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Dec 13, 2011

Iron Homeostasis Signatures Predict Immunosuppression Withdrawal for Liver Recipients

  • Scientists have identified gene expression signatures they suggest can help predict which liver transplant recipient patients will eventually be able to have their immunosuppressive therapy withdrawn without suffering immune damage to their grafts.  

    A team led by scientists at the Hospital Clinic Barcelona’s Liver Unit compared the gene expression profiles of liver specimens taken from immunosuppressed transplant recipients who were subsequently found to tolerate withdrawal of immunosuppression, and from those who required maintenance immunosuppressive therapy. There was relatively little difference in expression patterns before withdrawal of treatment, except for the intra-graft expression of genes involved in the regulation of iron homeostasis.

    Further studies showed that patients who went on to cope with the withdrawal of immunosuppression (operationally tolerant transplant recipients) exhibited higher serum levels of hepcidin and ferritin and increased hepatocyte iron deposition than their nontolerant counterparts. Alberto Sánchez-Fueyo, M.D., and colleagues say that the differential gene expression profiles could accurately predict which immunosuppressed patients would tolerate withdrawal of therapy. Writing in the Journal of Clinical Investigation, they claim, “These results point to a critical role for iron metabolism in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers to conduct drug-weaning trials in liver transplantation.” Their paper is titled “Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation.”

    Organ transplant recipients generally require lifelong immunosuppressive drugs, but studies in rodents, and observations in a few human patients, have demonstrated that some transplant recipients can develop spontaneous operational tolerance. In these cases stable graft function can be maintained with no evidence of adverse immunological effects, even in the absence of immunosuppression. What isn’t yet known, the authors point out, is how to identify these patients.

    To address this issue the team collected sequential blood and liver tissue samples from patients undergoing a prospective multicenter withdrawal clinical trial, so they could define differential blood and intra-graft transcription signatures in patients who were able to successfully discontinue immunosuppressive therapy, and those who demonstrated organ rejection when immunosuppressive therapy was withdrawn.

    Liver tissue specimens were collected before initiation of drug minimization in all patients, as well as at the time of rejection in nontolerant recipients, and 12 months after complete drug withdrawal in operationally tolerant patients. Peripheral blood mononuclear cells (PBMCs) and serum samples were sequentially collected from all patients. In all, 33 patients successfully discontinued all immunosuppressive drugs (operationally tolerant) while 42 rejected their allografts (nontolerant).

    The researchers carried out whole-genome transcriptional profiling on preminimization liver samples collected from 20 operationally tolerant and 28 nontolerant recipients, and on rejecting samples obtained from 18 nontolerant recipients. Interestingly, global gene expression patterns of samples collected before the initiation of drug minimization were broadly similar among immunosuppressed patients after immunosuppressive therapy minimization, whether they went on to demonstrate operational tolerance or not.

    However, there was a small difference in expression patterns. Closer comparison showed that the transcriptional profiles differ in their expression of HAMP (which encodes hepcidin) and TFRC, which are genes known to be critical for regulating iron homeostasis, and also in the expression of two ferritin-like pseudogenes FTHL12 and FTHLB. There were also less significant, but still evident, differences in the expression of other genes involved in iron metabolism. Overall, iron ion homeostasis was the biological pathway most significantly over-represented in the operational tolerance–associated expression profile, prior to reduction in immunosuppressive therapy.

    In contrast, at the time of rejection the nontolerant recipients exhibited marked overexpression of a range of immune-related genes, none of which were detectable in the same group of patients before the initiation of drug minimization. These pre- and post-withdrawal expression data were confirmed through a cross-platform validation experiment carried out on a selection of samples from operationally tolerant and nontolerant recipients.

    The next stage was to see whether biopsy-based gene expression classifiers could help predict the success of immunosuppression withdrawal. The search for gene-predictive models was carried out on data from the original set of samples together with an independent group of biopsies from operationally tolerant and nontolerant patients from other clinical centers.

