MicroRNAs as Biomarkers for Acute Graft Versus Host Disease Prediction Dianzheng Zhang, PhD Philadelphia College of Osteopathic of Medicine Philadelphia, PA, 19131

Allo-HSCT and aGVHD Hematopoietic stem cell transplantation (HSCT) Autologous hematopoietic stem cell transplantation Allogeneic hematopoietic stem cell transplantation (allo-HSCT) Acute graft-versus-host disease (aGVHD) occurs up to ~ 75% of allo-HSCT Cannot predict Who When Biomarkers!

MicroRNAs in aGVHD CD4+ T lymphocytes (8 of each) allo-HSCT with aGVHD allo-HSCT without aGVHD Healthy control qPCR with specific primers miR-155 miR-150 miR-181a miR-92b

MicroRNAs in CD4+ and Plasma The microRNAs levels in CD4+ cells and in the plasmas were estimated The microRNAs in plasma reflect that of the CD4+ cells Plasma microRNAs can be used for prediction of aGVDH Less invasive and easier

MicroRNAs in aGVHD Onset 27 patients with HSCT Blood draws every 7 Ds 5 without aGVHD 22 developed aGVHD At the day of aGVHD onset miR181a and 150 miR155 and 92b During the week before aGVHD onset NOTE: 19 of the 22 (86%) aGVHD patients showed microRNAs changes

Cytokines in aGVHD Onset 27 patients with HSCT Blood draws every 7 Ds 5 without aGVHD 22 developed aGVHD At day of aGVHD onset IFNγ, IL-2, TNF IL-10 In the week before the aGVHD onset, NONE of them changes MicroRNAs, but not cytokines, can predict aGVHD onset

MicroRNAs in aGVHD Severity Among the 22 aGVHD Grade II (9) Grade III (7) Grade IV (6) miR-150 and 181a reduced proportional to the severity miR-155 and 92b increased proportional to the severity The changes of miRNAs are proportional to the severity of aGVHD

IFNγ is A Potential MiR-181a Target Bioinformatics analysis identified IFNγ as a potential miR-181a target Wild type and mutant 3’-URT of IFNγ-Luc reporter + LV-mR-181a miR-181a repressed Luc-activity IFNγ a potential target of miR-181a

MiR-181a Downregulates IFNγ Human CD4+ lymphocytes Transfected with LV-Control LV-miR-181a miR-181a expression IFNγ mRNA levels unchanged IFNγ proteins Lymphocytes Culture media (Western blotting) (ELISA)

MiR-181a Downregulates IFNγ Human CD4+ lymphocytes Different amounts of miR-181a vector MiR-181a increase proportionally IFNγ mRNA levels unaffected IFNγ protein levels Cell Culture media MiR-181a downregulates INF γ in a dose-dependent manner

MiR-181a and T-cell Subsets Human CD4+ lymphocytes Transfected with LV-Control LV-miR-181a miR-181a upregulates Th1 and Th17 subsets IL-4 Foxp3 miR-181a downregulates Th2 and Treg subsets IFNγ IL-17A INFγ counteracts miR-181a

MiR-181a on Cytokines Secretion and T Cell Behavior ELISA of culture media Downregulating IFNγ and IL-17 Upregulating IL-4 Repressing T cell proliferation Enhancing T cell apoptosis

miR-181b Targeting IFNγ in Mouse Mouse miR-181b, a homolog of human miR181a miR-181b inhibits IFNγ-Luc (A) miR-181b downregulates IFNγ protein Without affecting mRNA (B) miR-181b represses released IFNγ (C)

MiR-181b Levels and aGVHD Onset Mouse aGVHD model in BABL/c MiR-181b levels monitored MiR-181b starts to drop 3 days prior onset (27/50) Cytokines unchanged till the onset MiR181b, not cytokines, can a predict aGVHD 3 days prior onset

Effects of MiR-181b or Dexamethasone on aGVHD Overexpressing miR-181b in mouse aGVHD model by transfection of splenocytes with LV MiR-181b is successfully expressed Or treating the animal with dexamethasone when miR-181b levels start dropping Survival rates increased MiR-181b expression Treating animals preemptively with dexamethasone MiRNA expression or preemptive treatment is beneficial

MiR-181b and Dexamethasone on aGVHD-related tissue damages At the end of the experiment, mice were sacrificed, liver and small intestine were examined by pathologists Either overexpression of miR181b or treat the animals with dexamethasone preemptively Damages to both liver and small intestine were reduced MiRNA expression or preemptive treatment is beneficial

Conclusion MiR-181a plays important role in aGVHD development By controlling the levels of different cytokines Sudden drop of miR-181a levels can predict aGVHD onset Providing a 3-day window for preemptive treatment The degree of miR-181a reduction also predicts aGVHD severity Upregulating miR-181a could be a preventive strategy for aGVHD

Future Research How miR-181a itself is upregulated by allo-HSCT How the other microRNAs were regulated What caused miR-181a downregulation prior aGVHD onset How the other cytokines were regulated by miR-181a How to prevent the miR-181a levels from dropping How do the other microRNAs function in aGVHD

Xuzhou Medical University Transplantation Immunology Philadelphia College of Medicine Dr. Wei Snag Xuzhou Medical University Transplantation Immunology Dr. Cong Zhang Dr. Thomas Loughran Dr. Kailin Xu University of Virginia Cancer Center