Isolation of mitochondrial mutation-specific T-cell receptors
It has been hypothesized that neoantigen-specific T cells play a major role in effective cancer immunotherapy, including neoantigen vaccines, immune checkpoint blockade (ICB) therapy, and adoptive T cell therapy using tumor-infiltrating lymphocytes (TILs). The major source of neoantigens are mutated proteins derived from non-synonymous mutations found in tumor genomic DNA. It's highly conceivable that non-synonymous mutations found in tumor mitochondrial DNA (mtDNA) can also generate neoantigens that can be recognized by T-cell receptors (TCRs). This project contains two types of single-cell data. One is single-cell CITEseq (5' mRNA plus antibody feature barcode plus TCR) seqeuncing to identify antigen-specific TCRs. The other is single-cell ATAC-seq to detect chromatin accessiblities and mtDNA. Overall design: For single-cell RNA/TCR-seq, unstimulated and stimulated T cells from donor D1 were labeled with anti-human TotalSeq-C Hashtag #4 and #5 antibody (Biolegend), respectively. Single-cell CITEseq (5' immune profing, 5' mRNA, antibody feature barcode and TCR) was performed for one pooled sample, according to manufacture's instruction (10X Genomics). The TCR and gene expression (GEX) libraries were sequenced by an Illumina Novaseq 6000 sequencer. The Feature Barcode library for hashtags was sequenced by an Illumina iSeq 100 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, tumor cells and PBL from 4 patients (P1, P2, P3 and P4) 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).