Ying Wang

"Id3 prevents graft-versus-host disease-causing T cells from terminal differentiation while promoting their persistence in the gastrointestinal tract"

Ying Wang, Tien Bui, Shan He, Gennaro Calendo, Jaroslave Jelinek, Jozef Madzo, Jiang Huang, Ran Reshef, Jean-Pierre Issa, and Yi Zhang

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is potentially a curative treatment for hematologic malignancies and non-malignant diseases. However, its applicability is limited by graft-versus-host disease (GVHD), which is induced by donor T cells. Data from our studies and others suggested that the persistence of host-reactive donor T cells is crucial for sustaining host tissue injury during GVHD. The molecular pathway(s) that maintains GVHD-causing alloreactive T cells remains largely unknown. Here we demonstrate that the transcription factor Id3 reduces terminal differentiation of GVHD-causing effector T cells while promoting their persistence in the gastrointestinal (GI) tract. We observed that the transfer of donor T cells derived from Id3 conditional knockout (Id3^-/-) B6 mice did not induce lethal GVHD in irradiated allogeneic BALB/C mice. Notably, Id3^-/- T cell recipients developed similar symptoms of GVHD (e.g. weight loss, hunched back) as compared to wild-type (WT) T cell recipients within 14 days after allo-HSCT.  However, while disease continually progressed in WT T cell recipients, which was evidenced by hair loss, severe diarrhea, skin ulceration and death, it was gradually diminished in mice receiving allogeneic Id3^-/- T-cells without apparent clinical signs of severe GVHD. As a result, all these Id3^-/- T-cell recipients survived over 70 days after allo-HSCT.  We found that by days 7 and 14 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 inflammatory cytokine IFN-g. Using 10X genomics drop-seq platform, we performed single cell RNA (scRNA)-sequencing analysis. Donor T cells were purified from the liver and spleen of mice receiving WT T cells and Id3-KO T cells, respectively, at day 22 after allo-HCT. As expected, WT CD4⁺ T cells derived from the liver produced high levels of effector molecules, cytokines and chemokines than those from the spleen. Since alloreactive CD4⁺ T cells in the target tissues caused tissue injury during GVHD, we focused on characterizing alloreactive CD4⁺ T cells in the liver based on scRNA-seq analysis. Ingenuity pathway analysis revealed that the cluster 1A CD4⁺ T cells expressed genes associated with Th1 effector cytokines (e.g., Csf2, Clcl1) and TNFR2 signaling activity, which are important for GVHD T cell survival and function. The cluster 3A CD4⁺ T cells expressed genes associated with Th1 effector cells with characteristics of cytolytic molecule granzyme B (Gmzb) and terminal differentiation marker KLRG1. Cluster 6 CD4⁺ T cells showed the genes involved in Th17 cell function and inflammation. Intriguingly, there was a reduction in proportion of the cluster 3A subset derived from the liver of GVHD mice receiving Id3^-/- CD4⁺ T cells compared to that of WT CD4⁺ T cells. These Id3^-/- cluster 3A CD4⁺ T cells produced higher levels of genes associated with terminally differentiated effector T cells than their WT counterparts, including Klrg1, Ikzf2, Cd74 and Gmzb. In addition, the absence of Id3 induced higher levels of genes encoding effector molecules in the cluster 1A CD4⁺ T cells (e.g., Csf2, Ccl1) while decreasing Il7r. Terminally differentiated CD4⁺ effector T cells express the phenotype of CD62L^loCD127^loKLRG1^hiCD39^hi. Using flow cytometric analysis, we confirmed that Id3 inhibition caused significant increase in frequency of terminally differentiated effector T cells in mice undergoing allo-HSCT. Furthermore, we found that deletion of Id3 led to significantly impaired persistence of IFN-g-producing alloreactive effector T cells in the GI tract. Our findings identify that Id3 is important for preventing terminal differentiation of effector T cells and for sustaining GVHD-causing effector T cells in the GI tract. Id3 and its-associated molecular pathways may be potentially targeted for GVHD prevention and treatment.