Mice were deeply anaesthetized with intraperitoneal injection of a ketamine (87 mg/kg), xylazine (13 mg/kg) mixture. Blood was collected from the right ventricle prior to transcardial perfusion with ice cold PBS, and further processed through red blood cell lysis. Single-cell suspensions from lymphoid organs were prepared by mechanical dissociation; single-cell suspensions from brain tissue were prepared by digestion for 30 minutes at 37ºC with 1 mg/ml collagenase IV (Thermo Fisher), 300 µg/ml hyaluronidase (Sigma-Aldrich) and 40 µg/ml DNase I (Sigma-Aldrich) in RPMI 1640 supplemented with 2 mM MgCl2, 2mM CaCl2, 20% FBS and 2 mM HEPES (Gibco), followed by mechanical disruption, filtration (through 100 µm mesh) and enrichment for leukocytes by gradient centrifugation (40% Percoll GE Healthcare, 600 x g, 10 min). Non-specific binding was blocked using 2.4G2 supernatant. To assess intracellular cytokine production, cells were cultured for 4h in the presence of phorbol mysristate acetate (1 µg/ml, Sigma-Aldrich), ionomycin (1 µg/ml, Sigma-Aldrich), and brefeldinA (BD). Cells were fixed and permeabilized with the eBioscience Foxp3 staining kit (eBioscience). Cellular phenotypes were assessed using high parameter flow cytometry panels, containing markers to identify cell types and markers to assess activation states. Data were acquired on a BD FACSymphony, with panels covering (i) CD45, CD4, CD8, CD3, CD19, NK1.1, Foxp3, eBioscience™ Fixable Viability Dye eFluor™ 780, CD103, CD62L, CD25, Neuropilin, ST2, PD-1, KLRG1, Helios, CD69, ICOS, CD44, and Ki67 or (ii) CCR6, CD80, TCRγδ, CD45, Foxp3, MHCII, eBioscience™ Fixable Viability Dye eFluor™ 780, IL1β, CD25, Ly6G, ST2, CX3CR1, PD-L1, TNF, CD44, Ki67, CD4, Ly6C, TrkB, CD19, CD69, CD8α, LAMP1, CD64, CD11b, CD3, or (iii) Foxp3, eBioscience™ Fixable Viability Dye eFluor™ 780, IL5, IL6, IL17, CD4, IFNγ, CD8α, TNFα, CD3, Amphiregulin, IL10, IL4, CD11b, CD19, GM-CSF, TCRγδ, pro-IL1β, TCRβ, IL2, NK1.1. For brain panels, cells obtain from the whole brain were analysed. Data was compensated using AutoSpill .
tSNE, FlowSOM and heatmap analysis were performed in RStudio (version 1.4.1717) using an in-house script . FlowSOM clusters are formed based on multi-marker similarity in a non-supervised manner. Clusters were annotated based on post-clustering comparison of marker expression, aligning the unique marker profile of each cluster to literature-based nomenclature. Key annotations for T cell clusters included CD4 naïve (CD3+CD4+CD62LhiCD44low), CD4 activated (CD3+CD4+CD62LlowCD44high), CD4 memory (CD3+CD4+CD62LhiCD44high), Tregs (CD3+CD4+Foxp3+), CD8 naïve (CD3+CD8+CD62LhiCD44low), CD8 activated (CD3+CD8+CD62LlowCD44high), and CD8 memory (CD3+CD8+CD62LhiCD44high).
Mice were deeply anaesthetized using intraperitoneal injection of a ketamine (87 mg/kg) / xylazine (13 mg/kg) mixture and transcardially perfused with PBS followed by 4% buffered formalin solution. The brain was removed and fixed in 10% buffered formalin solution overnight and stored in 30% sucrose until preservation in tissue freezing medium (Shandon™ Cryomatrix™ embedding resin, Thermo Scientific), and stored at -80°C. Sections (50 µm) were washed 15 minutes in 50 mM NH4Cl/PBS and pre-blocked with 10% normal donkey serum in 0.5% Triton-X-100/PBS for 1h at room temperature. Sections were incubated overnight at 4ºC with primary antibodies directed against Iba1 (1:1000, 014-19741, Wako) and 6E10 Abeta (1:1000, SIG-39320-1000, Covance Signet). Subsequently, the sections were incubated for 90 minutes at room temperature with appropriate fluorophore-conjugated secondary antibodies (Thermo Scientific). After each antibody incubation, slices were washed 3 times for 10 minutes with 0.1% Triton-X-100/PBS. All sections were incubated with DAPI (1:1000) for 15 min before final mounting on microslide slides using ProlongGold (Invitrogen). Images were obtained using a Nikon A1R Eclipse Ti confocal (Plan Apo 20X), or a Zeiss Axioscan Z.1 slide-scanner (20X Plan-Apochromat/NA 0.8) equipped with a Hamamatsu Orca Flash 4.0 V3 camera. Image processing was performed using ImageJ (https://imagej.nih.gov/ij/download.html).
Soluble and insoluble fractions of Aβ were extracted from cortex and hippocampus, as previously described  1368. The total protein content of the samples containing the soluble and insoluble Aβ fractions was determined using a modified Lowry-Peterson assay. The concentration of Aβ1−40 or Aβ1−42 in tissue samples was determined using standard sandwich ELISAs. Monoclonal antibodies JRFcAβ40/28 and JRFcAβ42/26, which recognize the C-terminal ends of Aβ species terminating at amino acids 40 or 42 respectively, were used for capture. HRP-conjugated JRFAβN/25 antibody, which recognizes the first seven N-terminal amino acids of human Aβ, was used as a detection antibody. Synthetic human Aβ1−40 and Aβ1−42 peptides were used to generate standard curves. Antibodies were kindly provided by Johnson and Johnson. Absorbance was measured at 450 nm in a Perkin Elmer EnVision 2103 Multilabel reader.