 min centrifugation. With CytoD treatment, 10 μM CytoD was pre-incubated with cells for 30 min at 37 °C and constantly present during the phagocytosis experiment. The phagocytosis was quantified using an iQue3 high throughput flow cytometer (Sartorius).

BV2 and HMC3 cell surface LILRB2 and TREM2 expression

BV2-LILRB2 or HMC3 cells were detached from culture plates by non-enzymatic dissociation buffer (Thermo Fisher Scientific). A total of 1 million LILRB2-BV2 or HMC3 cells were blocked by 0.1 mg/mL human Fc fragment in 1% BSA PBS for 30 min on ice to block Fc receptors. Then, primary antibodies (biotinylated anti-LILRB2, anti-TREM2, or control IgG) were added at a final concentration of 10 μg/mL and incubated for 30 min on ice. After centrifugation to remove unbound primary antibodies, Alexa Fluor 488-labeled streptavidin (Jackson ImmunoResearch) was added to 2 μg/mL in 1% BSA PBS for 30 min on ice. After removal of unbound streptavidin by centrifugation, cells were analyzed using an iQue3 high throughput flow cytometer (Sartorius) with at least 10,000 live cells collected.

HMGL migration in transwell assays

Cryopreserved iCell Microglia (R1131, FujiFilm) [30, 31] were seeded in a transwell insert (PET membrane, 8 μm pore size, Corning 3374) in basal medium supplied in the kit without cytokines. Corresponding treatments were added into both the migration and receiver chambers at designated concentrations. Only the receiver (bottom) chambers contain a 0.5 μM oAβ-lipid complex. Cells were cultured for 24 hours at 37 °C with 5% CO2. After incubation, cells were washed three times with DPBS, fixed in 4% PFA for 10 min, and then stained with 0.05% crystal violet for 10 min. Unbound crystal violet was removed by washing with DPBS, and the plate was allowed to air-dry. Cell number was quantified by eluting cell-bound crystal violet in 33% acetic acid in H2O (100 rpm shaking, 10 min) according to the manufacturer’s protocol and literature [32]. The amount of crystal violet was quantified by measuring absorbance at 590 nm using a plate reader. For quantifying migrated cells, unmigrated cells that remain inside the transwell insert were removed using moistened cotton swabs. Migration percentage was calculated by dividing OD values of migrated cells over OD values of total cells.

For imaging microglia migration, the assay was conducted similarly as mentioned above, except that the microglia cells were pre-labeled with 1 μM CFSE (Thermo) for 15 min at 37 °C. The migrated cells were imaged using Nikon Eclipse TE2000E Widefield Fluorescence Microscope.

Proximity ligation assays (PLA)

The PLA was performed according to the manufacturer’s protocol. HMGLs were seeded into 8-well chamber slides (Lab-Tek) in basal medium (as described in the method section “microglia phagocytosis assay”) overnight. Ligand oAβ-lipoprotein complex was diluted to a concentration equivalent to 100 nM FAM-oAβ with 1% BSA. The medium in the cell culture plate was replaced with the diluted oAβ-lipoprotein complex and incubated at 37 °C for 2 hours. If antibody treatment is applied, Ctrl IgG or Ab29 at 10 μg/mL final antibody concentration was diluted together with the oAβ-lipoprotein complex. After incubation, cells were fixed with 4% PFA for 10 min at room temperature and then permeabilized with 0.1% saponin for 10 min. Between all the incubation steps, cells were washed in 1% BSA in PBS with 0.05% Tween-20. PLA kit Duolink In Situ Red Starter Kit Mouse/Rabbit (DUO92101-1KT, Sigma) was applied to detect the clustering of LILRB2 and TREM2 according to the manufacturer’s protocol. Detection antibodies for LILRB2 and TREM2 were 42D1 (sc-53,594, rat isotype, compatible with the PLA kit, SCBT) and PA5-87933 (Thermo Fisher, rabbit isotype, compatible with the PLA kit), respectively. Saponin at 0.1% was always included in all the reagents and buffers during the PLA process. The LILRB2 and TREM2 antibodies were also pre-validated for not competing with ligands (oAβ and PS) or antibodies (Ctrl IgG and Ab29). The nucleus was stained with TO-PRO-3 (1 μM) in DPBS for 30 min and then mounted using ProLong Gold Antifade Mountant (Thermo Scientific). Slides were imaged using a Leica TCS SP5 confocal microscope.

