A novel immortalization method for immortalizing human primary CD8⁺ T cells by inserting a single copy of human...

Existing 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.

We developed a novel method utilizing a CRISPR/Cas9 system to replace 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. Human primary CD8⁺ cells from independent donors were immortalized and expanded more than 2.6 × 10⁷ times. Characterization of one of the immortalized CD8⁺ T-TERT cell lines revealed that the 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.

We established a stable immortalized cell lines using the novel immortalization method, and the immortalized CD8⁺ T cells had a phenotype consistent with T cells.