Can regulatory T cells improve outcomes of sensitised patients after HLA-Ab incompatible renal transplantation: study protocol for the Phase IIa GAMECHANgER-1 trial.

BACKGROUND: Kidney transplantation is the gold-standard treatment for patients with kidney failure. However, one-third of patients awaiting a kidney transplant are highly sensitized to human leukocyte antigens (HLA), resulting in an increased waiting time for a suitable kidney, more acute and chronic rejection, and a shorter graft survival compared to non-highly sensitised patients. Current standard immunosuppression protocols do not adequately suppress memory responses, and so alternative strategies are needed. Autologous polyclonally expanded regulatory T cells (Tregs) have been demonstrated to be safe in transplant settings and could be a potential alternative to modulate memory immune alloresponses. METHODS: The aim of this trial is to determine whether adoptive transfer of autologous Tregs into HLA sensitised patients can suppress memory T and B cell responses against specific HLA antigens. This is a two-part, multi-centre, prospective clinical trial, comprising an observational phase (Part 1) aiming to identify patients with unregulated cellular memory responses to HLA (Pure HLA Proteins) followed by an interventional phase (Part 2). The first 9 patients identified as being eligible in Part 1 will undergo baseline immune monitoring for 2 months to inform statistical analysis of the primary endpoint. Part 2 is an adaptive, open labelled trial based on Simon's two-stage design, with 21 patients receiving Good Manufacturing Practice (GMP)-grade polyclonally expanded Tregs to a dose of 5-10 × 106 cells/kg body weight. The primary EP is suppression of in vitro memory responses for 2 months post-infusion. 12 patients will receive treatment in stage 1 of Part 2, and 9 patients will receive treatment in stage 2 of Part 2 if ≥ 50% patients pass the primary EP in stage 1. DISCUSSION: This is a prospective study aiming to identify patients with unregulated cellular memory responses to Pure HLA Proteins and determine baseline variation in these patterns of response. Part 2 will be an adaptive phase IIa clinical trial with 21 patients receiving a single infusion of GMP-grade polyclonally expanded Tregs in two stages. It remains to be demonstrated that modulating memory alloresponses clinically using Treg therapy is achievable. TRIAL REGISTRATION: EudraCT Number: 2021-001,664-23. REC Number: 21/SC/0253. Trial registration number ISRCTN14582152.

Immune Exhaustion and Transplantation.

Exhaustion of lymphocyte function through chronic exposure to a high load of foreign antigen is well established for chronic viral infection and antitumor immunity and has been found to be associated with a distinct molecular program and characteristic cell surface phenotype. Although exhaustion has most commonly been studied in the context of CD8 viral responses, recent studies indicate that chronic antigen exposure may affect B cells, NK cells and CD4 T cells in a parallel manner. Limited information is available regarding the extent of lymphocyte exhaustion development in the transplant setting and its impact on anti-graft alloreactivity. By analogy to the persistence of a foreign virus, the large mass of alloantigen presented by an allograft in chronic residence could provide an ideal setting for exhausting donor-reactive T cells. The extent of T cell exhaustion occurring with various allografts, the kinetics of its development, whether exhaustion is influenced positively or negatively by different immunosuppressants, and the impact of exhaustion on graft survival and tolerance development remains a fertile area for investigation. Harnessing or encouraging the natural processes of exhaustion may provide a novel means to promote graft survival and transplantation tolerance.

Natural Killer Cells and Liver Transplantation: Orchestrators of Rejection or Tolerance?

Natural killer (NK) cells are highly heterogeneous innate lymphocytes with a diverse repertoire of phenotypes and functions. Their role in organ transplantation has been poorly defined due to conflicting clinical and experimental data. There is evidence that NK cells can contribute to graft rejection and also to tolerance induction. In most solid organ transplantation settings, the role of NK cells is only considered from the perspective of the recipient immune system. In contrast to other organs, the liver contains major resident populations of immune cells, particularly enriched with innate lymphocytes such as NK cells, NKT cells, and gamma-delta T cells. Liver transplantation therefore results in a unique meeting of donor and recipient immune systems. The unusual immune repertoire and tolerogenic environment of the liver may explain why this potentially inflammatory "meeting" often results in attenuated immune responses and reduced requirement for immunosuppression. Recent trials of immunosuppression withdrawal in liver transplant patients have identified NK cell features as possible predictors of tolerance. Here we propose that hepatic NK cells play a key role in the induction of tolerance post-liver transplant and examine potential mechanisms by which these cells influence liver transplant outcome.

