Abstract
Spontaneous operational tolerance to the allograft develops in a proportion of liver transplant (LTx) recipients weaned off immunosuppressive drugs (IS). Several previous studies have investigated whether peripheral blood gene expression profiles could identify operational tolerance in LTx recipients. However, the reported gene expression profiles differed greatly amongst studies, which could be caused by inadequate matching of clinical parameters of study groups. Therefore, the purpose of this study was to validate differentially expressed immune system related genes described in previous studies that identified tolerant LTx recipients after IS weaning. Blood was collected of tolerant LTx recipients (TOL), a control group of LTx recipients with regular IS regimen (CTRL), a group of LTx recipients with minimal IS regimen (MIN) and healthy controls (HC), and groups were matched on age, sex, primary disease, time after LTx, and cytomegalovirus serostatus after LTx. Quantitative Polymerase Chain Reaction was used to determine expression of twenty selected genes and transcript variants in PBMCs. Several genes were differentially expressed between TOL and CTRL groups, but none of the selected genes were differentially expressed between HC and TOL. Principal component analysis revealed an IS drug dosage effect on the expression profile of these genes. These data suggest that use of IS profoundly affects gene expression in peripheral blood, and that these genes are not associated with operational tolerance. In addition, expression levels of SLAMF7 and NKG7 were affected by prior cytomegalovirus infection in LTx recipients. In conclusion, we found confounding effects of IS regimen and prior cytomegalovirus infection, on peripheral blood expression of several selected genes that were described as tolerance-associated genes by previous studies.
Highlights
For end-stage liver disease a liver transplantation (LTx) is the sole treatment option
This data might imply that expression of KLRF1, SMAD2, CXCL8, and CD160 could have been influenced by viral infections, since the majority of minimal IS regimen (MIN) LTx recipients were transplanted for virus-related primary liver disease
Principal component analysis of the nine significantly different expressed genes between tolerant LTx recipients (TOL) and control group of stable LTx recipients (CTRL) revealed three components that separated CTRL from clustered healthy controls (HC) and TOL, with MIN clustered in-between the groups (Figure 1C). This suggests that gene expression of SMAD3, FOXP3, IL2RB, KLRB1, SLAMF7-1, SLAMF7-2, ZBTB21-1, ZBTB21-2 and CX3CR-2 in CTRL and MIN LTx recipients is affected by the height of the immunosuppressive drugs (IS) through levels
Summary
For end-stage liver disease a liver transplantation (LTx) is the sole treatment option. In the last fifteen years considerable efforts have been made to identify noninvasive biomarkers of operational tolerance in LTx. Several studies have investigated whether tolerant LTx recipients could be discriminated from a control group or a non-tolerant group of LTx recipients with regular IS regimen by examining gene expression in circulating peripheral blood mononuclear cells (PBMCs) [8,9,10,11,12,13]. It was suggested that certain gene profiles related to the general immune system, natural killer (NK) cells, gdT-cells and regulatory T-cells (Tregs) could identify tolerant LTx recipients. These gene profiles differed greatly amongst these studies. Afore mentioned studies have used microarray and/or polymerase chain reaction (PCR) to study gene expression, but it is unclear which splice variants of the studied genes have been detected
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