Generation of a Rag2 Il2rγ–/– knock out immune-deficient AD mouse model
All animal protocols were approved by the Methodist Research Institute's Institutional Animal Care and Use Committee in compliance with National Institutes of Health guidelines. Rag2 Il2rγ–/– double knock out mice were initially bred with purebred 5xFAD and strain-matched wild-type C57BL/6 mice to generate immune-deficient 5xFAD-Rag2KO and strain-matched WT-Rag2KO mice. The presence or absence of the Rag2 gene was determined by PCR using 250 ng of tail DNA and Eppendorf TaqDNA polymerase according to the manufacturer’s instructions. The following primers were used: Rag A) 5´-GGGAGGACACTCACTTGC-CAG-3´ and Rag B) 5´ AGTCAGGAGTCTCCATCTCAC-3´ and Neo C) 5´-CGGCGG-GAGAACCTGCGTGCAA-3´. Homozygotic mice will have one 350 bp band. Heterozygotic mice will have 350 and 263 bp bands. Wild-type mice will have one 263 bp band.
Ex vivo expansion of human Tregs and passive transfer to mice
Human CD4+CD25high T lymphocytes were isolated from peripheral blood of a healthy subject using the Regulatory T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Tregs were suspended at a concentration of 1x106 cells/ml in media containing 100nM of rapamycin (Miltenyi Biotec), 500 IU/ml IL-2 (Miltenyi Biotec) and DynabeadsTM Human Treg Expander (GibcoTM) at a 4:1 bead-to-cell ratio for 8 days (First stimulation). At day 8, beads were removed, and cells were resuspended in a culture medium containing 100 U/mL IL-2 and 100nM of rapamycin for 8 days. On day 16, Tregs were restimulated by adding Dynabeads expansion beads at a 1:1 bead-to-cell ratio for further 8 days. After the second stimulation, Tregs were harvested and washed on day 24. The Treg immunophenotype and suppressive function were assayed prior to injection to mice, as described previously (22) to confirm enhanced immunomodulatory function. 1x106 ex vivo expanded Treg cells were suspended in a 200 μl of phosphate-buffered saline (PBS) and were passively transferred into 5-month-old 5xFAD-Rag2 KO and WT-Rag2 KO mice via tail vein injections. This treatment was repeated every 28 days for a total of 5 months and the mice were sacrificed at age 10 month.
RNA sample preparation and transcriptome analysis
Using Trizol reagent, followed by Direct-zol RNA MiniPrep Kit (Zymo Research), messenger RNA was extracted from medial temporal cortex of mice. Quantitative PCR experiments were performed using a One-Step RT-PCR kit with SYBR Green and run on the Bio-Rad iQ5 Multicolor Real-Time PCR Detection Systems. For mouse neuroinflammation panel analysis, 770 transcripts were quantified with the NanoString nCounter multiplexed target platform (www.nanostring.com). nCounts of mRNA transcripts were normalized using the geometric means of 10 housekeeping genes (Csnk2a2, Ccdc127, Xpnpep1, Lars, Supt7l, Tada2b, Aars, Mto1, Tbp, and Fam104a).
Protein extraction and ELISA assay
The right hemisphere samples were homogenized in a 2% SDS lysis buffer (SDS, NaCl 150 mM and Triton™ 1%) containing phosphatase (Pierce) and protease (Roche) inhibitors. After centrifugation (60 min, 100,000 x g, 4°C), the supernatant was collected (SDS extract) and the protein concentration was quantified. 70% formic acid in water was added to the pellet and the supernatant was collected after sonication and centrifugation (FA extract). Soluble (SDS extract) and insoluble (FA extract) Aβ40 and Aβ42 were measured using Amyloid beta Human ELISA Kit (Invitrogen).
Splenocytes were isolated from spleens for flow cytometric analysis. Antibodies against the following surface markers were provided by: CD3 FITC (eBioscience™), CD4 PE (eBioscience™), CD8a Alexa Fluor 700 (eBioscience™), CD161 APC (eBioscience™) and CD19 PE-Cy5 (eBioscience™). Dead cells were stained by LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technology). For immunohistochemical brain analyses, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde in 0.1 M PBS for 2 days. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Sagittal brain sections were cut on a freezing microtome (Leica) and collected serially. Immunohistochemistry was performed on free-floating microtome-cut sections (10 μm in thickness). Sections were incubated with different antibodies: anti-mouse Iba1 (Polyclonal, 1:1000 Wako), anti-mouse CD68 (Clone FA-11, 1:200; BioRad), β-Amyloid (Clone 6E10, 1:1000; BioLegend), β-Amyloid 1-42 (polyclonal, 1:100; Millipore), anti-mouse GFAP (Clone GA-5, 1:100, Novus Biological) anti-human CD3 (Clone: CD3-12, 1:100, abcam) and anti-human Foxp3 (Clone 236A/E7, 1:100, Invitrogen). Appropriate secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen) were used followed by incubation with DAPI.
Confocal image quantification
After immunofluorescence staining, images were captured using a Nikon A1 laser scanning confocal microscope. The system uses a galvanometer scanner with a 20x Plan Apo objective, and a pinhole set to 1.2 Airy Unit. Laser power, numeric gain and magnification were kept constant between animals to avoid potential technical artefacts. NIS Elements Version 5.11.01 was used to quantify mean intensity of fluorescence, number of immunoreactive cells, and size of plaques. The area we analyzed were defined as an actual single image field. Since each image was 1024x1024 pixels with a resolution at 0.63um/pixel, we measured the amount of immunoreactive cells in a 416,179.81 um2 area of frontal cortex or dentate gyrus. Intensity and size thresholds were applied for Aβ, Iba1, CD68 or GFAP positive cell quantification. All absolute quantifications were performed at ×20 magnification.
Mice were grouped based on genotype and treatment. The analyses, including immunohistochemistry staining, ELISA and transcriptome analysis were performed by other independent investigators. Statistical analysis was performed using Prism 7.0 (GraphPad Software). The significance of group comparisons was tested using paired or unpaired student’s t-test (for two groups) or one-way ANOVA (for more than two groups). Data were expressed as Mean ± SEM and p values less than 0.05 were considered significant. For transcriptome analysis, nSolver software was used for background subtraction and normalization of data. Statistical analysis on the normalized expression profiles, including one-way ANOVA and multiple comparison using Tukey’s range test, were carried out using the Statistics and Machine Learning Toolbox in MATLAB R2020a. Volcano plots of differential expressed genes data were plotted using GraphPad Prism. Gene enrichment analysis was performed using Ingenuity pathway analysis (IPA).