RNAseq of tolerized auroreactive RBC-specific CD4 T cells, HODxOTII murine model of autoimmune hemolytic anemia (AIHA)

We have previously identified that T cell tolerance is the stopgap to red blood cell (RBC)-specific autoimmunity. We have also shown that while tolerance is robust when mice are young, it fails upon age and leads to development of RBC autoantibodies. Herein, we set out to determine which transcripts were associated with tolerization of RBC-autoreative CD4 T cells. For these studies, we used a murine model of autoimmune hemolytic anemia (AIHA), whereby HOD mice (expressing a known RBC-specific antigen) were bred with OTII animals (which react to the ovalbumin epitope contained within teh HOD antigen) [see PMCID PMC8634489]. To that end, splenocytes were collected from 10-12 week old HODxOTII F1 mice (n=4 each genotype) and stained with antibodies against Thy1.2, Ter119, CD19, and CD4. CD19+ B cells and Ter119+ RBCs cells were excluded from live singlets and CD4+Thy1.2+ T cells were sorted. Total RNA was harvested using RNeasy mini plus kit (Qiagen). Samples were sent to the Genome Technology Access Center in the Department of Genetics at Washington University School of Medicine for library preparation and RNAseq. Each sample was run on 3 lanes, with paired-end reads, and 2x150nts. 

Methods
Samples were prepared according to library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina HiSeq. Data analysis was performed by TAC Genomics (https://tacgenomics.com). The reads were first mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0 and the gene expression level was estimaed using RSEM v1.2.15. Differentially expressed genes were identified using the edgeR program. Genes showing altered expression with p < 0.05 and more than 1.5 fold changes were considered differentially expressed. Goseq was used to perform the GO enrichment analysis and Kobas was used to perform the pathway analysis.