PC-1 Genetic and pharmaceutical inactivation of hypoxia-inducible factor 1α reveals a molecular switch from graft vs host diseases to graft vs leukemia effect
Authors: Wang Yin , Liu Yan , Bailey Chris , Zheng Pan , Liu Yang 
Published: April 2019

Introduction:
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for hematopoietic malignancies and non-malignant conditions such as sickle cell anemia and severe combined immunodeficiency. However, graft-versus-host disease (GVHD) is a leading cause of morbidity and mortality associated with HSCT patients and limits the scope of HSCT applications. While the lymphocytes in donor bone marrow (BM) play a critical role in the prevention of tumor relapse, they are also responsible for the development of GVHD. Despite advances in preventing GVHD by the use of immunosuppressive drugs, GVHD remains a significant cause of morbidity and mortality following HSCT.
Hypoxia-inducible factor 1α (HIF-1α) plays a critical role in driving T cell differentiation, metabolism and cytotoxic activity. In addition, activation of T cells induces and stabilizes HIF-1α, and the stabilized HIF-1α increases the cytolytic activity of CD8+ T cells by stimulating expression and release of cytolytic molecules and costimulatory molecules. However, the function of HIF-1α in GVHD and graft-versus-leukemia (GVL) effect has not been investigated to our knowledge. Since targeting HIF-1α abrogated leukemia stem cells, it is of great interest to test the function of HIF-1α in the context of GVL and GVHD.

Results:
Our in silico analyses and experimental validations revealed that hypoxia-inducible factor 1α (HIF-1α) target genes were enriched in T cells of GVHD vs non-GVHD patients. Remarkably, targeted mutation of the Hif1a gene in donor T cells abrogated GVHD without affecting GVL. To explore translational potential of this finding, we developed a novel GVHD model comprising transplantation of human bone marrow into newborn NSG mice, and showed that Echinomycin, a known HIF-1α inhibitor with anti-leukemia properties, conferred significant protection against GVHD and leukemia. As an immunological basis for the molecular switch, we found that genetic and pharmacologic ablation of HIF-1α selectively expanded CD8 T cells while suppressing that of CD4 T cells, with concurrent induction of programmed death-ligand 1 (PD-L1) on T cells and host tissues. Taking advantage of the xenograft model, we showed that PD-L1 on T cells and non-T cells plays overlapping but distinct roles. While T cell-associated PD-L1 contributed to selective expansion of CD8 T cells in the lymphoid organs, PD-L1 expressed on non-T cell host cells limited GVHD in addition to supporting selective CD8 T cell expansion. Our data revealed HIF-1α as a molecular switch from GVL to GVHD and provided a therapeutic approach to preserve or even enhance GVL while abrogating GVHD.

Discussion:
Recent studies on mouse T cells have shown that HIF-1α plays a critical role in driving T cell differentiation, metabolism and cytotoxic activity. T cell activation both induces and stabilizes HIF-1α, leading to increased cytolytic activity of CD8+ T cells. Here our study provides a missing link between HIF-1α and GVHD pathogenesis and that genetic or therapeutic targeting of HIF-1α inhibits GVHD but preserves GVL. By showing convergence of genetic and pharmacology data, we established the central role for HIF-1α-PD-L1 antagonism as a critical switch between GVL and GVHD.
PD-1 negatively regulates T cell immune function through the interaction with its ligand PD-L1. PD-L1 is expressed on both hematopoietic and non-hematopoietic cells, and the expression can be induced by immune stimulation. PD-1/PD-L1 blockade with monoclonal antibodies exacerbates acute GVHD, and PD-L1 expression on parenchymal cells is critical for suppression of acute GVHD. CD8-intrinsic PD-L1 can promote GVL through its interaction with CD80, while host tissue-intrinsic PD-L1 can mediate CD8 T cell apoptosis and exhaustion in target tissues to reduce GVHD. We have shown that Hif1a mutation increased PD-L1 levels in CD8 T cells, making it possible for a robust CD80-PD-L1 interaction on CD8 T cells. We also observed increased IFN-γ levels in the serum due to elevated frequency of Tc1 cells, which was shown to be the underlying cause of PD-L1 expression in host tissues. Using anti-PD-L1 antibodies against either mouse or human PD-L1, we probed the function of PD-L1 on T cells and non-T cells. Our data showed that PD-L1 on non-T cells play a critical role in suppression of GVHD by Echinomycin, while PD-L1 on T cells is not critical for GVHD. Surprisingly, blocking either mouse or human PD-L1 is sufficient to cause altered CD4/CD8 ratio. These data make 2 interesting points. First, since blocking either mouse or human PD-L1 increased CD4/CD8 ratio, an increased CD4/CD8 ratio does not always lead to GVHD when the HIF activity is blocked by Echinomycin. Second, given the similar effect on T cells in the hematopoietic tissues, the differential effects of anti-mouse vs anti-human PD-L1 may relate to the induction of mouse PD-L1 in target tissues. In this regard, we demonstrated that PD-L1 expression correlates with T cell apoptosis in the target organs, including liver and kidney.

Conclusions:
In this study, we showed that the HIF-1α-PD-L1 antagonism is responsible for a molecular switch between GVL and GVHD. We further demonstrated that pharmacologic inhibition of HIF-1α can reverse this switch to treat GVHD, while preserving the GVL effect. We have demonstrated the therapeutic efficacy of Echinomycin in relapsed acute myeloid leukemia by targeting leukemia stem cells without adverse effects on host hematopoietic stem cells. Here we show that Echinomycin markedly diminishes GVHD in our humanized mouse model without interfering with GVL. Therefore, Echinomycin may be uniquely suited for transplantation in leukemia patients.