Regulatory T cells (Tregs) play a crucial role in balancing the immune system and show potential for treating autoimmune diseases and preventing transplant rejection. To date, clinical trials have primarily focused on adoptive cell transfer of patient-derived Treg. However, this autologous approach faces challenges including variability in Treg numbers among patients, impaired Treg function due to underlying pathology, lengthy and sometimes unsuccessful manufacturing processes as well as the related signficant costs. Here, we evaluated the effectiveness of unmatched, allogeneic Tregs as an 'off-the-shelf' immunomodulatory cell therapy. In a humanized mouse model with human skin transplants, we observed that allogeneic 'third-party' Tregs were significantly less effective in preventing graft rejection than autologous counterparts. Gene editing of B2M and CIITA in human Tregs allowed efficient silencing of MHC class I and II, respectively. A HLA-E-B2M fusion gene partially protected B2M-edited Tregs from NK cell lysis in vitro. Silencing of MHC class II increased the protection of Tregs from allo-specific T cell attack in vitro. Combined HLA-E-B2M knock-in and CIITA knockout significantly enhanced the suppressive activity of allogeneic, unmatched Treg in our humanized skin transplant model. In respect to long-term graft survival, allogeneic MHC-engineered Treg had comparable suppressive activity to autologous Treg. Peripheral blood analysis of humanized mice indicated circulating MHC-silenced Treg at day 20 of the experiment. We are conducting further in vivo tests to confirm these initial findings. Our study suggests that modifying MHC class I and II can facilitate successful allogeneic 'off-the-shelf' Treg therapy.