Computational protocol: Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens

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[…] Upon euthanasia, thymi were removed and placed immediately in iced buffer (Hank’s balanced salt solution [HBSS] [GIBCO, Waltham, MA, USA], 5% fetal bovine serum [FBS], 5 μg/mL DNase). All subsequent steps were performed on ice. Thymi and spleens were gently but thoroughly pressed between two ice-cold frosted glass slides to generate a single-cell suspension. Cells were counted using a hemacytometer; viable cells were determined by exclusion of trypan blue (Sigma-Aldrich, St Louis, MO, USA). Red blood cells were lysed in spleen samples using ACK lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA).After counting, cells were stained with the following antibodies from BioLegend: CD19-A594 (6D5), IgD-APCCy7 (11-26c.2a), and IgM-A647 (RMM-1). For surface IgG phenotyping, the following antibodies were used: IgG1-Biotin (RMG1-1), IgG2a-Biotin (RMG2a-62), IgG2b-Biotin (RMG2b-1), and IgG3-Biotin (RMG3-1), followed by incubation with Streptavidin-PerCp-Cy5.5. For ABC phenotyping, cells were stained with the following antibodies from BioLegend (unless otherwise noted): CD11c-BV510 (N418), CD43-A647 (1B11), CD93-PE (AA4.1; Molecular Probes), CD21-PeCy7 (8D9; eBioscience), and CD23-PerCp-eFluor710 (B3B4; eBioscience). For all phenotyping experiments, dead cells were excluded using DAPI (Molecular Probes) in fluorescence-activated cell sorting (FACS) buffer (HBSS, 5% FBS, 0.5% DNase [1 mg/mL, pH 7.2]). Flow cytometry analysis was performed using a BD LSRII flow cytometer, and data were analyzed using BD FACSDiva and FlowJo flow cytometry analysis software (Tree Star).Thymic single-cell suspensions from C57BL/6J mice (aged 5 weeks to 24 months) were stained with CD19-APC (MB19-1; eBioscience), followed by incubation with anti-APC magnetic MicroBeads (Miltenyi Biotec). CD19+ cells were subsequently magnetically enriched using a QuadroMACS separator with LS columns (Miltenyi Biotec). Enriched B cells were suspended in DAPI and sorted on CD19+ viable singlets using a BD FACSAria cell sorter (BD Biosciences) using the single-cell sorting mode. Post-sort analysis for sorted cell purity was accomplished using FACSDiva software. Sorted samples for RNA-seq analysis were selected to be at least 98.5% pure. RNA was isolated using the RNAqueous – Micro Kit (Invitrogen) as per manufacturer’s instructions. [...] These steps were carried out by the Genome Sequencing Core Facility. RNA quality was analyzed using an Agilent Bioanalyzer (Santa Clara, CA, USA) and/or Qubit fluorometer (Life Technologies). Approximately 20–50 ng total RNA was used for RNA-seq library preparation following the KAPA Stranded RNA-Seq Kit with RiboErase (HMR) according to the manufacturer’s instructions (catalog no. KR1151). cDNA libraries were sequenced using 50 bp single-read sequencing runs on an Illumina HiSeq 3000 sequencer. Reads were aligned to mouse genome (University of California, Santa Cruz [UCSC], mm9 build) or human genome (UCSC hg19 build) using TopHat (; ). The expression levels were extracted using HTSeq () with GENCODE annotation (GENCODE V24lift37; ) and converted to reads per kilobase per million mapped reads (RPKM) units according to . Data were then normalized and tested for differential expression by DESeq in R (). Data reported in this article were submitted to the NCBI’s GEO (GEO: GSE107112). Heatmap, hierarchical clustering (using Pearson correlation as distance function and average linkage as agglomeration method), and volcano plot were generated using R/Bioconductor (https://www.bioconductor.org). The mouse Aire-dependent B cell-specific list was mapped to human homologs using Homologene identifiers downloaded from MGI (http://www.informatics.jax.org). Genes from any list that did not map to gene symbols in our dataset were omitted from further analysis. [...] Splenic B cells from young (5 weeks) or aged (12 months) Adig mice were magnetically enriched using a mouse B cell Isolation Kit with a QuadroMACS separator and LS columns according to the manufacturer’s instructions. Enriched B cells were suspended in DAPI in FACS buffer and sorted on Aire-GFP− viable singlets. Subsequently, 2 × 105 sorted B cells were cultured for 3 days with or without agonistic anti-CD40 monoclonal antibody (FGK45; Bio X Cell) (10 μg/mL) and recombinant mouse IL-4 (PeproTech) (5 ng/mL) in flat-bottom 96-well plates. For qRT-PCR, RNA extraction, cDNA synthesis, and qPCR were performed on 2 × 105 cells using Taqman Gene Expression Probes (Thermo Fisher Scientific) for Aire and Hprt and gene expression values normalized as previously described.For flow cytometry analysis, 6 × 105 to 1 × 106 stimulated B cells were stained with CD19-A594 (6D5; BioLegend) and suspended in DAPI in FACS buffer. Aire-GFP expression was analyzed on CD19+ viable singlets using a BD LSRII flow cytometer with BD FACSDiva and FlowJo® flow cytometry analysis software. [...] RNA from sorted murine thymic B cells with a purity of at least 98.5% was extracted using the method described above, and cDNA synthesis was performed using a Superscript VILO cDNA synthesis kit (Invitrogen) per manufacturer’s instructions. qPCR (cycle 1, 95°C for 10 min; cycle 2 [×40], 95°C for 15 s and 60°C for 1 min) was performed using a Bio-Rad CFX96 Real-Time System and C1000 Touch Thermal Cycler (Bio-Rad) using presynthesized Taqman Gene Expression Assay Probes to amplify the following gene sequences: Tbx21 (assay ID: Mm00450960_m1), CD80 (Mm00711660_m1), CD40 (Mm00441891_m1), Tnfrsf11a (Mm00437132_m1), Aire (Mm00477457_m1), and Hprt (Mm00446968_m1). Gene expression values were analyzed using Bio-Rad CFX Manager software and normalized to Hprt. [...] p values for differences in gene expression between young and aged samples in RNA-seq data were calculated using base 2 log scale RPKM values for the two groups with the R package limma, using the empirical Bayes method to calculate p values (). p values for changes in the Aire-dependent gene lists during aging were calculated using the chi-square test. Scatterplots were generated in GraphPad Prism. p values for differences in surface phenotype (as in ) were calculated in GraphPad Prism using Student’s t test. […]

Pipeline specifications

Software tools CFX Manager, limma
Application RNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Autoimmune Diseases