Recipient Type-Sepcific Engineered Regulatory T Cells Prevent Graft-vs-Host Disease After Allogeneic Bone Marrow Transplantation in Mice

Abstract

Adoptive transfer of regulatory T cells (Tregs), in particular, recipient-type specific Tregs (sTregs), represents a promising approach to prevent graft-vs-host disease (GvHD) after allogeneic bone marrow transplantation. The objective of this study was to investigate whether engineered sTregs could prevent GvHD after bone marrow transplantation. Lentiviral vector forkhead box P3 (Foxp3)/pXZ9 containing Foxp3-IRES-GFP fragment and its mock control pXZ9 was constructed. Lentiviruses were produced via transient 3-plasmid transfection. BALB/c CD4+CD25−T cells were infected with lentiviruses and further stimulated using anti-CD3ε and anti-CD28 antibody (engineered irrelevant-Tregs [irTregs]) or C57BL/6 splenocytes (engineered sTregs). The expression of Tregs marks, production of cytokines, cell proliferation rate, and suppression function of Foxp3/pXZ9 infected cells were similar to natural Tregs. Irradiation BABL/c recipient were injected with C57BL/6 donor T cell–depleted bone marrow (TCD-BM) cells (1 × 10 7) and C57BL/6 splenocytes (1 × 10 7) together with engineered sTregs, irTregs, or natural Tregs (5 × 10 6). Irradiated BABL/c mice received TCD-BM cells only, TCD-BM cells plus splenocytes, or splenocytes and pXZ9-transduced cells (control). Recipient survival, short-term GvHD scores, and the Th1 subpopulation were monitored. Recipients of a combination of TCD-BM cells and splenocytes developed lethal GVHD. When engineered sTregs were added, 80% of recipients survived at least 60 days after transplantation; this survival rate was much higher than in any other group. The GvHD scores between the 3 Tregs groups did not demonstrate significance. Compared with other sources of Tregs in vivo, engineered sTregs strongly suppressed Th1 cell expansion. Therefore, a an in vitro strategy was developed to generate engineered sTregs. These cells demonstrated similar phenotypes and stable suppressive capacity as natural Tregs. Like natural Tregs, co-injection of engineered Tregs protected recipients from lethal GvHD in a murine model of GvHD. The engineered sTregs were superior to irTregs in minimizing murine GvHD.