Related Experiment Video
Updated: Dec 18, 2025
Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype
Published on: March 18, 2020
Generation, ex vivo expansion and safety of engineered PD1-knockout primary T cells from cynomolgus macaques
Chang-E Gao1, Qian Song2, Ming Zhang3
1Department of Medical Oncology, The First Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan, China; Department of Medical Oncology, Yunnan Cancer Hospital & The Third Affiliated Hospital of Kunming Medical University, Kunming 650032, Yunnan, China.
Abstract:
Programmed cell death protein 1 (PD1) is a cell-surface receptor that plays a vital regulatory role in suppressing inflammatory T cell activity; therefore, it is an ideal target for T cell-redirecting therapies. Here, we describe a cynomolgus macaque model for studying the transfer of PD1-modified T cells. We developed the first T cell engager targeting the disruption of PD1 by electroporation of plasmids encoding sgRNA and Cas9. There were no significant differences between mock T cells and PD1-knockout (PD1-KO) T cells in terms of cell viability, T cell signature marker expression, cell apoptosis, or cell cycling during prolonged in vitro culture. However, in a mixed lymphocyte reaction, PD1-KO T cells exhibited increased proliferation for both CD4+ and CD8+T cells and enhanced IFNγ release. We adoptively transferred autologous PD1-KO T cells into three cynomolgus monkeys. The PD1-KO T cells did not cause overt toxicity as measured by evaluating body weight, hematological parameters, and blood chemistry parameters. Histopathological analyses of tissues showed no lesions related to the infused PD1-KO T cells. Our findings demonstrate the utility of cynomolgus monkeys in expanding PD1-KO T cells and evaluating the safety of this immunotherapy and provide a new strategy for T cell-based adoptive cell therapies.
Insights
Researchers developed a novel method to knock out Programmed Cell Death protein 1 (PD1) in T cells using CRISPR-Cas9. This PD1-knockout T cell therapy showed enhanced function and safety in a cynomolgus macaque model.
Area of Science:
- Immunology
- Gene Therapy
- Primate Models
Background:
- Programmed Cell Death protein 1 (PD1) is a key regulator of T cell activity and a target for immunotherapy.
- Developing safe and effective T cell-modifying therapies requires robust preclinical models.
Purpose of the Study:
- To establish a cynomolgus macaque model for studying PD1-modified T cells.
- To develop and evaluate a novel CRISPR-Cas9-based strategy for PD1 disruption in T cells.
- To assess the safety and efficacy of adoptively transferred PD1-knockout (PD1-KO) T cells in non-human primates.
Main Methods:
- Utilized electroporation of plasmids encoding sgRNA and Cas9 to generate PD1-KO T cells.
- Assessed in vitro characteristics of PD1-KO T cells, including viability, marker expression, apoptosis, and cell cycling.
- Evaluated T cell function in mixed lymphocyte reactions (MLRs) by measuring proliferation and cytokine release (IFNγ).
- Conducted adoptive transfer of autologous PD1-KO T cells into cynomolgus monkeys.
- Monitored animal safety through body weight, hematological, and blood chemistry parameters.
- Performed histopathological analyses of tissues post-transfer.
Main Results:
- PD1-KO T cells showed no significant differences in viability, marker expression, apoptosis, or cell cycling compared to mock T cells in vitro.
- PD1-KO T cells demonstrated enhanced proliferation in both CD4+ and CD8+ T cell populations and increased IFNγ release in MLRs.
- Adoptive transfer of PD1-KO T cells in cynomolgus monkeys did not result in overt toxicity.
- No treatment-related lesions were observed in histopathological analyses.
Conclusions:
- CRISPR-Cas9-mediated PD1 disruption is a viable strategy for generating functional T cells.
- Cynomolgus macaques serve as a suitable model for evaluating the safety and expansion of PD1-KO T cells.
- This study provides a new strategy for T cell-based adoptive cell therapies and highlights the utility of non-human primate models in immunotherapy research.
