Expanded CD4+Foxp3+ Regulatory T Cells through DR3 Signaling Have a Distinct Immunophenotype and Abrogate the Lethal Acute-Graft and Host Disease after Allogeneic Transplantation

Background: CD4+Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain self-tolerance and enhance immune tolerance after transplantation. Several groups have demonstrated that donor-derived Treg prevent the development of lethal acute graft and host disease (GVHD) in murine allogeneic transplant models. However, the low frequency of Treg limits clinical translation. To overcome the paucity of Treg, several strategies have been developed for Treg expansion. However, the activation of other immune cells and the instability of Foxp3 expression in ex vivo culture are problematic for widescale clinical usage. Recently, we showed that a single dose of agonistic antibody to DR3 (Death receptor 3, also called tumor necrosis factor super family 25; TNFSF25) into donor mice resulted in the expansion of donor derived Treg and prevented acute GVHD (Blood. 2015). Although the treatment with DR3 antibodies can preferentially expand Treg in vivo, the precise role of DR3 signaling in Treg has not been fully elucidated. In this study, we investigated the immune phenotype, gene expression profiles, and function of Treg after activation with DR3 signaling.

Methods:To analyze the heterogeneous immunophenotype of Treg after DR3 signal activation, we comprehensively analyzed multicolor cytometry data using viSNE (visualization of stochastic neighbor embedding algorithm). For gene expression analysis using microarray (Affymetrix GeneChip 2.0 ST Array), CD4+Foxp3+ cells from Foxp3-GFP mice with or without DR3 activation were sorted by FACS. Normalized expression data was analyzed using TIGR Multi Experiment Viewer (MeV, version 4.9). To investigate the function of Treg after DR3 activation, CD4+CD25+Treg from wild type (WT) C57BL/6 mice (H2kb) with or without treatment of agonistic antibody to DR3 were isolated by FACS and then injected into lethally irradiated (8Gy in total) BALB/c mice (H2kd) together with 5x10⁶ T cell depleted bone marrow (from WT C57BL/6 mice) and 1x10⁶T cells (C57BL/6-luciferase mice). The transplanted mice were monitored by clinical GVHD score, weight, bioluminescence imaging (BLI) for donor T cell trafficking and survival.

Results: The results of viSNE showed the heterogenic elevated expression level of Nrp1, Helios (natural occurring Treg marker/transcription factor), CD103, KLRG1, CD44, ICOS, PD-1, Lag3, TIGIT (effector or inhibitory molecules), and Ki67 (proliferation marker) in Treg after DR3 activation. On the other hand, the expression of CD25, the receptor for IL-2 was down regulated. In the microarray data, a significant elevated level (>2 fold relative expression levels in DR3 activated Treg) of chemokine/cytokine (ccr3, cxcl10) and effector molecules (CD74, Gzmb) were observed. These data suggest that the effect of DR3 signaling in Treg results in not only the expansion of Treg but also their activation.  In transplantation experiments, the mice that received DR3 activated Treg (5X10⁵ /mouse) showed significantly lower donor T cell proliferation compared with the mice that received non-activated Tregs (n=5 in each group, P<0.01 on day 7 and 10 after transplant). Interestingly, even a smaller number (1x10⁵ /mouse) of DR3 treated Treg suppressed donor T cell proliferation in host mice (n=5 in each group, P<0.05 on day7 and day10), and the survival of the mice in the DR3 activated Treg group was also improved compared with control GVHD group (n=10 in each group, P<0.01 in Log-rank test). These data suggested that Treg isolated after DR3 activation were more functional for the prevention in GVHD.

Conclusion: In conclusion, our data demonstrate that the activation of DR3 signaling can induce Treg populations with enhanced function in vivo. These observations support for future clinical testing using human DR3 signal modulation.