Detection of mitochondrial DNA mutations in T cells following 5-FU or cisplatin exposure

T cells are pivotal to cancer immunotherapy, yet chemotherapy may erode their fitness. Using a single-cell technique, we show that exposure to two widely used chemotherapeutic agents, 5-FU (5-fluorouracil) and cisplatin, induces non-synonymous mitochondrial DNA mutations (mtDNA) in T cells. Notably, nearly all detected mtDNA mutations are transition mutations. Like the effects observed in genomic DNA mutations, the impacts of mtDNA mutations in T cells appear to be random. Some T cells with mtDNA mutations concentrate in clusters associated with gene markers, while others do not. Additionally, several mtDNA mutations are found in the fraction of treated T cells with low mitochondrial activity, suggesting their potential effect on mitochondrial function. Importantly, mtDNA mutations are detected in tumor-infiltrating T cells from patients with colorectal cancer who received chemotherapy. Our findings uncover an unappreciated consequence of chemotherapy on T cell mitochondria, and these results raise concerns about administering immunotherapy and chemotherapy concurrently. Overall design: 5-FU-treated and untreated (N) T cells from peripheral blood lymphocytes (PBL) from 3 donors (D1007, D2008, D6053) were studied using scRNAseq/scTCRseq. Single-cell RNA and TCR-seq libraries were prepared using the 10X Genomics 5' immune profing kit (including single-cell 5' mRNA and TCR sequencing). The scRNAseq gene expression (GEX) libraries and scTCRseq libraries were sequenced by an Illumina Novaseq 6000 sequencer. The sequencing data was processed by a Cell Ranger-multi pipeline (v7.1.0; 10X Genomics). The gene expression (GEX) sequencing data was mapped to the standard reference genome database (GRCh38-2020-A, 10X Genomics), and the TCR sequencing data was also mapped to the standard reference genome database (GRCh38-alts-ensembl-7.1.0, 10X Genomics). For single-cell ATAC-seq, the following samples were studied (1) 5-FU-treated and untreated (N) T cells from 4 donors (2) Cisplatin-treated and untreated (N) T cells from 3 donors, (3) Cisplatin-treated T cells from 3 samples were labeled with mitochondria dyes TMRM or MitoTracker-Green (labeled as MASS). Single-cell sequencing was performed on TMRM or MASS high (Hi) and low T cells. (4) Tumor infiltrating lymphocytes (TILs) and PBL from 3 patients with metastatic colorectal cancer (P1, P2, P3) were subjected to single-cell ATAC-seq sequencing (10X Genomics). The prepared samples were sequenced by an Illumina Novaseq 6000 sequencer. The sequencing data was processed a Cell Ranger-ATAC pipeline (v2.1.0; 10X Genomics) and mapped to reference genome database (GRCh38-r107).