DR3 Signaling Modulates the Function of Foxp3+ regulatory T Cells and the Severity of Acute Graft and Host Disease
Authors: Nishikii Hidekazu , Byung-Su Kim , Chen Yan , Baker Jeanette , Pierini Antonio , Alvarez Maite , Mavers Melissa , Maas-Bauer Kristina , Negrin Robert S. 
Published: October 2019

Abstract
Background :
CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft versus host disease (GVHD) in murine models of allogeneic hematopoietic cell transplantation (HCT). We recently demonstrated that a single treatment of the agonistic antibody to DR3 (Death receptor 3, aDR3) to donor mice resulted in the expansion/activation of donor derived Treg and prevented acute GVHD (Blood 126:546, 2015), although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we investigated the efficacy of αDR3 treatment to recipient mice in model of murine GVHD.
Methods
To analyze the DR3 expression in immune cells with or without TCR stimulation, we comprehensively analyzed the cells with multicolor cytometry using viSNE (visualization of stochastic neighbor embedding algorithm). In transplantation experiments, 5x10e6 T cell depleted bone marrow (from WT C57BL/6 mice, H2kb) and 1x10e6 T cells (C57BL/6-Luciferase mice, H2kb) were injected intravenously into lethally irradiated (8Gy in total) BALB/c recipient mice (H2kd). aDR3 was intraperitonealy injected at different time point after transplantation. The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking, and survival time. To investigate the role of donor or recipient derived Treg in this model, in vivo Treg depletion using B6-Foxp3DTR mice was also performed.
Results
viSNE analysis demonstrated that DR3 was preferentially expressed on resting-Treg (79%), although a subpopulation of CD4+Foxp3-T cells (59%), CD8+T cells (24%), and NK1.1+TCRb+NKT celsl (42%) also expressed DR3. However, DR3 expressions in CD4+Foxp3-T cells and CD8+T cells were elevated after TCR stimulation in vitro (p<0.01). These data suggested that activation of DR3 signaling would also affect the function of conventional T cell upon activation. In the mixed lymphocyte reaction using allogeneic T cells (from WT C57BL/6 mice) and irradiated splenocytes (from BALB/c mice), the activation of DR3 promoted allogeneic T cell proliferation (p<0.01). In transplantation experiments, aDR3 treatment (day 3 after transplant) to animals with ongoing GVHD failed to expand Treg and further promoted donor T cell activation/proliferation with worse outcomes (p<0.05 in BLI study, p<0.01 in survival).
However, the prophylactic treatment of animals with aDR3 (day 0 αDR3 and day 2 allogeneic T cells) resulted in the expansion of recipient derived Treg (H2kd+CD4+Foxp3+ cells, p<0.01) and reduced the severity of GVHD with markedly prolonged survival (p<0.001). These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. This survival benefit could be observed even if Treg were depleted from the donor allogeneic T cells (from diphtheria toxin treated B6-Foxp3DTR mice), suggesting that host derived Treg, rather than donor cells, play a critical role in abrogating GVHD in this model.
Conclusion
In conclusion, we demonstrated that activation through DR3 signaling not only expands and activates Treg, but also further activates conventional alloreactive T cells and has very different clinical impact depending upon the timing of administration. These data provide important information for future clinical translation using modification of DR3 signaling.

Disclosures
Negrin: Stanford University: Patents & Royalties.