Introduction: The induction of transplant tolerance or completely withdrawal of immunosuppressant is an ideal approach especially for the high risk patients with adverse event related immunosuppression, such as infection and cancer. To achieve the tolerance appropriate immune monitoring to prevent rejection is mandatory while weaning the immunosuppressant.  Here we have investigated new biopsy based monitoring system to predict rejection or diagnosis of tolerance.

Methods: The biopsy samples from liver transplant patients with clinical and pathological grade evidence of T cell mediated acute rejection(TCMR), chronic antibody mediated rejection(AMR) and operational tolerance were collected.  The panels of multi-immunohisto-chemistry were selected as follows; a panel of the balance between effector T cells and Treg by staining CD3, CD8, Tbet and FoxP3, a panel of clinical fibrosis by staining SMA, and a panel of M2 macrophage by CD68 and CD163 .  The relationship between the panels and clinicopathological evidence were investigated. Biopsy samples of 5mm were preserved in RNA later and RNA were extracted to create cDNA for RNA sequence. 

To confirm the preservation time for single cell RNA sequence from liver biopsy, each 10mm biopsy samples were preserved in UW solution for 0 hour 7 hours and 12hours after the procedure, and graft infiltrating CD45+ cells were sorted and quality of cDNA were analyzed.  

Results: Tbet+ CD8 Tcells were significantly infiltrated based on the severity of TCMR. The SMA expressed significantly more in the chronic AMR.  However standardization for the quality and intensity of the expressing molecules in the whole slide scan image and the analysis of  the validation by using more number of clinical samples were mandatory to proceed to clinical diagnosis.  

Enough volume of cDNA were successfully created from 5 mm biopsy samples. About a thousand of CD45+ single cell were sorted from 10mm samples and successfully analyzed in the single cell RNA sequence within 12 hours preservation time. 

Conclusion:  A new biopsy based diagnostic approach to predict rejection and tolerance is under investigation. We will proceed to validation cohort of this tools in the liver transplant tolerance clinical trial.