Evaluation of a gene expression biomarker to identify operationally tolerant liver transplant recipients: the LITMUS trial
Andrzej Chruscinski, Vanessa Rojas-Luengas, Sajad Moshkelgosha, Assaf Issachar, Jane Luo, Handy Yowanto, Leslie Lilly, Robert Smith, Eberhard Renner, Jianhua Zhang, Maor Epstein, David Grant, Caitriona M McEvoy, Ana Konvalinka, Atul Humar, Oyedele Adeyi, Sandra Fischer, Felix H Volmer, Richard Taubert, Elmar Jaeckel, Stephen Juvet, Nazia Selzner, Gary A Levy, Andrzej Chruscinski, Vanessa Rojas-Luengas, Sajad Moshkelgosha, Assaf Issachar, Jane Luo, Handy Yowanto, Leslie Lilly, Robert Smith, Eberhard Renner, Jianhua Zhang, Maor Epstein, David Grant, Caitriona M McEvoy, Ana Konvalinka, Atul Humar, Oyedele Adeyi, Sandra Fischer, Felix H Volmer, Richard Taubert, Elmar Jaeckel, Stephen Juvet, Nazia Selzner, Gary A Levy
Abstract
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 end point was the development of operational tolerance. Secondary end points 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 the 14 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. Clinical Trial Registration: ClinicalTrials.gov (LITMUS: NCT02541916).
Keywords: biomarker; gene expression; immunosuppression withdrawal; liver transplantation; tolerance.
© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Immunology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Figures
References
- Jain A, Reyes J, Kashyap R, et al. . Long-term survival after liver transplantation in 4,000 consecutive patients at a single center. Ann Surg 2000, 232, 490–500.
- Lechler RI, Sykes M, Thomson AW, Turka LA.. Organ transplantation—how much of the promise has been realized? Nat Med 2005, 11, 605–13.
- Thomson AW, Vionnet J, Sanchez-Fueyo A.. Understanding, predicting and achieving liver transplant tolerance: from bench to bedside. Nat Rev Gastroenterol Hepatol 2020, 17, 719–39.
- Mazariegos GV, Reyes J, Marino IR, et al. . Weaning of immunosuppression in liver transplant recipients. Transplantation 1997, 63, 243–9.
- Devlin J, Doherty D, Thomson L, et al. . Defining the outcome of immunosuppression withdrawal after liver transplantation. Hepatology 1998, 27, 926–33.
- Eason JD, Cohen AJ, Nair S, Alcantera T, Loss GE.. Tolerance: is it worth the risk? Transplantation 2005, 79, 1157–9.
- Tryphonopoulos P, Tzakis AG, Weppler D, et al. . The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation. Am J Transplant 2005, 5, 608–13.
- Tisone G, Orlando G, Cardillo A, et al. . Complete weaning off immunosuppression in HCV liver transplant recipients is feasible and favourably impacts on the progression of disease recurrence. J Hepatol 2006, 44, 702–9.
- Pons JA, Ramírez P, Revilla-Nuin B, et al. . Immunosuppression withdrawal improves long-term metabolic parameters, cardiovascular risk factors and renal function in liver transplant patients. Clin Transplant 2009, 23, 329–36.
- Benítez C, Londoño MC, Miquel R, et al. . Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients. Hepatology 2013, 58, 1824–35.
- de la Garza RG, Sarobe P, Merino J, et al. . Trial of complete weaning from immunosuppression for liver transplant recipients: factors predictive of tolerance. Liver Transplant 2013, 19, 937–44.
- Takatsuki M, Uemoto S, Inomata Y, et al. . Weaning of immunosuppression in living donor liver transplant recipients. Transplantation 2001, 72, 449–54.
- Appenzeller-Herzog C, Hartleif S, Vionnet J.. Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: a scoping review. Am J Transplant 2021, 21, 3312–23.
- Feng S, Ekong UD, Lobritto SJ, et al. . Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA 2012, 307, 283–93.
- Martínez-Llordella M, Puig-Pey I, Orlando G, et al. . Multiparameter immune profiling of operational tolerance in liver transplantation. Am J Transplant 2007, 7, 309–19.
- Li L, Wozniak LJ, Rodder S, et al. . A common peripheral blood gene set for diagnosis of operational tolerance in pediatric and adult liver transplantation. Am J Transplant 2012, 12, 1218–28.
- Bohne F, Martínez-Llordella M, Lozano JJ, et al. . Intra-graft expression of genes involved in iron homeostasis predicts the development of operational tolerance in human liver transplantation. J Clin Invest 2012, 122, 368–82.
