Current Tolerance-Associated Peripheral Blood Gene Expression Profiles After Liver Transplantation Are Influenced by Immunosuppressive Drugs and Prior Cytomegalovirus Infection

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.

The gray area between operational tolerance and overt rejection in kidney transplantation

Operational tolerance in kidney transplantation is characterized by stable serum creatinine < 1.7 mg/dL and proteinuria < 1 g/day in the absence of immunosuppression or immunodeficiency for over one year. However, simultaneous donor specific antibodies are common and serum creatinine is a poor surrogate of early lesions. Consequently, subclinical rejections will meet operational tolerance criteria if serum creatinine remains stable. We report a patient with operational tolerance criteria followed by biopsy-proven chronic active antibody mediated rejection, discussing the intricate challenges of immunosuppression management.

Evaluation of a Gene Expression Biomarker to Identify Operationally Tolerant Liver Transplant Recipients: The LITMUS Trial

LITMUS was a single-centre, Phase 2a study designed to investigate whether the gene biomarker FGL2/IFNG previously reported for the identification of tolerance in murine models could identify operationally tolerant liver transplant recipients. Multiplex RT-PCR was used to amplify eight immunoregulatory genes in peripheral blood mononuclear cells (PBMC) from 69 adult liver transplant recipients. Patients with PBMC FGL2/IFNG ≥ 1 and a normal liver biopsy underwent immunosuppression (IS) withdrawal. The primary endpoint was the development of operational tolerance. Secondary endpoints included correlation of tolerance with allograft gene expression and immune cell markers. Twenty-eight of 69 patients (38%) were positive for the PBMC tolerance biomarker and 23 proceeded to IS withdrawal. Nine of the 23 patients had abnormal baseline liver biopsies and were excluded. Of the14 patients with normal biopsies, eight (57%) have achieved operational tolerance and are off IS (range 12-57 months). Additional studies revealed that all of the tolerant patients and only one non-tolerant patient had a liver gene ratio of FOXP3/IFNG ≥1 prior to IS withdrawal. Increased CD4 + T regulatory T cells were detected both in PBMC and livers of tolerant patients following IS withdrawal. Higher expression of SELE (gene for E-selectin) and lower expression of genes associated with inflammatory responses (GZMB, CIITA, UBD, LSP1 and CXCL9) were observed in the pre-withdrawal liver biopsies of tolerant patients by RNA sequencing. These results suggest that measurement of PBMC FGL2/IFNG may enrich for the identification of operationally tolerant liver transplant patients, especially when combined with intragraft measurement of FOXP3/IFNG.

Defining a Methylation Signature Associated With Operational Tolerance in Kidney Transplant Recipients

Operational tolerance after kidney transplantation is defined as stable graft acceptance without the need for immunosuppression therapy. However, it is not clear which cellular and molecular pathways are driving tolerance in these patients. We performed genome-wide analysis of DNA methylation in peripheral blood mononuclear cells from kidney transplant recipients with chronic rejection and operational tolerance from the Genetic Analysis of Molecular Biomarkers of Immunological Tolerance (GAMBIT) study. Our results showed that both clinical stages diverge in 2737 genes, indicating that each one has a specific methylation signature associated with transplant outcome. We also observed that tolerance is associated with demethylation in genes involved in immune function, including B and T cell activation and Th17 differentiation, while in chronic rejection it is associated with intracellular signaling and ubiquitination pathways. Using co-expression network analysis, we selected 12 genomic regions that are specifically hypomethylated or hypermethylated in tolerant patients. Analysis of these genes in transplanted patients with low dose of steroids showed that these have a similar methylation signature to that of tolerant recipients. Overall, these results demonstrate that methylation analysis can mirror the immune status associated with transplant outcome and provides a starting point for understanding the epigenetic mechanisms associated with tolerance.

Clinical and Basic Research Progress on Treg-Induced Immune Tolerance in Liver Transplantation

Rejection after organ transplantation is a cause of graft failure. Effectively reducing rejection and inducing tolerance is a challenge in the field of transplantation immunology. The liver, as an immunologically privileged organ, has high rates of spontaneous and operational tolerance after transplantation, allowing it to maintain its normal function for long periods. Although modern immunosuppression regimens have serious toxicity and side effects, it is very risky to discontinue immunosuppression regimens blindly. A more effective treatment to induce immune tolerance is the most sought-after goal in transplant medicine. Tregs have been shown to play a pivotal role in the regulation of immune balance, and infusion of Tregs can also effectively prevent rejection and cure autoimmune diseases without significant side effects. Given the immune characteristics of the liver, the correct use of Tregs can more effectively induce the occurrence of operational tolerance for liver transplants than for other organ transplants. This review mainly summarizes the latest research advances regarding the characteristics of the hepatic immune microenvironment, operational tolerance, Treg generation in vitro, and the application of Tregs in liver transplantation. It is hoped that this review will provide a deeper understanding of Tregs as the most effective treatment to induce and maintain operational tolerance after liver transplantation.

Cell-Mediated Therapies to Facilitate Operational Tolerance in Liver Transplantation

Cell therapies using immune cells or non-parenchymal cells of the liver have emerged as potential treatments to facilitate immunosuppression withdrawal and to induce operational tolerance in liver transplant (LT) recipients. Recent pre-clinical and clinical trials of cellular therapies including regulatory T cells, regulatory dendritic cells, and mesenchymal cells have shown promising results. Here we briefly summarize current concepts of cellular therapy for induction of operational tolerance in LT recipients.