Prohibitin Is a Substrate of PRMT5 in T Cells and Is a Potential Therapeutic Target for Acute Graft-Versus-Host Disease

Introduction: Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation (HCT) recipients. In acute GVHD, donor T cells recognize host antigens as non-self and mount an immune response leading to end-organ damage, mainly in liver, skin, and gastrointestinal tract. However, obliteration of the alloreactive response is not the solution for treating and preventing acute GVHD as donor T cell alloreactivity is critical to prevent of relapse of the primary malignancy, a process known as graft-versus-leukemia (GVL). Current strategies to treating acute GVHD consist of broad immunosuppression which sacrifices GVL, increases vulnerability to infections, and exhibits suboptimal response rates, making acute GVHD morbidity and mortality the second leading cause of death in allogeneic HCT patients, superseded only by relapse of the primary malignancy. Previously, we showed that the enzyme protein arginine methyltransferase 5 (PRMT5) is upregulated in acute GVHD, and PRMT5 inhibition reduces T cell-mediated inflammation in the disorder without sacrificing GVL. Following this study, we identified prohibitin (PHB) as a binding partner of PRMT5 upon which PRMT5 exerts its enzymatic function - symmetric dimethylation of arginine residue(s). Here, we examine the enzymatic action of PRMT5 upon PHB as a means by which PRMT5 modulates T cell inflammatory response in acute GVHD.

Methods: For immunoprecipitation and Western blot experiments, T cells were lysed in RIPA buffer, followed by immunoprecipitation via magnetic beads approach. Western blotting was performed according to standard protocols. In murine experiments, lethally irradiated mice received T cell depleted bone marrow and allogeneic splenocytes intravenously. Pharmacological PRMT5 inhibitor (PRMT5i) was administered once weekly by oral gavage. Mice were monitored for disease progression. At days 25-28 post-transplant, blood, spleen, liver, and colonic intraepithelial lymphocytes (IEL) were harvested from recipients and analyzed by flow cytometry for expression of surface PHB within the donor T cell population. Further, splenocytes were stimulated with PMA/ionomycin (PMA/i) followed by brefeldin A, and donor T cells were evaluated for expression of PHB and inflammatory cytokines IFNγ and IL-17. In Th17 differentiation experiments, naïve murine CD4 T cells isolated from B6 splenocytes were cultured for 72 hr under Th0 conditions (CD3/CD28) or Th17 conditions (CD3/CD28, IL-1b, IL-6, IL-23, TGFb1, anti-IFNg, anti-IL-2, anti-IL-4) in the presence or absence of RocA. Cells were restimulated with PMA/i and brefeldin A followed by flow cytometry analysis of IL-17 secretion and evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR (qPCR). Murine PRMT5 floxed Cre+ (knock-out; KO) and Cre- littermate control T cells were and activated via CD3/CD28 for 72 hr and qPCR for Prmt5 and Phb was performed.

Results and Conclusion: We found that PHB is symmetrically dimethylated by PRMT5 in activated human T cells. We show increased surface expression of PHB in murine and human T cells upon activation in vitro, as well as in vivo from murine T cells in liver, spleen, colon, and peripheral blood in acute GVHD mice. Further, PHB-expressing splenic T cells secrete IFNγ and IL-17, inflammatory cytokines secreted by Th1 and Th17 cells, respectively, and PHB-inhibitor treatment of murine CD4 T cells impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Finally, PRMT5 KO murine T cells exhibit reduced Phb transcription upon activation compared to control T cells. Combined, these findings link PRMT5 and PHB, show relevancy of PHB in Th1- and Th17-mediated inflammation, and suggest a potential role for the PRMT5-PHB axis in acute GVHD.

Significance: As of 2019, nearly 10,000 patients undergo allogeneic HCT yearly, and this number is steadily rising. While this procedure is the only curative measure for hematological malignancies such as leukemia and lymphoma, development of GVHD severely hinders its success and is the leading cause of non-relapse mortality in recipients. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PRMT5/PHB inhibition to treat this deadly disease.

Ranganathan:Plexxikon Inc: Research Funding.