Immortalization of Human Primary CD8⁺ T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9

Background: Existing human cell immortalization methods made the cells obtain oncogenesis phenotype and/or caused the cells gain and/or lose chromosomes. Immortalized normal human T cells lines provide critical in vitro models for basic research and therapeutic products development.Methods: We utilized a CRISPR/Cas9 system to replace a single copy of the exon 2 of the cell cycle inhibitor gene CDKN2A (encoding p16 and p14 proteins) with a single copy of human telomerase reverse transcriptase (hTERT) to immortalize human primary CD8+ T cells (hCD8+T-TERT). By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8+T cells with a single copy of hTERT transgene, which also avoided uncontrolled insertion of Cas9 gene and guide RNA vector.Results: Human primary CD8+ cells were immortalized and expanded more than 2.6 × 107 times. Characterization of the cells revealed that the immortalized CD8+ T-TERT cells retained most of the cell surface markers and normal karyotype. The CD8+ T-TERT cells also retained the dependence of IL-2 and CD3/CD28 activator for survival and expansion.Conclusion: we established a stable immortalized CD8+ T cell line that had a phenotype consistent with T cells and will be a useful research model.