- Urbanellis P, Shyu W, Khattar R, et al. . The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts. Immunology 2015, 144, 91–106.
- Xie L, Ichimaru N, Morita M, et al. . Identification of a novel biomarker gene set with sensitivity and specificity for distinguishing between allograft rejection and tolerance. Liver Transpl 2012, 18, 444–54.
- Wood KJ. Regulatory T cells in transplantation. Transplant Proc 2011, 43, 2135–6.
- Demetris AJ, Bellamy C, Hübscher SG, et al. . 2016 Comprehensive update of the Banff working group on liver allograft pathology: introduction of antibody-mediated rejection. Am J Transplant 2016, 16, 2816–35.
- Banff Working Group on Liver Allograft P. Importance of liver biopsy findings in immunosuppression management: biopsy monitoring and working criteria for patients with operational tolerance. Liver Transplant 2012, 18, 1154–70.
- Livak KJ, Schmittgen TD.. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001, 25, 402–8.
- Streitz M, Miloud T, Kapinsky M, et al. . Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study. Transplant Res 2013, 2, 17.
- Taubert R, Hardtke-Wolenski M, Noyan F, et al. . Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies. J Hepatol 2014, 61, 1106–14.
- Taubert R, Danger R, Londoño MC, et al. . Hepatic infiltrates in operational tolerant patients after liver transplantation show enrichment of regulatory T cells before proinflammatory genes are downregulated. Am J Transplant 2016, 16, 1285–93.
- Chruscinski A, Sadozai H, Rojas-Luengas V, et al. . Role of regulatory T cells (Treg) and the Treg effector molecule fibrinogen-like protein 2 in alloimmunity and autoimmunity. Rambam Maimonides Med J 2015, 6, e0024.
- Morris AB, Farley CR, Pinelli DF, et al. . Signaling through the inhibitory Fc receptor FcγRIIB induces CD8+ T cell apoptosis to limit T cell immunity. Immunity 2020, 52, 136–50.e6.
- Sungnak W, Wagner A, Kowalczyk MS, et al. . T follicular regulatory cell-derived fibrinogen-like protein 2 regulates production of autoantibodies and induction of systemic autoimmunity. J Immunol 2020, 205, 3247–62.
- Hidalgo LG, Halloran PF.. Role of IFN-gamma in allograft rejection. Crit Rev Immunol 2002, 22, 317–49.
- Lui SL, Ramassar V, Urmson J, Halloran PF.. Mycophenolate mofetil reduces production of interferon-dependent major histocompatibility complex induction during allograft rejection, probably by limiting clonal expansion. Transpl Immunol 1998, 6, 23–32.
- Cherukuri A, Salama AD, Mehta R, et al. . Transitional B cell cytokines predict renal allograft outcomes. Sci Transl Med 2021, 13, eabe4929.
- Pons JA, Revilla-Nuin B, Baroja-Mazo A, et al. . FoxP3 in peripheral blood is associated with operational tolerance in liver transplant patients during immunosuppression withdrawal. Transplantation 2008, 86, 1370–8.
- Furukawa A, Wisel SA, Tang Q.. Impact of immune-modulatory drugs on regulatory T cell. Transplantation 2016, 100, 2288–300.
- Li W, Carper K, Liang Y, et al. . Anti-CD25 mAb administration prevents spontaneous liver transplant tolerance. Transplant Proc 2006, 38, 3207–8.
- McEver RP. Selectins: initiators of leucocyte adhesion and signalling at the vascular wall. Cardiovasc Res 2015, 107, 331–9.
- Abeynaike LD, Deane JA, Westhorpe CL, et al. . Regulatory T cells dynamically regulate selectin ligand function during multiple challenge contact hypersensitivity. J Immunol 2014, 193, 4934–44.
- Zhang N, Schröppel B, Lal G, et al. . Regulatory T cells sequentially migrate from inflamed tissues to draining lymph nodes to suppress the alloimmune response. Immunity 2009, 30, 458–69.
- Charrin S, Jouannet S, Boucheix C, Rubinstein E.. Tetraspanins at a glance. J Cell Sci 2014, 127, 3641–8.
- Levitsky J. Operational tolerance: past lessons and future prospects. Liver Transpl 2011, 17, 222–32.
- Dai H, Zheng Y, Thomson AW, Rogers NM.. Transplant tolerance induction: insights from the liver. Front Immunol 2020, 11, 1044.
- Goronzy JJ, Weyand CM.. Mechanisms underlying T cell ageing. Nat Rev Immunol 2019, 19, 573–83.
Source: PubMed