Hepatic Infiltrates in Operational Tolerant Patients After Liver Transplantation Show Enrichment of Regulatory T Cells Before Proinflammatory Genes Are Downregulated.

Immunosuppression can be discontinued from selected and stable patients after liver transplantation resulting in spontaneous operational tolerance (SOT), although the underlying mechanisms remain elusive. Thus, we analyzed serial liver biopsy specimens from adult liver recipients enrolled in a prospective multicenter immunosuppression withdrawal trial that used immunophenotyping and transcriptional profiling. Liver specimens were collected before the initiation of weaning, at the time of rejection, or at 1 and 3 years after complete drug discontinuation. Unexpectedly, the tolerated grafts developed portal tract expansion with increased T cell infiltration after immunosuppression withdrawal. This was associated with transient and preferential accumulation of CD4(+) FOXP3(+) cells and a trend toward upregulation of immune activation and regulatory genes, without signs of rejection. At the same time, no markers of endothelial damage or activation were noted. Portal infiltrates persisted at 3 years but were characterized by decreased expression of genes associated with chronic immunological damage. Further, SOT was not associated with a progressive liver fibrosis up to 5 years. These data suggest that SOT involves several mechanisms: a long-lasting local immune cell persistence with a transient regulatory T cells accumulation followed by a downregulation of immune-activated genes over years. These results have important implications for designs and follow-up of weaning trials.

Molecular Characterization of Acute Cellular Rejection Occurring During Intentional Immunosuppression Withdrawal in Liver Transplantation.

Acute cellular rejection occurs frequently during the first few weeks following liver transplantation. During this period, its molecular phenotype is confounded by peri- and postoperative proinflammatory events. To unambiguously define the molecular profile associated with rejection, we collected sequential biological specimens from 55 patients at least 3 years after liver transplantation who developed rejection during trials of intentional immunosuppression withdrawal. We analyzed liver tissue and blood samples obtained before initiation of drug withdrawal and at rejection, alongside blood samples collected during the weaning process. Gene expression profiling was conducted using whole-genome microarrays and real-time polymerase chain reaction. Rejection resulted in distinct blood and liver tissue transcriptional changes in patients who were either positive or negative for hepatitis C virus (HCV). Gene expression changes were mostly independent from pharmacological immunosuppression, and their magnitude correlated with severity of histological damage. Differential expression of a subset of genes overlapped across all conditions. These were used to define a blood predictive model that accurately identified rejection in HCV-negative, but not HCV-positive, patients. Changes were detectable 1-2 mo before rejection was diagnosed. Our results provide insight into the molecular processes underlying acute cellular rejection in liver transplantation and help clarify the potential utility and limitations of transcriptional biomarkers in this setting.

Immunoregulation: the interplay between metabolism and redox homeostasis.

Regulatory T cells are fundamental for the induction and maintenance of immune homeostasis, with their dysfunction resulting in uncontrolled immune responses and tissue destruction predisposing to autoimmunity, transplant rejection and several inflammatory and metabolic disorders. Recent discoveries have demonstrated that metabolic processes and mitochondrial function are critical for the appropriate functioning of these cells in health, with their metabolic adaptation, influenced by microenvironmental factors, seen in several pathological processes. Upon activation regulatory T cells rearrange their oxidation-reduction (redox) system, which in turn supports their metabolic reprogramming, adding a layer of complexity to our understanding of cellular metabolism. Here we review the literature surrounding redox homeostasis and metabolism of regulatory T cells to highlight new mechanistic insights of these interlinked pathways in immune regulation.

A need for biomarkers of operational tolerance in liver and kidney transplantation.

Both kidney and particularly liver recipients can occasionally discontinue all immunosuppressive drugs without undergoing rejection. These patients, who maintain stable graft function off immunosuppressive drugs without clinically significant detrimental immune responses and/or immune deficits, are conventionally termed operationally tolerant and offer a unique paradigm of tolerance in humans. The immune characterization of operationally tolerant transplant recipients has recently received substantial attention. Operationally tolerant patients might exhibit a signature of tolerance that could potentially be useful to select recipients amenable to drug minimization or withdrawal. Furthermore, elucidation of the molecular pathways associated with the operational tolerance phenotype could provide novel targets for therapy. Particular emphasis has been placed on the use of blood samples and high-throughput transcriptional profiling techniques. In liver transplantation, natural killer related transcripts seem to be the most robust markers of operational tolerance, whereas enrichment in B cell-related gene expression markers has been consistently found in blood samples from operationally tolerant kidney recipients, suggesting that different mechanisms operate in the two situations. In this minireview, we summarize the main achievements of recently published reports focused on the identification of transcriptional markers of operational tolerance, we highlight their mechanistic and clinical implications and describe their methodological limitations.

