A novel method for efficient murine regulatory T-cell expansion and retroviral transduction

Regulatory T-cell (Treg) infusions are an emerging therapy for the prevention or treatment of autoimmune, alloimmune and inflammatory disorders. Tregs that are modified to express a chimeric antigen receptor (CAR) are an additional promising method to expand the potential of Treg therapies. However, the development of CAR Treg is hampered by limitations in pre-clinical murine models, including poor murine Treg expansion and the need to use transgenic mouse strains to maintain pure Treg cultures. To address these issues and facilitate improved pre-clinical testing of CAR Treg products, we developed a novel protocol using wild-type (WT) mice.

Splenic CD4⁺CD8^-CD25^highCD62L⁺ murine T cells were flow cytometry sort-purified from WT mice to achieve >99% Foxp3 positivity. Tregs were activated by anti-CD3/CD28 beads and supported by exogenous human interleukin-2 for a total of 7 days. Retroviral transduction was performed on day 2 or day 3 of the culture. Tregs were analyzed by flow cytometry and used in in vitro and in vivo assays at the end of the 7-day culture.

Robust WT Treg expansion (>30 fold) was seen without a loss of Foxp3 expression and while maintaining high CD25 and CD62L levels. Expanded Tregs were highly suppressive in vitro and protective in vivo in an acute graft-versus-host disease model. Retroviral transduction on day 3 resulted in high transduction efficiency (>90%) without compromising overall expansion potential. When transduced with a CD19-CAR, CAR Tregs eliminated CD19⁺ malignant cells and maintained suppressive activity.

We developed a novel WT murine Treg culture protocol that produces highly functional CAR Treg with robust expansion, Foxp3 purity and retroviral transduction efficiency. This protocol may facilitate broader application of murine CAR Treg pre-clinical studies and may result in an expansion of future CAR Treg products for patients.