Induction of human CD8 T cell exhaustion via mitochondrial inhibition and ectopic TOX expression - transcriptional analysis

Human CD8⁺ T cells from healthy donors were treated with Azithromycin (AZM) to induce mitochondrial translation inhibition leading to exhaustion phenotypes in vitro, or with DMSO as a control. Cells were either analyzed in bulk or sorted into distinct T cell subsets based on PD-1 and CD127 expression. In a separate experiment, CD8⁺ T cells were retrovirally transduced with either a MIGR1 control plasmid or MIGR1-TOX and treated with DMSO or AZM. This assay was optionally combined with CRISPR-mediated KO of exhaustion-related transcription factors. All experiments were performed using cells from two independent donors. Human Library preparation for bulk-sequencing of poly(A)-RNA was done as described previously (Fischer et al., 2023). The library was sequenced on a NextSeq 500 (Illumina) with 65 cycles for the cDNA in read1 and 19 cycles for the barcodes and UMIs in read2. Data was processed using Drop-seq pipeline (v1.0) to generate sample- and gene-wise UMI tables (Macosko et al., 2015). Raw counts of mapped reads were processed in R with DESeq2 to determine differentially expressed genes and to generate normalized read counts.