Generation of non-human primate CAR Tregs using artificial antigen-presenting cells, simian tropic lentiviral vectors, and antigen-specific restimulation

• Published in: STAR protocols Vol. 3; no. 4; p. 101784
• Main Authors: Ellis, Gavin I., Deng, Mosha Z., Winn, Delaine W., Coker, Kimberly E., Shukla, Divanshu, Bhoj, Vijay, Milone, Michael C., Duran-Struuck, Raimon, Riley, James L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.12.2022; Elsevier
• Subjects: Animals; Antigen-Presenting Cells; Biotechnology and bioengineering; Cell culture; Cell isolation; Flow cytometry/Mass cytometry; Immunology; Model organisms; Primates; Protocol; Receptors, Chimeric Antigen - genetics; T-Lymphocytes, Regulatory; Tissue engineering; Transplantation Tolerance; Cell culture; Immunology; Biotechnology and bioengineering; Tissue engineering; Flow cytometry/Mass cytometry; Model organisms; Cell isolation
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101784

It is technically challenging to generate large doses of regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) in non-human primates (NHP). Here, we have optimized the manufacturing of CAR Tregs by stringent sorting of Tregs, stimulation by artificial antigen-presenting cells, transduction by simian tropic lentiviral vectors, and antigen-specific expansion. The result of this method is highly suppressive CAR Tregs for use in a pre-clinical, large animal model of transplant tolerance. For complete details on the use and execution of this protocol, please refer to Ellis et al. (2022). [Display omitted] •Non-human primates are ideal for pre-clinical development of CAR Treg cellular therapy•aAPC stimulation expands NHP CAR Tregs to clinical-sized doses•Simian tropic lentiviral vectors increase transduction relative to HIV-based vectors•Antigen-specific restimulation enriches CAR+ Treg population in vitro Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. It is technically challenging to generate large doses of regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) in non-human primates (NHP). Here, we have optimized the manufacturing of CAR Tregs by stringent sorting of Tregs, stimulation by artificial antigen-presenting cells, transduction by simian tropic lentiviral vectors, and antigen-specific expansion. The result of this method is highly suppressive CAR Tregs for use in a pre-clinical, large animal model of transplant tolerance.