A novel immortalization method for immortalizing human primary CD8 T cells by inserting a single copy of human telomerase reverse transcriptase via CRISPR/Cas9.

BACKGROUND

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.

METHODS

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 (hCD8T-TERT).

RESULTS

By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8T 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 × 10 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.

CONCLUSION

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.