Id3 Is Essential for Mediating Ezh2-Regulated Persistence of Alloreactive T Cells in Gvhd Target Tissues in Mice

Abstract: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative treatment for hematologic malignancies and non-malignant diseases. However, its application is limited by graft-versus-host disease (GVHD), which is induced by donor T cells that recognize and react to histocompatibilty differences. Despite pharmacological prophylaxis, acute and chronic GVHD still develop in 40-60% of allo-HSCT recipients. Data from our studies and others suggested critical roles of alloantigen-sensitized memory T cells in sustaining host tissue injury during GVHD. However, the molecular pathway(s) that are crucial for the development and maintenance of alloreactive memory T cells mediating persistent GVHD remain largely unknown. Id3 (Inhibitor of DNA Binding 3) is a transcription factor that negatively regulates the DNA-binding activity of E-protein. During response to acute viral infection, Id3 has been shown to regulate the generation of memory T cells, but is dispensable for primary effector response. Whether Id3 is responsible for persistent allo-immunity and tissue damage during GVHD has not been previously examined. Using experimental GVHD models, we demonstrate that Id3 functions as a key molecular gatekeeper for persistent generation of alloreactive T cells mediating tissue damage and GVHD. Infusion of donor T cells from wild-type (WT) B6 mice caused uniformly lethal GVHD in allogeneic BALB/C recipient mice. In contrast, transfer of donor T cells derived from Id3 conditional knockout (Id3^-/-) B6 mice did not induce lethal GVHD. Notably, Id3^-/- T cell recipients developed similar symptoms of GVHD (e.g. weight loss, hunched back and reduced mobility) as compared to WT T cell recipients within 20 days after allo-HSCT. However, while GVHD continually progressed in WT T cell recipients, which was evidenced by hair loss, severe diarrhea, skin ulceration, weight loss and death, it was gradually diminished in mice receiving allogeneic Id3^-/- T-cells without apparent skin inflammation and tissue damage. As a result, all Id3^-/- T-cell recipient mice survived over 70 days after allo-HSCT. We found that by 7 days and 14 days after transplantation, there were similar numbers of donor-derived CD4⁺ and CD8⁺ T cells in BALB/c recipients (e.g. spleen, liver, skin and gut) of Id3^-/- T cells compared with WT T cells. Furthermore, during this acute alloreactive response phase both Id3^-/- and WT T cells expressed high levels of activation markers (e.g. CD25, CD44, CD69, CD122) and had high IFN-g production. By 35 days after allo-HSCT, Id3^-/- T cell recipient mice showed significantly increased number of donor T cells in the spleen compared to WT T cell recipients, but had 10-fold fewer numbers of alloreactve T cells in GVHD target organs including gut, skin and liver. Analysis of samples from human patients undergoing allo-HSCT revealed that GVHD patients (n=8) had significantly higher frequency of T cells expressing high levels of ID3 compared to time-matched non-aGVHD patients (n=6). These findings suggest that Id3 plays critical roles in mediating the persistence and accumulation of tissue-infiltrating alloreactive T cells, while being dispensable for early activation, proliferation and effector differentiation of alloreactive T cells. Our recent studies have discovered a central role of the epigenetic regulator Ezh2 in controlling allogeneic T cell proliferation, differentiation and function (Blood 2012, Blood 2013 and Blood 2017). Using genome-wide profiling analysis and functional assessment, we found that Ezh2 directly activates Id3 transcription, independent of its canonical role in repressing gene transcription. Retroviral engineering of Ezh2-deficient T cells to express Id3 significantly enhanced the persistence of antigen-activated T cells in vivo, suggesting a critical role of Id3 in mediating Ezh2 regulation of activated T cell survival and maintenance. In summary, our findings identify the importance of Id3 in regulating persistent allo-immunity and tissue damage, and illuminate a novel molecular pathway of Ezh2-Id3 that may be potentially targeted for GVHD therapy.