    The analyses identified five gene expression signatures that the researchers claim were capable of predicting the outcome of weaning a patient off immunosuppression, with less than a 17.5% overall error rate and very high sensitivity (SN), specificity (SP), negative predictive value (NPV), and positive predictive value (PPV), regardless of the center in which recipients had been enrolled. “The predictive accuracy of the gene classifiers was also independent of recipient age, sex, time from transplantation, and type of baseline immunosuppression, and was more powerful than any combination of clinical parameters,” they state.

    Notably, for patients who were subsequently confirmed to be operationally tolerant, there was no difference in the expression profiles of liver tissue specimens collected before the initiation of drug withdrawal, and a year after complete drug discontinuation.  This suggested that the presence or absence of immunosuppression didn’t greatly alter the intra-graft gene expresson profiles of operationally tolerant recipients. Indeed, the five ‘predictive’ gene expression profiles classified both prediscontinuation, and 12-months postdiscontinuation liver tissue samples with similar accuracy.

    Looking more closely at the genes that were differentially expressed in the prewithdrawal patients who were subsequently found to be either operationally tolerant, or nontolerant, the researchers found a correlation between liver hepcidin expression and serum hepcidin levels. The nontolerant transplant recipients demonstrated lower serum hepcidin levels than operationally tolerant patients both before immunosuppressive treatment changes, and at the end of the study. In fact, nearly 30% of nontolerant individuals demonstrated very low or undetectable hepcidin levels. Low serum hepcidin levels correlated with low serum ferritin, an indicator of body iron stores, and upregulation of TFRC expression, which is commonly observed in patients with decreased hepatic iron stores.

    In contrast to the differences in serum hepcidin and ferritin levels, operationally tolerant and nontolerant recipients didn’t differ in terms of blood hemoglobin, soluble transferrin receptor, serum iron, or C-reactive protein levels, transferrin saturation rate, incidence of ferropenic anemia, or HFE gene polymorphisms.

    Interestingly, mild periportal hepatocyte iron deposition was observed in most grafts from operationally tolerant recipients, but only in a few liver biopsy samples from nontolerant recipients. These differences were exclusively due to hepatocyte, rather than mesenchymal (endothelial and Kupffer cell), iron accumulation.

    Prior work by the group had indicated that drug-free operationally tolerant liver recipients demonstrated different PBMC expression patterns to liver recipients who needed maintenance immunosuppression. A comparison of these patterns with the differentially expressed data set from the liver tissue samples showed no overlap, and the liver tissue-derived signature was actually found to be a better predictor of operational tolerance than the PBMC-based model.

    The team believes their research is the first to indicate that changes in hepcidin expression and redistribution of intra-graft iron stores may play a role in the ability of a liver allograft to resist alloreactive immune responses. However, they admit, “whether our findings constitute a cause or a consequence of the operationally tolerant state cannot be unambiguously established in the current study and will need to be formally demonstrated in future trials analyzing sequentially collected liver biopsy samples and addressing the immunologic outcome of iron metabolism therapeutic manipulations.”

    They note that regulation of iron homeostasis is unlikely to be the only mechanism contributing to operational tolerance in liver transplant patients. Indeed, their reported studies did find differences in the expression of new genes with cytoprotective and immunoregulatory function, including CD26, ADORA3, DAB2, MIF, HMOX1, and SOCS1.

    In short, the ability of the liver allograft to resist immune attack is likely to be a central mechanism in maintaining operational tolerance, the researchers conclude. “This is supported by the demonstration that liver allografts from operationally tolerant and nontolerant recipients differ in the expression of multiple genes potentially involved in the attenuation of intra-graft inflammatory responses, and by the absence of significant differences between the two groups of recipients in blood and liver tissue markers of adaptive immunity,” they write. “Our findings raise the prospect for therapeutic manipulations that could result in the widespread establishment of operational tolerance in liver transplantation.”


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