Comparison of transcriptional and blood cell-phenotypic markers between operationally tolerant liver and kidney recipients.

A proportion of transplant recipients can spontaneously accept their grafts in the absence of immunosuppression (operational tolerance). Previous studies identified blood transcriptional and cell-phenotypic markers characteristic of either liver or kidney tolerant recipients. However, the small number of patients analyzed and the use of different transcriptional platforms hampered data interpretation. In this study we directly compared samples from kidney and liver tolerant recipients in order to identify potential similarities in immune-related parameters. Liver and kidney tolerant recipients differed in blood expression and B-cell immunophenotypic patterns and no significant overlaps were detectable between them. Whereas some recipients coincided in specific NK-related transcripts, this observation was not reproducible in all cohorts analyzed. Our results reveal that certain immune features, but not others, are consistently present across all cohorts of operationally tolerant recipients. This provides a set of reproducible biomarkers that should be explored in future large-scale immunomonitoring trials.

ATG-Fresenius treatment and low-dose tacrolimus: results of a randomized controlled trial in liver transplantation.

We report the results of a prospective randomized controlled trial in liver transplantation assessing the efficacy and safety of antithymocyte globulin (ATG-Fresenius) plus tacrolimus monotherapy at gradually decreasing doses. Patients were randomized to either: (a) standard-dose tacrolimus plus steroids;or (b) peritransplant ATG-Fresenius plus reduced-dose tacrolimus monotherapy followed by weaning of tacrolimus starting 3 months after transplantation. The primary end-point was the achievement of very low-dose tacrolimus (every-other-day or once daily dose with <5 ng/mL trough levels) at 12 months after transplantation. Acute rejection occurring during the first 3 months after transplantation was more frequent in the ATG group (52.4% vs. 25%). Moreover, late acute rejection episodes occurred in all recipients in whom weaning was attempted and no recipients reached the primary end-point. This motivated the premature termination of the trial. Tacrolimus trough levels were lower in the ATG-Fresenius group but no benefits in terms of improved renal function, lower metabolic complications or increased prevalence of tolerance-related biomarkers were observed. In conclusion, the use of ATG-Fresenius and tacrolimus at gradually decreasing doses was associated with a high rate of rejection, did not allow for the administration of very low doses of tacrolimus and failed to provide detectable clinical benefits. ClinicalTrials.gov identifier: NCT00436722.

Comparative transcriptional and phenotypic peripheral blood analysis of kidney recipients under cyclosporin A or sirolimus monotherapy.

Due to its low level of nephrotoxicity and capacity to harness tolerogenic pathways, sirolimus (SRL) has been proposed as an alternative to calcineurin inhibitors in transplantation. The exact mechanisms underlying its unique immunosuppressive profile in humans, however, are still not well understood. In the current study, we aimed to depict the in vivo effects of SRL in comparison with cyclosporin A (CSA) by employing gene expression profiling and multiparameter flow cytometry on blood cells collected from stable kidney recipients under immunosuppressant monotherapy. SRL recipients displayed an increased frequency of CD4 + CD25highFoxp3 + T cells. However, this was accompanied by an increased number of effector memory T cells and by enrichment in NFkB-related pro-inflammatory expression pathways and monocyte and NK cell lineage-specific transcripts. Furthermore, measurement of a transcriptional signature characteristic of operationally tolerant kidney recipients failed to detect differences between SRL and CSA-treated recipients. In conclusion, we show here that the blood transcriptional profile induced by SRL monotherapy in vivo does not resemble that of operationally tolerant recipients and is dominated by innate immune cells and NFkB-related pro-inflammatory events. These data provide novel insights on the complex effects of SLR on the immune system in clinical transplantation.

Induction of tolerance in solid organ transplantation: the rationale to develop clinical protocols in liver transplantation.

