Background: we have previously shown that ex vivo Tregs from patients with DAIH demonstrate differentiation plasticity towards T_H17. To understand the mechanisms underlying this we evaluated: i) demethylation of the Treg specific demethylation region (TSDR); (ii) PD-L1 expression; & (iii) cytokine microenvironment of the DAIH liver.

Method: Tregs (CD127^–CD25^high) of total PBMC's from LT recipients with DAIH, without DAIH (LT CTRLS) & healthy children (HC) were FACS isolated. Sorted cells were harvested for genomic DNA & analyzed for demethylation of TSDR by qPCR (Epiontis). RNA was isolated from sorted Tregs for quantification of PD-L1 mRNA expression by qRT-PCR. PBMC's were stained for expression of PD-L1 on Foxp3⁺ cells. Paraffin embedded liver sections from DAIH patients were subjected to IF staining for IL-6, IL-1β, IL-17A, CD4 & Foxp3. Intrahepatic lymphocytes were isolated from DAIH liver biopsies for immunophenotyping for T_H1, T_H2 & T_H17 cells.

Results: IF staining and immunophenotyping reveals a pro-inflammatory microenvironment within the de novo liver, with a predominant T_H17 signature as evidenced by numerous IL-6, IL-17A positive cells within portal inflammatory infiltrates, occasional IL-1β positive cells; & the presence of T_H1 & T_H17 cells ; similar to findings in blood, Foxp3⁺/IL-17A double positive cells are present within the de novo liver.There is a significantly decreased expression of the negative regulatory pathway ligand, PD-L1 in DAIH Tregs compared to Tregs from LT CTRLS & HC (p=0.04). Demethylation of TSDR of Tregs however not different between DAIH patients & HC.

Conclusion: a pro-inflammatory, T_H[17 signature is present in the de novo liver. Decreased PD-L1 expression may play a role in mechanism of DAIH.

« Back to 2015 American Transplant Congress