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

2. 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!

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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)

10. 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

11. 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

12. 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

13. 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)

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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