Minimization or withdrawal of immunosuppressive treatments after organ transplantation represents a major objective for improving quality of life and long-term survival of grafted patients. Such a goal may be reached under some clinical conditions, particularly in liver transplantation, making these patients good candidates for tolerance trials. In this context in liver transplantation, the central questions are (1) how to promote the natural propensity of the liver graft to be accepted, (2) which type of immunosuppressive drug should be used for induction and maintenance, and (3) which biomarkers could be used to discriminate tolerant patients from those requiring long-term immunosuppression. Induction therapies using aggressive T-cell-depleting agents may favor graft acceptance. However, persistent and/or rapidly reemerging cell lines, such as memory-type cells or CD8(+) T cells, could represent a significant barrier for induction of tolerance. The type of maintenance drugs also remains questionable. Calcineurin inhibitors may be eventually deleterious in the context of tolerance protocols, through inhibitory effects on regulatory T cells, that are not observed with rapamycin. In conclusion, significant efforts must be made to achieve reliable strategies for immunosuppression minimization or withdrawal after organ transplantation into the clinics.

[Immunological tolerance and liver transplantation]. / Tolerancia inmunológica y trasplante de hígado.

The induction of tolerance to allografts has traditionally been one of the basic aims of transplantation research. Multiple data obtained in experimental models indicate that the outcome of transplantation (rejection versus acceptance/tolerance) depends on the balance between allo-reactive cytopathic lymphocytes and immunoregulatory lymphocytes. Thus, most tolerance-inducing treatments aim to reduce the number of allo-aggressive lymphocytes and, at the same time, to increase the population of regulatory lymphocytes, which ensure graft viability once drug therapy has been withdrawn. Liver allografts are singular in that they are accepted without the need for treatment in most experimental models. Likewise, in humans, liver grafts also show a lower susceptibility to rejection than any other organ and immunosuppressive treatment can be completely eliminated in approximately 25% of recipients. Many mechanisms have been proposed to explain the tolerogenic properties of the liver. Notable among these are the effects derived from the large number of passing leukocytes present in the liver and its peculiar anatomy that maximizes contact among blood lymphocytes and liver cells with tolerogenic potential. Although there are many cases of tolerance in human allograft recipients, therapeutic strategies that would allow predictable tolerance induction and without a high risk of adverse affects are still lacking. Therefore, most studies in humans have traditionally aimed to minimize doses of immunosuppressive drugs rather than eliminate them. However, recent results in preclinical models and pilot studies indicate that therapeutic protocols for tolerance induction may become available in the not too distant future.

Genetic evolution of GB virus C/hepatitis G virus (GBV-C/HGV) under interferon pressure.

The epidemiology and clinical features of chronic GBV-C/HGV infection have largely been explored, but there is little information about the mechanisms enabling GBV-C/HGV to cause persistent infection. Since analysis of the genomic variation of GBV-C/HGV under interferon pressure might provide some insight into this issue, we analyzed the nucleotide sequence variation of the 5'NC and NS3 regions in GBV-C/HGV isolates obtained sequentially from seven patients co-infected with HCV and treated with interferon. A reduction of GBV-C/HGV-RNA serum level below the detection limit of the RT-PCR assay was observed during treatment in all patients, but upon interferon withdrawal, viral RNA remained undetectable in only two patients. Among the five patients who did not clear GBV-C/HGV-RNA, viral strains emerging after treatment were identical to those present at baseline in three cases. In a further case, in whom GBV-C/HGV-RNA re-emerged during therapy (breakthrough episode), several mutations appeared in relapse samples. In the remaining patient, with a mixed infection before therapy, only one of the two GBV-C/HGV strains present at baseline was detected upon treatment withdrawal. These data raise the possibility that positive selection may act over GBV-C/HGV genome during interferon therapy, and contribute to persistence of infection with this virus.

Quantification of global and regional renal blood flow with electron beam computed tomography.

Alterations in renal blood flow distribution may occur in a variety of pathophysiologic situations; however, quantification of global and regional renal blood flows has been limited because of the lack of reliable, noninvasive techniques. To determine the feasibility of flow measurements with electron-beam computed tomography (EBCT), six anesthetized dogs were scanned by EBCT during basal conditions, after renal vasodilation, and at recovery. Flow (mL/min/cm3 tissue) was calculated from EBCT-derived time-density curves using three different algorithms and compared with simultaneously obtained electromagnetic flow (EMF) probe measurements after indexing to EBCT-derived renal volume. EBCT-determined flow correlated well with EMF measurements regardless of the algorithm used. An algorithm using the area under the time-density curve was concluded to be the most suitable for calculation of renal blood flow; it correlated with EMF as EBCT flow = 44.5 + 1.05 EMF (r = 0.885, SEE = 31.2 mL/min, P < .0001). Consistent overestimation of flow by EBCT resulted probably from retention of contrast media in the renal parenchyma. EMF showed an increase of 20 +/- 10% in renal blood flow after vasodilation. EBCT-derived global, cortical, and medullary flows increased by 33.8 +/- 10.3%, 24.8 +/- 17.8%, and 99.0 +/- 73.8%, respectively. In conclusion, EBCT was found feasible for credible quantitation of renal blood flow in the physiologic range studied.