Real-time quantitative PCR analysis for cytokines in hMGL

Cryopreserved iCell Microglia (R1131, FujiFilm) [30, 31] were seeded in a poly-D-lysine coated 24-well plate in basal medium supplied in the kit without cytokines. hMGLs were incubated with the designated treatments for 24 hours before being collected for qPCR studies. RNA was extracted by RNeasy Micro Kit (Qiagen) following the manufacturer’s protocol. RNA was reverse transcribed into the first-strand cDNA using iScript™ cDNA Synthesis Kit (Biorad) following the manufacturer’s protocol. Quantitative PCR was performed using SsoAdvanced Universal SYBR Green Supermix (Biorad). Quantitative PCR reactions were measured by ABI PRISM 7900HT Sequence Detection System (ThermoFisher) using the standard protocol recommended by the manufacturer (95 °C, 30s, 40x cycles with 95 °C 15 s, 60 °C 30s per cycle with instrument default melting curve analysis). The following primers were used in the qPCR (primers are written in 5′ to 3′ directions): IL-1β-Forward: AGCTACGAATCTCCGACCAC, IL-1β-Reverse: CGTTATCCCATGTGTCGAAGAA; IL-6-Forward: ACTCACCTCTTCAGAACGAATTG, IL-6-Reverse: CCATCTTTGGAAGGTTCAGGTTG; TNF-α-Forward: CCTCTCTCTAATCAGCCCTCTG, TNF-α -Reverse: GAGGACCTGGGAGTAGATGAG; CCL3-Forward: AGTTCTCTGCATCACTTGCTG, CCL3-Reverse: CGGCTTCGCTTGGTTAGGAA. GAPDH was used as the internal control with the following primers: GAPDH-Forward: ACAACTTTGGTATCGTGGAAGG, GAPDH-Reverse: GCCATCACGCCACAGTTTC. Fold-of-change was calculated using the standard ΔΔCt method [24].

5XFAD mice studies

The animal experiments were conducted according to the institutional guidelines with approved protocols. 5XFAD mice (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax, female, 8-week-old) were purchased from MMRRC and randomly grouped into 5 mice per group. Microglia implantation experiments started when the mice reached 5-mo-old age. To implant the microglia, 2 × 105 hMGLs mixed with antibodies were injected into the bilateral ventricles under anesthesia. Stereotaxic injection of microglia (2 μL on each side) was delivered through a Neuro syringe (Hamilton 1701RN) into the lateral ventricles at a controlled speed of 0.5 μL/min. The antibody dose was 10 mg/kg per mouse. The mice were sacrificed 4 days after injection, and the brain tissues were harvested after mice transcardial perfusion at 2 mL/min by DPBS for 10 min. Brains were collected with half flash-frozen in liquid nitrogen and another half prepared for cryo-sectioning. For immunofluorescence, the half mouse brains were dipped into 4% PFA for 1d, then 30% sucrose for 2d before being embedded into OCT medium (Sakura) and sectioned using Leica Cryostat CM1950 into 40 μm floating coronal sections. The floating sections were stored at 4 °C in PBS with 0.01% sodium azide until use.

For immunostaining, after blocking in 1% BSA PBS with 0.3% Triton X-100 for 2 hours, brain sections were stained with corresponding antibodies: IBA1 (1:1000, Abcam, ab178680 [31, 33]), 6E10 (1:500, Biolegend), CD68 (1:500 Biolegend 333,812), human nuclear antigen (1:200, Abcam ab191181) and LILRB2 (1:1000, Thermo Fisher, PA5-97929) in 1% BSA PBS with 0.3% Triton X-100 overnight at 4 °C with gentle rocking. After washing in PBS 0.3% Triton X-100, corresponding secondary antibodies with fluorescent labeling were incubated with brain slices for 2 hours at 4 °C with gentle rocking. The nucleus was stained with TO-PRO-3 (1 μM) in DPBS for 30 min and then mounted using ProLong Gold Antifade Mountant (Thermo Scientific). Brains slices were imaged using a Leica TCS SP5 confocal microscope. The quantification was done using ImageJ as previously described [34, 35]. For quantification of the fluorescent intensity of indicated markers in the mouse cortex and hippocampus, images were analyzed by ImageJ, and background was subtracted by the software for fluorescence images before quantification. For quantification of Aβ engulfed by microglial CD68+ phagosome, the co-localization function of LAS X 3.7 (Leica) was used on Aβ+, CD68+ and Aβ + CD68+ image data. The ratio of engulfed Aβ by phagosome was calculated as the total Aβ + CD68+ area divided by the total Aβ + area. All the quantifications were done by an independent researcher blinded to the group designs.

For measuring the concentration of antibodies in the brain, high-binding ELISA plates (Corning) were coated with anti-human Fc (Jackson Immunoresearch) at 2 μg/mL overnight at 4 °C. After blocking with 1% BSA PBS, individual brain lysates were incubated with coated capture antigen for 2 hours at room temperature. After washing with PBS + 0.05% Tween-20, anti-human F(ab’)2-HRP (Jackson Immunoresearch) was added at 1:5000 concentration and incubated for 1 hour at room temperature. After washing with PBS + 0.05% Tween-20, TMB substrate (Thermo Fisher Scientific) was added and incubated for 5 min before being stopped by 1 N H2SO4. OD values were read at 450 nm. Standard curves were established for individual antibodies using purified corresponding antibodies following the same method listed above.

Protein sequence analysis

Protein sequence alignment was performed using the T-Coffee multiple sequence alignment server and the alignment figures were generated in ESPript - http://espript.ibcp.fr [36]. The crystal structure was visualized using UCSF Chimera 1.15. The LILRB2 crystal structure was downloaded from the RCSB PDB database using entry 2gw5 [37].

Statistical analysis

GraphPad Prism (v8, GraphPad Software) was used to generate plots and perform statistical analysis. Statistical differences were determined to be significant at p < 0.05 using a two-tailed Student t-test. Data are presented as mean ± SD.

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