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

Published

April 25, 2025

Author(s)

Zhiyong He, Kenneth Cole, Hua-Jun He

Abstract

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 has a phenotype consistent with T cells and will be a useful research model.

Citation

International Journal of Molecular Sciences

Pub Type

Journals

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Keywords

Human CD8+ T cells, Immortalization, Telomerase reverse transcriptase (TERT), CRISPR/Cas9, CDKN2A, p16, Large DNA fragment insertion, Homologous recombination

Citation

He, Z. , Cole, K. and He, H. (2025), Immortalization of Human Primary CD8+ T cells by Inserting a Single Copy of Human Telomerase Reverse Transcriptase via CRISPR/Cas9, International Journal of Molecular Sciences, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=958070 (Accessed October 9, 2025)