Lentivirally transduced recipient-derived dendritic cells serve to ex vivo expand functional FcRγ-sufficient double-negative regulatory T Cells

αβTCR⁺CD4^-CD8^- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress antigen-specific immune responses mediated by CD8⁺ and CD4⁺ T cells in mice and humans. In this study, we developed a system to expand DN Treg cells for transplantation therapy that exclusively uses recipient-derived immune cells and confers a high degree of safety as the protocol does not involve the direct injection of lentiviral vectors. Recipient-derived dendritic cells (DCs) were transduced with lentiviral vectors that express major histocompatibility complex class I L^d antigen (LV-L^d), which is expressed by the donor graft but is allogeneic to the graft recipient. LV-L^d-transduced mature DCs (mDCs) were able to expand effectively both FcRγ^+/+ and FcRγ^-/- DN T cells. After expansion with LV-L^d-transduced mDCs, only the FcRγ^+/+ DN Treg cells maintained their ability to suppress CD8⁺ T cells in vitro. In addition, adoptive transfer of the FcRγ^+/+ ex vivo expanded DN Treg cells significantly prolonged the survival of L^d+ skin grafts. This study is the first description of successful ex vivo expansion of antigen-specific DN Treg cells using genetically modified syngeneic DCs for adoptive immunotherapy and demonstrates that although FcRγ^-/- DN T cells can be expanded, they do not gain regulatory ability.