RNAseq assessment of early IL-33-mediated signaling on donor T cells in mourse GVHD model

To understand pathways that IL-33 is controlling early after allogeneic hematopoietic cell transplantation (AlloHCT), we performed RNAseq on sorted splenic B6 ST2fl/fl and ST2WT donor CD4+ T cells co-transferred into the same BALB/c recipient on d5 post-alloHCT Overall design: B6 CD45.1+ (ST2WT) and B6 CD45.2+ CD4-CrexR26-LSL-YFPxSt2fl/fl (ST2fl/fl) donor splenic CD4+ T cells were enriched from CD90.1+ B6 or H2-Kd BALB/c recipient mice (n=4/group) using negative depletion with Dynabeads (Life Technologies). The cells were stained for CD3, CD4, CD90.1, H2-Kd CD45.1, CD45.2 and sorted for ST2WT and ST2fl/fl CD4+ populations on a FACSAria II, directly into SmartSeq low-input RNA kit lysis buffer for library prep and RNA sequencing. Libraries were prepared from isolated RNA using Nextera XT DNA library prep kits, and RNA sequencing was performed on Illumina NextSeq500 by the Health Sciences Sequencing Core at the University of Pittsburgh. Raw sequencing data were processed using CLC Genomics Workbench 20.0.3 (QIAGEN Inc., https://digitalinsights.qiagen.com) for quality control and aligned to the Mus musculus genome version GRCm38.p6. Reads assigned to each gene underwent TMM normalization and differential expression analysis was performed using edgeR within CLC Genomics to compare ST2WT versus ST2fl/fl donor T cells. The top differentially expressed genes were filtered by adjusted p-value q<0.05 and fold-change greater than 1.5 for subsequent downstream pathway analysis. The relative expression shown in heatmaps was calculated as the fragments per kilobase exon per million mapped reads value for each sample divided by the mean expression of that gene in all samples per each experiment. GSEA from the Broad Institute (http://www.broad.mit.edu/gsea) was used to calculate the enrichment of genes in each set.