Human CD4+ CD25+ Regulatory T Cells Effectively Control Xenogeneic-GvHD Induced by Autologous T Cells in Rag2−/− γc−/− Immune-Deficient Mice.
Authors: Mutis Tuna , van Rijn Rozemarijn S. , Simonetti Elles R. , Aarts Tineke , Emmelot Maarten , Ebeling Saskia B. 
Published: October 2019

Abstract
The curative Graft-versus-Leukemia (GvL) effect of allogeneic stem cell transplantation (SCT) and Donor Lymphocyte Infusions (DLI) is frequently complicated by Graft-versus-Host Disease (GvHD). To date, it is not possible to prevent GvHD without sacrificing the GvL effect. Recently, in a number of murine transplantation studies, administration of naturally occurring CD4+CD25+ regulatory T (Treg) cells in recipients of allogeneic bone marrow effectively prevented GvHD without abrogating GvL. If human (hu)CD4+CD25+ Treg cells also possess such properties, they may become new cellular immunotherapeutics for the prevention of GvHD. Therefore, we have started to investigate the impact of huTreg cells on GvHD in a recently developed, highly relevant xenogeneic(x)-GvHD model in immunodeficient Rag2−/− γc−/− mice. This model represents several features of human allo-GvHD, such as the involvement of both CD4 and CD8 T cells, the association of GvHD with a “cytokine storm” of several Th1/Th2 and inflammatory cytokines and the similarity of skin histopathology to the human allo-GvHD(1). As in this model the x-GvHD is induced by the i.v. injection of huPBMC and the severity of x-GvHD correlates with the number of T cells in the administered PBMC, we explored the impact of Treg cells on x-GvHD either by depletion of Treg cells from huPBMC at different administration doses of effector T cells (4-15 x106 CD25− T cells) and or co-injection of autologous Treg cells at high doses of effector T cells (12-15 x106 T cells). PBMC were isolated from the buffycoats of healthy blood bank donors. Part of the PBMC was used as effector cells, the remaining cells were fractionated into CD25+ and CD25− subsets, which contain Treg cells and conventional T cells, respectively. Different groups of mice were injected with low to high doses of Treg-cell-depleted-PBMC or with high doses PBMC supplemented with 4-6 x106 Treg cell-enriched CD25+ cells. Control mice received equivalent numbers of unmodified PBMC only. The development of x-GvHD was monitored weekly by determination of body weight, clinical scores (ruffled fur, alopecia, mobility) and survival. Peripheral blood obtained from orbital vein was analyzed for human T cell engraftment and expansion. In three independent experiments, depletion of Treg cells significantly exacerbated the x-GvHD signs and lethality. In striking contrast, the development of x-GvHD was significantly inhibited by the co-injection of Treg cell enriched cell fractions. In two independent experiments Treg cells completely protected mice from lethal x-GvHD. Phenotypical analyses of peripheral blood revealed that addition of Treg cells did not disturb huT cell engraftment, but inhibited the expansion of huT cells between 3-5 weeks of administration. These results demonstrate the effective control of x-GvHD in Rag2−/− γc−/− mice by huTreg cells. Studies are underway to reveal the mechanism of GvHD inhibition and the impact of huTreg cells on GvL. (1) R.S. van Rijn, E.R. Simonetti, M.C.H. Hogenes, G. Storm, A. Hagenbeek, H. Spits, K. Weijer, A. C. M. Martens, and S.B. Ebeling. A new in vivo model for graft-versus-host disease by intravenous transfer of human peripheral blood mononuclear cells in RAG2−/− γc−/− double mutant mice.