Prevention of recurrence of hepatitis B virus infection after liver transplantation.

Hepatitis B virus (HBV) infection is still an important cause of cirrhosis, hepatocellular carcinoma and acute liver failure worldwide, and orthotopic liver transplantation (OLT) is the only effective treatment for many of these conditions. Until recently, however, OLT in patients with HBV infection was associated with a high rate of graft loss due to viral recurrence. The use of long term passive immunoprophylaxis with high dosage human polyclonal immunoglobulin against HBV (HBIg) has, for the first time, allowed favourable outcomes after OLT in a selected group of patients, but this treatment is associated with high cost and questionable efficacy in patients with high viral loads. In the last few years alternative approaches have arisen, namely the use of the reverse transcriptase inhibitor lamivudine and the induction of anti-HBV seroconversion by HBV vaccination as a means of discontinuing HBIg treatment. In this article we review the advantages and drawbacks of each of these prophylactic strategies, particularly emphasising the viability of administering HBV vaccination to these patients.

How long should initiation of calcineurin inhibitors be delayed to protect renal function in liver transplantation?

BACKGROUND AND AIM: Delayed introduction of calcineurin inhibitors (CNI) in liver transplantation (OLT) seeks to protect renal function, although the optimal length of the delay is not well established. The aim of this study was to analyze the effects on renal function of CNI initiation on different days after OLT. METHODS: We reviewed the charts of 260 OLT recipients. Group D1-a (n = 36) underwent the standard initial immunosuppression at our center: namely, CNI introduction on day 1 with further daily administration to achieve target levels of 8 to 15 ng/mL for tacrolimus or 150 to 300 ng/mL for cyclosporine. Due to renal concerns, 126 patients (group D1-b) had CNI introduced on day 1 either not daily or at doses to achieve less than the target on at least two occasions. In 43 patients (group D2), CNI were introduced on day 2 in 23 on day 3 (group D3), in 12 on day 4 (group D4), and at least at day 5 in 20 others (group D5). In periods without CNI treatment, patients received mycophenolate mofetil. Steroids were administered to all patients. The study period included the first 3 months post-OLT. Renal function was estimated as creatinine clearance (CrCl) using the Cockcroft-Gault equation. RESULTS: Changes in CrCl from pre-OLT to month 3 were -19% ± 28% in group D1-a; -27% ± 19% in group D1-b; -29% ± 19% in group D2; -23% ± 26% in group D3; -4% ± 38% in group D4, and +4% ± 33% in group D5 (P < .05 vs groups D1-a, D1-b, D2, and D3). On multivariate analysis, CNI introduction at day ≥ 5 was protective for kidneys when adjusted for other variables that potentially influence renal function. CONCLUSION: CNI should be introduced at day 5 after OLT to protect renal function.

Multiparameter immune profiling of operational tolerance in liver transplantation.

Immunosuppressive drugs can be completely withdrawn in up to 20% of liver transplant recipients, commonly referred to as 'operationally' tolerant. Immune characterization of these patients, however, has not been performed in detail, and we lack tests capable of identifying tolerant patients among recipients receiving maintenance immunosuppression. In the current study we have analyzed a variety of biological traits in peripheral blood of operationally tolerant liver recipients in an attempt to define a multiparameter 'fingerprint' of tolerance. Thus, we have performed peripheral blood gene expression profiling and extensive blood cell immunophenotyping on 16 operationally tolerant liver recipients, 16 recipients requiring on-going immunosuppressive therapy, and 10 healthy individuals. Microarray profiling identified a gene expression signature that could discriminate tolerant recipients from immunosuppression-dependent patients with high accuracy. This signature included genes encoding for gammadelta T-cell and NK receptors, and for proteins involved in cell proliferation arrest. In addition, tolerant recipients exhibited significantly greater numbers of circulating potentially regulatory T-cell subsets (CD4+ CD25+ T-cells and Vdelta1+ T cells) than either non-tolerant patients or healthy individuals. Our data provide novel mechanistic insight on liver allograft operational tolerance, and constitute a first step in the search for a non-invasive diagnostic signature capable of predicting tolerance before undergoing drug weaning.